PLASTOCHRON1, a timekeeper of leaf initiation in rice, encodes cytochrome P450

Abstract

During postembryonic development of higher plants, the shoot apical meristem produces lateral organs in a regular spacing (phyllotaxy) and a regular timing (plastochron). Molecular analysis of mutants associated with phyllotaxy and plastochron would greatly increase understanding of the developmental mechanism of plant architecture because phyllotaxy and plastochron are fundamental regulators of plant architecture. pla1 of rice is not only a plastochron mutant showing rapid leaf initiation without affecting phyllotaxy, but also a heterochronic mutant showing ectopic shoot formation in the reproductive phase. Thus, pla1 provides a tool for analyzing the molecular basis of temporal regulation in leaf development. In this work, we isolated the PLA1 gene by map-based cloning. The identified PLA1 gene encodes a cytochrome P450, CYP78A11, which potentially catalyzes substances controlling plant development. PLA1 is expressed in developing leaf primordia, bracts of the panicle, and elongating internodes, but not in the shoot apical meristem. The expression pattern and mutant phenotype suggest that the PLA1 gene acting in developing leaf primordia affects the timing of successive leaf initiation and the termination of vegetative growth.

Fig. 1.

Phenotypes of wild-type, pla1 mutant, and transgenic pla1-2 plants. (a) Seedlings of wild-type (WT) and pla1-2 plants 17 days after germination, showing that many more leaves are formed in pla1-2 than in wild type. Arrowheads indicate lamina joint. (b) Panicles of wild type, pla1-1 with vegetative shoots instead of primary branches, and pla1-2 with truncated panicle, one shoot (arrowhead), and enlarged bract (arrow). (c) Scanning electron microscopy of a wild-type young panicle. (d) Scanning electron microscopy of a pla1-1 young panicle. Asterisks indicate ectopic vegetative shoots with an enlarged bract. (e) pla1-2-like transgenic plants carrying pBGH1 alone. Normal transgenic plants carrying pBGH1/P450 are shown to the right. (f) Panicles of transgenic plants: left, a pla1-2-like panicle with enlarged bract (arrowhead) in transgenic plant carrying pBGH1; right, normal panicle in transgenic plant carrying pBGH1/P450.

Fig. 2.

Structure of the PLA1 gene. (a) Exon/intron structure of PLA1. Two open boxes indicate protein coding regions composed of two exons split by a small intron. Mutations in four alleles are indicated as follows: (i) deletion of C at the position of 1,029 bp in pla1-1 resulting in frameshift of the ORF, (ii) nucleotide transition of C to T at position 1348 in pla1-2 resulting in the substitution of A to V at the position of 421 aa, (iii) transition of C to T at position 1687 in pla1-3 1-2 resulting in the substitution of P to S at position 496, and (iv) transition of G to A at position 1151 in pla1-4 resulting in the disruption of the splicing donor site. (b) Deduced amino acid sequence of PLA1 protein. In a and b, hydrophobic regions, oxygen binding motifs, and heme binding motifs are indicated by green, blue, and red, respectively. (c) Phylogenetic relationship among CYP78A proteins. The phylogenetic tree was generated based on the entire amino acid sequences by using the clustal w program (www.ddbj.nig.ac.jp/e-mail/clustalw-e.html).

Fig. 3.

In situ expression of PLA1 in vegetative and reproductive apex of wild-type plant. Dark blue stains represent PLA1 gene expression. (a) Median longitudinal section of shoot apex 1 month after germination. (b) Schematic representation of a.(c) Longitudinal section of shoot apex just after transition to reproductive phase. Two bracts of primary branches are formed. Arrows indicate PLA1 expression in the internodes of an elongating stem. (d) Longitudinal section of a young panicle at a slightly later stage of c.(e) Longitudinal section of a developing panicle in which spikelets are being formed. Arrows indicate PLA1 expression in the rachis internodes. (f) Longitudinal section of young spikelet. P0, plastochron0 leaf founder cells; P1, plastochron1 leaf; P2, plastochron2 leaf; P3, plastochron3 leaf; Br, bract; Ibr, incipient bract; Rm, rachis meristem; Pr, Primary branch primordium; Fm, floral meristem; Le, lemma primordium; Eg, empty glume primordium; Rg, rudimentary glume primordium.