MEETING REPORT

Cell Biology of Plant and Fungal Tip Growth—Getting to the Point

Tip growth is a process that has many similarities in diverse walled cells such as pollen tubes, root hairs, and hyphae. However, due to the diversity of the experimental systems, it is unusual for those working on the phenomenon to have the opportunity to get together and compare systems and concepts. From June 19 to 23, 2000, NATO, the European Commission and the Università di Siena sponsored a NATO Advanced Research Workshop which brought together 75 of the current tip growth investigators for a focused exchange of information and ideas on this fascinating topic. Much of the work will appear in a dedicated volume (Geitmann et al. 2000 ), and abstracts of the presentations can be viewed at: http://www.dpw.wageningen-ur.nl/epc/NATO-tipgrowth. The aim of this report is to identify trends in the field and bring together an introduction to a literature that is often widely dispersed.

ACTIN, NOT MICROTUBULES, DOES MUCH OF THE JOB
A clear consensus among meeting participants, consistent with a recent review (Geitmann and Emons 2000 ), was that an apical concentration of actin exists in all cell types examined. However, the organization of apical actin was not conserved, varying from diffuse fine filaments permeating the apical cytoplasm to coarser filaments, large patches, and smaller plaques, all predominantly associated with the apical plasma membrane. Apical actin was suggested to regulate exocytosis directly (Irene Lichtscheidl, University of Vienna, Austria), to set up subapical cables involved with organelle transport (Peter Hepler and Luis Vidali, University of Massachusetts, USA), and to be responsible for the regulation of tip extensibility (I. Brent Heath, York University, Canada). Supporting the latter were observations of tip swelling or deformation following actin disruption in many diverse cells (Frantisek Baluka, University of Bonn, Germany; Norbert de Ruijter, Wageningen University, The Netherlands; I.B. Heath; Tijs Ketelaar, Wageningen University, The Netherlands; L. Vidali). However, Orest Demkiv (Institute of Ecology of the Carpathians, Ukraine) showed an unexplained contrary result in moss cells, suggesting intercellular variability.

Alternative functions of actin included organizing morphogenically important accumulations of endoplasmic reticulum (Brigitte Buchen, University of Bonn, Germany) and orienting cell wall fibrils (Christos Katsaros, University of Athens, Greece) in algae, and positioning the translation apparatus in yeasts (Alison Adams, University of Arizona, USA; Jiri Haek, Czech Academy of Sciences, Czech Republic). The latter is likely to be even more important in longer and highly polarized cells, such as pollen tubes and fungal hyphae.

The repeated demonstrations of differential organization of apical actin, relative to subapical regions, and of its configurational changes upon exposure to external signals (N. de Ruijter; A. Geitmann, Wageningen University, The Netherlands) introduced the whole question of actin regulation. A number of actin binding proteins with physiologically relevant properties were localized to the appropriate regions of cells. These include villin (P. Hepler), profilin (L. Vidali), ARP 2/3 (F. Baluka; David Drubin, University of California at Berkeley, USA; Whitney Hable, University of Utah, USA) and ABP1 (D. Drubin). However, it is clear that this list is only just started. Hepler made a very compelling case for the integration of actin, actin binding proteins, and Ca²+ gradients in the organization of the apical cytoskeleton.

The factors instrumental in initiating and controlling the assembly of the tip growth apparatus were investigated in developing algal zygotes (D. Kropf, University of Utah, USA; W. Hable), yeast buds (D. Drubin) and hyphal branches (I.B. Heath; Sandra Jackson, University of Christchurch, New Zealand). In each system, assembly of characteristic actin arrays adjacent to the plasma membrane was one of the earliest events described. Both Hable and Drubin were able to add actin-related proteins (ARP2/3) as participants in this ensemble. The factors that precede the assembly of the actin arrays remain elusive. Heath presented evidence that a pulse of [Ca²+]_cyt was part of the early signaling system, but Jackson was unable to detect such pulses and presented evidence from the characteristics of micropipette-applied inducers that Ca²+ were not initiators.

In contrast to abundant evidence for a critical role for actin in regulating tip growth, it was repeatedly emphasized that microtubules have only an indirect role. Polarized tubular growth could be initiated or maintained in the absence of microtubules in diverse cells (F. Baluka; Giampiero Cai, University of Siena, Italy; D. Kropf). However, normal steering (see "Steering and Navigating") does involve microtubules in as yet undetermined ways.

"SPECTRIN" CONTRIBUTES TOO
In at least two phylogenetically very different organisms, Neurospora hyphae and Chara rhizoids and protonemata, proteins with substantial similarities to animal spectrins contribute to tip growth. In Neurospora, they form a cap concentrated at the growing plasma membrane (I.B. Heath) whereas in Chara, conspicuously and surprisingly, they are not associated with the plasma membrane but instead concentrate in the apical accumulation of endoplasmic reticulum, which plays a vital role in gravitropism (B. Buchen). These proteins are primarily identified by their size and their reaction with antibodies and have previously been reported in other hyphal and root hair tips (Kaminskyj and Heath 1995 ; de Ruijter et al. 1998 ). Their precise relationship to animal spectrins remains to be seen, but their location and currently known similarities make them very interesting to the tip growth process.

ION REGULATION
All tip-growing cells that have been examined generate a tip-high gradient of cytoplasmic [Ca²+], which apparently is obligatorily involved in active growth (Malho et al. 1994 ; Holdaway-Clarke et al. 1997 ; Hyde and Heath 1997 ; Wymer et al. 1997 ). This observation was reinforced throughout the meeting (José Feijó, Instituto Gulbenkian Ciencia, Portugal; I.B. Heath; P. Hepler; S. Jackson; Rui Malhó, University of Lisbon, Portugal; Kenneth Robinson, Purdue University, USA). A similar gradient of cytoplasmic [H⁺] has also been reported in some cells (Feijo et al. 1999 ; J. Feijó; K. Robinson). Exciting aspects of the ion studies were both the increased precision of correlation between the concentrations in the gradients, ion fluxes measured with vibrating probes and oscillating growth rates (see "Subtle Jackhammers"), and the addition of other ion fluxes to the equation. For example, Robinson showed that [Ca²+]_cyt resulting from apical Ca²+ influx follows a growth pulse, leading to a model in which extension results in elevated [Ca²+]_cyt and stimulated exocytosis, producing the necessary membrane and wall material for the next growth pulse. The Ca²+ stimulation of exocytosis probably also relates to regulation of both vesicle transport and the properties of the actin cytoskeleton, indicating the multi-functional roles of Ca²+, as emphasized by Hepler. The Ca²+ influx is followed by H⁺ and K⁺ influxes, the significance of which is unclear. Feijó noted that K⁺ fluxes tended to be highly variable in magnitude and direction, suggesting that they simply balance other ions. Nevertheless, Polydefkis Hatzopoulos (Agricultural University of Athens, Greece) showed that K⁺ transport is critical for root hair growth (but, surprisingly, apparently not pollen tubes) in Arabidopsis.

It generally has been assumed, and demonstrated, that the tip-high gradient of [Ca²+]_cyt is generated and maintained by Ca²+ influx through the apical plasma membrane, but this is not always so (Lew 1999 ), leading to the suggestion of some form of internal recycling, or "bootstrapping" system (Jackson and Heath 1993 ). Sara Torralba (York University, Canada) presented the first evidence for such a system by showing Ca²+ concentrated in the secretory vesicles in Neurospora hyphal tips, adding a new level of complexity to the ion regulatory systems in tip growth.

While most attention has previously focused on cations, it is emerging that anions are also important. Both Feijó and Laura Zonia (Institute of Experimental Botany, Czech Republic) showed pulsed apical efflux of Cl^- related to pulsed growth of pollen tubes and concluded that this efflux is important to water fluxes and thus turgor regulation. The other anions introduced were reactive oxygen species. Robinson showed that O₂ consumption at pollen tube tips increases following a growth pulse, but apparently not as a supplier of metabolic energy. He suggested that apical superoxide is converted to H₂O₂, which functions to regulate pectin cross-linking and thus cell wall extensibility, a suggestion supported by his induction of tip swelling with the antioxidant ascorbate.

KINASES AND G-PROTEINS
Given the ubiquity of signaling systems based on kinases and G-proteins, it is not surprising that there was substantial evidence for their involvement in diverse tip-growth systems. Baluka showed the accumulation of MAP kinases at root hair initiation sites, Malhó found calcium kinases at the plasma membrane and in the cytoplasm in tips of pollen tubes, and Drubin showed the importance of two other kinases in yeast bud cytoskeleton organization. The latter seem to be most important for endocytosis. The most direct evidence for kinase function in tip morphogenesis came from the severe morphological distortions that John Esseling (Wageningen University, The Netherlands) induced by antisense inhibition of several serine/threonine kinases in Arabidopsis root hairs.

Assorted G-proteins are specifically expressed and/or localized in pollen tubes (Victor ársk, Institute of Experimental Botany, Czech Republic), yeast buds (D. Drubin) and mating fungal hyphal tips (Marjatta Raudaskoski, University of Helsinki, Finland).

In none of the systems has it been possible to integrate the kinase and G-protein data with the ion and cytoskeletal data to produce a clear and coherent story, although certainly the yeast bud system is fast approaching that state.

HOW THE MEMBRANES COME AND GO
Because tip growing systems entail so much exocytotic activity, it is important that they accurately orchestrate both the production and placement of their membrane producing systems. Previous observations (Miller et al. 1995 ; Moscatelli et al. 1995 ; Steinberg 1998 ) have described a number of molecular motors that are involved in vesicle and organelle transport to the growing tips. However, additional complexities emerged. For example, Geoff Hyde (University of New South Wales, Australia) showed that in response to perturbations, the distributions of both endoplasmic reticulum and a tubular vacuolar system of hyphae were altered in specific ways, indicative of positioning systems for these membrane components. The basis for these rearrangements was not addressed, but in Chara cells a different, but highly organized, apical aggregation of endoplasmic reticulum was maintained with the involvement of a spectrin-like protein (B. Buchen).

At a later step in the exocytotic pathway, both Rosa-Maria Lopez-Franco (Instituto Technologico de Estudios Superiores de Monterrey, Mexico) and Salomon Bartnicki-Garcia (University of California at Riverside, USA) demonstrated the behavior of a highly concentrated apical aggregation of vesicles and actin (Bourett and Howard 1991 ) known as a Spitzenkörper. Spitzenkörpers represent an unexplained stage in the direction of the vesicles to the apical plasma membrane. SNARE proteins assist in ensuring correct vesicle fusions during exocytosis, and for the first time in a tip-growing system, Gagan Gupta (York University, Canada) showed a tip-high gradient of SNAREs in hyphae of Neurospora.

Another feature of membrane behavior that attracted considerable attention was retrieval by endocytosis. Both Lichtscheidl and Drubin showed evidence for fluid phase endocytosis (presumably with concomitant membrane internalization) in diverse cells, apparently generated by plasma membrane–associated actin. In yeast, Drubin also showed that there are clathrin-interacting proteins involved. Nick Read (University of Edinburgh, Scotland) presented impressive images of the time course of internalization of a membrane-specific dye in fungal hyphae and showed evidence for it passing through a membrane recycling system to become exocytosed back to the tip. However, in his data there was no direct evidence for endocytosis of membrane segments, as opposed to excision, internalization, and recycling of individual dye molecules, an equally plausible and interesting interpretation.

STEERING AND NAVIGATING
Tip growth is a mechanism for exploring the environment and getting cellular constituents to specific locations. It follows that cells need to be able to detect environmental cues (See "Environmental Sensing") and steer accordingly. Both algal zygotes and fungal hyphae retain a sense of direction. Kropf showed that unless other stimuli were applied, algal zygotes remembered the site of sperm entry and used this as the cue for tip growth initiation. In the same system, Sherryl Bisgrove (University of Utah, USA) showed that both microtubules and actin interact in "remembering" and responding to this site. Likewise, Lopez-Franco showed that laser tweezer positioning of the hyphal Spitzenkörper could steer hyphal growth, but only up to a point. Attempts to force hyphae to grow backward failed; they clearly "knew" which way they should go and were not going to be deviated too far from the straight and narrow! The mechanisms were not investigated but could involve microtubules interacting with actin, as in the algal zygotes, because hyphae steer poorly following microtubule disruption (Riquelme et al. 1998 ). Demkiv also showed that moss protonemata autotropism is guided by microtubules.

Perhaps the most dramatic demonstration of steering, or the lack thereof, was Hartmut Quader's (Universtät Hamburg, Germany) demonstration of helical growth in pollen tubes treated with methylxanthines and cyclopiazonic acid. Both microtubules and actin filaments were implicated in its regulation, although loss of microtubules did not suppress it. Perhaps the most fascinating but unexplained aspect of this and previous demonstrations of helical tip growth is that they are evidence for a rotary motor in the tips.

Given the indications of cytoskeletal function in navigation, it is perhaps no surprise that Malhó (Malho et al. 1994 ) was able to show that [Ca²+]_cyt is also part of the steering system in pollen tubes.

SUBTLE JACKHAMMERS AND TURGOR REGULATION
Pulsatile growth, with concomitant oscillations in ion fluxes and [Ca²+]_cyt, is a well-established phenomenon in pollen tubes (Pierson et al. 1995 ; Geitmann and Cresti 1997 ; P. Hepler; K. Robinson), although it emerged at the meeting that such is not always the case. There are variations in this behavior between species and with age in a single species. Pulsed growth is also reported in hyphae (Lopez-Franco et al. 1994 ), but Jackson showed that such can arise from technical artifacts and that some hyphae lack pulses.

A number of people suggested that the pulses are primarily generated by local oscillations/changes of turgor pressure (J. Feijó; K. Robinson; L. Zonia) and may be part of an oscillatory feedback mechanism (with regular frequency) that includes turgor, secretion, wall plasticity, and ions all as regulatory elements. However, Nicholas Money (Miami University, USA) showed that some hyphae actually grow faster with "zero" (or at least very low) turgor pressure, making the important point that turgor is not essential for the process of tip growth itself. Nevertheless, he did point out that normal turgor is essential for hyphal penetration of solid substrates, thus indicating that the analogy between pulsatile growth and a jackhammer may not be far fetched!

ENVIRONMENTAL SENSING
Tip-growing cells need to communicate in complex ways with their environment, both to receive and to send signals. Examples of this are plant pathogenic fungi, in which Harvey Hoch (Cornell University, USA) showed the ability both to sense a correct (e.g., leaf) surface before they adhere and germinate and to locate stomata with minimal directed tip growth. Hyphae and spores sense micrometer-sized surface features and respond either by secreting adhesives that will attach to hydrophobic surfaces (including Teflon) or by steering their growing tips. The mechanisms are unclear but involve both the cytoskeleton and Ca²+. A fascinating aspect of this sensing is that it traverses the cell wall to the cytoplasm, a process likely to involve plasma membrane to cell wall linkages. Such have been shown to be mediated by RGD-recognizing integrin-like proteins (Correa et al. 1996 ). Ashley Garrill (University of Christchurch, New Zealand) showed evidence for similar adhesions in non-sensing hyphae, suggesting that they are widespread.

One of the most critical sensing systems involving tip-growing cells is that mediating pollen–stigma interactions. In compatible interactions, the pollen tubes must establish and retain appropriate adhesions with cells of the transmitting tissues. Both Patricia Bedinger (Colorado State University, USA) and Elizabeth Lord (University of California at Riverside, USA) described secreted proteins likely to be involved in these interactions. Bedinger described an extensin-like pollen tube wall protein, disruption of which leads to irregular spiral tube growth in styles, and Lord described a small protein and a polygalacturonan, both secreted by pollen tubes, which combine to mediate adhesion of the tube to stylar tissues. Incompatible interactions entail the exchange of signaling molecules between pollen and stigma, the result of which is the failure of pollen tube growth. Both Anna Kalinina (St. Petersburg State University, Russia) and Geitmann presented very interesting indicators of what these molecules may be. Kalinina was able to show that the incompatibility reactions of rye plants could be altered with Ca²+ channel blockers. Geitmann was able to extend a comparable system to the cytoskeleton by showing major changes in actin organization in poppy pollen exposed to an incompatibility (S lo-cus) protein. Most interestingly, these changes did not mirror those elicited by actin-depolymerizing drugs. They are to some degree similar to cytoskeletal changes in apoptotic cells, introducing the exciting idea that incompatibility may represent another example of apoptotic cell death in plant biology.

WHERE TO NEXT?
In concluding remarks, Robinson summarized a number of outstanding questions that emerged from the meeting. These include: 1) greater effort to understand the similarities and differences between species and cell types; 2) more focus on in situ pollen studies to determine the validity of the currently emerging consensus from in vitro studies; 3) improved preservation methods to better understand the critical organization of the players in all systems; 4) more emphasis on the mechanisms of two-way communication across the apical plasma membrane; 5) greater effort on understanding the localized properties and changes in the apical cell wall, and interactions with cytoplasmic turgor pressure; 6) focus on directionality memory mechanisms that determine the direction of growth of tubular cells, and 7) identification and analysis of mechanosensitive channels in pollen tubes and other cells where they have yet to be described. No doubt the answers to some of these questions will make a solid base for a future tip growth meeting, hopefully in less than the 10 years since the last multidisciplinary work on the topic (Heath 1990 ). Ensuring that the location for such a meeting matches the ambiance of Siena remains a challenge for the next organizers!

REFERENCES

Bourett, T.M., and Howard, R.J. (1991) Ultrastructural immunolocalization of actin in a fungus. Protoplasma 163:199-202[CrossRef].

Corrêa, A., Staples, R.C., and Hoch, H.C. (1996) Inhibition of thigmostimulated cell differentiation with RGD-peptides in Uromyces germlings. Protoplasma 194:91-102[CrossRef].

de Ruijter, N.C., Brook, M.B., Bisseling, T., and Emons, A.M.C. (1998) Lipochito-oligosaccahrides re-initiate root hair tip growth in Vicia sativa with high calcium and spectrin-like antigen at the tip. Plant J. 13:341-350[CrossRef].

Feijó, J.A., Sainhas, J., Hackett, G.R., Kunkel, J.G., and Hepler, P.K. (1999) Growing pollen tubes possess a constitutive alkaline band in the clear zone and a growth-dependent acidic tip. J. Cell Biol. 144:483-496[Abstract/Free Full Text].

Geitmann, A., Cresti, M., and Heath, I.B. (2000). Cell Biology of Plant and Fungal Tip Growth. NATO Science Series. Amsterdam: IOS Press.

Geitmann, A., and Emons, A.M.C. (2000) The cytoskeleton in plant and fungal cell tip growth. J. Microscopy 198:218-245[ISI][Medline].

Geitmann, A., and Cresti, M. (1997) Ca²+ channels control the rapid expansions in pulsating growth of Petunia hybrida pollen tubes. J. Plant Physiol. 152:439-447.

Heath, I.B. (1990). Tip Growth in Plant and Fungal Cells. San Diego: Academic Press.

Holdaway-Clarke, T., Feijó, J., Hackett, G., Kunkel, J., and Hepler, P. (1997) Pollen tube growth and the intracellular cytosolic calcium gradient oscillate in phase while extracellular calcium influx is delayed. Plant Cell 9:1999-2010[Abstract].

Hyde, G.J., and Heath, I.B. (1997) Ca²+ gradients in hyphae and branches of Saprolegnia ferax. Fung. Genet. Biol. 21:238-251[CrossRef].

Jackson, S.L., and Heath, I.B. (1993) The roles of calcium ions in hyphal tip growth. Microbiol. Rev. 57:367-382[Abstract/Free Full Text].

Kaminskyj, S.G.W., and Heath, I.B. (1995) Integrin and spectrin homologues, and cytoplasm-wall adhesion in tip growth. J. Cell Sci. 108:849-856[Abstract].

Lew, R.R. (1999) Comparative analysis of Ca²+ and H⁺ flux magnitude and location along growing hyphae of Saprolegnia ferax and Neurospora crassa. Eur. J. Cell Biol. 78:892-902[Medline].

Lopez-Franco, R., Bartnicki-Garcia, S., and Bracker, C.E. (1994) Pulsed growth of fungal hyphal tips. Proc. Natl. Acad. Sci. USA 91:12228-12232[Abstract/Free Full Text].

Malhó, R., Read, N.D., Pais, M.S., and Trewavas, A.J. (1994) Role of cytosolic free calcium in the orientation of pollen tube growth. Plant Journal 5:331-341[CrossRef][ISI].

Miller, D.D., Scordilis, S.P., and Hepler, P.K. (1995) Identification and localization of three classes of mysins in pollen tubes of Lilium longiflorum and Nicotiana alata. J. Cell Sci. 108:2549-2563[Abstract].

Moscatelli, A., DelCasino, C., Lozzi, L., Cai, G., Scali, M., Tiezzi, A., and Cresti, M. (1995) High molecular weight polypeptides related to dynein heavy chains in Nicotiana tabacum pollen tubes. J. Cell Sci. 108:1117-1125[Abstract].

Pierson, E., Li, Y., Zhang, H., Willemse, M., Linskens, H., and Cresti, M. (1995) Pulsatory growth of pollen tubes: investigation of a possible relationship with the periodic distribution of cell wall components. Acta Bot. Neerl. 44:121-128.

Riquelme, M., Reynaga-Peña, C.G., Gierz, G., and Bartnicki-Garcia, S. (1998) What determines growth direction in fungal hyphae? Fung. Genet. Biol. 24:101-109.

Steinberg, G. (1998) Organelle transport and molecular motors in fungi. Fung. Genet. Biol. 24:161-177[CrossRef].

Wymer, C., Bibikova, T., and Gilroy, S. (1997) Cytoplasmic free calcium distributions during the development of root hairs of Arabidopsis thaliana. Plant J. 12:427-439[CrossRef][ISI][Medline].

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Heath, I. B. Articles by Geitmann, A. Search for Related Content PubMed PubMed Citation Articles by Heath, I. B. Articles by Geitmann, A. Agricola Articles by Heath, I. B. Articles by Geitmann, A.