Xiuying Kong

Molecular characterization of 60 isolated wheat MYB genes and analysis of their expression during abiotic stress

The proteins of the MYB superfamily play central roles in developmental processes and defence responses in plants. Sixty unique wheat MYB genes that contain full-length cDNA sequences were isolated. These 60 genes were grouped into three categories, namely one R1R2R3-MYB, 22 R2R3-MYBs, and 37 MYB-related members. The sequence composition of the R2 and R3 repeats was conserved among the 22 wheat R2R3-MYB proteins. Phylogenetic comparison of the members of this superfamily among wheat, rice, and Arabidopsis revealed that the putative functions of some wheat MYB proteins were clustered into the Arabidopsis functional clades. Tissue-specific expression profiles showed that most of the wheat MYB genes were expressed in all of the tissues examined, suggesting that wheat MYB genes take part in multiple cellular processes. The expression analysis during abiotic stress identified a group of MYB genes that respond to one or more stress treatments. The overexpression of a salt-inducible gene, TaMYB32, enhanced the tolerance to salt stress in transgenic Arabidopsis. This study is the first comprehensive study of the MYB gene family in Triticeae.

Evolutionary and functional study of the CDPK gene family in wheat (Triticum aestivum L.)

Calcium-dependent protein kinases (CDPKs) are crucial sensors of calcium concentration changes in plant cells under diverse endogenous and environmental stimuli. We identified 20 CDPK genes from bread wheat and performed a comprehensive study on their structural, functional and evolutionary characteristics. Full-length cDNA sequences of 14 CDPKs were obtained using various approaches. Wheat CDPKs were found to be similar to their counterparts in rice in genomic structure, GC content, subcellular localization, and subgroup classification. Divergence time estimation of wheat CDPK gene pairs and wheat–rice orthologs suggested that most duplicated genes already existed in the common ancestor of wheat and rice. The number of CDPKs in diploid wheat genome was estimated to be at least 26, a number close to that in rice, Arabidopsis, and poplar. However, polymorphism among EST sequences uncovered transcripts of all three homoeologous alleles for 13 out of 20 CDPKs. Thus, the hexaploid wheat should have 2–3 fold more CDPK genes expressing in their cells than the diploid species. Wheat CDPK genes were found to respond to various biotic and abiotic stimuli, including cold, hydrogen peroxide (H2O2), salt, drought, powdery mildew (Blumeria graminis tritici, Bgt), as well as phytohormones abscisic acid (ABA) and gibberellic acid (GA). Each CDPK gene often responded to multiple treatments, suggesting that wheat CDPKs are converging points for multiple signal transduction pathways. The current work represents the first comprehensive study of CDPK genes in bread wheat and provides a foundation for further functional study of this important gene family in Triticeae.

A wheat R2R3-MYB gene, TaMYB30-B, improves drought stress tolerance in transgenic Arabidopsis

The MYB-type proteins are involved in various processes of plant growth, development, and stress response. In a previous work, a polyethylene glycol (PEG) stress-induced gene, TaMYB30, which encodes a R2R3-type MYB protein was identified in wheat. In this study, the isolation and functional characterization of the TaMYB30 gene are reported. Three homologous sequences of TaMYB30 were isolated from hexaploid wheat and designated as TaMYB30-A, TaMYB30-B, and TaMYB30-D genes based on the localizations of these three sequences to chromosomes 2A, 2B, and 2D, respectively. The expression levels of these three genes were similar under PEG stress conditions, and TaMYB30-B was selected for further analysis. The TaMYB30-B protein was localized to the nucleus where it activated transcription. The detailed characterization of Arabidopsis transgenic plants that overexpress the TaMYB30-B gene revealed that the TaMYB30-B protein can improve drought stress tolerance during the germination and the seedling stages. It was also found that overexpression of TaMYB30-B resulted in altered expression levels of some drought stress-responsive genes and changes in several physiological indices, which allow plants to overcome adverse conditions. These results indicate that the TaMYB30-B protein plays important roles in plant stress tolerance, and modification of its expression may improve drought stress tolerance in crop plants.

Dominant and Pleiotropic Effects of a GAI Gene in Wheat Results from a Lack of Interaction between DELLA and GID1

Dominance, semidominance, and recessiveness are important modes of Mendelian inheritance. The phytohormone gibberellin (GA) regulates many plant growth and developmental processes. The previously cloned semidominant GA-insensitive (GAI) genes Reduced height1 (Rht1) and Rht2 in wheat (Triticum aestivum) were the basis of the Green Revolution. However, no completely dominant GAI gene has been cloned. Here, we report the molecular characterization of Rht-B1c, a dominant GAI allele in wheat that confers more extreme characteristics than its incompletely dominant alleles. Rht-B1c is caused by a terminal repeat retrotransposons in miniature insertion in the DELLA domain. Yeast two-hybrid assays showed that Rht-B1c protein fails to interact with GA-INSENSITIVE DWARF1 (GID1), thereby blocking GA responses and resulting in extreme dwarfism and pleiotropic effects. By contrast, Rht-B1b protein only reduces interaction with GID1. Furthermore, we analyzed its functions using near-isogenic lines and examined its molecular mechanisms in transgenic rice. These results indicated that the affinity between GID1 and DELLA proteins is key to regulation of the stability of DELLA proteins, and differential interactions determine dominant and semidominant gene responses to GA.

A novel wheat bZIP transcription factor, TabZIP60, confers multiple abiotic stress tolerances in transgenic Arabidopsis.

The basic region/leucine zipper (bZIP) transcription factors (TFs) play vital roles in the response to abiotic stress. However, little is known about the function of bZIP genes in wheat abiotic stress. In this study, we report the isolation and functional characterization of the TabZIP60 gene. Three homologous genome sequences of TabZIP60 were isolated from hexaploid wheat and mapped to the wheat homoeologous group 6. A subcellular localization analysis indicated that TabZIP60 is a nuclear-localized protein that activates transcription. Furthermore, TabZIP60 gene transcripts were strongly induced by polyethylene glycol, salt, cold and exogenous abscisic acid (ABA) treatments. Further analysis showed that the overexpression of TabZIP60 in Arabidopsis resulted in significantly improved tolerances to drought, salt, freezing stresses and increased plant sensitivity to ABA in seedling growth. Meanwhile, the TabZIP60 was capable of binding ABA-responsive cis-elements that are present in promoters of many known ABA-responsive genes. A subsequent analysis showed that the overexpression of TabZIP60 led to enhanced expression levels of some stress-responsive genes and changes in several physiological parameters. Taken together, these results suggest that TabZIP60 enhances multiple abiotic stresses through the ABA signaling pathway and that modifications of its expression may improve multiple stress tolerances in crop plants.

Characterization of a Wheat R2R3-MYB Transcription Factor Gene, TaMYB19, Involved in Enhanced Abiotic Stresses in Arabidopsis

MYB-type proteins have been shown to participate in multiple stress responses. In the present study, we identified a gene in wheat induced by multiple abiotic stresses, TaMYB19, which encodes a R2R3-type MYB protein. Three highly homologous sequences of TaMYB19 were isolated from hexaploid wheat. Using the nulli-tetrasomic (NT) lines of Chinese Spring wheat, the three sequences were localized to chromosomes 1A, 1B and 1D and designated as TaMYB19-A, TaMYB19-B and TaMYB19-D, respectively. The expression patterns of these three genes were similar under different stress conditions. The TaMYB19-B sequence was selected for further analysis. The TaMYB19-B protein localized to the nucleus. A detailed characterization of Arabidopsis transgenic plants overexpressing the TaMYB19-B gene revealed that the TaMYB19-B protein could improve tolerance to multiple stresses during the seedling stage. We also found that the overexpression of TaMYB19-B resulted in changes in several physiological indices and altered the expression levels of a number of abiotic stress-related genes, allowing the plants to overcome adverse conditions. These results indicate that the TaMYB19 protein plays an important role in plant stress tolerance and that modification of the expression of this protein may improve abiotic stress tolerance in crop plants.

The Novel Wheat Transcription Factor TaNAC47 Enhances Multiple Abiotic Stress Tolerances in Transgenic Plants

NAC transcription factors play diverse roles in plant development and responses to abiotic stresses. However, the biological roles of NAC family members in wheat are not well understood. Here, we reported the isolation and functional characterization of a novel wheat TaNAC47 gene. TaNAC47 encoded protein, localizing in the nucleus, is able to bind to the ABRE cis-element and transactivate transcription in yeast, suggesting that it likely functions as a transcriptional activator. We also showed that TaNAC47 is differentially expressed in different tissues, and its expression was induced by the stress treatments of salt, cold, polyethylene glycol and exogenous abscisic acid. Furthermore, overexpression of TaNAC47 in Arabidopsis resulted in ABA hypersensitivity and enhancing tolerance of transgenic plants to drought, salt, and freezing stresses. Strikingly, overexpression of TaNAC47 was found to activate the expression of downstream genes and change several physiological indices that may enable transgenic plants to overcome unfavorable environments. Taken together, these results uncovered an important role of wheat TaNAC47 gene in response to ABA and abiotic stresses.

Overexpression of a wheat MYB transcription factor gene, TaMYB56-B, enhances tolerances to freezing and salt stresses in transgenic Arabidopsis.

The MYB proteins play central roles in the stress response in plants. Our previous works identified a cold stress-related gene, TaMYB56, which encodes a MYB protein in wheat. In this study, we isolated the sequences of TaMYB56 genes, and mapped them to the wheat chromosomes 3B and 3D. The expression levels of TaMYB56-B and TaMYB56-D were strongly induced by cold stress, but slightly induced by salt stress in wheat. The detailed characterization of the Arabidopsis transgenic plants that overexpress TaMYB56-B revealed that TaMYB56-B is possibly involved in the responses of plant to freezing and salt stresses. The expression of some cold stress-responsive genes, such as DREB1A/CBF3 and COR15a, were found to be elevated in the TaMYB56-B-overexpressing Arabidopsis plants compared to wild-type. These results indicate that TaMYB56-B may act as a regulator in plant stress response.

Molecular evolution of two duplicated CDPK genes CPK7 and CPK12 in grass species: A case study in wheat (Triticum aestivum L.)

Gene duplication contributes to the expansion of gene families and subsequent functional diversification. Calcium-dependent protein kinases (CDPKs) are members of an important calcium sensor family involved in abiotic and biotic stress signaling in plants. We report here the molecular evolution and expression analysis of a pair of duplicated CDPK genes CPK7 and CPK12 that arose in the common ancestor of grass species. With higher nonsynonymous/synonymous ratios (dN/dS, or ω), CPK12 genes appear to diverge more rapidly than CPK7s, suggesting relaxed selection constraints on CPK12s. Sliding window analysis revealed increased dN and ω values at N-terminal regions and the calcium-binding EF hand loops. Likelihood analyses using various models in PAML 4.0 showed purifying selection on both CPK7 and CPK12 lineages. In addition to the divergence in cis-element combinations on their promoters, functional divergence of CPK7 and CPK12 genes was also observed in wheat where TaCPK7 was found to respond to drought (PEG), salt (NaCl), cold, and hydrogen peroxide (H(2)O(2)) while TaCPK12 responded only to the treatment of ABA, a feature that may complement or expand TaCPK7-mediated stress signaling networks of wheat. The contrasting expression patterns of CPK7 and CPK12 genes under stress conditions were also observed in rice, suggesting conservative functional evolution of these genes. Since no positive selection was detected between the two lineages, the divergence of CPK7 and CPK12 genes should be ascribed to subfunctionalization, rather than neofunctionalization. Thus, our work demonstrates another case of evolutionary employment of duplicated genes via subfunctionalization for better adaptation.

Characterization and mapping of novel chlorophyll deficient mutant genes in durum wheat

The yellow-green leaf mutant has a non-lethal chlorophyll-deficient mutation that can be exploited in photosynthesis and plant development research. A novel yellow-green mutant derived from Triticum durum var. Cappelli displays a yellow-green leaf color from the seedling stage to the mature stage. Examination of the mutant chloroplasts with transmission electron microscopy revealed that the shape of chloroplast changed, grana stacks in the stroma were highly variable in size and disorganized. The pigment content, including chlorophyll a, chlorophyll b, total chlorophyll and carotene, was decreased in the mutant. In contrast, the chla/chlb ratio of the mutants was increased in comparison with the normal green leaves. We also found a reduction in the photosynthetic rate, fluorescence kinetic parameters and yield-related agronomic traits of the mutant. A genetic analysis revealed that two nuclear recessive genes controlled the expression of this trait. The genes were designated ygld1 and ygld2. Two molecular markers co-segregated with these genes. ygld 1 co-segregated with the SSR marker wmc110 on chromosome 5AL and ygld 2 co-segregated with the SSR marker wmc28 on chromosome 5BL. These results will contribute to the gene cloning and the understanding of the mechanisms underlying chlorophyll metabolism and chloroplast development in wheat.