Gibberellin regulates pollen viability and pollen tube growth in rice

Abstract

Gibberellins (GAs) play many biological roles in higher plants. We collected and performed genetic analysis on rice (Oryza sativa) GA-related mutants, including GA-deficient and GA-insensitive mutants. Genetic analysis of the mutants revealed that rice GA-deficient mutations are not transmitted as Mendelian traits to the next generation following self-pollination of F1 heterozygous plants, although GA-insensitive mutations are transmitted normally. To understand these differences in transmission, we examined the effect of GA on microsporogenesis and pollen tube elongation in rice using new GA-deficient and GA-insensitive mutants that produce semifertile flowers. Phenotypic analysis revealed that the GA-deficient mutant reduced pollen elongation1 is defective in pollen tube elongation, resulting in a low fertilization frequency, whereas the GA-insensitive semidominant mutant Slr1-d3 is mainly defective in viable pollen production. Quantitative RT-PCR revealed that GA biosynthesis genes tested whose mutations are transmitted to the next generation at a lower frequency are preferentially expressed after meiosis during pollen development, but expression is absent or very low before the meiosis stage, whereas GA signal-related genes are actively expressed before meiosis. Based on these observations, we predict that the transmission of GA-signaling genes occurs in a sporophytic manner, since the protein products and/or mRNA transcripts of these genes may be introduced into pollen-carrying mutant alleles, whereas GA synthesis genes are transmitted in a gametophytic manner, since these genes are preferentially expressed after meiosis.

Figure 1.

Phenotype of the rpe1 Mutant. (A) Gross morphology of wild-type Fijiminori (left) and the rpe1 mutant (right) at the ripening stage. Bar = 30 cm. (B) Flowers of the wild type (left) and rpe1 (right). Cp, carpel; Le, lemma; Lo, lodicle; Pl, palea; St, stamen. Bar = 2 mm. (C) Mature stamens of the wild type (left) and rpe1 (right). An, anther; Fl, filament. Bar = 1 mm. (D) Pistils of the wild type (left) and rpe1 (right). Bar = 1 mm. (E) Pollen grains stained with I₂-KI solution. Wild type (left) and rpe1 (right). Pollen grains staining black were judged as viable, and those staining yellow or light red were judged as sterile. Bars = 100 μm.

Figure 2.

Germination and Elongation of rpe1 Pollen. (A) to (D) and (F) to (J) Growth of pollen of rpe1 ([A], [C], [F], [H], and [I]) and the wild type ([B], [D], [G], and [J]) on rpe1 ([A], [D], [F], [H], and [J]) or wild-type stigmas ([B], [C], [G], and [I]). Pollen grains were stained with aniline blue at 30 min ([A] to [D]), 2 h ([F], [G], [I], and [J]), or 4 h (H) after artificial pollination. Arrowheads indicate pollen tube elongation. Bars = 200 μm. (E) Germination and elongation frequencies of wild-type and rpe1 pollen. Open and closed bars represent the frequencies of wild-type and rpe1 pollen germination or elongation, respectively. Germination and elongation frequencies were estimated by staining with aniline blue at 30 min and 2 h after self-fertilization, respectively.

Figure 3.

Effect of GA on Pollen Tube Elongation. Growth of pollen from rpe1 ([A] to [D]) on its own stigmas that were left treated with mock ([A] and [C]) or with GA₄ ([B] and [D]). Pollen grains were stained with aniline blue at 30 min ([A] and [B]) or 2 h ([C] and [D]) after artificial pollination. Emasculated wild-type flowers with stamens removed were pretreated with GA₄ for 30 min before pollination. Arrowhead indicates pollen elongation. Bars = 200 μm.

Figure 4.

Dose-Dependent Effect of GA₄ on Pollen Germination and Elongation. Frequencies of pollen germination (A) and elongation (B) were estimated by counting the germinated and elongated pollen grains stained with aniline blue after 30 min or 2 h, respectively, on artificially pollinated wild-type stigmas treated with various concentrations of GA₄. The GA₄ treatment procedure was the same as in Figure 3. Open and closed bars represent the frequencies for wild type and rpe1 pollen, respectively. * and ** indicate significant differences at the 5 and 1% levels, respectively, as judged using the Student's t test.

Figure 5.

Phenotypes of GA-Insensitive Mutants with Different Levels of Impairment. Gross morphology at the ripening stage. Flower, stamen, and pistil of the wild type (T65), gid1-8, Slr1-d3, and gid1-7 (left to right). Wild-type and gid1-8 produce fertile flowers, Slr1-d3 produces semifertile flowers, and gid1-7 does not produce fertile flowers.

Figure 6.

Viability, Germination, and Elongation of Slr1-d3 Pollen. (A) and (D) Viability of wild-type T65 (A) and Slr1-d3 (D) pollen. Pollen grains were stained with I₂-KI solution. Pollen grains staining black were judged as viable, and those staining yellow or light red were considered sterile. (B), (C), (E), and (F) Growth of pollen of the wild type ([B] and [C]) and Slr1-d3 ([E] and [F]) on their own stigmas. Pollen was stained with aniline blue after 30 min ([B] and [E]) or 2 h ([C] and [F]). Artificial pollination was performed by hand. Arrowheads indicate pollen elongation. Bars = 200 μm.

Figure 7.

Expression Pattern of GA Synthesis and GA-Signaling Genes in the Process of Anther Development. (A) Anther development of wild-type rice at various stages. Each developmental stage was scored according to the length of the lemma: pollen mother cell stage (stage 1, lemma 2 mm), meiosis stage (stage 2, 2 to 3 mm), tetrad stage (stage 3, 3 to 5 mm), microspore stage (stage 4, 5 to 8 mm), and mature pollen stage (stage 5, ∼8 mm). Arrow indicates the timing of cytokinesis of meiosis. PMC, pollen mother cell; MC, meiocyte; Tds, tetrads; MS, microspore; MP, mature pollen. Bars = 25 μm. (B) Quantitative RT-PCR analysis of GA biosynthesis and GA-signaling genes at various anther developmental stages. The numbers 1 to 5 correspond to the anther developmental stages. Relative mRNA level was determined by normalizing the PCR threshold cycle number of each gene with that of the Actin1 reference gene, and data were the average of three replicates. The top panel represents the relative expression levels of all genes tested at stages 1 to 5. The bottom panel shows the enlarged view of the expression levels of genes with lower expression at stages 1 to 4. The striped and open boxes represent the GA synthesis and GA-signaling genes, respectively. Arrows indicate the timing of cytokinesis of meiosis. Error bars represent se of three biological replicates.

Figure 8.

Model to Explain Differences in the Transmission Frequencies of GA-Deficient and GA-Insensitive Mutations. Pollen carrying GA-deficient mutations, which are poorly transmitted, does not synthesize GA, which is essential for pollen tube germination and elongation, because the expression of these genes occurs after meiosis. By contrast, GA signaling functions normally in pollen carrying GA-insensitive mutations because the GA-signaling genes are actively expressed just before meiosis; therefore, their products should be transported into pollen carrying mutant alleles. +/− indicates a heterozygous genotype of pollen mother cells, whereas + and − indicate the wild-type and mutant alleles, respectively, of pollen cells. Gray shading indicates the expression of GA synthesis or GA-signaling genes in pollen mother cells or pollen.