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.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of <Emphasis Type="Italic">Lotus tenuis</Emphasis> and regeneration of transgenic lines

A protocol for the production of transgenic plants was developed for Lotus tenuis via Agrobacterium-mediated transformation of leaf segments. The explants were co-cultivated (for 3 days) with an A. tumefaciens strain harbouring either the binary vector pBi RD29A:oat arginine decarboxylase (ADC) or pBi RD29A:glucuronidase (GUS), which carries the neomycin phosphotransferase II (nptII) gene in the T-DNA region. Following co-cultivation, the explants were cultured in Murashige and Skoog medium supplemented with naphthalenacetic acid (NAA) and benzyladenine (BA) and containing kanamycin (30 μg ml⁻¹) and cefotaxime (400 μg ml⁻¹) for 45 days. The explants were subcultured several times (at 2-week intervals) to maintain the selection pressure during the entire period. About 40% of the explants inoculated with the pBiRD29:ADC strain produced eight to ten adventitious shoots per responsive explant through a direct system of regeneration, whereas 69% of the explants inoculated with the pBi RD29A:GUS strain produced 13–15 adventitious shoots per responsive explant. The selected transgenic lines were identified by PCR and Southern blot analysis. Three ADC transgenic lines were obtained from 30 infected explants, whereas 29 GUS transgenic lines were obtained from 160 explants, corresponding to a transformation efficiency of 10 and 18.1%, respectively. More than 90% of the in vitro plantlets were successfully transferred to the soil. The increase in the activity of arginine decarboxylase from stressed ADC- Lt19 lines was accompanied by a significant rise in the putrescine level. The GUS transgenic line driven by the RD29A promoter showed strong signals of osmotic stress in the leaves and stem tissues. All of the transgenic plants obtained exhibited the same phenotype as the untransformed controls under non-stress conditions, and the stability of the gene introduced into the cloned materials was established.     

Polymorphism of the <Emphasis Type="Italic">LEAFY</Emphasis> Gene in <Emphasis Type="Italic">Brassica</Emphasis> Plants

The arabidopsis gene LEAFY controls the induction of flowering and maintenance of the floral meristem identity. By comparing the primary structure of LEAFY and its homologs in other Brassicaceae species and beyond this family, we singled out four clusters corresponding to three systematically remote families of angiosperms, Brassicaceae, Solanaceae, and Poaceae, and to gymnosperms. Both structural and functional distinctions of LEAFY homologs from their arabidopsis prototype expanded in the range Brassicaceae—Solanaceae—Poaceae. A LEAFY homolog from B. juncea cloned in our laboratory was used as a hybridization probe to analyze the restriction fragment length polymorphism in six Brassica species comprising diploid (AA, BB, and CC) and allotetraploid (AABB, AACC, and BBCC) genomes. In this way we recognized LEAFY fragments specific of genomes A, B, and C; in contrast, the variations of the length and structure of the LEAFY intron 2 were not genome-specific. LEAFY polymorphism in the Brassica accessions comprising genome B was related to their geographic origin and apparently to the adaptation to day length.     

Overexpression and Inactivation of <Emphasis Type="Italic">UGT73B2</Emphasis> Modulate Tolerance to Oxidative Stress in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Glycosyltransferases (GTs) play an important role in modulating solubility, stability, bioavailability, and bioactivity of secondary metabolites, such as flavonoids. In Arabidopsis thaliana, at least 120 family 1 uridine diphosphate (UDP)-glycosyltransferases (UGTs) have been predicted. However, little is known about their substrates or their physiological roles in planta. To define the role of UGT73B2 in planta, we first characterized its expression pattern using transgenic Arabidopsis plants carrying the cis-elements of UGT73B2 fused to the GUS reporter. During vegetative phase, its expression was high in embryonic and postembryonic roots, where it may play a physiological role in the glycosylation of flavonoids. Loss of function of UGT73B2 alone or in conjunction with its closest homologs, UGT73B1 and UGT73B3, confers greater tolerance to oxidative stress, whereas overexpression of UGT73B2 increases sensitivity to oxidative stress. In addition, growth phenotypes of mutant and transgenic seedlings correlate well with ROS levels in planta. Our results suggest that the glycosylation of flavonoids by UGT73B2—and/or its closest homologs—modulate the response of plants to oxidative stress.     

Overexpression of Organellar and Cytosolic <Emphasis Type="Italic">AtHSP90</Emphasis> in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Impairs Plant Tolerance to Oxidative Stress

Three AtHSP90 isoforms, cytosol-localized AtHSP90.2, chloroplast-localized AtHSP90.5, and endoplasmic reticulum (ER)-localized AtHSP90.7 genes, were constitutively overexpressed in Arabidopsis thaliana to study their functional mechanisms under oxidative stress. Overexpression of AtHSP90 genes reduced germination of transgenic seeds under oxidative stress. When exposed to 10 mM H₂O₂, AtHSP90 transgenic seedlings displayed lower activities of superoxide dismutase, catalase, and peroxidase; higher content of malondialdehyde; and higher levels of protein damage than detected in the wild type. This indicated that overexpression of AtHSP90.2, AtHSP90.5, and AtHSP90.7 in Arabidopsis impaired plant tolerance to oxidative stress. Moreover, overexpression of chloroplast- and ER-localized AtHSP90 resulted in lower resistance to oxidative stress than that of cytosolic AtHSP90. This suggested that HSP90.2, HSP90.5, and HSP90.7 localized in different cellular compartments were involved in different functional mechanisms during oxidative stress.     

<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.     

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.     

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.     

Increase in Salt Tolerance of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> by <Emphasis Type="Italic">TaDi19</Emphasis>

The gene expression profile chip of salt-resistant wheat mutant RH8706-49 under salt stress was investigated. The overall length of the cDNA sequence of the probe was obtained using electronic cloning and RT-PCR. An unknown gene induced by salt was obtained, cloned, and named TaDi19 (Triticum aestivum drought-induced protein). No related report or research on the protein is available. qPCR analysis showed that gene expression was induced by many stresses, such as salt. Arabidopsis thaliana was genetically transferred using the overexpressing gene, which increased its salt tolerance. After salt stress, the transgenic plant demonstrated better physiological indicators (higher Ca²⁺ and lower Na⁺) than those of the wild-type plant. Results of non-invasive micro-test technology indicate that TaDi19-overexpressing A. thaliana significantly effluxed Na⁺ after salt treatment, whereas the wild-type plant influxed Na⁺. Chelating extracellular Ca²⁺ resulted in insignificant differences in salt tolerance between overexpressing and wild-type A. thaliana. Subcellular localization showed that the gene encoding protein was mainly located in the cell membrane and nucleus. TaDi19 was overexpressed in wild-type A. thaliana, and the transgenic lines were more salt-tolerant than the control A. thaliana. Thus, the wheat gene TaDi19 could increase the salt tolerance of A. thaliana.     

<Emphasis Type="Italic">Tolypothrix tenuis</Emphasis> stress response to nickel

Summary Metal ions are both essential and potentially toxic. The aim of this work was to demonstrate that diazotrophic cyanobacterium Tolypothrix tenuis N° 54 can tolerate toxic concentrations of Ni²⁺ in order to use the biomass in biofilters or as biofertilizer. For this purpose, growth, pigment and protein contents and catalase activity of T. tenuis growing in increasing concentrations of Ni²⁺ ranging from 10⁻¹⁰ to 10⁻⁴ M were assesed. The strain did not grow at Ni²⁺ concentration of 10⁻⁴ M, but at lower concentrations there were no significant differences with the control; it was tolerant at 10⁻¹⁰ and 10⁻⁸ M. Nickel concentration of 10⁻⁶ M is toxic for this cyanobacterial strain, because dry weight decreased by 30%; allophycocyanin and phycoerythrin decreased by 92% and 98%, respectively and protein content increased by 42%. Chlorophyll a concentration was more than double the control value in 10⁻¹⁰ and 10⁻⁸ M, but in 10⁻⁶ M it decreased by 19%. Catalase (E.C. 1.11.1.6) activity doubled the control value in the lowest nickel concentration whereas in 10⁻⁸ M there was no significant difference with the control and in 10⁻⁶, it decreased by 78%. The living biomass of this strain could be used as a step in the bioremediation process in waters contaminated with concentrations of nickel lower than 10⁻⁶ M and eventually as a biofertilizer.     

Overexpression of <Emphasis Type="Italic">IrlVHA</Emphasis>-<Emphasis Type="Italic">c</Emphasis>, A Vacuolar-Type H<Superscript>+</Superscript>-ATPase c Subunit Gene from <Emphasis Type="Italic">Iris lactea</Emphasis>, Enhances Salt Tolerance in Tobacco

Vacuolar-type H⁺-ATPase (V-ATPase), a multi-subunit endomembrane proton pump, plays an important role in plant growth and response to environmental stresses. In the present study, transgenic tobacco that overexpressed the V-ATPase c subunit gene from Iris lactea (IrlVHA-c) was used to determine the function of IrlVHA-c. Quantitative PCR analysis showed that IrlVHA-c expression was induced by salt stress in I. lactea roots and leaves. Subcellular localization of green fluorescent protein (GFP) as marker combined with FM4-64 staining showed that the IrlVHA-c-GFP was localized to the endosomal compartment in tobacco cells. Compared with the wild-type, the IrlVHA-c transgenic tobacco plants exhibited greater seed germination rates, root length, fresh weight, and higher relative water content (RWC) of leaves under salt stress. Furthermore, the IrlVHA-c transgenic tobacco leaves have lower stomatal densities and larger stomatal apertures than wild-type. Under salt stress, superoxide dismutase (SOD) activity in the transgenic tobacco was significantly enhanced. Moreover, the level of malondialdehyde (MDA) in the transgenic tobacco was significantly lower than that in wild-type plants under salt stress. Taken together, these results suggested that the IrlVHA-c plays an important role in salt tolerance in transgenic tobacco by influencing stomatal movement and physiological changes.     

Predation by <Emphasis Type="Italic">Nesidiocoris tenuis</Emphasis> on <Emphasis Type="Italic">Bemisia tabaci</Emphasis> and injury to tomato

Tomato is the most important vegetable crop in Spain. The mirid bug Nesidiocoris tenuis (Reuter) commonly appears in large numbers in protected and open-air tomato crops where little or no broad-spectrum insecticides are used. Nesidiocoris tenuis is known to be a predator of whiteflies, thrips and several other pest species. However, it is also considered a pest because it can feed on tomato plants, causing necrotic rings on stems and flowers and punctures in fruits. Our objectives were to evaluate predation by N. tenuis on sweetpotato whitefly Bemisia tabaci Gennadius under greenhouse conditions and establish its relationship to N. tenuis feeding on tomato. Two different release rates of N. tenuis were compared with an untreated control (0, 1 and 4 N. tenuis plant⁻¹) in cages of 8 m². Significant reductions of greater than 90% of the whitefly population and correspondingly high numbers of N. tenuis were observed with both release rates. Regression analysis showed that necrotic rings on foliage caused by N. tenuis were best explained by the ratio of B. tabaci nymphs:N. tenuis as predicted by the equation y = 15.086x − 0.6359.

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.