Wenhui Gu

Pyropia yezoensis, an economically valuable macroalga, occupies a pivotal position in evolutionary history as a red alga, making it an ideal model organism for investigating the evolution of photosynthesis. However, efficient genetic manipulation in P. yezoensis, particularly the stable expression of exogenous genes, presents substantial challenges. Major obstacles, including gene silencing, co-suppression, and unstable transgenerational inheritance, hinder progress in this field. Emerging evidence suggests that epigenetic modifications, including histone acetylation and deacetylation, play crucial roles in regulating endogenous gene expression. Nevertheless, whether these processes influence the expression of exogenous genes in P. yezoensis remains largely unexplored. In this study, we demonstrate that the expression of an exogenous gene can be enhanced by silencing the gene encoding Sin3-associated protein 30 (PySAP30), a critical component of the histone deacetylase (HDAC) complex. To evaluate exogenous gene expression in P. yezoensis, we employed the GUS reporter gene driven by the strong endogenous polyubiquitin gene promoter (PypUbi1). We designed a dual-ribozyme system, comprising hammerhead and hepatitis delta virus ribozymes, to generate antisense fragments targeting PySAP30 exons. This strategy effectively suppressed PySAP30 expression, resulting in a marked increase in both GUS transcript abundance and enzymatic activity. In contrast, transformants without PySAP30 interference showed low GUS activity and pronounced chimerism. Our findings confirm the repressive effect of epigenetic modification on exogenous gene expression in P. yezoensis and highlight a potential strategy for improving exogenous gene expression in macroalgae.

Free‐floating Ulva prolifera is one of the causative species of green tides. When green tides occur, massive mats of floating U. prolifera thalli accumulate rapidly in surface waters with daily growth rates as high as 56%. The upper thalli of the mats experience environmental changes such as the change in carbon source, high salinity, and desiccation. In this study, the photosynthetic performances of PSI and PSII in U. prolifera thalli exposed to different atmospheric carbon dioxide (CO₂) levels were measured. Changes in photosynthesis within salinity treatments and dehydration under different CO₂ concentrations were also analyzed. The results showed that PSII activity was enhanced as CO₂ increased, suggesting that CO₂ assimilation was enhanced and U. prolifera thalli can utilize CO₂ in the atmosphere directly, even when under moderate stress. In addition, changes in the proteome of U. prolifera in response to salt stress were investigated. Stress‐tolerance proteins appeared to have an important role in the response to salinity stress, whereas the abundance of proteins related to metabolism showed no significant change under low salinity treatments. These findings may be one of the main reasons for the extremely high growth rate of free‐floating U. prolifera when green tides occur.

Sporulation and spore release are essential phases of the life cycle in algae and land plants. Ulva prolifera, which is an ideal organism for studying sporulation and spore release, was used as the experimental material in the present study. The determination of photosynthetic parameters, combined with microscopic observation, treatment with photosynthetic inhibitors, limitation of carbon acquisition, and protein mass spectrometry, was employed in this experiment. Cycle electron transport (CEF) was found enhanced at the onset of sporangia formation. The inhibition effect of dibromothymoquinone (DBMIB) towards sporulation was always strong during the sporulation process whereas the inhibition effect of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU) was continuously declined accompanied with the progress of sporulation. The changes of photosynthesis resulted from the limitation of CO₂ acquisition could stimulate sporulation onset. Quantitative protein analysis showed that enzymes involved in carbon fixation, including RUBISCO and pyruvate orthophosphate dikinase, declined during sporogenesis, while proteins involved in sporulation, including tubulin and centrin, increased. These results suggest that enhanced cyclic electron flow (CEF) and oxidation of the plastoquinone pool are essential for sporangia formation onset, and changes in photosynthetic electron transport chain have significant impacts on sporulation of the green algae.

Summary: The development of microalgae on an industrial scale largely depends on the economic feasibility of mass production. High light induces productive suspensions during cultivation in a tubular photobioreactor. Herein, we report that high light, which inhibited the growth of Chlorella sorokiniana under autotrophic conditions, enhanced the growth of this alga in the presence of acetate. We compared pigments, proteomics and the metabolic flux ratio in C. sorokiniana cultivated under high light (HL) and under low light (LL) in the presence of acetate. Our results showed that high light induced the synthesis of xanthophyll and suppressed the synthesis of chlorophylls. Acetate in the medium was exhausted much more rapidly in HL than in LL. The data obtained from LC‐MS/MS indicated that high light enhanced photorespiration, the Calvin cycle and the glyoxylate cycle of mixotrophic C. sorokiniana. The results of metabolic flux ratio analysis showed that the majority of the assimilated carbon derived from supplemented acetate, and photorespiratory glyoxylate could enter the glyoxylate cycle. Based on these data, we conclude that photorespiration provides glyoxylate to speed up the glyoxylate cycle, and releases acetate‐derived CO₂ for the Calvin cycle. Thus, photorespiration connects the glyoxylate cycle and the Calvin cycle, and participates in the assimilation of supplemented acetate in C. sorokiniana under high light.

Under high light (HL) stress, astaxanthin-accumulating Haematococcus pluvialis and [bgr]-carotene-accumulating Dunaliella salina showed different responsive patterns. To elucidate cellular-regulating strategies photosynthetically and metabolically, thylakoid membrane proteins in H. pluvialis and D. salina were extracted and relatively quantified after 0[emsp14]h, 24[emsp14]h and 48[emsp14]h of HL stress. Proteomic analysis showed that three subunits of the cytochrome b6/f complex were greatly reduced under HL stress in H. pluvialis, while they were increased in D. salina. Additionally, the major subunits of both photosystem (PS) II and PSI reaction center proteins were first reduced and subsequently recovered in H. pluvialis, while they were gradually reduced in D. salina. D. salina also showed a greater ability to function using the xanthophyll-cycle and the cyclic photosynthetic electron transfer pathway compared to H. pluvialis. We propose a reoriented and effective HL-responsive strategy in H. pluvialis, enabling it to acclimate under HL. The promising metabolic pathway described here contains a reorganized pentose phosphate pathway, Calvin cycle and glycolysis pathway participating in carbon sink formation under HL in H. pluvialis. Additionally, the efficient carbon reorientation strategy in H. pluvialis was verified by elevated extracellular carbon assimilation and rapid conversion into astaxanthin.

Desiccation has significant effects on photosynthetic processes in intertidal macro‐algae. We studied an intertidal macro‐alga, Ulva sp., which can tolerate desiccation, to investigate changes in photosynthetic performance and the components and structure of thylakoid membrane proteins in response to desiccation. Our results demonstrate that photosystem II (PSII) is more sensitive to desiccation than photosystem I (PSI) in Ulva sp. Comparative proteomics of the thylakoid membrane proteins at different levels of desiccation suggested that there were few changes in the content of proteins involved in photosynthesis during desiccation. Interestingly, we found that both the PSII subunit, PsbS (Photosystem II S subunit) (a four‐helix protein in the LHC superfamily), and light‐harvesting complex stress‐related (LHCSR) proteins, which are required for non‐photochemical quenching in land plants and algae, respectively, were present under both normal and desiccation conditions and both increased slightly during desiccation. In addition, the results of immunoblot analysis suggested that the phosphorylation of PSII and LHCII increases during desiccation. To investigate further, we separated out a supercomplex formed during desiccation by blue native‐polyacrylamide gel electrophoresis and identified the components by mass spectrometry analysis. Our results show that phosphorylation of the complex increases slightly with decreased water content. All the results suggest that during the course of desiccation, few changes occur in the content of thylakoid membrane proteins, but a rearrangement of the protein complex occurs in the intertidal macro‐alga Ulva sp.