Activation of the NaCl- and drought-induced RD29A and RD29B promoters by constitutively active Arabidopsis MAPKK or MAPK proteins

Mitogen-activated protein (MAP) kinases mediate cellular responses to a wide variety of stimuli. Activation of a MAP kinase (MAPK) occurs after phosphorylation by an upstream MAP kinase kinase (MAPKK). The Arabidopsis thaliana genome encodes 10 MKKs, but few of these have been shown directly to activate any of the 20 Arabidopsis MAPKs (AtMPKs) and NaCl-, drought- or abscisic acid (ABA)-induced genes RD29A or RD29B. We have constructed the constitutively activated form for nine of the 10 AtMKK proteins, and tested their ability to activate the RD29A and RD29B promoters and also checked the ability of the nine activated AtMKK proteins to phosphorylate 11 of the AtMPK proteins in transient assays. The results show that three proteins, AtMKK1, AtMKK2 and AtMKK3, could activate the RD29A promoter, while these three and two additional AtMKK6/8 proteins could activate the RD29B promoter. Four other proteins, AtMKK7/AtMKK9 and AtMKK4/AtMKK5, can cause hypersensitive response (HR) in tobacco leaves using transient analysis. The activation of the RD29A promoter correlated with four uniquely activated AtMPK proteins. A novel method of activating AtMPK proteins by fusion to a cis-acting mutant of a human MAPK kinase MEK1 was used to confirm that specific members of the AtMPK gene family can activate the RD29A stress pathway.     

拟南芥干旱胁迫诱导型启动子RD29B驱动AtCKX1基因植物表达载体的构建

从拟南芥基因组中分别克隆AtCKX1基因和RD29B基因5′-侧翼1705bp启动子区域序列,生物信息学表明,AtCKX1含有黄素腺嘌呤二核苷酸(FAD)和细胞分裂素的结合位点;RD29B启动子片段中存在ABA响应元件(ABA response element;ABRE)、Myb结合位点、TATA-盒、CAAT-盒等顺式作用元件。分别将AtCKX1和RD29B插入载体pCAMBIA1390,构建了由RD29B驱动的AtCKX1的植物双元表达载体p1390RD29BAtCKX1。     

Differential interactions of promoter elements in stress responses of the Arabidopsis Adh gene.

The Adh (alcohol dehydrogenase, EC 1.1.1.1.) gene from Arabidopsis thaliana (L.) Heynh. can be induced by dehydration and cold, as well as by hypoxia. A 1-kb promoter fragment (CADH: -964 to +53) is sufficient to confer the stress induction and tissue-specific developmental expression characteristics of the Adh gene to a beta-glucuronidase reporter gene. Deletion mapping of the 5' end and site-specific mutagenesis identified four regions of the promoter essential for expression under the three stress conditions. Some sequence elements are important for response to all three stress treatments, whereas others are stress specific. The most critical region essential for expression of the Arabidopsis Adh promoter under all three environmental stresses (region IV: -172 to -141) contains sequences homologous to the GT motif (-160 to -152) and the GC motif (-147 to -144) of the maize Adh1 anaerobic responsive element. Region III (-235 to -172) contains two regions shown by R.J. Ferl and B.H. Laughner ([1989] Plant Mol Biol 12: 357-366) to bind regulatory proteins; mutation of the G-box-1 region (5'-CCACGTGG-3', -216 to -209) does not affect expression under uninduced or hypoxic conditions, but significantly reduces induction by cold stress and, to a lesser extent, by dehydration stress. Mutation of the other G-box-like sequence (G-box-2: 5'-CCAAGTGG-3', -193 to -182) does not change hypoxic response and affects cold and dehydration stress only slightly. G-box-2 mutations also promote high levels of expression under uninduced conditions. Deletion of region I (-964 to -510) results in increased expression under uninduced and all stress conditions, suggesting that this region contains a repressor binding site. Region II (-510 to -384) contains a positive regulatory element and is necessary for high expression levels under all treatments.     

DNA demethylases target promoter transposable elements to positively regulate stress responsive genes in Arabidopsis

Background

DNA demethylases regulate DNA methylation levels in eukaryotes. Arabidopsis encodes four DNA demethylases, DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1), DEMETER-LIKE 2 (DML2), and DML3. While DME is involved in maternal specific gene expression during seed development, the biological function of the remaining DNA demethylases remains unclear.

Results

We show that ROS1, DML2, and DML3 play a role in fungal disease resistance in Arabidopsis. A triple DNA demethylase mutant, rdd (ros1 dml2 dml3), shows increased susceptibility to the fungal pathogen Fusarium oxysporum. We identify 348 genes differentially expressed in rdd relative to wild type, and a significant proportion of these genes are downregulated in rdd and have functions in stress response, suggesting that DNA demethylases maintain or positively regulate the expression of stress response genes required for F. oxysporum resistance. The rdd-downregulated stress response genes are enriched for short transposable element sequences in their promoters. Many of these transposable elements and their surrounding sequences show localized DNA methylation changes in rdd, and a general reduction in CHH methylation, suggesting that RNA-directed DNA methylation (RdDM), responsible for CHH methylation, may participate in DNA demethylase-mediated regulation of stress response genes. Many of the rdd-downregulated stress response genes are downregulated in the RdDM mutants nrpd1 and nrpe1, and the RdDM mutants nrpe1 and ago4 show enhanced susceptibility to F. oxysporum infection.

Conclusions

Our results suggest that a primary function of DNA demethylases in plants is to regulate the expression of stress response genes by targeting promoter transposable element sequences.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0458-3) contains supplementary material, which is available to authorized users.     

Drought-avoidant soybean germplasm maintains nitrogen-fixation capacity under water stress

Inoculated soybeans (Glycine max L. (Merrill)) were grown in controlled environments to evaluate the relationship between genotype and plant water status on nodule function, nitrogen assimilation, growth rates, and seed yield. Plants were grown under well-watered (WW) and water-stressed (WS) conditions during the linear pod-filling growth stage in sand culture using N-free nutrient solution. Dry matter and N accumulation were greater for the drought-adapted Plant Introduction 416937 (PI) than for Forrest, a commercially adapted genotype of similar phenology. These differences are attributed to: (i) more favorable internal water balance throughout the pod-filling period (higher total leaf water potential), (ii) higher photosynthetic function (more total leaf area and higher net carbon exchange rates), and (iii) stronger nodule function (larger nodule mass, greater specific and total nodule activity, and thus more nitrogen assimilation) for the PI than for Forrest. While Forrest out yielded the PI under WW conditions, the percentage reduction in seed mass per plant was less for the PI than for Forrest when both genotypes were exposed to desiccating conditions. The inference is that soybean germplasm with the capacity to maintain tissue turgidity, and thus leaf and nodule function, during reproductively-imposed desiccation may reduce the extent to which yield is compromised during drought. These findings have implications for the role of symbiotic nitrogen fixation in conserving yield under dry weather conditions.Abbreviations DAE Days After Emergence - NCE Net CO₂ Exchange - PI PI 416937 - SNA Specific Nodule Activity - TNA Total Nodule Activity - WS Water Stressed - WW Well Watered     

拟南芥TCH4基因启动区转录调控元件的计算识别

TCH4基因在植物次生生长、疾病抵抗和逆境适应方面具有重要作用, 能被多种激素、环境和机械信号诱导表达。利用拟南芥TCH4的直系同源基因和芯片数据进行了启动子序列分析, 结果共识别出9个转录调控元件。它们均包含有已知元件序列, 并且在部分共表达基因和对应的直系同源基因启动子中排列顺序一致。根据已有TCH4基因启动子研究, 其中4个已被报道, 另5个为本研究新发现。根据预测结果进行知识整合, 构建了TCH4基因转录调控机制模型。     

拟南芥干旱诱导型启动子RD29A驱动花生AhNCED1基因双元表达载体的构建

从拟南芥基因组中克隆RD29A基因5'-侧翼520bp启动子区域序列,生物信息学分析表明,该启动子片段中存在脱水胁迫响应元件(DRE)、ABA响应元件(ABRE)、TATA-box、CAAT-box等顺式作用元件。构建了干旱诱导型启动子AtRD29Ap驱动花生AhNCED1基因的植物双元表达载体pAtRD29Ap::AhNCED1。     

Expression of the Zinnia TED3 promoter in developing tracheary elements of transgenic Arabidopsis

To determine the regulatory mechanism of gene expression in the early stages of tracheary element (TE) differentiation, we isolated and characterized a genomic fragment of TED3 whose mRNA is expressed preferentially in differentiating TEs 12–24 h before morphological changes in the in vitro Zinnia system. Transgenic Arabidopsis plants with a chimeric gene of the 537 bp TED3 promoter and the -glucuronidase (GUS) reporter gene indicated the strong expression of the GUS gene by the TED3 promoter in TEs, in particular in immature TEs as well as stipules and trichomes. GUS expression driven by the promoter was also induced in callus, in which GUS activity was localized in immature TEs and slender cells around TEs that may be TE precursor cells. The TED3 promoter was not significantly activated by wounding. This pattern of expression differed clearly from that of other vascular tissue-related genes such as PAL, 4CL, and GRP1.8. The nature of TED3 promoter enables us to use it to monitor TE differentiation in tissue and to introduce foreign genes preferentially into immature TE.     

干旱胁迫下活化水灌溉对大豆生长特征的影响

为了解在不同干旱条件下活化水灌溉对大豆生长特征的影响,探究活化水灌溉对大豆生长的影响机制。采用室内基质栽培大豆试验,在基质最大持水量(80%含水量)的95%～100%、75%～85%、55%～65%和35%～45% 4个水分条件下,分别用自来水、磁化水、增氧水和先磁化再增氧4种水灌溉,研究活化水对大豆苗期生长特征的影响。结果表明: 经过30 d的生长后,35%～45%重度干旱条件下磁化水灌溉的总生物量、叶面积、根冠比和根长与自来水灌溉相比分别增加了67.6%、23.5%、84.6%和122.8%,磁化增氧水灌溉分别增加了70.8%、24.0%、61.9%和162.3%,对叶绿素含量无显著影响;其余处理上述指标与自来水相比略有下降。表明重度干旱条件下,磁化水灌溉可以有效促进大豆根系生长、提高根冠比,提高大豆的水分利用效率,缓解干旱胁迫带来的负面效应。