Map-based cloning of PLA1, a heterochronic gene, in rice(Oryza sativa L.)

The rice PLASTOCHRON1 locus is considered to regulate the leaf initiation rate (plastochron) and the duration of vegetative phase without affecting reproductive phase (Itoh et al., 1998). To elucidate regulatory mechanism for the plastochron and the duration of vegetative phase, I have isolated the PLASTOCHRON1 gene by map-based cloning and investigated the in situ expression pattern during rice development. Molecular cloning of PLA1 gene gives a clue to understand the mechanism for the temporal regulation of developmental program in rice. A genetic and physical map was constructed to isolate the causal gene for pla1 syndrome. Small-scale mapping was carried out to determine approximate map position of the pla1 locus and then a high-resolution genetic mapping was performed for pla1-2, one of the pla1 alleles, using an F2 population comprising 578 pla1-2 homozygous plants. In a high-resolution genetic map, the pla1 locus was found to be mapped between RFLP markers C961 and R1738A on chromosome 10, within a 3.6cM genetic distance. A physical map encompassing the pla1 locus was constructed by overlapping Bacterial Artificial Chromosome (BAC) clones through chromosome walking. PCR-based RFLP markers from BAC-end clones were developed and mapped relative to the pla1 locus. Physical map construction using BAC clones indicated that a BAC clone, B44A10 (167Kb), contained the pla1 locus within 74Kb corresponding to a 0.52cM genetic distance. Gene prediction of 74Kb region carrying the pla1 locus was performed by using gene prediction program and designated several candidate genes for pla1 gene including cytochrome P450, putative GTPase regulator protein and transposable like element. Nucleotide comparison analysis between four alleles of pla1 mutants, pla1-1, pla1-2, pla1-3 and pla1-4, and wild type revealed that all alleles of pla1 mutant had mutations only in P450 gene among several candidate genes. Genetic complementation experiment confirmed that all transgenic plants transformed with binary vector pBGH1-PLA1 (6Kb), which contain P450 gene, to pla1-2 homozygous plants completely recovered to wild type phenotype, in contrast to no recovery by transformation with pBGH1 vector only. The PLA1 gene of P450 was designated as CYP78A11 in accordance with the guidelines for Cytochrome P450 by the nomenclature committee. This is the first protein belong to the CYP78A subfamily in plants. The predicted CYP78A11 was 1.8kb in size and consists of two exons and one short intron. The CYP78A11 codes 555 amino acid with a calculated molecular mass of 59KD. Similarity search showed that the PLA1 protein conserved significant sequences such as heme binding domain near C-terminus, potential oxygen binding domain and steroid binding domain, which were conserved in all cytochrome P450 superfamily. In situ hybridization experiments elucidated the spatial and temporal expression pattern of the PLA1 gene. PLA1 mRNA was localized in leaves but not at SAM through vegetative and reproductive phase. The mRNA localization was gradually changed during leaf development. Although pla1 mutants exhibited several defect in leaf and SAM, the leaf specific expression suggested that PLA1 gene functions in non cell-autonomous manner and affects leaf development and SAM activity. The decay pattern of PLA1 expression in leaves shows a correlation to the pattern of leaf development in monocotyledous plants, suggesting the possible role of PLA1 gene for regulating maturation schedule of leaf. In the future works, transgenic plants overexpressing the CYP78A11 gene may provide an additional clue to elucidate the functional role of the PLA1. The P450s, however, form the largest family of plant enzymes and have wide diversity of substrates and high degree of amino acid variation in the superfamily. Though the CYP78A11 seems to be functional in a given step of plant hormone biosynthesis or degradation pathway, analysis of changes of limited amount of plant hormones in the restricted regions would be very difficult. Even taking a time, biochemical analysis of CYP78A11 including a search of the substrates will give a clue to unravel the molecular mechanism of the phase change in plant growth and development controlled by PLA1