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

In response to the changing intertidal environment, intertidal macroalgae have evolved complicated Ci utilization mechanisms. However, our knowledge regarding the CO2 concentrating mechanism (CCM) of macroalgae is limited. Carbonic anhydrase (CA), a key component of CCM, plays essential roles in many physiological reactions in various organisms. While many genes encode CA in the Pyropia yezoensis genome, the exact function of specific CA in P. yezoensis remains elusive. To explore the particular function of chloroplast CA in intertidal macroalgae, we produced chloroplast-localized βCA1 knockdown mutants of P. yezoensis through RNA interference, and Pyβca1i mutants (hereinafter referred to as ca1i) 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, ca1i 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 the interference of 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 pentose phosphate 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 adaption mechanisms of intertidal macroalgae to their environment.