Potential sites of bioactive gibberellin production during reproductive growth in Arabidopsis

Abstract

Gibberellin 3-oxidase (GA3ox) catalyzes the final step in the synthesis of bioactive gibberellins (GAs). We examined the expression patterns of all four GA3ox genes in Arabidopsis thaliana by promoter-beta-glucuronidase gene fusions and by quantitative RT-PCR and defined their physiological roles by characterizing single, double, and triple mutants. In developing flowers, GA3ox genes are only expressed in stamen filaments, anthers, and flower receptacles. Mutant plants that lack both GA3ox1 and GA3ox3 functions displayed stamen and petal defects, indicating that these two genes are important for GA production in the flower. Our data suggest that de novo synthesis of active GAs is necessary for stamen development in early flowers and that bioactive GAs made in the stamens and/or flower receptacles are transported to petals to promote their growth. In developing siliques, GA3ox1 is mainly expressed in the replums, funiculi, and the silique receptacles, whereas the other GA3ox genes are only expressed in developing seeds. Active GAs appear to be transported from the seed endosperm to the surrounding maternal tissues where they promote growth. The immediate upregulation of GA3ox1 and GA3ox4 after anthesis suggests that pollination and/or fertilization is a prerequisite for de novo GA biosynthesis in fruit, which in turn promotes initial elongation of the silique.

Figure 1.

GA Biosynthetic and Catabolic Pathways in Plants. This is a simplified diagram only showing the pathways for the synthesis of non-13-hydroxylated GAs. Gray text indicates GA synthetic and catabolic enzymes catalyzing corresponding steps. GGDP, geranylgeranyl diphosphate; CDP, ent-copalyl diphosphate; CPS, ent-copalyl diphosphate synthase; KS, ent-kaurene synthase; KO, ent-kaurene oxidase; KAO, ent-kaurenoic acid oxidase.

Figure 2.

In Vitro Enzyme Assays of Wild-Type GA3ox3 and GA3ox4 Proteins. Gas chromatography–selected ion monitoring showing the conversion of GA₉ to GA₄ by MBP-GA3ox3 fusion protein (middle panel) and MBP-GA3ox4 fusion protein (bottom panel). Cell lysates containing MBP were used as a negative control (top panel). GA₉ and GA₄ were identified as GA₉-methyl ester (GA₉-Me) and GA₄-methyl ester-trimethylsilyl ether (GA₄-MeTMS), respectively, after derivatization. m/z, mass-to-charge ratio.

Figure 3.

Structures of GA3ox3 and GA3ox4 Promoter-GUS Reporter Gene Fusion Constructs and a GA3ox3 Genomic DNA Construct. Black boxes correspond to exons, and the gray boxes correspond to introns of GA3ox genes. p3ox3 contains a genomic GA3ox3 sequence that includes 1.8-kb 5′ promoter sequence and the entire coding region. Asterisks indicate the GUS constructs used for final expression studies. TC, transcriptional fusion; TL, translational fusion.

Figure 4.

Temporal and Spatial Expression of GA3ox Genes. (A) to (E) GA3ox3-GUS line. (A) Flower cluster. (B) Close-up of single flower. (C) Developing embryos between torpedo and upturned-U stages. Inset: close-up of torpedo stage embryo. (D) Close-up of walking stick stage embryo. (E) Close-up of upturned-U stage embryo. (F) to (M) GA3ox4-GUS line. (F) Seed, 6 h after imbibition. (G) Flower cluster. (H) Close-up of a young flower. (I) Close-up of flowers with young siliques. Silique age: 0 HAF (left) and 20 HAF (right). (J) Developing seeds in a silique at 7 DAF. (K) to (M) Clearing of developing GA3ox4-GUS seeds by Hoyer's medium. Bars = 50 μm. (N) Expression of GA3ox genes in 6- to 8-DAF wild-type siliques as gauged by qRT-PCR. Placenta contains septum, replums, and funiculi. (O) to (R) GA3ox1-GUS expression in carpel and developing siliques. (S) Temporal expression of GA3ox1, GA3ox3, and GA3ox4 during silique growth. The levels of gene expression in (N) and (S) are represented by the copies of cDNA per 10⁵ copies of ROC1. Means ± se of three technical replicates are shown in both (N) and (S). Each experiment was repeated once using independent samples with similar results. Silique stages were defined according to Bowman (1994) (see Supplemental Table 1 online).

Figure 5.

Cellular Location of GA3ox-GUS Expression during Flower Development. (A) to (C) Transverse sections of anthers expressing GA3ox3-GUS, GA3ox4-GUS, and GA3ox2-GUS, respectively. Numbers at the bottom left corner of each picture indicate the corresponding anther developmental stages according to Sanders et al. (1999). Arrowhead, tapetum; arrow, pollen grains. (D) Longitudinal section of inflorescence apex of GA3ox1-GUS. m, inflorescence meristem. 2, stage 2 flower primordia; 3, stage 3 flower; 7, stage 7 flower. Flower stages were determined according to Bowman (1994). All sections were viewed under dark-field microscopy, and the X-Gluc staining is pink. Bars = 50 μm.

Figure 6.

The ga3ox3 and ga3ox4 Mutant Lines. (A) Locations of mutations in the ga3ox3 and ga3ox4 mutants. T-DNA insertion sites are depicted as triangles. Numbers next to T-DNA insertions indicate the insertion sites relative to ATG. The black and gray asterisks indicate the positions of ga3ox4-1 and ga3ox4-4 missense mutations at 1094 and 1046 nucleotides after ATG, respectively. (B) Relative GA3ox4 transcript levels in siliques of ga3ox4-2 and ga3ox4-3 compared with the wild type. The means of three technical replicates of qRT-PCR ± se are shown. The expression level in the wild type was set to 1.0. Black asterisk indicates no wild-type GA3ox4 transcript was detected in ga3ox4-3. Similar results were obtained when qRT-PCR was performed using a second set of samples.

Figure 7.

Phenotypic Characterization of the ga3ox Mutants. (A) The 24-d-old rosettes of wild-type and homozygous mutants as labeled. (B) The 44-d-old plants as labeled. (C) Primary inflorescence stems of Category II ga3ox mutants. (D) Primary inflorescence stems of Category III ga3ox mutants. (E) Gradual improvement in the development of ga3ox1 ga3ox3 mutant flowers on primary inflorescence. Top row: wild-type and ga3ox1 flowers. Bottom row: ga3ox1 ga3ox3 flowers. Some of the floral organs were removed to show the anther phenotype. (F) Toluidine blue–stained transverse sections of anthers of the first three flowers on the primary inflorescence stem of ga3ox1 ga3ox3. Numbers at the bottom left corner of each picture in (E) and (F) indicate the positions of the flowers on the primary inflorescence stem. Bar = 50 μm.

Figure 8.

Endogenous GA levels in Rosette Leaves and Flowers of Wild-Type and Category II ga3ox Mutant Plants. (A) GA levels (nanograms per gram dry weight [ng/g d.w.]) in rosette leaves at anthesis of the first flower. The data for GA₄ are also shown in the inset with a different y axis scale for clarity. Data are means ± se of biological duplicates. (B) GA levels in early (top graph) and late (bottom graphs) flowers. Note that the y axis scale for GA₅₁ is different from that for other GAs. Means ± se of biological triplicates are shown. Results of 13-OH GAs are shown in Supplemental Figure 7 online.

Figure 9.

GA3ox1 and GA3ox4 Are Required for Normal Silique Growth. (A) to (E) Relationship between seed number per silique and final silique length for wild-type and ga3ox mutants either self-pollinated or cross-pollinated with pollen from wild-type or ga3ox1 plants. All data are from hand-pollinated flowers with at least 19 siliques for each combination. Some flowers were pollinated with a reduced amount of pollen to vary the number of seeds set. Data from self-pollinated wild-type, ga3ox1, and ga3ox1 ga3ox3 ga3ox4 flowers are shown in each panel to make comparisons easier. In (C) to (E), data from the pollination with wild-type and ga3ox1 pollen have been combined as both pollen genotypes gave very similar silique lengths. Lines of best fit are shown by solid lines of different colors (self-pollinated flowers) and by dotted black lines for flowers pollinated with wild-type and/or ga3ox1 pollen. (F) Dry siliques of self-pollinated wild-type and ga3ox mutants with the valve removed to reveal the seeds. Each carpel shown contains 23 to 24 seeds, which are more crowded in the mutants due to reduced silique growth. Siliques of the ga3ox1 ga3ox3 ga3ox4 triple mutant resemble those of the ga3ox1 ga3ox4 double mutant.