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Phosphorylation dynamics of LHCSR3 were investigated in Chlamydomonas reinhardtii by quantitative proteomics and genetic engineering. LHCSR3 protein expression and phosphorylation were induced in high light. Our data revealed synergistic and dynamic N-terminal LHCSR3 phosphorylation. Phosphorylated and nonphosphorylated LHCSR3 associated with PSII-LHCII supercomplexes. The phosphorylation status of LHCB4 was closely linked to the phosphorylation of multiple sites at the N-terminus of LHCSR3, indicating that LHCSR3 phosphorylation may operate as a molecular switch modulating LHCB4 phosphorylation, which in turn is important for PSII-LHCII disassembly. Notably, LHCSR3 phosphorylation diminished under prolonged high light, which coincided with onset of CEF. Hierarchical clustering of significantly altered proteins revealed similar expression profiles of LHCSR3, CRX, and FNR. This finding indicated the existence of a functional link between LHCSR3 protein abundance and phosphorylation, photosynthetic electron flow, and the oxidative stress response., (© 2019 The Authors The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

PsaI is the only subunit of PSI whose precise physiological function has not yet been elucidated in higher plants. While PsaI is involved in PSI trimerization in cyanobacteria, trimerization was lost during the evolution of the eukaryotic PSI, and the entire PsaI side of PSI underwent major structural remodelling to allow for binding of light harvesting complex II antenna proteins during state transitions. Here, we have generated a tobacco (Nicotiana tabacum) knockout mutant of the plastid-encoded psaI gene. We show that PsaI is not required for the redox reactions of PSI. Neither plastocyanin oxidation nor the processes at the PSI acceptor side are impaired in the mutant, and both linear and cyclic electron flux rates are unaltered. The PSI antenna cross section is unaffected, state transitions function normally, and binding of other PSI subunits to the reaction centre is not compromised. Under a wide range of growth conditions, the mutants are phenotypically and physiologically indistinguishable from wild-type tobacco. However, in response to high-light and chilling stress, and especially during leaf senescence, PSI content is reduced in the mutants, indicating that the I-subunit plays a role in stabilizing PSI complexes., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

In plants and algae, the serine/threonine kinase STN7/STT7, orthologous protein kinases in Chlamydomonas reinhardtii and Arabidopsis (Arabidopsis thaliana), respectively, is an important regulator in acclimation to changing light environments. In this work, we assessed STT7-dependent protein phosphorylation under high light in C. reinhardtii, known to fully induce the expression of light-harvesting complex stress-related protein3 (LHCSR3) and a nonphotochemical quenching mechanism, in relationship to anoxia where the activity of cyclic electron flow is stimulated. Our quantitative proteomics data revealed numerous unique STT7 protein substrates and STT7-dependent protein phosphorylation variations that were reliant on the environmental condition. These results indicate that STT7-dependent phosphorylation is modulated by the environment and point to an intricate chloroplast phosphorylation network responding in a highly sensitive and dynamic manner to environmental cues and alterations in kinase function. Functionally, the absence of the STT7 kinase triggered changes in protein expression and photoinhibition of photosystem I (PSI) and resulted in the remodeling of photosynthetic complexes. This remodeling initiated a pronounced association of LHCSR3 with PSI-light harvesting complex I (LHCI)-ferredoxin-NADPH oxidoreductase supercomplexes. Lack of STT7 kinase strongly diminished PSII-LHCII supercomplexes, while PSII core complex phosphorylation and accumulation were significantly enhanced. In conclusion, our study provides strong evidence that the regulation of protein phosphorylation is critical for driving successful acclimation to high light and anoxic growth environments and gives new insights into acclimation strategies to these environmental conditions., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

In Chlamydomonas reinhardtii, the LIGHT-HARVESTING COMPLEX STRESS-RELATED PROTEIN3 (LHCSR3) protein is crucial for efficient energy-dependent thermal dissipation of excess absorbed light energy and functionally associates with photosystem II-light-harvesting complex II (PSII-LHCII) supercomplexes. Currently, it is unknown how LHCSR3 binds to the PSII-LHCII supercomplex. In this study, we investigated the role of PHOTOSYSTEM II SUBUNIT R (PSBR) an intrinsic membrane-spanning PSII subunit, in the binding of LHCSR3 to PSII-LHCII supercomplexes. Down-regulation of PSBR expression diminished the efficiency of oxygen evolution and the extent of nonphotochemical quenching and had an impact on the stability of the oxygen-evolving complex as well as on PSII-LHCII-LHCSR3 supercomplex formation. Its down-regulation destabilized the PSII-LHCII supercomplex and strongly reduced the binding of LHCSR3 to PSII-LHCII supercomplexes, as revealed by quantitative proteomics. PHOTOSYSTEM II SUBUNIT P deletion, on the contrary, destabilized PHOTOSYSTEM II SUBUNIT Q binding but did not affect PSBR and LHCSR3 association with PSII-LHCII. In summary, these data provide clear evidence that PSBR is required for the stable binding of LHCSR3 to PSII-LHCII supercomplexes and is essential for efficient energy-dependent quenching and the integrity of the PSII-LHCII-LHCSR3 supercomplex under continuous high light., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

Light is essential for photosynthesis but excess light is hazardous as it may lead to the formation of reactive oxygen species. Photosynthetic organisms struggle to optimize light utilization and photosynthesis while minimizing photo-oxidative damage. Hereby light to heat dissipation via specialized proteins is a potent mechanism to acclimate toward excess light. In the green alga Chlamydomonas reinhardtii the expression of an ancient light-harvesting protein LHCSR3 enables cells to dissipate harmful excess energy. Herein we summarize newest insights into the function of LHCSR3 from C. reinhardtii.

Light and oxygen are factors that are very much entangled in the reactive oxygen species (ROS) stress response network in plants, algae and cyanobacteria. The first obligatory step in understanding the ROS network is to separate these responses. In this study, a LC-MS/MS based quantitative proteomic approach was used to dissect the responses of Chlamydomonas reinhardtii to ROS, light and oxygen employing an interlinked experimental setup. Application of novel bioinformatics tools allow high quality retention time alignment to be performed on all LC-MS/MS runs increasing confidence in protein quantification, overall sequence coverage and coverage of all treatments measured. Finally advanced hierarchical clustering yielded 30 communities of co-regulated proteins permitting separation of ROS related effects from pure light effects (induction and repression). A community termed redox(II) was identified that shows additive effects of light and oxygen with light as the first obligatory step. Another community termed 4-down was identified that shows repression as an effect of light but only in the absence of oxygen indicating ROS regulation, for example, possibly via product feedback inhibition because no ROS damage is occurring. In summary the data demonstrate the importance of separating light, O₂ and ROS responses to define marker genes for ROS responses. As revealed in this study, an excellent candidate is DHAR with strong ROS dependent induction profiles.

The fungus Fusarium fujikuroi causes "bakanae" disease of rice due to its ability to produce gibberellins (GAs), but it is also known for producing harmful mycotoxins. However, the genetic capacity for the whole arsenal of natural compounds and their role in the fungus' interaction with rice remained unknown. Here, we present a high-quality genome sequence of F. fujikuroi that was assembled into 12 scaffolds corresponding to the 12 chromosomes described for the fungus. We used the genome sequence along with ChIP-seq, transcriptome, proteome, and HPLC-FTMS-based metabolome analyses to identify the potential secondary metabolite biosynthetic gene clusters and to examine their regulation in response to nitrogen availability and plant signals. The results indicate that expression of most but not all gene clusters correlate with proteome and ChIP-seq data. Comparison of the F. fujikuroi genome to those of six other fusaria revealed that only a small number of gene clusters are conserved among these species, thus providing new insights into the divergence of secondary metabolism in the genus Fusarium. Noteworthy, GA biosynthetic genes are present in some related species, but GA biosynthesis is limited to F. fujikuroi, suggesting that this provides a selective advantage during infection of the preferred host plant rice. Among the genome sequences analyzed, one cluster that includes a polyketide synthase gene (PKS19) and another that includes a non-ribosomal peptide synthetase gene (NRPS31) are unique to F. fujikuroi. The metabolites derived from these clusters were identified by HPLC-FTMS-based analyses of engineered F. fujikuroi strains overexpressing cluster genes. In planta expression studies suggest a specific role for the PKS19-derived product during rice infection. Thus, our results indicate that combined comparative genomics and genome-wide experimental analyses identified novel genes and secondary metabolites that contribute to the evolutionary success of F. fujikuroi as a rice pathogen.

Background: Biogeochemical elemental cycling is driven by primary production of biomass via phototrophic phytoplankton growth, with 40% of marine productivity being assigned to diatoms. Phytoplankton growth is widely limited by the availability of iron, an essential component of the photosynthetic apparatus. The oceanic diatom Thalassiosira oceanica shows a remarkable tolerance to low-iron conditions and was chosen as a model for deciphering the cellular response upon shortage of this essential micronutrient., Results: The combined efforts in genomics, transcriptomics and proteomics reveal an unexpected metabolic flexibility in response to iron availability for T. oceanica CCMP1005. The complex response comprises cellular retrenchment as well as remodeling of bioenergetic pathways, where the abundance of iron-rich photosynthetic proteins is lowered, whereas iron-rich mitochondrial proteins are preserved. As a consequence of iron deprivation, the photosynthetic machinery undergoes a remodeling to adjust the light energy utilization with the overall decrease in photosynthetic electron transfer complexes., Conclusions: Beneficial adaptations to low-iron environments include strategies to lower the cellular iron requirements and to enhance iron uptake. A novel contribution enhancing iron economy of phototrophic growth is observed with the iron-regulated substitution of three metal-containing fructose-bisphosphate aldolases involved in metabolic conversion of carbohydrates for enzymes that do not contain metals. Further, our data identify candidate components of a high-affinity iron-uptake system, with several of the involved genes and domains originating from duplication events. A high genomic plasticity, as seen from the fraction of genes acquired through horizontal gene transfer, provides the platform for these complex adaptations to a low-iron world.

The plant-specific calcium binding protein CAS (calcium sensor) has been localized in chloroplast thylakoid membranes of vascular plants and green algae. To elucidate the function of CAS in Chlamydomonas reinhardtii, we generated and analyzed eight independent CAS knockdown C. reinhardtii lines (cas-kd). Upon transfer to high-light (HL) growth conditions, cas-kd lines were unable to properly induce the expression of LHCSR3 protein that is crucial for nonphotochemical quenching. Prolonged exposure to HL revealed a severe light sensitivity of cas-kd lines and caused diminished activity and recovery of photosystem II (PSII). Remarkably, the induction of LHCSR3, the growth of cas-kd lines under HL, and the performance of PSII were fully rescued by increasing the calcium concentration in the growth media. Moreover, perturbing cellular Ca(2+) homeostasis by application of the calmodulin antagonist W7 or the G-protein activator mastoparan impaired the induction of LHCSR3 expression in a concentration-dependent manner. Our findings demonstrate that CAS and Ca(2+) are critically involved in the regulation of the HL response and particularly in the control of LHCSR3 expression.