Overexpression of folate biosynthesis genes in rice (Oryza sativa L.) and evaluation of their impact on seed folate content.

Folate (vitamin B9) deficiency is a global health problem especially in developing countries where the major staple foods such as rice contain extremely low folates. Biofortification of rice could be an alternative complement way to fight folate deficiency. In this study, we evaluated the availability of the genes in each step of folate biosynthesis pathway for rice folate enhancement in the japonica variety kitaake genetic background. The first enzymes GTP cyclohydrolase I (GTPCHI) and aminodeoxychorismate synthase (ADCS) in the pterin and para-aminobenzoate branches resulted in significant increase in seed folate content, respectively (P < 0.01). Overexpression of two closely related enzymes dihydrofolate synthase (DHFS) and folypolyglutamate synthase (FPGS), which perform the first and further additions of glutamates, produced slightly increase in seed folate content separately. The GTPCHI transgene was combined with each of the other transgenes except ADCS to investigate the effects of gene stacking on seed folate accumulation. Seed folate contents in the gene-stacked plants were higher than the individual low-folate transgenic parents, but lower than the high-folate GTPCHI transgenic lines, pointing to an inadequate supply of para-aminobenzoic acid (PABA) precursor initiated by ADCS in constraining folate overproduction in gene-stacked plants.

A role for a dioxygenase in auxin metabolism and reproductive development in rice.

Indole-3-acetic acid (IAA), the natural auxin in plants, regulates many aspects of plant growth and development. Extensive analyses have elucidated the components of auxin biosynthesis, transport, and signaling, but the physiological roles and molecular mechanisms of auxin degradation remain elusive. Here, we demonstrate that the dioxygenase for auxin oxidation (DAO) gene, encoding a putative 2-oxoglutarate-dependent-Fe (II) dioxygenase, is essential for anther dehiscence, pollen fertility, and seed initiation in rice. Rice mutant lines lacking a functional DAO display increased levels of free IAA in anthers and ovaries. Furthermore, exogenous application of IAA or overexpression of the auxin biosynthesis gene OsYUCCA1 phenocopies the dao mutants. We show that recombinant DAO converts the active IAA into biologically inactive 2-oxoindole-3-acetic acid (OxIAA) in vitro. Collectively, these data support a key role of DAO in auxin catabolism and maintenance of auxin homeostasis central to plant reproductive development.

Transcriptome analysis of grain-filling caryopses reveals involvement of multiple regulatory pathways in chalky grain formation in rice.

BACKGROUND: Grain endosperm chalkiness of rice is a varietal characteristic that negatively affects not only the appearance and milling properties but also the cooking texture and palatability of cooked rice. However, grain chalkiness is a complex quantitative genetic trait and the molecular mechanisms underlying its formation are poorly understood. RESULTS: A near-isogenic line CSSL50-1 with high chalkiness was compared with its normal parental line Asominori for grain endosperm chalkiness. Physico-biochemical analyses of ripened grains showed that, compared with Asominori, CSSL50-1 contains higher levels of amylose and 8 DP (degree of polymerization) short-chain amylopectin, but lower medium length 12 DP amylopectin. Transcriptome analysis of 15 DAF (day after flowering) caryopses of the isogenic lines identified 623 differential expressed genes (P < 0.01), among which 324 genes are up-regulated and 299 down-regulated. These genes were classified into 18 major categories, with 65.3% of them belong to six major functional groups: signal transduction, cell rescue/defense, transcription, protein degradation, carbohydrate metabolism and redox homeostasis. Detailed pathway dissection demonstrated that genes involved in sucrose and starch synthesis are up-regulated, whereas those involved in non-starch polysaccharides are down regulated. Several genes involved in oxidoreductive homeostasis were found to have higher expression levels in CSSL50-1 as well, suggesting potential roles of ROS in grain chalkiness formation. CONCLUSION: Extensive gene expression changes were detected during rice grain chalkiness formation. Over half of these differentially expressed genes are implicated in several important categories of genes, including signal transduction, transcription, carbohydrate metabolism and redox homeostasis, suggesting that chalkiness formation involves multiple metabolic and regulatory pathways.

Two novel loci for pollen sterility in hybrids between the weedy strain Ludao and the Japonica variety Akihikari of rice (Oryza sativa L.).

Partial pollen sterility has been observed in hybrid progeny derived from a japonica cultivar, Akihikari and a weedy strain, Ludao, which naturally grows in Jiangsu province of east China. Cytological and histological analyses revealed that pollen abortion occurred largely at the bicellular pollen stage, primarily due to the gradual disaggregation of generative and vegetative cells. A genome-wide analysis was further carried out in a backcross population of Akihikari //Ludao/Akihikari using a total of 118 simple sequence repeat (SSR) markers and an expressed sequence tag (EST) marker distributed on the entire rice linkage map. Two loci controlling hybrid pollen sterility, designated as S33(t) and S34(t), were located on chromosomes 3 and 11, respectively. Both loci were putatively different from all the previously reported gametophyte genes and hybrid pollen sterility loci. Interaction between the Ludao and Akihikari alleles at each of the two loci resulted in reduction of fertility in the pollens carring the Ludao alleles. To map the precise location of the major locus, S33(t), we selected 165 plants of the backcross population with pollen fertility higher than 80.0%, and assayed the recombinant events surrounding the S33(t) locus using newly developed SSR markers. The S33(t) was delimited to an 86 kb region between SSR markers RM15621 and RM15627. Sequence analysis of this region indicated that there were ten open reading frames. These results will be valuable for cloning this gene and marker-assisted transferring of the corresponding neutral allele in rice breeding programs. Furthermore, the origin of the weedy strain Ludao is discussed.

[QTL analysis of low-temperature-sensitive pollen sterility in Indica-japonica hybrid rice (Oryza sativa L.)].

There existed a number of biological constraints in exploiting the heterosis of indica-japonica hybrid rice. The low-temperature-sensitive sterility (LTSS) of indica-japonica hybrid has become one of the major problems in indica-japonica hybrid rice breeding after the solution of poor fertility of the hybrids by the finding of wide-compatibility gene. Previous studies revealed that the LTSS might be caused by low-temperature-sensitive pollen sterility (LTSPS). However, the genetic basis of LTSPS remained unclear. To explore the genetic basis of LTSPS in indica-japonica hybrid rice, an F2 genetic population derived from 3037 (indica) and 02428 (japonica) was developed. At the booting stage, pollen fertility of F2 population together with parents were surveyed after the treatment with low temperature daily average of 21-23 degrees C. The linkage map was constructed containing 108 SSR markers distributed throughout the whole 12 chromosomes with average marker interval of 16.26 cM. Using software MapMaker/QTL, two putative QTLs, namely qLTSPS2 and qLTSPS5 on chromosomes 2 and 5 were detected by interval mapping, which could explain the phenotypic variation 15.6% and 11.9% respectively. The additive effects were 0.021 and 0.045, dominant effects were -0.246 and -0.215, and the degrees of dominance were 11.7 and 4.8, respectively for the two QTLs. Therefore, the mode of gene action in response to low-temperature stress was overdominance and LTSPS was mainly the result of interaction between the indica and japonica alleles within each locus. In addition, two-way ANOVA showed that the two QTLs acted essentially independent of each other in conditioning LTSPS.