Plauta (Berl.) 84, 97--121 (1969)
Cotton Embryogenesis : The Tissues of the Stigma and Style and their Relation to the Pollen Tube WILLIAM A. J ~ N S ~ a n d DONALD B. F I S H ~ Department of Botany, University of California, Berkeley Received September 12, 1968
Summary. The stigma of cotton (Gossypium hirsutum) is covered by unicellular hairs. The cytoplasm of these hairs degenerates before the stigma becomes receptive. The vacuole remains intact, but the hair cytoplasm becomes a mass of dark, amorphous material with only a few organelles still being visible. The rest of the stigma consists of thin-walled parenchyma cells with large vacuoles and large amounts of starch. The cells of the style are differentiated into a uniseriate epidermis, vascular tissue, a cortex of thin-walled, vacuolate parenchyma cells, and the transmitting tissue. This latter tissue occupies the center of the style and consists of thick-walled cells with few vacuoles. The cells are rich in starch, ribosomes, endoplasmic reticulum and dietyosomes. They also contain deposits of calcium salts in the form of druses. The pollen germinates on the stigmatic hairs, grows down the outside of the hair and between the cells of the stigma to the transmitting tissue of the style. There the tubes grow between the walls of the cells but do not enter the cells themselves. Some transmitting cells adjacent to the pollen tube degenerate after the tip of the pollen tube has grown past them. However, not all degenerate, and those that do show no fixed spatial relationship to one another. The cells which do degenerate follow a characteristic pattern of breakdown. No ultrastructural evidence was found for the secretion of hydrolytic enzymes by the pollen tube. Introduction The cellular o r g a n i z a t i o n of the s t i g m a a n d style of c o t t o n was first described b y BALLS (1904) a n d s t u d i e d in g r e a t e r detail b y IYE~CAR (1938). The style is solid, t h e center being occupied b y a g r o u p of cells t e r m e d t h e t r a n s m i t t i n g tissue t h r o u g h which t h e pollen t u b e grows. This p a p e r is a r e p o r t of t h e u l t r a s t r u c t u r e a n d composition of t h e cells of the s t i g m a a n d style a n d the r e l a t i o n of these cells to t h e g r o w t h of t h e pollen tube. The u l t r a s t r u c t u r e of t h e pollen t u b e in t h e s t i g m a a n d style will be d e a l t w i t h in a s e p a r a t e p a p e r (JE~sEN a n d FISHEI~, 1969). The u l t r a s t r u c t u r e a n d composition of t h e walls of t h e t r a n s m i t t i n g tissue a n d t h e pollen t u b e will be t r e a t e d in detail in a n o t h e r p a p e r (JE~sv, N e t al., 1969). Materials and Methods Stigma and styles of Gossypium hirsutum L., cv. 1V18949, were collected at various times after pollination. The tissue was fixed in cold 3 % glutaraldehyde in 7
P l a n t a (Berl.), Bd. 84
98
W.A. J E ~ S ~ and D. B. FISHER:
0.05 M cacodylate buffer, pH 6.8. I t was rinsed, then postfixed in 2% OsO4 for 2 hours. Following dehydration in acetone, it was embedded in Epon. The 70% acetone contained 1% uranyl nitrate. The sectioned tissue was stained with lead citrate and examined with a Zeiss EM-9 electronmicroscope. For observations with the light microscope the tissue was fixed in 3% glutaraldehyde in 0.05 M caeodylate buffer at pH 6.8 for 4 hours, dehydrated in acetone, embedded in Epon and sectioned at 1.5 ~. The periodic acid-Schiff (PAS) reaction was used for the localization of insoluble carbohydrates (JENsE~, 1961) and Aniline blue black for general protein (FIshER, 1969). Observations on callose were made on 6-tz frozen sections, using the aniline blue florescence procedure (JI~SEN, 1962). The stigma-style was also freeze-substituted and embedded in paraffin. 10-~ sections were stained with PAS to show the general organization of the tissue. Segments of style were freeze-substituted, differentially extracted for cell wall compounds using the method given by JE~SE~ (1962), dehydrated, and embedded in Epon. The extracted tissue was either sectioned at 1 ~ and stained with PAS or thin-sectioned and stained with periodic acid-silver (PA-silver) (J~NsE~ et al., 1968). Tissue was also stained with ruthenium red by adding it to the glutaraldehyde used in the fixation.
Results
The Stigma. The gross m o r p h o l o g y of t h e s t i g m a a n d style of c o t t o n can be seen in Figs. 1 a n d 2. The s t i g m a is 1 0 - - 1 2 m m in l e n g t h a n d is dissected into 3 - - 5 lobes. The t o p a n d o u t e r p o r t i o n of t h e lobes are covered w i t h long, unicellular hairs (Figs. 2 a n d 5a). W i t h increasing distance f r o m t h e t i p of t h e stigma, t h e hairs become s h o r t e r a n d more r e s t r i c t e d in their location (Fig. 5 b, c) u n t i l t h e y are no longer p r e s e n t (Fig. 5d). The hairs are closely p a c k e d a t their b a s a l ends b u t are loosely associated a t their apical ends, giving t h e s t i g m a a f e a t h e r y a p p e a r a n c e (Fig. 5a). W h e n t h e s t i g m a is r e c e p t i v e to pollen a t anthesis, t h e hair cells a p p e a r as in Figs. 3 a n d 4. The wall is r e l a t i v e l y t h i n a n d on t h e e x t e r i o r face is covered b y a d i s t i n c t cuticle which is closely a p p r e s s e d to t h e wall (Fig. 6). The cell is filled b y a large vacuole u s u a l l y containing electrondense material. The c y t o p l a s m is a n a l m o s t a m o r p h u s b l a c k mass of d e g e n e r a t i n g organel]es, some of which can still be t e n t a t i v e l y identified (Fig. 6 a). Small a m o u n t s of s t a r c h can be f o u n d in t h e hair cells.
Fig. 1. The cotton flower showing the arrangement of the anther sheath surrounding the style and base of the stigma. X 1 Fig. 2. The stigma, style, and ovary of the cotton flower. The lettered arrows correspond to the sections shown in Fig. 5 Fig. 3. Stigmatic hairs (H) and parenehyma (P) cells. GA fixed, Epon embedded, 1.5 ~ section, stained with Aniline blue black and PAS. X 450 Fig. 4. Stigmatic hairs. Preparation as in l~ig. 3 except that the tissue was stained only with Aniline blue black. Starch appears white. • 1,500
Cotton Embryogenesis: Stigma and Style
Figs. 7*
1--4
99
100
W.A. JENSEN and D. B. FISHER:
Fig. 5. Sections through the stigma and style. The position of the sections is indicated in Fig. 2. The transmitting tissue (TT) is the intensely stained cells in the section. l~rozen-substituted tissue, 10 ~ sections, stained with PAS. • 115 I m m e d i a t e l y below the hair cells are t h i n - w a l l e d p a r e n c h y m a cells which show no evidence of c y t o p l a s m i c d e g e n e r a t i o n (Fig. 7). These cells contain large vacuoles which o c c u p y a b o u t half of t h e cell (Fig. 8). T h e p l a s t i d s are large a n d each contains 5 - - 8 large s t a r c h grains (Fig. 8). Mitoehondria a n d d i e t y o s o m e s are numerous. B o t h ribosomes a n d endoplasmic r e t i c u l u m (ER) are a b u n d a n t , b u t m o s t of t h e ribosomes a p p e a r Fig. 6. Stigmatic hair fixed with GA-Os and stained with uranium and lead. The vacuole (V) is intact although the cytoplasm has degenerated. Starch (S) remains in the plastids. The cuticle is visible as the thin, dark layer covering the exterior surface of the wall. • 6,000. Insert. Portion of hair showing the condition of the cytoplasm at higher magnification, l~emains of a dictyosome (D) and possibly a mitochondrion (M) can be seen. • 22,750
Cotton Embryogenesis- Stigma and Style
Fig. 6
I01
102
W.A. JENSENand D. B. FISE~R:
unattached to the EI~ (Fig. 8). A few spherical protein accumulations have been seen in the E R which resemble those reported in the nucellar ceils of cotton (Js~sn~r 1965). The cells immediately below the hair cells are separated by numerous, large intercellular spaces (Fig. 7). Several cell layers beneath the hairs, the intercellular spaces decrease in size and the walls thicken (Fig. 9). Farther in, the intercellular spaces are no longer present and a thick wall, rich in pectic substances, is found (Fig. 9). The appearance of the sections suggests a transition from space filled with air to that filled with amorphous wall material. These cells lead to the thick-walled transmitting tissue of the style. They are distinct from the transmitting tissue and the cortical parenchyma cells of the style. From their position and cytology they appear to have a definite role in the growth of the pollen tube. The Style. The style is covered by a uniseriate epidermis which contains guard cells and stomata (Fig. 10a). The cortex is composed of thin-walled parenchyma cells. Small vascular bundles are present near the periphery of the cortical tissue. The center of the style is occupied by the transmitting tissue, the cells of which are arranged into groups (Fig. 10b). The arrangement and number of cell groups in the transmitting tissue appears related to the number of locules in the gynoecium; there are 3--5 in the variety of cotton used in this study. The transmitting tissue is a collection of thick-walled cells which are arranged in files (Figs. 11 and 12). The lateral walls are uniformly thick except for occasional pit fields (Fig. 11) while the transverse walls are relatively thin (Fig. 12). The lateral walls consist of several, distinct layers (Fig. 11) (J~ss~N et el., 1969). Against the plasma membrane there is a relatively uniformly textured wall, approximately 2 ~ thick (wall layer 1) that is composed primarily of pectic substances and hemicellulose. Surrounding this is a thinner, darker-appearing wall, 0.5 ~ thick (wall layer 2) similar in composition to wall layer 1 but richer in hemicellulose. This wall in turn is surrounded by a thick (4--6 ~) loose-textured wall that shows concentric rings of fibrous material (wall layer 3). This layer is rich in pectic substances, relatively poor in hemicellulose and is the only wall layer that contains detectable amounts of non-cellulosic polysaccharides and cellulose. Finally, there is the middle lamella region which is quite pronounced (wall layer 4), being 0.5--1
Fig. 7. The relation oi the base of the hair cells (H) to the underlying parenchyma cells of the stigma. Large intr~cellular spaces (IS) are characteristic of this region. GA-Os fixed tissue stained with ruthenium red to emphasize the pectic substances of the wall. This procedure reduces the general contrast of the cytoplasm. Large starch grains are present in the parenehyma cells. • 6,000
Cotton Embryogenesis: Stigma and Style
Fig. 7
103
Fig. 8. Parenchyma cells of the stigma. Large vacuoles (V) are present. The plasrids (P) are large and filled with starch. Intercellular spaces are numerous and large. GA-Os fixation. • 6,000
W. A. JENSEN and D. B. FlSgEI~: Cotton Embryogenesis: Stigma and Style
105
thick, distinctive in appearance and composed primarily of pectic substance. Both layers 3 and 4 stain with Aniline blue black for protein. The composition of the various wall layers based on the differential extraetion-PAS stain method (JExsE~, 1962) is shown in Fig. 13. This is a condensed summary of the composition of the wall and full details of the methods and results will be presented elsewhere ( J ~ s ~ et al., 1969). Embedded in the wall, primarily wall layer 3, are masses of small vesicles (Figs. 11 and 12), similar to those found in the walls of the stigma cells. The cells contain many mitoehondria and dictyosomes (Fig. 14). The latter appear particularly active in forming vesicles which can be seen in the cytoplasm and which presumably are fusing with the plasma membrane (Fig. 14). The plastids are large and contain numerous starch grains (Fig. 14) which sometimes look as if they fuse into a multilobed, single mass of starch (Fig. 14). Many of the cells contain 1 or 2 lipid bodies several microns in diameter. Polysomes are seen in the cytoplasm and the abundant E R has ribosomes associated with it. There are two types of vacuoles present in the transmitting cells. The first is usually spherical or slightly ellipsoidal in shape and is 7--9 ~ in diameter. Frequently the cell also contains a vacuole 20--40 Ix in diameter which contains a druse presumably composed of calcium salts (Fig. 15). The form of the drnse in Fig. 15 is characteristic of the EM preparation and is undoubtedly altered from the condition in the living cells. Not all the cells have wall-formed deposits, but many have what appear to be the remains of such crystals present in the large vacuole. The nuclei of the transmitting cells are large (Fig. 14) and frequently lobed (Fig. 12). The chromatin is usually condensed near the nuclear membrane (Fig. 14). Pockets of membrane appear toward the cell periphery sometimes adjacent to the wall itself (Fig. 14). They may take the form of concentric rings of membranes (Fig. 14), collections of tubes and vesicles, or intermediate forms with both tubes and concentric rings (Fig. 14). Relation o/ the Pollen Tube to the Cells o] the Stigma and Style. The pollen lands on the surface of the hairs of the stigma, germinates, and grows down the surface of the hair and penetrates the stigma (Figs. 16 and 17). When the pollen enters the tissue of the stigma, it grows between the cells in the intercellular spaces (Fig. 17). Some of the cells appear crushed by the pollen tube, but not all cells in contact with the tube are damaged (Fig. 1S). The tube grows from the outer, thin-wMled cells of the stigma into the transmitting tissue of the style. This tissue is composed of thickwalled cells as noted earlier and the pollen tube grows through the cell wall (Fig. 19). The pollen tube actually grows through wall layer 3 and
Fig. 9
W. A. J ~ s E ~ and D. B. FISHER: Cotton Embryogenesis: Stigma and Style
107
Fig. 10a and b. Tissues of the style, a Epidermis (E) with stomata and guard cells (arrow); cortex (C); vascular bundles (VA) and transmitting tissue (T). Tissue fixed with GA, 1.5 ~ Epon section, stained with PAS and aniline blue black. • 450. b Transmitting tissue (9") of style. This is one-half of the total transmitting tissue; the other half is a mirror image of that shown. Numerous pollen tubes are present in the transmitting tissue, and some are indicated by arrows. Tissue prepared and stained as in Fig. 10a. • 450
Fig. 9. The parenchyma cells (lower portion of figure) immediately below the parenchyma cells (upper portion of figure) adjacent to the hair cells are smaller and the intracellular spaces (IS) are filled with an amorphous material (arrows) believed to be pectins. These cells contain starch (S) and frequently have lobed nuclei (N). GA-Os fixation. • 6,000
108
W.A. JENSEX ~nd D. B. :FISttER:
Fig. 11
Cotton Embryogenesis: Stigma and Style
109
not through the middle lamella (Fig. 20). As the diameter of the tube increases distal to the tip, m a n y but not all of the adjacent transmitting cells are crushed. When a tube first grows through a portion of the style which has not yet been penetrated b y other pollen tubes, there are relatively few immediate changes in the transmitting cells (Fig. 21). At a given level in the style as the first tubes become older and more tubes grow through the transmitting tissue, more transmitting cells degenerate. The cells which do not actually degenerate are little affected b y the pollen tube even when immediately adjacent to it (Fig. 22). Starch is not depleted, and the lipid deposits are not lost. The calcium crystals m a y be dissolved, but this is difficult to establish because of the effects of the methods of tissue preparation. There is no change in ribosome number or aggregation and the E R remains the same. The dictyosomes appear more active, and the thickness of the wall increases (Fig. 22). Increased dictyosome activity and wall thickening also mark the beginning of the degeneration of the transmitting cells next to the pollen tubes (Fig. 20). The wall increases in thickness until the cytoplasm of the cell is compressed into a fraction of its former volume (Fig. 22). The nucleus becomes progressively more electron-dense, dark granules appear, and the chromatin becomes more condensed (Fig. 23). The cytoplasm also becomes darker as the ground cytoplasm fills with ribosomes and dark, amorphous material. The vacuoles collapse, and all of the membranes become black and lose their structure (Fig. 22). At no time does the starch break down. Cell degeneration appears related to the passage of the pollen tube as the cells which degenerate are always against or near the pollentube. However, not all cells close to or against the tube degenerate. The cells degenerate almost at random although only those near the pollen tube are affected. The cells in Fig. 22 are typical of the older styles : cells at all stages of degeneration appear near the pollen tube, but the stage of degeneration of the individual cells appears to be unrelated to the surrounding cells. The same condition holds for younger styles where degeneration is beginning (Fig. 20). The passage of the pollen tube results in the deposition of callose in the transmitting tissue. The callose appears in the pit fields of the cells, and only in extreme cases of degeneration was callose deposited over the entire surface of the wall. The callose reaction in the transmitting ceils
Fig. 11. Transverse section of the transmitting tissue of the stigma. The radial walls are thick and four layers can be identified morphologically and chemically (see Fig. 13). Plaslnodesmata are lacking except in Lhose localized portions of the wall Lhat are considerably thinner (arrows). Small vesicles (Ve) are present in wall layer 3. A portion of a pollen tube (PT) can be seen to be in wall layer 3. GA-Os fixation. • 6,000
Fig. 12. Cells of the transmitting tissue. Longitudinal section in which the thick radial walls and the thin end walls can be easily seen. No large vacuoles are found in the cells although small ones are present. GA-0s. • 6,000
increases with distance from the pollen t u b e tip. Near the pollen-tube tip (within a b o u t 50 ~) there were rarely a n y deposits of callose i n the t r a n s m i t t i n g tissue. F a r t h e r from the tip callose deposits were seen with
W. A. J ~ c s ~
and D. B. FIS~Er~: Cotton Embryogenesis: Stigma and Sgyle
111
L~yer PecficM(}terials Hemicellulose !.Non CelI.Poly. Cellulose l I
+
2
+
3
+ ++ + + + +
4
+
+
+
+
+
+ +
Fig. 13. The composition of the wall layers of the t,ransmit~ing tissue based on stainfi~g wit,h ruthenium red and differential extraction-PAS or PA-sitver staining
increasing frequency. The cells immediately adjacent to the pollen tube react first and then the cells farther away. Yet the reaction never spread more than abou~ three celt diameters from the pollen tube. Discussion Stigmas can be divided into two groups: those which are covered with secreted material at the time they are receptive, the "wet stigmas" ; and those which lack secreted material, the "dry stigmas ". Cotton is in the second group while Petunia, the only other plant the stigma of which has been examined ultrastructurally (KONA~ and L r s s x ~ s s , 19fi6 a), is in the first group. The differences in ultrastructure of the stigma and the relationship of the stigmatic hairs to the pollen tubes between the two plants are striking. In Petunia the cytoplasm of the stigmatic hairs does not degenerate, but copious amounts of material are secreted by the cell and accumulates between the wall and the cuticle. The secreted material is rich in amino acids, f a t t y acids, and sugars (Ko~A~ and LIssx~:c~s, 1966b). In the Cruci]erae the pollen tube grows in the space between the wall and the cuticle of the stigmatic hair cells (K~o~t, 1964).
112
W.A.
JE~s~
and D. B. FISHE~ :
Fig. 14
Cotton Embryogenesis: Stigma and Style
113
Fig. 15. Deposits of calcium salts in the large vacuote of a transmitting cell. The form of the crystal has probably been altered during fixation and dehydration. Tile small vacuoles show no evidence of crystal formation. The dense-appearing mitoehondria and the parallel rows of rough E R are characteristic of these cells. GA-Os fixation. • 22,750
Fig. 14. Cells of the transmitting tissue. The coils of membranes and collections of tubular elements (T) are characteristic of the conductive ceils. The ceils are richly cytoplasmic with many plastids (P), mitochonclria (M), and dictyosomes (D). GA-Os. • 14,600 8 Planta (Berl.), Bd. 84
114
W . A . JE~'SEN ~nd D. B. FIshER:
Figs. 16--19
Cotton Embryogenesis: Stigma and Style
115
In cotton the cytoplasm of the stigmatic hairs degenerates, no material is secreted, and the cuticle remains appressed to the wall The pollen tube grows down the outside of the stigmatic hairs and then between them at their bases. At no time does the pollen tube enter the cell or penetrate beneath the cuticle. The basis of this difference in stigmatic organization may well reside in the difference in pollen characteristics. Petunia pollen is relatively easy to germinate (SAss~, 1964) while cotton pollen is not. Only if moisture conditions are carefully regulated or certain aromatic compounds are present, can cotton pollen be induced to germinate off the s t i g m a (MII~AVALLE, 1965). The degenerate cytoplasm of the stigmatic hairs may influence the moisture content of the immediate environment of the pollen and liberate a variety of aromatic compounds. At least on morphological grounds it would appear that the pollen tube of cotton is not receiving metabolic snbstrates from the stigmatic hairs. Cotton pollen is large and unusually rich in storage materials of all types: carbohydrate, lipids and protein (FIsI~n et al., 1968). Petunia pollen appears less well supplied with its own reserves and the pollen tubes are known to absorb material during their development (SAssE~, 1964). Thus, it would appear possible that in cotton the functional significance of the condition of the stigmatic hair cells is related to the germination phase of pollen development while in petunia the condition of the stigmatic cells is related to the nutrition of the pollen tube. Once the pollen tube grows beyond the base of the stigmatic hairs it passes between the thin-walled cells of the stigma. The intercellular space between these cells is quite large, and the tubes appear to adapt themselves to this space. Relatively few of the stigma cells appear to be crushed by the growth of the pollen tube. There were no changes in the structure of the stigma cells related to the passage of the pollen tube other than the deposition of callose. The pollen tubes growing down the hair cells into the intercellular spaces between the stigma cells next reach the region where these spaces are replaced by a thick layer of pectic material From these cells the
Fig. 16. Pollen germinating on stigmatic hairs. The pollen tube on t h e right can be seen growing between the hairs (arrow). Tissue fixed in GA, 1.5 ~ Epon section, stained with PAS and Aniline blue black. • 100 Fig. 17. Pollen tube
(PT) growing between hairs of stigma. Tissue preparation as in Fig. 10. • 450
Fig. 18. Pollen tube ( P T ) at base of hairs. The tube has made a t u r n and has crushed some cells of the stigma. Tissue preparation as in Fig. 10. • 450 Fig. 19. Pollen tubes ( P T ) in the transmitting tissue of the style. The tubes are cut a t various distances from the tip a n d thus show varying cytoplasmic characteristics. Tissue preparation as in Fig. 10. • 300 8*
116
W . A . JE~sE~r and D. B. FISHER:
Fig. 20. Pollen tube in the transmitting tissue of the style. Several cells have been crushed by the pollen tube and appear black although the starch is grey. One uncrushed cell has begun to degenerate (DC) while several adjacent cells appear unchanged. The pollen tube grew through wall layer 3 of the transmitting cell at the top of the tube. GA-Os fixation. • 6,000
Cotton Embryogenesis: Stigma and Style
117
Fig. 21. Pollen tube in the transmitting tissue of the style, The vegetative nucleus (VAT), and the two sperm (S1 and $2) c~n be seen in the tube. Transmitting cells surrounding the tube are not changed by the immediate presence of the pollen t.ube, GA-Os. • 6,000
118
W.A.
J]~Ns]~ and D. ]3. FrSH~R:
pollen tube makes the transition to the thick wMls of the transmitting tissue. The wall layer through which the pollen tube grows is loosely organized, is rich in pectic material, and contains protein and numerous vesicles. These vesicles are the common morphological ]ink between the cells of the stigma and style and warrant further investigation. The impression gashed from the present data is that, once the pollen germinates and the pollen tube begins to grow down the hair, the p a t h of the tube is determined b y the morphology of the cells, particularly the cell walls, of the stigma and style. F r o m an intercellular space filled with air or aqueous medium to what is an essentially intercellular space filled with pectic material to a porous cell wall, the pollen tube appears to be developing down a preexisting path of least mechanical resistance. The file-like arrangement of the cells of the transmitting tissue and the grouping of these into units corresponding to the number of stigmatic lobes and locules result in a definite p a t h for the pollen tube and appear to minimize the need for a chemotropic response at this stage of pollen tube growth. The relationship of the pollen tube to the transmitting cell is a curious one in cotton. There is no degeneration of the transmitting tissue before the pollen tubes are present. The pollen tubes are not following a p a t h of degenerating cells; instead, degeneration of the transmitting cells follows in the wake of the tube. The passage of the pollen tube crushes some of the cells causing their immediate degeneration. Yet the mere passage of a tube near or adjacent to a transmitting cell is not enough to cause the immediate degeneration of t h a t cell. The uncrushed cells closest to the tube degenerate earliest but, of two adjacent cells both next to a tube, only one m a y degenerate. This can also be seen in the ca]lose reaction where not all the cells form callose and none more than 2 - - 3 cell diameters from the tube. The site of catlose formation is also puzzling. Presumably, the callose is formed as a wound response to the pollen tube and is related to the degeneration of the transmitting tissue. I n this context it might be postulated t h a t the passage of the pollen tube changes the permeability of the cells and t h a t callose is formed as a reaction against cell leakage. However, callose is not
Fig. 22. Pollen tube (p~r) in the transmitting t.issue of the style. The tip is much farther away than in Fig. 12 and the tube cytoplasm is highly vacuola~e. The transmitting cells are in various stages of degeneration, but this degeneration shows no pattern in relation to the tube. GA-Os. • 2,800 Fig. 23. Degenerating transmitting cell. The pyenotic appearance of the nucleus as well as the presence of clumps of particles in the nucleus are usual features at this stage of degeneration. The cytoplasm appears more dense and the wall greatly thickened. GA-Os. • 22,750
Cotton Embryogenesis: Stigma and Style
Figs. 22 and 23
119
120
W.A. JE~SEN and D. B. FISHER:
uniformly deposited over the surface of the cell but only at the pits. This may mean that the healthy or unaffected cells are depositing callose in the pits which acts to prevent transfer of material from these cells to the damaged cells. These considerations bear on the question of how much material the pollen tube acquires from the style. Clearly relatively little of the storage products of the transmitting cells themselves are used. Starch is not utilized in cotton nor are hpids or proteins visibly affected. The wall m a y contribute material but even here the case is not clear. I n view of the discussion of the role of ca]cinm in tube growth (MASCAI~E~gASand MACI~LIS, 1964) it is interesting that calcium deposits are found only in transmitting tissue of the style and may be changed by the passage of the pollen tube. The deposition of callose, however, in the degenerating cells without loss of starch m a y indicate a high level of low-molecularweight carbohydrates that could be utilized by the pollen tube. The impression gained from the present study of the ultrastructure and composition of the tissues of the stigma and style is that these tissues function more as a passive route for the growth of the tube rather than active agents in the control of tube development, although the presence of protein in the wall requires additional investigation. I n our discussion of the style we have employed the term transmitting tissue to designate the specialized cells between which the pollen tubes grow. This term was proposed by AXBEI~ (1937) and, as ESAIy (1965, p. 559) points out, is preferable to the term conducting tissue which can be confused with the vascular tissue. EsAIr prefers to refer to this tissue as the stigmatoid tissue on the basis of its similarity to the tissue of the stigma. While the cells lining the stylar canal in the case of open styles may be similar to those of the stigma, the present observations show how different the transmitting tissue of a closed style is from the stigma. We have therefore decided to retain the older term because we believe that for closed styles stigmatoid tissue does not accurately describe the condition of the cells. The research was supported by grants from the National Science Foundation (GB3460) and the I~ational Institutes of Health (5-1~0I-CA 03656-9) and carried out while one of us (WAJ) was a professor in the Miller Institute for Basic Science at the University of California, Berkeley and the other (DBF) was a Public Health postdoctoral fellow. We would like to express out thanks for the help we have received from Mrs. PAZILASTETLERand Miss MAI~YASI~TON. References
ARBER,A. : The interpretation of the flower: a study of some aspects of morphological thought. Biol. Res. 12, 157--184 (1937). BALLS,W. L. : The sexuality of cotton. In: Yearb. Khedivial Agric. Soc. (Cairo) 1905, p. 199--222.
Cotton Embryogenesis: Stigma and Style
121
EsAv, K.: Plant anatomy, 2nd edn. New York: J. Wiley & Sons 1965. FISEEa, D. B. : Protein staining of ribboned epon sections for light microscopy. tIistoehemie, in press (1969). - - W. A. J ~ S E ~ , and M. E. ASHTO~: I-Iistochemical studies of pollen: Storage pockets in the endoplasmie retieulum (ER). Histochemie 13, 169--182 (1968). IYENGAR,N. K.: Pollen tube studies in Gossypium. J. Genet. 37, 69--106 (1938). JENSEIg, W. A.: Botanical Histoehemistry. San Francisco: Freeman 1962. - - The composition and ultrastructure of the nucellus in cotton. J. Ultrastruct. Res. 111, 112--128 (]965). - - , and D. B. FISEE~: Cotton embryogenesis: The pollen tube in the stigma and style. Protoplasma (Wien), in press (1969). - - - - , and M. E. A s ~ o ~ : Cotton embryogenesis: Studies on the wall of the pollen tube and the transmitting tissue. J. Cell Sci., in press (1969). Ko~A~, R. N., and H. F. LINSKENS: The morphology and anatomy of the stigma of Petunia hybrida. Planta (Berl.) 71, 356--371 (1966a). - - - - Physiology and biochemistry of the stigmatic fluid of Petunia hybrida. Planta (Berl.) 71, 372--387 (1966b). KROH, M. : An electron microscopic study of the behavior of Cruei/erae pollen after pollination. In: Pollen physiology and fertilization ( H . F . LINSKENS, ed.), p. 221--224. Amsterdam:North-Holland Publ. Co. 1964. I)~ASCARENHAS,J.P., and L. MACHLIS: Chemotropie response of the pollen of Antirrhinum ma]us to calcium. Plant Physiol. 119, 70 77 (1964). MIRAVALLE, t~. J. : Germination of cotton pollen. Empire Cotton Growing gev. 42, 287--289 (1965). SaSSE~, M. M. A. : Fine structure of Petunia pollen grain and pollen tube. Aeta bot. neerl. 13, 175--181 (1964). Prof. WILLItSI A. JENSEN Department of Botany University of California Berkeley, California 94720, USA
Dr. DONALD B. FISheR Department of Botany University of Georgia Athens, Georgia 30601, USA
Cotton Embryogenesis : The Tissues of the Stigma and Style and their Relation to the Pollen Tube WILLIAM A. J ~ N S ~ a n d DONALD B. F I S H ~ Department of Botany, University of California, Berkeley Received September 12, 1968
Summary. The stigma of cotton (Gossypium hirsutum) is covered by unicellular hairs. The cytoplasm of these hairs degenerates before the stigma becomes receptive. The vacuole remains intact, but the hair cytoplasm becomes a mass of dark, amorphous material with only a few organelles still being visible. The rest of the stigma consists of thin-walled parenchyma cells with large vacuoles and large amounts of starch. The cells of the style are differentiated into a uniseriate epidermis, vascular tissue, a cortex of thin-walled, vacuolate parenchyma cells, and the transmitting tissue. This latter tissue occupies the center of the style and consists of thick-walled cells with few vacuoles. The cells are rich in starch, ribosomes, endoplasmic reticulum and dietyosomes. They also contain deposits of calcium salts in the form of druses. The pollen germinates on the stigmatic hairs, grows down the outside of the hair and between the cells of the stigma to the transmitting tissue of the style. There the tubes grow between the walls of the cells but do not enter the cells themselves. Some transmitting cells adjacent to the pollen tube degenerate after the tip of the pollen tube has grown past them. However, not all degenerate, and those that do show no fixed spatial relationship to one another. The cells which do degenerate follow a characteristic pattern of breakdown. No ultrastructural evidence was found for the secretion of hydrolytic enzymes by the pollen tube. Introduction The cellular o r g a n i z a t i o n of the s t i g m a a n d style of c o t t o n was first described b y BALLS (1904) a n d s t u d i e d in g r e a t e r detail b y IYE~CAR (1938). The style is solid, t h e center being occupied b y a g r o u p of cells t e r m e d t h e t r a n s m i t t i n g tissue t h r o u g h which t h e pollen t u b e grows. This p a p e r is a r e p o r t of t h e u l t r a s t r u c t u r e a n d composition of t h e cells of the s t i g m a a n d style a n d the r e l a t i o n of these cells to t h e g r o w t h of t h e pollen tube. The u l t r a s t r u c t u r e of t h e pollen t u b e in t h e s t i g m a a n d style will be d e a l t w i t h in a s e p a r a t e p a p e r (JE~sEN a n d FISHEI~, 1969). The u l t r a s t r u c t u r e a n d composition of t h e walls of t h e t r a n s m i t t i n g tissue a n d t h e pollen t u b e will be t r e a t e d in detail in a n o t h e r p a p e r (JE~sv, N e t al., 1969). Materials and Methods Stigma and styles of Gossypium hirsutum L., cv. 1V18949, were collected at various times after pollination. The tissue was fixed in cold 3 % glutaraldehyde in 7
P l a n t a (Berl.), Bd. 84
98
W.A. J E ~ S ~ and D. B. FISHER:
0.05 M cacodylate buffer, pH 6.8. I t was rinsed, then postfixed in 2% OsO4 for 2 hours. Following dehydration in acetone, it was embedded in Epon. The 70% acetone contained 1% uranyl nitrate. The sectioned tissue was stained with lead citrate and examined with a Zeiss EM-9 electronmicroscope. For observations with the light microscope the tissue was fixed in 3% glutaraldehyde in 0.05 M caeodylate buffer at pH 6.8 for 4 hours, dehydrated in acetone, embedded in Epon and sectioned at 1.5 ~. The periodic acid-Schiff (PAS) reaction was used for the localization of insoluble carbohydrates (JENsE~, 1961) and Aniline blue black for general protein (FIshER, 1969). Observations on callose were made on 6-tz frozen sections, using the aniline blue florescence procedure (JI~SEN, 1962). The stigma-style was also freeze-substituted and embedded in paraffin. 10-~ sections were stained with PAS to show the general organization of the tissue. Segments of style were freeze-substituted, differentially extracted for cell wall compounds using the method given by JE~SE~ (1962), dehydrated, and embedded in Epon. The extracted tissue was either sectioned at 1 ~ and stained with PAS or thin-sectioned and stained with periodic acid-silver (PA-silver) (J~NsE~ et al., 1968). Tissue was also stained with ruthenium red by adding it to the glutaraldehyde used in the fixation.
Results
The Stigma. The gross m o r p h o l o g y of t h e s t i g m a a n d style of c o t t o n can be seen in Figs. 1 a n d 2. The s t i g m a is 1 0 - - 1 2 m m in l e n g t h a n d is dissected into 3 - - 5 lobes. The t o p a n d o u t e r p o r t i o n of t h e lobes are covered w i t h long, unicellular hairs (Figs. 2 a n d 5a). W i t h increasing distance f r o m t h e t i p of t h e stigma, t h e hairs become s h o r t e r a n d more r e s t r i c t e d in their location (Fig. 5 b, c) u n t i l t h e y are no longer p r e s e n t (Fig. 5d). The hairs are closely p a c k e d a t their b a s a l ends b u t are loosely associated a t their apical ends, giving t h e s t i g m a a f e a t h e r y a p p e a r a n c e (Fig. 5a). W h e n t h e s t i g m a is r e c e p t i v e to pollen a t anthesis, t h e hair cells a p p e a r as in Figs. 3 a n d 4. The wall is r e l a t i v e l y t h i n a n d on t h e e x t e r i o r face is covered b y a d i s t i n c t cuticle which is closely a p p r e s s e d to t h e wall (Fig. 6). The cell is filled b y a large vacuole u s u a l l y containing electrondense material. The c y t o p l a s m is a n a l m o s t a m o r p h u s b l a c k mass of d e g e n e r a t i n g organel]es, some of which can still be t e n t a t i v e l y identified (Fig. 6 a). Small a m o u n t s of s t a r c h can be f o u n d in t h e hair cells.
Fig. 1. The cotton flower showing the arrangement of the anther sheath surrounding the style and base of the stigma. X 1 Fig. 2. The stigma, style, and ovary of the cotton flower. The lettered arrows correspond to the sections shown in Fig. 5 Fig. 3. Stigmatic hairs (H) and parenehyma (P) cells. GA fixed, Epon embedded, 1.5 ~ section, stained with Aniline blue black and PAS. X 450 Fig. 4. Stigmatic hairs. Preparation as in l~ig. 3 except that the tissue was stained only with Aniline blue black. Starch appears white. • 1,500
Cotton Embryogenesis: Stigma and Style
Figs. 7*
1--4
99
100
W.A. JENSEN and D. B. FISHER:
Fig. 5. Sections through the stigma and style. The position of the sections is indicated in Fig. 2. The transmitting tissue (TT) is the intensely stained cells in the section. l~rozen-substituted tissue, 10 ~ sections, stained with PAS. • 115 I m m e d i a t e l y below the hair cells are t h i n - w a l l e d p a r e n c h y m a cells which show no evidence of c y t o p l a s m i c d e g e n e r a t i o n (Fig. 7). These cells contain large vacuoles which o c c u p y a b o u t half of t h e cell (Fig. 8). T h e p l a s t i d s are large a n d each contains 5 - - 8 large s t a r c h grains (Fig. 8). Mitoehondria a n d d i e t y o s o m e s are numerous. B o t h ribosomes a n d endoplasmic r e t i c u l u m (ER) are a b u n d a n t , b u t m o s t of t h e ribosomes a p p e a r Fig. 6. Stigmatic hair fixed with GA-Os and stained with uranium and lead. The vacuole (V) is intact although the cytoplasm has degenerated. Starch (S) remains in the plastids. The cuticle is visible as the thin, dark layer covering the exterior surface of the wall. • 6,000. Insert. Portion of hair showing the condition of the cytoplasm at higher magnification, l~emains of a dictyosome (D) and possibly a mitochondrion (M) can be seen. • 22,750
Cotton Embryogenesis- Stigma and Style
Fig. 6
I01
102
W.A. JENSENand D. B. FISE~R:
unattached to the EI~ (Fig. 8). A few spherical protein accumulations have been seen in the E R which resemble those reported in the nucellar ceils of cotton (Js~sn~r 1965). The cells immediately below the hair cells are separated by numerous, large intercellular spaces (Fig. 7). Several cell layers beneath the hairs, the intercellular spaces decrease in size and the walls thicken (Fig. 9). Farther in, the intercellular spaces are no longer present and a thick wall, rich in pectic substances, is found (Fig. 9). The appearance of the sections suggests a transition from space filled with air to that filled with amorphous wall material. These cells lead to the thick-walled transmitting tissue of the style. They are distinct from the transmitting tissue and the cortical parenchyma cells of the style. From their position and cytology they appear to have a definite role in the growth of the pollen tube. The Style. The style is covered by a uniseriate epidermis which contains guard cells and stomata (Fig. 10a). The cortex is composed of thin-walled parenchyma cells. Small vascular bundles are present near the periphery of the cortical tissue. The center of the style is occupied by the transmitting tissue, the cells of which are arranged into groups (Fig. 10b). The arrangement and number of cell groups in the transmitting tissue appears related to the number of locules in the gynoecium; there are 3--5 in the variety of cotton used in this study. The transmitting tissue is a collection of thick-walled cells which are arranged in files (Figs. 11 and 12). The lateral walls are uniformly thick except for occasional pit fields (Fig. 11) while the transverse walls are relatively thin (Fig. 12). The lateral walls consist of several, distinct layers (Fig. 11) (J~ss~N et el., 1969). Against the plasma membrane there is a relatively uniformly textured wall, approximately 2 ~ thick (wall layer 1) that is composed primarily of pectic substances and hemicellulose. Surrounding this is a thinner, darker-appearing wall, 0.5 ~ thick (wall layer 2) similar in composition to wall layer 1 but richer in hemicellulose. This wall in turn is surrounded by a thick (4--6 ~) loose-textured wall that shows concentric rings of fibrous material (wall layer 3). This layer is rich in pectic substances, relatively poor in hemicellulose and is the only wall layer that contains detectable amounts of non-cellulosic polysaccharides and cellulose. Finally, there is the middle lamella region which is quite pronounced (wall layer 4), being 0.5--1
Fig. 7. The relation oi the base of the hair cells (H) to the underlying parenchyma cells of the stigma. Large intr~cellular spaces (IS) are characteristic of this region. GA-Os fixed tissue stained with ruthenium red to emphasize the pectic substances of the wall. This procedure reduces the general contrast of the cytoplasm. Large starch grains are present in the parenehyma cells. • 6,000
Cotton Embryogenesis: Stigma and Style
Fig. 7
103
Fig. 8. Parenchyma cells of the stigma. Large vacuoles (V) are present. The plasrids (P) are large and filled with starch. Intercellular spaces are numerous and large. GA-Os fixation. • 6,000
W. A. JENSEN and D. B. FlSgEI~: Cotton Embryogenesis: Stigma and Style
105
thick, distinctive in appearance and composed primarily of pectic substance. Both layers 3 and 4 stain with Aniline blue black for protein. The composition of the various wall layers based on the differential extraetion-PAS stain method (JExsE~, 1962) is shown in Fig. 13. This is a condensed summary of the composition of the wall and full details of the methods and results will be presented elsewhere ( J ~ s ~ et al., 1969). Embedded in the wall, primarily wall layer 3, are masses of small vesicles (Figs. 11 and 12), similar to those found in the walls of the stigma cells. The cells contain many mitoehondria and dictyosomes (Fig. 14). The latter appear particularly active in forming vesicles which can be seen in the cytoplasm and which presumably are fusing with the plasma membrane (Fig. 14). The plastids are large and contain numerous starch grains (Fig. 14) which sometimes look as if they fuse into a multilobed, single mass of starch (Fig. 14). Many of the cells contain 1 or 2 lipid bodies several microns in diameter. Polysomes are seen in the cytoplasm and the abundant E R has ribosomes associated with it. There are two types of vacuoles present in the transmitting cells. The first is usually spherical or slightly ellipsoidal in shape and is 7--9 ~ in diameter. Frequently the cell also contains a vacuole 20--40 Ix in diameter which contains a druse presumably composed of calcium salts (Fig. 15). The form of the drnse in Fig. 15 is characteristic of the EM preparation and is undoubtedly altered from the condition in the living cells. Not all the cells have wall-formed deposits, but many have what appear to be the remains of such crystals present in the large vacuole. The nuclei of the transmitting cells are large (Fig. 14) and frequently lobed (Fig. 12). The chromatin is usually condensed near the nuclear membrane (Fig. 14). Pockets of membrane appear toward the cell periphery sometimes adjacent to the wall itself (Fig. 14). They may take the form of concentric rings of membranes (Fig. 14), collections of tubes and vesicles, or intermediate forms with both tubes and concentric rings (Fig. 14). Relation o/ the Pollen Tube to the Cells o] the Stigma and Style. The pollen lands on the surface of the hairs of the stigma, germinates, and grows down the surface of the hair and penetrates the stigma (Figs. 16 and 17). When the pollen enters the tissue of the stigma, it grows between the cells in the intercellular spaces (Fig. 17). Some of the cells appear crushed by the pollen tube, but not all cells in contact with the tube are damaged (Fig. 1S). The tube grows from the outer, thin-wMled cells of the stigma into the transmitting tissue of the style. This tissue is composed of thickwalled cells as noted earlier and the pollen tube grows through the cell wall (Fig. 19). The pollen tube actually grows through wall layer 3 and
Fig. 9
W. A. J ~ s E ~ and D. B. FISHER: Cotton Embryogenesis: Stigma and Style
107
Fig. 10a and b. Tissues of the style, a Epidermis (E) with stomata and guard cells (arrow); cortex (C); vascular bundles (VA) and transmitting tissue (T). Tissue fixed with GA, 1.5 ~ Epon section, stained with PAS and aniline blue black. • 450. b Transmitting tissue (9") of style. This is one-half of the total transmitting tissue; the other half is a mirror image of that shown. Numerous pollen tubes are present in the transmitting tissue, and some are indicated by arrows. Tissue prepared and stained as in Fig. 10a. • 450
Fig. 9. The parenchyma cells (lower portion of figure) immediately below the parenchyma cells (upper portion of figure) adjacent to the hair cells are smaller and the intracellular spaces (IS) are filled with an amorphous material (arrows) believed to be pectins. These cells contain starch (S) and frequently have lobed nuclei (N). GA-Os fixation. • 6,000
108
W.A. JENSEX ~nd D. B. :FISttER:
Fig. 11
Cotton Embryogenesis: Stigma and Style
109
not through the middle lamella (Fig. 20). As the diameter of the tube increases distal to the tip, m a n y but not all of the adjacent transmitting cells are crushed. When a tube first grows through a portion of the style which has not yet been penetrated b y other pollen tubes, there are relatively few immediate changes in the transmitting cells (Fig. 21). At a given level in the style as the first tubes become older and more tubes grow through the transmitting tissue, more transmitting cells degenerate. The cells which do not actually degenerate are little affected b y the pollen tube even when immediately adjacent to it (Fig. 22). Starch is not depleted, and the lipid deposits are not lost. The calcium crystals m a y be dissolved, but this is difficult to establish because of the effects of the methods of tissue preparation. There is no change in ribosome number or aggregation and the E R remains the same. The dictyosomes appear more active, and the thickness of the wall increases (Fig. 22). Increased dictyosome activity and wall thickening also mark the beginning of the degeneration of the transmitting cells next to the pollen tubes (Fig. 20). The wall increases in thickness until the cytoplasm of the cell is compressed into a fraction of its former volume (Fig. 22). The nucleus becomes progressively more electron-dense, dark granules appear, and the chromatin becomes more condensed (Fig. 23). The cytoplasm also becomes darker as the ground cytoplasm fills with ribosomes and dark, amorphous material. The vacuoles collapse, and all of the membranes become black and lose their structure (Fig. 22). At no time does the starch break down. Cell degeneration appears related to the passage of the pollen tube as the cells which degenerate are always against or near the pollentube. However, not all cells close to or against the tube degenerate. The cells degenerate almost at random although only those near the pollen tube are affected. The cells in Fig. 22 are typical of the older styles : cells at all stages of degeneration appear near the pollen tube, but the stage of degeneration of the individual cells appears to be unrelated to the surrounding cells. The same condition holds for younger styles where degeneration is beginning (Fig. 20). The passage of the pollen tube results in the deposition of callose in the transmitting tissue. The callose appears in the pit fields of the cells, and only in extreme cases of degeneration was callose deposited over the entire surface of the wall. The callose reaction in the transmitting ceils
Fig. 11. Transverse section of the transmitting tissue of the stigma. The radial walls are thick and four layers can be identified morphologically and chemically (see Fig. 13). Plaslnodesmata are lacking except in Lhose localized portions of the wall Lhat are considerably thinner (arrows). Small vesicles (Ve) are present in wall layer 3. A portion of a pollen tube (PT) can be seen to be in wall layer 3. GA-Os fixation. • 6,000
Fig. 12. Cells of the transmitting tissue. Longitudinal section in which the thick radial walls and the thin end walls can be easily seen. No large vacuoles are found in the cells although small ones are present. GA-0s. • 6,000
increases with distance from the pollen t u b e tip. Near the pollen-tube tip (within a b o u t 50 ~) there were rarely a n y deposits of callose i n the t r a n s m i t t i n g tissue. F a r t h e r from the tip callose deposits were seen with
W. A. J ~ c s ~
and D. B. FIS~Er~: Cotton Embryogenesis: Stigma and Sgyle
111
L~yer PecficM(}terials Hemicellulose !.Non CelI.Poly. Cellulose l I
+
2
+
3
+ ++ + + + +
4
+
+
+
+
+
+ +
Fig. 13. The composition of the wall layers of the t,ransmit~ing tissue based on stainfi~g wit,h ruthenium red and differential extraction-PAS or PA-sitver staining
increasing frequency. The cells immediately adjacent to the pollen tube react first and then the cells farther away. Yet the reaction never spread more than abou~ three celt diameters from the pollen tube. Discussion Stigmas can be divided into two groups: those which are covered with secreted material at the time they are receptive, the "wet stigmas" ; and those which lack secreted material, the "dry stigmas ". Cotton is in the second group while Petunia, the only other plant the stigma of which has been examined ultrastructurally (KONA~ and L r s s x ~ s s , 19fi6 a), is in the first group. The differences in ultrastructure of the stigma and the relationship of the stigmatic hairs to the pollen tubes between the two plants are striking. In Petunia the cytoplasm of the stigmatic hairs does not degenerate, but copious amounts of material are secreted by the cell and accumulates between the wall and the cuticle. The secreted material is rich in amino acids, f a t t y acids, and sugars (Ko~A~ and LIssx~:c~s, 1966b). In the Cruci]erae the pollen tube grows in the space between the wall and the cuticle of the stigmatic hair cells (K~o~t, 1964).
112
W.A.
JE~s~
and D. B. FISHE~ :
Fig. 14
Cotton Embryogenesis: Stigma and Style
113
Fig. 15. Deposits of calcium salts in the large vacuote of a transmitting cell. The form of the crystal has probably been altered during fixation and dehydration. Tile small vacuoles show no evidence of crystal formation. The dense-appearing mitoehondria and the parallel rows of rough E R are characteristic of these cells. GA-Os fixation. • 22,750
Fig. 14. Cells of the transmitting tissue. The coils of membranes and collections of tubular elements (T) are characteristic of the conductive ceils. The ceils are richly cytoplasmic with many plastids (P), mitochonclria (M), and dictyosomes (D). GA-Os. • 14,600 8 Planta (Berl.), Bd. 84
114
W . A . JE~'SEN ~nd D. B. FIshER:
Figs. 16--19
Cotton Embryogenesis: Stigma and Style
115
In cotton the cytoplasm of the stigmatic hairs degenerates, no material is secreted, and the cuticle remains appressed to the wall The pollen tube grows down the outside of the stigmatic hairs and then between them at their bases. At no time does the pollen tube enter the cell or penetrate beneath the cuticle. The basis of this difference in stigmatic organization may well reside in the difference in pollen characteristics. Petunia pollen is relatively easy to germinate (SAss~, 1964) while cotton pollen is not. Only if moisture conditions are carefully regulated or certain aromatic compounds are present, can cotton pollen be induced to germinate off the s t i g m a (MII~AVALLE, 1965). The degenerate cytoplasm of the stigmatic hairs may influence the moisture content of the immediate environment of the pollen and liberate a variety of aromatic compounds. At least on morphological grounds it would appear that the pollen tube of cotton is not receiving metabolic snbstrates from the stigmatic hairs. Cotton pollen is large and unusually rich in storage materials of all types: carbohydrate, lipids and protein (FIsI~n et al., 1968). Petunia pollen appears less well supplied with its own reserves and the pollen tubes are known to absorb material during their development (SAssE~, 1964). Thus, it would appear possible that in cotton the functional significance of the condition of the stigmatic hair cells is related to the germination phase of pollen development while in petunia the condition of the stigmatic cells is related to the nutrition of the pollen tube. Once the pollen tube grows beyond the base of the stigmatic hairs it passes between the thin-walled cells of the stigma. The intercellular space between these cells is quite large, and the tubes appear to adapt themselves to this space. Relatively few of the stigma cells appear to be crushed by the growth of the pollen tube. There were no changes in the structure of the stigma cells related to the passage of the pollen tube other than the deposition of callose. The pollen tubes growing down the hair cells into the intercellular spaces between the stigma cells next reach the region where these spaces are replaced by a thick layer of pectic material From these cells the
Fig. 16. Pollen germinating on stigmatic hairs. The pollen tube on t h e right can be seen growing between the hairs (arrow). Tissue fixed in GA, 1.5 ~ Epon section, stained with PAS and Aniline blue black. • 100 Fig. 17. Pollen tube
(PT) growing between hairs of stigma. Tissue preparation as in Fig. 10. • 450
Fig. 18. Pollen tube ( P T ) at base of hairs. The tube has made a t u r n and has crushed some cells of the stigma. Tissue preparation as in Fig. 10. • 450 Fig. 19. Pollen tubes ( P T ) in the transmitting tissue of the style. The tubes are cut a t various distances from the tip a n d thus show varying cytoplasmic characteristics. Tissue preparation as in Fig. 10. • 300 8*
116
W . A . JE~sE~r and D. B. FISHER:
Fig. 20. Pollen tube in the transmitting tissue of the style. Several cells have been crushed by the pollen tube and appear black although the starch is grey. One uncrushed cell has begun to degenerate (DC) while several adjacent cells appear unchanged. The pollen tube grew through wall layer 3 of the transmitting cell at the top of the tube. GA-Os fixation. • 6,000
Cotton Embryogenesis: Stigma and Style
117
Fig. 21. Pollen tube in the transmitting tissue of the style, The vegetative nucleus (VAT), and the two sperm (S1 and $2) c~n be seen in the tube. Transmitting cells surrounding the tube are not changed by the immediate presence of the pollen t.ube, GA-Os. • 6,000
118
W.A.
J]~Ns]~ and D. ]3. FrSH~R:
pollen tube makes the transition to the thick wMls of the transmitting tissue. The wall layer through which the pollen tube grows is loosely organized, is rich in pectic material, and contains protein and numerous vesicles. These vesicles are the common morphological ]ink between the cells of the stigma and style and warrant further investigation. The impression gashed from the present data is that, once the pollen germinates and the pollen tube begins to grow down the hair, the p a t h of the tube is determined b y the morphology of the cells, particularly the cell walls, of the stigma and style. F r o m an intercellular space filled with air or aqueous medium to what is an essentially intercellular space filled with pectic material to a porous cell wall, the pollen tube appears to be developing down a preexisting path of least mechanical resistance. The file-like arrangement of the cells of the transmitting tissue and the grouping of these into units corresponding to the number of stigmatic lobes and locules result in a definite p a t h for the pollen tube and appear to minimize the need for a chemotropic response at this stage of pollen tube growth. The relationship of the pollen tube to the transmitting cell is a curious one in cotton. There is no degeneration of the transmitting tissue before the pollen tubes are present. The pollen tubes are not following a p a t h of degenerating cells; instead, degeneration of the transmitting cells follows in the wake of the tube. The passage of the pollen tube crushes some of the cells causing their immediate degeneration. Yet the mere passage of a tube near or adjacent to a transmitting cell is not enough to cause the immediate degeneration of t h a t cell. The uncrushed cells closest to the tube degenerate earliest but, of two adjacent cells both next to a tube, only one m a y degenerate. This can also be seen in the ca]lose reaction where not all the cells form callose and none more than 2 - - 3 cell diameters from the tube. The site of catlose formation is also puzzling. Presumably, the callose is formed as a wound response to the pollen tube and is related to the degeneration of the transmitting tissue. I n this context it might be postulated t h a t the passage of the pollen tube changes the permeability of the cells and t h a t callose is formed as a reaction against cell leakage. However, callose is not
Fig. 22. Pollen tube (p~r) in the transmitting t.issue of the style. The tip is much farther away than in Fig. 12 and the tube cytoplasm is highly vacuola~e. The transmitting cells are in various stages of degeneration, but this degeneration shows no pattern in relation to the tube. GA-Os. • 2,800 Fig. 23. Degenerating transmitting cell. The pyenotic appearance of the nucleus as well as the presence of clumps of particles in the nucleus are usual features at this stage of degeneration. The cytoplasm appears more dense and the wall greatly thickened. GA-Os. • 22,750
Cotton Embryogenesis: Stigma and Style
Figs. 22 and 23
119
120
W.A. JE~SEN and D. B. FISHER:
uniformly deposited over the surface of the cell but only at the pits. This may mean that the healthy or unaffected cells are depositing callose in the pits which acts to prevent transfer of material from these cells to the damaged cells. These considerations bear on the question of how much material the pollen tube acquires from the style. Clearly relatively little of the storage products of the transmitting cells themselves are used. Starch is not utilized in cotton nor are hpids or proteins visibly affected. The wall m a y contribute material but even here the case is not clear. I n view of the discussion of the role of ca]cinm in tube growth (MASCAI~E~gASand MACI~LIS, 1964) it is interesting that calcium deposits are found only in transmitting tissue of the style and may be changed by the passage of the pollen tube. The deposition of callose, however, in the degenerating cells without loss of starch m a y indicate a high level of low-molecularweight carbohydrates that could be utilized by the pollen tube. The impression gained from the present study of the ultrastructure and composition of the tissues of the stigma and style is that these tissues function more as a passive route for the growth of the tube rather than active agents in the control of tube development, although the presence of protein in the wall requires additional investigation. I n our discussion of the style we have employed the term transmitting tissue to designate the specialized cells between which the pollen tubes grow. This term was proposed by AXBEI~ (1937) and, as ESAIy (1965, p. 559) points out, is preferable to the term conducting tissue which can be confused with the vascular tissue. EsAIr prefers to refer to this tissue as the stigmatoid tissue on the basis of its similarity to the tissue of the stigma. While the cells lining the stylar canal in the case of open styles may be similar to those of the stigma, the present observations show how different the transmitting tissue of a closed style is from the stigma. We have therefore decided to retain the older term because we believe that for closed styles stigmatoid tissue does not accurately describe the condition of the cells. The research was supported by grants from the National Science Foundation (GB3460) and the I~ational Institutes of Health (5-1~0I-CA 03656-9) and carried out while one of us (WAJ) was a professor in the Miller Institute for Basic Science at the University of California, Berkeley and the other (DBF) was a Public Health postdoctoral fellow. We would like to express out thanks for the help we have received from Mrs. PAZILASTETLERand Miss MAI~YASI~TON. References
ARBER,A. : The interpretation of the flower: a study of some aspects of morphological thought. Biol. Res. 12, 157--184 (1937). BALLS,W. L. : The sexuality of cotton. In: Yearb. Khedivial Agric. Soc. (Cairo) 1905, p. 199--222.
Cotton Embryogenesis: Stigma and Style
121
EsAv, K.: Plant anatomy, 2nd edn. New York: J. Wiley & Sons 1965. FISEEa, D. B. : Protein staining of ribboned epon sections for light microscopy. tIistoehemie, in press (1969). - - W. A. J ~ S E ~ , and M. E. ASHTO~: I-Iistochemical studies of pollen: Storage pockets in the endoplasmie retieulum (ER). Histochemie 13, 169--182 (1968). IYENGAR,N. K.: Pollen tube studies in Gossypium. J. Genet. 37, 69--106 (1938). JENSEIg, W. A.: Botanical Histoehemistry. San Francisco: Freeman 1962. - - The composition and ultrastructure of the nucellus in cotton. J. Ultrastruct. Res. 111, 112--128 (]965). - - , and D. B. FISEE~: Cotton embryogenesis: The pollen tube in the stigma and style. Protoplasma (Wien), in press (1969). - - - - , and M. E. A s ~ o ~ : Cotton embryogenesis: Studies on the wall of the pollen tube and the transmitting tissue. J. Cell Sci., in press (1969). Ko~A~, R. N., and H. F. LINSKENS: The morphology and anatomy of the stigma of Petunia hybrida. Planta (Berl.) 71, 356--371 (1966a). - - - - Physiology and biochemistry of the stigmatic fluid of Petunia hybrida. Planta (Berl.) 71, 372--387 (1966b). KROH, M. : An electron microscopic study of the behavior of Cruei/erae pollen after pollination. In: Pollen physiology and fertilization ( H . F . LINSKENS, ed.), p. 221--224. Amsterdam:North-Holland Publ. Co. 1964. I)~ASCARENHAS,J.P., and L. MACHLIS: Chemotropie response of the pollen of Antirrhinum ma]us to calcium. Plant Physiol. 119, 70 77 (1964). MIRAVALLE, t~. J. : Germination of cotton pollen. Empire Cotton Growing gev. 42, 287--289 (1965). SaSSE~, M. M. A. : Fine structure of Petunia pollen grain and pollen tube. Aeta bot. neerl. 13, 175--181 (1964). Prof. WILLItSI A. JENSEN Department of Botany University of California Berkeley, California 94720, USA
Dr. DONALD B. FISheR Department of Botany University of Georgia Athens, Georgia 30601, USA
