Light-harvesting complex stress-related proteins play crucial roles in the acclimation of Physcomitrella patens under fluctuating light conditions.

Photosynthetic organisms have evolved photoprotective mechanisms to acclimate to light intensity fluctuations in their natural growth environments. Photosystem (PS) II subunit S (PsbS) and light-harvesting complex (LHC) stress-related proteins (LhcSR) are essential for triggering photoprotection in vascular plants and green algae, respectively. The activity of both proteins is strongly enhanced in the moss Physcomitrella patens under high-light conditions. However, their role in regulating photosynthesis acclimation in P. patens under fluctuating light (FL) conditions is still unknown. Here, we compare the responses of wild-type (WT) P. patens and mutants lacking PsbS (psbs KO) or LhcSR1 and 2 (lhcsr KO) to FL conditions in which the low-light phases were periodically interrupted with high-light pulses. lhcsr KO mutant showed a strong reduction in growth with respect to WT and psbs KO under FL conditions. The lack of LhcSR not only decreased the level of non-photochemical quenching, resulting in an over-reduced plastoquinone pool, but also significantly increased the PSI acceptor limitation values with respect to WT and psbs KO under FL conditions. Moreover, in lhcsr KO mutant, the abundance of PSI core and PSI-LHCI complex decreased greatly under FL conditions compared with the WT and psbs KO. We proposed that LhcSR in P. patens play a crucial role in moss acclimation to dynamic light changes.

Carbonic anhydrase isoforms of Neopyropia yezoensis: Intracellular localization and expression profiles in response to inorganic carbon concentration and life stage.

Macroalgae, 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), 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-, ß-, 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.

Co-suppression in Pyropia yezoensis (Rhodophyta) Reveals the Role of PyLHCI in Light Harvesting and Generation Switch.

The red macroalga Pyropia yezoensis is an economically important seaweed widely cultured in Asian countries and is a model organism for molecular biological and commercial research. This species is unique in that it utilizes both phycobilisomes and transmembrane light-harvesting proteins as its antenna system. Here, one of the genes of P. yezoensis (PyLHCI) was selected for introduction into its genome to overexpress PyLHCI. However, the co-suppression phenomenon occurred. This is the first documentation of co-suppression in algae, in which it exhibits a different mechanism from that in higher plants. The transformant (T1) was demonstrated to have higher phycobilisomes and lower LHC binding pigments, resulting in a redder color, higher sensitivity to salt stress, smaller in size, and slower growth rate than the wildtype (WT). The photosynthetic performances of T1 and WT showed similar characteristics; however, P700 reduction was slower in T1. Most importantly, T1 could release a high percentage of carpospores in young blades to switch generation during its life cycle, which was rarely seen in WT. The co-suppression of PyLHCI revealed its key roles in light harvesting, stress resistance, and generation alternation (generation switch from gametophytes to sporophytes, and reproduction from asexual to sexual).

RNA-seq between asexual archeospores and meiosis-related conchospores in Neopyropia yezoensis using Smart-seq2.

In the life cycle of Neopyropia yezoensis, a potential model system for marine macroalgae, both 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 up-regulated, indicating that complex life activities might be occurring 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) only in diploid stages (conchocelis, sporangial filament) and three archeospores specifically expressed DEGs only in haploid stages (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 sin N. yezoensis might require the inactivation of PyKNOX.

Slow zeaxanthin accumulation and the enhancement of CP26 collectively contribute to an atypical non-photochemical quenching in macroalga Ulva prolifera under high light.

Non-photochemical quenching (NPQ) is an important photoprotective mechanism in plants, which dissipates excess energy and further protects the photosynthetic apparatus under high light stress. NPQ can be dissected into a number of components: qE, qZ, and qI. In general, NPQ is catalyzed by two independent mechanisms, with the faster-activated quenching catalyzed by the monomeric light-harvesting complex (LHCII) proteins and the slowly activated quenching catalyzed by LHCII trimers, both processes depending on zeaxanthin but to different extent. Here, we studied the NPQ of the intertidal green macroalga, Ulva prolifera, and found that the NPQ of U. prolifera lack the faster-activated quenching, and showed much greater sensitivity to dithiothreitol (DTT) than to dicyclohexylcarbodiimide (DCCD). Further results suggested that the monomeric LHC proteins in U. prolifera included only CP29 and CP26, but lacked CP24, unlike Arabidopsis thaliana and the moss Physcomitrella patens. Moreover, the expression levels of CP26 increased significantly following exposure to high light, but the concentrations of the two important photoprotective proteins (PsbS and light-harvesting complex stress-related [LhcSR]) did not change upon the same conditions. Analysis of the xanthophyll cycle pigments showed that, upon exposure to high light, zeaxanthin synthesis in U. prolifera was gradual and much slower than that in P. patens, and could effectively be inhibited by DTT. Based on these results, we speculate the enhancement of CP26 and slow zeaxanthin accumulation provide an atypical NPQ, making this green macroalga well adapted to the intertidal environments.

Diluted seawater affects phytohormone receptors and maintains the protonema stage in Physcomitrella patens.

Due to its highly efficient homologous recombination ability and unusual evolutionary position, the moss Physcomitrella patens has begun to attract more attention in genetic and evolutionary studies. Protonema, the filament stage of the gametophyte, is of great significance in P. patens protoplast isolation. Moreover, protonema is widely used in genetic engineering. However, difficulties in the induction and state maintenance of protonema restrict its wider application. In this work, protonema was induced efficiently in a diluted seawater medium, and the filamentous state was maintained without further cell differentiation. The developmental process of the protonema resumed, progressing to bud assembly and gametophore formation after transfer to freshwater medium. In addition, a transcriptome analysis showed that plant hormone signal transduction pathways were downregulated when protonema was grown in diluted seawater medium. Consistent with the transcriptome results, the protonema failed to respond to the addition of indole-3-acetic acid and 6-benzylaminopurine to the diluted seawater medium. Based on these results, we concluded that diluted seawater medium blocks the differentiation of protonema. This result could provide a novel insight to benefit future protonema production.

A Supercomplex, of Approximately 720 kDa and Composed of Both Photosystem Reaction Centers, Dissipates Excess Energy by PSI in Green Macroalgae Under Salt Stress.

The thylakoid membranes of plants play a critical role in electron transfer and energy fixation, and are highly dynamic. So far, studies on the thylakoid membranes have mainly focused on microalgae and higher plants, yet very little information is available on the macroalgal thylakoids. Here, we studied the structure and organization of the thylakoid membranes in Ulva prolifera, a representative species of the green macroalgae. We found that U. prolifera had few but long loosely stacked membranes which lack the conventional grana found in higher plants. However, the thylakoid membrane complexes demonstrate lateral heterogeneity. Moreover, we found a supercomplex composed of PSII, light-harvesting complex II (LHCII) and PSI from U. prolifera under salt stress. The supercomplex is approximately 720 kDa, and includes the two important photoprotection proteins, the PSII S subunit (PsbS) and the light-harvesting complex stress-related protein (LhcSR), as well as xanthophyll cycle pigments (violaxanthin, antheraxanthin and zeaxanthin). Time-resolved fluorescence analysis suggested that, in the supercomplex, excitation energy could efficiently be transferred from PSII to PSI, even when PSII was inhibited, a function which disappeared when the supercomplex was incubated in mild detergent. We suggest that the supercomplex might be an important mechanism to dissipate excess energy by PSI in green macroalgae under salt stress.

High salt stress in the upper part of floating mats of Ulva prolifera, a species that causes green tides, enhances non-photochemical quenching.

Salt stress is a major abiotic stress factor that can induce many adverse effects on photosynthetic organisms. Plants and algae have developed several mechanisms that help them respond to adverse environments. Non-photochemical quenching (NPQ) is one of these mechanisms. The thalli of algae in the intertidal zone that are attached to rocks can be subjected to salt stress for a short period of time due to the rise and fall of the tide. Ulva prolifera causes green tides and can form floating mats when green tides occur and the upper part of the thalli is subjected to high salt stress for a long period of time. In this study, we compared the Ulva prolifera photosynthetic activities and NPQ kinetics when it is subjected to different salinities over various periods of time. Thalli exposed to a salinity of 90 for 4 d showed enhanced NPQ, and photosynthetic activities decreased from 60 min after exposure up to 4 d. This indicated that the induction of NPQ in Ulva prolifera under salt stress was closely related to the stressing extent and stressing time. The enhanced NPQ in the treated samples exposed for 4 d may explain why the upper layer of the floating mats formed by Ulva prolifera thalli were able to survive in the harsh environment. Further inhibitor experiments demonstrated that the enhanced NPQ was xanthophyll cycle and transthylakoid proton gradient-dependent. However, photosystem II subunit S and light-harvesting complex stress-related protein didn't over accumulate and may not be responsible for the enhanced NPQ.

Positive correlation between PSI response and oxidative pentose phosphate pathway activity during salt stress in an intertidal macroalga.

Studies have demonstrated that photosynthetic limitations and starch degradation are responses to stress; however, the relationship between the two is seldom described in detail. In this article, the effects of salt stress on photosynthesis, the levels of NADPH and total RNA, the starch content and the activities of glucose-6-phosphate dehydrogenase (G6PDH) and ribulose-5-phosphate kinase (RPK) were evaluated. In thalli that underwent salt treatments, the cyclic electron flow through PSI showed greater stress tolerance than the flow through PSII. Even though the linear electron flow was suppressed by DCMU, the cyclic electron flow still operated. The electron transport rate I (ETRI) increased as the salinity increased when the thalli recovered in seawater containing DCMU. These results suggested that PSI receives electrons from a source other than PSII. Furthermore, the starch content and RPK activity decreased, while the content of NADPH and total RNA, and the activity of G6PDH increased under salt stress. Soluble sugar from starch degradation may enter the oxidative pentose phosphate pathway (OPPP) to produce NADPH and ribose 5-phosphate. Data analysis suggests that NADPH provides electrons for PSI in Ulva prolifera during salt stress, the OPPP participates in the stress response and total RNA is synthesized in excess to assist recovery.

Photosystem I shows a higher tolerance to sorbitol-induced osmotic stress than photosystem II in the intertidal macro-algae Ulva prolifera (Chlorophyta).

The photosynthetic performance of the desiccation-tolerant, intertidal macro-algae Ulva prolifera was significantly affected by sorbitol-induced osmotic stress. Our results showed that photosynthetic activity decreased significantly with increases in sorbitol concentration. Although the partial activity of both photosystem I (PS I) and photosystem II (PS II) was able to recover after 30 min of rehydration, the activity of PS II decreased more rapidly than PS I. At 4 M sorbitol concentration, the activity of PS II was almost 0 while that of PS I was still at about one third of normal levels. Following prolonged treatment with 1 and 2 M sorbitol, the activity of PS I and PS II decreased slowly, suggesting that the effects of moderate concentrations of sorbitol on PS I and PS II were gradual. Interestingly, an increase in non-photochemical quenching occurred under these conditions in response to moderate osmotic stress, whereas it declined significantly under severe osmotic stress. These results suggest that photoprotection in U. prolifera could also be induced by moderate osmotic stress. In addition, the oxidation of PS I was significantly affected by osmotic stress. P700(+) in the thalli treated with high concentrations of sorbitol could still be reduced, as PS II was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), but it could not be fully oxidized. This observation may be caused by the higher quantum yield of non-photochemical energy dissipation in PS I due to acceptor-side limitation (Y(NA)) during rehydration in seawater containing DCMU.

G6PDH activity highlights the operation of the cyclic electron flow around PSI in Physcomitrella patens during salt stress.

Photosynthetic performances and glucose-6-phosphate dehydrogenase (G6PDH) activity in Physcomitrella patens changed greatly during salt stress and recovery. In P. patens, the cyclic electron flow around photosystem (PS) I was much more tolerant to high salt stress than PSII. After high salt stress, the PSII activity recovered much more slowly than that of PSI, which was rapidly restored to pretreatment levels even as PSII was almost inactivate. This result suggested that after salt stress the recovery of the cyclic electron flow around PSI was independent of PSII activity. In addition, G6PDH activity and NADPH content increased under high salt stress. When G6PDH activity was inhibited by glucosamine (Glucm, a G6PDH inhibitor), the cyclic electron flow around PSI and the NADPH content decreased significantly. Additionally, after recovery in liquid medium containing Glucm, the PSI activity was much lower than in liquid medium without Glucm. These results suggested the PSI activity was affected significantly by G6PDH activity and the NADPH content. Based on the above results, we propose that G6PDH in P. patens has a close relationship with the photosynthetic process, possibly providing NADPH for the operation of the cyclic electron flow around PSI during salt stress and promoting the restoration of PSI.