Changes in the shapes of leaves and flowers upon overexpression of cytochrome P450 in Arabidopsis

Abstract

In Arabidopsis, the two-dimensional expansion of leaves is regulated via the polarized elongation of cells. The ROTUNDIFOLIA3 (ROT3) protein, a member of the family of cytochromes P450, is involved in this process and regulates leaf length. Transgenic plants that overexpressed a wild-type ROT3 gene had longer leaves than parent plants, without any changes in leaf width. The shapes of floral organs were also altered, but elongation of the stem, roots, and hypocotyls was unaffected. To our knowledge, no similar specific regulation of leaf length has been reported previously. Transgenic plants overexpressing the rot3-2 gene had enlarged leaf blades but leaf petioles of normal length. Morphological alterations in such transgenic plants were associated with changes in shape of leaf cells. The ROT3 gene seems to play an important role in the polar elongation of leafy organs and should be a useful tool for the biodesign of plant organs.

Figure 1

Expression pattern of GUS encoded by the ROT3P∷GUS fusion gene. (A) Histochemical staining for GUS activity in four independent transgenic plants (left four) and a nontransgenic plant (right). (Bar = 1 cm.) (B–D) Magnified views of parts of a transgenic plant that carried the ROT3P∷GUS fusion gene. A young rosette (B), inflorescence (C), and young leaf (D) are shown. (E) A mature leaf stained for GUS activity shows ubiquitous expression of the ROT3P∷GUS fusion gene (Nomarski optics). (Bar = 100 μm.)

Figure 2

Phenotypes of wild-type (wt) plants, plants with one of two rot3 alleles, and transgenic rot3-1 plants that overexpressed ROT3. (A) The leaves in each row were taken from the following plants: (from left) wt, rot3-1, rot3-2, transgenic plants that overexpressed the wild-type ROT3 gene (A-1, A-3, and A-4), and a plant that expressed the ROT3^G80E gene (G80E). Upper and lower rows show the first leaves and the fifth leaves, respectively. (Bar = 10 mm.) Paradermal images of palisade cells in the fifth leaves of a wt plant (B), a rot3-1 mutant plant (C), and an A-3 transgenic plant (D) at the fully expanded stage. (Bar = 100 μm.) Flowers of a wt plant (E), a rot3-1 mutant plant (F), and an A-1 transgenic plant (G). (Bar = 1 mm.) Gross morphology of plants heterozygous for the transgene (InducibleP∷ROT3^G80E). Heterozygous 17-day-old plants (H) were transferred to medium plus 10 μM dexamethasone (I) or to control medium (J) and photographed 8 days later (I and J). (Bars = 10 mm.)

Figure 3

Amplification by RT-PCR of ROT3 mRNA. (From left) Nontransgenic wild type (wt), nontransgenic rot3-1 null mutant (rot3-1), and three independent transgenic lines that carry the InducibleP∷ROT3 chimeric gene are shown. Plants were cultivated with (+) or without (−) treatment with 10 μM dexamethasone (DEX). Total RNA (1 μg) was amplified for each strain. (Upper) The result of amplification by RT-PCR of the ROT3 cDNA. (Lower) The result for amplification by RT-PCR of β-tubulin 4 (TUB4) fragments is shown for standard controls.