S-Adenosylmethionine Synthetase 3 Is Important for Pollen Tube Growth

Abstract

S-Adenosylmethionine is widely used in a variety of biological reactions and participates in the methionine (Met) metabolic pathway. In Arabidopsis (Arabidopsis thaliana), one of the four S-adenosylmethionine synthetase genes, METHIONINE ADENOSYLTRANSFERASE3 (MAT3), is highly expressed in pollen. Here, we show that mat3 mutants have impaired pollen tube growth and reduced seed set. Metabolomics analyses confirmed that mat3 pollen and pollen tubes overaccumulate Met and that mat3 pollen has several metabolite profiles, such as those of polyamine biosynthesis, which are different from those of the wild type. Additionally, we show that disruption of Met metabolism in mat3 pollen affected transfer RNA and histone methylation levels. Thus, our results suggest a connection between metabolism and epigenetics.

Figure 1.

mat3 is a knockdown mutant and exhibits shorter siliques and reduced seed set. A, Structure of MAT3 (At2g36880). The exon is indicated by a black box, and untranslated regions are indicated by gray lines. The T-DNA in the 3′ untranslated region is indicated by the black triangle. UBIQUITIN5 (UBQ5) was used as an internal loading control. The numbers 1, 2, and 3 indicate primers used for RT-PCR. B, Representative 10-week-old plants of the wild type (WT) and mat3. Bars = 1 cm. C, Intact and opened siliques of the wild type and mat3. Bars = 0.5 cm (left) and 500 μm (right).

Figure 2.

mat3 exhibits defects in the male gametophyte. A, Representative images of pollen from wild-type (WT) and mat3 plants after in vitro germination for 6 h. Bars = 100 μm. B, Pollen germination percentage at 6 h. Data are means ± se of three experiments. For each independent replicate, 400 to 500 pollen grains were analyzed. C, Pollen tube lengths of the wild type and mat3 after germination for 6 h. Data are means ± se of three experiments. D, Semi in vivo pollen tube growth. Shown are wild-type and mat3 homozygous pistils 12 h (left) and 24 h (right) after pollination stained with decolorized Aniline Blue. E, Whole-mount images of mature siliques after clearing in a 0.2 n NaOH and 1% SDS solution.

Figure 3.

Complementation of the mat3 mutant and subcellular localization of MAT3. A, Representative wild-type (WT), mat3, and complemented immature siliques and mature siliques after clearing. Bars = 1 mm. B, Subcellular localization of MAT3 in a quartet, in which two of the four pollen grains are transgenic. Images (from left to right) show the 4′,6-diamino-2-phenylindole (DAPI) channel, GFP channel, bright field, and merged. C, Pollen tube lengths of qrt1/qrt1;mat3/mat3, qrt1/qrt1, and mat3/mat3 carrying pMAT3::MAT3-GFP measured after 6 h of germination. Data are means ± se of three experiments. For each independent replicate, 100 to 150 pollen tubes were analyzed.

Figure 4.

Wild type (WT) pollen tubes are more sensitive to ethionine and Met. A, In vitro pollen tube lengths after 6 h, with various concentrations of ethionine added to pollen germination medium. Data are means ± se of triplicate experiments. B, In vitro pollen tube lengths after 6 h, with various concentrations of Met added to pollen germination medium. Data are means ± se of triplicate experiments. Dashed lines mark I₅₀ of pollen tube length, black for the wild type and gray for mat3.

Figure 5.

Metabolite profiles of mat3 and wild-type (WT) mutant pollen and pollen tubes. Changes are represented as fold change between the level of a metabolite in mat3 and in the wild type and are listed in Supplemental Data S1. Solid arrows represent a single-step reaction between two metabolites, and dashed arrows indicate multiple steps. Metabolites in gray were not detected. FatA C14:0, Myristic acid; FatA C16:0, palmitic acid; FatA C18:0, stearic acid; FatA C16:1, palmitoleic acid; FatA C18:1, oleic acid; FatA C18:2, linoleic acid; F6P, fructose6-phosphate; Fru-1,6-bisP, fructose 2,6-bisphosphate; G6P, glucose 6-phosphate; GABA, γ-aminobutyric acid; Glu-cys, glutamyl-l-cysteine; Homo-cys, homocysteine; PEP, phosphoenolpyruvate; SAM, S-adenosyl-methionine.

Figure 6.

Immunofluorescence using specific antibodies against methylated histone H3 in wild-type (WT) and mat3 pollen. Mature pollen from wild-type and mat3 plants was incubated with anti-H3 (control), anti-H3K4m3, and anti-H3K27m3 antibodies. Representative examples show histone methylation in the vegetative nucleus. Quantitation of signal intensity is shown next to each image. Each x axis represents a 5-μm distance centered on the fluorescent focus, and each y axis represents relative fluorescence intensity. All images were acquired using the same exposure time. Forty to 50 pollen grains for each genotype were examined. DAPI, 4′,6-Diamino-2-phenylindole.

Figure 7.

Liquid chromatography-mass spectrometry analysis of total 5-methylcytidine (m⁵C) and 7-methylguanosine (m⁷G) levels in tRNA purified from wild-type (WT) and mat3 pollen. AU represents absorbance units.