Non-coding small RNAs responsive to abiotic stress in wheat (Triticum aestivum L.)

Plant small RNAs are emerging as significant components of epigenetic processes and of gene networks involved in development and homeostasis. In this paper, to identify small RNAs in wheat, 2,076 small RNAs were identified in a small RNA library from leaf, root, and spike. These small RNAs mapped to non-coding regions the CDS region of protein-coding genes and 5' UTR and 3' UTR regions. The expression of small RNAs in seedling leaves, roots, and spikes were analyzed by northern blot, which indicates that some small RNAs were responsive to abiotic stress treatments including heat, cold, salt and dehydration.     

Isolation and expression analysis of genes encoding DNA methyltransferase in wheat (Triticum aestivum L.)

DNA methylation of cytosine residues, catalyzed by DNA methyltransferases, is suggested to play important roles in regulating gene expression and plant development. In this study, we isolated four wheat cDNA fragments and one cDNA with open reading frame encoding putative DNA methyltransferase and designated TaMET1, TaMET2a, TaMET2b, TaCMT, TaMET3, respectively. BLASTX searches and phylogenetic analysis suggested that five cDNAs belonged to four classes (Dnmt1, Dnmt2, CMT and Dnmt3) of DNA methyltransferase genes. TaMET2a encoded a protein of 376 aa and contained eight of ten conserved motifs characteristic of DNA methyltransferase. Genomic sequence of TaMET2a was obtained and found to contain ten introns and eleven exons. The expression analysis of the five genes revealed that they were expressed in developing seed, during germination and various vegetative tissues, but in quite different abundance. It was interesting to note that TaMET1 and TaMET3 mRNAs were clearly detected in dry seeds. Moreover, the differential expression patterns of five genes were observed between wheat hybrid and its parents in leaf, stem and root of jointing stage, some were up-regulated while some others were down-regulated in the hybrid. We concluded that multiple wheat DNA methyltransferase genes were present and might play important roles in wheat growth and development.     

Identification of three Wx proteins in wheat (Triticum aestivum L.)

Nullisomic analysis of waxy (Wx) protein of hexaploid wheat (Triticum aestivum L.) cv. “Chinese Spring” using two-dimensional polyacrylamide gel electrophoresis revealed that threeWx loci,Wx-A1, Wx-B1, andWx-D1, located on chromosome arms 7AS, 4AL, and 7DS, produce three distinct Wx subunit groups, subunit group-A (SGA), SGB, and SGD, respectively. SGA has a higher molecular weight and a more basic isoelectric point (pI) than the other two. SGB and SGD have the same molecular weight but a slightly different pI range. Owing to the detection of these three subunit groups, we were able to identify the expression of three waxy genes in wheat endosperm and to find two types of mutants among Japanese wheat cultivars, one lacking SGA and the others SGB. These results suggest the possibility of breeding a waxy wheat.     

RELP markers linked to two Hessian fly-resistance genes in wheat (Triticum aestivum L.) from Triticum tauschii (coss.) Schmal

Summary Restriction fragment length polymorphism (RFLP) markers linked to genes controlling Hessian fly resistance from Triticum tauschii (Coss.) Schmal. were identified for two wheat (Triticum aestivum L.) germ plasm lines KS89WGRC3 (C3) and KS89WGRC6 (C6). Forty-six clones with loci on chromosomes of homoeologous group 3 and 28 clones on those of group 6 were surveyed for polymorphisms. Eleven and 12 clones detected T. tauschii loci in the two lines, respectively. Analysis of F₂ progenies indicated that the Hessian fly resistance gene H23 identified in C3 is linked to XksuH4 (6.9 cM) and XksuG48 (A) (15.6 cM), located on 6D. The resistance gene H24 in C6 is linked to XcnlBCD451 (5.9 cM), XcnlCD0482 (5.9 cM) and XksuG48 (B) (12.9 cM), located on 3DL.Paper No. 810 of the Cornell Plant Breeding Series     

Identification of leaf rust resistance genes in wheat (Triticum aestivum L.) cultivars using molecular markers

A collection of 68 cultivars of common wheat has been screened for leaf rust resistance genes with the use of molecular markers. Markers of genes Lr1, Lr9, Lr10, Lr19, Lr20, Lr21, Lr24, and Lr26 have been used. It has been suggested that allele Xgwm295 be used as a marker for identifying the Lr34 gene. The genes originating from Triticum aestivum L., as well as the Lr26 gene contained in rye translocation 1RS, are the most frequent. Genes originating from wild wheats were rarer in the cultivars studied.     

Nuclear genes affecting albinism in wheat (Triticum aestivum L.) anther culture

Summary Inheritance of the ability to respond in wheat anther culture was studied from 6×2 reciprocal crosses between six varieties with high and two varieties with low capacity for green plant formation and their parents, replicated in two environments. Effects of genotypes dominated embryo formation and percentages of green plants, accounting for 78.4% and 85.4% of total variation, respectively, while smaller genetic effects were indicated for regeneration. Nuclear genes could explain almost all the genotype effects in this material. Embryo formation showed heterosis over high parent for 5 of the 12 hybrids, while percentages of green plants from the hybrids were intermediate to the parents. General Combining Ability (GCA) could explain 78.8% of the variation for embryo formation among the hybrids, whereas differences in percentage of green plants were dominated by Specific Combining Ability (SCA), accounting for 67.9% of hybrid variation. A positive correlation (r=0.81^**) was observed between the genetic capacity for regeneration and green plant formation. Analysis of covariance indicated that effects causing GCA for green plant formation were mainly responsible for this correlation. A regression model with two parallel lines divided the six parent lines with high green plant formation into three groups with respect to their reactions with the two testers. The results are discussed with regard to possible involvement of two sets of nuclear genes affecting the percentage of green plants obtained in wheat anther culture: one set consisting of mainly additive effects affecting green plant percentage through an initial effect on regeneration ability, and another set of two or a few more major genes with dominance or epistatic effects uncorrelated with regeneration.     

Identification of leaf rust resistance genes in wheat (Triticum aestivum L.) cultivars using molecular markers

A collection of 68 cultivars of common wheat has been screened for leaf rust resistance genes with the use of molecular markers. Markers of genes Lr1, Lr9, Lr10, Lr19, Lr20, Lr21, Lr24, and Lr26 have been used. It has been suggested that allele Xgwm295 be used as a marker for identifying the Lr34 gene. The genes originating from Triticum aestivum L., as well as the Lr26 gene contained in rye translocation 1RS, are the most frequent. Genes originating from wild wheats were rarer in the cultivars studied.     

Plant regeneration from coleoptile tissue of wheat (Triticum aestivum L.)

Plant regeneration was achieved from coleoptile tissue of wheat (Triticum aestivum L. cv. Kharachia-65). Coleoptiles (1.0 - 3.5 cm long) were excised from 2- to 5-d-old seedlings and cultured on Murashige and Skoog's (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D - 0.5, 2.5, and 5.0 mg dm^-3). Cream, friable callus was obtained after 6 weeks of inoculation. This callus was sub-cultured on MS medium supplemented with 2,4-D (2.5 mg dm^-3) and 5 % coconut water. After 6 weeks of sub-culturing white, cream or pale, friable, nodular callus was obtained. Plant regeneration occurred when this callus was sub-cultured on MS medium supplemented with 0.2 mg dm^-3 1-naphthalene acetic acid + 1.0 mg dm^-3 6-benzylaminopurine. For rooting, regenerated shoots or plantlets were transferred on MS medium supplemented with 0.5 mg dm^-3 indole-3-acetic acid. Rooted plantlets were directly transferred into pots and grown under field conditions. Seed setting invariably occurred in all plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

Sequence and tissue-specific expression of a putative peroxidase gene from wheat (Triticum aestivum L.)

We have used a cDNA clone encoding a pathogen-induced putative wheat peroxidase to screen a genomic libary of wheat (Triticum aestivum L. cv. Cheyenne) and isolated one positive clone, lambda POX1. Sequence analysis revealed that this clone contains a gene encoding a putative peroxidase with a calculated pI of 8.1 which exhibits 58% and 83% sequence identity to the amino acid sequence of the turnip (Brassica rapa) peroxidase and a pathogen-induced putative wheat peroxidase, respectively. The two introns in the wheat gene are at the same positions as introns in the peroxidase genes of tomato and horseradish. Results of S1-mapping experiments suggest that this gene is neither pathogen-nor wound-induced in leaves but is constitutively expressed in roots.     

Identification of catalytically active groups of wheat (Triticum aestivum L.) germ lipase

The active site of wheat germ lipase was studied by the Dixon method and chemical modification. The profile of curve logV = f(pH), pK and ionization heat values, lipase photoinactivation, and lipase inactivation with diethylpyrocarbonate and dicyclohexylcarbodiimide led us to assume that the active site of the enzyme comprises the carboxylic group of aspartic or glutamic acid and the imidazole group of histidine. Apparently, the OH-group of serine plays a key role in catalysis: as a result of incubation for 1 h in the presence of phenylmethylsulfonyl fluoride, the enzyme activity decreased by more than 70%. It is shown that ethylenediamine tetraacetate is a noncompetitive inhibitor of lipase. Wheat germs are very healthful because they are rich in vitamins, essential amino acids, and proteins. For this reason, wheat germs are widely used in food, medical, and feed mill industries [1-3]. However, their use is limited by instability during storage, which is largely determined by the effect of hydrolytic and redox enzymes. Representative enzymes of this group are lipase (glycerol ester hydrolase, EC 3.1.1.3), which hydrolyzes triglycerides of higher fatty acids, and lipoxygenase (EC 1.13.11.13), which oxidizes polyunsaturated higher fatty acids.     

Ultraviolet-B-induced DNA lesions and their removal in wheat (Triticum aestivum L.) leaves

Monoclonal antibodies were used in an enzyme-linked immunosorbent assay (ELISA) to detect the induction and removal of cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts in DNA isolated from ultraviolet B (UV-B)-exposed primary wheat (Triticum aestivum L. cv. Chinese Spring) leaf tissue. The accumulation of lesions in the primary leaves of 6-d-old wheat seedlings was followed during the exposure of the leaf to an approximate dose of 3.6×10^?1 W m^?2 UV-B (Caldwell weighting). Significant increases in the levels of both CPDs and (6-4) photoproducts were detected in wheat leaves exposed to UV-B in the absence of other light However, only an increase in (6-4) photoproduct levels could be measured in wheat leaves exposed to the same UV-B source in the presence of supplemental white light. The removal of CPD antibody binding sites in the DNA after irradiation was rapid under conditions of high light intensity in contrast to the removal of (6-4) photoproduct antibody binding sites, which was significantly slower. The removal of CPDs appeared to be light dependent, this rate of removal decreasing with decreasing light fluences. The removal of (6-4) photoproducts also appeared light dependent, but to a lesser extent than the removal of CPDs, under the conditions studied here. Gene expression in the primary wheat leaf was measured and showed an up-regulation of chalcone synthase expression and a reduction in expression of chlorophyll a/b-binding protein (cab) in response to supplementary UV-B. No effect was seen on the expression of the other photosynthetic genes studied (the genes coding for the enzymes sedoheptu-lose 1,7-bisphosphatase and fructose 1,6-bisphosphatase). Measurement of the levels of DNA lesions in this same tissue showed that the observed changes in gene expression accompanied the appearance of UV-B induced lesions in the form of (6-4) photoproducts in the wheat leaf genome.     

Genetic mapping of new seed-expressed polyphenol oxidase genes in wheat (Triticum aestivum L.)

Polyphenol oxidase (PPO) enzymatic activity is a major cause in time-dependent discoloration in wheat dough products. The PPO-A1 and PPO-D1 genes have been shown to contribute to wheat kernel PPO activity. Recently a novel PPO gene family consisting of the PPO-A2, PPO-B2, and PPO-D2 genes has been identified and shown to be expressed in wheat kernels. In this study, the sequences of these five kernel PPO genes were determined for the spring wheat cultivars Louise and Penawawa. The two cultivars were found to be polymorphic at each of the PPO loci. Three novel alleles were isolated from Louise. The Louise X Penawawa mapping population was used to genetically map all five PPO genes. All map to the long arm of homeologous group 2 chromosomes. PPO-A2 was found to be located 8.9 cM proximal to PPO-A1 on the long arm of chromosome 2A. Similarly, PPO-D1 and PPO-D2 were separated by 10.7 cM on the long arm of chromosome 2D. PPO-B2 mapped to the long arm of chromosome 2B and was the site of a novel QTL for polyphenol oxidase activity. Five other PPO QTL were identified in this study. One QTL corresponds to the previously described PPO-D1 locus, one QTL corresponds to the PPO-D2 locus, whereas the remaining three are located on chromosome 2B.     

Molecular identification of powdery mildew resistance genes in common wheat (Triticum aestivum L.)

RFLP markers for the wheat powdery mildew resistance genes Pm1 and Pm2 were tagged by means of near-isogenic lines. The probe Whs178 is located 3 cM from the Pm1 gene. For the powdery mildew resistance gene Pm2, two markers were identified. The linkage between the Pm2 resistance locus and one of these two probes was estimated to be 3 cM with a F₂ population. Both markers can be used to detect the presence of the corresponding resistance gene in commercial cultivars. Bulked segregant analysis was applied to identify linkage disequillibrium between the resistance gene Pm18 and the abovementioned marker, which was linked to this locus at a distance of 4 cM. Furthermore, the RAPD marker OPH-11₁₉₀₀ (5-CTTCCGCAGT-3) was selected with pools created from a population segregating for the resistance of Trigo BR 34. The RAPD marker was mapped about 13 cM from this resistance locus.     

Accelerated production of transgenic wheat (Triticum aestivum L.) plants

We have developed a method for the accelerated production of fertile transgenic wheat (Triticum aestivum L.) that yields rooted plants ready for transfer to soil in 8–9 weeks (56–66 days) after the initiation of cultures. This was made possible by improvements in the procedures used for culture, bombardment, and selection. Cultured immature embryos were given a 4–6 h pre-and 16 h post-bombardment osmotic treatment. The most consistent and satisfactory results were obtained with 30 g of gold particles/bombardment. No clear correlation was found between the frequencies of transient expression and stable transformation. The highest rates of regeneration and transformation were obtained when callus formation after bombardment was limited to two weeks in the dark, with or without selection, followed by selection during regeneration under light. Selection with bialaphos, and not phosphinothricin, yielded more vigorously growing transformed plantlets. The elongation of dark green plantlets in the presence of 4–5 mg/l bialaphos was found to be reliable for identifying transformed plants. Eighty independent transgenic wheat lines were produced in this study. Under optimum conditions, 32 transformed wheat plants were obtained from 2100 immature embryos in 56–66 days, making it possible to obtain R3 homozygous plants in less than a year.     

Characterization of a common wheat (Triticum aestivum L.) TaSnRK2.7 gene involved in abiotic stress responses

Sucrose non-fermenting-1-related protein kinase 2 (SnRK2) plays a key role in the plant stress signalling transduction pathway via phosphorylation. Here, a SnRK2 member of common wheat, TaSnRK2.7, was cloned and characterized. Southern blot analysis suggested that the common wheat genome contains three copies of TaSnRK2.7. Subcellular localization showed the presence of TaSnRK2.7 in the cell membrane, cytoplasm, and nucleus. Expression patterns revealed that TaSnRK2.7 is expressed strongly in roots, and responds to polyethylene glycol, NaCl, and cold stress, but not to abscisic acid (ABA) application, suggesting that TaSnRK2.7 might participate in non-ABA-dependent signal transduction pathways. TaSnRK2.7 was transferred to Arabidopsis under the control of the CaMV-35S promoter. Function analysis showed that TaSnRK2.7 is involved in carbohydrate metabolism, decreasing osmotic potential, enhancing photosystem II activity, and promoting root growth. Its overexpression results in enhanced tolerance to multi-abiotic stress. Therefore, TaSnRK2.7 is a multifunctional regulatory factor in plants, and has the potential to be utilized in transgenic breeding to improve abiotic stress tolerance in crop plants.