Planta (Berl.) 71, 1--14 (1966)
I N C O R P O R A T I O N OF R A D I O A C T I V I T Y INTO W H E A T X Y L E M WALLS J. I). PICKETT-HEAPS Electron Microscope Unit, John Curtin School of l~Iedical Research, Canberra. A.C.T. Australia Received May 10, 1966
Summary. Studies on the fine structural changes accompanying xylem differentiation in wheat coleoptile have indicated that the microtubules are concerned with the inception of a regular wall thickening pattern, and later with wall deposit.ion at the thickening site. The endoplasmic reticulum is situated characteristically in continuous profiles between the thickenings. Radioautographie studies at the electron microscope level using labelled glucoses have shown that the endop]asmic reticulum, golgi bodies and the cytoplasm near the microtubules were often labelled during deposition into nearby thickenings of radioactive materials derived from the tritiated glucoses. Incorporation into the wall occurred mainly at the top of the thickenings. The plastids of the xylem cells were also often labelled, but only during the earlier stages of differentiation; when massive wall deposition was evident, such an incorporation was never observed. The fine structural and radioautographic results are briefly discussed in terms of the possible functions of the organelles in the plant cell. Introduction The xylem vessel is a plant cell in which wall deposition is organised to a very high degree, spiral or annular bands of wall material being formed along the length of the wall in a regular pattern of development. SI~OTT and BLOC~ (1945) noted cytoplasmic banding in Coleus xylem cells, and the location of the bands corresponded to the position of the developing wall thiekenings during rcdifferentiation. Using the same tissue for fine-structural investigations, I-IE~LE~ and NEWCOM~ (1963) equated the bands with an aggregation of cellular organelles, suggesting that this represented a locMised organisation of enhanced respiratory and synthetic activity in the cytoplasm. Both I-IEPLE~ and N~wco~B (1963) and W o o m N c and NOnTHCOTn (1964) noted the appearance of an increased golgi activity in these cells, indicating that the organelle might be involved in the synthesis of wall materials; WOODING and NOnTHCOTn observed apparent incorporation into the sides of developing thickenings of vesicles possibly derived from the golgi apparatus. The endoplasmie retieulum may also be involved in xylem wall formation. POtCTEI~ (1961) noted the appearance of endoplasmic reticulum between the maturing thiekenings of onion protoxylem vessels, and tIEsLoP-ttA~nISON (1963) reported "a striking association" in Pinguicula between the positioning of the wall thickenings and the 1
P l a n t a (BEN.), Bd. 71
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endoplasmic retieulum in the cytoplasm of the cells. Woo~)I~G and NOI~THCOTE (1964) and CRO~SHAW and BOUCK (1965) could find no specific orientation of the endoplasmic reticulum with respect to secondary wall growth in the xylem of Pinus and Avena coleoptile. However in Triticum coleoptile, Pm~ETT-HEA~S and NOI~TKCOTE (1966) showed that the endop]asmic reticulum was present in between the xylem thickenings as continuous circular lamellae. HEPLER and NEwco~B (1964) found fibrillar structures within the cisternae of the endoplasmic reticulum in regenerating Coleus xylem vessels; they suggested that this material was eventually incorporated into the wall. Since LEDBETTER and PORTER (1963) first demonstrated the existenee of microtubules in plant cells, particularly near the growing wall, several papers have described the association of these organelles with the developing thickening of the xylem wall. Groups of microtubules were found above the thickenings by HErLER and NEWCOMB (1964), WOODING and NOaTHCOTE (1964), CaONSHAW and BOUCK (1965) and Pm~ETT-HEAPS and NO~THCOTE (1966); these observations generally supported the hypothesis first proposed by LED~ETTER and PORTER (1963) that the microtubules might be concerned with the deposition of the highly organised cellulose phase in the wall. However, PICKETTHEAPS and NO~THCOTE (1966) have shown that the distribution of the microtubules along the xylem wall changed during the inception of wall differentiation. While initially distributed apparently at random along the undifferentiated wall, they were later found in groups between very immature thickenings, and later still, they were observable in the usual position above the more developed thickenings. During the course of investigations into wall development in wheat xylem cells, it was considered possible that radioautographic methods might elucidate the function of some cell organelles in wall formation. Materials and Methods Wheat seedlings (Triticum vulgate) were washed and germinated on damp filter paper in petrie dishes; they were grown at room temperature in ordinary daylight conditions. When the coleoptiles were about 1 cm long they were excised with a sharp razor blade and placed in the radioactive incubation solutions. D-glucose-6-Ha(l.3 C/raM, from the Radiochemical Center, Amersham, Bucks., England) and D-glucose-l-Ha(350--500 mC/mM, from the New England Nuclear Corp., Boston, Mass.) were made up in glass-distilled water to a concentration of 2 mC/ml and 0.5--1 mC/ml respectively. After a two hour incubation in the appropriate solutions, the coleoptiles were briefly rinsed, cut into 1 mm segments and fixed in a standard 6% glutaraldehyde/phosphate fixative, followed by osmium postfixation, the fixatives containing calcium chloride; fresh segments were similarly treated, or fixed in 4[% unbuffered potassium permanganate, containing sodium and calcium chlorides (PICKETT-HEA1)Sand NOI%THCOTE,1966). All material was then subjected to standard methods of embedding in araldite, sectioning and staining with uranium and/or lead; the radioactive sections were
Wall Incorporation in Xylem
3
coated with Ilford L-4 emulsion by a method based on that described by C~Ro and VAN TUBEI~GEN (1962) (see NORTHCOTE and PICKETT-HEAPS, 1966). After varying periods of photographic exposure (four weeks to four months) the autographic image was developed in Ilford ID-19 or Microdol-X for 45 sec.; the grids were then well washed and the emulsion fixed in Kodafix, diluted 1:4, for 45 see., fo]lowed by further washing and drying. Grids were examined in a Philips EM ]00 or EM 200 at 80 kV.
Results
Unlabelled Specimens Since no confs of a similar sequence of events has yet been presented for differentiating xylem vessels in other plants, extensive observations on wheat xylem vessels have been undertaken to confirm and extend the results reported previously (PICKETT-HEAPSand NORTHCOT~, 1966). For convenience, the sequence of development of the xylem cell has been divided into four stages. First Stage. In the youngest xylem vessels identifiable, the microtubules were evenly spread along the very thin wall with apparently small random variations in their distribution equivalent to those seen in other growing plant cells. Second Stage. The next distinct stage in the differentiation of the xylem wall recognisable in the thin sections, was often indicated by the appearance of a preliminary pattern of regular wall corrugations. The microtubules were organised into bands along the wall, often where no regularity in its structure was discernable in single sections of the cell (Fig. 1). At a slightly later stage, the groups of mierotubules were generally found between the corrugations (see PICKETT-HEAt'S and NOnT~COT~, 1966). Frequently at this stage, elements of the endoplasmic reticulum became closely applied to the plasmalemma in a characteristic fashion. I n a typical example, an area covered by seven photographs (Fig. 1 being one of them) showed thirty-six consecutive regularly-spaced microtubule bands along part of a xylem wall at this stage; with two exceptions, elements of the endoplasmic reticulum were found very close to or directly apposed to the plasmalemma between each group of microtnbules. This regular alternation in distribution of the two organelles is seen very clearly at Stage Three (below). Third Stage. During further differentiation of the xylem wall, massive secondary wall deposition occurs to give the final structure of the wall. Microtubules were found in their usual position above, and to a lesser extent, around the sides of the well-developed thickenings. Elements of the endoplasmie reticulum were found to lie in the cytoplasm in between nearly all of the thiekenings; this is most clearly shown in tangential sections of permanganate-fixed cells (Fig. 2; see PIC~.TT-It~APS and NORTHCOTE, 1966). Vesicular components, probably derived from the golgi apparatus, were numerous in the cytoplasm. 1"
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Fourth Stage. While the thickenings were reaching their final stages of growth, a progressive b r e a k d o w n of the cytoplasm was evident. The
Wall Incorporation in Xylem
5
tonoplast became disrupted and the cell lumen filled with the granular material of cell debris. Finally the end wall of the cell broke down and the ribbed vessel was incorporated into the conducting system of the vascular bundle.
Radioautographic Results The amount of incorporation into the xylem wall of derivatives of the labelled glucoses varied in different pieces of experimental tissue. These variations were probably related to two main variables: i) the proximity of the cell in question to the cut surface, and hence to the labelled metabolites (Fig. 5), and ii) the stage reached in the maturation of the cell. I n connection with the first point, previous experiments had indicated that small coleoptile segments (ca. 1--2 min long) did not give consistent, good radioautographie results. First and Second Stages. The amount of incorporation of labelled derivatives into the wall of these cells was always low, and equivalent to that observed into the walls of neighbouring cells. The labelling of cytoplasmic organelles was also low and it was impossible to correlate the two. Appreciable incorporation of labelled derivatives of both tritiated glucoses was often observed into the starch grains in the plastids of the coleoptile cells, including those of the xylem and occasionally of the sieve-tube cells. This incorporation into the starch of xylem was however, only found in cells during the first two stages of growth, when wall incorporation was very low (Fig. 4). The incorporation into the starch grains was greatest (as a rough fraction of the total radioactivity detected in the section) when glucose-l-tta(G-l-t{ ~) supplied the labelled atom. Fig. 11. Radial longitudinal section oi young xylem cell proceeding into Stage Two. ~iicrotubules are grouped in bands (black lines); alternating with the bands are elements of the endoplasmic retieulum (arrowed) closely applied to the wall. With a few exceptions, the pattern is characteristic of wheat xylem cells at this stage. • 20,000. E R endoplasmie reticulum, G golgi apparatus, M mitochondrion, _N nucleus, P plastid, T microtubules, T H xylem wall thickening, V vacuole, V S vesicles, W ceil wall Fig. 2. A tangential longitudinal section of a maturing xylem cell (Stage Three). The permanganatefixed image shows very clearly the endoplasmie reticulum near the wail lying in long profiles between ahnost all of the thickenings. Compare with radial sections (e.g. Figs. 3, 9, 1 l), where small or longer elements of endoplasmic reticulmn are seen between most of the wall thiekenings. Many vesicular components, probably derived from golgi bodies, are also visible, x 8,300 Fig. 3. Coleoptile tissue exposed to glucose-6-J:I a (G-6-tP) for two hours before fixation. The radial, longitudinal section shows the usual distribution of radio-active incorporation into the wall of a maturing xylem vessel (Stage Three); most of the incorporation has occurred into the top of the wall thickenings (compare with rig. 5). Cytoplasmic labelling is low in comparison to the amount in the walls; the smaller "dwarf" thiekenings generally show a reduced level of radioactivity. • 5,700 1 Glutaraldehyde/osmium
fixation was employed
o n all t i s s u e s e x c e p t t h a t
from which Fig. 2 was prepared; in this case, permanganate fixation was employed. All sections are oriented longitudinally. The radioautographs were developed in ID-19, except for Fig. 12 which was developed in Microdol-X.
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J.D. PIOKEr~-H~ArS:
Wall Incorporation in Xylem
7
Third Stage. During this stage in the development of the xylem cell, massive wall deposition occurs. This was evident radioautographieally, when very large amounts of radioactivity compared with that present elsewhere in the section, were detected in the wall thickenings. The plastids in these cells were never labelled (c.f. above) whilst in adjacent younger xylem vessels, and in the surrounding eambial and parenchyma tissue, the reverse situation was readily visible (Fig. 6). As in the case of root-cap cells ( N O R T H C O T E and P I C K E T T - H E A P S , 1966), far greater overall labelling in the sections was observed when glucose-6-H3 (G-6-H3), rather than G-1-H 3 was used as the precursor. The distribution of labelled material over the wall thiekenings themselves varied somewhat with their shape. Most of the radioactivity was confined to the top of the thiekenings when these were less developed and had moderately flat sides (Fig. 3). I-Iowever, at slightly later stages in the maturation of the wall, there was more tendency towards some incorporation into the sides of the thiekenings (top cell, Fig. 5). The overall space distribution of the labelling along the wall of any given cell was uniform. Often, dwarf or poorly developed thickenings were found amongst the others, and these were generally appreciably less labelled than the rest, or not at all (Fig. 3). The cytoplasm of these xylem cells always contained a small amount of labelling, particularly near the walls (though this was always far less than t h a t in the walls). The golgi bodies were often labelled (Fig. 10) but the proportion labelled to the total number of the organelles visible in the same section of the cell was always small. Occasionally, radioactivity was associated with collections of vesicles (Fig. ll). Fig. 4. Very y o u n g x y l e m cells (Stage One) exposed to glueose-l-H 3 (G-1-H E) for two hours. The microtubules are evenly distributed along the v e r y thin a n d undifferentiated wail. The incorporation of radioactive glucoses into the walls of such cells was always v e r y low, b u t appreciable accumulation of the label was generally present in the starch grains of the plastids. (Compare w i t h :Fig. 6.) • 14,500 ]~'ig. 5. The Radial, longitudinal section of two m a t u r i n g x y l e m cells (Stage Three) showing typical m a s s i v e incorporation of r a d i o a c t i v i t y into the wall thickenings; the eoleoptile h a d been incubated in G-6-H 3 for two hours. The difference in the total labelling in each cell was probably due to the upper cell b e i n g closer to the cut surface of the tissue, a n d hence to t h e labelled metabolite. Much of the incorporation has occurred into the sides of the thickenings (c.f. Fig. 3); in this autograph, it is impossible to ascertain whether the endopIasmic reticulum present between the thickenings, was also labelled. The end wall b e t w e e n the two cells is v i r t u a l l y unlabellcd. • 4,500 Fig. 6. A x y l e m cell (similar to those in Fig. 5) following the two hour incubation of the coleoptile in G - 6 - H h The x y l e m thickenings contain a f a r greater a m o u n t of the labelled derivatives t h a n t h a t in the n e i g h b o u r i n g walls of undifferentiated cells. H o w e v e r , in these latter cells, there is a m a r k e d a c c u m u l a t i o n of the label into the starch grains of the plastids; the plastids of the x y l e m at this point (Stage Three) were n e v e r labelled (c.f. Fig. 4). • 3,600 Fig. 7. X y l e m cell (Stage Three) f r o m a eoleoptilc incubated in G-1-}I 3 for two hours. There was always far less overall incorporation u s i n g this metabolite when c o m p a r e d with t h a t obtained w i t h G-6-Hh The elements of endoplasmic r e t i c u l u m between the thiekenings are the m o s t frequently labelled o r g a n d i e s found near x y l e m thickenings t h a t h a v e incorporated tritiated compounds derived f r o m glucose. • 23,500
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Wall Incorporation in Xylem
9
More consistent was the appearance of labelled material associated with the elements of endoplasmic reticulum that were present between most of the thiekenings (Fig. 7 and 9). This was observed with both tritiated glucoses. In general, a large proportion of the cytoplasmic radioactivity in the cells was found to be situated over these short profiles of the endoplasmic reticulum, and less frequently, some radioactivity was also associated with elements of the endoplasmic retieulum further removed from the wall (Fig. 9). Occasionally, cytoplasmic radioactivity was found over mitoehondria and other organelles (the amounts were always very small). Attempts were made to ascertain whether microtubules were labelled in conjunction with wall incorporation of the label. When the wall thiekenings were seen in profile, there was always radioactivity located in the region around plasmalemma (Figs. 9, 10 and 11). I n such sections, no loealisation of any labelled material specifically either in the microtubules just above the plasmalemma, or in the vesicles that were sometimes seen in these regions or in the wall itself was possible. However, in areas of the sections where the xylem cells and consequently the thickenings were approached tangentially, the bands of mierotubules were often present in the section clear of the plasmalemma and wall material. In these eases, small amounts (i.e. single silver grains) of activity were sometimes associated with the microtubules (Figs. 8 and 12). I t must be emphasised that this radioactivity could still not be specifically loealised "in" the microtubules. Such silver grains SOlnetimes appeared over the cytoplasm clear of the plasmalemma (the diffuse grey area), and all that can be said is t h a t this is the region also occupied by the mierotubules. Fourth Stage. By the time t h a t the thickenings had become well developed and rounded in profile, the incorporation of labelled materials into the wall decreased markedly. This coincided with the progressive breakdown of cytoplasmic organisation that preceeded the total emptying of the cell. Fig. 8. T a n g e n t i a l longitudinal section of a m a t u r i n g x y l e m vessel (Stage Three); tile coleoptile h a d been exposed to G-6-K s for two hours. Incorporation of radioactive d e r i v a t i v e s is visible on the i n n e r edges of the thickenings, v e r y close to the region in the cytoplasm occupied b y the microtubules. I n two eases (arrowed), silver grains are seen closely associated w i t h the mierotubules, a w a y f r o m the walls which were just out of the plane of the section a t this region. • 20,000 Fig. 9. S i m i l a r to Fig. 7, except t h a t the tissue h a d been exposed to G-6-tIS. The endoplasmic r e t i e u l u m is labelled between, close to, and f u r t h e r a w a y f r o m the wall thickenings. The surface of the endoplasmic r e t i c u l u m is characteristically covered w i t h spiral " p o l y s o m e s " . x 22,500 Figs. 10 a n d 11. F r o m the s a m e ceil as t h a t shown in Fig. 9. I n Fig. 10, the edge of the golgi a p p a r a t u s is labelled, a n d in Fig. 11, r a d i o a c t i v i t y is associated w i t h a collection of vesicles, these a h n o s t certainly b e i n g representative of a golgi a p p a r a t u s sectioned at its edge. Fig. 10 • 30,000. Fig. 11 • 22,500
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J.D.
PICKETT-HEAPS :
Fig. 12. The r a d i o a u t o g r a p h was developed in 5~icrodoI-X, to a t t e m p t a m o r e accurate localisation of the radioactivity. Several of the silver grains can be clearly seen over the c y t o p l a s m occupied b y the n u m e r o u s inierotubules, present on top of the thickenings. • 24,000
Discussion
Incorporation o/the Label into the Pla~tids I t has already been shown in the root-cap cells of the wheat seedling, t h a t the incorporation of tritiated glucose derivatives into the starch grains of the plastids can depend on both the metabolic activities of the cells and also the availability of externally supplied glucose (NonT}~COTE and PIC~:ETT-HEAPS, 1966). In young xylem cells, a somewhat similar phenomenon was observed. When the cells were young and wall synthesis was presumably occurring at a normal rate, there was little incorporation of the activity into the wall, but often quite a marked
Wall Incorporation in Xylem
11
accumulation of the label in the plastids of these cells. Later when wall synthesis was stimulated (in Stage Three), the situation was reversed with massive incorporation of labelled materials into the wall thickenings, but never into the plastids. This suggests that in the initial stages of growth of the cell, starch synthesis was probably occurring to some extent, but as wall synthesis increased, starch formation ceased and this storage polysaceharide was probably being broken down.
General Labelling Pattern Xylem vessels at Stage Three always showed massive incorporation of derivatives of labelled glucoses into the wall thiekenings, when compared to that observable into the cytoplasm (except for the plastids) and walls of all other cells in the section (Fig. 6). Furthermore, though the wall was generally very highly labelled, the rest of the cytoplasm in these xylem cells showed very little incorporation of the label, except near the wall (Fig. 3, 5, 6). This suggests that the labelled material near the wall was probably destined for incorporation into it. The nature of the labelled derivatives both in and near the wall thickening Js unknown; whilst some labelling might represent polysaeeharide material, many other high molecular weight components (e.g. proteins) must also be labelled to some extent. In these preliminary experiments, the primary concern has been to investigate how any material (polysaceharide, proteins, lipid etc.) might be incorporated into the growing thickening.
Overall Labelling Distribution in the Wall The young walls of the large xylem cells are initially very thin and presumably wall synthesis would be merely sufficient to allow for the wall expansion that occurs when the tissue is actively growing. This is suggested by the radioautographie results showing that the incorporation of labelled glucose into the young wall (i.e. during Stages One and Two) was equivalent to that observed into the walls of the other undifferentiated cells. Once the secondary thickening of the wall was well under way, deposition of comparatively very large amounts of tritiated derivatives (compared with the incorporation detected over all the section) into the thiekenings was found. Initially, these labelled materials were situated mainly on the top of the thickenings but later, when the sides of the thickening had become more rotund, some of the labelled wall materials were found in the sides. Cytoplasmic Organelle8 and Wall lncorToration The disposition of the endoplasmic retieulum with respect to the wall thiekenings observed in wheat xylem cells (Fig. 2) is possibly
12
J . D . PICKETT-HEAPS :
noticeable because the thiekenings are comparatively close together (Fig. 2; c.f. PICKETT-HEAPS and NOBT~ICOTE, 1966). In glutaraldehyde/ osmium fixed cells, these elements of the endoplasmic reticulum are often not readily diseernable. For example, close scrutiny of the micrographs is necessary to find them in Figs. 8, 9, l l and 12 (none are apparent in Fig. 10). The characteristic orientation of the endoplasmic retieulum is often apparent even at Stage Two (Fig. 1). The fact that the endoplasmie reticulum was found consistently labelled in the experiments described above, suggests that it may function in the synthesis and transport of various wall materials to the specific sites in the cytoplasm where these are required. The labelled materials were retained during fixation, dehydration and embedding and so these were probably of high molecular weight but their nature is unknown. No fibrillar material, equivalent to that described by HEPL]~I% and NEWCOMB (1964), has yet been seen in the lumen of the endoplasmie reticulum. In older, less highly differentiated cells of the wheat seedling (e.g. root cortical and vacuolated coleoptile parenchyma cells) radioautographic experiments similar to those described in this paper also showed that the only cytoplasmic organelle consistently labelled near such a heavily labelled wall was the endoplasmic reticulum (PIcKETT-HEAPS, 1966a). In several different types of plant cells, vesicles, apparently derived from the golgi apparatus, are evidently absorbed into the wall (MOLLEN~AUE~, W~AL~,Y and LEECH, 1961; SIEVEI%S, 1963; FI~EY-WYssLING, L6P~z-Ss and M~HLET~ALE]a, 1964; ROS~N, GAWLIK,I)ASHEK and SIEGESMV~D, 1964; GANTT and ARNOTT, 1965; LARSON, 1965; WOODING and I~OI~THCOTE,1965; PICKETT-HEAPSand NO~T~COTE, 1966). WOODING and NORTHCOTE (1964) considered this process likely to occur in the xylem cells of Acct. In the experiments described above, labelling of the golgi bodies was frequently observable, but the amount of activity associated with these organelles was always low. In general, the results lend some support to the hypothesis that the golgi body can supply wall material in these cells. The role of the microtubules in the formation and development of the xylem wall remains ambiguous and obscure. Microtnbules were associated with the highly labelled parts of the wall thickening and in some cases (Figs. 8 and 12), they were probably associated with labelled material in the cytoplasm immediately adjacent to the walh (No claim is entertained of having labelled the microtubules.) This might support the current hypothesis that they are involved in synthesis and/or deposition of cellulose in the wall. The author has some evidence that deposition of fibrillar wall material can occur in xylem cells in the absence of mierotubules (PICKETT-HEAPS, 1966b). They seem more
Wall Incorporation in Xylem
13
likely concerned w i t h t h e o r d e r l y direction of new wall m a t e r i a l s into t h e a p p r o p r i a t e regions of t h e c y t o p l a s m , since t h e i r removM from t h e x y l e m cell coincides with t h e a p p e a r a n c e of grossly m a l f o r m e d thiekenings (PICKETT-HEA~S, 1966b). I t is also considered r e l e v a n t t h a t t h e grouping of these organelles coincides with, a n d p o s s i b l y preeeeds t h e first sign of a regular wall d e v e l o p m e n t in t h e x y l e m of this tissue (Fig. 1). The fact t h a t t h e groups of m i e r o t u b u l e s were often seen in b e t w e e n v e r y y o u n g wall corrugations (PICKETT-HEAPS a n d NORTHCOTE, 1966) m i g h t i n d i c a t e t h a t these bulges along t h e wall were only a t e m p o r a r y p h e n o m e n o n , a p p e a r i n g before t h e full effects of t h e organelles were asserted. Thus, t h e y are considered i m p o r t a n t in establishing t h e p a t t e r n of r e g u l a r wall d e v e l o p m e n t (PIcKETT-HEAeS, 1966b), p o s s i b l y b y directing m a t e r i a l into t h e t o p a n d sides of t h e thiekenings.
Acknowledgement. I would like to thank Dr. E. H. MERCER for his pertinent suggestions and criticism of the manuscript. References CAiro, L.G., and R.P. vAN TU]~ERGEN: High resolution autoradiography. I. Methods. J. Cell Biol. 15, 173 (1962). C]~ONSHAW, J., and G.B. Boucle: The fine structure of differentiating xylem elements. J. Celt Biol. 24, 415 (1965). FREY-WYSSLING, A., J. F. L6PEz-SJ~EZ, and K. MUIzILETHALEE: Formation and development of the cell plate. J. Ultrastruct. l~es. I0, 422 (1964). GANTT, E., and H. J. AR~OTT: Spore germination and development of the young gametophyte of the ostrich fern (Matteuccia struthiopteris). Amer. J. Bot. 52, 82 (1965). HEPLER, P. K., and E. H. NEWCOMB: The fine structure of young tracheary xylem elements arising by redifferentiation of parenchym~ in wounded Coleus stem. J. exp. Bot. 14, 496 (1963). - - Microtubules and fibrils in the cytoplasm of Coleus cells undergoing secondary wall deposition. J. Cell Biol. 21), 529 (1964). HESLoP-HAR~mO~, J.: UltrastrueturM aspects of differentiation in sporogenous tissue. In: Symposium of the Society for Experimental Biology, "Cell differentiation" (G.E. FoGG, ed.), vol. i7, p. 315. Cambridge: Cambridge University Press 1963. LARSON, D. A. : Fine-structural changes in the cytoplasm of germinating pollen. Amer. J. Bot. 52, 139 (1965). LEDBETTER, M. C., and K. R. PORTER: A "microtubule" in plant cell fine strucurte. J. Cell Biol. 19, 239 (1963). MOLLENtIAUER, t-I. H., W. ~-. WHALE'g, and J. H. LEECH: A function of the golgi apparatus in outer rooteap cells. J. Ultrastruct. Res. 5, 193 (1961). NORT~eOTE, D. H., and J. D. PreKETT-HEArS: A function of the golgi apparatus in polysaccharide synthesis and transport in the root cap cells of the wheat seedling. Biochem. J. 98, 159 (1966).
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J.D. PICKETT-HEAPS: Wall Incorporation in Xylem
PICKETT-HEAPS, J. D. : Further observations on the golgi apparatus and its functions in cells of the wheat seedling (in preparation) (1966a). The effect of colchicine on mierotubule distribution and wall differentiation in wheat xylem cells (in preparation) (1966b). - - , and D. H. NORTHCOT~: The relationship of cellular organelles to the formation and development of the plant cell wall. J. exp. Bot. 17, 20 (1966). POgT~, K. g . : The endoplasmie reticulum: some current interpretations of its form and functions. In: Biological structure and function (GooDwI~ and LI:CDBEaO, editors), vol. 1, p. 127. New York: Academic Press, Inc. 1961. ROSEN, W. G., S. R. GAWLIK, W. V. D~SHEK, and K. A. SEm~SMV~D : Fine strutture and cytochemistry of Lilium pollen tubes. Amer. J. Bot. 51, 61 (1964). SIEVERS, A. : Beteiligung des Golgi-Apparates bei der Bildung der Zellwand von Wurzelhaaren. Protoplasma (Wien) ~6, 188 (1963). SIT:~I:~OTT, E . W . , and R. BLOCH: The cytoplasmic basis of intercellular patterns in vascular differentiation. Amer. J. Bot. 32, 151 (1945). WOODING, F. B. P., and D. H. NORTHeOTE: The development of the secondary wall of the xylem in Acer Pseudoplatanus. J. Cell Biol. 23, 327 (1964). - - The fine structure and development of the companion cell of the phloem of Acer pseudoplatanus. J. Cell Biol. 24, 117 (1965). Dr. J. D. PICKETT-HEAPS Elec%ron Microscope Unit, John Curtin School of Medical Research Canberra. A.C.T. Australia -
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I N C O R P O R A T I O N OF R A D I O A C T I V I T Y INTO W H E A T X Y L E M WALLS J. I). PICKETT-HEAPS Electron Microscope Unit, John Curtin School of l~Iedical Research, Canberra. A.C.T. Australia Received May 10, 1966
Summary. Studies on the fine structural changes accompanying xylem differentiation in wheat coleoptile have indicated that the microtubules are concerned with the inception of a regular wall thickening pattern, and later with wall deposit.ion at the thickening site. The endoplasmic reticulum is situated characteristically in continuous profiles between the thickenings. Radioautographie studies at the electron microscope level using labelled glucoses have shown that the endop]asmic reticulum, golgi bodies and the cytoplasm near the microtubules were often labelled during deposition into nearby thickenings of radioactive materials derived from the tritiated glucoses. Incorporation into the wall occurred mainly at the top of the thickenings. The plastids of the xylem cells were also often labelled, but only during the earlier stages of differentiation; when massive wall deposition was evident, such an incorporation was never observed. The fine structural and radioautographic results are briefly discussed in terms of the possible functions of the organelles in the plant cell. Introduction The xylem vessel is a plant cell in which wall deposition is organised to a very high degree, spiral or annular bands of wall material being formed along the length of the wall in a regular pattern of development. SI~OTT and BLOC~ (1945) noted cytoplasmic banding in Coleus xylem cells, and the location of the bands corresponded to the position of the developing wall thiekenings during rcdifferentiation. Using the same tissue for fine-structural investigations, I-IE~LE~ and NEWCOM~ (1963) equated the bands with an aggregation of cellular organelles, suggesting that this represented a locMised organisation of enhanced respiratory and synthetic activity in the cytoplasm. Both I-IEPLE~ and N~wco~B (1963) and W o o m N c and NOnTHCOTn (1964) noted the appearance of an increased golgi activity in these cells, indicating that the organelle might be involved in the synthesis of wall materials; WOODING and NOnTHCOTn observed apparent incorporation into the sides of developing thickenings of vesicles possibly derived from the golgi apparatus. The endoplasmie retieulum may also be involved in xylem wall formation. POtCTEI~ (1961) noted the appearance of endoplasmic reticulum between the maturing thiekenings of onion protoxylem vessels, and tIEsLoP-ttA~nISON (1963) reported "a striking association" in Pinguicula between the positioning of the wall thickenings and the 1
P l a n t a (BEN.), Bd. 71
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endoplasmic retieulum in the cytoplasm of the cells. Woo~)I~G and NOI~THCOTE (1964) and CRO~SHAW and BOUCK (1965) could find no specific orientation of the endoplasmic reticulum with respect to secondary wall growth in the xylem of Pinus and Avena coleoptile. However in Triticum coleoptile, Pm~ETT-HEA~S and NOI~TKCOTE (1966) showed that the endop]asmic reticulum was present in between the xylem thickenings as continuous circular lamellae. HEPLER and NEwco~B (1964) found fibrillar structures within the cisternae of the endoplasmic reticulum in regenerating Coleus xylem vessels; they suggested that this material was eventually incorporated into the wall. Since LEDBETTER and PORTER (1963) first demonstrated the existenee of microtubules in plant cells, particularly near the growing wall, several papers have described the association of these organelles with the developing thickening of the xylem wall. Groups of microtubules were found above the thickenings by HErLER and NEWCOMB (1964), WOODING and NOaTHCOTE (1964), CaONSHAW and BOUCK (1965) and Pm~ETT-HEAPS and NO~THCOTE (1966); these observations generally supported the hypothesis first proposed by LED~ETTER and PORTER (1963) that the microtubules might be concerned with the deposition of the highly organised cellulose phase in the wall. However, PICKETTHEAPS and NO~THCOTE (1966) have shown that the distribution of the microtubules along the xylem wall changed during the inception of wall differentiation. While initially distributed apparently at random along the undifferentiated wall, they were later found in groups between very immature thickenings, and later still, they were observable in the usual position above the more developed thickenings. During the course of investigations into wall development in wheat xylem cells, it was considered possible that radioautographic methods might elucidate the function of some cell organelles in wall formation. Materials and Methods Wheat seedlings (Triticum vulgate) were washed and germinated on damp filter paper in petrie dishes; they were grown at room temperature in ordinary daylight conditions. When the coleoptiles were about 1 cm long they were excised with a sharp razor blade and placed in the radioactive incubation solutions. D-glucose-6-Ha(l.3 C/raM, from the Radiochemical Center, Amersham, Bucks., England) and D-glucose-l-Ha(350--500 mC/mM, from the New England Nuclear Corp., Boston, Mass.) were made up in glass-distilled water to a concentration of 2 mC/ml and 0.5--1 mC/ml respectively. After a two hour incubation in the appropriate solutions, the coleoptiles were briefly rinsed, cut into 1 mm segments and fixed in a standard 6% glutaraldehyde/phosphate fixative, followed by osmium postfixation, the fixatives containing calcium chloride; fresh segments were similarly treated, or fixed in 4[% unbuffered potassium permanganate, containing sodium and calcium chlorides (PICKETT-HEA1)Sand NOI%THCOTE,1966). All material was then subjected to standard methods of embedding in araldite, sectioning and staining with uranium and/or lead; the radioactive sections were
Wall Incorporation in Xylem
3
coated with Ilford L-4 emulsion by a method based on that described by C~Ro and VAN TUBEI~GEN (1962) (see NORTHCOTE and PICKETT-HEAPS, 1966). After varying periods of photographic exposure (four weeks to four months) the autographic image was developed in Ilford ID-19 or Microdol-X for 45 sec.; the grids were then well washed and the emulsion fixed in Kodafix, diluted 1:4, for 45 see., fo]lowed by further washing and drying. Grids were examined in a Philips EM ]00 or EM 200 at 80 kV.
Results
Unlabelled Specimens Since no confs of a similar sequence of events has yet been presented for differentiating xylem vessels in other plants, extensive observations on wheat xylem vessels have been undertaken to confirm and extend the results reported previously (PICKETT-HEAPSand NORTHCOT~, 1966). For convenience, the sequence of development of the xylem cell has been divided into four stages. First Stage. In the youngest xylem vessels identifiable, the microtubules were evenly spread along the very thin wall with apparently small random variations in their distribution equivalent to those seen in other growing plant cells. Second Stage. The next distinct stage in the differentiation of the xylem wall recognisable in the thin sections, was often indicated by the appearance of a preliminary pattern of regular wall corrugations. The microtubules were organised into bands along the wall, often where no regularity in its structure was discernable in single sections of the cell (Fig. 1). At a slightly later stage, the groups of mierotubules were generally found between the corrugations (see PICKETT-HEAt'S and NOnT~COT~, 1966). Frequently at this stage, elements of the endoplasmic reticulum became closely applied to the plasmalemma in a characteristic fashion. I n a typical example, an area covered by seven photographs (Fig. 1 being one of them) showed thirty-six consecutive regularly-spaced microtubule bands along part of a xylem wall at this stage; with two exceptions, elements of the endoplasmic reticulum were found very close to or directly apposed to the plasmalemma between each group of microtnbules. This regular alternation in distribution of the two organelles is seen very clearly at Stage Three (below). Third Stage. During further differentiation of the xylem wall, massive secondary wall deposition occurs to give the final structure of the wall. Microtubules were found in their usual position above, and to a lesser extent, around the sides of the well-developed thickenings. Elements of the endoplasmie reticulum were found to lie in the cytoplasm in between nearly all of the thiekenings; this is most clearly shown in tangential sections of permanganate-fixed cells (Fig. 2; see PIC~.TT-It~APS and NORTHCOTE, 1966). Vesicular components, probably derived from the golgi apparatus, were numerous in the cytoplasm. 1"
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Fourth Stage. While the thickenings were reaching their final stages of growth, a progressive b r e a k d o w n of the cytoplasm was evident. The
Wall Incorporation in Xylem
5
tonoplast became disrupted and the cell lumen filled with the granular material of cell debris. Finally the end wall of the cell broke down and the ribbed vessel was incorporated into the conducting system of the vascular bundle.
Radioautographic Results The amount of incorporation into the xylem wall of derivatives of the labelled glucoses varied in different pieces of experimental tissue. These variations were probably related to two main variables: i) the proximity of the cell in question to the cut surface, and hence to the labelled metabolites (Fig. 5), and ii) the stage reached in the maturation of the cell. I n connection with the first point, previous experiments had indicated that small coleoptile segments (ca. 1--2 min long) did not give consistent, good radioautographie results. First and Second Stages. The amount of incorporation of labelled derivatives into the wall of these cells was always low, and equivalent to that observed into the walls of neighbouring cells. The labelling of cytoplasmic organelles was also low and it was impossible to correlate the two. Appreciable incorporation of labelled derivatives of both tritiated glucoses was often observed into the starch grains in the plastids of the coleoptile cells, including those of the xylem and occasionally of the sieve-tube cells. This incorporation into the starch of xylem was however, only found in cells during the first two stages of growth, when wall incorporation was very low (Fig. 4). The incorporation into the starch grains was greatest (as a rough fraction of the total radioactivity detected in the section) when glucose-l-tta(G-l-t{ ~) supplied the labelled atom. Fig. 11. Radial longitudinal section oi young xylem cell proceeding into Stage Two. ~iicrotubules are grouped in bands (black lines); alternating with the bands are elements of the endoplasmic retieulum (arrowed) closely applied to the wall. With a few exceptions, the pattern is characteristic of wheat xylem cells at this stage. • 20,000. E R endoplasmie reticulum, G golgi apparatus, M mitochondrion, _N nucleus, P plastid, T microtubules, T H xylem wall thickening, V vacuole, V S vesicles, W ceil wall Fig. 2. A tangential longitudinal section of a maturing xylem cell (Stage Three). The permanganatefixed image shows very clearly the endoplasmie reticulum near the wail lying in long profiles between ahnost all of the thickenings. Compare with radial sections (e.g. Figs. 3, 9, 1 l), where small or longer elements of endoplasmic reticulmn are seen between most of the wall thiekenings. Many vesicular components, probably derived from golgi bodies, are also visible, x 8,300 Fig. 3. Coleoptile tissue exposed to glucose-6-J:I a (G-6-tP) for two hours before fixation. The radial, longitudinal section shows the usual distribution of radio-active incorporation into the wall of a maturing xylem vessel (Stage Three); most of the incorporation has occurred into the top of the wall thickenings (compare with rig. 5). Cytoplasmic labelling is low in comparison to the amount in the walls; the smaller "dwarf" thiekenings generally show a reduced level of radioactivity. • 5,700 1 Glutaraldehyde/osmium
fixation was employed
o n all t i s s u e s e x c e p t t h a t
from which Fig. 2 was prepared; in this case, permanganate fixation was employed. All sections are oriented longitudinally. The radioautographs were developed in ID-19, except for Fig. 12 which was developed in Microdol-X.
6
J.D. PIOKEr~-H~ArS:
Wall Incorporation in Xylem
7
Third Stage. During this stage in the development of the xylem cell, massive wall deposition occurs. This was evident radioautographieally, when very large amounts of radioactivity compared with that present elsewhere in the section, were detected in the wall thickenings. The plastids in these cells were never labelled (c.f. above) whilst in adjacent younger xylem vessels, and in the surrounding eambial and parenchyma tissue, the reverse situation was readily visible (Fig. 6). As in the case of root-cap cells ( N O R T H C O T E and P I C K E T T - H E A P S , 1966), far greater overall labelling in the sections was observed when glucose-6-H3 (G-6-H3), rather than G-1-H 3 was used as the precursor. The distribution of labelled material over the wall thiekenings themselves varied somewhat with their shape. Most of the radioactivity was confined to the top of the thiekenings when these were less developed and had moderately flat sides (Fig. 3). I-Iowever, at slightly later stages in the maturation of the wall, there was more tendency towards some incorporation into the sides of the thiekenings (top cell, Fig. 5). The overall space distribution of the labelling along the wall of any given cell was uniform. Often, dwarf or poorly developed thickenings were found amongst the others, and these were generally appreciably less labelled than the rest, or not at all (Fig. 3). The cytoplasm of these xylem cells always contained a small amount of labelling, particularly near the walls (though this was always far less than t h a t in the walls). The golgi bodies were often labelled (Fig. 10) but the proportion labelled to the total number of the organelles visible in the same section of the cell was always small. Occasionally, radioactivity was associated with collections of vesicles (Fig. ll). Fig. 4. Very y o u n g x y l e m cells (Stage One) exposed to glueose-l-H 3 (G-1-H E) for two hours. The microtubules are evenly distributed along the v e r y thin a n d undifferentiated wail. The incorporation of radioactive glucoses into the walls of such cells was always v e r y low, b u t appreciable accumulation of the label was generally present in the starch grains of the plastids. (Compare w i t h :Fig. 6.) • 14,500 ]~'ig. 5. The Radial, longitudinal section of two m a t u r i n g x y l e m cells (Stage Three) showing typical m a s s i v e incorporation of r a d i o a c t i v i t y into the wall thickenings; the eoleoptile h a d been incubated in G-6-H 3 for two hours. The difference in the total labelling in each cell was probably due to the upper cell b e i n g closer to the cut surface of the tissue, a n d hence to t h e labelled metabolite. Much of the incorporation has occurred into the sides of the thickenings (c.f. Fig. 3); in this autograph, it is impossible to ascertain whether the endopIasmic reticulum present between the thickenings, was also labelled. The end wall b e t w e e n the two cells is v i r t u a l l y unlabellcd. • 4,500 Fig. 6. A x y l e m cell (similar to those in Fig. 5) following the two hour incubation of the coleoptile in G - 6 - H h The x y l e m thickenings contain a f a r greater a m o u n t of the labelled derivatives t h a n t h a t in the n e i g h b o u r i n g walls of undifferentiated cells. H o w e v e r , in these latter cells, there is a m a r k e d a c c u m u l a t i o n of the label into the starch grains of the plastids; the plastids of the x y l e m at this point (Stage Three) were n e v e r labelled (c.f. Fig. 4). • 3,600 Fig. 7. X y l e m cell (Stage Three) f r o m a eoleoptilc incubated in G-1-}I 3 for two hours. There was always far less overall incorporation u s i n g this metabolite when c o m p a r e d with t h a t obtained w i t h G-6-Hh The elements of endoplasmic r e t i c u l u m between the thiekenings are the m o s t frequently labelled o r g a n d i e s found near x y l e m thickenings t h a t h a v e incorporated tritiated compounds derived f r o m glucose. • 23,500
8
J. ]). PICKETT-HEAPS :
Wall Incorporation in Xylem
9
More consistent was the appearance of labelled material associated with the elements of endoplasmic reticulum that were present between most of the thiekenings (Fig. 7 and 9). This was observed with both tritiated glucoses. In general, a large proportion of the cytoplasmic radioactivity in the cells was found to be situated over these short profiles of the endoplasmic reticulum, and less frequently, some radioactivity was also associated with elements of the endoplasmic retieulum further removed from the wall (Fig. 9). Occasionally, cytoplasmic radioactivity was found over mitoehondria and other organelles (the amounts were always very small). Attempts were made to ascertain whether microtubules were labelled in conjunction with wall incorporation of the label. When the wall thiekenings were seen in profile, there was always radioactivity located in the region around plasmalemma (Figs. 9, 10 and 11). I n such sections, no loealisation of any labelled material specifically either in the microtubules just above the plasmalemma, or in the vesicles that were sometimes seen in these regions or in the wall itself was possible. However, in areas of the sections where the xylem cells and consequently the thickenings were approached tangentially, the bands of mierotubules were often present in the section clear of the plasmalemma and wall material. In these eases, small amounts (i.e. single silver grains) of activity were sometimes associated with the microtubules (Figs. 8 and 12). I t must be emphasised that this radioactivity could still not be specifically loealised "in" the microtubules. Such silver grains SOlnetimes appeared over the cytoplasm clear of the plasmalemma (the diffuse grey area), and all that can be said is t h a t this is the region also occupied by the mierotubules. Fourth Stage. By the time t h a t the thickenings had become well developed and rounded in profile, the incorporation of labelled materials into the wall decreased markedly. This coincided with the progressive breakdown of cytoplasmic organisation that preceeded the total emptying of the cell. Fig. 8. T a n g e n t i a l longitudinal section of a m a t u r i n g x y l e m vessel (Stage Three); tile coleoptile h a d been exposed to G-6-K s for two hours. Incorporation of radioactive d e r i v a t i v e s is visible on the i n n e r edges of the thickenings, v e r y close to the region in the cytoplasm occupied b y the microtubules. I n two eases (arrowed), silver grains are seen closely associated w i t h the mierotubules, a w a y f r o m the walls which were just out of the plane of the section a t this region. • 20,000 Fig. 9. S i m i l a r to Fig. 7, except t h a t the tissue h a d been exposed to G-6-tIS. The endoplasmic r e t i e u l u m is labelled between, close to, and f u r t h e r a w a y f r o m the wall thickenings. The surface of the endoplasmic r e t i c u l u m is characteristically covered w i t h spiral " p o l y s o m e s " . x 22,500 Figs. 10 a n d 11. F r o m the s a m e ceil as t h a t shown in Fig. 9. I n Fig. 10, the edge of the golgi a p p a r a t u s is labelled, a n d in Fig. 11, r a d i o a c t i v i t y is associated w i t h a collection of vesicles, these a h n o s t certainly b e i n g representative of a golgi a p p a r a t u s sectioned at its edge. Fig. 10 • 30,000. Fig. 11 • 22,500
10
J.D.
PICKETT-HEAPS :
Fig. 12. The r a d i o a u t o g r a p h was developed in 5~icrodoI-X, to a t t e m p t a m o r e accurate localisation of the radioactivity. Several of the silver grains can be clearly seen over the c y t o p l a s m occupied b y the n u m e r o u s inierotubules, present on top of the thickenings. • 24,000
Discussion
Incorporation o/the Label into the Pla~tids I t has already been shown in the root-cap cells of the wheat seedling, t h a t the incorporation of tritiated glucose derivatives into the starch grains of the plastids can depend on both the metabolic activities of the cells and also the availability of externally supplied glucose (NonT}~COTE and PIC~:ETT-HEAPS, 1966). In young xylem cells, a somewhat similar phenomenon was observed. When the cells were young and wall synthesis was presumably occurring at a normal rate, there was little incorporation of the activity into the wall, but often quite a marked
Wall Incorporation in Xylem
11
accumulation of the label in the plastids of these cells. Later when wall synthesis was stimulated (in Stage Three), the situation was reversed with massive incorporation of labelled materials into the wall thickenings, but never into the plastids. This suggests that in the initial stages of growth of the cell, starch synthesis was probably occurring to some extent, but as wall synthesis increased, starch formation ceased and this storage polysaceharide was probably being broken down.
General Labelling Pattern Xylem vessels at Stage Three always showed massive incorporation of derivatives of labelled glucoses into the wall thiekenings, when compared to that observable into the cytoplasm (except for the plastids) and walls of all other cells in the section (Fig. 6). Furthermore, though the wall was generally very highly labelled, the rest of the cytoplasm in these xylem cells showed very little incorporation of the label, except near the wall (Fig. 3, 5, 6). This suggests that the labelled material near the wall was probably destined for incorporation into it. The nature of the labelled derivatives both in and near the wall thickening Js unknown; whilst some labelling might represent polysaeeharide material, many other high molecular weight components (e.g. proteins) must also be labelled to some extent. In these preliminary experiments, the primary concern has been to investigate how any material (polysaceharide, proteins, lipid etc.) might be incorporated into the growing thickening.
Overall Labelling Distribution in the Wall The young walls of the large xylem cells are initially very thin and presumably wall synthesis would be merely sufficient to allow for the wall expansion that occurs when the tissue is actively growing. This is suggested by the radioautographie results showing that the incorporation of labelled glucose into the young wall (i.e. during Stages One and Two) was equivalent to that observed into the walls of the other undifferentiated cells. Once the secondary thickening of the wall was well under way, deposition of comparatively very large amounts of tritiated derivatives (compared with the incorporation detected over all the section) into the thiekenings was found. Initially, these labelled materials were situated mainly on the top of the thickenings but later, when the sides of the thickening had become more rotund, some of the labelled wall materials were found in the sides. Cytoplasmic Organelle8 and Wall lncorToration The disposition of the endoplasmic retieulum with respect to the wall thiekenings observed in wheat xylem cells (Fig. 2) is possibly
12
J . D . PICKETT-HEAPS :
noticeable because the thiekenings are comparatively close together (Fig. 2; c.f. PICKETT-HEAPS and NOBT~ICOTE, 1966). In glutaraldehyde/ osmium fixed cells, these elements of the endoplasmic reticulum are often not readily diseernable. For example, close scrutiny of the micrographs is necessary to find them in Figs. 8, 9, l l and 12 (none are apparent in Fig. 10). The characteristic orientation of the endoplasmic retieulum is often apparent even at Stage Two (Fig. 1). The fact that the endoplasmie reticulum was found consistently labelled in the experiments described above, suggests that it may function in the synthesis and transport of various wall materials to the specific sites in the cytoplasm where these are required. The labelled materials were retained during fixation, dehydration and embedding and so these were probably of high molecular weight but their nature is unknown. No fibrillar material, equivalent to that described by HEPL]~I% and NEWCOMB (1964), has yet been seen in the lumen of the endoplasmie reticulum. In older, less highly differentiated cells of the wheat seedling (e.g. root cortical and vacuolated coleoptile parenchyma cells) radioautographic experiments similar to those described in this paper also showed that the only cytoplasmic organelle consistently labelled near such a heavily labelled wall was the endoplasmic reticulum (PIcKETT-HEAPS, 1966a). In several different types of plant cells, vesicles, apparently derived from the golgi apparatus, are evidently absorbed into the wall (MOLLEN~AUE~, W~AL~,Y and LEECH, 1961; SIEVEI%S, 1963; FI~EY-WYssLING, L6P~z-Ss and M~HLET~ALE]a, 1964; ROS~N, GAWLIK,I)ASHEK and SIEGESMV~D, 1964; GANTT and ARNOTT, 1965; LARSON, 1965; WOODING and I~OI~THCOTE,1965; PICKETT-HEAPSand NO~T~COTE, 1966). WOODING and NORTHCOTE (1964) considered this process likely to occur in the xylem cells of Acct. In the experiments described above, labelling of the golgi bodies was frequently observable, but the amount of activity associated with these organelles was always low. In general, the results lend some support to the hypothesis that the golgi body can supply wall material in these cells. The role of the microtubules in the formation and development of the xylem wall remains ambiguous and obscure. Microtnbules were associated with the highly labelled parts of the wall thickening and in some cases (Figs. 8 and 12), they were probably associated with labelled material in the cytoplasm immediately adjacent to the walh (No claim is entertained of having labelled the microtubules.) This might support the current hypothesis that they are involved in synthesis and/or deposition of cellulose in the wall. The author has some evidence that deposition of fibrillar wall material can occur in xylem cells in the absence of mierotubules (PICKETT-HEAPS, 1966b). They seem more
Wall Incorporation in Xylem
13
likely concerned w i t h t h e o r d e r l y direction of new wall m a t e r i a l s into t h e a p p r o p r i a t e regions of t h e c y t o p l a s m , since t h e i r removM from t h e x y l e m cell coincides with t h e a p p e a r a n c e of grossly m a l f o r m e d thiekenings (PICKETT-HEA~S, 1966b). I t is also considered r e l e v a n t t h a t t h e grouping of these organelles coincides with, a n d p o s s i b l y preeeeds t h e first sign of a regular wall d e v e l o p m e n t in t h e x y l e m of this tissue (Fig. 1). The fact t h a t t h e groups of m i e r o t u b u l e s were often seen in b e t w e e n v e r y y o u n g wall corrugations (PICKETT-HEAPS a n d NORTHCOTE, 1966) m i g h t i n d i c a t e t h a t these bulges along t h e wall were only a t e m p o r a r y p h e n o m e n o n , a p p e a r i n g before t h e full effects of t h e organelles were asserted. Thus, t h e y are considered i m p o r t a n t in establishing t h e p a t t e r n of r e g u l a r wall d e v e l o p m e n t (PIcKETT-HEAeS, 1966b), p o s s i b l y b y directing m a t e r i a l into t h e t o p a n d sides of t h e thiekenings.
Acknowledgement. I would like to thank Dr. E. H. MERCER for his pertinent suggestions and criticism of the manuscript. References CAiro, L.G., and R.P. vAN TU]~ERGEN: High resolution autoradiography. I. Methods. J. Cell Biol. 15, 173 (1962). C]~ONSHAW, J., and G.B. Boucle: The fine structure of differentiating xylem elements. J. Celt Biol. 24, 415 (1965). FREY-WYSSLING, A., J. F. L6PEz-SJ~EZ, and K. MUIzILETHALEE: Formation and development of the cell plate. J. Ultrastruct. l~es. I0, 422 (1964). GANTT, E., and H. J. AR~OTT: Spore germination and development of the young gametophyte of the ostrich fern (Matteuccia struthiopteris). Amer. J. Bot. 52, 82 (1965). HEPLER, P. K., and E. H. NEWCOMB: The fine structure of young tracheary xylem elements arising by redifferentiation of parenchym~ in wounded Coleus stem. J. exp. Bot. 14, 496 (1963). - - Microtubules and fibrils in the cytoplasm of Coleus cells undergoing secondary wall deposition. J. Cell Biol. 21), 529 (1964). HESLoP-HAR~mO~, J.: UltrastrueturM aspects of differentiation in sporogenous tissue. In: Symposium of the Society for Experimental Biology, "Cell differentiation" (G.E. FoGG, ed.), vol. i7, p. 315. Cambridge: Cambridge University Press 1963. LARSON, D. A. : Fine-structural changes in the cytoplasm of germinating pollen. Amer. J. Bot. 52, 139 (1965). LEDBETTER, M. C., and K. R. PORTER: A "microtubule" in plant cell fine strucurte. J. Cell Biol. 19, 239 (1963). MOLLENtIAUER, t-I. H., W. ~-. WHALE'g, and J. H. LEECH: A function of the golgi apparatus in outer rooteap cells. J. Ultrastruct. Res. 5, 193 (1961). NORT~eOTE, D. H., and J. D. PreKETT-HEArS: A function of the golgi apparatus in polysaccharide synthesis and transport in the root cap cells of the wheat seedling. Biochem. J. 98, 159 (1966).
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J.D. PICKETT-HEAPS: Wall Incorporation in Xylem
PICKETT-HEAPS, J. D. : Further observations on the golgi apparatus and its functions in cells of the wheat seedling (in preparation) (1966a). The effect of colchicine on mierotubule distribution and wall differentiation in wheat xylem cells (in preparation) (1966b). - - , and D. H. NORTHCOT~: The relationship of cellular organelles to the formation and development of the plant cell wall. J. exp. Bot. 17, 20 (1966). POgT~, K. g . : The endoplasmie reticulum: some current interpretations of its form and functions. In: Biological structure and function (GooDwI~ and LI:CDBEaO, editors), vol. 1, p. 127. New York: Academic Press, Inc. 1961. ROSEN, W. G., S. R. GAWLIK, W. V. D~SHEK, and K. A. SEm~SMV~D : Fine strutture and cytochemistry of Lilium pollen tubes. Amer. J. Bot. 51, 61 (1964). SIEVERS, A. : Beteiligung des Golgi-Apparates bei der Bildung der Zellwand von Wurzelhaaren. Protoplasma (Wien) ~6, 188 (1963). SIT:~I:~OTT, E . W . , and R. BLOCH: The cytoplasmic basis of intercellular patterns in vascular differentiation. Amer. J. Bot. 32, 151 (1945). WOODING, F. B. P., and D. H. NORTHeOTE: The development of the secondary wall of the xylem in Acer Pseudoplatanus. J. Cell Biol. 23, 327 (1964). - - The fine structure and development of the companion cell of the phloem of Acer pseudoplatanus. J. Cell Biol. 24, 117 (1965). Dr. J. D. PICKETT-HEAPS Elec%ron Microscope Unit, John Curtin School of Medical Research Canberra. A.C.T. Australia -
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