Cloning and heterologous expression of nitrate reductase genes from <Emphasis Type="Italic">Dunaliella salina</Emphasis>

A cDNA corresponding to the nitrate reductase (NR) gene from Dunaliella salina was isolated by RT-PCR and (5′/3′)-RACE techniques. The full-length cDNA sequence of 3,694 bp contained an open reading frame of 2,703 bp encoding 900 amino acids, a 5′-untranslated region of 151 bp and a 3′-untranslated sequence of 840 bp with a poly (A) tail. The putative gene product exhibited 78%, 65%, 59% and 50% identity in amino acid sequence to the corresponding genes of Dunaliella tertiolecta, Volvox carteri, Chlamydomonas reinhardtii, and Chlorella vulgaris, respectively. Phylogenetic analysis showed that D. salina NR clusters together with known NR proteins of the green algae. The molecular mass of the encoded protein was predicted to be 99.5 kDa, with an isoelectric point of 8.31. This protein shares common structural features with NRs from higher plants and green algae. The full-length cDNA was heterologously expressed in Escherichia coli as a fusion protein, and accumulated to up to 21% of total bacteria protein. Recombinant NR protein was active in an enzyme assay, confirming that the cloned gene from D. salina is indeed NR.     

Distinct patterns of nitrate reductase activity in brown algae: light and ammonium sensitivity in Laminaria digitata is absent in Fucus species1

Fucus and Laminaria species, dominant seaweeds in the intertidal and subtidal zones of the temperate North Atlantic, experience tidal cycles that are not synchronized with light:dark (L:D) cycles. To investigate how nutrient assimilation is affected by light cycles, the activity of nitrate reductase (NR) was examined in thalli incubated in outdoor tanks with flowing seawater and natural L:D cycles. NR activity in Laminaria digitata (Huds.) Lamour. showed strong diel patterns with low activities in darkness and peak activities near midday. This diel pattern was controlled by light but not by a circadian rhythm. In contrast, there was no diel variation in NR activity in Fucus serratus L., F. vesiculosus (L.) Lamour., and F. spiralis L. either collected directly from the shore or maintained in the outdoor tanks. In laboratory cultures, transfer to continuous darkness suppressed NR activity in L. digitata, but not in F. vesiculosus; continuous light increased NR activity in L. digitata but decreased activity in F. vesiculosus. Furthermore, 4 d enrichment with ammonium (50 μmol · L^?1 pulses), resulted in NR activity declining by >80% in L. digitata, but no significant changes in F. serratus. Seasonal differences in maximum NR activity were present in both genera with activities highest in late winter and lowest in summer. This is the first report of NR activity in any alga that is not strongly regulated by light and ammonium. Because light and tidal emersion do not always coincide, Fucus species may have lost the regulation of NR by light that has been observed in other algae and higher plants.     

CHARACTERIZATION OF DIATOM (BACILLARIOPHYCEAE) NITRATE REDUCTASE GENES AND THEIR DETECTION IN MARINE PHYTOPLANKTON COMMUNITIES1

The complete assimilatory nitrate reductase (NR) gene from the pennate diatom Phaeodactylum triconutum Bohlin was sequenced from cDNA and compared with NR sequences from fungi, green algae, vascular plants, and the recently sequenced genome of the centric diatom Thalassiosira pseudonana Hasle and Heimdal CCMP1335. In all the major eukaryotic nitrate reductase (Euk‐NR) functional domains, diatom NR gene sequences are generally 50%–60% identical to plant and alga sequences at the amino acid level. In the less conserved N‐terminal, hinge 1, and hinge 2 regions, homology to other NR sequences is weak, generally<30%. Two PCR primer sets capable of amplifying Euk‐NR from plants, algae, and diatoms were designed. One primer set was used to amplify a 750‐base pair (bp) NR fragment from the cDNA of five additional diatom strains. The PCR amplicon spans part of the well‐conserved dimer interface region, the more variable hinge 1 region, and part of the conserved cytochrome b heme binding region. The second primer set, targeted to the dimer region, was used to amplify an approximately 400‐bp fragment of the NR gene from DNA samples collected in Monterey Bay, California and in central New Jersey inner continental shelf (LEO‐15 site) waters. Only diatom‐like NR sequences were recovered from Monterey Bay samples, whereas LEO‐15 samples yielded NR sequences from a range of photosynthetic eukaryotes. The prospect of using DNA‐ and RNA‐based methods to target the NR genes of diatoms specifically is a promising approach for future physiological and ecological experiments.     

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.     

茶树硝酸还原酶基因克隆及表达分析

利用茶树全器官转录组文库中硝酸还原酶(NR)的EST,通过RACE技术扩增出NR基因的cDNA,并利用实时荧光定量PCR检测了NR基因在不同茶树品种中的表达。结果表明:NR基因cDNA全长2 927bp,开放阅读框2 652bp,编码一个有884个氨基酸蛋白质,GenBank登录号为JX987133。经BlastX比对,与GenBank中登录的烟草NR相似性达到74%。茶树NR蛋白属于亲水性蛋白,可能为胞质蛋白。25个茶树品种叶片中NR表达水平差异明显,最高值是最低值的22.75倍。因NR是植物氮代谢过程中的关键限速酶,推测25个茶树品种间氮吸收利用能力存在差异。     

Peculiarities of growth of the monocellular green algae culture after the influence of electromagnetic field in deuterated water-containing media

Exposing the inoculum of monocellular green algae Dunaliella tertiolecta and Tetraselmis viridis to 50 Hz electromagnetic field for several hours resulted in a reduced growth rate in both cultures. It was ascertained that heavy water inhibited growth of algae Dunaliella tertiolecta. The light water activated growth of the culture in the exponential phase only.     

COMPARISONS OF NITRATE UPTAKE, STORAGE, AND REDUCTION IN MARINE DIATOMS AND FLAGELLATES

Diatoms, but not flagellates, have been shown to increase rates of nitrogen release after a shift from a low growth irradiance to a much higher experimental irradiance. We compared NO₃ ^? uptake kinetics, internal inorganic nitrogen storage, and the temperature dependence of the NO₃ ^? reduction enzymes, nitrate (NR) and nitrite reductase (NiR), in nitrogen‐replete cultures of 3 diatoms (Chaetoceros sp., Skeletonema costatum, Thalassiosira weissflogii) and 3 flagellates (Dunaliella tertiolecta, Pavlova lutheri, Prorocentrum minimum) to provide insight into the differences in nitrogen release patterns observed between these species. At NO₃ ^? concentrations <40 μmol‐N·L ^{? 1}, all the diatom species and the dinoflagellate P. minimum exhibited saturating kinetics, whereas the other flagellates, D. tertiolecta and P. lutheri, did not saturate, leading to very high estimated K _s values. Above ～60 μmol‐N·L ^{? 1}, NO₃ ^? uptake rates of all species tested continued to increase in a linear fashion. Rates of NO₃ ^? uptake at 40 μmol‐N·L ^{? 1}, normalized to cellular nitrogen, carbon, cell number, and surface area, were generally greater for diatoms than flagellates. Diatoms stored significant amounts of NO₃ ^? internally, whereas the flagellate species stored significant amounts of NH₄ ⁺ . Half‐saturation concentrations for NR and NiR were similar between all species, but diatoms had significantly lower temperature optima for NR and NiR than did the flagellates tested in most cases. Relative to calculated biosynthetic demands, diatoms were found to have greater NO₃ ^? uptake and NO₃ ^? reduction rates than flagellates. This enhanced capacity for NO₃ ^? uptake and reduction along with the lower optimum temperature for enzyme activity could explain differences in nitrogen release patterns between diatoms and flagellates after an increase in irradiance.     

THE PATH OF CARBON IN PHOTOSYNTHESIS BY MARINE PHYTOPLANKTON12

Using cultures of a number of different marine algae (diatoms Skeletonema costatum (Grev.) Cleve and Phaeodactylum tricornutum Bohlin, chrysophyte Isochrysis galbana Parke, green flagellate Dunaliella tertiolecta Butcher, dinoflagellate Gonyaulax tamarensis Lebour) the short-term, pattern of ¹⁴CO₂ assimilation has been investigated. In all except D. tertiolecta the labelling of amino acids and intermediates of the tricarboxylic acid (Krebs) cycle was significantly heavier than that of sugar phosphates. Over periods of 30–120 s labelling in amino acids and Krebs cycle intermediates accounted for 41–95% of the ¹⁴C fixed (depending on the alga). Over shorter times (< 10 s) the pattern in the 2 diatoms showed significant labelling of C₄ acids (and related com-pounds) and little labelling of sugar phosphates. The reverse wits seen with D. tertiolecta. Also, in the 2 diatoms and in G. tamarensis significant inhibition of photosynthesis by oxygen could only be achieved with 100% oxygen; atmospheric levels having little effect. Parallel measurements of 2 carboxylating enzymes showed that ribulose-1,5-diphosphate carboxylase (RuDPCase) was significantly greater than phospho (enol)pyruvate carboxylase (PEPCase) activity only in the green flagellate. It is suggested that photosynthesis in marine diatoms depends on an active PEPCase utilizing bicarbonate as a substrate and that a less active RuDPCase utilizes CO₂. In D. tertiolecta the pattern more closely resembles that of a “Calvin (C₃)” plant. The dinoflagellate and the chrysophyte appeared to show a mixed C₃ and C₄ photosynthesis.     

植物中硝酸还原酶和亚硝酸还原酶的作用

植物通过硝酸盐同化途径以硝酸盐和氨的形式吸收氮元素。硝酸盐的同化是一个受到严格控制的过程,其中两个先后参加反应的酶——硝酸还原酶(NR)和亚硝酸还原酶(NiR)对初级氮的同化起主要调控。在高等植物中,NR和NiR基因的转录及转录后加工受到各种内在和外在因素的影响,翻译后调控是消除亚硝酸盐积累的重要机制。随着分子生物学技术的发展,可以更容易地通过突变体和转基因方式来研究NR和NiR基因的调控。     

Cloning and expression study of a putative high-affinity nitrate transporter gene from Dunaliella salina

A nitrate transporter gene, named DsNRT2.1 (GeneBank accession number AY621079), from Dunaliella salina has been cloned by screening a cDNA library, which was constructed with mRNAs from D. salina after 60 min treatment with 5 mM nitrate, with a 342 bp NRT2 cDNA fragment from D. salina as a probe. DsNRT2.1 exhibits sequence similarity to those known nitrate transporters of the NRT2 family. A hydrophobicity blot indicated that DsNRT2.1 belongs to the major facilitator superfamily (MFS). Northern analysis showed that an mRNA species of 1.9 kb can be rapidly induced by NO^– ₃, but not by NH⁺ ₄. Northern analysis also showed that NaCl could significantly increase the expression of DsNRT2.1.