Abscisic acid-responsive proteins induce salinity tolerance in wheat seedlings

The response of wheat seedlings germinated under salinity stress to exogenous abscisic acid was discussed. Exogenous ABA acid induces two characteristic proteins of high molecular weights (109.4 and 84.0 kDa) in addition to the new protein set formed under high salinity levels. ABA, also induces synthesized forms of guaiacol peroxidase in seedlings treated with 200 mM NaCl. The induction and/or accumulation of peroxidase isoforms in ABA-treated seedlings, may reduce the active oxygen produced by salinity. The changes in gene expression and peroxidase isoforms may be selected to adaptation of wheat seedlings to NaCl-salinity.     

Repetitive, genome-specific probes in wheat (Triticum aestivum L. em Thell) amplified with minisatellite core sequences

The detection and analysis of DNA polymorphisms in crops is an essential component of marker-assisted selection and cultivar identification in plant breeding. We have explored the direct amplification of minisatellite DNA by PCR (DAMD-PCR) as a means for generating DNA probes that are useful for detecting DNA polymorphisms and DNA fingerprinting in wheat. This technique was facilitated by high-stringency PCR with known plant and animal minisatellite core sequences as primers on wheat genomic DNA. The products of DAMD-PCR from Triticum aestivum, T. durum, T. monococcum, T. speltoides and T. tauschii showed a high degree of polymorphism and the various genomes could be identified. Cloning of the DAMD-PCR products and subsequent Southern hybridization frequently revealed polymorphic probes showing a good degree of genome specificity. In addition, polymorphic, single locus, and moderately dispersed PCR products were cloned that may have a potential for DNA fingerprinting. Our experiments were limited primarily to diploid wheats and the results indicated that DAMD-PCR may isolate genome-specific probes from wild diploid wheat species that could be used to monitor genome introgression into hexaploid wheat.This paper reports the results of research only. Mention of a proprietary product does not constitute an endorsement or a recommendation for its use by the USDA or the University of Missouri. Contribution from the University of Missouri, the Agricultural Experimental Station and U.S. Department of Agriculture-Agricultural Research Service, Plant Genetics Research Unit, journal series No. 12523     

Identification and analysis of expressed resistance gene sequences in wheat

Forty-eight resistance (R) genes conferring resistance to various types of pests have been cloned from 12 plant species. Irrespective of the host or the pest type, most R genes share a strong protein sequence similarity especially for domains and motifs. The objective of this study was to identify expressed R genes of wheat, the fraction of which is expected to be very low in the genome. Using modified RNA fingerprinting and data mining approaches we identified 220 expressed R-gene candidates. Of these, 125 sequences structurally resembled known R genes. In addition to 25-87% protein sequence similarity with the known R genes, the sequence, order, and distribution of the domains and motifs were also the same. Among the remaining 95, 17 were probable R-related, 21 were a new class of nucleotide-binding kinases, 21 were probable kinases, and 36 were p-loop-containing unknown sequences. About 76% were rare including 73 novel sequences. Three new R-gene specific motifs were also identified. Physical mapping of the 164 best R-gene candidates on 339 deletion lines localized 121 mappable R-gene candidates to 26 small chromosomal regions encompassing about 16% of the genome. About 90 of the 110 phenotypically characterized wheat R genes corresponding to 18 different pests also mapped in these regions.     

Identification and mapping of polymorphic SSR markers from expressed gene sequences of barley and wheat

The growing availability of EST sequences from a range of crop plantsprovides a potentially valuable source of new DNA markers. We have examined theInternational Triticeae EST Cooperative database for the presence ofdinucleotide and trinucleotide simple sequence repeats. Analysis of 24,344 ESTsidentified 388 dinucleotide repeats and 978 trinucleotide repeats in ESTs,representing 1.6% and 4.0% of the total number of ESTs, respectively. To testthe utility and cross-species transferability of EST-derived SSR markers,primers were designed to the flanking regions of 41 barley SSRs and used toscreen 11 barley and 15 wheat varieties. Sixteen of the barley SSR markers werepolymorphic in barley and five were polymorphic in wheat. This represents arelatively high level of transferability of SSR markers between barley andwheat, which has important implications for the development of new markers andcomparative mapping of barley, wheat and other cereals. An additional 56 SSRsfrom wheat ESTs were tested in the same barley and wheat varieties. Four wheatEST SSR markers were polymorphic in wheat and one in barley. Chromosomallocations in barley and wheat were determined for the majority of polymorphicmarkers.     

Updating of transposable element annotations from large wheat genomic sequences reveals diverse activities and gene associations

Triticeae species (including wheat, barley and rye) have huge and complex genomes due to polyploidization and a high content of transposable elements (TEs). TEs are known to play a major role in the structure and evolutionary dynamics of Triticeae genomes. During the last 5 years, substantial stretches of contiguous genomic sequence from various species of Triticeae have been generated, making it necessary to update and standardize TE annotations and nomenclature. In this study we propose standard procedures for these tasks, based on structure, nucleic acid and protein sequence homologies. We report statistical analyses of TE composition and distribution in large blocks of genomic sequences from wheat and barley. Altogether, 3.8 Mb of wheat sequence available in the databases was analyzed or re-analyzed, and compared with 1.3 Mb of re-annotated genomic sequences from barley. The wheat sequences were relatively gene-rich (one gene per 23.9 kb), although wheat gene-derived sequences represented only 7.8% (159 elements) of the total, while the remainder mainly comprised coding sequences found in TEs (54.7%, 751 elements). Class I elements [mainly long terminal repeat (LTR) retrotransposons] accounted for the major proportion of TEs, in terms of sequence length as well as element number (83.6% and 498, respectively). In addition, we show that the gene-rich sequences of wheat genome A seem to have a higher TE content than those of genomes B and D, or of barley gene-rich sequences. Moreover, among the various TE groups, MITEs were most often associated with genes: 43.1% of MITEs fell into this category. Finally, the TRIM and copia elements were shown to be the most active TEs in the wheat genome. The implications of these results for the evolution of diploid and polyploid wheat species are discussed. Electronic Supplementary Material Supplementary material is available for this article at     

Abscisic acid induces the alcohol dehydrogenase gene in Arabidopsis.

Exogenous abscisic acid (ABA) induced the alcohol dehydrogenase gene (Adh) in Arabidopsis roots. Both the G-box-1 element and the GT/GC motifs (anaerobic response element) were required for Adh inducibility. Measurement of endogenous ABA levels during stress treatment showed that ABA levels increased during dehydration treatment but not following exposure to either hypoxia or low temperature. Arabidopsis ABA mutants (aba1 and abi2) displayed reduced Adh mRNA induction levels following either dehydration treatment or exogenous application of ABA. Low-oxygen response was slightly increased in the aba1 mutant but was unchanged in abi2. Low-temperature response was unaffected in both aba1 and abi2 mutants. Our results indicate that, although induction of the Adh gene by ABA, dehydration, and low temperature required the same cis-acting promoter elements, their regulatory pathways were at least partially separated in a combined dehydration/ABA pathway and an ABA-independent low-temperature pathway. These pathways were in turn independent of a third signal transduction pathway leading to low-oxygen response, which did not involve either ABA or the G-box-1 promoter element.     

Mapping of a retinoic acid-responsive element in the promoter region of the complement factor H gene.

Retinoic acid receptors are members of the steroid/thyroid hormone receptor superfamily. Pursuant to the discovery that dexamethasone increases complement factor H expression, we examined the effects of retinoic acid on this gene. Both H mRNA and protein levels are increased by retinoic acid in L cells. Using the luciferase reporter gene system we have identified a region of the H promoter required for the retinoic acid response. This region contains an imperfect palindrome of the TGACC motif, present in thyroid hormone and estrogen-responsive elements. We demonstrate specific binding of the retinoic acid receptor beta to this sequence of the H gene by DNA-protein gel retardation assay. Therefore, these studies extend the sphere of influence of the retinoids to complement, an intrinsic component of the humoral immune system.     

Abscisic acid-induced heat tolerance in Bromus inermis Leyss cell-suspension cultures. Heat-stable, abscisic acid-responsive polypeptides in combination with sucrose confer enhanced thermostability.

Increased heat tolerance is most often associated with the synthesis of heat-shock proteins following pre-exposure to a nonlethal heat treatment. In this study, a bromegrass (Bromus inermis Leyss cv Manchar) cell suspension cultured in a medium containing 75 microM abscisic acid (ABA) without prior heat treatment had a 87% survival rate, as determined by regrowth analysis, following exposure to 42.5 degrees C for 120 min. In contrast, less than 1% of the control cells survived this heat treatment. The heat tolerance provided by treatment with 75 microM ABA was first evidenced after 4 d of culture and reached a maximum tolerance after 11 d of culture. Preincubation with sucrose partially increased the heat tolerance of control cells and rendered ABA-treated cells tolerant to 45 degrees C for 120 min (a completely lethal heat treatment for control cells). Comparative two-dimensional polyacrylamide gel electrophoresis of cellular protein isolated from heat-tolerant cells identified 43 ABA-responsive proteins of which 26 were heat stable (did not coagulate and remained soluble after 30 min at 90 degrees C). Eight heat-stable, ABA-responsive proteins ranging from 23 to 45 kD had similar N-terminal sequences. The ABA-responsive (43-20 kD), but none of the control heat-stable, proteins cross-reacted to varying degrees with a polyclonal antibody directed against a conserved, lysine-rich dehydrin sequence. A group of 20- to 30-kD heat-stable, ABA-responsive proteins cross-reacted with both the anti-dehydrin antibody and an antibody directed against a cold-responsive winter wheat protein (Wcs 120). In ABA-treated cells, there was a positive correlation between heat- and pH-induced coagulation of a cell-free homogenate and the heat tolerance of these cells. At 50 degrees C, control homogenates coagulated after 8 min, whereas cellular fractions from ABA-treated cells showed only marginal coagulation after 15 min. In protection assays, addition of heat-stable, ABA-responsive polypeptides to control fractions reduced the heat-induced coagulation of cell-free homogenates. Sucrose (8%) alone and control, heat-stable fractions enhanced the thermostability of control fractions, but the most protection was conferred by ABA-responsive, heat-stable proteins in combination with sucrose. These data suggest that stress-tolerance mechanisms may develop as a result of cooperative interactions between stress proteins and cell osmolytes, e.g. sucrose. Hypotheses are discussed implicating the role of these proteins and osmolytes in preventing coagulation and denaturation of cellular proteins and membranes.     

Molecular genetic maps of the group 6 chromosomes of hexaploid wheat (Triticum aestivum L. em. Thell.).

Restriction fragment length polymorphism (RFLP) maps of chromosomes 6A, 6B, and 6D of hexaploid wheat (Triticum aestivum L. em. Thell.) have been produced. They were constructed using a population of F7-8 recombinant inbred lines derived from a synthetic wheat x bread wheat cross. The maps consist of 74 markers assigned to map positions at a LOD >= 3 (29 markers assigned to 6A, 24 to 6B, and 21 to 6D) and 2 markers assigned to 6D ordered at a LOD of 2.7. Another 78 markers were assigned to intervals on the maps. The maps of 6A, 6B, and 6D span 178, 132, and 206 cM, respectively. Twenty-one clones detected orthologous loci in two homoeologues and 3 detected an orthologous locus in each chromosome. Orthologous loci are located at intervals of from 1.5 to 26 cM throughout 70% of the length of the linkage maps. Within this portion of the maps, colinearity (homosequentiality) among the three homoeologues is strongly indicated. The remainder of the linkage maps consists of three segments ranging in length from 47 to 60 cM. Colinearity among these chromosomes and other Triticeae homoeologous group 6 chromosomes is indicated and a consensus RFLP map derived from maps of the homoeologous group 6 chromosomes of hexaploid wheat, tetraploid wheat, Triticum tauschii, and barley is presented. Key words : RFLP, wheat, linkage maps, molecular markers.     

Abscisic acid-induced secretory proteins in suspension-cultured cells of winter wheat

In suspension-cultured cells of winter wheat (Triticum aestivum L. cv. Chihokukomugi), the accumulation of soluble secretory proteins in the culture medium was promoted by ABA treatment in comparison with non-treated cells. The total amount of secretory proteins in ABA-treated cells was 1.7-fold higher than that in non-treated cells. The analysis of two-dimensional electrophoresis revealed that at least twelve secretory proteins were induced by ABA, and these were named WAS (wheat ABA-induced secretory) proteins 1 to 12. N-terminal amino acid sequence analysis of WAS proteins revealed the sequences of WAS-2 and WAS-3. Homology searches showed that WAS-2 had 55% identity with the N-terminus of the wheat chemically induced gene (WCI-5 gene) product. WAS-3 was also shown to have 93% identity with the N-terminus of the barley protein R, a typical member of thaumatin-like proteins (TLPs). Immunoblot analysis also suggested that WAS-3 was related to protein R. These results suggest that exogenous ABA induces some basic secretory proteins that are related to the plant defense system in wheat.     

Isolation of resistance gene analogues to powdery mildew resistance sequences in hexaploid wheat

This paper reports the characterization of the powdery mildew resistance homologous genes family of Triticum aestivum. Using degenerate primer pair for wheat resistance genes, we have cloned seven 3′ truncated powdery mildew resistance gene homologous fragments Tpc5a, Tp25a, Tp25b, Tp3a5a, Tp3a5b, Tp4b5a and Tp4b5b. These fragments were sequenced. The deduced amino acid sequences showed that six of them have premature stop codons. All these sequences had a very high level of similarity to known Pm resistance genes such as Pm3a, Pm3b, Pm3d and pm3f in hexaploid wheat. By ignoring the stop codons in the sequences, their deduced protein sequences were of coiled-coil (CC)-nucleotide binding site (NBS)-leucine repeat rich (LRR) structure. These results suggest that there are many powdery mildew resistance gene analogues in both resistant and susceptible wheat. Among them, small insertion/deletion events and point mutations can result in the diversity of wheat Pm resistance homologous genes.