Alleviation of Salt Stress by Enterobacter sp. EJ01 in Tomato and Arabidopsis Is Accompanied by Up-Regulation of Conserved Salinity Responsive Factors in Plants

Microbiota in the niches of the rhizosphere zones can affect plant growth and responses to environmental stress conditions via mutualistic interactions with host plants. Specifically, some beneficial bacteria, collectively referred to as Plant Growth Promoting Rhizobacteria (PGPRs), increase plant biomass and innate immunity potential. Here, we report that Enterobacter sp. EJ01, a bacterium isolated from sea china pink (Dianthus japonicus thunb) in reclaimed land of Gyehwa-do in Korea, improved the vegetative growth and alleviated salt stress in tomato and Arabidopsis. EJ01 was capable of producing 1-aminocy-clopropane-1-carboxylate (ACC) deaminase and also exhibited indole-3-acetic acid (IAA) production. The isolate EJ01 conferred increases in fresh weight, dry weight, and plant height of tomato and Arabidopsis under both normal and high salinity conditions. At the molecular level, short-term treatment with EJ01 increased the expression of salt stress responsive genes such as DREB2b, RD29A, RD29B, and RAB18 in Arabidopsis. The expression of proline biosynthetic genes (i.e. P5CS1 and P5CS2) and of genes related to priming processes (i.e. MPK3 and MPK6) were also up-regulated. In addition, reactive oxygen species scavenging activities were enhanced in tomatoes treated with EJ01 in stressed conditions. GFP-tagged EJ01 displayed colonization in the rhizosphere and endosphere in the roots of Arabidopsis. In conclusion, the newly isolated Enterobacter sp. EJ01 is a likely PGPR and alleviates salt stress in host plants through multiple mechanisms, including the rapid up-regulation of conserved plant salt stress responsive signaling pathways.     

Isolation and characterization of two sorbitol transporter gene promoters in micropropagated apple plants (Malus?×?domestica) regulated by drought stress

The role of polyol transporters in stress tolerance in plants have been elucidated by many studies. Sorbitol transporter genes MdSOT3, MdSOT4 and MdSOT5 in apple plants, which are important for sorbitol loading and unloading, are regulated by drought stress. To further confirm the role of sorbitol transporters in stress tolerance, the constructs harboring MdSOT3 and MdSOT5 genes were introduced into wild type Arabidopsis plants (Col-0) and the Arabidopsis transformed with MdSOT3 or MdSOT5 performed higher drought stress tolerance compared to WT. In order to further understand how sorbitol transporters are involved in drought tolerance in apple plants, upstream regions of sorbitol transporter genes were isolated from apple plant source leaves by Anchored PCR from genomic DNA obtained, and then were used to drive expression of the GUS reporter in tobacco plants. The results showed that the longest fragments of MdSOT3 and MdSOT5 promoters induced the highest GUS activity under drought stress conditions. Additionally, fragments of these promoters that contain cis-acting elements known to be involved in stress response also induced GUS activity under drought stress. Taken together, our data suggest that increased MdSOT3 and MdSOT5 activity, through cis-acting elements in the promoters of these genes, play important roles in imparting tolerance to drought in micropropagated apple plants.     

Cytokinin oxidase/dehydrogenase overexpression modifies antioxidant defense against heat,drought and their combination in Nicotiana tabacum plants

Cytokinins (CKs) as well as the antioxidant enzyme system (AES) play important roles in plant stress responses. The expression and activity of antioxidant enzymes (AE) were determined in drought, heat and combination of both stresses, comparing the response of tobacco plants overexpressing the main cytokinin degrading enzyme, cytokinin oxidase/dehydrogenase, under the control of root-specific WRKY6 promoter (W6:CKX1 plants) or constitutive promoter (35S:CKX1 plants) and the corresponding wild-type (WT). Expression levels as well as activities of cytosolic ascorbate peroxidase, catalase 3, and cytosolic superoxide dismutase were low under optimal conditions and increased after heat and combined stress in all genotypes. Unlike catalase 3, two other peroxisomal enzymes, catalase 1 and catalase 2, were transcribed extensively under control conditions. Heat stress, in contrast to drought or combined stress, increased catalase 1 and reduced catalase 2 expression in WT and W6:CKX1 plants. In 35S:CKX1, catalase 1 expression was enhanced by heat or drought, but not under combined stress conditions. Mitochondrial superoxide dismutase expression was generally higher in 35S:CKX1 plants than in WT. Genes encoding for chloroplastic AEs, stromatal ascorbate peroxidase, thylakoidal ascorbate peroxidase and chloroplastic superoxide dismutase, were strongly transcribed under control conditions. All stresses down-regulated their expression in WT and W6:CKX1, whereas more stress-tolerant 35S:CKX1 plants maintained high expression during drought and heat. The achieved data show that the effect of down-regulation of CK levels on AES may be mediated by altered habit, resulting in improved stress tolerance, which is associated with diminished stress impact on photosynthesis, and changes in source/sink relations.     

二氧化硫增强拟南芥植株对干旱的适应性

以模式植物拟南芥为材料,研究SO_2对植物干旱适应性的影响。采用分光光度法检测植物干旱生理指标的变化,并用半定量RT-PCR技术分析了拟南芥热激基因和干旱响应基因的转录水平。研究发现:4周龄拟南芥植株暴露于30mg/m3的SO_2后,6—72h间叶面气孔开度显著低于对照并逐渐减小,在暴露48h和72h时,热激转录因子HsfA2和热激基因Hsp17.7、Hsp17.6B、Hsp17.6C转录上调,干旱响应基因DREB2A、DREB2B和RD29A表达增强;在SO_2熏气72h后进行干旱胁迫,干旱期间SO_2预暴露植株的叶片相对含水量高于非熏气干旱处理组,植株萎蔫程度比后者明显减轻,且SO_2预暴露植株的地上组织中可溶性糖和脯氨酸含量升高,超氧化物歧化酶活性提高,丙二醛含量降低。结果表明:SO_2能降低气孔开度、提高抗氧化能力、上调热激基因和干旱响应基因转录,并能促进干旱期间植物细胞内渗透调节物质的合成和积累,促使抗氧化酶活性提高,从而降低干旱胁迫对植株造成的氧化损伤,增强拟南芥对干旱的适应性。植物通过基因转录应答、酶活性改变、渗透调节物质积累等,在适应环境高浓度SO_2的同时,提高了对干旱的适应性。     

A Role for Arabidopsis miR399f in Salt,Drought, and ABA Signaling

MiR399f plays a crucial role in maintaining phosphate homeostasis in Arabidopsis thaliana. Under phosphate starvation conditions, AtMYB2, which plays a role in plant salt and drought stress responses, directly regulates the expression of miR399f. In this study, we found that miR399f also participates in plant responses to abscisic acid (ABA), and to abiotic stresses including salt and drought. Salt and ABA treatment induced the expression of miR399f, as confirmed by histochemical analysis of promoter-GUS fusions. Transgenic Arabidopsis plants overexpressing miR399f (miR399f-OE) exhibited enhanced tolerance to salt stress and exogenous ABA, but hypersensitivity to drought. Our in silico analysis identified ABF3 and CSP41b as putative target genes of miR399f, and expression analysis revealed that mRNA levels of ABF3 and CSP41b decreased remarkably in miR399f-OE plants under salt stress and in response to treatment with ABA. Moreover, we showed that activation of stress-responsive gene expression in response to salt stress and ABA treatment was impaired in miR399f-OE plants. Thus, these results suggested that in addition to phosphate starvation signaling, miR399f might also modulates plant responses to salt, ABA, and drought, by regulating the expression of newly discovered target genes such as ABF3 and CSP41b.     

Overexpression of Arabidopsis Molybdenum Cofactor Sulfurase Gene Confers Drought Tolerance in Maize (Zea mays L.)

Abscisic acid (ABA) is a key component of the signaling system that integrates plant adaptive responses to abiotic stress. Overexpression of Arabidopsis molybdenum cofactor sulfurase gene (LOS5) in maize markedly enhanced the expression of ZmAO and aldehyde oxidase (AO) activity, leading to ABA accumulation and increased drought tolerance. Transgenic maize (Zea mays L.) exhibited the expected reductions in stomatal aperture, which led to decreased water loss and maintenance of higher relative water content (RWC) and leaf water potential. Also, transgenic maize subjected to drought treatment exhibited lower leaf wilting, electrolyte leakage, malondialdehyde (MDA) and H₂O₂ content, and higher activities of antioxidative enzymes and proline content compared to wild-type (WT) maize. Moreover, overexpression of LOS5 enhanced the expression of stress-regulated genes such as Rad 17, NCED1, CAT1, and ZmP5CS1 under drought stress conditions, and increased root system development and biomass yield after re-watering. The increased drought tolerance in transgenic plants was associated with ABA accumulation via activated AO and expression of stress-related gene via ABA induction, which sequentially induced a set of favorable stress-related physiological and biochemical responses.     

Overproduction of the Membrane-bound Receptor-like Protein Kinase 1, RPK1, Enhances Abiotic Stress Tolerance in Arabidopsis

RPK1 (receptor-like protein kinase 1) localizes to the plasma membrane and functions as a regulator of abscisic acid (ABA) signaling in Arabidopsis. In our current study, we investigated the effect of RPK1 disruption and overproduction upon plant responses to drought stress. Transgenic Arabidopsis overexpressing the RPK1 protein showed increased ABA sensitivity in their root growth and stomatal closure and also displayed less transpirational water loss. In contrast, a mutant lacking RPK1 function, rpk1-1, was found to be resistant to ABA during these processes and showed increased water loss. RPK1 overproduction in these transgenic plants thus increased their tolerance to drought stress. We performed microarray analysis of RPK1 transgenic plants and observed enhanced expression of several stress-responsive genes, such as Cor15a, Cor15b, and rd29A, in addition to H₂O₂-responsive genes. Consistently, the expression levels of ABA/stress-responsive genes in rpk1-1 had decreased compared with wild type. The results suggest that the overproduction of RPK1 enhances both the ABA and drought stress signaling pathways. Furthermore, the leaves of the rpk1-1 plants exhibit higher sensitivity to oxidative stress upon ABA-pretreatment, whereas transgenic plants overproducing RPK1 manifest increased tolerance to this stress. Our current data suggest therefore that RPK1 overproduction controls reactive oxygen species homeostasis and enhances both water and oxidative stress tolerance in Arabidopsis.     

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.