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Cytoskeletal organization and pollen tube growth
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reviews
Cytoskeletal organization and pollen tube growth GiampieroCa,, Alessandra MoscateHi and Maure Cresti The growth of pollen tubes is characterized by intense secretory activity in the tip region. This process of vesicle-mediated secretion and tip growth is strongly influenced by calcium gradients. The cytoskeletal apparatus is also critically involved, as it is required for the translocation of organelles along the tube (a prerequisite for tube extension) and for the transport of the generative/sperm cells. The microtubules and actin filaments probably have distinct functions that relate to different, but related, cytological events within the pollen tubes. Both systems, as well as cytoskeleton-based motor proteins, are necessary for the proper development and growth of the pollen tubes. Different approaches have allowed the roles of several cytoskeletal components to be deciphered, and it is now possible to speculate how they might interact. he pollen tube is a cellular extrusion of the pollen grain, and forms after germination of the pollen on the stigma of a receiving flower. The function of the pollen tube is to transmit gametes from the pollen grain to the ovary, and this can occur over long distances (many
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Fig. 1. Microtubulesin pollen tubes. The electron micrograph in (a) was obtained after chemicalfixation, and shows the arrangement of microtubules (arrowheads) near to the central region of pollen tubes ofSciIla bifolia. The image in (b) was obtained after physical fixation, and shows microtubules lying just beneath the plasma membrane in the pollen tube of tobacco. Abbreviation: CW, cell wall. Scale bars represent 0.5 t~m. Courtesy of Fabrizio Ciampolini (a) and Claudio Milanesi (b).
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centimetres in some cases) 1. Because of its fundamental importance to the process of fertilization in higher plants, the pollen tube has recently been subjected to intensive study, with the aim of understanding the cell biology involved and regulating it through biotechnology2. The pollen tube is one of several tip-growing cells others include fungal hyphae and root hairs - that show a high growth rate. Although pollen tube growth has been analyzed by a combination of techniques (including ultrastructural, cytological, biochemical, immunological and molecular approaches), many details of the elongation process are not yet known. This review focuses on recent insights into the molecular mechanisms of pollen tube growth, and considers those aspects that require further analysis,
The cytoskeleton during pollen tube growth: microtubules and actin filaments Like every other eukaryotic cell, the pollen tube contains an elaborate cytoskeletal apparatus, which mainly consists of microtubules and actin filaments. The pollen tube cytoskeleton has been extensively studied using fluorescence and electron microscopy following chemical or physical fixation methods. The microtubules extend along the main axis of pollen tubes and are generally more concentrated in the cortical region of the tubes, although this is not a feature of all species3 (Fig. 1). The orientation of actin filaments appears similar to that of microtubules, and the two systems sometimes clearly colocalize 4. However, the components responsible for such an alignment are presently unknown. Both cytoskeletal systems originate in the pollen grain and develop within the growing pollen tube 3. Unfortunately, few data are available on the assembly and dynamics of either microtubules or actin filaments. It is likely that the equilibrium between monomeric and filamentous actin is controlled by proteins related to profilinS; however, with the exception of two centrosomal-related polypeptides, the localization and structure of microtubule-organizing centres is unknown 6'7. The localization of one of the centrosomal antigens in the pollen tube cortex7 indicates that microtubule-organizing centres of pollen tubes may be associated with the plasma membrane, as in other plant cell types.
© 1997 Elsevier Science Ltd
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reviews Organelle movement and the cytoskeleton Observations using video microscopy have shown that pollen tubes possess intense organelle translocation activity (Fig. 2). This dynamic movement concerns the generative cell and the vegetative nucleus, and includes the larger organelles (such as mitochondria, plasrids, endoplasmic reticulum and dictyosomes) and the small secretory vesicles. The translocation activity is based on the cytoskeletal apparatus. Investigations with specific cytoskeletal inhibitors have shown that drugs that affect actin filaments, such as cytochalasins, disorganize the actin network and inhibit tube elongation 3. Colchicine (a microtubule-depolymerizing agent) has slightly different effects: tube growth is blocked, but only when the chemical is present at very high concentrations, and inhibition is not as marked as with cytochalasins 3. More recently, the inhibitory effects of microtubule drugs have been reexamined, either by making use of more efficient inhibitors (such as oryzalin), or by using in vivo conditions. These studies have shown that microtubules participate in the regulation of processes such as the translocation of the generative cell/ vegetative nucleus s, internal tube organization 9'1° and the pulsatory growth of pollen tubes n. Microtubules are also involved in the process of generative cell division, which produces two sperm cells 12.
Fig. 2. The active movement of organelles along the growing pollen tube of tobacco. Organelles were stained with the fluorescent dye DiOC~.The pollen tube was observed using a fluorescent microscope equipped with a charge-coupled device (CCD) camera combined with an image processor; images were taped with a video recorder. The two sets of sequences (a-d and e-h) contain frames from the video at intervals of 2 s. Although the tube cytoplasm is crowded, some organelles (arrowheads) can be taken as examples of the opposite movements occurring in the pollen tube. Note the cortical translocation of organelles towards the tip (white arrows) and the reverse movement in the central part of the tube (black arrows). The tube tip is indicated by the broken line in (a). Bar represents 10 ~m.
The role of actin filament-based motor proteins Inhibitory experiments with cytochalasins have suggested that the actin skeleton is principally responsible for cytoplasmic streaming and tube growth. The presence of myosin (an actin filament-based motor protein) in the pollen tube was first postulated following the observation that pollen organelles could move along Characean actin bundles in a Ca2+-dependent manner ~8. Subsequently, myosin has been identified using immunological approaches 14. Antibodies raised to the myosin heavy chain have revealed that a related polypeptide of 175 kD occurs in association with the generative cell and vegetative nucleus in pollen tubes. The polypeptide has also been found along the pollen tube, with a punctate pattern suggesting its association with pollen organelles 14. This evidence has been confirmed using immunogold labelling, which has shown a myosinimmunoreactive polypeptide of 174 kD localized in association with a variety of organelles, including small vesicles, mitochondria and the generative cell 1~. The myosin staining is found in defined patches along the outer membrane of the generative cell, suggesting that the myosin polypeptides are precisely organized on the cell surface. More recently, further information has been obtained on the role and structure of myosins from pollen tubes. A 170 kD polypeptide, which is able to translocate actin
filaments in in vitro motility assays, has been purified from lily pollen and shown to act as an actin filament-activated ATPase (Ref. 16). The polypeptide has been isolated as a soluble component, and used as an antigen to obtain specific antibodies directed to the supposed plant myosin. Immunolocalization studies have subsequently shown that the 170 kD myosin polypeptide resides mainly in the apical part of pollen tubes with a punctate pattern - the pattern resembles the distribution of organelles 17. The purification of pollen-specific myosin opens new perspectives for the fine analysis of the acto-myosin-based organelle translocation, as well as for the study of potential regulatory factors. A second significant result has been obtained using antibodies that are specific to some myosin classes. Putative members of myosin class I, II and V have been identified in pollen tubes of Lilium longiflorum and Nicotiana alata is. The three myosin polypeptides have different molecular masses, as well as dissimilar localization within the pollen tube. Consequently, it may be that each type of organelle is translocated by a different myosin molecule. This could account for the observation that pollen tube organelles move as singular, autonomous elements during tube elongation 19, and suggests that different motors are used as part of a general control system. The actin-myosin system appears to be responsible for the organeUe translocation activity along pollen tubes and, March 1997, Vol. 2, No. 3
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Microtubules and motor proteins The role of microtubules during pollen tube growth has often been con~AF yo troversial. Although the microtubule cytoskeleton is as abundant as that of actin filaments, the depolymerization of microtubules by specific inhibitors does C not inhibit the rate of pollen tube growth to the same extent ~. This suggests that microtubules are not strictly required for tube extension. Nevertheless, the chemical depolymerization of microtubules can generate at least three main effects: • First, the translocation rate of the generative cell and vegetative nucleus Fig. 8. Diagram showing the different roles that microtubules (MT) and actin filaments is decreased after the vegetative micro(AF) may perform in the translocation of the generative cell. It is suggested that myosin tubules have been disorganized s. molecules (Myo),which are likely to be located on the outer membrane of the generative Although the movement is not comcell (GC), may interact with actin filaments and promote the translocation of the generpletely blocked, this suggests that the ative cell. Microtubules do not seem to play a direct role in this movement. microtubules play a role in regulating Depolymerization of microtubules (dp MT) can induce a decrease in the translocation the movement of the male germ unit. rate of the generative cell, and this may change the organization of actin filaments This function could be carried out by around the generative cell in such a way as to modify their proper interaction with myosins and, consequently, to alter the generative cell translocation rate. Hypothetical assisting the alignment between the proteins involved in the microtubule-mediated arrangement of actin actin skeleton and the generative cellfilaments are indicated by question marks. Abbreviation: GN, generative nucleus. associated myosin molecules (Fig. 3). • Second, the disorganization of the microtubule apparatus in very old consequently, for pollen tube growth. However, some points pollen tubes (22 h after in vitro germination) causes the loss still remain unclear. For example, organelle movement of the internal tube organization and the cessation of pollen within pollen tubes is bidirectional between the grain and tube growth 9. These results have also been confirmed in vivo, the tip, but all myosins move towards the barbed end of actin where the role of the cytoskeleton could also be regulated by filaments. Consequently, the bidirectional translocation of external factors 1°. organelles requires actin filaments to be arranged in both • Third, the disarrangement of the microtubule cytoskeleton orientations within pollen tubes (although this has yet to be temporarily prevents rapid elongation in pulsating pollen demonstrated). There are also questions about whether the tubes ~1. acto-myosin system is responsible for the movement of secreThe involvement of microtubules might require the tory vesicles in the tube tip. Doubt about this has arisen presence of auxiliary components, such as microtubulefrom the conflicting results on the localization of actin fila- associated proteins and/or motors. No information is curments in the tip. Pollen tubes prepared with conventional rently available on microtubule-associated proteins, but comfixatives show actin cables in the tube tip (see Ref. 20). How- ponents related to the kinesin 24 and dynein 2~ families have ever, these results have not been confirmed in studies per- been identified in pollen tubes. In eukaryotic cells, members formed using physical fixation methods 2~. In contrast, these of the kinesin and dynein families are normally involved in suggest that an ordered and dense network of actin illa- the dynamic organization of the cytoplasm 26. Although the ments is found only in the subapical region of the tube, precise roles of the kinesin- and dynein-related polypeptides whereas the tip contains an unstructured mass of relatively in pollen tubes is not yet clearly established, they appear to few shorter actin filaments. Similar to other cell types, tip- be involved in organelle positioning and/or translocation 27. associated actin may also be linked to the plasma mere- The localization of the kinesin-related polypeptide suggests brane and regulate the organization of membrane proteins: that it is involved in events occurring in the tube cortex, some membrane constituents, such as Ca 2÷ channels, have especially in the apical region 2s. The pollen kinesin homobeen suggested to be constantly associated with the tip logue could have fimctions similar to those of yeast Smylp plasma membrane 22. It is implicit in this hypothesis that an (Refs 20 and 29), a kinesin-like protein probably active in intrinsic molecular mechanism exists to regulate the pos- focusing secretory vesicles to regions of polarized growth. ition of Ca 2÷ channels in the plasma membrane. In support Kinesin and dynein molecules in pollen tubes may serve of this possibility, spectrin-like molecules have been local- either to translocate organelles to a specific intracellular ized at the tube tip 2°, providing evidence for a structured position or to prevent their diffusion away from such a poscytoskeleton on the cytoplasmic side of the tip membrane ition. Alternatively, microtubule-based motors may have that may help to restrict the mobility of membrane proteins. a function in membrane recycling, which occurs at the As a second possibility, Ca 2÷ channels may be recycled in tube apex and is mediated by clathrin 23. Recently, different endocytotic vesicles and then reinserted in the tip mere- types of kinesin-like polypeptides have been identified in brane. This is supported by the localization of clathrin pollen tubes of Tradescantia and Nicotiana 8°. Using an 88
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reviews antibody against two peptides deduced from the Arabidopsis kinesin-like gene sequences, polypeptides of about 102-110 kD have been detected and appear to be involved in the division of the generative cell. A punctate staining pattern of variable intensity has also been reported in the vegetative cytoplasm. These results suggest that pollen tubes contain a set of kinesin-like polypeptides that occupy distinct locations and, consequently, perform different functions. Dynein-related polypeptides have been identified in pollen tubes by means of a polyclonal antibody raised against a peptide shown to be similar to dyneins in some biochemical properties (e.g. molecular mass, sedimentation coefficient and ATPase activity2~). Although the Fig. 4. Distribution of intracellular free Ca2÷in a growing pollen tube ofLilium longiflorum. The pollen tube was injected with fura-2-dextran (a fluorescent Ca2+indicatotal amount of dynein-related polytor) to obtain a pseudocolour-ratioimage. The Ca2÷concentration is given by reference peptides varies according to the germito the calibration bar. The red colour in the tube tip shows a very high concentration nation time of pollen tubes, their role is of Ca2÷,and the blue colour behind the tip represents a basal concentration. The tipstill uncertain. Evidence that dyneins focused Ca2+gradient is clearly evident. Bar represents 20 ~m. Modified, with perand kinesins usually translocate mission, from Ref. 44. organelles in opposite directions 26 led to speculation that the kinesin- and dynein-related components of pollen tubes act antagonistically to regulate organelle positioning. acid sequence to calmodulins from animal and other plant Further analysis, including the immunolocalization of cell types32. Immunohistochemical analysis suggests that the dyneins, is needed to determine whether such a system oper- calmodulin is probably involved in processes correlated with pollen tube extension 33, but its precise function is still ates in pollen tubes. It appears that microtubules and microtubule-based unclear. More information is available for Ca2÷, an essential commotors are not an absolute requirement for the growth ,of pollen tubes, but that they act to ensure'- that functional ponent for the growth of the pollen tube. Pollen tubes have growth is maintained. It is likely that the accumulation and been shown to contain an intracellular Ca2÷ gradient, which fusion of secretory vesicles in the tube tip does not require an is lilnited to the tip 34 (see Fig. 4). The Ca2÷ gradient is likely intact microtubule cytoskeleton or the presence of micro- to be maintained by both the influx of Ca 2÷ through Ca2÷ tubule-based motors. However, some features of the pollen channels located in the tip and the Ca2+-sequestering activity tube organization (such as the coupling of the tube growth of the endoplasmic reticulum 3~. The tip-focused Ca2÷gradient rate with the translocation velocity of the generative cell/ is thought to mark the precise area of the tip membrane vegetative nucleus and the distinct placement of organelles) where secretory vesicles fuse. The Ca2÷-dependent, phosphomay demand the existence of dynamic processes based on lipid-binding members of the annexin family of proteins microtubules. (which also localize to the tube apex8~) may mediate this process of vesicle fusion. The existence of a Ca2÷ gradient is fundamental to the polarized growth of pollen tubes, because The Ca 2. gradient and cytoskeleton activity during tip growth dissipation of the gradient can halt tube growth and a local Dynamic processes such as organelle translocation are increase in free Ca2÷ concentration precedes reorientation regulated so that, when needed, a specific organelle is moved and regrowth ~7. Although the role of the Ca 2÷gradient during the growth in the required direction. Consequently, the activity of motor proteins has to be individually adjusted according to the par- of pollen tubes is becoming clearer, very little is known ticular physiological status of the cell. Myosins are typically about any possible interaction between the Ca2÷ gradient, activated/repressed in a variety of ways, including phos- intracellular organization and the cytoskeleton. One of the phorylation of the heavy or light chains, and binding to Ca2÷- consequences of dissipation of the Ca 2÷ gradient is the loss regulated proteins 81. Members of the kinesin and dynein fam- of the internal tube organization $4, but how this is achieved ilies also seem to be regulated by Ca2÷, Ca2÷-binding proteins is unclear. The loss of pollen tube polarity after dissipation and phosphorylation 2~. Similar control systems are likely to of the Ca 2+ gradient may be the result of changes induced regulate the activity of motor proteins in pollen tubes. This in the arrangement of the cytoskeleton. However, further has already been suggested by evidence that Ca~÷ can influ- studies on the dynamics of actin filaments after Ca 2÷gradience the sliding of pollen tube organelles along actin fila- ent dissipation are required to understand the relationship ments 13. Both Ca2÷, and the Ca2+-binding protein, calmodulin, between the two systems 21. It has been proposed~s that the have been subjected to intense study in pollen tubes during high level of Ca 2÷ in the tip may be one part of a system of the past few years. Calmodulin has been purified and shown control for actin filaments, in which actin-binding proteins, to be almost identical in biochemical properties and amino such as profilin ~, may also be involved. March 1997, Vol. 2, No. 3
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tubes is currently poor, and an important research objective will be the identiActivated fication and characteriza,,m. ; ~ Ca2+ II Ca2+-channel tion of further motor pro+ teins. For example, the Annexin-like protein o,,-',-_~. • .~..'%\ • • • ooo o~,~t discovery of a myosin gene Calmodulin family and kinesin-like f . . . . ", ..t 1~-_-,I.'..:._':4~ -'...genes in Arabidopsis 42'4~ Calcium opens the way for searching Myosin for related gene sequences ~ ~ , . . . . . A--J ^ ~ 1,,C).~r-~¶..~. . ~ ~ ( I::::::n] . ~4)-%.~-t__~ o expressed during pollen ~ f - ~ , • r~ ...-~..~--~Ca~+ zzo::z~:::c~o2z:z~ ( I ?--v., "~ ~'.':'%7ell MT-based motor germination and growth. "r .\ 2%; . ~ 7?~ Many lines of evidence • .~. :..'..w ~ ~.) Microtubule ° °° eoee o°• suggest that the tube tip - ~ "." ~ ( J;-¢,41~"~ Q::Z::O Actin filament plays a role in modulating ~ .l_ .....:.y " Ca pollen tube growth by conSecretory vesicle "1"* ~ : ~ ' T •e °°•/ "." trolling the fusion rate of Clathrin secretory vesicles and the local organization of the Plasma membrane cytoskeleton. Consequently, another important area of Fig. 5. Diagram showing the principal components of a pollen tube and a hypothetical interpreresearch is the relationship tation of the involvement of these components in the growth process. The Ca2÷gradient may well play a central role in determining the polarized growth of pollen tubes. It is likely that this is between cytoskeletal dymaintained by the opposing activities of ion channels, which allow influx of Ca2+to the tip, and of nanlics, the Ca2+ gradient, Ca2+-sequestering organelles, such as the endoplasmic reticulum (ER). High Ca2÷levels in the tip cell wall formation and may facilitate the docking and fusion of secretory vesicles through the activity of annexin-like protube growth ~4. The identiteins. Clathrin molecules are likely to take part in membrane recycling. Also, Ca~+and Ca2÷-actification of kinesin-like vated proteins, such as calmodulin, may exert regulatory activity on both cytoskeletal fibrils and gene sequences with a motor proteins (open arrows). At the present time, however, this hypothesis is only speculative calmodulin-binding domain (indicated by question marks). The several distinct myosin molecules that have been identified in in potato and Arabidopsis pollen tubes are indicated as a single generic motor involved in the translocation of organelles suggests that Ca 2+ may (Org) and vesicles. Microtubule (MT) based motors are also shown interacting with generic regulate the activity of organ•lies (X), although this role remains to be clearly determined. For clarity, the cell wall of the pollen tube is not included. microtubule-based motors in plant cells 45. Figure 5 shows the main compo= Although it is not known whether Ca2+or the dissipation nents of the pollen tube apex and the hypothetical interacof a Ca 2+ gradient may affect the microtubutar organ- tions that may occur between them during pollen tube ization in pollen tubes, it is possible to speculate by com- growth. The recent identification of transmitting tissue-specific paring the pollen tube to other plant cells. For example, microtubules from carrot cells form a bundle following the arabinogalactan proteins that attract pollen tubes and addition of a plant homologue of elongation factor-la, and stimulate their growth in vitro 46 creates new possibilities for this activity is abolished by a Ca2+-calmodulin complex~9. the study of compounds that might regulate pollen tube There is no evidence for the presence of homologues of growth by acting on the cytoskeletal apparatus and the Ca 2+ elongation factor-la in pollen tubes, but it is tempting to gradient. The discovery that polypeptides related to Rho suppose that Ca2÷/calmodulin in the apex may prevent GTPases are present in the tube tip 47 suggests that the microtubules from forming bundles. According to this assembly of actin filaments in response to external signals view, the apical localization of centrosomal antigens 7 could in pollen tubes may use molecular mechanisms that are support a role for the tube apex as a putative microtubule- very similar to those present in animal cells. Proteins organizing site. related to integrins (transmembrane proteins that connect the cytoskeleton to extracellular matrix molecules, such as Future perspectives vitronectin) have not yet been identified in pollen tubes. The ability to use the pollen tube system for biotechno- However, integrin-like proteins have been identified and logical applications 4° depends on the ability to decode the linked to tip growth in the fungus Saprolegnia ts, and vitromolecular machinery required for pollen tube growth. nectin-like proteins occur in the stylar transmitting tissue 49. Although many details of pollen tube growth remain It is therefore possible that proteins that play a vital role in unclear, these are being clarified, and the number of known linking the cytoskeleton to the extracetlular matrix in other molecular components is steadily increasing. Recent studies eukaryotic cells may also serve in the growth and guidance have shown that cells can modulate dynamic processes of pollen tubes. using antagonist motor proteins, which could be involved in a 'tug-of-war' process. For example, several kinesin and Acknowledgements dynein molecules participate with different and antithetical The authors are grateful to Prof. Peter Hepler, Prof. roles in the organization of the spindle apparatus 41. Unfor- Antonio Tiezzi, Dr Yi-Qin Li and Dr Anja Geitmann for caretunately, the number of motor proteins identified in pollen ful reading of the manuscript, criticisms and suggestions. .
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reviews References 1 Cresti, M., Blackmore, S. and van Went, J.L. (1992)Atlas of Sexual Reproduction in Flowering Plants, Springer 2 Faure, J.E. et al. (1996) Emerging data on pollen tube growth and fertilization in flowering plants, 1990-1995, Protoplasma 193, 132-143 3 Pierson, E.S. and Cresti, M. (1992) Cytoskeleten and cytoplasmic *~ organization of pollen and pollen tubes, Int. Rev. Cytol. 140, 73-125 4 Lancelle, S.A. and Hepler, P.K. (1991)Association of actin with cortical microtubules revealed by immunogold localization in Nicotiana pollen tubes, Protoplasma 165, 167-172 5 Mittermann, I. et al. (1995)Molecular cloning and characterization of profilin from tobacco (Nicotiana tabacum): increased profilln expression during pollen maturation, Plant Mol. Biol. 27, 137-146 6 Palevitz, B.A.,Liu, B. and Joshi, C. (1994) ~-tubulin in tobaccopollen tubes: association with generative cell and vegetative microtubules, Sex. Plant Reprod. 7, 209-214 7 Cai, G. et al. (1996) The anti-centrosome mAb 6C6 reacts with a plasma membrane-associated polypeptide of 77-kDa from the Nicotiana tabacum pollen tubes, Protoplasma 190, 68-78 8/~_strSm, H., Sorri, O. and Randaskoski, M. (1995) Role of microtubules in the movement of the vegetative nucleus and generative cell in tobacco pollen tubes, Sex. Plant Reprod. 8, 61-69 9 Joos, U., van Aken, J. and Kristen, U. (1994) Microtubules are involved in maintainingthe cellular polarity in pollen tubes ofNicotlana sylvestris, Protoplasma 179, 5-15 10 Joos, U., van Aken, J. and Kristen, U. (1995) The anti-microtubule drug carbetamide stops Nicotiana sylvestris pollen tube growth in the style, Protoplasma 187, 182-191 11 Geitmann,A., Li, Y.Q. and Cresti, M. (1995) The role of cytoskeleten and dictyosome activity in the pulsatery growth ofNicotiana tabacum and Petunia hybrida pollen tubes, Bot. Acta 109, 102-109 12 Zhang, H.Q. et al. (1995)Microtubular organization during asymmetrical division of the generative cell in Gagea lutea, J. Plant Res. 108, 269-276 13 Khono, T. and Shimmen, T. (1988) Accelerated sliding of pollen tube organelles along Characeae actin bundles regulated by Ca2~,J. Cell Biol. 106, 1539-1543 14 Tang, X., Hepler, P.K. and Scordilis, S P. (1989) Immunochemical and immunocytochemicalidentification of a myosin heavy chain polypeptide in Nicotiana pollen tube, J. Cell Sci. 92, 569-574 15 Tirlapur, U. et al. (1995) Confocalimaging and immunogold electron microscopyof changes in distribution of myosin during pollen hydration, germination and pollen tube growth in Nicotiana tabacum L., Eur. J. Cell Biol. 67, 209-217 16 Yokota, E. and Shimmen, T. (1994) Isolation and characterization of plant myosin from pollen tubes of lily, Protoplasma 177, 153-162 17 Yokota, E. et al. (1995)Localization of a 170 kDa myosin heavy,chain in plant cells, Protoplasma 185, 178-187 18 Miller, D.D., Scordilis, S.P. and Hepler, P.K. (1995) Identification and localization of three classes of myosins in pollen tubes of Lilium longiflorum and Nicotiana alata, J. Cell Sci. 108, 2549-2563 19 Pierson, E.S., Lichtscheidl, I.L. and Derksen, J. (1990) Structure and behaviour of organelles in livingpollen tubes ofLilium longiflorum, J. Exp. Bot. 41, 1461-1468 20 Derksen, J. et al. (1995) Regulation of pollen tube growth, Acta Bot. Neerl. 44, 93-119 21 Miller, D.D., Lancelle, S.A. and Hepler, P.I~ (1996)Actin filaments do not form a dense meshwork in Lilium longiflorum pollen tube tips, Protoplasma 195, 123-132 22 Malhb, R. et al. (1995) Calcium channel activity during pollen tube growth and reorientation, Plant Cell 7, 1173-1184 23 Blackbourn, H.D. and Jackson, A.P. (1996) Plant clathrin heavy chain: sequence analysis and restricted localisation in growing pollen tubes, J. Cell Sci. 109, 777-786 24 Tiezzi,A~et aL (1992)An immm~oreactivehomologof mammalian kinesin in Nicotiana tabacum pollen tubes, Cell Motil. Cytoskeleton 21, 132-137 25 Moseatelli,A. et aL (1995)High molecular weight polypeptides related to dynein heavy chains in Nicotiana tabacum pollen tubes, J. Cell Sci. 108, 1117-1125 26 Cole, N.B. and Lippincott-Schwartz, J. (1995) Organization of organelles and membrane traffic by microtubules, Curr. Opin. Cell Biol. 7, 55-64
27 Liu, G.Q. et al. (1994)Kinesin-related polypeptide is associated with vesicles from Corylus avellana pollen, Cell MotE. Cytoskeleton 29, 155-166 28 Cai, G. et al. (1993)The kinesin-immunoreactive homologuefrom Nicotiana tabacum pollen tube: biochemical properties and subcellular localization,Planta 191, 496-506 29 Lillie, S.H. and Brown, S.S. (1994) Immunofluorescence localization of the unconventional myosin, Myo2p, and the putative kinesin-related protein, Smylp, to the same region of polarized growth in Saccharomyces cerevisiae, J. Cell Biol. 125, 825-842 30 Liu, B. and Palevitz, B.A. (1996) Localization ofa kinesin-like protein in the generative cells oftebacco, Protoplasma 195, 78-89 31 Hasson, T. and Mooseker, M.S. (1995) Molecular motors, membrane movements and physiology:emerging roles for myosins, Curr. Opin. Cell Biol. 7, 587-594 32 Scali, M. et al. (1994) Purification and biochemical characterization of calmodulin from Corylus avellana pollen, Plant Physiol. Biochem. 32, 831-838 33 Tirlapur, U.K. et aL (1994) Confocalimage analysis of spatial variations in immunocytochemicallyidentified calmodulin during pollen hydration, germination and pollen tube tip growth in Nicotiana tabacum L., Zygote 2, 63-68 34 Pierson, E.S. et al. (1994) Pollen tube growth is coupled to the extracellular calcium ion flux and the intracellular calcium gradient: effect of BAPTA-typebuffers and hypertonic media, Plant Cell 6, 1815-1828 35 Pierson, E.S. et al. (1996)Tip-localizedcalcium entry fluctuates during pollen tube growth, Dev. Biol. 174, 160-173 36 Blackbourn, H.D. et al. (1992)Properties and partial protein sequence of plant annexins, Plant Physiol. 99, 864-871 37 Malh6, R. et al. (1994) Role of cytesolicfree calcium in the reorientation of pollen tube growth, Plant J. 5, 331-341 38 Khono, T. and Shimmen, T. (1987) Ca2+-induced fragmentation of actin filaments in pollen tubes, Protoplasma 141, 177-179 39 Durso, N.A. and Cyr, R.J. (1994)A calmodulin-sensitive interaction between microtubules and a higher plant homologof elongation factor-l~, Plant Cell 6, 893-905 40 Dumas, C. and Mogensen, H.L. (1993) Gametes and fertilization: maize as a model system for experimental embryogenesis in flowering plants, Plant Cell 3, 1337-1348 41 Vernos, I. and Karsenti, E. (1996) Motors involved in spindle assembly and chromosomesegregation, Curr. Opin. Cell Biol. 8, 4-9 42 Kinkema, M., Wang, H.Y. and Schiefelbein,J. (1994) Molecular analysis of the myosin gene family in Arabidopsis thaliana, Plant Mol. Biol. 26, 1139-1153 43 Mitsul, H. et al. (1993) Identification of a gene family (kat) encoding kinesin-like proteins in Arabidopsis thaliana and the characterization of secondary structure of KatA, Mol. Gen. Genet. 238, 362-368 44 Li, Y.Q. et al. (1996) Enforced growth-rate fluctuation causes pectin ring formation in the cell wall ofLilium longiflorum pollen tubes, Planta 200, 41-49 45 Reddy, A.S.N. et al. (1996)A plant kinesin heavy chain-like protein is a calmodulin-bindingprotein, Plant J. 10, 9-21 46 Cheung, AX., Wang, H. and Wu, H. (1995) A floral transmitting tissuespecific glycoproteinattracts pollen tubes and stimulates their growth, Cell 82, 383-393 47 Lin, Y. et al. (1996)Localization ofa Rho GTPase implies a role in tip growth and movement of the generative cell in pollen tubes, Plant Cell 8, 293-303 48 Kaminskyj, S.G.W.and Heath, I.B. (1995) Integrin and spectrin homologues, and cytoplasm-walladhesion in tip growth, J. Cell Sci. 108, 849-856 49 Sanders, L.C. et al. (1991)A homologof the substrate adhesion molecule vitronectin occurs in four species of flowering plants, Plant Cell 3, 629-635 Giampiero Cai*, Nessandra M0scatefli and Mauro Crest are at the Dept of Environmental Biology, University of Siena, • Via Mattioli 4, 531 D0 Siena, Italy. *Author for correspondence (teN-39 577 298856; fax +39 577 298860: e-mail cai@unisLit),
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Cytoskeletal organization and pollen tube growth GiampieroCa,, Alessandra MoscateHi and Maure Cresti The growth of pollen tubes is characterized by intense secretory activity in the tip region. This process of vesicle-mediated secretion and tip growth is strongly influenced by calcium gradients. The cytoskeletal apparatus is also critically involved, as it is required for the translocation of organelles along the tube (a prerequisite for tube extension) and for the transport of the generative/sperm cells. The microtubules and actin filaments probably have distinct functions that relate to different, but related, cytological events within the pollen tubes. Both systems, as well as cytoskeleton-based motor proteins, are necessary for the proper development and growth of the pollen tubes. Different approaches have allowed the roles of several cytoskeletal components to be deciphered, and it is now possible to speculate how they might interact. he pollen tube is a cellular extrusion of the pollen grain, and forms after germination of the pollen on the stigma of a receiving flower. The function of the pollen tube is to transmit gametes from the pollen grain to the ovary, and this can occur over long distances (many
T
Fig. 1. Microtubulesin pollen tubes. The electron micrograph in (a) was obtained after chemicalfixation, and shows the arrangement of microtubules (arrowheads) near to the central region of pollen tubes ofSciIla bifolia. The image in (b) was obtained after physical fixation, and shows microtubules lying just beneath the plasma membrane in the pollen tube of tobacco. Abbreviation: CW, cell wall. Scale bars represent 0.5 t~m. Courtesy of Fabrizio Ciampolini (a) and Claudio Milanesi (b).
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centimetres in some cases) 1. Because of its fundamental importance to the process of fertilization in higher plants, the pollen tube has recently been subjected to intensive study, with the aim of understanding the cell biology involved and regulating it through biotechnology2. The pollen tube is one of several tip-growing cells others include fungal hyphae and root hairs - that show a high growth rate. Although pollen tube growth has been analyzed by a combination of techniques (including ultrastructural, cytological, biochemical, immunological and molecular approaches), many details of the elongation process are not yet known. This review focuses on recent insights into the molecular mechanisms of pollen tube growth, and considers those aspects that require further analysis,
The cytoskeleton during pollen tube growth: microtubules and actin filaments Like every other eukaryotic cell, the pollen tube contains an elaborate cytoskeletal apparatus, which mainly consists of microtubules and actin filaments. The pollen tube cytoskeleton has been extensively studied using fluorescence and electron microscopy following chemical or physical fixation methods. The microtubules extend along the main axis of pollen tubes and are generally more concentrated in the cortical region of the tubes, although this is not a feature of all species3 (Fig. 1). The orientation of actin filaments appears similar to that of microtubules, and the two systems sometimes clearly colocalize 4. However, the components responsible for such an alignment are presently unknown. Both cytoskeletal systems originate in the pollen grain and develop within the growing pollen tube 3. Unfortunately, few data are available on the assembly and dynamics of either microtubules or actin filaments. It is likely that the equilibrium between monomeric and filamentous actin is controlled by proteins related to profilinS; however, with the exception of two centrosomal-related polypeptides, the localization and structure of microtubule-organizing centres is unknown 6'7. The localization of one of the centrosomal antigens in the pollen tube cortex7 indicates that microtubule-organizing centres of pollen tubes may be associated with the plasma membrane, as in other plant cell types.
© 1997 Elsevier Science Ltd
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reviews Organelle movement and the cytoskeleton Observations using video microscopy have shown that pollen tubes possess intense organelle translocation activity (Fig. 2). This dynamic movement concerns the generative cell and the vegetative nucleus, and includes the larger organelles (such as mitochondria, plasrids, endoplasmic reticulum and dictyosomes) and the small secretory vesicles. The translocation activity is based on the cytoskeletal apparatus. Investigations with specific cytoskeletal inhibitors have shown that drugs that affect actin filaments, such as cytochalasins, disorganize the actin network and inhibit tube elongation 3. Colchicine (a microtubule-depolymerizing agent) has slightly different effects: tube growth is blocked, but only when the chemical is present at very high concentrations, and inhibition is not as marked as with cytochalasins 3. More recently, the inhibitory effects of microtubule drugs have been reexamined, either by making use of more efficient inhibitors (such as oryzalin), or by using in vivo conditions. These studies have shown that microtubules participate in the regulation of processes such as the translocation of the generative cell/ vegetative nucleus s, internal tube organization 9'1° and the pulsatory growth of pollen tubes n. Microtubules are also involved in the process of generative cell division, which produces two sperm cells 12.
Fig. 2. The active movement of organelles along the growing pollen tube of tobacco. Organelles were stained with the fluorescent dye DiOC~.The pollen tube was observed using a fluorescent microscope equipped with a charge-coupled device (CCD) camera combined with an image processor; images were taped with a video recorder. The two sets of sequences (a-d and e-h) contain frames from the video at intervals of 2 s. Although the tube cytoplasm is crowded, some organelles (arrowheads) can be taken as examples of the opposite movements occurring in the pollen tube. Note the cortical translocation of organelles towards the tip (white arrows) and the reverse movement in the central part of the tube (black arrows). The tube tip is indicated by the broken line in (a). Bar represents 10 ~m.
The role of actin filament-based motor proteins Inhibitory experiments with cytochalasins have suggested that the actin skeleton is principally responsible for cytoplasmic streaming and tube growth. The presence of myosin (an actin filament-based motor protein) in the pollen tube was first postulated following the observation that pollen organelles could move along Characean actin bundles in a Ca2+-dependent manner ~8. Subsequently, myosin has been identified using immunological approaches 14. Antibodies raised to the myosin heavy chain have revealed that a related polypeptide of 175 kD occurs in association with the generative cell and vegetative nucleus in pollen tubes. The polypeptide has also been found along the pollen tube, with a punctate pattern suggesting its association with pollen organelles 14. This evidence has been confirmed using immunogold labelling, which has shown a myosinimmunoreactive polypeptide of 174 kD localized in association with a variety of organelles, including small vesicles, mitochondria and the generative cell 1~. The myosin staining is found in defined patches along the outer membrane of the generative cell, suggesting that the myosin polypeptides are precisely organized on the cell surface. More recently, further information has been obtained on the role and structure of myosins from pollen tubes. A 170 kD polypeptide, which is able to translocate actin
filaments in in vitro motility assays, has been purified from lily pollen and shown to act as an actin filament-activated ATPase (Ref. 16). The polypeptide has been isolated as a soluble component, and used as an antigen to obtain specific antibodies directed to the supposed plant myosin. Immunolocalization studies have subsequently shown that the 170 kD myosin polypeptide resides mainly in the apical part of pollen tubes with a punctate pattern - the pattern resembles the distribution of organelles 17. The purification of pollen-specific myosin opens new perspectives for the fine analysis of the acto-myosin-based organelle translocation, as well as for the study of potential regulatory factors. A second significant result has been obtained using antibodies that are specific to some myosin classes. Putative members of myosin class I, II and V have been identified in pollen tubes of Lilium longiflorum and Nicotiana alata is. The three myosin polypeptides have different molecular masses, as well as dissimilar localization within the pollen tube. Consequently, it may be that each type of organelle is translocated by a different myosin molecule. This could account for the observation that pollen tube organelles move as singular, autonomous elements during tube elongation 19, and suggests that different motors are used as part of a general control system. The actin-myosin system appears to be responsible for the organeUe translocation activity along pollen tubes and, March 1997, Vol. 2, No. 3
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(
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heavy chains to the apical plasma membrane of pollen tubes 2~.
Microtubules and motor proteins The role of microtubules during pollen tube growth has often been con~AF yo troversial. Although the microtubule cytoskeleton is as abundant as that of actin filaments, the depolymerization of microtubules by specific inhibitors does C not inhibit the rate of pollen tube growth to the same extent ~. This suggests that microtubules are not strictly required for tube extension. Nevertheless, the chemical depolymerization of microtubules can generate at least three main effects: • First, the translocation rate of the generative cell and vegetative nucleus Fig. 8. Diagram showing the different roles that microtubules (MT) and actin filaments is decreased after the vegetative micro(AF) may perform in the translocation of the generative cell. It is suggested that myosin tubules have been disorganized s. molecules (Myo),which are likely to be located on the outer membrane of the generative Although the movement is not comcell (GC), may interact with actin filaments and promote the translocation of the generpletely blocked, this suggests that the ative cell. Microtubules do not seem to play a direct role in this movement. microtubules play a role in regulating Depolymerization of microtubules (dp MT) can induce a decrease in the translocation the movement of the male germ unit. rate of the generative cell, and this may change the organization of actin filaments This function could be carried out by around the generative cell in such a way as to modify their proper interaction with myosins and, consequently, to alter the generative cell translocation rate. Hypothetical assisting the alignment between the proteins involved in the microtubule-mediated arrangement of actin actin skeleton and the generative cellfilaments are indicated by question marks. Abbreviation: GN, generative nucleus. associated myosin molecules (Fig. 3). • Second, the disorganization of the microtubule apparatus in very old consequently, for pollen tube growth. However, some points pollen tubes (22 h after in vitro germination) causes the loss still remain unclear. For example, organelle movement of the internal tube organization and the cessation of pollen within pollen tubes is bidirectional between the grain and tube growth 9. These results have also been confirmed in vivo, the tip, but all myosins move towards the barbed end of actin where the role of the cytoskeleton could also be regulated by filaments. Consequently, the bidirectional translocation of external factors 1°. organelles requires actin filaments to be arranged in both • Third, the disarrangement of the microtubule cytoskeleton orientations within pollen tubes (although this has yet to be temporarily prevents rapid elongation in pulsating pollen demonstrated). There are also questions about whether the tubes ~1. acto-myosin system is responsible for the movement of secreThe involvement of microtubules might require the tory vesicles in the tube tip. Doubt about this has arisen presence of auxiliary components, such as microtubulefrom the conflicting results on the localization of actin fila- associated proteins and/or motors. No information is curments in the tip. Pollen tubes prepared with conventional rently available on microtubule-associated proteins, but comfixatives show actin cables in the tube tip (see Ref. 20). How- ponents related to the kinesin 24 and dynein 2~ families have ever, these results have not been confirmed in studies per- been identified in pollen tubes. In eukaryotic cells, members formed using physical fixation methods 2~. In contrast, these of the kinesin and dynein families are normally involved in suggest that an ordered and dense network of actin illa- the dynamic organization of the cytoplasm 26. Although the ments is found only in the subapical region of the tube, precise roles of the kinesin- and dynein-related polypeptides whereas the tip contains an unstructured mass of relatively in pollen tubes is not yet clearly established, they appear to few shorter actin filaments. Similar to other cell types, tip- be involved in organelle positioning and/or translocation 27. associated actin may also be linked to the plasma mere- The localization of the kinesin-related polypeptide suggests brane and regulate the organization of membrane proteins: that it is involved in events occurring in the tube cortex, some membrane constituents, such as Ca 2÷ channels, have especially in the apical region 2s. The pollen kinesin homobeen suggested to be constantly associated with the tip logue could have fimctions similar to those of yeast Smylp plasma membrane 22. It is implicit in this hypothesis that an (Refs 20 and 29), a kinesin-like protein probably active in intrinsic molecular mechanism exists to regulate the pos- focusing secretory vesicles to regions of polarized growth. ition of Ca 2÷ channels in the plasma membrane. In support Kinesin and dynein molecules in pollen tubes may serve of this possibility, spectrin-like molecules have been local- either to translocate organelles to a specific intracellular ized at the tube tip 2°, providing evidence for a structured position or to prevent their diffusion away from such a poscytoskeleton on the cytoplasmic side of the tip membrane ition. Alternatively, microtubule-based motors may have that may help to restrict the mobility of membrane proteins. a function in membrane recycling, which occurs at the As a second possibility, Ca 2÷ channels may be recycled in tube apex and is mediated by clathrin 23. Recently, different endocytotic vesicles and then reinserted in the tip mere- types of kinesin-like polypeptides have been identified in brane. This is supported by the localization of clathrin pollen tubes of Tradescantia and Nicotiana 8°. Using an 88
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reviews antibody against two peptides deduced from the Arabidopsis kinesin-like gene sequences, polypeptides of about 102-110 kD have been detected and appear to be involved in the division of the generative cell. A punctate staining pattern of variable intensity has also been reported in the vegetative cytoplasm. These results suggest that pollen tubes contain a set of kinesin-like polypeptides that occupy distinct locations and, consequently, perform different functions. Dynein-related polypeptides have been identified in pollen tubes by means of a polyclonal antibody raised against a peptide shown to be similar to dyneins in some biochemical properties (e.g. molecular mass, sedimentation coefficient and ATPase activity2~). Although the Fig. 4. Distribution of intracellular free Ca2÷in a growing pollen tube ofLilium longiflorum. The pollen tube was injected with fura-2-dextran (a fluorescent Ca2+indicatotal amount of dynein-related polytor) to obtain a pseudocolour-ratioimage. The Ca2÷concentration is given by reference peptides varies according to the germito the calibration bar. The red colour in the tube tip shows a very high concentration nation time of pollen tubes, their role is of Ca2÷,and the blue colour behind the tip represents a basal concentration. The tipstill uncertain. Evidence that dyneins focused Ca2+gradient is clearly evident. Bar represents 20 ~m. Modified, with perand kinesins usually translocate mission, from Ref. 44. organelles in opposite directions 26 led to speculation that the kinesin- and dynein-related components of pollen tubes act antagonistically to regulate organelle positioning. acid sequence to calmodulins from animal and other plant Further analysis, including the immunolocalization of cell types32. Immunohistochemical analysis suggests that the dyneins, is needed to determine whether such a system oper- calmodulin is probably involved in processes correlated with pollen tube extension 33, but its precise function is still ates in pollen tubes. It appears that microtubules and microtubule-based unclear. More information is available for Ca2÷, an essential commotors are not an absolute requirement for the growth ,of pollen tubes, but that they act to ensure'- that functional ponent for the growth of the pollen tube. Pollen tubes have growth is maintained. It is likely that the accumulation and been shown to contain an intracellular Ca2÷ gradient, which fusion of secretory vesicles in the tube tip does not require an is lilnited to the tip 34 (see Fig. 4). The Ca2÷ gradient is likely intact microtubule cytoskeleton or the presence of micro- to be maintained by both the influx of Ca 2÷ through Ca2÷ tubule-based motors. However, some features of the pollen channels located in the tip and the Ca2+-sequestering activity tube organization (such as the coupling of the tube growth of the endoplasmic reticulum 3~. The tip-focused Ca2÷gradient rate with the translocation velocity of the generative cell/ is thought to mark the precise area of the tip membrane vegetative nucleus and the distinct placement of organelles) where secretory vesicles fuse. The Ca2÷-dependent, phosphomay demand the existence of dynamic processes based on lipid-binding members of the annexin family of proteins microtubules. (which also localize to the tube apex8~) may mediate this process of vesicle fusion. The existence of a Ca2÷ gradient is fundamental to the polarized growth of pollen tubes, because The Ca 2. gradient and cytoskeleton activity during tip growth dissipation of the gradient can halt tube growth and a local Dynamic processes such as organelle translocation are increase in free Ca2÷ concentration precedes reorientation regulated so that, when needed, a specific organelle is moved and regrowth ~7. Although the role of the Ca 2÷gradient during the growth in the required direction. Consequently, the activity of motor proteins has to be individually adjusted according to the par- of pollen tubes is becoming clearer, very little is known ticular physiological status of the cell. Myosins are typically about any possible interaction between the Ca2÷ gradient, activated/repressed in a variety of ways, including phos- intracellular organization and the cytoskeleton. One of the phorylation of the heavy or light chains, and binding to Ca2÷- consequences of dissipation of the Ca 2÷ gradient is the loss regulated proteins 81. Members of the kinesin and dynein fam- of the internal tube organization $4, but how this is achieved ilies also seem to be regulated by Ca2÷, Ca2÷-binding proteins is unclear. The loss of pollen tube polarity after dissipation and phosphorylation 2~. Similar control systems are likely to of the Ca 2+ gradient may be the result of changes induced regulate the activity of motor proteins in pollen tubes. This in the arrangement of the cytoskeleton. However, further has already been suggested by evidence that Ca~÷ can influ- studies on the dynamics of actin filaments after Ca 2÷gradience the sliding of pollen tube organelles along actin fila- ent dissipation are required to understand the relationship ments 13. Both Ca2÷, and the Ca2+-binding protein, calmodulin, between the two systems 21. It has been proposed~s that the have been subjected to intense study in pollen tubes during high level of Ca 2÷ in the tip may be one part of a system of the past few years. Calmodulin has been purified and shown control for actin filaments, in which actin-binding proteins, to be almost identical in biochemical properties and amino such as profilin ~, may also be involved. March 1997, Vol. 2, No. 3
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tubes is currently poor, and an important research objective will be the identiActivated fication and characteriza,,m. ; ~ Ca2+ II Ca2+-channel tion of further motor pro+ teins. For example, the Annexin-like protein o,,-',-_~. • .~..'%\ • • • ooo o~,~t discovery of a myosin gene Calmodulin family and kinesin-like f . . . . ", ..t 1~-_-,I.'..:._':4~ -'...genes in Arabidopsis 42'4~ Calcium opens the way for searching Myosin for related gene sequences ~ ~ , . . . . . A--J ^ ~ 1,,C).~r-~¶..~. . ~ ~ ( I::::::n] . ~4)-%.~-t__~ o expressed during pollen ~ f - ~ , • r~ ...-~..~--~Ca~+ zzo::z~:::c~o2z:z~ ( I ?--v., "~ ~'.':'%7ell MT-based motor germination and growth. "r .\ 2%; . ~ 7?~ Many lines of evidence • .~. :..'..w ~ ~.) Microtubule ° °° eoee o°• suggest that the tube tip - ~ "." ~ ( J;-¢,41~"~ Q::Z::O Actin filament plays a role in modulating ~ .l_ .....:.y " Ca pollen tube growth by conSecretory vesicle "1"* ~ : ~ ' T •e °°•/ "." trolling the fusion rate of Clathrin secretory vesicles and the local organization of the Plasma membrane cytoskeleton. Consequently, another important area of Fig. 5. Diagram showing the principal components of a pollen tube and a hypothetical interpreresearch is the relationship tation of the involvement of these components in the growth process. The Ca2÷gradient may well play a central role in determining the polarized growth of pollen tubes. It is likely that this is between cytoskeletal dymaintained by the opposing activities of ion channels, which allow influx of Ca2+to the tip, and of nanlics, the Ca2+ gradient, Ca2+-sequestering organelles, such as the endoplasmic reticulum (ER). High Ca2÷levels in the tip cell wall formation and may facilitate the docking and fusion of secretory vesicles through the activity of annexin-like protube growth ~4. The identiteins. Clathrin molecules are likely to take part in membrane recycling. Also, Ca~+and Ca2÷-actification of kinesin-like vated proteins, such as calmodulin, may exert regulatory activity on both cytoskeletal fibrils and gene sequences with a motor proteins (open arrows). At the present time, however, this hypothesis is only speculative calmodulin-binding domain (indicated by question marks). The several distinct myosin molecules that have been identified in in potato and Arabidopsis pollen tubes are indicated as a single generic motor involved in the translocation of organelles suggests that Ca 2+ may (Org) and vesicles. Microtubule (MT) based motors are also shown interacting with generic regulate the activity of organ•lies (X), although this role remains to be clearly determined. For clarity, the cell wall of the pollen tube is not included. microtubule-based motors in plant cells 45. Figure 5 shows the main compo= Although it is not known whether Ca2+or the dissipation nents of the pollen tube apex and the hypothetical interacof a Ca 2+ gradient may affect the microtubutar organ- tions that may occur between them during pollen tube ization in pollen tubes, it is possible to speculate by com- growth. The recent identification of transmitting tissue-specific paring the pollen tube to other plant cells. For example, microtubules from carrot cells form a bundle following the arabinogalactan proteins that attract pollen tubes and addition of a plant homologue of elongation factor-la, and stimulate their growth in vitro 46 creates new possibilities for this activity is abolished by a Ca2+-calmodulin complex~9. the study of compounds that might regulate pollen tube There is no evidence for the presence of homologues of growth by acting on the cytoskeletal apparatus and the Ca 2+ elongation factor-la in pollen tubes, but it is tempting to gradient. The discovery that polypeptides related to Rho suppose that Ca2÷/calmodulin in the apex may prevent GTPases are present in the tube tip 47 suggests that the microtubules from forming bundles. According to this assembly of actin filaments in response to external signals view, the apical localization of centrosomal antigens 7 could in pollen tubes may use molecular mechanisms that are support a role for the tube apex as a putative microtubule- very similar to those present in animal cells. Proteins organizing site. related to integrins (transmembrane proteins that connect the cytoskeleton to extracellular matrix molecules, such as Future perspectives vitronectin) have not yet been identified in pollen tubes. The ability to use the pollen tube system for biotechno- However, integrin-like proteins have been identified and logical applications 4° depends on the ability to decode the linked to tip growth in the fungus Saprolegnia ts, and vitromolecular machinery required for pollen tube growth. nectin-like proteins occur in the stylar transmitting tissue 49. Although many details of pollen tube growth remain It is therefore possible that proteins that play a vital role in unclear, these are being clarified, and the number of known linking the cytoskeleton to the extracetlular matrix in other molecular components is steadily increasing. Recent studies eukaryotic cells may also serve in the growth and guidance have shown that cells can modulate dynamic processes of pollen tubes. using antagonist motor proteins, which could be involved in a 'tug-of-war' process. For example, several kinesin and Acknowledgements dynein molecules participate with different and antithetical The authors are grateful to Prof. Peter Hepler, Prof. roles in the organization of the spindle apparatus 41. Unfor- Antonio Tiezzi, Dr Yi-Qin Li and Dr Anja Geitmann for caretunately, the number of motor proteins identified in pollen ful reading of the manuscript, criticisms and suggestions. .
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reviews References 1 Cresti, M., Blackmore, S. and van Went, J.L. (1992)Atlas of Sexual Reproduction in Flowering Plants, Springer 2 Faure, J.E. et al. (1996) Emerging data on pollen tube growth and fertilization in flowering plants, 1990-1995, Protoplasma 193, 132-143 3 Pierson, E.S. and Cresti, M. (1992) Cytoskeleten and cytoplasmic *~ organization of pollen and pollen tubes, Int. Rev. Cytol. 140, 73-125 4 Lancelle, S.A. and Hepler, P.K. (1991)Association of actin with cortical microtubules revealed by immunogold localization in Nicotiana pollen tubes, Protoplasma 165, 167-172 5 Mittermann, I. et al. (1995)Molecular cloning and characterization of profilin from tobacco (Nicotiana tabacum): increased profilln expression during pollen maturation, Plant Mol. Biol. 27, 137-146 6 Palevitz, B.A.,Liu, B. and Joshi, C. (1994) ~-tubulin in tobaccopollen tubes: association with generative cell and vegetative microtubules, Sex. Plant Reprod. 7, 209-214 7 Cai, G. et al. (1996) The anti-centrosome mAb 6C6 reacts with a plasma membrane-associated polypeptide of 77-kDa from the Nicotiana tabacum pollen tubes, Protoplasma 190, 68-78 8/~_strSm, H., Sorri, O. and Randaskoski, M. (1995) Role of microtubules in the movement of the vegetative nucleus and generative cell in tobacco pollen tubes, Sex. Plant Reprod. 8, 61-69 9 Joos, U., van Aken, J. and Kristen, U. (1994) Microtubules are involved in maintainingthe cellular polarity in pollen tubes ofNicotlana sylvestris, Protoplasma 179, 5-15 10 Joos, U., van Aken, J. and Kristen, U. (1995) The anti-microtubule drug carbetamide stops Nicotiana sylvestris pollen tube growth in the style, Protoplasma 187, 182-191 11 Geitmann,A., Li, Y.Q. and Cresti, M. (1995) The role of cytoskeleten and dictyosome activity in the pulsatery growth ofNicotiana tabacum and Petunia hybrida pollen tubes, Bot. Acta 109, 102-109 12 Zhang, H.Q. et al. (1995)Microtubular organization during asymmetrical division of the generative cell in Gagea lutea, J. Plant Res. 108, 269-276 13 Khono, T. and Shimmen, T. (1988) Accelerated sliding of pollen tube organelles along Characeae actin bundles regulated by Ca2~,J. Cell Biol. 106, 1539-1543 14 Tang, X., Hepler, P.K. and Scordilis, S P. (1989) Immunochemical and immunocytochemicalidentification of a myosin heavy chain polypeptide in Nicotiana pollen tube, J. Cell Sci. 92, 569-574 15 Tirlapur, U. et al. (1995) Confocalimaging and immunogold electron microscopyof changes in distribution of myosin during pollen hydration, germination and pollen tube growth in Nicotiana tabacum L., Eur. J. Cell Biol. 67, 209-217 16 Yokota, E. and Shimmen, T. (1994) Isolation and characterization of plant myosin from pollen tubes of lily, Protoplasma 177, 153-162 17 Yokota, E. et al. (1995)Localization of a 170 kDa myosin heavy,chain in plant cells, Protoplasma 185, 178-187 18 Miller, D.D., Scordilis, S.P. and Hepler, P.K. (1995) Identification and localization of three classes of myosins in pollen tubes of Lilium longiflorum and Nicotiana alata, J. Cell Sci. 108, 2549-2563 19 Pierson, E.S., Lichtscheidl, I.L. and Derksen, J. (1990) Structure and behaviour of organelles in livingpollen tubes ofLilium longiflorum, J. Exp. Bot. 41, 1461-1468 20 Derksen, J. et al. (1995) Regulation of pollen tube growth, Acta Bot. Neerl. 44, 93-119 21 Miller, D.D., Lancelle, S.A. and Hepler, P.I~ (1996)Actin filaments do not form a dense meshwork in Lilium longiflorum pollen tube tips, Protoplasma 195, 123-132 22 Malhb, R. et al. (1995) Calcium channel activity during pollen tube growth and reorientation, Plant Cell 7, 1173-1184 23 Blackbourn, H.D. and Jackson, A.P. (1996) Plant clathrin heavy chain: sequence analysis and restricted localisation in growing pollen tubes, J. Cell Sci. 109, 777-786 24 Tiezzi,A~et aL (1992)An immm~oreactivehomologof mammalian kinesin in Nicotiana tabacum pollen tubes, Cell Motil. Cytoskeleton 21, 132-137 25 Moseatelli,A. et aL (1995)High molecular weight polypeptides related to dynein heavy chains in Nicotiana tabacum pollen tubes, J. Cell Sci. 108, 1117-1125 26 Cole, N.B. and Lippincott-Schwartz, J. (1995) Organization of organelles and membrane traffic by microtubules, Curr. Opin. Cell Biol. 7, 55-64
27 Liu, G.Q. et al. (1994)Kinesin-related polypeptide is associated with vesicles from Corylus avellana pollen, Cell MotE. Cytoskeleton 29, 155-166 28 Cai, G. et al. (1993)The kinesin-immunoreactive homologuefrom Nicotiana tabacum pollen tube: biochemical properties and subcellular localization,Planta 191, 496-506 29 Lillie, S.H. and Brown, S.S. (1994) Immunofluorescence localization of the unconventional myosin, Myo2p, and the putative kinesin-related protein, Smylp, to the same region of polarized growth in Saccharomyces cerevisiae, J. Cell Biol. 125, 825-842 30 Liu, B. and Palevitz, B.A. (1996) Localization ofa kinesin-like protein in the generative cells oftebacco, Protoplasma 195, 78-89 31 Hasson, T. and Mooseker, M.S. (1995) Molecular motors, membrane movements and physiology:emerging roles for myosins, Curr. Opin. Cell Biol. 7, 587-594 32 Scali, M. et al. (1994) Purification and biochemical characterization of calmodulin from Corylus avellana pollen, Plant Physiol. Biochem. 32, 831-838 33 Tirlapur, U.K. et aL (1994) Confocalimage analysis of spatial variations in immunocytochemicallyidentified calmodulin during pollen hydration, germination and pollen tube tip growth in Nicotiana tabacum L., Zygote 2, 63-68 34 Pierson, E.S. et al. (1994) Pollen tube growth is coupled to the extracellular calcium ion flux and the intracellular calcium gradient: effect of BAPTA-typebuffers and hypertonic media, Plant Cell 6, 1815-1828 35 Pierson, E.S. et al. (1996)Tip-localizedcalcium entry fluctuates during pollen tube growth, Dev. Biol. 174, 160-173 36 Blackbourn, H.D. et al. (1992)Properties and partial protein sequence of plant annexins, Plant Physiol. 99, 864-871 37 Malh6, R. et al. (1994) Role of cytesolicfree calcium in the reorientation of pollen tube growth, Plant J. 5, 331-341 38 Khono, T. and Shimmen, T. (1987) Ca2+-induced fragmentation of actin filaments in pollen tubes, Protoplasma 141, 177-179 39 Durso, N.A. and Cyr, R.J. (1994)A calmodulin-sensitive interaction between microtubules and a higher plant homologof elongation factor-l~, Plant Cell 6, 893-905 40 Dumas, C. and Mogensen, H.L. (1993) Gametes and fertilization: maize as a model system for experimental embryogenesis in flowering plants, Plant Cell 3, 1337-1348 41 Vernos, I. and Karsenti, E. (1996) Motors involved in spindle assembly and chromosomesegregation, Curr. Opin. Cell Biol. 8, 4-9 42 Kinkema, M., Wang, H.Y. and Schiefelbein,J. (1994) Molecular analysis of the myosin gene family in Arabidopsis thaliana, Plant Mol. Biol. 26, 1139-1153 43 Mitsul, H. et al. (1993) Identification of a gene family (kat) encoding kinesin-like proteins in Arabidopsis thaliana and the characterization of secondary structure of KatA, Mol. Gen. Genet. 238, 362-368 44 Li, Y.Q. et al. (1996) Enforced growth-rate fluctuation causes pectin ring formation in the cell wall ofLilium longiflorum pollen tubes, Planta 200, 41-49 45 Reddy, A.S.N. et al. (1996)A plant kinesin heavy chain-like protein is a calmodulin-bindingprotein, Plant J. 10, 9-21 46 Cheung, AX., Wang, H. and Wu, H. (1995) A floral transmitting tissuespecific glycoproteinattracts pollen tubes and stimulates their growth, Cell 82, 383-393 47 Lin, Y. et al. (1996)Localization ofa Rho GTPase implies a role in tip growth and movement of the generative cell in pollen tubes, Plant Cell 8, 293-303 48 Kaminskyj, S.G.W.and Heath, I.B. (1995) Integrin and spectrin homologues, and cytoplasm-walladhesion in tip growth, J. Cell Sci. 108, 849-856 49 Sanders, L.C. et al. (1991)A homologof the substrate adhesion molecule vitronectin occurs in four species of flowering plants, Plant Cell 3, 629-635 Giampiero Cai*, Nessandra M0scatefli and Mauro Crest are at the Dept of Environmental Biology, University of Siena, • Via Mattioli 4, 531 D0 Siena, Italy. *Author for correspondence (teN-39 577 298856; fax +39 577 298860: e-mail cai@unisLit),
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