小麦NO3-转运蛋白基因TaNRT2.3的克隆和表达分析

用RT-PCR和RACE技术在NO3-诱导处理的小麦(Triticum aestivum L.)根中克隆到一个硝酸根转运蛋白基因的cDNA,命名为TaNRT2.3(GenBank登录号AY053452).序列分析表明,TaNRT2.3全长1 744 bp,其中含有1 521bp的ORF,编码507个氨基酸,具有12个跨膜区,属于MFS超基因家族中的NNP家族.TaNRT2.3与其他植物中已知的NRT2具有很高的同源性.Northern杂交表明:TaNRT2具有在根中表达的组织特异性,而在叶中未检测到.TaNRT2的表达受NO3-诱导,在含NH4 介质中不表达.NO3-在低浓度(5～200μmol/L)和高浓度(2.0 mmol/L)时均起作用.通过研究小麦在0.2 mmol/LNO3-条件下TaNRT2的表达水平及对NO3-的吸收效率,表明TaNRT2在小麦高效吸收NO3-方面起着重要的作用.分根实验表明植物中N循环本身可以作为吸收N的调节信号.     

Identification and expression analyses of two genes encoding putative low-affinity nitrate transporters from Nicotiana plumbaginifolia

Higher plants have both high- and low-affinity nitrate uptake systems (HATS and LATS respectively). Here we report the isolation and characterization of two genes, NpNRT1.1 and NpNRT1.2, from Nicotiana plumbaginifolia whose structural features suggest that they both belong to the NRT1 gene family, which is involved in the LATS. Amino acid sequence alignment showed that the N. plumbaginifolia proteins have greater similarity to their corresponding tomato homologues than to each other. Genomic Southern blot analysis indicates that there are probably more than two members of this family in N. plumbaginifolia. Northern blot analysis shows that NpNRT1.2 expression is restricted strictly to roots, whereas NpNRT1.1, in addition to roots, is expressed at a basal level in all other plant organs. Likewise, differential expression in response to external treatments with various N sources was observed for these two genes: NpNRT1.1 can be considered as a constitutively expressed gene whereas NpNRT1.2 expression is dependent strictly on high nitrate concentrations. Finally, over-expression of a gene involved in the HATS does not lead to any modification of LATS gene expression.     

Gene structure and expression of the high-affinity nitrate transport system in rice roots

Rice has a preference for uptake of ammonium over nitrate and can use ammonium-N efficiently. Consequently, transporters mediating ammonium uptake have been extensively studied, but nitrate transporters have been largely ignored. Recently,some reports have shown that rice also has high capacity to acquire nitrate from growth medium, so understanding the nitrate transport system in rice roots is very important for improving N use efficiency in rice. The present study identified four putative NRT2 and two putative NAR2 genes that encode components of the high-affinity nitrate transport system (HATS) in the rice (Oryza sativa L. subsp, japonica cv. Nipponbare) genome. OsNRT2.1 and OsNRT2.2 share an identical coding region sequence, and their deduced proteins are closely related to those from monocotyledonous plants. The two NAR2 proteins are closely related to those from mono-cotyledonous plants as well. However, OsNRT2.3 and OsNRT2.4 are more closely related to Arabidopsis NRT2 proteins. Relative quantitative reverse tranecdption-polymerase chain reaction analysis showed that all of the six genes were rapidly upregulated and then downregulated in the roots of N-starved rice plants after they were re-supplied with 0.2 mM nitrate, but the response to nitrate differed among gene members.The results from phylogenetic tree, gene structure and expression analysis implied the divergent roles for the individual members of the rice NRT2 and NAR2 families. High-affinity nitrate influx rates associated with nitrate induction in rice roots were investigated and were found to be regulated by external pH. Compared with the nitrate influx rates at pH 6.5, alkaline pH (pH 8.0) inhibited nitrate Influx, and acidic pH (pH 5.0) enhanced the nitrate influx In I h nitrate induced roots, but did not significantly affect that in 4 to 8 h nitrate induced roots.     

Putative role of γ -aminobutyric acid (GABA) as a long-distance signal in up-regulation of nitrate uptake in Brassica napus L.

The relationship between nitrate influx, BnNrt2 nitrate transporter gene expression and amino acid composition of phloem exudate was investigated during N‐deprivation (short‐term experiment) and over a growth cycle (long‐term experiment) in Brassica napus L. The data showed a positive correlation between γ‐aminobutyric acid (GABA) in phloem exudate and nitrate uptake in the short‐ and the long‐term experiments. The hypothesis that this non‐protein amino acid could up‐regulate nitrate uptake via a long‐distance signalling pathway was tested by providing an exogenous GABA supply to the roots. The effect of GABA was compared with the effects of Gln, Glu and Asn, each known to be inhibitors of nitrate uptake. The results showed that GABA treatment induced a significant increase of BnNrt2 mRNA expression, but had less effect on nitrate influx. By contrast, Gln, Glu and Asn significantly reduced nitrate influx and BnNrt2 mRNA expression compared with the control plants. This study provides the first evidence that GABA may act as a putative long‐distance inter‐organ signal molecule in plants in conjunction with negative control exerted by Gln. The up‐regulation effect of GABA on nitrate uptake is discussed in the context of its role in N metabolism, nutritional stress and the recent discovery of a putative role of GABA as a signal molecule in plant development.     

Exogenous Nitrate as a Terminal Acceptor of Electrons in Rice (Oryza sativa) Coleoptiles and Wheat (Triticum aestivum) Roots under Strict Anoxia

To elucidate the physiological role of exogenous nitrate under anaerobic conditions, we studied the effect of 10 mM KNO₃ on the mitochondrial ultrastructure in rice (Oryza sativa L.) coleoptiles and in wheat (Triticum aestivum L.) roots, detached from four-day-old seedlings, under strict anoxia. In wheat roots, following 6-h-long anoxia in the absence of exogenous nitrate, the mitochondrial membranes were partially degraded and, after 9 h under anoxia, the mitochondrial membranes and the membranes of other organelles were completely destroyed. In rice coleoptiles, the partial membrane degradation was observed only after 24 h and their complete breakdown after 48 h of anaerobiosis. In the presence of exogenous nitrate, no membrane destruction was noticed even after 9 and 48 h of anaerobiosis in wheat roots and rice coleoptiles, respectively. These results indicate that exogenous nitrate exerts protective action as a terminal electron acceptor, alternative to the molecular oxygen. Our findings are compared with the results of other researchers concerning the adverse or favorable nitrate action on plant growth, metabolism, and energy status under anaerobic stress.     

Growth and development of seminal and crown root systems in N-limited barley,and their contributions to nitrate acquisition during vegetative and generative growth

Barley (Hordeum vulgare L., cvs Golf and Laevigatum) was grown under nitrogen limitation, controlled by the relative rate of nitrate-N addition (RA), in solution culture. The seminal and crown root systems were kept apart, but in contact with the same nutrient solution throughout culturing. Growth, nitrate uptake, and in vitro nitrate reductase (NR) activity in the different root parts were studied at plant ages from 40 (late vegetative stage) to 110 (mid grain-filling) days. The RA was during this time interval stepwise decreased from 0.08 day^–1 to 0.005 day^–1. The ratio between seminal root dry weight and total plant dry weight decreased drastically during post-anthesis growth, whereas the contribution by crown roots remained unchanged. Tissue nitrogen concentrations in seminal roots did not change with time, but decreased in crown roots after day 80. The NR activity decreased with age in both seminal and crown roots. The V_max for net nitrate uptake decreased throughout the experiment in the seminal root system, but not in the crown root system. The kinetic properties (V_max and K_M) were used to calculate the nitrate concentration required to maintain a relative rate of nitrate-N uptake that equals the relative addition rate. These concentrations (2 to 5 mmol m^–3) were found to closely match actually measured nitrate concentrations in the nutrient solution (1 to 6 mmol m^–3). From uptake kinetics, it was deduced that the contribution by seminal roots to total nitrate uptake at these concentrations decreased from more than 50% in vegetative plants, to about 20% just after main shoot anthesis, and to less than 5% during grain-filling. ei]Section editor: H Lambers     

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.     

Positive effects of continuous low nitrate levels on growth and photosynthesis of Alexandrium tamarense (Gonyaulacales, Dinophyceae)

The growth and photosynthesis of Alexandrium tamarense (Lebour) Balech in different nutrient conditions were investigated. Low nitrate level (0.0882 mmol/L) resulted in the highest average growth rate from day 0 to day 10 (4.58 × 10² cells mL^?1 d^?1), but the lowest cell yield (5420 cells mL^?1) in three nitrate level cultures. High nitrate‐grown cells showed lower levels of chlorophyll a‐specific and cell‐specific light‐saturated photosynthetic rate (P_m^{chl a} and P_m^cell), dark respiration rate (R_d^chla and R_d^cell) and chlorophyll a‐specific apparent photosynthetic efficiency (α^chla) than was seen for low nitrate‐grown cells; whereas the cells became light saturated at higher irradiance at low nitrate condition. When cultures at low nitrate were supplemented with nitrate at 0.7938 mmol/L in late exponential growth phase, or with nitrate at 0.7938 mmol/L and phosphate at 0.072 mmol/L in stationary growth phase, the cell yield was drastically enhanced, a 7–9 times increase compared with non‐supplemented control culture, achieving 43 540 cells mL^?1 and 52 300 cells mL^?1, respectively; however, supplementation with nitrate in the stationary growth phase or with nitrate and phosphate in the late exponential growth phase increased the cell yield by no more than 2 times. The results suggested that continuous low level of nitrate with sufficient supply of phosphate may facilitate the growth of A. tamarense.     

小麦叶内硝酸还原的代谢库

随营养液中No_3~-浓度升高,叶片内No_3~-总量、代谢库大小(NIPS)及硝酸还原酶(NR)活性均升高,其中MPS与NK活性呈同步变化;No_3~-浓度达2.0mmol/L时,两者趋于稳值;若再增加NO_3~-浓度,则被吸收的NO_3~-积累于液泡中,而代谢库中NO_3~-含量(MPS)与NO_3~-总量之比有一定程度降低。低氮(NO_3~-浓度为1.0 mmol/L)情况下,反应液中无NO_3~-时,叶片内NR活性品种间有差异,但在50 mmol/L NO_3~-反应液中则品种间无差异;NK活性高的品种鲁麦8号及品种321叶内有大的NO_3~-代谢库,反应液中NO_3~-对NR活性刺激程度低,代谢库NO_3~-含量与叶NO_3~-总量之比高,而叶组织长时间反应过程中其NR活性衰减速率低。     

Nitrate does not compete with abscisic acid as a substrate of AtNPF4.6/NRT1.2/AIT1 in Arabidopsis

We identified a member of the Arabidopsis NRT1/PTR FAMILY (NPF), AtNPF4.6, as an abscisic acid (ABA) transporter, AIT1. AtNPF4.6 was originally characterized as a low-affinity nitrate transporter NRT1.2. We hypothesized that the competition between nitrate and ABA as substrates for AtNPF4.6 might be involved in the interactions between nitrate and ABA signaling. However, the ABA transport activity of AtNPF4.6 was not inhibited by an excess amount of nitrate. In addition, the npf4.6 mutant was less sensitive to ABA than the wild type during germination irrespective of nitrate concentrations in the media. Furthermore, nitrate promoted germination of both wild type and npf4.6 in the presence of ABA. These results do not support the idea of a physiological linkage between nitrate and ABA signals through AtNPF4.6.     

A 150 kDa plasma membrane complex of AtNRT2.5 and AtNAR2.1 is the major contributor to constitutive high‐affinity nitrate influx in Arabidopsis thaliana

In plants that have been deprived of nitrate for a significant length of time, a constitutive high‐affinity nitrate transport system (cHATS) is responsible for initial nitrate uptake. This absorbed nitrate leads to the induction of the major nitrate transporters and enzymes involved in nitrate assimilation. By use of ¹³NO₃^– influx measurements and Blue Native polyacrylamide gel electrophoresis we examined the role of AtNRT2.5 in cHATS in wild type (WT) and various T‐DNA mutants of Arabidopsis thaliana. We demonstrate that AtNRT2.5 is predominantly expressed in roots of nitrate‐deprived WT plants as a 150 kDa molecular complex with AtNAR2.1. This complex represents the major contributor to cHATS influx, which is reduced by 63% compared with WT in roots of Atnrt2.5 mutants. The remaining cHATS nitrate influx in these mutants is due to a residual contribution by the inducible high‐affinity transporter encoded by AtNRT2.1/AtNAR2.1. Estimates of the kinetic properties of the NRT2.5 transporter reveal that its low K_m for nitrate makes this transporter ideally suited to detect and respond to trace quantities of nitrate in the root environment.