SummaryThe development of the flat morphology of leaf blades is dependent on the control of cell proliferation as well as cell expansion. Each process has a polarity with respect to the longitudinal and transverse axes of the leaf blade. However, only a few regulatory components of these processes have been identified to date. We have characterized two genes from Arabidopsis thaliana: ANGUSTIFOLIA3 (AN3), which encodes a homolog of the human transcription coactivator SYT, and GROWTH-REGULATING FACTOR5 (AtGRF5), which encodes a putative transcription factor. AN3 is identical to GRF-INTERACTING FACTOR1 (AtGIF1). The an3 and atgrf5 mutants exhibit narrow-leaf phenotypes due to decreases in cell number. Conversely, cell proliferation in leaf primordia is enhanced and leaves grow larger than normal when AN3 or AtGRF5 is overexpressed. Both genes are expressed in leaf primordia, and in the yeast two-hybrid assay, the gene products were found to interact with each other through their N-terminal domains. These results suggest that AN3 and AtGRF5 act together and are required for the development of appropriate leaf size and shape through the promotion and/or maintenance of cell proliferation activity in leaf primordia.

The plant leaf provides an ideal system to study the mechanisms of organ formation and morphogenesis. The key factors that control leaf morphogenesis include the timing, location and extent of meristematic activity during cell division and differentiation. We identified an Arabidopsis mutant in which the regulation of meristematic activities in leaves was aberrant. The recessive mutant allele blade-on-petiole1-1 (bop1-1)produced ectopic, lobed blades along the adaxial side of petioles of the cotyledon and rosette leaves. The ectopic organ, which has some of the characteristics of rosette leaf blades with formation of trichomes in a dorsoventrally dependent manner, was generated by prolonged and clustered cell division in the mutant petioles. Ectopic, lobed blades were also formed on the proximal part of cauline leaves that lacked a petiole. Thus, BOP1regulates the meristematic activity of leaf cells in a proximodistally dependent manner. Manifestation of the phenotypes in the mutant leaves was dependent on the leaf position. Thus, BOP1 controls leaf morphogenesis through control of the ectopic meristematic activity but within the context of the leaf proximodistality, dorsoventrality and heteroblasty.BOP1 appears to regulate meristematic activity in organs other than leaves, since the mutation also causes some ectopic outgrowths on stem surfaces and at the base of floral organs. Three class I knox genes,i.e., KNAT1, KNAT2 and KNAT6, were expressed aberrantly in the leaves of the bop1-1 mutant. Furthermore, the bop1-1 mutation showed some synergistic effect in double mutants with as1-1 oras2-2 mutation that is known to be defective in the regulation of meristematic activity and class I knox gene expression in leaves. Thebop1-1 mutation also showed a synergistic effect with thestm-1 mutation, a strong mutant allele of a class I knoxgene, STM. We, thus, suggest that BOP1 promotes or maintains a developmentally determinate state in leaf cells through the regulation of class I knox genes.

The polarized processes of cell elongation play a crucial role in morphogenesis of higher plants. We reported previously that the rotundifolia3 (rot3) mutant of Arabidopsis has a defect in the polar elongation of leaf cells. In the present study, we isolated two additional alleles with mutations in the ROT3 gene. The ROT3 gene was cloned by a T-DNA-tagging method and isolation of the gene was confirmed by a molecular analysis of three rot3 mutant alleles obtained from different mutagenesis. The ROT3 gene encodes a cytochrome P-450 (CYP90C1) with domains homologous to regions of steroid hydroxylases of animals and plants. Expression of the ROT3 gene was detected in all major plant organs. Especially, higher expression was detected in the tissues that had high activity of cell division. We confirmed that the ROT3 gene controls polar elongation specifically in leaf cells by an analysis of three rot3 mutants obtained from different mutagenesis experiments. Our results imply that the ROT3 protein is a member of a new class of cytochrome P-450 encoding putative steroid hydroxylases, which is required for the regulated polar elongation of cells in leaves of Arabidopsis.