CO2-dependent migration and relocation of LCIB, a pyrenoid-peripheral protein in Chlamydomonas reinhardtii

Abstract

Most microalgae overcome the difficulty of acquiring inorganic carbon (Ci) in aquatic environments by inducing a CO2-concentrating mechanism (CCM). In the green alga Chlamydomonas reinhardtii, two distinct photosynthetic acclimation states have been described under CO2-limiting conditions (low-CO2 [LC] and very low-CO2 [VLC]). LC-inducible protein B (LCIB), structurally characterized as carbonic anhydrase, localizes in the chloroplast stroma under CO2-supplied and LC conditions. In VLC conditions, it migrates to aggregate around the pyrenoid, where the CO2-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase is enriched. Although the physiological importance of LCIB localization changes in the chloroplast has been shown, factors necessary for the localization changes remain uncertain. Here, we examined the effect of pH, light availability, photosynthetic electron flow, and protein synthesis on the localization changes, along with measuring Ci concentrations. LCIB dispersed or localized in the basal region of the chloroplast stroma at 8.3–15 µM CO2, whereas LCIB migrated toward the pyrenoid at 6.5 µM CO2. Furthermore, LCIB relocated toward the pyrenoid at 2.6–3.4 µM CO2, even in cells in the dark or treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea and cycloheximide in light. In contrast, in the mutant lacking CCM1, a master regulator of CCM, LCIB remained dispersed even at 4.3 µM CO2. Meanwhile, a simultaneous expression of LCIC, an interacting protein of LCIB, induced the localization of several speckled structures at the pyrenoid periphery. These results suggest that the localization changes of LCIB require LCIC and are controlled by CO2 concentration with ∼7 µM as the boundary.

Comparative Phenotyping of Two Commonly Used Chlamydomonas reinhardtii Background Strains: CC-1690 (21gr) and CC-5325 (The CLiP Mutant Library Background)

“…Its genome has been sequenced multiple times by several laboratories [ 42 , 43 ]. It has also been used for several transcriptome analyses [ 12 , 23 ] and is an often-preferred background strain for photosynthesis- or stress-related experiments [ 25 , 44 , 45 , 46 ]. CC-5325 is relatively new, available to the Chlamydomonas community since 2014 [ 47 ], but has been frequently used because it is the background strain of the Chlamydomonas CLiP mutant Library [ 37 , 38 ].…”

New horizons for building pyrenoid-based CO2-concentrating mechanisms in plants to improve yields

“…Thus, LCIB helps to maintain an inward CO 2 diffusion gradient while increasing the overall stromal Ci concentration. In ambient CO 2 , LCIB is dispersed throughout the chloroplast stroma in a multimeric complex with low CO 2 -inducible C (LCIC) ( Yamano et al, 2010 , 2021 ). In support of its critical role, LCIB knockout mutants are unable to grow or assimilate CO 2 in ambient CO 2 , whereas knockout mutations in LCIC appear to have no identifiable impact on growth or photosynthesis (Spalding, unpublished).…”

“…Post-translational modification and Ca 2+ signaling are both implicated in regulating the transition between Ci uptake pathways at ambient or sub-ambient CO 2 , although current knowledge is somewhat fragmented. The migration of LCIB to the pyrenoid periphery is dependent on light and LCIC, and may be regulated by phosphorylation of itself, and/or LCIC within the LCIB/C complex ( Jin et al, 2016 ; Yamano et al, 2021 ). Recent work has also highlighted the importance of the starch sheath in facilitating the migration of LCIB and CCM function ( Toyokawa et al, 2020 ).…”

Physiology of microalgae and their application to sustainable agriculture: A mini-review

“…It has been suggested that the chloroplast protein limiting CO 2 inducible protein B (LCIB), which structurally resembles a β-type carbonic anhydrase ( Jin et al., 2016 ), is indispensable for the stimulation of CO 2 uptake under LC conditions ( Yamano et al., 2010 ; Wang and Spalding, 2014 ). It may be that LCIB captures CO 2 leaked from the pyrenoid by unidirectionally hydrating CO 2 to HCO 3 - under LC conditions ( Yamano et al., 2022 ), though recombinant LCIB did not show carbonic anhydrase activity ( Jin et al., 2016 ). LCIB proteins are dispersed uniformly in the chloroplast under LC conditions, whereas they migrate to the pyrenoid periphery under VLC conditions ( Yamano et al., 2022 ).…”

“…It may be that LCIB captures CO 2 leaked from the pyrenoid by unidirectionally hydrating CO 2 to HCO 3 - under LC conditions ( Yamano et al., 2022 ), though recombinant LCIB did not show carbonic anhydrase activity ( Jin et al., 2016 ). LCIB proteins are dispersed uniformly in the chloroplast under LC conditions, whereas they migrate to the pyrenoid periphery under VLC conditions ( Yamano et al., 2022 ). The starch sheath surrounding the pyrenoid is important in the localization of LCIB ( Toyokawa et al., 2020 ).…”