Expression profiling-based identification of CO2-responsive genes regulated by CCM1 controlling a carbon-concentrating mechanism in Chlamydomonas reinhardtii

Photosynthetic acclimation to CO2-limiting stress is associated with control of genetic and physiological responses through a signal transduction pathway, followed by integrated monitoring of the environmental changes. Although several CO2-responsive genes have been previously isolated, genome-wide analysis has not been applied to the isolation of CO2-responsive genes that may function as part of a carbon-concentrating mechanism (CCM) in photosynthetic eukaryotes. By comparing expression profiles of cells grown under CO2-rich conditions with those of cells grown under CO2-limiting conditions using a cDNA membrane array containing 10,368 expressed sequence tags, 51 low-CO2 inducible genes and 32 genes repressed by low CO2 whose mRNA levels were changed more than 2.5-fold in Chlamydomonas reinhardtii Dangeard were detected. The fact that the induction of almost all low-CO2 inducible genes was impaired in the ccm1 mutant suggests that CCM1 is a master regulator of CCM through putative low-CO2 signal transduction pathways. Among low-CO2 inducible genes, two novel genes, LciA and LciB, were identified, which may be involved in inorganic carbon transport. Possible functions of low-CO2 inducible and/or CCM1-regulated genes are discussed in relation to the CCM.     

CO2 limitation induces specific redox-dependent protein phosphorylation in Chlamydomonas reinhardtii

Acclimation of the green alga Chlamydomonas reinhardtii to limiting environmental CO2 induced specific protein phosphorylation at the surface of photosynthetic thylakoid membranes. Four phosphopeptides were identified and sequenced by nanospray quadrupole TOF MS from the cells acclimating to limiting CO2. One phosphopeptide originated from a protein that has not been annotated. We found that this unknown expressed protein (UEP) was encoded in the genome of C. reinhardtii. Three other phosphorylated peptides belonged to Lci5 protein encoded by the low-CO2-inducible gene 5 (lci5). The phosphorylation sites were mapped in the tandem repeats of Lci5 ensuring phosphorylation of four serine and three threonine residues in the protein. Immunoblotting with Lci5-specific antibodies revealed that Lci5 was localized in chloroplast and confined to the stromal side of the thylakoid membranes. Phosphorylation of Lci5 and UEP occurred strictly at limiting CO2; it required reduction of electron carriers in the thylakoid membrane, but was not induced by light. Both proteins were phosphorylated in the low-CO2-exposed algal mutant deficient in the light-activated protein kinase Stt7. Phosphorylation of previously unknown basic proteins UEP and Lci5 by specific redox-dependent protein kinase(s) in the photosynthetic membranes reveals the early response of green algae to limitation in the environmental inorganic carbon.     

Photosynthetic characteristics of a multicellular green alga Volvox carteri in response to external CO2 levels possibly regulated by CCM1/CIA5 ortholog

When CO(2) supply is limited, aquatic photosynthetic organisms induce a CO(2)-concentrating mechanism (CCM) and acclimate to the CO(2)-limiting environment. Although the CCM is well studied in unicellular green algae such as Chlamydomonas reinhardtii, physiological aspects of the CCM and its associated genes in multicellular algae are poorly understood. In this study, by measuring photosynthetic affinity for CO(2), we present physiological data in support of a CCM in a multicellular green alga, Volvox carteri. The low-CO(2)-grown Volvox cells showed much higher affinity for inorganic carbon compared with high-CO(2)-grown cells. Addition of ethoxyzolamide, a membrane-permeable carbonic anhydrase inhibitor, to the culture remarkably reduced the photosynthetic affinity of low-CO(2) grown Volvox cells, indicating that an intracellular carbonic anhydrase contributed to the Volvox CCM. We also isolated a gene encoding a protein orthologous to CCM1/CIA5, a master regulator of the CCM in Chlamydomonas, from Volvox carteri. Volvox CCM1 encoded a protein with 701 amino acid residues showing 51.1% sequence identity with Chlamydomonas CCM1. Comparison of Volvox and Chlamydomonas CCM1 revealed a highly conserved N-terminal region containing zinc-binding amino acid residues, putative nuclear localization and export signals, and a C-terminal region containing a putative LXXLL protein-protein interaction motif. Based on these results, we discuss the physiological and genetic aspects of the CCM in Chlamydomonas and Volvox.     

Identification of a new chloroplast carbonic anhydrase in Chlamydomonas reinhardtii

Carbonic anhydrases (CA) are zinc-containing metalloenzymes that catalyze the reversible hydration of CO2. The three evolutionarily unrelated families of CAs are designated alpha-, beta-, and gamma-CA. Aquatic photosynthetic organisms have evolved different forms of CO2 concentrating mechanisms (CCMs) to aid Rubisco in capturing CO2 from the surrounding environment. One aspect of all CCMs is the critical roles played by various specially localized extracellular and intracellular CAs. Five CAs have previously been identified in Chlamydomonas reinhardtii, a green alga with a well-studied CCM. Here we identify a sixth gene encoding a beta-type CA. This new beta-CA, designated Cah6, is distinct from the two mitochondrial beta-CAs in C. reinhardtii. Nucleotide sequence data show that the Cah6 cDNA contains an open reading frame encoding a polypeptide of 264 amino acids with a leader sequence likely targeting the protein to the chloroplast stroma. We have fused the Cah6 open reading frame to the coding sequence of maltose-binding protein in a pMal expression vector. The purified recombinant fusion protein is active and was used to partially characterize the Cah6 protein. The purified recombinant fusion protein was cleaved with protease Factor Xa to separate Cah6 from the maltose-binding protein and the purified Cah6 protein was used to raise an antibody. Western blots, immunolocalization studies, and northern blots collectively indicated that Cah6 is constitutively expressed in the stroma of chloroplasts. A possible role for Cah6 in the CCM of C. reinhardtii is proposed.     

Regulation of leaf morphology by microRNA394 and its target LEAF CURLING RESPONSIVENESS

The present study identified Arabidopsis miR394 and its target, an F-box (SKP1-Cullin/CDC53-F-box) gene At1g27340 (here referred to as LEAF CURLING RESPONSIVENESS, LCR), for regulation of leaf curling-related morphology. The loss-of-function lcr mutants exhibit pleiotropic defects with semi-dwarfism, altered leaf shape and a shorter stem. Overexpression of an miR394-resistant version of LCR under the 35S promoter (35S:m5LCR) and target mimicry MIM394 resulted in a curled-down leaf defect. Conversely, transgenic plants overexpressing 35S:MIR394a/b display a curled-up leaf phenotype. Detailed analyses show that there is a certain level of LCR that is optimal for leaf morphology, but lower or higher levels lead to abnormal leaf development, indicating that expression of miR394 in the leaf lamina is necessary for proper leaf morphology. Because the phytohormone auxin plays a crucial role in leaf morphogenesis and patterning, the DR5-GUS reporter gene was used to monitor the auxin response. We show that DR5 expression patterns in lcr and 35S::m5LCR plants were significantly different from those in the wild type. Also, overexpression of LCR in 35S::m5LCR plants drastically decreased the expression of the auxin-responsive genes IAA3, AXR3 and IAMT1, whereas increased expression of the genes was found in 35S::MIR394a plants. These results indicate that miR394 and its target LCR are involved in the regulation of leaf development.     

Identification and functional role of the carbonic anhydrase Cah3 in thylakoid membranes of pyrenoid of Chlamydomonas reinhardtii

The distribution of the luminal carbonic anhydrase Cah3 associated with thylakoid membranes in the chloroplast and pyrenoid was studied in wild-type cells of Chlamydomonas reinhardtii and in its cia3 mutant deficient in the activity of the Cah3 protein. In addition, the effect of CO(2) concentration on fatty acid composition of photosynthetic membranes was examined in wild-type cells and in the cia3 mutant. In the cia3 mutant, the rate of growth was lower as compared to wild-type, especially in the cells grown at 0.03% CO(2). This might indicate a participation of thylakoid Cah3 in the CO(2)-concentrating mechanism (CCM) of chloroplast and reflect the dysfunction of the CCM in the cia3 mutant. In both strains, a decrease in the CO(2) concentration from 2% to 0.03% caused an increase in the content of polyunsaturated fatty acids in membrane lipids. At the same time, in the cia3 mutant, the increase in the majority of polyunsaturated fatty acids was less pronounced as compared to wild-type cells, whereas the amount of 16:4ω3 did not increase at all. Immunoelectron microscopy demonstrated that luminal Cah3 is mostly located in the thylakoid membranes that pass through the pyrenoid. In the cells of CCM-mutant, cia3, the Cah3 protein was much less abundant, and it was evenly distributed throughout the pyrenoid matrix. The results support our hypothesis that CO(2) might be generated from HCO(3)(-) by Cah3 in the thylakoid lumen with the following CO(2) diffusion into the pyrenoid, where the CO(2) fixing Rubisco is located. This ensures the maintenance of active photosynthesis under CO(2)-limiting conditions, and, as a result, the active growth of cells. The relationships between the induction of CCM and restructuring of the photosynthetic membranes, as well as the involvement of the Cah3 of the pyrenoid in these events, are discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

A novel alpha-type carbonic anhydrase associated with the thylakoid membrane in Chlamydomonas reinhardtii is required for growth at ambient CO2.

A 29.5 kDa intracellular alpha-type carbonic anhydrase, designated Cah3, from the unicellular green alga Chlamydomonas reinhardtii is the first of this type discovered inside a photosynthetic eukaryote cell. We describe the cloning of a cDNA which encodes the protein. Immunoblot studies with specific antibodies raised against Cah3 demonstrate that the polypeptide is associated exclusively with the thylakoid membrane. The putative transit peptide suggests that Cah3 is directed to the thylakoid lumen, which is confirmed further by the presence of mature sized Cah3 after thermolysin treatment of intact thylakoids. Complementation of the high inorganic carbon concentration-requiring mutant, cia-3, with a subcloned cosmid containing the cah3 gene yielded transformants that grew on atmospheric levels of CO2 (0.035%) and contained an active 29.5 kDa alpha-type carbonic anhydrase. Although, cia-3 has reduced internal carbonic anhydrase activity, unexpectedly the level of Cah3 was similar to that of the wild-type, suggesting that the mutant accumulates an inactive Cah3 polypeptide. Genomic sequence analysis of the mutant revealed two amino acid changes in the transit peptide. Results from photosynthesis and chlorophyll a fluorescence parameter measurements show that the cia-3 mutant is photosynthetically impaired. Our results indicate that the carbonic anhydrase, extrinsically located within the chloroplast thylakoid lumen, is essential for growth of C.reinhardtii at ambient levels of CO2, and that at these CO2 concentrations the enzyme is required for optimal photosystem II photochemistry.     

The role of Corynebacterium glutamicum spiA gene in whcA-mediated oxidative stress gene regulation

The Corynebacterium glutamicum WhcA protein, which inhibits the expression of oxidative stress response genes, is known to interact with the SpiA protein. In this study, we constructed and analyzed spiA mutant cells with the goal of better understanding the function of the spiA gene. A C. glutamicum strain overexpressing the spiA gene showed retarded cell growth, which was caused by an increased sensitivity to oxidants. Expression of the spiA and whcA genes was repressed by oxidant diamide, indicating coordinate regulation and dispensability of the genes in cells under oxidative stress. In the spiA-overexpressing cells, the trx gene, which encodes thioredoxin reductase, was severely repressed. Deletion of whcA in spiA-overexpressing cells (or vice versa) produced phenotypes similar to the wild-type strain. Collectively, these data demonstrate a negative regulatory role of the spiA gene in whcA-mediated oxidative stress response and provide additional clues on the mechanism by which the whcA gene is regulated.