THE GROWTH OF THE POLLEN TUBE WALL IN OENOTHERA … · the style, most of the contents, including the starch, are drawn from the pollen grain. Once the tube has travelled the major

J. Cell Sci. 18, 519-S32 (i975) 519Printed in Great Britain

THE GROWTH OF THE POLLEN TUBE WALLIN OENOTHERA ORGANENSIS

H. G. DICKINSON AND JANICE LAWSON

Department of Botany, Plant Science Laboratories,University of Reading, Whiteknights, Reading RG6 2AS, England

SUMMARY

The growth of the pollen tube wall of Oenothera is effected by the expulsion of fibrillarmaterial from the cytoplasm into the developing wall. This material may also be seen in thecytoplasm, contained in membrane-bound vesicles. It is not clear how the content of thevesicles is discharged, but it appears not to involve the participation of microtubules.

The source of the cytoplasmic fibrillar bodies depends upon the stage of development of thepollen tube. The earliest growth is derived from the inclusion into the wall of vesicles containingpre-formed materials present in the grain on pollination. During the next stage of growth thewall is derived from the content of double-membraned inclusions also present in the pollen.The content of the former vesicles is not so similar to the wall as the latter, but intermediatesbetween the 2 types of vesicle may be seen in the cytoplasm, indicating that the former areformed from the latter. Most of the tube wall is derived from the products of dictyosomes inthe pollen grain or tube. These dictyosomes are few in number and they must be exceedinglyactive. This, and the observation that dictyosome vesicles are frequently associated with bankedcomplexes of mitochondria, indicates that some steps in the metabolism of the vesicular content,perhaps phosphorylation, take place distant from the dictyosomes.

These different sources of fibrillar material presumably permit the rapid starting of tubegrowth, without any attendant metabolism. However, it would be impossible to include enoughpre-formed wall material in the grain to enable the full growth of the tube, so once started, itseems that the tube then relies on the elaboration of simple reserves for the construction of itswall. These reserves are likely to be held in the pollen, and may be the large numbers of starchgrains characteristic of the pollen cytoplasm.

INTRODUCTION

The rapid growth of the pollen tube of higher plants must, of necessity, be accom-panied by an equally rapid synthesis of the tube wall. The source of materials for thiswall was recognized by Rosen (1964) to be the large, generally single-membrane-boundvesicles that form the major part of the cytoplasm of the growing tube.

Van Der Woude, Morr6 & Bracker (1971) examined the distribution and chemicalnature of these vesicles in Lilium, showing them to be derived from dictyosomes andto contain non-cellulosic glucans. At the plasma membrane, the content of the vesiclesappeared simply to be expelled into the wall without the participation of microtubules,or indeed any other cytoplasmic organizational structure. More recently Engels(1973) has demonstrated the presence of similar dictyosome vesicles in growing pollentubes of Petunia.

While the source and content of these vesicles appear now to be recognized, detailsof their organization within the cytoplasm and subsequent inclusion into the wall

520 H. G. Dickinson andj. Lawson

remain unknown. This is particularly true of genera such as Oenothera, where vesiclesproduced by the dictyosomes of the pollen tube are generally far smaller than, anddiffer strikingly in appearance from, the bodies finally included in the wall.

We report here ultrastructural aspects of pollen tube wall development in Oenotheraorganensis during germination and in the subsequent growth of the pollen tube.Development is described on stigmata following compatible intraspecific crosses,and in culture. The nature of tube growth following incompatible intraspecificpollinations is reported elsewhere (Dickinson & Lawson, 1974).

MATERIALS AND METHODS

Plants of Oenotliera organensis were kindly supplied by Professor D. Lewis (UniversityCollege, Gower Street, London W.C.i). Individual flowers were emasculated before anthesisto prevent contamination of the stigmata, and crosses were made only once the petals hadopened. At defined times following pollination, the styles were removed and both stigma andstyle cut into short sections {ca. 5 mm) under the surface of the fixative (3 % glutaraldehydein phosphate buffer at pH 7-2) at room temperature. A range of crosses was prepared in buffersvarying in molarity from 0-03 to i-o M. Fixation using 0-05 M buffer was found to produce thebest results.

Pollen grains were also cultured in vitro. Fresh pollen was incubated in a sucrose-basednutrient medium (Kwack, 1965) at 37 °C in hanging drops. Under these conditions tubes werefound to develop swiftly from pollen of all genotypes. After incubation, the germinated grainswere transferred to fixative (as used in the other preparations for electron microscopy, but withthe buffer molarity adjusted to that of the nutrient solution), at room temperature. Subsequentstages of preparation were identical with those used with the other specimens for electronmicroscopy, except in that following osmication the material was cast in blocks of 2 5 % agarto facilitate handling.

After fixation for 4 h, the material was rinsed in buffer at 4 °C and postfixed in 1 -5 % osmiumtetroxide for 3 h, also at 4 °C. Following rinsing in distilled water the tissue was dehydrated inan acetone series and embedded, via 1-2 epoxypropane, in Epon 812 (Shell, U.K.). The curedblocks were sectioned on a Reichert UM3 ultramicrotome, the sections post-stained in a7 % aqueous solution of uranyl acetate and in lead citrate (Reynolds, 1963), and finallyexamined in a JEOL 100B electron microscope.

Thick sections (1 -5 /tm) weie also cut on the ultramicrotome and stained for carbohydrate bytreatment with periodic acid and Schiff reagent (PAS) (Feder & O'Brien, 1968).

RESULTS

The germination of pollen on the stigma

Apart from usual cytoplasmic constituents, the pollen grain cytoplasm of Oenotheracontains large populations of double-membraned inclusions measuring between100 and 750 nm, and very irregular single-membraned inclusions, about 2600 nm inmaximum dimension (see Figs. 1, 2). The double-membraned inclusions containfibrogranular material, while those with the single membrane are filled with closelypacked fibrils, electron microscopically identical with those constituting the intine ofthe pollen grain. Profiles are also frequent indicating intermediate conditions betweenthese 2 types of fibrillar inclusion, either one of each type with membranous continuity,or a single body containing an intermediate matrix (see Fig. 1). Small single-mem-braned vesicles of constant dimension (ca. 60 nm) are also numerous in this cytoplasm,and may be seen associated with the small number of dictyosomes (see Fig. 3).

Growth of pollen tube wall 521

Other unusual components of the cytoplasm of these grains are large accumulationsof rough endoplasmic reticulum, unequally shaped bodies about 0-9 /*m in maximumdimension, which cytochemical tests show to contain acid phosphatase, and largefusiform starch grains (see Figs. 4, 5).

Closely following hydration of the grain on the stigma, associations are formedbetween the single-membraned fibrillar bodies and the intine over the colpal tip, thefibrils of the inclusion apparently merging with those of the intine (see Fig. 5). At thesame time, a striking increase in the numbers of small vesicles takes place, accom-panied by a change in their distribution in the cytoplasm. In addition to occurringrandomly throughout the vegetative cytoplasm, these vesicles also associate closelywith an unusual organellar complex. This appears to be formed of up to 20 double-membrane-bound sacs, banked in a manner reminiscent of dictyosomes (see Fig. 6).Between each sac lie layers of small vesicles. These vesicles are also frequent aroundthe peripheries of these complexes (see Fig. 6). The internal organization of the mem-branous sacs is electron-microscopically indistinguishable from that of mitochondriaelsewhere in the pollen cytoplasm. Each sac appears to be approximately discoid,measuring about 2 /tm in diameter and being about 200 nm in depth. While occasionalconnexions between the membranes of the sacs and the vesicles are observed, theyoccur rarely and, in general, the vesicular membrane appresses closely to the outermembrane of the sacs (see Fig. 7).

Treatment with PAS reveals the pollen cytoplasm and the young tube to containarge amounts of free carbohydrates (see Fig. 8).

As the pollen tube grows on to the stigmatic surface, changes occur in the numbersof some cytoplasmic inclusions. The small vesicles continue to increase in number,and profiles are frequently seen indicating their fusion with both the double- andsingle-membraned fibril-containing bodies (see Fig. 9). In addition a dramatic falloccurs in the numbers of single-membraned fibril-containing bodies and of thoseof the intermediate type.

Pollen tube growth into the outer layer of the stigma

Each arm of the stigma of Oenothera comprises an outer layer of secretory cells,investing loosely packed parenchymatous tissue and a thin strand of conductingelements. The outer layer of cells produces a viscous fluid in which, on pollination,he pollen becomes par tially immersed.

As the tube commences its growth through this fluid and between the cells of theouter layer, changes take place in the cytoplasmic structures associated with the tubewall. Very few of the irregularly shaped, single-membraned fibril-containing bodiesare present at this point, and instead, inclusions similar to the double-membranedbodies appear to be associated with the wall.

In the cytoplasm these bodies are also very frequently seen in apparent fusion withsmall vesicles (see Fig. 11). Adjacent to the areas of plasma membrane where associa-tions with the double-membraned bodies take place, crescent-shaped profiles appearin the tube wall (see Fig. 10). Their diameter is equivalent to that of the double-membraned bodies, and they are bounded by an electron-lucent periphery about


50 nm in width. These profiles are most common at the tube tip; further back theseimages become attenuated and eventually merge into a featureless fibrillar wall (seeFig. 12).

It is at this point, about the time when the tube grows into the alveolar parenchy-matous tissue, that the generative, and perhaps also the tube nucleus, passes downthe tube (see Fig. 13). Whereas the tube had previously grown between the cells of theouter layer, following the course of the middle lamella, in the alveolar layer it travelsin a film of moisture on the cell surfaces.

Subsequent growth of the pollen tube

As the tube grows from the outer layer of cells into the alveolar tissue the numberof double-membraned inclusions diminishes considerably. At the tip of the tube thegenerative nucleus is surrounded by mitochondria, small vesicles, and small 'vacuoles'containing varying amounts of fibres (see Fig. 15). These fibril-containing vacuolesfrequently associate with the small vesicles and, especially those containing largenumbers of fibres, are often seen fusing with the plasma membrane. Adjacent tothese regions crescent-shaped profiles are present in the tube wall. The boundary ofthese fibril-containing vacuoles is invariably indistinct and often appears to consist of2 unit membrane profiles, or even a nearly complete layer of small vesicles. Also stillpresent in the tube at this stage are starch grains and the enzyme-containing bodies.This organization of the cytoplasm of the pollen tube tip is maintained throughoutthe remainder of the growth of the pollen tube. As the tube grows down the length ofthe style, most of the contents, including the starch, are drawn from the pollen grain.Once the tube has travelled the major part of the distance down the style, plugs maybe formed well back in the tube, cutting off cytoplasmic continuity with the pollengrain. In these circumstances, the cytoplasm on the pollen grain side of the plug swiftlybecomes granular and necrotic (see Fig. 14).

Growth of the pollen tube in culture

Germination and subsequent growth of the pollen tube in culture are identical withsimilar events in vivo, except in that the tube walls are marginally thinner and thecytoplasm appears attenuated. This latter aspect of the cultured tubes reveals detailsof the relationship between the small vesicles and the masses of fibrils contained inwhat, in the previous section, are referred to as vacuoles.

In the cultured tubes, the small vesicles clearly fuse together to form these massesof fibrils, remains of the vesicle membrane frequently being visible embedded in thefibrillar matrix (see Fig. 16). Further, the small vesicles also appear to delimit theindividual fibrillar bodies by forming a single layer over their surfaces (see Fig. 17).The vesicle membranes on the cytoplasmic face and those on the face of the fibrillarinclusion then appear to become continuous, resulting in their 'diffuse double-membraned' boundaries.

Whether there is any involvement of other cytoplasmic components in the formationof these structures is not clear from this study, but the main bulk of the fibrils cer-tainly results from the amassing of vesicular content. While there is circumstantial


evidence from studies in vivo for such a mechanism of fibrillar body formation, thereis little doubt that, in the tips of these tubes, fibrils are also accumulated in genuinevacuoles.

DISCUSSION

As indicated by the previous studies (Rosen, Gawlik, Dashek & Siegesmund,1964; Van Der Woude et al. 1971; Engels, 1973) there is no doubt that the pollentube wall is derived from the content of cytoplasmic vesicles. While this material isalways fibrillar and the vesicles bounded by one, or perhaps two membranes, thesource of these inclusions appears to depend upon the stage of development of thepollen tube.

During the earliest stages of germination, irregular, single-membraned fibrillarbodies are included into the wall. Since associations are seen between these single-membraned bodies and those with double membranes, and since this latter class ofinclusion is a well established constituent of the cytoplasm of mature pollen inmany species (Fisher, Jensen & Ashton, 1968; Dickinson & Lewis, 1974), it is notunreasonable to propose that one be derived from the other. Certainly some of thesingle-membraned inclusions are present in the pollen grains before pollination, buttheir number is very variable.

In the next phase of tube growth the single-membraned inclusions are absent anddouble-membraned bodies themselves appear to be included in the wall. Theselatter inclusions differ from the former in more than their boundaries, for they manageto retain their shape after discharge into the wall, and vestiges of the space between the2 bounding membranes remains visible for some time. Certainly fusion between thesmall dictyosome vesicles and the larger double-membraned inclusions does take placein the cytoplasm, but there is little doubt that the bulk of the tube wall material duringthis growth phase is still derived from the content of the double-membraned inclusionspresent in the grains on pollination.

The main part of the pollen tube wall is, however, derived from neither of theseinclusions present in the pollen on release from the anther, but from the products ofdictyosomes contained in the pollen grain and tube. It is difficult to believe that thevast numbers of small vesicles are derived from the comparatively few dictyosomespresent in the cytoplasm at this time, but the membranous continuity of such vesicleswith these organelles, and the absence of any other cytoplasmic constituent thatmight generate them, leave no alternative conclusions. The association between thesedictyosome products and the banked organellar complexes may, in some way, berelated to this high rate of vesicle production. Van Der Woude et al. (1971) and Engels(1973) have shown the content of the vesicles included into the pollen tube wall to becomposed of non-cellulosic glucans. The formation of these glucans from reservebreakdown products (e.g. simple sugars) demands several phosphorylation stages,first by the formation of glucose-6-phosphate, and later may follow several phosphatetranslocations. It is possible that, in view of the high rate of vesicle production thesesmall dictyosomes are incapable of carrying out this metabolism to completion,leaving subsequent stages to take place in the cytoplasm. The mitochondrion is the

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major site of cellular phosphorylation and it is perhaps not unreasonable to proposethat the association between the dictyosome vesicles and the sacs of the organellarcomplex (which are indistinguishable in internal organization from mitochondriapresent elsewhere in the cell) constitutes evidence of cytoplasmic phosphorylation ofthe vesicular content, remote from the dictyosome.

Whatever change occurs in the small vesicles in association with the organellarcomplexes, at first sight there appear to be 2 mechanisms by which they aggregate toform fibrillar bodies before insertion into the wall. This apparent difference mayhowever result from biophysical phenomena. If, in the course of cyclosis a smallvesicle is brought near a vacuole such forces may cause its membrane to fuse with thatof the vacuole. If, on the other hand, no such vacuoles are present, as more oftenappears to be the case in cultured tubes, accumulation of vesicles would then continueuntil some threshold, or unstable state is reached, when the vesicles appear to fusetogether themselves.

Thus, this perplexing range of sources of the fibrillar bodies destined for the tubewall represent simply changes in the reserves utilized. The similarity of the contentof the single-membraned inclusions with the material of the wall, and the complexprocess of forming fibrillar bodies from dictyosome products indicates that growth is'primed' by the insertion of pre-formed wall on germination, and continues for a shortwhile on reserves held in the pollen, also closely akin to wall materials (the content ofthe double-membraned inclusions). The major part of growth, however, appears toinvolve nearly complete synthesis of the wall materials from simple carbohydrates.The identity of these reserve carbohydrates is not known; in culture sucrose from themedium may be used to manufacture wall materials, but this clearly cannot be thecase in the stigma and style, where only minimal amounts of carbohydrate could bederived from the tubes' environment. Lewis (1948) has shown that growth of thepollen tube is approximately proportional to the volume of the pollen grain, whichconstitutes indirect evidence that the major reserves for tube growth are held in thegrain, not derived from the stigma. The pollen of Oenothera contains large numbersof starch grains which pass down the tube during its growth, and it is tempting topropose that these are the reserves of carbohydrate utilized in tube wall manufacture.

Little has emerged from this study to indicate how the fibrillar bodies are finallyexpelled from the cytoplasm into the wall. Certainly microtubules have been seen inthe pollen tubes of Lilium (M. Davies, personal communication) and in Oenothera(H. G. Dickinson, unpublished), but they are too few in number to play any role inincluding the vesicles into the wall. However, simple examination of germinatingtubes in culture with the light microscope reveals the very active cyclosis of the tubecytoplasm. In addition, treatment of tubes with agents that disrupt cyclosis (Mascaren-has & Lafountain, 1972) results in changes in wall development. It thus seems possiblethat the cyclosis of the cytoplasm is enough to ensure that a steady stream of maturefibrillar bodies is brought to the cell surface, and simple biophysical forces then resultin the fusion of membrane and the expulsion of materials into the wall.

We would like to thank the Agricultural Research Council for financial support, and Mr. S. K.Irtiza AH and Mr. M. Crowder for valuable technical assistance.


REFERENCES

DICKINSON, H. G. & LAWSON, J. (1974). Pollen tube growth in the stigma of Oenothera or-ganensis following compatible and incompatible intraspecific pollinations. In Incompatibilityin Flowering Plants (Discussion meeting). Proc. R. Soc. B (in Press).

DICKINSON, H. G. & LEWIS, D. (1974). Changes in the pollen grain wall of Limim grandiflorumfollowing compatible and incompatible intraspecific pollinations. Ann. Bot. 38, 23-29.

ENGELS, F. M. (1973). Function of Golgi vesicles in relation to cell wall synthesis in germinatingPetunia pollen. I. Isolation of Golgi vesicles. Acta bot. neerl. 22, 6-13.

FEDER, N. & O'BRIEN, T. P. (1968). Plant microtechnique: some principles and new methods.Am. J. Bot. 55, 123-142.

FISHER, D. B., JENSEN, W. A. & ASHTON, M. E. (1968). Histochemical studies in pollen;storage pockets in the endoplasmic reticulum. Histochemie 11, 169-182.

KWACK, B. (1965). Stylar culture of pollen and physiological studies of self incompatibility inOenothera organensis. Physiologia PI. 18, 297-305.

LEWIS, D. (1948). Pollen tubes, incompatibility and osmotic pressure in plants. School Set.Rev. 108, 206-212.

MASCARENHAS, J. P. & LAFOUNTAIN, J. (1972). Protoplasmic streaming, cytochalasin B, and thegrowth of the pollen tube. Tissue & Cell 4, 11-14.

REYNOLDS, E. S. (1963). The use of lead citrate at a high pH as an electron-opaque stain inelectron microscopy J. Cell Biol. 17, 208-212.

ROSEN, W. G. (1964). Chemotropism and fine structure of pollen tubes. In Proc. int. Symp.Pollen Physiology and Fertilisation (ed H. F. Linskens), pp. 159-166. Amsterdam: North-Holland Publishing.

ROSEN, W. G., GAWLIK, S. R., DASHEK, W. V. & SIEGESMUND, K. A. (1964). Fine structureand cytochemistry of Lilium pollen tubes. Am.jf. Bot. 51, 61-71.

VAN DER WOUDE, W. J., MORRE, D. J. & BRACKER, C. E. (1971). Isolation and characterizationof secretory vesicles in germinated pollen of Lilium longiflorum.J. Cell Sci. 8, 331-335.

{Received 7 January 1975)

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Fig. 1. Intermediate inclusion (j) present in cytoplasm of ungerminated pollen.d, double-membraned inclusion; s, single-membraned inclusion, x 39000.Fig. 2. Pollen grain cytoplasm containing double-(d) and single-(s) membraned in-clusions, x 16500.Fig. 3. Dictyosome (d) in close association with small vesicles (v). x 13200.Fig. 4. Large mass of rough endoplasmic reticulum (rer) in pollen grain cytoplasm,x 14000.

Fig. 5. Association of the single-membraned inclusions (s) with the intine (i). Enzymebodies (e) and starch grains (sg) are present in the cytoplasm, x 14500.

Growth of pollen tube wall

- it;


Fig. 6. Small vesicles in apparent association with banks of double-membrane-boundsacs reminiscent of mitochondria, x 16000.Fig. 7. Detail of association between small vesicles and sacs of organellar complex suchas that shown in Fig. 6. s, organellar sac; v, vesicle, x 96000.Fig. 8. Light micrograph showing effects of PAS treatment of colpus (c) of germinatingpollen grain. The colpal region contains starch grains. Carbohydrate-containingvesicles (y) are present in the stigniatic exudate. s, stigma papilla, x 1500.Fig. 9. Fusion (arrows) between single-membraned fibrillar inclusion and smallvesicles, x 53600. Inset shows similar fusion (arrow) but between a small vesicle anda double-membraned inclusion, x 47250.Fig. 10. Pollen tube wall showing crescent-shaped profiles (e) of similar dimensionto the double-membraned inclusions, x 91 200.



Fig. 11. Apparent fusion of small vesicles with inclusions similar to double-membranedbodies (arrows), during pollen tube growth, x 51000.

Fig. 12. Older section of pollen tube wall showing fibrillar character and attenuatedcrescent shapes, x 60000.

Fig. 13. Generative nucleus (g) present in young pollen tube, x 15900.

Fig. 14. Old pollen tube behind the plug showing necrotic cytoplasm, x 20000.

Fig. 15. Pollen tube tip with generative nucleus (g) surrounded by mitochondria (m),and vacuolea (u) with fibrillar contents, x 22500.


532

"16

H. G. Dickinson andj. Latoson. •3- — - • - , — — : — . 1—x

Fig. 16. Small vesicles (n) aggregating to form a fibrillar mass (/). Remains of thevesicle membranes are visible (arrows), x 16800.Fig. 17. Delimitation of fibrillar bodies (/) in the fibrillar mass by a single layer ofvesicles (v). x 81000.

Documents

THE GROWTH OF THE POLLEN TUBE WALL IN OENOTHERA … · the style, most of the contents, including the starch, are drawn from the pollen grain. Once the tube has travelled the major