Constitutive expression of a meiotic recombination protein gene homolog, OsTOP6A1, from rice confers abiotic stress tolerance in transgenic Arabidopsis plants

Plant productivity is greatly influenced by various environmental stresses, such as high salinity and drought. Earlier, we reported the isolation of topoisomerase 6 homologs from rice and showed that over expression of OsTOP6A3 and OsTOP6B confers abiotic stress tolerance in transgenic Arabidopsis plants. In this study, we have assessed the function of nuclear-localized topoisomerase 6 subunit A homolog, OsTOP6A1, in transgenic Arabidopsis plants. The over expression of OsTOP6A1 in transgenic Arabidopsis plants driven by cauliflower mosaic virus-35S promoter resulted in pleiotropic effects on plant growth and development. The transgenic Arabidopsis plants showed reduced sensitivity to stress hormone, abscisic acid (ABA), and tolerance to high salinity and dehydration at the seed germination; seedling and adult stages as reflected by the percentage of germination, fresh weight of seedlings and leaf senescence assay, respectively. Concomitantly, the expression of many stress-responsive genes was enhanced under various stress conditions in transgenic Arabidopsis plants. Moreover, microarray analysis revealed that the expression of a large number of genes involved in various processes of plant growth and development and stress responses was altered in transgenic plants. Although AtSPO11-1, the homolog of OsTOP6A1 in Arabidopsis, has been implicated in meiotic recombination; the present study demonstrates possible additional role of OsTOP6A1 and provides an effective tool for engineering crop plants for tolerance to different environmental stresses. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Transgenic Plants of Creeping Bent Grass Harboring the Stress Inducible Gene, 9-cis-epoxycarotenoid dioxygenase, are Highly Tolerant to Drought and NaCl Stress

Transgenic lines of creeping bent grass were generated by Agrobacterium-mediated transformation with the VuNCED1 which was cloned from cow pea has a homology to 9-cis-epoxycarotenoid dioxygenase, which is supposed to be involved in abscisic acid (ABA) biosynthesis. ABA, a cleavage product of carotenoids, is involved in stress responses in plants. The limiting step of ABA biosynthesis in plants is presumably the cleavage of 9-cis-epoxycarotenoids, the first committed step of ABA biosynthesis. Molecular analyses of transgenic lines as performed by Southern hybridization genomic DNA-PCR revealed integration of the VuNCED1. Challenge studies performed with transgenic plants by exposure to salt stress (up to 10 dS m⁻¹) and water stress (up to 75%) for 10 weeks, revealed that more than 50% of the transgenic plants could survive NaCl and drought stress whereas wild-type was not. ABA levels were measured under drought and normal conditions, endogenous ABA was dramatically increased by drought and NaCl stress in transgenic plants. These results indicate that it is possible to manipulate ABA levels in plants by over expressing the key regulatory gene in ABA biosynthesis and that stress tolerance can be improved by increasing ABA levels. Chenna Reddy Aswath and Sun Hyung Kim - First two authors contributed equally to this work     

Over-expression of OsUGE-1 altered raffinose level and tolerance to abiotic stress but not morphology in Arabidopsis

OsUGE-1 is known to be induced by various abiotic stresses, but its exact function in plants is unclear. In the present study, OsUGE-1 was over-expressed in Arabidopsis, transgenic plants conferred tolerance to salt, drought and freezing stress without altering plant morphology. In addition, transgenic plants showed a higher level of the soluble sugar raffinose than did wild-type plants. Our results suggest that elevated level of raffinose with over-expressed OsUGE-1 resulted in enhanced tolerance to abiotic stress. Thus, the gene may be applied to improve tolerance to abiotic stress in crops.     

Enhanced drought tolerance in transgenic Leymus chinensis plants with constitutively expressed wheat TaLEA3

Leymus chinensis is an important grassland perennial grass. However, its drought tolerance requires to be improved. LEA (late embryogenesis abundant) genes are believed to confer resistance to drought and water deficiency. Using Agrobacterium-mediated transformation, a wheat LEA gene, TaLEA ₃ , was integrated into L. chinensis. The transgenic lines showed enhanced growth ability under drought stress during which transgenic lines had increased the relative water content, leaf water potential, relative average growth rate, but decreased the malondialdehyde content compared with the non-transgenic plant. Thus, transgenic breeding is an efficient approach to enhance drought tolerance in L. chinensis.     

Abiotic stress tolerance in transgenic eggplant (Solanum melongena L.) by introduction of bacterial mannitol phosphodehydrogenase gene

In the present work, the bacterial mannitol-1-phosphodehydrogenase(mtlD) gene was introduced into eggplant(Solanummelongena L.) by Agrobacteriumtumefaciens-mediated transformation. Several transformants weregenerated and the transgene integration was confirmed by PCR, dot blot andSouthern blot analysis. Transgenic lines of T₀ and T₁generations were examined for tolerance to NaCl-induced salt stress,polyethylene glycol-mediated drought and chilling stress under bothinvitro and in vivo growth conditions. Aconsiderable proportions of transgenic seeds germinated and seedlings grew wellon 200 mM salt-amended MS basal medium, whereas seeds ofuntransformed control plants failed to germinate. Further, leaf explants fromthe transgenics could grow and showed signs of shoot regeneration onsalt-amended MS regeneration medium, whereas wild type did not respond, and infact the explants showed necrosis and loss of chlorophyll after about one week.The transgenic leaves could also withstand desiccation, and transgenics couldgrow well under chilling stress, and hydroponic conditions with salt stress ascompared to wild type plants. Thus, the transgenic lines were found to betolerant against osmotic stress induced by salt, drought and chilling stress.The morphology of the transgenic plants was normal as controls, but thechlorophyll content was higher in some of the lines. These observations suggestthat mtlD gene can impart abiotic stress tolerance ineggplant.     

Overexpression of wheat dehydrin DHN-5 enhances tolerance to salt and osmotic stress in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Late Embryogenesis Abundant (LEA) proteins are associated with tolerance to water-related stress. A wheat (Triticum durum) group 2 LEA proteins, known also as dehydrin (DHN-5), has been previously shown to be induced by salt and abscisic acid (ABA). In this report, we analyze the effect of ectopic expression of Dhn-5 cDNA in Arabidopsis thaliana plants and their response to salt and osmotic stress. When compared to wild type plants, the Dhn-5 transgenic plants exhibited stronger growth under high concentrations of NaCl or under water deprivation, and showed a faster recovery from mannitol treatment. Leaf area and seed germination rate decreased much more in wild type than in transgenic plants subjected to salt stress. Moreover, the water potential was more negative in transgenic than in wild type plants. In addition, the transgenic plants have higher proline contents and lower water loss rate under water stress. Also, Na⁺ and K⁺ accumulate to higher contents in the leaves of the transgenic plants. Our data strongly support the hypothesis that Dhn-5, by its protective role, contributes to an improved tolerance to salt and drought stress through osmotic adjustment.     

Salt stress alleviation in transgenic Vigna mungo L. Hepper (blackgram) by overexpression of the glyoxalase I gene using a novel Cestrum yellow leaf curling virus (CmYLCV) promoter

A reproducible and efficient transformation system utilizing the nodal regions of embryonal axis of blackgram (Vigna mungo L. Hepper) has been established via Agrobacterium tumefaciens. This is a report of genetic transformation of Vigna mungo for value addition of an agronomic trait, wherein the gene of interest, the glyoxalase I driven by a novel constitutive Cestrum yellow leaf curling viral promoter has been transferred for alleviating salt stress. The overexpression of this gene under the constitutive CaMV 35S promoter had earlier been shown to impart salt, heavy metal and drought stress tolerance in the model plant, tobacco. Molecular analyses of four independent transgenic lines performed by PCR, Southern and western blot revealed the stable integration of the transgene in the progeny. The transformation frequency was ca. 2.25% and the time required for the generation of transgenic plants was 10–11 weeks. Exposure of T1 transgenic plants as well as untransformed control plants to salt stress (100 mM NaCl) revealed that the transgenic plants survived under salt stress and set seed whereas the untransformed control plants failed to survive. The higher level of Glyoxalase I activity in transgenic lines was directly correlated with their ability to withstand salt stress. To the best of our knowledge this is the only report of engineering abiotic stress tolerance in blackgram. Prasanna Bhomkar, Chandrama P. Upadhyay are contributed equally. An erratum to this article can be found at     

Increased glycine betaine synthesis and salinity tolerance in AhCMO transgenic cotton lines

Glycine betaine is an osmoprotectant that plays an important role and accumulates rapidly in many plants during salinity or drought stress. Choline monooxygenase (CMO) is a major catalyst in the synthesis of glycine betaine. In our previous study, a CMO gene (AhCMO) cloned from Atriplex hortensis was introduced into cotton (Gossypium hirsutum L.) via Agrobacterium mediation to enhance resistance to salinity stress. However, there is little or no knowledge of the salinity tolerance of the transgenic plants, particularly under saline-field conditions. In the present study, two transgenic AhCMO cotton lines of the T₃ generation were used to study the AhCMO gene expression, and to determine their salinity tolerance in both greenhouse and field under salinity stress. Molecular analysis confirmed that the transgenic plants expressed the AhCMO gene. Greenhouse study showed that on average, seedlings of the transgenic lines accumulated 26 and 131% more glycine betaine than those of non-transgenic plants (SM3) under normal and salt-stress (150 mmol l⁻¹ NaCl) conditions, respectively. The osmotic potential, electrolyte leakage and malondialdehyde (MDA) accumulation were significantly lower in leaves of the transgenic lines than in those of SM3 after salt stress. The net photosynthesis rate and Fv/Fm in transgenic cotton leaves were less affected by salinity than in non-transgenic cotton leaves. Therefore, transgenic cotton over-expressing AhCMO was more tolerant to salt stress due to elevated accumulation of glycine betaine, which provided greater protection of the cell membrane and photosynthetic capacity than in non-transgenic cotton. The seed cotton yield of the transgenic plants was lower under normal conditions, but was significantly higher than that of non-transgenic plants under salt-stressed field conditions. The results indicate that over-expression of AhCMO in cotton enhanced salt stress tolerance, which is of great value in cotton production in the saline fields.     

Expression of TERF1 in rice regulates expression of stress-responsive genes and enhances tolerance to drought and high-salinity

Drought and high-salinity are the important constraints that severely affect plant development and crop yield worldwide. It has been established that ethylene response factor (ERF) proteins play important regulatory roles in plant response to abiotic and biotic stresses. Our previous researches have revealed that transgenic tobacco over-expressing TERF1 (encoding a tomato ERF protein) showed enhanced tolerance to abiotic stress. Here, we further investigate the function of TERF1 in transgenic rice. Compared with the wild-type plants, overexpression of TERF1 resulted in an increased tolerance to drought and high-salt in transgenic rice. And the enhanced tolerance may be associated with the accumulation of proline and the decrease of water loss. Furthermore, TERF1 can effectively regulate the expression of stress-related functional genes Lip5, Wcor413-l, OsPrx and OsABA2, as well as regulatory genes OsCDPK7, OsCDPK13 and OsCDPK19 under normal growth conditions. Our analyses of cis-acting elements show that there exist DRE/CRT and/or GCC-box existing in TERF1 targeted gene promoters. Our results revealed that ectopic expression of TERF1 in rice caused a series of molecular and physiological alterations and resulted in the transgenic rice with enhanced tolerance to abiotic stress, indicating that TERF1 might have similar regulatory roles in response to abiotic stress in tobacco and rice. Shumei Gao, Haiwen Zhang and Yun Tian contributed equally to this work.     

Overexpression of a <Emphasis Type="Italic">Panax ginseng</Emphasis> tonoplast aquaporin alters salt tolerance,drought tolerance and cold acclimation ability in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> plants

Water movement across cellular membranes is regulated largely by a family of water channel proteins called aquaporins (AQPs). Since several abiotic stresses such as, drought, salinity and freezing, manifest themselves via altering water status of plant cells and are linked by the fact that they all result in cellular dehydration, we overexpressed an AQP (tonoplast intrinsic protein) from Panax ginseng, PgTIP1, in transgenic Arabidopsis thaliana plants to test its role in plant’s response to drought, salinity and cold acclimation (induced freezing tolerance). Under favorable conditions, PgTIP1 overexpression significantly increased plant growth as determined by the biomass production, and leaf and root morphology. PgTIP1 overexpression had beneficial effect on salt-stress tolerance as indicated by superior growth status and seed germination of transgenic plants under salt stress; shoots of salt-stressed transgenic plants also accumulated greater amounts of Na⁺ compared to wild-type plants. Whereas PgTIP1 overexpression diminished the water-deficit tolerance of plants grown in shallow (10 cm deep) pots, the transgenic plants were significantly more tolerant to water stress when grown in 45 cm deep pots. The rationale for this contrasting response, apparently, comes from the differences in the root morphology and leaf water channel activity (speed of dehydration/rehydration) between the transgenic and wild-type plants. Plants overexpressed with PgTIP1 exhibited lower (relative to wild-type control) cold acclimation ability; however, this response was independent of cold-regulated gene expression. Our results demonstrate a significant function of PgTIP1 in growth and development of plant cells, and suggest that the water movement across tonoplast (via AQP) represents a rate-limiting factor for plant vigor under favorable growth conditions and also significantly affect responses of plant to drought, salt and cold stresses.     

Overexpression of <Emphasis Type="Italic">AtHDG11</Emphasis> enhanced drought tolerance in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Drought is one of the major abiotic stresses restricting the yield of wheat (Triticum aestivum L.). Breeding wheat varieties with drought tolerance is an effective and durable way to fight against drought. Here we reported introduction of AtHDG11 into wheat via Agrobacterium-mediated transformation and analyzed the morphological and physiological characteristics of T2 generation transgenic lines under drought stress. With drought treatment for 30 days, transgenic plants showed significantly improved drought tolerance. Compared with controls, the transgenic lines displayed lower stomatal density, lower water loss rate, more proline accumulation and increased activities of catalase and superoxide dismutase. Without irrigation after booting stage, the photosynthetic parameters, such as net photosynthesis rate, water use efficiency and efficiency of excitation energy, were increased in transgenic lines, while transpiration rate was decreased. Moreover, the kernel yield of transgenic lines was also improved under drought condition. Taken together, our data demonstrate that AtHDG11 has great potential in genetic improvement of drought tolerance of wheat.     

A Medicago truncatula EF-Hand Family Gene,MtCaMP1, Is Involved in Drought and Salt Stress Tolerance

Background

Calcium-binding proteins that contain EF-hand motifs have been reported to play important roles in transduction of signals associated with biotic and abiotic stresses. To functionally characterize gens of EF-hand family in response to abiotic stress, an MtCaMP1 gene belonging to EF-hand family from legume model plant Medicago truncatula was isolated and its function in response to drought and salt stress was investigated by expressing MtCaMP1 in Arabidopsis.

Methodology/Principal Findings

Transgenic Arabidopsis seedlings expressing MtCaMP1exhibited higher survival rate than wild-type seedlings under drought and salt stress, suggesting that expression of MtCaMP1 confers tolerance of Arabidopsis to drought and salt stress. The transgenic plants accumulated greater amounts of Pro due to up-regulation of P5CS1 and down-regulation of ProDH than wild-type plants under drought stress. There was a less accumulation of Na⁺ in the transgenic plants than in WT plants due to reduced up-regulation of AtHKT1 and enhanced regulation of AtNHX1 in the transgenic plants compared to WT plants under salt stress. There was a reduced accumulation of H₂O₂ and malondialdehyde in the transgenic plants than in WT plants under both drought and salt stress.

Conclusions/Significance

The expression of MtCaMP1 in Arabidopsis enhanced tolerance of the transgenic plants to drought and salt stress by effective osmo-regulation due to greater accumulation of Pro and by minimizing toxic Na⁺ accumulation, respectively. The enhanced accumulation of Pro and reduced accumulation of Na⁺ under drought and salt stress would protect plants from water default and Na⁺ toxicity, and alleviate the associated oxidative stress. These findings demonstrate that MtCaMP1 encodes a stress-responsive EF-hand protein that plays a regulatory role in response of plants to drought and salt stress.     

MdVHP1 encodes an apple vacuolar H(+)-PPase and enhances stress tolerance in transgenic apple callus and tomato

Vacuolar H⁺-translocating inorganic pyrophosphatase (VHP, EC 3.6.1.1) is an electrogenic proton pump, which is related to growth as well as abiotic stress tolerance in plants. In this study, a VHP gene MdVHP1 was isolated from apple. The alignment of nucleotide and amino acid sequences showed that it encoded a type I VHP protein. It expressed in vegetative and reproductive organs, and its expression was induced by salt, PEG-mediated osmotic stress, cold and heat in apple in vitro shoot cultures. MdVHP1 expression showed a similar pattern in different apple tissues, but different change dynamics in response to abiotic stresses, compared with MdVHP2 (another MdVHP gene in apple). MdVHP1 overexpression enhanced tolerance to salt, PEG-mimic drought, cold and heat in transgenic apple calluses, which was related to an increased accumulation of proline and decreased MDA content compared with control calluses. In addition, MdVHP1 overexpression confers improved tolerance to salt and drought in transgenic tomato, along with an increased ion accumulation, high RWC and low solute potential compared with wild type. These results indicate that MdVHP1 is an important regulator for plant tolerance to abiotic stresses by modulating internal stores of ions and solutes.