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.     

Constitutive expression of abiotic stress-inducible hot pepper CaXTH3, which encodes a xyloglucan endotransglucosylase/hydrolase homolog, improves drought and salt tolerance in transgenic Arabidopsis plants

Xyloglucan endotransglucosylase/hydrolase (XTH) has been recognized as a cell wall-modifying enzyme, participating in the diverse physiological roles. From water-stressed hot pepper plants, we isolated three different cDNA clones (pCaXTH1, pCaXTH2, and pCaXTH3) that encode XTH homologs. RT-PCR analysis showed that three CaXTH mRNAs were concomitantly induced by a broad spectrum of abiotic stresses, including drought, high salinity and cold temperature, and in response to stress hormone ethylene, suggesting their role in the early events in the abiotic-related defense response. Transgenic Arabidopsis plants that constitutively expressed the CaXTH3 gene under the control of the CaMV 35S promoter exhibited abnormal leaf morphology; the transgenic leaves showed variable degrees of twisting and bending along the edges, resulting in a severely wrinkled leaf shape. Microscopic analysis showed that 35S-CaXTH3 leaves had increased numbers of small-sized cells, resulting in disordered, highly populated mesophyll cells in each dorsoventral layer, and appeared to contain a limited amount of starch. In addition, the 35S-CaXTH3 transgenic plants displayed markedly improved tolerance to severe water deficit, and to lesser extent to high salinity in comparison with the wild-type plants. These results indicate that CaXTH3 is functional in heterologous Arabidopsis cells, thereby effectively altering cell growth and also the response to abiotic stresses. Although the physiological function of CaXTHs is not yet clear, there are several possibilities for their involvement in a subset of physiological responses to counteract dehydration and high salinity stresses in transgenic Arabidopsis plants.     

Overexpression of a trehalose-6-phosphate synthase/phosphatase fusion gene enhances tolerance and photosynthesis during drought and salt stress without growth aberrations in tomato

Trehalose is a non-reducing disaccharide of glucose that confers tolerance against abiotic stresses in many diverse organisms, including higher plants. It was previously reported that overexpression of the yeast trehalose-6-phosphate synthase gene in tomato results in improved tolerance against abiotic stresses. However, these transgenic tomato plants had stunted growth and pleiotropic changes in appearance. In this study, transgenic tomato plants were generated by the introduction of a gene encoding a bifunctional fusion of trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase genes from Escherichia coli under the control of the CaMV35S promoter. Transgenic plants accumulated higher levels of trehalose in their leaves and exhibited enhanced drought and salt tolerance and photosynthetic rates under salt stress conditions than wild-type plants. All of the transgenic plants had normal growth patterns and appearances. Therefore, the system described in this study can be used for practical application of the gene in crop improvement.     

Constitutive expression of CaRma1H1, a hot pepper ER-localized RING E3 ubiquitin ligase, increases tolerance to drought and salt stresses in transgenic tomato plants

CaRma1H1, an endoplasmic reticulum (ER)-localized hot pepper really interesting new genes (RING) E3 Ub ligase, was previously reported to be a positive regulator of drought stress responses. To address the possibility that CaRma1H1 can be used to improve tolerance to abiotic stress in crop plants, CaRma1H1 was constitutively expressed in transgenic tomato (Solanum lycopersicum) plants. CaRma1H1-overexpressing tomato plants (35S:CaRma1H1) exhibited greatly enhanced tolerance to high-salinity treatments compared with wild-type plants. Leaf chlorophyll and proline contents in CaRma1H1 overexpressors were 4.3- to 8.5-fold and 1.2- to 1.5-fold higher, respectively, than in wild-type plants after 300?mM NaCl treatment. Transgenic cotyledons developed and their roots elongated in the presence of NaCl up to 200?mM. In addition, 35S:CaRma1H1 lines were markedly more tolerant to severe drought stress than were wild-type plants. Detached leaves of CaRma1H1 overexpressors preserved water more efficiently than did wild-type leaves during a rapid dehydration process. The ER chaperone genes LePDIL1, LeBIP1, and LeCNX1 were markedly up-regulated in 35S:CaRma1H1 tomatoes compared with wild-type plants. Therefore, overexpression of CaRma1H1 may enhance tomato plant ER responses to drought stress by effectively removing nonfunctional ubiquitinated proteins. Collectively, constitutive expression of CaRma1H1 in tomatoes conferrred strongly enhanced tolerance to salt- and water-stress. This raises the possibility that CaRma1H1 may be useful for developing abiotic stress-tolerant tomato plants. Key message CaRma1H1 increases drought tolerance in transgenic tomato plants.     

Overexpression of osmotin gene confers tolerance to salt and drought stresses in transgenic tomato (Solanum lycopersicum L.)

Abiotic stresses, especially salinity and drought, are major limiting factors for plant growth and crop productivity. In an attempt to develop salt and drought tolerant tomato, a DNA cassette containing tobacco osmotin gene driven by a cauliflower mosaic virus 35S promoter was transferred to tomato (Solanum lycopersicum) via Agrobacterium-mediated transformation. Putative T0 transgenic plants were screened by PCR analysis. The selected transformants were evaluated for salt and drought stress tolerance by physiological analysis at T1 and T2 generations. Integration of the osmotin gene in transgenic T1 plants was verified by Southern blot hybridization. Transgenic expression of the osmotin gene was verified by RT-PCR and northern blotting in T1 plants. T1 progenies from both transformed and untransformed plants were tested for salt and drought tolerance by subjecting them to different levels of NaCl stress and by withholding water supply, respectively. Results from different physiological tests demonstrated enhanced tolerance to salt and drought stresses in transgenic plants harboring the osmotin gene as compared to the wild-type plants. The transgenic lines showed significantly higher relative water content, chlorophyll content, proline content, and leaf expansion than the wild-type plants under stress conditions. The present investigation clearly shows that overexpression of osmotin gene enhances salt and drought stress tolerance in transgenic tomato plants.     

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.     

A comparative study on the protective role of trehalose and LEA proteins against abiotic stresses in transgenic Chinese cabbage (<Emphasis Type="Italic">Brassica campestris</Emphasis>) overexpressing<Emphasis Type="Italic">CaLEA</Emphasis> or<Emphasis Type="Italic">otsA</Emphasis>

Trehalose and LEA proteins, representative low MW chemicals that are synthesized under dehydration, are known to protect plants from drought stress. To compare their effectiveness on enhancing tolerance against various abiotic stresses, we generated transgenic Chinese cabbage plants overexpressingE. ctdi trehalose-6-phosphate synthase gene (otsA) or hot pepper (Capsicum annuum) LEA protein gene(CaLEA). Both transgenic plants exhibited altered phenotype including stunted growth and aberrant root development When subjected to drought, salt or heat stress, these plants showed remarkably improved tolerance against those stresses compared with nontransformants. After dehydration treatment, leaf turgidity and fresh weight was better maintained in both transgenic plants. GaUEA-plants performed somewhat better under dehydrated condition. When treated with 250 mM NaCI, both otsA-plants and CaLEA-plants remained equally healthier than nontransformants in maintaining leaf turgidity and delaying necrosis. Furthermore, leaf Chi content and Fv/Fm was maintained considerably higher in both transgenic plants than nontransformants. After heat-treatment at 45°C, both transgenic plants appeared much less damaged in external shape and PSII function, but LEA proteins were more protective. Our results indicate that although both trehalose and LEA proteins are effective in protecting plants against various abiotic stresses, LEA proteins seem to be more promising in generating stress-tolerant transgenic plants.     

Building stress tolerance through over-producing trehalose in transgenic plants

Trehalose is a rare sugar with unique abilities to protect biomolecules from environmental stresses and is present in many bacteria, fungi and some desiccation-tolerant higher plants. Increasing trehalose accumulation in crop plants could improve drought and salinity tolerance. Transgenic plants have been developed with trehalose biosynthetic genes--a recent study on the stress-inducible overexpression of the bifunctional TPSP fusion gene in transgenic rice could offer novel strategies for improving abiotic stress tolerance in crop plants.     

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.     

Expression of Arabidopsis CBF1 regulated by an ABA/stress inducible promoter in transgenic tomato confers stress tolerance without affecting yield

Modern‐day plants are subjected to various biotic and abiotic stresses thereby limiting plant productivity and quality. It has previously been reported that the use of a strong constitutive 35S cauliflower mosaic virus (CaMV) promoter to drive the expression of Arabidopsis CBF1 in tomato improved tolerance to cold, drought and salt loading, at the expense of growth and yield under normal growth conditions. Hence in the present study, the suitability of expressing the Arabidopsis CBF1 driven by three copies of an ABA‐responsive complex (ABRC1) from the barley HAV22 gene in order to improve the agronomic performance of the transgenic tomato plants was investigated. Northern blot analysis indicated that CBF1 gene expression was induced by chilling, water‐deficit and salt treatment in the transgenic tomato plants. Under these tested stress conditions, transgenic tomato plants exhibited enhanced tolerance to chilling, water‐deficit, and salt stress in comparison with untransformed plants. Under normal growing conditions the ABRC1‐CBF1 tomato plants maintained normal growth and yield similar to the untransformed plants. The results demonstrate the promise of using ABRC1‐CBF1 tomato plants in highly stressed conditions which will in turn benefit agriculture.     

The pepper extracellular peroxidase CaPO2 is required for salt, drought and oxidative stress tolerance as well as resistance to fungal pathogens

In plants, biotic and abiotic stresses regulate the expression and activity of various peroxidase isoforms. Capsicum annuum EXTRACELLULAR PEROXIDASE 2 (CaPO2) was previously shown to play a role in local and systemic reactive oxygen species bursts and disease resistance during bacterial pathogen infection. Here, we report CaPO2 expression patterns and functions during conditions of biotic and abiotic stress. In pepper plants, CaPO2 expression was strongly induced by abscisic acid, but not by defense-related plant hormones such as salicylic acid, ethylene and jasmonic acid. CaPO2 was also strongly induced by abiotic and biotic stress treatments, including drought, cold, high salinity and infection by the hemibiotrophic fungal pathogen Colletotrichum coccodes. Loss-of-function of CaPO2 in virus-induced gene silenced pepper plants led to increased susceptibility to salt- and osmotic-induced stress. In contrast, CaPO2 overexpression in transgenic Arabidopsis thaliana plants conferred enhanced tolerance to high salt, drought, and oxidative stress, while also enhancing resistance to infection by the necrotrophic fungal pathogen Alternaria brassicicola. Taken together, these results provide evidence for the involvement of pepper extracellular peroxidase CaPO2 in plant defense responses to various abiotic stresses and plant fungal pathogens.     

Plant salt tolerance

Soil salinity is a major abiotic stress in plant agriculture worldwide. This has led to research into salt tolerance with the aim of improving crop plants. However, salt tolerance might have much wider implications because transgenic salt-tolerant plants often also tolerate other stresses including chilling, freezing, heat and drought. Unfortunately, suitable genetic model systems have been hard to find. A recently discovered halophytic plant species, Thellungiella halophila, now promises to help in the detection of new tolerance determinants and operating pathways in a model system that is not limited to Arabidopsis traits or ecotype variations.