Plant Physiology Preview. Published on May 15, 2017, as DOI:10.1104/pp.17.00481
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NADPH thioredoxin reductase C and thioredoxins act concertedly in seedling
development
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Valle Ojeda1, Juan Manuel Pérez-Ruiz1*, Maricruz González1, Victoria A. Nájera1,
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Mariam Sahrawy2, Antonio J. Serrato2, Peter Geigenberger3 and Francisco Javier
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Cejudo1*
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Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla and Consejo
Superior de Investigaciones Científicas, 41092-Sevilla, Spain;
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Department of
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Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín,
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Consejo
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Maximilians-Universität München, Department Biologie I, 82152 Martinsried,
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Germany
Superior de Investigaciones
Científicas, Granada,
Spain;
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Ludwig-
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*Corresponding authors:
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Francisco Javier Cejudo ([email protected])
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Juan Manuel Pérez-Ruiz ([email protected])
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Short title: The concerted action of f- and x-type Trxs and NTRC
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One Sentence Summary
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NADPH thioredoxin reductase C plays a pivotal role required for the function of Trxs x
and f and is essential for early seedling development.
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Author contributions
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J.M.P-R. and F.J.C. designed the study. V.O. performed most of the experiments; M.G.
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and V.A.N. performed microscopy analyses; M.S. and A.J.S. performed FBPase in vitro
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studies. J.M.P-R., P.G. and F.J.C. analyzed data. F.J.C. wrote the paper. J.M.P-R. and
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P.G. revised the manuscript. All authors read and approved final manuscript.
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Copyright 2017 by the American Society of Plant Biologists
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Financial support
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This work was supported by European Regional Development Fund-cofinanced grant
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(BIO2013-43556-P) from the Spanish Ministry of Innovation and Competiveness
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(MINECO) and Grant CVI-5919 from Junta de Andalucía, Spain. V.O. was recipient of
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a pre-doctoral fellowship from MINECO. P.G. gratefully acknowledges support from
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the Deutsche Forschungsgemeinschaft (SFB-TR175 B02).
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Keywords: Chloroplast, NTRC, Thioredoxin f, Thiredoxin x, Redox regulation,
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Seedling
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Journal research area: Biochemistry and Metabolism
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ABSTRACT
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Thiol-dependent redox regulation of enzyme activity plays a central role in the rapid
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acclimation of chloroplast metabolism to ever fluctuating light availability. This
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regulatory mechanism relies on ferredoxin (Fdx) reduced by the photosynthetic electron
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transport chain, which fuels reducing power to thioredoxins (Trxs) via a Fdx-dependent
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Trx reductase (FTR). In addition, chloroplasts harbour an NADPH-dependent Trx
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reductase, which has a joint Trx domain at the C-terminus, termed NTRC. Thus, a
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relevant issue concerning chloroplast function is to establish the relationship between
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these two redox systems and its impact on plant development. To address this issue we
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generated Arabidopsis thaliana mutants combining the deficiency of NTRC with those
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of Trxs f, which participate in metabolic redox regulation, and Trx x, which has
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antioxidant function. The ntrc-trxf1f2 and, to a lower extent, the ntrc-trxx mutants
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showed severe growth retarded phenotypes, decreased photosynthesis performance and
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almost abolished light-dependent reduction of fructose-1,6-bisphosphatase. Moreover,
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the combined deficiency of both redox systems provokes aberrant chloroplast
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ultrastructure. Remarkably, both the ntrc-trxf1f2 and the ntrc-trxx mutants showed high
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mortality at the seedling stage, which was overcome by addition of an exogenous
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carbon source. Based on these results, we propose that NTRC plays a pivotal role in
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chloroplast redox regulation, being necessary for the activity of diverse Trxs with
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unrelated functions. The interaction between the two thiol-redox systems is
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indispensable to sustain photosynthesis performed by cotyledons chloroplasts, which is
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essential for early plant development.
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INTRODUCTION
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Photosynthesis is a central feature of chloroplasts allowing the use of light and water for
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the production of organic material, thus being the primary source of biomass and
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oxygen in the biosphere. Beside their essential function as factories of metabolic
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precursors, chloroplasts play also a relevant role harmonizing the growth of the different
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organs of the plant as well as its acclimation to changing environmental conditions.
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Therefore, chloroplast biogenesis and function are deeply integrated with plant growth
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and development (Inaba and Ito-Inaba, 2010; Jarvis and Lopez-Juez, 2013). A classic
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example of this integration occurs at the seedling stage, when true leaves are generated
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(Waters and Langdale, 2009; Pogson et al., 2015). To fulfil these functions, chloroplast
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metabolism needs to rapidly adjust to unpredictable changes of light availability. In this
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regard, thiol-dependent redox regulation of enzyme activity plays a relevant role
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(Couturier et al., 2013; Balsera et al., 2014), the disulphide reductase activity of
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thioredoxins (Trxs) being central for this regulatory mechanism (Meyer et al., 2012;
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Serrato et al., 2013).
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Trx-dependent redox regulation is universally found in any kind of organisms
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from bacteria and yeasts to plants and animals. In heterotrophic organisms and non-
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photosynthetic compartments of plant cells, the reducing power for Trx reduction is
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provided by NADPH in a reaction catalysed by an NADPH-dependent Trx reductase
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(NTR) (Jacquot et al., 2009). In contrast, redox regulation in plant chloroplasts has
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remarkable specific features. First, these organelles harbour a rather complex set of Trxs
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(Meyer et al., 2012; Balsera et al., 2014), while in heterotrophic organisms Trx and
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NTR are encoded by small gene families. Moreover, the source of reducing power for
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Trx reduction in chloroplasts is not NADPH, as in heterotrophic organisms, but reduced
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ferredoxin (Fdx), hence chloroplasts are equipped with a Fdx-dependent Trx reductase
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(FTR) (Schurmann and Buchanan, 2008), which is specific of these organelles. Based
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on the function of the FTR/Trx redox system, the classic view of redox regulation of
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chloroplast metabolism states that metabolic pathways such as carbon assimilation by
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the Calvin-Benson cycle are active during the day due to the reductive activation of
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enzymes of the cycle, which relies on Fdx reduced by the photosynthetic electron
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transport chain, thus linking metabolic regulation to light (Michelet et al., 2013; Serrato
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et al., 2013).
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The view of chloroplast redox regulation based on the action of the Fdx-
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dependent FTR/Trx system was modified after the discovery of a novel type of NTR
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with a joint Trx domain at the C-terminus, which was termed NTRC (Serrato et al.,
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2004). NTRC is exclusive of organisms that perform oxygenic photosynthesis and
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shows plastid localization in plants (Kirchsteiger et al., 2012). Moreover, biochemical
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analyses revealed that NTRC is able to conjugate both NTR and Trx activities to
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efficiently reduce the H2O2 scavenging enzyme 2-Cys peroxiredoxin (2-Cys Prx) (Moon
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et al., 2006; Perez-Ruiz et al., 2006; Alkhalfioui et al., 2007). Arabidopsis NTRC
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knockout mutants show a characteristic phenotype of retarded growth and pale green
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leaves, this phenotype being more severe under short-day conditions (Perez-Ruiz et al.,
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2006; Lepisto et al., 2009). Based on these results it was initially proposed that NTRC
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constitutes an NADPH-dependent redox system which might have a complementary
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function to the Fdx-dependent FTR/Trx system. Further evidence showed the
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participation of NTRC in redox regulation of enzymes which were previously known to
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be Trx-regulated. This is the case of ADP-glucose pyrophosphorylase (AGPase), a key
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enzyme of starch biosynthesis (Michalska et al., 2009; Lepisto et al., 2013), and of
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enzymes of the chlorophyll biosynthesis pathway (Richter et al., 2013; Perez-Ruiz et al.,
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2014). These results suggest that chloroplast redox regulation depends on the cross-talk
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of both Fdx/FTR/Trx and NTRC redox systems, hence being more complex than
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previously anticipated.
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It was recently reported that a double mutant of Arabidopsis devoid of NTRC
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and Trx f1, which is the most abundant f-type Trx, shows a very severe growth
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inhibition phenotype and an almost complete abolishment of light activation of fructose-
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1,6-bisphosphatase (FBPase), a redox-regulated enzyme of the Calvin-Benson cycle
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(Thormahlen et al., 2015). Furthermore, the Arabidopsis double mutant deficient in
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NTRC and FTR displays a lethal phenotype under autotrophic growth conditions
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(Yoshida and Hisabori, 2016) suggesting that NTRC and the Fdx/FTR/Trx systems act
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concertedly in redox regulation of common targets in planta. Bifluorescence
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complementation (BiFC) assays showing that NTRC interacts in vivo with well-
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established Trx-regulated enzymes (Nikkanen et al., 2016) further supports this notion.
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The question arising is whether the effect of NTRC is exerted only on Trxs such as
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those of the f type, which participate in redox regulation of metabolic pathways, or
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affects the activity of other types of Trxs. To address this issue, we have analysed the
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genetic interaction of NTRC with two functionally unrelated chloroplast Trxs: f-type, as
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representative of Trxs involved in redox regulation of metabolic pathways (Michelet et
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al., 2013), and x-type, as representative of Trxs involved in antioxidant defence (Collin
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et al., 2003). The Arabidopsis ntrc-trxx double mutant and the ntrc-trxf1f2 triple mutant
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were generated. While the Trx-deficient parental lines, trxf1f2 and trxx, showed a
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visible phenotype similar to the wild type plants when grown under long day conditions,
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these deficiencies caused a severe growth inhibition phenotype when combined with the
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lack of NTRC. Moreover, the ntrc-trxx mutant and, to a higher extent, the ntrc-trxf1f2
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mutant showed a massive mortality at the seedling stage. These results reveal that
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NTRC is essential for the activity of unrelated Trxs, thus playing a pivotal role in
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chloroplast redox regulation, which is critical during early stages of plant development.
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RESULTS
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The combined deficiencies of NTRC and f- or x-type Trxs cause severe growth
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inhibition phenotype and the impairment of light-dependent regulation of FBPase
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In our attempt to establish the function of NTRC in chloroplast redox regulation we
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have studied the genetic interaction of NTRC with functionally divergent chloroplast
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Trxs, such as Trxs f and Trx x. Previous results suggesting the concerted action of
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NTRC and Trx f1 were based on the severe growth retard phenotype of the Arabidopsis
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ntrc-trxf1 double mutant (Thormahlen et al., 2015). However, this mutant still contains
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Trx f2 which constitutes approx. 10% of the total content of f-type Trxs in wild type
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plants (Thormahlen et al., 2013). To avoid any effect of the residual Trx f2 in its
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relationship with NTRC, we generated the triple mutant ntrc-trxf1f2 devoid of NTRC
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and Trxs f. In addition, the ntrc mutant was crossed with the trxx single mutant (Pulido
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et al., 2010) and the ntrc-trxx double mutant was obtained. These lines were effectively
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knockout mutants as shown by the lack of transcripts of the corresponding genes in each
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of the mutants as determined by RT-qPCR analyses (Fig. 1A). As previously reported,
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the Trx-deficient lines trxf1f2 (Naranjo et al., 2016a) and trxx (Pulido et al., 2010)
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showed a visible phenotype very similar to the wild type plants when grown under long-
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day photoperiod (Fig. 1B, C), while the ntrc mutant showed growth retard and pale
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green phenotype (Fig. 1B, C), in agreement with previous results (Serrato et al., 2004;
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Lepisto et al., 2009). In contrast, the ntrc-trxx mutant, combining the deficiencies of
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NTRC and Trx x, showed a very severe growth retarded phenotype, which was even
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more severe in the case of the ntrc-trxf1f2 triple mutant (Fig. 1B). In line with their
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phenotypes, the ntrc-trxf1f2 and ntrc-trxx mutants showed significantly lower content of
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chlorophyll than their respective parent lines (Fig. 1C). It should be noted that despite
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their severe growth inhibition both the ntrc-trxx and the ntrc-trxf1f2 mutants were able
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to produce flowers and viable seeds, when grown under long-day photoperiod.
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Therefore, while the lack of Trxs f or Trx x have little effect on plant phenotype, when
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these deficiencies are combined with that of NTRC the effects become very severe.
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To establish the impact of the combined deficiencies of NTRC and Trxs f and x
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on redox regulation of chloroplast metabolic pathways, we analysed FBPase, a well-
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established redox regulated enzyme of the Calvin-Benson cycle. First, activity assays
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showed slightly lower FBPase activity in leaves of the ntrc, trxx and trxf1f2 mutants
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than in the wild type, while the ntrc-trxx mutant showed lower activity, which was even
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lower in the ntrc-trxf1f2 mutant (Fig. 2A). It should be taken into account that
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Arabidopsis contains three isoforms of FBPase, one localized in the cytosol (Cséke and
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Buchanan, 1980) and two in the chloroplast (Serrato et al., 2009), hence the FBPase
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activity here determined reflects the contribution of these isoforms. However, only one
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of the chloroplast-localized FBPase isoforms is redox regulated and Arabidopsis
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mutants lacking this isoform have a more severe dwarf phenotype than mutants lacking
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the cytosolic isoform (Rojas-González et al., 2015). Thus, to get more insight into the
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function of NTRC, Trxs f and Trx x in chloroplast redox regulation we analysed the
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light-dependent changes of the redox state of the plastid-localized FBPase isoform,
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which is known to be regulated by Trxs f, not by Trx x (Michelet et al., 2013). As
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previously reported (Thormählen et al., 2015; Naranjo et al., 2016a), thiol derivatization
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with the alkylating agent MM(PEG)24 showed that FBPase was fully oxidized in leaves
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from dark-adapted wild type plants and became reduced upon illumination, the level of
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reduction being dependent of light intensity (Fig. 2B, C). Light-dependent FBPase
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reduction was partially impaired in the trxf1f2 mutant and, surprisingly, also in the trxx
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mutant (Fig. 2B, C). Interestingly, the degree of FBPase photo-reduction was even
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lower in the ntrc mutant and almost abolished in mutants combining the deficiency of
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NTRC with those of Trx x or Trxs f (Fig. 2B, C), in agreement with previous studies
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with ntrc-trxf1 double mutants (Thormählen et al. 2015). Illumination with higher light
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intensity resulted in a higher level of enzyme reduction though the pattern of FBPase
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reduction of the different lines was similar to that obtained under growth light (Fig. 2B,
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C).
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These results are surprising since biochemical analyses have shown that FBPase
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is regulated by Trxs f but not by NTRC (Yoshida and Hisabori, 2016) or Trx x (Collin et
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al., 2003). Thus, to analyse further the role of these thiol redox systems in the regulation
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of FBPase, we performed in vitro assays with the purified enzymes. While almost full
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reduction of FBPase was accomplished with 0.5 μM concentration of either Trx f1 or
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Trx f2, the concentration of Trx x to obtain a similar level of FBPase reduction was at
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least ten-fold higher (Fig. 3), which is in contrast with the similar level of impairment of
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light-dependent FBPase reduction in the trxx and trxf1f2 mutants (Fig. 2B, C).
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Moreover, NTRC did not show any capacity to reduce FBPase in vitro (Fig. 3) despite
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the fact that the degree of light-dependent FBPase reduction was greatly decreased in
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the ntrc mutant (Fig. 2B, C). The severe impairment of light-dependent reduction of
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FBPase in the ntrc-trxx and ntrc-trxf1f2 mutants demonstrates the concerted action of
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both NTRC and f- or x-type Trxs on FBPase redox regulation, though these results
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suggest that NTRC affects FBPase redox regulation by an indirect mechanism.
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The combined deficiencies of NTRC and f- or x-type Trxs affect photosynthetic
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performance and chloroplast structure
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The effects of the combined deficiencies of NTRC and Trxs f or Trx x on plant
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growth (Fig. 1) and the activity (Fig. 2A) and redox regulation (Fig. 2B, C) of FBPase
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suggest that the impairment of these redox regulatory systems affects photosynthesis
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performance. Previous reports have shown that the deficiency of NTRC, but not of Trxs
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f or Trx x, provokes enhanced non-photochemical quenching (NPQ) at low light
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intensity (Naranjo et al., 2016a, b), hence causing a poor efficiency in the utilization of
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light energy by the ntrc mutant. Thus, as a first step to determine the effect of the
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combined deficiencies of NTRC and Trxs f or x on photosynthetic performance, we
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analysed NPQ in the different lines under study. In agreement with previous results, the
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ntrc mutant showed enhanced NPQ at light intensity as low as 42 μmol quanta m-2 s-1,
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while NPQ was only slightly enhanced in the trxx and trxf1f2 mutants (Fig. 4A). Similar
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to the ntrc mutant, ntrc-trxx and ntrc-trxf1f2 plants showed extended NPQ values during
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the whole illumination period, however, NPQ did not completely decay in the period of
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darkness as it did in ntrc plants (Fig. 4A), showing that light utilization in ntrc-trxx and
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ntrc-trxf1f2 mutants was even less efficient than in the ntrc mutant. In line with these
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results, the photosynthetic electron transport rate (ETR) was also impaired. The
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deficiency of NTRC affected the photosynthetic ETR more severely than the deficiency
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of Trxs x and f (Fig. 4B). Notably, both the ntrc-trxx and ntrc-trxf1f2 mutants showed
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dramatically reduced photosynthetic ETR at any of the light intensities analysed (Fig.
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4B). Moreover, the stability of photosystem II (PS II), determined as the Fv/Fm ratio,
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which was slightly affected in the ntrc and the trxx mutant, was also severely affected in
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the mutants combining the deficiencies of NTRC with those of x or f type Trxs (Table
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I). Finally, the net rate of CO2 fixation (AN) at growth light intensity (125 μE m-2 s-1),
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which was lower in the ntrc mutant but unaffected in the trxx and trxf1f2 mutants, as
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compared with the wild type, showed negative values in the ntrc-trxx and ntrc-trxf1f2
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mutants (Fig. 4C), hence indicating that the rates of respiration and photorespiration
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were higher than the rate of CO2 fixation in these plants. Moreover, the severe
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impairment of photosynthetic performance in mutants simultaneously devoid of NTRC
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and f- or x-type Trxs has a wider effect on chloroplast metabolism as shown by the
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content of starch, which was also severely decreased in these mutants (Table I).
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Therefore, these results show that the simultaneous deficiency of NTRC and Trxs f or x
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has a severe effect on photosynthesis performance, in line with the severe growth
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inhibition phenotype of these mutants.
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Such a defective photosynthetic performance of the ntrc-trxx and ntrc-trxf1f2
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mutants suggested as well alterations in chloroplast morphology. To test this possibility,
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the chloroplast structure in the different lines under study were analysed by
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transmission electron microscopy. The ntrc mutant contains chloroplasts with different
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levels of ultrastructural alterations, as previously reported (Perez-Ruiz et al., 2006;
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Lepisto et al., 2009), while chloroplasts in the trxx and trxf1f2 mutants showed similar
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morphology to those in the wild-type (Fig. 5). In contrast, the ntrc-trxx and, to a higher
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extent, the ntrc-trxf1f2 mutants showed heterogeneous chloroplast ultrastructure, with a
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number of chloroplasts showing clear alterations (Fig. 5). The most remarkable
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characteristic of chloroplasts from both the ntrc-trxx and the ntrc-trxf1f2 mutants was
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the increased number and size of plastoglobules (Fig. 5), which is indicative of
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oxidative stress and senescence (Austin et al., 2006).
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As an additional approach to determine the effect of the combined deficiencies
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of NTRC and Trxs f or Trx x on chloroplast function, selected proteins indicative of the
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different chloroplast compartments were analysed by Western blot. The ntrc-trxx and
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ntrc-trxf1f2 mutants showed decreased levels of D1, a core component of PSII, PsaC, a
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core component of PSI, lhca1, a component of PSI antenna, and the α and γ subunits of
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the ATPase (Fig. 6). However, no significant differences in the content of lhcb1, a
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component of the PSII antenna, and the stromal Rubisco large subunit was detected
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(Fig. 6). Altogether, these results show that the combined deficiency of NTRC and x- or
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f-type Trxs affect chloroplast structure and key components of the chloroplast
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photosynthetic electron transport chain, which is in agreement with the low
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photosynthetic performance and severe growth retarded phenotype of these plants. The
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increased number and size of plastoglobules in these chloroplasts suggest that the
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deficiency of the redox regulatory network of these organelles cause oxidative stress.
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The combined deficiencies of NTRC and f- or x-type Trxs cause high mortality at
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the seedling stage
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A remarkable feature of the ntrc-trxx and the ntrc-trxf1f2 mutants was the low
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number of individuals that reached the adult phase, indicating that the combined
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deficiency of these plastidial redox systems might be critical at early developmental
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stages. To investigate this possibility we analysed embryonic (seed germination) and
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postembryonic (production of roots and true leaves) growth and development. Of the
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Arabidopsis lines under study only the ntrc-trxf1f2 triple mutant showed decreased seed
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germination capacity (Fig. S1). At 5 days after germination (dag), all lines had produced
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cotyledons, though these were slightly smaller and showed paler green colour in the
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case of the ntrc-trxf1f2 mutant (Fig. 7A). At a later stage, 8 dag, seedlings of the wild
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type, as well as those of the ntrc, trxf1f2 and trxx mutants produced true leaves, a
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process that was delayed in the ntrc-trxx and, to a higher extent, in the ntrc-trxf1f2
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mutants (Fig. 7A, B). This delay was coincident with a sharp decrease of seedling
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viability in the case of the ntrc-trxf1f2 triple mutant, so that up to 95% of the seedlings
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of this line did not reach this stage of development (Fig. 7A, C). Though not as
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dramatic, the ntrc-trxx double mutant also showed a high decrease of viability at this
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stage of development (Fig. 7A, C). Seedlings of the ntrc-trxx and ntrc-trxf1f2 mutants
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that survived the true leaf stage were able to continue development though with
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impaired growth rate, which was more severe for the ntrc-trxf1f2 triple mutant, as
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shown above (Fig. 1B).
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The high mortality of the ntrc-trxx and the ntrc-trxf1f2 mutants at the transition
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of seedling to true leaves suggests that photosynthesis performed by cotyledons
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chloroplasts is critical for the development of vegetative organs. However, these assays
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were performed in soil and, thus, the nutritional conditions could not be strictly
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controlled. Therefore, we analysed in more detail the contribution of photosynthesis in
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cotyledons to seedling development. To this end, the generation of roots as a sink organ
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was studied in synthetic medium supplemented or not with sucrose under continuous
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light. Like the wild type, seedlings of the ntrc, trxx and trxf1f2 mutants produced roots
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regardless of the presence of sucrose in the medium (Fig. 8A-C). In contrast, root
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formation was completely abolished or severely impaired in seedlings of the ntrc-trxf1f2
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and ntrc-trxx mutants, respectively (Fig. 8A, B). Root elongation was restored by the
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addition of external sucrose, though the rate of root growth was still decreased in the
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ntrc-trxx and ntrc-trxf1f2 mutants, as compared to the other lines under analysis (Fig.
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8A, C). These results show the relevance of redox regulation in the photosynthetic
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activity of cotyledons chloroplasts for early plant development.
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DISCUSSION
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NTRC acts concertedly with different types of Trxs to sustain chloroplast function
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Chloroplasts play important roles in plant metabolism and development. A key
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component of the rapid adjustment of chloroplast metabolism to ever fluctuating light
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conditions is redox regulation of enzyme activity, which relies on two thiol redox
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systems, the NADPH-dependent NTRC and the Fdx-dependent FTR/Trx pathways
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(Spinola et al., 2008; Cejudo et al., 2012). Thus, a central issue to understand
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chloroplast redox regulation is to establish the functional relationship between these two
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redox systems and the relevance of chloroplast redox regulation to plant development.
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In this work we have addressed this issue by performing a comparative analysis of
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Arabidopsis mutants combining the deficiency of NTRC with that of two Trxs
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previously proposed to have unrelated functions such as Trxs f, which participate in
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redox regulation of metabolic pathways (Michelet et al., 2013), and Trx x, which was
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proposed to have antioxidant function (Collin et al., 2003). Based on our results we
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propose that NTRC exerts a pivotal role in chloroplast redox regulation. Moreover, the
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high mortality of the ntrc-trxx and, to a higher extent, the ntrc-trxf1f2 seedlings
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uncovers the essential function of cotyledon chloroplast redox regulation for early
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stages of plant development.
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372
Chloroplasts harbour a complex set of different Trxs (Meyer et al., 2012; Balsera
373
et al., 2014). Based on extensive biochemical analyses it was established that Trxs of
374
the types m and f are relevant in redox regulation of metabolic pathways (Buchanan and
375
Balmer, 2005; Michelet et al., 2013; Okegawa and Motohashi, 2015), while those of the
376
types x and y were proposed to perform antioxidant activity (Collin et al., 2003; Collin
377
et al., 2004). However, despite the major function proposed for Trxs f in redox
378
regulation of central metabolic pathways such as the Calvin-Benson cycle, Arabidopsis
379
mutants devoid of Trx f1 (Thormahlen et al., 2013) or Trx f1 and f2 (Yoshida et al.,
380
2015; Naranjo et al., 2016a) show a visible phenotype very similar to that of the wild
381
type, and so does an Arabidopsis mutant devoid of Trx x (Pulido et al., 2010). These
382
results indicate that alternative redox systems, either additional chloroplast Trxs or
383
NTRC, might compensate for the deficiency of these Trxs. In contrast, when the
384
deficiency of Trx f1 (Thormahlen et al., 2015), Trxs f1 and f2 or Trx x is combined with
385
the deficiency of NTRC a dramatic effect on plant phenotype is produced (Fig. 1B, C).
386
As previously reported (Thormahlen et al., 2015; Naranjo et al., 2016a, b), the
387
deficiency of NTRC causes more severe impairment on photosynthesis performance
388
than the deficiency of type f Trxs, as shown by the extensive NPQ at low light intensity,
389
the lower photosynthetic ETR and the rate of carbon fixation (Fig. 4A-C) in the ntrc
390
mutant. Remarkably, these parameters were dramatically affected in both the ntrc-trxx
391
and the ntrc-trxf1f2 mutants, thus showing the poor efficiency of light energy utilization
392
by these mutants, which has a wide effect on chloroplast metabolism as indicated by the
393
lower content of starch in these mutants (Table I).
394
The above discussed results provide an explanation for the severe growth
395
inhibition phenotype of the ntrc-trxx and the ntrc-trxf1f2 mutants and suggest the
396
concerted action of both systems in the redox regulation of common targets in vivo, a
397
notion further supported by the finding that NTRC interacts with well-established Trx
398
targets (Nikkanen et al., 2016). Indeed, NTRC has been shown to participate in the
399
regulation of enzymes such as AGPase (Michalska et al., 2009), and different enzymes
400
of the pathway of tetrapyrrole synthesis (Richter et al., 2013; Perez-Ruiz et al., 2014;
401
Yoshida and Hisabori, 2016), which were previously identified as Trx-regulated
402
(Ballicora et al., 2000; Geigenberger et al., 2005; Ikegami et al., 2007). Here we have
403
addressed this issue by analysing FBPase, a well-established redox regulated enzyme of
404
the Calvin-Benson cycle. The impairment of redox regulation caused by the combined
405
deficiencies of NTRC and Trxs f or x affected FBPase as shown by the lower level of
12
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
406
activity of this enzyme in leaves of the ntrc-trxx and the ntrc-trxf1f2 mutants (Fig. 2A).
407
Interestingly, the in-vivo level of FBPase reduction in response to light was lower in the
408
ntrc than in the trxf1f2 and trxx mutants (Fig. 2B). However, in vitro assays showed that
409
Trxs f1 and f2 are more efficient reductants of FBPase than Trx x, while NTRC is
410
unable to reduce the enzyme (Fig. 3). These results strongly support the notion that the
411
in vivo effect of NTRC on redox regulation of FBPase is indirect. The deficiency of
412
NTRC may cause an imbalance of the redox state of the chloroplast, which in turn
413
affects the regulatory function of Trxs f with respect of its targets.
414
Trxs f and x interact with different targets (Collin et al., 2003; Yoshida et al.,
415
2015) and, hence, are considered to have different functions in chloroplast redox
416
regulation (Meyer et al., 2012). Thus, an additional issue addressed in this work was to
417
establish whether NTRC is specifically required for the activity of Trxs involved in
418
regulation of metabolic pathways such as Trxs f, or is also required for Trxs involved in
419
antioxidant defence such as Trx x. The trxx mutant shows a phenotype indistinguishable
420
of the wild type plants (Pulido et al., 2010), whereas the ntrc-trxx double mutant shows
421
a severe growth retarded phenotype (Fig. 1B, C) and impairment of photosynthetic
422
performance (Fig. 4A-C and Table I), thus indicating that the function of Trx x is
423
dispensable in plants containing NTRC, but is not properly exerted in the absence of
424
NTRC. It should be noted that the level of light-dependent FBPase reduction was
425
similarly affected in the trxx and the trxf1f2 mutants (Fig. 2B, C). However, Trxs f,
426
which are about four-fold more abundant than Trx x in Arabidopsis chloroplasts
427
(Okegawa and Motohashi, 2015), are more efficient reductants of FBPase in vitro than
428
Trx x (Fig. 3). The phenotype of the ntrc-trxf1f2 mutant is more severe than that of the
429
ntrc-trxx mutant either at the stage of adult plant (Fig. 1B), seed germination capacity
430
(Fig. S1), or seedling viability (Fig. 7A-C), suggesting that the functions of Trxs f are
431
more relevant for plant growth than those of Trx x.
432
The deficiency of the redox regulatory network in the ntrc-trxx and ntrc-trxf1f2
433
mutants has a dramatic impact on photosynthetic performance as shown by the high
434
levels of NPQ at low light intensities (Fig. 4A), the corresponding decrease of the
435
photosynthetic ETR (Fig. 4B), PSII activity based on the Fv/Fm ratio (Table I) and the
436
rate of net photosynthesis (Fig. 4C). Previous reports have shown that the deficiency of
437
NTRC causes the impairment of light utilization and slightly affects the redox state of
438
the γ subunit of ATPase, hence affecting the qE component of NPQ (Carrillo et al.,
439
2016; Naranjo et al., 2016b). Therefore, the combined deficiency of NTRC and f- or x13
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
440
type Trxs affects the redox regulation of chloroplast metabolic pathways such as the
441
Calvin-Benson cycle but also the efficiency of light utilization, which ultimately might
442
be the reason of the severe growth retard phenotype shown by the ntrc-trxx and the ntrc-
443
trxf1f2 mutants. These results reveal the pivotal role of NTRC in the thiol redox
444
network of the chloroplast stroma integrating the feedback control of photochemical
445
reactions and regulation of metabolic pathways. Moreover, a combined deficiency in
446
NADPH and Fdx dependent chloroplast redox networks has severe effects on the
447
structure of these organelles as shown by the presence of chloroplasts with aberrant
448
ultrastructure in both the ntrc-trxx and the ntrc-trxf1f2 mutants (Fig. 5), and the lower
449
content of representative components of PSI, PSII and ATPase (Fig. 6).
450
Therefore, the severe growth phenotype of the ntrc-trxx and ntrc-trxf1f2 mutants
451
is the result of the effect of these mutations on a wide variety of chloroplast processes
452
from photochemical reactions to carbon metabolism. Our results indicate that the
453
regulation of these processes is coordinated and that NTRC exerts a central function in
454
this coordination. The question arising is the mechanistic basis of this central function
455
of NTRC in chloroplast redox regulation. A possibility is that the lower efficiency of
456
light energy utilization caused by the lack of NTRC (Fig. 4A-C) affects the availability
457
of reducing equivalents for Trxs, hence impairing the light-dependent redox regulation
458
of Calvin-Benson enzymes such as FBPase. This would provide an explanation for the
459
lower level of reduction of FBPase in NTRC-deficient plants (Fig. 2A-C) despite the
460
fact that NTRC is unable to reduce FBPase in vitro (Fig. 3). An additional aspect to be
461
taken into account is that the deficiency of NTRC may perturb chloroplast redox
462
balance affecting the Trx activity. In this regard, it should be noted that the chloroplasts
463
of the ntrc-trxx and ntrc-trxf1f2 mutants show higher number and size of plastoglobules
464
(Fig. 5), which are indicative of oxidative stress (Austin et al., 2006), hence suggesting
465
that the severe alteration of the redox regulatory network in these mutants has also
466
important effects on the antioxidant machinery of the chloroplast. Indeed, NTRC is an
467
efficient reductant of the peroxide scavenging enzyme 2-Cys Prx (Pérez-Ruiz et al.,
468
2006; Pulido et al., 2010).
469
470
Photosynthetic activity of cotyledon chloroplasts is essential for early plant
471
development
472
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473
Most studies on chloroplast redox regulation have been performed at the adult
474
plant stage, hence when leaves are fully developed. However, beside the severe growth
475
inhibition of the ntrc-trxx and ntrc-trxf1f2 mutants (Fig. 1B, C), a remarkable feature of
476
the phenotype of these mutants was the low number of individuals that reached the adult
477
phase (Fig. 7A-C), suggesting that the photosynthetic function becomes critical at
478
earlier stages of plant development. It is known that embryonic photosynthesis affects
479
seed vigour (Allorent et al., 2015), thus, the lower germination capacity of seeds of the
480
ntrc-trxf1f2 (Fig. S1) suggests the relevance of plastid redox regulation in seed
481
germination. It should be noted that the ntrc-trxx mutant shows germination capacity
482
similar to those of the wild type (Fig. S1), which is in line with the other phenotypic
483
parameters here analysed, most of them being more affected in the ntrc-trxf1f2 mutant
484
than in the ntrc-trxx mutant.
485
A critical stage in the plant life cycle occurs after seed germination when the
486
growing seedling depends on the heterotrophic use of seed storage compounds until
487
autotrophic growth is possible (Kircher and Schopfer, 2012). Once germinated, the
488
cotyledons of the ntrc-trxx and the ntrc-trxf1f2 mutants were slightly smaller and
489
showed yellowish green colour, as compared with cotyledons from wild type, ntrc, trxx
490
or trxf1f2 mutants (Fig. 7A). This indicates that the developmental program of
491
photomorphogenesis is not compromised by the impairment of chloroplast redox
492
regulation at this early stage of development. This notion was further supported by
493
growth tests in synthetic medium supplemented or not with sucrose (Fig. 8A). In
494
contrast with the wild type and the ntrc, trxx and trxf1f2 mutants, which produced roots
495
in the absence of sucrose, root production was severely defective in the ntrc-trxx
496
mutant, and completely abolished in the ntrc-trxf1f2 mutant unless sucrose was added to
497
the medium to compensate for decreased photosynthesis (Fig. 8A-C). These results
498
provide further evidence showing that the impairment of redox regulation caused by the
499
combined deficiency in NTRC and Trxs f or Trx x severely affects photosynthetic
500
performance of cotyledons chloroplasts. However, while in adult plants this failure
501
causes growth retardation but is not lethal, in the case of cotyledons it produces the
502
delay in the generation of new organs such as roots and true leaves, which provokes
503
increased seedling mortality (Fig. 7A-C). Photomorphogenesis, the reprograming of the
504
seedling growth promoted by light, has received much attention and a complex set of
505
light perception and signalling components have been identified (Huang et al., 2014;
506
Wu, 2014; Pogson et al., 2015). Less attention has been given to the contribution of
15
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
507
photosynthesis to this developmental process. The reduced viability of the ntrc-trxx and,
508
to a higher extent, the ntrc-trxf1f2 seedlings uncovers the essential function of redox
509
regulation of photosynthesis at this developmental stage.
510
EXPERIMENTAL PROCEDURES
511
512
Biological material and growth conditions
513
Arabidopsis thaliana wild type (ecotype Columbia) and mutant plants were
514
routinely grown in soil in growth chambers under long-day (16-h light/8-h darkness)
515
conditions at 22ºC during the light and 20ºC during the dark periods and a light intensity
516
of 125 µE m-2 s-1. For in vitro culture experiments, seeds were surface sterilized using
517
chlorine gas for 16 h, plated on Murashige and Skoog (MS) medium (DUCHEFA), pH
518
5.8 containing 0.35% Gelrite (DUCHEFA) in the presence or absence of 0.5% (w/v)
519
sucrose and stratified at 4ºC for 2 to 3 days. Mutant plants ntrc, trxx and trxf1f2 were
520
described previously (Serrato et al., 2004; Pulido et al., 2010; Naranjo et al., 2016a)
521
(Supplemental Table S1). The single ntrc mutant was manually crossed with the single
522
trxx mutant and the double trxf1f2 mutant. Seeds resulting from these crosses were
523
checked for heterozygosity of the T-DNA insertions in the NTRC, TRX x, TRX f1 and
524
TRX f2 genes, respectively. Plants were then self-pollinized and the ntrc-trxx and ntrc-
525
trxf1f2 mutants were identified in the progeny by PCR analysis of genomic DNA.
526
Primer sequences for PCR are listed in Supplemental Table S2.
527
528
RNA extraction and RT-qPCR analysis
529
530
Total RNA was extracted using Trizol reagent (Invitrogen). cDNA synthesis was
531
performed with 2 µg of total RNA using the Maxima first-strand cDNA synthesis kit
532
(Fermentas) according to manufacturer’s instructions. Real-time quantitative PCR (RT-
533
qPCR) was performed using an IQ5 real-time PCR detection system (Bio-Rad). A
534
standard thermal profile (95 °C, 3 min; 40 cycles at 95 °C for 10 s, and 60 °C for 30 s)
535
was used for all reactions. After the PCR, a melting curve analysis (55–94 °C at 0.5
536
°C/30 s) was performed to confirm the specificity of the amplicon and to exclude
537
primer-dimers or non-specific amplification. Oligonucleotides used for RT-qPCR
538
analyses are listed in Supplementary Table S3. Expression levels were normalized using
539
two reference genes: ACTIN and AT5G25760 (Czechowski et al., 2005).
540
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
541
Protein extraction, FBPase activity, alkylation assays and Western blot analysis
542
543
Plant tissues were ground with a mortar and pestle under liquid nitrogen. FBPase
544
activity was determined as previously reported (Rojas-González et al., 2015) from
545
leaves of plants grown for 4 weeks under long-day photoperiod and harvested at 12 h of
546
the day period. For the determination of the redox state of thiol-regulated enzymes in
547
vivo, alkylation assays using methyl-maleimide polyethylene glycol (MM(PEG)24,
548
Thermo Scientific) were performed as described previously (Naranjo et al., 2016a). To
549
study chloroplast protein levels, the Minute Chloroplast Isolation Kit (Invent
550
Biotechnologies) was used following the manufacturer’s instructions. Intact chloroplasts
551
were isolated from leaves of plants adapted to the dark during 30 min and total protein
552
content was quantified using the Bradford reagent (BIO-RAD).
553
Protein samples were subjected to SDS-PAGE under reducing or non-reducing
554
conditions using various acrylamide gel concentrations, as stated in the figure legends,
555
and transferred onto nitrocellulose membranes, which were probed with the indicated
556
antibody. Specific anti-NTRC antibodies were previously raised in our laboratory
557
(Serrato et al., 2004). Other antibodies (D1, lhcb1, PsaC, lhca1, α-ATPase, γ-ATPase,
558
RBC-L) were purchased from Agrisera (Sweden).
559
560
In vitro FBPase reduction assay
561
562
Arabidopsis chloroplast FBPase (2.5 μM) was incubated for 30 min at 22ºC with
563
increasing concentration (0-10 μM) of NTRC, Trxs f1 and f2 or Trx x in reaction
564
mixtures containing 100 mM Tris-HCl pH 8.0 and 0.1 mM DTT. In order to prevent
565
further oxidations, free thiol were alkylated by incubation in the dark for 30 min at 37
566
ºC in the presence of equals volumes of a solution containing 60 mM iodoacetamide
567
(IAM), 2% CHAPS and 100 mM Tris-HCl pH 8.0.
568
569
Determination of chlorophylls, starch and FBPase activity
570
571
Leaf discs were weighed and frozen in liquid nitrogen. After extraction in 1 mL
572
methanol for 16 h at 4 °C, chlorophyll levels were measured spectrophotometrically, as
573
described in Porra et al. (1989), and normalized to fresh weight. Starch contents were
574
determined as previously reported (Rojas-González et al., 2015) from rosette leaves of
17
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
575
plants grown for 4 weeks under long-day photoperiod and harvested at 12 h of the day
576
period.
577
578
Measurements of chlorophyll a fluorescence and rate of carbon assimilation (AN)
579
580
Room temperature chlorophyll fluorescence was measured using a pulse-
581
amplitude modulation fluorimeter (DUAL-PAM-100, Walz, Effeltrich, Germany). The
582
maximum quantum yield of PSII was assayed after incubation of plants in the dark for
583
30 min by calculating the ratio of the variable fluorescence, Fv, to maximal
584
fluorescence, Fm (Fv/Fm). Induction-recovery curves were performed using red (635 nm)
585
actinic light at 42 μE m−2 s−1 for 8 min. Saturating pulses of red light at 10000 μE m−2
586
s−1 intensity and 0.6 s duration were applied every 60 s and recovery in darkness was
587
recorded for up to 10 min. The parameters Y(II) and Y(NPQ) corresponding to the
588
respective quantum yields of PSII photochemistry and non-photochemical quenching
589
(NPQ) were calculated by the DUAL-PAM-100 software according to the equations in
590
(Kramer et al., 2004). Relative linear electron transport rates were measured in leaves of
591
pre-illuminated plants by applying stepwise increasing actinic light intensities up to
592
2000 μE m−2 s−1.
593
Net CO2 assimilation rate (AN) was measured in leaves from wild type and
594
mutant plants grown under long-day conditions for 4 weeks using an open gas exchange
595
system Li-6400 equipped with the chamber head (Li-6400–40). Leaves were dark-
596
adapted and then illuminated with photosynthetically active radiation (PAR) of 125 μE
597
m−2 s−1 until the rate of CO2 assimilation was stabilised. Six leaves were measured per
598
line. Measurements were performed by the Service for Photosynthesis, Instituto de
599
Recursos Naturales y Agrobiología, Sevilla (Spain).
600
601
Determination of seedling development and survival
602
603
The growth and development of the lines under study were monitored based on
604
two parameters: the formation of true (post-embryonic) leaves and seedling mortality.
605
For each line, three sets of at least 60 seeds were sown on soil and grown during 28
606
days under long-day conditions. Seedling survival and true leaves generation were
607
scored at 3-4 days intervals. Seedlings that were still green/greenish, considered as
18
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
608
survivors, were quantified to calculate the survival rate. Seedlings that exhibit true
609
leaves were quantified to determine the rate of seedlings reaching the true leaf stage.
610
611
Root growth and germination capacity measurements
612
613
For root growth assays, at least 30 seedlings were grown for 7 days on vertically
614
oriented MS plates in the presence or absence of 0.5% (w/v) sucrose. Root elongation
615
was scored at 1 day intervals from day 3 until day 7.
616
For germination studies, five sets of at least 100 seeds were sown on
617
horizontally oriented MS plates and the percentage of germinated seeds was recorded at
618
day 2 and day 7.
619
620
Transmission electron microscopy analysis
621
622
Fully expanded leaves from plants cultured under long day photoperiod were
623
collected at bolting time, approx. after 28 days of growth, just when the first floral bud
624
was visible. Since growth of ntrc-trxx and ntrc-trxf1f2 plants is delayed, the leaves of
625
these mutants were collected after approx. 60 days of growth. Small pieces (2 mm2) of
626
leaves were cut with a razor blade and immediately fixed in 4% glutaraldehyde in 0.1 M
627
Na-cacodylate buffer, pH 7.4 (3 h at 4 ºC, under vacuum). After fixation, samples were
628
rinsed three times for 30 min at 4 ºC with the same buffer. Samples were post-fixed in
629
1% osmium tetraoxide in cacodilate buffer (0.1 M, pH 7.4) for 1 h at 4 ºC. After
630
washing, samples were immersed in 2% uranyl acetate, dehydrated through a gradient
631
acetone series (50, 70, 90, 100%) and embedded in Spurr resin. Semi-thin sections (300
632
nm thickness) were obtained with a glass knife and stained with 1% toluidine blue for
633
cell localization and reorientation using a conventional optic microscope. Once a
634
suitable block face of the selected area was trimmed, several ultrathin sections (70 nm)
635
were obtained using an ultramicrotome (Leica UC7) equipped with diamond knife
636
(Diatome) and collected on 200 mesh cupper grids. Sections were examined in a Zeiss
637
Libra 120 transmission electron microscope and a digitized (2048 x 2048 x 16 bits)
638
using an on axis-mounted TRS camera. Sample post-fixation and cutting were
639
performed by the Microscopy Service at the Centro de Investigación, Tecnología e
640
Innovación (CITIUS) from the University of Seville.
641
19
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642
643
644
645
646
Table I. Effect of the combined deficiencies of NTRC and Trxs f or Trx x on PSII
647
stability and starch content
648
Plant line
Fv/Fm
WT
ntrc
trxx
trxf1f2
ntrc-trxx
ntrc-trxf1f2
0.78±0.03a
0.76±0.02b
0.75±0.03b
0.79±0.02a
0.62±0.11c
0.52±0.10d
8.11±2.06a
2.25±0.49cd
4.36±0.81b
3.57±1.65bc
0.82±0.34d
1.59±0.51d
Starch
(μg/mg FW)
649
650
The maximum PSII quantum yield was determined as variable fluorescence (Fv) to
651
maximal fluorescence (Fm), Fv/Fm, in dark adapted leaves of plants grown under long-
652
day conditions. The Fv/Fm values ± standard deviation (SD) is the average of at least 10
653
measurements. The content of starch was determined from rosette leaves of plants
654
grown under long-day for 31 days and harvested after 12 h of the light period. Values
655
represent mean ± SD of three independent samples. Letters indicate significant
656
differences with the Student´s t test (Fv/Fm) or the Tukey’s Least Significance Difference
657
test (starch content) and a confidence interval of 95%.
658
659
660
SUPPLEMENTAL DATA
661
662
663
664
665
666
Fig. S1. Effect of the deficiency in NTRC and x- or f-type Trxs on the germination
capacity.
Table S1. Arabidopsis mutants used in this study.
Table S2. Oligonucleotides used for determination of Arabidopsis mutant genotypes.
Table S3. Oligonucleotides used for RT-qPCR analyses.
667
Figure 1. The level of NTRC, Trx x, Trx f1 and Trx f2 transcripts and growth
668
phenotype of wild type and mutant plants.
669
A, The contents of transcripts of the genes NTRC, TRX x, TRX f1 and TRX f2 were
670
determined by RT-qPCR from total RNA samples, which were extracted from leaves of
671
Arabidopsis wild type and mutant lines, as indicated. The pairs of oligonucleotides used
20
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
672
for cDNA amplification are indicated in Supplementary Table S3. Transcript levels
673
were normalized to ACTIN and the AT5G25760 genes and referred to the level of each
674
of the genes in wild type plants. Determinations were performed three times and mean
675
values ± standard error (SE) are represented. B, Plants of wild type and mutant lines that
676
were grown under long-day conditions (16 h light / 8 h darkness, light intensity 125 μE
677
m-2 s-1) during 22 days. C, Chlorophyll content was determined from leaf discs (n=6)
678
and average values ±SE are represented. Letters indicate significant differences with the
679
Student’s t test and a confidence interval of 99%.
680
Figure 2. FBPase activity and in vivo redox state of FBPase in wild type and
681
mutant lines.
682
A, FBPase activity was assayed in leave extracts from plants grown for four weeks
683
under long-day photoperiod and harvested at 12 h of the day period. Results are the
684
mean ± standard error (SE) from three biological replicates. Different letters above each
685
bar represent significant difference (P < 0.05) determined by one-way analysis of
686
variance followed by Tukey’s post-test. B, The in vivo redox state of FBPase was
687
determined by derivatization of thiols with the alkylating agent MM(PEG)24, as
688
indicated in Methods. Plants of the wild type and mutant lines were grown under long
689
day conditions for four weeks. Samples were harvested at the end of the period of
690
darkness (D) and then illuminated for 30 min with light intensities of 125 μE m-2 s-1
691
(growth light, GL) or 500 μE m-2 s-1 (high light, HL). Protein extracts from leaves were
692
subjected to SDS-PAGE under non-reducing conditions, transferred onto nitrocellulose
693
filter and probed with an anti-FBPase antibody. C, Band intensities were quantified
694
(ScionImage) and the percentage of reduction was calculated as the ratio between the
695
shifted band (reduced form) and the sum of reduced and oxidized forms. Each value is
696
the mean of three independent experiments ± standard error (SE) and letters indicate
697
significant differences between mutants with the Student’s t test at 95% confidence
698
interval.
699
700
Figure 3. In vitro reduction of FBPase by NTRC and Trxs x or f.
701
702
The in vitro FBPase reduction assay was carried out by incubating a fixed concentration
703
of the purified enzyme (2.5 μM) in the presence of 0.1 mM DTT, with increasing
704
concentrations, as indicated, of NTRC, Trx f1, Trx f2 and Trx x. red, reduced; oxi,
705
oxidized.
21
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706
707
708
Figure 4. Light-dependent NPQ, linear photosynthetic electron transport and net
709
CO2 fixation rate in wild type and mutant lines.
710
Quantum yields of NPQ (A) and relative linear electron transport rates of photosystem
711
II, ETR (II) (B), were measured in pre-illuminated attached leaves of plants grown at
712
125 µE m-2 s-1 under long-day conditions. Chlorophyll fluorescence of photosystem II
713
was determined using a pulse-amplitude modulation fluorimeter. Determinations were
714
performed at least five times and each data point is the mean ± SE. White and black bars
715
indicate light and dark periods. C, Net CO2 assimilation rate (AN) was measured using
716
an open gas exchange system in leaves of plants grown during 4 weeks under long-day
717
photoperiod, which were dark-adapted and then illuminated with a PAR of 125 μE m−2
718
s−1. Six leaves were measured per line and mean mean ± SE are represented. Letters
719
indicate significant differences with the Tukey Test and a confidence interval of 99.9%.
720
721
Figure 5. Electron transmission microscopy analysis of chloroplast structure from
722
wild type and mutant lines.
723
Leaves of plants cultured under a 16-h light/8-h dark regime were collected after the
724
first symptoms of bolting. An increase in plastoglobule (white arrowheads) number, size
725
and association is observed in ntrc-trxx and ntrc-trxf1f2 mutant plants as compared to
726
wild-type and single mutant plants. Bars represent 1 µm. c, chloroplasts; cw, cell wall;
727
v, vacuoles; S, starch granules.
728
Figure 6. Level of representative chloroplast proteins in wild type and mutant
729
plants.
730
Western blot analysis of the content of representative components of photosynthetic
731
electron transport (D1, Lhcb1, PsaC and Lhca1), ATP synthesis (α-ATPase and γ-
732
ATPase) and carbon assimilation (RBC-L) in the lines under study. Chloroplasts of the
733
indicated lines were isolated from 5-week old long-day grown plants and fractionated
734
into thylakoid and stromal fractions as described in Methods. Protein samples
735
corresponding to thylakoids (0.5 μg of chlorophyll) or stroma (1 μg of protein) were
736
subjected to SDS-PAGE under reducing conditions, transferred to nitrocellulose filters
737
and probed with antibodies against thylakoid (D1, Lhcb1, PsaC, Lhca1, α-ATPase, γ22
Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
738
ATPase) and stromal (RBC-L) proteins. Additional amounts (3x and 1/4x) of wild type
739
extracts were loaded for comparison. The experiment was performed twice with similar
740
results.
741
742
Figure 7. Redox regulation impairment provokes high mortality at the seedling
743
stage.
744
A, Seeds of the wild type and mutant lines, as indicated, were allowed to germinate on
745
soil and representative seedlings at 5, 8 and 11 days after germination (dag) are shown.
746
Bars represent 0.5 cm. For each line, the percentage of seedlings with emerging true
747
leaves (B) and the percentage of seedlings that remained alive (C) at the indicated dag
748
were determined and represented. The experiment was performed twice and means
749
values ± SE from three replicates of each experiment are represented.
750
751
Figure 8. Effect of sucrose on root production from seedlings of the wild type and
752
mutant lines.
753
Seedlings were grown on MS synthetic medium supplemented or not with 0.5% (w/v)
754
sucrose. A, Seedlings of the different lines, as indicated, after 7 days of growth under
755
continuous light (125 μE m-2 s-1). Root growth in the absence (B) or presence (C) of
756
added sucrose was monitored and mean values ± SE from three replicates are
757
represented.
758
759
760
761
762
763
764
765
766
767
768
769
770
771
23
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772
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780
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782
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785
embryonic photosynthetic activity modulate seed fitness in Arabidopsis
786
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Austin II JR, Frost E, Vidi PA, Kessler F, Staehelin LA (2006) Plastoglobules are
788
lipoprotein subcompartments of the chloroplast that are permanently coupled to
789
thylakoid membranes and contain biosynthetic enzymes. Plant Cell 18: 1693-
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802
under low irradiance. Plant J 87: 654-663
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the NADPH thioredoxin reductase C-2-Cys peroxiredoxin system in plastid
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redox regulation and signalling. FEBS Lett 586: 2974-2980
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Downloaded from on June 17, 2017 - Published by www.plantphysiol.org
Copyright © 2017 American Society of Plant Biologists. All rights reserved.
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Figure 1. The level of NTRC, Trx x, Trx f1 and Trx f2 transcripts and growth
phenotype of wild type and mutant plants.
A, The contents of transcripts of the genes NTRC, TRX x, TRX f1 and TRX f2 were
determined by RT-qPCR from total RNA samples, which were extracted from leaves of
Arabidopsis wild type and mutant lines, as indicated. The pairs of oligonucleotides used
for cDNA amplification are indicated in Supplementary Table S3. Transcript levels
were normalized to ACTIN and the AT5G25760 genes and referred to the level of each
of the genes in wild type plants. Determinations were performed three times and mean
values ± standard error (SE) are represented. B, Plants of wild type and mutant lines that
were grown under long-day conditions (16 h light / 8 h darkness, light intensity 125 μE
m-2 s-1) during 22 days. C, Chlorophyll content was determined from leaf discs (n=6)
and average values ±SE are represented. Letters indicate significant differences with the
Student’s t test and a confidence interval of 99%.
1
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Figure 2. FBPase activity and in vivo redox state of FBPase in wild type and
mutant lines.
A, FBPase activity was assayed in leave extracts from plants grown for four weeks
under long-day photoperiod and harvested at 12 h of the day period. Results are the
mean ± standard error (SE) from three biological replicates. Different letters above each
bar represent significant difference (P < 0.05) determined by one-way analysis of
variance followed by Tukey’s post-test. B, The in vivo redox state of FBPase was
1
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
determined by derivatization of thiols with the alkylating agent MM(PEG)24, as
indicated in Methods. Plants of the wild type and mutant lines were grown under long
day conditions for four weeks. Samples were harvested at the end of the period of
darkness (D) and then illuminated for 30 min with light intensities of 125 μE m-2 s-1
(growth light, GL) or 500 μE m-2 s-1 (high light, HL). Protein extracts from leaves were
subjected to SDS-PAGE under non-reducing conditions, transferred onto nitrocellulose
filter and probed with an anti-FBPase antibody. C, Band intensities were quantified
(ScionImage) and the percentage of reduction was calculated as the ratio between the
shifted band (reduced form) and the sum of reduced and oxidized forms. Each value is
the mean of three independent experiments ± standard error (SE) and letters indicate
significant differences between mutants with the Student’s t test at 95% confidence
interval.
2
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Figure 3. In vitro reduction of FBPase by NTRC and Trxs x or f.
The in vitro FBPase reduction assay was carried out by incubating a fixed concentration
of the purified enzyme (2.5 μM) in the presence of 0.1 mM DTT, with increasing
concentrations, as indicated, of NTRC, Trx f1, Trx f2 and Trx x. red, reduced; oxi,
oxidized.
1
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
A
0.6
WT
ntrc
trxx
trxf1f2
ntrc-trxx
ntrc-trxf1f2
Y (NPQ)
0.4
0.2
0.0
0
200
400
600
time (s)
800
1000
1200
B
25
ETR (II)
20
15
10
5
0
0
400
200
600
Light intensity
C
AN (µmol m-2 s-1)
2.0
a
1.5
ac
1.0
ac
c
0.5
b
0.0
b
-0.5
Figure 4. Light-dependent NPQ, linear photosynthetic electron
transport and net CO2 fixation rate in wild type and mutant lines.
1
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Quantum yields of NPQ (A) and relative linear electron transport rates of photosystem
II, ETR (II) (B), were measured in pre-illuminated attached leaves of plants grown at
125 µE m-2 s-1 under long-day conditions. Chlorophyll fluorescence of photosystem II
was determined using a pulse-amplitude modulation fluorimeter. Determinations were
performed at least five times and each data point is the mean ± SE. White and black bars
indicate light and dark periods. C, Net CO2 assimilation rate (AN) was measured using
an open gas exchange system in leaves of plants grown during 4 weeks under long-day
photoperiod, which were dark-adapted and then illuminated with a PAR of 125 μE m−2
s−1. Six leaves were measured per line and mean mean ± SE are represented. Letters
indicate significant differences with the Tukey Test and a confidence interval of 99.9%.
2
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Figure 5. Electron transmission microscopy analysis of chloroplast structure from
wild type and mutant lines.
Leaves of plants cultured under a 16-h light/8-h dark regime were collected after the
first symptoms of bolting. An increase in plastoglobule (white arrowheads) number, size
and association is observed in ntrc-trxx and ntrc-trxf1f2 mutant plants as compared to
wild-type and single mutant plants. Bars represent 1 µm. c, chloroplasts; cw, cell wall;
v, vacuoles; S, starch granules.
1
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Figure 6. Level of representative chloroplast proteins in wild type and mutant
plants.
Western blot analysis of the content of representative components of photosynthetic
electron transport (D1, Lhcb1, PsaC and Lhca1), ATP synthesis (α-ATPase and γATPase) and carbon assimilation (RBC-L) in the lines under study. Chloroplasts of the
indicated lines were isolated from 5-week old long-day grown plants and fractionated
into thylakoid and stromal fractions as described in Methods. Protein samples
corresponding to thylakoids (0.5 μg of chlorophyll) or stroma (1 μg of protein) were
subjected to SDS-PAGE under reducing conditions, transferred to nitrocellulose filters
and probed with antibodies against thylakoid (D1, Lhcb1, PsaC, Lhca1, α-ATPase, γATPase) and stromal (RBC-L) proteins. Additional amounts (3x and 1/4x) of wild type
extracts were loaded for comparison. The experiment was performed twice with similar
results.
1
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Copyright © 2017 American Society of Plant Biologists. All rights reserved.
Figure 7. Redox regulation impairment provokes high mortality at the seedling
stage.
A, Seeds of the wild type and mutant lines, as indicated, were allowed to germinate on
soil and representative seedlings at 5, 8 and 11 days after germination (dag) are shown.
Bars represent 0.5 cm. For each line, the percentage of seedlings with emerging true
leaves (B) and the percentage of seedlings that remained alive (C) at the indicated dag
were determined and represented. The experiment was performed twice and means
values ± SE from three replicates of each experiment are represented.
1
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Figure 8. Effect of sucrose on root production from seedlings of the wild type and
mutant lines.
Seedlings were grown on MS synthetic medium supplemented or not with 0.5% (w/v)
sucrose. A, Seedlings of the different lines, as indicated, after 7 days of growth under
continuous light (125 μE m-2 s-1). Root growth in the absence (B) or presence (C) of
added sucrose was monitored and mean values ± SE from three replicates are
represented.
1
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