[Show abstract][Hide abstract] ABSTRACT: In order to understand the stress-responsive mechanism controlling OsDhn1 gene, promoter analysis of OsDhn1 gene was conducted. First, a 1.5 kb-long promoter region of OsDhn1 was isolated and characterized by β-glucuronidase (GUS) analysis. GUS assay of OsDhn1::gus plants showed that the GUS activity in the leaves was induced by drought stress but not by wound, cold, salt, or ABA treatment. Second, transactivation assay revealed that OsDhn1 promoter is activated by OsDREB1A or OsDREB1D as well as CBF1 but not by OsDREB1B or OsDREB1C. Taken together, these suggest that the drought-inducible OsDhn1 gene is regulated by abiotic stress signaling pathway involving CBF/DREB.
[Show abstract][Hide abstract] ABSTRACT: A full-length cDNA corresponding to ddOs319, previously isolated as a cold-responsive gene in the flowers by mRNA differential display (Plant Cell Rep 26:1097–1110, 2007), was obtained from the cold-treated flowers by reverse transcription and nested PCR. The cDNA encodes a putative class III peroxidase of 335 amino acids with 77–98% identity with rice peroxidases and named OsPOX1. To understand the regulation of OsPOX1 expression, a 1.8 kb promoter region of OsPOX1 was isolated and fused to β-glucuronidase (GUS) reporter gene. Transgenic rice plants expressing POsPOX1
-GUS showed minimal GUS activity in both shoots and roots at the vegetative stage. In the flowers at early young microspore stage, GUS activity was detected in the veins and anthers. Interestingly, at the later vacuolated pollen stage, the GUS activity was highly induced by cold stress, suggesting that OsPOX1 is a flower-preferential cold-responsive gene in rice.
[Show abstract][Hide abstract] ABSTRACT: During their reproductive stage, rice crops often are exposed to cold stress, which leads to sterility and reduced yields. To understand the cold response mechanism at that stage, we used an mRNA differential display method to isolate cold-responsive genes from pre-anthesis flowers. Approximately 5,000 transcripts were identified here, of which 123 were found to be displayed differentially between the control (30 degrees C) and cold-treated (12 degrees C) flowers. Among them, 26 were analyzed by northern analysis; 8 of those clones were confirmed as cold-responsive. OsLti6b, encoding a hydrophobic protein homologous to Arabidopsis RCI2, was analyzed in detail. RNA blot analysis revealed that its transcript is increased by cold, salt, drought, or ABA treatments. In situ hybridization indicated that this transcript is highly accumulated in the ovaries and stamens of cold-treated flowers, particularly in the anther walls and vascular tissues of the filaments. Over-expression of OsLti6b increased cold tolerance as revealed by seedling wilting rates and ion leakages of mature leaves, demonstrating that the extent of the tolerance correlates well with its expression level.
[Show abstract][Hide abstract] ABSTRACT: Anthocyanin is the major color pigment in plants. The apple anthocyanidin synthase gene(ANS) manifests fruit skin-preferential expression (Kim et al., 2003). To understand the regulatory mechanism for such expression,
we isolated and analyzed an appleANS genomic clone. Sequence analysis of ca. 1.4-kb of theANS promoter region predicted several cis-elements for MYB, light responsive GT-1, and the ABA Responsive Element (ABRE). Transgenic
tobacco plants carrying a chimeric fusion between theANS promoter and the β-glucuronidase gene(GUS) showed that GUS was expressed in the receptacles and immature seeds as well as in the floral buds, but not in the vegetative
[Show abstract][Hide abstract] ABSTRACT: Now that sequencing of the rice genome is nearly completed, functional analysis of its large number of genes is the next challenge. Because rice is easy to transform, T-DNA has been used successfully to generate insertional mutant lines. Collectively, several laboratories throughout the world have established at least 200,000 T-DNA insertional lines. Some of those carry the GUS or GFP reporters for either gene or enhancer traps. Others are activation tagging lines for gain-of-function mutagenesis when T-DNA is inserted in the intergenic region. A forward genetic approach showed limited success because of somaclonal variations induced during tissue culture. To utilize these resources more efficiently, tagged lines have been produced for reverse genetics approaches. DNA pools of the T-DNA-tagged lines have been prepared for polymerase chain reaction (PCR) screening of insertional mutants in a given gene. Appropriate T-DNA insertion sites are determined by sequencing the region flanking the T-DNA. This information is then used to make databases that are shared with the scientific community. International efforts on seed amplification and maintenance are needed to exploit these valuable materials efficiently.
[Show abstract][Hide abstract] ABSTRACT: To identify low-temperature-responsive genes in rice, we screened gus-trapped T-DNA tagging lines that had been subjected to cold stress at 5 °C. Out of 15,586 lines, 81 (0.52%) showed cold-responsive GUS activity. Among the 62 lines we selected for further study, 53 showed increased GUS activity while 9 showed less. In addition, 16 of those 62 lines were influenced by abscisic acid (ABA), suggesting an ABA-dependent cold response. We used inverse PCR or thermal asymmetric interlaced (TAIL) PCR to identify 37 tagged genes from those lines, and further characterized two of them at the molecular level. The OsRLK1 gene, which encodes a putative leucine rich repeat (LRR)-type receptor-like protein kinase, was inducible by cold and salt stresses. The OsDMKT1 gene, encoding a putative demethylmenaquinone methyltransferase (DMKT), was also inducible by low temperatures, but not by high salt or drought. The T-DNA-tagged OsDMKT1 gene also co-segregated with the cold-inducible gus gene. Our results indicate that the T-DNA tagging lines are useful in obtaining the stress-responsive genes in rice.
[Show abstract][Hide abstract] ABSTRACT: Anthocyanin is the major color pigment in plants. In order to understand anthocyanin biosynthesis mechanism in apple, the cDNAs encoding flavanone 3-hydroxylase (F3H), dihydroflavonol reductase (DFR), anthocyanidin synthase (ANS), and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) were isolated from cDNA libraries prepared from the skin tissues of apple (Malus domestica Borkh. cv. Fuji). Deduced amino acid sequences of the cDNAs showed high homology to the sequences from other plants. Each gene is a member of a multigene family. The mRNAs of anthocyanin biosynthetic genes were detected preferentially in the skin tissue and expression of the genes was coordinately induced by light. The transcripts were detected abundantly in the skins of cultivars with red skin, but rarely in that of a cultivar carrying non-red fruit, suggesting that these genes have major roles in determination of apple skin color.
[Show abstract][Hide abstract] ABSTRACT: Functioning of the thaumatin-like (TL) protein is known to be pathogenesis- or stress-related. Here we identified TL protein
cDNA, from an apple- skin library, that was nearly identical to that of theMdTl1 gene. Transcripts of this so-named gene,MdTL1a, were highly expressed in the fruit, but rarely in other tissue types. Expression was found in both the skin and the flesh
of the fruit We also examined the environmental or hormonal control ofMdTL1a expression. Exposing the fruit to light caused this gene to be induced in the skin tissues. Accumulation ofMdTL1a mRNA reached a peak between Days 1 and 5 after exposure. In the leaves,MdTL1a was induced by salicylic acid (SA), but was not significantly affected by any other stresses. Analysis of theMdTL1a genomic clone, λTL1, revealed that transcription ofMdTL1a begins 53 bp upstream of the start codon. Sequence analysis of the ca. 1.0-kbMdTL1a promoter region has enabled us to predict that it has a stress-related cis-element, such as the ABA responsive element (ABRE),
as well as a light-responsive GT-1 and l-box.