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

Abstract

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...

Characterization of photosynthetic ferredoxin from the Antarctic alga Chlamydomonas sp. UWO241 reveals novel features of cold adaptation

“…S4). All UWO241 Fds were predicted to contain the [2Fe‐2S] binding domain and clustered within previously defined classes in green algae (Yang et al ., ; Sawyer & Winkler, ) and plants (Fukuyama, ; Atkinson et al ., ) (Fig. ).…”

“…6), with the notable exception of Fd-2 and Fd-5. In C. reinhardtii, Fd-2 was shown to be involved in state transitions, while Fd-5 plays a role in maintaining thylakoid composition and functionality during growth in the dark ((Winkler et al, 2010;Yang et al, 2015). It appears that the composition of the Fd gene family in UWO241 is different from related mesophilic algae, owing to the extremophilic lifestyle of the psychrophile.…”

“…Although plants and green algae have multiple Fds, members of the Fd gene family usually exhibit significant differences in their primary amino acid sequence. For example, C. reinhardtii encodes 13 Fd genes, which show 30-67% similarity with one another, and only six of which (FDX1-6) have been characterized in some detail (Terauchi et al, 2009;Winkler et al, 2010;Peden et al, 2013;Yang et al, 2015;Boehm et al, 2016). Recently, a global interaction network was established for C. reinhardtii, providing putative roles for Fds in redox metabolism, carbohydrate modification, fatty acid biosynthesis, hydrogen production, nitrogen and sulfur metabolism, state transitions, and dark anoxia (Peden et al, 2013).…”

GreenCut protein CPLD49 of Chlamydomonas reinhardtii associates with thylakoid membranes and is required for cytochrome b6f complex accumulation

“…The CPLD49 gene from pENTR/D‐TOPO_ CPLD49 was introduced into pNX22‐DEST by LR cloning, while the CPLD38 gene from pENTR/D‐TOPO_ CPLD38 was introduced into pMetYC‐DEST by LR cloning. An interaction between CPLD49 and CPLD38 was assessed by the yeast mating‐based split ubiquitin system, as previously described (Yang et al ., ).…”

PGR5 is required for efficient Q cycle in the cytochromeb6fcomplex during cyclic electron flow

“…In yeast-based protein-protein interaction studies, candidates for PGR5 interaction were cytochrome b6 and Fd (34). The PGR5-dependent CEF effector proteins PETO and ANR1, which interact with the algal b6f (54-57), are part of the Fd interactome as well (58,59) and could have Fd shuttling functions. The yeast two-hybrid study also showed that PGRL1 interacts with PGR5 and FNR (34), and BiFC studies indicated protein-protein interaction between PGRL1 and ANR1 (56).…”