An Apoplastic Defensin of Wheat Elicits the Production of Extracellular Polysaccharides in Snow Mold.

TAD1 (Triticum aestivum defensin 1) is a plant defensin specifically induced by low temperature in winter wheat. In this study, we demonstrated that TAD1 accumulated in the apoplast during cold acclimation and displayed antifungal activity against the pink snow mold fungi Microdochium nivale. When M. nivale was treated with TAD1, Congo red-stainable extracellular polysaccharides (EPS) were produced. The EPS were degradable by cellulase treatment, suggesting the involvement of ß-1,4 glucans. Interestingly, when the fungus was treated with FITC-labeled TAD1, fluorescent signals were observed within the EPS layer. Taken together, these results support the hypothesis that the EPS plays a role as a physical barrier against antimicrobial proteins secreted by plants. We anticipate that the findings from our study will have broad impact and will increase our understanding of plant-snow mold interactions under snow.

A Causal Gene for Seed Dormancy on Wheat Chromosome 4A Encodes a MAP Kinase Kinase.

Seed germination under the appropriate environmental conditions is important both for plant species survival and for successful agriculture. Seed dormancy, which controls germination time, is one of the adaptation mechanisms and domestication traits [1]. Seed dormancy is generally defined as the absence of germination of a viable seed under conditions that are favorable for germination [2]. The seed dormancy of cultivated plants has generally been reduced during domestication [3]. Bread wheat (Triticum aestivum L.) is one of the most widely grown crops in the world. Weak dormancy may be an advantage for the productivity due to uniform emergence and a disadvantage for the risks of pre-harvest sprouting (PHS), which decreases grain quality and yield [4]. A number of quantitative trait loci (QTLs) controlling natural variation of seed dormancy have been identified on various chromosomes [5]. A major QTL for seed dormancy has been consistently detected on chromosome 4A [6-13]. The QTL was designated as a major gene, Phs1, which could be precisely mapped within a 2.6 cM region [14]. Here, we identified a mitogen-activated protein kinase kinase 3 (MKK3) gene (designated TaMKK3-A) by a map-based approach as a candidate gene for the seed dormancy locus Phs1 on chromosome 4A in bread wheat. Complementation analysis showed that transformation of a dormant wheat cultivar with the TaMKK3-A allele from a nondormant cultivar clearly reduced seed dormancy. Cultivars differing in dormancy had a single nonsynonymous amino acid substitution in the kinase domain of the predicted MKK3 protein sequence, which may be associated with the length of seed dormancy.

Mapping of a major locus controlling seed dormancy using backcrossed progenies in wheat (Triticum aestivum L.).

Seed dormancy is an important factor regulating preharvest sprouting (PHS) but is a complex trait for genetic analysis. We previously identified a major quantitative trait locus (QTL) controlling seed dormancy on the long arm of chromosome 4A (4AL) in common wheat. To transfer the QTL from the dormant lines 'OS21-5' and 'Leader' into the Japanese elite variety 'Haruyokoi', which has an insufficient level of seed dormancy, backcrossing was carried out through marker-assisted selection (MAS) using PCR-based codominant markers. Nineteen BC5F2 plants with homozygous alleles of 'OS21-5' or 'Haruyokoi' were developed and evaluated for seed dormancy under greenhouse conditions. The seeds harvested from plants with 'OS21-5' alleles showed a clearly high level of dormancy compared with seeds from plants with 'Haruyokoi' alleles. Additionally, the dormancy phenotype of BC3F3 seeds harvested from 128 BC3F2 plants with homozygous alleles of 'Leader' or 'Haruyokoi' showed a clear difference between these alleles. The QTL on 4AL confers a major gene, Phs1, which was mapped within a 2.6 cM region. The backcrossed lines developed in this study can be important sources for improving PHS resistance in Japanese wheat and for analyzing the mechanism of seed dormancy. MAS was useful for the development of near-isogenic lines in this complex trait, to facilitate the molecular dissection of genetic factors.

Diversification of the barley and wheat blt101/wpi6 promoters by the Xumet element without affecting stress responsiveness.

BLT101-family plasma membrane proteins are found in a wide range of organisms from bacteria to nematodes and are involved in the regulation of cellular cation concentration under stress conditions. A comparison of the promoter regions of barley blt101 and its wheat ortholog, wpi6, revealed highly conserved nucleotide sequences between both genes and a unique insertion of a Xumet element in the blt101 promoter. The Xumet insertion occurred between a putative abscisic acid-responsive element (ABRE) and the dehydration-responsive element/c-repeat (DRE/CRT) within the blt101 promoter. However, blt101 and wpi6 were induced similarly in response to ABA, drought and low temperature, suggesting that the insertion does not affect promoter functions. The Xumet insertion in the blt101/wpi6 promoter region was detected in five barley cultivars, but absent in two wheat cultivars tested, suggesting that the insertion is barley-specific. Genomic Southern blot analysis revealed a large number of Xumet sequences interspersed in the barley genome, whereas only one or very few copies are present in the wheat genome. The data suggested that an expansion in copy number of Xumet elements occurred in the barley genome through evolution.

Isolation and characterization of SSR sequences from the genome and TAC clones of common wheat using the PCR technique.

We have developed the 2-step PCR method, a kind of suppression PCR procedure, to isolate simple sequence repeats (SSRs) from common wheat (Triticum aestivum L.) in a more convenient manner. This system requires neither genomic library screening nor the SSR-enrichment procedure. As a result, we designed 131 primer pairs based on isolated SSRs from not only genomic DNA, but also transformation-competent artificial chromosome (TAC) clones. It has been demonstrated that 34 of the 131 SSR markers developed were polymorphic among 8 wheat lines. Four of 34 polymorphic SSR markers were derived from TAC clones, indicating that this method could be applied to the targeted development of unique SSR markers in large genomic DNA libraries such as those composed of bacterial artificial chromosomes (BACs). A considerable number of isolated SSR clones had similarities with part of several long terminal repeats of retrotransposons (LTR-RTs) identified in various Triticeae genome sequences. Most of those SSRs showed smear amplification profiles, suggesting that a considerable number of dysfunctional SSRs originating from repetitive DNA components, especially LTR-RTs, might exist in the common wheat genome.

SSR-based linkage map with new markers using an intraspecific population of common wheat.

Simple sequence repeats (SSRs) are valuable molecular markers in many plant species. In common wheat (Triticum aestivum L.), which is characteristic of its large genomes and alloploidy, SSRs are one of the most useful markers. To increase SSR marker sources and construct an SSR-based linkage map of appropriate density, we tried to develop new SSR markers from SSR-enriched genomic libraries and the public database. SSRs having (GA)n and (GT)n motifs were isolated from enriched libraries, and di- and tri-nucleotide repeats were mined from expressed sequence tags (ESTs) and DNA sequences of Triticum species in the public database. Of the 1,147 primer pairs designed, 842 primers gave accurate amplification products, and 478 primers showed polymorphism among the nine wheat lines examined. Using a doubled haploid (DH) population from an intraspecific cross between Kitamoe and Münstertaler (KM), we constructed an SSR-based linkage map that consisted of 464 loci: 185 loci from genomic libraries, 65 loci from the sequence database including ESTs, 213 loci from the SSR markers already reported, and 1 locus of morphological marker. Although newly developed SSR loci were distributed throughout all chromosomes, clustering of them around putative centromeric regions was found on several chromosomes. The total length of the KM map spanned 3,441 cM and corresponded to approximately 86% genome coverage. The KM map comprised of 23 linkage groups because two gaps of over 50 cM distance remained on chromosome 6A. This is a first report of SSR-based linkage map using single intraspecific population of common wheat. This mapping result suggests that it becomes possible to construct linkage maps with sufficient genome coverage using only SSR markers without RFLP markers, even in an intraspecific population of common wheat. Moreover, the new SSR markers will contribute to the enrichment of molecular marker resources in common wheat.

Molecular characterization of a cold-induced plasma membrane protein gene from wheat.

As a means to study the function of plasma membrane proteins during cold acclimation, we have isolated a cDNA clone for wpi6 which encodes a putative plasma membrane protein from cold-acclimated winter wheat. The wpi6 gene encodes a putative 5.9 kDa polypeptide with two predicted membrane-spanning domains, the sequence of which shows high sequence similarity with BLT101-family proteins from plants and yeast. Strong induction of wpi6 mRNA was observed during an early stage of cold acclimation in root and shoot tissues of both winter and spring wheat cultivars. In contrast to blt101 in barley, wpi6 mRNA was also induced by drought and salinity stresses, and exogenous application of ABA. Expression of wpi6 in a Deltapmp3 mutant of Saccharomyces cerevisiae, which is disturbed in plasma membrane potential due to the lack of a BLT101-family protein, partially complemented NaCl sensitivity of the mutant. Transient expression analysis of a WPI6::GFP fusion protein in onion epidermal cells revealed that WPI6 is localized in the plasma membrane. Taken together, these data suggested that WPI6 may have a protective role in maintaining plasma membrane function during cold acclimation in wheat.

A novel plant defensin-like gene of winter wheat is specifically induced during cold acclimation.

A novel cDNA clone, Tad1, was isolated from crown tissue of winter wheat after differential screening of cold acclimation-induced genes. The Tad1 cDNA encoded a 23kDa polypeptide with a potential N-terminal signal sequence. The putative mature sequence showed striking similarity to plant defensins or gamma-thionins, representing low molecular size antipathogenic polypeptides. High levels of Tad1 mRNA accumulation occurred within one day of cold acclimation in crown tissue and the level was maintained throughout 14 days of cold acclimation. Similar rapid induction was observed in young seedlings treated with low temperature but not with exogenous abscisic acid. In contrast to defensins from other plant species, neither salicylic acid nor methyl jasmonate induced expression of Tad1. The recombinant mature form of TAD1 polypeptide inhibited the growth of the phytopathogenic bacteria, Pseudomonas cichorii; however, no antifreeze activity was detected. Collectively, these data suggested that Tad1 is induced in cold-acclimated winter wheat independent of major defense signaling(s) and is involved in low temperature-induced resistance to pathogens during winter hardening.