Cytoplasmic calcium concentration ([Ca2+]i) and extracellular calcium (Ca2+o) influx has been studied in pollen tubes of Lilium longliflorum in which the processes of cell elongation and exocytosis have been uncoupled by use of Yariv phenylglycoside ((β-D-Glc)3). Growing pollen tubes were pressure injected with the ratio dye fura-2 dextran and imaged after application of (β-D-Glc)3, which binds arabinogalactan proteins (AGPs). Application of (β-D-Glc)3 inhibited growth but not secretion. Ratiometric imaging of [Ca2+]i revealed an initial spread in the locus of the apical [Ca2+]i gradient and substantial elevations in basal [Ca2+]i followed by the establishment of new regions of elevated [Ca2+]i on the flanks of the tip region. Areas of elevated [Ca2+]i corresponded to sites of pronounced exocytosis, as evidenced by the formation of wall ingrowths adjacent to the plasma membrane. Ca2+o influx at the tip of (β-D-Glc)3-treated pollen tubes was not significantly different to that of control tubes. Taken together these data indicate that regions of elevated [Ca2+]i, probably resulting from Ca2+o influx across the plasma membrane, stimulate exocytosis in pollen tubes independent of cell elongation.
The polarised growth of the pollen tube, which is essential for the delivery of the sperm cells to the egg apparatus, results from the continued fusion of Golgi vesicles at the extreme apex of the tube. The process provides new plasma membrane and cell wall components necessary for pollen tube elongation ( Heslop-Harrison 1987; Mascarenhas 1993; Steer & Steer 1989) and results from a delicate balance between exocytosis of cell wall components and cell wall assembly.
In lily we have previously shown that arabinogalactan proteins (AGPs) are secreted into the pollen tube tip via Golgi vesicles ( Jauh & Lord 1996; Roy et al. 1998 ). We have also shown that it is possible to uncouple exocytosis from pollen tube extension by blocking the incorporation of the newly synthesised cell wall components using Yariv phenylglycoside ((β- d-Glc)3), an agent that binds AGPs ( Nothnagel 1997; Roy et al. 1998 ). This subtle change in the balance between exocytosis and elongation not only causes a build up of cell wall components during arrest of lily pollen tube extension but also results in a surprising pattern of disorganized exocytosis that may be a consequence of changes in the dynamics of [Ca2+]i. In this study we have used fluorescent ratio imaging and the non-invasive ion-selective vibrating probe to decipher the patterns of [Ca2+]i and Ca2+o influx when pollen tube extension has been blocked but exocytosis still continues. Our results provide strong evidence that domains of high [Ca2+]i are correlated with secretion and may be responsible for triggering it. That binding of a cell surface glycoprotein can so dramatically affect [Ca2+]i and cell morphology is further indication that the cell wall-membrane interface is an active player in the process of polar growth.
Morphological response to (β- d-Glc)3Addition of 30 μm (β- d-Glc)3 to growing lily pollen tubes caused considerable inhibition of elongation rates, as previously reported ( Jauh & Lord 1996), with growth stopping within 10 min.
The clear zone, that is the vesicle-rich area at the tip of the pollen tube, was unchanged and no large organelles such as mitochondria, amyloplasts, dictyosomes or lipid bodies penetrated it (data not shown). Often the clear zone became enlarged and streaming decreased significantly (data not shown).
(β- d-Glc)3 modifies [Ca2+]i at the pollen tube tip
Fluorescence ratiometric analysis of growing pollen tubes loaded with fura-2 revealed a steep tip-focused gradient of [Ca2+]i, located in the first 20 μm of the apex, as previously reported ( Fig. 4, Pierson et al. 1994 ). Ratiometric ion imaging reveals that elevated [Ca2+]i followed treatment with 30 μm (β- d-Glc)3 in all pollen tubes observed (n = 10). The exact time between the treatment and the response varied but in all cases pollen tube growth had ceased within 10 min. This variation was probably due to differences in diffusion rates of the solution of (β- d-Glc)3 through the agarose in which the pollen tubes are embedded.
Figure 1 illustrates the typical change in [Ca2+]i at the tip of the pollen tube after treatment with 30 μm (β- d-Glc)3. The sequence includes images acquired every 10 s starting 90 s after treatment. In this particular example, growth became progressively slower and by 220 s had stopped. Treated pollen tubes initially still possessed a steep, tip-focused [Ca2+]i gradient ranging from 0.6 μm at the extreme tip to a basal level of 0.25 μm 20 μm back from the tip ( Figs 1a, 90 s and Fig. 2a, ♦), similar to the control (data not shown; for a previously published example see Fig. 4, Pierson et al. 1994 ). Within minutes pollen tubes exposed to (β- d-Glc)3 experience a global increase of [Ca2+]i in the tip-most 100 μm of the pollen tube. In addition to raised levels of basal [Ca2+]i (β- d-Glc)3 caused the region of elevated [Ca2+]i, normally confined to the very apex of the pollen tube, to be extended over a larger area of the tip ( Figs 1a, 100–140 s) within which discreet domains of high [Ca2+]i could also be seen on the flanks of the dome ( Fig. 1a, arrowheads, 140 s and 150 s).
[Ca2+]i fluctuates in (β- d-Glc)3 treated pollen tubes
The pollen tube imaged in Fig. 1(a), was measured again in a second sequence ( Fig. 1b). The viability of the pollen tube was checked before and after the second sequence and showed cytoplasmic streaming and an intact clear zone at the tip. The sequence of images acquired every 10 s starting 220 s after treatment with (β- d-Glc)3 reveal fluctuations in [Ca2+]i. Measurement of [Ca2+]i 220 s after application of (β- d-Glc) showed the treated pollen tube had an average concentration of 0.8 μm ( Fig. 1b, Figs 2b, 220 s). [Ca2+]i was elevated within 80 μm and perhaps further from the tip. It is possible that the modified gradient shown in Fig. 1(a) and 2(a) was still present with lower [Ca2+]i out of the field of view. Twenty seconds later the pollen tube showed an overall [Ca2+]i decrease ( Figs 1b, 240 s) followed by an increase of [Ca2+]i in the whole tube ( Figs 1b, 250 s and later images). Line scans ( Fig. 2b) illustrate an apparent reversal of the tip-focused gradient within the 15 μm behind the tip and fluctuations of [Ca2+]i. We did not attempt to evaluate the periodicity of the fluctuations in [Ca2+]i.
Domains of elevated [Ca2+]i outline the zone of secretion
Pollen tubes treated with (β- d-Glc)3 for 1 h or more showed a basal [Ca2+]i level of 0.8 μm, indicating that the ion concentration did not increase indefinitely (for example, Fig. 3). Treated cells are apparently able to regulate [Ca2+]i, however, discreet domains of very high [Ca2+]i were evident and usually associated with regions of marked accumulation of cell wall material ( Fig. 3, arrowheads). A [Ca2+]i signal higher than 4 μm was clearly detected along the plasma membrane outlining the accumulations of cell wall material ( Fig. 3b, arrowheads).
(β- d-Glc)3 does not stop the influx of Ca2+o at the tip
Ca2+o fluxes were measured in a growth medium that was low in Ca2+ and low in buffer ( Holdaway-Clarke et al. 1997 ). The non-invasive ion selective probe revealed a tip-directed inward Ca2+o current, as expected in growing pollen tubes ( Fig. 4). This influx was reduced when pollen tubes were treated with a growth-inhibiting solution of 3 m m caffeine as has been previously shown ( Pierson et al. 1996 ). In contrast, when pollen tubes were treated with (β- d-Glc)3 for less than 1 h the influx of Ca2+o persisted, despite the arrest of growth ( Fig. 4). The influx observed in (β- d-Glc)3-treated pollen tubes is not significantly different to that measured in control tubes, but is significantly different to the flux in tubes treated with caffeine (P > 0.05, F-ratio test).
(β- d-Glc)3 is the first agent known to us to arrest pollen tube elongation while simultaneously inducing elevated [Ca2+]i through a broad region of the tube apex and maintaining Ca2+o influx at the tip. Using (β- d-Glc)3 we have been able to uncouple pollen tube extension and the elevated [Ca2+]i at the tip, while the clear zone is maintained and secretion continues.
AGPs are selectively bound by (β- d-Glc)3 ( Nothnagel 1997; Yariv et al. 1962 ). Evidence from experiments using (β- d-Glc)3 indicates that these proteoglycans play a role in plant cell extension, cell division and in plant developmental processes ( Nothnagel 1997; Serpe & Nothnagel 1994; Thompson & Knox 1998; Willats & Knox 1996). AGPs have been found in stylar tissue ( Clarke et al. 1979 ) and may serve as a source of nutrition for growing pollen tubes, or as a signalling molecule for pollen tube guidance ( Cheung 1995). Although (β- d-Glc)3 arrests pollen tube elongation in Zea mays, Annona cherimoya and Lilium, Nicotiana tabacum is insensitive to the reagent (Lord, unpublished data). AGPs have been localised to the plasma membrane of in vivo grown lily pollen tubes ( Jauh & Lord 1996; Roy et al. 1998 ). It is not surprising that (β- d-Glc)3, which binds specifically to this component of the cell wall (for review see Nothnagel 1997), inhibits cell extension, since wall yield threshold is a major factor determining the growth rate of plant cells ( Cosgrove 1986). The effect of (β- d-Glc)3 on [Ca2+]i, however, was not anticipated, given that in most situations cessation of pollen tube elongation is correlated with collapse of the tip-focused [Ca2+]i gradient to basal levels ( Pierson et al. 1994 ).
Caffeine and (β- d-Glc)3 have markedly different effects on the ultrastructure and Ca2+ profile of pollen tubes despite the fact that both substances inhibit elongation. Pollen tube tips treated with caffeine for 1 h show collapse of the tip-focused [Ca2+]i gradient, decreased Ca2+o influx at the tip and a slightly thickened cell wall at the apex of the tube ( Lancelle et al. 1997 ). In contrast, tubes exposed to (β- d-Glc)3 retain apical Ca2+o influx, have elevated [Ca2+]i extending 100 μm back from the apex and large accumulations of cell wall material both at the tip and flanks of treated pollen tubes. ( Jauh & Lord 1996; Roy et al. 1998 ; Fig. 3). The large difference in wall volume at the pollen tube tip between the two treatments is compelling evidence that the rate of exocytosis is very much slower in caffeine than in (β- d-Glc)3. In pollen tubes treated with caffeine the zone of exocytosis appears to spread out from the apex indicating that there is a low basal level of exocytosis occurring constitutively ( Lancelle et al. 1997 ) and that the tip-focused [Ca2+]i gradient simply biases exocytosis to the pollen tube apex. The persistence of exocytosis in (β- d-Glc)3-treated pollen tubes ( Jauh & Lord 1996; Roy et al. 1998 ) supports an intimate association between the presence of elevated [Ca2+]i and secretion, a conclusion that holds for several other plant ( Carroll et al. 1998 ; Thiel et al. 1994 ; Zorec & Tester 1992), and animal systems (for review see Martin 1997).
The overall increase of [Ca2+]i in the (β- d-Glc)3-treated pollen tubes may explain why these tubes exhibit slower cytoplasmic streaming than controls and account for the unusual phenomenon of concurrent arrest of elongation, maintenance of the clear zone and exocytosis. First, when [Ca2+]i approaches 1 μm, as occurs in tubes exposed to (β- d-Glc)3 for more than a few minutes, cytoplasmic streaming is inhibited due to fragmentation of actin cables and the inhibition of myosin motor function ( Kohno & Shimmen 1987; Shimmen & Tazawa 1982). Second, the dissipation of the gradient resulting from arrested growth by caffeine is usually accompanied by the occlusion of the clear zone ( Pierson et al. 1996 ) due to the reformation of actin filaments at the tip ( Lancelle et al. 1997 ; Miller et al. 1996 ). In (β- d-Glc)3-treated pollen tubes, it is possible that a Ca2+-dependent fragmentation of actin cables results in an intact or enlarged clear zone. Third, high [Ca2+]i is also required for the aggregation, fusion and exocytosis of secretory vesicles with the plasma membrane ( Battey & Blackbourn 1993). The fact that elevated [Ca2+]i is antagonistic to actin filaments and stimulates exocytosis accounts for the observed correlation between the presence of a clear zone and secretion at regions of elevated [Ca2+]i gradient. The presence of high [Ca2+]i domains at the points where cell wall material accumulates ( Fig. 3) further links elevated [Ca2+]i and exocytosis and reveals that it is not the tip-focused gradient as such, that drives secretion but any domain of elevated [Ca2+]i. Clearly elevated [Ca2+]i at the tip of the pollen tube and indeed exocytosis itself are not sufficient to induce pollen tube elongation.
The observed pattern of [Ca2+]i elevation is difficult to explain by localised Ca2+ release from the ER in response to the interaction of (β- d-Glc)3 with the AGPs since ultrastructural analysis of cryofixed pollen tubes shows that the ER is evenly distributed throughout the tube cell ( Lancelle & Hepler 1992) and in situ visualisation of AGPs, to which (β- d-Glc)3 binds, shows that they outline the plasma membrane of the whole pollen tube ( Roy et al. 1998 ). The striking evidence of high [Ca2+]i domains outlining the secretion centers is consistent with the proposition that the elevated [Ca2+]i results from an increase of Ca2+o influx, and perhaps reduced extrusion via Ca2+ ATPases across the plasma membrane. The results of Serpe & Nothnagel (1994) indicate that (β- d-Glc)3 cross-links AGPs in the plasma membrane. It is possible that such cross-linking of plasma membrane elements may alter the function of membrane proteins directly, increasing the activity of Ca2+ influx channels or decreasing the efficiency of Ca2+-ATPases.
There is strong evidence that the tip-focused [Ca2+]i gradient in pollen tubes results from Ca2+ entry at the apical plasma membrane ( Jaffe et al. 1975 ; Malhóet al. 1994 ; Malhóet al. 1995 ; Messerli & Robinson 1997). In our results, a Ca2+o influx similar to the control is maintained at the pollen tube tip after treatment with (β- d-Glc)3 ( Fig. 4), indicating that the strength of the Ca2+‘sink’ at the pollen tube apex is not significantly altered by exposure to (β- d-Glc)3. Previously we calculated that in untreated pollen tubes the Ca2+o influx required to support the tip-focused gradient was only a small fraction of the influx measured at the tip and that the majority of the Ca2+o current could be accounted for by binding of Ca2+ to de-esterified pectins in the newly secreted cell wall ( Holdaway-Clarke et al. 1997 ). Thus even a twofold increase in Ca2+ influx across the plasma membrane may not significantly increase the total flux measured.
It has been suggested that Ca2+ influx across the apical plasma membrane is regulated by stretch-activated channels ( Feijóet al. 1995 ; Malhóet al. 1995 ; Pierson et al. 1996 ). We see such channels as likely mediators of (β- d-Glc)3-induced changes in [Ca2+]i. Serpe & Nothnagel (1994) have shown that (β- d-Glc)3 decreases the mobile fraction of probes that bind covalently to proteins and glycoconjugates of the plasma membrane, so reduced mobility of the plasma membrane proteins could result in tension in the lipid bilayer, which is known to open stretch-activated channels ( Ramahaleo et al. 1996 ). Elevated [Ca2+]i would then increase exocytosis which in turn would increase the number of Ca2+ channels. We suggest inward stretching of the plasma membrane caused by accumulation of unincorporated cell wall components in the enlarged extracellular matrix, could induce a non-reversible deformation of the plasma membrane and accordingly activate stretch-activated Ca2+ channels. A positive-feedback loop could then be completed by increased [Ca2+]i facilitating exocytosis which, in turn, provides more Ca2+ channels and cell wall material that would further stretch the membrane.
Fresh pollen of Lilium longiflorum (Easter lily, Ace) or Lilium formosanum collected from plants grown at the University of California, Riverside or at the University of Massachusetts, Amherst or lily pollen kept frozen in liquid nitrogen was used for this study. Pollen was germinated under constant agitation for 60–90 min in one of two different germination media. For most experiments we used regular medium containing 7% sucrose, 160 μm H3BO3, 15 m m 2N-morpholinoethane sulphonic acid (MES), 0.1 m m CaCl2 and 1 m m KCl adjusted to pH 5.5 with KOH. A low Ca2+, low buffer medium (LCLB) was devised for use with the extracellular Ca2+-selective vibrating probe and consisted of 7% sucrose, 1.6 m m H3BO3, 1.0 m m MES, 0.05 m m CaCl2, and 1 m m KCl, pH 6.0.
Pollen tubes were mounted for pressure injection or measurement with the non-invasive Ca2+-selective vibrating probe, by mixing a small drop of concentrated germinating pollen with a drop of low-temperature gelling agarose diluted 1.2% in culture medium (type VII, Sigma) on the cover slip of a microscope slide chamber. The mixture was cooled quickly to 4°C for a few seconds and flooded with the culture medium.
Microinjection of fluorescent dye
Pollen tubes about 300 μm in length were pressure-injected with fura-2-dextran (40 mg ml–1 in deionized water, Molecular Probes, Inc., Eugene, OR, USA) using micropipettes pulled from filamented 1.0 mm diameter glass using a vertical puller (Model 700D, David Kopf Instruments, Tujunga, CA, USA). The tip of the micropipette was filled with the dye and the remainder with deionized water. A Narishige MO-103R micromanipulator (Narishige Scientific Instruments, Tokyo, Japan) was used to position the micropipettes and impale the pollen tube 100 μm back from the tip.
Ratiometric imaging of [Ca2+]i[Ca2+]i distribution was determined by ratio ion imaging with the Ca2+ imaging system described in detail in Pierson et al. (1994) . Briefly, it is composed of a Nikon inverted microscope that includes a highly regulated Hg vapour lamp as a light source and a charge-coupled device camera as a fluorescence detector. The purpose-built filter slider was used to present the two excitation wavelengths, namely the Ca2+-insensitive or isosbestic wavelength (360 nm) and the Ca2+-sensitive wavelength (340 nm). Images were acquired with a 1 : 5–1 : 10 exposure time for the 360 and 340 nm excitation wavelengths, respectively. Images were collected approximately every 10 s using purpose-written macros on PMIS (GKR Computer Consulting, Boulder, CO) image acquisition software. Background images were acquired in the same way after moving the cell out of the field of view. The final ratio images (340 nm/360 nm) were calculated from fluorescence images after background subtraction and thresholding, in which pixels with background-subtracted fluorescence at the Ca2+-independent wavelength lower than a predefined threshold value, were displayed as black.
The absolute [Ca2+]i was determined according to well established methods ( Bolsover et al. 1993 ). Briefly, ratio values were related to standardised Ca2+ concentrations obtained by imaging fura-2-dextran (40 mg ml–1), 2.5 m m Hepes, pH 7.0, 100 m m KCl, 60% (w/w) sucrose in Ca2+-free, Ca2+-bound or equimix 2.5 m m BAPTA at the same exposure times used to acquire ratio images ( Miller et al. 1992 ; Pierson et al. 1994 , Pierson et al. 1996 ).
Quantitative information on the ratio values and thus on [Ca2+]i were determined by means of the line scan measurement function of the Image-1 program (Universal Imaging Corporation, West Chester, PA, USA).
Measurement of Ca2+o flux
A non-invasive vibrating Ca2+-specific microelectrode was used to measure Ca2+o flux at the tips of lily pollen tubes, as described by Holdaway-Clarke et al. (1997) . Briefly, electrodes were pulled from 1.5 mm glass capillaries, silanized with dimethyl-dichlorosilane (Sigma D-3879) and then backfilled with 100 m m CaCl2 to a length of 15 mm from the tip. A 10–15 μm column of Ca2+-selective liquid ion exchange cocktail (Fluka Chemie AG, Ca2+ Ionophore I, Cocktail A, 21048 Buchs, Switzerland) was then drawn into the tip of the electrode. Signals were measured with a purpose-built electrometer (Applicable Electronics, West Yarmouth, MA, USA), and data were acquired with the program 3DVIS (adapted by J.G. Kunkel from DVIS Version 6 described by Smith et al. 1994 ).
Ca2+o fluxes were measured at the very tip of pollen tubes by positioning the electrode normal to the tangent at the tip, and vibrating the electrode back and forth along this line. The probe oscillated at 0.3 Hz, with an excursion of 10 μm. The difference in the voltages recorded at these two points is a measure of the Ca2+ flux. Calibration solutions of known [Ca2+] were used to determine the actual slope of the electrode (Nernst slope for Ca2+ = 29 mV/decade) by measuring the static (not vibrating) voltage in each solution. The actual [Ca2+] at a pollen tube tip was determined from the calibration values by measuring the static voltage very close to the pollen tube tip. Once the actual [Ca2+]o at the tip is known, it is possible to convert voltage difference measurements (recorded in the vibrating mode) into Ca2+o flux units.
Growth inhibition of pollen tubes treated with (β- d-Glc)3 or caffeine
(β- d-Glc)3 was synthesised according to Serpe & Nothnagel (1994) and was a generous gift from G.Y. Jauh (University of California, Riverside, USA). In ratio-imaging experiments, pollen tubes that were actively growing after microinjection of fura-2–dextran were exposed to 30 μm (β- d-Glc)3 in culture medium. To visualise [Ca2+]i distribution in pollen tubes that have been treated with (β- d-Glc)3 for times longer than 1 h, we first treated the growing pollen tube with (β- d-Glc)3 then mounted them on the microscope slide chamber, and finally loaded them with fura-2–dextran. Ca2+ flux at the tip of pollen tubes 300–800 μm long were measured after the medium in the chamber was replaced with growth medium supplemented with either 30 μm (β-D-Glc)3 or 3 m m caffeine or growth medium alone (control).
This work was supported by National Science Foundation (NSF) grant MCB-93–04953 and MCB-96–01087 to P.K.H. and NSF grant IBN-92–06577 to E.M.L.