Rab geranylgeranyl transferase β subunit is essential for male fertility and tip growth in Arabidopsis

Abstract

Rab proteins, key players in vesicular transport in all eukaryotic cells, are post-translationally modified by lipid moieties. Two geranylgeranyl groups are attached to the Rab protein by the heterodimeric enzyme Rab geranylgeranyl transferase (RGT) αβ. Partial impairment in this enzyme activity in Arabidopsis, by disruption of the AtRGTB1 gene, is known to influence plant stature and disturb gravitropic and light responses. Here it is shown that mutations in each of the RGTB genes cause a tip growth defect, visible as root hair and pollen tube deformations. Moreover, FM 1-43 styryl dye endocytosis and recycling are affected in the mutant root hairs. Finally, it is demonstrated that the double mutant, with both AtRGTB genes disrupted, is non-viable due to absolute male sterility. Doubly mutated pollen is shrunken, has an abnormal exine structure, and shows strong disorganization of internal membranes, particularly of the endoplasmic reticulum system.

Keywords: Arabidopsis; Rab geranylgeranyl transferase; Rab protein; male fertility; pollen; pollen tube; protein prenylation; root hair; tip growth..

Fig. 1.

Expression of AtRGTB1 and AtRGTB2 in Atrgtb homozygous lines. Semi-quantitative RT–PCR in different organs of plants. L, leaf; F, flower; S, stem; R, root. Actin mRNA was used as normalization control. Results representative of three independent plant cultures are shown.

Fig. 2.

Morphology of generative organs in Atrgtb mutants. (A) Flowers, scale bar=1mm. (B) Siliques, scale bar=5mm; the arrowheads indicate the unfertilized ovules. (C) Number of seeds per silique after self-pollination, n>20. Bars represent the mean ±SE. Statistical significance analysed by Student t-test, *P<0.05, **P<0.01, ***P<0.001.

Fig. 3.

Male gametophyte morphology in Atrgtb mutants. (A) Morphological aberrations in pollen tubes germinated in vitro; scale bar=100 μm. (B) Frequency of pollen tube abnormalities; bars represent the mean ±SE. Statistical significance was analysed by Mann–Whitney test, ***P<0.001. (C) In vivo germination of mutant pollen on wt stigma. Alexander stain 16h after pollination; grey and pale pink pollen grains are germinated (arrows), dark purple grains are non-germinated (white arrowhead). Scale bar=100 μm. Note shrunken pollen grains deposited on stigma for Atrgtb1+/– mutant pollen (black arrowhead) and low frequency of germination for Atrgtb1–/– pollen. Representative images are shown.

Fig. 4.

Root hair morphology and functioning in Atrgtb mutants. (A) Aberrations in root hair morphology; scale bar=30 μm. (B) Frequency of abnormal root hairs; bars represent the mean ±SE. Statistical significance was analysed by Mann–Whitney test, **P<0.01, ***P<0.001. (C) Time course of FM 1-43 styryl dye uptake and recycling in root hairs, confocal microscopy. Z-stacks of ~20 images from each root hair are shown. Scale bar=30 μm. Note large dye aggregates in mutant cells (arrowhead). Representative images are shown.

Fig. 5.

Male gametophyte defect in the Atrgtb1Atrgtb2 double mutant. (A) Number of seeds per silique, n>20; bars represent mean ±SE. (B) Frequency of germination of seeds from self-pollination of Atrgtb1+/–Atrgtb2–/– plants, n>250. Statistical significance analysed by Student t-test, *P<0.05, ***P<0.001. (C) Siliques of an Atrgtb1+/–Atrgtb2–/– plant after self-pollination; scale bar=1mm. (This figure is available in colour at JXB online.)

Fig. 6.

Pollen grain development in single mutants in AtRGTB and in the Atrgtb1Atrgtb2 double mutant. (A) Alexander stain of mature anthers; scale bar=100 μm. (B) SEM images of mature pollen grains. Note that in pollen derived from Atrgtb1+/– some grains are hollow, but of normal length, but in pollen derived from Atrgtb1+/–Atrgtb2–/–, half of the grains are shrunken and many of them show abnormal exine structure; scale bar=10 μm for higher magnification and 20 μm for lower magnification. Representative images are shown. (This figure is available in colour at JXB online.)

Fig. 7.

Bicellular pollen grain ultrastructure; ultrathin section TEM images of wt, Atrgtb1– and Atrgtb1–Atrgtb2– pollen. (A) Cell wall and exine ultrastructure; scale bar=500nm. (B) General view of a pollen grain; scale bar=1 μm. Representative images are shown.