Hydrogen peroxide-induced salt tolerance in the <Emphasis Type="Italic">Arabidopsis</Emphasis> salicylate-deficient transformants <Emphasis Type="Italic">NahG</Emphasis>

The effect of hydrogen peroxide treatment on the salt tolerance of wild-type Arabidopsis thaliana L. plants (Col-0) and plants transformed with the bacterial salicylate hydroxylase gene (NahG) was studied. The base tolerance to salt stress caused by 200 mM of NaCl in solution culture was higher in plants with the NahG genotype in comparison with the wild-type plants. Growth inhibition was observed for wild-type plants under the action of exogenous hydrogen peroxide, which was not observed for the NahG transformants; salt tolerance increased in the both types of plants after treatment, which was assessed based on the growth indicators and the ability to preserve the chlorophyll pool following NaCl treatment. The content of endogenous Н2О2 in the leaves of wild-type plants increased significantly following exogenous hydrogen peroxide treatment and salt stress, while it practically did not change in the leaves of the NahG genotype. The SOD activity increased in both genotypes after treatment with exogenous hydrogen peroxide, and remained at an elevated level after salt stress in comparison with the nontreated plants. Furthermore, the catalase activity increased in leaves of the salicylate-deficient genotype but not in the Col-0 genotype. The guaiacol peroxidase activity increased in plants of both genotypes under the action of hydrogen peroxide and salt stress, with the NahG plants demonstrating a higher degree of increase. The Н₂О₂ treatment facilitated the increase of the proline content in leaves of the plants of both genotypes under conditions of salt stress. It was concluded that there were hydrogen peroxide signal transduction pathways in Arabidopsis plants that were salicylic acid independent and that the antioxidant system functioned more effectively in salicylate-deficient Arabidopsis plants.     

<Emphasis Type="Italic">Platycladus orientalis</Emphasis> PoKub3 is involved in abiotic stress responses in transgenic arabidopsis

Ku70-binding proteins associate with Ku70 and their expression levels can affect DSB repair efficiency via the DNA-PK-dependent repair pathway. However, how Ku70-binding proteins in plants exert a regulatory function under abiotic stress is poorly understood. Here, we cloned and characterized a PoKub3 gene from 500-year-old Platycladus orientalis. With increasing age, PoKub3 expression in P. orientalis increased gradually. The PoKub3 expression levels in leaves were upregulated under salt, heat, UV-C and abscisic acid treatments according to qRT-PCR. Moreover, PoKub3 overexpression in Arabidopsis thaliana improved tolerance to salt and drought stress compared with wild-type (WT) and vector control (VC) plants. High RAB18 and DREB2A expression and low JAZ1 and ABI2 expression provided strong evidence that salt tolerance was enhanced in the overexpression plants. Similarly, high RAB18 and DREB2A expression, accompanied by low JAZ1 and LOX1 expression and high DREB1A, CPK10, GSTF6 and APX1 expression, suggested the drought tolerance mechanism was associated with the abscisic acid pathway. In addition, lower malondialdehyde content, electrolyte leakage and stomatal conductance, and higher soluble sugar and relative water contents in PoKub3 overexpression lines than in WT and VC plants demonstrated its role in salt and drought tolerance. Together, these findings show that PoKub3 positively regulates salt and drought tolerance by regulating stress-related genes.     

Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> and K<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporters AtNHX1 and AtNHX3 from <Emphasis Type="Italic">Arabidopsis</Emphasis> improve salt and drought tolerance in transgenic poplar

The tonoplast and plasma membrane localized sodium (potassium)/proton antiporters have been shown to play an important role in plant resistance to salt stress. In this study, AtNHX1 and AtNHX3, two tonoplast Na⁺(K⁺)/H⁺ antiporter encoding genes from Arabidopsis thaliana, were expressed in poplar to investigate their biological functions in the resistance to abiotic stresses in woody plants. Transgenic poplar plants expressing either gene exhibited increased resistance to both salt and water-deficit stresses. Compared to the wild type (WT) plants, transgenic plants accumulated more sodium and potassium ions in the presence of 100 mM NaCl and showed reduced electrolyte leakage in the leaves under water stress. Furthermore, the proton-translocating and cation-dependent H⁺ (Na⁺/H⁺ or K⁺/H⁺) exchange activities in the tonoplast vesicles isolated from the leaves of transgenic plants were higher than in those isolated from WT plants. Therefore, constitutive expression of either AtNHX1 or AtNHX3 genetically modified the salt and water stress tolerance of transgenic poplar plants, providing a potential tool for engineering tree species with enhanced resistance to multiple abitotic stresses.     

Content of Osmolytes and Flavonoids under Salt Stress in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Plants Defective in Jasmonate Signaling

The effects of the salt stress (200 mM NaCl) and exogenous jasmonic acid (JA) on levels of osmolytes and flavonoids in leaves of four-week-old Arabidopsis thaliana L. plants of the wild-type (WT) Columbia-0 (Col-0) and the mutant jin1 (jasmonate insensitive 1) with impaired jasmonate signaling were studied. The increase in proline content caused by the salt stress was higher in the Col-0 plants than in the mutant jin1. This difference was especially marked if the plants had been pretreated with exogenous 0.1 μM JA. The sugar content increased in response to the salt stress in the JA-treated WT plants but decreased in the jin1 mutant. Treatment with JA of the WT plants but not mutant defective in jasmonate signaling also enhanced the levels of anthocyanins and flavonoids absorbed in UV-B range in leaves. The presence of JA increased salinity resistance of the Col-0 plants, since the accumulation of lipid peroxidation products and growth inhibition caused by NaCl were less pronounced. Under salt stress, JA almost did not render a positive effect on the jin1 plants. It is concluded that the protein JIN1/MYC2 is involved in control of protective systems under salt stress.     

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.     

Isolation and functional characterization of a novel stress inducible promoter from pigeonpea (<Emphasis Type="Italic">Cajanus cajan</Emphasis> L)

The present study deals with isolation and characterization of a novel hybrid-proline-rich protein gene (CcHyPRP) promoter from pigeonpea. Real time PCR analysis revealed that CcHyPRP expression was strongly induced by dehydration, salt, Abscisic acid (ABA) and Salicylic acid (SA) treatments. The CcHyPRP promoter, isolated by genome-walking method, contained 1112 bp and showed the presence of various cis -regulatory elements necessary for tissue specific expression and stress responsiveness. Different 5′ deletions of the promoter were generated and were used to drive the expression of β-glucuronidase reporter gene (gusA) in Arabidopsis thaliana. Histochemical and fluorometric assays confirmed that GUS expression driven by the full-length fragment (1112 bp) was higher when compared to different deletion fragments. Under normal conditions, GUS expression was predominantly detected in the roots and hypocotyls of transformants, while under mannitol, NaCl, ABA and SA treatment conditions higher GUS expression levels were observed in the roots and leaves. However, the GUS expression was mostly confined to the roots of transformants carrying 477 and 300 bp promoter regions. The results amply indicate that CcHyPRP promoter is regulated by different stress factors, and as such the promoter can be deployed in genetic engineering of crop plants for enhanced abiotic stress tolerance.     

Overexpression of <Emphasis Type="Italic">AtGRDP2</Emphasis> gene in common bean hairy roots generates vigorous plants with enhanced salt tolerance

Proteins with glycine-rich repeats have been identified in plants, mammalians, fungi, and bacteria. Plant glycine-rich proteins have been associated to stress response. Previously, we reported that the Arabidopsis thaliana AtGRDP2 gene, which encodes a protein with a glycine-rich domain, plays a role in growth and development of A. thaliana and Lactuca sativa. In this study, we generated composite Phaseolus vulgaris plants that overexpress the AtGRDP2 gene in hairy roots generated by Agrobacterium rhizogenes. We observed that hairy roots harboring the AtGRDP2 gene developed more abundant and faster-growing roots than control hairy roots generated with the wild type A. rhizogenes. In addition, composite common bean plants overexpressing the AtGRDP2 gene in roots were more tolerant to salt stress showing increments in their fresh and dry weight. Our data further support the role of plant GRDP genes in development and stress response.     

Salt tolerance conferred by expression of a global regulator IrrE from <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> in oilseed rape

As salinity is a major threat to sustainable agriculture worldwide, cultivation of salt-tolerant crops becomes increasingly important. IrrE acts as a global regulator and a general switch for stress resistance in Deinococcus radiodurans. In this study, to determine whether the irrE gene can improve the salt tolerance of Brassica napus, we introduced the irrE gene into B. napus by the Agrobacterium tumefaciens-mediated transformation method. Forty-two independent transgenic plants were regenerated. Polymerase chain reaction (PCR) analyses confirmed that the irrE gene had integrated into the plant genome. Northern as well as Western blot analyses revealed that the transgene was expressed at various levels in transgenic plants. Analysis for the T₁ progenies derived from four independent transformants showed that irrE had enhanced the salt tolerance of T₁ in the presence of 350 mM NaCl. Furthermore, under salt stress, transgenic plants accumulated more compatible solutes (proline) and a lower level of malondialdehyde (MDA), and they had higher activities of catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD). However, agronomic traits were not affected by irrE gene overexpression in the transgenic B. napus plants. This study indicates that the irrE gene can improve the salt tolerance of B. napus and represents a promising candidate for the development of crops with enhanced salt tolerance by genetic engineering.     

Salt tolerance mechanisms in three Irano-Turanian Brassicaceae halophytes relatives of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Salt tolerance mechanisms were studied in three Irano-Turanian halophytic species from the Brassicaceae ??(Lepidium latifolium, L. perfoliatum and Schrenkiella parvula) and compared with the glycophyte Arabidopsis thaliana. According to seed germination under salt stress, L. perfoliatum was the most tolerant species, while L. latifolium and S. parvula were rather susceptible. Contrastingly, based on biomass production L. perfoliatum was more salt sensitive than the other two species. In S. parvula biomass was increased up to 2.8-fold by 100 mM NaCl; no significant growth reduction was observed even when exposed to 400 mM NaCl. Stable activities of antioxidative defense enzymes, nil or negligible accumulation of superoxide anion and hydrogen peroxide, as well as stable membrane integrity in the three halophytes revealed that no oxidative stress occurred in these tolerant species under salt stress. Proline levels increased in response to salt treatment. However, it contributed only by 0.3?2.0% to the total osmolyte concentration in the three halophytes (at 400 mM NaCl) and even less (0.04%) in the glycophyte, A. thaliana (at 100 mM NaCl). Soluble sugars in all three halophytes and free amino acids pool in S. parvula decreased under salt treatment in contrast to the glycophyte, A. thaliana. The contribution of organic osmolytes to the total osmolyte pool increased by salt treatment in the roots, while decreased in halophyte and glycophyte, A. thaliana leaves. Interestingly, this reduction was compensated by a higher relative contribution of K in the leaves of the halophytes, but of Na in A. thaliana. Taken together, biomass data and biochemical indicators show that S. parvula is more salt tolerant than the two Lepidium species. Our data indicate that L. latifolium, as a perennial halophyte with a large biomass, is highly suitable for both restoration of saline habitats and saline agriculture.     

<Emphasis Type="Italic">OsNHX2</Emphasis>, an Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene,can enhance salt tolerance in rice plants through more effective accumulation of toxic Na<Superscript>+</Superscript> in leaf mesophyll and bundle sheath cells

The Na⁺/H⁺ antiporters play an important role in salt tolerance in plants. However, the functions of OsNHXs in rice except OsNHX1 have not been well studied. Using the gain- and loss-of-function strategies, we studied the potential role of OsNHX2 in salt tolerance in rice. Overexpression of OsNHX2 (OsNHX2-OE) in rice showed the significant tolerance to salt stress than wild-type plants and OsNHX2 knockdown transgenic plants (OsNHX2-KD). Under salt treatments of 300-mM NaCl for 5 days, the plant fresh weights, relative water percentages, shoot heights, Na⁺ contents, K⁺ contents, and K⁺/Na⁺ ratios in leaves of OsNHX2-OE transgenic plants were higher than those in wild-type plants, while no differences were detected in roots. K⁺/Na⁺ ratios in rice leaf mesophyll cells and bundle sheath cells were higher in OsNHX2-OE transgenic plants than in wild-type plants and OsNHX2-KD transgenic plants. Our data indicate that OsNHX2 plays an important role in salt stress based on leaf mesophyll cells and bundle sheath cells and can be served in genetically engineering crop plants with enhanced salt 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 Leucine-Rich Repeat Receptor-like Kinase from the Antarctic Moss <Emphasis Type="Italic">Pohlia nutans</Emphasis> Confers Salinity and ABA Stress Tolerance

Plant leucine-rich repeats receptor-like kinases (LRR-RLKs) play key roles in plant growth, development, and responses to environmental stresses. However, the functions of LRR-RLKs in bryophytes are still not well documented. Here, a putative LRR-RLK gene, PnLRR-RLK, was cloned and characterized from the Antarctic moss Pohlia nutans. Phylogenetic analysis revealed that PnLRR-RLK protein was clustered with the Arabidopsis thaliana LRR XI family proteins. Subcellular localization analysis of PnLRR-RLK revealed that it was mainly localized on plasma membrane. The expression of PnLRR-RLK was induced by mock high salinity, cold, drought, and exogenously supplied abscisic acid (ABA) and methyl jasmonate (MeJA). Meanwhile, the overexpression of PnLRR-RLK showed an increased tolerance of transgenic Arabidopsis to salt and ABA stresses than that of the wild type (WT) plants. Furthermore, the expression levels of several salt tolerance genes (AtHKT1, AtSOS3, AtP5CS1, and AtADH1) and an ABA negatively regulating gene AtABI1 were significantly increased in transgenic plants. Meanwhile, the expression levels of ABA biosynthesis genes (AtNCED3, AtABA1, and AtAAO3) and ABA early response genes (AtMYB2, AtRD22, AtRD29A, and AtDREB2A) were decreased in transgenic Arabidopsis after salt stress treatment. Therefore, these results suggested that PnLRR-RLK might involve in regulating salt stress-related and ABA-dependent signaling pathway, thereby contribute to the salinity tolerance of the Antarctic moss P. nutans.     

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.