INTRODUCTION

TOP INTRODUCTION Calcium Channels Needed for... Phosphoinositide Turnover and... Cytoplasmic Ca2+ Controls the... Calcium Oscillations G-Proteins Regulate Pollen Tube... Gametophytic Self-... A Stylar Glycoprotein Aids... Lipids Regulate Pollen... LITERATURE CITED

A novel reproductive feature of the seed plants that enabled them to colonize drier regions of the terrestrial environment was the pollen tube, a structure that serves to deliver sperm to the egg and central cell of the megagametophyte. Pollen, however, is not deposited directly on the megagametophytethat would be too easy. Each pollen grain must prove its mettle to the female, first by landing successfully on the stigmatic surface, then by germinating, and finally by elongating across the entire length of the long style. Three of the more interesting physiological questions surrounding this process include: how do pollen tubes achieve their phenomenal rates of growth, how do pollen tubes sense the location of the megagametophyte, and how do self-incompatibility reactions effectively remove some of the pollen grains from the race to the egg? This week's The Hot and the Classic summarizes 11 of the more highly cited pollen tube papers of the 1990s that bear upon these questions.

	Calcium Channels Needed for Pollen Tube Elongation

TOP INTRODUCTION Calcium Channels Needed for... Phosphoinositide Turnover and... Cytoplasmic Ca2+ Controls the... Calcium Oscillations G-Proteins Regulate Pollen Tube... Gametophytic Self-... A Stylar Glycoprotein Aids... Lipids Regulate Pollen... LITERATURE CITED

Pierson et al. (1994) provide a detailed spatial analysis of the steep Ca²⁺ gradient that exists in the tips of pollen tubes. They confirm that the high [Ca²⁺]_cyt at the tip arises from a highly localized influx of Ca²⁺ ions. Injection of intracellular Ca²⁺ buffers or application of elevated levels of Suc reversibly inhibits growth, destroys tip zonation of organelles, and modifies normal patterns of cytoplasmic streaming. These treatments also dissipate both the intracellular tip-focused gradient and the extracellular Ca²⁺ flux. These findings provide evidence that growing pollen tubes have open Ca²⁺ channels in their tip and that these channels become inactivated in non-growing tubes.

	Phosphoinositide Turnover and Pollen Tube Growth

TOP INTRODUCTION Calcium Channels Needed for... Phosphoinositide Turnover and... Cytoplasmic Ca2+ Controls the... Calcium Oscillations G-Proteins Regulate Pollen Tube... Gametophytic Self-... A Stylar Glycoprotein Aids... Lipids Regulate Pollen... LITERATURE CITED

Franklin-Tong et al. (1996) demonstrate that the pollen tubes of Papaver rhoeas have a Ca²⁺-dependent polyphosphoinositide-specific phospholipase C (PLC) activity that is inhibited by neomycin. The photolysis of caged inositol (1,4,5)-trisphosphate (IP₃) in pollen tubes reveals that IP₃ induces an intracellular release of Ca²⁺ ions, which is inhibited by heparin and neomycin. Mastoparan, which stimulates IP₃ production, also induces a rapid neomycin-sensitive increase in [Ca²⁺]_cyt. These data provide direct evidence for the involvement of a functional phosphoinositide signal-transducing system in pollen tubes.

	Cytoplasmic Ca2+ Controls the Axis of Pollen Tube Growth

TOP INTRODUCTION Calcium Channels Needed for... Phosphoinositide Turnover and... Cytoplasmic Ca2+ Controls the... Calcium Oscillations G-Proteins Regulate Pollen Tube... Gametophytic Self-... A Stylar Glycoprotein Aids... Lipids Regulate Pollen... LITERATURE CITED

Malho and Trewavas (1996) report that increasing [Ca²⁺]_cyt on one side of the pollen tube apex induces reorientation of the growth axis toward that side. Similarly, a decrease in [Ca²⁺]_cyt promoted bending toward the opposite side. These effects are mimicked by imposing localized external gradients of an ionophore (A23187) or a Ca²⁺ channel blocker (GdCl₃); the pollen tubes bend toward the highest concentration of A23187 and away from GdCl₃. Manipulation of [Ca²⁺]_cyt in regions farther back from the apical zone also induces changes in growth direction, but the new orientation is random.

	Calcium Oscillations

TOP INTRODUCTION Calcium Channels Needed for... Phosphoinositide Turnover and... Cytoplasmic Ca2+ Controls the... Calcium Oscillations G-Proteins Regulate Pollen Tube... Gametophytic Self-... A Stylar Glycoprotein Aids... Lipids Regulate Pollen... LITERATURE CITED

Pierson et al. (1996) report that a steep tip-focused gradient occurs in the elongating pollen tubes of all species examined. Analysis of Lilium longiflorum pollen tubes loaded with dextran-conjugated fura-2 reveals that the gradient derives from Ca²⁺ entry that is restricted to a small area of plasma membrane at the extreme apex of the tube dome. The authors propose that since the apical membrane is continually swept to the shanks during tube elongation, either Ca²⁺ channels are specifically retained at the extreme apex or the Ca²⁺ channels at the tip rapidly inactivate as new ones are inserted during vesicle fusion. The peak of the [Ca²⁺]_cyt gradient fluctuates in magnitude from 0.75 to above 3 µM, with the elevated points being correlated with an increased rate of tube growth (see also Franklin-Tong, 1996; Mahlo and Trewavas, 1996; Holdaway-Clarke et al., 1997). Inhibition of pollen tube growth caused by various treatments is correlated with the dissipation of both the tip-focused gradient and the Ca²⁺ influx.

	G-Proteins Regulate Pollen Tube Elongation

TOP INTRODUCTION Calcium Channels Needed for... Phosphoinositide Turnover and... Cytoplasmic Ca2+ Controls the... Calcium Oscillations G-Proteins Regulate Pollen Tube... Gametophytic Self-... A Stylar Glycoprotein Aids... Lipids Regulate Pollen... LITERATURE CITED

Pollen tube elongation depends on actin-dependent targeted secretion at the tip. Because small GTPases of the Rho family, which are homologs of Rac and Cdc42, have been implicated in the regulation of related processes in animal and yeast cells, two recent studies examined the possible role of Rho-type G-proteins in pollen tube elongation (Kost et al., 1999; Li et al., 1999).

Kost et al. (1999) demonstrated that the expression of a non-functional mutant Rho-type G-protein in Arabidopsis inhibited pollen tube elongation, whereas expression of a constitutively active Rho-type G-protein caused depolarized growth (see also Li et al., 1999). Rho-type G-protein was found to accumulate at the pollen tube tip plasma membrane and to be physically associated with phosphatidylinositol monophosphate kinase (PI P-K) and its product, phosphatidylinositol 4,5 bisphosphate (PtdIns 4,5-P₂). The expression of a PLC domain that binds specifically to PtdIns 4,5-P₂ also inhibited pollen tube elongation. The authors propose that Rho-type G-proteins may control the local activity of PI P-K in the tip of the pollen tube and that the product of PI P-K, PtdIns 4,5-P₂, may serve as a substrate for the production of IP₃ by PLC.

	Gametophytic Self-Incompatability

TOP INTRODUCTION Calcium Channels Needed for... Phosphoinositide Turnover and... Cytoplasmic Ca2+ Controls the... Calcium Oscillations G-Proteins Regulate Pollen Tube... Gametophytic Self-... A Stylar Glycoprotein Aids... Lipids Regulate Pollen... LITERATURE CITED

Gametophytic self-incompatibility provides a genetic barrier to self-fertilization, and in the simplest cases is controlled by the highly polymorphic S locus. Growth of a pollen tube in the style is arrested when the S allele carried by the pollen matches either one of the two S alleles carried by the pistil. Putative S allele proteins of the pistil, which are RNases, had been shown to co-segregate with S alleles, but there had been only correlative or indirect evidence for the claim that these S allele-associated proteins (S proteins) are involved in recognition and rejection of self-pollen. The study of Lee et al. (1994) demonstrated that the inhibition of synthesis of S2 and S3 proteins in Petunia inflata plants of S2S3 genotype by the antisense S3 gene resulted in failure of the transgenic plants to reject S3 and S2 pollen. The expression of the transgene encoding S3 protein in P. inflata plants of S1S2 genotype confers on the transgenic plants the ability to reject S3 pollen (see also Murfett et al., 1994). These findings provide direct in vivo evidence that S proteins control the self-incompatibility behavior of the pistil.

	A Stylar Glycoprotein Aids Pollen Tube Growth

TOP INTRODUCTION Calcium Channels Needed for... Phosphoinositide Turnover and... Cytoplasmic Ca2+ Controls the... Calcium Oscillations G-Proteins Regulate Pollen Tube... Gametophytic Self-... A Stylar Glycoprotein Aids... Lipids Regulate Pollen... LITERATURE CITED

When compared to pollen tubes that grow naturally (i.e. through stylar tissue), pollen tubes grown in vitro extend in random directions, exhibit reduced growth rates, and grow to shorter final lengths. Since pollen tubes elongate through the extracellular matrix of the stigma and the style, it has been proposed that the extracellular matrix of the pistil provides chemical and physical support as well as directional cues for the elongating pollen tube. Cheung et al. (1995) purified a glycoprotein, TTS, from tobacco (Nicotiana alata) stylar transmitting tissue, that supports pollen tube growth between the stigma and the ovary. TTS proteins belong to the arabinogalactan protein family, stimulate pollen tube growth in vitro, and attract pollen tubes grown in a semi-in vivo culture system. In vivo, the pollen tube growth rate is reduced in transgenic plants that have significantly reduced levels of TTS proteins as a result of either anti-sense suppression or sense co-suppression. These results identify the TTS protein as a pistil component that facilitates pollen tube growth.

	Lipids Regulate Pollen Penetration of Papillae

TOP INTRODUCTION Calcium Channels Needed for... Phosphoinositide Turnover and... Cytoplasmic Ca2+ Controls the... Calcium Oscillations G-Proteins Regulate Pollen Tube... Gametophytic Self-... A Stylar Glycoprotein Aids... Lipids Regulate Pollen... LITERATURE CITED

Pollen hydration, germination, and penetration of the stigma by pollen tubes are influenced by the exudate on wet stigmas. Wolters-Arts et al. (1998) tested selected compounds for their ability to act as functional substitutes for exudate in the initial stages of pollen-tube growth on transgenic stigmaless tobacco plants that did not produce exudate. They found that lipids are the essential factor needed for pollen tubes to penetrate the stigma and that, in the presence of these lipids, pollen tubes will also penetrate leaves. They propose that lipids direct pollen-tube growth by controlling the flow of water to pollen in species with wet stigmas.