Metabolomic response to high light from pgrl1 and pgr5 mutants of Chlamydomonas reinhardtii.

Chlamydomonas (C.) reinhardtii metabolomic changes in cyclic electron flow-dependent mutants are still unknown. Here, we used mass spectrometric analysis to monitor the changes in metabolite levels in wild-type, cyclic electron-deficient mutants pgrl1 and pgr5 grown under high-light stress. A total of 55 metabolites were detected using GC-MS analysis. High-light stress-induced selective anaplerotic amino acids in pgr5. In addition, pgr5 showed enhancement in carbohydrate, polyamine, and polyol metabolism by 2.5-fold under high light. In response to high light, pgr5 triggers an increase in several metabolites involved in regulating osmotic pressure. Among these metabolites are glycerol pathway compounds such as glycerol-3-phosphate and glyceryl-glycoside, which increase significantly by 1.55 and 3.07 times, respectively. In addition, pgr5 also enhanced proline and putrescine levels by 2.6- and 1.36-fold under high light. On the other hand, pgrl1-induced metabolites, such as alanine and serine, are crucial for photorespiration when subjected to high-light stress. We also observed a significant increase in levels of polyols and glycerol by 1.37- and 2.97-fold in pgrl1 under high-light stress. Both correlation network studies and KEGG pathway enrichment analysis revealed that metabolites related to several biological pathways, such as amino acid, carbohydrate, TCA cycle, and fatty acid metabolism, were positively correlated in pgrl1 and pgr5 under high-light stress conditions. The relative mRNA expression levels of genes related to the TCA cycle, including PDC3, ACH1, OGD2, OGD3, IDH3, and MDH4, were significantly upregulated in pgrl1 and pgr5 under HL. In pgr5, the MDH1 level was significantly increased, while ACS1, ACS3, IDH2, and IDH3 levels were reduced considerably in pgrl1 under high-light stress. The current study demonstrates both pgr5 and prgl1 showed a differential defense response to high-light stress at the primary metabolites and mRNA expression level, which can be added to the existing knowledge to explore molecular regulatory responses of prg5 and pgrl1 to high-light stress.

High light-induced changes in thylakoid supercomplexes organization from cyclic electron transport mutants of Chlamydomonas reinhardtii.

The localization of carotenoids and macromolecular organization of thylakoid supercomplexes have not been reported yet in Chlamydomonas reinhardtii WT and cyclic electron transport mutants (pgrl1 and pgr5) under high light. Here, the various pigments, protein composition, and pigment-protein interactions were analyzed from the cells, thylakoids, and sucrose density gradient (SDG) fractions. Also, the supercomplexes of thylakoids were separated from BN-PAGE and SDG. The abundance of light-harvesting complex (LHC) II trimer complexes and pigment-pigment interaction were changed slightly under high light, shown by circular dichroism. However, a drastic change was seen in photosystem (PS)I-LHCI complexes than PSII complexes, especially in pgrl1 and pgr5. The lutein and ß-carotene increased under high light in LHCII trimers compared to other supercomplexes, indicating that these pigments protected the LHCII trimers against high light. However, the presence of xanthophylls, lutein, and ß-carotene was less in PSI-LHCI, indicating that pigment-protein complexes altered in high light. Even the real-time PCR data shows that the pgr5 mutant does not accumulate zeaxanthin dependent genes under high light, which shows that violaxanthin is not converting into zeaxanthin under high light. Also, the protein data confirms that the LHCSR3 expression is absent in pgr5, however it is presented in LHCII trimer in WT and pgrl1. Interestingly, some of the core proteins were aggregated in pgr5, which led to change in photosynthesis efficiency in high light.

Autophagy Induced Accumulation of Lipids in pgrl1 and pgr5 of Chlamydomonas reinhardtii Under High Light.

Chlamydomonas (C.) reinhardtii is a potential microalga for lipid production. Autophagy-triggered lipid metabolism in microalgae has not being studied so far from a mutant of proton gradient regulation 1 like (PGRL1) and proton gradient regulation 5 (PGR5). In this study, C. reinhardtii cells (wild-type CC124 and cyclic electron transport dependant mutants pgrl1 and pgr5) were grown photoheterotrophically in high light 500 µmol photons m-2 s-1, where pgr5 growth was retarded due to an increase in reactive oxygen species (ROS). The lipid contents were increased; however, carbohydrate content was decreased in pgr5. Further, the Nile Red (NR) fluorescence shows many lipid bodies in pgr5 cells under high light. Similarly, the electron micrographs show that large vacuoles were formed in high light stress despite the grana stacks structure. We also observed increased production of reactive oxygen species, which could be one reason the cells underwent autophagy. Further, a significant increase of autophagy ATG8 and detections of ATG8-PE protein was noticed in pgr5, a hallmark characteristic for autophagy formation. Consequently, the triacylglycerol (TAG) content was increased due to diacylglycerol acyltransferases (DGAT) and phospholipid diacylglycerol acyl-transference (PDAT) enzymes' expression, especially in pgr5. Here the TAG synthesis would have been obtained from degraded membrane lipids in pgr5. Additionally, mono, polyunsaturated, and saturated fatty acids were identified more in the high light condition. Our study shows that the increased light induces the reactive oxygen species, which leads to autophagy and TAG accumulation. Therefore, the enhanced accumulation of TAGs can be used as feedstock for biodiesel production and aqua feed.

Role of cyclic electron transport mutations pgrl1 and pgr5 in acclimation process to high light in Chlamydomonas reinhardtii.

Light is crucial for photosynthesis, but the amount of light that exceeds an organism's assimilation efficacy can lead to photo-oxidative damage and even cell death. In Chlamydomonas (C). reinhardtii cyclic electron flow (CEF) is very important for the elicitation of non-photochemical quenching (NPQ) by controlling the acidification of thylakoid lumen. This process requires the cooperation of proton gradient regulation (PGR) proteins, PGRL1 and PGR5. Here, we compared the growth pattern and photosynthetic activity between wild type (137c, t222+) and mutants impaired in CEF (pgrl1 and pgr5) under photoautotrophic and photoheterotrophic conditions. We have observed the discriminative expression of NPQ in the mutants impaired in CEF of pgrl1 and pgr5. The results obtained from the mutants showed reduced cell growth and density, Chl a/b ratio, fluorescence, electron transport rate, and yield of photosystem (PS)II. These mutants have reduced capability to develop a strong NPQ indicating that the role of CEF is very crucial for photoprotection. Moreover, the CEF mutant exhibits increased photosensitivity compared with the wild type. Therefore, we suggest that besides NPQ, the fraction of non-regulated non-photochemical energy loss (NO) also plays a crucial role during high light acclimation despite a low growth rate. This low NPQ rate may be due to less influx of protons coming from the CEF in cases of pgrl1 and pgr5 mutants. These results are discussed in terms of the relative photoprotective benefit, related to the thermal dissipation of excess light in photoautotrophic and photoheterotrophic conditions.

Changes in the photosynthetic apparatus and lipid droplet formation in Chlamydomonas reinhardtii under iron deficiency.

The unicellular photosynthetic alga Chlamydomonas reinhardtii was propagated in iron deficiency medium and patterns of growth, photosynthetic efficiency, lipid accumulation, as well as the expression of lipid biosynthetic and photosynthesis-related proteins were analysed and compared with iron-sufficient growth conditions. As expected, the photosynthetic rate was reduced (maximally after 4 days of growth) as a result of increased non-photochemical quenching (NPQ). Surprisingly, the stress-response protein LHCSR3 was expressed in conditions of iron deficiency that cause NPQ induction. In addition, the protein contents of both the PSI and PSII reaction centres were gradually reduced during growth in iron deficiency medium. Interestingly, the two generations of Fe deficiency cells could be able to recover the photosynthesis but the second generation cells recovered much slower as these cells were severely in shock. Analysis by flow cytometry with fluorescence-activated cell sorting and thin layer chromatography showed that iron deficiency also induced the accumulation of triacylglycerides (TAG), which resulted in the formation of lipid droplets. This was most significant between 48 and 72 h of growth. Dramatic increases in DGAT2A and PDAT1 levels were caused by iron starvation, which indicated that the biosynthesis of TAG had been increased. Analysis using gas chromatography mass spectrometry showed that levels of 16:0, 18:0, 18:2 and 18:3Δ9,12,15 fatty acids were significantly elevated. The results of this study highlight the genes/enzymes of Chlamydomonas that affect lipid synthesis through their influence on photosynthesis, and these represent potential targets of metabolic engineering to develop strains for biofuel production.