Hiromi Higo

PLACE: a database of plant cis-acting regulatory DNA elements.

PLACE (http://www.dna.affrc.go.jp/htdocs/PLACE/) is a database of motifs found in plant cis -acting regulatory DNA elements, all from previously published reports. It covers vascular plants only. In addition to the motifs originally reported, their variations in other genes or in other plant species reported later are also compiled. The PLACE database also contains a brief description of each motif and relevant literature with PubMed ID numbers. This report summarizes the present status of this database and available tools.

Isolation of a Regulatory Gene of Anthocyanin Biosynthesis in Tuberous Roots of Purple-Fleshed Sweet Potato

Many transcriptional factors harboring the R2R3-MYB domain, basic helix-loop-helix domain, or WD40 repeats have been identified in various plant species as regulators of flavonoid biosynthesis in flowers, seeds, and fruits. However, the regulatory elements of flavonoid biosynthesis in underground organs have not yet been elucidated. We isolated the novel MYB genes IbMYB1 and IbMYB2s from purple-fleshed sweet potato (Ipomoea batatas L. Lam. cv Ayamurasaki). IbMYB1 was predominantly expressed in the purple flesh of tuberous roots but was not detected (or only scarcely) in other anthocyanin-containing tissues such as nontuberous roots, stems, leaves, or flowers. IbMYB1 was also expressed in the tuberous roots of other purple-fleshed cultivars but not in those of orange-, yellow-, or white-fleshed cultivars. Although the orange- or yellow-fleshed cultivars contained anthocyanins in the skins of their tuberous roots, we could not detect IbMYB1 transcripts in these tissues. These results suggest that IbMYB1 controls anthocyanin biosynthesis specifically in the flesh of tuberous roots. The results of transient and stable transformation experiments indicated that expression of IbMYB1 alone was sufficient for induction of all structural anthocyanin genes and anthocyanin accumulation in the flesh of tuberous roots, as well as in heterologous tissues or heterologous plant species.

Differential diurnal expression of rice catalase genes: the 5%-flanking region of CatA is not sufficient for circadian control

Three rice catalase genes (CatA, CatB, CatC) are expressed in a growth- and tissue-specific manner. In seedlings, the CatA, CatB and CatC genes were highly expressed in the leaf sheath, root and leaf blade, respectively. The expression of the CatA gene in the leaf sheath was modulated by a circadian rhythm with the maximum late in the light period. On the other hand, diurnal oscillations of CatC expression were detected in the leaf blade when plants were grown in the dark. The b-glucuronidase (GUS) gene driven by the 5%-flanking region of CatA was expressed with diurnal fluctuations, the pattern of which is different from that of the endogenous CatA mRNA, both at the pre-mRNA and at the mRNA level in transgenic plants. These results suggest that the circadian rhythmic expression of CatA is due to a transcriptional or post-transcriptional event such as modulation of pre-mRNA stability and requires some other region(s), in addition to the promoter region, exon-1 and intron-1.

Cloning and characterization of the rice CatA catalase gene, a homologue of the maize Cat3 gene

We isolated and sequenced a genomic clone (CatA) encoding CAT-A catalase, a homologue of the maize catalase isozyme 3 (CAT-3) from rice (Oryza sativa L.). The 5′-upstream non-coding region had very low similarity with the maize Cat3 gene and possible cis elements and sequence motifs in the maize Cat3 gene were not evident, except for TATA and CAAT motifs. Several sequence motifs found in the promoters of plant seed-specific genes were identified in the 5′-upstream non-coding region of the CatA gene. Northern blotting showed that the CatA gene is expressed at high levels in seeds during early development and also in young seedlings. Methyl viologen (paraquat) resulted in the 3-fold induction of the CatA gene in the leaves of young seedlings, whereas abscisic acid, wounding, salicylic acid, and hydrogen peroxide had no or only slight effects.The 1.9 kb 5′-upstream fragment (−1559 to +342) of the CatA gene was fused with the Escherichia coli β-glucuronidase (GUS) gene and introduced by electroporation into protoplasts prepared from rice suspension-cultured cells, then the transient expression of the GUS gene was examined. Deletion analysis of this chimeric gene suggested that a weak silencer is located in the region between −1564 to −699. Abscisic acid (ABA) at a final concentration of 10−6 M doubled GUS activity in protoplasts electroporated with the chimeric DNAs having 1.9 to 1.2 kb 5′-upstream regions. A sequence highly similar to the Sph box, a motif found in genes modulated by ABA, was found at −266 to −254. Deletion of this region however, did not eliminate the responsiveness to ABA. Expression of the chimeric gene in the protoplasts was not enhanced by stress such as low and high temperature, hydrogen peroxide, methyl viologen, salicylic acid, elicitor, and UV light.The chimeric CatA-GUS plasmid DNAs amplified in the methylation-positive strain, E. coli DH5α, showed GUS gene activities, whereas all the chimeric DNAs amplified in the methylation-deficient E. coli JM 110 were completely inactive in the presence or absence of ABA in the culture medium. DNA methylation, especially of either one or both of the deoxyadenosines at the two GATC motifs (one in the first exon and the other in the first intron of the rice CatA gene), appeared to be responsible for the CatA promoter activity identified in the transient assay.

Molecular cloning and characterization of a cDNA encoding a small GTP-binding protein related to mammalian ADP-ribosylation factor from rice

Abstract A cDNA clone encoding a small GTP-binding protein related to mammalian ADP-ribosylation factors (ARFs) was isolated from a cDNA library of cultured Oryza sativa cells. The cDNA encoded an open reading frame of 181 amino acids, which was highly similar to the ARFs from animals, yeast and Arabidopsis thaliana . The domains that were believed to be involved in the interaction with GTP were remarkably conserved. The estimated rate of amino acid changes in the ARF protein since the divergence of the plant and animal kingdoms was slower than most enzymes examined, but faster than histones H4 and H3, and calmodulin. Southern analysis of rice genomic DNAs suggested two copies of the ARF gene. Northern analysis of total RNA (or poly(A) + RNA) showed that ARF genes are highly expressed in young (2-week-old) seedlings, and in seeds at the early developmental stage (0–6 days after pollination), but to a lesser extent in cultured cells.

An Alfalfa Gene Similar to Glutathione S-Transferase Is Induced in Root by Iron Deficiency

Abstract In order to isolate genes influenced by the iron (Fe) status of plants, we constructed a cDNA library from the roots of Fe-starved alfalfa (Medicago sativa L.) plants. The library was then screened with a subtracted probe. Based on the results of hybridization, we selected a full-length clone cDNA, designated as Isr1 (iron starvation-responsive gene 1). The Isr1 consisted of 815 nucleotides containing an open reading frame of 642 bp and encoded a protein with 214 amino acids, which showed a homology to glutathione S-transferase (GST, EC 2.5.1.18). A significant increase in the amount of Isr1 mRNA was observed in roots after 8 h of Fe starvation. The amount of Isr1 transcript, however, decreased rapidly on the second day, then increased again after the third day. The amount of Isr1 transcript was not influenced by the resumption of the Fe for 1 d. Possible functions of the gene product are discussed.