Vegetative and sperm cell-specific aquaporins of Arabidopsis highlight the vacuolar equipment of pollen and contribute to plant reproduction

Abstract

The water and nutrient status of pollen is crucial to plant reproduction. Pollen grains of Arabidopsis (Arabidopsis thaliana) contain a large vegetative cell and two smaller sperm cells. Pollen grains express AtTIP1;3 and AtTIP5;1, two members of the Tonoplast Intrinsic Protein subfamily of aquaporins. To address the spatial and temporal expression pattern of the two homologs, C-terminal fusions of AtTIP1;3 and AtTIP5;1 with green fluorescent protein and mCherry, respectively, were expressed in transgenic Arabidopsis under the control of their native promoter. Confocal laser scanning microscopy revealed that AtTIP1;3 and AtTIP5;1 are specific for the vacuoles of the vegetative and sperm cells, respectively. The tonoplast localization of AtTIP5;1 was established by reference to fluorescent protein markers for the mitochondria and vacuoles of sperm and vegetative cells and is at variance with the claim that AtTIP5;1 is localized in vegetative cell mitochondria. AtTIP1;3-green fluorescent protein and AtTIP5;1-mCherry showed concomitant expression, from first pollen mitosis up to pollen tube penetration in the ovule, thereby revealing the dynamics of vacuole morphology in maturating and germinating pollen. Transfer DNA insertion mutants for either AtTIP1;3 or AtTIP5;1 showed no apparent growth phenotype and had no significant defect in male transmission of the mutated alleles. By contrast, a double knockout displayed an abnormal rate of barren siliques, this phenotype being more pronounced under limited water or nutrient supply. The overall data indicate that vacuoles of vegetative and sperm cells functionally interact and contribute to male fertility in adverse environmental conditions.

Figure 1.

Staining by neutral red of pollen vacuoles. Labeling of vacuoles in mature (A and C) and germinating (B) pollen grains using light transmission (A and B) or confocal laser scanning microscopy (C). Arrows indicate the labeling of small spherical structures that probably correspond to sperm cell vacuoles (as deduced from three-dimensional stacks of the respective pollen; Supplemental Movie S1). Note that the fluorescence image (C) represents an optical section, while the bright-field images (A and B) represent the totality of transmitted lights. Bar = 2 µm.

Figure 2.

Temporal expression pattern of AtTIP1;3-GFP in maturating pollen and germinating PT. The expression of AtTIP1;3-GFP (green) was analyzed during pollen grain maturation (A–D) and germination (E–H). DAPI staining of nuclei (blue) was used to define stages of pollen development as tetrad (A), released microspore (B), bicellular (C), and tricellular (D) pollen. Arrows indicate labeling by AtTIP1;3-GFP of a perinuclear ring (C) and of small vacuoles (D) in maturating pollen grains, as well as the labeling of the tonoplast (E and G) and vacuolar bulbs (F and H) in the vegetative cell and the PT of germinating pollen, respectively. The vegetative nucleus in D was detected with DAPI staining but at a different optical section. It is therefore not visible in this image. G and H represent the superposition of an AtTIP1;3-GFP fluorescence image (E and F) with the corresponding transmission image. Residual green fluorescence in the cytosol (A) and at the pollen walls (B–D) is due to autofluorescence, as it can be also found in nontransformed pollen (not shown). Bars = 10 µm.

Figure 3.

Colabeling of mature pollen grains with AtTIP1;3-GFP, AtTIP5;1-mCherry, and mitochondrial or vacuolar markers. A, Representative mature pollen grain coexpressing AtTIP1;3-GFP (left) and a vegetative cell-specific mitochondria fluorescent reporter (VC-mtRFP; middle). The merged image is shown on the right. B, Coexpression of AtTIP5;1-mCherry with a sperm cell-specific mitochondria fluorescent reporter (SC-mtGFP; middle). The merged image is shown on the right. C, Coexpression of AtTIP5;1-mCherry (left) with the E1 subunit of the vacuolar H⁺-ATPase fused to GFP (VHA-E1-GFP; middle). Note that the latter is specifically enriched in the two sperm cells. Same conventions as in B. D, Coexpression of AtTIP1;3-GFP (left) and AtTIP5;1-mCherry (middle) in mature pollen grains. The merged image is shown on the right. Arrows indicate the labeling of small spherical vesicles by AtTIP5;1-mCherry in the sperm cells. Bars = 10 µm.

Figure 4.

Temporal expression of AtTIP5;1-mCherry in maturating pollen and in germinating PT. The expression of AtTIP5;1-mCherry (red) was analyzed during pollen grain maturation as tetrad (A), released microspore (B), bicellular pollen (C), tricellular pollen (D), and during pollen germination (E and F). DAPI staining of nuclei (blue) was used to define stages of pollen development. Arrows indicate AtTIP5;1-mCherry labeling of a perinuclear ring (C) and small spherical structures in the sperm cells during pollen maturation (D). Similar structures are also labeled during PT growth (E) and in a released sperm cell (G; see arrows). F represents the superposition of a AtTIP5;1-mCherry fluorescence image (E) with the corresponding transmission image. Bars = 10 µm (A–F) and 1 µm (G).

Figure 5.

Molecular characterization and reproduction phenotype of Arabidopsis AtTIP1;3 and AtTIP5;1 T-DNA insertion lines. A, Schematic gDNA structure of AtTIP1;3 and AtTIP5;1 with indicated open reading frame (ATG to TAA). Exons and introns are displayed as black rectangles and black bars, respectively. Note the lack of introns in AtTIP1;3. The insertion site of the T-DNA (white rectangle) and its orientation is indicated for both tip1;3-1 and tip5;1-1. Horizontal arrows indicate the orientation and position of primers used for PCR analyses of the gDNA and cDNA as indicated below. LB, Left border; RB, right border. B, PCR analyses of gDNA of wild-type (WT), tip1;3-1, tip5;1-1, and tip1;3-1/tip5;1-1 plants using a pair of AtTIP1;3-specific primers (1;3fwd1/1;3rev; 1), a pair of AtTIP5;1-specific primers (5;1fwd2/5;1rev; 2), and primers specific for AtTIP1;3 (1;3fwd1) or the T-DNA (LBb1; 3) or specific for AtTIP5;1 (5;1rev) or the T-DNA (o8409; 4). C, Detection by reverse transcription-PCR of AtTIP transcripts on total RNA from flowers of wild-type, tip1;3-1, and tip5;1-1 plants. Expression of AtTIP1;3 and AtTIP5;1 cDNA was probed using gene-specific primer pairs (1;3fwd2/1;3rev [1], 5;1fwd2/5;1rev [3]). The amplification of EF1α cDNA (2 and 4) served as a control for cDNA integrity. D, Proportion (in percentage) of barren siliques in plants of the indicated phenotype grown in soil (See “Materials and Methods”). The number of plants studied is indicated in brackets. The inset shows the phenotypic appearance of the shoots of tip1;3-1/tip5;1-1 plants with short siliques, devoid of seeds (arrows).