Mutations in two putative phosphorylation motifs in the tomato pollen receptor kinase LePRK2 show antagonistic effects on pollen tube length

Abstract

The tip-growing pollen tube is a useful model for studying polarized cell growth in plants. We previously characterized LePRK2, a pollen-specific receptor-like kinase from tomato (1). Here, we showed that LePRK2 is present as multiple phosphorylated isoforms in mature pollen membranes. Using comparative sequence analysis and phosphorylation site prediction programs, we identified two putative phosphorylation motifs in the cytoplasmic juxtamembrane (JM) domain. Site-directed mutagenesis in these motifs, followed by transient overexpression in tobacco pollen, showed that both motifs have opposite effects in regulating pollen tube length. Relative to LePRK2-eGFP pollen tubes, alanine substitutions in residues of motif I, Ser(277)/Ser(279)/Ser(282), resulted in longer pollen tubes, but alanine substitutions in motif II, Ser(304)/Ser(307)/Thr(308), resulted in shorter tubes. In contrast, phosphomimicking aspartic substitutions at these residues gave reciprocal results, that is, shorter tubes with mutations in motif I and longer tubes with mutations in motif II. We conclude that the length of pollen tubes can be negatively and positively regulated by phosphorylation of residues in motif I and II respectively. We also showed that LePRK2-eGFP significantly decreased pollen tube length and increased pollen tube tip width, relative to eGFP tubes. The kinase activity of LePRK2 was relevant for this phenotype because tubes that expressed a mutation in a lysine essential for kinase activity showed the same length and width as the eGFP control. Taken together, these results suggest that LePRK2 may have a central role in pollen tube growth through regulation of its own phosphorylation status.

FIGURE 1.

Two-dimensional gel analysis suggests that LePRK2 is highly phosphorylated in tomato pollen. The acidic end of the gel (pH 4) is at the right of each panel. Asterisks indicate the position of LePRK2. A, membrane fractions from germinated pollen (200 μg) were separated by two-dimensional gels (pH 4–7) and immunoblotted with anti-LePRK2. B and C, membrane fractions from mature pollen (200 μg) incubated with [γ-³²P]ATP without (B) or with (C) pure fractions of Style interactor for LePRKs and then separated by two-dimensional gels (pH 4–7). In B and C, insets show Western blots of the same two-dimensional gels. The arrows showed two spots that did not change after incubation with Style interactor for LePRKs. These results were repeated three times.

FIGURE 2.

Schematic representation of selected LePRK2 mutants. A, diagram of LePRK2 protein domains. ECD, extracellular domain; TMD, transmembrane domain; JMD, juxtamembrane domain; KD, kinase domain; CD, C-terminal domain. B, alignment of putative homologues of LePRK2. LePRK2, S. esculentum; LpimpPRK2, S. pimpinellifolium; LpenPRK2, S. pennellii; StPRK2, S. tuberosum; NtPRK2, N. tabacum. Base numbers refer to the LePRK2 sequence. Selected residues are highlighted. Motifs I and II are shown by boxes. Subdomain I of the kinase domain is underlined. An asterisk highlights Lys³⁷², the residue mutated in the mLePRK2 construct. C, cartoon of LePRK2 showing the selected LePRK2 phosphorylation sites. The LePRK2 mutants are arranged in groups. The first eight mutants have mutations at Ser²⁷⁷, Ser²⁷⁹, and Ser²⁸² (AAA-WT, S277A/S279A/S282A; ASS-WT, S277A/S279/S282; SSA-WT, S277/S279/S282A; SAS-WT, S277/S279A/S282; DDD-WT, S277D/S279D/S282D; SDS-WT, S277/S279D/S282; DSS-WT, S277D/S279/S282; and SSD-WT, S277/S279/S282D). The second group contains mutations at Ser³⁰⁴, Ser³⁰⁷, and Thr³⁰⁸ (WT-AAA, S304A/S307A/T308A and WT-DDD, S304D/S307D/T308D), and the third group has two combinations of mutations of the first and second group (AAA-DDD, S277A/S279A/S282A/S304D/S307D/T308D; DDD-AAA, S277D/S279D/S282D/S304A/S307A/T308A).

FIGURE 3.

The increase in pollen tube length by the triple mutant AAA-WT is neutralized by the triple mutant DDD-WT. Data are means ± S.E. of three replicates. Different letters indicate significant differences. The arrow indicates the pollen tube tip. Scale bar represents 100 μm. A, AAA-WT tubes are significantly longer than in LePRK2-eGFP tubes (referred as WT-WT in the text) (p < 0.05). Inset shows a representative AAA-WT tube. B, AAA-WT induced phenotype is annulled by expression of DDD-WT (p < 0.05). Inset shows a representative DDD-WT tube. C, WT-AAA tubes are significantly shorter than LePRK2 (referred as WT-WT in the text) (p < 0.01). Inset shows a representative WT-AAA tube. D, WT-AAA inhibition of growth is annulled by WT-DDD (p < 0.05).

FIGURE 4.

Sextuple mutants showed no additive effects either in elongation (AAA-DDD) or shortening (DDD-AAA) of pollen tubes. Data are means ± S.E. of two replicates. Different letters indicate significant differences. A, AAA-DDD tubes were not significantly different in tube length when compared with WT-DDD or AAA-WT tubes. B, DDD-AAA tubes were not significantly different in tube length compared with DDD-WT tubes but were significantly different to WT-AAA tubes (p < 0.05).

FIGURE 5.

LePRK2 but not mLePRK2 reduced pollen tube growth and induced tip swelling. A, data are means ± S.E. of three replicates. Different letters indicate significant differences (p < 0.05). B–D, representative eGFP (B), LePRK2-eGFP (C), and mLePRK2-eGFP (D) pollen tubes are shown. Scale bar represents 100 μm. Arrows indicate pollen tube tips. The inset shows the pollen tube tip. Left and right panels, GFP channel and bright field images, respectively. Fig. 5A was also performed at 5 h of germination obtaining similar results. Scale bar represents 100 μm.

FIGURE 6.

LePRK2 but not mLePRK2 induced a stable actin network at the pollen tube tip. eGFP (A), LePRK2-eGFP (B), or mLePRK2-eGFP (C) were transiently co-expressed with mRFP-mTalin in tobacco pollen and visualized using confocal microscopy (RFP, left panels; eGFP, right panels). Scale bar represents 100 μm. Medial sections of the pollen tube are shown. Far right panels showed projection of the whole pollen tube with merged fluorescent emissions (eGFP+RFP). D, effect of latrunculin B (Lat B) on pollen tube length and tip width of pollen tubes overexpressing eGFP, LePRK2-eGFP, and mLePRK2-eGFP. *, p < 0.05; **, p < 0.01; and ***, p < 0.001.

FIGURE 7.

LePRK2 has a phosphorylation regulatory motif. Sequence logo was as derived from the MEME program for motif I and the list of the 15 close putative homologous LePRK2 proteins obtained under the MEME analysis. 3015486 corresponds to LePRK2. Motif I in the LePRK2 molecule is G271GSSLTSSSPTS (residues mutated in the analysis are underlined). Residue 1 of the MEME output corresponds to Leu²⁶⁸ in LePRK2 molecule. The obtained putative homologous proteins are as follows: 225424043 and 157338825 (Vitis vinifera); 15221403 and 14190425 (Arabidopsis thaliana); 224108443 (Populus trichocarpa), 77417493 (Malus domestica) and 12552430 (Oryza sativa var. Indica); 125568918, 115434366, and 53792169 (O. sativa var. Japonica); 13506904 (S. pimpinellifolium), 14279352 (Solanum peruvianum), 13506902 (S. pennellii), 168036577 (Physcomitrella patens), and 115458814 (O. sativa var. Japonica). SSC, small-sample correction.