Role of C4 carbon fixation in Ulva prolifera, the macroalga responsible for the world's largest green tides.

Most marine algae preferentially assimilate CO2 via the Calvin-Benson Cycle (C3) and catalyze HCO3- dehydration via carbonic anhydrase (CA) as a CO2-compensatory mechanism, but certain species utilize the Hatch-Slack Cycle (C4) to enhance photosynthesis. The occurrence and importance of the C4 pathway remains uncertain, however. Here, we demonstrate that carbon fixation in Ulva prolifera, a species responsible for massive green tides, involves a combination of C3 and C4 pathways, and a CA-supported HCO3- mechanism. Analysis of CA and key C3 and C4 enzymes, and subsequent analysis of δ13C photosynthetic products showed that the species assimilates CO2 predominately via the C3 pathway, uses HCO3- via the CA mechanism at low CO2 levels, and takes advantage of high irradiance using the C4 pathway. This active and multi-faceted carbon acquisition strategy is advantageous for the formation of massive blooms, as thick floating mats are subject to intense surface irradiance and CO2 limitation.

Fourfold daily growth rate in multicellular marine alga Ulva meridionalis.

Microalgae with high growth rates have been considered as promising organisms to replace fossil resources with contemporary primary production as a renewable source. However, their microscopic size makes it hard to be harvested for industrial applications. In this regard, multicellular macroalgae are more suitable for harvesting. Here, we show that Ulva meridionalis has the highest growth rate ever reported for a multicellular autotrophic plant. Contrasted to the known bloom-forming species U. prolifera growing at an approximately two-fold growth rate per day in optimum conditions, U. meridionalis grows at a daily rate of over fourfold. The high growth ability of this multicellular alga would provide the most effective method for CO2 fixation and biomass production.

Differential Photosynthetic Response of a Green Tide Alga Ulva linza to Ultraviolet Radiation, Under Short- and Long-term Ocean Acidification Regimes.

Both ocean acidification (OA) and solar ultraviolet (UV) radiation can bring about changes in macroalgal physiological performance. However, macroalgal responses to UV radiation when acclimatized to OA under different time scales are rare. Here, we investigate the response of Ulva linza, a green tide alga, to UV radiation in the form of photosynthetically active radiation (PAR) or PAB (PAR+UVA+UVB) radiation. Radiation exposures were assessed following long-term (from spore to adult stage, 1 month) and short-term (adult stage, 1 week) OA treatments. Results showed that increased CO2 decreased the damage rate (k) and repair rate (r) of thalli grown under short-term OA conditions with PAB treatment, the ratio of r:k was not altered. Following long-term OA conditions, r was not affected, although k was increased in thalli following PAB treatment, resulting in a reduced ratio of r:k. The relative level of UV inhibition increased and UV-absorbing compounds decreased when algae were cultured under long-term OA conditions. The recovery rate of thalli was enhanced when grown under long-term OA after UV radiation treatment. These results show that blooming algae may be more sensitive to UV radiation in marine environments, but it can develop effective mechanisms to offset the negative effects, reflecting acclimation to long-term OA conditions.

UV-A screening in Cladophora sp. lowers internal UV-A availability and photoreactivation as compared to non-UV screening in Ulva intestinalis.

In the Baltic Sea, two co-occurring green macroalgae Cladophora sp. and Ulva intestinalis grow in the upper eulittoral. Due to regular and high sunlight exposure in their habitat, both species need resistance mechanisms to protect themselves against ultraviolet-B (UV-B)-induced DNA damage. While Cladophora sp. possesses efficient screening of UV-B and ultraviolet-A (UV-A) radiation, U. intestinalis was recently shown to have higher DNA repair by UVA-driven photoreactivation than Cladophora sp. [F. Pescheck and W. Bilger, Mar. Biol., 2018, 165, 132]. In the present study, the hypothesis that the screening of UV-A radiation limits internal UV-A availability for photoreactivation in Cladophora sp. was tested. Both species had identical and much lower fractions of damaged DNA when sampled in situ under direct sunlight as expected based on a photophysical prediction. To quantify the effect of UV-A screening spectrally and physiologically, in vivo UV screening spectra were determined and the UV-A photon flux dependency of photoreactivation was investigated for both species. Identical intrinsic photoreactivation rates were revealed by the applied correction for internal UV-A photon flux density and under irradiation with visible radiation which is not screened by the UV absorbing compounds in Cladophora sp. Natural sunlight was weighted with in vivo action spectra for DNA damage induction and light-dependent repair. The resulting spectrum was further corrected for the apparent UV screening spectra of both species to calculate the species-specific internal ratios of DNA damaging and photoreactivating photons. This photophysical modelling improves the understanding of UV damage and tolerance mechanisms in the two co-occurring green macroalgae under solar irradiation.

Variations of dominant free-floating Ulva species in the source area for the world's largest macroalgal blooms, China: Differences of ecological tolerance.

Species composition and seasonal variations of free-floating Ulva species were investigated in the source area of the world's largest macroalgal blooms during 2009-2015. Based on a combination of a morphological analysis and sequences of nuclear-encoded ITS and 5S rDNA spacer regions, the dominant species in the free-floating Ulva community at the early stage of green tides were Ulva compressa, Ulva flexuosa, and Ulva linza. The first appearance of Ulva prolifera on the sea surface was in mid-May and it dominated the floating Ulva community in June from 2009 to 2011. From 2012-2015, U. prolifera was not only the first species to appear on the sea surface but also the dominant species during the whole early stage of green tides. To explain the successional mechanism, the effects of environmental factors on the growth of four Ulva species were examined in the laboratory under different combinations of light intensity and temperature. It was found that the highest growth rate of U. prolifera was 44.9%/d, which was much higher than the other three Ulva species. The strong tolerance of U. prolifera to extreme conditions also helps it survive and bloom in the Yellow Sea.

Exergy efficiency of light conversion into biomass in the macroalga Ulva sp. (Chlorophyta) cultivated under the pulsed light in a photobioreactor.

Marine macroalgae are a potential feedstock for biorefineries that can reduce dependence on fossil fuels and contribute to bioeconomy. New knowledge and technologies for efficient conversion of solar energy into macroalgae biomass are needed to increase biomass yields and energy conversion efficiency. In this work, we show that the green macroalgae from Ulva sp. can grow under the pulsed light in a photobioreactor with higher exergy conversion efficiency in comparison to cultivation under constant light with the same intensity. In the tested frequencies, 1-40 Hz and duty cycles (DC) 1-100%, DC has a stronger impact on the growth rate than frequency. The efficiency of light transformation into biomass increased with decreasing DC. Pulsating with DC 20% led to 60% of the biomass chemical energy yield for the respective constant light (DC 100%). Models of Ulva sp. growth rate and exergy conversion efficiency as a function of pulsating light parameters were developed. These results open new directions to enhance solar to chemical energy conversion through macroalgae by controlling the light distribution in the macroalgal biomass.

Growth, ammonium metabolism, and photosynthetic properties of Ulva australis (Chlorophyta) under decreasing pH and ammonium enrichment.

The responses of macroalgae to ocean acidification could be altered by availability of macronutrients, such as ammonium (NH4+). This study determined how the opportunistic macroalga, Ulva australis responded to simultaneous changes in decreasing pH and NH4+ enrichment. This was investigated in a week-long growth experiment across a range of predicted future pHs with ambient and enriched NH4+ treatments followed by measurements of relative growth rates (RGR), NH4+ uptake rates and pools, total chlorophyll, and tissue carbon and nitrogen content. Rapid light curves (RLCs) were used to measure the maximum relative electron transport rate (rETRmax) and maximum quantum yield of photosystem II (PSII) photochemistry (Fv/Fm). Photosynthetic capacity was derived from the RLCs and included the efficiency of light harvesting (α), slope of photoinhibition (ß), and the light saturation point (Ek). The results showed that NH4+ enrichment did not modify the effects of pH on RGRs, NH4+ uptake rates and pools, total chlorophyll, rETRmax, α, ß, Fv/Fm, tissue C and N, and the C:N ratio. However, Ek was differentially affected by pH under different NH4+ treatments. Ek increased with decreasing pH in the ambient NH4+ treatment, but not in the enriched NH4+ treatment. NH4+ enrichment increased RGRs, NH4+ pools, total chlorophyll, rETRmax, α, ß, Fv/Fm, and tissue N, and decreased NH4+ uptake rates and the C:N ratio. Decreased pH increased total chlorophyll content, rETRmax, Fv/Fm, and tissue N content, and decreased the C:N ratio. Therefore, the results indicate that U. australis growth is increased with NH4+ enrichment and not with decreasing pH. While decreasing pH influenced the carbon and nitrogen metabolisms of U. australis, it did not result in changes in growth.

Integration of deep transcriptome and proteome analyses of salicylic acid regulation high temperature stress in Ulva prolifera.

To investigate changes in transcript and relative protein levels in response to salicylic acid regulation of the thermotolerance in U. prolifera, complementary transcriptome and proteome analyses were performed with U. prolifera grown at 35 °C (UpHT) and with the addition of SA at high temperature (UpSHT). At mRNA level,12,296 differentially expressed genes (DEGs) were obtained from the comparison of UpSHT with UpHT. iTRAQ-labeling proteome analysis showed that a total of 4,449 proteins were identified and reliably quantified. At mRNA level, the up-regulated genes involved in antioxidant activity were thioredoxin,peroxiredoxin,FeSOD, glutathione peroxidase, partion catalase and MnSOD. The down-regulated genes were ascorbate peroxidase, glutathione S-transferase, catalase and MnSOD. In addition, the DEGs involved in plant signal transduction pathway (such as auxin response factors, BRI1 and JAZ) were down-regulated. At protein level, the up-regulated proteins involved in carbon fixation and the down-regulated protein mainly were polyubiquitin, ascorbate peroxidase. The expression of Ca2+-binding protein, heat shock protein and photosynthesis-related proteins, EDS1 were also significantly regulated both at mRNA and protein level. The results indicated that SA alleviated the high-temperature stimulus through partion antioxidant related proteins up-regulated, JA signal pathway enchanced, Ca2+-binding proteins, photosynthesis-related proteins significantly changed, antioxidant enzyme activities increased and photosynthesis index changed.

An Ocean Acidification Acclimatised Green Tide Alga Is Robust to Changes of Seawater Carbon Chemistry but Vulnerable to Light Stress.

Ulva is the dominant genus in the green tide events and is considered to have efficient CO2 concentrating mechanisms (CCMs). However, little is understood regarding the impacts of ocean acidification on the CCMs of Ulva and the consequences of thalli's acclimation to ocean acidification in terms of responding to environmental factors. Here, we grew a cosmopolitan green alga, Ulva linza at ambient (LC) and elevated (HC) CO2 levels and investigated the alteration of CCMs in U. linza grown at HC and its responses to the changed seawater carbon chemistry and light intensity. The inhibitors experiment for photosynthetic inorganic carbon utilization demonstrated that acidic compartments, extracellular carbonic anhydrase (CA) and intracellular CA worked together in the thalli grown at LC and the acquisition of exogenous carbon source in the thalli could be attributed to the collaboration of acidic compartments and extracellular CA. Contrastingly, when U. linza was grown at HC, extracellular CA was completely inhibited, acidic compartments and intracellular CA were also down-regulated to different extents and thus the acquisition of exogenous carbon source solely relied on acidic compartments. The down-regulated CCMs in U. linza did not affect its responses to changes of seawater carbon chemistry but led to a decrease of net photosynthetic rate when thalli were exposed to increased light intensity. This decrease could be attributed to photodamage caused by the combination of the saved energy due to the down-regulated CCMs and high light intensity. Our findings suggest future ocean acidification might impose depressing effects on green tide events when combined with increased light exposure.

Photosynthetic adaptation strategy of Ulva prolifera floating on the sea surface to environmental changes.

For 8 consecutive years, a green tide has originated in the southern Yellow Sea and spread to the Qingdao offshore area. The causative species, Ulva prolifera, always forms a very thick thallus mat that is capable of drifting long distances over long periods. During this process, although the thalli face disturbance by complex environmental factors, they maintain high biomass and proliferation. We hypothesized that some form of photosynthetic adaptation strategy must exist to protect the thalli. Therefore, we studied the different photosynthetic response characteristics of the surface and lower layers of the floating thallus mats, and investigated the physiological and molecular-level adaptation mechanisms. The results showed that: (1) U. prolifera has strong photosynthetic capability that ensures it can gain sufficient energy to increase its biomass and adapt to long-distance migration. (2) Surface layer thalli adapt to the complex environment by dissipating excess energy via photosynthetic quantum control (energy quenching and energy redistribution between PSII/PSI) to avoid irreversible damage to the photosynthetic system. (3) Lower layer thalli increase their contents of Chlorophyll a (Chl a) and Chlorophyll b (Chl b) and decrease their Chl a/Chl b ratio to improve their ability to use light energy. (4) U. prolifera has strong photosynthetic plasticity and can adapt to frequent exchange between the surface and lower layer environments because of wave disturbance. Pigment component changes, energy quenching, and energy redistribution between PSII/PSI contribute to this photosynthetic plasticity.

Evaluation of ultrasonic, acid, thermo-alkaline and enzymatic pre-treatments on anaerobic digestion of Ulva rigida for biogas production.

Pre-treatment of macroalgae has received considerable research globally due to its influence on the technical, economic and environmental sustainability of algae biogas production. Some of the most promising pre-treatment methods require the application of chemicals, enzymatic, and mechanical. This study focused on these pre-treatments of Ulva rigida for biogas production. The evaluation of different pre-treatment in terms of reducing sugar yields demonstrates that 3.62, 2.88, 2.53 and 7.3g/L of reducing sugar was obtained in acid catalysis, thermoalkaline, ultrasonication and enzymatic pre-treatment, respectively. However in crude macroalgae only 0.6g/L of reducing sugar was given. After anaerobic digestion, the enzymatic hydrolysis was demonstrated the best biogas yield than other pre-treatment which reached 626.5mL/gCODint with 62.65% of biodegradability. The best demonstrated method which uses crude broth of Aspergillus niger showed an effective and environmentally friendly strategy for enhancing the biogas production yields after the anaerobic digestion.

Vulnerability and acclimation to increased UVB radiation in three intertidal macroalgae of different morpho-functional groups.

The vulnerability and acclimation to increased UVB radiation in three macroalgae of different morpho-functional groups collected in the Mediterranean coastal waters were evaluated. The algae were submitted for 7 days to increased (PAB+) and decreased (PAB-) UVB radiation. The thickness and morphology influenced the response to increased UVB radiation, being Cystoseira tamariscifolia the less vulnerable algae followed by Ellisolandia elongata. The highest resistance to increased UVB radiation in C. tamariscifolia was related to the accumulation of polyphenols and high antioxidant activity, whereas E. elongata was due to its high reflectance. Finally, Ulva rigida suffered the highest photoinhibition under PAB+ culture. The latest species presented 10 times lower polyphenol content and antioxidant activity than C. tamariscifolia. The three species showed different acclimation patterns to the changes of UVB radiation related to the morphology, photosynthetic activity, accumulation of photoprotectors and antioxidant activities. The ecological implications of the UVB variations on macroalgae are discussed.

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.

Photoprotection in the green tidal alga Ulva prolifera: role of LHCSR and PsbS proteins in response to high light stress.

Ulva prolifera, an intertidal macroalga, has to adapt to wide variations in light intensity, making this species particularly rewarding for studying the evolution of photoprotective mechanisms. Intense light induced increased non-photochemical quenching (NPQ) and stimulated de-epoxidation of xanthophyll cycle components, while DTT-treated samples had lower NPQ capacity, indicating that the xanthophyll cycle must participate in photoprotection. In this work, we found that the PsbS-related NPQ was maintained in U. prolifera. According to analysed gene expression, both LhcSR and psbS were up-regulated in high light, suggesting that these two genes are light-induced. LHCSR and PsbS proteins were present at different light intensities and accumulated under high light conditions, and PsbS concentrations were higher than LHCSR, showing that the NPQ mechanism of U. prolifera is more dependent on PsbS protein concentration. Moreover, the level of both LHCSR and PsbS proteins was high even in the darkness, and neither the transcript level nor protein content of LhcSR and psbS genes varied significantly following short-term exposure to intense light. These findings suggest that this alga can modulate NPQ levels through regulation of the xanthophyll cycle and concentrations of PsbS and/or LHCSR.

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.

Differential gene expression in Ulva prolifera under low light and low temperature conditions.

The past several years witnessed the increasing global interest in the marine green macroalga Ulva prolifera as it is a key causative species of the massive green tides successively occurring in the Yellow Sea. Accurate localization of the 'seed' source is one of the principal scientific concerns to be solved before it is possible to manage these algal blooms. It has been suggested that somatic cells of Ulva prolifera which settled in cold benthic sediments might serve as one of the major propagule banks. To identify the molecular mechanisms underlying this hypothesis, PCR-based suppression subtractive hybridization was employed to analyze the differential gene expression of Ulva prolifera under low light and low temperature conditions (matching the cold benthic sediments conditions, 6 °C, 30 µmol photons m(-2) s(-1)). 137 ESTs representing 88 unigenes (80 singletons and 8 contigs) were detected as being over-expressed, whereas 109 unigenes (96 singletons and 13 contigs) in 130 ESTs were found to be down-regulated in this study. BLASTX analysis revealed that 65 % of the over-expressed and 59 % of the down-regulated genes did not belong to any documented functionally annotated or hypothetical proteins in the public database. However, analysis of the functional defined sequences displayed (1) an obvious sign of senescence, (2) enhancements of the photosynthesis system and the pentose phosphate pathway, (3) slow-down of activities in a wide range of processes including the DNA replication, the transcription, the translation, the glycolysis, the citrate cycle and the pyruvate metabolism in Ulva prolifera cells under low light and low temperature conditions. This work disclosed some basic information of the molecular mechanisms of Ulva prolifera cells under low light and low temperature conditions and provides useful clues for future studies on the "seed" source of the massive green tides.

Recruitment potential of a green alga Ulva flexuosa Wulfen dark preserved zoospore and its development.

The recruitment potential and the ability of Ulva flexuosa Wulfen zoospores to survive darkness were tested under different conditions in the present study. The dark preserved zoospore was cultured under a two-factor experimental design to test the effect of salinity and nitrate, effect of salinity and phosphate, effect of light and salinity, and effect of light and phosphate. The recruitment (germination and growth) of zoospores was significantly affected by light and salinity. The nitrate concentration of 20 µmol.l(-1) was found to initiate the process of germination and its subsequent growth and, its effect appeared greatest under 25 psu condition. While nitrate enhances the growth of biomass more than phosphate, both show a positive interactive effect on biomass increase when crossed with salinity. The combined effect of 25 psu salinity and 8 µmol.l(-1) phosphate exhibited higher biomass growth. There was a significant effect of light and salinity on the biomass of zoospore, though there was no significant interaction between the two factors. There was an increase in biomass of growing zoospores to increase in light intensity and 80 µmol.m(-2).s(-1) of light intensity was considered optimal. Similarly, high light intensity condition favored higher biomass growth and there was significant interaction between light (80 µmol. m(-2). s(-1)) and phosphate (4 µmol. l(-1)) in high salinity (35 psu) condition. The result of this study showed that dark preserved zoospores of U. flexuosa have the potential for recruitment and it gives us an understanding how different factors play a role in the process of recruitment.

Nitric oxide up-regulates the expression of methionine sulfoxide reductase genes in the intertidal macroalga Ulva fasciata for high light acclimation.

Nitric oxide (NO) has emerged as a fundamental signal molecule involved in the responses of plant to stress. A role for NO in the regulation of methionine sulfoxide reductase (MSR) mRNA expression and high light acclimation was studied in a green macroalga Ulva fasciata Delile. Transfer from darkness to high light (≥1,200 µmol photons m(-2) s(-1)) inhibited photosynthesis and growth but increased NO production and UfMSRA and UfMSRB transcripts. Treatment with an NO scavenger, 2-(4-carboxy- phenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (cPTIO), at 1,200 µmol photons m(-2) s(-1) caused a further growth inhibition accompanied by an inhibition of the increase of UfMSRA and UfMSRB transcripts by high light, while treatment with an NO generator, sodium nitroprusside (SNP), alleviated the growth inhibition and enhanced UfMSRA and UfMSRB expression. Exposure to moderate light (300 µmol photons m(-2) s(-1)) conditions also increased UfMSRA and UfMSRB transcripts, which were not affected by cPTIO treatment but were enhanced by SNP treatment. So, NO does not mediate the up-regulation of UfMSR genes by transfer to moderate light possibly as a precautionary mechanism in the sense of increasing light intensities in the daytime. In conclusion, NO production can be induced in U. fasciata upon exposure to high light for up-regulation of UfMSRA and UfMSRB expression but the level of NO production is not sufficient for acquisition of full tolerance to high light stress. Enhanced NO production by an exogenously applied NO generator can effectively trigger the high light acclimation process, including UfMSRA and UfMSRB expression.

Modulation of gene expression of carotene biosynthesis-related protein by photosynthetic electron transport for the acclimation of intertidal macroalga Ulva fasciata to hypersalinity and excess light.

A gene (UfCBR) encoding carotene biosynthesis-related (CBR) protein that potentially functions for the dissipation of excessive energy has been cloned from the intertidal green macroalga Ulva fasciata Delile. Hypersalinity and high light ≥300 µmol m(-2) s(-1) increased both UfCBR mRNA level and non-photochemical quenching (NPQ). The increase of UfCBR mRNA level and NPQ by high light was inhibited by treatment of photosynthetic electron transport inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea or 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, but not by stigmatellin, an inhibitor that blocks electron transfer from quinol oxidase to iron-sulfur protein in cytochrome b(6) f complex. Treatment of dimethylthiourea, an H(2) O(2) scavenger, under 1200 µmol m(-2) s(-1) condition inhibited H(2) O(2) accumulation but did not affect UfCBR mRNA level, while treatment of H(2) O(2) in 150 µmol m(-2) s(-1) condition decreased UfCBR mRNA level. Thus, an reactive oxygen species-independent redox control via a more reduced state downstream the cytochrome b(6) f complex is involved in high light up-regulation of UfCBR expression in U. fasciata. The expression of UfCBR in U. fasciata against oxidative stress occurring in high light or high salinity in relation to NPQ is discussed.

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.