Chloroplast-mediated regulation of CO ₂ -concentrating mechanism by Ca ²⁺ -binding protein CAS in the green alga Chlamydomonas reinhardtii

Abstract

Aquatic 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.

A Spatial Interactome Reveals the Protein Organization of the Algal CO2-Concentrating Mechanism

“…Under very low CO 2 conditions, the LCIB/LCIC complex, whose role is still uncertain (Jin et al, 2016), is known to form puncta around the pyrenoid periphery (Yamano et al, 2010). Recently, a Ca 2+ -binding protein, CAS, has been shown to specifically localize to the pyrenoid tubules at low CO 2 (Wang et al, 2016). Here, we identify seven additional pyrenoid-localized components and three previously un-described sub-pyrenoid localization patterns (Figure 4B–D).…”

“…Recent work has shown that Ca 2+ signalling is key for proper regulation of the CCM, with the Ca 2+ -binding protein CAS1 transcriptionally regulating HLA3 and other components (Wang et al, 2016). One HCIP of HLA3 is an EF-hand-containing Ca 2+ /calmodulin-dependent protein kinase (Cre13.g571700), which could potentially regulate HLA3 post-translationally.…”

Advances and current challenges in calcium signaling

“…In Arabidopsis, CAS crucially functions in the generation of cytosolic and stromal Ca 2+ transients as well as in the modulation of MAPK signaling (Guo et al, 2016). Moreover, it is involved in photo-acclimation in Chlamydomonas reinhardtii by contributing to the control of expression of LHCSR3 and to the expression of proteins related to the carbon-concentrating mechanism (Petroutsos et al, 2011;Wang et al, 2016b). Also the activity of the cyclic electron flow (CEF) is modulated via CAS and the availability of Ca 2+ in C. reinhardtii and in Arabidopsis (Terashima et al, 2012;Takahashi et al, 2013;Strand et al, 2015).…”

The singular properties of photosynthetic cytochrome c550 from the diatom Phaeodactylum tricornutum suggest new alternative functions

“…Actually, PsbP is considered to be evolutionarily recruited as a replacement of Cc 550 in the photosynthetic green lineage, playing a similar role stabilizing the binding of Ca 2+ , and thus increasing PSII affinity for this ion (Frazão et al , Roncel et al ). Moreover, a Ca 2+ ‐binding protein (CAS) has been also recently shown to specifically localize into the intrapyrenoid thylakoids of C. reinhardtii (Wang et al ). Thus, the presence of Cc 550 in the pyrenoid of P. tricornutum could be related to its role as a calcium‐binding stabilizing protein.…”