Terena L. Holdaway-Clarke

Pollen Tube Growth and the Intracellular Cytosolic Calcium Gradient Oscillate in Phase while Extracellular Calcium Influx Is Delayed.

Ratio images of cytosolic Ca2+ (Ca2+i) in growing, fura-2-dextran-loaded Lilium longiflorum pollen tubes taken at 3- to 5-sec intervals showed that the tip-focused [Ca2+]i gradient oscillates with the same period as growth. Similarly, measurement of the extracellular inward current, using a noninvasive ion-selective vibrating probe, indicated that the tip-directed extracellular Ca2+ (Ca2+o) current also oscillates with the same period as growth. Cross-correlation analysis revealed that whereas the [Ca2+]i gradient oscillates in phase with growth, the influx of Ca2+o lags by ~11 sec. Ion influx thus appears to follow growth, with the effect that the rate of growth at a given point determines the magnitude of the ion influx ~11 sec later. To explain the phase delay in the extracellular inward current, there must be a storage of Ca2+ for which we consider two possibilities: either the inward current represents the refilling of intracellular stores (capacitative calcium entry), or it represents the binding of the ion within the cell wall domain.

Cellular oscillations and the regulation of growth: the pollen tube paradigm

The occurrence of oscillatory behaviours in living cells can be viewed as a visible consequence of stable, regulatory homeostatic cycles. Therefore, they may be used as experimental windows on the underlying physiological mechanisms. Recent studies show that growing pollen tubes are an excellent biological model for these purposes. They unite experimental simplicity with clear oscillatory patterns of both structural and temporal features, most being measurable during real‐time in live cells. There is evidence that these cellular oscillators involve an integrated input of plasma membrane ion fluxes, and a cytosolic choreography of protons, calcium and, most likely, potassium and chloride. In turn, these can create positive feedback regulation loops that are able to generate and self‐sustain a number of spatial and temporal patterns. Other features, including cell wall assembly and rheology, turgor, and the cytoskeleton, play important roles and are targets or modulators of ion dynamics. Many of these features have similarities with other cell types, notably with apical‐growing cells. Pollen tubes may thus serve as a powerful model for exploring the basis of cell growth and morphogenesis. BioEssays 23:86–94, 2001.

Calcium signalling in pollen of Papaver rhoeas undergoing the self-incompatibility (SI) response

Abstract Self-incompatibility (SI) is a genetically controlled system used by many flowering plants to prevent self-pollination. We established, using calcium imaging, that the SI response in Papaver rhoeas L. (poppy) pollen involves a Ca2+-mediated intracellular signalling pathway. Here we review what is known about the signalling components and cascades implicated in the SI response in poppy pollen. We present some studies using calcium green (CG-1) that show SI-induced alterations in CG-1 fluorescence and localization. We have begun to examine potential sources of Ca2+ involved in the responses induced by SI. This work presents preliminary data showing that influx of extracellular Ca2+ at the ”shank” of the pollen tube is possible. This is the first evidence suggesting that influx at this localization may play a role in the SI response. We also describe preliminary studies that begin to investigate whether the phosphoinositide signalling pathway is implicated in the SI response.

Arabinogalactan-Proteins in Pollen Tube Growth

Arabinogalactan-proteins (AGPs) were associated with reproductive tissues in the early days of their discovery (Nothnagel 1997). In particular, the stigmas and styles of flowering plants were abundant sources of AGPs, as revealed by Yariv phenylglycoside staining [(β-D-glucosyl) Yariv phenylglycoside [(β-D-G1C)3]], a red-colored reagent thought to bind AGPs (Yariv et al 1962). Several species were examined in detail using the (β-D-Glc)3 to precipitate the stigma and stylar AGPs (Clarke et al 1979, Gleeson and Clarke 1979 1980, Fincher et al 1983, Miki-Hirosige et al 1987). Only recently has DNA sequencing provided us with knowledge of the protein component of some of these large proteoglycans, and the first AGPs sequenced were from stylar transmitting tract tissues (Du et al 1994, Mau et al 1995). The carbohydrate branches of AGPs consist mainly of arabinose and galactose, and the protein core is typically rich in Hyp/Pro, Ala and Ser, but the entire molecular structure of any AGP is unknown (Nothnagel 1997). The exciting recent discovery that some plasma membrane AGPs have glycosylphosphatidylinositol anchors has suggested the possibility of their involvement in signaling cascades at the cell surface (Youl et al 1998, Schultz et al 1998, Svetek et al 1999). Polyclonal and monoclonal antibodies to AGPs have been produced with polysaccharide epitopes of mostly unknown composition (Pennell et al 1991, Knox 1992). There is good circumstantial evidence that they bind AGPs. These studies have confirmed that AGPs are abundant in stigmas, styles, and pollen tubes of many species. The antibodies have revealed the diversity of expression patterns for AGPs in development and have ignited interest in their roles in plant biology.