Expression of the Arabidopsis AtAux2-11 auxin-responsive gene in transgenic plants

Five constructions containing deletions of the promoter from an auxin-inducible gene of Arabidopsis thaliana, AtAux2-11, were fused to the coding region of the reporter gene LacZ, which encodes -galactosidase, and a polyadenylation 3-untranslated nopaline synthase sequence from Agrobacterium. These chimeric genes were introduced into Arabidopsis by Agrobacterium tumefaciens-mediated transformation, and expression of the gene was examined by spectrophotometric and histochemical analyses. A 600 bp fragment from the AtAux2-11 promoter conferred histochemical patterns of staining similar to the longest 5 promoter tested, a 3.0 kb fragment. Localization of AtAux2-11/LacZ activity in the transgenic plants revealed spatial and temporal expression patterns that correlated with tissues and cells undergoing physiological processes modulated by auxin. LacZ activity was expressed in the elongating region of roots, etiolated hypocotyls, and anther filaments. Expression was detected in the vascular cylinder of the root and the vascular tissue, epidermis, and cortex of the hypocotyl, and filament. The AtAux2-11/LacZ gene was preferentially expressed in cells on the elongating side of hypocotyls undergoing gravitropic curvature. Expression of the chimeric gene in the hypocotyls of light-grown seedlings was less than that in etiolated seedling hypcotyls. The AtAux2-11/LacZ gene was active in the root cap, and expression in the root stele increased at sites of lateral root initiation. Staining was evident in cell types that develop lignified cell walls, e.g. trichomes, anther endothecial cells, and especially developing xylem. The chimeric gene was not expressed in primary meristems. While the magnitude of expression increased after application of exogenous auxin (2,4-D), the histochemical localization of AtAux2-11/LacZ remained unchanged.Transgenic plants with a 600 bp promoter construct (–0.6 kb AtAux2-11/LacZ) had higher levels of basal and auxin-inducible expression than plants with a 3.0 kb promoter construct. Transgenic plants with a –500 bp promoter had levels of expression similar to the –3.0 kb construct. The –0.6 kb AtAux2-11/LacZ gene responded maximally to a concentration of 5 × 10^–6 to 5 × 10^–5 M 2,4-D and was responsive to as little as 5 × 10^–8 M. The evidence presented here suggests that this gene may play a role in several auxin-mediated developmental and physiological processes.co-first authors     

Meristem-specific gene expression directed by the promoter of the S-phase-specific gene,cyc07, in transgenic Arabidopsis

A genomic clone for the cyc07 gene, which is expressed specifically at the S phase during the cell cycle in synchronous cultures of periwinkle (Catharanthus roseus) cells, was isolated. Determination of the nucleotide sequence of the clone revealed that the cyc07 gene consists of seven exons separated by six introns. Genomic Southern analysis indicated that the cyc07 gene is present as a single copy per haploid genome in periwinkle. Expression of related genes was detected in a wide range of other plants. Transgenic Arabidopsis plants were generated that expressed the gene for -glucuronidase (GUS) under the control of the promoter of the cyc07 gene. The tissue-specific pattern of expression directed by the promoter was investigated by analysis of GUS activity. Histochemical tests demonstrated that 589 bp of the 5-upstream sequence of the cyc07 gene could direct specifical expression of the GUS reporter gene in meristematic tissues in transgenic plants. The spatial pattern of expression directed by the promoter was closely correlated with meristematic activity and cell proliferation, suggesting an association between the function of the cyc07 gene and cell proliferation.     

Isolation,expression, and evolution of the gene encoding mitochondrial elongation factor Tu in Arabidopsis thaliana

We have characterized a second nuclear gene (tufM) in Arabidopsis thaliana that encodes a eubacterial-like protein synthesis elongation factor Tu (EF-Tu). This gene does not closely resemble the previously described Arabidopsis nuclear tufA gene, which encodes the plastid EF-Tu, and does not contain sequence elements found in all cyanobacterial and plastid tufA genes. However, the predicted amino acid sequence includes an N-terminal extension which resembles an organellar targeting sequence and shares three unique sequence elements with mitochondrial EF-Tu's, from Saccharomyces cerevisiae and Homo sapiens, suggesting that this gene encodes the Arabidopsis mitochondrial EF-Tu. Consistent with this interpretation, the gene is expressed at a higher level in flowers than in leaves. Phylogenetic analysis confirms the mitochondrial character of the sequence and indicates that the human, yeast, and Arabidopsis tufM genes have undergone considerably more sequence divergence than their cytoplasmic counterparts, perhaps reflecting a cross-compartmental acceleration of gene evolution for components of the mitochondrial translation apparatus. As previously observed for tufA, the tufM gene is present in one copy in Arabidopsis but in several copies in other species of crucifers.     

Tissue-specific expression of the rolA gene mediates morphological changes in transgenic tobacco

The spatial and temporal activity of the entire and individual promoter domains of the rolA gene of Agrobacterium rhizogenes was investigated and correlated with the distinctive features of the phenotypes of transgenic tobacco plants. The GUS assay was performed in the presence of an oxidative catalyst during the development of transgenic plants expressing chimeric genes containing the -glucuronidase coding sequence under the control of the different promoter domains. In situ hybridization was also used on transgenic plants harbouring rolA under the control of the entire or deleted promoter. This paper demonstrates for the first time that the entire rolA promoter, composed of domains, A, B and C, is silent in seeds, then activated at the onset of germination in the cotyledons and in the elengation zone of the radicle and is finally expressed throughout the vegetative and floral phases. Domains B+C, which were sufficient to induce wrinkled leaves and short internodes, were active in all the stem tissues, but only in the companion cells of the phloem strands of the leaves. Domain C, which specified a dwarf phenotype with normal leaves, was weakly expressed in the stem vascular bundles and in the leaf internal phloem. These results indicate that the vascular bundles are the primary targets for the generation of the short internode phenotype. Furthermore, the local expression of rolA in the stem vascular bundles induced a size reduction of the surrounding parenchyma cells, suggesting the existence of some diffusible factor(s) associated with the expression of the rolA gene.     

Calmodulin isoforms in Arabidopsis encoded by multiple divergent mRNAs

Three new, unique cDNA sequences encoding isoforms of calmodulin (CaM) were isolated from an Arabidopsis cDNA library cloned in gt10. These sequences (ACaM-4, -5, and -6) represent members of the Arabidopsis CaM gene family distinct from the three DNA sequences previously reported. ACaM-4 and -6 encode full-length copies of CaM mRNAs of ca. 0.75 kb. The ACaM-5 sequence encodes a partial length copy of CaM mRNA that is lacking sequences encoding the amino-terminal 10 amino acids of mature CaM and the initiator methionine. The derived amino acid sequence of ACaM-5 is identical to the sequences encoded by two of the previously characterized ACaM cDNAs, and is identical to TCH-1 mRNA, whose accumulation was increased by touch stimulation. The polypeptides encoded by ACaM-4 and -6 differ from that encoded by ACaM-5 by six and two amino acid substititions, respectively. Most of the deduced amino acid sequence substitutions in the Arabidopsis CaM isoforms occurred in the fourth Ca²⁺-binding domain. Polymerase chain reaction amplification assays of ACaM-4, -5 and -6 mRNA sequences indicated that each accumulated in Arabidopsis leaf RNA fractions, but only ACaM-4 and -5 mRNAs were detected in silique total RNA. The six different CaM cDNA sequences each hybridize with unique Eco RI restriction fragments in genomic Southern blots of Arabidopsis DNA, indicating that these sequences were derived from distinct structural genes. Our results suggest that CaM isoforms in Arabidopsis may have evolved to optimize the interaction of this Ca²⁺-receptor protein with specific subsets of response elements.     

Tissue-specific and constitutive expression of a hypothetical gene At2g37610 in transgenic Arabidopsis

Hypothetical genes should play important roles in plant growth and development, although their biological functions await elucidation. One of these genes, namely At2g37610, caught our attention during the gene cloning of several salt-tolerant mutants. Promoter-GUS fusion analysis indicated a unique tissue-specific expression pattern of At2g37610 in Arabidopsis. Constitutive expression of the gene under 35S promoter caused obvious morphological changes in transgenic Arabidopsis plants, such as curled rosette leaves and bushy phenotype at maturity. Phenotypic characterization revealed that the cause of the bushy phenotype was the enhanced lateral bud outgrowth at the bottom region of the primary inflorescence, which is different from that of reported mutant plants (bushy or branched) such as max, axr1, and bus mutants. Together, these data suggest that At2g37610 is a possible novel gene related to the regulation of leaf development and shoot patterning.     

Developmentally regulated expression of a sunflower 11S seed protein gene in transgenic tobacco

Summary The trifunctional TRP1 gene from Neurospora crassa (N-TRP1) was subcloned into the yeast-Escherichia coli shuttle vector YEp13 and expressed in Saccharomyces cerevisiae. The three activities of the N-TRP1 gene product were detected in yeast mutants that lacked either N-(5-phosphoribosyl) anthranilate (PRA) isomerase or both the glutamine amidotransferase function of anthranilate synthase and indole-3-glycerol phosphate (InGP) synthase. The protein was detected on immunoblots only as the full length 83 kda product indicating that the trifunctional gene product was expressed in yeast primarily in a fully active, undegraded form. By placing the subcloned N-TRP1 gene under the control of the inducible PHO5 promoter from yeast, the expression of all three activities was increased to more than ten fold that of wild-type yeast and the overproduced protein could be visualized by SDS-polyacrylamide gel electrophoresis of crude extract and Coomassie Blue staining. Using the expression system described the effect of selective deletion of regions of the coding sequence of the N-TRP1 gene on expression of the three activities was tested. Expression of either the F- or C-domains, catalyzing respectively the PRA isomerase or InGP synthase activities, did not depend on the presence of the other domain in the active polypeptide. Furthermore, normal dimer formation occurred with a protein active for InGP synthase in a deletion derivative lacking most of the PRA isomerase domain, ruling out the hypothesis that interaction between the active site regions for PRA isomerase and InGP synthase accounted for dimer formation in the trifunctional product.Abbreviations PRA N-(5'-phosphoribosyl)anthranilate - InGP indole-3-glycerol phosphate - SDS sodium dodecyl sulfate     

Drought tolerance through over-expression of the expansin gene TaEXPB23 in transgenic tobacco

Expansins are proteins that are the key regulators of wall extension during plant growth. To investigate the role of TaEXPB23, a wheat expansin gene, we analyzed TaEXPB23 mRNA expression levels in response to water stress in wheat and examined the drought resistance of transgenic tobaccos over-expressing TaEXPB23. We found that the expression of TaEXPB23 corresponded to wheat coleoptile growth and the response to water stress. The results also indicated that the transgenic tobacco lines lost water more slowly than the wild-type (WT) plants under drought stress; their cells could sustain a more integrated structure under water stress than that of WT. Other physiological and biochemical parameters under water stress, such as electrolyte leakage, malondialdehyde (MDA) level, photosynthetic rate, F_v/F_m and ΦPSII, also suggested that the transgenic tobaccos were more drought resistant than WT plants.     

北美鹅掌楸CPP转录因子家族LtTCX2基因的克隆与分析

CPP(cystein-richpolycomb-likeproteinor Tesmin/TOS1-like)家族属于成员数目较少的一类转录因子基因家族,含有保守的富含Cystein的CRC结构域,在植物发育进程中,主要参与花发育、细胞分裂、分子进化等。为了探索CPP转录因子家族在北美鹅掌楸花发育中的作用,该文以北美鹅掌楸(Liriodendron tulipifera)为材料,采用RACE技术克隆出1个CPP-like家族基因,命名为LtTCX2,全长2 866 bp。通过NCBI网站在线分析,ORF长2 424 bp,编码了807个氨基酸,含2个保守的TSO1-like CXC结构域,分子量为88 699.25 Da,理论等电点为5.83,不稳定系数为62.38,疏水性平均值为-0.619,预测为亲水性蛋白、非跨膜蛋白、核蛋白,不含信号肽及切割位点。氨基酸比对及系统进化分析结果显示,LtTCX2与其他物种的CPP家族TCX蛋白具有较高的同源性,与亚洲莲(Nelumbo nucifera)的NnTCX2、胡杨(Populus euphratica)的PeTCX2进化关系最近。荧光定量PCR结果显示,LtTCX2基因在叶片中表达量最高,在萼片、花瓣中几乎不表达,表达量由高至低如下:叶片、花芽、雌蕊、雄蕊、茎、根、花瓣、萼片。以上结果说明,LtTCX2属于较古老、保守的一类基因,可为从分子生物学层面研究鹅掌楸属植物系统进化提供一定的理论依据。