Interaction between two cis-acting elements,ABRE and DRE,in ABA-dependent expression of Arabidopsis rd29A gene in response to dehydration and high-salinity stresses

Many abiotic stress-inducible genes contain two cis-acting elements, namely a dehydration-responsive element (DRE; TACCGACAT) and an ABA-responsive element (ABRE; ACGTGG/TC), in their promoter regions. We precisely analyzed the 120 bp promoter region (-174 to -55) of the Arabidopsis rd29A gene whose expression is induced by dehydration, high-salinity, low-temperature, and abscisic acid (ABA) treatments and whose 120 bp promoter region contains the DRE, DRE/CRT-core motif (A/GCCGAC), and ABRE sequences. Deletion and base substitution analyses of this region showed that the DRE-core motif functions as DRE and that the DRE/DRE-core motif could be a coupling element of ABRE. Gel mobility shift assays revealed that DRE-binding proteins (DREB1s/CBFs and DREB2s) bind to both DRE and the DRE-core motif and that ABRE-binding proteins (AREBs/ABFs) bind to ABRE in the 120 bp promoter region. In addition, transactivation experiments using Arabidopsis leaf protoplasts showed that DREBs and AREBs cumulatively transactivate the expression of a GUS reporter gene fused to the 120 bp promoter region of rd29A. These results indicate that DRE and ABRE are interdependent in the ABA-responsive expression of the rd29A gene in response to ABA in Arabidopsis.     

DREB1A 转录因子蛋白的原核表达

DREB1A是DREB转录因子的一员,它们都含有一段与DNA结合的保守区域,由58个氨基酸组成,称为EREBP/AP2结构域(EREBP/AP2 domain)。一个DREB转录因子可以调控多个与植物干旱、高盐及低温耐性有关的功能基因表达。从拟南芥中克隆了DREB1A转录因子基因,成功构建了DREB1A基因的原核表达载体,转化E.coliBL21(DE3),经1 mmol/L IPTG诱导表达获得了DREB1A蛋白,但以包涵体形式存在。为以后深入研究DREB转录因子与DRE顺式作用元件相互作用的具体机制奠定了基础。     

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。     

DRE顺式作用元件dsDNA芯片制备

DRE顺式作用元件能与DREB转录因子特异结合,在诱导逆境（干旱、高盐、低温）基因表达过程中起重要作用。dsDNA（double strand DNA）微阵列芯片技术能够有效地检测序列特异性DNA结合蛋白质（转录因子）与大量DNA靶点（顺式作用元件）的特异性结合,可有效分析生物分子结合作用。根据DRE顺式作用元件核心序列设计并化学合成含发夹结构的单链DNA探针,采用Taq DNA聚合酶在片延伸,并对其在片延伸体系的反应温度、Mg^2＋浓度以及单链探针是否变性等条件进行了优化。结果表明,50％的反应温度,2．5mmol／L的Mg^2＋浓度和单链不变性是TaqDNA聚合酶在片延伸的最佳条件。优化方法制备的dsDNA芯片将更有利于DRE顺式作用元件与DREB抗逆转录因子相互作用的研究。     

DREB转录因子研究进展

DREB转录因子即干旱应答元件结合蛋白质,它能特异结合启动子中含有 DRE/CRT 顺式元件,激活许多逆境诱导基因的表达,增强植物对逆境的忍耐力。介绍DREB转录因子与DRE顺式作用元件的关系,DREB 转录因子与 DRE 元件的结合特异性,DREB 的结构特点和功能,DREB 转录因子的表达调控,DREB 转录因子的克隆及鉴定等方面的研究进展,简述 DREB 转录因子对调控逆境诱导基因的表达具有非常重要的作用,在提高植物综合抗逆性方面将有巨大的应用前景。同时,指出 DREB 转录因子在信号转导、作用机理及基因表达等方面的复杂性。      

Abscisic acid induces the alcohol dehydrogenase gene in Arabidopsis.

Exogenous abscisic acid (ABA) induced the alcohol dehydrogenase gene (Adh) in Arabidopsis roots. Both the G-box-1 element and the GT/GC motifs (anaerobic response element) were required for Adh inducibility. Measurement of endogenous ABA levels during stress treatment showed that ABA levels increased during dehydration treatment but not following exposure to either hypoxia or low temperature. Arabidopsis ABA mutants (aba1 and abi2) displayed reduced Adh mRNA induction levels following either dehydration treatment or exogenous application of ABA. Low-oxygen response was slightly increased in the aba1 mutant but was unchanged in abi2. Low-temperature response was unaffected in both aba1 and abi2 mutants. Our results indicate that, although induction of the Adh gene by ABA, dehydration, and low temperature required the same cis-acting promoter elements, their regulatory pathways were at least partially separated in a combined dehydration/ABA pathway and an ABA-independent low-temperature pathway. These pathways were in turn independent of a third signal transduction pathway leading to low-oxygen response, which did not involve either ABA or the G-box-1 promoter element.     

Hyperosmotic stress induces a rapid and transient increase in inositol 1,4,5-trisphosphate independent of abscisic acid in Arabidopsis cell culture

Phospholipid metabolism is involved in hyperosmotic-stress responses in plants. To investigate the role of phosphoinositide-specific phospholipase C (PI-PLC)-a key enzyme in phosphoinositide turnover-in hyperosmotic-stress signaling, we analyzed changes in inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) content in response to hyperosmotic shock or salinity in Arabidopsis thaliana T87 cultured cells. Within a few s, a hyperosmotic shock, caused by mannitol, NaCl, or dehydration, induced a rapid and transient increase in Ins(1,4,5)P3. However, no transient increase was detected in cells treated with ABA. Neomycin and U73122, inhibitors of PI-PLC, inhibited the increase in Ins(1,4,5)P3 caused by the hyperosmotic shock. A rapid increase in phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in response to the hyperosmotic shock also occurred, but the rate of increase was much slower than that of Ins(1,4,5)P3. These findings indicate that the transient Ins(1,4,5)P3 production was due to the activation of PI-PLC in response to hyperosmotic stress. PI-PLC inhibitors also inhibited hyperosmotic stress-responsive expression of some dehydration-inducible genes, such as rd29A (lti78/cor78) and rd17 (cor47), that are controlled by the DRE/CRT cis-acting element but did not inhibit hyperosmotic stress-responsive expression of ABA-inducible genes, such as rd20. Taken together, these results suggest the involvement of PI-PLC and Ins(1,4,5)P3 in an ABA-independent hyperosmotic-stress signal transduction pathway in higher plants.