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Aegilops tauschii is the diploid progenitor of the bread wheat D-genome. It originated from Iran and is a source of abiotic stress tolerance genes. However, little is known about the molecular events of salinity tolerance in Ae. tauschii. This study investigates the leaf transcriptional changes associated with long-term salt stress. Total RNA extracted from leaf tissues of control and salt-treated samples was sequenced using the Illumina technology, and more than 98 million high-quality reads were assembled into 255,446 unigenes with an average length of 1398 bp and an N50 of 2269 bp. Functional annotation of the unigenes showed that 93,742 (36.69%) had at least a significant BLAST hit in the SwissProt database, while 174,079 (68.14%) showed significant similarity to proteins in the NCBI nr database. Differential expression analysis identified 4506 salt stress-responsive unigenes. Bioinformatic analysis of the differentially expressed unigenes (DEUs) revealed a number of biological processes and pathways involved in the establishment of ion homeostasis, signaling processes, carbohydrate metabolism, and post-translational modifications. Fine regulation of starch and sucrose content may be important features involved in salt tolerance in Ae. tauschii. Moreover, 82% of DEUs mapped to the D-subgenome, including known QTL for salt tolerance, and these DEUs showed similar salt stress responses in other accessions of Ae. tauschii. These results could provide fundamental insight into the regulatory process underlying salt tolerance in Ae. tauschii and wheat and facilitate identification of genes involved in their salt tolerance mechanisms.

We produced transgenic Arabidopsis plants that express chimeric genes for transcription factors converted to dominant repressors, using Chimeric REpressor gene-Silencing Technology (CRES-T), and evaluated the salt tolerance of each line. The seeds of the CRES-T lines for ADA2b, Msantd, DDF1, DREB26, AtGeBP, and ATHB23 exhibited higher germination rates than Wild type (WT) and developed rosette plants under up to 200 mM NaCl or 400 mM mannitol. WT plants did not grow under these conditions. In these CRES-T lines, the expression patterns of stress-related genes such as RD29A, RD22, DREB1A, and P5CS differed from those in WT plants, suggesting the involvement of the six transcription factors identified here in the stress response pathways regulated by the products of these stress-related genes. Our results demonstrate additional proof that CRES-T is a superior tool for revealing the function of transcription factors.

We have previously reported that expression of salt-responsive genes, including Bruguiera gymnorhiza ankyrin repeat protein 1 (BgARP1), enhances salt tolerance in both Agrobacterium tumefaciens and Arabidopsis. In this report, we further characterized BgARP1-expressing Arabidopsis to elucidate the role of BgARP1 in salt tolerance. BgARP1-expressing plants exhibited more vigorous growth than wild-type plants on MS plates containing 125–175 mM NaCl. Real-time PCR analysis showed enhanced induction of osmotin34 in the 2-week-old transformants under 125 mM NaCl. It was also showed that induction of typical salt-responsive genes, including RD29A, RD29B, and RD22, was blunted and delayed in the 4-week-old transformants during 24 h after 200 mM NaCl treatment. Ion content analysis showed that transgenic plants contained more K+, Ca2+, and NO3 −, and less NH4 +, than wild-type plants grown in 200 mM NaCl. Our results suggest that BgARP1-expressing plants may reduce salt stress by up-regulating osmotin34 gene expression and maintaining K+ homeostasis and regulating Ca2+ content. These results indicate that BgARP1 is functional on a heterogeneous background.

Genes involved in the fertility restoration (Rf) of wild-abortive (WA)-type cytoplasmic male sterility (CMS) in rice have not been clearly identified, because of the inconsistency of their number and map position. Although the Rf-1 genes, which restore Chinsurah Boro (BT)-type CMS, have been recently isolated, their effects on WA-type CMS are still unclear. In the present study, near-isogenic lines (NILs) of japonica rice, differing at Rf loci, were developed using marker-assisted selection (MAS) to determine the effect of each Rf gene on WAor BT-cytoplasm in a japonica background. The NILs were crossed with a japonica CMS line, then the pollen and seed fertility of the hybrid plants was analyzed. The Rf-1 locus consisting of two Rf genes from IR36 was sufficient to restore fertility in the hybrids with BT-type CMS but conferred very little fertility to those with WA-type CMS. Rf-3 on chromosome 1, a weak Rf gene, and Rf-6(t) on chromosome 10 were unable to confer seed fertility to plants with BT-type or WA-type CMS. Both Rf genes, however, could accelate the development of abortive pollen in the case of WA-type CMS and their effect was additive. Crossing of CMS rice with chromosome segment substitution lines carrying Rf loci from IR24 resulted in similar pollen development and seed fertility.

An antisense gene strategy was used to suppress the expression of rice allergen (RA) genes that comprise a multigene family. We produced transgenic rice plants harboring antisense genes that consisted of cDNA for a major RA driven by promoters of the RA, rice starch branching enzyme, rice prolamine and rice glutelin genes, which are specifically expressed in developing seeds. Among more than 100 transformants, the RA content in some transformants was reduced to no more than 20 % of that in non-transformant. However, an allergen-free transformant was not obtained. Since the transcribed antisense mRNAs are not fully used for suppression of the RA genes in transformants, we examined here the possibility of a spatial or temporal difference in the expression of the introduced antisense and endogenous RA genes. However, we found no remarkable difference in the place or timing of their expression. We also examined the effect of antisense genes on each of the genes in the RA family in transformants by twodimensional electrophoresis and immunoblotting. The results showed that the contents of the major RA and other RAs with nucleotide sequences that are highly homologous to the antisense sequence were markedly lowered, while the contents of other RAs with sequences showing relatively low homology to the major RA were hardly affected. These results suggest that the effect of the antisense gene on the RA gene family depends on the homology between the antisense sequence used and the target sequence.

Probenazole (3-allyloxy-1,2-benzisothiazole-1,1-dioxide) is an agricultural chemical primarily used to prevent rice blast disease. Probenazole-treated rice acquires resistance to blast fungus irrespective of the rice variety. The chemical is applied prophylactically, and is thought to induce or bolster endogenous plant defenses. However, the mechanisms underlying this effect have not been established. To understand the mode of the chemical's action, we screened for novel probenazole-responsive genes in rice by means of differential display and identified a candidate gene, RPR1. RPR1 contains a nucleotide binding site and leucine-rich repeats, thus sharing structural similarity with known disease resistance genes. The expression of RPR1 in rice can be up-regulated by treatment with chemical inducers of systemic acquired resistance (SAR) and by inoculation with pathogens. RPR1-related sequences in rice varieties seem to be varied in sequence and/or expression, indicating that RPR1 itself is not a crucial factor for induced resistance in rice. However, Southern blot analysis revealed the existence of homologous sequences in all varieties examined. While the role of RPR1 has yet to be clarified, this is the first report of the identification of a member of this gene class and its induction during the systemic expression of induced disease resistance.

An antisense gene strategy was applied to suppress the 14–16 kDa allergen gene expression in maturing rice seeds. Gene constructs producing antisense RNAs of the 16 kDa allergen under the control of some rice seed-specific promoters were introduced into rice by electroporation. Immunoblot and RNA blot analyses of the seeds from the transgenic rice plants using the allergen-specific monoclonal antibody and a sequence-specific antisense RNA probe demonstrated that the 14–16 kDa allergen proteins and their transcripts of the seeds from several transgenic lines were present in much lower in amounts than those of the seeds from parental wild-type rice. The high levels of reduction observed were stably inherited in at least three generations.

A translational initiation sequence, conserved among soybean lipoxygenase genes, was fused with the β-glucuronidase (GUS) gene, placed under 35S cauliflower mosaic virus promoter control, then electroporated into suspension-cultured protoplasts of tobacco and rice. These constructs produced 11- and 7-fold increases in GUS activity, respectively, compared with the original GUS construct. The transcriptional activity of the modified GUS gene fused with the TATA box from a soybean lipoxygenase gene was also detected with great sensitivity. The fusion construct is useful for detectingcis-regulatory element effects on transcription, in particular, silencers.

The beta-glucuronidase (GUS) gene linked to the promoter of rice light harvesting chlorophyll a/b-binding protein of photosystem II (LHCPII) was introduced into rice by electroporation. Expression of the GUS gene with the LHCP promoter in transformed protoplasts and callus was much lower than with the cauliflower mosaic virus (CaMV) 35S promoter. In green organs, however, LHCP-GUS activity with the LHCP promoter was 10-fold higher than that with the CaMV 35S promoter. Expression of the LHCP-GUS gene was detected in leaves, stems and floral organs but not in roots. Histochemical analysis of leaves showed that the GUS gene was expressed specifically in green tissues. In selfed seeds of the transformants, GUS gene expression was detectable only in the germinated embryos after greening. Northern blot analysis revealed that transcription of the GUS gene with the LHCP promoter was not detectable in dark-grown seedlings of transformants but was induced by exposure to light. These results show that regulation of the introduced chimeric gene in transgenic plants was characteristic of the LHCP gene itself.

We have developed a method for reproducibly obtaining transgenic rice at a high frequency (10(-4)): electroporation with a buffer in which chloride ions are replaced with organic acids. Co-transformation frequencies of the β-glucuronidase (GUS) and hygromycin phosphotransferase (HPT) genes located on two separate plasmids were higher than 50%. Transgenic rice plants contained multiple copies of introduced genes integrated into their genomes in a complex manner. GUS enzyme activity was not proportional to gene copy number. Introduced HPT genes were detected and expressed in the progeny of transformants.

Transgenic rice plants (Oryza sativa cv. Nipponbare) carrying 1 or 2 copies of a rice homeobox gene, OSH1, under the control of the CaMV 35S promoter were generated. The transgene caused altered morphology of leaf, such as ligule-replacement and abnormal division of sclerenchyma cells. The phenotype of these leaves resembles that of maize leaf morphological mutant, Knotted 1, which is caused by duplication of the KN1 gene (Veit et al., 1990). The in situ hybridization analysis has revealed that the expression of endogenous OSH1 is mainly localized in developing vascular strands of stem. We have discussed the biological roles of OSH1 in rice based on these results.

In angiosperms, the expression of the cab gene that encodes the chlorophyll a/b-binding protein of PSII is light-regulated. However, the pine cab gene is expressed in a light-independent but cell-type-specific manner. In the present study, the cab-6 promoter (1.7 kbp) from pine was fused to a beta-glucuronidase (GUS) reporter gene and the chimeric gene was introduced into rice protoplasts by electroporation. The GUS expression was studied in the resultant transgenic rice plants. Expression of GUS at a substantial level was confirmed in primary leaves of dark-germinated rice seedlings, and no obvious effect of light on the GUS activity was observed. The expression of GUS was restricted to photosynthetic tissues. The pine cab-6 promoter is, thus, sufficient for induction of light-independent but cell-type-specific expression in cells of a monocot, as is the case in the original pine cells.

Pyruvate,orthophosphate dikinase (PPDK; EC 2.7.9.1) activity is abundant in leaves of C4 plants, while it is difficult to detect in leaves of C3 plants. Recent studies have indicated that C3 plants have a gene encoding PPDK, with a structure similar to that of PPDK in C4 plants. However, low expression makes PPDK detection difficult in C3 plants. This finding suggests that high PPDK expression in C4 plants is due to regulatory mechanisms which are not operative in C3 plants. We have introduced a chimeric gene consisting of the gene encoding beta-glucuronidase (GUS; EC 3.2.1.31) controlled by the PPDK promoter from a C4 plant, maize, into a C3 cereal, rice. The chimeric gene was exclusively expressed in photosynthetic organs, leaf blades and sheaths, and not in roots or stems. Histochemical analysis of GUS activity demonstrated high expression of the chimeric gene in photosynthetic organs, localized in mesophyll cells, and no or very low activity in other cells. GUS expression was also regulated by light in that it was low in etiolated leaves and was enhanced by illumination. These observations indicate that the mechanisms responsible for cell-specific and light-inducible regulation of PPDK observed in C4 plants are also present in C3 plants. We directly tested whether rice has DNA-binding protein(s) which interact with a previously identified cis-acting element of the C4-type gene. Gel retardation assays indicate the presence in rice of a protein which binds this element and is similar to a maize nuclear protein which binds PPDK in maize. Taken together, these results indicate that the regulatory system which controls PPDK expression in maize is not unique to C4 plants.

We have isolated a cDNA clone encoding a homeobox sequence from rice. DNA sequence analysis of this clone, which was designated as Oryza sativa homeobox 1 (OSH1), and a genomic clone encoding the OSH1 sequence have shown that the OSH1 gene consists of five exons and encodes a polypeptide of 361 amino acid residues. Restriction fragment length polymorphism analysis has shown that OSH1 is a single-copy gene located near the phytochrome gene on chromosome 3. Introduction of the cloned OSH1 gene into rice resulted in altered leaf morphology, which was similar to that of the maize morphological mutant Knotted-1 (Kn1), indicating that OSH1 is a rice gene homologous to the maize Kn1 gene. RNA gel blot analysis has shown that the gene is primarily expressed in the shoot apices of young rice seedlings. This finding is supported by results of transformation experiments in which the 5' flanking region of the gene directed expression of a reporter gene in the shoot apex, particularly in stipules, of transgenic Arabidopsis. To elucidate the biological function of the OSH1 gene product, the coding region was introduced into Arabidopsis under the control of the cauliflower mosaic virus 35S promoter. Almost all transformants showed abnormal morphology. The typical phenotype was the formation of clumps of abundant vegetative and reproductive shoot apices containing meristems and leaf primordia, which did not form elongated shoots. Some transformants with a less severe phenotype formed elongated shoots but had abnormally shaped leaves and flowers with stunted sepals, petals, and stamens. The abnormal phenotypes were inherited, and the level of expression of the introduced OSH1 correlates with the severity of the phenotype. These findings indicate that the abnormal morphologies of the transgenic plants are caused by the expression of the OSH1 gene product and, therefore, that OSH1 is related to the plant development process.

Plants absorb and metabolize formaldehyde, a C-1 compound that is one of the main indoor air pollutants. To elucidate the molecular mechanism of formaldehyde metabolism in plants, we isolated formaldehyde-responsive genes from golden pothos by means of GeneFishing PCR. We focused on the immediate-early response genes following formaldehyde treatment. Two full length cDNA sequences corresponding to a putative class II chitinase and a hypothetical novel protein, which we termed DEG2, were generated by rapid amplification of cDNA ends (RACE). The chitinase, which we designated EaCHI1, was up-regulated in the leaves and stems, whereas DEG2 was up-regulated in the leaves and roots of formaldehyde-treated golden pothos. Phylogenetic analysis showed that putative class II chitinases were split into two groups, monocot and dicot. EaCHI1 belonged to the former group, occupying the most basal level among the monocot chitinases analyzed. The identification of chitinase as a formaldehyde-responsive gene suggests a novel physiological role for this enzyme in plant carbon metabolism and environmental responses. The DEG2 sequence was not similar to any known protein sequence.