Forcing expression of a soybean root glutamine synthetase gene in tobacco leaves induces a native gene encoding cytosolic enzyme

Glutamine synthetase (GS; EC 6.3.1.2) is present in different subcellular compartments in plants. It is located in the cytoplasm in root and root nodules while generally present in the chloroplasts in leaves. The expression of GS gene(s) is enhanced in root nodules and in soybean roots treated with ammonia. We have isolated four genes encoding subunits of cytosolic GS from soybean (Glycine max L. cv. Prize). Promoter analysis of one of these genes (GS15) showed that it is expressed in a root-specific manner in transgenic tobacco and Lotus corniculatus, but is induced by ammonia only in the legume background. Making the GS15 gene expression constitutive by fusion with the CaMV-35S promoter led to the expression of GS in the leaves of transgenic tobacco plants. The soybean GS was functional and was located in the cytoplasm in tobacco leaves where this enzyme is not normally present. Forcing this change in the location of GS caused concomitant induction of the mRNA for a native cytosolic GS in the leaves of transgenic tobacco. Shifting the subcellular location of GS in transgenic plants apparently altered the nitrogen metabolism and forced the induction in leaves of a native GS gene encoding a cytosolic enzyme. The latter is normally expressed only in the root tissue of tobacco. This phenomenon may suggest a hitherto uncharacterized metabolic control on the expression of certain genes in plants.     

Overexpression of a soybean cytosolic glutamine synthetase gene linked to organ-specific promoters in pea plants grown in different concentrations of nitrate

A glutamine synthetase gene ( GS15) coding for soybean cytosolic glutamine synthetase (GS1) fused to a constitutive promoter (CaMV 35S), a putative nodule-specific promoter (LBC(3)) and a putative root-specific promoter (rolD) was transformed into Pisum sativum L. cv. Greenfeast. Four lines with single copies of GS15 (one 35S-GS15 line, one LBC (3) -GS15 line, and two rolD-GS15 lines) were tested for the expression of GS15, levels of GS1, GS activity, N accumulation, N(2) fixation, and plant growth at different levels of nitrate. Enhanced levels of GS1 were detected in leaves of three transformed lines (the 35S-GS15 and rolD-GS15 transformants), in nodules of three lines (the LBC (3) -GS15 and rolD-GS15 transformants), and in roots of all four transformants. Despite increased levels of GS1 in leaves and nodules, there were no differences in GS activity in these tissues or in whole-plant N content, N(2) fixation, or biomass accumulation among all the transgenic lines and the wild-type control. However, the rolD-GS15 transformants, which displayed the highest levels of GS1 in the roots of all the transformants, had significantly higher GS activity in roots than the wild type. In one of the rolD-GS15 transformed lines (Line 8), increased root GS activity resulted in a lower N content and biomass accumulation, supporting the findings of earlier studies with Lotus japonicus (Limami et al. 1999 ). However, N content and biomass accumulation was not negatively affected in the other rolD-GS15 transformant (Line 9) and, in fact, these parameters were positively affected in the 0.1 mM treatment. These findings indicate that overexpression of GS15 in various tissues of pea does not consistently result in increases in GS activity. The current study also indicates that the increase in root GS activity is not always consistent with decreases in plant N and biomass accumulation and that further investigation of the relationship between root GS activity and growth responses is warranted.     

Overexpression of a soybean gene encoding cytosolic glutamine synthetase in shoots of transgenic Lotus corniculatus L. plants triggers changes in ammonium assimilation and plant development

A soybean cytosolic glutamine synthetase gene (GS15) was fused with the constitutive 35S cauliflower mosaic virus (CaMV) promoter in order to direct overexpression in Lotus corniculatus L. plants. Following transformation with Agrobacterium rhizogenes, eight independent Lotus transformants were obtained which synthesized additional cytosolic glutamine synthetase (GS) in the shoots. To eliminate any interference caused by the T-DNA from the Ri plasmid, three primary transformants were crossed with untransformed plants and progeny devoid of T_L- and T_R-DNA sequences were chosen for further analyses. These plants had a 50–80% increase in total leaf GS activity. Plants were grown under different nitrogen regimes (4 or 12 mM NH₄ ⁺) and aspects of carbon and nitrogen metabolism were examined. In roots, an increase in free amino acids and ammonium was accompanied by a decrease in soluble carbohydrates in the transgenic plants cultivated with 12 mM NH₄ ⁺ in comparison to the wild type grown under the same conditions. Labelling experiments using ¹⁵NH₄ ⁺ were carried out in order to monitor the influx of ammonium and its subsequent incorporation into amino acids. This experiment showed that both ammonium uptake in the roots and the subsequent translocation of amino acids to the shoots was lower in plants overexpressing GS. It was concluded that the build up of ammonium and the increase in amino acid concentration in the roots was the result of shoot protein degradation. Moreover, following three weeks of hydroponic culture early floral development was observed in the transformed plants. As all these properties are characteristic of senescent plants, these findings suggest that expression of cytosolic GS in the shoots may accelerate plant development, leading to early senescence and premature flowering when plants are grown on an ammonium-rich medium. Received: 17 July 1996 / Accepted: 16 October 1996     

The sucrose synthase-1 promoter from Citrus sinensis directs expression of the β-glucuronidase reporter gene in phloem tissue and in response to wounding in transgenic plants

Interest in phloem-specific promoters for the engineering of transgenic plants has been increasing in recent years. In this study we isolated two similar, but distinct, alleles of the Citrus sinensis sucrose synthase-1 promoter (CsSUS1p) and inserted them upstream of the β-glucuronidase (GUS) gene to test their ability to drive expression in the phloem of transgenic Arabidopsis thaliana and Nicotiana tabacum. Although both promoter variants were capable of conferring localized GUS expression in the phloem, the CsSUS1p-2 allele also generated a significant level of expression in non-target tissues. Unexpectedly, GUS expression was also instigated in a minority of CsSUS1p::GUS lines in response to wounding in the leaves of transgenic Arabidopsis. Deletion analysis of the CsSUS1p suggested that a fragment comprising nucleotides −410 to −268 relative to the translational start site contained elements required for phloem-specific expression while nucleotides −268 to −103 contained elements necessary for wound-specific expression. Interestingly, the main difference between the two CsSUS1p alleles was the presence of a 94-bp insertion in allele 2. Fusion of this indel to a minimal promoter and GUS reporter gene indicated that it contained stamen and carpel-specific enhancer elements. This finding of highly specific and separable regulatory units within the CsSUS1p suggests that this promoter may have a potential application in the generation of constructs for the use in the development of transgenic plants resistant to a wide variety of target pests.     

Does root glutamine synthetase control plant biomass production in Lotus japonicus L.?

To investigate the contribution of root cytosolic glutamine synthetase (GS) activity in plant biomass production, two different approaches were conducted using the model legume Lotus japonicus. In the first series of experiments, it was found that overexpressing GS activity in roots of transgenic plants leads to a decrease in plant biomass production. Using ¹⁵N labelling it was shown that this decrease is likely to be due to a lower nitrate uptake accompanied by a redistribution to the shoots of the newly absorbed nitrogen which cannot be reduced due to the lack of nitrate reductase activity in this organ. In the second series of experiments, the relationship between plant growth and root GS activity was analysed using a series of recombinant inbred lines issued from the crossing of two different Lotus ecotypes, Gifu and Funakura. It was confirmed that a negative relationship exists between root GS expression and plant biomass production in both the two parental lines and their progeny. Statistical analysis allowed it to be estimated that at least 13% of plant growth variation can be accounted for by variation in GS activity. Received: 24 September 1998 / Accepted: 14 April 1999     

Isolation and Characterization of OsGSTL1 Promoter from Rice

OsGSTL1 gene was isolated from the rice genomic library. Semi-quantitative RT-PCR analysis demonstrated that the expression of the OsGSTL1 in rice was not induced by chlorsulfuron, ethylene, abscisic acid, salicylic acid, and methyl jasmonate. In order to investigate the cis-elements of OsGSTL1 promoter, the promoter regions with different lengths were fused to the β-glucuronidase (GUS) reporter gene. All constructs were transformed into onion epidermal cells or A. thaliana plants to detect the expression patterns. In onion epidermal cells, the 160 bp fragment and longer ones were functional for directing GUS expression. In transgenic A. thaliana, the 2?155 bp upstream region of OsGSTL1 gene directed the GUS expression only in cotyledon after germination, but not in the root of young seedlings. In the later seedling, the 2?155 bp upstream region of OsGSTL1 gene directed GUS expression in roots, stems, and leaves. However, the GUS gene directed by a 1?224 bp upstream fragment is expressed in all the checked tissues. These results suggest that the spatiotemporal expression response elements of OsGSTL1 existed in the 5′-upstream region between −2?155 and −1?224 bp.     

水稻胁迫相关基因OsPM1启动子的克隆与分析

依据NCBI数据库OsPM1的序列信息,采用PCR技术扩增获取OsPM1的2 100bp的启动子序列。利用PLACE预测启动子的顺式作用元件分析表明,启动子内含有大量与胁迫相关的顺式作用元件,主要有ABA响应相关元件、脱水响应元件、低温响应元件、热激响应元件和转录因子结合元件。构建OsPM1的启动子和GUS基因融合表达载体,转入拟南芥。组织化学染色分析结果显示,非生物胁迫处理前,幼苗中GUS基因表达水平很低;干旱、低温、高盐等胁迫处理后,GUS基因表达量显著升高。研究表明,OsPM1的启动子能够显著提高在干旱、高盐和低温处理后下游基因的表达水平。     

Molecular characterization of the PpMADS1 gene from peach

PpMADS1, a member of the euAP1 clade of the class A genes, was previously cloned from peach. In this study, PpMADS1 was constitutively expressed in Arabidopsis thaliana to study its function in plant development. The transgenic A. thaliana plants containing 35S::PpMADS1 showed severe phenotype variation including early flowering, conversion of inflorescence branches to solitary flowers, formation of terminal flowers, production of higher number of carpels, petals, and stamens than non-transgenic plants, and prevention of pod shatter. Significantly, the transgenic plants produced more than one silique from a single flower. The results obtained by using cDNA microarray and real-time PCR analyses in the transgenic Arabidopsis indicated that PpMADS1 might play dual roles in regulating the floral meristem development by activating or repressing different sets of genes that would determine the different fate of a floral meristem. In addition, the PpMADS1 gene promoter was further cloned, and deletion analyses were conducted by using fused GUS as a reporter gene in transgenic A. thaliana. Histochemical staining of different organs from transgenic plants revealed the region between ?197 and ?454?bp was specific for GUS expression in flower primordium, and the region between ?454 and ?678?bp was specific for GUS expression in sepals and petals. In contrast, a negative regulatory element present between ?678 and ?978?bp could suppress GUS expression in filament.     

玉米胚胎发生后期丰富蛋白基因的启动子克隆与功能验证

该研究在生物信息学分析的基础上,克隆玉米胚胎发生后期丰富蛋白基因(MGL3)的启动子序列(pMGL3),进行非生物逆境应答元件分析以及实时定量PCR验证其非生物逆境胁迫响应特性,构建了pMGL3启动子驱动报告基因(GUS)表达载体,基因枪法转化玉米愈伤组织,通过GUS染色验证pMGL3启动子在非生物逆境胁迫下的驱动活性。再根据启动子序列分析结果,去除不同的顺式作用元件,构建不同长度pMGL3启动子驱动报告基因GUS表达载体,农杆菌介导法转化烟草叶盘,以确定pMGL3启动子的最短活性序列。结果显示:pMGL3启动子长1 554bp,存在多种与非生物逆境胁迫应答相关的调控元件,在干旱、高盐、低温胁迫及脱落酸、乙烯诱导下驱动MGL3基因增量表达,用以驱动GUS基因转化玉米愈伤组织,在高渗、高盐、低温胁迫及脱落酸诱导下具有驱动活性,且截短至325bp仍可保持驱动活性。研究表明,pMGL3启动子的确有非生物逆境诱导启动活性,进一步验证其作用机理后可运用于玉米抗逆转基因研究。     

The Promoter of the Gene for Plastidic Glutamine Synthetase (GS2) fromRice Is Developmentally Regulated and Exhibits Substrate-Induced Expression in Transgenic Tobacco Plants

A 1.3-kb fragment from the 5'-flanking region of the RGS-38gene, which encodes the plastidic glutamine synthetase in Oryzasativa L., was fused to a ß-glucuronidase (GUS) reportergene and introduced into Nicotiana tabacum by Agrobacterium-mediatedtransformation. The promoter directed GUS expression, both inleaves and in roots, and the expression of GUS was regulatedby light. The GUS activity was high in the mature leaves ofthe transgenic tobacco plants, in marked contrast to the activityof the GS1 promoter. The GS2 promoter also responded to externallyapplied ammonia, as is the case for the GS1 promoter. Theseresults suggest that the cis-acting regulatory elements thatcontrol the response to ammonia, a substrate for glutamine synthetase,are located within a 1.3-kb region of the promoter. (Received October 1, 1991; Accepted January 20, 1992)