Retrograde regulation of nuclear gene expression in CW-CMS of rice

The CW-cytoplasmic male sterility (CMS) line has the cytoplasm of Oryza rufipogon Griff, and mature pollen is morphologically normal under an optical microscope but lacks the ability to germinate; restorer gene Rf17 has been identified as restoring this ability. The difference between nuclear gene expression in mature anthers was compared for the CW-CMS line, [cms-CW] rf17rf17, and a maintainer line with normal cytoplasm of Oryza sativa L., [normal] rf17rf17. Using a 22-k rice oligoarray we detected 58 genes that were up-regulated more than threefold in the CW-CMS line. Expression in other organs was further investigated for 20 genes using RT-PCR. Five genes, including genes for alternative oxidase, were found to be preferentially expressed in [cms-CW] rf17rf17 but not in [normal] rf17rf17 or [cms-CW] Rf17Rf17. Such [cms-CW] rf17rf17-specific gene expression was only observed in mature anthers but not in leaves, stems, or roots, indicating the presence of anther-specific mitochondrial retrograde regulation of nuclear gene expression, and that Rf17 has a role in restoring the ectopic gene expression. We also used a proteomic approach to discover the retrograde regulated proteins and identified six proteins that were accumulated differently. These results reveal organ-specific induced mitochondrial retrograde pathways affecting nuclear gene expression possibly related to CMS. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Sequence and expression analysis of the thioredoxin protein gene family in rice

Thioredoxin (Trx) proteins play important biological functions in cells by changing redox via thioldisulfied exchange. This system is especially widespread in plants. Through database search, we identified 30 potential Trx protein-encoding genes (OsTrx) in rice (Oryza sativa L.). An analysis of the complete set of OsTrx proteins is presented here, including chromosomal location, conserved motifs, domain duplication, and phylogenetic relationships. Our findings suggest that the expansion of the Trx gene family in rice, in large part, occurred due to gene duplication. A comprehensive expression profile of Trx genes family was investigated by analyzing the signal data of this family extracted from the whole genome microarray analysis of Minghui 63 and Zhenshan 97, two indica parents, and their hybrid Shanyou 63, using 27 different tissues representing the entire life cycle of rice. Results revealed specific expression of some members at germination transition as well as the 3-leaf stage during the vegetative growth phase of rice. OsTrx genes were also found to be differentially up- or down-regulated in rice seedlings subjected to treatments of phytohormones and light/dark conditions. The expression levels of the OsTrx genes in the different tissues and under different treatments were also checked by RT-PCR analysis. The identification of OsTrx genes showing differential expression in specific tissues among different genotypes or in response to different environmental cues could provide a new avenue for functional analyses in rice.     

Differential regulation of waxy gene expression in rice endosperm

Summary In order to examine the effects of different alleles on the gene expression at the waxy locus, the Wx gene product which controls the synthesis of amylose was isolated from endosperm starch of rice plants and analysed by electrophoretic techniques. The major protein bound to starch granules was absent in most of waxy strains and increased with the number of Wx alleles in triploid endosperms, suggesting that the major protein is the Wx gene product. In addition to wx alleles which result in the absence or drastic reduction of the Wx gene product and amylose, differentiation of Wx alleles seemed to have occurred among nonwaxy rice strains. At least two Wx alleles with different efficiencies in the production of the major protein as well as amylose were detected. These alleles are discussed in relation to regulation of the gene expression.     

A complete sequence of the rice sucrose synthase-1 (RSs1) gene

Using a fragment of the maize sucrose synthase gene Sh-1 as probe, the rice genome was shown to contain at least three genes encoding sucrose synthase. One of these genes was isolated from a genomic library, and its full sequence, including 1.7 kb of 5 flanking sequence and 0.9 kb of 3 flanking sequence, is reported. The new rice gene, designated RSs1, is highly homologous to maize Sh-1 (approx. 94% identity in derived amino acid sequence), and contains an identical intron-exon structure (16 exons and 15 introns). Both RSs1 and maize Sh-1 show similar sequence homologies to a second rice sucrose synthase gene described recently (designated RSs2, Yu et al. (1992) Plant Mol Biol 18: 139–142), although both the rice genes predict an extra 6 amino acids at the C-terminus of the protein when compared to the maize gene. The RSs1 5 flanking sequence contains a number of promoter-like sequences, including putative protein-binding regions similar to maize zein genes.     

Accessing genes in the tertiary gene pool of rice by direct introduction of total DNA fromZizania palustris (wild rice)

Transfer of useful genes from wild relatives of crop plants has relied upon successful conventional crossing or the availability of the cloned gene. Co-bombardment of rice callus with total genomic DNA from wild rice (Zizania palustris) and a plasmid containing a gene confirming hygromycin resistance allowed recovery under selection of transgenic plants with grain characteristics from wild rice. Amplified Fragment Length Polymorphism (AFLP) analysis suggested that a significant amount of DNA fromZizania was introduced by this procedure. One plant had 16 of a possible 122Zizania specific AFLP markers detected with the primers used. This approach may have potential for introgression of genes from wild relatives in other cases where highly efficient transformation methods are available.     

cDNA cloning and gene expression of the major prolamins of rice

A full-length cDNA (pS 18) encoding the 16 kDa rice prolamin composed of 158 amino acids was sequenced. Analysis of N-terminal amino acid sequence of a major rice prolamin indicated that an 18 amino acid signal peptide was removed from 16 kDa precursor prolamin to form the 14 kDa prolamin during seed development. Synthesis of the 16 kDa precursor prolamin began around 8 days after flowering (DAF), increased remarkably at 8–11 DAF and gradually reached maximum levels with the maturation of rice seeds.     

Molecular cloning, expression and characterization of a glycosyltransferase from rice

Secondary plant metabolites undergo several modification reactions, including glycosylation. Glycosylation, which is mediated by UDP-glycosyltransferase (UGT), plays a role in the storage of secondary metabolites and in defending plants against stress. In this study, we cloned one of the glycosyltransferases from rice, RUGT-5 resulting in 40–42% sequence homology with UGTs from other plants. RUGT-5 was functionally expressed as a glutathione S-transferase fusion protein in Escherichia coli and was then purified. Eight different flavonoids were used as tentative substrates. HPLC profiling of reaction products displayed at least two peaks. Glycosylation positions were located at the hydroxyl groups at C-3, C-7 or C-4′ flavonoid positions. The most efficient substrate was kaempferol, followed by apigenin, genistein and luteolin, in that order. According to in vitro results and the composition of rice flavonoids the in vivo substrate of RUGT-5 was predicted to be kaempferol or apigenin. To our knowledge, this is the first time that the function of a rice UGT has been characterized.     

DNA markers tightly linked to a gall midge resistance gene (Gm2) are potentially useful for marker-aided selection in rice breeding

We have developed a polymerase chain reaction (PCR)-based assay that could effectively reduce the time period required to screen and select for Gall Midgeresistant rice lines under field conditions. The primers for the assay were designed on the basis of sequence information of two phenotype specific random amplified polymorphic DNA fragments which were found to be tightly linked to Gall Midge biotype-1 resistance gene (Gm2). The two RAPD fragments, F8₁₇₀₀ in the susceptible parent ARC6650 and F10₆₀₀ in the resistant parent Phalguna, were identified after screening 5450 loci using 520 random primers on genomic DNAs of ARC6650 and Phalguna. These primers, when used in a multiplexed PCR, amplified specifically a 1.7-kb and 0.6-kb fragment in the susceptible and resistant parents, respectively. When this assay was performed on genomic DNAs of 44 recombinant inbred lines derived from ARC6650 x Phalguna and 5 lines derived from other crosses where one of the parents was Phalguna, ARC6650 or their derivatives, the primers amplified a 1.7-kb fragment in all of the susceptible lines or a 0.6-kb fragment in all of the resistant ones. These markers can be of potential use in the marker-aided selection of Gall Midge biotype-1 resistant phenotypes. As screening for resistance can now be conducted independent of the availability of insects, the breeding of resistant varieties can be hastened.     

An improved technique for culture of rice panicles

An improved technique for long-term culture of rice caryopses is necessary for physiological and genetic studies. Panicles of three rice (Oryza sativa) cultivars `Lemont', `Gummo-byeu' and `Hwasung-byeu' were cultured in liquid media with combinations of light versus dark, panicle position, nitrogen level (5-40 mM), and sucrose level (29–351 mM). Grain growth was increased when panicles were positioned horizontally, partially submerged in the media, owing to greater media contact and apoplastic uptake as observed by fluorescent dyes. The optimal media assimilate supply included 175 mM sucrose and 5 mM nitrogen. Grain fill occurred for up to four weeks; grain dry weight reached 80% of that on intact plants, with 50% germination. This technique should allow for future physiological studies with rice or other panicle-bearing species.