Gibberellins are required for seed development and pollen tube growth in Arabidopsis

Abstract

Gibberellins (GAs) are tetracyclic diterpenoids that are essential endogenous regulators of plant growth and development. GA levels within the plant are regulated by a homeostatic mechanism that includes changes in the expression of a family of GA-inactivating enzymes known as GA 2-oxidases. Ectopic expression of a pea GA 2-oxidase2 cDNA caused seed abortion in Arabidopsis, extending and confirming previous observations obtained with GA-deficient mutants of pea, suggesting that GAs have an essential role in seed development. A new physiological role for GAs in pollen tube growth in vivo also has been identified. The growth of pollen tubes carrying the 35S:2ox2 transgene was reduced relative to that of nontransgenic pollen, and this phenotype could be reversed partially by GA application in vitro or by combining with spy-5, a mutation that increases GA response. Treatment of wild-type pollen tubes with an inhibitor of GA biosynthesis in vitro also suggested that GAs are required for normal pollen tube growth. These results extend the known physiological roles of GAs in Arabidopsis development and suggest that GAs are required for normal pollen tube growth, a physiological role for GAs that has not been established previously.

Figure 1.

RT-PCR Analysis of 2ox2 and GA5 Expression. (A) Semiquantitative RT-PCR analysis of 2ox2 transcript levels (top gel) in RNA from seedlings of nontransgenic control Ler and 35S:2ox2 lines in the Ler background. Pea 2ox2 cDNA was used as a positive control. RT-PCR (bottom gel) of UBIQUITIN3 (Ubi-3) transcripts, which were detected consistently in all of the plant samples used for the 2ox2 RT-PCR experiment. (B) Semiquantitative RT-PCR analysis of GA5 (GA 20-oxidase) transcript levels (top gel) in RNA from seedlings of nontransgenic control Ler and 35S:2ox2 lines in the Ler background. RT-PCR (bottom gel) of β-tubulin transcripts, which were detected consistently in all of the plant samples used for the GA5 RT-PCR experiment.

Figure 2.

The 35S:2ox2 Transgene Is Transmitted Poorly to the Next Generation. Seeds from self-pollinated hemizygous 35S:2ox2 Arabidopsis plants were collected and scored as either transgenic (kanamycin-resistant seedlings; black sectors) or nontransgenic (kanamycin-sensitive seedlings; white sectors) by germinating on Murashige and Skoog (1962) medium containing 15 or 25 μg/mL kanamycin. For comparison, the circle marked 35S:GFP (green fluorescent protein) represents the ratio of transgenic to nontransgenic seedlings observed when heterozygous 35S:GFP plants containing a single transgene locus were allowed to self-pollinate. As expected if the 35S:GFP construct does not alter seed or pollen development, ∼75% of the progeny were transgenic. The total numbers of seeds analyzed were 605 for 35S:GFP (χ² _[3:1] = 0.005, P > 0.90) and 159 to 858 for each of the 35S:2ox2 lines (for all lines, P < 0.001 from the χ² test for agreement with the expected 3:1 transgenic-to-nontransgenic ratio).

Figure 3.

Phenotypic Analysis of 35S:2ox2 Plants. (A) 35S:2ox2 causes early and late seed abortion, as shown in a typical silique from a self-pollinated 35S:2ox2/29c plant. Only 3% of surviving seeds carry the 35S:2ox2 transgene. (B) Siliques developing on self-pollinated homozygous 35S:2ox2/28c plants are only approximately half the size of siliques on self-pollinated wild-type Ler plants, and seeds are present only in the part of the silique nearest the stigma. (C) A typical silique from a self-pollinated homozygous 35S:2ox2/28c plant showing a number of unfertilized ovules at the base of the silique, distal to the fertilized seed farthest from the stigma (arrow). (D) The 35S:2ox2/28c pollen tube phenotype is partially rescued in the 35S:2ox2/28c spy-5 double mutant.

Figure 4.

Pollen Grain Germination and Pollen Tube Growth. (A) Scanning electron microscopy images of germinated pollen grains on stigmas at 5 h after self-pollination of wild-type Ler and homozygous 35S:2ox2/28c flowers. (B) Aniline blue staining at 5 h of wild-type Ler self-pollinated pistils (left), homozygous 35S:2ox2/28c self-pollinated pistils (center), and 35S:2ox2/28c pistils pollinated with 28c spy-5 pollen (right).

Figure 5.

Partial Suppression of the 35S:2ox2/28c Pollen Tube Phenotype by spy-5. Plants were grown at day/night temperatures of 19/17°C to minimize the effects of the spy-5 mutation on anther development and pollen production. Data represent the largest fruit from the first 10 flowers on the main stem of at least 11 self-pollinated plants of each genotype. (A) Final silique length. (B) Mean number of fertilized seeds per silique. (C) Mean number of unfertilized ovules at the base of each silique (see Figure 3C).

Figure 6.

Changing GA Levels of Pollen Tubes Growing in Vitro. Pollen tube length after 24 h on pollen germination medium. (A) Pollen from Ler or homozygous 35S:2ox2/28c plants was germinated in vitro with or without 1 × 10⁻⁵ M GA₁ or GA₄. (B) Pollen from Ler was germinated in vitro in the presence of 1 × 10⁻⁵ M uniconazole (Uni), a chemical inhibitor of GA biosynthesis, with or without GA₁.