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Hybrid necrosis and hybrid chlorosis are sometimes observed in interspecific hybrids between the tetraploid wheat cultivar Langdon and diploid wild wheat Aegilops tauschii. Many WRKY transcription factor genes are dramatically upregulated in necrosis and chlorosis wheat hybrids. Here, we isolated cDNA clones for four wheat WRKY transcription factor genes, TaWRKY49, TaWRKY92, TaWRKY112, and TaWRKY142, that were commonly upregulated in the hybrid necrosis and hybrid chlorosis and belonged to the same clade of the WRKY gene family. Expression patterns of the four TaWRKY genes in response to several stress conditions were similar in wheat seeding leaves. The four TaWRKY-GFP fusion proteins were targeted to the nucleus in onion epidermal cells. The TaWRKY gene expression levels were increased by high salt, dehydration, darkness, and blast fungus treatment in common wheat. Expression of either of the TaWRKY genes increased salinity and osmotic stress tolerance accompanied with overexpression of STZ/Zat10, and induced overexpression of the salicylic acid-signal pathway marker gene AtPR1 in transgenic Arabidopsis. TaWRKY142 expression also induced the jasmonic acid-pathway marker gene AtPDF1.2 and enhanced resistance against the fungal pathogen Colletotrichum higginsianum in transgenic Arabidopsis. These results suggest that the four TaWRKY genes act as integrated hubs of multiple stress signaling pathways in wheat and play important roles in autoimmune response-inducing hybrid necrosis and hybrid chlorosis.

Wheat and its relatives include some allopolyploid species such as Aegilops juvenalis (Thell.) Eig (DDMMUU) and Aegilops vavilovii (Zhuk.) Chenn. (DDMMSS). Here, we successfully produced for the first time three allodecaploid lines with the AABBDDMMUU and AABBDDMMSS genomes through interspecific crosses between a tetraploid wheat cultivar and the two Aegilops species. Spike architecture of the synthetic lines showing a brittle rachis phenotype strongly resembled that of the parental hexaploid Aegilops species, but not that of the parental tetraploid wheat. Phenotypic differences of the spike and spikelet morphology were caused by one of the parental Aegilops species. Glumes of the synthetic decaploids were extremely hard, and all three synthetic lines had a soft texture with a smooth starch surface in endosperm cells. The soft-textured grains of the synthetic decaploids were due to accumulation of the puroindoline proteins derived from the genomes of parental allohexaploid Aegilops species. Therefore, the nascent synthetic decaploid lines highly reflect the phenotypic characteristics of the pollen parents. With further wheat breeding, the synthetic decaploids could have great potential to improve various traits.

Cuticular wax on the aerial surface of plants has a protective function against many environmental stresses. The bluish-whitish appearance of wheat leaves and stems is called glaucousness. Most modern cultivars of polyploid wheat species exhibit the glaucous phenotype, while in a wild wheat progenitor, Ae. tauschii, both glaucous and non-glaucous accessions exist. Iw2, a wax inhibitor locus on the short arm of chromosome 2D, is the main contributor to this phenotypic variation in Ae. tauschii, and the glaucous/non-glaucous phenotype of Ae. tauschii is usually inherited by synthetic hexaploid wheat. However, a few synthetic lines show the glaucous phenotype although the parental Ae. tauschii accessions are non-glaucous. Molecular marker genotypes indicate that the exceptional non-glaucous Ae. tauschii accessions share the same genotype in the Iw2 chromosomal region as glaucous accessions, suggesting that these accessions have a different causal locus for their phenotype. This locus was assigned to the long arm of chromosome 3D using an F2 mapping population and designated W4, a novel glaucous locus in Ae. tauschii. The dominant W4 allele confers glaucousness, consistent with phenotypic observation of Ae. tauschii accessions and the derived synthetic lines. These results implied that glaucous accessions of Ae. tauschii with the W2W2iw2iw2W4W4 genotype could have been the D-genome donor of common wheat.

Aegilops tauschii, a wild wheat relative, is the D-genome donor of common wheat. Subspecies and varieties of Ae. tauschii are traditionally classified based on differences in their inflorescence architecture. However, the genetic information for their diversification has been quite limited in the wild wheat relatives. The variety anathera has no awn on the lemma, but the genetic basis for this diagnostic character is unknown. Wide variations in awn length traits at the top and middle spikes were found in the Ae. tauschii core collection, and the awn length at the middle spike was significantly smaller in the eastward-dispersed sublineage than in those in other sublineages. To clarify loci controlling the awnless phenotype of var. anathera, we measured awn length of an intervariety F2 mapping population, and found that the F2 individuals could be divided into two groups mainly based on the awn length at the middle of spike, namely short and long awn groups, significantly fitting a 3:1 segregation ratio, which indicated that a single locus controls the awnless phenotype. The awnless locus, Anathera (Antr), was assigned to the distal region of the short arm of chromosome 5D. Quantitative trait locus analysis using the awn length data of each F2 individual showed that only one major locus was at the same chromosomal position as Antr. These results suggest that a single dominant allele determines the awnless diagnostic character in the variety anathera. The Antr dominant allele is a novel gene inhibiting awn elongation in wheat and its relatives.

Alternative splicing produces multiple transcripts from a single gene, and RNA sequencing using next-generation sequencing apparatus has contributed to comprehensive detection of splicing variants in higher plants. Here, we completed a large-scale survey of alternatively spliced transcripts from RNA samples derived from seedling leaves and young spikes of two distinct accessions of wild diploid wheat Aegilops tauschii under normal growth conditions. Alternative splicing for 23,778 loci were analyzed in this study, and at least two splicing variants were found in 4712 genes. The proportion of events for intron retention was much smaller in Ae. tauschii than in other plant species, whereas the proportions of exon skipping and splice donor site events were higher. In at least some genes, alternative splicing patterns were clearly distinct between the two Ae. tauschii accessions. Distinct splicing patterns were transmitted from the parental Ae. tauschii accessions into their synthetic hexaploid wheat lines. Alternative splicing patterns in one of the two synthetic hexaploid wheat lines analyzed appeared to be generally similar to patterns intermediate between the parental lines under normal growth conditions, and seemed to be slightly altered in the other synthetic wheat.

Hybrid necrosis has been observed in many interspecific hybrids from crosses between tetraploid wheat and the wheat D-genome donor Aegilops tauschii. Type II necrosis is a kind of hybrid incompatibility that is specifically characterized by low-temperature induction and growth suppression. Two complementary genes, Net1 on the AB genome and Net2 on the D genome, putatively control type II necrosis in ABD triploids and synthetic hexaploid wheat. Toward map-based cloning of Net2, a fine map around the Net2 region on 2DS was constructed in this study. Using the draft genome sequence of Ae. tauschii and the physical map of the barley genome, the Net2 locus was mapped within a 0.6 cM interval between two closely linked markers. Although local chromosomal rearrangements were observed in the Net2-corresponding region between the barley/Brachypodium and Ae. tauschii genomes, the two closely linked markers were significantly associated with type II necrosis in Ae. tauschii. These results suggest that these markers will aid efficient selection of Net2 non-carrier individuals from the Ae. tauschii population and intraspecific progeny, and could help with introgression of agriculturally important genes from Ae. tauschii to common wheat.

Photosystem I (PSI) photoinhibition suppresses plant photosynthesis and growth. However, the mechanism underlying PSI photoinhibition has not been fully clarified. In this study, in order to investigate the mechanism of PSI photoinhibition in higher plants, we applied repetitive short-pulse (rSP) illumination, which causes PSI-specific photoinhibition in chloroplasts isolated from spinach leaves. We found that rSP treatment caused PSI photoinhibition, but not PSII photoinhibition in isolated chloroplasts in the presence of O2. However, chloroplastic superoxide dismutase and ascorbate peroxidase activities failed to protect PSI from its photoinhibition. Importantly, PSI photoinhibition was largely alleviated in the presence of methyl viologen, which stimulates the production of reactive oxygen species (ROS) at the stromal region by accepting electrons from PSI, even under the conditions where CuZn-superoxide dismutase and ascorbate peroxidase activities were inactivated by KCN. These results suggest that the ROS production site, but not the ROS production rate, is critical for PSI photoinhibition. Furthermore, we found that not only superoxide (O2−) but also singlet oxygen (1O2) is involved in PSI photoinhibition induced by rSP treatment. From these results, we suggest that PSI photoinhibition is caused by both O2− and 1O2 produced within the thylakoid membranes when electron carriers in PSI become highly reduced. Here, we show, to our knowledge, new insight into the PSI photoinhibition in higher plants.

Aegilops umbellulata is a wild diploid wheat species with the UU genome that is an important genetic resource for wheat breeding. To exploit new synthetic allohexaploid lines available as bridges for wheat breeding, a total of 26 synthetic hexaploid lines were generated through crossing between the durum wheat cultivar Langdon and 26 accessions of Ae. umbellulata. In nascent synthetic hexaploids with the AABBUU genome, the presence of the set of seven U-genome chromosomes was confirmed with U-genome chromosome-specific markers developed based on RNA-seq-derived data from Ae. umbellulata. The AABBUU synthetic hexaploids showed large variations in flowering- and morphology-related traits, and these large variations transmitted well from the parental Ae. umbellulata accessions. However, the variation ranges in most traits examined were reduced under the AABBUU hexaploid background compared with under the diploid parents. The AABBUU and AABBDD synthetic hexaploids were clearly discriminated by several morphological traits, and an increase of plant height and in the number of spikes and a decrease of spike length were commonly observed in the AABBUU synthetics. Thus, interspecific differences in several morphological traits between Ae. umbellulata and A. tauschii largely affected the basic plant architecture of the synthetic hexaploids. In conclusion, the AABBUU synthetic hexaploid lines produced in the present study are useful resources for the introgression of desirable genes from Ae. umbellulata to common wheat.

Background Aegilops umbellulata Zhuk. (2n = 14), a wild diploid wheat relative, has been the source of trait improvement in wheat breeding. Intraspecific genetic variation of Ae. umbellulata, however, has not been well studied and the genomic information in this species is limited. Results To develop novel genetic markers distributed over all chromosomes of Ae. umbellulata and to evaluate its genetic diversity, we performed RNA sequencing of 12 representative accessions and reconstructed transcripts by de novo assembly of reads for each accession. A large number of single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) were obtained and anchored to the pseudomolecules of Ae. tauschii and barley (Hordeum vulgare L.), which were regarded as virtual chromosomes of Ae. umbellulata. Interestingly, genetic diversity in Ae. umbellulata was higher than in Ae. tauschii, despite the narrow habitat of Ae. umbellulata. Comparative analyses of nucleotide polymorphisms between Ae. umbellulata and Ae. tauschii revealed no clear lineage differentiation and existence of alleles with rarer frequencies predominantly in Ae. umbellulata, with patterns clearly distinct from those in Ae. tauschii. Conclusions The anchored SNPs, covering all chromosomes, provide sufficient genetic markers between Ae. umbellulata accessions. The alleles with rarer frequencies might be the main source of the high genetic diversity in Ae. umbellulata. Electronic supplementary material The online version of this article (10.1186/s12870-018-1498-8) contains supplementary material, which is available to authorized users.

Blast, caused by Pyricularia oryzae, is one of the major diseases of wheat in South America. We identified a new gene for resistance to Triticum isolates of P. oryzae in common wheat ‘S-615’, and designated it “resistance to Magnaporthe grisea 8” (Rmg8). Rmg8 was assigned to chromosome 2B through molecular mapping with simple-sequence repeat markers. To identify an avirulence gene corresponding to Rmg8, Triticum isolate Br48 (avirulent on S-615) was crossed with 200R29 (virulent on S-615), an F1 progeny derived from a cross between an Eleusine isolate (MZ5-1-6) and Br48. Segregation analysis of their progeny revealed that avirulence of Br48 on S-615 was conditioned by a single gene, which was designated AVR-Rmg8. AVR-Rmg8 was closely linked to AVR-Rmg7, which corresponded to Rmg7 located on chromosome 2A of tetraploid wheat.

Hybrid necrosis is a well-known reproductive isolation mechanism in plant species, and an autoimmune response is generally considered to trigger hybrid necrosis through epistatic interaction between disease resistance-related genes in hybrids. In common wheat, the complementary Ne1 and Ne2 genes control hybrid necrosis, defined as type I necrosis. Two other types of hybrid necrosis (type II and type III) have been observed in interspecific hybrids between tetraploid wheat and Aegilops tauschii. Another type of hybrid necrosis, defined here as type IV necrosis, has been reported in F1 hybrids between Triticum urartu and some accessions of Triticum monococcum ssp. aegilopoides. In types I, III and IV, cell death occurs gradually starting in older tissues, whereas type II necrosis symptoms occur only under low temperature. To compare comprehensive gene expression patterns of hybrids showing growth abnormalities, transcriptome analysis of type I and type IV necrosis was performed using a wheat 38k oligo-DNA microarray. Defense-related genes including many WRKY transcription factor genes were dramatically up-regulated in plants showing type I and type IV necrosis, similarly to other known hybrid abnormalities, suggesting an association with an autoimmune response. Reactive oxygen species generation and necrotic cell death were effectively inhibited by ZnCl2 treatment in types I, III and IV necrosis, suggesting a significant association of Ca(2+) influx in upstream signaling of necrotic cell death in wheat hybrid necrosis.

A cold-responsive wheat gene, WLT10, encodes a member of the cereal-specific low temperature-responsive/cold-responsive protein family, which contains a hydrophobic N-terminal 20 amino acid sequence that corresponds to signal peptides associated with extracellular trafficking. To verify the subcellular localization of WLT10 and the function of its putative signal peptide, we constructed three chimeric genes in which either the WLT10 signal peptide, a signal peptide with only 6 additional amino acids, or the full-length WLT10 polypeptide was fused to the N-terminus of green fluorescent protein (GFP). These fusion constructs were transiently introduced into onion epidermal cells by particle bombardment. GFP signals were observed not only in the extracellular space (ECS) but also in the endoplasmic reticulum (ER) and Golgi apparatus. The time course of GFP signal localization suggests the movement of WLT10 through the ER/Golgi pathway and into the ECS. Thus, WLT10 is a cold-responsive secreted protein, and its N-terminal 20 amino acid region is important for transport to the ECS.

The phytohormone abscisic acid (ABA) plays important roles in response to environmental stress as well as in seed maturation and dormancy. In common wheat, quantitative trait loci (QTLs) for ABA responsiveness at the seedling stage have been reported on chromosomes 1B, 2A, 3A, 6D and 7B. In this study, we identified a novel QTL for ABA responsiveness on chromosome 5A using an F2 population derived from a cross between the common wheat cultivar Chinese Spring (CS) and a chromosome substitution line of CS with chromosome 5A of cultivar Hope (Hope5A). This QTL was found in a similar chromosomal region to previously reported QTLs for drought tolerance and seed dormancy. Physiological characterization of the QTL revealed a small effect on dehydration tolerance and seed dormancy. The rate of water loss from leaves during dehydration was lower, and transcript accumulation of the cold responsive (COR)/late embryogenesis abundant (LEA) genes Wrab18 and Wdhn13 tended to be higher under dehydration stress in F2 individuals carrying the Hope allele of the QTL, which also showed higher ABA responsiveness than the CS allele-carrying individuals. Seed dormancy of individuals carrying the Hope allele also tended to be lower than those carrying the CS allele. Our results suggest that variation in ABA responsiveness among common wheat cultivars is at least partly determined by the 5A QTL, and that this QTL contributes to development of dehydration and preharvest sprouting tolerance.

Light is an important factor for determining photosynthetic performance in land plants. At high light intensity, land plants develop photosynthetic activity by increasing electron sinks, such as the Calvin cycle and photorespiration and photoprotective mechanisms in photosystem II (PSII), to effectively utilize light and protect them from photoinhibition. In addition to PSII, photosystem I (PSI) has a risk of undergoing photoinhibition under high light intensity because of the reactive oxygen species (ROS) produced within PSI. However, the acclimation response has hardly been evaluated in the relationship of PSI photoprotection to growth light. In this study, we studied the effect of growth light intensity on the photoprotective mechanisms in PSI using six wheat cultivars. To evaluate the susceptibility of PSI to its photoinhibition, we used the repetitive short-pulse (rSP) illumination method to cause O2-dependent PSI photoinhibition. We found that PSI photoinhibition induced by rSP illumination was much more alleviated in wheat cultivars grown under high-light conditions compared to those grown under low-light conditions. Here, we observed that wheat plant grown under high-light conditions lowered the susceptibility of PSI to its photoinhibition compared to those grown under low-light conditions. Furthermore, the acclimation response toward PSI photoinhibition was significantly different among the studied wheat cultivars, although the quantum yields both of PSII and PSI were increased by high-light acclimation in all wheat cultivars as reported previously. Interestingly, we observed that total chlorophyll content in leaves correlated with the susceptibility of PSI to its photoinhibition. On the basis of these results, we suggest that high-light acclimation induces protection mechanisms against PSI photoinhibition in land plants, and the increase in the leaf chlorophyll content relates to the susceptibility of PSI photoinhibition in wheat plants.

Synthetic hexaploid wheat is an effective genetic resource for transferring agronomically important genes from Aegilops tauschii to common wheat. Wide variation in grain size and shape, one of the main targets for wheat breeding, has been observed among Ae. tauschii accessions. To identify the quantitative trait loci (QTLs) responsible for grain size and shape variation in the wheat D genome under a hexaploid genetic background, six parameters related to grain size and shape were measured using SmartGrain digital image software and QTL analysis was conducted using four F2 mapping populations of wheat synthetic hexaploids. In total, 18 QTLs for the six parameters were found on five of the seven D-genome chromosomes. The identified QTLs significantly contributed to the variation in grain size and shape among the synthetic wheat lines, implying that the D-genome QTLs might be at least partly functional in hexaploid wheat. Thus, synthetic wheat lines with diverse D genomes from Ae. tauschii are useful resources for the identification of agronomically important loci that function in hexaploid wheat.

Wheat grain size and shape are associated not only with yield but also with product and milling quality. A subspecies of cultivated tetraploid wheat, Triticum turgidum ssp. polonicum, is characterized by elongated glumes. To elucidate morphological effects of the subspecies differentiation-related gene, we conducted QTL analysis for grain and spikelet shape using a mapping population between two tetraploid wheat subspecies, polonicum and durum. P1, the gene controlling the elongated glumes, was located on chromosome 7A, and the polonicum-type allele acted in an incomplete dominance manner to express the elongated glume phenotype. The polonicum allele of the P1 locus significantly affected not only glume length but also grain shape, spike shape, awn length and seed fertility in tetraploid wheat. The elongated glume phenotype was correlated with an increase in spike length, grain length and grain weight, and with a decrease in fertility, grain number and awn length. Thus, the subspecies differentiation-related gene in subspecies polonicum dramatically affects grain shape accompanied by alteration of spikelet shape in tetraploid wheat.

The gene pool of Aegilops tauschii, the D-genome donor of common wheat (Triticum aestivum L.), can be easily accessed in wheat breeding, but remains largely unexplored. In our previous studies, many synthetic hexaploid wheat lines were produced through interspecific crosses between the tetraploid wheat cultivar Langdon and various A. tauschii accessions. The synthetic hexaploid wheat lines showed wide variation in many characteristics. To elucidate the genetic basis of variation in flowering-related traits, we analyzed quantitative trait loci (QTL) affecting time to heading, flowering and maturity, and the grain-filling period using four different F2 populations of synthetic hexaploid wheat lines. In total, 10 QTLs located on six D-genome chromosomes (all except 4D) were detected for the analyzed traits. The QTL on 1DL controlling heading time appeared to correspond to a flowering time QTL, previously considered to be an ortholog of Eps-A m 1 which is related to the narrow-sense earliness in einkorn wheat. The 5D QTL for heading time might be a novel locus associated with wheat flowering, while the 2DS QTL appears to be an allelic variant of the photoperiod response locus Ppd-D1. Some of the identified QTLs seemed to be novel loci regulating wheat flowering and maturation, including a QTL controlling the grain filling period on chromosome 3D. The exercise demonstrates that synthetic wheat lines can be useful for the identification of new, agriculturally important loci that can be transferred to, and used for the modification of flowering and grain maturation in hexaploid wheat.

Low temperature induces expression of Cor (cold-responsive)/Lea (late embryogenesis-abundant) gene family members through C-repeat binding factor (CBF) transcription factors in common wheat. However, the relationship between the genetic loci controlling cold-responsive gene expression and freezing tolerance is unclear. In expression quantitative trait locus (eQTL) analysis, accumulated transcripts of Cor/Lea and CBF genes were quantified in recombinant inbred lines derived from a cross between two common wheat cultivars with different levels of freezing tolerance. Four eQTLs controlling five cold-responsive genes were found, and the major eQTL with the greatest effect was located on the long arm of chromosome 5A. At least the 1D and 5A eQTLs played important roles in development of freezing tolerance in common wheat. The chromosomal location of the 5A eQTL, controlling four cold-responsive genes, coincided with a region homoeologous to a frost-tolerance locus (Fr-A m 2) reported as a CBF cluster region in einkorn wheat. The 5A eQTL plays a significant role through Cor/Lea gene expression in cold acclimation of wheat. In addition, our results suggest that one or more CBF copies at the Fr-2 region positively regulate other copies, which might amplify the positive effects of the CBF cluster on downstream Cor/Lea gene activation.

Wheat landraces carry abundant genetic variation in heading and flowering times. Here, we studied flowering-related traits of two Nepalese varieties, KU-4770 and KU-180 and a Japanese wheat cultivar, Shiroganekomugi (SGK). These three wheat varieties showed similar flowering time in a common garden experiment. In total, five significant quantitative trait loci (QTLs) for three examined traits, the heading, flowering and maturation times, were detected using an F2 population of SGK/KU-4770. The QTLs were found at the Ppd-1 loci on chromosomes 2B and 2D and the 2B QTL was also confirmed in another F2 population of SGK/KU-180. The Ppd-D1 allele from SGK and the Ppd-B1 alleles from the two Nepalese varieties might be causal for early-flowering phenotype. The SGK Ppd-D1 allele contained a 2-kb deletion in the 5' upstream region, indicating a photoperiod-insensitive Ppd-D1a allele. Real-time PCR analysis estimating the Ppd-B1 copy number revealed that the two Nepalese varieties included two intact Ppd-B1 copies, putatively resulting in photoperiod insensitivity and an early-flowering phenotype. The two photoperiod-insensitive Ppd-1 homoeoalleles could independently contribute to segregation of early-flowering individuals in the two F2 populations. Therefore, wheat landraces are genetic resources for discovery of alleles useful for improving wheat heading or flowering times.

Intracellular signaling pathways between the mitochondria and the nucleus are important in both normal and abnormal development in plants. The homeotic transformation of stamens into pistil-like structures (a phenomenon termed pistillody) in cytoplasmic substitution (alloplasmic) lines of bread wheat (Triticum aestivum) has been suggested to be induced by mitochondrial retrograde signaling, one of the forms of intracellular communication. We showed previously that the mitochondrial gene orf260 could alter the expression of nuclear class B MADS-box genes to induce pistillody. To elucidate the interactions between orf260 and nuclear homeotic genes, we performed a microarray analysis to compare gene expression patterns in the young spikes of a pistillody line and a normal line. We identified five genes that showed higher expression levels in the pistillody line. Quantitative expression analysis using real-time PCR indicated that among these five genes, Wheat Calmodulin-Binding Protein 1 (WCBP1) was significantly upregulated in young spikes of the pistillody line. The amino acid sequence of WCBP1 was predicted from the full-length cDNA sequence and found to encode a novel plant calmodulin-binding protein. RT-PCR analysis indicated that WCBP1 was preferentially expressed in young spikes at an early stage and decreased during spike maturation, indicating that it was associated with spikelet/floret development. Furthermore, in situ hybridization analysis suggested that WCBP1 was highly expressed in the pistil-like stamens at early to late developmental stages. These results indicate that WCBP1 plays a role in formation and development of pistil-like stamens induced by mitochondrial retrograde signaling.

In the tetrapyrrole biosynthetic pathway of higher plants, 5-aminolevulinic acid (ALA) is metabolized by ALA dehydratase (ALAD). Here, we isolated ALAD1 cDNA from common wheat (Triticum aestivum L.) and its diploid progenitors, and produced transgenic tobacco plants expressing the wheat ALAD1 gene. The ALAD1 genes were highly conserved among wheat relatives, and three homoeologous loci of wheat ALAD1 (TaALAD1) were equally transcribed in common wheat. A transient expression assay of a TaALAD1-GFP (green fluorescent protein) fusion protein suggested that TaALAD1 is localized in chloroplasts. Overexpression of TaALAD1 in transgenic tobacco resulted in a significant increase in ALAD activity in leaves. Moreover, the transgenic tobacco showed vigorous growth and increased survival rate on medium containing ALA at herbicidal concentrations. These results indicate that wheat ALAD1 has catalytic activity in metabolizing ALA in plastids, and that ectopic expression of TaALAD1 in transgenic plants increases their tolerance to ALA application at high concentrations.

Common wheat is an allohexaploid species originating from a naturally occurring inter-specific cross between tetraploid wheat and the diploid wild wheat Aegilops tauschii Coss. Artificial allopolyploidization can produce synthetic hexaploid wheat. However, synthetic triploid hybrids show four types of hybrid growth abnormalities: type II and III hybrid necrosis, hybrid chlorosis, and severe growth abortion. Of these hybrid abnormalities, type II necrosis is induced by low temperature. Under low temperature, elongation of stems and expansion of new leaves is repressed in type II necrosis lines, which later exhibit necrotic symptoms. Here, we characterize type II necrosis in detail. Comparative transcriptome analysis showed that a number of defense-related genes were highly up-regulated in seedling leaves that showed type II necrosis. Transmission electron microscopy revealed extensive cell death in the leaves under low-temperature conditions, accompanied by abundant generation of reactive oxygen species. In addition, down-regulation of cell cycle-related genes was observed in shoot apices of type II necrosis lines under low-temperature conditions. Quantitative RT-PCR and in situ hybridization showed repression of accumulation of histone H4 transcripts in the shoot apical meristem of type II necrosis lines. These results strongly suggest that an autoimmune response-like reaction and repression of cell division in the shoot apical meristem are associated with the abnormal growth phenotype in type II necrosis lines.

The D-genome progenitor of hexaploid wheat, Aegilops tauschii Coss., has a wide natural species range in central Eurasia and possesses wide natural variation in heading and flowering time. Here, we report identification of two Ae. tauschii accessions insensitive to short day length. Similarly to a loss or reduced degree of vernalization requirement, the photoperiod-insensitive mutations were found only in the early flowering sublineage (TauL1b) of Ae. tauschii. Quantitative trait locus (QTL) analyses using two F2 mapping populations showed that a QTL for heading time on the long arm of chromosome 5D was related to the early heading phenotype of the photoperiod-insensitive accessions under short-day conditions. In the photoperiod-insensitive accession, expression patterns of two flowering-related genes were altered under short-day conditions compared with the patterns in photoperiod-sensitive accessions. This study indicates that analysis of natural variations in the Ae. tauschii population is useful to find novel genetic loci controlling agronomically important traits.

Bread wheat (Triticum aestivum) is a hexaploid species with A, B and D genomes. Therefore, most breadwheat genes are present in the genome as triplicated homoeologous genes (homoeologs) derived from the an-cestral A, B, and D genome diploid species. Maturing-time, which is associated with flowering-time and thegrain-filling period, is one of the most important agronomic traits for wheat breeding. Here, the effects ofhomoeologs derived from D genome diploid species on maturing-time in bread wheat were examined in syn-thetic hexaploid wheats obtained by crossing tetraploid durum wheat T. turgidum ssp. durum cv. Langdonand three accessions of the D genome diploid species (Aegilops tauschii). After vernalization, the synthetichexaploid wheat derived from an early-flowering D genome donor showed an early-flowering phenotypeamong the synthetic hexaploids, whereas the synthetic wheat derived from a late-flowering D genome donorwas late-flowering among the synthetic hexaploids. This suggests that the early-flowering phenotype inhexaploid wheat is affected by the homoeolog for early-flowering in the D genome donor. In contrast,maturing-time and grain-filling period in the synthetic hexaploids did not correspond with those of the Dgenome donors, suggesting that these traits are controlled by the interaction between homoeologs on the A,B and D genomes in hexaploid wheat.Key Words: maturing-time, grain-filling period, hexaploid, wheat, Triticum aestivum, Aegilops tauschii,homoeolog.IntroductionIn cereal crops such as wheat and barley, maturing-time,which includes flowering-time and the grain-filling period,is an important character because of its influence on adapt-ability to different environmental conditions. Bread wheat(Triticum aestivum, 2n=6x =42, genome constitutionAABBDD) is grown in a wide range of environments allover the world, and its wide adaptability results from thevariation in maturing-time among cultivars (reviewed inWorland and Snape 2001).Many genetic studies have been performed to investigatethe genetic control of heading/flowering-time in wheat andthree component factors have been identified: vernalizationrequirement, photoperiod sensitivity, and narrow-senseearliness (earliness per se) (Yasuda and Shimoyama 1965,reviewed in Murai et al. 2005). Vernalization requirement isconcerned with the sensitivity of the plant to cold tempera-ture for accelerating spike primordium formation. The ver-nalization insensitivity (spring habit) genes, Vrn-A1, Vrn-B1and Vrn-D1, have been shown to be located on chromo-somes 5A, 5B and 5D, respectively, of bread wheat. The Agenome of hexaploid bread wheat came from T. urartu, theB genome from Aegilops speltoides or another speciesclassified in the Sitopsis section, and the D genome fromAe. tauschii (Feldman 2001). As a consequence of itsorigin, the hexaploid wheat genome contains triplicatedhomoeologous genes (homoeologs) derived from the ances-tral diploid species. Vrn-A1, Vrn-B1 and Vrn-D1 are thehomoeologs of the Vrn-1 gene. Vrn-A1 of the diploideinkorn wheat T. monococcum (2n =2x =14, A

It was recently shown that allopolyploidy brings novel epistatic interactions to genes belonging to different genomes. However, systematic studies of the phenotypic relationships between synthetic hexaploid wheats and their parental lines have not been conducted. In this study, 27 synthetic hexaploid wheats were produced by crossing the tetraploid wheat cultivar ‘Langdon’ with 27 accessions of Aegilops tauschii. Variations in 20 morphological and flowering traits were analysed in both the synthetic wheat lines and the parental Ae. tauschii accessions. The 20 traits exhibited large variations in the wheat lines. For many of the traits, the degree of variation in the parental accessions was reduced in the hexaploid derivatives. Principal component analysis of floret-related traits divided the Ae. tauschii accessions into two subspecies, ssp. tauschii and ssp. strangulata, but this parental pattern of subspecific division was not detectable in the hexaploids. Our results suggest that the ‘Langdon’ genome may have an alleviating effect on the expression of D-genome-derived variations in derived synthetics.

The genealogical and geographic structure of variation in spikelet morphology was analyzed for central Eurasian wild wheat Aegilops tauschii Coss. using a diverse array of 203 sample accessions that represented the entire species range. In this sample set, two subspecies were identified on the basis of sensu-stricto criteria: only the accessions having markedly moniliform spikes were assigned to Ae. tauschii Coss. subspecies strangulata (Eig) Tzvel., whereas those having mildly moniliform and cylindrical spikes to Ae. tauschii Coss. subspecies tauschii. In a graph of the first two axes from a principal component analysis based on nine spikelet traits, the plots of the two subspecies formed separate clusters, indicating that subspecies strangulata sens. str. is a practically usable taxon. Chloroplast-DNA-based genealogical analyses suggested that subspecies strangulata diverged from an ancestor that carried a specific chloroplast DNA type, whereas, after divergence, this subspecies became polyphyletic, likely through hybridization. Geographically, significant longitudinal and latitudinal clines were detected for spikelet size, with spikelets tending to be small in the eastern and southern regions. These results shed some light on the patterns of subspecies divergence and spikelet-shape diversification in the course of Ae. tauschii’s long-distance dispersal from the Transcaucasus to China.

Homeotic transformation of stamens into pistil-like structures (pistillody) can occur in cytoplasmic substitution (alloplasmic) lines of bread wheat (Triticum aestivum) that have the cytoplasm of the related species, Aegilops crassa. Previously we showed that pistillody results from altered patterns of expression of class B MADS-box genes mediated by mitochondrial gene(s) in the Ae. crassa cytoplasm. The wheat cultivar Chinese Spring does not show pistillody when Ae. crassa cytoplasm is introduced. The absence of an effect is due to a single dominant gene (designated Rfd1) located on the long arm of chromosome 7B. To identify the mitochondrial gene involved in pistillody induction, we performed a subtraction analysis using cDNAs derived from young spikes of a pistillody line and a normal line. We found that mitochondrial cDNA clone R04 was abundant in the young spikes of the pistillody line but was down-regulated in the normal line that carried nuclear Rfd1. Sequencing of the full-length cDNA corresponding to clone R04 showed that two genes were present, cox I (cytochrome c oxidase subunit I) and orf260(cra). orf260(cra) shows high sequence similarity to orf256, the T. timopheevii mitochondrial gene responsible for cytoplasmic male sterility (CMS). orf260(cra) was also present in the cytoplasms of Ae. juvenalis and Ae. vavilovii, which induce pistillody, but not in the cytoplasms of other species not associated with pistillody. Furthermore, Western blot analysis revealed that the ORF260cra protein was more abundant in the pistillody line than in the normal line. We suggest therefore that orf260(cra) is associated with pistillody induction.

ABA-responsive element binding protein (AREB) and ABA-responsive element binding factor (ABF), members of the basic region/leucine zipper (bZIP)-type protein family, act as major transcription factors in ABA-responsive gene expression under abiotic stress conditions in Arabidopsis. Barley HvABI5 and rice transcription factor responsible for ABA regulation 1 (TRAB1) are homologues of AREB/ABF and are expressed in drought- and ABA-treated seedlings. However, no direct evidence has shown an association of an AREB/ABF-type transcription factor with stress tolerance in cereals. To understand the molecular basis of abiotic stress tolerance through a cereal AREB/ABF-type transcription factor, a wheat HvABI5 ortholog, Wabi5, was isolated and characterized. Wabi5 expression was activated by low temperature, drought and exogenous ABA treatment, and its expression pattern differed between two wheat accessions with distinct levels of stress tolerance and ABA sensitivity. Wabi5-expressing transgenic tobacco plants showed a significant increase in tolerance to abiotic stresses such as freezing, osmotic and salt stresses and a hypersensitivity to exogenous ABA in the seedling stage compared with wild-type plants. Expression of a GUS reporter gene under the control of promoters of three wheat cold-responsive/late embryogenesis abundant (Cor/Lea) genes, Wdhn13, Wrab18 and Wrab19, was enhanced by ectopic Wabi5 expression in wheat callus and tobacco plants. These results clearly indicated that WABI5 functions as a transcriptional regulator of the Cor/Lea genes in multiple abiotic stress responses in common wheat.

Cold acclimation is an adaptive process for acquiring cold/freezing tolerance in wheat. To clarify the cultivar difference of freezing tolerance, we compared mitochondrial respiration activity and the expression profile of alternative oxidase (AOX) genes under low-temperature conditions using two common wheat cultivars differing in freezing tolerance. During cold acclimation, the respiration capacity of the alternative pathway significantly increased in a freezing-tolerant cultivar compared with a freezing-sensitive cultivar. More abundant accumulation of the AOX and uncoupling protein gene transcripts was also observed under the low-temperature conditions in the tolerant cultivar than in the sensitive cultivar. These results suggest that the mitochondrial alternative pathway might be partly associated with the cold acclimation and freezing tolerance in wheat.

Cereal lip19 genes encoding bZIP-type transcription factors are assumed to play a regulatory role in gene expression during the cold acclimation process. However, no direct evidence shows an association of LIP19-type bZIPs with stress tolerance or activation of stress-responsive Cor/Lea genes. To understand the molecular basis of development of abiotic stress tolerance through the LIP19 transcription factor, a wheat lip19 homologue, Wlip19, was isolated and characterized. Wlip19 expression was activated by low temperature in seedlings and was higher in a freezing-tolerant cultivar than in a freezing-sensitive one. Wlip19 also responded to drought and exogenous ABA treatment. Wlip19-expressing transgenic tobacco showed a significant increase in abiotic stress tolerance, especially freezing tolerance. Expression of a GUS reporter gene under the control of promoter sequences of four wheat Cor/Lea genes, Wdhn13, Wrab17, Wrab18, and Wrab19, was enhanced by Wlip19 expression in wheat callus and tobacco plants. These results indicate that WLIP19 acts as a transcriptional regulator of Cor/Lea genes in the development of abiotic stress tolerance. Moreover, direct protein-protein interaction between WLIP19 and a wheat OBF1 homologue TaOBF1, another bZIP-type transcription factor, was observed, suggesting that this interaction is conserved in cereals.

The wheat (Triticum aestivum L.) CBF gene family is assumed to play important roles in development of low-temperature and freezing tolerance through activation of the downstream Cor/Lea genes. However, no direct evidence shows association of the wheat CBF genes with stress tolerance or any interaction between wheat CBF transcription factors and Cor/Lea gene activation. Here, we introduced Wcbf2, one of the wheat CBF genes, into the tobacco (Nicotiana tabacum L.) genome. Expression of Wcbf2 significantly increased the level of freezing tolerance in the transgenic tobacco plants without phenotypic retardation, and altered the expression patterns of tobacco genes, including cold-responsive genes. A transgenic tobacco plant expressing Wcbf2 was crossed to other transgenic plants expressing a GUS reporter gene under control of the wheat Cor/Lea gene promoter. Analysis of the F1 plants showed that the WCBF2 protein positively regulated at least the expression of Wdhn13 and Wrab17. These results strongly indicate that WCBF2 functions as a transcription factor in the development of freezing tolerance in common wheat.

Freezing tolerance and vernalization requirement are important traits for adaptation of wheat to growing in a high-latitude area. Fr-1 and Vrn-1, tightly linked on homoeologous group 5 chromosomes, are major loci for controlling the freezing tolerance and vernalization requirement, respectively. It was previously supposed that winter-habit wheat should possess a winter-type Fr-1 allele guaranteeing winter survival, but that such allele is unnecessary for spring-habit cultivars. To clarify such allelic linkage between the Vrn-1 and Fr-1 loci in the D genome, we studied freezing tolerance and vernalization requirement, and compared cold-responsive expression patterns of Cor (cold responsive)/Lea (late-embryogenesis-abundant), their putative transcription factor genes and three Vrn-1 homoeologs in Japanese common wheat cultivars. Wide variation of freezing tolerance and two alleles of Vrn-D1 were observed in the Japanese cultivars, whereas the accumulation levels of the Cor/Lea transcripts were not be correlated with the levels of freezing tolerance. The allelic linkage between Vrn-1 and Fr-1 loci well known in the A genome was not observed in the D genome of Japanese cultivars possessing Vrn-D1 or vrn-D1 allele.

Homeotic transformation of stamens into pistil-like structures (called pistillody) has been reported in cytoplasmic substitution (alloplasmic) lines of bread wheat (Triticum aestivum) having the cytoplasm of a wild relative species, Aegilops crassa. Our previous studies indicated that pistillody is caused by alterations of the class B MADS-box gene expression pattern associated with mitochondrial gene(s) in the Ae. crassa cytoplasm. To elucidate the nuclear gene involved in the cross-talk between pistillody-related mitochondrial gene(s) and nuclear homeotic genes, we performed cDNA subtraction analysis using cDNAs derived from young spikes of a pistillody line and a normal line. As a result, we identified a protein kinase gene, WPPK1 (wheat pistillody-related protein kinase 1), which is upregulated in the young spikes of the pistillody line. RT-PCR analysis indicated that WPPK1 is strongly expressed in pistils and pistil-like stamens in the pistillody line, suggesting that it is involved in the formation of pistil-like stamens as well as pistils. The full-length cDNA sequence for WPPK1 showed high similarity with a flowering plant PVPK-1 protein kinase, and phylogenetic analysis indicated that it is a member of AGC group protein kinases. Furthermore, a phosphorylation assay indicated that it has protein kinase activity. In situ hybridization analysis revealed that WPPK1 is expressed in developing pistils and pistil-like stamens as well as in their primordia. These indicate that in the alloplasmic line, WPPK1 plays a role in formation and development of pistil-like stamens.

Heading time in bread wheat (Triticum aestivum L.) is determined by three characters - vernalization requirement, photoperiodic sensitivity and narrowsense earliness (earl iness per se) - which are involved in the phase transition from vegetative to reproductive growth. The wheat APETALA 1(AP1)-like MADS-box gene, wheat API (WAP1, identical with VRN1), has been identified as an integrator of vernalization and photoperiod flowering promotion pathways. A MADS-box gene, SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) is an integrator of flowering pathways in Arabidopsis. In this study, we isolated a wheat ortholog of SOC1, wheat SOC1 (WSOC1), and investigated its relationship to WAP1 in the flowering pathway. WSOC1 is expressed in young spikes but preferentially expressed in leaves. Expression starts before the phase transition and is maintained during the reproductive growth phase. Overexpression of WSOC1 in transgenic Arabidopsis plants caused early flowering under short-day conditions, suggesting that WSOC1 functions as a flowering activator in Arabidopsis. WSOC1 expression is affected neither by vernalization nor photoperiod, whereas it is induced by gibberellin at the seedling stage. Furthermore, WSOC1 is expressed in transgenic wheat plants in which WAP1 expression is cosuppressed. These findings indicate that WSOC1 acts in a pathway different from the WAP1-related vernalization and photoperiod pathways.

Bread wheat (Triticum aestivum) is a hexaploid species with A, B, and D ancestral genomes. Most bread wheat genes are present in the genome as triplicated homoeologous genes (homoeologs) derived from the ancestral species. Here, we report that both genetic and epigenetic alterations have occurred in the homoeologs of a wheat class E MADS box gene. Two class E genes are identified in wheat, wheat SEPALLATA (WSEP) and wheat LEAFY HULL STERILE1 (WLHS1), which are homologs of Os MADS45 and Os MADS1 in rice (Oryza sativa), respectively. The three wheat homoeologs of WSEP showed similar genomic structures and expression profiles. By contrast, the three homoeologs of WLHS1 showed genetic and epigenetic alterations. The A genome WLHS1 homoeolog (WLHS1-A) had a structural alteration that contained a large novel sequence in place of the K domain sequence. A yeast two-hybrid analysis and a transgenic experiment indicated that the WLHS1-A protein had no apparent function. The B and D genome homoeologs, WLHS1-B and WLHS1-D, respectively, had an intact MADS box gene structure, but WLHS1-B was predominantly silenced by cytosine methylation. Consequently, of the three WLHS1 homoeologs, only WLHS1-D functions in hexaploid wheat. This is a situation where three homoeologs are differentially regulated by genetic and epigenetic mechanisms.

Abscisic acid (ABA) plays important role in mediating stress responses and in acquiring desiccation tolerance and dormancy of plant seeds. To study roles of ABA in cold acclimation and freezing tolerance in wheat, expression profiles of Cor/Lea and their putative transcription factor (TF) genes were analysed using a dominant mutation line of common wheat EH47-1 lacking seed dormancy. The mutant line was less sensitive to exogenous ABA than the original line as judged by the magnitude of ABA inhibition of seedling growth. Expression analysis of Cor/Lea and TF genes however, showed that more transcripts were present in ABA-treated seedlings of the mutant line. In developing caryopses, the same tendency was observed. The mutant line showed no changes in the cold acclimation ability, but it showed a higher level of freezing tolerance than the original line without cold acclimation. No significant differences were observed in the expression profiles of Cor/Lea and TF genes during cold acclimation between the two lines. Our results imply the presence of an unknown ABA-dependent cold responsive pathway, which enhances the basal level of freezing tolerance by a dominant mutation in EH47-1.

Cold acclimation, an adaptive process for developing freezing tolerance in over-wintering plants, is associated with increased expression levels of a series of cold-responsive (Cor)/late embryogenesis abundant (Lea) genes. To investigate the function of Wcor15, a member of the wheat Cor/Lea gene family, for improvement of freezing tolerance, two types of transgenic tobacco lines expressing Wcor15-containing chimeric genes were produced and characterized. Immunoblot and gene expression analyses of a transgenic tobacco line expressing the Wcor15-GFP fusion gene under control of the CaMV35S promoter showed transport and abundant accumulation of the WCOR15 protein in the stromal compartment of the chloroplasts. The 5' upstream region of Wcor15 induced expression of the GFP reporter gene under low-temperature conditions in the transgenic tobacco. Both transgenic lines expressing the Wcor15-GFP fusion gene showed a similar and significantly improved level of freezing tolerance compared with the wild-type tobacco plants. Our results demonstrate that the induced expression of the wheat Wcor15 gene positively contributes to the development of freezing tolerance in the heterologous tobacco plants.

Vrn-1/Fr-1 chromosomal regions of common wheat possess major QTLs for both winter hardiness (Fr )a nd vernalization requirement (Vrn). The Vrn-1/Fr-1 intervals are assigned to long arms of the homoeologous group 5 chromosomes. To investigate the role of the Vrn-1/Fr-1 intervals on the low-temperature (LT) inducibility of wheat Cor/Lea genes and its putative transcription factor gene Wcbf2, LT response of these genes was monitored using near-isogenic lines (NILs) for the Vrn-1 loci. The Wcbf2 transcript accumulated rapidly after LT treatment and remained at a high level in lines without any dominant Vrn-1 alleles. By contrast, the Wcbf2 transcript level was greatly reduced in lines carrying the Vrn-1 alleles. The Vrn-1 NILs accumulated much lower amounts of Cor/Lea transcripts and COR/LEA proteins than the non-carrier line. The observed patterns and levels of gene expression, particularly in the Vrn-A1 NIL, agreed with the higher sensitivity to freezing damage in this line than in the noncarrier line. Up-regulation of the expression of the WAP1 gene, a candidate of the Vrn-1 loci, was much delayed in the non-carrier line than all the NILs carrying the Vrn-1 loci. Neither positive nor negative relationships were found between the WAP1 expression and the Cbf2/Cor/ Lea expression. These results support the intimate relationship between the Cbf2/Cor/Lea expression and the level of freezing tolerance, and suggest that a functional Fr-A1 allele linked to the vrn-A1 allele, instead of the vernalization gene itself, plays a major role in regulating the CBF-mediated Cor/lea gene expression in wheat.

Expression profiles of a set of Cor/Lea genes were assessed during early stages of cold acclimation in seedlings of two wheat cultivars, which showed contrasting levels of freezing tolerance. These Cor/Lea family members consisted of three EST clones and 13 previously identified cDNA clones of wheat and rye. Northern blot analysis using RNA extracted from seedling leaves and roots showed that most of the genes exhibited a quite similar time-course of expression, although with different expression levels: They rapidly responded to low temperature and their transcript levels reached high plateaus within 3-5 days. The overall gene expression profiles were correlated with the time-dependent development and the level of freezing tolerance under low temperature in the two cultivars. Western blot analysis of protein accumulation further verified this observation. Abscissic acid response was proved for at least four genes. Light was stimulatory to most of the genes, and this positive light response associated with low temperature occurred not only in leaf-specific genes but also in leaf/root-expressed genes. Taken together, the present results suggest that the Cor/Lea gene family represents a major group of downstream genes involved in the ABA-dependent and -independent signal pathways and that most of them are co-regulated in determining freezing tolerance in wheat seedlings.

Class-B floral homeotic genes are involved in specifying petal and stamen identity during flower development in plant species. Homeotic transformation of stamens into pistil-like structures (called pistillody) has been observed in alloplasmic lines of bread wheat (Triticum aestivum L.) having the cytoplasm of a wild relative species, Aegilops crassa Boiss. To obtain information about the molecular mechanism underlying pistillody induction, we isolated two PISTILLATA (PI)-type class-B MADS-box genes, WPI1 (wheat PI STILLATA # 1) and WPI2, from wheat. Phylogenetic reconstruction indicated that WPI1 is orthologous to OsMADS4 and that WPI2 is probably an ortholog of OsMADS2. Both OsMADS4 and OsMADS2 genes were suggested to be PI orthologs in rice, and the function of OsMADS4 as a class-B gene was proven by the transgenic study. An in situ hybridization study demonstrated that the WPI1 gene is expressed in primordia of lodicules and stamens in developing florets in wheat. In the alloplasmic wheat line exhibiting pistillody, the WPI1 transcripts were not detected in the primordia of pistil-like stamens, whereas WPI1 was expressed in the lodicules. The wheat APETALA3 (AP3)-type class-B MADS-box gene WAP3 (wheat AP3)/TaMADS#82 showed an expression pattern similar to that of WPI1. These results suggest that pistillody in alloplasmic wheats is caused by alterations to the expression pattern of class-B MADS-box genes.

Heading time in bread wheat (Triticum aestivum L.) is determined by three characters: vernalization requirement, photoperiodic sensitivity and narrow-sense earliness, which are involved in the phase transition from vegetative to reproductive growth. We identified and characterized the APETALA1 (AP1)-like MADS box gene in wheat (WAP1) as an activator of phase transition. Its expression starts just before the phase transition and is maintained during the reproductive phase. Inhibition of WAP1 expression in the transgenic plants by co-suppression affected neither vernalization requirement nor photoperiodic sensitivity, but resulted in delayed narrow-sense earliness, indicating that WAP1 accelerates autonomous phase transition. Analyses of the WAP1 expression in the near-isogenic lines (NILs) for spring habit genes (Vrn) revealed that WAP1 transcripts were induced by vernalization strongly in the NILs with Vrn dominant alleles and weakly with the recessive alleles. Furthermore, WAP1 expression was up-regulated by a long photoperiod in both NILs with and those without a photoperiod-insensitive gene (Ppd). These results suggest that WAP1 is a key gene in the regulatory pathway controlling the phase transition from vegetative to reproductive growth in wheat.

Transcript profile and post-transcriptional processing of the mitochondrial orf156 were compared among the nucleus-cytoplasm (NC) hybrids of tetraploid and hexaploid wheat and their parental lines. These NC hybrids, in which the plasmon from the maternal Aegilops squarrosa parent is combined with the nuclear genomes of the paternal parents, and their parental lines were previously shown to have heteroplasmic copies of the polycistronic transcriptional unit nad3-orf156 in different sub-stoichiometries. Despite the heteroplasmic conditions, only single transcript types were detected among reverse transcription-PCR products of the orf156 mRNA in the respective lines. In the NC hybrids only a major maternal copy was transcribed and edited, likewise in the parental lines only single major copies were transcribed and edited. RNA gel blot analysis using the orf156 probe showed that the NC hybrids had the transcripts similar to the parental lines but unlike the maternal line. Primer extension analysis further revealed the orf156 transcripts with different 5' termini, which most probably resulted from the alternative post-transcriptional cleavages of the precursor mRNA. These cleaved transcripts were polymorphic not only among the lines but also among four different tissues. The cleavages were highly selective and the paternal-type cleavages predominated in the NC hybrids, irrespective of the tissues studied. This seemingly neutral polymorphisms in wheat could make this system convenient for further analysis of mitochondrial gene expression in plants.

Pistillody, homeotic transformation of stamens into pistil-like structures, has been reported in cytoplasmic substitution (alloplasmic) lines of common wheat (Triticum aestivum) with Aegilops crassa cytoplasm. The induction of pistillody is suppressed by the Rfd1 gene detected on the long arm of chromosome 7B in wheat cultivar 'Chinese Spring' (CS). Because of the absence of Rfd1, the alloplasmic line CS ditelosomic 7BS [(cr)-CSdt7BS] lacking the long arm of chromosome 7B exhibits pistillody in all florets, whereas the euplasmic CS ditelosomic 7BS (CSdt7BS) with normal cytoplasm forms normal stamens. To study the molecular mechanism of pistillody caused by nuclear-cytoplasm interaction in alloplasmic wheat, we cloned and characterized a wheat AGAMOUS homolog, WAG. The WAG gene copy number in the wheat genome was estimated at three, located on the homoeologous group 1 chromosomes. Northern blot analysis with a normal wheat line revealed that the transcription level of WAG was lower in young spikes and increased during spike development. The highest expression was observed in spikes at the booting to heading stage. The 1.1 kb WAG transcript accumulated in both the reproductive (pistil and stamen) and non-reproductive (palea and lemma) portions of spikes at the heading stage, whereas an extra transcript of different molecular size (1.3 kb) was observed in pistil-like stamens of line (cr)-CSdt7BS as well as in pistils of CSdt7BS and (cr)-CSdt7BS. These results suggest that the product of the 1.3 kb WAG transcript is involved in pistil development and is associated with pistillody caused by a nuclear-cytoplasm interaction in alloplasmic wheat.

We have constructed a linkage map of the rice brown planthopper (BPH)resistance gene, Bph1. RFLP and AFLP markers were selected by thebulked segregant analysis and used in the mapping study of 262 F2sthat were derived from a cross of `Tsukushibare', a susceptible japonica cultivar, and `Norin-PL3', an authentic japonicaBph1-introgression line. Twenty markers were mapped within a 28.9-cMregion containing the Bph1 locus on the long arm of rice chromosome12. Combining the result of segregation analysis of BPH resistance by themass seedling test and that of the markers, the Bph1 locus wasmapped within a 5.8-cM region between two flanking markers. The closestAFLP markers, em5814N and em2802N, was at 2.7 cM proximal to theBph1 locus. Together with the previously constructed high-resolutionmap of bph2 locating the locus at ca. 10 cM proximal to the Bph1 locus, this improved version of the linkage map would facilitatepyramiding these two important BPH resistance genes.

We studied the kinetics of induction of the wheat (Triticum aestivum L.) gene Wdhn 13, which encodes a predominantly cold-responsive protein belonging to the LEA D-11 dehydrin family. The deduced polypeptide WDHN 13 (MW = 12.8 kDa) represented the smallest dehydrin member in cereals with three lysine-rich K-segments. Purified WDHN 13 was boiling-stable due to its high hydrophilicity. Western blot analysis using the polyclonal anti-WDHN 13-antibody revealed a number of cross-reacting proteins in mature embryos and endosperms and in seedling leaves under normal temperature (25 degrees C), while a single major protein and a transcript were induced in response to low temperature (4 degrees C) in the leaves. An increase in the amount of mRNA was temporary with a peak occurring at day 3 to 5 of the low temperature treatment, while the protein accumulation proceeded with a significant time lag and continued until the end of the experiment (day 10). Steady-state levels of the transcript and protein were much higher in the leaves than in the roots of low temperature-treated seedlings and were apparently modulated by light/dark conditions. The light/dark modulation of the transcript and protein levels suggested stabilization of the mRNA under the low temperature condition. Genomic Southern blot analysis showed that Wdhn 13 is located on the homoeologous group 7 chromosomes, unlike all other wheat Dhn genes that are located on the group 6 chromosomes.

Homeotic transformation of stamens into pistil-like structures (pistillody) has been observed in a cytoplasmic substitution (alloplasmic) line of wheat (Triticum aestivum L.) cv. Norin 26, which has the cytoplasm of a wild relative species, Aegilops crassa L. On the other hand, an alloplasmic line of wheat cv. Chinese Spring (CS) with Ae. crassa cytoplasm has normal flowers. This is due to the presence in the CS nucleus of a fertility-restoring gene, Rfd1. Deletion mapping analysis revealed that Rfd1 is located on the middle part of the long arm of chromosome 7B. To investigate the function of the Rfd1 gene by a loss-of-function strategy, we produced alloplasmic lines of CS ditelosomic 7BS [(cr)-CSdt7BS] and CS monotelodisomic 7BS [(cr)-CSmd7BS] with the Ae. crassa cytoplasm, and characterized their phenotypes. The line (cr)-CSdt7BS without Rfd1 exhibited pistillody in all florets, and also female sterility. Scanning electron microscopy of the young spikes revealed that the pistillody was induced at an early stage of stamen development. The pistillate stamens often developed incomplete ovule-like structures with integuments instead of tapetum and pollen grains. It is possible that MADS box genes are associated with the induction of pistillody, because the expression of wheat APETALA3 homologue (WAP3) was reduced in the young spikes of (cr)-CSdt7BS. In addition, a histological study indicated that the female sterility in (cr)-CSdt7BS is due to the abnormality of the ovule, which fails to form an inner epidermis and integuments in the chalaza region. The line (cr)-CSmd7BS, hemizygous for Rfd1, showed partial pistillody (51%) and restored female fertility up to 72%. These results suggest that the induction of both pistillody and ovule deficiency caused by the Ae. crassa cytoplasm is inhibited by the Rfd1 gene in a dose-dependent manner.

In common wheat, cultivar differences in freezing tolerance are considered to be mainly due to allelic differences at two major loci controlling freezing tolerance. One of the two loci, Fr-2, is coincident with a cluster of genes encoding C-repeat binding factors (CBFs), which induce downstream Cor/Lea genes during cold acclimation. Here, we conducted microarray analysis to study comprehensive changes in gene expression profile under long-term low-temperature (LT) treatment and to identify other LT-responsive genes related to cold acclimation in leaves of seedlings and crown tissues of a synthetic hexaploid wheat line. The microarray analysis revealed marked up-regulation of a number of Cor/Lea genes and fructan biosynthesis-related genes under the long-term LT treatment. For validation of the microarray data, we selected four synthetic wheat lines that contain the A and B genomes from the tetraploid wheat cultivar Langdon and the diverse D genomes originating from different Aegilops tauschii accessions with distinct levels of freezing tolerance after cold acclimation. Quantitative RT-PCR showed increased transcript levels of the Cor/Lea, CBF, and fructan biosynthesis-related genes in more freezing-tolerant lines than in sensitive lines. After a 14-day LT treatment, a significant difference in fructan accumulation was observed among the four lines. Therefore, the fructan biosynthetic pathway is associated with cold acclimation in development of wheat freezing tolerance and is another pathway related to diversity in freezing tolerance, in addition to the CBF-mediated Cor/Lea expression pathway.

Time-courses of the development of freezing tolerance and the expression of a cold-responsive gene wlt10 were monitored during cold acclimation in wheat (Triticum aestivum L.). Bioassay showed that cold acclimation conferred much higher freezing tolerance on a winter cultivar than a spring cultivar. Northern blot analysis showed that the expression of wlt10 encoding a novel wheat member of a cereal-specific LT-COR protein family was specifically induced by low temperature. A freezing-tolerant winter cultivar accumulated the mRNA more rapidly and for a longer period than a susceptible spring cultivar. The increase in the amount of mRNA was temporary but the peak occurred at the time when the maximum level of freezing tolerance was attained. The mRNA accumulated more in the leaves than in the roots, and different light/dark regimes modulated the level of mRNA accumulation. Genomic Southern blot analyses using the nulli-tetrasomic series showed that the wlt10 homologues were located on the homologous group 2 chromosomes.

A Sri Lankan indica rice (Oryza sativa L.) cultivar Babawee harboring a brown planthopper (BPH)(Nilaparvata lugens Stal) resistance gene bph4 was crossed as a male parent with two susceptible cultivars, either indica IR24 or japonica Tsukushibare. Segregation of the BPH resistance in the two crosses was studied by directly assaying the F2 phenotypes and by determining the F2 genotypes based on the F3 phenotypes. In both cross combinations, the segregation of the BPH resistance significantly deviated from the ratio expected for the single recessive gene model. Using bulked DNAs of homozygous resistant and susceptible F2s and the parents, seven out of 214 RFLP markers were selected as linked markers, of which six on chromosome 6 were common in the two cross combinations. Two microsatellite markers on chromosome 6 were also linked to bph4. Although the map position of bph4 could not be determined, the gene was assigned to the distal region of the short arm of rice chromosome 6 based on the bulked segregant analysis and linkage analysis.

Libraries of plasmid clones covering the entire chloroplast (cp) genome of the common wheat,Triticum aestivum cv. Chinese Spring were constructed and assembled into contig-clones. From these, we obtained the complete nucleotide sequence of wheat chloroplast DNA—a 134,540 bp circular DNA (DDBJ accession no. AB042240) containing four species of ribosomal RNA, 30 genes for 20 species of transfer RNA, and 71 protein coding genes. Additionally, we detected five unidentified open reading frames conserved among grasses. Plasmid clones are available on request.