Conchosporangia maturation is crucial for the yield of Pyropia/Porphyra. However, the molecular mechanisms underlying this process are poorly understood. In this study, we selected two strains of Pyropia haitanensis that show significant differences in conchosporangia maturation as materials to produce RNA-Seq libraries. Then, we identified key molecular pathways and genes involved in conchosporangia maturation by conducting a weighted gene co-expression network analysis. Two specific modules were identified, and included functions such as phosphorus metabolism, lipid metabolism, and the phosphatidylinositol signaling system. The hub genes that responded positively during conchosporangia maturation encoded diacylglycerol kinase (DGK) and phosphatidylinositol-3-phosphate-5-kinase, which are involved in the synthesis of phosphatidic acid, a key component of lipid metabolism. A full-length DGK sequence of P. haitanensis, designated as PhDGK1, was obtained by rapid-amplification of cDNA ends. Conserved motif and phylogenetic tree analyses showed that PhDGK1 belongs to DGK Cluster II. The transcript level of PhDGK1 increased during conchosporangia maturation in both strains, but increased earlier, and to higher levels, in the early-maturing strain than in the late-maturing strain. This pattern of gene expression was consistent with the patterns of maturity and changes in pigment contents. These results indicate that lipid metabolism plays a key role in regulating conchosporangia maturation in Pyropia spp., and that PhDGK1 might be a useful molecular marker for breeding new early-maturing strains.

Global warming is one of the key limiting factors affecting the cultivation of Pyropia haitanensis which is an economically important macroalgae species grown in southern China. However, the mechanism underlying the high-temperature tolerance of P. haitanensis remains largely unknown. In a previous study, we showed that the expression of the small heat shock protein 22 gene (Hsp22) is upregulated in P. haitanensis in response to high-temperature stress, but the associated regulatory mechanism was not fully elucidated. In this study, a transgenic Chlamydomonas reinhardtii expression system was used to functionally characterize P. haitanensis Hsp22. Our analyses indicated that the C-terminal of PhHsp22 is highly conserved and contains an A-crystal structure domain. A phylogenetic analysis revealed PhHsp22 is not closely related to small heat shock protein genes in other species. Additionally, PhHsp22 expression significantly increased at 3 and 6 h after initiating 33 °C treatment, which improved the survival rate of transgenic C. reinhardtii during the early stage of high-temperature treatment. The further transcriptome analysis revealed that PhHsp22 expression can promote pathways related to energy metabolism, metabolites metabolism, and protein homeostasis in transgenic C. reinhardtii cells exposed to high temperatures. Therefore, PhHsp22 may be crucial for the response of Pyropia species to high-temperature stress. Furthermore, this gene may be useful for breeding new high-temperature algal strains.

Floridean starch and floridoside are the main storage carbohydrates of red algae. However, their complete metabolic pathways and the origin, function, and regulatory mechanism of their pathway genes have not been fully elucidated. In this study, we identified their metabolic pathway genes and analyzed the changes in related gene expression and metabolite content in Neoporphyra haitanensis under continuous dark conditions. Our results showed that genes from different sources, including eukaryotic hosts, cyanobacteria, and bacteria, were combined to construct floridean starch and floridoside metabolic pathways in N. haitanensis. Moreover, compared with those in the control, under continuous dark conditions, floridean starch biosynthesis genes and some degradation genes were significantly upregulated with no significant change in floridean starch content, whereas floridoside degradation genes were significantly upregulated with a significant decrease in floridoside content. This implies that floridean starch content is maintained but floridoside is consumed in N. haitanensis under dark conditions. This study elucidates the “floridean starch–floridoside” metabolic network and its gene origins in N. haitanensis for the first time.

In plants under hypersaline stress, the main transporter that extrudes sodium ions (Na⁺) is the Na⁺/H⁺ antiporter SOS1. Different from land plants, the intertidal macroalgae, Neopyropia/Neoporphyra contains an animal-type Na⁺/K⁺-ATPase as well as the SOS1 system. However, the contribution of Na⁺/K⁺-ATPase to the K⁺/Na⁺ homeostasis of intertidal macroalgae remains unclear. In this study, we analyzed the function of Na⁺/K⁺-ATPase in the response of Neoporphyra haitanensis to salt stress from the perspective of ion transport dynamics. Both the transcript level of NhNKA2 and enzyme activity of Na⁺/K⁺-ATPase increased in the early response of N. haitanensis thalli to hypersaline stress. Addition of ouabain, an inhibitor of Na⁺/K⁺-ATPase, resulted in Na⁺ accumulation in the cells, severe K⁺ leakage from the thalli, and then remarkably disturbed the K⁺/Na⁺ homeostasis in N. haitanensis thalli. This disruption might induce a significant decrease in photosynthesis and a severe oxidative damage in thalli. Accordingly, these results suggested that the important role of Na⁺/K⁺-ATPase in the resistance of intertidal macroalgae to hypersaline stress, and shed light on the diversity of K⁺/Na⁺ homeostasis maintenance mechanisms in plants.

Increasing global temperatures have seriously affected the sustainable development of Neoporphyra haitanensis cultivation. Although several pathways are reported to be involved in the response of N. haitanensis to heat stress, it is unknown which ones are activated by signal transduction. Previously, we detected a large influx of calcium ions (Ca²⁺) in N. haitanensis under heat stress. In this study, we further investigated the specificity of Ca²⁺ signaling and how is it transduced. Transmission electron microscopy and Fv/Fm analyses showed that the Ca²⁺ signal derived from extracellular Ca²⁺ formed at the early stage of the response to heat stress, and the signal was recognized and decoded by N. haitanensis calmodulin (NhCaM). In yeast two-hybrid assays, DnaJ, a voltage-dependent anion channel, and a bromodomain-containing protein interacted with PhCaM1 in vivo. The transcript levels of the genes encoding these proteins increased significantly in response to heat stress, but decreased upon inhibition of NhCaM1, indicating that these interacting factors were positively related to NhCaM1. Additionally, a comparative transcriptome analysis indicated that Ca²⁺ signal transduction is involved in phosphatidylinositol, photosystem processes, and energy metabolism in N. haitanensis under heat stress. Our results suggest that Ca²⁺-CaM plays important roles in signal transduction in response to heat stress in N. haitanensis.