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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.

Common wheat represents a typical allohexaploid species and provides a good experimental system for studying genomic alterations associated with allopolyploidization. We studied three homoeologous loci of wheat Wknox1 gene, which is highly homologous to the knotted1 (kn1)-like homeobox (KNOX) genes functioning at shoot apical meristems (SAM). Wknox1 transcripts were detected in SAM, and its overexpression caused abnormal leaf morphology with occasional ectopic leaves in transgenic tobacco plants. A comparative study of the three Wknox1 genomic sequences revealed accumulation of a large number of mutations including insertions and deletions, particularly in the fourth intron and the 5'-upstream region. Some structural mutations including MITE-insertions were allocated in the evolutionary lineage of the wheat genome, suggesting that they occurred at all stages of wheat evolution. A mutation rate was the highest in the Wknox1b locus, which is consistent with the known highest degree of differentiation in the B genome. Despite the structural differentiation, all three Wknox1 homoeologs showed an identical expression pattern in wheat and their promoter regions induced the conserved expression pattern in transgenic tobacco plants. A potential of the intragenic diversity in homoeologs of essential genes as a tool for studying the genome evolution associated with allopolyploidization was discussed.

Vrn-1/Fr-1 chromosomal regions of common wheat possess major QTLs for both winter hardiness (Fr) and vernalization requirement (Vrn). The Vrn-1/Fr-1 intervals are assigned to long arms of the homologous 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 non-carrier 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.

Interaction between nucleus and cytoplasm has essential roles in plant development, including that of floral organs. We isolated a wheat homolog Whlp of Arabidopsis HUELLENLOS PARALOG (HLP) gene encoding a mitochondrial (mt) ribosomal protein L14. Transient expression analysis using the green fluorescent protein (GFP) fusion protein showed that 50 amino residues located on the N-terminal of the wheat HLP homolog (WHLP) protein acted as a mt-targeting signal (MTS). Expression patterns of the Whlp gene were compared among floral organs of alloplasmic lines of wheat, in which intrinsic cytoplasms were replaced by the cytoplasm of a wild relative Aegilops crassa. In these alloplasmic lines, pistillody (homeotic transformation of stamens into pistil-like organs) is induced by the alien cytoplasm in the absence of nuclear restorer genes. The Whlp transcripts preferentially accumulated in stamens compared with pistils, leaves, and roots. The expression level of Whlp in the pistillate stamens of the alloplasmic lines was similar to that in genuine pistils of both euplasmic lines and fertile alloplasmic lines. The result suggested that the elevated expression of the Whlp gene plays a role in aiding the development of male reproductive organ but not in the determination of its whorl identity. A comparable expression pattern was observed in another nuclear-encoded mt ribosomal protein gene but not in a mt-encoded gene. The different expression patterns of different mt ribosomal protein genes suggest that the abundance of mt ribosomal proteins is differentially regulated in the organ/tissue development in wheat.

Japanese rice ( Oryza sativa L.) cultivars that are strictly used for the brewing of sake (Japanese rice wine) represent a unique and traditional group. These cultivars are characterized by common traits such as large grain size with low protein content and a large, central white-core structure. To understand the genetic diversity and phylogenetic characteristics of sake-brewing rice, we performed amplified fragment length polymorphism and simple sequence repeat analyses, using 95 cultivars of local and modern sake-brewing rice together with 76 cultivars of local and modern cooking rice. Our analysis of both nuclear and chloroplast genome polymorphisms showed that the genetic diversity in sake-brewing rice cultivars was much smaller than the diversity found in cooking rice cultivars. Interestingly, the genetic diversity within the modern sake-brewing cultivars was about twofold higher than the diversity within the local sake-brewing cultivars, which was in contrast to the cooking cultivars. This is most likely due to introgression of the modern cooking cultivars into the modern sake-brewing cultivars through breeding practices. Cluster analysis and chloroplast haplotype analysis suggested that the local sake-brewing cultivars originated monophyletically in the western regions of Japan. Analysis of variance tests showed that several markers were significantly associated with sake-brewing traits, particularly with the large white-core structure.

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.

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.

Brown planthopper (BPH) (Nilaparvata lugens Stål) is a significant insect pest of rice (Oryza sativa L.). We constructed a gene-pyramided japonica line, in which two BPH resistance genes Bph1 and Bph2 on the long arm of chromosome 12 independently derived from two indica resistance lines were combined through the recombinant selection. The gene-pyramiding was achieved based on the previously constructed high-resolution linkage maps of the two genes. Two co-dominant and four dominant PCR-based markers flanking the loci were used to select for a homozygous recombinant line in a segregating population that was derived from a cross between the parental homozygous single-gene introgression lines. BPH bioassay showed that the resistance level of the pyramided line was equivalent to that of the Bph1-single introgression line, which showed a higher level of resistance than the Bph2-single introgression line. The pyramid line should provide a useful experimental means for studying the fine structure of the chromosomal region covering these two major BPH resistance 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.

Wcor15, a member of the wheat cold-responsive (Cor) gene family, has been isolated and characterized. The deduced polypeptide WCOR15 (MW=14.7 kDa) showed high homology to the previously identified wheat and barley COR proteins. Southern blot analysis using diploid, tetraploid and hexaploid wheat and diploid Aegilops species showed that the wheat and related wild genomes possessed multiple copies of Wcor15 homologues. Five copies were assigned to the homologous group 2 chromosomes by nulli-tetrasomic analysis. Northern blot analysis showed that expression of Wcor15 was specifically induced by low-temperature. Homologous transcripts accumulated in leaves, and light markedly increased their steady-state levels. Bombardment-mediated transient expression analysis of a chimeric CaMV 35S::Wcor15-GFP construct showed protein-targeting to epidermal guard cell chloroplasts in excised spiderwort leaves. A promoter of Wcor15 contained at least three CRT/DRE-like sequence motifs found in Arabidopsis Cor genes and induced the reporter GUS gene expression in leaves of transgenic tobacco plants under low-temperature and light conditions. These results suggest that the functional Cor gene system involving the CRT/DRE cis-element is conserved in both monocotyledonous and dicotyledonous plants.

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.

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.

A heterotrimeric GTP-binding protein (G protein) plays a number of important roles in the signal-transduction pathways of eukaryotic cells. The allotetraploid tobacco genome has two alpha-subunit genes, NtGA1 and NtGA2, of the heterotrimeric G protein. In this study, we determined the nucleotide sequences and the exon-intron structures of the NtGA loci in tobacco and its ancestral diploid species. The genomic sequences of the NtGA loci were interrupted by 13 introns. The sizes of most exons (12 of 14) were completely conserved among the NtGA genes and the Arabidopsis alpha-subunit gene (GPA1), but most introns (11 of 13) in the NtGA genes were longer than those in GPA1. In comparison with the genomic sequences of the NtGA orthologues of ancestral Nicotiana sylvestris and Nicotiana tomentosiformis, the tobacco NtGA1 and NtGA2 were concluded to be homoeologous and assigned to the S and T genomes, respectively. More than 300 mutations including insertions-deletions (indels) and nucleotide substitutions were found in the intron regions between the NtGA1 and NtGA2 loci, whereas the exon sequences were highly conserved among these and GPA1. The structural comparison revealed larger divergence at the NtGA2 locus than at NtGA1.

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.

A mitochondrial (mt) transcriptional unit, nad3-orf156, was studied in the nucleus-cytoplasm hybrids of wheat with D/D2 plasmons from Aegilops species and their parental lines. A comparative RFLP analysis and sequencing of the random PCR clones revealed the presence of seven sequence types and their polymorphic sites were mapped. All the hybrids possessed the paternal copies besides the maternal copies. More paternal copies were present in the D2 plasmon hybrids, whereas more maternal copies were present in the D plasmon hybrids. Two major copies were present with different stoichiometries in the maternal Aegilops parents. However, only a major D plasmon copy was detected in the hybrids, irrespective of their plasmon types. The hexaploid wheat parent (AABBDD genome) possessed the major D plasmon copy in approximately 5% stoichiometry, while no D plasmon-homologous copies were detected in the tetraploid wheat parent (AABB genome). The results suggest that the observed mtDNA heteroplasmy is due to paternal contribution of mtDNA. The different copy stoichiometry suggests differential amplification of the heteroplasmic copies among the hybrids and the parental lines. All editing sites and their editing frequencies were conserved among the lines, and only the maternal pattern of editing occurred in the hybrids.

Structural features of the wheat plastome were clarified by comparison of the complete sequence of wheat chloroplast DNA with those of rice and maize chloroplast genomes. The wheat plastome consists of a 134,545-bp circular molecule with 20,703-bp inverted repeats and the same gene content as the rice and maize plastomes. However, some structural divergence was found even in the coding regions of genes. These alterations are due to illegitimate recombination between two short direct repeats and/or replication slippage. Overall comparison of chloroplast DNAs among the three cereals indicated the presence of some hot-spot regions for length mutations. Whereas the region with clustered tRNA genes and that downstream of rbcL showed divergence in a species-specific manner, the deletion patterns of ORFs in the inverted-repeat regions and the borders between the inverted repeats and the small single-copy region support the notion that wheat and rice are related more closely to each other than to maize.

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.

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.

Mitochondrial (mt) DNA structures were studied in 12 nucleus-cytoplasm (NC) hybrids of tetraploid and hexaploid wheat in which nuclear genomes of the recurrent paternal wheat parents were combined with D and D2 plasmons from Aegilops species. RFLP analysis of 15 mtDNA regions indicated the presence of the paternal sequences in six regions encompassing 11 structural genes in all the NC hybrids. PCR-RFLP analysis showed that one region (a nad3-orf156 region) consisted of a mixture of the maternal, paternal and novel paternal-like sequences. The presence of unexpectedly high levels of the paternal sequences was confirmed by random PCR cloning and sequencing of this region. PCR-RFLP analysis of the random clones further showed that the relative stoichiometry of the maternal and paternal sequences varied depending on the plasmons from the maternal parents and the nuclear backgrounds of the paternal parents. Our results suggest that the differential amplification of the paternal mtDNA sequences is under the control of NC interaction in these NC hybrids.

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.

To investigate the excision of a maize transposable element in wheat cells, plasmid DNAs containing a Dissociation (Ds) element located between a rice actin 1 gene promoter and a beta-glucuronidase (GUS) gene (gus) were introduced into wheat callus lines by microprojectile bombardment, and transient GUS expression was assayed. The gus-expressing cells after Ds excision were detected only when the Activator (Ac) transposase gene was co-transformed. To further examine a relationship between the amount of Ac mRNA and the Ds excision frequency, the Ds-containing plasmids were introduced into 15 independent transgenic callus lines transformed with the Ac transposase gene. Ten lines expressed the Ac transposase gene under the control of either the cauliflower mosaic virus 35S promoter or the Ac native promoter. The gus gene expression that indicated the Ds excision was observed only in the transgenic callus lines stably expressing the Ac transposase gene. The number of blue spots reflecting the frequency of Ds excision was variable among them. Northern-blot analysis also showed a large variability in the amount of Ac transposase transcripts among the lines. It was however noted that the excision frequency was decreased at a high level of the Ac transposase transcripts, supporting the hypothesis that Ds excision is inhibited above a certain level of the Ac transposase as observed in maize and transgenic tobacco.

A method using particle bombardment and beta-glucuronidase (GUS) assay was applied to rice callus for detecting the excision of the maize Ds element trans-activated by the Ac transposase source. Excision of Ds biolistically introduced into rice callus resulted in the restoration of the interrupted gus gene expression, allowing visual detection of trans-activation two days after bombardment. Only the transgenic callus lines expressing the Ac transposase gene and the wild-type callus co-transformed with Ac and Ds revealed GUS activity. Frequency of excision, estimated based on the relative GUS activity, ranged from 0.3% to 2.2%. Callus lines showing different levels of Ac transcripts revealed varying excision frequencies. At the later stages of callus growth after selection for the Ac/Ds transformed lines, excision events were detected by GUS assay and confirmed by PCR and sequence analyses of the excision sites in individual colonies. GUS activity was also demonstrated in the primary regenerants from the Ac/Ds-transformed callus colonies. The method described in this study may be used as an approach for rapid detection of excision events and assessment of various factors limiting Ac/Ds activity in rice cells.

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.

To investigate the activation and transposition of maize transposable elements in wheat cultured cells, plasmid DNAs containing the maize Ac/Ds elements located between the CaMV 35S promoter and a hygromycin B resistance gene (hph) were introduced into two wheat (Triticum aestivum and Triticum monococcum) cultured cell lines by microprojectile bombardment. In the first experiment, hph was activated by excision of the Ac element, which encodes transposase, in the two wheat cell lines. In the second experiment, the Ds element was excised by a stabilized Ac element, lacking inverted repeats of the Ac element and located on another plasmid, and therefore leading to activation of hph. After selection of bombarded cells by hygromycin B, many resistant calli were recovered in both wheat cell lines. The integration of hph and the Ac transposase gene was confirmed by PCR and genomic Southern analysis. The stable expression of hph and the transposase gene was also assessed by Northern blot and reverse transcriptase PCR analysis, respectively. Moreover, characteristic sequence alterations were found at Ac/Ds excision sites. These findings indicate that the maize Ac/Ds transposable elements are activated and excised by expression of the Ac transposase gene in both diploid and hexaploid wheat cells.