Arthur R. Grossman

A pigment-binding protein essential for regulation of photosynthetic light harvesting.

Photosynthetic light harvesting in plants is regulated in response to changes in incident light intensity. Absorption of light that exceeds a plants capacity for fixation of CO2 results in thermal dissipation of excitation energy in the pigment antenna of photosystem II by a poorly understood mechanism. This regulatory process, termed nonphotochemical quenching, maintains the balance between dissipation and utilization of light energy to minimize generation of oxidizing molecules, thereby protecting the plant against photo-oxidative damage. To identify specific proteins that are involved in nonphotochemical quenching, we have isolated mutants of Arabidopsis thaliana that cannot dissipate excess absorbed light energy. Here we show that the gene encoding PsbS, an intrinsic chlorophyll-binding protein of photosystem II, is necessary for nonphotochemical quenching but not for efficient light harvesting and photosynthesis. These results indicate that PsbS may be the site for nonphotochemical quenching, a finding that has implications for the functional evolution of pigment-binding proteins.

Arabidopsis mutants define a central role for the xanthophyll cycle in the regulation of photosynthetic energy conversion.

A conserved regulatory mechanism protects plants against the potentially damaging effects of excessive light. Nearly all photosynthetic eukaryotes are able to dissipate excess absorbed light energy in a process that involves xanthophyll pigments. To dissect the role of xanthophylls in photoprotective energy dissipation in vivo, we isolated Arabidopsis xanthophyll cycle mutants by screening for altered nonphotochemical quenching of chlorophyll fluorescence. The npq1 mutants are unable to convert violaxanthin to zeaxanthin in excessive light, whereas the npq2 mutants accumulate zeaxanthin constitutively. The npq2 mutants are new alleles of aba1, the zeaxanthin epoxidase gene. The high levels of zeaxanthin in npq2 affected the kinetics of induction and relaxation but not the extent of nonphotochemical quenching. Genetic mapping, DNA sequencing, and complementation of npq1 demonstrated that this mutation affects the structural gene encoding violaxanthin deepoxidase. The npq1 mutant exhibited greatly reduced nonphotochemical quenching, demonstrating that violaxanthin deepoxidation is required for the bulk of rapidly reversible nonphotochemical quenching in Arabidopsis. Altered regulation of photosynthetic energy conversion in npq1 was associated with increased sensitivity to photoinhibition. These results, in conjunction with the analysis of npq mutants of Chlamydomonas, suggest that the role of the xanthophyll cycle in nonphotochemical quenching has been conserved, although different photosynthetic eukaryotes rely on the xanthophyll cycle to different extents for the dissipation of excess absorbed light energy.

An ancient light-harvesting protein is critical for the regulation of algal photosynthesis

Light is necessary for photosynthesis, but its absorption by pigment molecules such as chlorophyll can cause severe oxidative damage and result in cell death. The excess absorption of light energy by photosynthetic pigments has led to the evolution of protective mechanisms that operate on the timescale of seconds to minutes and involve feedback-regulated de-excitation of chlorophyll molecules in photosystem II (qE). Despite the significant contribution of eukaryotic algae to global primary production, little is known about their qE mechanism, in contrast to that in flowering plants. Here we show that a qE-deficient mutant of the unicellular green alga Chlamydomonas reinhardtii, npq4, lacks two of the three genes encoding LHCSR (formerly called LI818). This protein is an ancient member of the light-harvesting complex superfamily, and orthologues are found throughout photosynthetic eukaryote taxa, except in red algae and vascular plants. The qE capacity of Chlamydomonas is dependent on environmental conditions and is inducible by growth under high light conditions. We show that the fitness of the npq4 mutant in a shifting light environment is reduced compared to wild-type cells, demonstrating that LHCSR is required for survival in a dynamic light environment. Thus, these data indicate that plants and algae use different proteins to dissipate harmful excess light energy and protect the photosynthetic apparatus from damage.

Stable nuclear transformation of the diatom Phaeodactylum tricornutum.

A nuclear transformation system has been developed for the diatomPhaeodactylum tricornutum using microparticle bombardment to introduce thesh ble gene fromStreptoalloteichus hindustanus into cells. Thesh ble gene encodes a protein that confers resistance to the antibiotics Zeocin and phleomycin. Chimeric genes containing promoter and terminator sequences from theP. tricornutum fcp genes were used to drive expression ofsh ble. Between 10–100 transformants were recovered/108 cells. Transformants were able to grow on at least 500 µg/ml of Zeocin, which is 10 times the amount necessary to kill wild-type cells. Based on Southern hybridizations thesh ble gene was present in 1–3 copies/transformant. Relative levels of correctly processed transcripts were correlated with the abundance of the Sh ble protein (present at 0.1–2.0 µg/mg total protein). Thecat reporter gene fused to afcp promoter could also be introduced by microparticle bombardment and was found to be highly expressed (average of 7.1 U/mg total protein). This work demonstrates that heterologous genes can be readily expressed inP. tricornutum. The development of selectable marker and reporter gene constructs provides the tools necessary for dissecting gene structure and regulation, and introducing novel functions into diatoms.

Similarity of a Chromatic Adaptation Sensor to Phytochrome and Ethylene Receptors

Complementary chromatic adaptation in cyanobacteria acts through photoreceptors to control the biosynthesis of light-harvesting complexes. The mutant FdBk, which appears black, cannot chromatically adapt and may contain a lesion in the apparatus that senses light quality. The complementing gene identified here, rcaE, encodes a deduced protein in which the amino-terminal region resembles the chromophore attachment domain of phytochrome photoreceptors and regions of plant ethylene receptors; the carboxyl- terminal half is similar to the histidine kinase domain of two-component sensor kinases.

Chlamydomonas Xanthophyll Cycle Mutants Identified by Video Imaging of Chlorophyll Fluorescence Quenching.

The photosynthetic apparatus in plants is protected against oxidative damage by processes that dissipate excess absorbed light energy as heat within the light-harvesting complexes. This dissipation of excitation energy is measured as nonphotochemical quenching of chlorophyll fluorescence. Nonphotochemical quenching depends primarily on the [delta]pH that is generated by photosynthetic electron transport, and it is also correlated with the amounts of zeaxanthin and antheraxanthin that are formed from violaxanthin by the operation of the xanthophyll cycle. To perform a genetic dissection of nonphotochemical quenching, we have isolated npq mutants of Chlamydomonas by using a digital video-imaging system. In excessive light, the npq1 mutant is unable to convert violaxanthin to antheraxanthin and zeaxanthin; this reaction is catalyzed by violaxanthin de-epoxidase. The npq2 mutant appears to be defective in zeaxanthin epoxidase activity, because it accumulates zeaxanthin and completely lacks antheraxanthin and violaxanthin under all light conditions. Characterization of these mutants demonstrates that a component of nonphotochemical quenching that develops in vivo in Chlamydomonas depends on the accumulation of zeaxanthin and antheraxanthin via the xanthophyll cycle. However, observation of substantial, rapid, [delta]pH-dependent nonphotochemical quenching in the npq1 mutant demonstrates that the formation of zeaxanthin and antheraxanthin via violaxanthin de-epoxidase activity is not required for all [delta]pH-dependent nonphotochemical quenching in this alga. Furthermore, the xanthophyll cycle is not required for survival of Chlamydomonas in excessive light.

Transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes

A general purpose transformation vector, designated pPha‐T1, was constructed for use with the diatom Phaeodactylum tricornutum Bohlin. This vector harbors the sh ble cassette for primary selection on medium containing the antibiotic zeocin, and a multiple cloning site flanked by the P. tricornutum fcpA promoter. pPha‐T1 was used to establish the utility of three selectable marker genes and two reporter genes for P. tricornutum transformation. The nat and sat‐1 genes confer resistance to the antibiotic nourseothricin, and nptII confers resistance to G418. Each of these genes was effective as a selectable marker for identifying primary transformants. These markers could also be used for dual selections in combination with the sh ble gene. The reporter genes uidA and gfp were also introduced into P. tricornutum using pPha‐T1. Gus expression in some transformants reached 15 μg·μg−1 of total soluble protein and permitted excellent cell staining, while GFP fluorescence was readily visible with standard fluorescence microscopy. The egfp gene, which has optimal codon usage for expression in human cells, was the only version of gfp that produced a strong fluorescent signal in P. tricornutum. The codon bias of the egfp gene is similar to that of P. tricornutum genes. This study suggests that codon usage has a significant effect on the efficient expression of reporter genes in P. tricornutum. The results presented here demonstrate that a variety of selectable markers and reporter genes can be expressed in P. tricornutum, enhancing the potential of this organism for exploring basic biological questions and industrial applications.

Three Acyltransferases and Nitrogen-responsive Regulator Are Implicated in Nitrogen Starvation-induced Triacylglycerol Accumulation in Chlamydomonas

Background: Nitrogen-starvation and other stresses induce triacylglycerol (TAG) accumulation in algae, but the relevant enzymes and corresponding signal transduction pathways are unknown. Results: RNA-Seq and genetic analysis revealed three acyltransferases that contribute to TAG accumulation. Conclusion: TAG synthesis results from recycling of membrane lipids and also by acylation of DAG. Significance: The genes are potential targets for manipulating TAG hyperaccumulation. Algae have recently gained attention as a potential source for biodiesel; however, much is still unknown about the biological triggers that cause the production of triacylglycerols. We used RNA-Seq as a tool for discovering genes responsible for triacylglycerol (TAG) production in Chlamydomonas and for the regulatory components that activate the pathway. Three genes encoding acyltransferases, DGAT1, DGTT1, and PDAT1, are induced by nitrogen starvation and are likely to have a role in TAG accumulation based on their patterns of expression. DGAT1 and DGTT1 also show increased mRNA abundance in other TAG-accumulating conditions (minus sulfur, minus phosphorus, minus zinc, and minus iron). Insertional mutants, pdat1-1 and pdat1-2, accumulate 25% less TAG compared with the parent strain, CC-4425, which demonstrates the relevance of the trans-acylation pathway in Chlamydomonas. The biochemical functions of DGTT1 and PDAT1 were validated by rescue of oleic acid sensitivity and restoration of TAG accumulation in a yeast strain lacking all acyltransferase activity. Time course analyses suggest than a SQUAMOSA promoter-binding protein domain transcription factor, whose mRNA increases precede that of lipid biosynthesis genes like DGAT1, is a candidate regulator of the nitrogen deficiency responses. An insertional mutant, nrr1-1, accumulates only 50% of the TAG compared with the parental strain in nitrogen-starvation conditions and is unaffected by other nutrient stresses, suggesting the specificity of this regulator for nitrogen-deprivation conditions.

Anaerobic acclimation in Chlamydomonas reinhardtii: anoxic gene expression, hydrogenase induction, and metabolic pathways.

Both prokaryotic and eukaryotic photosynthetic microbes experience conditions of anoxia, especially during the night when photosynthetic activity ceases. In Chlamydomonas reinhardtii, dark anoxia is characterized by the activation of an extensive set of fermentation pathways that act in concert to provide cellular energy, while limiting the accumulation of potentially toxic fermentative products. Metabolite analyses, quantitative PCR, and high density Chlamydomonas DNA microarrays were used to monitor changes in metabolite accumulation and gene expression during acclimation of the cells to anoxia. Elevated levels of transcripts encoding proteins associated with the production of H2, organic acids, and ethanol were observed in congruence with the accumulation of fermentation products. The levels of over 500 transcripts increased significantly during acclimation of the cells to anoxic conditions. Among these were transcripts encoding transcription/translation regulators, prolyl hydroxylases, hybrid cluster proteins, proteases, transhydrogenase, catalase, and several putative proteins of unknown function. Overall, this study uses metabolite, genomic, and transcriptome data to provide genome-wide insights into the regulation of the complex metabolic networks utilized by Chlamydomonas under the anaerobic conditions associated with H2 production.

A small polypeptide triggers complete degradation of light-harvesting phycobiliproteins in nutrient-deprived cyanobacteria

Phycobilisomes are the multiprotein complexes predominantly responsible for harvesting light energy in cyanobacteria and some eukaryotic algae. When the cyanobacterium Synechococcus sp. strain PCC 7942 is deprived of an essential nutrient, the phycobilisomes are specifically and rapidly degraded. Degradation may be either partial (after phosphorus deprivation) or complete (after sulfur or nitrogen deprivation). We have developed a visual screen to obtain mutants unable to degrade their phycobilisomes upon nutrient starvation. Complementation of one of these mutants led to the identification of a gene, designated nblA, that encodes a 59 amino acid polypeptide essential for phycobilisome degradation. Transcription of nblA increases dramatically in sulfur‐ or nitrogen‐deprived cells and moderately in phosphorus‐deprived cells. Using the phosphorus‐regulated alkaline phosphatase (phoA) promoter as a tool, we engineered constructs from which we could control the expression of either sense or antisense nblA. Increased expression of sense nbLA caused complete phycobilisome degradation during phosphorus deprivation, while expression of antisense nblA prevented phycobilisome degradation. Hence, nblA is necessary, and may be sufficient, for the degradation of phycobilisomes under adverse environmental conditions. Further investigation of the mechanism by which nblA causes phycobilisome destruction may reveal general principles that govern the specificity of macromolecular complex degradation.