Photosynthetic activity and proteomic analysis highlights the utilization of atmospheric CO2 by Ulva prolifera (Chlorophyta) for rapid growth

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.     

Restricted use of nitrate and a strong preference for ammonium reflects the nitrogen ecophysiology of a light‐limited red alga

Ammonium and nitrate are important sources of inorganic nitrogen for coastal primary producers. Nitrate has higher energy requirement for uptake and assimilation, compared with ammonium, suggesting that it might be a more efficient nitrogen source for slow‐growing, light‐limited macroalgae. To address this hypothesis, we examined the nitrogen ecophysiology of Anotrichium crinitum, a rhodophyte macroalgae common in low‐light habitats in New Zealand. We measured seasonal changes in seawater nitrate and ammonium concentrations and the concentration of nitrate and ammonium stored internally by A. crinitum. We determined the maximal uptake rates of nitrate and ammonium seasonally and grew A. crinitum in the laboratory with these nitrogen sources under two ecologically relevant saturating light levels. Our results show that field‐harvested A. crinitum has a high affinity for ammonium and although it will grow when supplied exclusively with nitrate, internal nitrate pools are low and it is unable to take up nitrate without several days of acclimation to saturating light. Our data predict that A. crinitum would be able to sustain growth with ammonium as the sole source of nitrogen, a strategy that would help it survive under low‐light conditions that prevail in the field.     

High Nitrogen and Phosphorous Acquisition by Belowground Parts of Caulerpa prolifera (Chlorophyta) Contribute to the Species' Rapid Spread in Ria Formosa Lagoon,Southern Portugal

Despite worldwide proliferation of the genus Caulerpa and subsequent effects on benthic communities, little is known about the nutritional physiology of the Caulerpales. Here, we investigated the uptake rates of ammonium, nitrate, amino acids, and phosphate through the fronds and rhizoids + stolon, the internal translocation of nitrogen, and developed a nitrogen budget for the rapidly spreading Caulerpa prolifera in Ria Formosa lagoon, southern Portugal. Caulerpa prolifera acquired nutrients by both aboveground and belowground parts at similar rates, except nitrate, for which fronds showed 2-fold higher uptake rates. Ammonium was the preferential nitrogen source (81% of the total nitrogen acquisition), and amino acids, which accounted for a significant fraction of total N acquisition (19%), were taken up at faster rates than nitrate. Basipetal translocation of ¹⁵N incorporated as ammonium was nearly 3-fold higher than acropetal translocation, whereas ¹⁵N translocation as nitrate and amino acids was smaller but equal in either direction. The estimated total nitrogen acquisition by C. prolifera was 689 μmol · m⁻² · h⁻¹, whereas the total nitrogen requirement for growth was 672 μmol · m⁻² · h⁻¹. The uptake of ammonium and amino acids by belowground parts accounted for the larger fraction of the total nitrogen acquisition of C. prolifera and is sufficient to satisfy the species nitrogen requirements for growth. This may be one reason explaining the fast spreading of the seaweed in the bare sediments of Ria Formosa where it does not have any macrophyte competitors and the concentration of nutrients is high.     

Rapid and slow modulation of nitrate reductase activity in the red macroalga <Emphasis Type="Italic">Gracilaria chilensis</Emphasis> (Gracilariales,Rhodophyta): influence of different nitrogen sources

Nitrate is one of the most important stimuli in nitrate reductase (NR) induction, while ammonium is usually an inhibitor. We evaluated the influence of nitrate, ammonium or urea as nitrogen sources on NR activity of the agarophyte Gracilaria chilensis. The addition of nitrate rapidly (2 min) induced NR activity, suggesting a fast post-translational regulation. In contrast, nitrate addition to starved algae stimulated rapid nitrate uptake without a concomitant induction of NR activity. These results show that in the absence of nitrate, NR activity is negatively affected, while the nitrate uptake system is active and ready to operate as soon as nitrate is available in the external medium, indicating that nitrate uptake and assimilation are differentially regulated. The addition of ammonium or urea as nitrogen sources stimulated NR activity after 24 h, different from that observed for other algae. However, a decrease in NR activity was observed after the third day under ammonium or urea. During the dark phase, G. chilensis NR activity was low when compared to the light phase. A light pulse of 15 min during the dark phase induced NR activity 1.5-fold suggesting also fast post-translational regulation. Nitrate reductase regulation by phosphorylation and dephosphorylation, and by protein synthesis and degradation, were evaluated using inhibitors. The results obtained for G. chilensis show a post-translational regulation as a rapid response mechanism by phosphorylation and dephosphorylation, and a slower mechanism by regulation of RNA synthesis coupled to de novo NR protein synthesis.     

Effects of nitrogen and phosphate enrichment on the activity of nitrate reductase of <Emphasis Type="Italic">Ulva prolifera</Emphasis> in coastal zone

As one of the main species causing “green tides”, Ulva prolifera always inhabits in estuarine areas with changes in salinity and nutrients. Reduced salinity may affect directly or indirectly the processes of uptake and assimilation of nitrate, in which the nitrate reductase (NR) activity play the crucial roles. In this experiment, we investigated the different effects of enriched nitrogen and phosphate on NR activity of Ulva prolifera at salinity 30, 15, and 5 psu. The results showed that when salinity being lowered NR activity decreased under no enrichment (CT) or PO₄ ^3? enrichment condition. NO₃ ^? or combination with PO₄ ^3? could significantly enhance NR activity at three salinities, among which the highest value occurred at 15 psu. Enrichment of NH₄ ⁺ significantly decreased NR activity at 30 and 15 psu, but not at 5 psu. The results suggested NR of Ulva prolifera could be triggered by NO₃ ^?, especially at middle salinity, and keep low when exposed under hyposaline or NH₄ ⁺ enrichment for long term to rapidly respond to pulse of NO₃ ^? in estuarine areas.     

Discrimination of the common macroalgae (Ulva and Blidingia) in coastal waters of Yellow Sea,northern China,based on restriction fragment-length polymorphism (RFLP) analysis

Since 2007, reoccurring large-scale green algae blooms have caused deleterious effects to the estuarine ecosystem of Yellow Sea, northern China and subsequent economical losses. Previous surveys indicated the green tides were initiated in the coastal water of southern Jiangsu province where Porphyra farming was intensively conducted; however, the main ‘seed source’ of floating green algae is still under debate. Ulva prolifera was confirmed to be the major causative species of green tides. The multiple sympatric ulvoid species in the natural environment has complicated species identification in both field surveys and laboratory studies due to their morphological plasticity. Thus, we developed a genetic identification key based on restriction fragment length polymorphism (RFLP) analysis of the ITS nuclear marker to discriminate the common Ulva and Blidingia species in the Yellow Sea. Ten genetic lineages (1 in Blidingia, 9 in Ulva) were detected along the coast of China through phylogenetic analysis of ITS sequences. They can be separated by virtual restriction digestion using the four selected restriction enzymes (BspT107 I, EcoO109 I, Hin1 I and VpaK11B I). With additional PCR amplification of the 5S spacer region, we were able to discriminate U. prolifera from Ulva linza. Using this genetic key, we screened macroalgal samples collected from the coast of the Yellow Sea, and the results indicated 6 common lineages (U. prolifera, U. linza, Ulva compressa, Ulva pertusa, Clade 6 and Blidingia sp.) in this region, which could be explicitly distinguished by a single enzyme (BspT107 I) coupled with 5S spacer polymorphism. U. prolifera was confirmed to be present on the Porphyra aquaculture rafts with seasonal variation in the species composition. This genetic key will facilitate our long-term field surveys to investigate the origin of the floating U. prolifera and furthermore to explore its bloom dynamics along the coast of the Yellow Sea. It also provided a framework for the future inclusion of more Ulva species, which will expand the usage of this key.     

Seasonal variation of dominant free-floating and attached Ulva species in Rudong coastal area,China

In this paper, species compositions and seasonal variations of attached Ulva species on Porphyra aquaculture rafts and free floating Ulva species at Rudong coastal area, Jiangsu Province of China were investigated during 2010–2011. Based on the sequences analysis of nuclear-encoded ITS (including 5.8S rDNA regions) and 5S rDNA spacer regions, dominant species of both attached and free-floating Ulva samples were identified as Ulva compressa, Ulva linza, Ulva prolifera and Ulva flexuosa. Phylogenetic tree based on sequences of ITS and 5S rDNA spacer regions for attached and free-floating Ulva species was constructed, respectively. Species compositions of the Ulva population attached on aquaculture rafts varied with seasons, and U. prolifera was only found on aquaculture rafts in March 2011 during the 2010–2011 Porphyra yezoensis cultivation season, which had the same sequences of ITS and 5S rDNA spacer regions as that of the dominant species bloomed in the Yellow Sea of China in 2008. Dominant species of the free-floating Ulva population at the early stage of the green tide were U. compressa, U. flexuosa, and U. linza. Free-floating U. prolifera appeared in the middle of May, 2011. ITS sequence similarity rates of U. compressa and U. flexuosa between the attached and free-floating species were 100%. And ITS and 5S rDNA spacer sequences of the attached and the free-floating U. prolifera population also showed no differences. Further study showed that there were two types of free-floating U. prolifera population (Type 5S-A and Type 5S-B) based on 5S rDNA spacer sequences. The present study would provide some useful information for clarifying the outbreak mechanism of green tides occurred in the Yellow Sea, China.     

Competition between the green alga Scenedesmus and the cyanobacterium Synechococcus under different modes of inorganic nitrogen supply

In this study, we evaluated growth responses of the green alga Scenedesmus and the cyanobacterium Synechococcus supplied with inorganic nitrogen in different ways. A competitive situation in which nitrogen was limiting was created in mixed cultures as well as in cultures growing in the same vessel but separated by a permeable dialysis membrane. Supplying inorganic nitrogen in small pulses at a high frequency favoured the cyanobacterium Synechococcus, whereas batch additions favoured the green alga Scenedesmus. When using a large-pulse/low-frequency supply mode, the yield of the green alga was higher when ammonium was added as nitrogen source compared to when nitrate was added. By contrast, the yield of the cyanobacterium was higher in the nitrate regime. However, uptake experiments using unialgal cultures showed that both organisms depleted the medium of ammonium more rapidly than they depleted the medium of nitrate; i.e. the higher yield of the cyanobacterium in the nitrate regime than in the ammonium regime can be attributed to the effects of competition with the green alga. Since nitrate assimilation involves the consumption of reductive power, we suggest that the outcome of competition was governed by the fact that green alga was light limited and therefore better able to compete for ammonium than for nitrate. The results from the laboratory studies are discussed in relation to results from an enclosure experiment performed in Lake Erken, Sweden. In that field experiment, in which additions of both phosphate and ammonium were applied every second day to 350-l enclosures, the green algal biomass increased exponentially during an incubation period of 22 days.     

Characterization of the Nitrate-Nitrite Transporter of the Major Facilitator Superfamily (the nrtP Gene Product) from the Cyanobacterium Nostoc punctiforme Strain ATCC 29133

The products of the NpR1527 and NpR1526 genes of the filamentous, diazotrophic, fresh-water cyanobacterium Nostoc punctiforme strain ATCC 29133 were identified as a nitrate transporter (NRT) and nitrate reductase (NR) respectively, by complementation of nitrate assimilation mutants of the cyanobacterium Synechococcus elongatus strain PCC 7942. While other fresh-water cyanobacteria, including S. elongatus, have an ATP-binding cassette (ABC)-type NRT, the NRT of N. punctiforme belongs to the major facilitator superfamily, being orthologous to the one found in marine cyanobacteria (NrtP). Unlike the ABC-type NRT, which transports both nitrate and nitrite with high affinity, Nostoc NrtP transported nitrate preferentially over nitrite. NrtP was distinct from ABC-type NRT also in its insensitivity to ammonium-promoted regulation at the post-translational level. The nitrate reductase of N. punctiforme was, on the other hand, inhibited upon addition of ammonium to medium, lending ammonium sensitivity to nitrate assimilation.     

Ulva diversity in the Yellow Sea during the large‐scale green algal blooms in 2008–2009

In the Yellow Sea of China, large‐scale green tides have broken out for three consecutive years from 2007 to 2009. As part of the efforts to localize the algal source, two cruises were conducted in the early stage and the outbreak stage of the bloom in 2009. We analyzed the morphological and genetic diversity of drifting Ulva specimens and culture‐derived isolates from seawater sampled in different localities. For phylogenetic analyses, the nuclear encoded ribosomal DNA internal transcribed spacer region (ITS nrDNA) and the plastid encoded large subunit of ribulose‐1, 5‐bisphosphate carboxylase/oxgenase gene (rbcL) were used. Our molecular and morphological data indicate that the dominant free‐floating Ulva species in 2008 and 2009 possibly belonged to a single strain of the U. linza‐procera‐prolifera (LPP) clade. The ITS sequences from bloom‐forming algal samples with dense branches were identical to those from U. linza‐like specimens without branches derived from the Yellow Sea. Microscopic individuals of the dominant Ulva strain were detected in eight stations, revealing that spore dispersal in the water helped to enlarge biomass in the water during the outbreak stage of green tide in the Yellow Sea.     

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.     

Nitrate regulation of nitrate uptake and nitrate reductase expression in barley grown at different nitrate:ammonium ratios at constant relative nitrogen addition rate

Barley (Hordeum vulgare L. cv. Golf) was cultured using the relative addition rate technique, where nitrogen is added in a fixed relation to the nitrogen already bound in biomass. The relative rate of total nitrogen addition was 0.09 day^?1 (growth limiting by 35%), while the nitrate addition was varied by means of different nitrate: ammonium ratios. In 3- to 4-week-old plants, these ratios of nitrate to ammonium supported nitrate fluxes ranging from 0 to 22 μmol g^?1 root dry weight h^?1, whereas the total N flux was 21.8 ± 0.25 μmol g^?1 root dry weight h^?1 for all treatments. The external nitrate concentrations varied between 0.18 and 1.5 μM. The relative growth rate, root to total biomass dry weight ratios, as well as Kjeldahl nitrogen in roots and shoots were unaffected by the nitrate:ammonium ratio. Tissue nitrate concentration in roots were comparable in all treatments. Shoot nitrate concentration increased with increasing nitrate supply, indicating increased translocation of nitrate to the shoot. The apparent V_max for net nitrate uptake increased with increased nitrate fluxes. Uptake activity was recorded also after growth at zero nitrate addition. This activity may have been induced by the small, but detectable, nitrate concentration in the medium under these conditions. In contrast, nitrate reductase (NR) activity in roots was unaffected by different nitrate fluxes, whereas NR activity in the shoot increased with increased nitrate supply. NR-mRNA was detected in roots from all cultures and showed no significant response to the nitrate flux, corroborating the data for NR activity. The data show that an extremely low amount of nitrate is required to elicit expression of NR and uptake activity. However, the uptake system and root NR respond differentially to increased nitrate flux at constant total N nutrition. It appears that root NR expression under these conditions is additionally controlled by factors related to the total N flux or the internal N status of the root and/or plant. The method used in this study may facilitate separation of nitrate-specific responses from the nutritional effect of nitrate.