Genetic evidence for functions of Chloroplast CA in Pyropia yezoensis: decreased CCM but increased starch accumulation

To adapt to changes in the intertidal environment, intertidal macroalgae have evolved complicated Ci utilization mechanisms. However, our knowledge regarding the CO 2 concentrating mechanism (CCM) of macroalgae is limited. Carbonic anhydrase (CA), a key component of CCM, plays important roles in many physiological reactions in various organisms. While there are a large number of genes encoding CA in the Pyropia yezoensis genome, the exact function of specific CA in P. yezoensis remains elusive. To explore the specific function of chloroplast CA in intertidal macroalgae, we produced chloroplast-localized βCA1 knockdown mutants of P. yezoensis through RNA interference, and Pyca1i mutants showed a notable decrease in leaf area and overall biomass, as well as decreased soluble protein and unsaturated fatty acid content under different DIC conditions. However, Pyca1i mutants showed relatively higher starch content compared to the wild type. The activity of enzymes involved in the Calvin cycle, photorespiration, pentose-phosphate pathway and floridean starch synthesis of P. yezoensis indicated an effective starch accumulation pathway after interference with βCA1. All results suggest that the decreased activity of PyβCA1 impaired the CCM and development of thalli of P. yezoensis but stimulated starch accumulation in the cytoplasm through feedback to the photorespiration pathway and PP pathway to replenish intermediates for the Calvin cycle. This study is the first to explore the specific function of chloroplast CA in intertidal macroalgae using genomic technology. The results provide valuable insights into the adaptation mechanisms of intertidal macroalgae to their environment.