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Articles Delivery in Flowering : The Control of Tube Growth Downloaded from https://academic.oup.com/bioscience/article/57/10/835/232382 by guest on 02 October 2021 KATHLEEN L. WILSEN AND PETER K. HEPLER

Although most people think of pollen merely as an , its true biological function is to facilitate sexual in flowering plants. The angiosperm pollen grain, upon arriving at a receptive , germinates, producing a tube that extends through the style to deliver its cargo to the , thereby fertilizing the egg, and completing the cycle of the . The grows rapidly, exclusively at its tip, and produces a that is highly polarized both in its outward shape and its internal cytoplasmic organization. Recent studies reveal that the growth oscillates in rate. Many underlying physiological processes, including ionic fluxes and energy levels, also oscillate with the same periodicity as the growth rate, but usually not with the same phase. Current research focuses on these phase relationships in an attempt to decipher their hierarchical sequence and to provide a physiological explanation for the factors that govern pollen tube growth.

Keywords: , dynamics, molecular switches, oscillatory growth, pollen tube

n , produces that fuse to do the sperm of protists, , , and some gymno- Iform a during . The life cycle of . Instead, the pollen grain may travel a great distance, flowering plants, referred to as an alternation of generations, transported by or an carrier (e.g., bird, , is more complex. Here, a multicellular diploid generation, ), before alighting on a receptive stigma and germi- known as the generation, alternates with a multi- nating. The sperm cells then travel in the of the cellular haploid generation, known as the gen- large vegetative cell of the pollen tube to their target. A eration. Meiosis does not directly produce gametes. Rather, pollen tube is thus the tricellular male gametophyte gener- cells of the sporophyte generation undergo meiosis to produce ation that emerges from a pollen grain to bring about haploid , which in turn divide mitotically to give rise . to the -producing gametophyte generation. The em- Pollen tube growth is fast and highly polarized, with new bryo sac, which bears the ovule and is embedded in the material being added at the tip, which is called the apex. , constitutes the female gametophyte generation. The Secretory vesicles inside the pollen tube transport the cellu- pollen grain is the male gametophyte generation produced lar building blocks required for growth to the apex, where they from in the anthers of flowering plants, and are incorporated into the extending pollen tube by exocyto- consists of a vegetative cell and a generative cell. Either in the sis (Hepler et al. 2006). This process is so efficient that a grain or during pollen tube growth, depending on the species, pollen tube from Zea mays (corn) can attain growth rates of the generative cell undergoes to give rise to two sperm up to 1 centimeter (cm) per hour, or approximately 3 micro- cells (Raven et al. 1999). meters (µm) per second, making it one of the fastest-grow- Double fertilization is a hallmark of flowering plants ing cells known. With the added realization that cell elongation (Dresselhaus 2006). In addition to the fusing of one sperm cell can be visualized under carefully controlled in vitro conditions, with an egg cell to give rise to an , the second sperm the pollen tube becomes an excellent model system for stud- cell fuses with the two polar nuclei in the central cell of the ies of growth. Clearly, it is ideal for enhancing our female gametophyte to produce the nutritive triploid understanding of other cells undergoing tip growth, such as of the . The embryo and endosperm are packaged hairs, fungal hyphae, and and moss protonemata, as a , which becomes encased in a formed from the but the pollen tube may also serve as an effective model for and, in some instances, from additional floral parts. It will be apparent, therefore, that double fertilization is not only necessary for sexual reproduction in flowering plants but Kathleen L. Wilsen (e-mail: [email protected]) works in the School also essential for the production of much of the that we of Biological Sciences at the University of Northern Colorado in Greeley. eat, including nuts, , grains, and . Peter K. Hepler (e-mail: [email protected]) works in the Biology and Plant Because the sperm of flowering plants have no flagella, they Biology Graduate Program at the University of Massachusetts in Amherst. do not depend on to transport them to the ovule, as © 2007 American Institute of Biological Sciences. www.biosciencemag.org November 2007 / Vol. 57 No. 10 • BioScience 835 Articles the many plant cells that exhibit diffuse growth. In defense of so-called clear zone (figure 1). Although lacking certain in- this assertion, we note the similarity in structure and clusions, the extreme apex contains numerous secretory vesi- composition and membrane trafficking machinery between cles, as well as elements of the endoplasmic reticulum (ER), pollen tubes and other plant cells. mitochondria, and Golgi dictyosomes (figure 2). These vesi- In this article we describe the structure and of cles are of particular interest because they contain the cell wall a growing pollen tube, focusing on several processes believed precursors, which are delivered to the apex of the pollen to be key in the regulation of growth. We exploit the fact that tube, where their contents are secreted to the wall, allowing pollen tube growth oscillates, as do many underlying physi- the cell perimeter to extend. Although mitochondria, Golgi ological processes and structural elements. Through an analy- dictyosomes, and elements of the ER are abundant in the clear sis of the temporal and spatial ordering of these many factors, zone, they are not confined to this region; they are present we provide insight into the basic regulatory events that con- throughout the pollen-tube shank (Parton et al. 2003, Lovy- Downloaded from https://academic.oup.com/bioscience/article/57/10/835/232382 by guest on 02 October 2021 trol pollen tube growth. Although we include data from Wheeler et al. 2007). pollen tubes of different species (e.g., and Nico- Cytoskeletal elements, including both actin tiana), we focus in particular on those from the lily family (e.g., and , are ubiquitous components of the pollen the Easter lily, Lilium longiflorum), which, because of their tube (Hepler et al. 2001). In lily pollen tubes, microtubules relatively large size and their readiness to germinate in vitro form a prominent collar at the base of the clear zone (Lovy- and grow at in vivo rates, have been the subject of many Wheeler et al. 2005); their function, however, is not clear. The insightful studies. depolymerization of microtubules with oryzalin has no effect on pollen tube growth or organelle positioning in vitro (Lovy- Growing pollen tubes are highly organized Wheeler et al. 2007). Nevertheless, recent studies show that Whereas the diameter of a pollen grain is between 10 and 100 some cytoplasmic components, including mitochondria and µm (lily pollen is about 60 µm), the length of a pollen tube Golgi vesicles, move slowly along microtubules, indicating that is much greater, and can reach several centimeters (approx- these cytoskeletal elements may indeed contribute to the imately 10 cm in lily) as it grows through the tissues of the style control of pollen tube growth (Romagnoli et al. 2007). to deliver the sperm cells to the embryo sac. The growing lily In contrast to microtubules, a dynamic actin cytoskeleton pollen tube is around 15 µm in diameter (figure 1) and has is widely acknowledged to be essential for pollen tube growth an apical dome that is roughly hemispherical in shape. As the (Hepler et al. 2001). Although there is agreement that the pollen tube grows, plugs are laid down in the shank shank of the pollen tube contains longitudinal bundles of actin in such a manner that the cytoplasm remains in the apical end parallel to the axis of growth, the organization of these mi- of the growing tube. Because of this mechanism, pollen tubes crofilaments in the clear zone has been widely debated. have been described as moving cells, in that a fixed plug of Lovy-Wheeler and colleagues (2005) resolved this issue by em- cytoplasm containing floating sperm cells moves forward as ploying an improved method of fixation, followed by label- the cell wall extends (Sanders and Lord 1992). ing with antiactin antibodies, and analysis with confocal Both and electron reveal that organelles microscopy. This study confirmed that a cortical fringe of actin have a particular arrangement inside a pollen tube (figures 1, is a consistent feature of the clear zone, and that the extreme 2; Hepler et al. 2001). Most of the pollen tube is granular in apex contains relatively few actin filaments (figure 3; Lovy- appearance because of a rich supply of -containing Wheeler et al. 2005). , or (figure 1). However, amyloplasts and The actin cytoskeleton serves three important functions in are excluded from the extreme apex, creating the growing pollen tubes. First, actin microfilaments, together with the motor (myosin XI in lily), drive cyto- plasmic streaming. This process is thought to transport the secretory vesicles from their point of origin to the apical end of the pollen tube, where they ultimately empty their contents into the expanding cell wall (Yokota and Shimmen 2006). Like the secretory vesicles, all organelles, including amyloplasts, Figure 1. A living lily pollen tube observed using No- dictyosomes, ER, mitochondria, and vacuoles, are propelled marski differential interference contrast optics. Many by the actomyosin system and are in constant motion. In starch-containing amyloplasts give the shank a granular angiosperm pollen tubes, cytoplasmic streaming exhibits a “re- appearance. However, these amyloplasts are excluded verse fountain” pattern in which the lanes move forward from the apex, giving rise to the clear zone. All of the cyto- along the edge of the cell and then reverse direction in the clear plasmic inclusions undergo continuous cytoplasmic zone, creating a massive central retrograde flow. streaming, in which they flow forward along the edge of A second function of the actin cytoskeleton is controlling the cell and rearward through the central core. This pat- the position of organelles within the pollen tube. Under tern has been called “reverse fountain” streaming, and normal conditions, the polarized distribution of organelles, gives rise to the funnel-shaped appearance, which is evi- including notably the detailed morphology of the clear zone, dent at the base of the clear zone. Bar = 10 micrometers. is maintained despite the fact that the contents are constantly

836 BioScience • November 2007 / Vol. 57 No. 10 www.biosciencemag.org Articles

dome, esterified pectins are enzymatically demethyl- ated, and the cell wall is greatly reinforced when cal- cium cross-links the acidic residues on the pectin chains (Bosch and Hepler 2005). Thus, the pollen tube wall is stronger and more rigid in the shank of the tube than in the apex. This arrangement means that only the apical wall is plastic and able to un- dergo strain deformation in response to internal (Bosch and Hepler 2005). These conditions contribute to the polarity of pollen tube growth. Downloaded from https://academic.oup.com/bioscience/article/57/10/835/232382 by guest on 02 October 2021 and protons are essential for pollen tube growth For more than 40 years it has been recognized that calcium ions are required for pollen and pollen tube growth (reviewed in Hepler et al. Figure 2. An electron micrograph of a lily pollen tube, prepared by rapid 2006). In addition to calcium being an essential freeze fixation and freeze substitution, shows the clear zone. The apex component of the surrounding extracellular envi- contains numerous small vesicles; these fuse with the plasma membrane ronment, an intracellular calcium gradient and ex- and contribute material to the expanding cell wall. Mitochondria and tracellular fluxes associated with the pollen tube elements of the endoplasmic reticulum are also abundant. Note that the are essential for growth. Like most other cells, plant cell wall is thickest at the extreme apex, and that it becomes substantially or animal, the pollen tube experiences a relatively thinner along the flanks of the dome. It is thought that this transition in high external calcium concentration, where it ranges thickness reflects the conversion of the cell wall pectins from methyl esters between 10 micromolar (µM) and 10 millimolar. to acids together with calcium cross-linking. Source: Lancelle and Hepler However, within the cell the free concentration of (1992). Bar = 1 micrometer. this ion is substantially lower, with basal con- centrations from 150 to 300 nanomolar (nM) in motion. However, this polarized distribution of organelles (Holdaway-Clarke and Hepler 2003). The underlying reason is quickly and dramatically disrupted by antimicrofilament for the low concentration of intracellular calcium may derive agents (e.g., latrunculin B; Gibbon et al. 1999, Vidali et al. from the physiological incompatibility of millimolar con- 2001). By some process that is not well understood, the actin centrations of phosphate and calcium, at which point these cytoskeleton appears to create a filter that allows certain or- two ions would react to form an insoluble precipitate of cal- ganelles such as the vesicles, mitochondria, Golgi dictyosomes, cium phosphate, and destroy phosphate-based energy me- and ER to flow into the clear zone while preventing the in- tabolism.Virtually all living cells thus evolved mechanisms for cursion of amyloplasts and vacuoles. reducing the intracellular calcium ion concentration, leading A third function of actin relates to its direct role in the con- to the enormous concentration difference between the cytosol trol of tube elongation. Researchers initially assumed that actin contributed to growth indirectly, through the control of cyto- plasmic streaming and the consequent delivery of secretory vesicles to the apex. However, studies with agents that control actin polymerization (e.g., latrunculin B, cytochalasin D, DNAse, ) indicate that growth can be inhibited at concentrations of these agents that are significantly lower Figure 3. A confocal fluorescence image of actin labeled than those required to block cytoplasmic streaming (Vidali with an antiactin antibody. The cell was first subjected to et al. 2001). These results strongly support the idea that actin rapid freeze fixation and freeze substitution. It was then polymerization contributes directly to pollen tube growth. rehydrated and probed with the antibody. This figure, Although much more work is needed to completely unravel which is a projection of several confocal image slices, the mechanisms of actin’s contribution to , we shows the prominent actin fringe that starts about 5 increasingly realize that actin has a profound effect on cell micrometers (µm) back from the apex and extends elongation. basally for another 7 µm. Three-dimensional analysis The cell wall is another important structural feature of indicates that the fringe is restricted to the cortex of the growing pollen tubes (Geitmann and Steer 2006). The freshly apical dome. Behind the fringe, the actin is evenly dis- secreted pollen tube wall in the apex consists of methyl- tributed throughout the thickness of the pollen tube in esterified pectins, which are displaced to the shank of the finely articulated actin bundles. Source: Lovy-Wheeler pollen tube as the cell grows. On the flanks of the apical and colleagues (2005). Bar = 10 µm. www.biosciencemag.org November 2007 / Vol. 57 No. 10 • BioScience 837 Articles Downloaded from https://academic.oup.com/bioscience/article/57/10/835/232382 by guest on 02 October 2021

Figure 4. A composite image showing calcium and pH distribution in living lily pollen tubes. These are selected image pairs from oscillating pollen tubes that show high (top left) and low (top right) calcium, and high (bottom left) and low (bottom right) pH. indicates high calcium concentration and high pH. For calcium detection, the cell was injected with fura-2-dextran; for pH detection, the cell was injected with BCECF-dextran. The cells were subjected to ratiometric ion imaging. Source: Hepler and colleagues (2006). and the extracellular environment. It seems likely that and position of the calcium gradient, and growth stops when during the process of , this concentration difference the gradient dissipates (Malhó and Trewavas 1996, Pierson et was exploited for signaling purposes. For example, a primary al. 1996). messenger (e.g., , light, gravity) relays its presence The observation that pollen tubes as well as virtually all to the cell through an interaction that causes the opening of other cells have low basal levels of calcium means that there a calcium-selective pore or channel. Calcium flows into the are compensatory systems that respond to the ion, sequestering cell, generating a large local increase in its intracellular con- it or extruding it from the cell. Indeed, within the pollen centration. Calcium becomes the second messenger because tube the region of elevated calcium is restricted to the apical it will now bind to and activate (e.g., ) portion of the clear zone, indicating that the removal system that are poised to carry the signal forward to the next step. must be located there. The localized ER and mitochondria are In the pollen tube, there is direct evidence for a localized strong candidates for the sequestering system because they domain of elevated calcium (see below), which we presume occur at high density at the base of the clear zone; preliminary behaves as a second messenger and activates growth- evidence also shows that the ER takes up calcium and trans- dependent events, without compromising phosphate-based ports it basally (Wilsen 2005). Studies directed at the flow of energy metabolism. ER and mitochondria in living pollen tubes reveal that they Several investigators report a region of high intracellular continually move into the clear zone, providing a fresh sup- calcium in the apex of the pollen tube, immediately adjacent ply of uncharged elements that remove calcium ions and re- to the plasma membrane, where growth is maximal (figure strict the basal spread of the tip-focused gradient (Lovy- 4, top row; Holdaway-Clarke and Hepler 2003, Hepler et al. Wheeler et al. 2007). There are also calcium adenosine- 2006). The concentration of calcium in this localized region triphosphatases (ATPases) on the plasma membrane; these are may reach 10 µM (Messerli et al. 2000), and is more than an membrane-bound that use the energy of adenosine order of magnitude greater than that in the shank, where triphosphate (ATP) to move calcium against its concentration basal values from 150 to 300 nM are observed (Holdaway- gradient (Sze et al. 2006). Taken together, these lines of evi- Clarke and Hepler 2003). This gradient is referred to as dence make it clear that the pollen tube has overlapping “tip-focused” because the calcium concentration is greatest components that respond to elevated calcium and restore the in the extreme apex and declines sharply with distance from basal concentration. the apex. Thus calmodulin, a ubiquitous calcium-binding Extracellular calcium, as previously noted, is at much protein that binds to and regulates numerous cellular targets, higher concentrations than are concentrations within the or key enzymes, such as the calcium-dependent protein cell. For this reason, an influx of extracellular calcium requires kinases (CDPKs) commonly found in plants, may be saturated only the opening of selected pores or channels on the plasma with calcium and maximally active in the apex of growing membrane. Early predictions of an inward flow of calcium ions pollen tubes but switched off in the region just behind the apex into the pollen tube apex from the surrounding environ- (Holdaway-Clarke and Hepler 2003). Pollen tube growth ment were confirmed by high-resolution studies using the rate and direction are positively correlated with the slope calcium-specific vibrating probe. This is an electrode that

838 BioScience • November 2007 / Vol. 57 No. 10 www.biosciencemag.org Articles oscillates between two points close to the pollen tube, mea- Signaling are essential suring the local calcium ion concentration. The difference in for pollen tube growth ion concentration between these two points indicates both the Pollen tube growth is also regulated by a complex network of direction and magnitude of flux. Studies on the growing signaling molecules and pathways. Two categories of mole- pollen tube demonstrated that calcium flows in only at its cules and their associated enzymes and cofactors have been extreme apex (Kühtreiber and Jaffe 1990, Pierson et al. 1994), localized to the apex of the pollen tube and appear to play fueling ideas that stretch-activated calcium channels were central roles in regulating growth; these include the phos- responding to the growth-dependent deformation of the phoinositides and the small G-proteins, which are guanosine apical plasma membrane. More recently, these stretch- triphosphate (GTP)-binding proteins, such as ROP (Rho activated calcium channels have been identified in proto- family GTPases of plants). The phosphoinositides are mem- plasts from Lilium longiflorum pollen tube tips and from brane of which phosphatidyl inositol 4,5 bisphosphase Downloaded from https://academic.oup.com/bioscience/article/57/10/835/232382 by guest on 02 October 2021 regions of the pollen grain associated with the site of germi- (PIP2) has received the most attention. It is a multifaceted mol- nation (Dutta and Robinson 2004). ecule that associates with certain actin-binding proteins, in- Although it seems clear that calcium ions flow into the cell cluding actin depolymerizing factor (ADF), profilin, and from the extracellular environment, it must also be recognized villin (Martin 1998), which are common in pollen tubes. In that a substantial amount of this apparent flow may be addition, PIP2 is enzymatically cleaved by phospholipase C associated with binding to cell wall components. The bulk of (PLC) to produce two more signaling agents, diacylglycerol the cell wall material is secreted as methyl-esterified pectins. and inositol 1,4,5 trisphosphate. The former remains in the As previously noted, these are demethylated by the plasma membrane, and can be converted to phosphatidic acid, pectin methyl esterase, exposing acidic groups which then which itself is a regulatory that can affect actin dy- bind calcium in a process that cross-links these pectic residues namics (Žárský et al. 2006). The latter diffuses in the cytoplasm and strengthens the cell wall (Bosch and Hepler 2005). and can release calcium from internal stores (Franklin-Tong

Calculations indicate that more than 90% of the calcium et al. 1996). It is noteworthy, therefore, that PIP2 localizes to flux observed with the ion-selective vibrating electrode is the apical plasma membrane of the pollen tube (figure 5; Kost for wall binding, and thus does not represent ions that cross et al. 1999). the plasma membrane (Holdaway-Clarke and Hepler 2003). Observations support the importance of these phospho- Nevertheless, the small amount that does cross the plasma inositides to pollen tube growth. For example, an increase in membrane creates the apical gradient and is essential for PIP2 at the apex is associated with an increase in pollen tube pollen tube growth. growth (Dowd et al. 2006). Further studies on PLC reveal that Although receiving much less attention than calcium, modulation of its activity also modulates growth; thus in- protons are also essential for pollen germination and pollen hibition of PLC activity markedly blocks pollen tube growth tube growth. In experiments with pollen tubes, the culture (Helling et al. 2006). There is evidence that PLC activity may medium must be acidic, with growth being optimal at pH 5 impinge most directly on the actin cytoskeleton, because to pH 6 (Holdaway-Clarke et al. 2003). However, intracellu- cells compromised with an inactive form of the enzyme can lar pH has proved to be more challenging to gauge than be partially rescued with the actin polymerization inhibitor intracellular calcium because protons, which are considerably latruculin B (Dowd et al. 2006). Both of these recent studies more mobile than calcium ions, are easily perturbed when support the idea that PLC cleaves PIP2 that might migrate away probed by an indicator . Nonetheless, it has been shown, from the apex. PLC activity thus restricts PIP2 to the extreme using low dye concentrations, that growing pollen tubes ex- apex of the tube and in so doing contributes centrally to the hibit an intracellular pH gradient (figure 4, bottom row). The control of pollen tube growth polarity (Dowd et al. 2006, apex of the pollen tube is slightly acidic (pH 6.8), whereas the Helling et al. 2006). rest of the clear zone is characterized by the presence of an al- The small G-proteins are also potential central regulators kaline band, with a pH of 7.5 (Feijó et al. 1999). In addition of pollen tube growth (Hwang and Yang 2006). ROPs con- to the intracellular expression of pH noted above, studies with stitute a subfamily of molecular switches, unique to plants, extracellular ion-selective electrodes reveal a pattern of fluxes that function in developmental and signaling events. In the that correlates with the pattern of intracellular pH. These ob- cell, ROP toggles between an active GTP-bound form and servations indicate that there is an influx of protons at the ex- an inactive guanosine diphosphate–bound form, under the treme apex of the tube, precisely at the intracellular acidic influence of guanine nucleotide exchange factors, GTPase- domain. Moreover, there is an efflux along the sides of the clear activating proteins, and guanine nucleotide dissociation zone, which corresponds to the position of the intracellular inhibitors. It has been shown that certain ROPs localize to the alkaline band (Feijó et al. 1999). This pattern of extracellu- apical region of the plasma membrane of pollen tubes, the site lar proton fluxes provides evidence for the presence of a cur- of growth (figure 6; Kost et al. 1999, Hwang and Yang 2006). rent loop in the apical dome of the pollen tube, which we As with PIP2, the structural studies on ROPs reveal the pres- presume is driven by a plasma membrane–associated proton- ence of regulating factors that inactivate these agents if they ATPase. The regulation of pH emerges as a key factor in the migrate from the extreme apex to the flank of the apical control of pollen tube growth. dome (Gu et al. 2006, Klahre and Kost 2006, Klahre et al. 2006). www.biosciencemag.org November 2007 / Vol. 57 No. 10 • BioScience 839 Articles

reach a length of about 700 µm, they begin to exhibit regu- lar oscillations, and at this point their overall growth speeds up (Pierson et al. 1996). Oscillatory growth thus is more rapid than nonoscillatory growth. Because only one pollen tube will fertilize each egg cell and thereby perpetuate its DNA, speed of growth is a trait that enjoys a selective ad- Figure 5. Phosphatidyl inositol 4,5, bisphosphate (PIP2) vantage (Snow and Spira 1991). localizes to the apical plasma membrane. PIP2 has been identified through its binding to the pleckstrin homology As the rate of growth oscillates, so do several underlying domain of phospholipase C, which is fused to fluo- physiological processes, typically with the same periodicity as rescent protein. This medial confocal image clearly shows growth, but usually not with the same phase. To identify how key processes are connected to pollen tube growth, investi- that PIP2 resides in the apical plasma membrane. Source: Downloaded from https://academic.oup.com/bioscience/article/57/10/835/232382 by guest on 02 October 2021 Kost and colleagues (1999). Bar = 10 micrometers. gators determine the phase relationships between oscillating physiological events and pollen tube growth. To do this, we These conditions ensure that the active species will be have used cross-correlation analysis, which is a mathemati- confined to the extreme apex of the tube, where they may cal procedure that provides a detailed comparison of the re- participate in the control of pollen tube growth polarity. It lated oscillatory data and makes it possible to infer whether seems important that both ROPs and PIP2 localize to the a physiological process precedes or follows growth (Hepler et same extreme apical domain and may act together in a al. 2006). common pathway (Kost et al. 1999). Because oscillating processes that lag growth are unlikely to initiate it, the emphasis has been on identifying oscillating Pollen tube growth oscillates processes that peak before growth does. However, the signif- An exciting finding that has emerged in the last 15 years is that icance of events that follow growth should not be overlooked, pollen tubes do not extend uniformly but exhibit pronounced because they may be involved in terminating growth. Thus, oscillations in their growth rate. For example, in lily pollen the events preceding growth may be viewed as the accelera- tubes the growth rate oscillates between 100 and 500 nano- tor and those following growth as the brakes: as with a speed- meters per second, with a periodicity of about 20 to 50 ing car, to quickly negotiate a tortuous path, brakes together seconds (Holdaway-Clarke and Hepler 2003). Inevitably the with an accelerator are essential components of the regula- question arises as to why pollen tube growth oscillates. tory machinery. Ultimately, we would like to determine what Although the answer cannot be stated with certainty, there are sets the oscillations in motion, and how these oscillations some important considerations that help us understand the translate into growth of the pollen tube. matter. First, many vital processes exhibit oscillatory behavior Because oscillatory growth is known almost entirely from (Goldbeter 1996, Feijó et al. 2001). It has been known for many studies of pollen tubes grown in vitro, questions are fre- years that cultures of yeast cells generate oscillations in reduced quently raised about whether this process occurs in vivo.The nicotinamide adenine dinucleotide phosphate (NAD[P]H), answer is yes; Iwano and colleagues (2004) have demon- a coenzyme essential in numerous energetic reactions within strated growth oscillations in Arabidopsis pollen tubes grow- the cell (Goldbeter 1996). To the extent that pollen tubes ing in styles. Interestingly, the in vivo pollen tubes grew faster produce and use NAD(P)H, we can expect similar oscillations and oscillated more quickly than their in vitro counterparts. in those cells. But beyond these general considerations there In addition, using transgenic Arabidopsis plants expressing the are issues that pertain more specifically to pollen tube growth. yellow cameleon calcium indicator, they demonstrated os- In our studies of lily, we note that when the pollen grains first cillations in the tip-focused calcium gradient similar to those germinate, the emerging tubes exhibit fluctuations in their observed in pollen tubes grown in vitro (Iwano et al. 2004). growth rate but not regular oscillations. However, once they Setting the pace for growth: Available energy oscillates Given the rapidity of cell elongation and the necessary syn- thesis, transport, and of cell wall components, it can be appreciated that an abundant supply of energy is essential for pollen-tube growth. ATP is the fuel for key events, such Figure 6. Rac/ROP localizes to the apical plasma mem- as actin polymerization, proton pumping, exocytosis, and brane. The small G-protein Rac2, which is a member of kinase activity. From these brief considerations, it follows ROP family, has been identified as the fusion construct: that ATP plays a key role in pollen tube growth. GFP-At-Rac2. Although more broadly spread along the Cárdenas and colleagues (2006) recently demonstrated apical plasma membrane than PIP2 (phosphatidyl inosi- fluctuations in NAD(P)H fluorescence in growing pollen tol 4,5, biphosphate), the distribution of Rac2 neverthe- tubes. This essential coenzyme drives many biosynthetic less is maximally centered at the apex of the tube. Source: processes, including ATP production in mitochondria. The re- Kost and colleagues (1999). Bar = 10 micrometers. sults show that the dominant signal colocalizes with mito-

840 BioScience • November 2007 / Vol. 57 No. 10 www.biosciencemag.org Articles chondria at the base of the clear zone, and further that faster pollen tube growth. However, when protons flow into NAD(P)H fluorescence oscillates with the same periodicity the apex, the pH in this region is lowered, which results in as growth rate, with peaks following growth by approxi- decreased actin turnover and slower growth. Thus, the os- mately 95° (i.e., by 95 out of 360, or 26.4%, of a growth cillations in growth rate may be a direct response to the oscillation) and troughs anticipating growth by approxi- oscillations in pH in the pollen tube apex, which in turn mately –80°. Cárdenas and colleagues (2006) focus on the cause their effect by modulating the actin cytoskeleton. troughs because they anticipate growth and also because they show as strong a correlation with growth as do the Accelerating growth: ROPs oscillate peaks. The suggestion is made that the changes in fluorescence Recent studies on ROP1 reveal that it forms an apical gradi- represent the periodic oxidation of NAD(P)H to form ent in growing pollen tubes and that its activity oscillates with NAD(P)+, with the concomitant production of ATP.Oxida- the same periodicity as growth rate (Hwang et al. 2005). The Downloaded from https://academic.oup.com/bioscience/article/57/10/835/232382 by guest on 02 October 2021 tion of NAD(P)H may therefore precede and regulate pollen observation that ROP1 activity leads growth by approxi- tube growth by fueling ATP production. If ATP output does mately –90° supports the idea that it also plays a role in indeed oscillate, the activity of many ATP-dependent processes promoting pollen tube growth. It has been argued that will also oscillate. ROPs control pollen tube growth through their regulation of both calcium and actin cytoskeletal dynamics (Hwang and Accelerating growth: Alkalinity oscillates Yang 2006). More specifically, Gu and colleagues (2005) Sequential examination of appropriately stained pollen tubes suggest that ROP1 brings about its effects on pollen tube reveals that pH oscillates with the same period as growth rate growth by altering the activity of RICs (ROP-interactive (figure 7). of the phase relationship reveals that CRIB-motif-containing proteins). With a focus on RIC3 and the alkaline band is most alkaline approximately –95° before RIC4, Gu and colleagues (2005) report that overexpression a growth surge, whereas acidity peaks approximately 70° of RIC4 results in the formation of a dense network of actin after growth (Lovy-Wheeler et al. 2006). Phase analysis of the in the apex of the pollen tube. Thus, through an interaction extracellular proton influx shows that it too lags growth, and with RIC4, ROPs may be responsible for establishing the by a magnitude similar to the intracellular peak in acidity (ap- correct configuration of actin in the apex of growing pollen. proximately 100°; Messerli et al. 1999). The results further in- By contrast, overexpression of RIC3 results in an elevated dicate that the formation of the alkaline band and the increase intracellular calcium concentration, together with the inhi- in acidity at the apex are driven by two independent events. bition of growth and the formation of a swollen apical domain. It seems likely that the alkaline band derives from activity of a proton-ATPase on the plasma membrane that pumps protons out of the cell (figure 8; Feijó et al. 1999). These conclusions are consistent with recent observations by Lefeb- vre and colleagues (2005) showing a proton-ATPase that is lo- cated along the shank of the tube, but not at the extreme apex. By contrast, the acidic domain at the apex may arise through the growth-dependent opening of stretch-activated chan- nels at the apex, in a manner similar to that shown for calcium (Dutta and Robinson 2004). The observation that the pH of the alkaline band peaks before growth does (figure 7; Lovy-Wheeler et al. 2006) means that alkalinity becomes a candidate for an event or process that might serve as a growth initiator. One important entity that could be regulated by pH is the formation and activity of the actin cytoskeleton. Given that the actin fringe colocalizes with the alkaline band, it is possible that the band Figure 7. A graph showing the oscillation in growth rate helps organize actin in this region (Lovy-Wheeler et al. 2006). (dark blue) together with the oscillation in pH (pink) for ADF and pollen-specific actin-interacting protein also the same lily pollen tube. It is clear that the pH oscilla- localize to the actin fringe (Lovy-Wheeler et al. 2006) and are tions exhibit the same periodicity as those for growth; known to enhance depolymerization of F-actin at alkaline pH however, from visual inspection it is not possible to (reviewed in Hepler et al. 2006). Although it may seem that determine the phase relationship. Application of cross- ADF would only degrade actin microfilaments, it is im- correlation analysis reveals that the alkaline domain portant to emphasize that during the process of F-actin increases in pH in anticipation of an increase in growth fragmentation, free plus (fast-growing) ends of actin micro- rate, whereas the decline in pH at the apex follows the filaments are exposed. These can then serve as templates increase in growth rate. Source: Lovy-Wheeler and upon which new actin microfilaments can grow. ADF thus colleagues (2006). Abbreviations: µm, micrometer; sec, promotes the turnover of F-actin, which may translate into second. www.biosciencemag.org November 2007 / Vol. 57 No. 10 • BioScience 841 Articles

Because the effects of RIC3 are suppressed in the presence of signal and stimulate processes that would promote growth. a calcium channel blocker, it has been suggested that the The results show instead that it is the growth process that normal function of RIC3 is to regulate the influx of calcium, controls the calcium response. As the pollen tube surges which stimulates F-actin disassembly (Gu et al. 2005). forward, calcium rushes in through stretch-activated chan- nels. Elevated calcium may then retard growth. Applying the brakes: Calcium oscillates Calcium is thought to bring about its effects through Apical calcium ion concentration is yet another component intermediary proteins, including calmodulin and CDPKs. that oscillates in growing pollen tubes; changes in concen- Although calmodulin is evenly distributed throughout the tration from 0.75 µM to more than 3 µM during a single pollen tube, its activity is greatly enhanced in the apex (Rato growth oscillation have been reported (Pierson et al. 1996). et al. 2004). A CDPK with elevated activity in the pollen tube Once again, although these oscillations show the same period apex was found to be redistributed prior to changes in growth Downloaded from https://academic.oup.com/bioscience/article/57/10/835/232382 by guest on 02 October 2021 as the growth rate, they do not show the same phase. Analy- orientation (Moutinho et al. 1998). More recently two different sis of the phase relationship between intracellular calcium and CDPK isoforms have been identified in Petunia pollen tubes; growth rate using cross-correlation analysis has yielded an one (PiCDPK1) affects pollen tube polarity, whereas the unexpected result, namely, that the intracellular calcium other (PiCDPK2) affects the overall tube growth (Yoon et al. gradient follows, rather than leads, the peak in growth rate by 2006). Whereas calmodulin is known to interact with myosin 38° (Messerli et al. 2000). This conclusion has come as a sur- and villin, and possibly also regulate ACA9, a plasma mem- prise because it had been thought that calcium, as an intra- brane calcium ATPase (Sze et al. 2006), the downstream cellular signaling molecule, would appear as an anticipatory targets of CDPKs in pollen tubes are unknown. Several structures and related processes are mod- ulated by calcium, which may have a profound effect on oscillatory pollen tube growth. The cyto- skeleton is a prime example. Actin-binding proteins that are found commonly in pollen tubes include profilin, villin, and gelsolin-like protein; they all re- spond to calcium or to calcium plus calmodulin, and either prevent actin polymerization or fragment existing bundles of F-actin (Yokota et al. 2005, Yokota and Shimmen 2006). Another important cellular process that is regulated by calcium is cyto- plasmic streaming (Yokota and Shimmen 2006). Myosin XI, a involved in actin-based motility, is commonly found in pollen tubes and is blocked by elevated levels of calcium (Yokota and Shimmen 2006), accounting for the observations that cytoplasmic streaming is inhibited in the tube apex, but is prominent in the shank, where cal- cium is present at basal levels. A conclusion from these studies is that the local calcium concentration Figure 8. Summary diagram of a pollen-tube apex showing structural ele- plays a key role in determining the structure and ments and physiological processes that participate in pollen-tube growth. activity of the actin cytoskeleton. In oscillatory cell At the apex of the tube, calcium (Ca2+) and protons (H+) enter the cell. elongation, the decrease in actin polymerization and The local high calcium concentration facilitates secretion of the vesicles increase in F-actin fragmentation brought about by (V). The excess calcium is taken up by the endoplasmic reticulum (ER), elevated calcium prevent the surge in growth caused and also most likely extruded to the outside (not shown). Mitochondria by actin polymerization (Gibbon et al. 1999,Vidali (Mito) metabolize , which have been obtained from starch (ST)- et al. 2001). containing amyloplasts (AP), and generate adenosine triphosphate The oscillating calcium gradient may have a (ATP). ATP, through its coupled hydrolysis to adenosine diphosphate profound effect on the activity of the proton- (ADP), powers many key processes of pollen tube growth. Some of these ATPase in the apical domain. As Feijó and col- include the H+-ATPase at the cell surface, which creates the alkaline band leagues (1999) noted, high calcium inhibits (OH–), and the extracellular proton (H+) flux, polymerization of actin proton-ATPase activity in other plant cell systems. and transport along microfilaments (Actin), the uptake of calcium by If this holds for the pollen tube, the elevated calcium the ER, and the trafficking and fusion of vesicles in the apex. Certain key in the apex may explain why this enzyme is not signaling molecules, including, notably, Rho family GTPases of plants observed in the extreme apical domain. In addition,

(ROP) and phosphatidyl inositol 4,5 bisphosphase (PIP2), are localized oscillatory increases in calcium together with those to the apical plasma membrane and contribute to cell polarity. in acidity may spread sufficiently and block proton-

842 BioScience • November 2007 / Vol. 57 No. 10 www.biosciencemag.org Articles

ATPase activity on the plasma membrane bordering the clear as candidate agents that serve a primary role in controlling zone. This would cause a decrease in the alkaline band and growth initiation. However, it is still unclear what the perti- in the promotive effect that it would have on growth. nent pathways are, both upstream and down, through which Membrane trafficking, including exocytosis (secretion) these small G-proteins bring about and coordinate the myr- and endocytosis, is another process that is modulated by iad processes required for pollen tube growth. high intracellular calcium concentrations (Battey et al. 1999). Energy is fundamental to biological processes, and we Thus, the apical calcium gradient promotes fusion of secre- anticipate that ATP synthesis occupies a position very close tory vesicles, which is necessary for growth and which accounts to the central pacemaker of pollen tube growth. Because for the close correlation between the location of the gradient mitochondrial metabolism possesses the properties of an and the site of maximal growth. However, it remains a ques- endogenous oscillator in other cells, notably yeast cells (Gold- tion whether the oscillation in the calcium gradient generates beter 1996), it seems plausible that similar events take place Downloaded from https://academic.oup.com/bioscience/article/57/10/835/232382 by guest on 02 October 2021 an equivalent oscillation in secretion. Preliminary data, sum- in the pollen tube. Thus through the endogenous oscillation marized in a recent review (Holdaway-Clarke and Hepler of the pollen mitochondrial electron transport chain, the 2003), fail to note a similar phase relationship between the in- ATP concentration oscillates, and this underpins all other crease in calcium and the increase in secretion (wall thickness). oscillatory events. For example, through nucleoside diphos- The explanation may derive from the observation that the phate kinase, ATP drives the synthesis of GTP, which is es- tip-focused calcium gradient, even at its low point, is close to sential for all G-proteins, including ROPs discussed above. or above the threshold for stimulation of secretion. Sutter and Although key processes such as an oscillatory production of colleagues (2000) have shown in corn protoplasts that ATP have not been shown, there are experimental proce- secretion saturates at 1.5 µM and is half maximal at 0.9 µM. dures available with which these fundamental questions can Oscillations from 0.75 to above 3 µM have been reported for be probed. We are encouraged, therefore, that the knowl- pollen tubes (Pierson et al. 1996). However, it is important to edge gained from the examination of oscillatory processes will realize that these values are probably underestimates for the lead to new studies that will provide an even greater insight true concentration of calcium at the membrane surface, into the basic mechanism of pollen tube growth. where secretion occurs. Where the ion enters the cell, the local concentration will be very high but restricted to an ex- Acknowledgments tremely small volume of cytoplasm, which is below the limit We thank Benedikt Kost for providing figures 5 and 6. We also of microscopic resolution. The diffusing indicator dye does thank our colleagues Colleen Parris, Alex Bannigan, Alenka not accurately sample this tiny volume; rather, its response is Lovy-Wheeler, and Sylvester McKenna for help in the prepa- dominated by nearby domains where the ion concentration ration of the figures. Our research has been supported by a is much reduced. It is possible, therefore, that the local calcium grant from the National Science Foundation (MCB-0516852). concentration is always above the threshold for secretion, and that oscillatory increases in the ion do not further stim- References cited ulate this process. Battey NH, James NC, Greenland AJ, Brownlee C. 1999. Exocytosis and endocytosis. Plant Cell 14: 2915–2927. Summary Bosch M, Hepler PK. 2005. Pectin methylesterases and pectin dynamics in Much has been discovered about the structure and physiol- pollen tubes. Plant Cell 17: 3219–3226. Cárdenas L, McKenna ST, Kunkel JG, Hepler PK. 2006. NAD(P)H oscillates ogy of the pollen tube, and considerable insight has been in pollen tubes and is correlated with tip growth. 142: gained concerning the characterization and interaction of the 1460–1468. many processes that control tube growth. However, several Dowd PE, Coursol S, Skirpan AL, Kao T, Gilroy S. 2006. Petunia phospho- issues remain to be elucidated, including, notably, the iden- lipase C1 is involved in pollen tube growth. Plant Cell 18: 1438–1453. tity of the central pacemaker and the pathways through Dresselhaus T. 2006. Cell-cell communication during double fertilization. which it generates oscillatory growth. Figure 8 is a summary Current Opinion in Plant Biology 9: 41–47. Dutta R, Robinson KR. 2004. Identification and characterization of stretch- diagram of the apical domain of the pollen tube that at- activated ion channels in pollen protoplasts. Plant Physiology 135: tempts to show the major components and where they par- 1398–1406. ticipate. It is clear that calcium and protons play essential roles Feijó JA, Sainhas J, Hackett GR, Kunkel JG, Hepler PK. 1999. Growing pollen in pollen tube growth. The high level of calcium in the apex tubes possess a constitutive alkaline band in the clear zone and a growth- inhibits cytoplasmic streaming and fragments the actin cyto- dependent acidic tip. Journal of 144: 483–496. Feijó J A, Sainhas J, Holdaway-Clarke T, Cordeiro MS, Kunkel JG, Hepler PK. skeleton, whereas the zone of high pH in the region of the actin 2001. Cellular oscillations and the regulation of growth: The pollen fringe is thought to promote actin turnover, and thereby tube paradigm. Bioessays 23: 86–94. serves as an initiator of pollen tube growth. The discovery that Franklin-Tong VE, Drøbak BK, Allan AC, Watkins RAC, Trewavas AJ. 1996. Growth of pollen tubes of is regulated by a slow-moving certain key signaling factors, including PIP2 and ROPs, localize on the plasma membrane at the apex of the growing pollen calcium wave propagated by inositol 1,4,5-trisphosphate. Plant Cell 8: tube has focused our attention on these agents as central 1305–1321. Geitmann A, Steer M. 2006. The architecture and properties of the pollen tube switches in pollen tube growth. The ROPs, with their antic- cell wall. Pages 177–200 in Malhó R, ed. The Pollen Tube: A Cellular and ipatory increase in activity during oscillatory growth, emerge Molecular Perspective. Berlin: Springer. www.biosciencemag.org November 2007 / Vol. 57 No. 10 • BioScience 843 Articles

Gibbon BC, Kovar DR, Staiger CJ. 1999. Latrunculin B has different effects Martin TFJ. 1998. Phosphoinositide lipids as signaling molecules: on pollen germination and tube growth. Plant Cell 11: 2349–2364. Common themes for signal transduction, cytoskeletal regulation, and Goldbeter A. 1996. Biochemical Oscillations and Cellular Rhythms. Cambridge membrane trafficking. Annual Review of Cell and Developmental (UK): Cambridge University Press. Biology 14: 231–264. Gu Y, Fu Y, Dowd P, Li S, Vernoud V, Gilroy S, Yang Z. 2005. A Rho family Messerli M, Danuser G, Robinson K. 1999. Pulsatile influxes of H+,K+ and GTPase controls actin dynamics and tip growth via two counteracting Ca2+ lag growth pulses of Lilium longiflorum pollen tubes. Journal of Cell downstream pathways in pollen tubes. Journal of Cell Biology 169: Science 112: 1497–1509. 127–138. Messerli M, Creton R, Jaffe LF, Robinson KR. 2000. Periodic increases in Gu Y, Li S, Lord EM, Yang Z. 2006. Members of a novel class of Arabidopsis elongation rate precede increases in cytosolic Ca2+ during pollen tube Rho guanine nucleotide exchange factors control Rho GTPase-depen- growth. 222: 84–98. dent polar growth. Plant Cell 18: 366–381. Moutinho A, Love J, Trewavas AJ, Malhó R. 1998. Distribution of Helling D, Possart A, Cottier S, Klahre U, Kost B. 2006. Pollen tube tip calmodulin protein and mRNA in growing pollen tubes. Sexual Plant growth depends on plasma membrane polarization mediated by Reproduction 11: 131–139. tobacco PLC3 activity and endocytotic membrane recycling. Plant Cell Parton RM, Fischer-Parton S, Trewavas AJ, Watahiki MK.2003. Pollen tubes Downloaded from https://academic.oup.com/bioscience/article/57/10/835/232382 by guest on 02 October 2021 18: 3519–3534. exhibit regular periodic membrane trafficking events in the absence of Hepler PK,Vidali L, Cheung AY.2001. Polarized cell growth in higher plants. apical extension. Journal of Cell Science 116: 2707–2719. Annual Review of Cell and Developmental Biology 17: 159–187. Pierson ES, Miller DD, Callaham DA, Shipley AM, Rivers BA, Cresti M, Hepler PK, Lovy-Wheeler A, McKenna ST, Kunkel JG. 2006. Ions and pollen Hepler PK. 1994. Pollen tube growth is coupled to the extracellular tube growth. Pages 47–69 in Malhó R, ed. The Pollen Tube: A Cellular calcium ion flux and the intracellular calcium gradient: Effect of BAPTA- and Molecular Perspective. Berlin: Springer. type buffers and hypertonic media. Plant Cell 6: 1815–1828. Holdaway-Clarke TL, Hepler PK. 2003. Control of pollen tube growth: Role Pierson ES, Miller DD, Callaham DA, van Aken J, Hackett G, Hepler PK. of ion gradients and fluxes. New Phytologist 159: 539–563. 1996. Tip-localized calcium entry fluctuates during pollen tube Holdaway-Clarke TL, Weddle NM, Kim S, Robi A, Parris C, Kunkel JG, growth. Developmental Biology 174: 160–173. Hepler PK. 2003. Effect of extracellular calcium, pH and borate on Rato C, Monteiro D, Hepler PK, Malhó R. 2004. Calmodulin activity and growth oscillations in Lilium formosanum pollen tubes. Journal of cAMP signalling modulate growth and apical secretion in pollen tubes. Experimental 54: 65–72. Plant Journal 38: 887–897. Hwang J-U, Yang Z.2006. Small GTPases and spatiotemporal regulation of Raven PH, Evert RF,Eichhorn SE. 1999. Biology of Plants. New York: Worth. pollen tube growth. Pages 95–116 in Malhó R, ed. The Pollen Tube: A Romagnoli S, Cai G, Faleri C, Yokota E, Shimmen T, Cresti M. 2007. Cellular and Molecular Perspective. Berlin: Springer. - and actin filament-dependent motors are distributed on Hwang J-U, Gu Y,Lee Y-J,Yang Z.2005. Oscillatory ROP GTPase activation pollen tube mitochondria and contribute differently to the their move- leads the oscillatory polarized growth of pollen tubes. Molecular ment. Plant and 48: 345–361. doi:10.1093/pcp/pcm001 Biology of the Cell 16: 5385–5399. Sanders L, Lord E. 1992. A dynamic role for the stylar matrix in pollen tube Iwano M, Shiba H, Miwa T, Che F-S, Takayama S, Nagai T, Miyawaki A, extension. International Review of Cytology 140: 297–318. Isogai A. 2004. Ca2+ dynamics in a pollen grain and papilla cell during Snow AA, Spira TP. 1991. Pollen vigour and the potential for sexual of Arabidopsis. Plant Physiology 136: 3562–3571. selection in plants. Nature 352: 796–797. Klahre U, Kost B. 2006. Tobacco RhoGTPase ACTIVATING PROTEIN1 Sutter JU, Homann U, Thiel G. 2000. Ca2+-stimulated exocytosis in spatially restricts signaling of RAC/Rop to the apex of pollen tubes. coleoptile cells. Plant Cell 12: 1127–1136. Plant Cell 18: 3033–3046. Sze H, Frietsch S, Li X, Bock K, Harper J. 2006. Genomic and molecular analy- Klahre U, Becker C, Schmitt AC, Kost B. 2006. Nt-RhoGDI2 regulates ses of transporters in the male gametophyte. Pages 71–94 in Malhó R, Rac/Rop signaling and polar cell growth in tobacco pollen tubes. Plant ed. The Pollen Tube: A Cellular and Molecular Perspective. Berlin: Journal 46: 1018–1031. Springer. Kost B, Lemichez E, Spielhofer P,Hong Y,Tolias K,Carpenter C, Chua N-H. Vidali L, McKenna ST, Hepler PK. 2001. Actin polymerization is essential for 1999. Rac homologues and compartmentalized phosphatidylinositol 4, pollen tube growth. of the Cell 12: 2534–2545. 5-bisphosphate act in a common pathway to regulate polar pollen tube Wilsen KL. 2005. Monitoring the actin cytoskeleton and calcium dynamics growth. Journal of Cell Biology 145: 317–330. in growing pollen tubes. PhD dissertation. University of Massachusetts, Kühtreiber W,Jaffe L. 1990. Detection of extracellular calcium gradients with Amherst. a calcium-specific vibrating electrode. Journal of Cell Biology 110: Yokota E, Shimmen T. 2006. The actin cytoskeleton in pollen tubes: Actin and 1565–1573. actin binding proteins. Pages 139–155 in Malhó R, ed. The Pollen Tube: Lancelle SA, Hepler PK. 1992. Ultrastructure of freeze-substituted pollen tubes A Cellular and Molecular Perspective. Berlin: Springer. of Lilium longiflorum. Protoplasma 167: 215–230. Yokota E, Tominaga M, Mabuchi I, Tsuji Y, Staiger CJ, Oiwa K, Shimmen T. Lefebrve B, Arango M, Oufattole M, Crouzet J, Purnelle B, Boutry M. 2005. Plant villin, lily P-135-ABP,possesses G-actin binding activity and 2005. Identification of a Nicotiana plumbaginifolia plasma membrane accelerates the polymerization and depolymerization of actin in a Ca2+- G+-ATPase gene expressed in the pollen tube. Plant Molecular Biology sensitive manner. Plant and Cell Physiology 46: 1690–1703. 58: 775–787. Yoon GM, Dowd PE, Gilroy S, McCubbin AG. 2006. Calcium-dependent Lovy-Wheeler A, Wilsen KL, Baskin TI, Hepler PK. 2005. Enhanced fixation protein kinase isoforms in Petunia have distinct functions in pollen reveals the apical cortical fringe of actin filaments as a consistent feature tube growth, including regulating polarity. Plant Cell 18: 867–878. of the pollen tube. Planta 221: 95–104. Lovy-Wheeler A, Kunkel JG, Allwood EG, Hussey PJ, Hepler PK. 2006. Žárský V, Potocky M, Baluška F, Cvrčková F. 2006. metabolism, Oscillatory increases in alkalinity anticipate growth and may regulate actin compartmentalization and signalling in the regulation of pollen tube growth. dynamics in pollen tubes of lily. Plant Cell 18: 2182–2193. Pages 117–138 in Malhó R, ed. The Pollen Tube: A Cellular and Molecular Lovy-Wheeler A, Cárdenas L, Kunkel JG, Hepler PK. 2007. Differential organelle movement on the actin cytoskeleton in lily pollen tubes. Cell Perspective. Berlin: Springer. Motility and the Cytoskeleton 64: 217–232. Malhó R, Trewavas AJ. 1996. Localized apical increases of cytosolic free doi:10.1641/B571006 calcium control pollen tube orientation. Plant Cell 8: 1935–1949. Include this information when citing this material.

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