Fumitaka Abe

A Wheat Homolog of MOTHER OF FT AND TFL1 Acts in the Regulation of Germination

Among the environmental signals affecting seed development, temperature is the most influential in the formation of seed dormancy in wheat. In this study, transcriptional profiling of the effects of temperature on seed dormancy formation identified MFT as a candidate gene for seed dormancy regulation. Seed dormancy is an adaptive mechanism and an important agronomic trait. Temperature during seed development strongly affects seed dormancy in wheat (Triticum aestivum) with lower temperatures producing higher levels of seed dormancy. To identify genes important for seed dormancy, we used a wheat microarray to analyze gene expression in embryos from mature seeds grown at lower and higher temperatures. We found that a wheat homolog of MOTHER OF FT AND TFL1 (MFT) was upregulated after physiological maturity in dormant seeds grown at the lower temperature. In situ hybridization analysis indicated that MFT was exclusively expressed in the scutellum and coleorhiza. Mapping analysis showed that MFT on chromosome 3A (MFT-3A) colocalized with the seed dormancy quantitative trait locus (QTL) QPhs.ocs-3A.1. MFT-3A expression levels in a dormant cultivar used for the detection of the QTL were higher after physiological maturity; this increased expression correlated with a single nucleotide polymorphism in the promoter region. In a complementation analysis, high levels of MFT expression were correlated with a low germination index in T1 seeds. Furthermore, precocious germination of isolated immature embryos was suppressed by transient introduction of MFT driven by the maize (Zea mays) ubiquitin promoter. Taken together, these results suggest that MFT plays an important role in the regulation of germination in wheat.

Ethylene and reactive oxygen species are involved in root aerenchyma formation and adaptation of wheat seedlings to oxygen-deficient conditions

Ethylene-mediated reactive oxygen species signalling is involved in adaptive responses of wheat seedlings to waterlogged conditions through controlling formation of lysigenous aerenchyma and expression of genes encoding ethanol fermentation enzymes in roots

Ectopic expression of wheat and barley DOG1-like genes promotes seed dormancy in Arabidopsis.

To develop strategies for manipulating the level of crop seed dormancy, it is necessary to search for the genes that control dormancy. In this study, we investigated whether wheat and barley homologues of the Arabidopsis dormancy gene DOG1 (Delay of Germination 1), TaDOG1L1 and HvDOG1L1 (respectively), also induce seed dormancy. Because their sequence similarity to DOG1 is low and the tissue-specific expression pattern of DOG1 was not conserved in either of these genes, these genes do not appear to retain the function of DOG1. However, ectopic overexpression of either of these DOG1 homologues in transgenic Arabidopsis markedly increased seed dormancy. Furthermore, dormancy release during dry seed storage in the transgenic Arabidopsis overexpressing the Triticeae genes occurred similarly to that in a transgenic line overexpressing DOG1. This evidence demonstrates conservation of the function of DOG1 in both TaDOG1L1 and HvDOG1L1. Thus, these DOG1-like genes in wheat and barley are good candidate transgenes for reducing pre-harvest germination in wheat.

Characterization of the rice blast resistance gene Pik cloned from Kanto51

To study similar, but distinct, plant disease resistance (R) specificities exhibited by allelic genes at the rice blast resistance locus Pik/Pikm, we cloned the Pik gene from rice cultivar Kanto51 and compared its molecular features with those of Pikm and of another Pik gene cloned from cv. Kusabue. Like Pikm, Pik is composed of two adjacent NBS-LRR (nucleotide-binding site, leucine-rich repeat) genes: the first gene, Pik1-KA, and the second gene, Pik2-KA. Pik from Kanto51 and Pik from Kusabue were not identical; although the predicted protein sequences of the second genes were identical, the sequences differed by three amino acids within the NBS domain of the first genes. The Pik proteins from Kanto51 and Kusabue differed from Pikm in eight and seven amino acids, respectively. Most of these substituted amino acids were within the coiled-coil (CC) and NBS domains encoded by the first gene. Of these substitutions, all within the CC domain were conserved between the two Pik proteins, whereas all within the NBS domain differed between them. Comparison of the two Pik proteins and Pikm suggests the importance of the CC domain in determining the resistance specificities of Pik and Pikm. This feature contrasts with that of most allelic or homologous NBS-LRR genes characterized to date, in which the major specificity determinant is believed to lie in the highly diverged LRR domain. In addition, our study revealed high evolutionary flexibility in the genome at the Pik locus, which may be relevant to the generation of new R specificities at this locus.

Mapping a diploid wheat abscisic acid 8′-hydroxylase homologue in the seed dormancy QTL region on chromosome 5Am

In Arabidopsis, two genes of abscisic acid (ABA) 8′-hydroxylase (cytochrome P450 (CYP) 707A1 and A2) have been found to play important roles in seed dormancy through the regulation of ABA content in seeds. In order to examine the role of wheat ABA 8′-hydroxylase gene in seed dormancy, a diploid wheat ABA 8′-hydroxylase gene was cloned that showed high similarity to a barley ABA8′-hydroxylase gene (HvABA8′OH-2), and the cloned gene was designated as TmABA8′OH-2. Using recombinant inbred lines derived from a cross between diploid wheat Triticum boeoticum L. (Tb) and Triticum monococcum L. (Tm), TmABA8′OH-2 has been mapped to near the centromeric region of the long arm of chromosome 5Am, where the major seed dormancy QTL has been previously found. Comparison of the deduced amino acid sequences of TmABA8′OH-2 between Tb and Tm revealed five amino acid residue substitutions. These amino acid residues have distinctly different characteristics, and one of the substitutions occurs in the highly conserved amino acid residues in CYP707A family, indicating that these substitutions may have effects on the enzyme activities. Moreover, hexaploid wheat TmABA8′OH-2 homologue revealed that the level of its expression during seed development peaks at mid-maturation stage. This resembles the expression pattern of the ArabidopsisCYP707A1, which was shown to control seed dormancy. These results imply a possibility that TmABA8′OH-2 might be involved in seed dormancy, and associated with the QTL on chromosome 5Am.

A transgenic approach to controlling wheat seed dormancy level by using Triticeae DOG1-like genes

Seed dormancy is an important agronomic trait: low levels can cause premature germination, while too much can inhibit uniform germination. As an approach to controlling the seed dormancy level in crops, we used Triticeae DOG1-like genes as transgenes. DOG1 is an Arabidopsis gene that underlies natural variation in seed dormancy. We previously showed that although their sequence similarities to DOG1 were low, some cereal DOG1-like genes enhanced seed dormancy in Arabidopsis. Here, we introduced two DOG1-like genes, TaDOG1L4 from wheat and HvDOG1L1 from barley, individually into the wheat cultivar Fielder. Their overexpression under the control of a maize ubiquitin promoter enhanced the seed dormancy level while leaving other traits unchanged. TaDOG1L4 was more effective than HvDOG1L1, which accords with the previously revealed difference in the effectiveness of these two genes in Arabidopsis seed dormancy. Knockdown of endogenous TaDOG1L4 in Fielder using double-strand RNA interference decreased the seed dormancy level by several tens of percent. This result indicates that some degree of seed dormancy inherent in wheat is imparted by DOG1-like genes.

Adventitious roots of wheat seedlings that emerge in oxygen-deficient conditions have increased root diameters with highly developed lysigenous aerenchyma

Exposing roots of plants to hypoxic conditions is known to greatly improve their anoxic stress tolerance. We previously showed that pre-treatment of wheat seedlings with an ethylene precursor, 1-aminocyclopropanecarboxylic acid (ACC), enhanced their tolerance of oxygen-deficient conditions. Although ACC-pretreated seminal roots of wheat seedlings grown under oxygen-deficient conditions avoided root tip death, they elongated very little. In the present study, we assessed the effects of ethylene on the responses of adventitious roots of wheat seedlings to oxygen-deficient conditions. Lysigenous aerenchyma formation in the adventitious roots of wheat seedlings pretreated with ACC appeared to reduce tip death under oxygen-deficient conditions, but the adventitious roots, like the seminal roots, hardly elongated. We also found that adventitious roots that emerge in oxygen-deficient conditions continued to elongate even under such conditions. The adventitious roots emerged in oxygen-deficient conditions were found to have thicker root diameters than those emerged in aerated conditions. These results suggest that the adventitious roots with thicker root diameters can better cope with oxygen-deficient conditions. Measurements of the area of the lysigenous aerenchyma confirmed that the increased root diameters have a greater amount of air space generated by lysigenous aerenchyma formation.

Characterization of a wheat pathogenesis-related protein, TaBWPR-1.2, in seminal roots in response to waterlogging stress.

We examined the role of pathogenesis-related protein TaBWPR-1.2 in the context of molecular and physiological responses of wheat (Triticum aestivum) seminal roots under waterlogging stress. Two cDNAs corresponding to the TaBWPR-1.2 gene, TaBWPR-1.2#2 and TaBWPR-1.2#13 were cloned from seminal roots. These cDNAs were predicted to encode proteins of 173 and 172 amino acids, respectively. In a time-course experiment, TaBWPR-1.2 gene expression was highest in whole seminal roots after 1 day of waterlogging treatment and higher than the control for at least 10 days; significantly increased protein abundance was observed after 7 days of waterlogging. Drought, another abiotic stress, did not influence TaBWPR-1.2 gene expression in wheat seminal roots at 5-d-old seedlings. Tissue-specific studies revealed that the highest TaBWPR-1.2 gene expression and protein levels were in the aerenchymatous root zone. TaBWPR-1.2 expression in seminal roots was also increased by the signalling molecules 1-aminocyclopropane-1-carboxylic acid (ACC; an ethylene precursor), H2O2, jasmonic acid (JA), and nitric oxide (NO); however, treatment with abscisic acid (ABA), salicylic acid (SA), and ethanol did not alter its expression. Interestingly, aerenchyma formation in the seminal root cortex was induced only by ACC and H2O2. Taken together, these results indicate that TaBWPR-1.2 is a waterlogging-responsive gene that might be associated with root cortex tissue alteration in wheat plants through ACC and/or H2O2 regulatory mechanisms.

DOG1-like genes in cereals: investigation of their function by means of ectopic expression in Arabidopsis.

The Arabidopsis gene DOG1 (AtDOG1) functions in seed dormancy and in sugar signaling. Little is known about the structural and functional features of plant genes homologous to AtDOG1, except for one type (clade 1) of Triticeae AtDOG1-like genes, which was previously demonstrated to be functionally orthologous to AtDOG1. Here, through phylogenetic, structural, and functional analyses of cereal AtDOG1-like genes, we characterized their features: these genes exist as a gene family that can be classified into five distinct clades (1-5). Of these, AtDOG1-like genes in clades 1-4 have a similar architecture to AtDOG1: they encode proteins with three conserved regions. In contrast, the clade 5 genes are distinct; their encoded proteins lack these conserved regions, but harbor domains that interact with DNA. Ectopic expression of the cereal AtDOG1-like genes of clades 2-4 in Arabidopsis demonstrated that like the clade 1 genes, they performed the same function as AtDOG1. The correlation between the depth of seed dormancy and the efficiency of sugar signaling in transgenic Arabidopsis conferred by genes in clades 1-4 suggests a close link in the underlying mechanisms between the seed dormancy and sugar signaling functions of AtDOG1.

The cold-induced defensin TAD1 confers resistance against snow mold and Fusarium head blight in transgenic wheat.

TAD1 (Triticum aestivum defensin 1) is induced during cold acclimation in winter wheat and encodes a plant defensin with antimicrobial activity. In this study, we demonstrated that recombinant TAD1 protein inhibits hyphal growth of the snow mold fungus, Typhula ishikariensis in vitro. Transgenic wheat plants overexpressing TAD1 were created and tested for resistance against T. ishikariensis. Leaf inoculation assays revealed that overexpression of TAD1 confers resistance against the snow mold. In addition, the TAD1-overexpressors showed resistance against Fusarium graminearum, which causes Fusarium head blight, a devastating disease in wheat and barley. These results indicate that TAD1 is a candidate gene to improve resistance against multiple fungal diseases in cereal crops.