Plant Cell Advance Publication. Published on September 21, 2016, doi:10.1105/tpc.16.00630
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RESEARCH ARTICLE
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A role for TIC55 as a hydroxylase of phyllobilins, the products of chlorophyll
breakdown during plant senescence.
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Mareike Hauenstein, Bastien Christ1, Aditi Das, Sylvain Aubry and Stefan Hörtensteiner2
Institute of Plant Biology, University of Zurich, Zollikerstrasse 107, CH-8008 Zurich, Switzerland
Present address: Whitehead Institute, Massachusetts Institute of Technology, Cambridge, MA 021394307
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Address correspondence to [email protected]
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Short title: TIC55 functions in chlorophyll breakdown
One-sentence summary: TIC55, a Rieske-type oxygenase was previously described as component of the
chloroplast protein import machinery; TIC55 also functions as a chlorophyll catabolite hydroxylase.
The author responsible for distribution of materials integral to the findings presented in this article in
accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Stefan
Hörtensteiner ([email protected])
ABSTRACT
Chlorophyll degradation is the most obvious hallmark of leaf senescence. Phyllobilins, linear
tetrapyrroles that are derived from opening of the chlorin macrocycle by the Rieske-type oxygenase
PHEOPHORBIDE a OXYGENASE (PAO), are the end products of chlorophyll degradation. Phyllobilins carry
defined modifications at several peripheral positions within the tetrapyrrole backbone. While most of
these modifications are species-specific, hydroxylation at the C32 position is commonly found in all
species analyzed to date. We demonstrate that this hydroxylation occurs in senescent chloroplasts of
Arabidopsis thaliana. Using bell pepper (Capsicum annuum) chromoplasts, we establish that phyllobilin
hydroxylation is catalyzed by a membrane-bound, molecular oxygen- and ferredoxin-dependent activity.
As these features resemble the requirements of PAO, we considered membrane-bound Rieske-type
oxygenases as potential candidates. Analysis of mutants of the two Arabidopsis Rieske-type oxygenases
(besides PAO), uncovered that phyllobilin hydroxylation depends on TRANSLOCON AT THE INNER
CHLOROPLAST ENVELOPE 55 (TIC55). Our work demonstrates a catalytic activity for TIC55, which in the
past has been considered as a redox sensor of protein import into plastids. Given the wide evolutionary
distribution of both PAO and TIC55, we consider that chlorophyll degradation likely co-evolved with land
plants.
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©2016 American Society of Plant Biologists. All Rights Reserved
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INTRODUCTION
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Chlorophyll is the main pigment of the photosynthetic apparatus of plants and is responsible for the
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absorption of the energy of sunlight. In the process of photosynthesis, this energy is converted to
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chemical energy used for carbon fixation. During leaf senescence, however, when the photosynthetic
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machinery is dismantled in order to retrieve nutrients, in particular nitrogen, to sink organs such as
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seeds (Hörtensteiner and Feller, 2002), the photodynamic properties of chlorophyll become potentially
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cell toxic. As a consequence, the pigment is detoxified to uncolored and photodynamically inactive
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products that are termed phyllobilins (Kräutler, 2014). Chlorophyll degradation to phyllobilins is
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catalyzed by a multistep process, named the “PAO/phyllobilin” pathway (Hörtensteiner and Kräutler,
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2011; Kräutler and Hörtensteiner, 2014), to acknowledge PAO (pheophorbide a oxygenase), a Rieske-
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type oxygenase that is the key component of this pathway (Pružinská et al., 2003). PAO, in concert with
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red chlorophyll catabolite reductase (RCCR), catalyzes the ring-opening reaction of pheophorbide a, the
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phytol- and Mg-free intermediate of the early part of chlorophyll breakdown, to a primary fluorescent
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chlorophyll catabolite (pFCC), the primary (fluorescent) phyllobilin that is the precursor of all
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subsequently formed phyllobilins. The direct product of the PAO reaction, red chlorophyll catabolite
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(RCC), does not accumulate, but is immediately reduced at the C15/C16-double bond by RCCR in an
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intriguing stereospecific manner (Wüthrich et al., 2000; Pružinská et al., 2007) (for atom and pyrrole
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numbering of phyllobilins, see the structure of pFCC in Figure 1B). Thus, depending on the source of the
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enzyme, two C16-stereoisomers, pFCC or epi-pFCC, may occur (Mühlecker et al., 1997; Mühlecker et al.,
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2000). For example, the RCCR of Arabidopsis thaliana (Arabidopsis) (At-RCCR) produces pFCC, while in
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Capsicum annuum (bell pepper) or Solanum lycopersicum (tomato) epi-pFCC is formed (Hörtensteiner et
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al., 2000). It was shown in vitro that the stereospecificity of Arabidopsis RCCR is defined by a single
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amino acid residue, Phe219, which when mutated to Val changes the specificity (Pružinská et al., 2007).
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Likewise, complementation of the accelerated cell death2 (acd2) mutant of Arabidopsis that is deficient
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in RCCR with an RCCR version ‘X’, where six amino acids of the wild-type sequence including Phe219 were
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replaced by the respective residues of the tomato RCCR (acd2-2+At-RCCR-X), caused the exclusive
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accumulation during senescence of C16-isomerized phyllobilins (Pružinská et al., 2007).
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The end products of chlorophyll degradation are categorized into two types (Kräutler, 2014): (C1-)
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formyl-(C19-) oxobilins, also termed nonfluorescent chlorophyll catabolites (NCCs), are the ultimate
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products of PAO activity, while the activity of a cytochrome P450 monooxygenase, as shown for
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Arabidopsis (Christ et al., 2013), gives rise to (C1,19-) dioxobilins, also named dioxobilin-type
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nonfluorescent chlorophyll catabolites (DNCCs). NCCs and DNCCs are found in the vacuoles of senescent
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cells (Matile et al., 1988; Christ et al., 2012) and have been shown to be derived from the respective
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fluorescent precursor phyllobilins (FCCs and DFCCs) as the result of non-enzymatic isomerization inside
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the acidic vacuolar sap (Oberhuber et al., 2003; Christ et al., 2013). Since the first identification of an
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NCC in senescent leaves of Hordeum vulgare (barley) in 1991 (Kräutler et al., 1991), phyllobilins were
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found in senescent leaves from more than 20 different angiosperm species (Kräutler, 2016), but are also
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formed during fruit ripening (Müller et al., 2007; Moser et al., 2009). Although the principal linear
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tetrapyrrole backbone of all phyllobilins is identical, they exhibit rather great structural diversity among
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the investigated species. Thus, for example, 90% of the phyllobilins that accumulate in senescent
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Arabidopsis leaves are DNCCs (Christ et al., 2013), while Cercidiphyllum japonicum exclusively produces
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NCCs (Oberhuber et al., 2003). Furthermore, some species, like Arabidopsis, accumulate an array of up
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to eight different phyllobilins simultaneously (Christ et al., 2016), while others, like C. japonicum,
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produce only two (Oberhuber et al., 2003). The molecular basis for these differences is enzyme activities
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that modify pFCC in a species-specific manner at different peripheral positions of the tetrapyrrole
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backbone structure. Best investigated in this respect is Arabidopsis where, besides CYP89A9, the already
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mentioned cytochrome P450 monooxygenase that is responsible for DNCC formation (Christ et al.,
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2013), methylesterase 16 (MES16) has been identified (Christ et al., 2012). This enzyme demethylates
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the C82 carboxymethyl ester present in chlorophyll with high efficiency. Again, other species, such as C.
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japonicum, exclusively produce phyllobilins that carry the intact carboxymethyl group, indicating that
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they lack a MES16 ortholog. Interestingly, these modifying reactions are localized in the cytosol (MES16)
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(Christ et al., 2012) and at the endoplasmic reticulum (CYP89A9) (Christ et al., 2013), while all enzymes
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identified to date that are required for the conversion of chlorophyll to pFCC are located within the
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plastid (Sakuraba et al., 2012). This led to the concept that reactions that commonly occur during
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chlorophyll breakdown take place in senescing chloroplasts, while species-specific modifications occur
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outside. Consequently, pFCC is believed to be exported from plastids, but the nature of pFCC transport
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at the plastid envelope is unknown (Christ and Hörtensteiner, 2014). An intriguing common modification
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of phyllobilins found in all species that have been analyzed so far is the specific hydroxylation at the C32
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ethyl side chain (Christ and Hörtensteiner, 2014; Kräutler, 2014). This indicates that, distinct from other
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species-specific modifications, phyllobilin hydroxylation may be a reaction commonly occurring in
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angiosperms and that the activity may be localized in the plastid.
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The aim of this work was to identify the molecular nature of the hydroxylating activity. We could show
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that, indeed, senescent chloroplasts (gerontoplasts) contained both pFCC and hydroxylated pFCC
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(hydroxy-pFCC), indicating the respective activity to be a chloroplast protein. Using bell pepper
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chromoplasts as starting material, we successfully established an in vitro enzyme assay that allowed us
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to biochemically characterize the properties of the enzyme in question. Based on these analyses and on
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Rieske-type oxygenases: TRANSLOCON AT THE INNER CHLOROPLAST ENVELOPE (TIC) 55 (TIC55) and
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PROTOCHLOROPHYLLIDE-DEPENDENT TRANSLOCON AT THE INNER CHLOROPLAST ENVELOPE (PTC) 52
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(PTC52), which both have been implicated in chloroplast protein import. Analysis of respective T-DNA
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insertion mutants verified that tic55 mutants are unable to produce hydroxylated phyllobilins, while in a
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ptc52 mutant hydroxylation was unaffected. Thus, TIC55 is responsible for phyllobilin hydroxylation
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during chlorophyll breakdown. TIC55 orthologs are widely distributed in higher plants, but are
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phylogenetically distinct to PAO orthologs. This indicates that phyllobilin hydroxylation likely appeared
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with the evolution of land plants.
O2 labeling and CO inhibition studies we could narrow down the number of likely candidates to two
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RESULTS
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Hydroxy-pFCC is Present in Gerontoplasts of Arabidopsis.
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The steps of chlorophyll to pFCC conversion have been shown to take place in gerontoplasts (Sakuraba et
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al., 2012). In agreement with this, fluorescent HPLC fractions corresponding to pFCC and to epi-pFCC
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have been identified in extracts of gerontoplasts of Brassica napus (oilseed rape) and barley
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(Schellenberg et al., 1993; Ginsburg et al., 1994; Moser and Matile, 1997). However, these early analyses
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indicated additional, more-polar fluorescent fractions to be present in plastids. To investigate which, if
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any, chlorophyll catabolites are found in Arabidopsis plastids, isolated gerontoplasts were analyzed by
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liquid-chromatography (LC) – tandem mass spectrometry (MS/MS). Indeed, besides pFCC that produced
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a pseudomolecular ion of [M+H]+ = C35H41N4O7 (m/z 629.2977) in positive ionization mode, we identified
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a second, more-polar fraction with a mass of m/z 645.2929 ([M+H]+ = C35H41N4O8), i.e. containing one
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additional oxygen atom (Figure 1A). In tandem MS experiments (Figure 1B and C), both ions fragmented
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in an identical manner that earlier had been shown to be characteristic for pFCC fragmentation
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(Mühlecker et al., 1997; Mühlecker et al., 2000). Thus, pyrrole ring A (cleavage ‘d’ shown in Figure 1B and
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C) and/or the C83 methoxy group (cleavage ‘a’ shown in Figure 1B and C; loss as methanol) were lost
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with high efficiency, while pyrrole ring D was stable in both ions. To confirm the identity of these
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compounds as FCCs that are distinct from the respective NCCs, we performed isomerization experiments
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to experimentally convert an FCC to its respective NCC isomer (Oberhuber et al., 2003). For this, we used
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epi-pFCC that we produced from pheophorbide a in a well-established in vitro system using bell pepper
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chromoplasts (Christ et al., 2012). On reversed-phase HPLC, epi-pFCC has a longer retention time than
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pFCC (see Figure 2C) (Rodoni et al., 1997), but the fragmentation behavior in tandem MS experiments
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with a stable ring D was identical to that of pFCC (Supplemental Figure 1A online). When incubating at
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pH 5, epi-pFCC isomerized to a more-polar compound (Supplemental Figure 1B online), which when
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analyzed by tandem MS fragmented as a typical NCC (Christ et al. 2016), i.e. with high probability of ring
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D loss (Supplemental Figure 1A online), confirming the identity of the catabolites isolated from senescent
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Arabidopsis chloroplasts as FCCs, specifically as pFCC and hydroxy-pFCC, which solely differ by the
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attachment in the latter of an additional oxygen atom at ring A, likely at the C32 ethyl side chain.
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To verify that hydroxy-pFCC was present – and thus also likely formed – inside the intact plastids and to
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exclude the possibility that the identified FCCs were derived from extraplastidial contaminations of our
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chloroplast preparations, we took advantage of the fact that MES16 demethylates FCCs (Christ et al.,
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2012). Thus, we incubated plastid preparations before and after mechanical rupture with MES16 and
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analyzed the formation of the respective demethylated forms of pFCC and hydroxy-pFCC by LC-MS/MS
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(Figure 1A). Only after chloroplast rupture, pseudomolecular ions ([M+H]+) corresponding to desmethyl-
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pFCC (m/z 615.282; C34H39N4O7) and desmethyl-hydroxy-pFCC (m/z 631.277; C34H39N4O8) were produced,
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indicating that indeed, hydroxy-pFCC formation from pFCC takes place within intact senescent plastids.
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pFCC Hydroxylation Localizes in Plastid Membranes and Requires Molecular Oxygen and Ferredoxin.
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The above-mentioned in vitro assay that uses bell pepper chromoplasts as the source of PAO and RCCR
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for the production of epi-pFCC from pheophorbide a yielded, as a by-product, a second, more-polar,
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fluorescing fraction (Figure 2A) that exhibited a typical FCC absorption spectrum (Supplemental Figure
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2A), which we reckoned could be hydroxy-epi-pFCC, indicating that the chromoplast extracts used for
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epi-pFCC synthesis may also contain the activities required for catabolite hydroxylation. Indeed, in an
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identical assay, but using epi-pFCC instead of pheophorbide a as substrate, the polar FCC fraction
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increased in a time-dependent manner (Figure 2B). To confirm identity of this compound as hydroxy-epi-
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pFCC, we compared its retention time in HPLC with the retention times of the FCCs accumulating in
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gerontoplasts of Col-0 plants and of acd-22+At-RCCR-X plants (Pružinská et al., 2007) that were
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complemented with the epi-pFCC-producing ‘X’ version of Arabidopsis RCCR (see Introduction). Co-
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injection experiments indeed confirmed that the product of in vitro hydroxylation of epi-pFCC is identical
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to the polar FCC fraction from acd2-2+At-RCCR-X chloroplasts (Figure 2C). In addition, the product of in
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vitro hydroxylation was analyzed by LC-MS/MS (Supplemental Figure 2B and C), demonstrating it to be
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hydroxy-epi-pFCC with an identical mass and fragmentation pattern as shown for hydroxy-pFCC (Figure
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1B).
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The C32 hydroxylation reaction introduces one oxygen atom into the respective phyllobilins. To analyze
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whether this oxygen was derived from molecular oxygen, we dark-incubated Arabidopsis wild type
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leaves in the presence of 18O2 and analyzed phyllobilin labeling by LC-MS. Several phyllobilins known to
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be C32 hydroxylated, such as DNCC_618 and NCC_630 (Christ et al., 2016) carried up to two labeled
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oxygen atoms, while only one 18O-label was found in non-hydroxylated phyllobilins, such as DNCC_602
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and NCC_614 (Figure 3). In all cases the labeled oxygen atoms were exclusively bound to pyrrole ring A.
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In the case of NCCs, one label was derived from PAO activity (Hörtensteiner et al., 1998), while CYP89A9
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activity, ultimately leading to DNCCs, replaced the PAO-derived label by another labeled oxygen (Christ
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et al., 2013). This explained the presence of one 18O-label in non-hydroxylated phyllobilins, implying that
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the second label that was present in hydroxylated ones was derived by the activity of the hydroxylase,
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which introduced the oxygen atom at C32 from molecular oxygen.
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These data indicated that the hydroxylating activity could be a cytochrome P450 monooxygenase.
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Although most of these enzymes reside in the endoplasmic reticulum, the Arabidopsis genome encodes
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several P450 monooxygenases that likely are directed to the chloroplast (Schuler et al., 2006). P450
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monooxygenases are highly sensitive to CO (Schuler, 1996), which we used in a mixture with 50% (v/v)
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ambient air to test for inhibition in the hydroxylation assay. However, CO did not inhibit hydroxy-epi-
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pFCC formation as compared to an atmosphere containing a 1:1 (v/v) mixture of ambient air and
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nitrogen gas, while incubation in 100% nitrogen atmosphere largely inhibited the activity (Figure 4A).
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Thus, the responsible enzyme was unlikely a P450 monooxygenase.
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The bell pepper chromoplast-based in vitro assay for epi-pFCC production from pheophorbide a was
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shown to require both chromoplast membranes, as the source of PAO, and a soluble chromoplast
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fraction, as the source of RCCR. To further dissect the requirements for in vitro epi-pFCC hydroxylation,
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we fractionated total chromoplasts into soluble and membrane fractions. After two washing steps using
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ultracentrifugation, the activity remained in the membrane fraction, indicating that the responsible
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enzyme(s) are tightly associated with chloroplast membranes (Figure 4B). Similarly, the in vitro epi-pFCC
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hydroxylation assay was analyzed for the requirement for the additional cofactors present in the original
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PAO/RCCR assay, i.e. ferredoxin (Fd) and a Fd-reducing system, consisting of NADPH, Fd-NADPH
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oxidoreductase (FNR), glucose-6-phosphate (G-6-P) and G-6-P dehydrogenase (GDH). Using 2x
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ultracentrifugation-washed membranes to reduce carry over of potential cofactors, respective assays for
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the production of hydroxy epi-pFCC demonstrated the requirement of both Fd and the Fd-regenerating
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system (Figure 4C). Some activity was obtained without the addition of Fd, which likely was caused by
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some residual Fd remaining attached to the chromoplast membranes during the washing procedure.
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pFCC Hydroxylation is catalyzed by TIC55, a Rieske-Type Monooxygenase.
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Taking together, the properties for the in vitro epi-pFCC hydroxylating activity determined above, were
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similar to the requirements of PAO, a Rieske-type monooxygenase (Schellenberg et al., 1993;
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Hörtensteiner et al., 1995; Hörtensteiner et al., 1998), i.e. activity was attached to plastid membranes,
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was dependent on Fd, was not inhibited by CO, and the incorporated oxygen atom was derived from
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molecular oxygen. The Arabidopsis thaliana genome encodes five Rieske-type oxygenases (Gray et al.,
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2004), all of which have been shown to localize to chloroplasts (Rathinasabapathi et al., 1997; Tanaka et
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al., 1998; Bartsch et al., 2008). In addition, three of them, PAO (At3g44880), TIC55 (At2g24820) and
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PTC52 (At4g25650) possess predicted C-terminally located transmembrane domains (Figure 5A) that
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likely anchor these proteins to plastid membranes, while the other two Rieske-type oxygenases in
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Arabidopsis, chlorophyll a oxygenase (At1g44446) and choline monooxygenase (CMO)-like (At4g29890)
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are predicted to be soluble proteins (Tusnády and Simon, 2001). Both TIC55 and PTC52 have been
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considered to be components of distinct protein import complexes of the chloroplast, i.e. the TIC and
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PTC complex, respectively; however the role of the PTC complex in protein import in true leaves has
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been questioned (Kim and Apel, 2014). Nevertheless, we considered both proteins as likely candidates
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for FCC hydroxylation. First, we re-analyzed their predicted localization in the envelope (Bartsch et al.,
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2008) by transiently expressing GFP fusion proteins in mesophyll protoplasts isolated from senescent
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Arabidopsis wild-type leaves (Figure 5B). Indeed, GFP signals for both TIC55 and PTC52 surrounded the
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chloroplasts, i.e. indicating envelope localization like the envelope positive control, TIC110, while PAO
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co-localized with chlorophyll autofluorescence, i.e. confirming thylakoid localization (Sakuraba et al.,
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2012).
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To investigate whether one (or both) of these candidates catalyze FCC hydroxylation in vivo, we analyzed
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respective T-DNA insertion lines for both genes. As shown previously (Boij et al., 2009), none of the lines
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showed growth differences compared to wild type. In addition and similar to mutants in phyllobilin-
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modifying enzymes (Christ et al., 2012; Christ et al., 2013), we did not detect a visible phenotype during
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senescence. However, LC-MS analysis of senescent leaf extracts uncovered major phyllobilin differences
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between the three analyzed tic55 alleles and ptc52, which looked like wild type (Supplemental Figure 3
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online). Although the relative abundance of DNCCs and NCCs was unaltered in all investigated lines
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(Figure 6A), two of the three tic55 mutants, tic55-2 and tic55-3, lacked hydroxylated phyllobilins (Figure
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6B). The tic55-1 mutants had a largely reduced proportion of hydroxylated phyllobilins, implying some
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residual activity to be present in this allele. By contrast, ptc52 was comparable to Col-0 with about 60%
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of all phyllobilins being hydroxylated. Furthermore, glucosylated phyllobilins were absent from tic55
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mutants (Figure 6C) showing that phyllobilin glucosylation depends on prior C32 hydroxylation. These
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results demonstrated that phyllobilin hydroxylation is catalyzed by TIC55, but not by PTC52. To our
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surprise, tic55 mutants accumulated a smaller proportion of O84 demethylated phyllobilins (Figure 6D),
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indicating that MES16, catalyzing demethylation in Arabidopsis (Christ et al., 2012), might preferentially
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accept hydroxylated catabolites as substrate. Furthermore, the proportion of hydroxymethylated
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phyllobilins was higher in tic55 mutants (Figure 6E), which was in agreement with the suggestion that C2
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or C4 hydroxymethylation and C32 hydroxylation exclude each other (Süssenbacher et al., 2015).
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We additionally confirmed the absence of pFCC hydroxylation in tic55-3 by analyzing the FCC pattern of
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isolated plastids. As expected, hydroxy-pFCC was absent from gerontoplasts, which contained relatively
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more pFCC (Figure 7).
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TIC32 and TIC62 are Not Involved in the Redox-Cycle of TIC55.
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TIC55 had been considered to exhibit a redox regulatory role in protein import together with TIC32 and
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TIC62, two additional proposed components of the protein import machinery at the inner chloroplast
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envelope (Küchler et al., 2002; Hörmann et al., 2004). Both proteins were shown to possess NADPH
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binding domains and, in addition, TIC62 is able to interact with FNR (Bölter et al., 2015). This implies that
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one or both of these additional TIC components could be involved in delivering electrons to the Rieske
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and/or the mononuclear iron center of TIC55 and, thus, drive the redox cycle required for TIC55 activity.
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To test this possibility, we analyzed phyllobilin accumulation in tic32 and tic62 T-DNA insertion mutants
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during leaf senescence. As seen in Figure 6F, mutations in these genes did not affect phyllobilin
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hydroxylation, indicating that electron supply towards TIC55 is independent from TIC62 or TIC32.
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DISCUSSION
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Phyllobilin Hydroxylation by TIC55
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Most of the phyllobilins that have been identified to date as degradation products of chlorophyll in
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higher plants carry a hydroxyl group at the C32 position (Christ and Hörtensteiner, 2014; Kräutler, 2014,
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2016; Scherl et al., 2016). Furthermore, hydroxylated phyllobilins have been found in each species
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analyzed so far (Figure 8A), and in many cases have been shown to represent the major phyllobilin
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fraction (Oberhuber et al., 2003; Christ et al., 2013). This implies that chlorophyll catabolite
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hydroxylation is a common and important reaction, which, together with the upstream reactions that
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lead to the formation of pFCC, forms the core part of the PAO/phyllobilin pathway of chlorophyll
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breakdown in higher plants. Here we identified TIC55, an envelope-localized member of the Rieske-type
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oxygenases, as the phyllobilin hydroxylase in Arabidopsis. Our biochemical characterization and
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subsequent analysis of mutants defective in likely candidates unequivocally demonstrates that TIC55 is
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solely responsible for the formation of hydroxylated phyllobilins during leaf senescence-related
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chlorophyll breakdown. More specifically, based on the localization of TIC55 in the chloroplast and the
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fact that other phyllobilin-modifying activities are located outside plastids (Christ et al., 2012; Christ et
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al., 2013), pFCC is the likely sole in vivo substrate for hydroxylation by TIC55.
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This finding is rather surprising, because TIC55 was originally identified as a potential component of the
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protein import machinery at the chloroplast inner envelope (Calibe et al., 1997). Specifically, it was
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proposed that TIC55 together with TIC62 and TIC32 functions as redox sensor that links protein import to
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the redox state of the chloroplast (Balsera et al., 2010). As such, TIC55, TIC62, and TIC32 would play a
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regulatory role rather than being core components of the inner envelope import machinery itself, the
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exact composition of which is still under debate (Bölter et al., 2015). The rationale behind this proposed
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function is the presence in TIC55 of redox-active cysteine residues at the C-terminus that have been
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shown to be regulated by the thioredoxin system (Bartsch et al., 2008). Furthermore, the other two
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components of the so-called redox regulon (Stengel et al., 2007), i.e. TIC62 and TIC32, possess NADPH-
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binding sites and could exert their regulatory role depending on changes of the NADP+/NADPH ratio, a
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well-known read-out of the redox state of the chloroplast. Indeed, TIC62 was shown to shuttle between
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thylakoids and the inner envelope in a NADP+/NADPH ratio-dependent manner (Stengel et al., 2008),
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which together with its additional ability to bind FNR (Benz et al., 2009), could allow electron fluxes at
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these membranes to be highly dynamically regulated (Bölter et al., 2015). Metabolic NADP+/NADPH
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ratios directly influenced import efficiency of a subset of preproteins in Pisum sativum (Stengel et al.,
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2009). In addition, inhibition of protein import by ionomycin and ophiobolin A, two drugs that disrupt
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calcium signaling, has been attributed to inhibition of TIC32, which possesses a calmodulin binding site
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(Chigri et al., 2006). However, mutant analysis of redox regulon components did not show clear
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phenotypes, implying that under standard conditions this redox control may be not essential for plant
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viability and/or chloroplast protein import (Benz et al., 2009; Boij et al., 2009; Balsera et al., 2010).
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Here we show that TIC55 exhibits a defined role as phyllobilin hydroxylase; however, ferredoxin-derived
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electrons that are required to drive the redox cycle of this Rieske-type oxygenase (Ferraro et al., 2005)
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unlikely involve TIC62 and/or TIC62-bound FNR, because respective mutants did not exhibit phyllobilin
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hydroxylation deficiency.
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Rieske-type Oxygenases Play a Dominant Role in Chlorophyll Metabolism
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As stated above, the role of TIC55 in protein import has not been firmly established. The Rieske-type
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iron-sulfur cluster and the mononuclear iron site present in this protein were considered as parts of a
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hypothetical electron transport chain at the inner envelope with potential final transfer of electrons to
310
oxygen, thus integrating protein import to the redox regulatory network of chloroplasts (Bölter et al.,
311
2015). However, potential substrates for TIC55 that, as a genuine Rieske-type oxygenase (Ferraro et al.,
312
2005), catalyzes activation and incorporation of molecular oxygen have neither been considered nor
313
identified. Our
314
demonstrate that TIC55 is a monooxygenase that incorporates one oxygen atom derived from molecular
315
oxygen into pFCC.
316
The Arabidopsis genome encodes five Rieske-type oxygenases (Gray et al., 2002). With the identification
317
of TIC55 as a phyllobilin hydroxylase reported here, four out of these five have been attributed to
318
chlorophyll metabolism. Two of them are involved in chlorophyll biosynthesis, i.e. CAO that catalyzes the
319
oxidation of chlorophyll(ide) a to chlorophyll(ide) b (Tanaka et al., 1998), and PTC52 that has been
320
proposed to carry out the same oxidation, but using protochlorophyllide a as substrate; however, an in
321
vivo role of protochlorophyllide b has been questioned (Scheumann et al., 1999; Armstrong et al., 2000).
322
The other two Rieske-type oxygenases are involved in chlorophyll degradation, i.e. PAO, which is
323
responsible for porphyrin ring opening of pheophorbide a (Pružinská et al., 2005), and TIC55. The fifth
324
Rieske-type oxygenase, CMO-like, has high sequence similarity to CMO of Spinacia oleracea (spinach)
325
that catalyzes the first step of glycine betaine biosynthesis (Rathinasabapathi et al., 1997). However,
326
Arabidopsis does not produce glycine betaine and Arabidopsis CMO-like was unable to promote glycine
327
betaine formation (Hibino et al., 2002).
328
Phylogenetic analyses had revealed that for all five Rieske-type oxygenases highly homologous proteins
329
are present in higher plants (Gray et al., 2004). Re-evaluation of the homologies of PAO, PTC52, and
330
TIC55 incorporating sequences from Klebsormidium flaccidum (Hori et al., 2014) and from the
331
Phytozome database (https://phytozome.jgi.doe.gov/pz/portal.html), which currently includes 52
332
sequenced Viridiplantae species, showed that these three Rieske-type oxygenase cluster into three
333
distinct clades (Figure 8B). TIC55 and PAO orthologs are present in all land plants, including Selaginella
10
18
O2 labeling experiments of senescent leaves combined with in vitro activity assays
334
moellendorffii, a member of the Lycopodiopsida, the oldest lineage of vascular plants, and the bryophyte
335
Physcomitrella patens. The wide distribution of all three homologs within land plant species, but
336
apparent absence from chlorophyte green algae for which genomes are available, such as
337
Chlamydomonas reinhardtii, Micromonas pusilla, Volvox carteri, and Ostreococcus lucimarinus, suggests
338
that TIC55 in higher plants most likely originated from a recent gene duplication and subsequent
339
neofunctionalization to play a potential role in the adaptation to a terrestrial growth habit and/or high
340
light adaptation. Interestingly, the genome of the early charophyte species Klebsormidium flaccidum,
341
which is considered to be a close ancestor of land plants (Hori et al. 2014) also encodes TIC55- and PAO-
342
like proteins that exhibit rather high sequence similarities to the respective proteins from land plants.
343
This is in support of the idea that chlorophyll degradation co-evolved with the colonization of land by
344
plants.
345
346
Spatial Distribution of Chlorophyll Catabolic Enzymes within the Plastid
347
The common reactions of the PAO/phyllobilin pathway leading to pFCC localize to the chloroplast. This
348
has been substantiated by numerous investigations using different approaches, among them GFP-fusion
349
protein analysis, subcellular fragmentation studies, and enzyme activity measurements on isolated
350
chloroplast fractions (for recent reviews on the biochemistry of chlorophyll breakdown, see
351
(Hörtensteiner, 2013a, b). However, the sub-chloroplast location of respective reactions remained
352
largely unclear until recently, in particular because of the inconsistency with regard to the presence or
353
absence of transmembrane domains in these proteins. Thus, for example, PAO is an integral membrane
354
protein with two predicted transmembrane domains (Figure 5A) (Sakuraba et al., 2012), while RCCR,
355
with which it physically interacts during catalysis (Rodoni et al., 1997; Pružinská et al., 2007), is a soluble
356
protein (Wüthrich et al., 2000). Recently, based on protein interaction studies, we proposed the model
357
that all chloroplast-located chlorophyll catabolic enzymes form a highly dynamic multiprotein complex at
358
the thylakoid membrane with interaction with light harvesting complex II proteins (Sakuraba et al.,
359
2012).
360
The identification of the envelope-bound TIC55 as the phyllobilin hydroxylase is intriguing in this respect
361
(see Figure 9 for a topographical model of the PAO/phyllobilin pathway integrating TIC55). Nevertheless,
362
one can speculate that there is no requirement of including TIC55 in the above-mentioned thylakoid
363
membrane-bound degradative complex, because the proposed rationale behind this complex, i.e.
364
metabolic channeling of degradation intermediates to prevent potential phototoxicity (Sakuraba et al.,
365
2012), is reached with the formation of pFCC. A pFCC-derivative was recently shown to be able to
366
efficiently produce singlet oxygen in vitro when excited with UV light (Jockusch et al., 2014).
11
367
Nevertheless, when compared with pheophorbide, which absorbs a much wider spectral range and is
368
known as being highly cell phototoxic (Xodo et al., 2012), the in vivo capacity of pFCC to produce reactive
369
oxygen species and, thus, to act as a phototoxin is most likely rather limited. Envelope-localization of
370
TIC55 implies the requirement for pFCC movement from the thylakoid to the envelope, but it remains
371
unclear whether this is achieved by simple diffusion or is protein-guided. Instead of being important for
372
intermediate detoxification, like the upstream reactions, the localization of TIC55 at the envelope rather
373
implies a link to catabolite export from the plastid. The molecular nature of the chloroplast exporter(s)
374
for chlorophyll catabolites is unknown to date, but it has been speculated to be driven by an active
375
transport mechanism (Matile et al., 1992). The former link of TIC55 with protein import at the inner
376
envelope (Küchler et al., 2002) implies the intriguing possibility for phyllobilin export through the same
377
protein complex; however, this is highly speculative and export through the TIC/TOC system has never
378
been described. Whether TIC55 may directly interact with potential catabolite exporters remains
379
unclear, because phyllobilin export does not absolutely require previous reaction by TIC55, as
380
demonstrated by the presence of a substantial proportion of non-hydroxylated phyllobilins in senescent
381
wild-type leaves (Figure 6B) (Christ et al., 2016)
382
383
What is the Biological Role of Phyllobilin Modification?
384
None of the Arabidopsis mutants that are deficient in one of the known phyllobilin modifying reactions,
385
i.e. CYP89A9, MES16 or TIC55, show an obvious visible phenotype or are compromised in chlorophyll
386
breakdown during senescence (Christ et al., 2012; Christ et al., 2013). Furthermore, deformylation
387
(through CYP89A9 in Arabidopsis) and demethylation (through MES16 in Arabidopsis), but also C18-
388
dihydroxylation and C32-OH group conjugation are species-specific reactions (Hörtensteiner and Kräutler,
389
2011; Kräutler, 2014). Thus, seemingly these are non-essential reactions in chlorophyll degradation.
390
Nevertheless, the fact that all three, i.e. TIC55, CYP89A9, and MES16, are members of protein
391
subfamilies with 3, 7, and 3 members, respectively, in Arabidopsis (Gray et al., 2004; Bak et al., 2011;
392
Christ et al., 2012), suggests that they have been specifically recruited during evolution to function in
393
chlorophyll breakdown. This implies that phyllobilins could possibly play a role beyond being merely
394
degradation products. In line with this, phyllobilins have been shown to be efficient antioxidants and
395
may act as light filters or optical brighteners (Kräutler, 2016). To what extend such physicochemical
396
properties may serve for a biological role and to what extend phyllobilin modification is important in this
397
respect, remains unclear so far. The identification of TIC55 in this work offers the tool of producing a
398
cyp89a9 mes16 tic55 triple mutant in the future, for a more in-depth analysis about the role of
12
399
phyllobilin modification during chlorophyll breakdown and to investigate whether absence of these
400
modifications might have an impact on plant fitness, for example, during stress conditions.
401
402
403
METHODS
404
405
Plant Material and Senescence Induction
406
407
Arabidopsis thaliana ecotype Columbia-0 (Col-0) was used as the wild type. T-DNA insertion lines for the
408
following genes were used: TIC55 (AT2G24820), PTC52 (AT4G25650), TIC32 (At4g23430), and TIC62
409
(At3g18890). They were from the following collections: SALK lines (Alonso et al., 2003): tic55-1,
410
SALK_137948; tic55-2, SALK_086048C; ptc52, SALK_006984; GABI-KAT lines (Rosso et al., 2003): tic32,
411
GK_117H08; tic62, GK_439H04; SAIL lines (Sessions et al., 2002): tic55-3, SAIL_167_G08. These lines
412
were obtained from the European Arabidopsis Center, Nottingham, UK. Homozygous plants were
413
identified by PCR using T-DNA- and gene-specific primers as listed in Supplemental Table 1 online. Two
414
Arabidopsis acd2-2 complementation lines that express RCCR with either stereospecificity (acd2-2+At-
415
RCCR, producing pFCC; and acd2-2+At-RCCR-X, producing epi-pFCC) (Pružinská et al., 2007) were used as
416
reference for HPLC analysis.
417
For biochemical and physiological experiments, plants were either grown under short-day conditions (16
418
h dark/8 h light) or under long-day conditions (16 h light/8 h dark) under fluorescent light of 60 to 120
419
μmol photons m-2 s-1 at 22°C. Plants for seed production and DNA isolation were grown in a greenhouse
420
with rates of 100 to 200 μmol photons m-2 s-1 at 22°C. Leaf senescence was inducted by incubating
421
detached leaves on wet filter paper for up to 8 d in permanent darkness. For dark incubation under 18O2
422
atmosphere, leaves from short-day-grown Col-0 plants were excised and placed on wet filter paper in a
423
glass desiccator. After evacuation with a vacuum pump, the desiccator was aerated with 18O2 (97.1%,
424
Campro Scientific GmbH, Germany) and leaves senesced in the dark for 5 d. Red bell pepper fruits used
425
for chromoplast isolation were bought from a local market.
426
427
Biocomputational Methods
428
429
Orthologs of TIC55, PAO and PTC52 were identified using BLASTP searches (Altschul et al., 1997) at the
430
Phytozome database of the Joint Genome Institute (https://phytozome.jgi.doe.gov). Klebsormidium
431
flaccidum sequences were likewise obtained from the National Center for Biotechnology Information
13
432
(http://ncbi.nlm.nih.gov). For phylogenetic analysis, a multiple sequence alignment was generated with
433
the neighborhood joining method using MEGA7 (Kumar et al., 2016). Bootstrap analysis was performed
434
with 1000 replicates.
435
436
GFP Fusion Protein Production and Confocal Microscopy
437
438
TIC55 (BX819909) and PTC52 (pda06002) full-length cDNAs were, respectively, obtained from the Centre
439
National de Ressources Génomiques Végétales, France, and the RIKEN Tsukuba Institute
440
BioResourceCenter, Japan. TIC55 was amplified by PCR with KAPA HiFi HotStart ReadyMix (Kapa
441
Biosystems) using the primers listed in Supplemental Table 1. The resulting PCR fragment was cut with
442
XmaI and SpeI and ligated to XmaI/SpeI-digested pUC18-spGFP6 (Meyer et al., 2006) to create a fusion
443
between TIC55 and GFP at the C-terminus. Likewise, PTC52 was amplified by PCR using the primers listed
444
in Supplemental Table 1 and cloned into pUC18-spGFP6 via BamHI. Correctness of cDNA sequences and
445
orientation was verified by sequencing. As references for localization, GFP fusion proteins of TIC110
446
(chloroplast inner envelope membrane) and PAO (thylakoid membrane) (Sakuraba et al., 2012) were
447
used. The constructs were used to transform Arabidopsis protoplasts isolated from 6-week-old plants
448
grown in short-day conditions. Isolation of protoplasts was performed according to a published protocol
449
(Christ et al., 2012 and references therein). GFP fluorescence was imaged with a laser scanning confocal
450
microscope (TCS SP5; Leica Microsystems) at an excitation wavelength of 488 nm and signals were
451
detected between 495 nm and 530 nm. Chlorophyll autofluorescence was recorded at 643-730 nm.
452
453
Arabidopsis Chloroplast Isolation and MES16 Incubation
454
455
4-6-week-old plants grown in short-day conditions were used for chloroplast isolation in a two-step
456
purification procedure. A published protocol (Schelbert et al., 2009) was adapted to optimize isolation of
457
senescent protoplasts and chloroplasts. Leaves were detached and dark incubated for 4 d. After
458
isolation, mesophyll protoplasts were lysed by the addition of breakage buffer [containing 300 mM
459
sorbitol, 20 mM Tricine-KOH pH 8.4, 5 mM EDTA, 5 mM EGTA, 10 mM NaHCO3 and 0.1% (w/v) BSA] and
460
gerontoplasts purified on a discontinuous [85% and 40% (v/v)] gradient of Percoll containing the same
461
salts as the breakage buffer. After washing the chloroplasts obtained from the 85%/40% interphase
462
twice with HEPES-sorbitol buffer (330 mM sorbitol and 50 mM Hepes-KOH pH 8) they were analyzed for
463
colorless catabolites by HPLC or by LC-MS/MS (see below). For this, chloroplast fractions were
464
supplemented with one volume of methanol (v/v), mixed by vortexing and twice centrifuged (16,000g; 2
14
465
min). Alternatively, isolated chloroplasts were incubated for 10 min with MES16. For this, recombinant
466
MES16 was produced as described (Christ et al., 2012). A twenty-fifth volume of MES16-containing crude
467
protein extract (1 µg µL-1) was either added directly to intact chloroplasts or to chloroplasts that were
468
broken-up by a freezing in liquid N2 prior to the addition of the enzyme. Reactions were stopped by the
469
addition of one volume of methanol (v/v) and vortexing. After centrifugation twice (16,000g; 2 min) the
470
formation of demethylated hydroxy-pFCC was analyzed by LC-MS/MS (see below).
471
472
epi-pFCC Preparation
473
474
In order to produce epi-pFCC as substrate for the hydroxylation assay, PAO and RCCR were extracted
475
from red bell pepper chromoplasts according to a published protocol (Christ et al., 2012). The extracted
476
enzymes were then used to convert pheophorbide a (Hörtensteiner et al., 1995) to epi-pFCC according to
477
an established assay (Pružinská et al., 2007). epi-pFCC was concentrated on C18-SepPak columns
478
(Waters, Baden-Dättwil, Switzerland) and purified by HPLC (see below) for later use in hydroxylation
479
assays.
480
481
Isolation of the Hydroxylating Activity From Bell Pepper Chromoplasts and Activity Determination
482
483
Total chromoplasts isolated from red bell pepper fruits were also used as source of the hydroxylating
484
enzyme. Essentially the same protocol as for the extraction of PAO and RCCR (Christ et al., 2012) was
485
used, but with some modifications. Briefly, red bell pepper exocarp was blended with a fruit juicer in
486
chromoplast isolation buffer (1.33 mL g-1 tissue; Christ et al., 2012), filtered through Miracloth and then
487
centrifuged for 10 min at 12,000 g. The pellet was carefully resuspended in chromoplast isolation buffer
488
(0.33 mL g-1 tissue; Christ et al., 2012) and re-centrifuged. The pellet was finally resuspended in 8 ml of
489
25 mM Tris-MES pH 8 (25 µL g-1 tissue) and chromoplasts were broken by pressing them 10 times
490
through a 0.6 mm syringe needle. The resulting Total Chromoplast Protein fraction was either frozen in
491
liquid nitrogen and stored at -80°C for later use or centrifuged (15,000 g, 10 min) to separate
492
Supernatant and Pellet. The supernatant was further fractionated by ultra-centrifugation (UC) at
493
150,000g for 60 min into 1xUC-Membrane and 1xUC-Stroma. The obtained pellet was resuspended in
494
the start volume of 25 mM Tris-MES pH 8 and 10 times pushed through a needle as above. The
495
ultracentrifugation step was repeated, yielding 2xUC-Membrane and 2xUC-Stroma fractions,
496
respectively.
15
497
The standard hydroxylation assays (total volume 50 µL) consisted of 28 µL of respective chromoplast
498
fractions, 15 µM of epi-pFCC, 10 µg of Fd (Sigma Aldrich), and a Fd-reducing system consisting of 10 mM
499
G-6-P, 2.5 mM NADPH, 50 milliunits GDH, and 5 milliunits of FNR (Sigma Aldrich). After incubation at
500
25°C in darkness for up to 40 min, the reactions were terminated by adding methanol to a final
501
concentration of 50% (v/v). After centrifugation for 2 min at 16,000 g, samples were analyzed by HPLC or
502
by LC-MS/MS as described below. For reactions under defined atmospheric conditions, assays were set
503
up on ice in glass bulbs. After removal of the ambient air by vacuum suction, bulbs were flushed with the
504
desired gas mixtures, and assays performed as described above.
505
506
Colorless Chlorophyll Catabolites.
507
508
Chlorophyll catabolites were either analyzed by HPLC or by LC-MS/MS.
509
510
HPLC
511
For HPLC analysis, plant material was ground in liquid nitrogen and colorless catabolites were extracted
512
with 3 volumes (w/v) of 50 mM phosphate buffer pH 7/methanol (1:3, v/v). Plant extracts or
513
hydroxylation assay reactions were analyzed on a reverse-phase HPLC system as described (Christ et al.,
514
2012). The column was developed with a gradient (flow rate 1 mL min-1) of solvent A (25 mM potassium
515
phosphate pH 7.0), solvent B (methanol) and solvent C (H2O) as follows [%A/%B/%C (v/v/v)]: from
516
80/20/0 to 40/60/0 in 35 min, to 0/60/40 in 12 min, to 0/100/0 in 2 min, back to 0/60/40 in 7 min, and
517
finally back to 80/20/0 in 2 min. Peak detection was performed with sequential monitoring using a PDA-
518
100 photodiode array detector (200–700 nm; ThermoFisher Scientific) and a RF2000 fluorescence
519
detector (excitation at 320 nm, emission at 450 nm; Shimadzu). Chlorophyll catabolites were identified
520
by their absorption (FCCs, NCCs and DNCCs) and fluorescence (FCCs) properties. Relative amounts of
521
hydroxy-epi-pFCC obtained in hydroxylation assays were determined by peak areas at 320 nm.
522
523
LC-MS/MS
524
For LC-MS/MS analysis, plant material was collected in 2-mL microfuge tubes containing 500 µL of 1.25-
525
1.65 mm glass beads. The tissue was ground in liquid nitrogen using a MM300 Mixer Mill (Retsch,
526
Germany) at 30 Hz for 5 min. Chlorophyll catabolites were extracted with 5 volumes (w/v) of ice-cold
527
80% methanol, 20% H2O and 0.1% formic acid (v/v/v) containing 1 µg mL-1 ampicillin as internal standard,
528
sonicated for 2 min and twice centrifuged at 16,000 g for 2 min. Total plant extracts, chloroplast extracts
529
or reactions of hydroxylation assays were analyzed by LC-MS/MS (UltiMate 3000 RSLC system Thermo
16
530
Fisher Scientific, coupled to a Compact Q-TOF mass spectrometer with electron spray ionization, Bruker
531
Daltonics) as described (Christ et al., 2016). Two different LC programs were employed. In the short
532
program (Christ et al., 2016) the column was developed with a gradient (flow rate of 0.3 mL min-1) of
533
solvent B [acetonitrile with 0.1% (v/v) formic acid] in solvent A [water with 0.1% (v/v) formic acid] as
534
follows (all v/v): 30% for 0.5 min, 30% to 70% in 7.5 min, 70% to 100% in 0.1 min and 100% for 3.9 min.
535
In the long program, identical solvents were used, but the column was developed as follows (all v/v): 5%
536
B for 0.5 min, 30% B to 100% B in 11 min and 100% B for 4 min. Phyllobilins were identified and
537
quantified using an in-house-built spectrum library (Christ et al., 2016).
538
539
540
Accession Numbers
541
The following genes were used: TIC55 (AT2G24820), PTC52 (AT4G25650), TIC32 (At4g23430), and TIC62
542
(At3g18890).
543
Sequence data from Klebsormidium flaccidum can be found in GenBank/EMBL database
544
(http://www.ncbi.nlm.nih.gov) under the following accession numbers: Kfla_1 (GAQ77973.1), Kfla_2
545
(GAQ78701.1), Kfla_3 (GAQ92150.1).
546
All other sequences can be found in the Phytozome database (https://phytozome.jgi.doe.gov) under the
547
following accession numbers: Aquilegia coerulea: Acor_1, 22029521; Acor_2, 22030025; Acor_3,
548
22030648; Acor_4, 22035222; Acor_5, 22062530; Arabidopsis lyrata: Alyr_1, 16058352; Alyr_2,
549
16049304; Alyr_3, 16052333; Arabidopsis thaliana: Atha_PAO (At3g44880), 19664072; Atha_PTC52
550
(At4g25650), 19646489; Atha_TIC55, 19643456 (At2g24820); Amborella trichopoda: Atri_1, 31571943;
551
Atri_2, 31573466; Atri_3, 31569219; Brachypodium distachon: Bdis_1, 32798942; Bdis_2, 32805471;
552
Bdis_3, 32814680; Brassica rapa: Brap_1, 30640093; Brap_2, 30639463; Brap_3, 30615756;
553
Brachypodium stacei: Bsta_1, 32873310; Bsta_2, 32877401; Bsta_3, 32863896; Boechera stricta: Bstr_1,
554
30657466; Bstr_2, 30657466; Bstr_3, 30677801; Citrus clementina: Ccle_1, 20794617; Ccle_2, 20794921;
555
Ccle_3, 20814974; Ccle_4, 20817945; Ccle_5, 20814172; Capsella grandiflora: Cgra_1, 28896530;
556
Cgra_2., 28912086; Cgra_3., 28914744; Carica papaya: Cpap_1, 16406577; Cpap_2, 16406579; Cpap_3,
557
16409268; Cpap_4, 16420643; Capsella rubella: Crub_1, 20894180; Crub_2, 20886114; Crub_3,
558
20902616; Cucumis sativus: Csat_1, 16963640; Csat_2, 16963642; Csat_3, 16968781; Csat_4, 16973514;
559
Citrus sinensis: Csin_1, 18104396; Csin_2, 18110147; Csin_3, 18110062; Csin_4, 18109308; Csin_5,
560
18109304; Eucalyptus grandis: Egra_1, 32059933; Egra_2, 32036721; Egra_3, 32033828; Eutrema
561
salsugineum: Esal_1, 20208666; Esal_2, 20196368; Esal_3, 20194144; Fragaria vesca: Fves_1, 27274244;
562
Fves_2, 27249443; Fves_3, 27248027; Fves_4, 27263237; Fves_5, 27276459; Fves_6, 27275924; Glycine
17
563
max: Gmax_1, 30490036; Gmax_2, 30523624; Gmax_3, 30553241; Gmax_4, 30540276; Gmax_5,
564
30528498; Gmax_6, 30548653; Gossypium raimondii: Grai_1, 26820373; Grai_2, 26773144; Grai_3,
565
26773258; Grai_4, 26773690; Grai_5, 26765361; Kalanchoe marnieriana: Kmar_1, 32592111; Kmar_2,
566
32568622; Kmar_3, 32585756; Kmar_4, 32565330; Kmar_5, 32587661; Kmar_6, 32537639; Linum
567
usitatissimum: Lusi_1, 23170108; Lusi_10, 23152413; Lusi_11, 23179897; Lusi_12, 23153476; Lusi_2,
568
23178747; Lusi_3, 23178807; Lusi_4, 23178789; Lusi_5, 23178765; Lusi_6, 23150802; Lusi_7, 23150844;
569
Lusi_8, 23150790; Lusi_9, 23156438; Musa acuminata: Macu_1, 32305345; Macu_2, 32298593; Macu_3,
570
32299412; Macu_4, 32301167; Malus domestica: Mdom_1, 22648216; Mdom_2, 22680745; Mdom_3,
571
22673860; Mdom_4, 22657421; Mdom_5, 22669270; Mdom_6, 22681531; Mdom_7, 22646192;
572
Mdom_8, 22657424; Manihot esculenta: Mesc_1, 32328964; Mesc_2, 32352206; Mesc_3, 32352768;
573
Mesc_4, 32351848; Mesc_5, 32351381; Mesc_6, 32343636; Mimulus guttatus: Mgut_1, 28939106;
574
Mgut_2, 28929299; Mgut_3, 28927831; Mgut_4, 28928063; Medicago truncatula: Mtru_1, 31054512;
575
Mtru_2, 31108864; Mtru_3, 31076177; Mtru_4, 31075891; Mtru_5, 31071508; Oryza sativa: Osat_1,
576
33136036; Osat_2, 33129257; Osat_3, 33130101; Osat_4, 33126987; Osat_5, 33128763; Panicum hallii:
577
Phal_1, 32526147; Phal_2, 32510109; Physcomitrella patens: Ppat_1, 32958411; Ppat_2, 32971600;
578
Ppat_3, 32943162; Ppat_4, 32923430; Ppat_5, 32934884; Prunus persica: Pper_1, 32111912; Pper_2,
579
32118911; Pper_3, 32087889; Pper_4, 32086408; Populus trichocarpa: Ptri_1, 27044612; Ptri_2,
580
27044125; Ptri_3, 26998722; Ptri_4, 26997353; Ptri_5, 26997432; Ptri_6, 26992599; Ptri_7, 27007363;
581
Ptri_8, 27010289; Panicum virgatum: Pvir_1, 30249395; Pvir_2, 30232924; Pvir_3, 30210200; Pvir_4,
582
30208474; Pvir_5, 30224983; Phaseolus vulgaris: Pvul_1, 27170687; Pvul_2, 27168327; Pvul_3,
583
27147757; Pvul_4, 27151382; Ricinus communis: Rcom_1, 16802948; Rcom_2, 16802951; Rcom_3,
584
16803313; Rcom_4, 16811159; Sorghum bicolor: Sbic_1, 32753731; Sbic_2, 32754631; Sbic_3,
585
32744056; Sphagnum fallax: Sfal_1, 32628626; Sfal_2, 32612026; Sfal_3, 32611292; Sfal_4, 32631199;
586
Sfal_5, 32631202; Sfal_6, 32631211; Sfal_7, 32615809; Setaria italica: Sita_1, 32697922; Sita_2,
587
32693755; Sita_3, 32689656; Solanum lycopersicum: Slyc_1, 27278224; Slyc_2, 27297008; Slyc_3,
588
27308810; Selaginella moellendorffii: Smoe_1, 15421380; Smoe_2, 15420984; Spirodela polyrhiza:
589
Spol_1, 31503412; Spol_2, 31521506; Spol_3, 31512183; Spol_4, 31510559; Salix purpurea: Spur_1,
590
31446831; Spur_2, 31444937; Spur_3, 31408599; Spur_4, 31425200; Spur_5, 31434385; Spur_6,
591
31420119; Spur_7, 31420805; Spur_8, 31420645; Solanum tuberosum: Stub_1, 24409796; Stub_2,
592
24385728; Stub_3, 24377864; Setaria viridis: Svir_1, 32663617; Svir_2, 32653526; Svir_3, 32653709;
593
Theobroma cacao: Tcac_1, 27426931; Tcac_2, 27427235; Tcac_3, 27426954; Vitis vinifera: Vvin_1,
594
17833437; Vvin_2, 17833438; Vvin_3, 17837646; Vvin_4, 17841070; Zea mays: Zmay_1, 30990042;
595
Zmay_2, 31009998; Zmay_3, 31016834.
18
596
597
598
Supplemental Data
599
Supplemental Figure 1. Confirmation of epi-pFCC and pFCC Identity and epi-pFCC-to-epi-pNCC
600
Isomerization.
601
Supplemental Figure 2. LC-MS Confirmation of Hydroxy-epi-pFCC.
602
Supplemental Figure 3. LC-MS Analysis of Phyllobilins from Col-0 and Rieske-type Oxygenase
603
Mutants.
604
Supplemental Table 1. List of Primers Used in This Study.
605
Supplemental File 1. Text file of the alignment corresponding to the phylogenetic analysis in
606
Figure 8.
607
608
609
ACKNOWLEDGMENTS
610
This work was supported by the Swiss National Science Foundation (grant no. 31003A_149389) and
611
CropLife (to S.H.).
612
613
AUTHOR CONTRIBUTIONS
614
615
S.H. designed research; M.H., B.C., and A.D. performed research; M.H., B.C., S.A. and S.H. analyzed data;
616
and M.H., and S.H. wrote the paper.
617
618
619
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811
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812
807.
813
814
815
25
816
FIGURE LEGENDS
817
818
Figure 1. pFCC and Hydroxy-pFCC Accumulate in Gerontoplasts of Arabidopsis.
819
820
(A) Extracted ion chromatograms (EICs) of intact and broken gerontoplasts incubated with MES16. In
821
intact gerontoplasts, pFCC (EIC 629.2977±0.01) and hydroxy-pFCC (EIC 645.2929±0.01) are not
822
demethylated by the added MES16. However, in broken gerontoplasts, MES16 can access both as
823
substrate, leading to the formation of demethylated products (EIC 615.2827 and EIC 631.2777,
824
respectively). Thus, pFCC hydroxylation occurs inside the chloroplast.
825
(B) and (C) MS (top panel) and MS/MS spectra (bottom panel) of epi-pFCC [EIC 629.2980±0.01 (B)] and
826
hydroxy-epi-pFCC [EIC 645.2929±0.01 (C)]. Constitutional formulae and MS/MS fragmentation sites are
827
shown. [M+H]+ indicates the precursor ion.
828
829
830
Figure 2. In Vitro Formation of Hydroxy-epi-pFCC from epi-pFCC in Bell Pepper Chromoplast Extracts.
831
832
(A) In vitro assay for the production of epi-pFCC from pheophorbide a using a bell pepper chromoplast
833
extract. Besides epi-pFCC, the HPLC chromatogram shows a small, polar peak that was identified as
834
hydroxy-epi-pFCC.
835
(B) Time-dependent synthesis of hydroxy-epi-pFCC from epi-pFCC in an assay that is identical to the one
836
used in (A) for the production of epi-pFCC from pheophorbide a.
837
(C). HPLC-based confirmation of the product of the in vitro hydroxylation assay as hydroxy-epi-pFCC.
838
Chromatograms from top: gerontoplast extracts from acd2-2+At-RCCR (Pružinská et al., 2007) that
839
produces pFCC and hydroxy-pFCC; gerontoplast extracts from acd2-2+At-RCCR-X (Pružinská et al., 2007)
840
that produces epi-pFCC and hydroxy-epi-pFCC; in vitro assay after 40 min of incubation; co-injection of
841
acd2-2+At-RCCR-X and the assay, yielding a single polar peak that corresponds to hydroxy-epi-pFCC.
842
843
844
Figure 3. 18O2-Labeling of Phyllobilins during Col-0 Leaf Senescence.
845
846
MS spectra of selected DNCCs (A) and NCCs (B) produced in the presence of ambient air (16O2; top
847
spectra) or in an 18O2 atmosphere (bottom spectra). Note that in the presence of 18O2 C32-hydroxylated
848
phyllobilins (e.g. DNCC_618 and NCC_630) carry two labeled oxygens, while non-hydroxylated
26
849
phyllobilins (e.g. DNCC_602 and NCC_614) only carry one. This confirms that the C32 hydroxyl group-
850
oxygen is derived from molecular oxygen.
851
852
853
Figure 4. Biochemical Characterization of epi-pFCC Hydroxylation.
854
855
(A). In vitro hydroxylation assays with epi-pFCC as substrate were performed in different atmospheric
856
conditions as indicated. Note that the absence of oxygen significantly inhibits hydroxy epi-pFCC
857
formation (inset), thus confirming the molecular oxygen incorporation shown in Figure 3. Note also that
858
CO treatment does not affect hydroxy epi-pFCC formation (inset), likely excluding the respective enzyme
859
to be a cytochrome P450 monooxygenase. Inset, relative abundance of formed hydroxy epi-pFCC. Ratios
860
of peak areas of hydroxy epi-pFCC and epi-pFCC are shown.
861
(B) Fractionation of the epi-pFCC hydroxylating activity in bell pepper extracts. Fractionation at low
862
centrifugation speed (15,000 g) into a soluble (Supernatant) and membrane (Pellet) fraction was unable
863
to clearly separate the activity; however, subsequent twofold ultracentrifugation (UC) (150,000g) of the
864
Supernatant fraction uncovered the activity to reside in chromoplast membranes (1xUC- and 2xUC-
865
Membrane), while no activity was found in respective supernatants (1xUC- and 2xUC-Stroma).
866
(C) epi-pFCC hydroxylation depends on ferredoxin. Co-factor requirement was analyzed in assays using
867
the 2xUC-Membrane fraction of (B) and added factors as indicated. All cofactors are ferredoxin (Fd),
868
NADPH, glucose-6-phosphate (G-6-P) and G-6-P dehydrogenase (GDH). Note that in the absence of Fd
869
little activity was retained, likely because of the presence of residual amounts of Fd in the 2xUC-
870
Membrane fraction. Data are the mean of three replicates. Error bars indicate SD.
871
872
873
Figure 5. Protein Alignment, and Analysis of Subcellular Localization of the Arabidopsis Rieske-type
874
Oxygenases TIC55, PTC52 and PAO.
875
876
(A) Sequence alignment of Arabidopsis TIC55, PTC52, and PAO. Sequences were aligned using the
877
program DIALIGN. Dark gray shading highlights residues identical between all three proteins, light gray
878
shading highlights residues identical between two proteins, with Blosum62 similarity groups enabled.
879
The following colored domains are boxed: green, Rieske center; blue, mononuclear iron site; purple,
880
predicted transmembrane domains. The predicted cleavage sites of chloroplast transit peptides are
881
indicated with red lines.
27
882
(B) Localization of Rieske-type oxygenase-green fluorescent protein (GFP) fusion proteins in protoplasts
883
isolated from senescent Arabidopsis wild-type leaves. GFP fluorescence (column 1) and chlorophyll
884
autofluorescence (column 2) were examined by confocal laser scanning microscopy. Column 3, merge of
885
GFP and chlorophyll fluorescence; column 4, bright field image. Bars = 20 μm.
886
887
888
Figure 6. Phyllobilin Analysis of tic and ptc52 Mutants.
889
890
Abundances of phyllobilins were determined by LC-MS/MS analysis according to a published method
891
(Christ et al., 2016) and are shown as relative values. Data are the mean of four biological replicates.
892
Error bars indicate SD.
893
(A)-(E) Phyllobilin abundance in short-day-grown tic55 and ptc52 mutants after senescence induction for
894
5 d. Note the different scale used in (C) and (D) because of the low abundance of glucosylated and
895
demethylated phyllobilins.
896
(F) Abundance of hydroxylated phyllobilins in a tic32 and tic62 mutant.
897
898
899
Figure 7. Hydroxy-pFCC is Absent from tic55-3 Gerontoplasts.
900
901
Extracted ion chromatograms (EICs) of pFCC (EIC 629.2977±0.01) and hydroxy-pFCC (EIC 645.2929±0.01)
902
of gerontoplasts of wild type (Col-0) and tic55-3 are shown. Note the absence of hydroxy-pFCC from the
903
mutant.
904
905
906
Figure 8. Phyllobilin Distribution and Phylogenetic Analysis of Rieske-type Oxygenases in Plants.
907
908
(A) Phyllobilin hydroxylation (R1) is a common modification, which is found in DNCCs (red square) and
909
NCCs (green square) of all 20 species analyzed so far for structures of phyllobilins. By contrast, other
910
modifications (R2-R4; see DNCC and NCC structures for the position of these modifications) occur in a
911
species-specific manner.
912
(B) Phylogenetic analysis of Arabidopsis TIC55 (yellow asterisk), PAO (blue asterisk) and PTC52 (red
913
asterisk) homologs in plants. Note that except a few (black lines), most of the 204 protein sequences
914
compared in this analysis cluster in one of three distinct clades. Also note that for each species at least
28
915
one Rieske-type oxygenase protein is located within each clade. The tree was generated with the
916
neighborhood joining method using MEGA7 (Kumar et al., 2016). Important nodes separating the three
917
clades are labeled with bootstrap values (% of 1,000 replicates). Acor, Aquilegia coerulea; Alyr,
918
Arabidopsis lyrata; Atha, Arabidopsis thaliana; Atri, Amborella trichopoda; Bdis, Brachypodium
919
distachon; Brap, Brassica rapa; Bsta, Brachypodium stacei; Bstr, Boechera stricta; Ccle, Citrus clementina;
920
Cgra, Capsella grandiflora; Cpap, Carica papaya; Crub, Capsella rubella; Csat, Cucumis sativus; Csin, Citrus
921
sinensis; Egra, Eucalyptus grandis; Esal, Eutrema salsugineum; Fves, Fragaria vesca; Gmax, Glycine max;
922
Grai, Gossypium raimondii; Kfla, Klebsormidium flaccidum; Kmar, Kalanchoe marnieriana; Lusi, Linum
923
usitatissimum; Macu, Musa acuminata; Mdom, Malus domstica; Mesc, Manihot esculenta; Mgut,
924
Mimulus guttatus; Mtru, Medicago truncatula; Osat, Oryza sativa; Phal, Panicum hallii; Ppat,
925
Physcomitrella patens; Pper, Prunus persica; Ptri, Populus trichocarpa; Pvir, Panicum virgatum; Pvul,
926
Phaseolus vulgaris; Rcom, Ricinus communis; Sbic, Sorghum bicolor; Sfal, Sphagnum fallax; Sita, Setaria
927
italica; Slyc, Solanum lycopersicum; Some, Selaginella moellendorffii; Spol, Spirodela polyrhiza; Spur, Salix
928
purpurea; Stub, Solanum tuberosum; Svir, Setaria viridis; Tcac, Theobroma cacao; Vvin, Vitis vinifera;
929
Zmay, Zea mays.
930
931
932
Figure 9. Topographical Model of the Chlorophyll Breakdown Pathway Integrating the Findings of This
933
Work.
934
935
The model shows the subcellular localization of pFCC-modifying activities, i.e. CYP89A9 (deformylation),
936
MES16 (demethylation) and TIC55 (hydroxylation). Note, that all the reactions involved in converting
937
chlorophyll to pFCC, including pheophorbide a oxygenase (PAO) the name-giving key enzyme of the
938
PAO/phyllobilin pathway, are not shown, but have been considered to form a highly dynamic complex at
939
the thylakoid membrane to prevent release into the stroma of potentially phototoxic breakdown
940
intermediates upstream of pFCC (Sakuraba et al., 2012). Note also that due to the fact that non-
941
hydroxylated phyllobilin occur in wild type Arabidopsis, a fraction of pFCC likely leaves the chloroplast
942
without hydroxylation. As indicated by the question marks, the molecular identity of phyllobilin
943
transporters at the chloroplast envelope and the tonoplast is unknown. DFCC, dioxobilin-type
944
fluorescent chlorophyll catabolites; DNCC, dioxobilin-type nonfluorescent chlorophyll catabolites; ER,
945
endoplasmic
946
formyloxobilin-type nonfluorescent chlorophyll catabolites; pFCC, primary fluorescent chlorophyll
947
catabolite.
reticulum;
FCC,
formyloxobilin-type
29
fluorescent
chlorophyll
catabolites;
NCC,
A
Intens. Intact chloroplasts
4
x10
hydroxy-pFCC
pFCC
0.75
EIC 629.297±0.01
EIC 645.292±0.01
0.50
EIC 615.282±0.01
0.25
EIC 631.277±0.01
0
4
x10
1.00
0.75
Broken chloroplasts
desmethylhydroxy-pFCC
hydroxy-pFCC
pFCC
desmethyl-pFCC
EIC 629.297±0.01
0.50
EIC 645.292±0.01
EIC 615.282±0.01
0.25
EIC 631.277±0.01
0
4
B
5
6
7
Intens.
epi-pFCC
4
x10
3
8
9
Retention time (min)
H2C
O
18
H3C
c 16
D
MS
CH3
2
O
1
19
NH
A
NH 4 d
32
15
2
N
629.2980
NH
B
C
H3C
CH3
82
O
1
0
4
x10
O
83
O
OH
O
a
CH3
460.1868 [M+H]+-[a+d]
0.5
MS/MS
492.2133 [M+H]+-[d]
0.4
0.3
506.2319 [M+H]+-[c]
0.2
597.2647 [M+H]+-[a]
0.1
[M+H]+
0.0
100
C
200
300
400
500
Intens.
4
Hydroxy-epi-pFCC
x10
4
H2C
600
O
OH
H3C
NH
NH
N
NH
[m/z]
MS
CH3
O
c
700
645.2927
d
3
H3C
2
CH3
O
O
O
OH
1
a
O
CH3
0
4
x10
MS/MS
492.2133 [M+H]+-[d]
1.0
460.1889 [M+H] -[a+d]
+
0.5
613.2598 [M+H]+-[a]
522.2266 [M+H]+-[c]
[M+H]+
0.0
100
200
300
400
500
600
700
[m/z]
Figure 1. pFCC and Hydroxy-pFCC Accumulate in Gerontoplasts of Arabidopsis.
(A) Extracted ion chromatograms (EICs) of intact and broken gerontoplasts incubated with MES16. In intact gerontoplasts, pFCC (EIC 629.2977±0.01) and
hydroxy-pFCC (EIC 645.2929±0.01) are not demethylated by the added MES16. However, in broken gerontoplasts, MES16 can access both as substrate, leading to
the forma on of demethylated products (EIC 615.2827 and EIC 631.2777, respec vely). Thus, pFCC hydroxyla on occurs inside the chloroplast.
(B) and (C) MS (top panel) and MS/MS spectra (bo om panel) of pFCC [EIC 629.2980±0.01 (B)] and hydroxy-pFCC [EIC 645.2929±0.01 (C)]. Cons tu onal formulae
and MS/MS fragmenta on sites are shown. [M+H]+ indicates the precursor ion.
Relative fluorescence (320/450 nm)
A
epi-pFCC
hydroxy-epi-pFCC
25
30
B
35
40
45
50
Retention time (min)
hydroxy-epi-pFCC
epi-pFCC
Relative fluorescence (320/450 nm)
0 min
10 min
20 min
40 min
25
C
30
hydroxy-pFCC
35
hydroxy-epi-pFCC
40
45
50
Retention time (min)
epi-pFCC
pFCC
Relative fluorescence (320/450 nm)
Chloroplasts
acd2-2+At-RCCR
Chloroplasts
acd2-2+At-RCCR-X
Hydroxylation assay
with epi-pFCC
Co-injection: assay/
acd2-2+At-RCCR-X
25
30
35
40
45
50
Retention time (min)
Figure 2. In Vitro Forma on of Hydroxy-epi-pFCC from epi-pFCC in Bell Pepper Chromoplast Extracts.
(A) In vitro assay for the produc on of epi-pFCC from pheophorbide a using a bell pepper chromoplast extract. Besides epi-pFCC, the HPLC chromatogram shows a small, polar peak
that was iden fied as hydroxy-epi-pFCC.
(B) Time-dependent synthesis of hydroxy-epi-pFCC from epi-pFCC in an assay that is iden cal to the one used in (A) for the produc on of epi-pFCC from pheophorbide a.
(C) HPLC-based confirma on of the product of the in vitro hydroxyla on assay as hydroxy-epi-pFCC. Chromatograms from top: gerontoplast extracts from acd2-2+At-RCCR
(Pružinská et al., 2007) that produces pFCC and hydroxy-pFCC; gerontoplast extracts from acd2-2+At-RCCR-X (Pružinská et al., 2007) that produces epi-pFCC and hydroxy-epi-pFCC;
in vitro assay a er 40 min of incuba on; co-injec on of acd2-2+At-RCCR-X and the assay, yielding a single polar peak that corresponds to hydroxy-epi-pFCC.
A
H2C
H2C
CH3
H3C
DNCC_618
16
O2
NH
NH
NH
NH
O
DNCC_602
16
18
618
619
620
621
O
622
619
620
621
622
624
623
H2C
625
624
O
625
[m/z]
O2
602
603
604
605
606
607
608
[m/z]
601
602
603
604
605
606
607
608
[m/z]
O2
[m/z]
H2C
18
632
633
634
NH
NH
NH
16
637
638
633
634
635
636
637
638
NH
NH
O
OH
OH
636
NH
CH3
O
O
[m/z]
O2
632
O2
NH
H3C
18
631
CH3
CH3
NCC_614
CH3
O
635
O
O
H3C
NH
OH
631
O
OH
OH
H3C
O
CH3
601
H3C
16
NH
O
CH3
O
NCC_630
NH
OH
OH
18
618
NH
CH3
O
O
623
NH
H3C
O2
617
O2
CH3
OH
617
H3C
OH
H3C
B
CH3
O O
O O
[m/z]
O
OH
614
615
616
617
618
619
620
621
[m/z]
614
615
616
617
618
619
620
621
[m/z]
O2
Figure 3. 18O2-Labelling of Phyllobilins during Col-0 Leaf Senescence.
MS spectra of selected DNCCs (A) and NCCs (B) produced in the presence of ambient air (16O2; top spectra) or in an 18O2 atmosphere (bo om spectra). Note that
in the presence of 18O2 C32-hydroxylated phyllobilins (e.g. DNCC_618 and NCC_630) carry two labeled oxygens, while non-hydroxylated phyllobilins (e.g.
DNCC_602 and NCC_614) only carry one. This confirms that the C32 hydroxyl group-oxygen is derived from molecular oxygen.
A
Relative
abundance
16
epi-pFCC
12
8
4
Relative fluorescence (320/450 nm)
0
Air
N2 CO/Air N2/Air
hydroxy-epi-pFCC
Ambient air
100% N2
50%CO:50%Air (v/v)
50%N2:50%Air (v/v)
25
30
35
45
50
Retention time (min)
150,000 g
6
4
2
0
12
10
8
6
4
2
n.d.
s
n.d.
Co
fac
tor
No
ofa
cto
r
lC
Al
t
lle
Pe
1x
UC
-S
1x
tro
UC
ma
-M
em
bra
2x
ne
UC
S
2x
tr o
UC
ma
-M
em
bra
ne
Su
pe
rna
tan
t
0
s
+
GD NA
D
H, PH
G- ,
6P
+F
NR
,F
d
15,000 g
8
Relative abundance of hydroxy-epi-pFCC
C
Relative abundance of hydroxy-epi-pFCC
B
40
Figure 4. Biochemical Characteriza on of epi-pFCC Hydroxyla on.
(A) In vitro hydroxyla on assays with epi-pFCC as substrate were performed in different atmospheric condi ons as indicated. Note that absence of oxygen
significantly inhibits hydroxy epi-pFCC forma on (inset), thus confirming the molecular oxygen incorpora on shown in Figure 3. Note also that CO treatment does
not affect hydroxy epi-pFCC forma on (inset), likely excluding the respec ve enzyme to be a cytochrome P450 monooxygenase. Inset, rela ve abundance of
formed hydroxy epi-pFCC. Ra os of peak areas of hydroxy epi-pFCC and epi-pFCC are shown.
(B) Frac ona on of the epi-pFCC hydroxyla ng ac vity in bell pepper extracts. Frac ona on at low centrifuga on speed (15,000g) into a soluble (Supernatant)
and membrane (Pellet) frac on was unable to clearly separate the ac vity; however, subsequent twofold ultracentrifuga on (UC) (150,000g) of the Supernatant
frac on uncovered the ac vity to reside in chromoplast membranes (1xUC- and 2xUC-Membrane), while no ac vity was found in respec ve supernatants (1xUCand 2xUC-Stroma).
(C) epi-pFCC hydroxyla on depends on ferredoxin. Co-factor requirement was analyzed in assays using the 2xUC-Membrane frac on of (B) and added factors as
indicated. All cofactors are ferredoxin (Fd), NADPH, glucose-6-phosphate (G-6-P) and G-6-P dehydrogenase (GDH). Note that in the absence of Fd li le ac vity
was retained, likely because of the presence of residual amounts of Fd in the 2xUC-Membrane frac on. Data are the mean of three replicates. Error bars indicate
SD.
A
*
20
*
40
*
60
*
80
*
100
TIC55 : MAVPFLSSSLQLTPTSPILFTKVTPTPIIHNHRSTCTIPTKPRLRLLRRSAVAGTA--------------------------VSDQTEGGGDVLLNPEEEKRV-- :
PTC52 : MEAALAACALPSLRILNTKPRFRCSFSNP-------SLPISPNSLITRKSSRFTTA--------------------------VSSPPSSSAATSTNSPPEPEALF :
PAO
: MSVVLLS---------------------------------STSATITKSQSKKIPFLSPTTKFPLKVSISPSRSKLFHNPLRVAAPPSVPTSDST---EEKRIEE :
77
72
69
*
120
*
140
*
160
*
180
*
200
*
TIC55 : --------EVADYDWTEEWYPLYLTKNVPEDAPLGLTVYDRQIVLYKDGEGTLRC-YEDRCPHRLAKLSEGQLI-DGRLECLYHGWQFEGEGKCVKIPQLP---- : 168
PTC52 : ------EPGSDKFDWYANWYPVMPICDLDKKVPHGKKVMGIDLVVWWDRNEKQWKVMDDTCPHRLAPLSDGRIDQWGRLQCVYHGWCFNGSGDCKLIPQAPPDGP : 171
PAO
: EYGGDKEEEGSEFKWRDHWYPVSLVEDLDPNVPTPFQLLGRDLVLWFDRNDQKWAAFDDLCPHRLAPLSEGRLDENGHLQCSYHGWSFGGCGSCTRIPQAATSGP : 174
220
*
240
*
260
*
280
*
300
*
TIC55 : -ASAKIPKAACVKTYEVKDSQGVVWVWMSTKTPPNPEKLPWF---E-NFARPGFFDISTTHELPYDHSILLENLMDPAHVPISHDRTDFTAK------REDAQPL : 262
PTC52 : PVHTF--KQACVAVYPSTVQHEIIWFWPNSDPKYKNIIETNKPPYIPELEDPSFTKLMGNRDIPYGYDVLVENLMDPAHVPYAHYGLMRFPKPKEKIDREGGKPL : 274
PAO
: EARAVKSPRACAIKFPTMVSQGLLFVWPDENGWDRANSIEPPRLPD-DFDKPEFSTVTIQRDLFYGYDTLMENVSDPSHIDFAHHKV--TGR------RDRAKPL : 270
320
*
340
*
360
*
380
*
400
*
420
TIC55 : VFEVTERSNRGFAGTWGREKEGGKGSNLLRFDAPCVLQNNREFEGKDGV--------------KNYFSGLFLCRPTGQGKSMLIVRFGVTKRSPLV------SVL : 347
PTC52 : EINVKKLDNKGFFSKQEWGYSN--------FIAPCVYRSSTDPLPEQEHEYPAPAASDKAALSKRRLSLIFICIPVSPGRSRLIWTFPRNFGVFID------KIV : 365
PAO
: PFKVESSGPWGFQGANDDSPRITA-----KFVAPCYSMNKIELDAKLPIVGNQKWVIWICSFN----------IPMAPGKTRSIVCSARNFFQFSVPGPAWWQVV : 360
*
440
*
460
*
480
*
500
*
520
TIC55 : PQWFWHQNACKVFEQDMGFLSSQNEVLMKEKVPTKDLYLNLKS---------SDTWVAEYRKWMDKVGHGMPYHFGHRTIS-LPKVPPVVEHAPAGLIAALSASY : 442
PTC52 : PRWVFHIGQNTILDSDLHLLHVEERKILERGPEN--------WQKACFIPTKSDANVVTFRRWFNKYSEARVDWRGKFDPFLLPPTPP---RE------------ : 447
PAO
: PRWYEHWTSNLVYDGDMIVLQGQEKVFLAKSMESPDYDVNKQYTKLTFTPTQADRFVLAFRNWLRRHGKSQPEWFGSTPSN-QPLPSTVLTKR------------ : 452
*
540
*
560
*
580
*
600
*
620
TIC55 : PAKGGIGTMHAPNLANRYFRHIIHCRSCSNVIKSFELWKNILSATAVALTALAILVVSR-----QWKAVLLGSAALCSAAAYTCLRAINLNTNNFIRTHRRL : 539
PTC52 : ------------QLFDRYWSHVENCSSCKKAHKYLNALEVILQIASVAMIGVMAVLKQTTMSNVARIAVLVAAVLSFAASKWLSHFIYKTFHYHDYNHAVV- : 536
PAO
: ------------QMLDRFDQHTQVCSSCKGAYNSFQILKKFLVGATVFWAATAGVP-----SDVQIRLVLAGLSLISAASAYALHEQEKNFVFRDYVHSEIE : 537
B
chlorophyll
merge
bright field
TIC110-GFP
PAO-GFP
PTC52-GFP
TIC55-GFP
GFP
Figure 5. Protein Alignment, and Analysis of Subcellular Localiza on of the Arabidopsis Rieske-type Oxygenases TIC55, PTC52 and PAO.
(A) Sequence alignment of Arabidopsis TIC55, PTC52 and PAO. Sequences were aligned using the program DIALIGN. Dark gray shading highlights residues iden cal
between all three proteins, light gray shading highlights residues iden cal between two proteins, with Blosum62 similarity groups enabled. The following colored
domains are boxed: green, Rieske center; blue, mononuclear iron site; purple, predicted transmembrane domains. The predicted cleavage sites of chloroplast
transit pep des are indicated with red lines.
(B) Localiza on of Rieske-type oxygenase-green fluorescent protein (GFP) fusion proteins in protoplasts isolated from senescent Arabidopsis wild type leaves. GFP
fluorescence (column 1) and chlorophyll autofluorescence (column 2) were examined by confocal laser scanning microscopy. Column 3, merge of GFP and
chlorophyll fluorescence; column 4, bright field image. Bars = 20 μm.
A
DNCCs
NCCs
60
40
20
C
Col-0 ptc52 tic55-1 tic55-2 tic55-3
Glucosylated phyllobilins
92
88
84
Hydroxymethylated phyllobilins
20
0
Col-0 ptc52 tic55-1 tic55-2 tic55-3
96
92
88
84
80
F
Col-0 ptc52 tic55-1 tic55-2 tic55-3
Hydroxylated phyllobilins
100
Relative amount (%)
Relative amount (%)
40
Demethylated phyllobilins
Col-0 ptc52 tic55-1 tic55-2 tic55-3
100
80
60
40
20
0
60
100
96
E
80
D
Relative amount (%)
Relative amount (%)
100
80
Hydroxylated phyllobilins
100
80
0
B
Relative amount (%)
Relative amount (%)
100
Col-0 ptc52 tic55-1 tic55-2 tic55-3
80
60
40
20
0
Col-0 tic55-3
tic32
tic62
Figure 6. Phyllobilin Analysis of c and ptc52 Mutants.
Abundances of phyllobilins were determined by LC-MS/MS analysis according to a published method (Christ et al., 2016) and are shown as rela ve values. Data
are the mean of four biological replicates. Error bars indicate SD.
(A)-(E) Phyllobilin abundance in short-day-grown c55 and ptc52 mutants a er senescence induc on for 5 d. Note the different scale used in (C) and (D) because
of the low abundance of glucosylated and demethylated phyllobilins.
(F) Abundance of hydroxylated phyllobilins in a c32 and c62 mutant.
Intens.
4
x10
4
Col-0
hydroxy-pFCC
pFCC
3
2
1
EIC 629.297
±0.01
EIC 645.292
0
Intens.
4
x10 3
±0.01
tic55-3
2
1
EIC 629.297
±0.01
EIC 645.292
0
±0.01
5
6
7
8 Time [min]
Figure 7. Hydroxy-pFCC is absent from c55-3 Gerontoplasts.
Extracted ion chromatograms (EICs) of pFCC (EIC 629.2977±0.01) and hydroxy-pFCC (EIC 645.2929±0.01) of gerontoplasts of wild type (Col-0) and c55-3 are
shown. Note the absence of hydroxy-pFCC from the mutant.
R4
Liquidambar styraciflua
Malus domestica
Musa acuminata
Nicotiana rustica
Prunus domestica
Pyrus communis
Spinacia oleracea
Spathiphyllum wallisii
Tilia cordata
Ulmus glabra
Vitis vinifera
Zea mays
Hordeum vulgare
Eriobotrya japonica
Cerecidiphyllum japonicum
Cydonia oblonga
Brassica oleracea
Brassica napus
Arabidopsis thaliana
Acer platanoides
A
DNCCs
O
OH
R4
NCCs
*
Mesc_3
Mesc_4
Mesc_2
Ptri_2
Spur_
Rcom_62
Ptri_
Spur_5
2
P
Ptri_ tri_3
1
Sp
Sp ur_7
Lus ur_8
Lus i_5
i_8
Lus
i_1
Lus
i_4
L
Lu Lusi_usi_6
Gr Gr
si_ 7
2
Tc ai_4ai_3
ac
Lu
_ G
si_
Cp C 2 rai_
3
pa
2
a
Br p_ p_
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Cs c_3
at
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Figure 8. Phyllobilin Distribu on and Phylogene c Analysis of Rieske-type Oxygenases in Plants.
(A) Phyllobilin hydroxyla on (R1) is a common modifica on, which is found in DNCCs (red square) and NCCs (green square) of all 20 species analyzed so far for structures of
phyllobilins. By contrast, other modifica ons (R2-R4; see DNCC and NCC structures for the posi on of these modifica ons) occur in a species-specific manner.
(B) Phylogene c analysis of Arabidopsis TIC55 (yellow asterisk), PAO (blue asterisk) and PTC52 (red asterisk) homologs in plants. Note that except a few (black lines), most of the
204 protein sequences compared in this analysis cluster in one of three dis nct clades. Also note that for each species at least one Rieske-type oxygenase protein is located within
each clade. The tree was generated with the neighborhood joining method using MEGA7 (Kumar et al., 2016). Important nodes separa ng the three clades are labeled with
bootstrap values (% of 1,000 replicates). Acor, Aquilegia coerulea; Alyr, Arabidopsis lyrata; Atha, Arabidopsis thaliana; Atri, Amborella trichopoda; Bdis, Brachypodium distachon;
Brap, Brassica rapa; Bsta, Brachypodium stacei; Bstr, Boechera stricta; Ccle, Citrus clemen na; Cgra, Capsella grandiflora; Cpap, Carica papaya; Crub, Capsella rubella; Csat,
Cucumis sa vus; Csin, Citrus sinensis; Egra, Eucalyptus grandis; Esal, Eutrema salsugineum; Fves, Fragaria vesca; Gmax, Glycine max; Grai, Gossypium raimondii; Kfla, Klebsormidium flaccidum; Kmar, Kalanchoe marnieriana; Lusi, Linum usita ssimum; Macu, Musa acuminata; Mdom, Malus doms ca; Mesc, Manihot esculenta; Mgut, Mimulus gu atus;
Mtru, Medicago truncatula; Osat, Oryza sa va; Phal, Panicum hallii; Ppat, Physcomitrella patens; Pper, Prunus persica; Ptri, Populus trichocarpa; Pvir, Panicum virgatum; Pvul,
Phaseolus vulgaris; Rcom, Ricinus communis; Sbic, Sorghum bicolor; Sfal, Sphagnum fallax; Sita, Setaria italica; Slyc, Solanum lycopersicum; Some, Selaginella moellendorffii; Spol,
Spirodela polyrhiza; Spur, Salix purpurea; Stub, Solanum tuberosum; Svir, Setaria viridis; Tcac, Theobroma cacao; Vvin, Vi s vinifera; Zmay, Zea mays.
Chloroplast
Chlorophyll a / b
Cytosol
Thylakoids
pFCC
Hydroxylation
hydroxy-pFCC
?
pFCC/hydroxy-pFCC
Deformylation
ER
TIC55
Vacuole
Demethylation
MES16
CYP89A9
FCCs
Demethylation
DFCCs
FCCs
?
Isomerization
DFCCs
NCCs
DNCCs
Figure 9. Topographical Model of the Chlorophyll Breakdown Pathway Integra ng the Findings of this Work.
The model shows the subcellular localiza on of pFCC-modifying ac vi es, i.e. CYP89A9 (deformyla on), MES16 (demethyla on) and TIC55 (hydroxyla on).
Note, that all the reac ons involved in conver ng chlorophyll to pFCC, including pheophorbide a oxygenase (PAO) the name-giving key enzyme of the
PAO/phyllobilin pathway are not shown, but have been considered to form a highly dynamic complex at the thylakoid membrane to prevent release into
the stroma of poten ally phototoxic breakdown intermediates upstream of pFCC (Sakuraba et al., 2012). Note also that due to the fact that
non-hydroxylated phyllobilin occur in wild type Arabidopsis, a frac on of pFCC likely leaves the chloroplast without hydroxyla on. As indicated by ques on
marks, the molecular iden ty of phyllobilin transporters at the chloroplast envelope and the tonoplast is unknown. DFCC, dioxobilin-type fluorescent
chlorophyll catabolites; DNCC, dioxobilin-type nonfluorescent chlorophyll catabolites; ER, endoplasmic re culum; FCC, formyloxobilin-type fluorescent
chlorophyll catabolites; NCC, formyloxobilin-type nonfluorescent chlorophyll catabolites; pFCC, primary fluorescent chlorophyll catabolite.
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A role for TIC55 as a hydroxylase of phyllobilins, the products of chlorophyll breakdown during
plant senescence
Mareike Hauenstein, Bastien Christ, Aditi Das, Sylvain Aubry and Stefan Hörtensteiner
Plant Cell; originally published online September 21, 2016;
DOI 10.1105/tpc.16.00630
This information is current as of June 18, 2017
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