A tumor suppressor homolog, AtPTEN1, is essential for pollen development in Arabidopsis

Abstract

Although it is well known that Tyr phosphatases play a critical role in signal transduction in animal cells, little is understood of the functional significance of Tyr phosphatases in higher plants. Here, we describe the functional analysis of an Arabidopsis gene (AtPTEN1) that encodes a Tyr phosphatase closely related to PTEN, a tumor suppressor in animals. The recombinant AtPTEN1 protein, like its homologs in animals, is an active phosphatase that dephosphorylates phosphotyrosine and phosphatidylinositol substrates. RNA gel blot analysis and examination of promoter-reporter constructs in transgenic Arabidopsis plants revealed that the AtPTEN1 gene is expressed exclusively in pollen grains during the late stage of development. Suppression of AtPTEN1 gene expression by RNA interference caused pollen cell death after mitosis. We conclude that AtPTEN1 is a pollen-specific phosphatase and is essential for pollen development.

Figure 1.

Sequence Analysis of AtPTEN1. (A) AtPTEN1 protein sequence deduced from the cDNA. (B) Sequence alignments of Arabidopsis PTEN-like sequences and animal PTENs. (C) Alignment of AtPTEN1 and a wild tomato pollen PTEN deduced from an EST clone. In (B) and (C), the PTEN sequences shown are AtPTEN1, AtPTEN2, and AtPTEN3 from Arabidopsis, DmPTEN from Drosophila, HsPTEN from human, MmPTEN from rat, and LpPTEN from wild tomato. In (A) and (B), the numbers at right indicate the amino acid residue positions in the respective proteins, and the active site signature motif is underlined. In (B) and (C), the shaded boxes indicate identical amino acid residues. In (B), dashes represent gaps introduced to optimize the alignments.

Figure 2.

AtPTEN1 Is an Active Tyr and PIP3 Phosphatase. (A) Tyr phosphatase activity of AtPTEN1 is proportional to enzyme concentration. The activity of AtPTEN1 (closed squares), AtPTEN1 in the presence of sodium vanadate (closed circles), and the AtPTEN1C152S mutant protein (closed diamonds) was measured as described in Methods. Relative activity is presented as a percentage of substrate dephosphorylation in 1 h. (B) Dephosphorylation of PIP3 by AtPTEN1 and HsPTEN. The activity of wild-type or the Cys→Ser mutant of AtPTEN1 and HsPTEN against PIP3 was measured by the amount of free phosphate released from the substrate, as described in Methods.

Figure 3.

AtPTEN1 Is an Anther- and Pollen-Specific Gene. RNA gel blot analysis of AtPTEN1 mRNA levels in various plant organs (A), at different stages of flower development (B), and in separate floral parts (C) of Arabidopsis. In (A), the bottom gel shows ethidium bromide–stained rRNA as a loading control.

Figure 4.

AtPTEN1 Promoter Activity in Transgenic Arabidopsis Plants. Transgenic plants harboring the AtPTEN1 promoter–GUS reporter construct were generated and analyzed histochemically for GUS activity in various floral organs. GUS activity (indicated by blue) was detected in the anthers of the whole flower (A), pollen grains on the stigma (B), pollen in the anther (C), and pollen grains released from the anther (D).

Figure 5.

Silencing of AtPTEN1 by the RNAi Procedure. (A) Schemes of AtPTEN1 RNAi constructs. Transcription cassettes contain a partial AtPTEN1 cDNA (AtPTEN1), a partial GUS gene (GUS), the AtPTEN1 promoter or tomato Lat52 promoter (LeLat52 promoter), and the nopaline synthetase terminator (NOS). Shaded boxes depict the inverted repeat of AtPTEN1 cDNA. The hatched box in the bottom construct depicts the internal deletion in AtPTEN1 cDNA used to disrupt the open reading frame. Arrows point in the 5′-to-3′ direction. (B) RNA gel blot analysis of AtPTEN1 transcripts from open flowers of control (lane 1) and AtPTEN1 RNAi lines containing different RNAi constructs (lanes 2 to 7). The top gel shows ethidium bromide–stained rRNA as a loading control. The bottom gel shows the RNA gel blot probed with AtPTEN1 cDNA. (C) RT-PCR analysis of AtPTEN1 transcripts from open flowers in control and RNAi plants. Total RNAs isolated from open flowers of six independent AtPTEN1 RNAi lines from different constructs (lanes 2 to 7) and the empty vector line (lane 1) were used as templates in RT-PCR with either AtPTEN1-specific primers (bottom gel) or actin2-specific primers (top gel). M, 1-kb DNA ladder.

Figure 6.

Phenotypic Analysis of Pollen from Control and AtPTEN1 RNAi Plants. (A) Bright-field view of hydrated mature pollen from a control plant. (B) Bright-field view of hydrated mature pollen from an AtPTEN1 RNAi plant. (C) AtPTEN1 RNAi binuclear pollen. (D) Propidium iodide–stained mature pollen from a control plant. (E) to (G), (I), and (J) Rhodamine 123–stained mature pollen from control (E) and AtPTEN1 RNAi plants ([F], [G], [I], and [J]). (H) Propidium iodide–stained mature pollen from an AtPTEN1 RNAi plant. (I), (J), and (M) to (O) Enlarged views of pollen from AtPTEN1 RNAi plants show signs of cellular degeneration. Bright-field images in (M) and (N) are of the same pollen grain shown in (I) and (J), respectively. Arrows in (J) and (N) indicate the lesion in intine. (K) and (O) Alexander vitality staining of pollen from a control plant (K) and an RNAi plant (O). (L), (P), and (Q) Tinopal/DAPI double-stained mature pollen from a control plant (L) and RNAi plants ([P] and [Q]). Enlarged views of pollen from AtPTEN1 RNAi plants show signs of disintegration ([P] and [Q]).

Figure 7.

Analysis of Pollen from Control and AtPTEN1 RNAi Plants by Scanning Electron Microscopy. Whole anthers from control (A) and RNAi (B) plants were examined by scanning electron microscopy. Pollen grains from a small area of anther surface are shown. Pollen grains from control (C) and AtPTEN1 RNAi (D) plants were photographed at a higher magnification to show invagination in RNAi pollen. The AtPTEN1 RNAi pollen also showed disruption in exine pattern ([E] and [F]). In (D) to (F), the arrows point to invaginations and bacula junctions. Bar in (A) = 10 μm for (A) and (B); bar in (C) = 5 μm for (C) to (E); bar in (F) = 2 μm.