The alpha-amylase induction in endosperm during rice seed germination is caused by gibberellin synthesized in epithelium

We recently isolated two genes (OsGA3ox1 and OsGA3ox2) from rice (Oryza sativa) encoding 3beta-hydroxylase, which catalyzes the final step of active gibberellin (GA) biosynthesis (H. Itoh, M. Ueguchi-Tanaka, N. Sentoku, H. Kitano, M. Matsuoka, M. Kobayashi [2001] Proc Natl Acad Sci USA 98: 8909-8914). Using these cloned cDNAs, we analyzed the temporal and spatial expression patterns of the 3beta-hydroxylase genes and also an alpha-amylase gene (RAmy1A) during rice seed germination to investigate the relationship between GA biosynthesis and alpha-amylase expression. Northern-blot analyses revealed that RAmy1A expression in the embryo occurs before the induction of 3beta-hydroxylase expression, whereas in the endosperm, a high level of RAmy1A expression occurs 1 to 2 d after the peak of OsGA3ox2 expression and only in the absence of uniconazol. Based on the analysis of an OsGA3ox2 null mutant (d18-Akibare dwarf), we determined that 3beta-hydroxylase produced by OsGA3ox2 is important for the induction of RAmy1A expression and that the OsGA3ox1 product is not essential for alpha-amylase induction. The expression of OsGA3ox2 was localized to the shoot region and epithelium of the embryo, strongly suggesting that active GA biosynthesis occurs in these two regions. The synthesis of active GA in the epithelium is important for alpha-amylase expression in the endosperm, because an embryonic mutant defective in shoot formation, but which developed epithelium cells, induced alpha-amylase expression in the endosperm, whereas a mutant defective in epithelium development did not.     

Amino acid replacement studies of human cytochrome c by a complementation system using CYC1 deficient yeast

Various in vitro mutated human cytochrome c genes which encode displaced amino acid residues at the 14th, 17th, 28th, 37th, 38th, 56th, and/or 84th residues were constructed, and their degrees of complementation of yeast CYC1 deficiency were examined. Invariant Cys-17 and Arg-38 could not be replaced by alanine and tryptophan, respectively, without function impairment. Cytochrome c containing Ala-14 instead of conserved Cys-14, Gly-38 or Lys-38 instead of Arg-38, and Ser-84 instead of invariant Gly-84 were partly functional. These results indicate that these invariant or conserved residues are important. Cytochromes c containing Cys-56 instead of native Gly-56 was partly functional. Cytochrome c containing Arg-37 and Gly-38 instead of Gly-37 and Arg-38 was slightly functional. Replacement of variable Thr-28 and Gly-37 by Ile-28 and Arg-37, respectively, produced no effects. Our results are as a whole consistent with the view that conserved residues are important and variable residues are less important for cytochrome c to function.     

Pure and mixed genetic lines of Saccharomyces bayanus and Saccharomyces pastorianus and their contribution to the lager brewing strain genome

The yeast species Saccharomyces bayanus and Saccharomyces pastorianus are of industrial importance since they are involved in the production process of common beverages such as wine and lager beer; however, they contain strains whose variability has been neither fully investigated nor exploited in genetic improvement programs. We evaluated this variability by using PCR-restriction fragment length polymorphism analysis of 48 genes and partial sequences of 16. Within these two species, we identified "pure" strains containing a single type of genome and "hybrid" strains that contained portions of the genomes from the "pure" lines, as well as alleles termed "Lager" that represent a third genome commonly associated with lager brewing strains. The two pure lines represent S. uvarum and S. bayanus, the latter a novel group of strains that may be of use in strain improvement programs. Hybrid lines identified include (i) S. cerevisiae/S. bayanus/Lager, (ii) S. bayanus/S. uvarum/Lager, and (iii) S. cerevisiae/S. bayanus/S. uvarum/Lager. The genome of the lager strains may have resulted from chromosomal loss, replacement, or rearrangement within the hybrid genetic lines. This study identifies brewing strains that could be used as novel genetic sources in strain improvement programs and provides data that can be used to generate a model of how naturally occurring and industrial hybrid strains may have evolved.     

Vacuolar proton pumps and aquaporins involved in rapid internode elongation of deepwater rice

Rapid growth of the submerged shoots of deepwater rice is essential for survival during the rainy season. We investigated changes in the expression of vacuolar H(+)-ATPase (V-ATPase), H(+)-pyrophosphatase (V-PPase), and aquaporins under submerged conditions. The amounts of vacuolar proton pumps, which support the active transport of ions into the vacuoles, were maintained on a membrane protein basis in the developing vacuoles. Among the six isogenes of V-PPase, OsVHP1;3 was markedly enhanced by submersion. The gene expression of efficient water channels, OsTIP1;1, OsTIP2;2, OsPIP1;1, OsPIP2;1, and OsPIP2;2, was markedly enhanced by submersion. The increase in aquaporin expression might support quick elongation of internodes. The mRNA levels of OsNIP2;2 and OsNIP3;1, which transport silicic and boric acids respectively, clearly decreased. The present study indicates that internodes of deepwater rice upregulate vacuolar proton pumps and water channel aquaporins and downregulate aquaporins that allow permeation of the substrates that suppress internode growth.     

Engineering of the rose flavonoid biosynthetic pathway successfully generated blue-hued flowers accumulating delphinidin

Flower color is mainly determined by anthocyanins. Rosa hybrida lacks violet to blue flower varieties due to the absence of delphinidin-based anthocyanins, usually the major constituents of violet and blue flowers, because roses do not possess flavonoid 3',5'-hydoxylase (F3'5'H), a key enzyme for delphinidin biosynthesis. Other factors such as the presence of co-pigments and the vacuolar pH also affect flower color. We analyzed the flavonoid composition of hundreds of rose cultivars and measured the pH of their petal juice in order to select hosts of genetic transformation that would be suitable for the exclusive accumulation of delphinidin and the resulting color change toward blue. Expression of the viola F3'5'H gene in some of the selected cultivars resulted in the accumulation of a high percentage of delphinidin (up to 95%) and a novel bluish flower color. For more exclusive and dominant accumulation of delphinidin irrespective of the hosts, we down-regulated the endogenous dihydroflavonol 4-reductase (DFR) gene and overexpressed the Irisxhollandica DFR gene in addition to the viola F3'5'H gene in a rose cultivar. The resultant roses exclusively accumulated delphinidin in the petals, and the flowers had blue hues not achieved by hybridization breeding. Moreover, the ability for exclusive accumulation of delphinidin was inherited by the next generations.     

Interaction of two recessive genes, <Emphasis Type="Italic">hbd2</Emphasis> and <Emphasis Type="Italic">hbd3</Emphasis>, induces hybrid breakdown in rice

Reproductive barriers are important for the maintenance of species identity. We discovered a reproductive barrier via hybrid breakdown among the progeny of a cross between the japonica rice cultivar Koshihikari and the indica rice cultivar Habataki. Genetic analysis indicated that the hybrid breakdown is regulated by the interaction of two recessive genes: hbd2 in Habataki and hbd3 in Koshihikari. Linkage mapping showed that hbd2 is located near the 100 cM region of chromosome 2 in Habataki, whereas hbd3 is located near the 60 cM region of chromosome 11 in Koshihikari. Construction of nearly isogenic lines for hbd2 and Hbd3 (NIL-hbd2 and NIL-Hbd3), as well as a pyramiding line (NIL-hbd2 + Hbd3), confirmed that the hybrid breakdown is induced by the interaction of these two recessive genes. Our results indicate that these genes are novel for the induction of hybrid breakdown in rice.     

Secretion of chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase from cultured chick embryo chondrocytes.

We found that chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase were released into the culture medium from the cultured chick embryo chondrocytes. Since the release of the sulfotransferases was observed not only in serum-supplemented medium but also in serum-free medium, the released sulfotransferases were unlikely to be derived from serum. Addition of ascorbate to the serum-free medium supported the continuous release of the sulfotransferases. Monensin, which is known to cause dilatation of the Golgi apparatus and to inhibit sulfation of proteoglycan, was found to affect the release of the sulfotransferases. In the presence of 10(-6) M monensin, chondroitin 6-sulfotransferase activity in the cell layer was decreased to less than one tenth of the control, and the rate of the release of the activity became much smaller than the control after the initial rapid release. The activity of chondroitin 4-sulfotransferase was also affected by monensin, but the reduction of the chondroitin 4-sulfotransferase activity in the cell layer was not so great as the reduction of chondroitin 6-sulfotransferase activity. Unlike to the microsomal sulfotransferases, both chondroitin 6-sulfotransferase and chondroitin 4-sulfotransferase released into the culture medium were retained in the soluble fraction after centrifugation at 100,000 x g for 60 min, and were not activated by detergent. pH optimum and requirements for sulfhydryl compounds of the released sulfotransferases were similar to those observed previously in the chondroitin sulfotransferases from chick embryo cartilage and from cultured chick embryo chondrocytes. These results suggest that chondroitin sulfotransferases, which are localized in the Golgi apparatus, may be secreted to the extracellular space in a soluble form under the culture conditions.     

The Rice brassinosteroid-deficient dwarf2 mutant, defective in the rice homolog of Arabidopsis DIMINUTO/DWARF1, is rescued by the endogenously accumulated alternative bioactive brassinosteroid, dolichosterone

We have identified a rice (Oryza sativa) brassinosteroid (BR)-deficient mutant, BR-deficient dwarf2 (brd2). The brd2 locus contains a single base deletion in the coding region of Dim/dwf1, a homolog of Arabidopsis thaliana DIMINUTO/DWARF1 (DIM/DWF1). Introduction of the wild-type Dim/dwf1 gene into brd2 restored the normal phenotype. Overproduction and repression of Dim/dwf1 resulted in contrasting phenotypes, with repressors mimicking the brd2 phenotype and overproducers having large stature with increased numbers of flowers and seeds. Although brd2 contains low levels of common 6-oxo-type BRs, the severity of the brd2 phenotype is much milder than brd1 mutants and most similar to d2 and d11, which show a semidwarf phenotype at the young seedling stage. Quantitative analysis suggested that in brd2, the 24-methylene BR biosynthesis pathway is activated and the uncommon BR, dolichosterone (DS), is produced. DS enhances the rice lamina joint bending angle, rescues the brd1 dwarf phenotype, and inhibits root elongation, indicating that DS is a bioactive BR in rice. Based on these observations, we discuss an alternative BR biosynthetic pathway that produces DS when Dim/dwf1 is defective.     

Crown rootless1, which is essential for crown root formation in rice, is a target of an AUXIN RESPONSE FACTOR in auxin signaling

Although the importance of auxin in root development is well known, the molecular mechanisms involved are still unknown. We characterized a rice (Oryza sativa) mutant defective in crown root formation, crown rootless1 (crl1). The crl1 mutant showed additional auxin-related abnormal phenotypic traits in the roots, such as decreased lateral root number, auxin insensitivity in lateral root formation, and impaired root gravitropism, whereas no abnormal phenotypic traits were observed in aboveground organs. Expression of Crl1, which encodes a member of the plant-specific ASYMMETRIC LEAVES2/LATERAL ORGAN BOUNDARIES protein family, was localized in tissues where crown and lateral roots are initiated and overlapped with beta-glucuronidase staining controlled by the DR5 promoter. Exogenous auxin treatment induced Crl1 expression without de novo protein biosynthesis, and this induction required the degradation of AUXIN/INDOLE-3-ACETIC ACID proteins. Crl1 contains two putative auxin response elements (AuxREs) in its promoter region. The proximal AuxRE specifically interacted with a rice AUXIN RESPONSE FACTOR (ARF) and acted as a cis-motif for Crl1 expression. We conclude that Crl1 encodes a positive regulator for crown and lateral root formation and that its expression is directly regulated by an ARF in the auxin signaling pathway.