Constitutive expression of CaXTH3, a hot pepper xyloglucan endotransglucosylase/hydrolase, enhanced tolerance to salt and drought stresses without phenotypic defects in tomato plants (Solanum lycopersicum cv. Dotaerang)

The hot pepper xyloglucan endo-trans-gluco-sylase/hydrolase (CaXTH3) gene that was inducible by a broad spectrum of abiotic stresses in hot pepper has been reported to enhance tolerance to drought and high salinity in transgenic Arabidopsis. To assess whether CaXTH3 is a practically useful target gene for improving the stress tolerance of crop plants, we ectopically over-expressed the full-length CaXTH3 cDNA in tomato (Solanum lycopersicum cv. Dotaerang) and found that the 35S:CaXTH3 transgenic tomato plants exhibited a markedly increased tolerance to salt and drought stresses. Transgenic tomato plants exposed to a salt stress of 100?mM NaCl retained the chlorophyll in their leaves and showed normal root elongation. They also remained green and unwithered following exposure to 2?weeks of dehydration. A high proportion of stomatal closures in 35S:CaXTH3 was likely to be conferred by increased cell-wall remodeling activity of CaXTH3 in guard cell, which may reduce transpirational water loss in response to dehydration stress. Despite this increased stress tolerance, the transgenic tomato plants showed no detectable phenotype defects, such as abnormal morphology and growth retardation, under normal growth conditions. These results raise the possibility that CaXTH3 gene is appropriate for application in genetic engineering strategies aimed at improving abiotic stress tolerance in agriculturally and economically valuable crop plants.     

Erratum to: Constitutive expression of CaXTH3, a hot pepper xyloglucan endotransglucosylase/hydrolase, enhanced tolerance to salt and drought stresses without phenotypic defects in tomato plants (Solanum lycopersicum cv. Dotaerang)

The hot pepper xyloglucan endo-trans-gluco-sylase/hydrolase (CaXTH3) gene that was inducible by a broad spectrum of abiotic stresses in hot pepper has been reported to enhance tolerance to drought and high salinity in transgenic Arabidopsis. To assess whether CaXTH3 is a practically useful target gene for improving the stress tolerance of crop plants, we ectopically over-expressed the full-length CaXTH3 cDNA in tomato (Solanum lycopersicum cv. Dotaerang) and found that the 35S:CaXTH3 transgenic tomato plants exhibited a markedly increased tolerance to salt and drought stresses. Transgenic tomato plants exposed to a salt stress of 100 mM NaCl retained the chlorophyll in their leaves and showed normal root elongation. They also remained green and unwithered following exposure to 2 weeks of dehydration. A high proportion of stomatal closures in 35S:CaXTH3 was likely to be conferred by increased cell-wall remodeling activity of CaXTH3 in guard cell, which may reduce transpirational water loss in response to dehydration stress. Despite this increased stress tolerance, the transgenic tomato plants showed no detectable phenotype defects, such as abnormal morphology and growth retardation, under normal growth conditions. These results raise the possibility that CaXTH3 gene is appropriate for application in genetic engineering strategies aimed at improving abiotic stress tolerance in agriculturally and economically valuable crop plants.     

Heterologous expression and molecular and cellular characterization of CaPUB1 encoding a hot pepper U-Box E3 ubiquitin ligase homolog

The U-box motif is a conserved domain found in the diverse isoforms of E3 ubiquitin ligase in eukaryotes. From water-stressed hot pepper (Capsicum annuum L. cv Pukang) plants, we isolated C. annuum putative U-box protein 1 (CaPUB1), which encodes a protein containing a single U-box motif in its N-terminal region. In vitro ubiquitination and site-directed mutagenesis assays revealed that CaPUB1 possessed E3 ubiquitin ligase activity and that the U-box motif was indeed essential for its enzyme activity. RNA gel-blot analysis showed that CaPUB1 mRNA was induced rapidly by a broad spectrum of abiotic stresses, including drought, high salinity, cold temperature, and mechanical wounding, but not in response to ethylene, abscisic acid, or a bacterial pathogen, suggesting its role in the early events in the abiotic-related defense response. Because transgenic work was extremely difficult in hot pepper, in this study we overexpressed CaPUB1 in Arabidopsis (Arabidopsis thaliana) to provide cellular information on the function of this gene in the development and plant responses to abiotic stresses. Transgenic Arabidopsis plants that constitutively expressed the CaPUB1 gene under the control of the cauliflower mosaic virus 35S promoter had markedly longer hypocotyls and roots and grew more rapidly than the wild type, leading to an early bolting phenotype. Microscopic analysis showed that 35S::CaPUB1 roots had increased numbers of small-sized cells, resulting in disordered, highly populated cell layers in the cortex, endodermis, and stele. In addition, CaPUB1-overexpressing plants displayed increased sensitivity to water stress and mild salinity. These results indicate that CaPUB1 is functional in Arabidopsis cells, thereby effectively altering cell and tissue growth and also the response to abiotic stresses. Comparative proteomic analysis showed that the level of RPN6 protein, a non-ATPase subunit of the 26S proteasome complex, was significantly reduced in 35SCaPUB1 seedlings as compared to the wild type. Pull-down and ubiquitination assays demonstrated that RPN6 interacted physically with CaPUB1 and was ubiquitinated in a CaPUB1-dependent manner in vitro. Although the physiological function of CaPUB1 is not yet clear, there are several possibilities for its involvement in a subset of physiological responses to counteract dehydration and high-salinity stresses in transgenic Arabidopsis seedlings.     

The immunolocation of a xyloglucan endotransglucosylase/hydrolase specific to elongating tissues in Cicer arietinum suggests a role in the elongation of vascular cells

In a previous work, a Cicer arietinum cDNA clone (CaXTH1) encoding a xyloglucan endotransglucosylase/hydrolase (XTH1) protein was isolated and characterized. CaXTH1 showed an expression pattern specific to growing tissue: mostly epicotyls and the upper growing internodes of adult stems. CaXTH1 mRNA was not detected in any other organs of either seedlings or adult plants, suggesting an involvement of the putative XTH encoded by CaXTH1 in the chickpea cell expansion process. After the generation of polyclonal antibodies by using the XTH1 recombinant protein and the analysis of the specificity of the antibodies for XTH proteins, here the specific location of the chickpea XTH1-cross-reacting protein in cell walls of epicotyls, radicles, and stems is reported, evaluated by western blot and immunocytochemical studies. The results indicate a function for this protein in the elongation of parenchyma cells of epicotyls and also in developing vascular tissue, suggesting a role in the elongation of vascular cells.     

Enhancement of abiotic stress tolerance in poplar by overexpression of key Arabidopsis stress response genes, <Emphasis Type="Italic">AtSRK2C</Emphasis> and <Emphasis Type="Italic">AtGolS2</Emphasis>

Recent environmental issues have increased the demand for woody biomass as a renewable resource for industry and energy. For a stable supply of woody biomass, it is critical to decrease the effects of abiotic stresses, such as drought and salinity, which hinder plant growth. For the goal to develop practical stress-tolerant trees, we generated transgenic poplar plants (P. tremula × tremuloides), in which a key Arabidopsis regulatory factor involved in stress responses, SNF1-related protein kinase 2C (AtSRK2C), or galactinol synthase 2 (AtGolS2), was overexpressed. Both types of transgenic poplar plants displayed higher tolerance to abiotic stresses, in comparison with nontransgenic plants, indicating that AtSRK2C and AtGolS2 can function in the abiotic stress response pathway of poplar. We also examined the expression profiles of ten poplar genes putatively homologous to well-known Arabidopsis stress response genes and found that several of the poplar genes showed different responses to abiotic stress from their Arabidopsis counterparts. Whereas the overexpression of AtSRK2C in transgenic Arabidopsis plants was reported to upregulate the expression of endogenous genes, the overexpression of AtSRK2C or AtGolS2 in transgenic poplar did not. Taken together, our findings suggest that the details of the underlying molecular mechanisms of the abiotic stress response may differ, but that the key regulatory factors in Arabidopsis and poplar have common features and are effective molecular targets for further breeding to enhance abiotic stress tolerance in poplar.     

拟南芥AtGLK-N基因的过量表达对代谢物质积累的影响

AtGLK-N（At2g01060）是拟南芥转录因子Golden2-like基因家族的一个成员,仅存在于植物中。本文通过启动子一GUS和Northem检测分析TAtGLK-N的表达模式,结果显示：AtGLK-N在愈伤组织、球形胚、心形胚、鱼雷胚中表达,在幼叶（真叶）及侧根分生组织中也有表达。此外,Nonhern检测结果还表明AtGLK-N可被高盐胁迫所诱导。通过农杆菌介导转化拟南芥,得到过表达AtGLK-N的转基因植株,其表现为矮小。为弄清AtGLK-N的功能,对过表达AtGLK-N以南芥进行代谢谱分析,结果显示：转基因植物的脯氨酸、棉籽糖和海藻糖以及催化上述代谢产物的蛋白酶的基因表达水平明显比对照植株高。上述结果表明AtGLK-N表达主要分布在分裂细胞或组织,并可能与高盐胁迫反应相关。     

XVSAP1 from Xerophyta viscosa improves osmotic-, salinity- and high-temperature-stress tolerance in Arabidopsis

XVSAP1, a gene isolated from a dehydrated Xerophyta viscosa cDNA library, was transformed into Arabidopsis thaliana by Ti plasmid-mediated transformation under the control of a cauliflower mosaic virus 35S promoter, a nos terminator and bar gene selection. Expression of XVSAP1 in Arabidopsis led to constitutive accumulation of the corresponding protein in the leaves. Transgenic Arabidopsis grown in tissue culture maintained higher growth rates during osmotic, high-salinity and high temperature stress, respectively. Non-transgenic plants had shorter roots, leaf expansion was inhibited and leaves were more chlorotic than those of the transgenic plants. This study demonstrates that XVSAP1 has a significant impact on dehydration, salinity and high-temperature stress tolerance in Arabidopsis.     

Two cysteine proteinase inhibitors from Arabidopsis thaliana, AtCYSa and AtCYSb, increasing the salt, drought, oxidation and cold tolerance

Two cysteine proteinase inhibitors (cystatins) from Arabidopsis thaliana, designated AtCYSa and AtCYSb, were characterized. Recombinant GST-AtCYSa and GST-AtCYSb were expressed in Escherichia coli and purified. They inhibit the catalytic activity of papain, which is generally taken as evidence for cysteine proteinase inhibitor function. Northern blot analyses showed that the expressions of AtCYSa and AtCYSb gene in Arabidopsis cells and seedlings were strongly induced by multiple abiotic stresses from high salt, drought, oxidant, and cold. Interestingly, the promoter region of AtCYSa gene contains a dehydration-responsive element (DRE) and an abscisic acid (ABA)-responsive element (ABRE), which identifies it as a DREB1A and AREB target gene. Under normal conditions, AtCYSa was expressed in 35S: DREB1A and 35S: AREB1 plants at a higher level than in WT plants, while AtCYSa gene was expressed in 35S: DREB2A plants at the same level as in WT plants. Under stress conditions (salt, drought and cold), AtCYSa was expressed more in all three transgenic plants than in WT plants. Over-expression of AtCYSa and AtCYSb in transgenic yeast and Arabidopsis plants increased the resistance to high salt, drought, oxidative, and cold stresses. Taken together, these data raise the possibility of using AtCYSa and AtCYSb to genetically improve environmental stresses tolerance in plants.     

An alfalfa (Medicago sativa L.) ethylene response factor gene, MsERF11, enhances salt tolerance in transgenic Arabidopsis

A novel orthologue of ethylene response factor gene, MsERF11, was isolated from alfalfa in this study. It has an open reading frame of 807?bp, encoding a predicted polypeptide of 268 amino acids. Sequence similarity analysis clearly suggested that MsERF11 encoded an ethylene response factor protein. The results of transient expression of MsERF11 in onion epidermal cells indicated that MsERF11 is a nuclear protein. The expression pattern of MsERF11 gene was analyzed by real-time quantitative PCR and a higher level of expression was observed in leaves than was observed in roots, stems, flower buds and flowers. Furthermore, the expression was induced by PEG6000, NaCl, Al(2)(SO(4))(3) and six different hormones. Over-expressing MsERF11 resulted in enhanced tolerances to salt stress in transgenic Arabidopsis plants. This research indicates that MsERF11 has the potential to be used for improving crop's salt tolerance in areas where salinity is a limiting factor for agricultural productivity. Key message MsERF11 was isolated from alfalfa. Its expression was induced by different abiotic stresses and hormones. Over-expressing MsERF11 resulted in enhanced salt tolerance in transgenic Arabidopsis plants.     

Arabidopsis SAP5 functions as a positive regulator of stress responses and exhibits E3 ubiquitin ligase activity

AtSAP5, one of approximately 14 members of the Stress Associated Protein gene family in Arabidopsis, was identified by its expression in response to salinity, osmotic, drought and cold stress. AtSAP5 shows strong homology to OSISAP1, an A20/AN1-type zinc finger protein implicated in stress tolerance in rice. To evaluate the function of AtSAP5 in the regulation of abiotic stress responses, transgenic Arabidopsis plants that over-express AtSAP5 (35S::AtSAP5) were characterized, along with wild-type and T-DNA knock-down plants. Plants that over-express AtSAP5 showed increased tolerance to environmental challenges including salt stress, osmotic stress and water deficit. Comparison of gene expression patterns between 35S::AtSAP5 transgenic plants and wild-type plants under normal conditions and water deficit stress indicated that over-expression of AtSAP5 correlates with up-regulation of drought stress responsive gene expression. Analysis of transgenic plants that express GFP-AtSAP5 showed that it is localized primarily in nuclei of root cells and recombinant AtSAP5 has E3 ubiquitin ligase activity in vitro. These results indicate that AtSAP5 has E3 ligase activity and acts as a positive regulator of stress responses in Arabidopsis.     

The pepper late embryogenesis abundant protein CaLEA1 acts in regulating abscisic acid signaling,drought and salt stress response

As sessile organisms, plants are constantly challenged by environmental stresses, including drought and high salinity. Among the various abiotic stresses, osmotic stress is one of the most important factors for growth and significantly reduces crop productivity in agriculture. Here, we report a function of the CaLEA1 protein in the defense responses of plants to osmotic stress. Our analyses showed that the CaLEA1 gene was strongly induced in pepper leaves exposed to drought and increased salinity. Furthermore, we determined that the CaLEA1 protein has a late embryogenesis abundant (LEA)_3 homolog domain highly conserved among other known group 5 LEA proteins and is localized in the processing body. We generated CaLEA1‐silenced peppers and CaLEA1‐overexpressing (OX) transgenic Arabidopsis plants to evaluate their responses to dehydration and high salinity. Virus‐induced gene silencing of CaLEA1 in pepper plants conferred enhanced sensitivity to drought and salt stresses, which was accompanied by high levels of lipid peroxidation in dehydrated and NaCl‐treated leaves. CaLEA1‐OX plants exhibited enhanced sensitivity to abscisic acid (ABA) during seed germination and in the seedling stage; furthermore, these plants were more tolerant to drought and salt stress than the wild‐type plants because of enhanced stomatal closure and increased expression of stress‐responsive genes. Collectively, our data suggest that CaLEA1 positively regulates drought and salinity tolerance through ABA‐mediated cell signaling.     

Transgenic Analysis Reveals 5′ Abbreviated <Emphasis Type="Italic">Os</Emphasis>RGLP2 Promoter(s) as Responsive to Abiotic Stresses

Germins and germin-like proteins are ubiquitous, expressed at various developmental stages and in response to various abiotic and biotic stresses. In this study, to functionally validate the OsRGLP2 promoter, 5′ deletion analysis of the promoter sequences was performed and the deletion fragments fused with the β-glucuronidase (GUS) and green fluorescent protein reporter genes were used for transient expression in tobacco as well as for generating stable transgenic Arabidopsis plants. Very high level of GUS activity was observed in agroinfiltrated tobacco leaves by the construct carrying the P-1063 and P-565 when subjected to abiotic stresses. Histochemical analysis of transgenic Arabidopsis plants revealed expression of reporter gene in root, leaf and stem sections of plants harboring P-1063 and P-565. Real-time qPCR analysis of transiently expressed tobacco leaves and transgenic Arabidopsis plants subjected to several abiotic stresses supported histochemical data and showed that P-565 responded to all the stresses to which the full-length promoter was responsive. The data suggest that P-565 may be a good alternative to full-length promoter region that harbors the necessary cis-elements in providing stable and high level of expression in response to wound, salt and temperature stresses.