Phage Infection Benefits Marine Diatom Phaeodactylum tricornutum by Regulating the Associated Bacterial Community.

The interaction between marine phyto- and bacterioplankton is regulated by multiple environmental and biological factors. Among them, phages as the major regulators of bacterial mortality are considered to have important impacts on algae-associated bacteria and algae-bacteria relationship. However, little is currently known about the actual impact of phages from this perspective. Here, we revealed that phage infection improved the maximum quantum efficiency of photosystem II of Phaeodactylum tricornutum by regulating the associated bacterial community. Specifically, phage infection weakened bacterial abundance and eliminated their negative effects on the diatom. Unexpectedly, the structure of the bacterial community co-cultured with the diatom was not significantly affected, likely because the shaping effect of the diatom on the bacterial community structure can far outcompete or mask the impact of phage infection. Our results established a link between algae, bacteria, and phages, suggesting that phage infection benefits the diatom by regulating the associated bacterial community.

A Novel Phage Indirectly Regulates Diatom Growth by Infecting a Diatom-Associated Biofilm-Forming Bacterium.

Algae and heterotrophic bacteria have close and intricate interactions, which are regulated by multiple factors in the natural environment. Phages are the major factor determining bacterial mortality rates. However, their impacts on the alga-associated bacteria and thus on the alga-bacterium interactions are poorly understood. Here, we obtained a diatom-associated bacterium, Stappia indica SNL01, that could form a biofilm and had an inhibitory effect on the growth of the diatom Thalassiosira pseudonana. Meanwhile, phage SI01, with a double-stranded circular DNA genome (44,247 bp), infecting S. indica SNL01 was isolated. Phylogenetic analysis revealed that phage SI01 represents a novel member of the Podoviridae family. The phage contained multiple lysis genes encoding cell wall-lysing muramidase and spore cortex-lysing SleB, as well as depolymerase-like tail spike protein. By lysing the host bacterium and inhibiting the formation of biofilm, this phage could indirectly promote the growth of the diatom. Our results provide new insights into how phages indirectly regulate algal growth by infecting bacteria that are closely associated with algae or in the phycosphere. IMPORTANCE The impact of phage infection on the alga-bacterium relationship in the ocean is poorly understood. Here, a novel phage infecting the diatom-associated bacterium Stappia indica SNL01 was isolated. This bacterium could form a biofilm and had a negative effect on diatom growth. We revealed that this phage contained multiple lysis genes and could inhibit the formation of the bacterial biofilm, thus indirectly promoting diatom growth. This study suggests that phages not only are important regulators of bacteria but also have substantial indirect effects on algae and the alga-bacterium relationship.

A dark-tolerant diatom (Chaetoceros) cultured from the deep sea.

Although the extreme conditions of the deep sea are typically not suitable for the growth of photosynthetic algae, accumulating evidence indicates that there are diverse healthy phytoplankton living in this environment. However, living phytoplankton from the deep sea have rarely been isolated and cultivated, and so our understanding of where they come from and how they adapt to (or tolerate) the extreme deep-sea environment is limited. Here, under long-term dark stress and subsequent light treatment, we successfully isolated a diatom from a depth of 1,000 m in the Western Pacific Ocean. Morphological observations and molecular phylogenetic analysis revealed that it is affiliated to the genus Chaetoceros, and thus, we tentatively named it Chaetoceros sp. DS1. We observed that the chloroplast genome of this species, is most closely related to that of Chaetoceros simplex. It was shown to have a strong tolerance to darkness in that it maintained its morphological integrity and vitality for up to 3 months in complete darkness at room temperature. We also demonstrated that Chaetoceros sp. DS1 presented a facultative heterotrophic function. Its growth was promoted by many organic carbon sources (e.g., glycerine, ethanol, and sodium acetate) under low light conditions. However, under dark and high light conditions, the growth promotion effect of organic carbon was not obvious. Indeed, Chaetoceros sp. DS1 grew best under low light conditions, indicating that it likely came from the deeper layer of the euphotic zone. The facultative heterotrophic function of this diatom and tolerance to darkness may help it survive in these conditions or enter a dormant period in the deep sea.

Effects of CO2 and seawater acidification on the early stages of Saccharina japonica development.

In this paper, we demonstrated that ocean acidification (OA) had significant negative effects on the microscopic development of Saccharina japonica in a short-term exposure experiment under a range of light conditions. Under elevated CO2, the alga showed a significant reduction in meiospore germination, fecundity, and reproductive success. Larger female and male gametophytes were noted to occur under high CO2 conditions and high light magnified these positive effects. Under conditions of low light combined with high PCO2, the differentiation of gametophytes was delayed until the end of the experiment. In contrast, gametophytes were able to survive after having been subjected to a long-term acclimation period, of 105 days. Although the elevated PCO2 resulted in a significant increase in sporophyte length, the biomass abundance (expressed as individual density attached to the seed fiber) was reduced significantly. Further stress resistance experiments showed that, although the acidified samples had lower resistance to high light and high temperature conditions, they displayed higher acclimation to CO2-saturated seawater conditions compared with the control groups. These combined results indicate that OA has a severe negative effect on S. japonica, which may result in future shifts in species dominance and community structure.

Long-term experiment on physiological responses to synergetic effects of ocean acidification and photoperiod in the Antarctic sea ice algae Chlamydomonas sp. ICE-L.

Studies on ocean acidification have mostly been based on short-term experiments of low latitude with few investigations of the long-term influence on sea ice communities. Here, the combined effects of ocean acidification and photoperiod on the physiological response of the Antarctic sea ice microalgae Chlamydomonas sp. ICE-L were examined. There was a general increase in growth, PSII photosynthetic parameters, and N and P uptake in continuous light, compared to those exposed to regular dark and light cycles. Elevated pCO2 showed no consistent effect on growth rate (p=0.8) and N uptake (p=0.38) during exponential phrase, depending on the photoperiod but had a positive effect on PSII photosynthetic capacity and P uptake. Continuous dark reduced growth, photosynthesis, and nutrient uptake. Moreover, intracellular lipid, mainly in the form of PUFA, was consumed at 80% and 63% in low and high pCO2 in darkness. However, long-term culture under high pCO2 gave a more significant inhibition of growth and Fv/Fm to high light stress. In summary, ocean acidification may have significant effects on Chlamydomonas sp. ICE-L survival in polar winter. The current study contributes to an understanding of how a sea ice algae-based community may respond to global climate change at high latitudes.

Analysis of ΔpH and the xanthophyll cycle in NPQ of the Antarctic sea ice alga Chlamydomonas sp. ICE-L.

Non-photochemical fluorescence quenching (NPQ) is mainly associated with the transthylakoid proton gradient (ΔpH) and xanthophyll cycle. However, the exact mechanism of NPQ is different in different oxygenic photosynthetic organisms. In this study, several inhibitors were used to study NPQ kinetics in the sea ice alga Chlamydomonas sp. ICE-L and to determine the functions of ΔpH and the xanthophyll cycle in the NPQ process. NH4Cl and nigericin, uncouplers of ΔpH, inhibited NPQ completely and zeaxanthin (Z) was not detected in 1 mM NH4Cl-treated samples. Moreover, Z and NPQ were increased in the samples containing N,N'-dicyclohexyl-carbodiimide (DCCD) under low light conditions. We conclude that ΔpH plays a major role in NPQ, and activation of the xanthophyll cycle is related to ΔpH. In dithiothreitol (DTT)-treated samples, no Z was observed and NPQ decreased. NPQ was completely inhibited when NH4Cl was added suggesting that part of the NPQ process is related to the xanthophyll cycle and the remainder depends on ΔpH. Moreover, lutein and ß-carotene were also essential for NPQ. These results indicate that NPQ in the sea ice alga Chlamydomonas sp. ICE-L is mainly dependent on ΔpH which affects the protonation of PSII proteins and de-epoxidation of the xanthophyll cycle, and the transthylakoid proton gradient alone can induce NPQ.

De novo sequencing and analysis of the Ulva linza transcriptome to discover putative mechanisms associated with its successful colonization of coastal ecosystems.

BACKGROUND: The green algal genus Ulva Linnaeus (Ulvaceae, Ulvales, Chlorophyta) is well known for its wide distribution in marine, freshwater, and brackish environments throughout the world. The Ulva species are also highly tolerant of variations in salinity, temperature, and irradiance and are the main cause of green tides, which can have deleterious ecological effects. However, limited genomic information is currently available in this non-model and ecologically important species. Ulva linza is a species that inhabits bedrock in the mid to low intertidal zone, and it is a major contributor to biofouling. Here, we presented the global characterization of the U. linza transcriptome using the Roche GS FLX Titanium platform, with the aim of uncovering the genomic mechanisms underlying rapid and successful colonization of the coastal ecosystems. RESULTS: De novo assembly of 382,884 reads generated 13,426 contigs with an average length of 1,000 bases. Contiguous sequences were further assembled into 10,784 isotigs with an average length of 1,515 bases. A total of 304,101 reads were nominally identified by BLAST; 4,368 isotigs were functionally annotated with 13,550 GO terms, and 2,404 isotigs having enzyme commission (EC) numbers were assigned to 262 KEGG pathways. When compared with four other full sequenced green algae, 3,457 unique isotigs were found in U. linza and 18 conserved in land plants. In addition, a specific photoprotective mechanism based on both LhcSR and PsbS proteins and a C4-like carbon-concentrating mechanism were found, which may help U. linza survive stress conditions. At least 19 transporters for essential inorganic nutrients (i.e., nitrogen, phosphorus, and sulphur) were responsible for its ability to take up inorganic nutrients, and at least 25 eukaryotic cytochrome P450s, which is a higher number than that found in other algae, may be related to their strong allelopathy. Multi-origination of the stress related proteins, such as glutamate dehydrogenase, superoxide dismutases, ascorbate peroxidase, catalase and heat-shock proteins, may also contribute to colonization of U. linza under stress conditions. CONCLUSIONS: The transcriptome of U. linza uncovers some potential genomic mechanisms that might explain its ability to rapidly and successfully colonize coastal ecosystems, including the land-specific genes; special photoprotective mechanism based on both LhcSR and PsbS; development of C4-like carbon-concentrating mechanisms; muti-origin transporters for essential inorganic nutrients; multiple and complex P450s; and glutamate dehydrogenase, superoxide dismutases, ascorbate peroxidase, catalase, and heat-shock proteins that are related to stress resistance.

Evaluation of putative internal reference genes for gene expression normalization in Nannochloropsis sp. by quantitative real-time RT-PCR.

Quantitative real-time reverse transcription PCR (RT-qPCR), a sensitive technique for quantifying gene expression, depends on the stability of the reference gene(s) used for data normalization. To date, few studies on reference genes have been undertaken for Nannochloropsis sp. In this study, 12 potential reference genes were evaluated for their expression stability using the geNorm and NormFinder statistical algorithms by RT-qPCR. The results showed that the best reference genes differed depending on the treatments: different light intensities (DL), the diurnal cycle (DC), high light intensity (HL) and low temperature treatments (LT). A combination of ACT1, ACT2 and TUA would be appropriate as a reference panel for normalizing gene expression data across all the treatments. ACT2 showed the most stable expression across all tested samples but was not the most stable one for individual treatments. Though 18S showed the least stable expression considering all tested samples, it is the most stable one for LT using geNorm. The expression of Lhc confirmed that the appropriate reference genes are crucial. These results provide a foundation for more accurate use of RT-qPCR under different experimental conditions in Nannochloropsis sp. gene analysis.

The validity of a reference gene is highly dependent on the experimental conditions in green alga Ulva linza.

Normalization based on inappropriate reference gene may lead to the reduction of the accuracy of RT-qPCR. Although determination of suitable reference genes is essential to RT-qPCR studies, reports on the evaluation of reference genes in Ulva linza, a ubiquitous green-tide forming alga, are lacking. The expression levels of ten candidate reference genes were analyzed in U. linza across different experimental treatments, and the best-ranked reference genes differed across the treatments. The most suitable reference genes were tubulin2 (TUB2) among different salinity and UV treatments. Histone 2 (H2) was stably expressed in different temperature and desiccation stress treatments. 18S rRNA exhibited better expression stability in different light intensity treatments. While all tested samples were considered, none of single gene was widely applicable as a reference gene. Moreover, using a combination of two genes as reference genes might improve the reliability of gene expression by RT-qPCR, and the combination of TUB1 and TUB2 was selected as ideal for all tested samples. The results suggest that assessing the stability of reference gene expression patterns, determining candidates, and testing their suitability are required for each experimental investigation. The results will guide the selection of reference genes for gene expression studies in U. linza.

Cloning and expression analysis of two different LhcSR genes involved in stress adaptation in an Antarctic microalga, Chlamydomonas sp. ICE-L.

Light-harvesting complexes (LHCs) play essential roles in light capture and photoprotection. Although the functional diversity of individual LHCs in many plants has been well described, knowledge regarding the extent of this family in the majority of green algal groups is still limited. In this study, two different LhcSR genes, LhcSR1 and LhcSR2 from Chlamydomonas sp. ICE-L, were cloned from the total cDNA and characterized in response to high light (HL), low light (LL), UV-B radiation and high salinity. The lower F (v)/F (m) as well as the associated induction of non-photochemical quenching (NPQ), observed under those conditions, indicated that Chlamydomonas sp. ICE-L was under stress. Under HL stress, the expression of LhcSR1 and LhcSR2 increased rapidly from 0.5 h HL and reached a maximum after 3 h. In LL, LhcSR2 expression was up-regulated during the first 0.5 h after which it decreased, while the expression of LhcSR1 decreased gradually from the beginning of the experiment. In addition, the transcript levels of LhcSR1 and LhcSR2 increased under UV-B radiation and high salinity. These results showed that both genes were inducible and up-regulated under stress conditions. A higher NPQ was accompanied by the up-regulated LhcSR genes, suggesting that LhcSR plays a role in thermal energy dissipation. Overall, the results presented here suggest that LhcSR1 and LhcSR2 play a primary role in photoprotection in Chlamydomonas sp. ICE-L under stress conditions and provide an important basis for investigation of the adaptation mechanism of LhcSR in Antarctic green algae.

Insight Into the Pico- and Nano-Phytoplankton Communities in the Deepest Biosphere, the Mariana Trench.

As photoautotrophs, phytoplankton are generally present in the euphotic zone of the ocean, however, recently healthy phytoplankton cells were found to be also ubiquitous in the dark deep sea, i.e., at water depths between 2000 and 4000 m. The distributions of phytoplankton communities in much deeper waters, such as the hadal zone, are unclear. In this study, the vertical distribution of the pico- and nano-phytoplankton (PN) communities from the surface to 8320 m, including the epipelagic, mesopelagic, bathypelagic, and hadal zones, were investigated via both 18S and p23S rRNA gene analysis in the Challenger Deep of the Mariana Trench. The results showed that Dinoflagellata, Chrysophyceae, Haptophyta, Chlorophyta, Prochloraceae, Pseudanabaenaceae, Synechococcaceae, and Eustigmatophyceae, etc., were the predominant PN in the Mariana Trench. Redundancy analyses revealed that depth, followed by temperature, was the most important environmental factors correlated with vertical distribution of PN community. In the hadal zone, the PN community structure was considerably different from those in the shallower zones. Some PN communities, e.g., Eustigmatophyceae and Chrysophyceae, which have the heterotrophic characteristics, were sparse in shallower waters, while they were identified with high relative abundance (94.1% and 20.1%, respectively) at the depth of 8320 m. However, the dinoflagellates and Prochloraceae Prochlorococcus were detected throughout the entire water column. We proposed that vertical sinking, heterotrophic metabolism, and/or the transition to resting stage of phytoplankton might contribute to the presence of phytoplankton in the hadal zone. This study provided insight into the PN community in the Mariana Trench, implied the significance of phytoplankton in exporting organic matters from the euphotic to the hadal zone, and also hinted the possible existence of some undetermined energy metabolism (e.g., heterotrophy) of phytoplankton making themselves adapt and survive in the hadal environment.

Cyclobutane pyrimidine dimers photolyase from extremophilic microalga: remarkable UVB resistance and efficient DNA damage repair.

Bacteria living in the Antarctic region have developed several adaptive features for growth and survival under extreme conditions. Chlamydomonas sp. ICE-Lis well adapted to high levels of solar UV radiation. A putative photolyase was identified in the Chlamydomonas sp. ICE-L transcriptome. The complete cDNA sequence was obtained by RACE-PCR. This PHR encoding includes a polypeptide of 579 amino acids with clear photolyase signatures belonging to class II CPD-photolyases, sharing a high degree of homology with Chlamydomonas reinhardtii (68%). Real-time PCR was performed to investigate the potential DNA damage and responses following UVB exposure. CPD photolyase mRNA expression level increased over 50-fold in response to UVB radiation for 6h. Using photolyase complementation assay, we demonstrated that DNA photolyase increased photo-repair more than 116-fold in Escherichia coli strain SY2 under 100µw/cm(2) UVB radiation. To determine whether photolyase is active in vitro, CPD photolyase was over-expressed. It was shown that pyrimidine dimers were split by the action of PHR2. This study reports the unique structure and high activity of the enzyme. These findings are relevant for further understanding of molecular mechanisms of photo-reactivation, and will accelerate the utilization of photolyase in the medical field.

Occurrence of the PsbS and LhcSR products in the green alga Ulva linza and their correlation with excitation pressure.

To avoid photoinhibition, plants have developed diverse photoprotection mechanisms. One of the short-term high light protection mechanisms in plants is non-photochemical quenching (NPQ), which dissipates the absorbed light energy as thermal energy. In the green alga, Ulva linza, the kinetics of NPQ starts with an initial, quick rise followed by a decline, and then a second and higher rise at longer time periods. During the whole phase, NPQ is triggered and controlled by ΔpH, then strengthened and modulated by zeaxanthin. Light-harvesting complex (LHC) family members are known to play crucial roles in this mechanism. The PSBS protein, a member of the LHC family that was thought to be present exclusively in higher plants, has been identified for the first time in U. linza. The expression of both PSBS and LHCSR was up-regulated during high light conditions, and LHCSR increased more than PSBS. Both LHCSR and PSBS-dependent NPQ may be important strategies for adapting to the environment, and they have undoubtedly played a role in their evolution.

Expression of fatty acid desaturase genes and fatty acid accumulation in Chlamydomonas sp. ICE-L under salt stress.

The Antarctic ice microalgae Chlamydomonas sp. ICE-L which is highly resistant to salt stress holds promise in providing an alternative species for the production of microalgal oil. We studied the effects of the alga in confrontation with NaCl stress on the growth, oil yield and expression of fatty acid desaturase genes. The growth rate of Chlamydomonas sp. ICE-L decreased with the gradual increase in NaCl concentration. Interestingly, we found that the highest lipid content was achieved at 16‰ NaCl, reaching 23% (w/w). Meanwhile, the expression of Δ9ACPCiFAD increased rapidly while Δ12CiFAD, ω3CiFAD2 and Δ6CiFAD showed a delayed elevation in response to altered salt stress. C18:3 was the dominant PUFA, which account for about 75% TFA in Chlamydomonas sp. ICE-L. Under 96‰ and 128‰ NaCl stress, the content of C20:5 almost approached that of C18:3. In contrast, low salinity enhanced the dominance of C18:3 at the expense of C20:3 and C20:5.

Temperature regulates fatty acid desaturases at a transcriptional level and modulates the fatty acid profile in the Antarctic microalga Chlamydomonas sp. ICE-L.

Chlamydomonas sp. ICE-L which can thrive in extreme environments of the Antarctic is a major biomass producer. The FAD genes in Chlamydomonas sp. ICE-L were obtained and sequence alignment showed that these genes are homologous to known FADs with conserved histidine motifs. In this study, we analyzed the transcription of five FADs and FA compositions at different temperatures. The results showed that the expressions of Δ9CiFAD, ω3CiFAD1 and ω3CiFAD2 were apparently up-regulated at 0°C, however, the up-regulation of Δ6CiFAD intensified with rising temperature. Meanwhile, analysis of the FA compositions showed that PUFAs were dominant compositions, accounting for more than 75% TFA in Chlamydomonas sp. ICE-L. Furthermore, PUFAs were significantly increased at 0 and 5°C, which may be attributed to higher proportions of C18:3 and C20:3. Moreover, PUFAs were significantly decreased at 15°C whereas SFAs were significantly increased.

Detection and quantitation of lipid in the microalga Tetraselmis subcordiformis (Wille) Butcher with BODIPY 505/515 staining.

BODIPY 505/515, a lipophilic bright green fluorescent dye was tested for lipid detection in the microalga Tetraselmis subcordiformis. A concentration of 0.28 µg ml(-1) and staining for 6 min was optimal. Lipid bodies stained with BODIPY505/515 had a characteristic green fluorescence. Their volumes were determined using the sphere volume formula. Lipid accumulation under different nitrogen concentrations was analyzed. With an increase in NaNO(3) concentration from 0 to 240 mg L(-1), the maximum algal concentration increased from 8.23 ± 0.62 (× 10(5) cells ml(-1)) to 1.61 ± 0.13 (×10(6) cells ml(-1)), while the maximum volume of intracellular neutral lipid decreased from 9.78 ± 1.77 µm(3) cell(-1) to 6.00 ± 0.59 µm(3) cell(-1). A comparison of the lipid contents measured by BODIPY 505/515 staining and the gravimetric method showed a positive correlation coefficient of R(2) = 0.93. BODIPY 505/515 staining is a promising method in lipid quantitation in T. subcordiformis.

A transthylakoid proton gradient and inhibitors induce a non-photochemical fluorescence quenching in unicellular algae Nannochloropsis sp.

Non-photochemical quenching (NPQ) of chlorophyll fluorescence is thought to be an indicator of an essential regulation and photoprotection mechanism against high-light stress in photosynthetic organisms. In this report, special chemicals were used to perturb the kinetics of the ΔpH build-up and the xanthophyll cycle (XC) in Nannochloropsis sp. We found that NPQ was stimulated rapidly on exposure to high light and relaxed rapidly in darkness. The ΔpH could be obligatory for NPQ and ΔpH alone was not sufficient to induce NPQ. The XC, being strictly mediated by ΔpH, was also essential for NPQ. The results demonstrate that the mechanism of NPQ in Nannochloropsis sp. resembled that of diatoms.

Complete mitochondrial genome of the sea slug, Placida sp., contains unusual noncoding regions.

The complete sequence (14,751 bp) of the mitochondrial DNA (mtDNA) of the opisthobranch gastropod Placida sp. was determined using long PCR and genome-walking techniques. The genome contains 13 protein-coding genes (PCG), 2 ribosomal RNA (rRNA), 22 transfer RNA (tRNA) genes that are typical of metazoan mtDNA, and 2 lengthy noncoding regions with a total length of 1441 bp. All the tRNA genes have general secondary structures without reduced T or D stems. The mitochondrial gene arrangement of Placida sp. is almost identical to that of sea slugs, whereas the tRNA gene arrangement is different from other animals outside Sacoglossa in the Euthyneura. A phylogenetic analysis based on the mitochondrial genome of Euthyneura indicated that Placida sp. and Placida dendritica are closely related to Elysia chlorotica as a sister taxon, which strongly supports their close phylogenetic affinity.

Activities of principal photosynthetic enzymes in green macroalga Ulva linza: functional implication of C4 pathway in CO2 assimilation.

The green-tide-forming macroalga Ulva linza was profiled by transcriptome sequencing to ascertain whether the alga carries both C3 and C4 photosynthesis genes. The key enzymes involved in C4 metabolism including pyruvate orthophosphate dikinase (PPDK), phosphoenolpyruvate carboxylase (PEPC), and phosphoenolpyruvate carboxykinase (PCK) were found. When measured under normal and different stress conditions, expression of rbcL was higher under normal conditions and lower under the adverse conditions, whereas that of PPDK was higher under some adverse conditions, namely desiccation, high salinity, and low salinity. Both ribulose-1, 5-biphosphate carboxylase (RuBPCase) and PPDK were found to play a role in carbon fixation, with significantly higher PPDK activity across the stress conditions. These results suggest that elevated PPDK activity alters carbon metabolism in U. linza leading to partial operation of the C4 carbon metabolism, a pathway that, under stress conditions, probably contributes to the hardy character of U. linza and thus to its wide distribution.

Evidence of coexistence of C3 and C4 photosynthetic pathways in a green-tide-forming alga, Ulva prolifera.

Ulva prolifera, a typical green-tide-forming alga, can accumulate a large biomass in a relatively short time period, suggesting that photosynthesis in this organism, particularly its carbon fixation pathway, must be very efficient. Green algae are known to generally perform C3 photosynthesis, but recent metabolic labeling and genome sequencing data suggest that they may also perform C4 photosynthesis, so C4 photosynthesis might be more wide-spread than previously anticipated. Both C3 and C4 photosynthesis genes were found in U. prolifera by transcriptome sequencing. We also discovered the key enzymes of C4 metabolism based on functional analysis, such as pyruvate orthophosphate dikinase (PPDK), phosphoenolpyruvate carboxylase (PEPC), and phosphoenolpyruvate carboxykinase (PCK). To investigate whether the alga operates a C4-like pathway, the expression of rbcL and PPDK and their enzyme activities were measured under various forms and intensities of stress (differing levels of salinity, light intensity, and temperature). The expression of rbcL and PPDK and their enzyme activities were higher under adverse circumstances. However, under conditions of desiccation, the expression of rbcL and ribulose-1, 5-biphosphate carboxylase (RuBPCase) activity was lower, whereas that of PPDK was higher. These results suggest that elevated PPDK activity may alter carbon metabolism and lead to a partial operation of C4-type carbon metabolism in U. prolifera, probably contributing to its wide distribution and massive, repeated blooms in the Yellow Sea.