Developmental and pathogen-induced activation of an msr gene, str246C, from tobacco involves multiple regulatory elements

A family of genes, the so-called msr genes (multiple stimulus response), has recently been identified on the basis of sequence homology in various plant species. Members of this gene family are thought to be regulated by a number of environmental or developmental stimuli, although it is not known whether any one member responds more specifically to one stimulus, or whether each gene member responds to various environmental stimuli. In this report, we address this question by studying the tobacco msr gene str246C. Using transgenic tobacco plants containing 2.1 kb of 5 flanking DNA sequence from the str246C gene fused to the -glucuronidase (GUS) coding region, the complex expression pattern of the str246C promoter has been characterized. Expression of the str246C promoter is strongly and rapidly induced by bacterial, fungal and viral infection and this induction is systemic. Elicitor preparations from phytopathogenic bacteria and fungi activate the str246C promoter to high levels, as do wounding, the application of auxin, auxin and cytokinin, salicylic acid or copper sulfate, indicating the absence of gene specialization within the msr gene family, at least for str246C. In addition, GUS activity was visualized. histochemically in root meristematic tissues of tobacco seedlings and is restricted to roots and sepals of mature plants. Finally, analysis of a series of 5 deletions of the str246C promoter-GUS gene fusion in transgenic tobacco plants confirms the involvement of multiple regulatory elements. A region of 83 by was found to be necessary for induction of promoter activity in response to Pseudomonas solanacearum, while auxin inducibility and root expression are apparently not controlled by this element, since its removal does not abolish either response. An element of the promoter with a negative effect on promoter activation by P. solanacearum was also identified.Joint first authors     

Cloning,characterization, and expression of the <Emphasis Type="Italic">BjEXPA1</Emphasis> gene and its promoter region from <Emphasis Type="Italic">Brassica juncea</Emphasis> L.

To explore the regulation of expansin expression under heavy metal stresses, a novel expansin gene, BjEXPA1, and its upstream region were obtained from Indian mustard (Brassica juncea L.). BjEXPA1 encodes a cell wall protein of the α-subgroup of the expansin family. The expression of BjEXPA1 is regulated by exogenous phytohormones, NaCl, and heavy metals. Sequence analysis of its upstream region revealed the presence of several putative cis-acting elements, including phytohormone response, abiotic stress response, and tissue-specific elements. GUS activity under the control of the BjEXPA1 promoter allowed observation of BjEXPA1 spatial and temporal expression patterns. The reporter construct indicated that BjEXPA1 is induced by exogenous gibberellin and auxin, and inhibited by abscisic acid, CdCl₂ and PEG6000. Transgenic tobacco seedlings overexpressing BjEXPA1 showed an altered phenotype and enhanced sensitivity to cadmium. Higher H₂O₂ accumulation and extensive plasma membrane damage were detected in the leaves of transgenic seedlings when exposed to Cd, compared with wild-type plants. Our findings support the conclusion that overexpression of BjEXPA1 in tobacco renders plants susceptible to Cd stress, probably due to disruption of the growth process and/or the breakdown of cell walls.     

CYP72B1基因和AUR3基因响应光、生长素和油菜素内酯的转录调控机制研究

为研究光、生长素和油菜素内酯在基因层次上的互作机制,开发了转录调控元件识别工具OCMMat,其中,在对共表达基因信息和直系同源基因信息进行整合时,利用了转录调控元件在直系同源基因启动子中的富集性.利用该方法发现,CYP7281基因和AUR3基因启动子含有3个相同的调控模序GAGACA、AAGAAAAA、ATCATG,它们分别承担了AuxRE元件、GT元件和GT辅助元件的功能.其中,ATCATG模序是目前尚未报道过的调控元件,与AAGAAAAA模序的距离相对恒定.基于调控元件识别结果,构建了CYP7281基因和AUR3基因响应光、生长素和油菜素内酯的转录调控模型,模型显示:光信号和生长素、油菜素内酯信号在CYP72B1基因和AUR3基因的转录调控元件上相互交叠,而生长素和油菜素内酯信号则在转录因子ARF水平上相交.     

Analysis of the Promoter of the Auxin-Inducible Gene, parC, of Tobacco

The auxin-responsive region (AuxRR) in the promoter of the parCgene was analyzed in transgenic tobacco plants in which the5' flanking region of the parC promoter was placed upstreamof the gene for rß-glucuronidase (GUS). The AuxRRwas located between nucleotides (nt) –226 and –54.Detailed dissection of this segment revealed that the presenceof the non-contiguous sequences from nt –226 to –151and from nt –84 to –54 was required for the expressionof the auxin responsiveness of the parC promoter. The sequencefrom nt –226 to –151 was found to contain a sequencewhich resembles the as-1 element in the 35S promoter of cauliflowermosaic virus (CaMV). Although it has been reported that theas-1 element is involved in auxin responsiveness [Liu and Lam(1994) J. Biol. Chem. 269: 668], we showed that introductionof a point mutation into the as-1-like sequence completely eliminatedauxin responsiveness, a result that suggests that the sequenceis indispensable for auxin responsiveness. However, the presenceof the as-1-like sequence alone was not sufficient for auxinresponsiveness, since the segment (nt –226 to –84)that included the as-1-like sequence failed to confer auxinresponsiveness on the core promoter. It is possible that thetwo separately located sequences play specific roles in interactionswith trans-factors that are required for the expression of theauxin responsiveness of the parC promoter. (Received March 11, 1996; Accepted July 9, 1996)     

The ocs element in the soybean GH2/4 promoter is activated by both active and inactive auxin and salicylic acid analogues

The octopine synthase (ocs or ocs-like) element has been previously reported to be responsive to the plant hormones, auxin, salicylic acid, and methyl jasmonate. Using transient assays with carrot protoplasts, we have demonstrated that an ocs element from the soybean auxin-inducible GH2/4 promoter is not only activated by strong auxins (i.e, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, -naphthalene acetic acid) and salicylic acid, but also by weak auxin analogues (-naphthalene acetic acid), inactive auxin analogs (i.e., 2,3-dichlorophenoxyacetic acid, 2,4,6-trichlorophenoxyacetic acid), and inactive salicylic acid analogs (3-hydroxybenzoic acid and 4-hydroxybenzoic acid). Our results indicate that the ocs element in the GH2/4 promoter is not selectively induced by plant hormones and might function similarly to tandem AP-1 sites in some animal glutathione S-transferase (GST) genes. The ocs element, like the AP-1 sites in animal GST promoters, may be induced not only by certain hormones but also by some non-hormonal stress-inducing or electrophilic agents.Abbreviations GST glutathione S-transferase - MUG 4-methyl-umbelliferyl-glucuronide - GUS -glucuronidase - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,3-D 2,3-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - 2,4,6-T 2,4,6-trichlorophenoxyacetic acid - NAA naphthalene acetic acid - SA salicylic acid - SARE putative salicylic acid-responsive element - BA benzoic acid - UTR untranslated region - nos nopaline synthase - ocs octopine synthase - mas mannopine synthase - ocs element-(–)46 CaMV 35S promoter-GUS reporter gene: the ocs element fused to a minimal –46 cauliflower mosaic virus 35S promoter fused to a GUS reporter gene with a 3 nos untranslated region     

Promoter analysis of the auxin-regulated tobacco glutathione S-transferase genes Nt103-1 and Nt103-35

We have analysed the promoter regions of two closely related auxin-regulated glutathione S-transferase genes. All active deletion constructs tested showed expression of the reporter gene -glucuronidase (gusA) in root tips of young seedlings and newly developing lateral roots. Auxin treatment greatly enhanced the level of expression. The Nt103-1 promoter region –370/–276 was found to be necessary, at least as a quantitative element to confer auxin-responsiveness to a reporter gene, and sequences responsible for the auxin-responsiveness must be located downstream of –370. The region –651/–370 contains sequence information necessary for uninduced expression. The Nt103-35 promoter manifested its auxin-responsiveness within the –504/–310 region. Electrophoretic mobility shift analysis, using nuclear extracts from tobacco leaves and suspension cells, identified a factor binding to a sequence (ap103, TGAGTCT) at position –560 of the Nt103-1 promoter, which shows homology to the mammalian AP-1 site. A second factor was found to bind a sequence (as103, ATAGCTAAGTGCTTACG) with homology to the CaMV 35S promoter as-1 element. The as103 element is present in both promoters and positioned around –360, so within the region determined to be indispensable for the response to auxin. A third factor was found binding to the –276/–190 region of both promoters. Combined, these data point to the relevance of a 90 bp region for auxin-induced activity of both tobacco genes. The ASF-1 like factor binding to the as103 element within this region might be involved in mediating the auxin response.     

Construction of an inducible transposon,INAc, to develop a gene tagging system in higher plants

An inducible transposable element, termed INAc (inducible Activator), was constructed for development of a gene tagging system in higher plants. The advantage of such an inducible element is that, unlike the native transposon, its excision can be induced at any time during plant development and the resulting mutants are stable after removal of the inducer. A fusion of the SA inducible promoter (PR-1a) with the Ac transposase gene was inserted together with a hygromycin resistance gene between ca. 400 bp sequences from each end of the maize Ac element, yielding INAc. The INAc element was introduced into tobacco and tomato plants. A high frequency of spontaneous transposition was apparent in primary transformed tomato calli but not in tobacco calli. Treatment of tobacco plants with salicylic acid induced transposition of INAc in both somatic and germinal tissue, with germinal transposition events being revealed by characterization of the progeny of transformed plants whose flowers were exposed to SA. The INAc element thus exhibits potential for development of an inducible transposon system suitable for gene isolation in heterologous plant species.     

檀香NDH脱氢酶基因的克隆、定位与启动子分析

为研究檀香NDH脱氢酶基因的功能和调控机制,该文以檀香心材为材料,利用RACE技术克隆SaNDH6基因的全长序列,利用实时荧光定量PCR(RT-qPCR)技术分析其组织和激素处理后的表达模式,在拟南芥原生质体观测其亚细胞定位,利用PlantCARE分析SaNDH6起始密码子ATG上游2 kb的启动子序列,同时运用PlantRegMap预测可能与其结合的转录因子。结果表明:(1)SaNDH6编码303个氨基酸,为疏水蛋白,亚细胞定位于叶绿体。(2)进化树分析表明,檀香SaNDH6与木本植物NDH6进化关系较近。(3)PlantCARE分析发现,SaNDH6启动子中除含有ACE、AE-box、Box 4、G-Box和GT1-motif等大量光响应元件外,同时还有茉莉酸甲酯(MeJA)反应元件CGTCA-motif和TGACG-motif,赤霉素(GA₃)响应元件P-box,以及防御和胁迫响应元件TC-rich repeats等。(4)PlantRegMap分析发现,有76个转录因子可能与SaNDH6启动子结合,其中ERF家族最多,达40个。(5)SaNDH6在檀香的根、心材、叶片和愈伤组织中均有表达,其中在叶片中的表达量较高; 用1×10^-4 mol·L^-1的MeJA和GA₃分别处理檀香愈伤组织后,与处理前(0 h)相比,SaNDH6的表达均在3 h后显著升高。综上结果表明,檀香SaNDH6为核基因编码的蛋白,受光和激素等诱导表达,SaNDH6可能参与檀香逆境胁迫反应的过程。