与高等植物吸收(NO_3)~-/(NH_4)~+、(PO_4)~(3-)和K~+有关的膜转运蛋白编码基因的分子生物学研究现状

高等植物对土壤中营养元素的吸收是其一切生命活动过程的基础,尤其在营养元素缺乏的状态下,更与其抗营养饥饿等特性息息相关。兼于土壤中Ｎ、Ｐ、Ｋ元素缺乏的严重性与普遍性,以及Ｎ、Ｐ、Ｋ对高等植物生长和发育的重要性,有关高等植物吸收营养元素的膜转运蛋白编码基因的分子生物学研究已引起有关学者的高度重视。ＮＯ－３／ＮＨ＋４、ＰＯ３－４与Ｋ＋膜转运蛋白均有低亲和力和高亲和力系统（ＬｏｗＡｆｉｎｉｔｙＴｒａｎｓｐｏｒｔｅｒ＆ＨｉｇｈＡｆｉｎｉｔｙＴｒａｎｓｐｏｒｔｅｒ）。对ＰＯ４３－和Ｋ＋而言,低亲和力系统是组成性表达的系统,在正常营养状态下对根系吸收营养起重要作用。而高亲和力系统是受营养缺乏而诱导表达的系统,对于植物的抗逆性、耐营养饥饿至关重要。迄今为止,与之有关的基因的全长ｃＤＮＡ或全基因已在几种植物中被克隆。此外,对基因的表达特性亦有广泛研究。本文简要概述这三大营养元素的膜转运蛋白编码基因的分子生物学研究现状。     

Conservation and divergence of both phosphate- and mycorrhiza-regulated physiological responses and expression patterns of phosphate transporters in solanaceous species

Here, orthologous genes of six phosphate transporter (PiT) genes, which are members of the Pht1 and Pht2 families in tomato and potato, have been cloned from the solanaceous species pepper, eggplant and tobacco. Overall, expressions of these genes in pepper, eggplant and tobacco showed similar patterns to those in tomato and potato: P-starvation enhancement in both leaves and roots for Pht1;1, P-depletion induction exclusively in roots for Pht1;2, mycorrhizal enhancement for Pht1;3, and mycorrhizal induction for both Pht1;4 and Pht1;5. In the roots of nonmycorrhizal eggplant, SmPht1;3, SmPht1;4 and SmPht1;5 were also expressed under extreme P starvation. Mycorrhizal symbiosis under high-P supply conditions reduced plant growth, with concurrent enhancement of Pht1;2 expression in the roots of pepper as well as eggplant. In addition, the mycorrhizal symbiosis down-regulated the expression of Pht2;1 genes greatly in the leaves of pepper and tobacco. The discrepancies between the evolutionary distances of the PiT genes and their expression patterns among the five species suggest greater complexity in function of PiT in plants than previously expected.     

Transgenic Tobacco with αα Mutant Gene Has Higher Tolerance to Heavy Metals

Metallothionein (MT) has two domains, α and β domain. α domain preferred to bind Cd2+and Hg2+. Mouse metallothionein mutant αα has been constructed and expressed in E.coli, which has the same stability as the nature one but has stronger affinity to heavy metals. To testify the result in vivo, αα mutant gene was cloned into plant expression vector pE3 under the CaMV 35S promoter. A transgenic tobacco was obtained by using leaf discs of tobacco (Nicotiana tabacum L. cv. NC89) to Agrobacterium-mediated ααgene transfer. Southern blotting analysis indicated that the αα mutant gene was indeed integrated into the tobacco genome; Western blot indicated that the αα mutant gene was expressed in transgenic tobacco. It was also demonstrated that the transgenic tobacco with αα mutant gene have a little higher tolerance to heavy metals than that with natural MT gene. Moreover, the transgenic tobacco can accumulate more Cd2+ in its roots than natural, so that, it can decrease the concentration of Cd2+ in its leaves.     

The sink-specific plastidic phosphate transporter PHT4;2 influences starch accumulation and leaf size in Arabidopsis

Nonphotosynthetic plastids are important sites for the biosynthesis of starch, fatty acids, and amino acids. The uptake and subsequent use of cytosolic ATP to fuel these and other anabolic processes would lead to the accumulation of inorganic phosphate (Pi) if not balanced by a Pi export activity. However, the identity of the transporter(s) responsible for Pi export is unclear. The plastid-localized Pi transporter PHT4;2 of Arabidopsis (Arabidopsis thaliana) is expressed in multiple sink organs but is nearly restricted to roots during vegetative growth. We identified and used pht4;2 null mutants to confirm that PHT4;2 contributes to Pi transport in isolated root plastids. Starch accumulation was limited in pht4;2 roots, which is consistent with the inhibition of starch synthesis by excess Pi as a result of a defect in Pi export. Reduced starch accumulation in leaves and altered expression patterns for starch synthesis genes and other plastid transporter genes suggest metabolic adaptation to the defect in roots. Moreover, pht4;2 rosettes, but not roots, were significantly larger than those of the wild type, with 40% greater leaf area and twice the biomass when plants were grown with a short (8-h) photoperiod. Increased cell proliferation accounted for the larger leaf size and biomass, as no changes were detected in mature cell size, specific leaf area, or relative photosynthetic electron transport activity. These data suggest novel signaling between roots and leaves that contributes to the regulation of leaf size.     

Increased expression of OsPT1, a high-affinity phosphate transporter, enhances phosphate acquisition in rice

Most high-affinity phosphate transporter genes (OsPTs) in rice were highly induced in roots when phosphate was depleted. OsPT1, however, was highly expressed in primary roots and leaves regardless of external phosphate concentrations. This finding was confirmed histochemically using transgenic rice plants that express the GUS reporter gene under the control of the OsPT1 promoter, which exhibited high GUS activity even in the phosphate sufficient condition. Furthermore, transgenic rice plants overexpressing the OsPT1 gene accumulated almost twice as much phosphate in the shoots as did wild-type plants. As a result, transgenic plants had more tillers than did wild-type plants, which is a typical physiological indicator for phosphate status in rice.     

Mutation of a nitrate transporter, AtNRT1:4, results in a reduced petiole nitrate content and altered leaf development

Unlike nitrate uptake of plant roots, less is known at the molecular level about how nitrate is distributed in various plant tissues. In the present study, characterization of the nitrate transporter, AtNRT1:4, revealed a special role of petiole in nitrate homeostasis. Electrophysiological studies using Xenopus oocytes showed that AtNRT1:4 was a low-affinity nitrate transporter. Whole-mount in situ hybridization and RT-PCR demonstrated that AtNRT1:4 was expressed in the leaf petiole. In the wild type, the leaf petiole had low nitrate reductase activity, but a high nitrate content, indicating that it is the storage site for nitrate, whereas, in the atnrt1:4 mutant, the petiole nitrate content was reduced to 50-64% of the wild-type level. Moreover, atnrt1:4 mutant leaves were wider than wild-type leaves. This study revealed a critical role of AtNRT1:4 in regulating leaf nitrate homeostasis, and the deficiency of AtNRT1:4 can alter leaf development.     

Understanding the differential nitrogen sensing mechanism in rice genotypes through expression analysis of high and low affinity ammonium transporter genes

Two rice genotypes, Kalanamak 3119 (KN3119) and Pusa Basmati 1(PB1) differing in their optimum nitrogen requirements (30 and 120 kg/ha, respectively) were undertaken to study the expression of both high and low affinity ammonium transporter genes responsible for ammonium uptake. Exposing the roots of the seedlings of both the genotypes to increasing (NH₄)₂SO₄ concentrations revealed that all the three families of rice AMT genes are expressed, some of which get altered in a genotype and concentration specific manner. This indicates that individual ammonium transporter genes have defined contributions for ammonium uptake and plant growth. Interestingly, in response to increasing nitrogen concentrations, a root specific high affinity gene, AMT1;3, was repressed in the roots of KN3119 but not in PB1 indicating the existence of a differential ammonium sensing mechanism. This also indicates that not only AMT1;3 is involved not only in ammonium uptake but may also in ammonium sensing. Further, if it can differentiate and could be used as a biomarker for nitrogen responsiveness. Expression analysis of low affinity AMT genes showed that, both AMT2;1 and AMT2;2 have high levels of expression in both roots and shoots and in KN3119 are induced at low ammonium concentrations. Expressions of AMT3 family genes were higher shoots than in the roots indicating that these genes are probably involved in the translocation and distribution of ammonium ions in leaves. The expression of the only high affinity AMT gene, AMT1;1, along with six low affinity AMT genes in the shoots suggests that low affinity AMTs in the shoots leaves are involved in supporting AMT1;1 to carry out its activities/function efficiently.     

Cloning and expression of distinct nitrite reductases in tobacco leaves and roots

Summary Three tobacco nitrite reductase (NiR) cDNA clones were isolated using spinach NiR cDNA as a probe. Sequence analysis and Southern blot hybridization revealed four genes in tobacco. Two of these genes presumably derived from the ancestral species Nicotiana tomentosiformis, the other two from the ancestor N. sylvestris. Northern blot analysis showed that one gene from each ancestral genome was expressed predominantly in leaves, whilst RNA from the other was detected mostly in roots. The accumulation of both leaf and root NiR mRNAs was induced by nitrate and repressed by nitrate- or ammonium-derived metabolites. In addition, the expression of the root NiR gene was detectable in leaves of a tobacco nitrate reductase (NR)-deficient mutant. Thus, the regulation of expression of tobacco NiR genes is comparable to the regulation of expression of barley NR genes.     

Characterization of a selenate-resistant Arabidopsis mutant. Root growth as a potential target for selenate toxicity

Screening an Arabidopsis (Arabidopsis thaliana) T-DNA mutant library for selenate resistance enabled us to isolate a selenate-resistant mutant line (sel1-11). Molecular and genetic characterization showed that the mutant contained a lesion in the SULTR1;2 gene that encodes a high affinity root sulfate transporter. We showed that SULTR1;2 is the only gene among 13 mutated genes of the Arabidopsis sulfate transporter family whose mutation conferred selenate resistance to Arabidopsis. The selenate resistance phenotype of the sel1-11 mutant was mirrored by an 8-fold increase of root growth in the presence of selenate as shown by the calculated lethal concentration values. The impairment of SULTR1;2 activity in sel1-11 resulted in a reduced (35)S-sulfate uptake capacity by both roots and calli and a reduced sulfate and selenate content in root, shoot, and calli. Comparing sulfate-to-selenate ratios instead of absolute sulfate and selenate contents in roots and shoots enabled us to gain better insight into the mechanism of selenate toxicity in Arabidopsis. Roots of the sel1-11 mutant line showed a higher sulfate to selenate ratio than that of wild-type roots, while there were no significant differences in sulfate to selenate ratios in shoots of wild-type and mutant lines. These results indicated that the mechanism that confers the selenate resistance phenotype to the sel1-11 line takes place rather in the roots. It might be in part the result of a lower selenate uptake and of a protective effect of sulfate against the toxic effects of selenate on root growth. These results revealed in plants a central and specific role of the transporter SULTR1;2 in selenate sensitivity; they further suggested that root growth and potentially the root tip activity might be a specific target of selenate toxicity in Arabidopsis.     

Characterization of two functional phosphoenolpyruvate/phosphate translocator (PPT) genes in Arabidopsis--AtPPT1 may be involved in the provision of signals for correct mesophyll development

The Arabidopsis thaliana chlorophyll a/b-binding protein underexpressed 1 (cue1) mutant shows a reticulate leaf phenotype and is defective in a plastidic phosphoenolpyruvate (PEP)/phosphate translocator (AtPPT1). A functional AtPPT1 providing plastids with PEP for the shikimate pathway is therefore essential for correct leaf development. The Arabidopsis genome contains a second PPT gene, AtPPT2. Both transporters share similar substrate specificities and are therefore able to transport PEP into plastids. The cue1 phenotype could partially be complemented by ectopic expression of AtPPT2 but obviously not by the endogeneous AtPPT2. Both genes are differentially expressed in most tissues: AtPPT1 is mainly expressed in the vasculature of leaves and roots, especially in xylem parenchyma cells, but not in leaf mesophyll cells, whereas AtPPT2 is expressed ubiquitously in leaves, but not in roots. The expression profiles are corroborated by tissue-specific transport data. As AtPPT1 expression is absent in mesophyll cells that are severely affected in the cue1 mutant, we propose that the vasculature-located AtPPT1 is involved in the generation of phenylpropanoid metabolism-derived signal molecules that trigger development in interveinal leaf regions. This signal probably originates from the root vasculature where only AtPPT1, but not AtPPT2, is present.     

Transcriptional regulation and functional properties of <Emphasis Type="Italic">Arabidopsis</Emphasis> Pht1;4, a high affinity transporter contributing greatly to phosphate uptake in phosphate deprived plants

Phosphate mobilization into the plant is a complex process requiring numerous transporters for absorption and translocation of this major nutrient. In the genome of Arabidopsis thaliana, nine closely related high affinity phosphate transporters have been identified but their specific roles remain unclear. Here we report the molecular, histological and physiological characterization of Arabidopsis pht1;4 high affinity phosphate transporter mutants. Using GUS-gene trap and in situ hybridization, Pht1;4 was found mainly expressed in inorganic phosphate (Pi) limiting medium in roots, primarily in the epidermis, the cortex and the root cap. In addition to this, expression was also observed at the lateral root branch points on the primary root and in the stele of lateral roots, suggesting a role of Pht1;4 in phosphate absorption and translocation from the growth medium to the different parts of the plant. Pi-starved pht1;4 plantlets exhibited a strong reduction of phosphate uptake capacity (40). This phenotype appears only related to the pht1;4 mutation as there were no obvious changes in the expression of other Pht1 family members in the mutants background. However, after 10 days of growth on phosphate deficient or sufficient medium, the Pi content in the mutants was not significantly different from that of the corresponding wild type controls. Furthermore, the mutants did not display any obvious growth defects or visible phenotypes when grown on a low phosphate containing medium. The work described here offers a first step in the complex genetic dissection of the phosphate transport system in planta.     

Tomato roots have a functional silicon influx transporter but not a functional silicon efflux transporter

Silicon (Si) accumulation in shoots differs greatly with plant species, but the molecular mechanisms for this interspecific difference are unknown. Here, we isolated homologous genes of rice Si influx (SlLsi1) and efflux (SlLsi2) transporter genes in tomato (Solanum lycopersicum L.) and functionally characterized these genes. SlLsi1 showed transport activity for Si when expressed in both rice lsi1 mutant and Xenopus laevis oocytes. SlLsi1 was constitutively expressed in the roots. Immunostaining showed that SlLsi1 was localized at the plasma membrane of both root tip and basal region without polarity. Furthermore, overexpression of SlLsi1 in tomato increased Si concentration in the roots and root cell sap but did not alter the Si concentration in the shoots. By contrast, two Lsi2-like proteins did not show efflux transport activity for Si in Xenopus oocytes. However, when functional CsLsi2 from cucumber was expressed in tomato, the Si uptake was significantly increased, resulting in higher Si accumulation in the leaves and enhanced tolerance of the leaves to water deficit and high temperature. Our results suggest that the low Si accumulation in tomato is attributed to the lack of functional Si efflux transporter Lsi2 required for active Si uptake although SlLsi1 is functional.