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To adapt to the change of intertidal environment, intertidal macroalgae have evolved complicated Ci utilization mechanism. However, our knowledge regarding the CO2 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 Calvin cycle, photorespiration, Pentose-phosphate pathway and floridean starch synthesis of P.yezoensis indicated an effective starch accumulation pathway after 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 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 adaption mechanisms of intertidal macroalgae to their environment.

In the life cycle of Neopyropia yezoensis, a potential model system for marine macroalgae, both of asexual archeospores and meiosis-related conchospores develop into thalli (gametophyte). To understand this special life phenomenon in macroalgae, we picked out the two kinds of spores (10-30 cells in each sample) and conducted RNA-seq using Smart-seq2. Comparative analysis showed that light capture and carbon fixation associated differentially expressed genes (DEGs) were upregulated in archeospores, thus indicating that archeospores are in a state of rapid vegetative growth. In conchospores, protein synthesis and degradation, especially molecular chaperone, associated DEGs were upregulated, indicating the complex life activities might be occurred in conchospores. There were 68 genes related to DNA replication and repair expressed in conchospores, showing that active DNA replication might occur in conchospores. Moreover, we found that one conchospore specifically expressed DEG (py04595: DNA helicase) was only expressed in diploid stage (conchocelis, sporangial filament) and three archeospore specifically expressed DEGs were only expressed in haploid stage (thalli). These molecular level results indicated that conchospores were closer to diploid, and might be the meiotic mother cells of N. yezoensis. In addition, we found that the knotted-like homeobox gene (PyKNOX), which might relate to the transition of gametophyte from sporophyte, was only expressed in sporophyte generation but not expressed in conchospores, archeospores and thalli, indicating the morphogenesis of gametophyte in N. yezoensis might require the inactivation of PyKNOX.

Macroalgae, particularly commercially grown seaweed, substantially contribute to COsub2/subremoval and carbon storage. However, knowledge regarding the COsub2/subconcentrating mechanism (CCM) of macroalgae is limited. Carbonic anhydrase (CA), a key component of the biophysical CCM, plays important roles in many physiological reactions in various organisms. Few characteristics of CA in Neopyropia yezoensis are known, particularly its intracellular location and responses to different concentrations of Ci. We identified, amplified, and characterized 11 putative genes encoding N. yezoensis CA. The predicted corresponding proteins clustered into three subfamilies: α-, β-, and γ-type. The intracellular localization of seven CA isoforms-one in the chloroplasts, three in the cytoplasm, and three in the mitochondria-was elucidated with fusion proteins. Higher NyCA expression, particularly of certain chloroplastic, cytosolic, and mitochondrial CAs, is observed more often during the foliose stage, thus suggesting that CAs play important roles in development in N. yezoensis.

BackgroundMacroalgae, particularly commercially grown seaweed, substantially contribute to CO2 removal and carbon storage. However, knowledge regarding the CO2 concentrating mechanism (CCM) of macroalgae is limited. Carbonic anhydrase (CA), the key component of biophysical CCM, plays important roles in many physiological reactions in various organisms. CA has been widely studied in microalgae and higher plants. However, few characteristics of CA in Pyropia yezoensis are known, particularly its intracellular location and responses to different concentrations of Ci.ResultsWe retrieved transcriptomic and genomic data for P. yezoensis and identified, amplified and characterized 11 putative genes encoding CA. The predicted corresponding proteins clustered into three subfamilies: α-, β- and γ-type. Transcriptomic and qRT-PCR results revealed different expression levels of these PyCA genes in the two life stages of P. yezoensis, and their expression levels varied under different Ci conditions. The intracellular localization of eight CA isoforms—one in the chloroplasts, four in the cytoplasm and three in the mitochondria—were elucidated with fusion proteins. One γCA isoform (PyCA10) was shown to be localized in the cytosol, and its localization is discussed. We also discuss the potential roles of the CA isoforms in development and the CCM in the two life stages. ConclusionsCA isoforms in P. yezoensis are widely distributed within cells, and higher PyCA expression, particularly of certain chloroplastic, cytosolic and mitochondrional CAs, is observed more often during the leafy stage. This expression results in stronger carbon sequestration ability in leafy thalli than filamentous thalli.