Functional Analysis Of OsAS2 Gene In Arabidopsis And Rice

Leaf is the important lateral organs of higher plants. The development of leaf is under the control of both environmental factors and internal genetic factors. In addition to the differences of leaf morphology and structure between the monocots and dicots, the mechanism of leaf development may be different. In recent years, the studies on mutants of Arabidopsis and Antirrhinum have provided important clues to understand the molecular mechanism controlling leaf development of dicots. However, litter is known about the regulation of leaf development in monocots. Rice is a monocot and an important cereal. So it is important to determine the molecular mechanism of its leaf development. The genes containing LATERAL ORGAN BOUNDARIES (LOB)-domain, found in plants only, played an important role in plant development. For example, many LOB-domain genes of dicot had been identified and their function in determining polarity and development of lateral organs including leaf was revealed. In this study, we isolated two LOB-domain genes, OsAS2 and OsLOB1, from rice (Oryza sativa) and analyzed their expression patterns and functions in plant development. Our data provide some important information to understand the molecular mechanism of rice leaf development. The results and conclusions are as follows:1. Isolation and characterization of OsAS2To investigate the regulation mechanism of leaf abaxial-adaxial polarity in rice, we isolated full length cDNAs of OsAS2 from the young leaves The full length of OsAS2 cDNA was 824bp, encoding a predicted protein with 270 amino acid residues. In its'N terminus, the proteins had LOB domain which containing C motif and GAS motif. Sequences analysis revealed that OsAS2 belong to the CLASS I LOB domain family. Sequencing and phylogenetic analysis showed that OsAS2 was closer to Arabidopsis AS2 suggesting that OsAS2 were the orthologs of AS2. 2. Analysis of OsAS2 expression patternThe expression pattern of OsAS2 was determined by reverse transcriptase-mediated (RT) PCR and in situ hybridization. OsAS2 gene transcripts were detected in rice vegetative and reproductive organs. The expression of OsAS2 was higher in young organs than in mature organs. In situ hybridization revealed that different from AS2 the expression of OsAS2 was detected in shoot apical meristem.but was lower than that in leaf primordium, young leaf and reproductive organ. The OsAS2 mRNA accumulated in the abaxial part of leaf primordia. These results indicated that OsAS2 were involved in the determination of shoot apical meristem and the regulation of lateral organ development.3. Function analysis of OsAS2 in Arabidopsis and RiceThe overexpression of AS2 functioned in lateral organ development and polarity determination of transgenic plants. Cotyledons and leaves of Arabidopsis expressed 35S::AS2 were narrow and curled toward the adaxial side, and finger-like outgrowth on the abaxial side of leaf. The elongation of inflorescence was seriously restrained and the short peduncle, protrusive calyces on the abaxial side and sterile flower were found in some transgenic plants. Further analysis of the rosette leaves of transgenic plants revealed the polarity abnormality of the internal tissues in leaf mesophyll.To compare the functions between OsAS2 and AS2, we transferred 35S::OsAS2 into Arabidopsis and analyzed the phenotype of transgenic plants. Compared with the wild type, the transgenic Arabidopsis exhibited dwarfing plant, narrower leaf, shorter and wider leafstalk and smaller leaf. The inflorescence stems of 35S:OsAS2 plants did not elongate. In addition, the transgenic plant had abnormal floral organs, such as sterile flower and shorter filament and ovary than those of the wild type. Further analysis revealed that the abaxial cells of mesophyll of transgenic plants owned some traits of the adaxial cells, indicating the abnormal polarity of mesophyll of transgenic plants. Our results indicated that OsAS2 was involved in the regulation of later organ development which was similar to Arabidopsis AS. But they also have some different functions in regulate lateral organs development. OsAS2 affected the shoot apical meristem and lateral organ development.To determine OsAS2 functions in rice development, the ubi::OsAS2 and ubi::anti-OsAS2 were transferred into rice. Although green tissues were found in the callus overexpressed OsAS2, the differentiation of transgenic callus was later than that of the control. Few green tissues could produce abnormal shoots with leaf-like tissue, however, the growth of seedlings was arrested afterwards. Histology analysis revealed that the cell division capacity of transgenic callus kept longer than that of the control, however, the differentiation of these cells was inhibited. In addition, the expression of Oryza;CYCD3;1, a gene involved in promoting cell division, was upregulated in callus. And the upregulated expression of Oryza sativa homeobox1 (OSH1), a meristem-characteristic gene, and Oryza sativa WUS-like homeobox3 (OsWOX3), a gene involved in differentiation of lateral organs, were detected in the transgenic callus of overexpressing OsAS2. The transgenic callus expressing ubi::anti-OsAS2 could differentiate into plants, which is similar to that of the control. The phenotype of ubi::anti-OsAS2 plants is being analyzed. These results suggested that different from the function of Arabidopsis AS2, OsAS2 not only was involved in lateral organ development but also played an important role in shoot differentiation and development.OsAS2 might play its roles in shoot differentiation and development by regulating the expression levels of the genes which involved in cell division and meristem formation.4. Isolation and expression pattern analysis of OsLOB1We isolated full length cDNAs of OsLOB1 from the root tips. The cDNA length and the predicted protein of OsLOB1 were 774bp and 253 amino acid residues, respectively. The protein had LOB domain which containing C motif and GAS motif. Sequences analysis revealed that OsLOB1 belong to the CLASS I LOB domain family. Sequencing and phylogenetic analysis showed that OsLOB1 was closer to Arabidopsis LOB, suggesting that OsLOB1 were the orthologs of LOB.As OsAS2, the expression of OsLOB1 mainly was detected in shoot apical meristem, leaf primordium, young leaf and floral organ. However, no apparent polarity of OsLOB1 mRNA distribution was found in young leaf. These results indicated that OsLOB1 were involved in the determination of shoot apical meristem and the regulation of lateral organ development.