The Search for Pollen Tube Receptors

How do pollen tubes recognize chemotropic guidance cues and transduce them into intracellular responses that result in changes in the direction of the tip? One hypothesis is that tubes express transmembrane receptors that specifically interact with guidance cues like those described above. Interaction between receptor and ligand is expected to initiate a signal transduc-tion cascade within the tube that results in tip re-orientation and guidance (Malho, this volume). It has been shown that localised [Ca2+]c changes in the pollen tube apex control tube directioning (Malho and Trewavas 1996). These changes may arise through asymmetric activity of putative Ca2+-channels (Malho et al. 1995; Sze et al., this volume). Preliminary evidence suggested the existence of low-voltage stretch-activated channels (Geitmann and Cresti 1998; Malho et al. 1995) that could play a key role in the perception of guidance cues because of the multiple signalling pathways to which they respond (Ding and Pickard 1993).

Tremendous progress has been made in identifying intracellular factors that are crucial for tip growth (Malho, this volume). For example, ROP proteins seem to act as a molecular switch that determines the site of cellular extension (Gu et al. 2005; Hwang and Yang, this volume). ROP modulates F-actin assembly and interferes with the dynamics of the tip-focused Ca2+ gradient, both of which are essential for tip growth and are thought to direct the flow of vesicles that ultimately results in polar tip extension (Malho, this volume; Hepler et al., this volume; Yokota and Shimmen, this volume). Thus, changing the direction of tube growth may simply be a matter of changing the position/activity of certain proteins on the pollen tube plasma membrane. This has been demonstrated for several components like protein kinases, calmodulin, phosphoinositides and ROPs (Malho et al. 2000; Malho, this volume; Hwang and Yang, this volume; Zarsky et al., this volume). Further characterization of these receptors for guidance cues and their modes of signal transduction within the pollen tube represent a very exciting challenge.

Genetic analysis of pollen tube-expressed genes required for guidance is an attractive experimental approach because it is not biased toward a pre conceived notion about how the tube perceives guidance cues and a thorough genetic analysis has the potential to identify genes that mediate multiple steps in signal transduction pathways. A loss of function mutation in a pollen tube-expressed guidance cue receptor putatively results in defective tube guidance that phenocopies loss of guidance cue production. For example, a tube that lacks the receptor for a micropylar guidance cue (perhaps EA1) would be expected to fail to enter the micropyle when grown in a wild type pistil the way wild type pollen tubes do when they grow toward an ea1- FG. Ideally, multiple mutants would be collected with defects in each of the specific phases of tube guidance described above and by identifying the disrupted genes in each group of mutants, hypotheses could be made about how the tube perceives cues that direct each phase of guidance. However, it is conceivable that such receptors act simultaneously as structural components of the tip growth machinery, and thus a mutation in their genes could result in the failure for pollen tubes to develop.

A large number of pollen tube mutants have now been characterized through a combination of reverse and forward genetic approaches (Twell et al., this volume; Guermonprez et al., this volume). Many of these affect tube germination and early stages of growth resulting in short tubes that fail to enter the ovary (Johnson et al. 2004; Lalanne et al. 2004; Guermonprez et al., this volume). These mutants have been useful for defining new mechanisms responsible for tip growth revealing a diverse set of biochemical processes that underlie tube extension. Several pollen mutants have also been characterized that do not affect germination, but disrupt tube growth; often these mutants have reduced growth rates that result in an inability to compete with wild-type tubes for access to ovules (Goubet et al. 2003; Johnson et al. 2004; Lalanne et al. 2004; Mouline et al. 2002; Schiott et al. 2004). Again, this group of mutants identified a diverse set of genes required for tube growth that include ion channels (Mouline et al. 2002; Schiott et al. 2004) and cell wall biosynthetic enzymes (Goubet et al. 2003). Despite growth defects, these mutant pollen tubes can target the ovules and are therefore able to respond to guidance cues along their growth path; thereby genetically separating the growth and guidance processes.

Pollen tube guidance mutants that have wild-type germination and growth rates, yet fail to target ovules, are rare. The hapless (hap) mutants are tagged with a pollen-specific marker gene that facilitates analysis of mutant pollen tube growth phenotypes within the ovary (Johnson et al. 2004). Out of 30 pollen mutants that were analyzed, 10 appeared to extend tubes that could grow the length of the pistil, yet these mutants showed reduced ability to target ovules. hap1, hap18, and hap22 tubes failed to exit the transmitting tract; hap11, hap26, and hap30 had relatively normal tube growth paths but were less likely than wild-type to enter the micropyle; and hap2, hap4, hap24, and hap27 showed a chaotic growth path in the ovary and were often found growing on ovule surfaces where wild-type tubes do not grow (Johnson et al.

2004). So far, only a few of the genes disrupted in hap mutants have been identified and none of them appear to be similar to known receptors. Future work will be aimed at identifying more hap genes so that gene functions can be associated with specific defects in individual guidance steps.

Interestingly, pollen tube guidance mutants have yet not been found that completely block the ability of tubes to target ovules. This may suggest that there are redundant signaling mechanisms responsible for each phase of tube guidance and that no single gene mutation will completely disrupt guidance. Support for this idea comes from analysis of ea1 mutants of maize and myb98 and pop2 mutants of Arabidopsis (Kasahara et al. 2005; Marton et al. 2005; Palanivelu et al. 2003; Wilhelmi and Preuss 1996). These mutants are proposed to disrupt production of chemotropic guidance cues, and all of them disrupt guidance: however, none of them completely block tube guidance.

A directed approach to identifying receptors for guidance cues is to study genes expressed by the tube that are homologous to known receptors. Mi-croarray analysis of the 612-member receptor-like kinase (RLK) gene family in Arabidopsis showed that 10% are pollen expressed and that ~ 90% of these are pollen specific (Honys and Twell 2003). This represents just one of several known receptor types expressed by pollen and illustrates the staggering potential diversity of signal transduction mechanisms in this cell. Functional characterization of members of the RLK gene family in tomato is well underway. LePRK1 and LePRK2 are pollen-specific RLKs that are localized to the surface of growing tubes (Muschietti et al. 1998). They both encode active kinases (Muschietti et al. 1998) and interact with each other at the pollen tube membrane; interestingly, this interaction and the phosphorylation of LePRK2 can be disrupted by incubation with a style extract in vitro (Muschietti et al. 1998; Wengier et al. 2003). This suggests that LePRKs may be involved in perception of extracellular growth regulators expressed by cells along the tube growth path.

Proteins that interact with the extracellular domain of LePRKs and are therefore putative ligands have been identified by yeast two hybrid screening of pollen [LAT52 (Tang et al. 2002)] and stigma cDNA libraries [LeSTIG1 and LeSHY (Tang et al. 2004)]. Loss of LAT52 function leads to pollen tube growth defects and LeSTIG1 has been shown to stimulate tube growth in vitro, suggesting that these two proteins may regulate growth via interactions with LePRKs. One interesting hypothesis is that the LePRK1/2 dimer switches ligands from LAT52 to LeSTIG1 upon contact with the stigma (Tang et al. 2004) and that this switch is critical for initiation of tube growth. One possibility to be tested is that LeSTIG1 is the active component of the stigma extract that causes LePRK1 and LePRK2 to dissociate and LePRK2 to become dephosphorylated.

To determine how signaling events at the pollen tube surface mediated by LePRKs are transduced into changes in tube growth, proteins that interact with intracellular domains of LePRKs were identified (Kaothien et al. 2005).

KPP is pollen-specific, phosphorylated in pollen, associated with pollen tube membranes, and its overexpression leads to defects in tip-localized actin dynamics that result in loss of apical polarity (Kaothien et al. 2005). All of these features implicate KPP in signal transduction events that regulate tube growth. Interestingly, KPP interacts with the cytoplasmic domains for LePRK1 and LePRK2 in vitro and this interaction does not require the kinase domains of either LePRK1or LePRK2. It is not yet known whether LePRK1 and/or LePRK2 mediate phosphorylation of KPP in pollen. In addition, it will be interesting to determine whether incubation with stigma extracts or LeSTIG1 alters the LePRK:KPP interaction or the phosphorylation status of KPP.

Analysis of LePRKs shows that taking a candidate-gene approach to the identification of receptors that sit atop pollen signal transduction pathways can be very productive. LePRKs have candidate ligands expressed by the pollen and stigma suggesting an autocrine/paracrine system for regulation of tip growth. Given the large number of candidate receptor genes expressed by pollen, more directed approaches will have to be taken to identify the specific receptor molecules that perceive guidance cues.

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