Control of axillary bud initiation and shoot architecture in Arabidopsis through the SUPERSHOOT gene

Abstract

The aerial architecture of flowering plants is determined to a large extent by shoot growth and shoot branching arising from the initiation and growth of axillary meristems. We have identified an Arabidopsis mutant, supershoot (sps), which is characterized by a massive overproliferation of shoots, such that a single plant can generate 500 or more inflorescences. Analysis of the mutant plants shows that the primary defect is because of an increase in the number of meristems formed in leaf axils, together with release of bud arrest, resulting in reiterative branch formation from rosette and cauline leaves. The SPS gene is shown here to encode a cytochrome P450, and together with a 3- to 9-fold increase in levels of Z-type cytokinins in sps mutant plants, indicate a role for SPS in modulating hormone levels. The expression pattern of SPS, with strong expression at the leaf axils, correlates well with the phenotypic defects. Our results indicate that control of shoot branching in Arabidopsis may be accomplished in part by suppression of axillary meristem initiation and growth through the localized attenuation of cytokinin levels at sites of bud initiation.

Figure 1

Shoot branching patterns of sps mutants. (A) Comparison of wild-type (left) and sps mutant (right) showing axillary inflorescences from the rosette leaves. (B) Multiple axillary inflorescences have developed from each single cauline leaf of sps mutant. (C) A single sps mutant plant at 4 mo. (D) Axillary meristem emerging from the axil of a sps cotyledon (bottom), wild-type plant (top) for comparison. (E) Cauline leaf axil from wild-type plant (top) with single arrested axillary meristem, and from sps mutant (bottom) with multiple axillary meristems. The first inflorescences formed in the cauline leaf axils have been excised for convenience in observation. (F) sps mutant plant carrying Ac transposase showing a revertant wild-type sector because of excision of the Ds element.

Figure 2

Patterns of axillary inflorescence development in the rosette leaves after bolting. R1–R7 represent rosette leaves 1–7. R1 is the oldest rosette leaf. Cot represents a cotyledon. (A–C) Wild-type axillary buds in the axils of rosette leaves: R7 (A), R5 (B), and R3 (C). (D–F) sps axillary buds in the axils of rosette leaves: R7 (D), R3 (E), and R1 (F). The photographs were taken at the same magnification for the comparison. Arrows indicate the developing axillary inflorescence. (G) Diagrammatic representation of the axillary buds developing in each leaf axil and shown as dotted bars. Relative sizes of the axillary buds are shown as relative sizes of bars. Eight plants from wild type and an sps mutant allele (see text) were used for the analysis. As all of the wild-type plants show a similar pattern of axillary inflorescence development, a representative wild-type plant is shown here (wt), whereas eight individual sps plants are shown (sps: 1–8).

Figure 3

Other developmental defects in the sps mutants. (A,B) Details of the leaf veins in wild-type and sps plants. (A) Prominent dark green veins on the leaf surface of the sps adult leaf (right); veins are not apparent in the wild-type leaf (left). (B) Pattern of leaf vasculature in wild-type and sps plants observed after clearing. The sps mutant (right) has less complexity in vascular patterning compared to the wild type (left). (C,D) Floral morphology and development: sps flower with a reduced number of organs and is not fully developed (D), compared to wild type (C).

Figure 4

Positions of the donor T-DNA and insertion sites of Ds gene trap elements in different sps alleles. Positions of the SPS/CYP79F1 gene and the duplicated gene, CYP79F2, on the BAC clone accession number AC006341, are shown. Boxes represent exons. Insertion sites of Ds elements in the SPS gene are indicated as small arrowheads; position of the donor T-DNA is indicated as big arrowhead. Insertion sites of the Ds element in sps-1 to sps-5 are in the exons, whereas in sps-6, it is in the 5′ untranslated leader sequence.

Figure 5

SPS expression patterns monitored by GUS expression from the Ds gene trap insertion in sps-2. (A–D) GUS staining patterns of heterozygous sps plants. (A) Staining detected at the vascular junction of a 7-d-old seedling. (B) Extensive staining in the vascular tissue of a 10-d-old seedling. (C) Strong SPS expression at the cauline leaf axil. (D) Staining at the receptacle of flowers. (E) GUS staining patterns of heterozygous (left) and homozygous sps (right) plants.

Figure 6

Cytokinin levels in wild-type and sps plants. Cytokinin bases (free and released) and cytokinin glucosides (free and released) were measured using scintillation proximity immnoassay after hydrolysis and purification (including the HPLC step to resolve different cytokinins). Cytokinin bases: zeatin (Z), dihydrozeatin (DZ), and N⁶-(2-isopentenyl) adenine (iP); cytokinin O-glucosides (zeatin O-glucoside [OGZ] and dihydrozeatin O-glucoside [OGDZ]); cytokinin N-glucosides (zeatin-7-glucoside [Z-7-G] and zeatin-9-glucoside [Z-9-G]). Data were derived from five samples.

Figure 7

Double mutant analysis with sps and amp1. (A) Phenotypes of 4-wk-old seedlings of wild type, amp1, sps, and amp1sps mutants. (B) 6-wk-old amp1sps plant.