Yaping Fu

Identification and characterization of HTD2: a novel gene negatively regulating tiller bud outgrowth in rice

Tiller number is highly regulated by controlling the formation of tiller bud and its subsequent outgrowth in response to endogenous and environmental signals. Here, we identified a rice mutant htd2 from one of the 15,000 transgenic rice lines, which is characterized by a high tillering and dwarf phenotype. Phenotypic analysis of the mutant showed that the mutation did not affect formation of tiller bud, but promoted the subsequent outgrowth of tiller bud. To isolate the htd2 gene, a map-based cloning strategy was employed and 17 new insertions-deletions (InDels) markers were developed. A high-resolution physical map of the chromosomal region around the htd2 gene was made using the F2 and F3 population. Finally, the gene was mapped in 12.8xa0kb region between marker HT41 and marker HT52 within the BAC clone OSJNBa0009J13. Cloning and sequencing of the target region from the mutant showed that the T-DNA insertion caused a 463xa0bp deletion between the promoter and first exon of an esterase/lipase/thioesterase family gene in the 12.8xa0kb region. Furthermore, transgenic rice with reduced expression level of the gene exhibited an enhanced tillering and dwarf phenotype. Accordingly, the esterase/lipase/thioesterase family gene (TIGR locus Os03g10620) was identified as the HTD2 gene. HTD2 transcripts were expressed mainly in leaf. Loss of function of HTD2 resulted in a significantly increased expression of HTD1, D10 and D3, which were involved in the strigolactone biosynthetic pathway. The results suggest that the HTD2 gene could negatively regulate tiller bud outgrowth by the strigolactone pathway.

Identification and fine mapping of a thermo-sensitive chlorophyll deficient mutant in rice (Oryza sativa L.)

A thermo-sensitive chlorophyll deficient mutant was isolated from more than 15,000 transgenic rice lines. The mutant displayed normal phenotype at 23°C or lower temperature (permissive temperature). However, when grown at 26°C or higher (nonpermissive temperature) the plant exhibited an abnormal phenotype characterized by yellow green leaves. Genetic analysis revealed that a single nuclear-encoded recessive gene is responsible for the mutation, which is tentatively designed as cde1(t) (chlorophyll deficient 1, temporally). PCR analysis and hygromycin resistance assay indicated the mutation was not caused by T-DNA insertion. To isolate the cde1(t) gene, a map-based cloning strategy was employed and 15 new markers (five SSR and ten InDels markers) were developed. A high-resolution physical map of the chromosomal region around the cde1(t) gene was made using F2 and F3 population consisting of 1,858 mutant individuals. Finally, the cde1(t) gene was mapped in 7.5xa0kb region between marker ID10 and marker ID11 on chromosome 2. Sequence analysis revealed only one candidate gene, OsGluRS, in the 7.5xa0kb region. Cloning and sequencing of the target region from the cde1(t) mutant showed that a missense mutation occurred in the mutant. So the OsGluRS gene (TIGR locus Os02xa0g02860) which encode glutamyl-tRNA synthetase was identified as the Cde1(t) gene.

Phosphomannose-isomerase (pmi) gene as a selectable marker for rice transformation via Agrobacterium

A new selectable marker system was adapted for use in Agrobacterium-mediated transformation of rice (Oryza sativa L.). This selection system utilizes the pmi gene encoding for phosphomannose-isomerase that converts mannose-6-phosphate to fructose-6-phosphate. Only transformed cells were capable of utilizing mannose as a carbon source. Transgenic rice plants regenerated from selected transformed immature embryo-derived calli on media containing various concentrations of mannose. The highest transformation frequency of 6.0% was obtained when a combination of 25 g/l mannose and 5 g/l sucrose was used. Molecular and genetic analyses showed that the plants contained the pmi gene and the gene was transmitted to the progeny in Mendelian fashion. The results indicated that the mannose selection system, which was devoid of the disadvantages of antibiotic or herbicide selection, could be used for rice Agrobacterium-mediated immature embryo transformation.

Isolation and characterization of a rice mutant with narrow and rolled leaves

Appropriate leaf shape has proved to be useful in improving photosynthesis and increasing grain yield. To understand the molecular mechanism of leaf morphogenesis, we identified a rice mutant nrl1, which was characterized by a phenotype of narrow and rolled leaves. Microscopic observation showed that the mutation significantly decreased the number of vascular bundles of leaf and stem. Genetic analysis revealed that the mutation was controlled by a single nuclear-encoded recessive gene. To isolate the nrl1 gene, 756 F2 and F3 mutant individuals from a cross of the nrl1 mutant with Longtepu were used and a high-resolution physical map of the chromosomal region around the nrl1 gene was made. Finally, the gene was mapped in 16.5xa0kb region between marker RL21 and marker RL36 within the BAC clone OSJNBa0027H05. Cloning and sequencing of the target region from the mutant showed that there was a 58xa0bp deletion within the second exon of the cellulose synthase-like D4 gene (TIGR locus Os12g36890). The nrl1 mutation was rescued by transformation with the wild-type cellulose synthase-like D4 gene. Accordingly, the cellulose synthase-like D4 gene was identified as the NRL1 gene. NRL1 was transcribed in various tissues and was mainly expressed in panicles and internodes. NAL7 and SLL1 were found to be upregulated, whereas OsAGO7 were downregulated in the nrl1 mutant. These findings suggested that there might be a functional association between these genes in regulating leaf development.

The Effect of the Crosstalk between Photoperiod and Temperature on the Heading-Date in Rice

Photoperiod and temperature are two important environmental factors that influence the heading-date of rice. Although the influence of the photoperiod on heading has been extensively reported in rice, the molecular mechanism for the temperature control of heading remains unknown. This study reports an early heading mutant derived from tissue culture lines of rice and investigates the heading-date of wild type and mutant in different photoperiod and temperature treatments. The linkage analysis showed that the mutant phenotype cosegregated with the Hd1 locus. Sequencing analysis found that the mutant contained two insertions and several single-base substitutions that caused a dramatic reduction in Hd1mRNA levels compared with wild type. The expression patterns of Hd1 and Hd3a were also analyzed in different photoperiod and temperature conditions, revealing that Hd1 mRNA levels displayed similar expression patterns for different photoperiod and temperature treatments, with high expression levels at night and reduced levels in the daytime. In addition, Hd1 displayed a slightly higher expression level under long-day and low temperature conditions. Hd3a mRNA was present at a very low level under low temperature conditions regardless of the day-length. This result suggests that suppression of Hd3a expression is a principle cause of late heading under low temperature and long-day conditions.

Genetic analysis and identifi- cation of a large leaf angles (lla) mutant in rice

Mutants are essential genetic materials to elucidation of biological functions of genes involved. Characterization and isolation of genes in mutants is one of the research tasks in functional genomic era. T-DNA insertional mutagenesis has provided an efficient way to identify genes in plant species, in which the mutated genes could be rapidly isolated once the mutant was confirmed by T-DNA insertion. About 40% of 620 cloned Arabidopsis genes with mutant phenotypes were determined by T-DNA tagging. In recent years, a large number of T-DNA insertional rice mutant pools were established. Moreover, three genes were successfully isolated from rice. Leaf angles, the angles between main culms and leaves, are one of the important features of the plant types. Investigation on the mechanism of leaf angle formation will probably provide the basic knowledge of plant breeding on plant architectures. Several QTLs of leaf and flag leaf angles in rice were identified, which would provide the useful materials for manipulating these QTLs in a marker-assisted selection program. However, the major genes, which control leaf angles, have not been reported yet and very little is known about the mechanism of leaf angle formation. Based on T-DNA inserted rice (Oryza sativa L. subsp. Japonica cv. Zhonghua11) mutant pool, which contains about 10000 insertional mutant lines, a large leaf angles (lla) mutant T429 was found in T1 lines. At seeding stage, the leaf angles of lla mutant were larger than those of the wild type. In addition, the mutant seedlings showed semi-dwarf. At heading stage, the abnormal phenotypes of lla mutants were observed in plant height, leaf angel, leaf blade length and leaf blade width. The plant of lla was shorter than the wild type (Fig. 1). The length and width of the lla mutant leaves were shorter and wider than those of the wild type (unpublished data). Under natural long day conditions, the heading date of lla was about two weeks later than that of the wild type. Under winter natural short day conditions in greenhouse, the heading date of lla was about one week later than that of the wild type while the plant height and leaf angles remained significantly different from the control. This indicated that the mutant phenotype was slightly influenced by environmental conditions such as photoperiod and temperature. Different concentrations of GA3 were used to treat the plants at both seeding and heading stages; the results showed that the plant heights of wild type and the lla mutant were all sensitive to exogenous GA3 at different concentrations. But plant heights and leaf angles of GA3 treated mutant plants differed significantly from the corresponding wild types. At the same time, leaf angles of GA3 treated wild type and lla did not show any visible differences from the corresponding untreated plants.

OsCOL16, encoding a CONSTANS-like protein, represses flowering by up-regulating Ghd7 expression in rice

Flowering time is an important agronomic trait that coordinates the plant life cycle with regional adaptability and thereby impacts yield potentials for cereal crops. The CONSTANS (CO)-like gene family plays vital roles in the regulation of flowering time. CO-like proteins are typically divided into four phylogenetic groups in rice. Several genes from groups I, III, and IV have been functionally characterized, though little is known about the genes of group II in rice. We report the functional characterization in rice of a constitutive floral inhibitor, OsCOL16, encoding a group-II CO-like protein that delays flowering time and increases plant height and grain yield. Overexpression of OsCOL16 resulted in late heading under both long-day and short-day conditions. OsCOL16 expression exhibits a diurnal oscillation and serves as a transcription factor with transcriptional activation activity. We determined that OsCOL16 up-regulates the expression of the floral repressor Ghd7, leading to down-regulation of the expression of Ehd1, Hd3a, and RFT1. Moreover, genetic diversity and evolutionary analyses suggest that remarkable differences in flowering times correlate with two major alleles of OsCOL16. Our combined molecular biology and phylogeographic analyses revealed that OsCOL16 plays an important role in regulating rice photoperiodic flowering, allowing for environmental adaptation of rice.