Genetic diversity and phylogeny of Japanese sake-brewing rice as revealed by AFLP and nuclear and chloroplast SSR markers.

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.

Cold-specific and light-stimulated expression of a wheat (Triticum aestivum L.) Cor gene Wcor15 encoding a chloroplast-targeted protein.

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.

Structural features of a wheat plastome as revealed by complete sequencing of chloroplast DNA.

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.

Expression of a cold-responsive Lt-Cor gene and development of freezing tolerance during cold acclimation in wheat (Triticum aestivum L.).

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.

New members of a cold-responsive group-3 Lea/Rab-related Cor gene family from common wheat (Triticum aestivum L.).

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.

Nicotiana tabacum cDNAs encoding alpha and beta subunits of a heterotrimeric GTP-binding protein isolated from hairy root tissues.

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.

Identification of paternal mitochondrial DNA sequences in the nucleus-cytoplasm hybrids of tetraploid and hexaploid wheat with D and D2 plasmons from Aegilops species.

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.

Molecular cloning of three homoeologous cDNAs encoding orthologs of the maize KNOTTED1 homeobox protein from young spikes of hexaploid wheat.

The plant knotted1 (kn1)-like homeobox genes are known to play important roles in the maintenance of shoot apical meristem (SAM), determination of cell fate and differentiation of vegetative tissues. To study structural diversity of the three homologous loci encoding a KN1-like homeobox protein in the hexaploid wheat genome, we isolated clones from a cDNA library of young spikes of Japanese common wheat cultivar 'Norin 26'. Three different but highly homologous cDNAs were isolated and their sequences were determined. The mean homology of the deduced amino acid sequences was 96% as compared to the barley ortholog KNOX3. The wheat kn1-like homeobox proteins named WKNOX1 are encoded by a single set of homologous genes on the homologous group 4 chromosomes in the three component genomes of common wheat, i.e. 4A, 4B and 4D. The nucleotide sequence data and the Southern blot pattern suggested that the three homologous loci of wknox1 genes are highly conserved through polyploid evolution of wheat. They were expressed in SAM-containing shoots and young spikes but not in developed leaves, glumes and lemmas and callus tissues. The ectopic expression of the wknox1 was observed in lemma of wheat Hooded (Hd) mutants. The result suggested that the Hd gene is a dominant allele of the wknox1 locus on chromosome 4A.

A cold-responsive wheat (Triticum aestivum L.) gene wcor14 identified in a winter-hardy cultivar 'Mironovska 808'.

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.

Variations in the maize Ac transposase transcript level and the Ds excision frequency in transgenic wheat callus lines

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.

Ac-mediated trans-activation of the Ds element in rice (Oryza sativa L.) cells as revealed by GUS assay.

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.

Does Decrease in Ribulose-1,5-Bisphosphate Carboxylase by Antisense RbcS Lead to a Higher N-Use Efficiency of Photosynthesis under Conditions of Saturating CO2 and Light in Rice Plants?

Rice (Oryza sativa L.) plants with decreased ribulose-1,5-bisphosphate carboxylase (Rubisco) were obtained by transformation with the rice rbcS antisense gene under the control of the rice rbcS promoter. The primary transformants were screened for the Rubisco to leaf N ratio, and the transformant with 65% wild-type Rubisco was selected as a plant set with optimal Rubisco content at saturating CO2 partial pressures for photosynthesis under conditions of high irradiance and 25[deg]C. This optimal Rubisco content was estimated from the amounts and kinetic constants of Rubisco and the gas-exchange data. The R1 selfed progeny of the selected transformant were grown hydroponically with different N concentrations. Rubisco content in the R1 population was distributed into two groups: 56 plants had about 65% wild-type Rubisco, whereas 23 plants were very similar to the wild type. Although the plants with decreased Rubisco showed 20% lower rates of light-saturated photosynthesis in normal air (36 Pa CO2), they had 5 to 15% higher rates of photosynthesis in elevated partial pressures of CO2, (100-115 Pa CO2) than the wild-type plants for a given leaf N content. We conclude that the rice plants with 65% wild-type Rubisco show a higher N-use efficiency of photosynthesis under conditions of saturating CO2 and high irradiance.

Variation in transformation frequencies among six common wheat cultivars through particle bombardment of scutellar tissues.

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.

Hygromycin-resistant calli generated by activation and excision of maize Ac/Ds transposable elements in diploid and hexaploid wheat cultured cell lines.

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.