Planta (Berl.) 84, 239--249 (1969)
A Comparative Study of the Uhrastructure of Resting and Active Cambium of Salix fragilis, L. A. W. ROBAI~DS a n d 1)ARVEEI~ KIDWAI Department of Biology, University of York t~eceived October 21, 1968
Summary. Cells of the resting cambium contMn vesiculate smooth endoplasmic reticuhim, free ribosomes, oil droplets, and protein bodies. There are comparatively few vacuoles, and these are small. The nucleus is fairly central within the cell and is surrounded by a cluster of plastids and mitochondria. Active cambiM cells and young differentiating xylem elements are highly vacuolate, contain rough endoplasmic reticulum and polyribosomes, the Golgi apparatus is active in the production of vesicles, and the distribution of org~nelles is a function of the vacuolation of the cell. It is suggested that the lipid droplets and protein bodies are storage materials which are required during the first stages of differentiation at the beginning of the growing period. Introduction R e c e n t c o n t r i b u t i o n s r e l e v a n t to the u l t r a s t r u c t u r e of the vascular c a m b i u m (BVVAT, 1964; C~ONSEAW a n d BovcK, 1965; C~O~SHAW a n d WA~DnOP, 1964; CZA~I~SKL 1966; ESAV et al., 1966; PICKETT-IliAdS, 1967; I~O~A~DS, 1968; SnIVASTAVA, 1966; SnrVASTAVA a n d O ' B R I ~ , 1966) have g r e a t l y e n h a n c e d our u n d e r s t a n d i n g of this tissue; but, except for a comparison b e t w e e n active a n d resting c a m b i a of ash (SRIvASTAVA, 1966) a n d pine (S~IvASTAVA a n d O'BRIE~, 1966), v e r y little has been p u b l i s h e d a b o u t the differences i n the u l t r a s t r u c t u r e of resting a n d active states w i t h i n the same species. Accordingly, it seemed i m p o r t a n t to describe i n some detail such differences as have been f o u n d i n the c a m b i u m of crack willow (Salix/ragilis, L.).
Materials and Methods Thin radial slices of stem including cambium were cut from pollarded willow growing out of doors during January. The stem was immersed in the fixation solution while the segments were excised. Various fixatives were used, including glutaraldehyde, potassium permanganate, chi'ome-acetic-acid, formalin-acetic-Mcohol, and acrolein. For ghitaraldehyde fixation, a 3% solution in 0.05M cacodylate or 0.1 M phosphate buffer at pit 7.2 with the addition of sucrose to 0.2 or 0.4 M and added sodium, calcium, and potassium chlorides to a combined molarity of about 0.1 M was used. As actively differentiating xylem is extremely difficult to fix adequately, different mo]arities of sucrose were added to the fixation solutions: 0.4 M was found most effective in the present study, thus giving a sohitio~ of very high osmolMity
240
A . W . RORAI~DS and P. KIDWAI:
(in excess of 1,000 milliosmols - - see MASER et al., 1967). More detailed information concerning the effect of different fixative solutions on the cambium will be reported elsewhere. Fixation was carried out for 4h either a t room temperature or ar 4 ~ C, after which the specimens were washed in four changes of wash solution (as for the fixation solution b u t with glutaraldehyde replaced b y sucrose) allowing a t least 30 rain in each change. The tissue was transferred to 1% osmium tetroxide in 0.2 M cacodylate or phosphate buffer a t p H 7.2 with t h e addition of enough sucrose to give a final concentration of 0.2 or 0.4 M: fixation was for 4h at 4 ~ C. After further washing over 2h, specimens were dehydrated through a graded acetone series with 1% uranyl acetate at the 70% stage (or, occasionally, using the method of G~ZCEZMARTIN et al., 1966 for double staining). The segments were passed through propylene oxide into a graded propylene oxide/final resin series. Infiltration was carried out over 4 to 15 days, changing the resin (MoLLEN~ALTER, 1964) daily, and evacuating intermittently. Polymerization was at 40 and 60 ~ C. Sections were cut using diamond knives on LKB Ultrotome I or III ultramierotomes, and were mounted on 200 mesh copper grids coated with carbon or carbon and formvar, or on uncoated 400 mesh grids. Sections were stained on the grids in lead citrate (P~EY~OLDS, 1963), and were observed and the image recorded in a n A E I EM6B electron microscope. Material for light microscopy was passed after fixation through a graded tertiary butyl alcohol series and embedded in Ester W a x 1960 (STEEDI~IA~,1960). Sections were cut using steel knives on an AO-Spencer 820 rotary microtome, a n d were stained with ]ignin pink and chlorazol black E (ROBARDS and PURWS, 1964) or safranin a n d light green. Thick sections from resin embedded material were also cut on an AO-Spencer microtome which h a d been modified to accept glass knives. Such sections were stained in alkaline toluidine blue. Photomicrographs were recorded on 35 m m film using a Zeiss photomicroscope. Stem segments containing active cambium were obtained in the following m a n n e r : 25 em segments were cut from 1--2 yr old internodes a n d were placed with their lower ends in a diluted Hoagland's medium. Desiccation was prevented by coating the cut upper end with lanolin paste. These segments were placed in a growth chamber a t 25 ~ C a n d fairly high relative humidity. Young shoots grew out from t h e internodal segments, a n d were processed for light or electron microscopy as described previously. This method of obtaining actively dividing cambium cells avoids the dependence upon the time of year otherwise limiting such experiments. F u r t h e r material was obtained from shoots growing out of doors during April a n d June. Histochemica] localization of protein was carried out using a modification of the ninhydrin-Schiff's reaction (JENSEN, 1962). W h e n acrolein fixation had been employed, aldehyde groups were blocked b y soaking the de-waxed sections in a saturated solution of dimedone (5.5 dimethylcyelohexane 1 : 3 dione) a t room temperature for 16 to 24 h. Subsequently the sections were passed t h r o u g h 0.5% ninh y d r i n in absolute alcohol at 37 ~ C (24 h); rinsed in alcohol and t h e n water; stained in Sehiff's reagent (30 min); rinsed in water a n d t h e n 2% sodium bisulphite; dehydrated a n d mounted.
Observations
Resting cambium.
The non-active cambium comprises two to four l a y e r s of cells b e t w e e n m a t u r e ( f u l l y d i f f e r e n t i a t e d ) s e c o n d a r y x y l e m a n d s e c o n d a r y p h l o e m (Fig. 1). T h e r a d i a l w a l l s of t h e c a m b i a l cells a r e
Ultrastructure of Besting and Active Cambium
241
Fig. 1. Phase contrast photomicrograph of transverse section of glutaraldehyde fixed, resin embedded, resting cambium. Protein bodies (arrowed) and oil droplets (circled) are abundant. • 1,500 Fig. 2. Transverse section from active cambium processed as for :Fig. 1. Note the highly vacuoIa~e ceils with a thin layer of parietal cytoplasm (arrowed), and the rather less vacuolate ray cells. • 1,500 All scale marks are equivalent to 1.0 ~ with the exception of Fig. 6 (0.5 ~). Large arrows indicate the direction of xylem differentiation. Abbreviations used: D dictyosome, E R endoplasmic reticulum, L lipid droplet (or spherosome), M mitochondrion, N nucleus, P plastid, Pb protein body, R ray cell, St starch, V vacuole, ve vesicles.
242
ROBARDSand I~DWAI : Ultrastructure of Resting and Active Cambium
t h i c k as c o m p a r e d to t h e i r t a n g e n t i a l walls (Fig. 3). F u s i f o r m a n d r a y initials h a v e m o r e or less t h e s a m e u l t r a s t r u c t u r a l components, b u t can be d i s t i n g u i s h e d on t h e basis of t h e i r r e l a t i v e size, frequency, a n d direction of growth. F u r t h e r , t h e p r o t e i n bodies (described later) a r e m o r e a b u n d a n t in ray initials. Plasmodesmata are present through tangential
walls of ray and fusiform initials. The cells are rich in cytoplasm, and the few vacuoles present are generally small. Nuclei are large and almost spherical or ovoid in shape; they occupy a more or less central position within the cell. The nuclear envelope has pores of about 550--650 diameter. The cytoplasm is rich in smooth endoplasmic reticulum (ER), but there are numerous free ribosomes (generally not aggregated as polyribosomes) present in the cells. The ER is in the form of vesicles, rather than the parallel configuration of membranes more typical in active cells. Dictyosomes of the Golgi apparatus are present, although not abundant, and it is interesting that the cisternal number (5--7) is the same as that in active cells. The dietyosomes do not, however, generally appear to be producing vesicles. Mitochondria show inpushings of the inner membrane more characteristic of tubuli than cristae. The plastids are of variable structure : some have elongated lameUae (although mature grana are not developed), as well as large starch grains (Fig. 4) and plastoglobuli; others are less elaborated and are more typical of proplastids. The nucleus, with a length considerably less than 10 % that of the resting fusiform initials, is often not seen in transverse sections. This, together with the fact that the mitochondria and plastids show a preferential aggregation around the nucleus, means that very many cells in transverse section fail to reveal these three organelles: an adequate survey can only be obtained by using longitudinal sections as well. Microtubules, which are often longitudinally orientated, are present in the peripheral cytoplasm. Other structures characteristic of the resting cambium are single membrane bound bodies of various types (Fig. 6). One type appears as a circle of about 0.75--2.0 ~ in transverse section, and has an irregular appearance in longitudinal section (Figs. I, 3, 4 and 5). The contents are finely granular and, in some cases, appear to be unevenly distributed within the b o d y . A l t h o u g h histochemieal t e s t s for p r o t e i n gave a s t r o n g positive
Fig. 3. Transverse section of resting cambium prepared as for Fig. 1, but examined in an electron microscope. The large protein bodies, lipid droplets, and smooth endoplasmic reticulum are the characteristic features of the ceils. • 9,000 Fig. 4. Part of a resting fusiform initial sectioned longitudinally to show the nucleus with its associated cluster of starch-laden plastids and small mitochondria. The perinuclear area is bounded by protein bodies. • 9,500
Figs. 3 and 4
Figs. 5--7
I~O]3ARDSand KIDWAr: Ultrastructure of t~esting and Active Cambium
245
reaction, there was sometimes a reaction in control sections. However, this is probably due to the large quantities of protein involved in these bodies, and we are confident that they are, in fact, protein bodies. Other, smaller, membrane bound bodies about 0.5--1.25 fz in diameter are also present: their contents are more homogeneous and osmiophilic. I n addition, the electron opacity of these bodies increased as the fixation time in osmium tetroxide increased. I t is therefore considered that these bodies are oil droplets or spherosomes.
Active cambium. There are more rows of cells in the active (Fig. 2) cambium than in the resting cambium and, although the cambium proper is often regarded as a single layer of cells, it is impossible to distinguish between the cambium and the young differentiating vascular elements on either side of it. Beyond this eambial zone there is a gradual change through all stages of development to mature phloem and xylem. The main difference between the active and resting eambia, so far as ultrastrueture is concerned, is the marked vaeuolation of the former. This is particularly so in the fusiform initials (Fig. 7). Cells are rich in rough E R (:Fig. 8) which increases as the aei~ivity of the cell increases: there are coneomitantly fewer free ribosomes, and cytoplasmic ribosomes form polyribosome aggregations. I n active cells the dictyosomes appear to be producing vesicles which often seem to fuse with the plasmalemma. A more detailed account of vesicular incorporation is in preparation. Microtubules are present close to the plasmalemma : they have a diameter of about 250 A, and are more commonly seen in material fixed in glutaraldehyde at room temperature than at 4 ~ C. I t is common to see microtubules adjacent to the nucleus in these active ceils (Fig. 9). Plastids and mitochondria do not show the restricted distribution apparent in resting cambium, although they, like all other organelles, have their distribution determined by the vacuolation of the ceil. The plastids may contain starch, plastoglobuli, and phytoferritin (see also ROBARDS and Hv~P~m~so~, 1967; RO]~AgDS and RoBInson, i968). The protein bodies, so frequent in the resting cambium, are completely absent from active cells, although a few lipid droplets or spherosomes are present. Fig. 5. Longitudinal section of resting cambium treated as in Fig. 1. The irregularly elongated protein bodies and plentiful lipid droplets (circled) are packing the e~mbial cells. • 1,300 Fig. 6. The boundary of a protein body showing the unit membrane surrounding it. • 60,000 Fig. 7. Transverse section of active cambium as seek in the electron microscope after glutaraldehyde/osmium tetroxide fixation. The cells are highly vaeuolate, although the thin outer layer of cytoplasm is welt endowed with organelles, espeeially dictyosomes producing vesicles and multivesicular bodies. • 19,000
Figs. 8 and 9
RO]3ARDS a n d KI])wAI: Ultrastructure of Resting a n d Active Cambium
247
Discussion In contrast to the resting cambium - - which is relatively easy to fix for electron microscopic observation - - actively dividing and differentiating cells in the eambial zone are virtually impossible to fix so that all types are well preserved. This problem is apparently brought about by the rapid vacuolation during differentiation and is, presumably, related to the nature of the vacuolar contents as well as to the contemporary dynamics of the tonoplast. This means that, in general, one must be satisfied to obtain reasonable preservation of the eambial cells at the expense of the youngest differentiating xylem cells (the most difficult to fix), or vice versa. While the walls of the active cambial cells are thin (tangential, 0.06--0.3 ~z; radial, 0.3--1.0 ~z), those of the resting cells are comparatively thick (0.1--0.7 ix and 2.0--3.0 ~z respectively). Although we have not seen it, the first division at the beginning of the growing season must produce an odd, unequally thickened, cell. The most striking feature of the resting cambium is the large amount of space occupied by protein bodies, lipid droplets and, to a lesser extent, vesiculate smooth ER. S~IVASTAV~ (1966) has also reported protein bodies and lipid droplets from the resting cambium of Fraxinus, although the protein bodies were less welt developed than those described here. This is interesting, as S~rVAST~VA makes the point that, although the cells in which they were found were quiescent, they were not 'winter' cells. In our material, there is no doubt that the cells were completely dormant. I t seems, therefore that the most probable function of the protein bodies - - and of the lipid droplets - - is a storage role. I t may be that those protein bodies with an uneven distribution of contents represent either incompletely filled bodies or the removal of protein prior to the grox~4ng season. Such a hypothesis would be in keeping with the absence of such bodies from the active cambium. Although the resting cambium is not highly vaeuolate, the differentiating cells rapidlybeeome so. This means that all cell reactions taking place in the cytoplasm (and including the many and complex processes of wall formation) must take place in the thin parietal layer of cytoplasm. As pointed out in another paper (RoBA~DS, 1968), even wall synthesis would require a supply of many enzymes, some of which could be required for transport into the Fig. 8. P a r t of a n actively differentiating cambial cell. The ribosomes are attached to the endoplasmic reticulum or form polyribosomaI arrays. Small vesicles or tubules of E R are present as well as a multivesicular body. • 23,000 Fig. 9. A cell from the active cambium showing mierotubules adjacent to b o t h nucleus and ptasmalemma (arrowed). Vesicles of unknown origin are present a n d show similarities to the coated vesicles reported b y other workers. • 40,000 17
Planta (Berl.),Bd. 84
248
A.W. ROU.~DS and P. KIDWAI:
wall itself. Further, the outermost layer of cytoplasm in cells actively forming cell walls is often rich in rough E R and/or polyribosomes. These facts suggest that a plentiful supply of protein could be more important than a superficial examination might lead us to suspect. The localization of plastids and the rather small mitochondria around the nucleus of inactive cells is of interest because it has been found so commonly in our material. We have no evidence of significance for this distribution; nor for the ontogeny of the organelles in question although it is common to obtain aspects of plastids and mitochondria which suggest that they may be dividing. In some way these organelles must replicate, and it appears that they are present in comparatively large numbers during the resting period. I t is interesting to recall the observations of BELL and MOH~ETHALE~ (1962) who described the degeneration of organelles in Pteridium aquilinum prior to their reformation from initials budded off from the nuclear envelope. The ontogeny of plastids and mitochondria in cambial cells is clearly worthy of further consideration and experimentation. The presence of dictyosomes of similar appearance in resting and active cambia further supports the view that the Golgi complex is a reasonably stable entity. Indeed, it is in the very active cells that the number of eisternae per dietyosome is reduced, and it may be that this represents intermediate stages in the replication of the dictyosomes: something that would need to take place as there are far fewer dictyosomes in resting than in active cambium. The nature of actively differentiating xylem elements as well as of mature xylem fibres themselves have been described previously (RoBARDS, 1968; ROBA~DS, 1967). From the results recorded here it appears that the cytoplasmic content of the resting cambium is similar to that of the active cambium and differing in degree rather than nature. The characteristics peculiar to the resting cells alone are their reduced vacuolation; the presence of apparent storage materials as protein and lipid in membrane bound bodies, and starch in plastids; and the quiescent nature of the other organelles. This is especially noticeable when considering the membranes themselves which appear so dynamic in the active state (and particularly the plasmalemma with its fusing vesicles and paramural b o d i e s - MAI~CItANTand I~OBARDS,1968), and yet are so static and continuous in the resting cells. Of particular interest now will be not only to advance work on the processes occurring during the differentiation of vascular tissues, but also to study more closely the events taking place during the transition from the resting to the active state. We should like to thank Miss E. DAWDSONand Mr. P. CROS~r for skilled technical assistance. This work was carried out under a grant from the Natural Environment t~esearch Council.
Ultrastrueture of Resting and Active Cambium
249
References
BELL, P. 1:~., and K. MtiHLETJ~AJ~E~:The fine structure of the cells taking part in oogenesis in Pteridium aquilinum (L.) Kuhn. J. Ultrastruct. Res. 7, 452--466 (1962). BCVAT, R. : Comportement des membranes plasmiques lors de la diff~renciation des parois lat4rales des vaisseaux (m6taxyleme de Cucurbita pep@ C. R. Acad. Sci. (Paris) 258, 5511--5514 (1964). CRo~s~Aw, J., and J. B. BOUCK: The fine structure of differentiating xylem elements. J. Cell Biol. 24, 4 1 5 - 4 3 2 (1965). - - , and A. B. W~RDROP: The organisation of cytoplasm in differentiating xylem. Aust. J. Bot. 12, 15--23 (1964). CZA~INSKI, Y. : Aspects infrastructuraux de cellules contigues aux vaisseaux dans le xyl~me de Robinia pseudoacacia. C. R. Acad. Sci. (Paris) 262, 2336--2339 (1966). EsAv, K., V. I. C~EADL~, and R. H. GILL: Cytology of differentiating tracheary elements. I. Organelles and membrane systems. Amer. J. Bot. 53, 756--771 (1966). GIM~Ez-M~T~N, G., M. C. RISUE~O, and J. F. L6r~z-S~]~z: A simple staining technique for electron microscopy with lead-uranyl acetate. Experientia (Basel) 23, 316--317 (1967). JENSEN, W. A. : Botanical histochemistry, Principles and practice. San Francisco and London: Freemen and Company 1962. MARcm~N~, t~., and A. W. ROBAgDS : Membrane systems associated with the plasmalemma of plant cells. Ann. Bot., N.S. 32, 97--117 (1968). MASER, M. D., T. E. POWELL, and C. W. PHILrOTT: Relationships among pH, osmolMity, and concentration of fixative solutions. Stain Technol. 42, 175--182 (1967). MOLnENm~VE~, H. H. : Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39, 111--114 (1964). PICKETT-HEAPS,J. D.: The effect of colchicine on the ultrastructure of dividing plant cells, xylem wall differentiation, and distribution of cytoplasmic microtubules. Develop. Biol. 15, 206--236 (1967). REYNOLDS, E. S.: The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208--212 (1963). ROBA~DS, A. W. : The xylem fibres of Salix/ragilis, L. J. roy. micr. Soc. 87, 329--352 (1967). - - On the ultrastructure of differentiating secondary xylem in willow. Protoplasma 65, 449--464 (1968). - - , and P. G. HUMP~ERS0~ : Phytoferritin in plastids of the cambial zone of willow. Ptanta (Berl.) 76, 169--178 (1967). - - , and M. J. PuRws : Chlorazol black E as a stain for tension wood. Stain Technol. 39, 309--315 (1964). - - , and C. L. ROmNSON: Further studies on phytoferritin.Planta (Bcrl.) 82, 179--188 (1968). SRIVASTAVA,L. : On the fine structure of the cambium of Fraxinus americana, L. J. Cell Biol. 31, 79--93 (1966). - - , and T. P. O'B~IEN: On the ultrastructure of the cambium and its vascular derivatives. I. Cambium of Pinus strobu~ L. Protoplasma 61, 257--276 (1966). S~EED~AN, H. F. : Section cutting in microscopy. Oxford: Blackwell 1960. Dr. A. W. ROB)-RDS Department of Biology, University of York IIeslington, York, England 17"
A Comparative Study of the Uhrastructure of Resting and Active Cambium of Salix fragilis, L. A. W. ROBAI~DS a n d 1)ARVEEI~ KIDWAI Department of Biology, University of York t~eceived October 21, 1968
Summary. Cells of the resting cambium contMn vesiculate smooth endoplasmic reticuhim, free ribosomes, oil droplets, and protein bodies. There are comparatively few vacuoles, and these are small. The nucleus is fairly central within the cell and is surrounded by a cluster of plastids and mitochondria. Active cambiM cells and young differentiating xylem elements are highly vacuolate, contain rough endoplasmic reticulum and polyribosomes, the Golgi apparatus is active in the production of vesicles, and the distribution of org~nelles is a function of the vacuolation of the cell. It is suggested that the lipid droplets and protein bodies are storage materials which are required during the first stages of differentiation at the beginning of the growing period. Introduction R e c e n t c o n t r i b u t i o n s r e l e v a n t to the u l t r a s t r u c t u r e of the vascular c a m b i u m (BVVAT, 1964; C~ONSEAW a n d BovcK, 1965; C~O~SHAW a n d WA~DnOP, 1964; CZA~I~SKL 1966; ESAV et al., 1966; PICKETT-IliAdS, 1967; I~O~A~DS, 1968; SnIVASTAVA, 1966; SnrVASTAVA a n d O ' B R I ~ , 1966) have g r e a t l y e n h a n c e d our u n d e r s t a n d i n g of this tissue; but, except for a comparison b e t w e e n active a n d resting c a m b i a of ash (SRIvASTAVA, 1966) a n d pine (S~IvASTAVA a n d O'BRIE~, 1966), v e r y little has been p u b l i s h e d a b o u t the differences i n the u l t r a s t r u c t u r e of resting a n d active states w i t h i n the same species. Accordingly, it seemed i m p o r t a n t to describe i n some detail such differences as have been f o u n d i n the c a m b i u m of crack willow (Salix/ragilis, L.).
Materials and Methods Thin radial slices of stem including cambium were cut from pollarded willow growing out of doors during January. The stem was immersed in the fixation solution while the segments were excised. Various fixatives were used, including glutaraldehyde, potassium permanganate, chi'ome-acetic-acid, formalin-acetic-Mcohol, and acrolein. For ghitaraldehyde fixation, a 3% solution in 0.05M cacodylate or 0.1 M phosphate buffer at pit 7.2 with the addition of sucrose to 0.2 or 0.4 M and added sodium, calcium, and potassium chlorides to a combined molarity of about 0.1 M was used. As actively differentiating xylem is extremely difficult to fix adequately, different mo]arities of sucrose were added to the fixation solutions: 0.4 M was found most effective in the present study, thus giving a sohitio~ of very high osmolMity
240
A . W . RORAI~DS and P. KIDWAI:
(in excess of 1,000 milliosmols - - see MASER et al., 1967). More detailed information concerning the effect of different fixative solutions on the cambium will be reported elsewhere. Fixation was carried out for 4h either a t room temperature or ar 4 ~ C, after which the specimens were washed in four changes of wash solution (as for the fixation solution b u t with glutaraldehyde replaced b y sucrose) allowing a t least 30 rain in each change. The tissue was transferred to 1% osmium tetroxide in 0.2 M cacodylate or phosphate buffer a t p H 7.2 with t h e addition of enough sucrose to give a final concentration of 0.2 or 0.4 M: fixation was for 4h at 4 ~ C. After further washing over 2h, specimens were dehydrated through a graded acetone series with 1% uranyl acetate at the 70% stage (or, occasionally, using the method of G~ZCEZMARTIN et al., 1966 for double staining). The segments were passed through propylene oxide into a graded propylene oxide/final resin series. Infiltration was carried out over 4 to 15 days, changing the resin (MoLLEN~ALTER, 1964) daily, and evacuating intermittently. Polymerization was at 40 and 60 ~ C. Sections were cut using diamond knives on LKB Ultrotome I or III ultramierotomes, and were mounted on 200 mesh copper grids coated with carbon or carbon and formvar, or on uncoated 400 mesh grids. Sections were stained on the grids in lead citrate (P~EY~OLDS, 1963), and were observed and the image recorded in a n A E I EM6B electron microscope. Material for light microscopy was passed after fixation through a graded tertiary butyl alcohol series and embedded in Ester W a x 1960 (STEEDI~IA~,1960). Sections were cut using steel knives on an AO-Spencer 820 rotary microtome, a n d were stained with ]ignin pink and chlorazol black E (ROBARDS and PURWS, 1964) or safranin a n d light green. Thick sections from resin embedded material were also cut on an AO-Spencer microtome which h a d been modified to accept glass knives. Such sections were stained in alkaline toluidine blue. Photomicrographs were recorded on 35 m m film using a Zeiss photomicroscope. Stem segments containing active cambium were obtained in the following m a n n e r : 25 em segments were cut from 1--2 yr old internodes a n d were placed with their lower ends in a diluted Hoagland's medium. Desiccation was prevented by coating the cut upper end with lanolin paste. These segments were placed in a growth chamber a t 25 ~ C a n d fairly high relative humidity. Young shoots grew out from t h e internodal segments, a n d were processed for light or electron microscopy as described previously. This method of obtaining actively dividing cambium cells avoids the dependence upon the time of year otherwise limiting such experiments. F u r t h e r material was obtained from shoots growing out of doors during April a n d June. Histochemica] localization of protein was carried out using a modification of the ninhydrin-Schiff's reaction (JENSEN, 1962). W h e n acrolein fixation had been employed, aldehyde groups were blocked b y soaking the de-waxed sections in a saturated solution of dimedone (5.5 dimethylcyelohexane 1 : 3 dione) a t room temperature for 16 to 24 h. Subsequently the sections were passed t h r o u g h 0.5% ninh y d r i n in absolute alcohol at 37 ~ C (24 h); rinsed in alcohol and t h e n water; stained in Sehiff's reagent (30 min); rinsed in water a n d t h e n 2% sodium bisulphite; dehydrated a n d mounted.
Observations
Resting cambium.
The non-active cambium comprises two to four l a y e r s of cells b e t w e e n m a t u r e ( f u l l y d i f f e r e n t i a t e d ) s e c o n d a r y x y l e m a n d s e c o n d a r y p h l o e m (Fig. 1). T h e r a d i a l w a l l s of t h e c a m b i a l cells a r e
Ultrastructure of Besting and Active Cambium
241
Fig. 1. Phase contrast photomicrograph of transverse section of glutaraldehyde fixed, resin embedded, resting cambium. Protein bodies (arrowed) and oil droplets (circled) are abundant. • 1,500 Fig. 2. Transverse section from active cambium processed as for :Fig. 1. Note the highly vacuoIa~e ceils with a thin layer of parietal cytoplasm (arrowed), and the rather less vacuolate ray cells. • 1,500 All scale marks are equivalent to 1.0 ~ with the exception of Fig. 6 (0.5 ~). Large arrows indicate the direction of xylem differentiation. Abbreviations used: D dictyosome, E R endoplasmic reticulum, L lipid droplet (or spherosome), M mitochondrion, N nucleus, P plastid, Pb protein body, R ray cell, St starch, V vacuole, ve vesicles.
242
ROBARDSand I~DWAI : Ultrastructure of Resting and Active Cambium
t h i c k as c o m p a r e d to t h e i r t a n g e n t i a l walls (Fig. 3). F u s i f o r m a n d r a y initials h a v e m o r e or less t h e s a m e u l t r a s t r u c t u r a l components, b u t can be d i s t i n g u i s h e d on t h e basis of t h e i r r e l a t i v e size, frequency, a n d direction of growth. F u r t h e r , t h e p r o t e i n bodies (described later) a r e m o r e a b u n d a n t in ray initials. Plasmodesmata are present through tangential
walls of ray and fusiform initials. The cells are rich in cytoplasm, and the few vacuoles present are generally small. Nuclei are large and almost spherical or ovoid in shape; they occupy a more or less central position within the cell. The nuclear envelope has pores of about 550--650 diameter. The cytoplasm is rich in smooth endoplasmic reticulum (ER), but there are numerous free ribosomes (generally not aggregated as polyribosomes) present in the cells. The ER is in the form of vesicles, rather than the parallel configuration of membranes more typical in active cells. Dictyosomes of the Golgi apparatus are present, although not abundant, and it is interesting that the cisternal number (5--7) is the same as that in active cells. The dietyosomes do not, however, generally appear to be producing vesicles. Mitochondria show inpushings of the inner membrane more characteristic of tubuli than cristae. The plastids are of variable structure : some have elongated lameUae (although mature grana are not developed), as well as large starch grains (Fig. 4) and plastoglobuli; others are less elaborated and are more typical of proplastids. The nucleus, with a length considerably less than 10 % that of the resting fusiform initials, is often not seen in transverse sections. This, together with the fact that the mitochondria and plastids show a preferential aggregation around the nucleus, means that very many cells in transverse section fail to reveal these three organelles: an adequate survey can only be obtained by using longitudinal sections as well. Microtubules, which are often longitudinally orientated, are present in the peripheral cytoplasm. Other structures characteristic of the resting cambium are single membrane bound bodies of various types (Fig. 6). One type appears as a circle of about 0.75--2.0 ~ in transverse section, and has an irregular appearance in longitudinal section (Figs. I, 3, 4 and 5). The contents are finely granular and, in some cases, appear to be unevenly distributed within the b o d y . A l t h o u g h histochemieal t e s t s for p r o t e i n gave a s t r o n g positive
Fig. 3. Transverse section of resting cambium prepared as for Fig. 1, but examined in an electron microscope. The large protein bodies, lipid droplets, and smooth endoplasmic reticulum are the characteristic features of the ceils. • 9,000 Fig. 4. Part of a resting fusiform initial sectioned longitudinally to show the nucleus with its associated cluster of starch-laden plastids and small mitochondria. The perinuclear area is bounded by protein bodies. • 9,500
Figs. 3 and 4
Figs. 5--7
I~O]3ARDSand KIDWAr: Ultrastructure of t~esting and Active Cambium
245
reaction, there was sometimes a reaction in control sections. However, this is probably due to the large quantities of protein involved in these bodies, and we are confident that they are, in fact, protein bodies. Other, smaller, membrane bound bodies about 0.5--1.25 fz in diameter are also present: their contents are more homogeneous and osmiophilic. I n addition, the electron opacity of these bodies increased as the fixation time in osmium tetroxide increased. I t is therefore considered that these bodies are oil droplets or spherosomes.
Active cambium. There are more rows of cells in the active (Fig. 2) cambium than in the resting cambium and, although the cambium proper is often regarded as a single layer of cells, it is impossible to distinguish between the cambium and the young differentiating vascular elements on either side of it. Beyond this eambial zone there is a gradual change through all stages of development to mature phloem and xylem. The main difference between the active and resting eambia, so far as ultrastrueture is concerned, is the marked vaeuolation of the former. This is particularly so in the fusiform initials (Fig. 7). Cells are rich in rough E R (:Fig. 8) which increases as the aei~ivity of the cell increases: there are coneomitantly fewer free ribosomes, and cytoplasmic ribosomes form polyribosome aggregations. I n active cells the dictyosomes appear to be producing vesicles which often seem to fuse with the plasmalemma. A more detailed account of vesicular incorporation is in preparation. Microtubules are present close to the plasmalemma : they have a diameter of about 250 A, and are more commonly seen in material fixed in glutaraldehyde at room temperature than at 4 ~ C. I t is common to see microtubules adjacent to the nucleus in these active ceils (Fig. 9). Plastids and mitochondria do not show the restricted distribution apparent in resting cambium, although they, like all other organelles, have their distribution determined by the vacuolation of the ceil. The plastids may contain starch, plastoglobuli, and phytoferritin (see also ROBARDS and Hv~P~m~so~, 1967; RO]~AgDS and RoBInson, i968). The protein bodies, so frequent in the resting cambium, are completely absent from active cells, although a few lipid droplets or spherosomes are present. Fig. 5. Longitudinal section of resting cambium treated as in Fig. 1. The irregularly elongated protein bodies and plentiful lipid droplets (circled) are packing the e~mbial cells. • 1,300 Fig. 6. The boundary of a protein body showing the unit membrane surrounding it. • 60,000 Fig. 7. Transverse section of active cambium as seek in the electron microscope after glutaraldehyde/osmium tetroxide fixation. The cells are highly vaeuolate, although the thin outer layer of cytoplasm is welt endowed with organelles, espeeially dictyosomes producing vesicles and multivesicular bodies. • 19,000
Figs. 8 and 9
RO]3ARDS a n d KI])wAI: Ultrastructure of Resting a n d Active Cambium
247
Discussion In contrast to the resting cambium - - which is relatively easy to fix for electron microscopic observation - - actively dividing and differentiating cells in the eambial zone are virtually impossible to fix so that all types are well preserved. This problem is apparently brought about by the rapid vacuolation during differentiation and is, presumably, related to the nature of the vacuolar contents as well as to the contemporary dynamics of the tonoplast. This means that, in general, one must be satisfied to obtain reasonable preservation of the eambial cells at the expense of the youngest differentiating xylem cells (the most difficult to fix), or vice versa. While the walls of the active cambial cells are thin (tangential, 0.06--0.3 ~z; radial, 0.3--1.0 ~z), those of the resting cells are comparatively thick (0.1--0.7 ix and 2.0--3.0 ~z respectively). Although we have not seen it, the first division at the beginning of the growing season must produce an odd, unequally thickened, cell. The most striking feature of the resting cambium is the large amount of space occupied by protein bodies, lipid droplets and, to a lesser extent, vesiculate smooth ER. S~IVASTAV~ (1966) has also reported protein bodies and lipid droplets from the resting cambium of Fraxinus, although the protein bodies were less welt developed than those described here. This is interesting, as S~rVAST~VA makes the point that, although the cells in which they were found were quiescent, they were not 'winter' cells. In our material, there is no doubt that the cells were completely dormant. I t seems, therefore that the most probable function of the protein bodies - - and of the lipid droplets - - is a storage role. I t may be that those protein bodies with an uneven distribution of contents represent either incompletely filled bodies or the removal of protein prior to the grox~4ng season. Such a hypothesis would be in keeping with the absence of such bodies from the active cambium. Although the resting cambium is not highly vaeuolate, the differentiating cells rapidlybeeome so. This means that all cell reactions taking place in the cytoplasm (and including the many and complex processes of wall formation) must take place in the thin parietal layer of cytoplasm. As pointed out in another paper (RoBA~DS, 1968), even wall synthesis would require a supply of many enzymes, some of which could be required for transport into the Fig. 8. P a r t of a n actively differentiating cambial cell. The ribosomes are attached to the endoplasmic reticulum or form polyribosomaI arrays. Small vesicles or tubules of E R are present as well as a multivesicular body. • 23,000 Fig. 9. A cell from the active cambium showing mierotubules adjacent to b o t h nucleus and ptasmalemma (arrowed). Vesicles of unknown origin are present a n d show similarities to the coated vesicles reported b y other workers. • 40,000 17
Planta (Berl.),Bd. 84
248
A.W. ROU.~DS and P. KIDWAI:
wall itself. Further, the outermost layer of cytoplasm in cells actively forming cell walls is often rich in rough E R and/or polyribosomes. These facts suggest that a plentiful supply of protein could be more important than a superficial examination might lead us to suspect. The localization of plastids and the rather small mitochondria around the nucleus of inactive cells is of interest because it has been found so commonly in our material. We have no evidence of significance for this distribution; nor for the ontogeny of the organelles in question although it is common to obtain aspects of plastids and mitochondria which suggest that they may be dividing. In some way these organelles must replicate, and it appears that they are present in comparatively large numbers during the resting period. I t is interesting to recall the observations of BELL and MOH~ETHALE~ (1962) who described the degeneration of organelles in Pteridium aquilinum prior to their reformation from initials budded off from the nuclear envelope. The ontogeny of plastids and mitochondria in cambial cells is clearly worthy of further consideration and experimentation. The presence of dictyosomes of similar appearance in resting and active cambia further supports the view that the Golgi complex is a reasonably stable entity. Indeed, it is in the very active cells that the number of eisternae per dietyosome is reduced, and it may be that this represents intermediate stages in the replication of the dictyosomes: something that would need to take place as there are far fewer dictyosomes in resting than in active cambium. The nature of actively differentiating xylem elements as well as of mature xylem fibres themselves have been described previously (RoBARDS, 1968; ROBA~DS, 1967). From the results recorded here it appears that the cytoplasmic content of the resting cambium is similar to that of the active cambium and differing in degree rather than nature. The characteristics peculiar to the resting cells alone are their reduced vacuolation; the presence of apparent storage materials as protein and lipid in membrane bound bodies, and starch in plastids; and the quiescent nature of the other organelles. This is especially noticeable when considering the membranes themselves which appear so dynamic in the active state (and particularly the plasmalemma with its fusing vesicles and paramural b o d i e s - MAI~CItANTand I~OBARDS,1968), and yet are so static and continuous in the resting cells. Of particular interest now will be not only to advance work on the processes occurring during the differentiation of vascular tissues, but also to study more closely the events taking place during the transition from the resting to the active state. We should like to thank Miss E. DAWDSONand Mr. P. CROS~r for skilled technical assistance. This work was carried out under a grant from the Natural Environment t~esearch Council.
Ultrastrueture of Resting and Active Cambium
249
References
BELL, P. 1:~., and K. MtiHLETJ~AJ~E~:The fine structure of the cells taking part in oogenesis in Pteridium aquilinum (L.) Kuhn. J. Ultrastruct. Res. 7, 452--466 (1962). BCVAT, R. : Comportement des membranes plasmiques lors de la diff~renciation des parois lat4rales des vaisseaux (m6taxyleme de Cucurbita pep@ C. R. Acad. Sci. (Paris) 258, 5511--5514 (1964). CRo~s~Aw, J., and J. B. BOUCK: The fine structure of differentiating xylem elements. J. Cell Biol. 24, 4 1 5 - 4 3 2 (1965). - - , and A. B. W~RDROP: The organisation of cytoplasm in differentiating xylem. Aust. J. Bot. 12, 15--23 (1964). CZA~INSKI, Y. : Aspects infrastructuraux de cellules contigues aux vaisseaux dans le xyl~me de Robinia pseudoacacia. C. R. Acad. Sci. (Paris) 262, 2336--2339 (1966). EsAv, K., V. I. C~EADL~, and R. H. GILL: Cytology of differentiating tracheary elements. I. Organelles and membrane systems. Amer. J. Bot. 53, 756--771 (1966). GIM~Ez-M~T~N, G., M. C. RISUE~O, and J. F. L6r~z-S~]~z: A simple staining technique for electron microscopy with lead-uranyl acetate. Experientia (Basel) 23, 316--317 (1967). JENSEN, W. A. : Botanical histochemistry, Principles and practice. San Francisco and London: Freemen and Company 1962. MARcm~N~, t~., and A. W. ROBAgDS : Membrane systems associated with the plasmalemma of plant cells. Ann. Bot., N.S. 32, 97--117 (1968). MASER, M. D., T. E. POWELL, and C. W. PHILrOTT: Relationships among pH, osmolMity, and concentration of fixative solutions. Stain Technol. 42, 175--182 (1967). MOLnENm~VE~, H. H. : Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39, 111--114 (1964). PICKETT-HEAPS,J. D.: The effect of colchicine on the ultrastructure of dividing plant cells, xylem wall differentiation, and distribution of cytoplasmic microtubules. Develop. Biol. 15, 206--236 (1967). REYNOLDS, E. S.: The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208--212 (1963). ROBA~DS, A. W. : The xylem fibres of Salix/ragilis, L. J. roy. micr. Soc. 87, 329--352 (1967). - - On the ultrastructure of differentiating secondary xylem in willow. Protoplasma 65, 449--464 (1968). - - , and P. G. HUMP~ERS0~ : Phytoferritin in plastids of the cambial zone of willow. Ptanta (Berl.) 76, 169--178 (1967). - - , and M. J. PuRws : Chlorazol black E as a stain for tension wood. Stain Technol. 39, 309--315 (1964). - - , and C. L. ROmNSON: Further studies on phytoferritin.Planta (Bcrl.) 82, 179--188 (1968). SRIVASTAVA,L. : On the fine structure of the cambium of Fraxinus americana, L. J. Cell Biol. 31, 79--93 (1966). - - , and T. P. O'B~IEN: On the ultrastructure of the cambium and its vascular derivatives. I. Cambium of Pinus strobu~ L. Protoplasma 61, 257--276 (1966). S~EED~AN, H. F. : Section cutting in microscopy. Oxford: Blackwell 1960. Dr. A. W. ROB)-RDS Department of Biology, University of York IIeslington, York, England 17"
