Chloroplast-mediated regulation of CO2-concentrating mechanism by Ca2+-binding protein CAS in the green alga Chlamydomonas reinhardtii
Sunday, 2016/11/06 | 06:52:39
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Lianyong Wang, Takashi Yamano, Shunsuke Takane, Yuki Niikawa, Chihana Toyokawa, Shin-ichiro Ozawa, Ryutaro Tokutsu, Yuichiro Takahashi, Jun Minagawa, Yu Kanesaki, Hirofumi Yoshikawa, and Hideya Fukuzawa SignificanceCa2+ and CO2 are fundamental biological signaling molecules in microbes, animals, and plants. Although Ca2+ was proposed to act as a second messenger in CO2 signaling in guard cells of terrestrial plants, the role of Ca2+ in CO2 signal transduction pathways in aquatic photosynthetic organisms remains largely unknown. We show here that a chloroplast Ca2+-binding protein, CAS, changes its localization in response to environmental CO2 conditions and regulates the expression of nuclear-encoded limiting-CO2–induced genes, including two key bicarbonate transporters. These findings led us to propose a model for the participation of Ca2+ signals in chloroplast-regulated CO2 signal transduction of aquatic photosynthetic organisms and help us to further understand the role of Ca2+ in CO2 signal transduction in eukaryotes. AbstractAquatic photosynthetic organisms, including the green alga Chlamydomonas reinhardtii, induce a CO2-concentrating mechanism (CCM) to maintain photosynthetic activity in CO2-limiting conditions by sensing environmental CO2 and light availability. Previously, a novel high-CO2–requiring mutant, H82, defective in the induction of the CCM, was isolated. A homolog of calcium (Ca2+)-binding protein CAS, originally found in Arabidopsis thaliana, was disrupted in H82 cells. Although Arabidopsis CAS is reported to be associated with stomatal closure or immune responses via a chloroplast-mediated retrograde signal, the relationship between a Ca2+ signal and the CCM associated with the function of CAS in an aquatic environment is still unclear. In this study, the introduction of an intact CAS gene into H82 cells restored photosynthetic affinity for inorganic carbon, and RNA-seq analyses revealed that CAS could function in maintaining the expression levels of nuclear-encoded CO2-limiting–inducible genes, including the HCO3– transporters high-light activated 3 (HLA3) and low-CO2–inducible gene A (LCIA). CAS changed its localization from dispersed across the thylakoid membrane in high-CO2 conditions or in the dark to being associated with tubule-like structures in the pyrenoid in CO2-limiting conditions, along with a significant increase of the fluorescent signals of the Ca2+ indicator in the pyrenoid. Chlamydomonas CAS had Ca2+-binding activity, and the perturbation of intracellular Ca2+ homeostasis by a Ca2+-chelator or calmodulin antagonist impaired the accumulation of HLA3 and LCIA. These results suggest that Chlamydomonas CAS is a Ca2+-mediated regulator of CCM-related genes via a retrograde signal from the pyrenoid in the chloroplast to the nucleus.
See: http://www.pnas.org/content/113/44/12586.abstract.html?etoc PNAS November 8 2016; vol.113; no.44: 12586–12591
Fig. 1. Characterization of CAS insertion mutant H82 and its complemented strain C-1. (A) Inorganic carbon (Ci) affinity of WT, H82, C-1, and transgenic H82 cells containing inducible genes of HLA3 and LCIA (AH-1) grown in medium with NH+4 or NO−3 as nitrogen sources in low-CO2 (LC) conditions for 12 h. Photosynthetic O2-evolving activity was measured in external dissolved Ci concentrations at pH 7.8, and the Ci concentration required for half maximal velocity [K0.5 (Ci)] was calculated. Data in all experiments are mean values ± SD from three biological replicates. *P < 0.01 by Student’s t test. (B and C) Accumulation (B) and fixation (C) of Ci in WT, H82, and C-1 cells. Cells were grown in LC conditions for 12 h, and the intracellular Ci concentration and carbon fixation were measured using a silicone-oil layer method. SIS, sorbitol impermeable space. (D) Accumulation of CAS, LCIA, HLA3, and LCIB in WT, H82, and C-1 cells. Cells were grown in high-CO2 (HC) or LC conditions for 12 h. Histone H3 was used as a loading control. |
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