Guangmin Xia

A Proteomic Study of the Response to Salinity and Drought Stress in an Introgression Strain of Bread Wheat

The effect of drought and salinity stress on the seedlings of the somatic hybrid wheat cv. Shanrong No. 3 (SR3) and its parent bread wheat cv. Jinan 177 (JN177) was investigated using two-dimensional gel electrophoresis and mass spectrometry. Of a set of 93 (root) and 65 (leaf) differentially expressed proteins (DEPs), 34 (root) and six (leaf) DEPs were cultivar-specific. The remaining DEPs were salinity/drought stress-responsive but not cultivar-specific. Many of the DEPs were expressed under both drought and salinity stresses. The amounts of stress-responsive DEPs between SR3 and JN177 were almost equivalent, whereas only some of these DEPs were shared by the two cultivars. Overall, the number of salinity-responsive DEPs was greater than the number of drought-responsive DEPs. And most of the drought-responsive DEPs also responded to salinity. There are both similarities and differences in the responses of wheat to salinity and drought. A parallel transcriptomics analysis showed that the correlation between transcriptional and translational patterns of DEPs was poor. The enhanced drought/salinity tolerance of SR3 appears to be governed by a superior capacity for osmotic and ionic homeostasis, a more efficient removal of toxic by-products, and ultimately a better potential for growth recovery.

Proteomic analysis on a high salt tolerance introgression strain of Triticum aestivum/Thinopyrum ponticum

Soil salinity is a major abiotic constraint to agricultural productivity. We successfully bred a new common wheat (Triticum aestivum L.) introgression variety (Shanrong No. 3) with high salt‐tolerance via asymmetric somatic hybridization between common wheat cultivar (Jinan 177) and UV‐irradiated Agropyron elongatum (Thinopyrum ponticum Podp). We report here a comparative proteomic analysis to investigate variety‐specific and salt‐responsive proteins between seedling‐roots of Shanrong No. 3 and Jinan 177. In total, 114 spots reproducibly presented differential expression patterns on 2‐DE maps. Of them, 34 were variety‐specific and 49 were salt‐responsive. We identified 110 spots by MALDI‐TOF MS and partially confirmed by MALDI‐TOF‐TOF MS, and functionally classified them into signal transduction, transcription and translation, transporting, chaperones, proteolysis and detoxification, etc. Meanwhile, we also found the alteration of protein expression of Shanrong No. 3 through inhibition of old proteins and production of novel ones, change in abundance and sensitivity of some nonsalt‐responsive and salt‐responsive proteins, as well as PTMs. Furthermore, comparison between proteome and transcripteome using cDNA microarray showed that there were only 20 proteins with abundances correlative to signal densities of corresponding EST probes. This study gives us a global insight into proteomic difference between Shanrong No. 3 and Jinan 177 in constitute and to salt‐response.

Asymmetric somatic hybridization between wheat (Triticum aestivum L.) and Agropyron elongatum (Host) Nevishi

Abstract Suspension-derived protoplasts of Agropyron elongatum irradiated by ultra-violet light (UV) were fused with the suspension-derived protoplasts of Triticum astivum using PEG. Fertile intergeneric somatic hybrid plants were produced and various hybrid lines have been selected and propagated in successive generations. Their hybrid nature was confirmed by analysis of profiles of isozymes, RAPDs, and 5S rDNA spacer sequences, and via GISH analysis. By the procedure described, the phenotype and chromosome number of wheat could be maintained besides transfer of a few chromosomes and chromosomal fragments from the donor A. elongatum. The results above indicated that highly asymmetric fertile hybrid plants and hybrid progenies of wheat were produced via somatic hybridization.

Asymmetric somatic hybridization between wheat ( Triticum aestivum ) and Avena sativa L.

Protoplasts from cell suspensions ofyoung-embryo-derived calli, which were nonregenerable for long-term subculture and protoplasts from embryogenic calli with the regeneration capacity of 75% ofthe same wheat Jinan 177, were mixed as recipient. Protoplasts from embryogenic calli of Avena sativa (with the regeneration capacity ofless than 10%) irradiated with UV at an intensity of 300 μW/cm2 for 30 s, 1 min, 2 min, 3 min, 5 min were used as the donor. Protoplasts ofthe recipient and the donor were fused by PEG method. Many calli and normal green plants were regenerated at high frequency, and were verified as somatic hybrids by chromosome counting, isozyme, 5S rDNA spacer sequence analysis and GISH (genomic in situ hybridization). Fusion combination between protoplasts either from the cell suspensions or from the calli and UV-treated Avena sativa protoplasts could not regenerate green plants.

Over-expression of TaMYB33 encoding a novel wheat MYB transcription factor increases salt and drought tolerance in Arabidopsis

Salt and drought stresses often adversely affect plant growth and productivity, MYB transcription factors have been shown to participate in the response to these stresses. Here we identified a new R2R3-type MYB transcription factor gene TaMYB33 from wheat (Triticum aestivum). TaMYB33 was induced by NaCl, PEG and ABA treatments, and its promoter sequence contains putative ABRE, MYB and other abiotic stress related cis-elements. Ectopic over-expression of TaMYB33 in Arabidopsis thaliana remarkably enhanced its tolerance to drought and NaCl stresses, but not to LiCl and KCl treatments. The expressions of AtP5CS and AtZAT12 which mirror the activities of proline and ascorbate peroxidase synthesis respectively were induced in TaMYB33 over-expression lines, indicating TaMYB33 promotes the ability for osmotic pressure balance-reconstruction and reactive oxidative species (ROS) scavenging. The up-regulation of AtAAO3 along with down-regulation of AtABF3, AtABI1 in TaMYB33 over-expression lines indicated that ABA synthesis was elevated while its signaling was restricted. These results suggest that TaMYB33 enhances salt and drought tolerance partially through superior ability for osmotic balance reconstruction and ROS detoxification.

Ectopic expression of a wheat MYB transcription factor gene, TaMYB73, improves salinity stress tolerance in Arabidopsis thaliana

MYB transcription factors (TFs) play pivotal roles in the abiotic stress response in plants, but their characteristics and functions in wheat (Triticum aestivum L.) have not been fully investigated. A novel wheat MYB TF gene, TaMYB73, is reported here based on the observation that its targeting probe showed the highest salinity-inducibility level among all probes annotated as MYB TFs in the cDNA microarray. TaMYB73 is a R2R3 type MYB protein with transactivation activity, and binds with types I, II, and IIG MYB binding motifs. The gene was induced by NaCl, dehydration, and several phytohormones, as well as some stress-, ABA-, and GA-responsive cis-elements present in its promoter region. Its over-expression in Arabidopsis enhanced the tolerance to NaCl as well as to LiCl and KCl, whereas it had no contribution to mannitol tolerance. The over-expression lines had superior germination ability under NaCl and ABA treatments. The expression of many stress signalling genes such as AtCBF3 and AtABF3, as well as downstream responsive genes such as AtRD29A and AtRD29B, was improved in these over-expression lines, and TaMYB73 can bind with promoter sequences of AtCBF3 and AtABF3. Taken together, it is suggested that TaMYB73, a novel MYB transcription factor gene, participates in salinity tolerance based on improved ionic resistance partly via the regulation of stress-responsive genes.

Arabidopsis DREB1A / CBF3 bestowed transgenic tall fescue increased tolerance to drought stress

In order to improve drought tolerance of tall fescue (Festuca arundinacea Schreb.), an important perennial cool-season grass, we introduced Arabidopsis DREB1A/CBF3 driven by the inducible rd29A promoter into tall fescue mediated by Agrobacterium tumefaciens strains AGL1. PCR and Southern blot analysis confirmed that DREB1A/CBF3 gene had been integrated into the genome of tall fescue. AtDREB1A gene was stably inherited and expressed in T1 plants, as indicated by PCR, RT-PCR and Western blotting analysis. The transgenic plants also showed an increased expression of AtP5CS2, which was confirmed to be a downstream target gene of DREB in Arabidopsis. We found that the transgenic tall fescue showed increased resistance to drought and accumulated high level of proline, indicating ability of the CBF3 gene to induce stress related response in tall fescue. The result here provides evidence for drought improvement of tall fescue via transformation with stress-related transcription factor and stress-induced promoter.

From Model to Crop: Functional Analysis of a STAY-GREEN Gene in the Model Legume Medicago truncatula and Effective Use of the Gene for Alfalfa Improvement

Medicago truncatula has been developed into a model legume. Its close relative alfalfa (Medicago sativa) is the most widely grown forage legume crop in the United States. By screening a large population of M. truncatula mutants tagged with the transposable element of tobacco (Nicotiana tabacum) cell type1 (Tnt1), we identified a mutant line (NF2089) that maintained green leaves and showed green anthers, central carpels, mature pods, and seeds during senescence. Genetic and molecular analyses revealed that the mutation was caused by Tnt1 insertion in a STAY-GREEN (MtSGR) gene. Transcript profiling analysis of the mutant showed that loss of the MtSGR function affected the expression of a large number of genes involved in different biological processes. Further analyses revealed that SGR is implicated in nodule development and senescence. MtSGR expression was detected across all nodule developmental zones and was higher in the senescence zone. The number of young nodules on the mutant roots was higher than in the wild type. Expression levels of several nodule senescence markers were reduced in the sgr mutant. Based on the MtSGR sequence, an alfalfa SGR gene (MsSGR) was cloned, and transgenic alfalfa lines were produced by RNA interference. Silencing of MsSGR led to the production of stay-green transgenic alfalfa. This beneficial trait offers the opportunity to produce premium alfalfa hay with a more greenish appearance. In addition, most of the transgenic alfalfa lines retained more than 50% of chlorophylls during senescence and had increased crude protein content. This study illustrates the effective use of knowledge gained from a model system for the genetic improvement of an important commercial crop.

TaCHP: A Wheat Zinc Finger Protein Gene Down-Regulated by Abscisic Acid and Salinity Stress Plays a Positive Role in Stress Tolerance

The plant response to abiotic stresses involves both abscisic acid (ABA)-dependent and ABA-independent signaling pathways. Here we describe TaCHP, a CHP-rich (for cysteine, histidine, and proline rich) zinc finger protein family gene extracted from bread wheat (Triticum aestivum), is differentially expressed during abiotic stress between the salinity-sensitive cultivar Jinan 177 and its tolerant somatic hybrid introgression cultivar Shanrong No.3. TaCHP expressed in the roots of seedlings at the three-leaf stage, and the transcript localized within the cells of the root tip cortex and meristem. TaCHP transcript abundance was higher in Shanrong No.3 than in Jinan 177, but was reduced by the imposition of salinity or drought stress, as well as by the exogenous supply of ABA. When JN17, a salinity hypersensitive wheat cultivar, was engineered to overexpress TaCHP, its performance in the face of salinity stress was improved, and the ectopic expression of TaCHP in Arabidopsis (Arabidopsis thaliana) also improved the ability of salt tolerance. The expression level of a number of stress reporter genes (AtCBF3, AtDREB2A, AtABI2, and AtABI1) was raised in the transgenic lines in the presence of salinity stress, while that of AtMYB15, AtABA2, and AtAAO3 was reduced in its absence. The presence in the upstream region of the TaCHP open reading frame of the cis-elements ABRE, MYBRS, and MYCRS suggests that it is a component of the ABA-dependent and -independent signaling pathways involved in the plant response to abiotic stress. We suggest that TaCHP enhances stress tolerance via the promotion of CBF3 and DREB2A expression.

Generation of novel high quality HMW-GS genes in two introgression lines of Triticum aestivum/Agropyron elongatum

BackgroundHigh molecular weight glutenin subunits (HMW-GS) have been proved to be mostly correlated with the processing quality of common wheat (Triticum aestivum). But wheat cultivars have limited number of high quality HMW-GS. However, novel HMW-GS were found to be present in many wheat asymmetric somatic hybrid introgression lines of common wheat/Agropyron elongatum.ResultsTo exploit how these new subunits were generated, we isolated HMW-GS genes from two sib hybrid lines (II-12 and 11-4-6) and compared them with those from their parents. The result shows that two genes of hybrid (H11-3-3 and H11-4-3) are directly introgressed from the donor parent Agropyron elongatum; one hybrid gene (H1Dx5) comes from point mutation of a parental wheat gene (1Dx2.1); two other hybrid genes (H1By8 and H1By16) are likely resulting from unequal crossover or slippage of a parental wheat gene (1By9.1); and the sixth novel hybrid gene (H1Dy12) may come from recombination between two parental genes.ConclusionTherefore, we demonstrate that novel HMW-GS genes can be rapidly created through asymmetric somatic hybridization in a manner similar with the evolution mechanism of these genes supposed before. We also described gene shuffling as a new mechanism of novel HMW-GS gene formation in hybrids. The results suggest that asymmetric somatic hybridization is an important approach for widening HMW-GS genebank of wheat quality improvement.