Plant Physiology Preview. Published on March 27, 2017, as DOI:10.1104/pp.17.00222
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Short Title: Pipecolate biosynthesis by ALD1 and SARD4/ORNCD1
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Corresponding author:
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Jürgen Zeier
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Institute for Molecular Ecophysiology of Plants
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Department of Biology
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Heinrich Heine University
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Universitätsstraße 1, D-40225 Düsseldorf, Germany
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[email protected]
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+49-211-81-14733
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Copyright 2017 by the American Society of Plant Biologists
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Biochemical principles and functional aspects of pipecolic acid
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biosynthesis in plant immunity
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Michael Hartmann1, Denis Kim1, Friederike Bernsdorff1, Ziba Ajami-Rashidi1,2,
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Nicola Scholten1, Stefan Schreiber1, Tatyana Zeier1, Stefan Schuck1,2, Vanessa
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Reichel-Deland1, and Jürgen Zeier1,2, *
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1
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University, Universitätsstraße 1, D-40225 Düsseldorf, Germany
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Universitätsstraße 1, D-40225 Düsseldorf, Germany
Institute for Molecular Ecophysiology of Plants, Department of Biology, Heinrich Heine
Cluster of Excellence on Plant Sciences (CEPLAS), Heinrich Heine University,
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One sentence summary
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The immune regulator pipecolic acid is synthesized by ALD1-mediated conversion of
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L-Lys to 2,3-dehydropipecolic acid and consecutive reduction to which Arabidopsis
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SARD4/ORNCD1 largely contributes.
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Footnotes
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Author contributions: M.H., D.K., F.B., Z.A.-R., N.S., S.Sr., T.Z., S.Sk., V.R.-D., and
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J.Z. performed the experiments and analysed the data; M.H. assisted in project
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supervision, design of experiments, and manuscript writing; J.Z. conceived the
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project, supervised the research, and wrote the article.
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This work was supported by the Deutsche Forschungsgemeinschaft (DFG grant
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ZE467/6-1).
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* author for correspondence: [email protected]
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ABSTRACT
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The non-protein amino acid pipecolic acid (Pip) regulates plant systemic acquired
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resistance (SAR) and basal immunity to bacterial pathogen infection. In Arabidopsis
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thaliana, the Lys aminotransferase AGD2-LIKE DEFENSE RESPONSE PROTEIN1
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(ALD1) mediates the pathogen-induced accumulation of Pip in inoculated and distal
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leaf tissue. Here we show that ALD1 transfers the α-amino group of L-Lys to
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acceptor oxoacids. Combined mass spectrometric and infrared spectroscopic
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analyses of in vitro assays and plant extracts indicate that the final product of the
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ALD1-catalysed reaction is enaminic 2,3-dehydropipecolic acid (2,3-DP), whose
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formation involves consecutive transamination, cyclization and isomerization steps.
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Besides L-Lys, recombinant ALD1 transaminates L-Met, L-Leu, diaminopimelate and
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several other amino acids to generate oxoacids or derived products in vitro.
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However, detailed in planta analyses suggest that the biosynthesis of 2,3-DP from
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L-Lys is the major in vivo function of ALD1. Since ald1 mutant plants are able to
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convert exogenous 2,3-DP into Pip, their Pip-deficiency relies on the incapability to
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form
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cyclodeaminase/μ-crystallin (ORNCD1) alias SAR-DEFICIENT4 (SARD4) converts
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ALD1-generated 2,3-DP into Pip in vitro. SARD4 significantly contributes to the
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production of Pip in pathogen-inoculated leaves but is not the exclusive reducing
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enzyme involved in Pip biosynthesis. Functional SARD4 is required for proper basal
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immunity to the bacterial pathogen Pseudomonas syringae. Although SARD4
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knockout plants show greatly reduced accumulation of Pip in leaves distal to P.
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syringae inoculation, they display a considerable SAR response. This suggests a
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triggering function of locally accumulating Pip for systemic resistance induction.
the
2,3-DP
intermediate.
The
Arabidopsis
reductase
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ornithine
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INTRODUCTION
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Plants are able to recognize conserved molecular patterns of potentially pathogenic
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microorganisms and thereupon induce a basal immune program that is generally
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designated as pattern-triggered immunity (PTI) or basal resistance (Macho and
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Zipfel, 2014). Basal resistance is able to attenuate the invasive spread of virulent
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pathogens in plant tissue but is often not strong enough to fully prevent disease
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development (Spoel and Dong, 2012). However, plants can reinforce their defensive
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capacities to virulent pathogen invasion after a previous microbial attack. A
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precedent localized leaf infection can thus activate a state of enhanced immunity to
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biotrophic and hemibiotrophic pathogens systemically in the entire foliage, a
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resistance phenomenon termed systemic acquired resistance (SAR) (Fu and Dong,
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2013).
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Plants with activated SAR have undergone massive transcriptional and
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metabolic reprogramming at the systemic level and are primed for more rapid and
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effective defense activation during pathogen challenge (Jung et al., 2009; Návarová
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et al., 2012; Gruner et al., 2013; Bernsdorff et al., 2016). In Arabidopsis thaliana,
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several genes required for proper SAR activation have been previously identified
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(reviewed in Shah and Zeier, 2013). Two of the Arabidopsis genes implicitly required
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for SAR are AGD2-LIKE DEFENSE RESPONSE PROTEIN1 (ALD1) and FLAVIN-
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DEPENDENT-MONOOXYGENASE1 (FMO1). Both genes are systemically up-
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regulated in the plant upon localized pathogen inoculation (Song et al., 2004a;
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Mishina and Zeier, 2006; Návarová et al., 2012).
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Plant metabolites are important regulatory components of plant basal
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resistance and SAR (Zeier, 2013). While the oxylipin jasmonic acid (JA) primarily
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promotes plant defenses directed against necrotrophic pathogens and herbivorous
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insects (Memelink, 2009), the phenolic salicylic acid (SA) is a key regulator of plant
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basal immunity to biotrophic and hemibiotrophic pathogens (Vlot et al., 2009). Upon
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pathogen attack, chorismate-derived SA accumulates in free and glycosidic forms
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systemically in the foliage and induces expression of a set of SAR-related genes
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(Wildermuth et al., 2001; Attaran et al., 2009). Since more than two decades, SA is
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considered as an important mediator of SAR in plants (Vernooij et al., 1994; Nawrath
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and Métraux, 1999; Wildermuth et al., 2001).
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More recently, our own studies have revealed that the Lys-derived non-protein
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amino acid pipecolic acid (Pip) acts as a crucial regulator of plant SAR (Návarová et
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al., 2012; Vogel-Adghough et al., 2013; Zeier, 2013; Bernsdorff et al., 2016). Upon
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inoculation of Arabidopsis with the hemibiotrophic bacterial pathogen Pseudomonas
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syringae, Pip accumulates to high levels in the inoculated leaves and to considerable
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amounts also in the leaves distal from the site of attack (Návarová et al., 2012). Pip
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is thereby biosynthesized in dependence of a functional ALD1 gene (Návarová et al.,
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2012), which encodes an aminotransferase with in vitro substrate preference for Lys
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(Song et al., 2004b). Pip-deficient ald1 mutant plants are fully defective in SAR, in
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the induced systemic priming phenomenon associated with SAR, and also exhibit
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reduced basal resistance towards bacterial infection (Song et al., 2004a; Návarová
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et al., 2012). The immune defects of ald1 can be rescued by exogenous Pip,
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demonstrating that Pip accumulation in the plant is necessary for SAR, defense
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priming, and a complete basal resistance program (Návarová et al., 2012). In
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addition, elevations of the in planta levels of Pip to physiological relevant amounts by
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exogenous application is sufficient to induce SAR-like resistance, to amplify
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pathogen-induced defense responses such as SA biosynthesis and defense gene
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expression, and to establish a primed state in wild-type plants (Návarová et al.,
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2012). Pip requires a functional FMO1 gene to exert its defense-amplifying and
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resistance-enhancing activities, indicating that the flavin monooxygenase FMO1 acts
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downstream of Pip in SAR activation (Návarová et al., 2012; Bernsdorff et al., 2016).
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Moreover, SAR induction and the establishment of a primed defense state in plants
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proceed by SA-dependent and SA-independent signaling pathways that both require
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Pip accumulation and functional FMO1. In addition, Pip and SA act both
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synergistically and independently from each other in Arabidopsis basal resistance to
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P. syringae attack (Bernsdorff et al., 2016).
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It is known since the 1950s that L-Pip is widely distributed in angiosperms
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(Morrison, 1953; Zacharius et al., 1954; Broquist, 1991). Beside its P. syringae-
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inducible accumulation in Arabidopsis, Pip was found to be biosynthesized to high
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levels in response to bacterial, fungal, or viral infection in several mono- and
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dicotyledonous plant species, including rice, potato, tobacco, and soybean (Pálfi and
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Dézsi, 1968; Vogel-Adghough et al., 2013; Aliferis et al., 2014). Moreover,
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Arabidopsis plants with constitutively activated defenses and autophagy mutants that
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exhibit stress-related phenotypes exhibit constitutively elevated Pip levels (Návarová
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et al., 2012; Masclaux-Daubresse et al., 2014). Exogenously applied Pip also
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increases resistance of tobacco plants to P. syringae pv. tabaci infection and primes
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tobacco for early SA accumulation (Vogel-Adghough et al., 2013). Furthermore,
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transgenic rice plants overexpressing the Pip biosynthesis gene ALD1 exhibited
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increased resistance towards infection by the fungus Magnaporthe oryzae (Jung et
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al., 2016). Together, these findings suggest a conserved regulatory role for Pip in
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plant immunity. In addition, Pip might contribute to the control of root nodulation in
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legumes, since Lotus japonicus ALD1 is strongly expressed in nodules induced by
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the symbiotic rhizobium Mesorhizobium loti. A proper function of the ALD1 gene
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proved necessary for the normal nodule development and the containment of the
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number of bacterial infection threads (Chen et al., 2014).
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L-Pip is an endogenous metabolite in both plants and animals (Broquist,
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1991). Soon after Pip was first identified as a plant natural product (Zacharius et al.,
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1954), its biosynthetic origin from L-Lys was uncovered by the finding that rats
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metabolise 14C-radiolabelled L-Lys to Pip (Rothstein and Miller, 1954). Notably, when
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administering Lys labelled with the heavy nitrogen isotope
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ε-amino group, Rothstein and Miller found that the α-NH2 group was abstracted and
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the ε-NH2 group was retained in the conversion of Lys to Pip. These experiments
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supported the hypothesis that the metabolic pathway from Lys to Pip in mammals
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includes an aminotransferase reaction to the α-keto acid ε-amino-α-ketocaproic acid
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(KAC) which is in chemical equilibrium with the dehydrated, cyclized ketimine
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compound Δ1-piperideine-2-carboxylic acid (Δ1-P2C) alias 1,2-dehydropipecolic acid
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(1,2-DP). A subsequent reduction of the imine bond in the 1,2-DP molecule would
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then be required to generate L-Pip. Later, partially purified enzyme preparations from
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different mammalian tissue with imine reductase activities towards several cyclic
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ketimines and the abilities to convert 1,2-DP to Pip were obtained (Meister and
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Buckley, 1957; Nardini et al., 1988; Hallen et al., 2013). More recently, the
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mammalian protein µ-crystallin (CRYM) was characterized as a NAD(P)H-dependent
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ketimine reductase that is able to reduce 1,2-DP to the supposed product L-Pip
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(Hallen et al., 2011; Hallen et al., 2015).
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N at either the α- or the
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Feeding studies with radioisotope-labeled Lys also showed that, like animals,
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plants synthesize L-Pip from Lys (Schütte and Selig, 1967; Gupta and Spenser,
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1969; Fujioka and Sakurai, 1997). However, contradicting results were reported with
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respect to the biochemical mechanism of Lys to Pip conversion in plants. For
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example, Schütte and Selig (1967) administered α-15NH2- and ε-15NH2-labelled Lys
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variants to bean plants and observed a preferred incorporation of the α-amino group
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into Pip. This would favor a pathway that includes the formation of α-aminoadipate
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semialdehyde (AAS) and the cyclized imine Δ1-piperideine-6-carboxylic acid
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(Δ1-P6C) alias 1,6-dehydropipecolic acid (1,6-DP). AAS and 1,6-DP have been
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described as biosynthetic intermediates of another Lys-derived amino acid, α-amino
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adipic acid (Aad) (Galili et al., 2001; Zeier, 2013). By contrast, after feeding δ-3H-, 6-
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14
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Spenser (1969) found that the 3H:14C-ratio of the administered Lys precursor and the
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Pip metabolic product were similar, consistent with the assumption that in plants and
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animals, the ε-amino group of Lys is incorporated into Pip via the formation of KAC
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and 1,2-DP.
C-radiolabelled Lys to bean plants, Sedum acre plants or intact rats, Gupta and
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The results of above-described isotope-tracer studies strongly suggest that
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the first step in the Pip biosynthetic pathway represents a transamination of L-Lys.
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Since the above-mentioned, SAR-essential ALD1 protein was characterized as plant
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aminotransferase with substrate preference for L-Lys (Song et al., 2004b), and
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because our own findings demonstrated that functional ALD1 is necessary for Pip
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biosynthesis (Návarová et al., 2012), we previously suggested that ALD1 catalyzes
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this first transamination step in the biosynthesis of Pip from L-Lys (Návarová et al.,
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2012; Zeier, 2013). The presumed ALD1-derived dehydropipecolic acid product
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could then be reduced to Pip in a second step (Zeier, 2013), since ketimine
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reductase activities similar to those in animals have been described (Meister and
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Buckley, 1957). In addition, an Arabidopsis gene initially annotated as ornithine
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cyclodeaminase/μ-crystallin (ORNCD1) and later designated as SAR-DEFICIENT4
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(SARD4) shows high sequence similarity to the mammalian ketimine reductase
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CRYM (Zeier, 2013; Ding et al., 2016).
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In the present work, we show that ALD1 directly transfers the α-amino group
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of L-Lys to acceptor oxoacids such as pyruvate or α-ketoglutarate. Both in vitro
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assays with recombinant ALD1 protein and in planta studies with wild-type and ald1
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mutant plants suggest that the end product of the ALD1-catalysed transamination of
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L-Lys is the enamine Δ2-piperideine-2-carboxylic acid (Δ2-P2C) alias 2,3-
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dehydropipecolic acid (2,3-DP). A plausible mechanism of 2,3-DP formation includes
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ALD1-mediated transamination of L-Lys to KAC, dehydrative cyclization to 1,2-DP,
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and isomerization to 2,3-DP. Although ALD1 is able to accept several other amino
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acid substrates to generate corresponding transamination products in vitro, in planta
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analyses suggest that the dominant function for ALD1 in vivo is the conversion of
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L-Lys to 2,3-DP. Our findings also indicate that Arabidopsis SARD4 reduces
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dehydropipecolic acid intermediates to Pip in vitro and in planta. The accumulation of
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Pip in sard4 knockout plants is significantly attenuated but still considerable in
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P. syringae-inoculated leaves, and markedly reduced in the distal leaf tissue.
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Bacterial growth assays indicate that SARD4-generated Pip contributes to basal
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resistance to P. syringae infection and to the resistance level of SAR-induced plants.
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Nevertheless, a wild-type-like resistance increase is observed after SAR induction in
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sard4 knockout plants. These findings refine our previous model of SAR
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establishment, because they suggest that locally accumulating Pip substantially
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contributes to resistance induction in the systemic tissue.
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RESULTS
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The bacterial strain P. syringae pv. maculicola (Psm) robustly activates SAR in wild-
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type Arabidopsis plants (Mishina and Zeier, 2007). Two days after an inducing
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inoculation with Psm in lower (1°) leaves has occurred, SAR manifests itself by the
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accumulation of the immune signals Pip and SA both in inoculated (1°) and in upper,
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distal (2°) leaves and protects plants to subsequent pathogen challenge (Attaran et
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al., 2009; Návarová et al., 2012; Bernsdorff et al., 2016).
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ALD1-mediated L-Lys conversion: abstraction of the α-NH2 group leads to the
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formation of enaminic 2,3-dehydropipecolic acid in vitro and in planta
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To experimentally verify the proposed scheme of Pip biosynthesis and identify
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possible ALD1-derived pathway intermediates (Zeier, 2013), we first performed
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comparative GC-MS-based metabolite profiling of leaf extracts from Psm-inoculated
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and mock-treated wild-type Col-0 and ald1 mutant leaves. The applied analytical
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procedure (“procedure A”) includes a final derivatization step that converts free
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carboxylic acids into their methyl esters to optimize GC separation (Schmelz et al.,
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2004; Mishina and Zeier, 2006). By comparatively analysing individual ion
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chromatograms (m/z between 50 and 300) of extract samples from leaves of Psm-
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inoculated Col-0, Psm-inoculated ald1, mock-treated Col-0, and mock-treated ald1
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plants, we identified a molecular species (1a) that was present in the samples from
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Psm-treated Col-0 leaves, but barely detectable in the other samples (Fig. 1A).
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Hence, the plant compound associated with this substance peak accumulated in
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Col-0 wild-type, but not in the ald1 mutant upon pathogen treatment, which parallels
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the accumulation characteristics of Pip (Návarová et al., 2012).
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The mass spectrum of compound 1a showed a molecular ion of m/z 141 and
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a main fragment at m/z 82, which is characteristic for molecules with a one-fold
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unsaturated piperideine ring (Fig. 1C). Overall, the mass spectral fragmentation
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pattern suggested that the chemical nature of 1a is the methyl ester of a piperideine
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carboxylic acid isoform (Fig. 1C), such as the ketimine 1,2-DP, the enamine 2,3-DP,
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or 1,6-DP (Fig. 2). 1,2-DP represents the previously proposed product of the ALD1-
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catalyzed L-Lys transamination (Zeier, 2013). The methyl group of 1a is thereby
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introduced after tissue extraction by derivatization, since 1a was not detected in non-
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derivatized samples.
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As a next step, recombinant ALD1 protein lacking the putative plastidial
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targeting sequence (Song et al., 2004b; Cecchini et al., 2015), and carrying a C-
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terminal His6-Tag was overexpressed in Escherichia coli and then purified via
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immobilized metal ion affinity chromatography (IMAC), followed by a desalting step
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to adjust the buffer conditions.
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recombinant ALD1 enzyme in the presence of various amino acceptors and
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pyridoxal-5′-phosphate (PLP) as a cofactor, using assay conditions similar to those
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employed previously by Song and colleagues (for details compare material and
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methods section; Song et al., 2004b). After stopping the reaction either by addition of
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0.1 M HCl, equal amounts of MeOH or heating of the reactions mixture (85°C for 10
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min), substrates and ALD1 reactions products were derivatized to produce their
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respective methyl esters and analysed by GC-MS as described before. In the
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presence of an oxoacid, such as pyruvate or α-ketoglutarate, ALD1 converted L-Lys
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to an in vitro reaction product whose GC retention time and mass spectrum were
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identical to the plant-derived substance 1a after derivatization (Fig. 1B, Fig. 1C, and
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Supplemental Fig. S1). Together, these data indicated that ALD1 catalyses the
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conversion of L-Lys to the same dehydropipecolic acid (DP) isoform in vitro and in
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planta.
L-Lys was tested in vitro as a substrate of
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Next, the highly substrate-specific L-Lysine α-oxidase (LysOx) from
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Trichoderma viridae (Kusakabe et al., 1980) was used to generate 1,2-DP and
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compare it directly to the reaction product formed by ALD1 and the metabolite peak
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missing in ald1 plant extracts (Fig. 1A, B, C). As other members of the L-amino acid
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oxidases (EC 1.4.3.2.), LysOx from T. viridae is a flavoprotein with non-covalently-
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bound flavin adenine dinucleotide (FAD) and catalyses the stereospecific oxidative
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deamination of an L-amino acid, thereby producing ammonia and hydrogen peroxide
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as by-products (Lukasheva and Berezov, 2002; Pollegioni et al., 2013, Campillo-
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Brocal et al., 2015). The formal product of the oxygen-consuming and H2O2-
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generating oxidation of the α-carbon atom of L-Lys is KAC which is described to
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further cyclize to 1,2-DP, if the reaction is conducted in the presence of catalase
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(Kusakabe et al., 1980; Supplemental Fig. S2). Biochemical assays with LysOx and
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L-Lys as a substrate yielded, in the presence of catalase, a reaction product that was
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identical with 1a after derivatization (Fig. 1B, Fig. 1C). Therefore, the ALD1 and the
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LysOx/catalase biochemical assay both yielded the same Lys-derived piperideine
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carboxylic acid isoform as the one detected in plant extracts.
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To further elucidate the biochemical mechanism and identify the end product
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of the ALD1-catalysed Lys conversion reaction, we used isotope-labelled Lys
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precursors in the in vitro bioassays. Use of L-Lys-6-13C,ε-15N as an ALD1 substrate
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promised to clarify whether the transamination involves abstraction of the α- or ε-N,
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and thus to discriminate between KAC and AAS as oxoacid intermediates and the
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thereof derived dehydropipecolic acid cyclization products (Fig. 2). With L-Lys-6-
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13
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mass spectral fragmentation pattern that was incremented by two mass units
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compared to assays with unlabelled L-Lys, indicating that the ε-nitrogen is retained in
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the piperideine carboxylic acid product (Fig. 1D). This suggests that the ALD1-
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catalysed aminotransferase reaction proceeds via abstraction of the α−amino group
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of Lys to form KAC, which in turn can cyclize to 1,2-DP, and that 1,6-DP is
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consequently not the reaction product (Fig. 2). Furthermore, ketimine compounds
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can rearrange to enamine isomers (Nardini et al., 1988), and the ketimine 1,2-DP
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might in this way form enaminic 2,3-DP as the ultimate ALD1-derived product (Fig.
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2). In any case, the so far observed mass spectral fragmentation patterns are
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consistent with the structure of 1,2-DP, 2,3-DP, or potentially, another piperideine-2-
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carboxylic acid isoform finally generated from KAC.
C,ε-15N as the substrate, the product of the ALD1-catalysed reaction showed a
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We then used tetradeuterated L-Lys-4,4,5,5-d4 as an ALD1 substrate in the in
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vitro assays. This yielded a product with a mass spectral fragmentation pattern that
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was incremented by four mass units compared to unlabelled L-Lys (Fig. 1E). This
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shift indicated that the carbons 4 and 5 of the piperedeine ring are still saturated in
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the reaction product, essentially excluding that a piperideine-2-carboxylic acid
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isoform other than 1,2-DP or 2,3-DP was formed (Fig. 1E; Fig. 2). Notably, when
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L-Lys-6-13C,ε-15N or L-Lys-4,4,5,5-d4 were co-applied with Psm to Col-0 wild-type
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plants, we observed in addition to substance 1a derived from endogenous Lys, the in
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planta formation of isotope-labelled versions of 1a that are identical to those
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detected in the ALD1 in vitro assay (Supplemental Fig. S3). This indicates that the
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mechanism of the ALD1-catalyzed reaction in vitro and in planta is the same.
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To discriminate between the ketimine 1,2-DP and the enamine 2,3-DP as
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possible end products, we analysed the ALD1 assay mixture with L-Lys as the
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substrate by GC-Fourier Transform Infrared (FTIR) spectroscopy to obtain the
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infrared (IR) spectrum of 1a (Fig. 1F). If the substance was an enamine, the IR
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spectrum would exhibit an N-H stretching band at wavenumbers of about 3400 cm-1,
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whereas a spectrum of a ketiminic substance would not. A distinct band at 3441 cm-1
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in the IR spectrum of 1a demonstrated the presence of the N-H vibration (Fig. 1F),
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indicating, together with the above mass spectral information, that the substance is
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the methyl ester of the enamine 2,3-DP and not of the ketimine 1,2-DP. Another
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distinctive feature of the IR spectrum of 1a is a strong band at 1734 cm-1, which is
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indicative of a carbonyl stretching vibration (Fig. 1F). The absorption of the C=O
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vibration in aliphatic carboxylic acid methyl esters such as methyl butyrate or methyl
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valerate occurs at approximately 1760 cm-1 (Supplemental Fig. S4). The C=O band
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of α,β-unsaturated carbonyl compounds is usually shifted to lower wavenumbers
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compared to the respective non-conjugated forms. For example, the carbonyl
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vibrations of methyl crotonate and methyl benzoate occur at 1749 cm-1 and 1746
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cm-1, respectively (Supplemental Fig. S4). The observed carbonyl absorption at 1734
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cm-1 of 1a is therefore consistent with the occurrence of an unsaturated ester group
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with a conjugated C=C double bond in α,β-position, as it is the case for 2,3-DP.
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Moreover, the medium absorption at 1648 cm-1 can be assigned to the stretching
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vibration of the C=C double bond in the 2,3-DP methylester (Fig. 1F). In sum, the IR
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spectral analysis of substance 1a is consistent with the enaminic structure of 2,3-DP
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but not with the structure of the ketimine 1,2-DP. Together, our data indicates that
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the enamine 2,3-DP is the final end product of the ALD1-catalysed conversion of L-
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Lys, both in vitro and in planta. We propose a plausible mechanism for 2,3-DP
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333
formation which includes consecutive transamination of L-Lys to KAC, dehydrative
334
cyclization to 1,2-DP, and isomerization to 2,3-DP (Fig. 2).
335
Previous studies show that LysOx also catalyses the formation of a
336
dehydropipecolic acid-dimer from L-Lys in the presence of catalase (Hope et al.,
337
1967; Supplemental Fig. S2A). Indeed, when L-Lys was used as a substrate, we
338
detected a substance with a mass spectrum identical to the one of the
339
spectroscopically
well-characterized
dimer
in
the
LysOx/catalase
assay
13
340
(Supplemental Fig. S5; Hope et al., 1967). When using L-Lys-6- C,ε-15N and L-Lys-
341
4,4,5,5-d4 as substrates, the mass spectral fragmentation pattern of the isotope-
342
labelled DP-dimer variants were shifted by four and eight mass units, respectively,
343
consistent with a previous assumption that the DP-dimer can be generated from
344
L-Lys via KAC-derived DP monomers (Supplemental Fig. S5; Hope et al., 1967). In
345
addition to 2,3-DP, formation of the DP-dimer was also observed in L-Lys conversion
346
assays with recombinant ALD1, but only if the reaction mixtures were incubated
347
overnight (> 12 h). Moreover, the DP-dimer was not detected in plant extracts.
348
In the absence of catalase, LysOx catalyzed the formation of valerolactam
349
from L-Lys (Supplemental Fig. S2A, Supplemental Fig. S6). This is consistent with
350
the previously reported LysOx-catalysed generation of 5-aminovaleric acid from
351
L-Lys (Kusakabe et al., 1980; Pukin et al., 2010), since 5-aminovaleric acid is able to
352
spontaneously cyclize to valerolactam (Bird et al., 2012). The fragmentation patterns
353
of the isotope-labelled valerolactam derivatives also suggest that, as 2,3-DP,
354
valerolactam is produced from LysOx by abstraction of the α-NH2 group of Lys
355
(Supplemental Fig. S6). 2,3-DP, the DP dimer, and valerolactam all exhibit a
356
secondary amino group in their ring structures. Moreover, the dimer and 2,3-DP both
357
contain enamine moieties. Consistent with these structural similarities, all three
358
substances exhibit a very similar N-H stretching vibration around 3430 to 3440 cm-1
359
(Supplemental Fig. S7).
360
In addition to the analytical procedure A that includes a derivatization step to
361
convert carboxylic acids into corresponding methyl esters (Schmelz et al., 2004;
362
Mishina and Zeier, 2006), we applied an alternative procedure to prepare plant
363
extracts and in vitro assays for GC-MS analysis. This method (“procedure B”) is
364
based on propyl chloroformate derivatization which converts amino groups into
365
propyl carbamate and carboxyl groups into propyl ester derivatives (Kugler et al.,
12
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
366
2006; Návarová et al., 2012). When applying the analytical procedure B to L-Lys
367
conversion assays with ALD1 or LysOx/catalase, we also identified a single product
368
peak (1b) in the resulting GC-MS chromatograms (Fig. 3A). The substance
369
corresponding to peak 1b accumulated in extract samples from Psm-inoculated wild-
370
type leaves, but not in samples derived from Psm-inoculated ald1 leaves or in
371
samples from the leaves of mock-treated plants (Fig. 3B, F). The mass spectrum of
372
compound 1b exhibited a molecular ion of m/z 255 and a fragmentation pattern that
373
was consistent with the structure of the enamine 2,3-DP that is propyl carbamylated
374
at its amino group and propyl esterified at its carboxyl group (Fig. 3C). When L-Lys-
375
6-13C,ε-15N or L-Lys-4,4,5,5-d4 were used instead of L-Lys as substrates in the ALD1
376
in vitro assays, or when the isotope-labelled L-Lys versions were co-applied with
377
Psm to wild-type leaves, we detected the isotope-labelled variants of 1b (Fig. 3D;
378
Fig. 3E). For L-Lys-6-13C,ε-15N as the precursor, we found an increment in the mass
379
spectral pattern of two mass units whereas a shift of four mass units was observed
380
for L-Lys-4,4,5,5-d4. As outlined before for methylated 1a, the now observed labelling
381
patterns are consistent with an ALD1-catalysed deamination of L-Lys to KAC and
382
subsequent formation of 2,3-DP as an end product (Fig. 3D; Fig. 3E; Fig. 2). A
383
derivatized 1,2-DP ketimine structure for the detected compound 1b can be excluded
384
because the imine nitrogen is not prone to carbamylation, and a hypothetical propyl
385
esterified 1,2-DP product would generate a molecular ion of m/z 169 and not the
386
observed M+ of m/z 255 in the mass spectrometric analysis.
387
As a next step, we monitored the ALD1-mediated L-Lys transamination
388
reaction from the perspective of the ketoacid that is supposed to accept the α-NH2
389
group of Lys to form the corresponding amino acid and KAC (Fig. 2). When pyruvate
390
and α-ketoglutarate were used as amino group acceptors in the ALD1 bioassays, we
391
observed, as expected, Ala and Glu as the product amino acids (Fig. 4A, C). When
392
L-Lys-α-15N was used as the amino acid substrate instead of L-Lys, α-15N-labelled
393
Ala and Glu were formed, as indicated by shifts in the mass spectral patterns of the
394
propyl chloroformate-derivatized amino acid products by one mass unit (Fig. 4B, D).
395
This directly shows that ALD1 transfers the α-NH2 group from L-Lys to acceptor
396
ketoacids and confirms that the ε-NH2 group of L-Lys is retained in the ALD1-
397
catalysed aminotransferase reaction (Fig. 2). Moreover, we compared the relative
398
activities of ALD1 to transaminate L-Lys in the presence of difference acceptor
399
oxoacids, namely glyoxylate, pyruvate, oxaloacetate, α-ketoglutarate (Fig. 4E). This
13
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
400
comparison revealed that pyruvate is the preferred ketoacid (100 % relative activity),
401
followed by oxaloacetate (48.0 %), α-ketoglutarate (6.5 %), and glyoxylate (4.5 %).
402
403
ALD1 catalyzes the transamination of various amino acids other than L-Lys in
404
vitro, but the resulting products are either not detectable in plants or not
405
relatable to an in planta function of ALD1
406
It has been previously shown that the ALD1 protein exerts in vitro amino
407
transferase activity towards several amino acids other than Lys in the presence of
408
pyruvate or β-ketoglutarate as amino group acceptors (Song et al., 2004b). However,
409
the resulting oxoacid or oxoacid-derived products of such alternative ALD1-catalysed
410
transamination reactions have not been characterized so far. Although it is now
411
evident that the conversion of L-Lys to the Pip precursor 2,3-DP is a major in vivo
412
function of ALD1 (Figs. 1 to 4; Návarová et al., 2012), it is not clear whether a
413
possible activity of ALD1 towards other amino acids has biochemical and functional
414
relevance in planta. To shed light onto these unknown aspects of ALD1
415
biochemistry, we performed in vitro conversion assays with ALD1 protein and L-Lys
416
or other possible amino acid substrates using pyruvate as the acceptor oxoacid
417
(Table 1). We assessed the in vitro activities of ALD1 towards the different amino
418
acids by monitoring the formation of Ala from pyruvate. Moreover, we attempted to
419
identify the corresponding oxoacid or oxoacid-derived in vitro reaction products by
420
GC-MS analysis using either procedure A or procedure B (Table1).
421
The in vitro assays with 500 nmol L-Lys and 500 nmol pyruvate as ALD1
422
substrates indicated that more than ¾ of the employed L-Lys was consumed within
423
30 min of incubation time, resulting in the equimolar formation of the product amino
424
acid Ala (Table 1). As described above, 2,3-DP was detected as the Lys-derived
425
product (Fig.1, Fig. 3, Table 1). The transamination activity of ALD1 towards L-Lys
426
was higher than towards any of the other employed substrate amino acids and set to
427
100 % relative activity. By contrast, ALD1 did not accept the α-enantiomer D-Lys as
428
a substrate, showing a relative in vitro activity of only 0.1 % of the value for L-Lys
429
(Table 1). The observed residual activity might be attributed to impurities within the
430
employed D-Lys (≥ 98 %; Sigma L8021).
431
L,L-diaminopimelate (L,L-DAP) and meso-diaminopimelate (meso-DAP) are
432
diastereomeric Lys biosynthetic pathway intermediates. They only differ from L-Lys
14
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
433
by the presence of an additional carboxyl group at the ε-carbon (Fig. 5A; Jander and
434
Joshi, 2009). ALD1 accepted both L,L- and meso-DAP as substrates, with relative
435
activities of 16.3 % and 12.9 % compared to L-Lys (Table 1). With both DAP
436
diastereomers as substrates, we detected a transamination product that was
437
identified as the 6-carboxylated variant of 2,3-DP (Fig. 5A, B; Table 1). The identity
438
of the product as 6-carboxy-2,3-DP was supported by the mass spectral
439
fragmentation pattern of the di-methylated derivative 2a (procedure A), which
440
differed from those of methylated 2,3-DP (1a) by shifts of the M+ ion and main
441
fragments by 58 mass units, indicating the presence of the additional methylated
442
carboxyl moiety (Fig. 5C). In analogy, when applying the analytical procedure B, we
443
detected chloroformate-derivatized 2b whose mass spectrum differed from the
444
spectrum of the corresponding 2,3-DP derivate 1b by 86 mass units, reflecting the
445
additional propylated carboxyl group (Fig. 5D). The IR spectrum of di-methylated 6-
446
carboxy-2,3-DP (2a) was strikingly similar to the IR spectrum of methylated 2,3-DP
447
(1a), exhibiting an N-H stretching vibration at 3429 cm-1, a C=C stretching bond at
448
1650 cm-1, and a C=O bond at 1739 cm-1 that was assigned to the methyl ester
449
carbonyl in conjugation with the enaminic C=C double bond (Supplemental Fig.
450
S7D). Together, this emphasizes the enaminic structure of 2a. The additional non-
451
conjugated carboxyl ester group of 2a compared to 1a discriminates both IR spectra,
452
yielding a second carbonyl maximum at 1755 cm-1 and an additional C-O stretching
453
vibration at 1210 cm-1 (Supplemental Fig. S7D; Supplemental Fig. S4). These
454
analyses show that ALD1 catalyses the in vitro conversion of L,L- or meso-DAP to
455
enaminic 6-carboxy-2,3-DP (Fig. 5A), which essentially parallels the L-Lys to 2,3-DP
456
conversion (Fig. 2). The use of a non-chiral GC column in our analyses did not allow
457
discriminating between enantiomers. However, it is reasonable to assume that the S-
458
enantiomer of 6-carboxy-2,3-DP is formed from L,L-DAP, whereas the R-form is
459
supposed to be generated from meso-DAP (Fig. 5A).
460
We then analysed plant extracts from Psm-inoculated Col-0 leaves for the
461
occurrence of the in vitro observed ALD1 products in planta. To this end, we closely
462
inspected the ion chromatograms of three characteristic fragment ions of a particular
463
in vitro product at its specific retention time (Table 1). Whereas we observed in the
464
extract samples, as documented above (Fig. 1, Fig. 3), distinct peaks with expected
465
ratios of abundance in the characteristic ion chromatograms for the derivatized 2,3-
466
DP forms (e.g. 1a; Fig. 1C) at the specific retention time (Fig. 5E), no 6-carboxy-2,3-
15
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
467
DP (2a; Fig. 5C) could be detected (Fig. 5E). Therefore, albeit ALD1 is able to
468
substantially convert DAP into 6-carboxy-2,3-DP in vitro, the conversion does not
469
take place in planta to detectable levels.
470
In addition to L-Lys, we employed two other ω-NH2-substituted α-amino acids,
471
L-Orn and 2,4-diaminobutyric acid (2,4-DABA), as possible ALD1 in vitro substrates.
472
With 52.9 % relative conversion, L-Orn was readily accepted by recombinant ALD1
473
as a substrate (Table 1). Moreover, an L-Orn-derived in vitro product was identified,
474
and the mass spectrum of the propyl chloroformate-derivatized compound (3b)
475
strongly suggested its nature as Δ2-pyrroline-2-carboxylic acid (Δ2-Pyr2C) (3), the
476
pyrrolidine homolog of 2,3-DP (Table 1; Supplemental Fig. 8A, B). However,
477
Δ2-Pyr2C was not detected in plant extracts of Psm- and mock-treated wild-type or
478
ald1 plants (Table1). With 2.0 % relative activity, ALD1 showed only a modest
479
tendency to transaminate 2,4-DABA, and a possible in vitro transamination product
480
could not be identified (Table 1).
481
ALD1 also exhibited considerable in vitro transamination capacities for L-Met,
482
L-Leu, and L-Arg, showing relative activities between 78.5 % and 57.0 % towards
483
these amino acids (Table 1; Supplemental Table S1). For L-Met and L-Leu, we
484
identified the corresponding oxoacids α-ketomethionine and α-ketoleucine as the in
485
vitro transamination products (Table 1; Supplemental Fig. S8C, D, E, F). Again,
486
these ketoacids were not detectable in extracts of Psm- or mock-treated plants
487
(Table 1). For L-Arg, an in vitro transamination product could not be identified with
488
the employed analytical methods, most likely because the highly basic nature of the
489
guanidine group of the putative product prevents gas chromatographic analyses.
490
ALD1 also showed a medium in vitro transamination activity (between 6.1%
491
and 12.4 % relative activity) towards L-Phe, L-Trp, L-Glu, L-Aad, L-Asn, and L-His
492
(Table 1; Supplemental Table S1). For all of these amino acid substrates except
493
L-His, we identified the corresponding oxoacids as in vitro ALD1 products by GC-MS
494
analyses. As expected, the L-Glu transamination product and common primary
495
metabolite α-ketoglutarate was detected to high levels in plant extracts. However, no
496
differences between Col-0 wild-type and ald1 mutant plants were observed
497
(Table 1). Interestingly, L-Phe-derived phenylpyruvic acid and L-Aad-derived
498
α-ketoadipic acid were detectable in plant extracts to moderate amounts, and
499
tendencies towards two- to three-fold higher levels in Psm-treated compared to
16
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
500
mock-control leaves were registered (Table 1; Supplemental Fig. S8G, H, K, L).
501
However, both Col-0 and ald1 plants contained similar amounts of these substances,
502
indicating that ALD1 does not contribute to their formation in planta (Table 1).
503
Moreover, L-Trp-derived indole-3-pyruvic acid (Supplemental Fig. S8I, J) and L-Asn-
504
derived 2-oxosuccinamic acid were not found in plant extracts (Table 1). Finally,
505
amino acids for which ALD1 showed either a very low or substantially no in vitro
506
transamination activity included L-Ile, L-Val, L-Ala, L-Asp, L-Tyr, L-Ser, L-Thr, L-Cys,
507
Gly, L-Pro, and L-Pip (Table 1; Supplemental Table S1).
508
Together, these in vitro analyses show that L-Lys is the preferred amino acid
509
substrate of the ALD1 aminotransferase. However, considerably high in vitro
510
enzymatic activities also exist towards L-Met, L-Arg, L-Leu, and L-Orn, and clearly
511
observable but more moderate conversion activities were measured for L,L-DAP,
512
meso-DAP, L-Phe, L-Glu, L-Aad, L-Asn, and 2,4-DABA. Although most of the
513
corresponding ketoacids or ketoacid-derived products could be identified in in vitro
514
assays, they were, in contrast to the L-Lys derived product 2,3-DP, generally not
515
detectable in plant extracts. In the few cases we had detected transamination
516
products in planta, they were - in contrast to 2,3-DP - equally produced in Col-0 wild-
517
type and ald1 mutant plants. These results suggest that the transamination of L-Lys
518
leading to 2,3-DP and Pip production is the major in vivo function of ALD1.
519
520
Arabidopsis SARD4 (alias ORNCD1) is able to catalyse the reductive step from
521
dehydropipecolic acid to Pip
522
Our data so far indicate that ALD1 catalyses the first transamination step of
523
Pip biosynthesis from L-Lys which results in the generation of 2,3-DP in planta.
524
However, as postulated before (Návarová et al., 2012; Zeier, 2013), ALD1-initiated
525
Pip generation requires a further reduction step that converts the ALD1-derived DP
526
intermediate into Pip (Fig. 2). The ald1 mutant is unable to accumulate Pip upon
527
P. syringae inoculation (Návarová et al., 2012; Bernsdorff et al., 2016; Fig. 6A),
528
potentially because of its inability to generate the DP intermediate necessary for Pip
529
production (Fig. 1, Fig. 3). To examine whether exogenously applied 2,3-DP can
530
restore the ability of ald1 to biosynthesize Pip, we co-infiltrated Psm and 2,3-DP, that
531
was produced in vitro by the ALD1 bioassay, into ald1 leaves (Fig. 5A). As a
532
consequence, the ald1 leaves were able to accumulate Pip (Fig. 6A, B). By contrast,
17
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
533
Pip accumulation did not occur when a blank assay mixture lacking 2,3-DP was co-
534
infiltrated with Psm (Fig. 6A). Further, we conducted the same co-infiltration
535
experiment with tetradeuterated d4-2,3-DP that was produced from L-Lys-4,4,5,5-d4
536
in the ALD1 bioassays (Fig. 1E, Fig. 3F). In the presence of d4-2,3-DP, Psm-
537
challenged ald1 plants synthesized a compound whose mass spectrum contained a
538
fragmentation pattern shifted by four mass units compared to the pattern of the Pip
539
spectrum, indicating that tetradeuterated d4-Pip was produced in the leaves that
540
were supplemented with d4-2,3-DP (Fig. 6C, D). These data show that P. syringae-
541
inoculated leaves of ald1 mutant plants are able to synthesize Pip, provided external
542
addition of 2,3-DP to the plants.
543
In addition, we performed a similar experiment with the Col-0 wild-type which
544
is able to accumulate both 2,3-DP and Pip after Psm-challenge (Fig. 1; Fig. 3;
545
Návarová et al., 2012). After co-application of L-Lys-4,4,5,5-d4 and Psm to Col-0
546
leaves, we observed not only the production of d4-2,3-DP (Fig. 1E), but also a strong
547
accumulation of d4-labelleled Pip (Fig. 5D). The isotope-labelled compound thereby
548
exhibited a mass spectrum identical to the spectrum of the d4-Pip detected in the
549
d4-2,3-DP-supplemented ald1 plants (Fig. 5E). In sum, this set of experiments
550
indicates that the inability of ald1 plants to generate 2,3-DP upon pathogen
551
inoculation is causative for the failure of Pip biosynthesis in this mutant.
552
Nevertheless, ald1 mutant plants possess the capacity to reduce 2,3-DP to Pip.
553
Arabidopsis At5g52810 (SARD4 alias ORNCD1) represents the Arabidopsis
554
gene with the highest sequence similarity to CRYM (30.5% amino acid identity,
555
51.8% amino acid similarity; EMBOSS Needleman-Wunsch pairwise alignment,
556
version 6.3.1, EMBL-EBI; http:// www.ebi.ac.uk/Tools/psa/; Zeier, 2013), the ketimine
557
reductase that is able to reduce 1,2-DP to Pip (Hallen et al., 2011; Hallen et al.,
558
2015). Similar to CRYM, SARD4 possesses a putative NAD(P) binding domain with
559
a predicted Rossmann-fold, a structural motif frequently found in nucleotide-binding
560
dehydrogenases (Rao and Rossmann, 1973; Zeier, 2013). To test our previous
561
hypothesis that SARD4 is involved in catalyzing the reductive step in Pip
562
biosynthesis (Zeier, 2013), recombinant c-terminally His-tagged SARD4 enzyme was
563
purified via IMAC and tested for reductase activity in a coupled assay setting with
564
purified ALD1 enzyme. As positive control, CRYM protein was also expressed in
565
E.coli, purified and tested analogously to SARD4, as described in detail in the
566
material and method section.
18
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
567
We hereby examined whether enzyme combinations of ALD1 and CRYM or
568
ALD1 and SARD4 would be able to biosynthesize Pip from L-Lys in vitro. As outlined
569
before, ALD1 protein alone was able to transaminate L-Lys in the presence of
570
pyruvate to produce 2,3-DP in the bioassay (Fig. 7). When in addition to L-Lys,
571
ALD1, the acceptor oxoacid and the cofactor PLP necessary for the transamination
572
step, CRYM protein and NADPH (or NADH) were present in the reaction mixture, the
573
levels of 2,3-DP were markedly reduced compared to the ALD1 only-assay, and
574
substantial amounts of Pip were generated (Fig. 7). Similarly, when ALD1 and
575
SARD4 were co-applied in the bioassay, Pip was detected to high amounts in the
576
enzyme mixture, and the levels of 2,3-DP decreased compared to an individual
577
ALD1 incubation (Fig. 7). The combinatory assays therefore support the existence of
578
a Pip biosynthetic pathway that involves both an ALD1-mediated transamination step
579
and an SARD4-catalysed reductive step (Fig. 2).
580
581
sard4 mutant plants show attenuated Pip biosynthesis and over-accumulate
582
2,3-DP
583
Consistent with a role for SARD4 in pathogen-inducible biosynthesis of Pip,
584
SARD4 transcripts accumulated in both in inoculated (1°) and in distal (2°) leaves of
585
Col-0 plants upon Psm treatment (Fig. 8A, B). To functionally investigate the
586
relevance of SARD4 for in planta Pip production and plant immunity, we searched for
587
Arabidopsis T-DNA insertion lines with defects in the At5g52810 gene. Using PCR-
588
based genotyping (Alonso et al., 2003), we identified two lines, GK_696E11 (initially
589
named orncd1-1, later renamed sard4-5 according to the nomenclature proposed by
590
Ding et al., 2016) and GK_428E01 (sard4-6, initially orncd1-2), with T-DNA insertions
591
in the single exon of the SARD4 gene (Supplemental Fig. S9). Whereas the T-DNA
592
insertion in the sard4-5 line leads to a full loss of gene function (knockout) as
593
asserted by a complete lack of transcripts, the sard4-6 line still showed clearly
594
detectable SARD4 transcript levels that increased in the 1° inoculated and in the
595
distal leaves of P. syringae-treated sard4-6 mutant plants, albeit to markedly lower
596
amounts than in the Col-0 wild-type (Fig. 8A, B).
597
A possible explanation for this at first sight surprising difference of the mutant
598
lines provide the predicted sites of T-DNA insertions. The predicted insertion is
599
relatively central within the exon for sard4-5 and within the first 100 bp downstream
19
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
600
of
601
http://signal.salk.edu/cgi-bin/tdnaexpress). This is consistent with the sizes of bands
602
resulting from PCR analyses with genomic DNA from sard4-5 and sard4-6 plants,
603
respectively, and a combination of T-DNA left border primers and gene specific
604
primers annealing in the 3’-untranslated regions (Supplemental Fig. S9B). The
605
second ATG and possible start codon in frame with the coding sequence is located
606
at position +166 of the nucleotide sequence, suggesting the possibility that a shorter
607
but potentially functional version of SARD4 might be expressed in sard4-6.
the
start
codon
(ATG,
+1)
for
sard4-6
(Supplemental
Fig.
S9A;
608
We next determined the levels of Pip in the leaves of Col-0, sard4-5, and
609
sard4-6 plants upon Psm-inoculation or mock-control treatments. The contents of Pip
610
in the control plants were generally in the range between 0.1 to 0.6 µg g-1 fresh
611
weight (FW) (Fig. 8C). In the Psm-inoculated leaves of the Col-0 wild-type, Pip
612
strongly accumulated to levels of about 35 µg g-1 FW at 24 hours post inoculation
613
(hpi) and to levels of 125 µg g-1 FW at 48 hpi, respectively (Fig. 8C). By contrast,
614
inoculated leaves of sard4-5 showed a strongly diminished early increase of Pip at
615
24 hpi to levels of 1.3 µg g-1 FW only. At 48 hpi, a considerable accumulation of Pip
616
in the Psm-inoculated sard4-5 leaves to levels of about 55 µg g-1 FW was detected
617
(Fig. 8C), which accounted for about half of the values of the wild-type at this later
618
infection stage. At inoculation sites, the sard4-6 mutant showed a still marked but
619
compared to the wild-type attenuated Pip accumulation to 20 µg g-1 FW at 24 hpi,
620
which increased to wild-type-like levels of about 120 µg g-1 FW at 48 hpi (Fig. 8C).
621
The Pip levels in the distal 2° leaves increased in the wild-type to 12 µg g-1 FW at 48
622
h after inoculation of 1° leaves. This systemic increase of Pip at 48 hpi was absent in
623
the sard4-5 mutant, and sard4-6 showed a diminished accumulation to 4 µg g-1 FW
624
in the distal leaves at this point of time (Fig. 8C). To test whether the systemic
625
accumulation of Pip was fully compromised in sard4-5, or - as the local accumulation
626
patterns suggested - only delayed, we performed a time course analyses of Psm-
627
induced Pip accumulation between 24 and 96 hpi in the systemic leaves of the plant
628
lines under investigation (Fig. 8E). In the Col-0 wild-type and the sard4-6 mutant, the
629
systemic levels of Pip were significantly increased at 48 hpi onwards in response to
630
Psm inoculation, whereby Pip again accumulated to higher levels in the wild-type
631
than in the sard4-6 mutant. Notably, Psm-inoculation triggered systemic increases in
632
the sard4-5 mutant at 72 hpi and 96 hpi. However, the degree of systemic Pip
20
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
633
accumulation was greatly reduced in sard4-5, since Pip levels stayed below 2 µg g-1
634
FW until 96 hpi (Fig. 8E).
635
As a next step, we analyzed the basal and P. syringae-induced levels of the
636
Pip precursor 2,3-DP in the leaves of Col-0 and sard4 mutant plants, which can be
637
converted into Pip in vitro by recombinant SARD4 protein (Fig. 7). The Col-0 wild-
638
type showed a significant increase of 2,3-DP both in the inoculated 1° and in the
639
distal 2° leaves upon Psm treatment (Fig. 8D). Remarkably, the levels of 2,3-DP in
640
inoculated Col-0 plants were 1 to 2 orders of magnitude lower than the levels of Pip
641
(Fig. 8C, D). Compared to the wild-type, both sard4-5 and sard4-6 mutants over-
642
accumulated 2,3-DP in the locally inoculated and/or in the systemic leaf tissue,
643
suggesting that they harbor defects in the SARD4-mediated 2,3-DP to Pip
644
conversion (Fig. 8D).
645
Together, these metabolite analyses indicate that the gene knockout line
646
sard4-5 has a reduced but not fully abrogated capacity to synthesize Pip after Psm
647
attack in the locally inoculated and systemic leaf tissue. SARD4 therefore
648
significantly contributes to pathogen-induced Pip production, but appears to be not
649
the only reducing enzyme involved in Pip biosynthesis (Fig. 2). The sard4-6 mutant
650
showed a pronouncedly weaker metabolic phenotype than the knockout line sard4-5,
651
because it exhibited an attenuated but still marked ability to produce Pip in Psm-
652
inoculated and in systemic leaves. This confirms the notion that sard4-6 represents a
653
knockdown line for the SARD4 gene.
654
655
The sard4-5 mutant displays attenuated basal resistance to P. syringae but is
656
competent to systemically enhance resistance in response to precedent attack
657
We next compared basal resistance towards attack of virulent P. syringae
658
bacteria between Col-0, sard4-5, sard4-6, and ald1 plants. The ald1 mutant is fully
659
blocked in the accumulation of 2,3-DP or Pip upon P. syringae attack (Fig. 3C;
660
Návarová et al., 2012; Bernsdorff et al., 2016). The growth of a bioluminescent,
661
virulent Psm strain that expresses the Photorhabdus luminescens luxCDABE operon
662
(Psm lux) in inoculated leaves was thereby taken as a parameter to assess basal
663
resistance. The use of the Psm lux strain allows a rapid quantification of bacterial
664
numbers in leaves via luminescence measurements (Fan et al., 2008), which was
665
performed at 60 hpi. After inoculating leaves with Psm lux suspensions of optical
21
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
666
densities at 600 nm (OD600) equal to 0.001, we determined significantly higher
667
bacterial numbers in the leaves of sard4-5 compared to the leaves of the wild-type at
668
60 hpi, indicating a higher susceptibility to bacterial attack of the mutant (Fig. 9A).
669
Moreover, the ald1 mutant was more susceptible to Psm lux than sard4-5, and
670
sard4-6 exhibited a resistance phenotype intermediate to those of Col-0 and sard4-5
671
(Fig. 9A). Therefore, the efficiency of basal resistance to P. syringae appeared to
672
correlate with the amount of Pip produced during the plant-bacterial interaction (Fig.
673
8C; Návarová et al., 2012). In addition, we measured the levels of the phenolic
674
defense hormone SA, whose accumulation in inoculated leaves is required for
675
effective basal resistance to P. syringae (Wildermuth et al., 2001; Bernsdorff et al.,
676
2016), at 24 h post Psm inoculation. At this point of time, SA accumulated to similar
677
levels in the leaves of Psm-inoculated Col-0, sard4-5 and sard4-6 plants (Fig. 9C).
678
The ability of Arabidopsis plants to synthesize Pip upon bacterial attack is a
679
prerequisite for the establishment of SAR (Zeier, 2013). Since sard4-5 mutant plants
680
accumulated markedly reduced levels of Pip in the 2° tissue (Fig. 8C, E), we
681
hypothesized that they might be, just as ald1, fully compromised in SAR. We hence
682
conducted comparative SAR bioassays with Col-0 and both of the sard4 mutant lines
683
(Fig. 9B). Therefore, plants were Psm-inoculated or mock-treated in three 1° leaves,
684
and two days later, challenge-infected in three distal, 2° leaves with Psm lux. SAR
685
establishment is characterized by a significant lower bacterial multiplication in the
686
course of the 2°-challenge infection in Psm-pre-treated compared to mock-pre-
687
treated plants, and this is generally accompanied with a distinctly visible reduction of
688
chlorotic disease symptoms (Mishina and Zeier, 2007). In Col-0 plants, inducing
689
inoculations with Psm limited the growth of Psm lux in challenged distant leaves by a
690
factor of about 15 at 60 hpi, indicating strong SAR establishment in the wild-type
691
(Fig. 9B). This was associated with a marked reduction of disease symptom severity
692
(Supplemental Fig. S10). Mock-pre-treated sard4-5 mutant plants suffered from a
693
higher bacterial multiplication compared to wild-type plants, again indicating that
694
basal resistance is attenuated in sard4-5 (Fig. 9C). Remarkably, in response to Psm-
695
pre-treatment, the sard4-5 mutant was able to establish a significant SAR response,
696
which manifested itself by a wild-type-like factor of growth attenuation in challenge-
697
infected 2° leaves (Mock/Psm = 16; Fig. 9C), and by a pronounced reduction of the
698
disease symptoms of 2° leaves (Supplemental Fig. S10). Consequently, however,
699
the absolute resistance levels of SAR-induced wild-type plants were higher than
22
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
700
those of SAR-induced sard4-5 plants (Fig. 9B). The sard4-6 mutant again displayed
701
an intermediate phenotype: the resistance levels of mock-treated control plants were
702
less strongly compromised compared to sard4-5, and induction of SAR by 1°-Psm
703
treatment enhanced the resistance levels of sard4-6 to almost wild-type-like levels
704
(Fig. 9B; Supplemental Fig. S10).
705
Finally, since SA accumulation in the 2° leaf tissue is one of the hallmarks of
706
SAR (Vlot et al., 2009), we analysed the levels of SA in 1°-mock-treated and SAR-
707
induced Col-0 and sard4 mutant plants. As expected, SA strongly accumulated in the
708
non-treated 2° leaves of wild-type plants following a 1° Psm-inoculation (Fig. 9C).
709
Notably, the systemic accumulation of SA was markedly reduced albeit not totally
710
compromised in sard4-5 (Fig. 9C). Further, sard4-6 mutants accumulated SA in the
711
systemic tissue to relatively high levels that reached about 80% of the respective
712
wild-type values (Fig. 9C).
713
Taken together, the SAR assays show that sard4-5 mutant plants display a
714
considerable SAR response following Psm-treatment, although Pip accumulation in
715
the systemic tissue is delayed and strongly reduced. Nevertheless, just as naïve
716
plants, SAR-induced sard4-5 plants possess a significantly lower resistance level to
717
bacterial infection than the corresponding wild-type plants. This goes hand in hand
718
with a distinctly reduced systemic accumulation of SA after SAR activation.
719
720
Exogenous feeding with 2,3-DP increases plant basal resistance to bacterial
721
infection
722
Exogenous application of Pip to plants increases resistance to leaf infection
723
by P. syringae in the Col-0 wild-type, and overrides the defects in basal resistance in
724
Pip-deficient ald1 mutants (Návarová et al., 2012). Since sard4 mutants show
725
attenuated Pip generation upon pathogen inoculation (Fig. 8C), we tested whether
726
exogenously applied Pip would restore their basal resistance capacity to wild-type-
727
like levels. Indeed, plants watered with Pip one day before Psm lux inoculation
728
significantly increased resistance to bacterial infection in Col-0, sard4-5 and sard4-6
729
plants (Fig. 9D). The bacterial growth in the leaves of the normally more susceptible
730
sard4-5 line was thereby restricted to the same absolute values than the wild-type
731
and the sard4-6 line (Fig. 9D). This indicates that the resistance defects of sard4
23
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
732
mutants are attributable to their attenuated biosynthesis of Pip during the plant-
733
bacterial interaction (Fig. 8C).
734
We next examined the effect of exogenous treatment with the Pip precursor
735
2,3-DP on plant basal resistance towards P. syringae infection. When 2,3-DP
736
generated by the ALD1 in vitro assay was co-infiltrated into leaves with Psm lux,
737
bacterial growth was reduced by a factor of about three in both Col-0 and ald1 plants
738
at 60 hpi compared to co-application of Psm lux and a control solution which did not
739
contain 2,3-DP (Fig. 9E). This suggests that elevated levels of the Pip precursor 2,3-
740
DP in leaves are sufficient to trigger an increased resistance response in
741
Arabidopsis.
742
743
DISCUSSION
744
The accumulation of pipecolic acid in leaves in response to an inducing pathogen
745
inoculation is a prerequisite for the establishment of SAR in plants. Pip amplifies
746
pathogen-induced signals and promotes plants to a primed state to ensure effective
747
activation of defense responses (Návarová et al., 2012; Vogel-Adghough et al.,
748
2013). Pip-mediated SAR induction and defense priming thereby occur by signalling
749
mechanisms that have both SA-dependent and SA-independent character
750
(Bernsdorff et al., 2016).
751
752
ALD1-mediated Lys conversion: formation of enaminic 2,3-DP
753
The biosynthetic precursor of Pip in plants and animals is L-Lys (Rothstein
754
and Miller, 1954; Fujioka and Sakurai, 1997), and a first necessary step for the
755
formation of Pip from L-Lys is a transamination of the precursor amino acid (Gupta
756
and Spenser, 1969). Previous studies by Song and colleagues identified the
757
Arabidopsis ALD1 protein as an L-Lys aminotransferase (Song et al., 2004b).
758
Moreover, our own findings demonstrated that functional ALD1 is required for the
759
pathogen-inducible biosynthesis of Pip in plants (Návarová et al., 2012). Together,
760
this strongly suggested that ALD1 is the plant aminotransferase responsible for Pip
761
biosynthesis (Zeier, 2013). L-Lys carries amino groups at both the α- and the ε-
762
positions, leaving the possibilities that the transamination reaction required for Pip
763
biosynthesis might either involve abstraction of the α- or the ε-amino group, which
24
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
764
would result in the formation of KAC or AAS as oxoacid intermediates, respectively.
765
KAC can cyclize by water loss to form the ketiminic dehydropipecolic acid derivative
766
1,2-DP, whereas AAS is able to form the ketimine 1,6-DP in an analogous manner
767
(Gupta and Spenser, 1969; Galili et al., 2001; Fig. 2). Previous studies based on
768
supplementation of plant tissue with isotope-labelled L-Lys precursors and analysis
769
of the distribution of the isotope labels in the Pip product report controversial results
770
about the mechanism of the Lys transamination reaction that lead to Pip formation.
771
For instance, whereas Schütte and Seelig (1967) proposed a pathway via
772
abstraction of the ε-amino group and the formation of AAS and 1,6-DP as
773
intermediates, the findings of Gupta and Spenser (1969) were consistent with a
774
reaction scheme that involves abstraction of the α-amino group and subsequent
775
generation of KAC and 1,2-DP.
776
In this study, we used two different approaches to examine the ALD1-
777
catalysed conversion reaction of L-Lys involved in Pip biosynthesis. On one hand,
778
we performed comparative metabolite profiling of leaf extracts from wild-type Col-0
779
and ald1 knockout plants to identify ALD1-derived products in planta. On the other
780
hand, we studied the biochemistry of purified, recombinant ALD1 protein in vitro.
781
Using a combination of GC-MS and GC-FTIR analysis techniques, we identified
782
enaminic 2,3-DP (1) as the final L-Lys-derived conversion product of ALD1 in vitro
783
and in planta (Fig. 2). The use of the isotope-labelled L-Lys variant L-Lys-6-13C,ε-15N
784
and L-Lys-α-15N in plant feeding experiments and in in vitro assays showed that
785
ALD1 abstracts the α-amino group of L-Lys and directly transfers it to acceptor
786
oxoacids. The ε-amino group, by contrast, remains in the dehydropipecolic acid
787
product (Fig.1, Fig. 3, Fig. 4, Supplemental Fig. S3). Our analyses are consistent
788
with the reaction scheme previously proposed by Gupta and Spenser (1969), in
789
which transamination of the α-position of L-Lys forms KAC as an oxoacid
790
intermediate
791
aminotransferase reaction, we propose that a dehydrative cyclization of KAC to the
792
ketimine 1,2-DP and a subsequent isomerization to the detected 2,3-DP end product
793
take place (Fig. 2). The cyclization and isomerization steps in this reaction sequence
794
might occur independently of the ALD1 protein in a spontaneous manner or be part
795
of the enzymatic mechanism.
in
the
biosynthesis
of
Pip.
Following
this
25
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
ALD1-mediated
796
In addition to the
15
N-isotope-labelling studies mentioned above, several
797
experimental data indicate that the in vitro and in planta detected ALD1-derived
798
product is enaminic 2,3-DP (1). First, the mass spectral fragmentation patterns of
799
methylated 1a and propyl chloroformate-derivatized 1b are consistent with the
800
piperideine-2-carboxylate structure (Fig. 1C, 3C). Second, the experiments in which
801
L-Lys-4,4,5,5-d4 was fed to plants or used as an in vitro substrate exclude the
802
formation of DP isomers with double bonds in the 3,4-, the 4,5-, or the 5,6-position of
803
the piperideine ring (Fig. 1E, Fig. 3E). Third, the IR spectrum of 1a substantiates the
804
enamine structure and rules out the ketimine form (Fig. 1F). Notably, the spectrum
805
shows a distinct band at 3441 cm-1, which indicates the presence of an N-H bond in
806
the molecule. Similar N-H bonds and corresponding stretching vibrations are present
807
in the IR spectra of valerolactam, the in vitro detected DP-dimer and dimethylated 6-
808
carboxy-2,3-DP (2a), the compound detected in ALD1 in vitro assays with DAP as
809
the substrate amino acid (Supplemental Fig. S7). The IR spectrum of 1a further
810
exhibits an absorption at 1648 cm-1 which supports the presence of an enaminic
811
C=C double bond. A band at nearly the same wavenumber (1646 cm-1) was
812
observed in Resonance Raman spectra of 2,3-DP obtained as the enzymatic product
813
of D-Lysine and D-amino acid oxidase from hog-kidney, and has been assigned to
814
the enaminic C(2)=C(3) stretching mode (Nishina et al, 1991). The enaminic C=C
815
vibrations in IR spectra of differently substituted piperideine enamines have been
816
recorded between 1640 and 1673 cm-1 (Leonard and Hauck, 1957). In addition, the
817
absorption of the C=O stretching bond in the IR spectrum of 1a occurs at lower
818
wavenumbers (1734 cm-1) than those of aliphatic, saturated carboxylic acid methyl
819
esters and is therefore consistent with the occurrence of an enaminic C=C double
820
bond in conjugation with the methyl carboxylate group in 1a (Fig. 1F, Supplemental
821
Fig. S4). Fourth, propyl chloroformate-derivatization of plant extracts and in vitro
822
assay samples generated the N-propyl carbamate product 1b (Fig. 3C), which is
823
supposed to be generated from nucleophilic attack of amine groups to the
824
derivatisation reagent propyl chloroformate. This further supports the notion that the
825
amine 2,3-DP, but not the imine 1,2-DP, constitutes the ALD1-derived product.
826
Hence, two different analytical methods and complementary mass spectral and IR
827
spectroscopic information suggest that 2,3-DP is the final product generated from
828
L-Lys by the action of the ALD1 protein.
26
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
829
The biochemical investigation of the ALD1-mediated L-Lys conversion by
830
LC-MS/MS analysis recently published by Ding and colleagues coincides with the
831
present work in respect to the stereospecific abstraction of the α-amino group of
832
L-Lys and the formation of an ALD1-generated dihydropipecolic acid end product via
833
KAC as the oxoacid (Ding et al., 2016). Remarkably, KAC was neither detected by
834
the LC-MS protocol of Ding et al. (2016) nor in the two GC-MS-based analytical
835
procedures that we have applied here. On the basis of MS/MS fragmentation
836
patterns after positive ionization, Ding et al. (2016) proposed 1,2-DP as the end
837
product of the reaction. Our findings however indicate that the in vitro and in planta
838
generated ALD1-derived product is 2,3-DP. As we have outlined for our own MS
839
analyses of compound 1a (Fig. 1), the mass spectrometric information alone is not
840
sufficient to distinguish between 1,2-DP and 2,3-DP forms (Fig. 1), and only the
841
additional IR spectroscopic data gave direct evidence for the presence of the N-H
842
group and the enaminic C=C double bound, discriminating the actual 2,3-DP end
843
product from the 1,2-DP isomer. In our opinion, the MS/MS-derived information
844
presented by Ding et al. (2016) alone might not be sufficient to allow the
845
discrimination between both DP isomers, because the [M+H]+ ions generated from
846
1,2-DP and 2,3-DP after positive ionization would possess identical masses and
847
could both yield the observed mass fragments, including the “C4H7”-fragment that
848
was quoted as an indicator of 1,2-DP formation (Ding et al., 2016).
849
Chemical interconversions between 1,2-DP and 2,3-DP and structurally-
850
related ketimine/enamine tautomeric pairs can occur, and the relative stability of
851
corresponding ketimine and enamine tautomers might depend on several factors,
852
such as the pH in free solution or complexation with cofactors in an enzymatic
853
environment (Nardini et al., 1988; Nishina et al., 1991). Alkaline conditions have
854
been proposed to favour enamine formation (Nardini et al., 1988). Subcellular
855
localization studies in Arabidopsis and rice show that ALD1 fusion proteins localize to
856
chloroplasts (Cecchini et al., 2015; Jung et al., 2016), indicating that the site of ALD1
857
action is the organelle in which the substrate L-Lys is biosynthesized (Mazelis et al.,
858
1976). With pH values around 7 and 8 in the stroma of dark-exposed and illuminated
859
chloroplasts, respectively (Werdan et al., 1975), the site of ALD1-mediated L-Lys
860
conversion is neutral to moderately alkaline which might favour the formation of the
861
2,3-DP enamine. Our standard assay buffer for in vitro conversion studies was also
862
adjusted to slightly basic conditions (pH 7.5 to 8). However, enamine formation by
27
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
863
ALD1 in in vitro studies proved rather pH-independent in our hands, because
864
variations of the pH of the assay buffer between pH 5 and pH 9 always yielded the
865
same products 1a and 1b in the two employed analytical procedures (data not
866
shown). While analytical “procedure A”, that produced the methylated derivative 1a,
867
involves a short acidic extraction step, analytical “procedure B”, yielding
868
chloroformate-derivatized 1b, involves an acidic extraction step, followed by an
869
alkaline washing step and derivatization under neutral conditions. Despite of these
870
methodological differences, both procedures detected derivatives of enaminic 2,3-
871
DP as the end product of the ALD1-mediated reaction.
872
L-Lysine oxidase (LysOx) from T. viride in combination with catalase is
873
reported to convert L-Lys to ketiminic 1,2-DP, which is characterized by
874
complexation with o-aminobenzaldehyde and UV/VIS spectroscopy of the yellow
875
adduct in previous studies (Vogel and Davis, 1952; Misono et al., 1971; Kusakabe et
876
al., 1980). We aimed at generating 1,2-DP by the LysOx/catalase reaction to directly
877
compare it to the L-Lys-derived ALD1 product (Supplemental Fig. S2). Both
878
LysOx/catalase and ALD1 assays yielded a yellow substance with the reported
879
absorption maximum at 450 nm (Soda et al., 1968; Kusakabe et al., 1980), which
880
sets it clearly apart from the spectra of the described o-aminobenzaldehyde
881
complexes formed by the isomer 1,6-DP (λmax = 465 nm) or the five-ring analogue
882
Δ1-Pyr2C (λmax = 436 nm). Hence, these analyses suggest the ketimine tautomer
883
1,2-DP as the product of both the LysOx/catalase- and ALD1-catalysed reactions. By
884
contrast, as detailed above, our GC-MS and GC-FTIR analyses indicate that 2,3-DP
885
is the final product of L-Lys conversion by LysOx/catalase and ALD1 (Fig. 1, Fig. 3).
886
Yet the conclusiveness of the UV/VIS-spectroscopic detection method is not
887
unambiguous, because heterocyclic enamines such as 2,3-DP can also undergo
888
condensation with o-aminobenzaldehyde (Cervinka, 1988). Moreover, evidence has
889
been provided that heterocyclic imines such as Δ1-piperideine which builds the core
890
structural element of 1,2-DP can indeed react as their heterocyclic enamine
891
tautomers in the o-aminobenzaldehyde reaction (Harada et al., 1970; Hickmott,
892
1982). Together, this strongly argues against a possibility to discriminate between
893
1,2-DP and 2,3-DP by the o-aminobenzaldehyde reaction and subsequent UV/VIS
894
spectroscopic analysis.
895
28
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
896
Do ALD1-mediated reactions beyond the Pip biosynthetic pathway possess
897
physiological relevance in planta?
898
Recombinant ALD1 was previously characterized as an aminotransferase with
899
substrate preference for L-Lys, but the protein also accepted several other amino
900
acids as substrates in vitro. The corresponding transamination products, however,
901
had not been identified (Song et al., 2004b). Since leaf petiole exudates of ALD1
902
overexpressing plants induced stronger defense responses than such from wild-type
903
plants and Pip levels were similar in both kinds of exudates, it was proposed that
904
ALD1 could activate defense responses by “non-Pip metabolites” (Cecchini et al.,
905
2015). To shed light on possible alternative roles of ALD1 beyond Lys catabolism,
906
we examined the relative in vitro activity of ALD1 protein towards a series of other
907
amino acids and attempted to identify the respective conversion products in vitro.
908
Furthermore, we analysed whether in vitro identified conversion products would be
909
detectable in plant extracts, accumulate upon P. syringae inoculation in the wild-type,
910
and fail to do so in the ald1 mutant (Table 1, Supplemental Table S1). These
911
features are common to the Lys-derived metabolites 2,3-DP and Pip (Fig. 1, Fig. 3;
912
Návarová et al., 2012).
913
Besides L-Lys being the preferred substrate, recombinant ALD1 exhibited
914
marked relative aminotransferase activities towards L-Met, L-Leu, L-Arg, L-Orn, and
915
a more moderate but clearly detectable relative activities towards L,L-DAP, meso-
916
DAP, L-Phe, L-Glu, L-Aad, L-Asn, and L-His (Table 1, Supplemental Table S1). The
917
here observed activity range overlaps largely with the set of ALD1-converted amino
918
acid substrates previously described by Song and colleagues (Song et al., 2004b). A
919
common feature of the accepted α-amino acid substrates is a side chain extending
920
to at least a δ-position, whereby the chemical characteristics of individual side chain
921
positions vary considerably. A branching of the side chain at the β-position, as it is
922
the case for L-Ile, appears to reduce the substrate affinity of ALD1 (Table 1).
923
For all of the above-mentioned amino acid substrates except L-Arg and L-His,
924
we were able to detect in vitro transamination products by GC-MS analyses.
925
Whereas the corresponding α-ketoacids were identified as in vitro products for
926
L-Met, L-Leu, L-Phe, L-Glu, L-Aad, L-Asn as ALD1 substrates, cyclized products
927
derivable from 2-ketoacid precursors were detected as DAP- and L-Orn-derived
928
ALD1 conversion products (Table 1, Supplemental Fig. S8). Out of products
29
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
929
detected in vitro, only L-Glu-derived α-ketoglutaric acid, a common primary
930
metabolite involved in numerous metabolic pathways (Lancien et al., 2000), occurred
931
to higher levels in plant extracts. Moreover, moderate levels of the L-Phe-derived
932
phenylpyruvic acid and L-Aad-derived α-ketoadipic acid were identified (Table 1).
933
However, these transamination products occurred to similar amounts in wild-type
934
and ald1 mutant plants, indicating that the in vivo biosynthesis of these compounds
935
is not related to functional ALD1. The other identified in vitro products were not
936
detected in Col-0 leaf extracts, irrespective of whether plants were P. syringae-
937
inoculated or not (Table 1). This suggests that the corresponding catabolic pathways
938
are not activated in plants to detectable levels. From dilution experiments with
939
standard substances, we conservatively estimate that the detection limits of the
940
applied GC-MS methods are well below 0.5 nmol g-1 FW for the relevant compounds.
941
Our results thus indicate that the transamination of L-Lys to 2,3-DP leading to Pip
942
production is a major in vivo function of ALD1. Since possible transamination
943
products of L-Arg and L-His were not amenable to our analyses, we cannot
944
completely exclude a role for ALD1 in L-Arg and L-His catabolism in the plant.
945
However, since exogenous application of Pip is virtually sufficient to complement the
946
defects of the ald1 mutant in basal resistance to P. syringae and SAR (Návarová et
947
al., 2012; Bernsdorff et al., 2016), we consider it unlikely that the L-Arg- or L-His
948
metabolism significantly contributes to ALD1-controlled immune responses.
949
Why is L-Lys catabolism to 2,3-DP the supposedly dominant if not the only in
950
planta function of ALD1, although the enzyme exerts considerable in vitro
951
transaminase activity towards other substrates? First, the relative activity towards
952
L-Lys is higher than towards any of the other amino acids under investigation (Table
953
1). Second, ALD1 resides in the chloroplasts (Cecchini et al., 2015), the subcellular
954
compartment in which L-Lys biosynthesis takes place (Mazelis et al., 1976). Third,
955
the levels of Lys strongly increase upon pathogen inoculation in parallel to the ALD1
956
transcript abundance (Návarová et al., 2012). Hence, ALD1-mediated L-Lys
957
catabolism to 2,3-DP in planta is obviously favoured by the spatial and temporal
958
concurrence of enzyme and substrate. However, the biosyntheses of the readily
959
accepted ALD1 substrates L-Met, L-Leu, DAP, and L-Orn are supposed to take
960
place in chloroplasts as well (Jander and Joshi, 2009; Binder, 2010; Winter et al.,
961
2015),
962
6-carboxy-2,3-DP, and Δ2-Pyr2C, respectively, could be detected in vitro ALD1
and
their
transamination
products
α-ketomethionine,
30
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
α-ketoleucine,
963
assays but not in planta (Table 1, Fig. 5, Supplemental Table S1; Supplemental Fig.
964
S8). The levels of Met, DAP, and Orn in P. syringae-inoculated leaves are markedly
965
lower than the levels of Lys or not detectable at all (Návarová et al., 2012;
966
Supplemental Table S3), indicating that their availabilities as in planta ALD1
967
substrates lag significantly behind the availability of Lys. However, just as Lys, Leu
968
strongly accumulates in the P. syringae-infected leaves (Návarová et al., 2012),
969
making it another good candidate for an ALD1 in vivo substrate, in theory. The main
970
organelle of Leu catabolism in plants is the mitochondrium (Binder, 2010), and
971
possibly, rapid and efficient transport of Leu out of the chloroplast might account for
972
our finding that ALD1-mediated transamination in the chloroplast does not occur to
973
detectable levels.
974
L,L- and meso-DAP, the 6-carboxylated variants of L-Lys, were converted into
975
6-carboxy-2,3-DP (Fig. 5), and it is reasonable to assume that the underlying
976
reaction series conforms the proposed L-Lys to 2,3-DP conversion sequence (Fig.
977
2). The enaminic 6-carboxy-2,3-DP (2a and 2b) and 2,3-DP derivatives (1a and 1b)
978
show strikingly overlapping mass and IR spectral properties, reflecting the fact that
979
the two compounds only differ from each other by the presence or absence of the
980
6-carboxyl substitution (Fig. 1, Fig. 3, Fig. 5, and Supplemental Fig. S7). Both L,L-
981
and meso-DAP are intermediates in the biosynthetic pathway of L-Lys (Jander and
982
Joshi, 2009). The aminotransferase with the closest sequence similarity to ALD1 in
983
Arabidopsis is ABERRANT GROWTH AND DEATH2 (AGD2) (Song et al., 2004b),
984
and recombinant AGD2 has been reported to catalyze the interconversion of
985
L-tetrahydrodipicolinate to L,L-DAP in vitro (Hudson et al., 2006). Since
986
L-tetrahydrodipicolinate and 6-carboxy-2,3-DP are ketimine/enamine tautomers, this
987
previous data and our results indicate that AGD2 and ALD1 both have in vitro
988
transamination activities towards L,L-DAP. Nevertheless, the crystal structures of
989
AGD2 and ALD1 show differences in their presumed substrate binding sites
990
suggesting different biochemical functions in vivo (Watanabe et al., 2007; Sobolev et
991
al., 2013).
992
993
SARD4-catalysed generation of Pip from 2,3-DP
994
Although ald1 mutant plants fail to accumulate 2,3-DP and Pip after
995
P. syringae inoculation, they are able to convert exogenously applied 2,3-DP into Pip
31
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
996
in the presence of a pathogen stimulus (Fig. 6A, B). Therefore, the capacity to
997
reduce a dehydropipecolic acid intermediate to Pip is intact in the ald1 mutant. On
998
the basis of protein sequence comparison, we have previously proposed that
999
Arabidopsis SARD4 (alias ORNCD1) might be involved in the reduction step required
1000
for Pip formation (Zeier, 2013). SARD4 is the closest homologue of the mammalian
1001
ketimine reductase CRYM that utilizes either NADH or NADPH as cofactor (Hallen et
1002
al., 2011). CRYM preferentially catalyses the reduction of cyclic ketimine
1003
monocarboxylate substrates such as 1,2-DP or its five-membered ring analogue
1004
Δ1-Pyr2C (Hallen et al., 2015). Subcellular localization experiments indicate that, just
1005
as ALD1, Arabidopsis SARD4 resides in plastids (Sharma et al., 2013). Moreover,
1006
SARD4 transcripts accumulate both in inoculated (1°) and in distal (2°) leaves of Col-
1007
0 plants upon P. syringae attack (Fig. 8A, B). These features made SARD4 a likely
1008
candidate reductase for a possible function in pathogen-inducible Pip biosynthesis
1009
that could act in combination with ALD1 in the chloroplast.
1010
As outlined above, enzyme assays in which recombinant ALD1, the amino
1011
acid substrate L-Lys, the acceptor oxoacid pyruvate, and the cofactor PLP necessary
1012
for the transamination step were present, resulted in the formation of 2,3-DP from L-
1013
Lys (Fig. 1, Table 1). The same was true when the reducing co-factor NADPH (or
1014
NADH) was additionally present in these ALD1-only assays (Fig. 7). However, in
1015
coupled assays in which both ALD1 and SARD4 protein were combined with PLP,
1016
pyruvate, and reducing equivalents, substantial amounts of Pip were generated from
1017
L-Lys, and the detected levels of 2,3-DP were markedly lower than in the ALD1-only
1018
assay (Fig. 7). This indicates that the ALD1 product 2,3-DP is converted to Pip by
1019
SARD4 and NAD(P)H as the reducing cofactor in the enzyme mixture. Likewise,
1020
after co-application of ALD1 and the mammalian ketimine reductase CRYM in the
1021
bioassay, Pip was formed to significant amounts in the enzyme mixture, and the
1022
levels of 2,3-DP decreased compared to an individual ALD1 incubation (Fig. 7). This
1023
confirms our hypothesis that the combined action of ALD1 and SARD4 is sufficient to
1024
realize L-Lys to Pip conversion in vitro.
1025
Interestingly, we also detected trace amounts of Pip in the ALD1 control
1026
assays lacking any of the reductase enzymes in the reaction mixture, but containing
1027
all of the co-factors relevant for the coupled assay (Fig. 7). An explanation for this at
1028
first-sight surprising observation is given by earlier studies showing that
32
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1029
dehydropipecolic acid can be non-enzymatically reduced to Pip by pyridine
1030
nucleotides such NADH (Meister et al., 1957).
1031
Our results further suggest that SARD4 and its mammalian homologue CRYM
1032
have comparable biochemical characteristics because the ALD1/CRYM and the
1033
ALD1/SARD4 combinations provided similar results. Since CRYM has been
1034
characterized as a reductase that accepts ketiminic substrates such as 1,2-DP
1035
(Hallen et al., 2015), we assume that the 2,3-DP detected in our assays and plant
1036
extracts is not the direct substrate for SARD4 (and CRYM). Instead, an equilibrium
1037
between 2,3-DP and 1,2-DP that is largely shifted to the detectable enaminic form
1038
could exist. Reductive conversion of the minor equilibrium component 1,2-DP to Pip
1039
by SARD4 (or CRYM) might constantly re-establish low amounts of 1,2-DP ketimine
1040
through an equilibrium shift (Fig. 2). The here-suggested mechanism involving
1041
enaminic 2,3-DP as the dominant cyclic dehydropipecolic acid form and a rapid
1042
enamine-ketimine tautomerism in the course of reduction could indirectly favour Pip
1043
generation, because ketimines are prone to spontaneous hydrolysis to the
1044
corresponding open oxoacid (Nardini et al., 1988), which would destabilize the
1045
piperideine ring structure and hence disfavour a subsequent reduction to the
1046
saturated piperidine ring of Pip.
1047
It is noteworthy in this context that in coupled assays with ALD1 and SARD4
1048
and with L-Orn as substrate, the formation of significant amounts of Pro could be
1049
detected, which suggests that SARD4 also possesses Pyr2C reductase activity in
1050
vitro (Supplemental Fig. S11). Mammalian CRYM possesses a similar Pyr2C
1051
reductase activity (Hallen et al., 2015; Supplemental Fig. S11). Meister et al. (1957)
1052
already observed a Pyr2C to proline reducing activity in plant extracts. Meister
1053
(1965) later hypothesized that this activity may reflect a lack of specificity of the
1054
enzyme catalysing the reduction step in Pip formation. The commonly accepted core
1055
Pro biosynthetic pathway in planta, however, is operating via pyrroline-5-carboxylate
1056
(Verslues and Sharma, 2010). Since the Δ2-Pyr2C metabolite was not detected in
1057
planta in our study and the leaf levels of Pro do not rise in response to P. syringae
1058
infection (Table 1; Supplemental Fig. S8; Supplemental Table S3), it is unlikely that
1059
SARD4-mediated Pyr2C to Pro reduction or coupled ALD1/SARD4-mediated Orn to
1060
Pro conversion has biochemical or functional relevance in planta.
1061
To examine the biochemical and physiological function of SARD4 in planta,
1062
we examined the metabolite profiles and immune responses of the knockout line
33
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1063
sard4-5 that completely lacks SARD4 transcription (Fig. 8A, B). The local and
1064
systemic accumulation of Pip in P. syringae-inoculated plants is markedly delayed in
1065
this mutant (Fig. 8C, E). Whereas at inoculation sites, still considerable levels of Pip
1066
are generated at 2 dpi in sard4-5, the systemic accumulation characteristically
1067
observed in the wild-type at 2 dpi is absent. At later stages (3 and 4 dpi), however, a
1068
modest systemic increase is detectable (Fig. 8C, E). In addition, 2,3-DP over-
1069
accumulated in the P. syringae-inoculated sard4-5 plants (Fig. 8D). This data and
1070
the outcome of the coupled ALD1/SARD4 assays suggest that SARD4 significantly
1071
contributes to the pathogen-inducible biosynthesis of Pip in Arabidopsis leaves by
1072
reducing dehydropipecolic acid intermediates to Pip. However, since the ald1
1073
knockout mutant completely lacks Pip accumulation but sard4-5 does not (Návarová
1074
et al., 2012; Fig. 8C, E), another reducing activity downstream of ALD1 supposedly
1075
exists that contributes to Pip formation from DP precursors (Fig. 2). This suggests
1076
the necessity to investigate the relevance of alternative plant dehydrogenases
1077
involved in Pip biosynthesis in future studies.
1078
Parallel to sard4-5, the knockdown line sard4-6, which exhibited residual and
1079
pathogen-inducible expression of SARD4, was analysed (Fig. 8A, B). Together with
1080
the qPCR-based transcriptional analysis, PCR-based genotyping suggested that a
1081
truncated SARD4 transcript version is expressed in sard4-6 (Fig. 8A, B;
1082
Supplemental Fig. S9). The metabolite analyses showed that the ability to
1083
accumulate Pip in the local and systemic leaf tissue was still strong in sard4-6
1084
though moderately reduced compared to the wild-type (Fig. 8C, E). This suggests
1085
that sard4-6 expresses a still active, albeit a presumably truncated version of
1086
SARD4. In line with this interpretation, a study by Wang and colleagues collecting
1087
data on the effect of T-DNA insertions in the exon region of Arabidopsis genes on
1088
transcript levels showed that in 8% of all documented cases reduced transcript levels
1089
could still be detected, and in 2 % of the cases a truncated version of the protein was
1090
expressed (Wang et al., 2008). However, the sard4-6 mutant also over-accumulated
1091
2,3-DP in the leaves of P. syringae-inoculated plants, which might be a result of
1092
reduced SARD4 gene expression or attenuated enzymatic activity (Fig. 8A, B, D).
1093
Although the sard4-6 mutant is a weaker allele than sard4-5, sard4-6 over-
1094
accumulated 2,3-DP to even a greater extent than sard4-5 in the systemic, 2° leaves
1095
(Fig. 8C). As exemplified by the higher systemic accumulation of SA in sard4-6
1096
compared to sard4-5, the overall systemic response is assumingly stronger in the
34
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1097
sard4-6 mutant than in sard4-5 (Fig. 9C). Since increased expression of ALD1 is an
1098
integral part of this systemic response (Bernsdorff et al., 2016), this would result in a
1099
significantly stronger systemic ALD1 expression and concomitant systemic
1100
biosynthesis of 2,3-DP in sard4-6 compared to sard4-5. Together with the markedly
1101
attenuated SARD4 levels in sard4-6 (Fig. 8B), this would then contribute to the
1102
observed strong over-accumulation of 2,3-DP in the systemic tissue of sard4-6.
1103
In parallel to the current study, the above-mentioned work of Ding and
1104
colleagues has characterized the role of SARD4 in Pip biosynthesis and SAR (Ding
1105
et al., 2016). A forward genetic screen identified Arabidopsis sard4 mutants with
1106
defects in SAR to the oomycete pathogen Hyaloperonospora arabidopsidis, and the
1107
mutated SARD4 gene was shown to be allelic with SARD4. Using the sard4-5
1108
mutant (GK_428E01; alias orncd1-1) for in planta analyses and in vitro biochemical
1109
characterization of the SARD4 protein, the results of Ding et al. (2016) essentially
1110
coincide with the here-described function of SARD4 as a reductase that significantly
1111
contributes to Pip biosynthesis in Arabidopsis. Notably, based on leaf samples
1112
collected at 2 d post P. syringae inoculation, Ding et al. (2016) reported the full
1113
absence of systemic Pip increases in sard4-5 plants, and consequently emphasized
1114
the strict necessity of functional SARD4 for the systemic accumulation of Pip. By
1115
contrast, our more extended time course analysis identified a significant pathogen-
1116
induced systemic increase of Pip at later infection stages in sard4-5, even though we
1117
also did not observe systemic increases at 2 dpi and detected greatly reduced levels
1118
at 3 and 4 dpi (Fig. 8C, E). Thus, although SARD4 provides a predominant
1119
contribution to the systemic accumulation of Pip, a delayed SARD4-independent
1120
pathway exists for this process.
1121
1122
Relevance of SARD4-mediated Pip biosynthesis for basal plant immunity and
1123
SAR
1124
Naïve ald1 mutant plants are not able to accumulate Pip in response to
1125
pathogens and are more susceptible to P. syringae infection than wild-type plants.
1126
This resistance defect of ald1 is abolished after exogenous treatment of plants with
1127
Pip, indicating a significant function for Pip in plant basal immunity (Fig. 9B; Song et
1128
al., 2004a; Návarová et al., 2012; Bernsdorff et al., 2016). The P. syringae-
1129
inoculated sard4-5 plants exhibited a marked delay in local Pip accumulation, and
35
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1130
naïve or mock-pretreated sard4-5 mutants also showed significantly attenuated
1131
basal resistance to P. syringae in different experiments (Fig. 8B; Fig. 9A, B and D).
1132
Non- or mock-pretreated sard4-6 plants, that are only moderately compromised in
1133
local Pip biosynthesis, allowed marginally higher bacterial multiplication in their
1134
leaves than the wild-type after a single infection; a mild tendency, however, not
1135
statistically significant in most of the experiments (Fig. 9A, B and D). Together, these
1136
data indicate that the effectivity of basal resistance to P. syringae correlates with the
1137
magnitude of Pip accumulation in Col-0, ald1, sard4-5, and sard4-6 plants (Fig. 8C;
1138
Návarová et al., 2012). In addition, sard4-5 and sard4-6 mutant plants exogenously
1139
supplemented with Pip, showed enhanced basal resistance to P. syringae similar to
1140
the resistance level observed in Col-0 wild-type (Fig. 9D). In sum, these data
1141
suggest that the accumulation of SARD4-derived Pip in infected leaves adds a
1142
significant contribution to the basal resistance capacity of these leaves to bacterial
1143
infection. In this context it should be noted that Ding et al. (2016) observed no
1144
difference in basal resistance to P. syringae between the sard4-5 line and the wild-
1145
type. As outlined above, we cannot confirm this finding because sard4-5 showed
1146
significant defects in basal resistance to P. syringae in every of our conducted
1147
experiments (e.g. Fig. 9A, B, and D).
1148
The defect of sard4-5 in basal resistance, however, was not associated with a
1149
weakening of pathogen-induced SA biosynthesis, as documented by a similar
1150
accumulation of the phenolic defense hormone in P. syringae-inoculated Col-0 and
1151
sard4-5 leaves at 24 hpi (Fig. 9C). The relation of SA and Pip in resistance induction
1152
was recently investigated, and it was established that Pip-mediated amplification of
1153
SA-inducible defense gene expression such as PATHOGENESIS-RELATED1 (PR1)
1154
is an important component of Pip/SA cross-talk (Bernsdorff et al., 2016). Therefore,
1155
the diminished production of Pip in inoculated sard4-5 leaves, which is especially
1156
pronounced at earlier infection stages (24 hpi; Fig. 8C), might cause attenuated
1157
defense gene expression and, as a consequence, reduced disease resistance.
1158
The ability of Arabidopsis plants to synthesize Pip upon pathogen attack is a
1159
prerequisite for the establishment of SAR (Shah and Zeier, 2013). Evidence for the
1160
necessity of elevated Pip levels for SAR comprises the findings that Pip strongly
1161
accumulates in the pathogen-inoculated 1° leaves and in distant 2° leaves. Further,
1162
Pip-deficient ald1 mutants are fully compromised in SAR as well as in the systemic
1163
transcriptional response associated with SAR. Exogenous Pip, in turn, restores the
36
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1164
ability of ald1 to establish SAR upon localized P. syringae inoculation (Návarová et
1165
al., 2012; Bernsdorff et al., 2016). The chemical complementation assays used in
1166
these previous studies were based on exogenous pre-treatments of individual plants
1167
with a dose of 10 µmol Pip via the root system, which resulted in an uptake of Pip
1168
into the shoot and an increase of the Pip levels in the leaves to values of about 8
1169
µg g-1 fresh weight (Návarová et al., 2012). The increases of Pip levels in leaves by
1170
this treatment were thus similar to the rises of Pip observed in the systemic (2°)
1171
leaves after biological SAR induction with P. syringae. Moreover, the resistance
1172
effects caused by biological SAR induction and by exogenous Pip treatments were
1173
quantitatively comparable (Fig. 8C, E; Návarová et al., 2012; Bernsdorff et al., 2016).
1174
On this basis, we have previously proposed a working model that highlighted a
1175
central role for the Pip that accumulates in the systemic (2°) leaf tissue for the
1176
induction of SAR (Návarová et al., 2012; Shah and Zeier, 2013; Zeier, 2013;
1177
Bernsdorff et al., 2016). However, both the metabolic phenotype of the ald1 mutant
1178
lacking Pip biosynthesis in the 1°-inoculated and the non-inoculated 2° leaf tissue
1179
and the mode of the chemical complementation assay via the root that resulted in
1180
the rise of Pip in the whole shoot (and therefore in both 1° and 2° leaves), cannot
1181
rule out a decisive function for accumulating Pip in the 1°-inoculated leaves from
1182
which SAR signalling to the systemic tissue is initiated. The question of whether the
1183
SAR-inducing action of Pip primarily takes place in the inoculated leaves, in the
1184
distant, systemic leaves, or to a similar extent in both types of leaf tissue is not trivial
1185
to address in our experience. For example, feeding experiments that include
1186
localized infiltration of Pip specifically to the 1° or to the 2° leaves are difficult to
1187
interpret, because the infiltration of Pip solutions into leaves did not result in
1188
resistance induction within the treated leaves (Návarová et al., 2012). Possibly, in
1189
contrast to root tissue, leaf cells lack a proper uptake system for the polar amino acid
1190
across the plasma membrane (Tegeder, 2014).
1191
The metabolic phenotype of the sard4-5 mutant, however, is informative in
1192
this context. Albeit in a markedly delayed manner, Pip accumulated to high levels in
1193
the P. syringae-inoculated leaves of sard4-5. By contrast, the systemic increase in
1194
the 2° leaves are absent at 2 dpi and rather weak at later infection stages (Fig. 8C,
1195
E). Nevertheless, sard4-5 plants were able to induce a significant SAR response to
1196
P. syringae (Fig. 9B), and actually established a partial but diminished SAR also
1197
towards the oomycete pathogen H. arabidopsidis (Ding et al., 2016). In terms of
37
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1198
relative bacterial growth attenuation between mock-pre-treated and P. syringae-
1199
pretreated plants, the SAR effect in sard4-5 was similar to the effect in the wild-type
1200
in our bacterial growth assays (Fig. 9B). However, the absolute resistance levels of
1201
both mock-treated plants (“basal resistance”) and of SAR-induced plants were lower
1202
for sard4-5 mutant than for the wild-type plants (Fig. 9B). A possible interpretation of
1203
these findings is that Pip locally accumulating in the inoculated 1° leaves already
1204
exerts a significant triggering function for systemic resistance induction. The
1205
systemic Pip that accumulates in the 2° leaves after induction would then contribute
1206
to the absolute strength of acquired resistance in the systemic tissue. The sard4-5
1207
mutants also accumulated markedly reduced amounts of SA in their systemic leaves
1208
compared to the wild-type (Fig. 9C). This is consistent with our previous hypothesis
1209
that the systemic increases of Pip observed upon SAR induction in the wild-type is
1210
necessary for a strong systemic accumulation of SA (Návarová et al., 2012;
1211
Bernsdorff et al., 2016). The phenotype of sard4-5, however, allows now to refine our
1212
previous model and to introduce a spatial aspect of the action of Pip during SAR.
1213
This includes a SAR-inducing function of locally accumulating Pip in the inoculated
1214
(1°) leaves, and, as worked out previously (Návarová et al., 2012; Bernsdorff et al.,
1215
2016), a role in defense amplification in the distant (2°) leaves.
1216
Exogenous application of the Pip precursor 2,3-DP proved sufficient to induce
1217
a moderate resistance response in Arabidopsis leaves (Fig. 9E). It is not entirely
1218
clear whether this effect is related to a direct resistance-enhancing function of 2,3-
1219
DP or to the in planta production of immune-active Pip from the dehydropipecolic
1220
acid precursor (Fig. 6A, B). In the wild-type, a marked contribution of 2,3-DP to the
1221
overall pipecolate resistance pathway is unlikely, because the local and systemic
1222
accumulation of signalling active Pip quantitatively exceeds the accumulation of the
1223
2,3-DP intermediate by about 2 orders of magnitude (Fig. 8C, D). However, 2,3-DP
1224
strongly over-accumulates in the sard4-5 and the sard4-6 mutants and might
1225
therefore add a significant contribution to the local and systemic resistance
1226
responses in these plants (Fig. 8D). As mentioned before, enhanced defense-
1227
stimulating activity of leaf exudates from ALD1-overexpressing plants were reported
1228
recently, albeit the collected exudates were not enriched in Pip (Cecchini et al.,
1229
2015). Considering the consecutive ALD1/SARD4 mechanism for Pip production
1230
described in this study and by Ding and colleagues (Fig. 2; Ding et al., 2016), it is
38
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1231
conceivable that 2,3-DP accumulating in the ALD1 over-expressing lines studied by
1232
Cecchini et al. (2015) might have mediated the observed resistance effects.
1233
1234
SUMMARY AND CONCLUSIONS
1235
Our study has identified a pathogen-inducible pipecolic acid biosynthesis pathway
1236
starting from L-Lys in Arabidopsis and involving the consecutive action of the L-Lys
1237
aminotransferase ALD1 and the NAD(P)H-dependent reductase SARD4. These
1238
findings are consistent with results from a parallel study by Ding et al. (2016). Both
1239
studies show that ALD1 abstracts the α-amino group of L-Lys in vitro. On top of that,
1240
our data demonstrate this L-Lys-α-aminotransferase function for ALD1 also directly
1241
in planta. In addition, we show by in vitro analyses that the α-N of L-Lys is directly
1242
transferred to acceptor oxoacids (e.g. pyruvate or α-ketoglutarate) to produce the
1243
corresponding amino acids (Ala or Glu) within the ALD1-mediated transamination
1244
reaction. These findings are consistent with earlier work that propose an L-Pip
1245
biosynthesis pathway via the ketoacid KAC as an intermediate in plants and in
1246
animals (Rothstein and Miller, 1954; Gupta and Spenser, 1969), but inconsistent with
1247
a suggested plant metabolic pathway that involves abstraction of the ε-N of L-Lys
1248
and the formation of AAS as a biosynthetic intermediate (Schütte and Selig, 1967)
1249
(Fig. 2).
1250
However, the ketoacid KAC is not the final product of the ALD1-catalysed
1251
transamination of L-Lys. Whereas Ding et al. (2016), solely based on mass
1252
spectrometric information, suggested the cyclic ketimine 1,2-DP as the detectable
1253
product, our study indicates by combined mass spectrometric and infrared
1254
spectroscopic evidence and the application of two different analytical procedures that
1255
the final in vitro and in planta product of the ALD1-catalysed reaction is enaminic 2,3-
1256
DP. As a plausible mechanism of 2,3-DP formation, we propose ALD1-mediated
1257
transamination of L-Lys to KAC, dehydrative cyclization to 1,2-DP, and isomerization
1258
to the 2,3-DP tautomer (Fig. 2). Quantitative analyses of 2,3-DP in plant extracts
1259
showed that the Pip precursor 2,3-DP accumulates in both P. syringae-inoculated
1260
and in systemic leaves of wild-type plants, but the absolute levels of 2,3-DP are
1261
about two orders of magnitude lower than those of Pip.
1262
Our study also addresses the question of whether ALD1-mediated
1263
transamination of amino acids other than L-Lys would have biochemical and
39
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1264
physiological relevance in plants. Although ALD1 is able to transaminate several
1265
amino acids such as L-Met, L-Leu, L-Orn, and diaminopimelate to corresponding
1266
oxoacids or derived products in vitro, detailed metabolite analyses of plant extracts
1267
suggest that such transamination reactions do not occur in planta, indicating that
1268
L-Lys catabolism to 2,3-DP is the predominant if not the only in vivo function of
1269
ALD1. In planta analyses also show that ald1 knockout plants, albeit fully defective in
1270
Pip accumulation, have the capacity to reduce exogenous 2,3-DP into Pip, indicating
1271
that their Pip-deficiency relies on the incapability to form the 2,3-DP enamine.
1272
Our combined in vitro assays with recombinant ALD1 and SARD4 and
1273
comparable assays conducted by Ding et al. (2016) reveal that SARD4 functions as
1274
a dehydropipecolate reductase and converts DP intermediates into Pip. Analyses of
1275
sard4 knockout plants further show that SARD4 significantly contributes to the
1276
pathogen-inducible formation of Pip in Arabidopsis, but that another reducing activity
1277
must exist in the plant that accounts for an SARD4-independent Pip accumulation. In
1278
line with the data of Ding et al. (2016), we find substantial, but delayed and reduced
1279
accumulation of Pip in P. syringae-inoculated leaves of sard4 knockout plants.
1280
However, whereas Ding et al. (2016) proposed a strict necessity of SARD4 for the
1281
systemic accumulation of Pip on the basis of samples from knockout plants collected
1282
at 2 dpi, we performed a more extended time course analysis and identified a
1283
markedly reduced but significant pathogen-induced systemic increase of Pip in these
1284
plants later on at 3 and 4 dpi, indicating that a residual SARD4-independent Pip
1285
biosynthesis in Arabidopsis is not restricted to inoculation sites. Furthermore, our
1286
comparative in vitro assays with Arabidopsis SARD4 and the mammalian reductase
1287
homolog CRYM directly illustrate the biochemical similarity of both enzymes.
1288
Interestingly, both reductases have not only the in vitro capacity to reduce the six-
1289
membered heterocycle 2,3-DP into Pip but also can convert five-membered Pyr2C
1290
into Pro, albeit in planta analyses suggest that the latter reaction is not related to an
1291
in vivo function of SARD4.
1292
The results of both the current work and the study of Ding et al. (2016) suggest
1293
that functional SARD4 is necessary for a proper, wild-type like SAR response.
1294
However, while Ding et al. (2016) report a full SAR defect of sard4 knockout plants,
1295
we observe a significant SAR response in these mutants. We show that sard4 plants
1296
exhibit a reproducibly lower basal resistance to P. syringae than the wild-type but
1297
increase systemic resistance upon a localized inoculation to a wild-type like degree
40
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1298
(as expressed by fold-changes of bacterial growth in the challenge-infected 2° leaves
1299
upon 1°-mock vs. 1°-Psm-treatment). This ultimately results in SAR-induced sard4
1300
plants with a reduced absolute resistance level compared to SAR-induced wild-type
1301
plants, accompanied with markedly reduced systemic responses (such as SA
1302
accumulation) in the mutants. Finally, on the bases of the metabolic and immune
1303
phenotypes of sard4 knockout plants, we propose that a local action of Pip which
1304
accumulates in the 1°-inoculated leaves exerts a significant triggering function
1305
contributing to systemic resistance induction.
1306
1307
MATERIALS AND METHODS
1308
1309
Plant materials and growth conditions
1310
Arabidopsis thaliana plants were grown in individual pots containing a mixture of soil
1311
(Klasmann-Deilmann, Substrat BP3), vermiculite, and sand (8:1:1) under strictly
1312
controlled conditions inside of an growth chamber with a 10 h day cycle (9 AM to 7
1313
PM; photon flux density 100 µmol m-2 s-1) and a 14 h night cycle. The relative
1314
humidity in the chambers was adjusted to 60% and the temperatures during the light
1315
and the dark period were set to 21 °C and to 18 °C, respectively. For experiments,
1316
five to six-week-old plants with an unstressed, uniform appearance were used. The
1317
SARD4 (alias ORNCD1; At5g52810) T-DNA insertion lines sard4-5 (orncd1-1;
1318
GK_428E01) and sard4-6 (orncd1-2; GK_696E11) were obtained from the European
1319
Arabidopsis Stock Centre (NASC). Identification of homozygous individual mutants
1320
was performed according to the protocol of Alonso et al. (2003) using gene-specific
1321
and T-DNA left border primers (Supplemental Fig. S9; Supplemental Table S2). In
1322
addition, the SAR-deficient Arabidopsis ald1 mutant (ald1_T2; Salk_007673) was
1323
used in this study (Song et al., 2004a; Návarová et al., 2012). All mutant lines are in
1324
the Arabidopsis Col-0 background.
1325
1326
Cultivation of bacteria and plant inoculation
1327
Pseudomonas syringae pv. maculicola strain ES4326 (Psm) and Psm carrying the
1328
Photorhabdus luminescens luxCDABE operon (Psm lux) were grown in King´s B
1329
medium supplemented with the appropriate antibiotics at 28°C under permanent
41
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1330
shaking (Zeier et al., 2004; Fan et al., 2008). Overnight log phase cultures were
1331
washed four times with 10 mM MgCl2 solution and diluted to different final optical
1332
densities at 600 nm (OD600) for leaf inoculation. The diluted bacterial solutions as
1333
well as mixtures thereof with more complex solutions, containing metabolites of
1334
interest, were carefully pressure-infiltrated from the abaxial side of the leaves using a
1335
needleless syringe, covering the whole surface of the respective leaf.
1336
1337
Assessment of basal resistance and SAR
1338
To induce SAR, plants were infiltrated into three full-grown lower (1°) leaves with a
1339
suspension of Psm at OD600 = 0.005 between 10 and 11 AM. Plants infiltrated with a
1340
10 mM MgCl2 solution served as mock-treated controls. For the determination of
1341
systemic responses, upper (2°) leaves were harvested 48 h after the primary
1342
treatment.
1343
For SAR assessment, three upper (2°) leaves of all pre-treated plants were
1344
infiltrated with Psm lux at OD600 = 0.001 48 hours after the primary treatment.
1345
Bacterial growth was quantified by measuring the bacterial bioluminescence in leaf
1346
discs (Ø 10 mm) of 2° leaves (one disc per leaf, 3 discs per plant) with a Serius
1347
FB12 luminometer (Berthold Detection Systems GMBH, Pforzheim/Germany).
1348
Bacterial growth rates were expressed as relative light units (rlu) per cm2 leaf area
1349
(Fan et al., 2008). At least six to seven replicate plants per treatment and plant
1350
genotype were measured before a statistic analysis of the resulting values was
1351
performed.
1352
To assess bacterial growth in naive 5-week old plants (basal resistance),
1353
three full-grown leaves per plant were infiltrated with a suspension of Psm lux at
1354
OD600 = 0.001 between 10 and 11 AM. 60 h later, the bacterial growth in the
1355
infiltrated leaves was determined by measuring the bacterial bioluminescence in leaf
1356
discs as described above. The analysis of the resulting data was performed as
1357
explained for the SAR assessment.
1358
1359
Determination of 2,3-DP, SA, and other relevant metabolites in leaf material
1360
and in vitro enzyme assays (analytical “procedure A”)
1361
Levels of free SA, 2,3-DP, and other metabolites listed in Table 1 in Arabidopsis leaf
1362
samples or enzyme assays were determined by solvent extraction of leaf material or
42
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1363
enzyme assays followed by a vapour-phase extraction-based work up of the extracts
1364
and subsequent analysis of the resulting samples via GC-MS. In detail, shock-frozen
1365
leaf material (approximately 100-200 mg originating from up to six leaves) was
1366
pulverized in liquid N2 using a ball mill and homogenized with 600 µl of extraction
1367
buffer, consisting of H2O:1-propanol:HCl (1:2:0.005). In the case of enzyme assays,
1368
50-100 µl per assay were used and extracted as described above. Following the
1369
addition of 100 ng of D4-salicylic acid as an internal standard and 1 ml of methylene
1370
chloride, the mixture was thoroughly re-homogenized and centrifuged at 14000 x g
1371
for 1 min for optimal phase separation. For the analyses of free SA and 2,3-DP, the
1372
lower organic phase was removed, dried with Na2SO4, and incubated with 2 µl of 2 M
1373
trimethylsilyl-diazomethane
1374
temperature, driving the conversion of carboxylic acids into the corresponding methyl
1375
esters. To stop the methylation reaction, an excess of acetic acid (2 µl of a 2 M
1376
solution in hexane) was added to the vials, and the sample was then subjected to a
1377
vapour phase extraction procedure at 70 °C and 200 °C using a steady stream of
1378
nitrogen and a volatile collector trap packed with Porapak-Q absorbent (VCT-1/4X3-
1379
POR-Q; Analytical Research Systems) according to Schmelz et al. (2004). The
1380
volatilized and trapped metabolites were then eluted from the absorbent with 1 ml
1381
methylene chloride, and the sample volume was reduced to 30 µl in a stream of
1382
nitrogen prior to GC-MS analysis.
in
hexane
(Sigma-Aldrich)
for
5
min
at
room
1383
4 µl of the processed sample mixture were then separated on a gas
1384
chromatograph (GC 7890A; Agilent Technologies) equipped with a fused silica
1385
capillary column (ZB5 MS, Zebron) and mass spectra were recorded with a 5975C
1386
mass spectrometric detector (Agilent Technologies) in the electron ionization (EI)
1387
mode as described before (Návarová et al., 2012). The quantitative determination of
1388
metabolites was performed by integrating peaks originating from selected ion
1389
chromatograms (for m/z values compare Table 1) and relating the areas of a
1390
substance peak to the peak area of D4-SA (ion: m/z 124). Correction factors
1391
experimentally determined for each substance by use of authentic substances were
1392
considered. The correction factor for the quantification of 2,3-DP (ion: m/z 108) was
1393
approximated by measuring the consumption of the amino acceptor α-ketoglutarate
1394
in the ALD1 assay reaction mix at distinct concentrations. Metabolite levels were
1395
either related to leaf fresh weight or total volume of an in vitro assay.
1396
43
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1397
Determination of amino acids (analytical “procedure B”)
1398
Levels of pipecolic acid and other relevant amino acids were determined using the
1399
EZ:faast free amino acid analysis kit for GC-MS (Phenomenex), which is based on
1400
the separation and identification of propyl chloroformate–derivatized amino acids by
1401
GC-MS (Kugler et al., 2006). To assess amino acids levels from Arabidopsis leaves,
1402
50 mg of homogenized leaf material were extracted, derivatized and analyzed as
1403
previously described in detail (Návarová et al., 2012). For the identification and
1404
quantification of amino acids from in vitro enzyme assays, the work-up procedure
1405
was identical, with the exception that instead of 50 mg of leaf material, 50 µl of an
1406
individual assay were processed and analyzed. A correction factor for the
1407
quantification of 2,3-DP (ion: m/z 255) was experimentally established in reference to
1408
the internal standard L-Norvaline by measuring the consumption of L-Lys and the
1409
formation of Ala from the amino-acceptor pyruvate in the ALD1 assay reaction mix at
1410
distinct substrate concentrations.
1411
1412
GC-FTIR analysis of metabolites
1413
GC-FTIR spectra were acquired using a Hewlett-Packard 6890 Series gas
1414
chromatograph coupled with an IRD3 infrared detector manufactured by ASAP
1415
analytical (Analytical Solutions and Providers, Covington, KY, USA). Infrared spectra
1416
were recorded in the mid-IR range from 4000-600 cm-1 (wavenumber = reciprocal
1417
value of wavelength) with a resolution of 16 cm-1 and a scan rate of 8 scans/second.
1418
The
1419
Apodization=Triangle;
1420
temperature of the flow cell and the transfer lines were both set to 250 °C. Nitrogen
1421
was used as sweep gas. The GC was operated in splitless mode using helium as
1422
carrier gas, with a flow rate of 2 ml min-1 and a column head pressure of 9.54 psi.
1423
The column used in the GC–FTIR studies was a fused silica capillary column
1424
(Zebron ZB-5; 30m x 0.32 mm x 0.25 µm) purchased from Phenomenex Corporation
1425
(Aschaffenburg, Germany). The GC oven temperature program consisted of an initial
1426
temperature of 50°C for 3 min, ramped up to 240°C for 8 min, followed by a ramping
1427
step to 320°C over the course of 20 min (Total time: 33.75 min). Depending on the
1428
nature of the sample, 1 to 4 µl were injected using an Agilent 7863 Series auto-
1429
injector. Please note that the derivatization of the samples (procedure A), the
IRD
method
parameters
Phase
in
detail
were
correction=Mertz;
as
follows:
Zero-Fill=1;
Resolution=16;
Co-Add=2.
44
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
The
1430
column, as well as the temperature program used in our GC-FTIR studies were all
1431
identical to the setup used in the complementing GC-MS studies with the goal to
1432
simplify the identification of relevant target peaks.
1433
1434
Plant treatment with L-Lys and isotope-labelled lysine varieties
1435
For in planta labelling experiments, three to four mature leaves of 5-week old soil-
1436
grown Arabidopsis wild-type Col-0 and ald1 mutant plants were infiltrated in the
1437
morning with Psm (OD600nm = 0.005) or MgCl2 (mock controls) as described before.
1438
After 4 hours, the same leaves were infiltrated with 5 mM solutions of L-Lysine or
1439
one of the isotope labelled varieties L-Lysine-6-13C-ε-15N (CAS: 204451-46-7 -
1440
Sigma) and L-Lysine-4,4,5,5-d4 (Cambridge Isotope Laboratories) prepared in
1441
HPLC-grade water. H2O-infiltrations served as control treatments. Infiltrated leaves
1442
were harvested at 48 hpi (counting from the first infiltration event with Psm or mock)
1443
and subsequently extracted, derivatized and analysed by GC-MS according to the
1444
analytical procedures A and B.
1445
1446
Plant treatments with pipecolic acid
1447
Treatments with Pip were essentially performed as detailed in Návarová et al.
1448
(2012). 10 ml of an aqueous 1 mM D,L-Pip solution (equates to 10 µmol) or 10 ml
1449
H2O as a control treatment were exogenously applied to individual, five-week-old
1450
plants. For resistance assays, inoculation with Psm lux (OD600nm = 0.001) was
1451
performed 24 h after Pip or H2O applications, and bacterial growth assessed as
1452
outlined above. 48 hours later, leaf samples were harvested and defense responses
1453
quantified as described (please refer to section: Assessment of basal resistance and
1454
SAR).
1455
1456
Plant treatment with 2,3-DP
1457
Application of exogenous 2,3-DP to Arabidopsis Col-0 and ald1 plants was achieved
1458
by co-infiltrating mature leaves of 5-week-old plants with Psm and 2,3-DP obtained
1459
from ALD1 or LysOx/catalase in vitro assays. With regard to ALD1 assays, mixtures
1460
containing 20 mM L-Lys as substrate were run to completion (> 99 % of lysine used)
1461
as described in detail in the respective methods section, heat-inactivated, filtered to
45
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1462
eliminate remaining protein (Vivaspin 6 centrifugal concentrator - MWCO 10,000 Da;
1463
Sigma) and diluted six-fold in 10 mM MgCl2. As a control, the same assay containing
1464
boiled, inactive ALD1 enzyme (no enzyme control) was used in the co-infiltration
1465
assays. With respect to LysOx/catalase assays, LysOx- and catalase-containing
1466
reaction mixtures were run to completion, stopped, filtered as described above and
1467
diluted. Controls consisted of full assays that lacked LysOx (catalase only). The
1468
bacterial suspension was added into ALD1 (or LysOx/catalase) assay mixture to a
1469
final OD600 of 0.005, and infiltrated into three leaves of five-week-old plants. 48 hours
1470
later, infiltrated leaf samples were harvested for subsequent metabolite analysis and
1471
processed by analytical procedure B. Additional controls consisted of Psm- and
1472
mock-infiltrations without any of the ALD1 or LysOx/catalase assay components.
1473
For basal resistance assays, Psm lux (OD600 = 0.001) and enzymatically
1474
synthesized 2,3-DP were co-infiltrated into three mature leaves of 5-6 week old Col-0
1475
or ald1 plants. The final concentration of 2,3-DP in those infiltration experiments was
1476
approximately 2 mM. Control experiments were performed as described in the main
1477
text. After 60 hours, bacterial growth was assessed as outlined above.
1478
1479
Data analyses
1480
HPLC data was analyzed using ChemStation for LC 3D systems software version
1481
Rev.B.04.02 SP1 [208] (Agilent Technologies Inc.). GC-MS data was analyzed using
1482
MSD ChemStation software version E.02.01.1177 (Agilent Technologies Inc.). For
1483
analysis of FTIR data, Essential FTIR software v3.10.037 was used (Operant LLC;
1484
Madison WI - USA).
1485
1486
Analysis of gene expression
1487
Gene expression was assessed by quantitative real-time PCR analysis, essentially
1488
as described in Návarová et al. (2012) and Bernsdorff et al. (2016) with the
1489
exception that an absolute standard curve was used (Pfaffl et al., 2004). In brief, 1
1490
µg total RNA was extracted with peqGOLD TriFast™ reagent (PeqLab, Erlangen,
1491
Germany) and used for cDNA synthesis using the GoScript reverse transcriptase
1492
(Promega, Mannheim, Germany) in combination with Oligo(dT)18 primers according
1493
to the manufacturer’s instructions. Quantification of the targeted amplified cDNA
46
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1494
templates was carried out with GoTaq qPCR master mix (Promega) according to the
1495
manufacturer´s
1496
Supplemental Table S2. The qPCR was performed in triplicates using a Rotor-
1497
Gene™ Q system (Qiagen, Hilden, Germany) with the following cycling program:1
1498
minute at 95°C (enzyme activation); followed by 40 cycles of 20 seconds at 95°C
1499
(denaturation), 20 seconds at 60°C (annealing/extension); and a final extension step
1500
at 72°C for 20 seconds. Data analysis was performed with Rotor-Gene™ Q software
1501
version 2.0.2 via absolute quantification of transcripts using an external standard
1502
curve. The standard curve was obtained using recombinant plasmid DNA, more
1503
precisely the SARD4 coding sequence cloned into the commercial pET32b vector
1504
(Invitrogen), quantified by multiple measurements in various dilutions and
1505
concentrations (0.02 ng – 20 ng). Unknown sample amounts were determined from
1506
the respective calibration data by interpolating the PCR signals into the external
1507
standard curve. The values for the correlation coefficient R2 for the standard curves
1508
had to be greater than 0.99 to be considered reliable. The resulting quantification
1509
data was depicted as a concentration in ng µl-1.
instructions
using
the
gene-specific
primer
pairs
listed
in
1510
1511
L-Lysine oxidase assays and biosynthesis of 2,3-DP
1512
L-Lysine oxidase (LysOx) from T. viride (EC 1.4.3.14) was purchased from Sigma-
1513
Aldrich as lyophilized powder (CAS: 70132-14-8) and used according to the
1514
manufacturers protocol (Kusakabe et al., 1980) with a few minor modifications.
1515
Lyophilized powder was re-suspended in either H2O or 50 mM sodium phosphate
1516
buffer (pH 8.0) to obtain a stock solution with approximately 5 units ml-1 enzyme. The
1517
solution was homogenized, aliquoted and immediately stored at -20°C until further
1518
use. The enzyme stock solution could be stored for several months without any
1519
significant loss in activity. As described in great detail before (Supplemental figure
1520
S2), the substrate-specific LysOx from T. viride catalyzes the conversion of L-Lysine
1521
to α-keto-ε-aminocaproic acid (KAC) and if conducted in the presence of catalase to
1522
remove H2O2 formed during the oxidation step, 2,3-DP is formed as main product of
1523
the reaction due to the spontaneous intramolecular cyclization of the KAC
1524
intermediate. Catalase from bovine liver was purchased as lyophilized powder from
1525
Sigma (CAS number: 9001-05-2) and had to be prepared immediately before use in
1526
cold deionized H2O (stock solution with 3500 units ml-1 enzyme). At first, LysOx
1527
assays were carried out following the exact protocol provided by the manufacturer.
47
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1528
However, to minimize the generation of two undesired by-products, 5-aminovaleric
1529
acid and the P2C dimer (compare Supplemental figure S2), various adjustments to
1530
this protocol were made. At the end of the optimization process, the following
1531
conditions were chosen. Reactions were carried out with a final concentration of L-
1532
Lys of 10 mM in 25 mM sodium phosphate buffer (pH 8.0) in the presence of 0.5
1533
units of LysOx and 875 units of catalase per ml reaction mix. Assays were allowed to
1534
run to completion at 37°C (1h to O/N). 1 mM CaCl2 was added to the reaction mix to
1535
improve relative activity (Kusakabe et al.,1980). Each set of assays included at least
1536
a blank control (no enzyme), as well as controls lacking one of the two enzymes of
1537
the reaction mixture, catalase and LysOx, respectively, incubated under the same
1538
conditions. Depending on the next steps, the assays were either stopped by addition
1539
of MeOH (equal amount), 0.1 M HCl (1/10th of the volume) or by heating the reaction
1540
mixture at 85°C for 15 min. Full assays and controls were usually run as replicates of
1541
three. TLC analysis was regularly used to rapidly assess the success of an assay
1542
before proceeding to a consecutive step of an experiment such as coupled assays or
1543
feeding experiments. In this case, 2 µl of an O/N assay series was spotted onto a
1544
TLC silica gel 60 F254 plate (20x20 cm; Merck, Germany).
1545
consisted of either of acetone/MeOH/H2O at 10:2:2 or of butanol/acetic acid/H2O
1546
(4:1:1). To visualize the consumption of L-Lys, the plates were sprayed with
1547
ninhydrin reagent (0.5 % ninhydrin in ethanol) and heated to 100°C in an oven until
1548
individual spots were seen. The post-chromatographic derivatization with ninhydrin
1549
stained L-Lys dark red/orange, whereas pipecolic acid was stained purple.
1550
Quantitative measurements of the enzyme activity were performed in all cases by
1551
both, following the formation of the 2,3-DP product and the consumption of the
1552
substrate Lys via GC-MS procedures A or B.
The mobile phase
1553
1554
Cloning of ALD1, SARD4 (ORNCD1) and CRYM
1555
cDNA/DNA fragments corresponding to ALD1 (At2g13810), SARD4 (ORNCD1;
1556
At5g52810) and human CRYM (EC 1.5.1.25) were amplified using high fidelity
1557
Phusion polymerase (NEB, Frankfurt, Germany) according to the manufacturers
1558
recommendations. NCBI Reference Sequence accession numbers are as follows:
1559
ALD1 (NM_126957.1) and SARD4 (NM_124659.1). The full length human CRYM
1560
ORF bacterial expression clone (Entrez gene ID: 1428 transcript variant 1, longer
1561
isoform – Clone ID: HsCD00333103) was purchased from Harvard Medical School
48
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1562
Plasmid stocks (Boston - MA, USA). Presumable targeting sequences within the
1563
gene sequences were identified via TargetP, SignalP, and related tools
1564
(Emanuelsson et al., 2007) and taken into account during the primer design. For
1565
ALD1, the nucleotides coding for the plastidial N-terminal targeting (-20 AA: Δ20-
1566
ALD1) sequence have been omitted as described in prior studies (Song et al. 2004b;
1567
Hudson et al., 2006; Sobolev et al., 2013). The gene sequences of Δ20-ALD1,
1568
SARD4 and CRYM were introduced into the target vector pET-32b(+) (Novagen) via
1569
sticky-end cloning (Zeng, 1998), using NdeI and XhoI restriction sites. The
1570
recombinant proteins thus contained eight non-native residues (LEHHHHHH) at the
1571
C-terminus, including the poly-His tag. Primer sequences can be found in
1572
Supplemental Table S2. Plasmids containing the respective genes were transformed
1573
into chemically competent into E. coli BL21 Rosetta™ 2(DE3) pLysS cells and plated
1574
on LB-Agar plates containing the appropriate selection markers. Transformants
1575
carrying the gene of interest were identified by colony PCR using the same gene-
1576
specific primers used for the initial amplification and were verified by plasmid
1577
sequencing.
1578
1579
Expression and purification of recombinant enzymes
1580
All genes expressed in this study were cloned into the pET-32b vector system,
1581
putting the genes under the control of the phage-derived IPTG-inducible T7 promoter
1582
(LaVallie et al., 1993). For expression of the recombinant protein(s) described in this
1583
study, a single colony was picked and cultured overnight in 3 ml of lysogeny broth
1584
(LB) medium supplemented with the appropriate selection markers at 37 °C and with
1585
constant shaking on an orbital shaker before inoculating and growing a 500 ml
1586
culture under the same conditions until the OD600 reached 0.5-0.8. At this point the
1587
culture was briefly cooled down and the transgene expression was induced with 1
1588
mM isopropyl-β-D-1-thiogalactopyranoside (IPTG), followed by incubation overnight
1589
at 16°-25°C with constant shaking (240 rpm). It should be noted that best results
1590
were obtained with freshly transformed cells and incubation at reduced temperatures
1591
(16°C) after induction of transgene expression to minimize the precipitation of
1592
recombinant enzymes as insoluble inclusion bodies. The bacterial pellets were
1593
collected by centrifugation at 6000 x g for 15 min at 4°C (Eppendorf R5810). Cells
1594
were re-suspended in a minimum of extraction/binding buffer (50 mM sodium
49
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1595
phosphate, pH 8.0, 500 mM NaCl, 10% glycerol, 20 mM imidazole, 5 mM β-
1596
mercaptoethanol, 1mM PMSF). The homogenate was transferred to a pre-cooled
1597
mortar and ground in liquid nitrogen with a pestle until a homogenous powder was
1598
obtained. The powder was transferred to 2 ml Eppendorf tubes and allowed to thaw
1599
on ice. The resulting homogenate was then precipitated using a centrifuge at 16000
1600
x g for 30 min at 4°C. The cell-free supernatant containing the majority of soluble
1601
recombinant proteins was collected and filtered through a low-protein binding nylon
1602
filter (0.22µm) before being applied to a pre-equilibrated immobilized immune affinity
1603
chromatography (IMAC) column, such as a nickel-charged His GraviTrap™affinity
1604
column (GE Healthcare, Germany) or cobalt-charged HisTALON™ Gravity column
1605
(Takara Bio, USA) (1 ml). After the binding step, the column was successively
1606
washed according to the respective manufacturers recommendations. The proteins
1607
were then eluted with 50 mM sodium phosphate buffer, pH 8.0, containing up to 500
1608
mM NaCl and 200 mM imidazole and collected in fractions of 0.75 ml. Under those
1609
conditions, most of the enzyme activity was found to be in 4 to 5 consecutive
1610
fractions, which were combined and desalted using a 5 ml desalting PD10 column
1611
(GE Healthcare) equilibrated with the respective buffers, 50 mM Tris, pH 8 and 100
1612
mM potassium phosphate buffer (pH 8), both containing 10% (v/v) glycerol and 1
1613
mM DTT for ALD1 and the reductases, respectively. Aliquots of the purified proteins
1614
were used for the quantification of total protein content by the Bradford method
1615
(Kruger et al., 1994) using the Bradford Assay reagent (Bio-Rad, Düsseldorf,
1616
Germany) according to the manufacturers protocol. Bovine serume albumin was
1617
used for the standard curve. Target enzyme purity was determined by SDS-
1618
polyacrylamide gel electrophoresis on a 12% gel in accordance with Laemmli’s
1619
method (Laemmli, 1970) (results not shown). The purified recombinant protein
1620
stocks were used directly for enzyme assays and the remaining protein was flash
1621
frozen in liquid nitrogen and stored at -80 °C in 20 % glycerol supplemented with 1%
1622
PMSF until further use.
1623
1624
ALD1 enzyme assays
1625
ALD1 enzyme assays were performed based on the protocol from Song et al.
1626
(2004b) with a few minor modifications. In general, reactions were carried out in an
1627
assay mix (50 or 200 µl) containing purified recombinant Δ20-ALD1 enzyme at a final
50
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1628
concentration of 20-100 µg ml-1 with 10-40 mM of a respective amino-donor (e.g. L-
1629
Lys), 10-40 mM of an amino-acceptor (e.g. pyruvate), 5 mM MgCl2, 100 µM
1630
pyridoxal-5′-phosphate (PLP) in 50 mM Tris buffer, pH 8.0, supplemented with 5%
1631
glycerol. Depending on the purpose of the experiment, such as maximizing the
1632
formation of possible ALD1 products with alternative amino acid substrates, assays
1633
were incubated at 37°C for up to 16 h. In general, the reaction was initiated by the
1634
addition of purified Δ20-ALD1 enzyme which had usually been diluted in the
1635
corresponding assay buffer. At the end of the incubation period, reactions were
1636
stopped by one of the following methods: addition of an equal volume of MeOH,
1637
addition of 10% (v/v) 0.1M HCl to the reaction mixture or by boiling of the assays for
1638
10 min at 85°C. In general, the reaction was stopped by inactivating the enzyme at
1639
85°C for 10 min. These different methods for stopping the reaction were all tested to
1640
determine the effects of pH and solvents on the composition of the reaction products.
1641
Reactions
1642
systematically performed as controls. Quantitative measurements of ALD1 activity
1643
were made by either following the formation of ALD1 reaction products such as 2,3-
1644
DP or the transamination side-products like L-Ala (from pyruvate), or by measuring
1645
the consumption of the respective substrates via GC-MS-based procedures A and B.
1646
All assays were repeated at least in triplicates.
without
ALD1
enzyme
or
with
heat-inactivated
enzyme
were
1647
1648
Coupled assays with recombinant ALD1 enzyme and CRYM/SARD4
1649
Coupled enzyme were performed by incubating freshly purified recombinant ALD1
1650
enzyme alone or in combination with one of the two reductases CRYM or SARD4 at
1651
a final concentration of 100 µg ml-1 per enzyme in a reaction mix containing 20 mM
1652
L-Lys (or 20 mM L-Orn), 20 mM pyruvate, 5 mM MgCl2, 100 µM PLP and 200 µM
1653
NADH (or NADPH) in 20 mM Tris buffer, pH 8.0. All reaction mixtures contained 5 %
1654
glycerol for additional enzyme stability and were incubated at 37°C for 16 h.
1655
Reactions were stopped by inactivating the enzymes at 85°C for 10 min. The
1656
formation of 2,3-DP and Pip was monitored using GC-MS after derivatization of the
1657
assays with trimethylsilyl-diazomethane (procedure A) or propyl chloroformate
1658
(procedure B), respectively. Control assays included reaction mixtures not containing
1659
any substrates as well as reaction mixtures incubating all three enzymes separately
1660
in the presence of all assay components. Again, all assays were repeated at least in
1661
triplicates.
51
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1662
1663
Statistical analyses
1664
Statistical significances were assessed by analysis of variance (ANOVA) with type II-
1665
sum of squares using the R statistical package (https://www.r-project.org/), the
1666
command “aov (Phenotype ~ Treatment + Genotype + Treatment * Genotype,
1667
data=object1)” (with object1 being the data table loaded into the R workspace), and
1668
subsequent post-hoc Tukey HSD test (Brady et al., 2015; Bernsdorff et al., 2016).
1669
1670
1671
Supplemental Data
1672
The following materials are available in the online version of this article.
1673
Supplemental Figure S1. The dehydropipecolic acid isomer identified in plant
1674
extracts and the in vitro ALD1 DP product have identical GC retention times.
1675
Supplemental Figure S2. Synthesis of 2,3-DP by L-Lysine oxidase from
1676
Trichoderma viride.
1677
Supplemental
1678
dehydropipecolic acid versions. The products are identical to those detected in ALD1
1679
in vitro assays.
1680
Supplemental Figure S4. Wavenumbers of the infrared absorption bands of the
1681
C=O stretching vibrations occurring in different carboxylic acid methyl esters, as
1682
determined by GC-FTIR analysis.
1683
Supplemental Figure S5. In addition to 2,3-DP, the L-Lys-derived DP-dimer can be
1684
detected in L-Lysine oxidase assays carried out in the presence of catalase.
1685
Supplemental Figure S6. Valerolactam is the main L-Lys-derived reaction product
1686
in L-Lysine oxidase assays performed in the absence of catalase.
1687
Supplemental Figure S7. Infrared spectra of valerolactam, DP-dimer, 2,3-DP
1688
methyl ester, and 6-carboxy-2,3-DP dimethyl ester, as determined by GC-FTIR
1689
analyses.
1690
Supplemental Figure S8. Structures of non-derivatized and derivatized ALD1
1691
transamination products and their mass spectra (as determined by GC-MS analysis).
Figure
S3.
In
planta
formation
of
isotope-labelled
52
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
2,3-
1692
Supplemental Figure S9. Genomic organization of the SARD4 mutant alleles
1693
sard4-5 (orncd1-1; GK_428E01) and sard4-6 (orncd1-2; GK-696E11), and
1694
characterization of sard4 mutant lines.
1695
Supplemental Figure S10. Systemic acquired resistance (SAR) bioassay with the
1696
Col-0 wild-type, the sard4-5 mutant, and the sard4-6 mutant. Disease symptomology
1697
of representative plants.
1698
Supplemental Figure S11. Coupled in vitro assays with Arabidopsis ALD1 and
1699
SARD4, or with ALD1 and the human reductase CRYM yield proline with L-Orn as
1700
substrate.
1701
Supplemental Table S1. ALD1 in vitro activity towards additional amino acids.
1702
Supplemental Table S2. List of primers used in this study.
1703
Supplemental Table S3. Levels of free amino acids in the leaves of Psm-inoculated
1704
and mock-treated Arabidopsis Col-0 plants.
1705
1706
ACKNOWLEDGMENTS
1707
We thank Karin Kiefer and Holger Schnell for excellent technical assistance.
1708
53
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1709
Tables
ALD1 in vitro assay
amino
acid
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1710
L-Lys
rel. vitro
activity
(%)
% transamination
(30 min)
100
77.75
D-Lys
0.1
L,L-DAP
16.3
meso-DAP
12.9
chemical analyses
possible
product oxoacid
(or derived product)
2,3-DP
± 6.58
0.05
in vitro
detection
of product
1
2,3-DP
1
6-carboxy-2,3-DP
2
± 0.01
12.64
± 1.19
10.01
± 1.76
36.31
2
Δ -pyrroline-2carboxylic acid
4-amino-2-oxobutyric acid
yes
GC/MS
procedure
M+
RT
(m/z) (min)
in planta analyses
ion fragments
(m/z)
in planta
detection
Col-0
mock
Col-0
Psm
41
1557*
75
1253*
± 119
± 28
± 12
ald1
mock
39
ald1
Psm
47
A
1a
141
11.6
141, 126, 108
yes
B
1b
255
13.8
255, 169, 127
yes
-
-
-
-
-
-
-
-
±7
± 25
± 240
±4
79
±9
104
no
A, B
yes
A
2a
199
17.3
199, 140, 108
no
nd
nd
nd
nd
yes
B
2b
341
17.3
239, 168, 108
no
nd
nd
nd
nd
yes
B
3b
241
13.0
241, 155, 113
no
nd
nd
nd
nd
no
B
-
-
-
-
-
-
-
-
L-Orn
52.9
2,4-DABA
2.0
L-Met
78.5
61.04
α-ketomethionine
4
yes
A
4a
162
12.0
162, 103, 61
no
nd
nd
nd
nd
L-Leu
57.0
44.34
α-ketoleucine
5
yes
A
5a
144
6.9
144,102, 85
no
nd
nd
nd
nd
L-Ile
0.5
0.36
α-ketoisoleucine
no
A
144
6.8
144, 85, 57
no
nd
nd
nd
nd
L-Val
0.3
0.23
α-ketovaline
yes
A
130
4.9
130, 71, 59
no
nd
nd
nd
nd
L-Phe
10.6
8.25
phenylpyruvic acid
6
yes
A
6a
178
14.2
178, 119, 91
yes
L-Trp
7.6
6.09
indole-3-pyruvic acid
7
yes
A
7a
217
23.3
217, 130, 77
no
L-Glu
12.4
9.61
α-ketoglutaric acid
yes
A
174
12.2
143, 115, 87
yes
L-Aad
11.9
yes
A
188
13.9
157, 129, 101
yes
yes
A
145
15.0
144, 115, 69
no
L-Asn
6.1
± 8.89
1.58
± 0.21
± 7.41
± 1.53
± 0.04
± 0.02
± 0.42
± 0.63
± 0.96
9.23
α-ketoadipic acid
4.72
2-oxosuccinamic acid
± 1.68
± 0.21
3
8
8a
30
79*
37
87*
±8
± 22
±6
± 23
nd
nd
nd
nd
5981
8826*
6507
7806
± 817
± 1064
± 725
± 784
86
147*
± 15
±5
91
181*
nd
nd
nd
nd
± 15
± 39
1711
Table 1. ALD1 catalyzes the transamination of various amino acids other than L-Lys
1712
in vitro but these ALD1-mediated reactions are not detected in planta.
1713
Left: The in vitro activities of the ALD1 aminotransferase towards the indicated amino
1714
acids were assessed by incubation of ALD1 protein with the amino acid substrate
1715
and pyruvate as an acceptor oxoacid for 30 min. The molar concentrations of the
1716
product amino acid Ala and the given substrate amino acid were determined by GC-
1717
MS, and the percentage of transamination calculated by forming the quotient
1718
[Ala]/([substrate amino acid]+[Ala]). The percentage values represent the mean ± SD
1719
of the values from three replicate assays. Relative in vitro activities were calculated
1720
from the transamination percentages by relating the mean transamination values to
1721
the one determined for L-Lys.
1722
Middle: The in vitro formation of product oxoacids or oxoacid-derived compounds
1723
was determined by the indicated GC-MS procedure. Retention times, molecular ions,
1724
and specific ion fragments in the mass spectra of the derivatized products are
1725
indicated. Underlined m/z values represent dominant masses in mass spectra, and
1726
ion chromatograms for the m/z-values indicated in bold have been used for
1727
substance quantification in planta. The molecular structures of several of the non-
1728
derivatized and derivatized products and the mass spectra of the latter are given in
1729
Supplemental Fig. S8, Fig. 1C, Fig. 3C, Fig. 5C, and Fig. 5D.
1730
Right: The detectability of oxoacid-products in leaf extracts from Psm-inoculated or
1731
mock-treated Col-0 and ald1 plants (48 hpi) is indicated. The level of a detected
1732
product in leaf tissue is given in ng g-1 FW (mean ± SD, n = 3); nd: not detected.
1733
Asterisks denote statistically significant differences between Psm- and mock-
1734
samples of a genotype (P < 0.05; two-tailed t test).
1735
The results for further tested amino acid substrates are summarized in Supplemental
1736
Table S1.
1737
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1738
Figure legends
1739
Figure 1. ALD1-mediated L-Lys conversion: formation of 2,3-dehydropipecolic acid
1740
(2,3-DP) by abstraction of the α-NH2 group of L-Lys. The GC-MS (GC-FTIR) analysis
1741
of plant extracts or assay samples included methylation of carboxyl groups as
1742
derivatisation strategy (“procedure A”).
1743
A, Segment of overlaid ion chromatograms (m/z = 108) of GC-MS-analysed extracts
1744
from mock- or Psm-inoculated leaves of Col-0 wild-type and ald1 mutant plants. Leaf
1745
samples were harvested 48 h after inoculation. A molecular species (1a) with m/z
1746
108 (or m/z 126, or m/z 141) is exclusively present in the Col-0 – Psm samples.
1747
B, Segment of overlaid ion chromatograms (m/z = 108) of GC-MS-analysed L-Lys
1748
conversion assays with ALD1 protein, L-Lys conversion assays with LysOx in the
1749
presence of catalase (Supplemental Fig. 2), and leaf extracts from Psm-inoculated
1750
Col-0 plants. Retention times of the molecular species with m/z 108 (or m/z 126, or
1751
m/z 141) in the different samples are identical.
1752
C, Left: mass spectrum of the compound 1a derived from extracts of Psm-inoculated
1753
Col-0 plants (green), ALD1 in vitro assays (blue), and LysOx/catalase in vitro assays
1754
(red) are identical. Right: chemical structure of L-Lys and plausible structures for 1a,
1755
as deduced from the so far collected mass spectrometric information. The molecular
1756
ion (M+) and plausible ion fragments are indicated. The methyl group (red) is
1757
introduced by derivatisation.
1758
D, Use of L-Lys-6-13C,ε-15N as substrate in the ALD1 in vitro assay reveals retention
1759
of the ε-nitrogen and abstraction of the α-nitrogen in the transamination reaction
1760
leading to DP products. Left: mass spectrum of the isotope-labelled product with
1761
shifts in fragmentation pattern by two mass units compared to unlabeled 1a. Right:
1762
isotope-labelled 2,3-DP-methylester is depicted as plausible structure. The same
1763
result was observed for the LysOx/catalase assay.
1764
E, Use of L-Lys-4,4,5,5-d4 as substrate in the ALD1 in vitro assay excludes formation
1765
of DP isomers with double bonds in the 3,4-, 4,5-, or 5,6-position. Left: mass
1766
spectrum of the isotope-labeled product with shifts in fragmentation pattern by four
1767
mass units compared to unlabeled 1a. Right: isotope-labelled 2,3-DP-methylester is
1768
depicted as plausible structure. The same result was observed for the
1769
LysOx/catalase assay.
56
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1770
F, GC-FTIR analysis of 1a indicates its enaminic structure, supports its identity as
1771
methylated 2,3-DP-, and excludes the ketiminic 1,2-DP derivate as a possible
1772
structure. The IR spectrum of 1a is depicted (wavenumbers from 4000 to 600 cm-1).
1773
Assignments of IR absorption bands to functional groups are given in the box on the
1774
right.
1775
1776
Figure 2. Summarized scheme of pipecolic acid biosynthesis from L-Lys in
1777
Arabidopsis.
1778
The biochemical pathway supported by the experimental data of this study is
1779
represented by arrows with solid lines. Detected Lys-derived compounds in extracts
1780
and in vitro assays are framed. Arrows with dashed lines represent likely biochemical
1781
scenarios, supported from literature findings.
1782
Arabidopsis ALD1 first catalyses a transamination step that transfers the α-amino
1783
group of L-Lys to an acceptor oxoacid (preferentially pyruvate). Thereby, ε-amino-α-
1784
ketocaproic acid (KAC) and alanine are formed. A subsequent dehydrative
1785
cyclization of KAC produces the ketimine 1,2-dehydropipecolic acid (1,2-DP) which
1786
isomerizes to the enamine 2,3-dehydropipecolic acid (2,3-DP). 2,3-DP is detected in
1787
plant extracts and in vitro assays as the ALD1-derived product. Arabidopsis SARD4
1788
(alias ORNCD1) then reduces a dehydropipecolic acid isomer to pipecolic acid.
1789
Since the human SARD4 homolog CRYM has been described as a ketimine
1790
reductase, it is possible that the reduction takes place via 1,2-DP which is supposed
1791
to be in chemical equilibrium with 2,3-DP. Alternatively, a direct reduction of the
1792
detected intermediate 2,3-DP might take place. Since the sard4-5 knockout line is
1793
still able to biosynthesize (reduced amounts of) Pip, an alternative reductive
1794
mechanism capable of generating Pip from DP intermediates supposedly exists in
1795
plants. The hypothetical pathway illustrated with dotted arrows (top) proceeding via
1796
abstraction of the ε-amino group, and the formation of α-aminoadipate semialdehyde
1797
(AAS) and 1,6-dehydropipecolic acid (1,6-DP) can be ruled out on the basis of our
1798
data.
1799
57
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1800
Figure 3. ALD1-mediated L-Lys conversion: formation of 2,3-dehydropipecolic acid
1801
as determined by GC-MS analyses using propyl chloroformate derivatization
1802
(“procedure B”).
1803
A, Segment of overlaid ion chromatograms (m/z = 255) of GC-MS-analysed L-Lys
1804
conversion assays with ALD1 protein showing the propyl chloroformate-derivatized
1805
transamination product 1b. Red: ALD1 enzyme assay with L-Lys as amino acid
1806
substrate and pyruvate as acceptor oxoacid. Green: control assay lacking ALD1
1807
protein. Blue: control assay lacking L-Lys as substrate.
1808
B, Segment of overlaid ion chromatograms (m/z = 255) from extract samples of
1809
mock- or Psm-inoculated Col-0 wild-type and ald1 mutant leaves. Leaf samples were
1810
harvested 48h after inoculation. Substance (1b) accumulates in the Col-0 – Psm
1811
samples only.
1812
C, Mass spectrum and molecular structure of substance 1b derived from ALD1-
1813
mediated L-Lys conversion assays. The mass spectral information is consistent with
1814
a 2,3-DP derivative that is propyl carbamylated at its amino group and propyl
1815
esterified at its carboxyl group. The groups introduced by propyl chloroformate
1816
derivatization are labelled in red. The molecular ion (M+) and plausible ion fragments
1817
are indicated.
1818
D, Use of L-Lys-6-13C,ε-15N as substrate in ALD1 in vitro assays results in the
1819
formation of an isotope-labelled variant of 1b with shifts in the mass spectral
1820
fragmentation pattern by two mass units compared to unlabeled 1b.
1821
E, Use of L-Lys-4,4,5,5-d4 as substrate in ALD1 assays results in the formation of an
1822
isotope-labelled variant of 1b with shifts in the fragmentation pattern by four mass
1823
units compared to unlabeled 1b.
1824
F, Quantification of 2,3-DP in leaf extracts from Col-0 and ald1 plants. Leaves were
1825
mock (MgCl2)-infiltrated or Psm-inoculated and harvested at 48 hpi. Bars represent
1826
the mean ± SD of three biological replicate samples, each consisting of six leaves. A
1827
correction factor for the absolute quantification of 2,3-DP was estimated as
1828
described in the method section. Different letters above the bars denote statistically
1829
significant differences (P < 0.001, ANOVA and post-hoc Tukey HSD test).
1830
1831
Figure 4. ALD1 transfers the α-NH2 group from L-Lys to acceptor oxoacids.
58
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1832
A, ALD1 in vitro assay with L-Lys as the substrate amino acid and pyruvate as the
1833
acceptor oxoacid results in the formation of Ala as the product amino acid. The mass
1834
spectrum of Ala after propyl chloroformate-derivatization and the molecular structure
1835
of the product are shown. The M+ ion and the main fragment ion are indicated (dark
1836
red). The groups introduced by propyl chloroformate derivatization are labelled in
1837
blue.
1838
B, ALD1 assay with isotope-labelled L-Lys-α-15N as the amino acid substrate and
1839
pyruvate as the acceptor oxoacid results in the formation of α-15N-labelled Ala. See
1840
A for further details.
1841
C, ALD1 assay with L-Lys as the substrate amino acid and α-ketoglutarate as the
1842
acceptor oxoacid results in the formation of Glu as the product amino acid. See A for
1843
further details.
1844
D, ALD1 assay with isotope-labelled L-Lys-α-15N as the amino acid substrate and
1845
α-ketoglutarate as the acceptor oxoacid results in the formation of α-15N-labelled
1846
Glu. See A for further details.
1847
E, Relative activities of ALD1 towards L-Lys and either glyoxylate (glyo), pyruvate
1848
(pyr), oxaloacetate (oxalo), or α-ketoglutarate (α-KG) as the acceptor oxoacid. The
1849
formation of the corresponding product amino acid (Gly for glyoxylate, Ala for
1850
pyruvate, Asp for oxaloacetate, and Glu for α-ketoglutarate) were determined after
1851
30 min of incubation time for activity assessments.
1852
1853
Figure 5. ALD1 catalyses the conversion of diaminopimelate (DAP) to 6-carboxy-
1854
2,3-dehydropipecolic acid in vitro, but the conversion does not occur to detectable
1855
levels in planta.
1856
A, Reaction scheme of the in vitro conversion of L,L- or meso-DAP to 6-carboxy-2,3-
1857
DP (2) by ALD1.
1858
B, In vitro ALD1 activity assays with L-Lys as the amino acid substrate (red), L,L-
1859
DAP as the amino acid substrate (black), or no amino acid substrate (blue). Pyruvate
1860
served as the acceptor oxoacid in all cases. Overlaid ion chromatograms (m/z 108)
1861
are depicted after applying “workup procedure A”. The m/z 108 ion occurs both in the
1862
L-Lys-derived 2,3-DP product (1a) and in the L,L-DAP-derived 6-carboxy-2,3-DP
1863
product (2a).
59
Downloaded from on June 16, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1864
C, Mass spectrum and chemical structure of 2a obtained from 6-carboxy-2,3-DP (2)
1865
by procedure A. The two methyl groups (blue) are introduced by derivatisation. The
1866
molecular ion (M+), plausible ion fragments and fragment losses are indicated.
1867
D, Mass spectrum and chemical structure of 2b obtained from 6-carboxy-2,3-DP (2)
1868
by procedure B. The propyl- and propyl carbamate-groups introduced by
1869
derivatisation are depicted in blue. The molecular ion (M+), plausible ion fragments
1870
and fragment losses are indicated.
1871
E, Segments of overlaid ion chromatograms from extract samples of Psm-inoculated
1872
Col-0 wild-type leaves, as analysed by GC-MS procedure A. Leaf samples were
1873
harvested 48 h after inoculation. Whereas the presence of L-Lys-derived 2,3-DP (1a)
1874
in the extracts evokes characteristic ion chromatograms (m/z 141, 126, and 108)
1875
with the expected ratios of abundance (Fig. 1C) at a retention time of 12.0 min (left
1876
figure), 6-carboxy-2,3-DP (2a; m/z 199, 140, and 108; Fig. 5C) is not detected at the
1877
supposed retention time of 17.65 min (right figure).
1878
1879
Figure 6. The ald1 mutant is able to convert exogenously supplied 2,3-DP to Pip in
1880
response to P. syringae inoculation.
1881
A, Segment of overlaid ion chromatograms (m/z = 170) from leaf extract samples of
1882
differently treated ald1 mutant plants. Leaves were co-infiltrated with Psm and 2,3-
1883
DP obtained from ALD1 in vitro assays (black; Fig. 3C)). The assays containing
1884
20 mM L-Lys as substrate were run to completion (>99% of lysine used), enzyme-
1885
inactivated, and diluted six-fold with 10 mM MgCl2. The mixture was then supplied
1886
with the bacterial suspension to a final OD600 of 0.005, and infiltrated into three
1887
leaves of five-week-old ald1 plants. 48 h later, the infiltrated leaves were harvested
1888
and processed by analytical procedure B. Co-infiltration of the assay mixture not
1889
containing ALD1 enzyme (green), MgCl2 (mock)-infiltration alone (blue), and Psm-
1890
treatment alone (red) served as the control treatments. The peak at 11.8 min is only
1891
observed in the Psm/2,3-DP sample (black) and corresponds to derivatized Pip.
1892
Similar results were obtained when 2,3-DP generated by the LysOx/catalase assay
1893
was used.
1894
B, Mass spectrum of propyl chloroformate-derivatized Pip (compare Návarová et al.,
1895
2012) derived from 2,3-DP-supplemented and Psm-inoculated ald1 leaves (peak at
1896
11.8 min in Fig. 6A). The groups introduced by derivatization are labelled in red.
60
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1897
C, Segment of overlaid ion chromatograms (m/z = 174) from leaf extract samples of
1898
differently treated ald1 mutant plants. Leaves were co-infiltrated with Psm and d4-
1899
labelled 2,3-DP obtained from ALD1 in vitro assays (black; Fig. 3E), co-infiltrated
1900
with Psm and the assay mixture not containing 2,3-DP (green), infiltrated with mock
1901
(10 mM MgCl2) solution alone (blue), or infiltrated with Psm alone. Further
1902
experimental details are identical those described in A. The peak at 11.76 min only
1903
observed in the Psm/d4-2,3-DP sample (black) corresponds to derivatized d4-Pip.
1904
D, Mass spectrum of propyl chloroformate-derivatized D4-Pip derived from D4-2,3-
1905
DP-supplemented and Psm-inoculated ald1 leaves (peak at 11.76 min in Fig. 6C).
1906
E, Segment of overlaid ion chromatograms (m/z = 174) from leaf extract samples of
1907
differently treated Col-0 wild-type plants. Plants were co-infiltrated in leaves with
1908
5 mM L-Lys-4,4,5,5-d4 (d4-Lys) and Psm (OD600 = 0.005) (black), co-infiltrated with
1909
5 mM d4-Lys and mock (10 mM MgCl2) solution (green), infiltrated with mock solution
1910
alone (blue), or infiltrated with Psm alone. The peak at 11.76 min accumulating in the
1911
Psm/d4-Lys sample (black) corresponds to derivatized d4-Pip. It is noteworthy that, in
1912
addition to labelled Pip, Psm/d4-Lys-treated Col-0 plants also accumulate
1913
endogenous, unlabelled Pip.
1914
F, Mass spectrum of propyl chloroformate-derivatized D4-Pip derived from D4-Lys-
1915
supplemented and Psm-inoculated Col-0 leaves (Fig. 6E).
1916
1917
Figure 7. Coupled assays with ALD1 and the human reductase CRYM or its
1918
Arabidopsis orthologue SARD4 (ORNCD1) yield pipecolic acid in vitro with L-Lys as
1919
substrate.
1920
Reactions were carried out in an assay mix (200 µl) containing purified recombinant
1921
enzymes (ALD1, CRYM and SARD4) at a final concencentration of 100 µg ml-1 with
1922
20 mM L-lysine, 20 mM pyruvate, 5 mM MgCl2, 100 µM pyridoxal-5′-phosphate
1923
(PLP) and 200 µM NADH in 20 mM Tris buffer, pH 8.0. All reactions contained 5%
1924
glycerol for additional enzyme stability and were incubated at 37°C for 16 h. At the
1925
end of the incubation period, the reaction was stopped by inactivating the enzymes
1926
at 85°C for 10 min. The formation of 2,3-DP and Pip was monitored using GC-MS
1927
after derivatization of the assays with trimethylsilyl-diazomethane (procedure A) or
1928
propyl chloroformate (procedure B), respectively. From left to right: no enzyme
1929
(control); ALD1 single enzyme assay, CRYM single enzyme assay, ALD1 and CRYM
61
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1930
co-incubation, SARD4 single enzyme assay; ALD1 and SARD4 co-incubation. The
1931
amounts generated 2,3-P and Pip products are given in nmol. Bars represent the
1932
mean ± SD of three replicate incubations.
1933
1934
Figure 8. Transcript levels of SARD4, and metabolite levels of 2,3-DP and Pip in
1935
inoculated (1°) and distal (2°) leaves of wild-type Col-0, sard4-5, and sard4-6 mutant
1936
plants in response to P. syringae- and mock-treatments.
1937
A, SARD4 transcript levels in 1° leaves of Col-0, sard4-5, and sard4-6 plants
1938
infiltrated with Psm (OD600 = 0.005) or with 10 mM MgCl2 (mock-treatment) at 24 h
1939
post treatment. Transcript values are given in µg µl-1 and represent the mean ± SD of
1940
at least three biological replicate samples from different plants, each replicate
1941
consisting of six leaves. Values for biological replicates were calculated as the mean
1942
of two technical replicates. Different letters above the bars denote statistically
1943
significant differences (P < 0.002, ANOVA and post-hoc Tukey HSD test); n.d. = no
1944
transcripts detected. The results were confirmed in two independent experiments.
1945
B, SARD4 transcript levels in untreated distal (2°) leaves of Col-0, sard4-5, and
1946
sard4-6 plants infiltrated in 1° leaves with Psm (OD600 = 0.005) or 10 mM MgCl2
1947
(mock-treatment) at 48 h post treatment. Other experimental details as described for
1948
Fig. 8A. Different letters above the bars denote statistically significant differences (P
1949
< 0.002, ANOVA and post-hoc Tukey HSD test); n.d. = no transcripts detected. The
1950
results were confirmed in two independent experiments.
1951
C, Levels of Pip in treated 1° leaves of Col-0, sard4-5, and sard4-6 plants infiltrated
1952
with Psm (OD600 = 0.005) or 10 mM MgCl2 (mock-treatment) at 24 h (left graph) and
1953
48 h (middle graph) post treatment, and in untreated distal (2°) leaves at 48 h post
1954
treatment of 1° leaves (right graph). Data represent the mean ± SD of at least three
1955
biological replicates from different plants, each replicate consisting of six leaves from
1956
two plants. Different letters above the bars denote statistically significant differences
1957
(P < 0.05, ANOVA and post-hoc Tukey HSD test). The results were confirmed in two
1958
independent experiments.
1959
D, Levels of 2,3-DP in inoculated (1°) and distal (2°) leaves of Col-0, sard4-5, and
1960
sard4-6 plants. Experimental details as described for Fig. 8C. The results were
1961
confirmed in two independent experiments.
62
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1962
E, Levels of Pip in untreated 2° leaves of Col-0, sard4-5 (s4-5), and sard4-6 (s4-6)
1963
plants infiltrated with Psm (OD600 = 0.005) or 10 mM MgCl2 at 24h, 48h, 72h, and
1964
96h after treatment. Data represent the mean ± SD of three biological replicates.
1965
Asterisks denote statistically significant differences between Psm- and mock-treated
1966
samples of a given plant genotype and point of time (P < 0.01; two-tailed t test).
1967
1968
Figure 9. Basal resistance responses, SAR, Pip-induced resistance and 2,3-DP-
1969
induced resistance in wild-type Col-0, sard4-5, sard4-6 and/or ald1 mutant plants.
1970
A, Basal resistance to P. syringae of Col-0, sard4-5, sard4-6 and ald1 plants. Naïve
1971
plants (three leaves each) were inoculated with bioluminescent Psm lux (OD600 =
1972
0.001), and bacterial growth was assessed 60 h later by luminescence and
1973
expressed as relative light units (rlu) per cm2 leaf area. Data represent the mean ±
1974
SD of the growth values of at least 15 leaf replicates. Different letters above the bars
1975
denote statistically significant differences (P < 0.05, ANOVA and post-hoc Tukey
1976
HSD test). The results were confirmed in three other independent experiments.
1977
B, Systemic acquired resistance in Col-0, sard4-5, and sard4-6 plants.
1978
Three lower, 1° leaves per plant were infiltrated with either 10 mM MgCl2 or Psm
1979
(OD600 = 0.005), and three upper, 2° leaves were challenge-infected with Psm lux
1980
(OD600 = 0.001) two days later. Growth of Psm lux in 2° leaves was assessed 60 h
1981
post 2° challenge-inoculation by luminescence measurements. Experimental details
1982
and statistical analyses as described for Fig. 9A. The results were confirmed in two
1983
other independent experiments.
1984
C, Levels of salicylic acid (SA) in treated 1° leaves of Col-0, sard4-5, and sard4-6
1985
plants infiltrated with Psm (OD 0.005) or 10 mM MgCl2 (mock-treatment) at 24 h (left
1986
graph) and in untreated distal (2°) leaves at 48 h post treatment of 1° leaves (right
1987
graph). Data represent the mean ± SD of at least three biological replicates from
1988
different plants, each replicate consisting of six leaves from two plants. Different
1989
letters above the bars denote statistically significant differences (P < 0.05, ANOVA
1990
and post-hoc Tukey HSD test). The results were confirmed in two other independent
1991
experiments.
1992
D, Pipecolic acid-induced resistance in Col-0, sard4-5, and sard4-6 plants.
63
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
1993
Plants were each supplied with 10 ml of 1 mM Pip (= dose of 10 µmol) or with 10 ml
1994
of water (control treatment) via the root system (Návarová et al., 2012), and three
1995
leaves per plant challenge-infected 1 d later with Psm lux (OD600 = 0.001). Bacterial
1996
growth was assessed 60 h after inoculation and analysed as described for Fig. 9A.
1997
The results were confirmed in two other independent experiments.
1998
E, 2,3-DP-induced resistance in Col-0 and ald1 plants.
1999
Three leaves per plants were co-infiltrated with 2,3-DP (~ 2 mM) obtained by the
2000
ALD1 in vitro assay and Psm lux (OD600 = 0.001), and bacterial growth was
2001
assessed 60 h later. Determination of bacterial growth and statistical analysis were
2002
performed as described above (Fig. 9A). The results were confirmed in two other
2003
independent experiments.
2004
64
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
2005
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1a
abundance (*10 3)
700
B
m/z 108
Col-0 - mock
600
Col-0 - Psm
500
ald1 - mock
400
ald1 – Psm
300
200
plant extract
ALD1 assay
1600
LysOx assay
1200
800
400
0
11.5
11.6 11.7 11.8 11.9 12.0
retention time (min)
C
82
54
100
11.7
12.1
1a
141
126
L-Lys
α
H2N
ε
H2N
108
CO2H
relative abundance (%)
0
54
100
ALD1 assay
82
126
50
141
82 126
108
0
54
100
82
LysOx assay
50
50
D
60
70
relative
abundance
(%)
15
H2 N
90 100 110 120 130 140 150 m/z
80
130
D
D
60
70
80
H 2N
CO2H
ν (cm-1)
3441:
2955:
2854:
1734:
1648:
1264:
O
CH3
O
CH3
O
D
D
86 130
D
90 100 110 120 130 140 150 m/z
1a
O
N
H
M+: 143
D
L-Lys-4,4,5,5-d4
50
100
50
CO2H
D
H 2N
112
N
H
D
145
13
H2 C 15
CH2
H2N
L-Lys-6-13C,ε-15N
86
50
0
84 128
110
100
2,3-DPmethylester
143
13
56
CH3
O
M+: 141
84
50
50
O
plausible structures for 1a
90 100 110 120 130 140 150 m/z
80
N
H
CH3
1,2-DPmethylester
128
0
relative
abundance
(%)
70
55
100
E
60
82 126
or
O
M+: 141
141
108
0
O
N
126
relative absorbance (%)
11.9
retention time (min)
plant extract
50
F
m/z 108
2000
100
0
1a
2400
abundance (*10 3)
A
N
H
O
M+: 145
O
CH3
assignment
N-H (secondary amine, stretching)
C-H (methyl, sym. stretching)
C-H (methylene, sym. stretching)
C=O (ester,conjugated, stretching)
C=C (stretching)
C-O (stretching)
0
4000
3000
2000
1000
wavenumber (cm-1)
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Fig. 1
Figure 1. ALD1-mediated L-Lys conversion: formation of 2,3-dehydropipecolic acid (2,3-DP) by abstraction of the
α-NH2 group of L-Lys. The GC-MS (GC-FTIR) analysis of plant extracts or assay samples included methylation of
carboxyl groups as derivatisation strategy (“procedure A”).
A, Segment of overlaid ion chromatograms (m/z = 108) of GC-MS-analysed extracts from mock- or Psm-inoculated
leaves of Col-0 wild-type and ald1 mutant plants. Leaf samples were harvested 48 h after inoculation. A molecular
species (1a) with m/z 108 (or m/z 126, or m/z 141) is exclusively present in the Col-0 – Psm samples.
B, Segment of overlaid ion chromatograms (m/z = 108) of GC-MS-analysed L-Lys conversion assays with ALD1
protein, L-Lys conversion assays with LysOx in the presence of catalase (Supplemental Fig. 2), and leaf extracts
from Psm-inoculated Col-0 plants. Retention times of the molecular species with m/z 108 (or m/z 126, or m/z 141)
in the different samples are identical.
C, Left: mass spectrum of the compound 1a derived from extracts of Psm-inoculated Col-0 plants (green), ALD1 in
vitro assays (blue), and LysOx/catalase in vitro assays (red) are identical. Right: chemical structure of L-Lys and
plausible structures for 1a, as deduced from the so far collected mass spectrometric information. The molecular ion
(M+) and plausible ion fragments are indicated. The methyl group (red) is introduced by derivatisation.
D, Use of L-Lys-6-13C,ε-15N as substrate in the ALD1 in vitro assay reveals retention of the ε-nitrogen and
abstraction of the α-nitrogen in the transamination reaction leading to DP products. Left: mass spectrum of the
isotope-labelled product with shifts in fragmentation pattern by two mass units compared to unlabeled 1a. Right:
isotope-labelled 2,3-DP-methylester is depicted as plausible structure. The same result was observed for the
LysOx/catalase assay.
E, Use of L-Lys-4,4,5,5-d4 as substrate in the ALD1 in vitro assay excludes formation of DP isomers with double
bonds in the 3,4-, 4,5-, or 5,6-position. Left: mass spectrum of the isotope-labeled product with shifts in
fragmentation pattern by four mass units compared to unlabeled 1a. Right: isotope-labelled 2,3-DP-methylester is
depicted as plausible structure. The same result was observed for the LysOx/catalase assay.
F, GC-FTIR analysis of 1a indicates its enaminic structure, supports its identity as methylated 2,3-DP-, and
excludes the ketiminic 1,2-DP derivate as a possible structure. The IR spectrum of 1a is depicted (wavenumbers
from 4000 to 600 cm-1). Assignments of IR absorption bands to functional groups are given in the box on the right.
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O
ε
H2N
α
H2N
CO2H
H2N
1
CO2H
N
1,6-DP (Δ1-P6C)
AAS
transamination
isomerization
cyclization
ALD1
L-Lys
6
CO2H
- H2O
CO2H
CO2H
O
pyruvate
NH2
H2N
O
KAC
CO2H
1
N
3
1
2
2
N
H
CO2H
1,2-DP (Δ1-P2C)
“ketimine form“
CO2H
2,3-DP (Δ2-P2C)
“enamine form“
Ala
SARD4 (alias ORNCD1)
CRYM
further plant reductase(s)
reduction
NAD(P)H
4
3
5
6
1
N
H
2
CO2H
pipecolic acid
Figure 2. Summarized scheme of pipecolic acid biosynthesis from L-Lys in Arabidopsis.
The biochemical pathway supported by the experimental data of this study is represented by arrows with
solid lines. Detected Lys-derived compounds in extracts and in vitro assays are framed. Arrows with dashed
lines represent likely biochemical scenarios, supported from literature findings.
Arabidopsis ALD1 first catalyses a transamination step that transfers the α-amino group of L-Lys to an
acceptor oxoacid (preferentially pyruvate). Thereby, ε-amino-α-ketocaproic acid (KAC) and alanine are
formed. A subsequent dehydrative cyclization of KAC produces the ketimine 1,2-dehydropipecolic acid (1,2DP) which isomerizes to the enamine 2,3-dehydropipecolic acid (2,3-DP). 2,3-DP is detected in plant
extracts and in vitro assays as the ALD1-derived product. Arabidopsis SARD4 (alias ORNCD1) then reduces
a dehydropipecolic acid isomer to pipecolic acid. Since the human SARD4 homolog CRYM has been
described as a ketimine reductase, it is possible that the reduction takes place via 1,2-DP which is supposed
to be in chemical equilibrium with 2,3-DP. Alternatively, a direct reduction of the detected intermediate 2,3DP might take place. Since the sard4-5 knockout line is still able to biosynthesize (reduced amounts of) Pip,
an alternative reductive mechanism capable of generating Pip from DP intermediates supposedly exists in
plants. The hypothetical pathway illustrated with dotted arrows (top) proceeding via abstraction of the εamino group, and the formation of α-aminoadipate semialdehyde (AAS) and 1,6-dehydropipecolic acid (1,6DP) can be ruled out on the basis of our data.
Fig. 2
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B
ALD1 full assay with L-Lys
control assay (no substrate)
control assay (no enzyme)
400
MgCl2
1b
20
abundance (*10 3)
200
m/z 255
a
10
1.2
1.0
0.8
0.6
0.4
0.2
0
0
13.2
Psm
1.6
1.4
1b
m/z 255
abundance (*10 3)
F
Col-0 - mock
Col-0 - Psm
ald1 - mock
ald1 - Psm
2,3-DP (µg g-1 FW)
A
13.4
13.6
b
b
0.0
13.3 13.4 13.5 13.6
13.8
b
Col-0
ald1
retention time (min)
C
82
relative abundance (%)
+H
100
50
0
127
82
169
169
+H
154
O
109
54
60
140
80
100
120
182
140
O
N
160 180
O
1b
O
Pr
Pr
M+
255
196
200
220
D
260 m/z
240
84
relative abundance (%)
+H
171
100
129
84
156
+H
111
50
55
0
13
60
142
80
100
120
140
160 180
relative abundance (%)
173
131
158
220
D
O
Pr 257
260 m/z
240
86
+H
N
+H
O
O
O
Pr
O
Pr
M+
259
240
260 m/z
113
56
60
O
D
D
173
86
0
O
200
D
50
Pr
M+
184 198
E
100
O
H2 C 15
N
171
144
80
100
120
140
186 200
160 180
200
220
Fig. 3
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Figure 3. ALD1-mediated L-Lys conversion: formation of 2,3-dehydropipecolic acid as determined by GC-MS
analyses using propyl chloroformate derivatization (“procedure B”).
A, Segment of overlaid ion chromatograms (m/z = 255) of GC-MS-analysed L-Lys conversion assays with ALD1
protein showing the propyl chloroformate-derivatized transamination product 1b. Red: ALD1 enzyme assay with LLys as amino acid substrate and pyruvate as acceptor oxoacid. Green: control assay lacking ALD1 protein. Blue:
control assay lacking L-Lys as substrate.
B, Segment of overlaid ion chromatograms (m/z = 255) from extract samples of mock- or Psm-inoculated Col-0
wild-type and ald1 mutant leaves. Leaf samples were harvested 48h after inoculation. Substance (1b) accumulates
in the Col-0 – Psm samples only.
C, Mass spectrum and molecular structure of substance 1b derived from ALD1-mediated L-Lys conversion assays.
The mass spectral information is consistent with a 2,3-DP derivative that is propyl carbamylated at its amino group
and propyl esterified at its carboxyl group. The groups introduced by propyl chloroformate derivatization are
labelled in red. The molecular ion (M+) and plausible ion fragments are indicated.
D, Use of L-Lys-6-13C,ε-15N as substrate in ALD1 in vitro assays results in the formation of an isotope-labelled
variant of 1b with shifts in the mass spectral fragmentation pattern by two mass units compared to unlabeled 1b.
E, Use of L-Lys-4,4,5,5-d4 as substrate in ALD1 assays results in the formation of an isotope-labelled variant of 1b
with shifts in the fragmentation pattern by four mass units compared to unlabeled 1b.
F, Quantification of 2,3-DP in leaf extracts from Col-0 and ald1 plants. Leaves were mock (MgCl2)-infiltrated or
Psm-inoculated and harvested at 48 hpi. Bars represent the mean ± SD of three biological replicate samples, each
consisting of six leaves. A correction factor for the absolute quantification of 2,3-DP was estimated as described in
the method section. Different letters above the bars denote statistically significant differences (P < 0.001, ANOVA
and post-hoc Tukey HSD test).
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A
relative abundance (%)
relative abundance (%)
B
relative abundance (%)
relative abundance (%)
C
D
130
100
E
O
H3C
N
O
0
88
70
50
70
90
116
Pr
M+
217
158
110 130 150 170 190 210
131
100
O
H 3C
15
O
50
O
71
0
50
70
90
117
15
N
L-Lys-α-15N H
+
pyruvate
H
Pr
110 130 150 170 190 210
230
O
Pr
O
O
O
230
142 170
N
O
O
M+
317
258
60
100
100
140
180
220
260
O
O
15
O
O
259
0
60
100
140
180
220
260
O
231
143 171
50
Pr
O
Pr
L-Lys
+
α-ketoglutarate
300
231
85
Pr
H
50
0
N H
M+
218
159
84
100
CO2H
H2N
Pr
O
131
89
L-Lys
+
pyruvate
H
50
O
Pr
O
130
M+
318
N
Pr
H
L-Lys-α-15N
+
α-ketoglutarate
Pr
300
relative activity with ALD1 [%]
120
100
80
60
40
20
0
glyo
pyr
oxalo
α-KG
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Fig. 4
Figure 4. ALD1 transfers the α-NH2 group from L-Lys to acceptor oxoacids.
A, ALD1 in vitro assay with L-Lys as the substrate amino acid and pyruvate as the acceptor oxoacid results in the
formation of Ala as the product amino acid. The mass spectrum of Ala after propyl chloroformate-derivatization and
the molecular structure of the product are shown. The M+ ion and the main fragment ion are indicated (dark red).
The groups introduced by propyl chloroformate derivatization are labelled in blue.
B, ALD1 assay with isotope-labelled L-Lys-α-15N as the amino acid substrate and pyruvate as the acceptor oxoacid
results in the formation of α-15N-labelled Ala. See A for further details.
C, ALD1 assay with L-Lys as the substrate amino acid and α-ketoglutarate as the acceptor oxoacid results in the
formation of Glu as the product amino acid. See A for further details.
D, ALD1 assay with isotope-labelled L-Lys-α-15N as the amino acid substrate and α-ketoglutarate as the acceptor
oxoacid results in the formation of α-15N-labelled Glu. See A for further details.
E, Relative activities of ALD1 towards L-Lys and either glyoxylate (glyo), pyruvate (pyr), oxaloacetate (oxalo), or αketoglutarate (α-KG) as the acceptor oxoacid. The formation of the corresponding product amino acid (Gly for
glyoxylate, Ala for pyruvate, Asp for oxaloacetate, and Glu for α-ketoglutarate) were determined after 30 min of
incubation time for activity assessments.
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A
HO2C
CO2H
NH2
ALD1 (in vitro)
HO2C
NH2
B
relative abundance (%)
DAP
(L,L- or meso-)
100
2,3-DP
(1a)
relative abundance (%)
11
relative abundance (%)
12
13
100
14
15
16
140
108
17
O
H3C
- CH3OH
80
100 120
140
160 180
200 m/z
168
Pr
108
- PrOH
254
0
126
O
254
100
140
180
2,3-DP
(1a)
140
220
O
O
O
Pr
2b
Pr
M+
341
281
260
O
N
- PrOH
53
60
O
239
197
80
2a
199
+H
50
CH3
M+
168
100
O
167 184
124
60
O
N
H
O
53
RT (min)
19
140
80
50
18
184
- CH3OH
E
abundance (*10 3)
6-carboxy2,3-DP
(2a)
m/z 108
50
0
D
6-carboxy-2,3-DP (2)
L-Lys
L,L-DAP
no substrate
0
C
in planta
CO2H
N
H
300
340 m/z
140
120
m/z
120
m/z
100
141
126
108
100
199
140
108
80
60
80
60
40
40
20
20
0
11.6
12.0
12.4
0
expected
6-Carboxy-2,3-DP
17.2
17.6
18.0
18.4 RT (min)
Fig. 5
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Figure 5. ALD1 catalyses the conversion of diaminopimelate (DAP) to 6-carboxy-2,3-dehydropipecolic acid in vitro,
but the conversion does not occur to detectable levels in planta.
A, Reaction scheme of the in vitro conversion of L,L- or meso-DAP to 6-carboxy-2,3-DP (2) by ALD1.
B, In vitro ALD1 activity assays with L-Lys as the amino acid substrate (red), L,L-DAP as the amino acid substrate
(black), or no amino acid substrate (blue). Pyruvate served as the acceptor oxoacid in all cases. Overlaid ion
chromatograms (m/z 108) are depicted after applying “workup procedure A”. The m/z 108 ion occurs both in the LLys-derived 2,3-DP product (1a) and in the L,L-DAP-derived 6-carboxy-2,3-DP product (2a).
C, Mass spectrum and chemical structure of 2a obtained from 6-carboxy-2,3-DP (2) by procedure A. The two
methyl groups (blue) are introduced by derivatisation. The molecular ion (M+), plausible ion fragments and fragment
losses are indicated.
D, Mass spectrum and chemical structure of 2b obtained from 6-carboxy-2,3-DP (2) by procedure B. The propyland propyl carbamate-groups introduced by derivatisation are depicted in blue. The molecular ion (M+), plausible
ion fragments and fragment losses are indicated.
E, Segments of overlaid ion chromatograms from extract samples of Psm-inoculated Col-0 wild-type leaves, as
analysed by GC-MS procedure A. Leaf samples were harvested 48 h after inoculation. Whereas the presence of LLys-derived 2,3-DP (1a) in the extracts evokes characteristic ion chromatograms (m/z 141, 126, and 108) with the
expected ratios of abundance (Fig. 1C) at a retention time of 12.0 min (left figure), 6-carboxy-2,3-DP (2a; m/z 199,
140, and 108; Fig. 5C) is not detected at the supposed retention time of 17.65 min (right figure).
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A
B
100
relative abundance (%)
abundance (*10 3)
ald1 – Psm + 2,3-DP
ald1 – Psm + control
ald1 – mock
ald1 – Psm
150
50
0
11.7
170
100
m/z 170
11.9
O
D
M+
257
198
60
80 100 120 140 160 180 200 220 240 260
50
174
100
relative abundance (%)
abundance (*10 3)
ald1 – Psm + d4-2,3-DP
ald1 – Psm + control
ald1 – mock
ald1 – Psm
100
D
D
D
D
O
N
50
132
O
O
11.7
11.9
M+
0
12.1
60
m/z
F
m/z 174
174
100
Col-0 – mock + d4-Lys
Col-0 – Psm + d4-Lys
Col-0 – mock
Col-0 – Psm
150
100
50
relative abundance (%)
abundance (*10 3)
250
200
D
11.7
11.9
12.1
D
D
D
O
N
132
50
O
88
O
Pr
O
Pr
M+
202
0
261
80 100 120 140 160 180 200 220 240 260
retention time (min)
E
Pr
O
Pr
88
202
0
O
Pr
m/z
m/z 174
150
Pr
84
retention time (min)
C
O
128
50
0
12.1
O
N
0
60
261
80 100 120 140 160 180 200 220 240 260
retention time (min)
Fig. 6
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Figure 6. The ald1 mutant is able to convert exogenously supplied 2,3-DP to Pip in response to P. syringae
inoculation.
A, Segment of overlaid ion chromatograms (m/z = 170) from leaf extract samples of differently treated ald1 mutant
plants. Leaves were co-infiltrated with Psm and 2,3-DP obtained from ALD1 in vitro assays (black; Fig. 3C)). The
assays containing 20 mM L-Lys as substrate were run to completion (>99% of lysine used), enzyme-inactivated,
and diluted six-fold with 10 mM MgCl2. The mixture was then supplied with the bacterial suspension to a final OD600
of 0.005, and infiltrated into three leaves of five-week-old ald1 plants. 48 h later, the infiltrated leaves were
harvested and processed by analytical procedure B. Co-infiltration of the assay mixture not containing ALD1
enzyme (green), MgCl2 (mock)-infiltration alone (blue), and Psm-treatment alone (red) served as the control
treatments. The peak at 11.8 min is only observed in the Psm/2,3-DP sample (black) and corresponds to
derivatized Pip. Similar results were obtained when 2,3-DP generated by the LysOx/catalase assay was used.
B, Mass spectrum of propyl chloroformate-derivatized Pip (compare Návarová et al., 2012) derived from 2,3-DPsupplemented and Psm-inoculated ald1 leaves (peak at 11.8 min in Fig. 6A). The groups introduced by
derivatization are labelled in red.
C, Segment of overlaid ion chromatograms (m/z = 174) from leaf extract samples of differently treated ald1 mutant
plants. Leaves were co-infiltrated with Psm and d4-labelled 2,3-DP obtained from ALD1 in vitro assays (black; Fig.
3E), co-infiltrated with Psm and the assay mixture not containing 2,3-DP (green), infiltrated with mock (10 mM
MgCl2) solution alone (blue), or infiltrated with Psm alone. Further experimental details are identical those
described in A. The peak at 11.76 min only observed in the Psm/d4-2,3-DP sample (black) corresponds to
derivatized d4-Pip.
D, Mass spectrum of propyl chloroformate-derivatized D4-Pip derived from D4-2,3-DP-supplemented and Psminoculated ald1 leaves (peak at 11.76 min in Fig. 6C).
E, Segment of overlaid ion chromatograms (m/z = 174) from leaf extract samples of differently treated Col-0 wildtype plants. Plants were co-infiltrated in leaves with 5 mM L-Lys-4,4,5,5-d4 (d4-Lys) and Psm (OD600 = 0.005)
(black), co-infiltrated with 5 mM d4-Lys and mock (10 mM MgCl2) solution (green), infiltrated with mock solution
alone (blue), or infiltrated with Psm alone. The peak at 11.76 min accumulating in the Psm/d4-Lys sample (black)
corresponds to derivatized d4-Pip. It is noteworthy that, in addition to labelled Pip, Psm/d4-Lys-treated Col-0 plants
also accumulate endogenous, unlabelled Pip.
F, Mass spectrum of propyl chloroformate-derivatized D4-Pip derived from D4-Lys-supplemented and Psminoculated Col-0 leaves (Fig. 6E).
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nmol of generated product
100
90
2,3-DP
80
Pip
70
60
50
40
30
20
10
0
n.d.
control
n.d.
ALD1
CRYM
n.d.
ALD1 +
CRYM
SARD4
ALD1 +
SARD4
Figure 7. Coupled assays with ALD1 and the human reductase CRYM or its Arabidopsis orthologue SARD4
(ORNCD1) yield pipecolic acid in vitro with L-Lys as substrate.
Reactions were carried out in an assay mix (200 µl) containing purified recombinant enzymes (ALD1, CRYM and
SARD4) at a final concencentration of 100 µg ml-1 with 20 mM L-lysine, 20 mM pyruvate, 5 mM MgCl2, 100 µM
pyridoxal- 5′- phosphate (PLP) and 200 µM NADH in 20 mM Tris buffer, pH 8.0. All reactions contained 5% glycerol
for additional enzyme stability and were incubated at 37°C for 16 h. At the end of the incubation period, the reaction
was stopped by inactivating the enzymes at 85°C for 10 min. The formation of 2,3-DP and Pip was monitored using
GC-MS after derivatization of the assays with trimethylsilyl-diazomethane (procedure A) or propyl chloroformate
(procedure B), respectively. From left to right: no enzyme (control); ALD1 single enzyme assay, CRYM single
enzyme assay, ALD1 and CRYM co-incubation, SARD4 single enzyme assay; ALD1 and SARD4 co-incubation.
The amounts generated 2,3-P and Pip products are given in nmol (n.d.: not detected). Bars represent the mean ±
SD of three replicate incubations.
Fig. 7
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E
B
distal (2°) leaves
48 hpi
inoculated (1°) leaves
24 hpi
a
MgCl2
Psm
9
8
7
6
5
4
b
3
2
c
1
c
n.d.
Col-0
sard
4-5
sard
4-6
1°: MgCl2
1°: Psm
4
3
2
1
c
0
n.d.
Col-0
sard
4-5
c
b
s4-6 – 1° Psm *
8
*
6
*
4
*
2
sard
4-6
*
*
*
*
0
24 hpi
C
48hpi
72 hpi
96 hpi
inoculated (1°) leaves
45
24 hpi
a
40
b
25
20
15
10
5
0
d
e
Col-0
c
sard
4-5
D
sard
4-6
a
12
100
10
80
60
6
40
4
0
Col-0
1°: MgCl2
1°: Psm
8
b
d
a
14
120
20
d
16
a
140
30
distal (2°) leaves, 48 hpi
48 hpi
160
35
Pip (µg g-1 FW)
10
Pip in 2° leaves (µg g-1 FW)
a
10
0
2
c
d
sard
4-5
sard
4-6
0
b
c,d
d c,d
Col-0
sard
4-5
c
sard
4-6
inoculated (1°) leaves
24 hpi
16
a
14
10
6
8
4
c
Col-0
1.5
4
2
c
d
sard
4-5
sard
4-6
0
a,b
1.0
6
b
1°: MgCl2
1°: Psm
2.0
12
8
a
2.5
16
10
0
3.0
a
18
12
2
distal (2°) leaves, 48 hpi
48 hpi
20
a
14
2,3-DP (µg g-1 FW)
12
SARD4 transcript levels (ng µl-1)
SARD4 transcript levels (ng µl-1)
A
Col-0 – 1° MgCl2
Col-0 – 1° Psm
s4-5 – 1° MgCl2
s4-5 – 1° Psm
s4-6 – 1° MgCl2
b
b
c
Col-0
d
c
sard
4-5
sard
4-6
b,c
0.5
d
0.0
Col-0
c
sard
4-5
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
d
sard
4-6
Fig. 8
Figure 8. Transcript levels of SARD4, and metabolite levels of 2,3-DP and Pip in inoculated (1°) and distal (2°)
leaves of wild-type Col-0, sard4-5, and sard4-6 mutant plants in response to P. syringae- and mock-treatments.
A, SARD4 transcript levels in 1° leaves of Col-0, sard4-5, and sard4-6 plants infiltrated with Psm (OD600 = 0.005) or
with 10 mM MgCl2 (mock-treatment) at 24 h post treatment. Transcript values are given in µg µl-1 and represent the
mean ± SD of at least three biological replicate samples from different plants, each replicate consisting of six
leaves. Values for biological replicates were calculated as the mean of two technical replicates. Different letters
above the bars denote statistically significant differences (P < 0.002, ANOVA and post-hoc Tukey HSD test); n.d. =
no transcripts detected. The results were confirmed in two independent experiments.
B, SARD4 transcript levels in untreated distal (2°) leaves of Col-0, sard4-5, and sard4-6 plants infiltrated in 1°
leaves with Psm (OD600 = 0.005) or 10 mM MgCl2 (mock-treatment) at 48 h post treatment. Other experimental
details as described for Fig. 8A. Different letters above the bars denote statistically significant differences (P <
0.002, ANOVA and post-hoc Tukey HSD test); n.d. = no transcripts detected. The results were confirmed in two
independent experiments.
C, Levels of Pip in treated 1° leaves of Col-0, sard4-5, and sard4-6 plants infiltrated with Psm (OD600 = 0.005) or 10
mM MgCl2 (mock-treatment) at 24 h (left graph) and 48 h (middle graph) post treatment, and in untreated distal (2°)
leaves at 48 h post treatment of 1° leaves (right graph). Data represent the mean ± SD of at least three biological
replicates from different plants, each replicate consisting of six leaves from two plants. Different letters above the
bars denote statistically significant differences (P < 0.05, ANOVA and post-hoc Tukey HSD test). The results were
confirmed in two independent experiments.
D, Levels of 2,3-DP in inoculated (1°) and distal (2°) leaves of Col-0, sard4-5, and sard4-6 plants. Experimental
details as described for Fig. 8C. The results were confirmed in two independent experiments.
E, Levels of Pip in untreated 2° leaves of Col-0, sard4-5 (s4-5), and sard4-6 (s4-6) plants infiltrated with Psm
(OD600 = 0.005) or 10 mM MgCl2 at 24h, 48h, 72h, and 96h after treatment. Data represent the mean ± SD of three
biological replicates. Asterisks denote statistically significant differences between Psm- and mock-treated samples
of a given plant genotype and point of time (P < 0.01; two-tailed t test).
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A
B
1°: MgCl2
1°: Psm
a
100
10
Col-0
C
sard
4-5
inoculated (1°) leaves
24 hpi
7
a
a
a
SA (µg g-1 FW)
SA (µg
g-1 FW)
4
3
2
0
b
soil: H2O
soil: Pip
a
1.5
1.0
c
d
c
c
10
Col-0
sard
4-5
d
d
Col-0
sard
4-5
sard
4-6
E
sard
4-6
bacterial numbers at 60 hpi
Psm lux (rlu cm-2 * 10-3)
bacterial numbers at 60 hpi
c
1
1°: MgCl2
1°: Psm
2.0
control
2,3-DP
a
1000
b
sard
4-6
b
b
100
sard
4-5
Col-0
distal (2°) leaves
48 hpi
a
0.0
sard
4-6
D
1000
10
1
0.5
b
sard
4-5
Col-0
e
2.5
5
b
e
3.0
6
1
d
100
ald1
sard
4-6
b
c
Psm lux (rlu cm-2 * 10-3)
1
a
1000
Psm lux (rlu cm-2 * 10-3)
bacterial numbers, 2° leaves, 60 hpi
Psm lux (rlu cm-2 * 10-3)
bacterial numbers at 60 hpi
b,c
c
18
15
b
1000
16
b
b
c
100
10
1
Col-0
ald1
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Fig. 9
Figure 9. Basal resistance responses, SAR, Pip-induced resistance and 2,3-DP-induced resistance in wild-type
Col-0, sard4-5, sard4-6 and/or ald1 mutant plants.
A, Basal resistance to P. syringae of Col-0, sard4-5, sard4-6 and ald1 plants. Naïve plants (three leaves each)
were inoculated with bioluminescent Psm lux (OD600 = 0.001), and bacterial growth was assessed 60 h later by
luminescence and expressed as relative light units (rlu) per cm2 leaf area. Data represent the mean ± SD of the
growth values of at least 15 leaf replicates. Different letters above the bars denote statistically significant
differences (P < 0.05, ANOVA and post-hoc Tukey HSD test). The results were confirmed in three other
independent experiments.
B, Systemic acquired resistance in Col-0, sard4-5, and sard4-6 plants.
Three lower, 1° leaves per plant were infiltrated with either 10 mM MgCl2 or Psm (OD600 = 0.005), and three upper,
2° leaves were challenge-infected with Psm lux (OD600 = 0.001) two days later. Growth of Psm lux in 2° leaves was
assessed 60 h post 2° challenge-inoculation by luminescence measurements. Experimental details and statistical
analyses as described for Fig. 9A. The results were confirmed in two other independent experiments.
C, Levels of salicylic acid (SA) in treated 1° leaves of Col-0, sard4-5, and sard4-6 plants infiltrated with Psm (OD
0.005) or 10 mM MgCl2 (mock-treatment) at 24 h (left graph) and in untreated distal (2°) leaves at 48 h post
treatment of 1° leaves (right graph). Data represent the mean ± SD of at least three biological replicates from
different plants, each replicate consisting of six leaves from two plants. Different letters above the bars denote
statistically significant differences (P < 0.05, ANOVA and post-hoc Tukey HSD test). The results were confirmed in
two other independent experiments.
D, Pipecolic acid-induced resistance in Col-0, sard4-5, and sard4-6 plants.
Plants were each supplied with 10 ml of 1 mM Pip (= dose of 10 µmol) or with 10 ml of water (control treatment) via
the root system (Návarová et al., 2012), and three leaves per plant challenge-infected 1 d later with Psm lux (OD600
= 0.001). Bacterial growth was assessed 60 h after inoculation and analysed as described for Fig. 9A. The results
were confirmed in two other independent experiments.
E, 2,3-DP-induced resistance in Col-0 and ald1 plants.
Three leaves per plants were co-infiltrated with 2,3-DP (~ 2 mM) obtained by the ALD1 in vitro assay and Psm lux
(OD600 = 0.001), and bacterial growth was assessed 60 h later. Determination of bacterial growth and statistical
analysis were performed as described above (Fig. 9A). The results were confirmed in two other independent
experiments.
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