Overexpression of maize mitogen-activated protein kinase gene, ZmSIMK1 in Arabidopsis increases tolerance to salt stress

Mitogen-activated protein kinase (MAPK) cascades play a remarkably crucial role in plants. It has been studied intensively in model plants Arabidopsis, tobacco and rice. However, the function of MAPKs in maize (Zea mays L.) has not been well documented. ZmSIMK1 (Zea mays salt-induced mitogen-activated protein kinase 1) is a previously identified MAPK gene in maize. In this research, we charactered ZmSIMK1 and showed that ZmSIMK1 was involved in Arabidopsis salt stress. The genomic organization of ZmSIMK1 gene and its expression in maize have been analyzed. In order to investigate the function of ZmSIMK1, we generated transgenic Arabidopsis constitutively overexpressing ZmSIMK1. Ectopic expression of ZmSIMK1 in Arabidopsis resulted in increased resistance against salt stress. Importantly, ZmSIMK1-overexpressing Arabidopsis exhibited constitutive expression of stress-responsive marker genes, RD29A and P5CS1. Furthermore, RD29A and P5CS1 were upregulated under salt stress. These results suggest that ZmSIMK1 may play an important role in plant salt stress.     

A CIPK protein kinase targets sucrose transporter MdSUT2.2 at Ser254 for phosphorylation to enhance salt tolerance

Soil salinity is one of the major abiotic stressors that negatively affect crop growth and yield. Salt stress can regulate antioxidants and the accumulation of osmoprotectants. In the study, a sucrose transporter MdSUT2.2 was identified in apple. Overexpression of MdSUT2.2 gene increased salt tolerance in the transgenic apple, compared with the WT control “Gala.” In addition, it was found that protein MdSUT2.2 was phosphorylated at Ser²⁵⁴ site in response to salt. A DUAL membrane yeast hybridization system through an apple cDNA library demonstrated that a protein kinase MdCIPK13 interacted with MdSUT2.2. A series of transgenic analysis in apple calli showed that MdCIPK13 was required for the salt‐induced phosphorylation of MdSUT2.2 protein and enhanced its stability and transport activity. Finally, it was found that MdCIPK13 improved salt resistance in an MdSUT2.2‐dependent manner. These findings had enriched our understanding of the molecular mechanisms underlying abiotic stress.     

CIPK6, a CBL-interacting protein kinase is required for development and salt tolerance in plants

Calcineurin B-like proteins (CBL) and CBL-interacting protein kinases (CIPK) mediate plant responses to a variety of external stresses. Here we report that Arabidopsis CIPK6 is also required for the growth and development of plants. Phenotype of tobacco plants ectopically expressing a homologous gene ( CaCIPK6 ) from the leguminous plant chickpea ( Cicer arietinum ) indicated its functional conservation. A lesion in AtCIPK6 significantly reduced shoot-to-root and root basipetal auxin transport, and the plants exhibited developmental defects such as fused cotyledons, swollen hypocotyls and compromised lateral root formation, in conjunction with reduced expression of a number of genes involved in auxin transport and abiotic stress response. The Arabidopsis mutant was more sensitive to salt stress compared to wild-type, while overexpression of a constitutively active mutant of CaCIPK6 promoted salt tolerance in transgenic tobacco. Furthermore, tobacco seedlings expressing the constitutively active mutant of CaCIPK6 showed a developed root system, increased basipetal auxin transport and hypersensitivity to auxin. Our results provide evidence for involvement of a CIPK in auxin transport and consequently in root development, as well as in the salt-stress response, by regulating the expression of genes.     

Overexpression of SOD2 increases salt tolerance of Arabidopsis

The yeast (Schizosaccharomyces pombe) SOD2 (Sodium2) gene was introduced into Arabidopsis under the control of the cauliflower mosaic virus 35S promoter. Transformants were selected for their ability to grow on medium containing kanamycin. Southern- and northern-blot analyses confirmed that SOD2 was transferred into the Arabidopsis genome. There were no obvious morphological or developmental differences between the transgenic and wild-type (wt) plants. Several transgenic homozygous lines and wt plants (control) were evaluated for salt tolerance and gene expression. Overexpression of SOD2 in Arabidopsis improved seed germination and seedling salt tolerance. Analysis of Na+ and K+ contents of the symplast and apoplast in the parenchyma cells of the root cortex and mesophyll cells in the spongy tissue of the leaf showed that transgenic lines accumulated less Na+ and more K+ in the symplast than the wt plants did. The photosynthetic rate and the fresh weight of the transgenic lines were distinctly higher than that of wt plants after NaCl treatment. Results from different tests indicated that the expression of the SOD2 gene promoted a higher level of salt tolerance in vivo in transgenic Arabidopsis plants.     

MdSOS2L1 phosphorylates MdVHA-B1 to modulate malate accumulation in response to salinity in apple

Key message

Salt-induced phosphorylation of MdVHA-B1 protein was mediated by MdSOS2L1 protein kinase, and thereby increasing malate content in apple.

Abstract

Salinity is an important environmental factor that influences malate accumulation in apple. However, the molecular mechanism by which salinity regulates this process is poorly understood. In this work, we found that MdSOS2L1, a novel AtSOS2-LIKE protein kinase, interacts with V-ATPase subunit MdVHA-B1. Furthermore, MdSOS2L1 directly phosphorylates MdVHA-B1 at Ser³⁹⁶ site to modulate malate accumulation in response to salt stress. Meanwhile, a series of transgenic analyses in apple calli showed that the MdSOS2L1–MdVHAB1 pathway was involved in the regulation of malate accumulation. Finally, a viral vector-based transformation approach demonstrated that the MdSOS2L1–MdVHAB1 pathway also modulated malate accumulation in apple fruits with or without salt stress. Collectively, our findings provide a new insight into the mechanism by which MdSOS2L1 phosphorylates MdVHA-B1 to modulate malate accumulation in response to salinity in apple.

     

Overexpression of SlSOS2 (SlCIPK24) confers salt tolerance to transgenic tomato

The Ca(2+)-dependent SOS pathway has emerged as a key mechanism in the homeostasis of Na(+) and K(+) under saline conditions. We have identified and functionally characterized the gene encoding the calcineurin-interacting protein kinase of the SOS pathway in tomato, SlSOS2. On the basis of protein sequence similarity and complementation studies in yeast and Arabidopsis, it can be concluded that SlSOS2 is the functional tomato homolog of Arabidopsis AtSOS2 and that SlSOS2 operates in a tomato SOS signal transduction pathway. The biotechnological potential of SlSOS2 to provide salt tolerance was evaluated by gene overexpression in tomato (Solanum lycopersicum L. cv. MicroTom). The better salt tolerance of transgenic plants relative to non-transformed tomato was shown by their faster relative growth rate, earlier flowering and higher fruit production when grown with NaCl. The increased salinity tolerance of SlSOS2-overexpressing plants was associated with higher sodium content in stems and leaves and with the induction and up-regulation of the plasma membrane Na(+)/H(+) (SlSOS1) and endosomal-vacuolar K(+), Na(+)/H(+) (LeNHX2 and LeNHX4) antiporters, responsible for Na(+) extrusion out of the root, active loading of Na(+) into the xylem, and Na(+) and K(+) compartmentalization.     

Overexpression of the AtSTK gene increases salt, PEG and ABA tolerance in Arabidopsis

AtSTK (At5g02800), which is a serine-threonine protein kinase gene of Arabidopsis thaliana, was cloned, and its function was studied. The study found that the overexpression of AtSTK could significantly improve the ability of A. thaliana to tolerate salt, PEG, and ABA stresses. RT-PCR analysis revealed that the expression of the AtSTK gene could be obviously induced by salt, PEG, and ABA. The examination of the physiological characteristics showed that the overexpression of AtSTK in Arabidopsis significantly reduced the plasma membrane permeability, significantly increased the proline content, and decreased the MDA content. These changes may reflect the physiological mechanisms through which AtSTK overexpression improves stress resistance in Arabidopsis. In addition, the overexpression of the AtSTK gene significantly antagonised the inhibitory effect of high concentrations of exogenous ABA on Arabidopsis seed germination. The subcellular localisation results showed that AtSTK is located in both the cytosol and the nucleus. The examination of its tissue-specific expression showed that AtSTK is expressed in various Arabidopsis tissues and is particularly strongly expressed in the vessels. The signalling pathway analysis indicated that AtSTK might transfer the salt stress signal in Arabidopsis through the MAPK pathway.     

Overexpression of osmotin gene confers tolerance to salt and drought stresses in transgenic tomato (Solanum lycopersicum L.)

Abiotic stresses, especially salinity and drought, are major limiting factors for plant growth and crop productivity. In an attempt to develop salt and drought tolerant tomato, a DNA cassette containing tobacco osmotin gene driven by a cauliflower mosaic virus 35S promoter was transferred to tomato (Solanum lycopersicum) via Agrobacterium-mediated transformation. Putative T0 transgenic plants were screened by PCR analysis. The selected transformants were evaluated for salt and drought stress tolerance by physiological analysis at T1 and T2 generations. Integration of the osmotin gene in transgenic T1 plants was verified by Southern blot hybridization. Transgenic expression of the osmotin gene was verified by RT-PCR and northern blotting in T1 plants. T1 progenies from both transformed and untransformed plants were tested for salt and drought tolerance by subjecting them to different levels of NaCl stress and by withholding water supply, respectively. Results from different physiological tests demonstrated enhanced tolerance to salt and drought stresses in transgenic plants harboring the osmotin gene as compared to the wild-type plants. The transgenic lines showed significantly higher relative water content, chlorophyll content, proline content, and leaf expansion than the wild-type plants under stress conditions. The present investigation clearly shows that overexpression of osmotin gene enhances salt and drought stress tolerance in transgenic tomato plants.     

Overexpression of SlGMEs leads to ascorbate accumulation with enhanced oxidative stress, cold, and salt tolerance in tomato

GDP-Mannose 3′,5′-epimerase (GME; EC 5.1.3.18) catalyses the conversion of GDP-d-mannose to GDP-l-galactose, an important step in the ascorbic acid (AsA) biosynthesis pathway in higher plants. In this study, two members of the GME gene family were isolated from tomato (Solanum lycopersicum). Both SlGME genes encode 376 amino acids and share a 92% similarity with each other. Semi-quantitative RT-PCR indicated that SlGME1 was constantly expressed in various tissues, whereas SlGME2 was differentially expressed in different tissues. Transient expression of fused SlGME1-GFP (green fluorescent protein) and SlGME2-GFP in onion cells revealed the cytoplasmic localisation of the two proteins. Transgenic plants over-expressing SlGME1 and SlGME2 exhibited a significant increase in total ascorbic acid in leaves and red fruits compared with wild-type plants. They also showed enhanced stress tolerance based on less chlorophyll content loss and membrane-lipid peroxidation under methyl viologen (paraquat) stress, higher survival rate under cold stress, and significantly higher seed germination rate, fresh weight, and root length under salt stress. The present study demonstrates that the overexpression of two members of the GME gene family resulted in increased ascorbate accumulation in tomato and improved tolerance to abiotic stresses.     

Overexpression of Arabidopsis thaliana LTL1, a salt-induced gene encoding a GDSL-motif lipase, increases salt tolerance in yeast and transgenic plants

Genes involved in the mechanisms of plant responses to salt stress may be used as biotechnological tools for the genetic improvement of salt tolerance in crop plants. This would help alleviate the increasing problem of salinization of lands cultivated under irrigation in arid and semi-arid regions. We have isolated a novel halotolerance gene from Arabidopsis thaliana, A. thaliana Li-tolerant lipase 1 (AtLTL1), on the basis of the phenotype of tolerance to LiCl conferred by its expression in yeast. AtLTL1 encodes a putative lipase of the GDSL-motif family, which includes bacterial and a very large number of plant proteins. In Arabidopsis, AtLTL1 expression is rapidly induced by LiCl or NaCl, but not by other abiotic stresses. Overexpression of AtLTL1 increases salt tolerance in transgenic Arabidopsis plants, compared to non-transformed controls, allowing germination of seeds in the presence of toxic concentrations of LiCl and NaCl, and stimulating vegetative growth, flowering and seed set in the presence of NaCl. These results clearly point to a role of AtLTL1 in the mechanisms of salt tolerance. In addition, we show that AtLTL1 expression is also activated, although only transiently, by salicylic acid (SA), suggesting that the lipase could also be involved in defence reactions against pathogens.     

Exogenous catechin increases antioxidant enzyme activity and promotes flooding tolerance in tomato (Solanum lycopersicum L.)

We studied the extent to which catechin applied as a soil drench modifies the effects of soil waterlogging on plant growth, the functioning of the free radical scavenging system and on oxidative stress levels. Forty-day-old tomato plants (Solanum lycopersicum L.) were treated with 0 and 2?mM catechin 48 h prior to 5 d waterlogging followed by a 4 d drainage period. Exogenous catechin increased total fresh and dry weight of flooded plants, reduced membrane damage, maintained chlorophyll concentrations, promoted photosynthesis and increased ATP concentration in the leaves, and raised sucrose synthase and alcohol dehydrogenase activities in the roots. Catechin pre-treatment also reduced hydrogen peroxide and superoxide radical concentration and increased various components of the antioxidative system in leaves. Catechin treatment affected superoxide dismutase and catalase activities in close coordination with ascorbate peroxidases and glutathione reductase. Exogenous catechin can markedly reduce the waterlogging injury in leaves and roots of tomato by enhancing free radical scavenging system sufficiently to lower hydrogen peroxide and superoxide concentrations.     

过表达TaLEA1和TaLEA2基因提高转基因拟南芥的耐盐性

我国土壤盐碱化日益严重,对我国的粮食安全造成了严重威胁。耐盐基因挖掘对作物耐盐育种非常重要。LEA蛋白家族是一个多基因家族,在植物应对非生物胁迫中发挥重要作用。本课题组前期研究阐明小麦TaLEA1基因在拟南芥中过表达可以提高转基因植物的耐盐性和抗旱性。本研究系统分析了小麦TaLEA2基因表达蛋白的理化性质、基因表达模式及启动子功能区域,并在拟南芥中过表达TaLEA2基因及共表达TaLEA1和TaLEA2基因,分析TaLEA2基因的抗逆功能及2个LEA基因的抗逆效果。结果表明,TaLEA2基因的表达产物属于第3组LEA蛋白,是稳定的亲水蛋白,富含α-螺旋、β-转角等结构。TaLEA2基因在小麦根、茎、叶、花、种子等不同组织中均有表达,盐胁迫条件诱导其高表达。在拟南芥中过表达TaLEA2基因,或过表达TaLEA1和TaLEA2基因都能够提高转基因拟南芥的耐盐性和抗旱性,转基因株系的种子萌发率、根长及叶绿素含量显著高于野生型,且双基因过表达的转基因植物的抗逆能力高于单个基因过表达株系。本研究结果为LEA基因抗逆机理的研究和多基因共转提高植物抗逆性提供了重要信息。     

Overexpression of a rice gene encoding a small C2 domain protein OsSMCP1 increases tolerance to abiotic and biotic stresses in transgenic Arabidopsis

Plant growth and crop production are limited by environmental stress. We used a large population of transgenic Arabidopsis expressing rice full-length cDNAs to isolate the rice genes that improve the tolerance of plants to environmental stress. By sowing T2 seeds of the transgenic lines under conditions of salinity stress, the salt-tolerant line R07047 was isolated. It expressed a rice gene, OsSMCP1, which encodes a small protein with a single C2 domain, a Ca²⁺-dependent membrane-targeting domain. Retransformation of wild-type Arabidopsis revealed that OsSMCP1 is responsible for conferring the salt tolerance. It is particularly interesting that R07047 and newly constructed OsSMCP1-overexpressing Arabidopsis showed enhanced tolerance not only to high salinity but also to osmotic, dehydrative, and oxidative stresses. Furthermore, R07047 showed improved resistance to Pseudomonas syringae. The OsSMCP1 expression in rice is constitutive. Particle-bombardment-mediated transient expression analysis revealed that OsSMCP1 is targeted to plastids in rice epidermal cells. It induced overexpression of several nuclear encoded genes, including the stress-associated genes, in transgenic Arabidopsis. No marked morphological change or growth retardation was observed in R07047 or retransformants. For molecular breeding to improve the tolerance of crops against environmental stress, OsSMCP1 is a promising candidate.     

Overexpression of the transcription factor MdbHLH33 increases cold tolerance of transgenic apple callus

As cold stress greatly affects plant growth and development, understanding the mechanisms underlying cold tolerance in plants is important. In this study, we analyzed the expression levels of apple (Malus domestica) MdbHLH33 and MdCBF1–5 by semi-quantitative PCR after exposure to 4 °C for different amounts of time and generated evolutionary trees for MdbHLH33 and the MdCBFs. Overexpressing MdbHLH33 pro-GUS in ‘Orin’ callus, indicated that transgenic callus had higher GUS activity and was more deeply stained at 4 °C than at 25 °C. Subcellular localization showed that MdbHLH33 was located in the nucleus. Overexpressing MdbHLH33 in ‘Orin’ callus increased the expression level of MdCBF2, MdCOR15A-1, and MdCOR15A-2, and resulted in increased cold tolerance. EMSA and Chip-PCR analysis showed that MdbHLH33 could bind the LTR cis-acting element found in the MdCBF2 promoter. Overexpressing MdCBF2 in ‘Orin’ callus indicated that MdCBF2 could also increase the expression level of MdCOR15A-1 and MdCOR15A-2 and improve cold tolerance; we also found that transgenic callus overexpressing MdCBF2 had reduced MdCBF1 and MdCBF5 expression and increased MdCBF3 and MdCBF4 expression. Overall, these results show that MdbHLH33 can regulate the expression of MdCBF2 and improve the cold tolerance of transgenic callus.     

Overexpression of protein kinase C-delta increases tight junction permeability in LLC-PK1 epithelia

The Ca²⁺-independent-isoform of protein kinase C (PKC-) was overexpressed inLLC-PK₁ epithelia and placed undercontrol of a tetracycline-responsive expression system. In the absenceof tetracycline, the exogenous PKC- is expressed. Westernimmunoblots show that the overexpressed PKC- is found in thecytosolic, membrane-associated, and Triton-insoluble fractions.Overexpression of PKC- produced subconfluent and confluentepithelial morphologies similar to that observed on exposure ofwild-type cells to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. Transepithelialelectrical resistance(R_T) in cellsheets overexpressing PKC- was only 20% of that in cell sheetsincubated in the presence of tetracycline, in which the amount ofPKC- and R_Twere similar to those in LLC-PK₁ parental cell sheets. Overexpression of PKC- also elicited a significant increase in transepithelial flux ofD-[¹⁴C]mannitoland a radiolabeled 2 × 10⁶-molecular-weight dextran,suggesting with theR_T decrease that overexpression increased paracellular, tight junctional permeability. Electron microscopy showed that PKC- overexpression results in amultilayered cell sheet, the tight junctions of which are almost uniformly permeable to ruthenium red. Freeze-fracture electron microscopy indicates that overexpression of PKC- results in a moredisorganized arrangement of tight junctional strands. As withLLC-PK₁ cell sheets treated with12-O-tetradecanoylphorbol-13-acetate, the reducedR_T, increasedD-mannitol flux, and tightjunctional leakiness to ruthenium red that are seen with PKC-overexpression suggest the involvement of PKC- in regulation oftight junctional permeability.