Production and characterization of auxin-insensitive rice by overexpression of a mutagenized rice IAA protein

Since auxin was first isolated and characterized as a plant hormone, the underlying molecular mechanism of auxin signaling has been elucidated primarily in dicot plants represented by Arabidopsis. In monocot plants, the molecular mechanism of auxin signaling has remained unclear, despite various physiological experiments. To understand the function and mechanism of auxin signaling in rice ( Oryza sativa ), we focused on the IAA gene, a well-studied gene in Arabidopsis that serves as a negative regulator of auxin signaling. We found 24 IAA gene family members in the rice genome. OsIAA3 is one of these family members whose expression is rapidly increased in response to auxin. We produced transgenic rice harboring m OsIAA3 - GR , which can overproduce mutant OsIAA3 protein containing an amino acid change in domain II to cause a gain-of-function phenotype, by treatment with dexamethasone. The transgenic rice was insensitive to auxin and gravitropic stimuli, and exhibited short leaf blades, reduced crown root formation, and abnormal leaf formation. These results suggest that , in rice, auxin is important for development and its signaling is mediated by IAA genes.     

A rice tryptophan deficient dwarf mutant, tdd1, contains a reduced level of indole acetic acid and develops abnormal flowers and organless embryos

Indole-3-acetic acid (IAA) plays a critical role in many aspects of plant growth and development; however, complete pathways of biosynthesis, localization and many aspects of functions of IAA in rice remain unclear. Here, we report the analysis of a rice tryptophan- (Trp-) and IAA-deficient mutant, tryptophan deficient dwarf1 ( tdd1 ) , which is embryonic lethal because of a failure to develop most organs during embryogenesis. Regenerated tdd1 plants showed pleiotropic phenotypes: dwarfing, narrow leaves, short roots and abnormal flowers. TDD1 encodes a protein homologous to anthranilate synthase β-subunit, which catalyses the first step of the Trp biosynthesis pathway and functions upstream of Trp-dependent IAA biosynthesis. TDD1-uidA and DR5-uidA expression overlapped at many sites in WT plants but was lacking in tdd1 , indicating that TDD1 is involved in auxin biosynthesis. Both Trp and IAA levels in flowers and embryos were much lower in tdd1 than in wild type (WT). Trp feeding completely rescued the mutant phenotypes and moderate expression of OsYUCCA1 , which encodes a key enzyme in Trp-dependent IAA biosynthesis, also rescued plant height and root length, indicating that the abnormal phenotypes of tdd1 are caused predominantly by Trp and IAA deficiency. In tdd1 embryos, the expression patterns of OSH1 and OsSCR , which mark the presumptive apical region and the L2 layer, respectively, are identical to those in WT, suggesting a possibility either that different IAA levels are required for basic pattern formation than for organ formation or that an orthologous gene compensates for TDD1 deficiency during pattern formation.     

Tea catechins and related polyphenols as anti-cancer agents

Epigallocatechin gallate (EGCg) and theaflavins, a major constituent of green tea infusion and the constituents of black tea, respectively, were found to inhibit matrix metalloproteinases (MMPs) which are intimately associated with tumor invasion and metastasis. EGCg and related polyphenols exhibited apoptosis-inducing activity for several cancer cell lines including human stomach and colon cancer cells. Comparison of the activity of these compounds revealed the importance of the number and the steric disposition of hydroxyl groups. A pyrogallol-type structure in a molecule is a minimum requirement for apoptosis induction of catechin compounds and that in the B ring has an important role in the activity. These data would provide useful information for designing anti-cancer agents on the basis of anti-inhibitory activity for MMPs and/or apoptosis-inducing activity.     

Auxin biosynthesis by the YUCCA genes in rice

Although indole-3-acetic acid (IAA), the predominant auxin in plants, plays a critical role in various plant growth and developmental processes, its biosynthesis and regulation have not been clearly elucidated. To investigate the molecular mechanisms of IAA synthesis in rice (Oryza sativa), we identified seven YUCCA-like genes (named OsYUCCA1-7) in the rice genome. Plants overexpressing OsYUCCA1 exhibited increased IAA levels and characteristic auxin overproduction phenotypes, whereas plants expressing antisense OsYUCCA1 cDNA displayed defects that are similar to those of rice auxin-insensitive mutants. OsYUCCA1 was expressed in almost all of the organs tested, but its expression was restricted to discrete areas, including the tips of leaves, roots, and vascular tissues, where it overlapped with expression of a beta-glucuronidase reporter gene controlled by the auxin-responsive DR5 promoter. These observations are consistent with an important role for the rice enzyme OsYUCCA1 in IAA biosynthesis via the tryptophan-dependent pathway.     

Analysis of the rice mutant dwarf and gladius leaf 1. Aberrant katanin-mediated microtubule organization causes up-regulation of gibberellin biosynthetic genes independently of gibberellin signaling

Molecular genetic studies of plant dwarf mutants have indicated that gibberellin (GA) and brassinosteroid (BR) are two major factors that determine plant height; dwarf mutants that are caused by other defects are relatively rare, especially in monocot species. Here, we report a rice (Oryza sativa) dwarf mutant, dwarf and gladius leaf 1 (dgl1), which exhibits only minimal response to GA and BR. In addition to the dwarf phenotype, dgl1 produces leaves with abnormally rounded tip regions. Positional cloning of DGL1 revealed that it encodes a 60-kD microtubule-severing katanin-like protein. The protein was found to be important in cell elongation and division, based on the observed cell phenotypes. GA biosynthetic genes are up-regulated in dgl1, but the expression of BR biosynthetic genes is not enhanced. The enhanced expression of GA biosynthetic genes in dgl1 is not caused by inappropriate GA signaling because the expression of these genes was repressed by GA3 treatment, and degradation of the rice DELLA protein SLR1 was triggered by GA3 in this mutant. Instead, aberrant microtubule organization caused by the loss of the microtubule-severing function of DGL1 may result in enhanced expression of GA biosynthetic genes in that enhanced expression was also observed in a BR-deficient mutant with aberrant microtubule organization. These results suggest that the function of DGL1 is important for cell and organ elongation in rice, and aberrant DGL1-mediated microtubule organization causes up-regulation of gibberellin biosynthetic genes independently of gibberellin signaling.     

Expression of DRG during murine embryonic development.

We had previously characterised a cDNA which encodes a novel GTP-binding protein DRG. The expression of drg gene is down-regulated during the embryonic development of murine central nervous system. Further analysis of drg mRNA and protein in adult mouse tissues and various cell lines of different origins indicated that it is expressed widely, albeit at low and variable levels. In situ hybridisation analysis of mRNA expression in sections of mouse embryos indicated that drg is expressed strongly in various embryonic tissues. The expression of drg mRNA is greatly reduced in newborn animals. At cellular level, DRG protein can be detected in the cytoplasm. These observations suggest that DRG may play multiple roles in development and normal cell metabolism.     

Positional cloning of <Emphasis Type="Italic">ds1</Emphasis>, the target leaf spot resistance gene against <Emphasis Type="Italic">Bipolaris sorghicola</Emphasis> in sorghum

Target leaf spot is one of the major sorghum diseases in southern Japan and caused by a necrotrophic fungus, Bipolaris sorghicola. Sorghum resistance to target leaf spot is controlled by a single recessive gene (ds1). A high-density genetic map of the ds1 locus was constructed with simple sequence repeat markers using progeny from crosses between a sensitive variety, bmr-6, and a resistant one, SIL-05, which allowed the ds1 gene to be genetically located within a 26-kb region on the short arm of sorghum chromosome 5. The sorghum genome annotation database for BTx623, for which the whole genome sequence was recently published, indicated a candidate gene from the Leucine-Rich Repeat Receptor Kinase family in this region. The candidate protein kinase gene was expressed in susceptible plants but was not expressed or was severely reduced in resistant plants. The expression patterns of ds1 gene and the phenotype of target leaf spot resistance were clearly correlated. Genomic sequences of this region in parental varieties showed a deletion in the promoter region of SIL-05 that could cause reduction of gene expression. We also found two ds1 alleles for resistant phenotypes with a stop codon in the coding region. The results shown here strongly suggest that the loss of function or suppression of the ds1 protein kinase gene leads to resistance to target leaf spot in sorghum.     

The rice mutant dwarf bamboo shoot 1: a leaky mutant of the NACK-type kinesin-like gene can initiate organ primordia but not organ development

That plant dwarfism is caused by hormonal defects related to gibberellin and brassinosteroid has been well documented. Other contributing elements, however, have not been elucidated. Here, we report on one of the most severe dwarf mutants of rice, dwarf bamboo shoot 1 (dbs1). Most mutant plants died within 1 month after sowing, but a few (5.2%) survived and grew. Vacuolation enlarged cells in the leaf primordia and seminal root before abortion, which disrupted the organized cell files in these organs. Relative to the severe defects in shoot and root growth, the overall structure of the dbs1 embryo was almost normal. Similarly, initiation and organogenesis of the leaf primordia at the shoot apical meristem and those of the lateral root primordia at the root elongation zone occurred normally. These observations suggest that DBS1 is involved in the growth and development of organs but not in organ initiation or organogenesis. Positional cloning of DBS1 revealed that it encoded a NACK-type kinesin-like protein (OsNACK), homologous to the essential components of a mitogen-activated protein kinase cascade during plant cytokinesis. A BLAST search indicated that DBS1 was the only gene encoding the OsNACK-type protein in the rice genome, and the dbs1 mutant produced only small amounts of the translatable DBS1 mRNA. Thus, we conclude that the dbs1 mutation causes a severe defect in DBS1 function but does not completely shut it down. We discuss the leaky phenotype of dbs1 under the restricted functioning of OsNACK.     

Apoptosis-inducing activity of a driselase digest fraction of green tea residue

We enzymatically digested green tea residue with Driselase, a crude preparation containing cellulase, pectinase and proteases, in order to examine the potential usefulness of the residue. A fraction of the digest soluble in 70% ethanol was found to induce the death of U937 human histiocytic lymphoma cells by apoptosis. Other enzyme preparations gave similar products with cell death-inducing activity of varing potency. The green tea residue may therefore be a useful source of potential agents with anti-cancer activity.