Overexpression of cinnamyl alcohol dehydrogenase gene from sweetpotato enhances oxidative stress tolerance in transgenic Arabidopsis

Cinnamyl alcohol dehydrogenase (CAD) is the enzyme in the last step of lignin biosynthetic pathway and is involved in the generation of lignin monomers. IbCAD1 gene in sweetpotato (Ipomoea batatas) was identified, and its expression was induced by abiotic stresses based on promoter analysis. In this study, transgenic Arabidopsis plants overexpressing IbCAD1 directed by CaMV 35S promoter were developed to determine the physiological function of IbCAD1. IbCAD1-overexpressing transgenic plants exhibited better plant growth and higher biomass compared to wild type (WT), under normal growth conditions. CAD activity was increased in leaves and roots of transgenic plants. Sinapyl alcohol dehydrogenase activity was induced to a high level in roots, which suggests that IbCAD1 may regulate biosynthesis of syringyl-type (S) lignin. Lignin content was increased in stems and roots of transgenic plants; this increase was in S lignin rather than guaiacyl (G) lignin. Overexpression of IbCAD1 in Arabidopsis resulted in enhanced seed germination rates and tolerance to reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂). Taken together, our results show that IbCAD1 controls lignin content by biosynthesizing S units and plays an important role in plant responses to oxidative stress.

     

Regulation of durum wheat Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger TdSOS1 by phosphorylation

We have identified a plasma membrane Na⁺/H⁺ exchanger from durum wheat, designated TdSOS1. Heterologous expression of TdSOS1 in a yeast strain lacking endogenous Na⁺ efflux proteins showed complementation of the Na⁺- and Li⁺-sensitive phenotype by a mechanism involving cation efflux. Salt tolerance conferred by TdSOS1 was maximal when co-expressed with the Arabidopsis protein kinase complex SOS2/SOS3. In vitro phosphorylation of TdSOS1 with a hyperactive form of the Arabidopsis SOS2 kinase (T/DSOS2∆308) showed the importance of two essential serine residues at the C-terminal hydrophilic tail (S1126, S1128). Mutation of these two serine residues to alanine decreased the phosphorylation of TdSOS1 by T/DSOS2∆308 and prevented the activation of TdSOS1. In addition, deletion of the C-terminal domain of TdSOS1 encompassing serine residues at position 1126 and 1128 generated a hyperactive form that had maximal sodium exclusion activity independent from the regulatory SOS2/SOS3 complex. These results are consistent with the presence of an auto-inhibitory domain at the C-terminus of TdSOS1 that mediates the activation of TdSOS1 by the protein kinase SOS2. Expression of TdSOS1 mRNA in young seedlings of the durum wheat variety Om Rabia3, using different abiotic stresses (ionic and oxidative stress) at different times of exposure, was monitored by RT–PCR.     

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.     

Overexpression ofENA1 from yeast increases salt tolerance inArabidopsis

In yeast, the plasma membrane Na⁺/H⁺ antiporter and Na⁺-ATPase are key enzymes for salt tolerance.Saccharomyces cerevisiae Na⁺-ATPase (Enalp ATPase) is encoded by theENA1/PMR2A gene; expression ofENA1 is tightly regulated by Na⁺ and depends on ambient pH. Although Enalp is active mainly at alkaline pH values inS. cerevisiae, no Na⁺-ATPase has been found in flowering plants. To test whether this yeast enzyme would improve salt tolerance in plants, we introducedENA1 intoArabidopsis (cv. Columbia) under the control of the cauliflower mosaic virus 35S promoter. Transformants were selected for their ability to grow on a medium containing kanamyin. Southern blot analyses confirmed thatENA1 was transferred into theArabidopsis genome and northern blot analyses showed thatENA1 was expressed in the transformants. Several transgenic homozygous lines and wild-type (WT) plants were evaluated for salt tolerance. No obvious morphological or developmental differences existed between the transgenic and WT plants in the absence of stress. However, overexpression ofENA1 inArabidopsis improved seed germination rates and salt tolerance in seedlings. Under saline conditions, transgenic plants accumulated a lower amount of Na⁺ than did the wild type, and fresh and dry weights of the former were higher. Other experiments revealed that expression ofENA1 promoted salt tolerance in transgenicArabidopsis under both acidic and alkaline conditions. These authors contributed equally to this article.     

Chloroplast-targeted BrMT1 (<Emphasis Type="Italic">Brassica rapa</Emphasis> type-1 metallothionein) enhances resistance to cadmium and ros in transgenic<Emphasis Type="Italic">atabidopsis</Emphasis> plants

Metallothioneins (MTs) are low-molecular-weight, cysteine-rich proteins that bind to heavy metals. Type-1 MTs function under various abiotic stresses, including exposure to the cadmium ion. We have now isolated theBrassica rapa type-1 metallothioneirt gene (BrMT1)using yeast systems, and have found that it confers resistance to Cd in otherwise Cd-sensitive yeast. Using a constitutive CaMV35S promoter and an RbsS transit peptide, we successfully targeted BrMT1 to the chloroplastsof Arabidopsis. Overexpression in either the chloroplasts or the cytosol effectively detoxified cadmium and H₂O₂ stresses in transgenicArabidopsis. in particular, the chloropfast-targeted BrMTl was associated with a significant reduction in paraquat-induced chlorosis and the accumulation of H₂O₂. This is the first report regarding the effects of type-1 MT1 targeted to chloroplasts. Our results suggest that this may be applicable to the development of plants with enhanced tolerance against environmental stresses.     

RhEXPA4, a rose expansin gene, modulates leaf growth and confers drought and salt tolerance to Arabidopsis

Drought and high salinity are major environmental conditions limiting plant growth and development. Expansin is a cell-wall-loosening protein known to disrupt hydrogen bonds between xyloglucan and cellulose microfibrils. The expression of expansin increases in plants under various abiotic stresses, and plays an important role in adaptation to these stresses. We aimed to investigate the role of the RhEXPA4, a rose expansin gene, in response to abiotic stresses through its overexpression analysis in Arabidopsis. In transgenic Arabidopsis harboring the Pro _RhEXPA4 ::GUS construct, RhEXPA4 promoter activity was induced by abscisic acid (ABA), drought and salt, particularly in zones of active growth. Transgenic lines with higher RhEXPA4 level developed compact phenotypes with shorter stems, curly leaves and compact inflorescences, while the lines with relatively lower RhEXPA4 expression showed normal phenotypes, similar to the wild type (WT). The germination percentage of transgenic Arabidopsis seeds was higher than that of WT seeds under salt stress and ABA treatments. Transgenic plants showed enhanced tolerance to drought and salt stresses: they displayed higher survival rates after drought, and exhibited more lateral roots and higher content of leaf chlorophyll a under salt stress. Moreover, high-level RhEXPA4 overexpressors have multiple modifications in leaf blade epidermal structure, such as smaller, compact cells, fewer stomata and midvein vascular patterning in leaves, which provides them with more tolerance to abiotic stresses compared to mild overexpressors and the WT. Collectively, our results suggest that RhEXPA4, a cell-wall-loosening protein, confers tolerance to abiotic stresses through modifying cell expansion and plant development in Arabidopsis.     

Characterization and expression analysis of the <Emphasis Type="Italic">SNF2</Emphasis> family genes in response to phytohormones and abiotic stresses in rice

The function of SNF2 ATPases, the major catalytic subunits of chromatin remodeling complexes, in plants is not sufficiently understood. Here we identified 39 putative SNF2 genes of rice (Oryza sativa L.) by homology analyses and analyzed the expression profiles of eight of them in response to phytohormones and abiotic stresses. Our results indicated that expression of the SNF2 genes was affected by auxin, gibberellin, cytokinin, abscisic acid, ethylene, and some abiotic stresses such as heat, chilling, darkness, drought and salinity. It suggests that, like Arabidopsis SNF2s, rice SNF2 proteins may function in phytohormone signaling pathways and/or be associated with the resistance to abiotic stresses, but in distinct manners from their Arabidopsis orthologs. Some SNF2 proteins in rice may be involved in cross-talk of the signaling pathways between phytohormones and abiotic stresses.     

Ectopic overexpression of <Emphasis Type="Italic">Arabidopsis AtmiR393a</Emphasis> gene changes auxin sensitivity and enhances salt resistance in tobacco

To characterize the biological function of microRNA miR393 in tobacco, AtmiR393a gene was isolated from Arabidopsis using PCR and fused downstream to CaMV 35S promoter to make a plant expression construct 35S::AtmiR393a. The resultant construct was then introduced into tobacco with Agrobacterium-mediated transformation. Transgenic tobacco lines ectopically overexpressing AtmiR393a were successfully obtained. Transgenic lines L1 (a weak line), L2 (a middle line), and L3 (a strong line) were confirmed using stem-loop RT-PCRs and used to characterize the function of miR393 in tobacco. The results showed that L1, L2, and L3 exhibited reduced plant size and root length related to the WT control. In addition, seedling growth was less sensitive to IAA treatment and NaCl stress in three transgenic lines than the non-transgenic WT control. Furthermore, L1, L2, and L3 showed reduced phototropism relative to WT. Therefore, the biological function of miR393 is conserved in tobacco, just like in Arabidopsis. It regulates plant growth and development as well as the responses to environmental cues by influencing auxin sensitivity.