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Knockdown of limiting-CO2-induced gene HLA3 decreases HCO3- transport and photosynthetic Ci affinity in Chlamydomonas reinhardtii.

ABSTRACT: The CO(2)-concentrating mechanism (CCM) of Chlamydomonas reinhardtii and other microalgal species is essential for photosynthetic growth in most natural settings. A great deal has been learned regarding the CCM in cyanobacteria, including identification of inorganic carbon (Ci; CO(2) and HCO(3)(-)) transporters; however, specific knowledge of analogous transporters has remained elusive in eukaryotic microalgae such as C. reinhardtii. Here we investigated whether the limiting-CO(2)-inducible, putative ABC-type transporter HLA3 might function as a HCO(3)(-) transporter by evaluating the effect of pH on growth, photosynthetic Ci affinity, and [(14)C]-Ci uptake in very low CO(2) conditions following RNA interference (RNAi) knockdown of HLA3 mRNA levels in wild-type and mutant cells. Although knockdown of HLA3 mRNA alone resulted in only modest but high-pH-dependent decreases in photosynthetic Ci affinity and Ci uptake, the combination of nearly complete knockdown of HLA3 mRNA with mutations in LCIB (which encodes limiting-Ci-inducible plastid-localized protein required for normal Ci uptake or accumulation in low-CO(2) conditions) and/or simultaneous, apparently off-target knockdown of LCIA mRNA (which encodes limiting-Ci-inducible plastid envelope protein reported to transport HCO(3)(-)) resulted in dramatic decreases in growth, Ci uptake, and photosynthetic Ci affinity, especially at pH 9, at which HCO(3)(-) is the predominant form of available Ci. Collectively, the data presented here provide compelling evidence that HLA3 is directly or indirectly involved in HCO(3)(-) transport, along with additional evidence supporting a role for LCIA in chloroplast envelope HCO(3)(-) transport and a role for LCIB in chloroplast Ci accumulation.

SUBMITTER: Duanmu D

PROVIDER: S-EPMC2667020 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Knockdown of limiting-CO2-induced gene HLA3 decreases HCO3- transport and photosynthetic Ci affinity in Chlamydomonas reinhardtii.

Duanmu Deqiang D Miller Amy R AR Horken Kempton M KM Weeks Donald P DP Spalding Martin H MH

Proceedings of the National Academy of Sciences of the United States of America 20090324 14

The CO(2)-concentrating mechanism (CCM) of Chlamydomonas reinhardtii and other microalgal species is essential for photosynthetic growth in most natural settings. A great deal has been learned regarding the CCM in cyanobacteria, including identification of inorganic carbon (Ci; CO(2) and HCO(3)(-)) transporters; however, specific knowledge of analogous transporters has remained elusive in eukaryotic microalgae such as C. reinhardtii. Here we investigated whether the limiting-CO(2)-inducible, put ...[more]

PMID: 19321421

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Project description:BackgroundThe cellular proteome and metabolome are underlying dynamic regulation allowing rapid adaptation to changes in the environment. System-wide analysis of these dynamics will provide novel insights into mechanisms of stress adaptation for higher photosynthetic organisms. We applied pulsed-SILAC labeling to a photosynthetic organism for the first time and we established a method to study proteome dynamics in the green alga Chlamydomonas reinhardtii, an emerging model system for plant biology. In addition, we combined the analysis of protein synthesis with metabolic profiling to study the dynamic changes of metabolism and proteome turnover under salt stress conditions.ResultsTo study de novo protein synthesis an arginine auxotroph Chlamydomonas strain was cultivated in presence of stable isotope-labeled arginine for 24 hours. From the time course experiment in 3 salt concentrations we could identify more than 2500 proteins and their H/L ratio in at least one experimental condition; for 998 protiens at least 3 ratio counts were detected in the 24 h time point (0 mM NaCl). After fractionation we could identify 3115 proteins and for 1765 of them we determined their de novo synthesis rate. Consistently with previous findings we showed that RuBisCO is among the most prominent proteins in the cell; and similar abundance and turnover for the small and large RuBisCO subunit could be calculated. The D1 protein was identified among proteins with a high synthesis rates. A global median half-life of 45 h was calculated for Chlamydomonas proteins under the chosen conditions.ConclusionTo investigate the temporal co-regulation of the proteome and metabolome, we applied salt stress to Chlamydomonas and studied the time dependent regulation of protein expression and changes in the metabolome. The main metabolic response to salt stress was observed within the amino acid metabolism. In particular, proline was up-regulated manifold and according to that an increased carbon flow within the proline biosynthetic pathway could be measured. In parallel the analysis of abundance and de novo synthesis of the corresponding enzymes revealed that metabolic rearrangements precede adjustments of protein abundance.

Dataset Information

Knockdown of limiting-CO2-induced gene HLA3 decreases HCO3- transport and photosynthetic Ci affinity in Chlamydomonas reinhardtii.

Publications

Knockdown of limiting-CO2-induced gene HLA3 decreases HCO3- transport and photosynthetic Ci affinity in Chlamydomonas reinhardtii.

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