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CELL BIOLOGY AND SIGNAL TRANSDUCTION

The Pollen Receptor Kinase LePRK2 Mediates Growth-Promoting Signals and Positively Regulates Pollen Germination and Tube Growth^1,[W],[OA]

Shanghai Institutes for Biological Sciences-University of California at Berkeley Center of Molecular Life Sciences, National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China (D.Z., C.-P.G., W.-H.T.); Instituto de Ingeniería Genética y Biología Molecular, CONICET Departamento de Fisiología y Biología Molecular y Celular FCEN, Universidad de Buenos Aires, Obligado 2490, 1428 Buenos Aires, Argentina (D.W., J.M.); and Plant Gene Expression Center, United States Department of Agriculture/Agricultural Research Service, and Department of Plant and Microbial Biology, University of California at Berkeley, Albany, California 94710 (D.W., B.S., S.M., W.-H.T.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
In flowering plants, the process of pollen germination and tube growth is required for successful fertilization. A pollen receptor kinase from tomato (Solanum lycopersicum), LePRK2, has been implicated in signaling during pollen germination and tube growth as well as in mediating pollen (tube)-pistil communication. Here we show that reduced expression of LePRK2 affects four aspects of pollen germination and tube growth. First, the percentage of pollen that germinates is reduced, and the time window for competence to germinate is also shorter. Second, the pollen tube growth rate is reduced both in vitro and in the pistil. Third, tip-localized superoxide production by pollen tubes cannot be increased by exogenous calcium ions. Fourth, pollen tubes have defects in responses to style extract component (STIL), an extracellular growth-promoting signal from the pistil. Pollen tubes transiently overexpressing LePRK2-fluorescent protein fusions had slightly wider tips, whereas pollen tubes coexpressing LePRK2 and its cytoplasmic partner protein KPP (a Rop-GEF) had much wider tips. Together these results show that LePRK2 positively regulates pollen germination and tube growth and is involved in transducing responses to extracellular growth-promoting signals.

The pollen receptor kinase LePRK2 is one of the receptor-like kinases localized in the plasma membrane of tomato (Solanum lycopersicum) pollen tubes (Muschietti et al., 1998; Kim et al., 2002). Several pieces of evidence suggested a signaling role for LePRK2 during pollen germination and tube growth. Before pollen germination, the extracellular portion of LePRK2, which mainly contains five Leu-rich repeats, can interact with an extracellular protein from pollen, LAT52 (Tang et al., 2002). Tomato pollen expressing antisense LAT52 RNA appeared to be normal at the mature pollen stage, but could not hydrate and germinate normally when cultured in vitro, and formed aberrant, twisted tubes on the stigma, failing to reach the embryo sac (Muschietti et al., 1994). LePRK2 can switch from interacting with LAT52 to interact with an extracellular protein from the stigma, LeSTIG1 (Tang et al., 2004). The addition of LeSTIG1 to germination medium promoted pollen tube growth in vitro (Tang et al., 2004). In the cytoplasm, the intracellular portion of LePRK2 interacts with KPP (Kaothien et al., 2005). KPP belongs to a family of plant-specific guanine nucleotide exchange factors (Berken et al., 2005) for Rop small GTPases. Pollen tubes that overexpressed nearly full-length KPP (missing the first eight amino acids at the N terminus) were wider, especially at the tip (Kaothien et al., 2005), probably due to the partial loss of polarity, which is maintained by local activation of Rop (Zhang and McCormick, 2007). Furthermore, the phosphorylated form of LePRK2, which is present in the plasma membranes of pollen tubes, could be specifically dephosphorylated by a 3- to 10-kD fraction of style extracts (Wengier et al., 2003), showing that LePRK2 on the pollen tube responds to signals from the style. The biological functions of these extracellular and cytosolic binding partners suggest that LePRK2 plays a role in pollen germination and tube growth, but how LePRK2 regulates pollen germination and tube growth was not known.

Here, using antisense constructs that reduce the expression level of LePRK2 in pollen and pollen tubes, we provide evidence that LePRK2 positively regulates pollen germination and tube growth rate. Antisense LePRK2 pollen tubes have mispositioned large vacuoles and reduced spacing between callose plugs, suggesting a problem with turgor pressure. Furthermore, they do not increase reactive oxygen species (ROS) production or their lengths in response to exogenous calcium ions. We also show that the style extract component (STIL) that dephosphorylates LePRK2 (Wengier et al., 2003) and can increase pollen tube lengths (D. Wengier, S. McCormick, and J. Muschietti, unpublished data) cannot increase the lengths of antisense LePRK2 pollen tubes grown in vitro, indicating that this increase is dependent on LePRK2.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Generation of Transgenic Tomato Plants with Specific Reductions in LePRK2 Expression in Mature Pollen and Pollen Tubes

To directly address the biological function of LePRK2 in pollen, we generated transgenic tomato plants with a construct including a full-length LePRK2 antisense DNA driven by a pollen-specific promoter (LAT52), a separate GFP gene driven by the LAT52 promoter, and a kanamycin resistance gene driven by the cauliflower mosaic virus 35S promoter. We used the LAT52 promoter to drive antisense LePRK2, because both LePRK2 and LAT52 are expressed in mature pollen and in pollen tubes, and in immature pollen LAT52 is expressed slightly earlier (Twell et al., 1989b) than LePRK2 (Muschietti et al., 1998). Furthermore, LAT52 mRNA is about 10-fold more abundant than LePRK2 mRNA in mature pollen and pollen tubes (real-time PCR, data not shown), and therefore the LAT52 promoter is likely stronger than the LePRK2 promoter. Last, the LAT52 promoter was used in generating antisense Shy in petunia (Petunia hybrida) pollen (Guyon et al., 2004) and in generating antisense LAT52 in tomato pollen (Muschietti et al., 1994).

Seeds of 19 self-pollinated T0 plants were collected and germinated on kanamycin medium. Judging by the kanamycin resistance to sensitive ratio, four lines had multiple insertions and were not further characterized. Thirteen lines showed a 3:1 ratio, while two lines showed a 1:1 ratio. We further analyzed one line with a 1:1 ratio (line 1) and five lines with a 3:1 ratio (lines 2–6; Supplemental Table S1). We grew six kanamycin-resistant T1 seedlings of each of these lines to flowering.

We confirmed the presence of the LePRK2 antisense construct in T1 plants of all six lines by genomic DNA PCR (data not shown). For each line, most of the T1 plants had about 50% GFP-expressing pollen, as expected for heterozygotes, but in at least one plant in each line, all pollen expressed GFP, consistent with these plants being homozygotes (Supplemental Table S1). We self-pollinated heterozygous T1 plants of four lines, and counted the kanamycin resistance to sensitive ratio of their progenies. Lines 2, 4, and 6 again gave a 3:1 ratio, and line 1 again gave a 1:1 ratio (Supplemental Table S1). We also pollinated wild-type tomato pistils with pollen from heterozygous lines 2 and 4, but didn't see any male transmission defects (Supplemental Table S1). We pollinated wild-type tomato pistils with pollen from the putative homozygous antisense LePRK2 lines 1 to 6 and germinated the resulting seeds on medium containing kanamycin. All seedlings were resistant to kanamycin, confirming that these plants were homozygous for the construct. We obtained homozygous transgenic plants for all six lines and all subsequent experiments used homozygous plants.

To determine whether LePRK2 expression was reduced in these plants, we performed quantitative reverse transcription-PCR with LePRK2-specific primers, using total RNA of mature pollen as templates. Figure 1A shows that the LePRK2 expression level was significantly reduced in all six lines, to 20% to 30% of wild-type levels in lines 1 and 6 and to less than 5% in lines 2 to 5. LePRK1 is the closest homolog for LePRK2 among known tomato genes, with 54% identity in overall protein sequence, and 64% identity in overall nucleotide sequence (Kim et al., 2002). The longest identical DNA fragment between LePRK1 and LePRK2 is 26 nucleotides. To address the specificity of the antisense construct, we checked whether the LePRK1 expression level was also altered in LePRK2 antisense plants. Figure 1A shows that the LePRK1 mRNA level varied from 80% to 105% of the levels in wild-type pollen. We therefore concluded that LePRK1 expression was not affected significantly, and that LePRK2 expression was specifically reduced in mature pollen of lines 1 to 6.

View larger version (24K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. LePRK2 expression is reduced in antisense LePRK2 pollen. Quantitative reverse transcription-PCR of LePRK2 and LePRK1 mRNA levels, using total RNA of mature pollen (A) or in vitro germinated pollen (B) as templates. WT, Wild type; 1 to 6, antisense LePRK2 lines 1 to 6. The tomato actin gene was used as a control. In vitro germinated pollen was harvested 6 h after culturing in optimized germination medium. Experiments were performed in triplicate. Error bars = SE.

View larger version (70K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Protein levels of LePRK2 are reduced in antisense LePRK2 pollen. A and B, Immunoblots showing LePRK2 (A) and LePRK1 (B) protein levels in germinated pollen of wild-type (WT), GFP-expressing, and antisense LePRK2 lines 1 to 4. C, The LePRK2 protein level was normalized against the LePRK1 protein level. The mean intensity average of two experiments, as measured with ImageJ. Two bands are detected by anti-LePRK2 antibodies (Wengier et al., 2003).

We examined the pollen phenotypes of all six transgenic lines. Mature pollen of each line showed no obvious differences from pollen of wild type or of plants transformed with a pLAT52::GFP construct (data not shown). When placed in germination medium, antisense LePRK2 pollen hydrated and germinated normally shaped tubes. Although the germination percentage of wild-type or GFP-expressing pollen varied from day to day (within a range between 40%–90%), the germination percentage of antisense LePRK2 pollen was always 10% to 30% lower than the percentage for wild-type or GFP-expressing pollen germinated on the same day. To further understand why this was so, we plotted pollen germination percentage against time. Wild-type, GFP-expressing, and antisense LePRK2 pollen (lines 2, 3, and 4) all started to germinate approximately 30 min after transfer to germination medium, and within the first hour of germination the increase in germinating grains was similar for all samples. However, the numbers of wild-type and GFP-expressing pollen with tubes continued to increase during the first 120 to 150 min of incubation, while the numbers of antisense LePRK2 pollen with tubes did not increase after 70 to 100 min (Fig. 3A shows two representative experiments). Thus the reduced germination percentage of antisense LePRK2 pollen is not due to a delay in starting germination, but instead to a reduced time window for germination competence.

View larger version (39K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Germination percentage and growth rate of antisense LePRK2 pollen. A, Germination percentage of tomato pollen plotted against culture time. Wild-type VF36 (WT1 and WT2) and homozygous transgenic plants carrying pLAT52::GFP (GFP) are controls; 2, 3, and 4 are antisense LePRK2 lines 2, 3, and 4. A combination of two independent experiments is shown. WT1 and antisense LePRK2 line 2 were measured in experiment 1. WT2, GFP, and antisense LePRK2 lines 3 and 4 were measured in experiment 2. B, Pollen tube length measured after 3 h in vitro culture. Numbers 1 to 6 are antisense LePRK2 lines 1 to 6. C, Tomato pollen tubes from wild type (left) or from antisense LePRK2 line 2 (right), after 10 h in germination medium. In each plate, pollen was added at 1 mg/mL. Arrows point to the mats of pollen tubes.

Besides the reduction in germination, antisense LePRK2 pollen tubes grown in vitro were shorter than wild-type pollen tubes, when observed at either 3 h (Fig. 3B) or 10 h after germination (Fig. 3C). The length difference at 3 h was statistically significant in double-blinded assays. After 10 h the difference was readily apparent with the naked eye. Wild-type pollen formed long tubes and overnight growth resulted in interlocked pollen tubes resembling a mat of fungal hyphae, whereas the antisense LePRK2 pollen tubes formed only small clumps (Fig. 3C). Figure 3B shows that pollen tubes of lines 1 and 6, which express approximately 10% of wild-type levels of LePRK2 (Fig. 1B), were slightly longer than pollen tubes of lines 2 to 4, which express less than 5% of wild-type levels of LePRK2 (Fig. 1B). The correlation between LePRK2 expression level and pollen tube length supports a positive role of LePRK2 in regulating pollen tube growth. These results were confirmed in the T2 and T3 generations.

To determine whether the reduced tube length of antisense LePRK2 pollen was due to a slower growth rate, to early termination of tube growth, or both, we measured pollen tube lengths after 2 to 4 h, and calculated an average growth rate. The growth rate of line 4 pollen tubes was approximately 0.11 mm/h, half of the rate (approximately 0.22 mm/h) for wild-type pollen tubes (Supplemental Fig. S1). Although many tubes of antisense LePRK2 pollen continued to grow after 10 h, many stopped growth much earlier. The reduction in growth rate is at least one of the causes for the reduction of tube length, but earlier termination of tube growth might also contribute.

To determine whether antisense LePRK2 pollen tubes also grew slower in pistils, we pollinated wild-type pistils with pollen from lines 2 and 4 as well as with wild-type pollen, then checked pollen germination status on the stigmas and recorded the time pollen tubes arrived at the ovary. On the stigmas, the wild-type pollen and antisense LePRK2 pollen germinated 3 to 5 h after pollination (Fig. 4, A–D ). Wild-type or GFP pollen tubes arrived at ovaries 7 to 9 h after pollination (growth rate estimated at 1.2 mm/h), while pollen tubes of lines 2 and 4 did not reach the ovaries until 10 to 12 h after pollination, with growth rates estimated at 0.9 mm/h (Table I ; Fig. 4). Thus antisense LePRK2 pollen tubes grew slower than wild-type tubes both in vitro and in vivo, although the difference was smaller in vivo.

View larger version (93K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Pollen tubes of antisense LePRK2 plants are delayed in reaching ovaries. Wild-type tomato pistils hand-pollinated with either wild-type pollen (WT), GFP-expressing pollen (GFP), or antisense LePRK2 pollen (line 2 and line 4) were dissected and stained with decolorized aniline blue to reveal pollen tubes. A to D, Germination of indicated pollen tubes on stigmas 5 h after pollination. E to L, Ovaries were dissected after pollination at the times indicated to reveal the presence or absence of pollen tubes. Scale bars = 200 µm; arrows point to pollen tubes.

To determine what downstream processes might account for the slower growth of antisense LePRK2 pollen tubes, we observed the subcellular morphology of growing pollen tubes. Vacuoles perform multiple functions in plant cells, including the storage and degradation of cellular components, osmoregulation, and modulation of turgor (Bassham and Raikhel, 2000; Lew, 2004; MacRobbie, 2006). Growing wild-type pollen tubes usually have large vacuoles (>5 µm in diameter) at the very rear, while only thin tubular vacuoles are seen near the tip (Lovy-Wheeler et al., 2007). Many of the antisense LePRK2 pollen tubes had large vacuoles near the tip (Fig. 5A ) and sometimes the front edge of these vacuoles were as near as 10 µm from the tip. These large vacuoles were not present in early stages of tube growth, but were seen in about 50% of the tubes, starting as early as 5 h after germination. Upon further observation of such pollen tubes, larger and larger vacuoles moving closer to the tip were noted. If they reached the tip they should disrupt the normal organelle distribution of the tip and the tube should stop growth. However, continued observations of these pollen tubes showed that the large vacuoles sometimes moved away from the tip—this backward and forward movement kept the vacuoles near the front but away from the clear zone, allowing such pollen tubes to grow for quite a while (Supplemental Movies S1–S3).

View larger version (56K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Antisense LePRK2 pollen tubes have large vacuoles near the tip region and shorter intervals between callose plugs. A, The tip regions of a representative GFP-expressing pollen tube (control) and antisense LePRK2 pollen tube of lines 2 and 4. The pollen tubes were imaged after 5 h in vitro culture. Scale bars = 10 µm. DIC, Differential interference contrast. B, Representative pictures of pollen tubes with callose plugs stained by decolorized aniline blue. Wild-type and antisense LePRK2 pollen tubes after 10 and 12 h in vitro culturing, respectively. Insets show enlargements of the callose plugs. Scale bars = 200 µm. G, Pollen grain; T, tip of pollen tube. C, The average interval lengths between callose plugs in pollen tubes of wild-type, GFP-expressing, or antisense LePRK2 lines after 12 h in vitro culturing. Error bars = SE (n = 3).

Antisense LePRK2 Pollen Tubes Have Impaired Responses to Ca²⁺

In tobacco (Nicotiana tabacum), ROS production was detected at the pollen tube tip, and the ROS level was increased with exogenous Ca²⁺ (Potock et al., 2007). To determine whether there was a difference in ROS levels between wild-type and antisense LePRK2 pollen tubes, we stained growing pollen tubes with nitroblue tetrazolium (NBT), which reacts with O₂^– and forms a blue precipitate. NBT staining of wild-type tomato pollen tubes was darker in a medium with 1 mM CaCl₂ than in a medium without CaCl₂, indicating an increase in the ROS level with exogenous Ca²⁺ (Fig. 6, A and B ). However, NBT staining for lines 1, 2, and 4 didn't change significantly upon adding Ca²⁺, indicating that the antisense LePRK2 pollen tubes had a diminished response to Ca²⁺ for ROS production.

View larger version (33K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Responses of antisense LePRK2 pollen tubes to 1 mM CaCl2 treatment. A and B, Tip-localized ROS production of pollen tubes detected by NBT staining. A, Average of minimum intensities of NBT-stained tubes of wild-type (WT), GFP-expressing pollen (GFP), and antisense LePRK2 lines 1, 2, and 4 after 6 h in vitro culturing with or without 1 mM CaCl2. The results for individual experiments are shown in separate sections. For each experiment an average of 30 tubes per well and three wells per treatment were measured. Error bars = SE. Asterisks indicate significant differences at the same data point (P < 0.05; t test). B, Representative NBT-staining pictures for pollen tubes of wild-type and antisense LePRK2 lines 2 and 4. Scale bar = 10 µm. C, Pollen tube lengths for wild-type (WT), GFP-expressing pollen (GFP), and antisense LePRK2 lines 2 and 4 after 3 h culture with or without 1 mM CaCl2. Error bars = SE (n = 3).

A Style Component Promotes Pollen Tube Growth of Wild-Type Pollen, But Not of Antisense LePRK2 Pollen

LePRK2 is specifically dephosphorylated by a component of style extract and this component can also cause dissociation of a complex that includes LePRK1 and LePRK2 (Wengier et al., 2003). We speculated that the style component triggers LePRK2 signaling during pollen tube growth through dephosphorylation and complex dissociation, but the nature and the biological function of this style component remained unknown. Recently STIL, the factor in the style component that is responsible for LePRK2 dephosphorylation, was purified and shown to stimulate pollen tube growth (D. Wengier, S. McCormick, and J. Muschietti, unpublished data). Figure 7 confirms that adding STIL to pollen germination medium increased the length of wild-type and GFP-expressing pollen tubes. However, the lengths of antisense LePRK2 pollen tubes were not increased in the presence of STIL, suggesting that this stimulatory effect depends on LePRK2 expression.

View larger version (10K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. The effects of STIL on tomato pollen tube growth. The graph shows average pollen tube length after 3 h germination from antisense LePRK2, wild-type, and GFP-expressing pollen tubes. The data are pooled from two experiments; each experiment contained three replicates and at least 30 pollen tubes were measured in each replicate. Error bars = SE.

Overexpression phenotypes are sometimes informative (Li et al., 1999; Kaothien et al., 2005). We attempted to obtain transgenic plants that overexpressed LePRK2, but were unsuccessful. Therefore we used microprojectile bombardment of pollen (Twell et al., 1989a) to transiently overexpress a LePRK2-GFP fusion protein. The green pollen tubes were not shorter than untransformed pollen tubes, but their tips were 20% wider than tips of wild-type or GFP-expressing pollen tubes (Fig. 8, A and B ). A similar swollen tip phenotype was seen with LePRK2-RFP-expressing pollen tubes (Fig. 8, C and D); GFP can form dimers but mRFP is a monomer (Campbell et al., 2002), so these similar phenotypes suggest that the swollen tip phenotype is not due to dimerization of the fusion protein via the fluorescent protein. The LePRK2-GFP and LePRK2-RFP proteins were mainly localized at the plasma membrane of the pollen tube, although some fluorescent protein aggregates, possibly due to overexpression, were also seen in the cytosol (Fig. 8B, middle tube).

View larger version (45K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 8. Transient overexpression of LePRK2 and/or KPP fusion proteins in pollen tubes. A and B, Tomato pollen tubes overexpressing GFP (A) or LePRK2-GFP (B). C to F, Tobacco pollen tubes overexpressing RFP (C), LePRK2-RFP (D), GFP (E), or full-length KPP-GFP (F) individually. G, Tobacco pollen tubes coexpressing LePRK2-RFP and KPP-GFP. All constructs used the LAT52 promoter. Pictures were taken using DIC, GFP, or RFP channels, as indicated. Pollen tubes visible in the DIC channel but invisible in either the GFP or RFP channels are not transformed. Scale bars = 50 µm.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Homozygous plants were readily obtained from all six antisense LePRK2 lines. This was surprising, especially from line 1, which had a 1:1 ratio for kanamycin resistance to sensitivity, and from the lines with greatly reduced levels of LePRK2 mRNA. These results indicate that pollen with less than 5% of the normal level of LePRK2 expression still can deliver sperm for successful fertilization. Nonetheless, pollen grains of the homozygous plants had reduced chances for germination (Fig. 3A) and had a reduced tube growth rate (Fig. 3, B and C; Supplemental Fig. S1). Furthermore, the antisense LePRK2 pollen tubes had morphological defects (Fig. 5) and were deficient in responses to some (Ca²⁺ and STIL; Figs. 6 and 7) but not all (boric acid; Supplemental Fig. S2) exogenous promotion factors. Therefore the main function for LePRK2 is to transduce specific external growth-promoting signals for the growing pollen tube.

Cytosolic turgor has to be maintained for pollen tube growth (Benkert et al., 1997). Among other functions, vacuoles modulate turgor during cell growth (Hicks et al., 2004; Lew, 2004). In normal-growing pollen tubes only thin thread-like vacuoles are usually seen in the front, while large vacuoles are usually seen in the rear, close to callose plugs (Hicks et al., 2004; Lovy-Wheeler et al., 2007). As growth proceeded in the antisense LePRK2 pollen tubes larger vacuoles were seen more often closer to the tip region. The phenomenon might be interpreted as compensation for reduced cytosolic turgor in the front. The periodic formation of callose plugs that separate the growing front from the evacuated regions is thought to be useful for maintaining a manageable cytosolic volume for pollen tubes, because pollen tubes need to extend many times the grain diameter to reach ovules (Nishikawa et al., 2005). The antisense LePRK2 pollen tubes had shorter intervals between callose plugs, giving a smaller cytosolic volume for the growing tube cell. Although the callose plug interval difference can be interpreted in many different ways, it might be a compensation for or a result of the reduced cytosolic turgor, because less turgor would be required to maintain the force needed for a smaller cell volume to grow forward. The presence of large vacuoles in front of the tube and the more frequent callose plugs both suggest that cytosolic turgor in the antisense LePRK2 pollen tubes was reduced, and might explain why antisense LePRK2 pollen tubes cannot grow as fast as wild-type tubes.

Consistent with the idea that pollen tubes with reduced LePRK2 expression have less cytosolic turgor, wild-type pollen tubes grew as slowly as antisense LePRK2 pollen tubes when cultured in germination medium containing 32% polyethylene glycol (data not shown), which increases the external osmotic pressure. Pollen germination also requires the accumulation of cytosolic turgor to a threshold level to start protruding from the pollen grain (Taylor and Hepler, 1997). In antisense LePRK2 pollen fewer pollen grains might have enough turgor to start germination, perhaps explaining the reduced germination percentage. Consistent with the idea that LePRK2 might regulate cytosolic turgor, overexpression of LePRK2-GFP caused slightly swollen tips, which could be interpreted as higher turgor. Swollen tips are also seen in depolarized growth, such as when a constitutively active ROP (Li et al., 1999) is overexpressed, but such depolarized growth is usually more extreme (so-called balloon tips) and also causes slower growth or arrest of pollen tube growth. The cell wall at the tip of pollen tubes is thin as it lacks callose or cellulose and contains mainly esterified pectins (Krichevsky et al., 2007). Under slightly higher cytolic turgor, the tip will be the first place to swell. In summary, we interpret the multiple morphological defects caused by altering LePRK2 expression as affects on cytosolic turgor.

Antisense LePRK2 pollen tubes did not increase production of extracellular O₂^– upon exogenous Ca²⁺, while wild-type pollen tubes did, suggesting that LePRK2 might participate in sensing extracellular Ca²⁺ and regulating O₂^– production. In plants, ROS, including O₂^–, have been shown to play roles in mediating multiple physiological responses (Mori and Schroeder, 2004). Extracellular O₂^– can be monitored by NBT staining (Rossetti and Bonatti, 2001) and in plants is thought to be produced by plasma membrane-localized NADPH oxidases (Mittler, 2002). Plant NADPH oxidases are homologs of the mammalian neutrophil NADPH oxidase gp91^phox, which catalyzes cytosolic NADPH and extracellular O₂ to cytosolic NADP⁺, H⁺, and extracellular O₂^– (Taylor et al., 1993; Keller et al., 1998). Root hairs and pollen tubes both grow by tip growth and O₂^– plays important roles in root hair development (Foreman et al., 2003). In root hairs, localized ROS, along with a plasma membrane-localized NADPH oxidase, ROOT HAIR DEFECTIVE2, and Ca²⁺, have been shown to form a positive feedback loop to maintain polarity during root hair growth (Takeda et al., 2008). A similar mechanism might also work in pollen tubes, considering that tobacco pollen tubes produce tip-localized O₂^– that was increased by extracellular Ca²⁺, and conversely, RNA interference of a tobacco NADPH oxidase gene impaired pollen tube growth (Potock et al., 2007). Furthermore, cytosolic NADPH has been reported to oscillate in lily (Lilium formosanum) pollen tubes and to be correlated with tip growth (Cárdenas et al., 2006). In addition, the activity of NADPH oxidase can be regulated by extracellular Ca²⁺. Although plant NADPH oxidases contain two Ca²⁺-binding EF-hand motifs, they are located in the cytoplasmic N-terminal portion of this transmembrane protein (Wong et al., 2007), and so extracellular Ca²⁺ would need to be sensed and transduced across the plasma membrane to increase NADPH oxidase activity. Our results support a role for LePRK2 in this transduction.

We showed that antisense LePRK2 pollen tubes don't increase growth upon addition of calcium ions. Exogenous calcium ions of appropriate concentration increase pollen tube growth, while higher concentrations inhibit pollen tube growth (Steer and Steer, 1989). However, how the exogenous Ca²⁺ signal is transduced and affects pollen tube growth is not clear. Our results suggest that LePRK2 is involved in transducing the extracellular Ca²⁺ signal.

For the antisense LePRK2 pollen, the average reduction of in vitro tube growth (50%–70% less than wild type) was very significant, but the delay in arriving at ovaries was small (2–3 h delay for a journey that normally takes 7–9 h) and, except for line 1, the transmission ratio of the transgene was not significantly distorted. Considering that there is always more pollen on the stigma than the number of ovaries to be fertilized, only the earliest-arriving pollen tubes will successfully deliver sperm. Given the variation of tube growth rate among pollen of same genetic background, it is possible that the fastest-growing antisense LePRK2 pollen tubes had growth rates comparable to those of wild-type pollen. In self-pollinated flowers, pollen does not land on the stigma at the same time, and this might have further diminished the difference in average tube growth. It is also possible that other growth-promoting factors from the pistil helped pollen tube growth in ways that are independent of LePRK2. Furthermore, LePRK1, a similar pollen receptor-like kinase that can interact with LePRK2 (Muschietti et al., 1998), might compensate for LePRK2 when LePRK2 is knocked down.

Mutations in ligands and receptors in the same signaling pathway have similar phenotypes, for example, Clavata 1, 2, and 3 (for review, see Williams and Fletcher, 2005). We showed here that antisense LePRK2 pollen had a lower germination percentage and slower tube growth, and similar phenotypes were seen when two extracellular partners for LePRK2 were down-regulated. LAT52 is an extracellular partner of LePRK2 before pollen germination (Tang et al., 2002), and antisense LAT52 pollen cannot germinate at all in vitro (Muschietti et al., 1994). The tomato homolog of SHY is also an extracellular partner for LePRK2 (Tang et al., 2004), and in petunia antisense SHY pollen has a lower germination percentage and slower tube growth (Guyon et al., 2004). Our results suggest that other factors (Ca²⁺, STIL) and processes (turgor modulation) also act through LePRK2.

Pollen overexpressing a nearly full-length version of KPP (missing eight amino acids at the N terminus) showed depolarized tube growth (Kaothien et al., 2005). However, overexpression of a full-length AtRopGEF12, the closest Arabidopsis (Arabidopsis thaliana) homolog for KPP, only resulted slightly increase on pollen tube width (Zhang and McCormick, 2007), suggesting that those eight amino acids were important for the phenotypic differences. Here we confirmed that, since overexpressing a full-length version KPP caused only a slight increase of pollen tube width (Fig. 8F). Coexpressing AtRopGEF12 and a LePRK2 homolog in Arabidopsis (AtPRK2a) gave much wider pollen tube tips than when overexpressing AtRopGEF or AtPRK2a alone (Zhang and McCormick, 2007). Here, we showed similar situation in tomato for KPP and LePRK2 (Fig. 8G). This supports the model proposed by Zhang and McCormick (2007) that pollen-enriched RopGEFs serve as a link between pollen receptor kinases and Rop-mediated intracellular responses. It has been reported that ROP1 activation controls the polar accumulation and exocytosis of vesicles at pollen tube tips, through RIC3 and/or RIC4 pathways (Lee et al., 2008). It is possible that antisense LePRK2 reduced the activation of KPP, which in turn reduced the activation of Rop, thus leading to reduced exocytosis, which slowed tube growth. It was also reported that a rice (Oryza sativa) Rop (OsRac1) can interact and regulate OsRboHB, which is a plasma membrane-localized NADPH oxidase that produces extracellular O₂^– (Wong et al., 2007). It is plausible that the defects of antisense LePRK2 pollen tubes in extracellular O₂^– production might be mediated by Rop.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

In Vitro Pollen Germination and Measurements

Open flowers of tomato (Solanum lycopersicon ‘VF36’) were picked in the afternoons. Mature pollen was obtained by vibrating anthers of open flowers with a biovortexer (BioSpec Products). Pollen was used directly for germination or bombardment experiments, or was stored at –80°C for protein extraction. Pollen was germinated in optimized pollen germination medium [20 mM MES, pH 6.0, 3 mM Ca(NO₃)₂, 1 mM KCl, 0.8 mM MgSO₄, 1.6 mM boric acid, 2.5% (w/v) Suc, and 24% (w/v) polyethylene glycol 4000], in dishes that were rotated horizontally at 60 rpm, at a concentration of 1 mg pollen/mL medium, unless otherwise specified. Pollen tube images were captured with a digital camera attached to an epifluorescence microscope. Pollen tube length and width were measured using ImageJ (Rasband, 1997–2007). For measuring tube lengths, pollen was cultured in five individual wells for 3 h and fixed in FAA (10% formaldehyde, 5% glacial acetic acid, 50% ethanol [v/v]) before images were taken.

Pollen Bombardment Assay

The pollen-specific LAT52 promoter (Twell et al., 1991) was used to replace the cauliflower mosaic virus 35S promoter in a 35S::GFP plasmid (pPK100, a kind gift from Robert Blanvillain) to obtain a pLAT52::GFP plasmid. Full-length LePRK2 cDNA was inserted in frame at the 5' end of the GFP coding sequence in the pLAT52::GFP plasmid. The mRFP coding region was amplified from the pMT-mRFP1 plasmid (Toews et al., 2004) to replace GFP in the pLAT52::GFP and pLAT52::LePRK2-GFP plasmids to obtain pLAT52::RFP and pLAT52::LePRK2-RFP. The plasmids pLAT52::GFP, pLAT52::LePRK2-GFP, pLAT52::RFP, and pLAT52::LePRK2-RFP were used for pollen bombardment. Tomato pollen bombardment assays were as described by Kaothien et al. (2005) for tobacco (Nicotiana tabacum) pollen bombardment except that pollen was cultured with shaking at 60 rpm. We observed pollen tubes 4 to 7 h after bombardment.

Transgenic Tomato Development

A GFP expression cassette and an antisense full-length LePRK2, both driven by the LAT52 promoter, were individually inserted into pCAMBIA2300 to obtain the pCAMBIA-antisense LePRK2 plasmid. Agrobacterium tumefaciens strain LBA4404 (Hoekema et al., 1983) carrying this plasmid was used to transform tomato (cv VF36) as described (McCormick, 1991).

NBT and Decolorized Aniline Blue Staining of Pollen Tubes

Production of O₂^– was determined by its ability to reduce NBT (Rossetti and Bonatti, 2001). Pollen tube staining was slightly modified from the method described in Potock et al. (2007). To avoid nitrate interference with the NBT reaction, tomato pollen was cultured in a simplified medium (20 mM MES, pH 6.0, 13% [w/v] Suc, 20% [w/v] polyethylene glycol 4000, 1.6 mM boric acid, 1 mM KCl) with or without 1 mM CaCl₂ as indicated, for 1 to 4 h before the addition of 1 mM NBT. Each treatment had at least three replicates. Images were captured using an Olympus BX51 microscope fitted with an Olympus DP71 digital camera. The intensity of formazan precipitation in pollen tube tips was quantified using ImageJ (Rasband, 1997–2007). Minimal pixel intensity was calculated within each single image and inside regions of interest fitted to the outline of the pollen tube tip. Less intensity indicates more NBT stain. At least 15 pollen tubes from each replicate were measured. Callose of pollen tubes in the pistil was stained with decolorized aniline blue directly after dissection of pistils without fixation, as described (Johnson-Brousseau and McCormick, 2004).

RNA Extraction and Quantitative PCR

Total RNA extraction from mature or germinated pollen of tomato was according to a modification of the Qiagen protocol (http://www.pgec.usda.gov/McCormick/McCormick/mclab.html). We performed quantitative real-time PCR reaction of reverse-transcribed RNA with SYBR Green detection on an ABI PRISM 7000 sequence detector (Applied Biosystems) as described by Tang et al. (2006). The primers used to amplify a 100-bp fragment of LePRK2 were 5'-CTTGCCTGATTTGTTGAAAGCC-3' and 5'-AACAACCATGACAGGGCCA-3'. The primers used to amplify a 122-bp fragment of LePRK1 were 5'-GGCCTGAAGTACAAGCAGTACAACA-3' and 5'-CGAACCAAACACACCGCTA-3'. The primers used to amplify a 123-bp fragment of a tomato actin gene (BG140412) were 5'-GCGAGAAATTGTCAGGGACGT-3' and 5'-TGCCCATCTGGGAGCTCAT-3'.

Pollen Protein Extraction and Immunoblotting

Tomato pollen protein extraction was as described by Tang et al. (2002) with a TED buffer (50 mM Tris-HCl, pH 7.6, 1 mM EDTA, 1 mM dithiothreitol, and 1x Protease Inhibitor Cocktail Complete [Boehringer Mannheim]). The P₁₀₀ (membrane-associated) protein fraction was used for immunoblots with polyclonal anti-ECD1 or anti-ECD2 antibodies (Muschietti et al., 1998).

Style Component Preparation and Treatment

STIL was purified as in D. Wengier, S. McCormick, and J. Muschietti (unpublished data). Briefly, a pistil exudate was obtained by cutting 100 tobacco styles and stigmas transversely in 5 mm segments and incubating overnight in 50 mM ammonium bicarbonate (25 mL) at 4°C with gentle agitation. The pistil exudate was filtered through miracloth and filter paper and then subjected to chloroform-methanol extraction. The aqueous phase was dried by rotary evaporation and the pellet was dissolved in water. The dissolved pellet was centrifuged 10 min at 10,000g in a tabletop centrifuge and the supernatant was fractionated by FPLC in a Mono Q 5/50 GL Monobead column (GE Healthcare Life Sciences). The presence of eluted STIL in fractions was assayed by LePRK2-specific dephosphorylation in mature pollen protein extracts, as in Muschietti et al. (1998). Fractions that showed LePRK2 dephosphorylation were pooled, freeze dried, and subjected to solid-phase extraction in a Sep-Pak Plus C18 cartridge (Waters). The percolate was freeze dried to eliminate acetonitrile and the STIL fraction was obtained by dissolving the pellet in MilliQ water.

For germination assays, freshly collected pollen from each line (wild type, GFP expressing, antisense LePRK2 lines 2 and 3) was prehydrated in pollen germination medium (PGM) without Suc for 30 min at room temperature with occasional gentle agitation. After incubation, the pollen suspension was centrifuged for 5 min at 3,000g and resuspended to a final concentration of 1 mg pollen/mL in complete PGM (no STIL treatment) or supplemented with 0.0003 Abs units (280 nm) of STIL/µL of PGM (+STIL treatment). In every experiment, each line included three replicates for each treatment. Pollen germination was carried out on a rotating shaker (50 rpm) for 3 h at 28°C in 24-well microplates and each well contained 400 µL of the pollen suspension. After germination, the pollen suspension was transferred to 1.5 mL microtubes and 10x fixing solution (5.6% formaldehyde, 0.5% glutaraldehyde, and 25% polyethylene glycol 3350) was added to a final concentration of 1x. Samples were incubated with gentle agitation at 50 rpm for 30 min at 4°C. Fixed pollen tubes were observed using an Axiovert microscope (Zeiss) and 50 pictures were taken for each replicate with a digital camera (Diagnostic Instruments). Fifteen pictures were randomly selected and the lengths of all the pollen tubes in each picture were determined using AxioVision software (Zeiss) and averaged. Pollen tube lengths for each replicate were calculated as the average from all 15 values previously obtained. Control and STIL treatments were analyzed with the Student's t test using Prism version 4.03 for Windows (GraphPad). Germination experiments were repeated twice and since variances did not differ, data were pooled.

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers U58473 (LePRK2), U58474 (LePRK1), and AY730762 (KPP).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Growth rate of pollen tubes germinated in vitro for 2 to 4 h.

Supplemental Figure S2. Pollen tube lengths for wild-type (WT) and antisense LePRK2 lines 2 and 4 after 3 h culture, with indicated concentrations of boric acid (BA).

Supplemental Figure S3. Relative tip widths of tobacco pollen tubes transiently expressing either RFP alone, LePRK2-RFP alone, KPP-GFP alone, or coexpressing LePRK2-RFP and KPP-GFP.

Supplemental Table S1. Summary of antisense LePRK2 plants.

Supplemental Movie S1. The growth of a wild-type tomato pollen tube.

Supplemental Movie S2. The growth of a pollen tube of antisense LePRK2 line 2; note the vacuole moving towards the tip.

Supplemental Movie S3. The growth of the pollen tube shown in movie 2, but taken 2 min later; note the vacuole moving away from the tip.

	ACKNOWLEDGMENTS

 
We thank undergraduates Xin Huang, Wei-Jie Huang, Hai-Kuan Liu, Vivian Ng, Yuan-Sheng Yu, and Lin Zhang for assistance. We thank the Fungal Genomics Stock Center for the pMT-mRFP1 plasmid.

Received June 11, 2008; accepted September 10, 2008; published September 17, 2008.

	FOOTNOTES

 
¹ This work was supported by the U.S. Department of Agriculture Current Research Information System (grant no. 5335–21000–030–00D to S.M.). W.-H.T. was supported by the Ministry of Science and Technology of China (grant nos. 2007CB108700, 2007AA10Z187, and 2006AA10A102), the Knowledge Innovation Program of the Chinese Academy of Sciences (grant no. KSCX2–YW–N–058), and the National Natural Science Foundation of China (grant no. 30770196).

² These authors contributed equally to the article.

³ Present address: Department of Biological Sciences, Wichita State University, Wichita, KS 67260.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Wei-Hua Tang (whtang{at}sibs.ac.cn).

^[W] The online version of this article contains Web-only data.

^[OA] Open Access articles can be viewed online without a subscription.

www.plantphysiol.org/cgi/doi/10.1104/pp.108.124420

^* Corresponding author; e-mail whtang{at}sibs.ac.cn.

	LITERATURE CITED

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Bassham DC, Raikhel NV (2000) Unique features of the plant vacuolar sorting machinery. Curr Opin Cell Biol 12: 491–495[CrossRef][Web of Science][Medline]

Benkert R, Obermeyer G, Bentrup FW (1997) The turgor pressure of growing lily pollen tubes. Protoplasma 198: 1–8[CrossRef][Web of Science]

Berken A, Thomas C, Wittinghofer A (2005) A new family of RhoGEFs activates the Rop molecular switch in plants. Nature 436: 1176–1180[CrossRef][Web of Science][Medline]

Campbell RE, Tour O, Palmer AE, Steinbach PA, Baird BS, Zacharias DA, Tsien RY (2002) A monomeric red fluorescent protein. Proc Natl Acad Sci USA 99: 7877–7882[Abstract/Free Full Text]

Cárdenas L, Lovy-Wheeler A, Kunkel JG, Hepler PK (2008) Pollen tube growth oscillations and intracellular calcium levels are reversibly modulated by actin polymerization. Plant Physiol 146: 1611–1621[Abstract/Free Full Text]

Cárdenas L, McKenna ST, Kunkel JG, Hepler PK (2006) NAD(P)H oscillates in pollen tubes and is correlated with tip growth. Plant Physiol 142: 1460–1468[Abstract/Free Full Text]

Foreman J, Demidchik V, Bothwell JHF, Mylona P, Miedema H, Torres MA, Linstead P, Costa S, Brownlee C, Jones JDG, et al (2003) Reactive oxygen species produced by NADPH oxidase regulate plant cell growth. Nature 422: 442–446[CrossRef][Web of Science][Medline]

Guyon V, Tang W, Monti M, Raiola A, DeLorenzo G, McCormick S, Taylor L (2004) Antisense phenotypes reveal a role for SHY, a pollen-specific leucine-rich repeat protein, in pollen tube growth. Plant J 39: 643–654[CrossRef][Web of Science][Medline]

Hicks G, Rojo E, Hong S, Carter D, Raikhel N (2004) Geminating pollen has tubular vacuoles, displays highly dynamic vacuole biogenesis, and requires VACUOLELESS1 for proper function. Plant Physiol 134: 1227–1239[Abstract/Free Full Text]

Hoekema A, Hirsch PR, Hooykaas P, Schilperoort RA (1983) A binary plant vector strategy based on separation of vir and T-region of Agrobacterium tumefaciens Ti plasmid. Nature 303: 179–180[CrossRef][Web of Science]

Johnson-Brousseau S, McCormick S (2004) A compendium of methods useful for characterizing Arabidopsis pollen mutants and gametophytically-expressed genes. Plant J 39: 761–775[CrossRef][Web of Science][Medline]

Johri BM, Vasil IK (1961) Physiology of pollen. Bot Rev 27: 325–381[CrossRef]

Kaothien P, Ok SH, Shuai B, Wengier D, Cotter R, Kelley D, Kiriakopolos S, Muschietti J, McCormick S (2005) Kinase partner protein interacts with the LePRK1 and LePRK2 receptor kinases and plays a role in polarized pollen tube growth. Plant J 42: 492–503[CrossRef][Web of Science][Medline]

Keller T, Damude H, Werner D, Doerner P, Dixon R, Lamb C (1998) A plant homolog of the neutrophil NADPH oxidase gp91^phox subunit gene encodes a plasma membrane protein with Ca²⁺ binding motifs. Plant Cell 10: 255–266[Abstract/Free Full Text]

Kim HY, Cotter R, Johnson S, Senda M, Dodds P, Kulikauskas R, Tang W, Ezcurra I, Herzmark P, McCormick S (2002) New pollen-specific receptor kinases identified in tomato, maize and Arabidopsis: the tomato kinases show overlapping but distinct localization patterns on pollen tubes. Plant Mol Biol 50: 1–16[CrossRef][Web of Science][Medline]

Krichevsky A, Kozlovsky SV, Tian GW, Chen MH, Zaltsman A, Citovsky V (2007) How pollen tubes grow. Dev Biol 303: 405–420[CrossRef][Web of Science][Medline]

Lee YJ, Szumlanski A, Yang Z (2008) Rho-GTPase-dependent filamentous actin dynamics coordinate vesicle targeting and exocytosis during tip growth. J Cell Biol 181: 1155–1168[Abstract/Free Full Text]

Lew R (2004) Osmotic effects on the electrical properties of Arabidopsis root hair vacuoles in situ. Plant Physiol 134: 352–360[Abstract/Free Full Text]

Li H, Lin Y, Heath RM, Zhu MX, Yang Z (1999) Control of pollen tube tip growth by a Rop GTPase-dependent pathway that leads to tip-localized calcium influx. Plant Cell 11: 1731–1742[Abstract/Free Full Text]

Lovy-Wheeler A, Cárdenas L, Kunkel JG, Hepler PK (2007) Differential organelle movement on the actin cytoskeleton in lily pollen tubes. Cell Motil Cytoskeleton 64: 217–232[CrossRef][Web of Science][Medline]

MacRobbie E (2006) Osmotic effects on vacuolar ion release in guard cells. Proc Natl Acad Sci USA 103: 1135–1140[Abstract/Free Full Text]

McCormick S (1991) Transformation of tomato with Agrobacterium tumefaciens. In K Lindsey, ed, Plant Tissue Culture Manual, Fundamentals and Applications, Vol B. Kluwer, Amsterdam, pp 1–9

Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7: 405–410[CrossRef][Web of Science][Medline]

Mori IC, Schroeder JI (2004) Reactive oxygen species activation of plant Ca²⁺ channels: a signaling mechanism in polar growth, hormone transduction, stress signaling, and hypothetically mechanotransduction. Plant Physiol 135: 702–708[Free Full Text]

Muschietti JP, Dircks L, Vancanneyt G, McCormick S (1994) LAT52 protein is essential for tomato pollen development: pollen expressing antisense LAT52 RNA hydrates and germinates abnormally and cannot achieve fertilization. Plant J 6: 321–338[CrossRef][Web of Science][Medline]

Muschietti JP, Eyal Y, McCormick S (1998) Pollen tube localization implies a role in pollen-pistil interactions for the tomato receptor-like protein kinases LePRK1 and LePRK2. Plant Cell 10: 319–330[Abstract/Free Full Text]

Nishikawa S, Zinkl GM, Swanson RJ, Maruyama D, Preuss D (2005) Callose (beta-1,3 glucan) is essential for Arabidopsis pollen wall patterning, but not tube growth. BMC Plant Biol 5: 22[Medline]

Potock M, Jones MA, Bezvoda R, Smirnoff N, Zársk V (2007) Reactive oxygen species produced by NADPH oxidase are involved in pollen tube growth. New Phytol 174: 742–751[CrossRef][Web of Science][Medline]

Rossetti S, Bonatti PM (2001) In situ histochemical monitoring of ozone- and TMV-induced reactive oxygen species in tobacco leaves. Plant Physiol Biochem 39: 433–442[CrossRef][Web of Science]

Steer M, Steer J (1989) Pollen tube tip growth. New Phytol 111: 323–358[CrossRef][Web of Science]

Takeda S, Gapper C, Kaya H, Bell E, Kuchitsu K, Dolan L (2008) Local positive feedback regulation determines cell shape in root hair cells. Science 319: 1241–1244[Abstract/Free Full Text]

Tang W, Coughlan S, Crane E, Beatty M, Duvick J (2006) The application of laser microdissection to in planta gene expression profiling of the maize anthracnose stalk rot fungus Colletotrichum graminicola. Mol Plant Microbe Interact 19: 1240–1250[CrossRef][Web of Science][Medline]

Tang W, Ezcurra I, Muschietti J, McCormick S (2002) A cysteine-rich extracellular protein, LAT52, interacts with the extracellular domain of the pollen receptor kinase LePRK2. Plant Cell 14: 2277–2287[Abstract/Free Full Text]

Tang W, Kelley D, Ezcurra I, Cotter R, McCormick S (2004) LeSTIG1, an extracellular binding partner for the pollen receptor kinases LePRK1 and LePRK2, promotes pollen tube growth in vitro. Plant J 39: 343–353[CrossRef][Web of Science][Medline]

Taylor LP, Hepler P (1997) Pollen germination and tube growth. Annu Rev Plant Physiol Plant Mol Biol 48: 461–491[CrossRef][Web of Science]

Taylor WR, Jones DT, Segal AW (1993) A structural model for the nucleotide binding domains of the flavocytochrome b-₂₄₅ β-chain. Protein Sci 2: 1675–1685[Web of Science][Medline]

Toews MW, Warmbold J, Konzack S, Rischitor P, Veith D, Vienken K, Vinuesa C, Wei H, Fischer R (2004) Establishment of mRFP1 as a fluorescent marker in Aspergillus nidulans and construction of expression vectors for high-throughput protein tagging using recombination in vitro (GATEWAY). Curr Genet 45: 383–389[CrossRef][Web of Science][Medline]

Twell D, Klein TM, Fromm ME, McCormick S (1989a) Transient expression of chimeric genes delivered into pollen by microprojectile bombardment. Plant Physiol 91: 1270–1274[Abstract/Free Full Text]

Twell D, Wing R, Yamaguchi J, McCormick S (1989b) Isolation and expression of an anther-specific gene from tomato. Mol Gen Genet 217: 240–245[CrossRef][Web of Science][Medline]

Twell D, Yamaguchi J, Wing RA, Ushiba J, McCormick S (1991) Promoter analysis of genes that are coordinately expressed during pollen development reveals pollen-specific enhancer sequences and shared regulatory elements. Genes Dev 5: 496–507[Abstract/Free Full Text]

Wengier D, Valsecchi I, Cabanas ML, Tang W, McCormick S, Muschietti J (2003) The receptor kinases LePRK1 and LePRK2 associate in pollen and when expressed in yeast, but dissociate in the presence of style extract. Proc Natl Acad Sci USA 100: 6860–6865[Abstract/Free Full Text]

Williams L, Fletcher JC (2005) Stem cell regulation in the Arabidopsis shoot apical meristem. Curr Opin Plant Biol 8: 582–586[CrossRef][Web of Science][Medline]

Wong HL, Pinontoan R, Hayashi K, Tabata R, Yaeno T, Hasegawa K, Kojima C, Yoshioka H, Iba K, Kawasaki T, et al (2007) Regulation of rice NADPH oxidase by binding of Rac GTPase to its N-terminal extension. Plant Cell 19: 4022–4034[Abstract/Free Full Text]

Zhang Y, McCormick S (2007) A distinct mechanism regulating a pollen-specific guanine nucleotide exchange factor for the small GTPase Rop in Arabidopsis thaliana. Proc Natl Acad Sci USA 104: 18830–18835[Abstract/Free Full Text]

Zonia L, Munnik T (2008) Vesicle trafficking dynamics and visualization of zones of exocytosis and endocytosis in tobacco pollen tubes. J Exp Bot 59: 861–873[Abstract/Free Full Text]

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