The Cloning And Function Analysis Of A Gene Corresponding For A Plant Architecture Mutant(Pad) In Rice

As one of the crucial staple food crops, rice feeds nearly half the world's population. Among several factors related to rice grain yields, plant architecture plays a vital role. Plant height is an important trait that affects plant architecture and is determined by the number and lengths of internodes as well as environmental conditions. Plant height is primarily regulated by autologous phytohormones, including gibberellins (GAs) and brassinosteroids (BRs). When GA biosynthesis or signal transduction pathways were interupted, plant growth and development would be influenced and finally affects plant architecture, especially plant height.In this study, we characterized a recessive mutant, plant architecture determinant (pad), derived from the Oryza sativa ssp. indica cultivar MH86. The mutant exhibited obvious dwarf phenotype. A gene corresponding to pad was isolated via a map-based cloning strategy and verified by the genetic complementation assay. Meanwhile, we investigated the PAD function and molecular mechanism of affecting plant architecture defect. The primary results were showed as follows:1. The pad mutant exhibits a visibly abnormal phenotype during the vegetative and reproductive growth stages, including dwarf architecture, smaller shoot, curly and wrinkled leaf, smaller panicle and reduced plant height.2. The characterization of grain morphologies of the mutant indicated that no matter the seed or brown rice, the length and thickness of the seeds reduced in the mutant compared with in the wild type, while the grain width is significant difference between in the mutant and wild type. However, but extremely significant difference of grain thickeness of the brown rice between the mutant and the wild type. Obvioulsy, the mutant affects the grain filling.3. Cytological studies revealed that the cell sizes in the leaves and roots of the pad mutant were smaller than in those of WT. In particular, the uppermost internode was significantly longer in WT than that in the mutant. This result suggests that cell expansion is severely disrupted in the mutant.4. The genetic analysis of the mutant indicated that the phenotype in the F1exhibits wild-type phenotype, implying that the mutant locus is recessive, and exhibited Mendel's single gene segregation in the F2population of MH86×pad and58N×pad crosses. These data indicate that the phenotypes of pad mutant are controlled by a single recessive locus.5. The pad locus was mapped to the long arm of chromosome3by Bulk Segregant Analysis (BSA). Then, the pad locus finally was fine-mapped to located within an8.7kb region using470and3869homozygous recessive plants, respectively. Only one open reading frame (ORF), Os03g0157300, was found in this region. Sequencing of the genomic DNA in this region revealed that the mutant allele harbored a G to C substitution at nucleotide position131in the predicted exon2of Os03g0157300. Os03g0157300was primarily considered as the candidate gene of pad locus.6. The genetic complementation verified all the mutant phenotypes, including dwarf shape (vegetative phase and ripening phase), shorter stems, aberrant leaves, smaller panicles, and partial grain filling, were rescued in all independent T1transgenic plants carrying the candidate PAD gene.7. We found the phenotype of reduced plant height in RNAi plants. Furthermore, RNAi plants significantly reduced the transcript levels of OsMCA I/PAD compared with the wild type Nongken58. However, the RNAi lines did not display dwarf phneotype although the plant architecter morphology had some change than the control. These results imply that the abnormal phenotype of the mutant mainly might be caused the change of protein function.8. The expression profiles of OsMCA1/PAD gene were done by quantitative real-time PCR (QRT-PCR) analyses and GUS activity assay. The results showed the expression of OsMCAl/PAD can be detected in all tissues, including shoot, seedling, root, stem, node, young panicle and husk.9. Subcellular distribution analysis of the GFP-OsMCA1fusion protein indicated that PAD located on the plasma membrane, whch was confirmed by plasmolysis that treated with1M sucrose.10. High levels of expression of the mutated OsMCA1/PAD (OsmMCA1/pad) protein were obtained in E. coli, however, the protein became insoluble and formed inclusion bodies. Whereas the expression of the WT protein was very low with detectable level by western blot only. These results indicated the pad protein might have distinct physicochemical properties from the PAD protein.11. The phylogenetic evolution of OsMCA1/PAD showed that OsMCA1/PAD homologous proteins were catorized into two clades, one belonging to monocots and the other belonging to dicots. These data suggest that the OsMCA1/PAD protein may exhibit a highly conserved biological function in higher plants.12. The sensitive assay responding to GA3of the pad seedling indicated the pad mutant exhibited weak sensitivity to exogenous GA3no matter treated with uniconazol pretreatment. The effects for GA3exhibited in a concentration-dependent manner compared with WT. Furthermore, the difference in the length of the second sheath became more obvious with increasing GA3concentrations.13. We analyzed endogenous GAs contents in the third internode of WT plants and pad mutants. Results showed the levels of the precursors of GA1, GA53, GA44and GA19in the pad mutant were decreased approximately one-third to one-half compared with WT. Furthermore, bioactive GA (GA1) contents in pad mutant significantly decreased up to one-third of WT levels.14. The expression levels of the genes involved in GA metabolism were upregulated in the internodes of pad mutant. In particular, the genes for GA deactivitation were largely upregulated. These results indicated that the OsMCA1/PAD involved in regulation of GA metabolism in rice.In summary, we characterized a recessive mutant, plant architecture determinant (pad), derived from the Oryza sativa ssp. indica cultivar MH86. The mutant exhibited severe dwarf phenotypes, including shorter and stunted leaves, fewer secondary branches during the entire growth and developmental stage. Cytological studies revealed that pad mutant growth defects are primarily due to the inhibition of cell expansion. The PAD gene was isolated using a map-based cloning strategy. It encodes a plasma membrane protein OsMCAl, a homolog of AtMCAl in Arabidopsis. A SNP of G to C substitution in the predicted exon2of OsMCA1led to the amino acid change R44P. Genetic complementation experiment demonstrated the SNP in OsMCA1/PAD caused pad dwarf phenotypes. Tissue-specific expression profile showed OsMCA1/PAD was universally expressed in rice tissues from the vegetative to reproductive growth stages. Quantitative real-time PCR analysis revealed that the most of the genes responding to gibberellin (GA) metabolism were up-regulated in pad mutant internodes. The endogenous GA content measurement revealed that the levels of GA1were significantly decreased in the third internode of pad mutants. Moreover, a GA response assay suggested that OsMCA1/PAD might be involved in the regulation of GA metabolism and signal transduction. Our further results revealed the pad is a loss-of-function mutant of the OsMCAl/PAD, leading to upregulation of genes related to GA deactivation, which decreased bioactive GA levels. This finding will provide insight to further understanding of the mechanism of GA metabolism regulation and the function of kind of OsMCA1/PAD genes in higher plants.