Overexpression of <Emphasis Type="Italic">Zm-HINT1</Emphasis> Confers Salt and Drought Tolerance in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Histidine triad nucleotide-binding protein 1 (HINT1) is highly conserved in many species and plays important roles in various biological processes. However, little is known about the responses of HINT1 to abiotic stress in plants. Salt and drought stress are major limiting factors for plant growth and development, and their negative effects on crop productivity may threaten the world’s food supply. Previously, we identified a maize gene, Zm-HINT1, which encodes a 138-amino-acid protein containing conserved domains including the HIT motif, helical regions, and β-strands. Here, we demonstrate that overexpression of Zm-HINT1 in Arabidopsis confers salt and drought tolerance to plants. Zm-HINT1 significantly regulated Na⁺ and K⁺ accumulation in plants under salt stress. The improve tolerance characteristics of Arabidopsis plants that were overexpressing Zm-HINT1 led to increased survival rates after salt and drought treatments. Compared with control plants, those plants that overexpressed Zm-HINT1 showed increased proline content and superoxide dismutase activity, as well as lower malondialdehyde and hydrogen peroxide accumulation under salt and drought treatments. The expression patterns of stress-responsive genes in Arabidopsis plants that overexpressed Zm-HINT1 significantly differed from those in control lines. Taken together, these results suggest that Zm-HINT1 has potential applications in breeding and genetic engineering strategies that are designed to produce new crop varieties with improved salt and drought tolerance.     

Over-Expression of <Emphasis Type="Italic">PmHSP17.9</Emphasis> in Transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Confers Thermotolerance

Small heat shock proteins (sHSPs) have been shown to be involved in stress tolerance. However, their functions in Prunus mume under heat treatment are poorly characterized. To improve our understanding of sHSPs, we cloned a sHSP gene, PmHSP17.9, from P. mume. Sequence alignment and phylogenetic analysis indicated that PmHSP17.9 was a member of plant cytosolic class III sHSPs. Besides heat stress, PmHSP17.9 was also upregulated by salt, dehydration, oxidative stresses and ABA treatment. Leaves of transgenic Arabidopsis thaliana that ectopically express PmHSP17.9 accumulated less O₂ ^? and H₂O₂ compared with wild type (WT) after 42 °C treatment for 6 h. Over-expression of PmHSP17.9 in transgenic Arabidopsis enhanced seedling thermotolerance by decreased relative electrolyte leakage and MDA content under heat stress treatment when compared to WT plants. In addition, the induced expression of HSP101, HSFA2, and delta 1-pyrroline-5-carboxylate synthase (P5CS) under heat stress was more pronounced in transgenic plants than in WT plants. These results support the positive role of PmHSP17.9 in response to heat stress treatment.     

A Phytocystatin Gene from <Emphasis Type="Italic">Malus prunifolia</Emphasis> (Willd.) Borkh., <Emphasis Type="Italic">MpCYS5</Emphasis>, Confers Salt Stress Tolerance and Functions in Endoplasmic Reticulum Stress Response in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Cystatins, or phytocystatins (PhyCys), comprise a family of plant-specific inhibitors of cysteine proteinases. They are thought to help regulate endogenous processes and protect plants against biotic or abiotic stresses, such as heat, salinity, cold, water deficit, chilling, and abscisic acid (ABA) treatment. We isolated and identified a novel cystatin gene from Malus prunifolia, MpCYS5. Its expression was typically induced by salt stress treatment; ectopic expression in Arabidopsis enhanced salt tolerance. Physiological parameters confirmed this phenotype, with the transgenics having remarkably lower electrolyte leakage (EL) values, higher chlorophyll concentrations, and lower levels of malondialdehyde (MDA) upon salt treatment. In addition, the accumulation of reactive oxygen species was markedly regulated by MpCYS5 under stress conditions, as shown by fluctuations in the concentrations of hydrogen peroxide and superoxide radicals and the activities of antioxidant enzymes. We also noted that this gene modulated tunicamycin (TM)-induced endoplasmic reticulum (ER) stress tolerance and functioned in the unfolded protein response (UPR)-signaling pathway in Arabidopsis. This was confirmed by the expression of eight ER stress-responsive genes. All marker genes examined were strongly induced in the wild type, while most of them maintained relatively stable over time in the transgenics. These results demonstrated that ectopic expression of a cystatin gene is associated with salt-tolerant and TM-tolerant phenotypes. Therefore, the discovery of MpCYS5 from M. prunifolia might establish a molecular link between the ER stress response and salt tolerance in plants.     

<Emphasis Type="Italic">SnRK2</Emphasis> Homologs in <Emphasis Type="Italic">Gossypium</Emphasis> and <Emphasis Type="Italic">GhSnRK2.6</Emphasis> Improved Salt Tolerance in Transgenic Upland Cotton and <Emphasis Type="Italic">Arabidopsis</Emphasis>

SnRK2s are a large family of plant-specific protein kinases, which play important roles in multiple abiotic stress responses in various plant species. But the family in Gossypium has not been well studied. Here, we identified 13, 10, and 13 members of the SnRK2 family from Gossypium raimondii, Gossypium arboreum, and Gossypium hirsutum, respectively, and analyzed the locations of SnRK2 homologs in chromosomes based on genome data of cotton species. Phylogenetic tree analysis of SnRK2 proteins showed that these families were classified into three groups. All SnRK2 genes were comprised of nine exons and eight introns, and the exon distributions and the intron phase of homolog genes among different cotton species were analogous. Moreover, GhSnRK2.6 was overexpressed in Arabidopsis and upland cotton, respectively. Under salt treatment, overexpressed Arabidopsis could maintain higher biomass accumulation than wild-type plants, and GhSnRK2.6 overexpression in cotton exhibited higher germination rate than the control. So, the gene GhSnRK2.6 could be utilized in cotton breeding for salt tolerance.     

Pollen-Specific Expression of <Emphasis Type="Italic">Oryza sativa Indica</Emphasis> Pollen Allergen Gene (<Emphasis Type="Italic">OSIPA</Emphasis>) Promoter in Rice and <Emphasis Type="Italic">Arabidopsis</Emphasis> Transgenic Systems

Earlier, a pollen-specific Oryza sativa indica pollen allergen gene (OSIPA), coding for expansins/pollen allergens, was isolated from rice, and its promoter—upon expression in tobacco and Arabidopsis—was found active during the late stages of pollen development. In this investigation, to analyze the effects of different putative regulatory motifs of OSIPA promoter, a series of 5′ deletions were fused to β-glucuronidase gene (GUS) which were stably introduced into rice and Arabidopsis. Histochemical GUS analysis of the transgenic plants revealed that a 1631 bp promoter fragment mediates maximum GUS expression at different stages of anther/pollen development. Promoter deletions to −1272, −966, −617, and −199 bp did not change the expression profile of the pollen specificity. However, the activity of promoter was reduced as the length of promoter decreased. The region between −1567 and −199 bp was found adequate to confer pollen-specific expression in both rice and Arabidopsis systems. An approximate 4-fold increase in the GUS activity was observed in the pollen of rice when compared to that of Arabidopsis. As such, the OSIPA promoter seems promising for generation of stable male-sterile lines required for the production of hybrids in rice and other crop plants.     

Transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> Plants Expressing Grape Glutathione S-Transferase Gene (<Emphasis Type="Italic">VvGSTF13</Emphasis>) Show Enhanced Tolerance to Abiotic Stress

Although glutathione S-transferase (GST, EC 2.5.1.18) is thought to play important roles in abiotic stress, limited information is available regarding the function of its gene in grapes. In this study, a GST gene from grape, VvGSTF13, was cloned and functionally characterized. Transgenic Arabidopsis plants containing this gene were normal in terms of growth and maturity compared with control plants but had enhanced resistance to salt, drought, and methyl viologen stress. The increased tolerance of the transgenic plants correlated with changes in activities of antioxidative enzymes. Our results indicate that the gene from grape plays a positive role in improving tolerance to salinity, drought, and methyl viologen stresses in Arabidopsis.     

Overexpression of <Emphasis Type="Italic">OsVP1</Emphasis> and <Emphasis Type="Italic">OsNHX1</Emphasis> Increases Tolerance to Drought and Salinity in Rice

Drought and salinity are major abiotic stresses affecting rice production. To improve plant tolerance to salinity and drought, we overexpressed rice Na⁺/H⁺ exchangers (OsNHX1) and H⁺-pyrophosphatase in tonoplasts (OsVP1) in a japonica elite rice cultivar, Zhonghua 11. Compared with our wild-type control, transgenic plants overexpressing both genes incurred less damage when exposed to long-term treatment with 100 mM NaCl or water deprivation. Under high-saline conditions, the transformants accumulated less Na⁺ and malondialdehyde in the leaves, thereby allowing the plants to maintain a low level of leaf water potential and reduce stress-induced damage. Those transgenics also had higher photosynthetic activity during the stress period. Under those conditions, they also showed an increase in root biomass, which enabled more water uptake. These results suggest that OsVP1 and OsNHX1 improve the tolerance of rice crops against drought and salt by employing multiple strategies in addition to osmotic regulation.     

Transgenic Rice Plants Expressing a Modified <Emphasis Type="Italic">cry1Ca1</Emphasis> Gene are Resistant to <Emphasis Type="Italic">Spodoptera litura</Emphasis> and <Emphasis Type="Italic">Chilo</Emphasis><Emphasis Type="Italic">suppressalis</Emphasis>

Nucleotide sequence encoding the truncated insecticidal Cry1Ca1 protein from Bacillus thuringiensis was extensively modified based on the codon usage of rice genes. The overall G + C contents of the synthetic cry1Ca1 coding sequence were raised to 65% with an additional bias of enriching for G and C ending codons as preferred by monocots. The synthetic gene was introduced into the Chinese japonica variety, Xiushui 11, by Agrobacterium-mediated transformation. Transgenic rice plants harboring this gene were highly resistant to Chilo suppressalis and Spodoptera litura larvae as revealed by insect bioassays. High levels of Cry1Ca1 protein were obtained in the leaves of transgenic rice, which were effective in achieving 100% mortality of S. litura and C. suppressalis larvae. The levels of Cry1Ca1 expression in the leaves of these transgenic plants were up to 0.34% of the total soluble proteins. The larvae of C. suppressalis and S. litura could consume a maximum of 1.89  and 4.89 mm² of transgenic leaf area whereas the consumption of non-transgenic leaves by these larvae was significantly higher; 58.33 and 61.22 mm², respectively. Analysis of R₁ transgenic plants indicated that the cry1Ca1 was inherited by the progeny plants and provided complete protection against C. suppressalis and S. litura larvae.