Overexpression of the transcription factor MdWRKY115 improves drought and osmotic stress tolerance by directly binding to the MdRD22 promoter in apple

Abiotic stress reduces plant yield and quality.WRKY transcription factors play key roles in abiotic stress responses in plants,but the molecular mechanisms by which WRKY transcription factors mediate responses to drought and osmotic stresses in apple(Malus×domestica Borkh.)remain unclear.Here,we functionally characterized the apple GroupⅢWRKY gene MdWRKY115.qRT-PCR analysis showed that MdWRKY115 expression was up-regulated by drought and osmotic stresses.GUS activity analysis revealed that the promoter activity of MdWRKY115 was enhanced under osmotic stress.Subcellular localization and transactivation assays indicated that MdWRKY115 was localized to the nucleus and had a transcriptional activity domain at the N-terminal region.Transgenic analysis revealed that the overexpression of MdWRKY115 in Arabidopsis plants and in apple callus markedly enhanced their tolerance to drought and osmotic stresses.DNA affinity purification sequencing showed that MdWRKY115 binds to the promoter of the stress-related gene MdRD22.This binding was further verified by an electrophoretic mobility shift assay.Collectively,these findings suggest that MdWRKY115 is an important regulator of osmotic and drought stress tolerance in apple.

The CBL-interacting protein kinase OsCIPK1 phosphorylated by SAPK10 positively regulates responses to ABA and osmotic stress in rice

SubclassⅢsucrose nonfermenting1-related protein kinase 2s(SnRK2s)function in ABA and abiotic stress responses by unknown mechanisms.We found that osmotic stress/ABA-activated protein kinase 10(SAPK10),a member of rice SnRK2s,physically interacted with CBL-interacting protein kinase 1(OsCIPK1).OsCIPK1 expression was up-regulated by ABA and PEG treatment,and overexpression increased the ABA sensitivity of seed germination and root growth and plant osmotic stress tolerance.Osmotic stress or ABA-induced activation of OsCIPK1 is dependent on SAPK10.SAPK10 phosphorylates Thr-24 of OsCIPK1 in vitro,and this phosphorylation increases the activity of OsCIPK1 and positively regulates the function of OsCIPK1 in ABA responses and plant osmotic stress tolerance.This study suggests that OsCIPK1 is a direct phosphorylated substrate of SAPK10,and SAPK10-mediated phosphorylation of OsCIPK1 functions in ABA signaling and increases rice osmotic stress tolerance.

JA-mediated MYC2/LOX/AOS feedback loop regulates osmotic stress response in tea plant

Osmotic stress caused by low-temperature,drought and salinity was a prevalent abiotic stress in plant that severely inhibited plant development and agricultural yield,particularly in tea plant.Jasmonic acid(JA)is an important phytohormone involving in plant stress.However,underlying molecular mechanisms of JA modulated osmotic stress response remains unclear.In this study,high concentration of mannitol induced JA accumulation and increase of peroxidase activity in tea plant.Integrated transcriptome mined a JA signaling master,MYC2 transcription factor is shown as a hub regulator that induced by mannitol,expression of which positively correlated with JA biosynthetic genes(LOX and AOS)and peroxidase genes(PER).CsMYC2 was determined as a nuclei-localized transcription activator,furthermore,ProteinDNA interaction analysis indicated that CsMYC2 was positive regulator that activated the transcription of CsLOX7,CsAOS2,CsPER1 and CsPER3via bound with their promoters,respectively.Suppression of CsMYC2 expression resulted in a reduced JA content and peroxidase activity and osmotic stress tolerance of tea plant.Overexpression of CsMYC2 in Arabidopsis improved JA content,peroxidase activity and plants tolerance against mannitol stress.Together,we proposed a positive feedback loop mediated by CsMYC2,CsLOX7 and CsAOS2 which constituted to increase the tolerance of osmotic stress through fine-tuning the accumulation of JA levels and increase of POD activity in tea plant.

Adaptive strategy of Nitraria sibirica to transient salt,alkali and osmotic stresses via the alteration of Na+/K+fluxes around root tips

Nitraria sibirica Pall.is an important shrub with a strong salt-alkali tolerance,but the mechanism underlying this tolerance remains obscure.In this study,N.sibirica,with salt-sensitive Vigna radiata(Linn.)Wilczek as the control,was subjected to transient salt stress(100 mM NaCl),alkali stress(50 mM Na_(2)CO_(3)),and osmotic stress(175 mM mannitol).The ionic fluxes of Na^(+)and K^(+)in the root apical region were measured.Results show that,under salt and alkali stress,N.sibirica roots exhibited higher capacities to limit Na+influx and reduce K+efflux,thereby resulting in lower Na^(+)/K^(+)ratios compared with V.radiata roots.Alkali stress induced stronger Na^(+)influx and K+efflux in the root salt stress treatment;Na^(+)influx was mainly observed in the root cap,while K^(+)efflux was mainly observed in the elongation zone.While under osmotic stress,N.sibirica roots showed stronger Na+efflux and weaker K+efflux than V.radiata roots.Na+efflux was mainly observed in the root elongation zone,while K+efflux was in the root cap.These results reveal the ionic strategy of N.sibirica in response to transient salt,alkali,and osmotic stresses through the regulation of Na+/K+flux homeostasis.

Effects of Nitric Oxide on the Germination of Wheat Seeds and Its Reactive Oxygen Species Metabolisms Under Osmotic Stress

Effects of sodium nitroprusside (SNP), a nitric oxide (NO) donor, on the germination and metabolism of reactive oxygen species were surveyed in wheat (Triticum aestivum L.) seeds. Germination of wheat seeds and even the elongation of radicle and plumule were dramatically promoted by SNP treatments during the germination under osmotic stress. Meanwhile, activities of amylase and EP were enhanced, thus leading to the degradation of storage reserve in seeds. After osmotic stress was removed, higher viability of wheat seeds was also maintained. In addition, the activities of CAT, APX and the content of proline were increased by SNP treatment simultaneously, but activities of LOX were inhibited, and both of which were beneficial for improving the antioxidant capacity during the germination of wheat seeds under osmotic stress. It was also shown that the increase of the activity of amylase induced by SNP in embryoless half-seeds of wheat in the beginning period of germination (6 h) might be indirectly related to GA(3).

Effects of Polyamine on Leaf Physiological and Biochemical Indexes of Maize Seedlings under Osmotic Stress

[Objective]The research aimed to probe into the mechanism of polyamine enhancing the tolerance of plants to drought stress and provide theoretical basis for application of polyamine in process of maize drought resistance. [Method]With PEG-6000 simulating natural drought,the change in content of soluble protein and relative water content were investigated in seedling leaves of two maize cultivars,Nongda 108 and Yedan 13 under osmotic stress with exogenous Spd treatment. [Result]On the 7th day,leaf relative water content and the content of soluble protein decreased more significantly in leaves of Yedan 13 （drought-sensitive） than in Nongda 108 （drought-tolerant）. Exogenous Spd treatment not only obviously inhibited the decrease of leaf relative water content,but also increased the content of soluble protein. [Conclusion]Exogenous Spd treatment could enhance the tolerance of maize seedlings to osmotic stress,via improving the content of soluble protein in seedling leaves.

Effect of Osmotic Stress with PEG6000 on Osmotic Adjustment Solute in Seedling Leaves of Chenopodium album L.

[ Objective] The purpose was to discuss drought resistance mechanism of Chenopodium album L. and supply theoretical basis and practical guidance for artificial cultivation and popularization of C. album. [ Method] C. album seedlings grown to 6th leaf stage were conducted osmotic stress treatment with PEG6000 osmotic whose concentration was set up as 0, 5%, 10% and 20% and the various physiological indices of the 3rd -5th function leaves in upper plant were determined after being treated for 0, 1,3, 5, 7 and 9 d. [ Result] Under osmotic stress with 5% PGE, the relative water content （RWC） of C. album reduced less. Under osmotic stress with 10%, the RWC in seedling leaves of C. album decreased to 62% on the fifth day and the leaves began to wither. Under osmotic stress with 20%, the RWC in seedling leaves of C. album decreased to 61.9% on the third day and the leaves appeared withering, and the RWC decreased to 48.6% on the 7th day and the leaves were dry and yellow. Proline contents in seedling leaves of C. album under osmotic stress with 5%, 10% and 20% PEG were 7.64, 10.9 and 29.4 times of CK on the 7th day. [ Conclusion] C. album hed some adaptability to moderate osmotic stress, but the PEG osmotic stress with high concentration and long time might lead to severe damage on C. album.

OSMOTIC STRESS DECREASES THE ACTIVITY OF ATPASE ASSOCIATED WITH THE ROOT CAP PLASMODESMATAIN ZEA MAYS

With light and electron microscopy the substructural change and the ATPase activity of corn (Zea mays L.) root cap cells after short-term osmotic stress were studied. Some spoke-like fine strands originating from the departed periplasm and stretching towards cell wall could be observed even after plasmolysis. By observing the precipitation of ATPase activity product (lead phosphate) at plasma membrane and plasmodesmata, it was found that the fine strands were plasma membrane-lined channels surrounding the cytoplasm and that they still firmly connected to the plasmodesmata during plasmolysis. Compared with the control (unstressed), a sharp decrease of ATPase activity in the plasmodesmata of the stressed cells was observed. Inhibition of energy metabolism in these limited locales would affect the physiological activity, maybe including the regulation of permeability and the change of size exclusion limit (SEL) of plasmodesmata.

Relationship between ATPase activity and conjugated polyamines in mito-chondrial membrane from wheat seedling roots under osmotic stress

The effects of osmotic stress on the ATPase activity, the contents of —SH group and conjugated polyamines in mitochondrial membrane from wheat seedling [Triticum aestivum L. cv. Yumai No.18(drought-tolerant) and cv. Yumai No.9(drought-sensitive)] roots were investigated. The results showed that ATPase activity and —SH group content decreased with polyethylene glycol(PEG) 6000(-0.55 MPa) treatment for 7 d, in concert with the decrease of the ratio of noncovalently conjugated spermidine(NCC-Spd)/noncovalently conjugated putrescine(NCC-Put) and increase of the covalently conjugated putrescine(CC-Put). Osmotic stress injury to Yangmai No.9 seedlings was alleviated greatly with 1 mmol/L exogenous spermidine(Spd), in concert with marked increases of the ratio of NCC-Spd/NCC-Put, —SH group contents and ATPase activity in mitochondrial membrane. Under osmotic stress, the concomitant treatment of Yumai No.18 seedlings with methylglyoxyl bis(guanylhydrazone) (MGBG), an inhibitor of S-adenosyl methionine decarboxylase(SAMDC), and phenanthrolin (o-Phen), an inhibitor of transglutaminase(TGase), caused a significant decrease of the ratio of NCC-Spd / NCC-Put, CC-Put contents, respectively, in concert with the marked decreases of ATPase activity, —SH group content and its tolerance to osmotic stress. All the results above suggested that osmotic stress tolerance of wheat seedlings was associated with the ATPase activity, the contents of —SH group, NCC-Spd and CC-Put in mitochondrial membrane.

Effects of osmotic stress on antioxidant enzymes activities in leaf discs of P_(SAG12)-IPT modified gerbera

Leaf senescence is often caused by water deficit and the chimeric gene PSA612-1PT is an auto-regulated gene delaying leaf senescence. Using in vitro leaf discs culture system, the changes of contents of chlorophylls, carotenoids, soluble protein and thiobarbituric acid reactive substance （TBARS） and antioxidant enzymes activities were investigated during leaf senescence of PSA612-1PT modified gerbera induced by osmotic stress compared with the control plant （wild type）. Leaf discs were incubated in 20%, 40% （w/v） polyethylene glycol （PEG） 6 000 nutrient solution for 20 h under continuous light [ 130 μmol/（m^2·s）]. The results showed that the contents of chlorophylls, carotenoids and soluble protein were decreased by osmotic stress with the decrease being more pronounced at 40% PEG, but that, at the same PEG concentration the decrease in the transgenic plants was significantly lower than that in the control plant. The activities of superoxide dismutase （SOD）, catalases （CAT）, ascorbate peroxidase （APX）, guaiacol peroxidase （GPX） and dehydroascorbate reductase （DHAR） were stimulated by PEG treatment. However, the increases were higher in PSA612-IPT transgenic plants than in the control plants, particularly at 40% PEG treatment. Lipid peroxidation （TBARS content） was increased by PEG treatment with the increase being much lower in transgenic plant than in the control plant. It could be concluded that the increases in the activities ofantioxidant enzymes including SOD, CAT, APX, GPX and DHAR were responsible for the delay of leaf senescence induced by osmotic stress.

Interactive effect of shade and PEG-induced osmotic stress on physiological responses of soybean seedlings

Intensively farmed crops used to experience numerous environmental stresses.Among these,shade and drought significantly influence the morpho-physiological and biochemical attributes of plants.However,the interactive effect of shade and drought on the growth and development of soybean under dense cropping systems has not been reported yet.This study investigated the interactive effect of PEG-induced osmotic stress and shade on soybean seedlings.The soybean cultivar viz.,C-103 was subjected to PEG-induced osmotic stress from polyethylene glycol 6000(PEG-6000)under shading and non-shading conditions.PEG-induced osmotic stress significantly reduced the relative water contents,morphological parameters,carbohydrates and chlorophyll contents under both light environments.A significant increase was observed in osmoprotectants,reactive oxygen species and antioxidant enzymes in soybean seedlings.Henceforth,the findings revealed that,seedlings grown under non-shading conditions produced more malondialdehyde and hydrogen peroxide contents as compared to the shade-treated plants when subjected to PEG-induced osmotic stress.Likewise,the shaded plants accumulated more sugars and proline than non-shaded ones under drought stress.Moreover,it was found that nonshaded grown plants were more sensitive to PEG-induced osmotic stress than those exposed to shading conditions,which suggested that shade could boost the protective mechanisms against osmotic stress or at least would not exaggerate the adverse effects of PEG-induced osmotic stress in soybean seedlings.

Changes induced by osmotic stress in the morphology, biochemistry, physiology, anatomy and stomatal parameters of almond species(Prunus L. spp.) grown in vitro

We investigated the influence of different levels of osmotic stress on growth and development in selected wild almond species （eight Prunus spp.） grown in vitro. The study, while endorsing the efficacy of in vitro screening of auxiliary buds of wild almond for osmotic stress tolerance, showed species variability in its response to osmotic stress. Osmotic stress reduced growth and development of all the species. How-ever, the putative tolerant Prunus spp. showed better performance than the putative susceptible genotypes. On average there was an 80% de-crease in shoot dry weight at -1.2 MPa. Reduction in shoot weight was more common in osmotic stress-susceptible species in the section labeled‘Euamygdalus’. The tolerant Prunus species produced smaller changes in biochemical responses than the sensitive cultivars for malondialdehyde content, catalase activity, relative permeability of protoplast membranes, and net photosynthetic rate. The tolerant species maintained cell integrity better than drought sensitive species. Wild almond species in the section labeled ‘Spartioides’ （Prunus arabica （Olivier） Neikle, Prunus glauca＆amp;nbsp;（Browicz） A.E. Murray, Prunus scoparia Spach） and ‘Lycioides’ （Pru-nus lycioides Spach, Prunus reuteri Bossi. et Bushe） were best adapted to osmotic stress. Increase in chlorophyll concentration and leaf thickness under high osmotic stress can be considered as preliminary selection parameters for osmotic stress tolerance in Prunus spp. The study con-firmed the efficacy of the in vitro method for screening of large number of genotypes for osmotic stress tolerance in wild almond species.

Loss of Responsiveness to Osmotic Stress in Maize Root:the Effect of Water Channel Blocker HgCl_2

In order to investigate the effect of water channel blocker HgCl2 on the hydraulic resistance in roots of maize seedlings, a xylem pressure probe was used to monitor the changes in root xylem pressure in response to NaCl- or mannitol-induced osmotic stresses before and after the application of HgCl2. When the maize roots were subjected to 500 umol L-1 HgCl2 in root bathing solution, not only a considerable decline in xylem pressure （increase in xylem tension） was observed, but the loss of responsiveness of the plant to both salt- and mannitol-induced osmotic stresses in terms of xylem pressure change was seen as well when the transpiration rate of the plant was not significantly changed. The results are similar but different from the reversed osmosis by the Fenton reaction in the internodes of Chara coralline, showing that the mechanisms of water transport across cell membrane in plant roots are far more complicated than expected.

GmGPDH12,a mitochondrial FAD-GPDH from soybean, increases salt and osmotic stress resistance by modulating redox state and respiration

In plants,glycerol-3-phosphate dehydrogenase(GPDH)catalyzes the interconversion of glycerol-3-phosphate(G3P)and dihydroxyacetone phosphate(DHAP)coupled to the reduction/oxidation of the nicotinamide adenine dinucleotide(NADH)pool,and plays a central role in glycerolipid metabolism and stress response.Previous studies have focused mainly on the NAD+-dependent GPDH isoforms,neglecting the role of flavin adenine dinucleotide(FAD)-dependent GPDHs.We isolated and characterized three mitochondrialtargeted FAD-GPDHs in soybean,of which one isoform(GmGPDH12)showed a significant transcriptional response to NaCl and mannitol treatments,suggesting the existence of a major FAD-GPDH isoform acting in soybean responses to salt and osmotic stress.An enzyme kinetic assay showed that the purified GmGPDH12 protein possessed the capacity to oxidize G3P to DHAP in the presence of FAD.Overexpression and RNA interference of GmGPDH12 in soybean hairy roots resulted in elevated tolerance and sensitivity to salt and osmotic stress,respectively.G3P contents were significantly lower in GmGPDH12-overexpressing hair roots and higher in knockdown hair roots,indicating that GmGPDH12 was essential for G3P catabolism.A significant perturbation in redox status of NADH,ascorbic acid(ASA)and glutathione(GSH)pools was observed in GmGPDH12-knockdown plants under stress conditions.The impaired redox balance was manifested by higher reactive oxygen species generation and consequent cell damage or death;however,overexpressing plants showed the opposite results for these traits.GmGPDH12 overexpression contributed to maintaining constant respiration rates under salt or osmotic stress by regulating mRNA levels of key mitochondrial respiratory enzymes.This study provides new evidence for the roles of mitochondria-localized GmGPDH12 in conferring resistance to salt or osmotic stress by maintaining cellular redox homeostasis,protecting cells and respiration from oxidative injury.

Proteomic analysis of salt and osmotic-drought stress in alfalfa seedlings

Alfalfa is widely grown and is one of the most important forage crops in the world, but its growth and biomass production are markedly reduced under salt and drought stress, particularly during the early seedling stage. To identify the mechanisms behind salt and drought responsiveness at the alfalfa seedling stage, the proteins expressed were analyzed under no-treatment, 200 mol L–1 Na Cl and 180 g L–1 PEG treatment conditions during the seedling stage. Out of more than 800 protein spots detected on two-dimensional electrophoresis（2-DE） g els, 35 proteins showed statistically significant responses（P〈0.05） to Na Cl and PEG stress, which were selected for tandem mass spectrometric identification, owing to their good resolution and abundance levels, and 32 proteins were positively identified. The identified proteins were divided into seven functional categories： photosynthetic metabolism, protein biosynthesis, folding and assembly, carbohydrate metabolism-associated proteins, stress defense related protein, metabolism of nucleic acid, other function categories and unknown proteins. Our results suggested that these proteins may play roles in alfalfa adaptation to salt and drought stress. Further study of these proteins will provide insights into the molecular mechanisms of abiotic stress and the discovery of new candidate markers in alfalfa.

Relationship between H_(2)O_(2) accumulation and NO synthesis during osmotic stress:promoted somatic embryogenesis of Fraxinus mandshurica

Osmotic stress promotes somatic embryogenesis of Fraxinus mandshurica,which leads to accumulation of reactive oxygen species(ROS).The single pieces of cotyledons of F.mandshurica were used as explants to induce somatic embryogenesis in osmotic-stress medium.Furthermore,the hydrogen peroxide H_(2)O_(2) content of explanted cells was varied by adding exogenous H_(2)O_(2) or catalase solution to assess the effects of the exogenous H_(2)O_(2)on somatic embryogenesis,intracellular H_(2)O_(2)accumulation,and the relationship between signaling mediated by ROS or reactive nitrogen species.The results revealed that exogenous H_(2)O_(2)(100?300μmol L^(–1))increased the number of somatic embryos.On 60th day of exogenous H_(2)O_(2)(200μmol L^(–1))treatment,the number of somatic embryos of explants treated,which was 136.54%,was higher than the control.Moreover,exogenous H_(2)O_(2)(100μmol L^(–1))significantly increased the intracellular H_(2)O_(2)content and enhanced the activities of superoxidase dismutase and peroxidase.Finally,exogenous H_(2)O_(2)(100μmol L^(–1))activated the intracellular non-enzymatic pathway for nitric oxide(NO)synthesis.The somatic embryogenesis in broadleaf trees increases with the change of endogenic ROS content,and depends on the upregulation of antioxidant enzymes.Both H_(2)O_(2)and NO,as signaling molecules,were found to be involved in the process of somatic embryogenesis in broadleaf trees.In the process of exogenous H_(2)O_(2)promoting somatic embryogenesis,NO synthesis depended on non-enzymatic reactions.These results provide a scientific basis for resolving the mechanism by which ROS levels are regulated during somatic embryogenesis of broadleaf trees and establish a reasonable and efficient technology system for regulating somatic embryogenesis of trees.

Effect of NdCl_3 on the Injury of Membrane and ABA Content under Osmotic Stress

Wheat seedlings grown in 0. 67 MPa Polyethylene glycol(PEG) , the relative water content(RWC) of leaves declined, the malondialdehyde(MDA) content and the permeability of plasma-membrane increased. The activity of superoxide dismutase (SOD) decreased, but that of peroxidase (POD) increased. At 3 h and 15 h osmotic treatment the abscisic acid(ABA) content showed two peaks respectively. When 0. 67 MPa PEG contained 35 4mol/L NdCl3 the RWC was higher than that of the control, the permeability of the plasma membrane and the MDA content were decreased. The activity of SOD of POD all increased by adding NdCl3. The ABA content showed only one peak at 24 h osmotic treatment, and the value of that peak declined obviously when treated with NdCl3.

Effects of short-term osmotic stress on leaf hydraulic conductivity and ZmPIPs mRNA accumulation in maize seedlings

Plants maintain water balance by varying hydraulic properties, and plasma membrane intrinsic proteins（PIPs） may be involved in this process. Leaf xylem and root hydraulic conductivity and the m RNA contents of four highly expressed Zm PIP genes（Zm PIP1;1, Zm PIP1;2, Zm PIP2;2, and Zm PIP2;5） in maize（Zea mays） seedlings were investigated. Under well-watered conditions, leaf hydraulic conductivity（K_（leaf）） varied diurnally and was correlated with whole-plant hydraulic conductivity. Similar diurnal rhythms of leaf transpiration rate（E）, K_（leaf） and root hydraulic conductivity（K_（root）） in well-watered plants are important for maintaining whole-plant water balance. After 2 h of osmotic stress treatment induced by 10% polyethylene glycol 6000, the K_（root） of stressed plants decreased but K_（leaf） increased, compared with well-watered plants. The m RNA contents of four Zm PIPs were significantly up-regulated in the leaves of stressed plants, especially for Zm PIP1;2. Meanwhile, Zm PIP2;5 was significantly down-regulated in the roots of stressed plants. After 4 h of osmotic stress treatment, the E and leaf xylem water potentials of stressed plants unexpectedly increased. The increase in K_（leaf） and a partial recovery of K_（root） may have contributed to this process. The m RNA content of Zm PIP1;2 but not of the other three genes was up-regulated in roots at this time. In summary, the m RNA contents of these four Zm PIPs associated with K_（leaf） and K_（root） change in maize seedlings during short-term osmotic stress, especially for Zm PIP1;2 and Zm PIP2;5, which may help to further reveal the hydraulic resistance adjustment role of Zm PIPs.

Selection of Maize Genotypes with Tolerance to Osmotic Stress Associated with Salinity

Thirteen different inbred lines in relation to the type of grain and life cycles were characterized by testing for osmotic stress associated with salinity. The identification of tolerant genotypes would be an effective strategy to overcome the saline stress. Osmotic stress reduces immediately the expansion of the roots and young leaves which determine a reduction in the size of the plant. A completely randomized design was adopted to test seedlings under controlled conditions of light and temperature. Two treatments were used: 0 mM NaCl (as control) and 100 mM NaCl. After 15 days of complete salinization, the seedlings were harvested and several morphological traits were measured. The morphological traits of growth were leaf growth (Ar1, Ar2, Ar3 and Ar4), dry masses of shoot and root (SDM and RDM, respectively). Also, traits associated with water economy were registered: leaf water loss (LWL) and relative water content (RWC). The morphological traits were expressed in relative terms, while the traits associated with the economy of water were expressed in absolute terms. Uni and multivariate techniques were applied to identify genotypes with divergent behaviors to osmotic stress tolerance. Also, a Tolerance Index was employed to identify superior genotypes. Four clusters were obtained after applying a Cluster Analysis and Principal Component Analysis (PCA). The genotypes were compared to each other with a test of DMS. The results obtained with different statistical techniques converged. Some variables presented a differential weight classification of genotypes. The morphological traits like RDM, SDM, Ar3, Ar4 and Ar5 were the most discriminating. Tolerance Index allowed to classify genotypes, thus SC2 and AD3 lines were that reached highest value of the index and therefore would be tolerant lines, while AF3 and LP3 had a low index and were seen as sensible.

Osmotic Stress Resistance of Transgenic Tobacco Expressed Soybean Lysine-rich Protein Gene

PM2 gene （accession number： M80664） with high lysine content from soybean （Glycine max） was found in GenBank by changing three BLASTp parameters. Amino acid composition analysis of PM2 showed that Lys content was on the high level of 18.22%. Protein encoded by PM＇2 also belonged to the family of late embryogenesis abundant （LEA） proteins, which was considered that it had a strong relation with the abiotic stress resistance. In this experiment, PM2 gene was obtained from dry soybean seeds by RT-PCR, plant expression vector pEMTPM2 was constructed, and then transformed into tobacco by using agrobacterium-mediated method. Eight salt and drought tolerant lines were obtained from 31 differentiated lines. Real-time PCR showed that PM2 gene overexpressed in all four PCR positive lines with the osmotic stress resistance. These results confirmed that the overexpression of PM2 gene enhanced the osmotic stress resistance of transgenic tobacco.