Novel Thylakoid Membrane GreenCut Protein CPLD38 Impacts Accumulation of the Cytochrome b6f Complex and Associated Regulatory Processes

Heinnickel, Mark; Alric, Jean; Wittkopp, Tyler M.; Yang, Wenqiang; Catalanotti, Claudia; Dent, Rachel M.; Niyogi, Krishna; Wollman, Françis-André; Grossman, Arthur R.

doi:10.1074/jbc.m112.427476

Cited by 24 publications

(29 citation statements)

References 54 publications

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“…Growth was analyzed in liquid and solid medium, with or without various antibiotics. Samples from liquid cultures were collected and cells counted and/or quantified based on Chl levels (36,37).…”

Section: Methodsmentioning

confidence: 99%

Critical role of Chlamydomonas reinhardtii ferredoxin-5 in maintaining membrane structure and dark metabolism

Yang

Wittkopp

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Photosynthetic microorganisms typically have multiple isoforms of the electron transfer protein ferredoxin, although we know little about their exact functions. Surprisingly, a Chlamydomonas reinhardtii mutant null for the ferredoxin-5 gene (FDX5) completely ceased growth in the dark, with both photosynthetic and respiratory functions severely compromised; growth in the light was unaffected. Thylakoid membranes in dark-maintained fdx5 mutant cells became severely disorganized concomitant with a marked decrease in the ratio of monogalactosyldiacylglycerol to digalactosyldiacylglycerol, major lipids in photosynthetic membranes, and the accumulation of triacylglycerol. Furthermore, FDX5 was shown to physically interact with the fatty acid desaturases CrΔ4FAD and CrFAD6, likely donating electrons for the desaturation of fatty acids that stabilize monogalactosyldiacylglycerol. Our results suggest that in photosynthetic organisms, specific redox reactions sustain dark metabolism, with little impact on daytime growth, likely reflecting the tailoring of electron carriers to unique intracellular metabolic circuits under these two very distinct redox conditions. ferredoxin | dark growth | thylakoid lipids | triacylglycerol | redox regulation F erredoxins (FDXs) are soluble, iron-sulfur proteins that mediate electron transfer in a variety of essential metabolic reactions (1-3) (SI Appendix, Fig. S1). The Chlamydomonas genome encodes 13 FDXs (SI Appendix, Table S1) with localization and redox properties that suggest involvement in specific redox reactions (4). Using a yeast two hybrid approach, a global FDX interaction network was established for Chlamydomonas, suggesting putative roles for FDX1 (originally designated Fd) in redox metabolism, carbohydrate modification, and fatty acid biosynthesis (5); this FDX was already known to function in both linear and cyclic photosynthetic electron flow (4, 6). FDX1 also accepts electrons from FDX-NADP oxidoreductase (FNR) (7) and donates electrons to HYDA hydrogenases (5,8,9). Other FDXs may be involved in state transitions, nitrogen metabolism, cellular responses to reactive oxygen species (ROS), and dark anoxia (4, 5, 10-13).The major lipids in thylakoid membranes are monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), phosphatidylglycerol (PG), and sulfoquinovosyldiacylglycerol (SQDG) (14-17), with MGDG being the most abundant, followed by DGDG (14, 17). In Arabidopsis, three enzyme systems are involved in MGDG and DGDG synthesis (18). In contrast, there is only a single copy each of an MGDG and DGDG synthase gene in Chlamydomonas (19). Chlamydomonas uses the prokaryotic pathway for thylakoid lipid synthesis (20) in which the MGDG species synthesized in chloroplasts contain predominantly C18:3 Δ9,12,15 -sn-1 with the unusual hexadeca-4, 7, 10, 13-tetraenoic acid C16:4 Δ4,7,10,13 at the sn-2 position (20). Desaturation of the sn-2 acyl group of Chlamydomonas MGDG requires the CrΔ4FAD desaturase, which is not present in Arabidopsis (21), and MGDG accumulation in...

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Section: Methodsmentioning

confidence: 99%

Critical role of Chlamydomonas reinhardtii ferredoxin-5 in maintaining membrane structure and dark metabolism

Yang

Wittkopp

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Sodium bicarbonate (1 mM) was added as a terminal electron acceptor. Chlorophyll fluorescence was measured using a DUAL-PAM 100 (Walz) on the light curve setting, with 60-s steps at each light fluence rate (Heinnickel et al, 2013). Saturating pulses were given immediately following each intensity step to determine F v /F m and FPSII values.…”

Section: Chlorophyll Fluorescencementioning

confidence: 99%

Bilin-Dependent Photoacclimation in Chlamydomonas reinhardtii

et al. 2017

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In land plants, linear tetrapyrrole (bilin)-based phytochrome photosensors optimize photosynthetic light capture by mediating massive reprogramming of gene expression. But, surprisingly, many green algal genomes lack phytochrome genes. Studies of the heme oxygenase mutant (hmox1) of the green alga Chlamydomonas reinhardtii suggest that bilin biosynthesis in plastids is essential for proper regulation of a nuclear gene network implicated in oxygen detoxification during dark-to-light transitions. hmox1 cannot grow photoautotrophically and photoacclimates poorly to increased illumination. We show that these phenotypes are due to reduced accumulation of photosystem I (PSI) reaction centers, the PSI electron acceptors 59-monohydroxyphylloquinone and phylloquinone, and the loss of PSI and photosystem II antennae complexes during photoacclimation. The hmox1 mutant resembles chlorophyll biosynthesis mutants phenotypically, but can be rescued by exogenous biliverdin IXa, the bilin produced by HMOX1. This rescue is independent of photosynthesis and is strongly dependent on blue light. RNA-seq comparisons of hmox1, genetically complemented hmox1, and chemically rescued hmox1 reveal that tetrapyrrole biosynthesis and known photoreceptor and photosynthesis-related genes are not impacted in the hmox1 mutant at the transcript level. We propose that a bilin-based, bluelight-sensing system within plastids evolved together with a bilin-based retrograde signaling pathway to ensure that a robust photosynthetic apparatus is sustained in light-grown Chlamydomonas.

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“…All three of its genomes (nuclear, chloroplast, and mitochondrial) have been sequenced and can be transformed (Boynton et al, 1988;Kindle et al, 1989;RandolphAnderson et al, 1993;Maul et al, 2002;Grossman et al, 2003;Merchant et al, 2007). This alga is particularly suitable for studies of photosynthesis because it can grow heterotrophically with acetate as a carbon and energy source, allowing the isolation of mutants deficient in photosynthesis (Sager and Zalokar, 1958;Levine, 1960;Kates and Jones, 1964;Grossman et al, 2010;Karpowicz et al, 2011;Calderon et al, 2013;Heinnickel et al, 2013;Goodenough, 2015).…”

Section: Introductionmentioning

confidence: 99%

An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii

et al. 2016

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The green alga Chlamydomonas reinhardtii is a leading unicellular model for dissecting biological processes in photosynthetic eukaryotes. However, its usefulness has been limited by difficulties in obtaining mutants in specific genes of interest. To allow generation of large numbers of mapped mutants, we developed high-throughput methods that (1) enable easy maintenance of tens of thousands of Chlamydomonas strains by propagation on agar media and by cryogenic storage, (2) identify mutagenic insertion sites and physical coordinates in these collections, and (3) validate the insertion sites in pools of mutants by obtaining >500 bp of flanking genomic sequences. We used these approaches to construct a stably maintained library of 1935 mapped mutants, representing disruptions in 1562 genes. We further characterized randomly selected mutants and found that 33 out of 44 insertion sites (75%) could be confirmed by PCR, and 17 out of 23 mutants (74%) contained a single insertion. To demonstrate the power of this library for elucidating biological processes, we analyzed the lipid content of mutants disrupted in genes encoding proteins of the algal lipid droplet proteome. This study revealed a central role of the long-chain acyl-CoA synthetase LCS2 in the production of triacylglycerol from de novo-synthesized fatty acids.

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Novel Thylakoid Membrane GreenCut Protein CPLD38 Impacts Accumulation of the Cytochrome b6f Complex and Associated Regulatory Processes

Cited by 24 publications

References 54 publications

Critical role of Chlamydomonas reinhardtii ferredoxin-5 in maintaining membrane structure and dark metabolism

Critical role of Chlamydomonas reinhardtii ferredoxin-5 in maintaining membrane structure and dark metabolism

Bilin-Dependent Photoacclimation in Chlamydomonas reinhardtii

An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii

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