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Wheat straw is one of the major attractive resources for low-cost raw materials for renewable energy, biofuels and biochemicals. However, like other sources of lignocellulosic biomass, straw is a heterogeneous material due to its mixed origin from different tissue and cell types. Here, to examine the genotypic effects on biorefinery usage of wheat straw, straw obtained from different wheat cultivars and experimental lines was pretreated with dilute acid. Significant differences between cultivars were observed in the concentrations of glucose and toxic by-products of the liquid hydrolysates. A higher content of xylose than glucose was found in liquid hydrolysates from wheat straw, and the xylose content appeared to be affected by both environmental and genetic factors. Analysis using chromosome substitution lines of the common wheat cultivar Chinese Spring showed that chromosomes 2A and 3A from other wheat cultivars, Hope and Timstein, significantly increased the xylose content. However, no significant relationship was observed between the liquid hydrolysate xylose content and the glucose content obtained from enzymatic saccharification of the acid-insoluble residue. These results highlight the potential of wheat breeding to improve biomass-related traits in wheat straw.

The consensus model for floral organ formation in higher plants, the so-called ABCDE model, proposes that floral whorl-specific combinations of class A, B, C, D, and E genes specify floral organ identity. Class A, B, C, D and E genes encode MADS-box transcription factors; the single exception being the class A gene APETALA2. Bread wheat (Triticum aestivum) is a hexaploid species with a genome constitution AABBDD; the hexaploid originated from a cross between tetraploid T. turgidum (AABB) and diploid Aegilops tauschii (DD). Tetraploid wheat is thought to have originated from a cross between the diploid species T. urartu (AA) and Ae. speltoides (BB). Consequently, the hexaploid wheat genome contains triplicated homoeologous copies (homoeologs) of each gene derived from the different ancestral diploid species. In this study, we examined the expression patterns of homoeologs of class B, C and D MADS-box genes during floral development. For the class B gene wheat PISTILLATA2 (WPI2), the homoeologs from the A and D genomes were expressed, while expression of the B genome homoeolog was suppressed. For the class C gene wheat AGAMOUS1 (WAG1), the homoeologs on the A and B genomes were expressed, while expression of the D genome homoeolog was suppressed. For the class D gene wheat SEEDSTICK (WSTK), the B genome homoeolog was preferentially expressed. These differential patterns of homoeolog expression were consistently observed among different hexaploid wheat varieties and synthetic hexaploid wheat lines developed by artificial crosses between tetraploid wheat and Ae. tauschii. These results suggest that homoeolog-specific regulation of the floral MADS-box genes occurs in allopolyploid wheat.

Flowering time is an important trait for Japanese wheat breeding. Aegilops tauschii, the D-genome donor of hexaploid wheat, is a useful resource to enlarge the D-genome diversity of common wheat. Previously, we identified flowering-related QTLs in F2 populations of synthetic hexaploid wheat lines between the tetraploid wheat cultivar Langdon and Ae. tauschii accessions. Here, to evaluate the usefulness of the early-flowering alleles from Ae. tauschii for Japanese wheat breeding, QTL analyses were conducted in two F2 populations derived from crosses between Japanese wheat cultivars and early-flowering lines of synthetic hexaploid wheat. Only two chromosomal regions controlling flowering-related traits were identified, on chromosomes 2DS and 5AL in the mapping populations, and no previously identified QTLs were found in the synthetic hexaploid lines. The strong effect of the 2DS QTL, putatively corresponding to Ppd-D1, was considered to hide any significant expression of other QTLs with small effects on flowering-related traits. When F2 individuals carrying Ae. tauschii-homozygous alleles around the 2DS QTL region were selected, the Ae. tauschii-derived alleles of the previously identified flowering QTLs partly showed an early-flowering phenotype compared with the Japanese wheat-derived alleles. Thus, some early-flowering alleles from Ae. tauschii may be useful for production of early-flowering Japanese wheat cultivars.

Hybrid chlorosis, one of the reproductive barriers between tetraploid wheat and its D-genome progenitor, Aegilops tauschii, inhibits normal growth of synthetic wheat hexaploids. Hybrid chlorosis appears to be due to an epistatic interaction of two loci from the AB and D wheat genomes. Our previous study assigned the causal D-genome gene for hybrid chlorosis, Hch1, to the short arm of chromosome 7D. Here, we constructed a fine map of 7DS near Hch1 using 280 F2 individuals from a cross of two wheat synthetic lines, one showing normal growth and the other showing hybrid chlorosis. The hybrid chlorosis phenotype was controlled by a single dominant allele of the Hch1 locus in the synthetic hexaploids. Hch1 was closely linked to four new markers within 0.2 cM, and may be localized near or within the two Ae. tauschii scaffolds containing the linked markers on 7DS. Comparative analysis of the Hch1 chromosomal region for Ae. tauschii, barley and Brachypodium showed that a local inversion occurred in the region proximal to Hch1 during the divergence between barley and Ae. tauschii, and that the Hch1 region on wheat 7DS is syntenic to Brachypodium chromosome 1. These observations provide useful information for further studies toward map-based cloning of Hch1.

Introgression between related species with different ploidy levels has played important roles in wheat subspecies differentiation. Persian wheat, a cultivated tetraploid wheat subspecies (Triticum turgidum subsp. carthlicum), is postulated to have evolved through interploidy hybridization between tetraploid and hexaploid wheats. Here, we report evidence for the origin of subsp. carthlicum based on the discovery of a new allele for the 5th-to-6th exon region of the Wknox1bKNOTTED1-type homeobox gene in a common wheat subspecies (T. aestivum subsp. carthlicoides). In this Wknox1b region, subsp. carthlicoides contains an inverted duplication mutation in the 3' flanking region of a 157-bp MITE insertion site. This structural mutation resulted in the suppression of Wknox1b expression in subsp. carthlicoides, but no structural mutation was observed in the same region of subsp. carthlicum. In addition, the carthlicum allele for the Wknox1b 5th-to-6th exon region exhibited the same sequence as that in the wild emmer wheat subsp. dicoccoides. These observations support an alternative hypothesis that subsp. carthlicum evolved by interploidy hybridization between subsp. carthlicoides and tetraploid wheat.

In the course of reconstructing Aegilops caudata from its own genome (CC) and its plasmon, which had passed half a century in common wheat (genome AABBDD), we produced alloplasmic Ae. cylindrica (genome CCDD) with the plasmon of Ae. caudata. This line, designated (caudata)-CCDD, was found to express male sterility in its second substitution backcross generation (SB2) of (caudata)-AABBCCDD pollinated three times with the Ae. cylindrica pollen. We repeatedly backcrossed these SB2 plants with the Ae. cylindrica pollen until the SB5 generation, and SB5F2 progeny were produced by self-pollination of the SB5 plants. Thirteen morphological and physiological characters, including pollen and seed fertilities, of the (caudata)-CCDD SB5F2 were compared with those of the euplasmic Ae. cylindrica. The results indicated that the male sterility expressed by (caudata)-CCDD was due to genetic incompatibility between the Ae. cylindrica genome and Ae. caudata plasmon that did not affect any other characters of Ae. cylindrica. Also, we report that the genome integrity functions in keeping the univalent transmission rate high.

Domestication-related changes that govern a spike morphology suitable for seed harvesting in cereals have resulted from mutation and selection of the genes. A synthetic hexaploid wheat (S-6214, genome AABBDD) produced by a cross between durum wheat (AABB) and wild goat grass (DD) showed partial non-domestication-related phenotypes due to genetic effects of the wild goat grass genome. Quantitative trait loci (QTLs) affecting wheat domestication-related spike characters including spike threshability, rachis fragility and spike compactness were investigated in F2 progeny of a cross between Chinese Spring (CS) wheat (AABBDD) and S-6214. Of 15 relevant QTLs identified, eight seemed to be consistent with peaks previously reported in wheat, while four QTL regions were novel. Four QTLs that affected spike threshability were localized to chromosomes 2BS, 2DS, 4D and 5DS. The QTL on 2DS probably represents the tenacious glume gene, Tg-D1. Based on its map position, the QTL located on 2BS coincides with Ppd-B1 and seems to be a homoeolocus of the soft glume gene. Two novel QTLs were detected on 4D and 5DS, and their goat grass alleles increased glume tenacity. Three novel QTLs located on 2DL, 3DL and 4D for rachis fragility were found. Based on the map position, the QTL on 3DL seems different from Br1 and Br2 loci and its CS allele appears to promote the generation of barrel-type diaspores. Three disarticulation types of spikelets were found in F2 individuals: wedge-type, barrel-type and both types. Among eight QTL peaks that governed spike morphology, six, located on 2AS, 2BS, 2DS, 4AL and 5AL, coincided with ones previously reported. A QTL for spike compactness on 5AL was distinct from the Q gene. A novel QTL that controls spike length was detected on 5DL. Complex genetic interactions between genetic background and the action of each gene were suggested.

Available information on genetically assigned molecular markers is not sufficient for efficient construction of a high-density linkage map in wheat. Here, we report on application of high resolution melting (HRM) analysis using a real-time PCR apparatus to develop single nucleotide polymorphism (SNP) markers linked to a hybrid necrosis gene, Net2, located on wheat chromosome 2D. Based on genomic information on barley chromosome 2H and wheat expressed sequence tag libraries, we selected wheat cDNA sequences presumed to be located near the Net2 chromosomal region, and then found SNPs between the parental Ae. tauschii accessions of the synthetic wheat mapping population. HRM analysis of the PCR products from F(2) individuals' DNA enabled us to assign 44.4% of the SNP-representing cDNAs to chromosome 2D despite the presence of the A and B genomes. In addition, the designed SNP markers were assigned to chromosome 2D of Ae. tauschii. The order of the assigned SNP markers in synthetic hexaploid wheat was confirmed by comparison with the markers in barley and Ae. tauschii. Thus, the SNP-genotyping method based on HRM analysis is a useful tool for development of molecular markers at target loci in wheat.

Common wheat (Triticum aestivum L.) is an allohexaploid that originated from natural hybridization between tetraploid wheat (Triticum turgidum) and diploid Aegilops tauschii. Ae. tauschii is considered one of the potential sources of new genetic variation in abiotic stress tolerance for improving common wheat. Abscisic acid (ABA) plays an important role in plant adaptation to environmental stresses. In this study, ABA responsiveness of 67 Ae. tauschii accessions and their synthetic hexaploid wheat lines, derived from crosses between T. turgidum cv. Langdon and the Ae. tauschii accessions, was evaluated based on growth inhibition by 20 µM ABA. Wide variation was found in ABA responsiveness for both synthetic wheat lines and their parental Ae. tauschii accessions. The variations due to D-genome found at the diploid level were also expressed in a hexaploid genetic background. Two pairs of synthetic wheat lines differing in ABA responsiveness were then selected for gene expression analysis and to test abiotic stress tolerance, because their parental Ae. tauschii accessions similarly exhibited the differential response to ABA. Gene expression of ABA inducible transcription factor, WABI5, and the downstream Cor/Lea genes (Wrab17, Wdhn13 and Wrab18) were analysed. In one pair, the highly responsive line exhibited higher induction of Wrab17 by ABA treatment, but no significant difference in dehydration or salinity tolerance was observed between these lines. In contrast, in the second pair, the highly ABA-responsive line showed higher levels of Wdhn13 expression and dehydration and salinity tolerance. In synthetic wheat lines, the difference in the ABA responsiveness of the lines appeared to be determined by the different sets of D-genome genes. Our findings suggest that highly ABA-responsive Ae. tauschii accessions should be valuable genetic resources for improving the abiotic stress tolerance of common wheat.

Grain shape and size are involved in the main components of the domestication syndrome in cereals. Wheat grain shape has been dramatically altered at each stage of the domestication of tetraploid wheat and through common wheat speciation. To elucidate the evolutionary change of wheat grain shape, principal component (PC) analysis of grain shape-related traits was first conducted using wild and cultivated tetraploid, synthetic hexaploid, and common wheat accessions. The synthetic hexaploid wheat lines were previously produced through interspecific crosses between two common wheat progenitors, tetraploid wheat and Aegilops tauschii, and produced grains similar to those of cultivated tetraploid wheat. To identify genetic loci related to the difference in grain shape between common wheat and the synthetic wheat, the 15 traits related to grain and spikelet shape were measured in 108 F(2) individuals between Norin 61 and a synthetic wheat line, and the first three PC values for the 15 traits, PC1, PC2 and PC3, were mapped as quantitative traits in the F(2) population. In total, six QTLs, found on chromosomes 1A, 5A, 1D, 2D and 7D, showed significant LOD scores. Among them, a QTL for PC2, located on the 2DS chromosomal region near the Ppd-D1 locus, mainly contributed to the phenotypic difference in grain shape. Tg-D1, controlling tenacious glume phenotype, was located at a similar region to the 2DS QTL, which suggested that the Tg-D1 locus pleiotropically affects not only glume toughness but also spikelet and grain shape in hexaploid wheat. Therefore, it was predicted that wheat grains were rapidly improved toward a shorter and rounder phenotype accompanied with free-threshing wheat formation.

Vernalization promotes flowering in winter wheat cultivars, whereas spring wheat cultivars are able to transition from vegetative to reproductive phase without vernalization. The wheat vernalization requirement is mainly controlled by the major locus Vrn-1, an APETALA1/FRUITFULL MADS-box gene homolog. To study natural variation of the vernalization requirement in a wild progenitor of common wheat, we sequenced the Vrn-D(t)1 locus in four accessions of Aegilops tauschii Coss. Some structural mutations were found in the promoter and first intron regions of Vrn-D(t)1, and haplotype analysis was conducted to examine the distribution of each identified mutation within 211 accessions of Ae. tauschii germplasm. Out of the total, nine accessions, which were originally collected in Afghanistan and Pakistan, contained deletions of a 5.4-kb sequence in the critical region of the Vrn-D(t)1 first intron. The 5.4-kb deletion mutation appeared independently of the dominant allele of the common wheat Vrn-D1 locus. The large deletion was absolutely associated with a lack of vernalization requirement for flowering under long-day conditions, but had no influence on heading date under field growth conditions. The levels of Vrn-1 and WFT transcript increased in the Ae. tauschii accessions having the large deletion. Identification of natural mutant accessions with a loss of vernalization requirement indicates the agricultural significance of Ae. tauschii as a genetic resource for wheat breeding.

Maize MuDR/Mu elements are one of the most active Class II transposons and are widely used for transposon tagging for gene cloning. The autonomous MuDR encodes a transposase, while diverse non-autonomous elements share similarity to MuDR only within their ~215 bp terminal inverted repeats (TIRs). Four independent Mu-induced mutable alleles of the anthocyanin pigment pathway Bronze2 (Bz2) locus have been sequenced; bz2-mu1, bz2-mu2, and bz2-mu3 contain Mu1 element insertions while bz2-mu4 contains a MuDR insertion. Somatic excision activity can be monitored for each allele as a purple spotted phenotype on the otherwise beige epidermal layer of the kernel. To study epigenetic silencing of Mu elements, we investigated inheritance of somatic transposition of these four reporter alleles, and using samples from leaves just below the ear and the tassel, DNA methylation status and mudrA expression were quantified through three consecutive generations. Percentages of spotted kernels when crossed into bz2 tester as pollen parent were lower than those when crossed with bz2 tester as ear parent in all bz2-mu alleles. The propensity for silencing in kernels with different frequencies of spotting was investigated. In the inactive lines, both MuDR and Mu1 elements were de novo methylated. The transposition frequency was negatively correlated with the level of Mu methylation and positively correlated with the level of mudrA transcript. The most reliable indicator of incipient silencing was a decrease in mudrA transcript levels in the leaf below the tassel, and this transcriptional silencing could precede methylation of Mu elements.

Mitochondrial alternative oxidase (AOX) is the terminal oxidase responsible for cyanide-insensitive and salicylhydroxamic acid-sensitive respiration in plants. AOX is a key enzyme of the alternative respiration pathway. To study the effects of necrotic cell death on the mitochondrial function, production of reactive oxygen species (ROS), respiration capacities and accumulation patterns of mitochondria-targeted protein-encoding gene transcripts were compared between wild-type, lesion-mimic mutant and hybrid necrosis wheat plants. Around cells with the necrosis symptom, ROS accumulated abundantly in the intercellular spaces. The ratio of the alternative pathway to the cytochrome pathway was markedly enhanced in the necrotic leaves. Transcripts of a wheat AOX gene, Waox1a, were more abundant in a novel lesion-mimic mutant of common wheat than in the wild-type plants. An increased level of the Waox1a transcripts was also observed in hybrid plants containing Ne1 and Ne2 genes. These results indicated that an increase of the wheat AOX transcript level resulted in enhancement of respiration capacity of the alternative pathway in the necrotic cells.

The einkorn wheat (Triticum monococcum) mutant, maintained vegetative phase (mvp), was induced by nitrogen ion-beam treatment and was identified by its inability to transit from the vegetative to reproductive phase. In our previous study, we showed that WAP1 (wheat APETALA1) is a key gene in the regulatory pathway that controls phase transition from vegetative to reproductive growth in common wheat. WAP1 is an ortholog of the VRN1 gene that is responsible for vernalization insensitivity in einkorn wheat. The mvp mutation resulted from deletion of the VRN1 coding and promoter regions, demonstrating that WAP1/VRN1 is an indispensable gene for phase transition in wheat. Expression analysis of flowering-related genes in mvp plants indicated that wheat GIGANTIA (GI), CONSTANS (CO) and SUPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) genes either act upstream of or in a different pathway to WAP1/VRN1.

Under low temperature conditions, the cytochrome pathway of respiration is repressed and reactive oxygen species (ROS) are produced in plants. Mitochondrial alternative oxidase (AOX) is the terminal oxidase responsible for the cyanide-insensitive and salicylhydroxamic acid-sensitive respiration. To study functions of wheat AOX genes under low temperature, we produced transgenic Arabidopsis by introducing Waox1a expressed under control of the cauliflower mosaic virus (CaMV) 35S promoter in Arabidopsis thaliana. The enhancement of endogenous AOX1a expression via low temperature stress was delayed in the transgenic Arabidopsis. Recovery of the total respiration activity under low temperature occurred more rapidly in the transgenic plants than in the wild-type plants due to a constitutively increased alternative pathway capacity. Levels of ROS decreased in the transgenic plants under low temperature stress. These results support the hypothesis that AOX alleviates oxidative stress when the cytochrome pathway of respiration is inhibited under abiotic stress conditions.

A number of cold responsive (Cor)/late embryogenesis abundant (Lea) genes are induced by both low temperature (LT) and dehydration. To understand the molecular basis of cold acclimation and its relationship with drought stress response in wheat seedlings, we isolated a DREB2 homolog Wdreb2, which is the candidate gene for a transcription factor of the Cor/Lea genes. The Wdreb2 expression was activated by cold, drought, salt and exogenous ABA treatment. Detailed expression studies of Wdreb2 indicated the involvement of two distinct pathways of its activation, a drought and salt stress-responsive pathway and a cold-responsive pathway. The transient expression analysis showed that the Wrab19 expression was directly activated by the WDREB2 transcription factor in wheat cells. Three transcript forms of Wdreb2 (Wdreb2alpha, Wdreb2beta and Wdreb2gamma) were produced through alternative splicing. Under drought and salt stress conditions, the amount of the Wdreb2beta form remained fairly constant during 24-hour treatment, while those of the Wdreb2alpha and Wdreb2gamma forms showed transient increases. On the other hand, the LT treatment resulted in increased transcript levels of all three forms of Wdreb2. Thus, under the LT and drought/salt stress conditions the amount of the WDREB2 transcription factors in wheat is differentially controlled by the level of transcription and alternative splicing.

FLORICAULA (FLO) of Antirrhinum and LEAFY (LFY) of Arabidopsis encode plant-specific transcription factors, which are necessary and sufficient to specify floral meristem identity. We isolated WFL, a wheat FLO/LFY ortholog, and analyzed its expression pattern. RT-PCR analysis indicated that WFL is expressed predominantly in young spike. The WFL expression pattern during reproductive development was analyzed in more detail by using in situ hybridization technique. WFL transcripts were observed in all layers of the young spike excepting spikelet initiation sites as axillary meristem. In the double-ridge stage, WFL transcripts were localized in the lower ridge but were absent in the upper ridge, where spikelet meristem initiates. The WFL expression pattern indicated that WFL is associated with spikelet formation rather than floral meristem identity in wheat. As development of floret proceeds, the WFL transcripts were detectable in the developing palea, but not in other floral organs, suggesting that WFL may play a novel role in developing palea in the wheat floret.

The cold acclimation process in plants is primarily regulated through the signal transduction pathways that lead to the induction and enhancement of expression of different sets of Cor/Lea genes. Winter wheat 'Mironovskaya 808' (M808) exhibited a much higher level of freezing tolerance than spring wheat 'Chinese Spring' (CS), and the difference became clearer after the long-term cold acclimation. To understand the molecular basis of this cultivar difference, we isolated two CBF/DREB1 homologs, Wcbf2, which are the candidate gene for a transcription factor of the Cor/Lea genes. Expression of the Wcbf2 gene was induced rapidly by low temperature (LT) and drought but not by abscisic acid (ABA). The gene expression was temporal and at least twice up-regulated by LT. The first up-regulation occurred within 1-4 h, which might correspond to the rapid response to LT, while the second up-regulation occurred during 2-3 weeks of cold acclimation. After the second up-regulation, the amount of Wcbf2 transcript greatly decreased in CS, while it increased again in M808 after 4 weeks until 9 weeks (end of the test period). The maintenance of this high level of the Wcbf2 transcript might represent the long-term effect of cold acclimation. The activation of Cor/Lea genes followed the accumulation of Wcbf2 transcript suggested direct involvement of the Wcbf2 gene in the induction and enhancement of the Cor/Lea gene expression. The cultivar difference in freezing tolerance developed during different stages of cold acclimation can be at least partly explained by the differential and coordinated regulation of the predicted Cor/Lea gene signal transduction pathway that is mediated by the CBF/DREB1 transcription factors in common wheat.

To understand the origin and evolution of common wheat, chloroplast (ct) and nuclear DNA variations were studied in five hexaploid and three tetraploid wheat subspecies. Based on chloroplast simple sequence repeats at 24 loci, they were classified into two major plastogroups. Plastogroup I consisted of 11 plastotypes, including the major plastotype H10 that occurred at the highest frequency (59%) in common wheat. Plastogroup II consisted of five plastotypes and occurred in eight out of 27 accessions of T. aestivum ssp. spelta and one accession of ssp. aestivum. As for nuclear DNA variations, AFLP data using 10 primer sets revealed two major clades of a phylogenetic tree constructed by UPGMA (unweighted pair-group method with arithmetic mean), one consisting of common wheat and the other of emmer wheat. The clade of common wheat was further divided into two major and six minor subclades. One of the major subclades consisted only of non-free-threshing ssp. spelta accessions, which were grouped into two clusters, one consisting only of accessions with plastogroup I ctDNA and the other with both plastogroups I and II. T. aestivum ssp. macha, another non-free-threshing common wheat, formed the other cluster. Taken together, our data indicate the existence of at least two maternal lineages in common wheat and support the hypothesis that European spela wheat originated in Europe separately from other groups of common wheat.

The genome of common wheat has evolved through allopolyploidization of three ancestral diploid genomes. A previously identified restriction fragment length polymorphism (RFLP) marker, pTag546, has the unique feature of showing hypervariability among closely related common wheat cultivars. To understand the origin and the mode of dispersal of this hypervariable sequence in the wheat genome, the distribution and structure of the homologous sequences were studied using ancestral diploid species, tetraploid disomic substitution lines and synthetic hexaploid lines. Comparative Southern blot and PCR analyses suggested that pTag546 homologs in the tetraploid and hexaploid wheat were derived from the S genome of Aegilops speltoides. Some pTag546 homologs were found to have transposed to A and D genomes in polyploid wheat. Evidence of transposition and elimination in some synthetic hexaploid lines was also obtained by comparing their copy numbers with those in the parental lines. Southern blot analysis of a genomic clone using a contiguous subset of sequences as probes revealed a core region of hypervariability that coincided with the region containing pTag546. No obvious structural characteristics that could explain the hypervariability, however, were found around the pTag546 sequence, except for accumulation of small repetitive sequences at one border. It was concluded that pTag546 increased its copy number through yet unknown mechanism(s) of transposition to various chromosomal locations over the period of allopolyploid evolution and during the artificial genome manipulation in wheat.

Structural heterogeneity depicted as heteroplasmy of the mitochondrial (mt) transcriptional unit of nad3-orf156 (atp8) was studied in a nucleus-cytoplasm (NC) hybrid of Triticum timopheevi with the D plasmon from the maternal Aegilops squarrosa and compared with that of the parental lines. The tetraploid NC hybrid and the parental lines both showed varying degrees of heteroplasmy in this mtDNA region. The G plasmon of the paternal T. timopheevi possessed five sequence types, while two sequence types were detected in the D plasmon of Ae. squarrosa. The NC hybrid possessed all the five sequence types identical to those of the paternal parent in a 30% relative stochiometry. The remaining 70% comprised only one of the two maternal sequence types, suggestive of strong and selective NC interaction. No novel sequence types were detected and the relative stoichiometries of the paternal sequence types were conserved in the NC hybrid. No paternal-identical or -related sequences were detected in the maternal D plasmon. These results provide evidence of the paternal transmission of the mtDNA and possibly account for the origin of the observed mtDNA heteroplasmy in the NC hybrid.

Mitochondrial alternative oxidase (AOX) is the terminal oxidase responsible for cyanide-insensitive and salicylhydroxamic acid-sensitive respiration. We have isolated two non-homoeologous genes (Waox1a and Waox1c) encoding AOX proteins from common wheat (Triticum aestivum L.). These two genes were orthologous to rice AOX1a and AOX1c, and their exon/intron structure was conserved, as it is in most other plant AOX genes. Southern blot analysis indicated that both Waox1a and Waox1c were located in at least three homoeologous loci and that additional AOX genes with lower homology were present in the genome of common wheat. The Waox1a and Waox1c loci were respectively assigned to the homoeologous group 2 and 6 chromosomes. The steady-state level of Waox1a and Waox1c transcripts increased under cold stress, while only that of Waox1a was increased by cyanide treatment.

Heterotrimeric GTP-binding proteins (G-proteins) play important roles in signal transduction pathways in eukaryotic cells. Through differential screening of a hairy root cDNA library of tobacco (Nicotiana tabacum L.) against transcripts from non-root tissues of normal cuttings, we obtained a partial cDNA clone that showed abundant expression and high homology to the alpha subunit gene of plant G-protein. After RACE-PCR, a full-length cDNA clone was obtained, which was 1,677-bp in length and contained an open reading frame encoding a protein of 384 amino acids. A cDNA clone encoding a beta subunit of G-protein was also isolated from the same cDNA library based on PCR amplification and library screening. The clone was 1,600-bp in length and contained an open reading frame encoding 377 amino acids. The deduced amino acid sequences of these clones showed high homology (75.5 to 99.8% amino acid identity) with alpha and beta subunits of other plant G-proteins. Genomic Southern blot analysis showed that the amphidiploid tobacco genome possessed two major copies of both alpha and beta subunit genes and some minor homologous copies. Northern blot analysis showed that the transcript of alpha subunit gene was abundant in the root tissues, particularly in the hairy root tissues. In contrast, the level of expression of the beta subunit gene was equivalent in all the tissues studied. Possible function of tobacco G-protein was discussed.

A Cor (cold-responsive) cDNA that belongs to the group-3 Lea (late embryogenesis abundant)/Rab (responsive to abscisic acid, ABA) family was isolated from a winter-hardy cultivar of common wheat (Triticum aestivum L.). Screening of a cold-acclimated cDNA library was performed using an ABA- and other stress-responsive barley cDNA clone, Hva1, as a probe. A wheat cDNA clone (designated as Wrab19) putatively encoded a basic (pI = 10.3) and hydrophobic protein with 179 amino acids. The deduced protein showed characteristics of the group-3 LEA/RAB protein family. In contrast to the single copy barley Hva1, Wrab19 belonged to a multigene family in the hexaploid wheat genome and six loci were assigned to the homoeologous group 1 chromosomes. Using Wrab19 as a probe, four homologous cDNAs (designated as Wrab17) were isolated that encoded acidic (pI = 4.6-4.7) and hydrophobic proteins, all with 166 amino acids. The deduced proteins showed high homology (a mean of 84% identity) with a barley gibberellic acid (GA3)-inducible protein, ES2A, and several other group-3 LEA/RAB proteins. Wrab17 was considered to be a three-copy gene and each copy was assigned to chromosome 5A, 4B or 4D of hexaploid wheat. Transcripts of both Wrab19 and Wrab17 accumulated within 1 day of cold acclimation at 4 degrees C. They were responsive to ABA and/or GA3, but showed some cultivar differences in their response to these plant hormones. We conclude that the two genes are new members of the group-3 Lea/Rab-related Cor gene family in wheat.

A cDNA library was constructed from a cold-acclimated winter-hardy common wheat (Triticum aestivum L.) cultivar 'Mironovska 808'. Using this library and a cold- and light-responsive barley cDNA clone cor14b as a probe, cDNAs of a homologous wheat gene wcor14 were isolated. Two identical cDNAs designated as wcor14a had an open reading frame encoding an acidic (pI = 4.71) and hydrophobic polypeptide with 140 amino acids (MW = 13.5 kDa). The deduced WCOR14a polypeptide showed 70% identity with the barley chloroplast-imported COR14b and had a nearly identical N-terminal, putative chloroplast transit peptide of 51 amino acid residues. Another cDNA clone wcor14b was assumed to encode a polypeptide WCORb which had 5 substitutions and a frame shift in the C-terminal region as compared with WCOR14a. RACE PCR, genomic PCR and Southern blot analyses suggested that wcor14 and its related sequences constitute a small multigene family with and without an intron in the hexaploid wheat genome. Northern blot analysis showed that transcripts of wcor14 accumulated within 3-6 hours of cold acclimation at 4 degrees C and the level reached a maximum at day 3. The transcripts became non-detectable within 3 hours after de-acclimation at room temperature. Contrary to the barley cor14b, a similar level of wcor14 transcripts was detected under the continuous darkness. Neither treatment with NaCl, ABA nor dehydration induced its expression. Based on these results we conclude that wcor14 is a wheat orthologue of the barley cor14b and specifically induced by low temperature.

The transformation technique in common wheat has already been established by using microprojectile bombardment and scutellar tissues of immature embryos. In this study, in vitro culture response of immature embryos and the production of transgenic wheat plants were examined in six common wheat cultivars, i.e., Chinese Spring, Akadaruma, Haruhikari, Shiroganekomugi, Norin 12, and Norin 61. In all genotypes, more than seven hundred immature embryos were bombarded with a plasmid containing a bialaphos-resistant gene under control of the rice actin 1 gene. (Act1) promoter. Although the transient expression of the reporter gene encoding beta-glucuronidase following the rice Act1 promoter was similar in five of the six cultivars tested, the frequency of stable transformation varied with the genotype. The frequency of transformation was the highest in Akadaruma and Norin 12 of the six wheat cultivars; independently transformed plants were produced from 1.4% and 1.7% of bombarded embryos, respectively. On the other hand, the immature embryos of Norin 61 and Shiroganekomugi showing low efficiency of in vitro culture generated no transgenic plants. This variation of the transformation frequency was generally caused by the difference in the in vitro culture response with the genotype, rather than the efficiency of the introduction of the transgene into wheat cells by particle bombardment.