[Show abstract][Hide abstract] ABSTRACT: The ubiquitin-conjugating enzyme, named TaE2, was obtained as the candidate interaction protein of TaMAPK2 by yeast-two-hybrid system. Bioinformatic analysis showed that TaE2 was a member of ubiquitin-conjugating enzyme E2 family. Semi-quantitative RT-PCR showed that TaE2 was expressed in root, stem, leaves and seeds. TaE2 was up-regulated under drought, high-salt and ABA treatments. The fusion protein GST-TaE2 was expressed in Escherichia.coli and purified by GST-Trap HP. TaE2 gene reported in this study lay a foundation for further studying the function and mechanisms of the ubiquitin proteasome pathway response to stresses.
[Show abstract][Hide abstract] ABSTRACT: Phosphoinositides are involved in regulation of recruitment and activity of signalling proteins in cell membranes. Phosphatidylinositol (PI) 4-kinases (PI4Ks) generate PI4-phosphate the precursor of regulatory phosphoinositides. No type II PI4K research on the abiotic stress response has previously been reported in plants. A stress-inducible type II PI4K gene, named TaPI4KIIγ, was obtained by de novo transcriptome sequencing of drought-treated wheat (Triticum aestivum). TaPI4KIIγ, localized on the plasma membrane, underwent threonine autophosphorylation, but had no detectable lipid kinase activity. Interaction of TaPI4KIIγ with wheat ubiquitin fusion degradation protein (TaUDF1) indicated that it might be hydrolysed by the proteinase system. Overexpression of TaPI4KIIγ revealed that it could enhance drought and salt stress tolerance during seed germination and seedling growth. A ubdkγ7 mutant, identified as an orthologue of TaPI4KIIγ in Arabidopsis, was sensitive to salt, polyethylene glycol (PEG), and abscisic acid (ABA), and overexpression of TaPI4KIIγ in the ubdkγ7 mutant compensated stress sensitivity. TaPI4KIIγ promoted root growth in Arabidopsis, suggesting that TaPI4KIIγ might enhance stress resistance by improving root growth. Overexpression of TaPI4KIIγ led to an altered expression level of stress-related genes and changes in several physiological traits that made the plants more tolerant to stress. The results provided evidence that overexpression of TaPI4KIIγ could improve drought and salt tolerance.
[Show abstract][Hide abstract] ABSTRACT: TaAIP was obtained as the interaction protein of wheat stress-related TaMAPK2, which was used as bait protein to screen the wheat cDNA library by yeast-two-hybrid system. TaAIP, containing a wali domain, was similar to some aluminum-induced proteins. Real-time PCR showed that the expression of TaAIP was up-regulated by the imposition of aluminum, high-salt and drought stress. Semi-quantitative RT-PCR analysis indicated TaAIP was a stem-specific gene, not expressed in root, leaves and flower. The subcellular localization assay indicated that TaAIP located on plasma membrane. These results provided the foundation for further analysis of TaAIP resistance mechanisms.
[Show abstract][Hide abstract] ABSTRACT: MAPKs are of importance in stress signal transduction process in plant. In order to investigate the function of wheat MAPKs, a MAPK gene named TaMAPK2, was isolated from wheat. To obtain the polyclonal antibody of TaMAPK2, the non-conservation fraction of TaMAPK2 gene was constructed into the prokaryotic expression vector pET-28a-(+). Under the condition of 1 mmol/L of IPTG, the expression of the fusion protein His-antiMAPK2 was up to the peak. The fusion protein His-antiMAPK2 was purified by HisTrapTMHP and used to prepare antibody. The titer of the rabbit`s anti-serum was measured by ELISA method. The rabbit` antiserum with high titer (>80000) was obtained. The polyclonal antibodies can be used for further investigation, which establish the foundation for investigating the function of the MAPK2 gene in protein level.
[Show abstract][Hide abstract] ABSTRACT: Protein kinases play crucial roles in response to external environment stress signals. A putative protein kinase, W55a, belonging to SNF1-related protein kinase 2 (SnRK2) subfamily, was isolated from a cDNA library of drought-treated wheat seedlings. The entire length of W55a was obtained using rapid amplification of 5' cDNA ends (5'-RACE) and reverse transcription-polymerase chain reaction(RT-PCR). It contains a 1,029 -bp open reading frame (ORF) encoding 342 amino acids. The deduced amino acid sequence of W55a had eleven conserved catalytic subdomains and one Ser/Thr protein kinase active-site that characterize Ser/Thr protein kinases. Phylogenetic analysis showed that W55a was 90.38% homologous with rice SAPK1, a member of the SnRK2 family. Using nullisomic-tetrasomic and ditelocentric lines of Chinese Spring, W55a was located on chromosome 2BS. Expression pattern analysis revealed that W55a was upregulated by drought and salt, exogenous abscisic acid, salicylic acid, ethylene and methyl jasmonate, but was not responsive to cold stress. In addition, W55a transcripts were abundant in leaves, but not in roots or stems, under environmental stresses. Transgenic Arabidopsis plants overexpressing W55a exhibited higher tolerance to drought. Based on these findings, W55a encodes a novel dehydration-responsive protein kinase that is involved in multiple stress signal transductions.
[Show abstract][Hide abstract] ABSTRACT: Leucine-rich repeat proteins (LRRs) play important roles in signal perception and activation of defense responses. We isolated two new LRR genes, NtLRR1 and NtLRR2, in tobacco (Nicotiana tabacum). NtLRR1 and NtLRR2 are characterized by 9 and 3 LRR domains, respectively. The phylogenetic relationships show that NtLRR1 and NtLRR2 belong to different subfamilies of the polygalacturonase inhibitor proteins (PGIPs) and LRP-related proteins (LRPs), respectively. NtLRR1 and NtLRR2 are responsive to pathogen attack and salt stress, but display differential expression patterns in tobacco. NtLRR1 is activated rapidly by infection with the tobacco wildfire pathogen (Pseudomonas syringae pv. tabaci), but slowly by tobacco mosaic virus (TMV). In contrast, NtLRR2 transcripts rapidly accumulate after infection with TMV, and only sluggishly with infection by the wildfire pathogen. In addition, NtLRR1 transcripts abundantly accumulate in stems, whereas NtLRR2 appears mainly in the roots. Isolation of the NtLRR2 promoter revealed some cis-acting elements responding to stresses and defense signal molecules. Subcellular localization indicated that NtLRR1 and NtLRR2 proteins localize in the cell walls and plasma membranes, respectively. It was concluded that NtLRR1 and NtLRR2 are important proteins having different functions in response to different stresses and mediating in binding interactions in a wide variety of biological processes.
[Show abstract][Hide abstract] ABSTRACT: A gene encoding Hordeum vulgare dehydration-responsive element binding protein 1 (HvDREB1), a member of the A-2 subgroup of the DREB subfamily, was isolated from barley seedlings. A subcellular localization assay revealed accumulation of HvDREB1 protein in the nucleus. As a trans-acting factor, HvDREB1 was able to bind to DRE/CRT elements and transactivate reporter gene expression in yeast cells. A study of various deletion mutants of HvDREB1 proteins indicated that the transactivation activity was localized to the N-terminal region. Expression of the HvDREB1 gene in barley leaves was significantly induced by salt, drought, and low-temperature. In contrast to most A-2 subgroup members in Arabidopsis thaliana, HvDREB1 also responded to exogenous ABA. Overexpression of HvDREB1 activated a downstream gene, RD29A, under normal growth conditions and led to increased tolerance to salt stress in Arabidopsis plants. These results suggest that HvDREB1 produces a DRE-/CRT-binding transcription factor that may have an important role in improving salt tolerance in plants.
Journal of Plant Research 01/2009; 122(1):121-30. DOI:10.1007/s10265-008-0195-3 · 1.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An ethylene responsive factor (ERF) gene W6, which belongs to ERF IV subfamily, was isolated from the cDNA library of wheat (Triticum aestivum L.) landrace Xiaobaimai. The full-length cDNA of W6 encoded an ERF protein containing a conserved ERF DNA-binding motif, 2 putative nuclear localization sequences, and a C-terminal acidic transcription activation domain. The expression of W6 in wheat was induced by various treatments, such as cold, drought, salt, abscisic acid (ABA), and fungal pathogens. To identify the function of the W6 gene, a transgenic vector (35S::pBI121::W6) containing CaMV 35S promoter was constructed and the W6-overexpresed tobacco (Nicotiana tabacum) plants by Agrobacterium-mediated transformation was obtained. Under the treatment with 200 mmol L−1 NaCl for 50 d, the transgenic plants grew well but the control plants almost died. The root length of the transgenic tobacco plants ranged from 1.40 to 3.93 cm but that of control was only 0.20 cm. The root weight of the transgenic tobacco plants ranged from 2.41 to 7.79 g compared with the control of 0.06 g. The superoxide dismutase activity and chlorophyll content in the transgenic plants were obviously higher than those of the control. The results showed that the overexpression of W6 improved the salt tolerance of tobacco, and W6 probably acted as a connector among different signal transduction pathways. The overexpression of W6 activated the expression of GCC box-containing genes PR2, PR3, PR5, and drought-responsive element/C-repeat (DRE/CRT) genes (NtERD10A and NtERD10C) under normal growth conditions. This improvement of the transgenic tobacco plants to salt stress suggested that W6 regulates osmotic tolerance by activation of downstream gene expression through interaction with the GCC box or DRE elements.
[Show abstract][Hide abstract] ABSTRACT: The study aims to detect the subcellular localization of ERF (ethylene-responsive element binding factor) transcription factor W17 protein, the interaction between W17 and cis-acting regulatory elements GCC-box and DRE in vitro, the binding and transactivating ability in vivo, and the role of W17 in higher plant stress-signal pathway. Recombinant plasmid W17/163hGFP was introduced into onion epidermal cells by the particle bombardment method with a PDS1000/He. Transformed cells were incubated for 24 h at 22°C in the dark and green fluorescence was monitored under a confocal microscope. The gene W17 was fused N-terminus of GST (glutathione-S-transferase) in prokaryotic expression vector pGEX-4T-1 and then transformed into E. coli strain BL21 (DE3). IPTG (0.5 mmol L−1) was added to induce the expression of recombinant GST/W17 for 3 h. The fused proteins were purified by GST purification columns, and then subjected to gel retardation assay with a 32P-labeled GCC or DRE sequence. The different reporter and effector plasmids were introduced into tobacco leaves through agroinfiltration, then transformed leaves stained by X-Gluc, faded with 75% alcohol and monitored under a Stereozooming microscope. The GFP fused with W17 protein was localized in the nuclei; SDS-PAGE assay demonstrated that the fused protein GST/W17 could be induced and purified with molecular weight at around 42.2 kD under the induction of IPTG. Purified fused protein was able to specifically bind to both the wild-type GCC-box and DRE element, but had no interaction with either the mutant DRE or GCC-box; W17 protein can bind to GCC-box and transactive downstream GUS gene in vivo. W17 can localize into the nuclei, and it may be involved not only in biotic stresses controlled by GCC-box, but also in abiotic stresses (e.g., salt-) induced signaling pathway.
Agricultural Sciences in China 06/2008; DOI:10.1016/S1671-2927(08)60098-X · 0.82 Impact Factor