- Email: [email protected]
Cytodifferentiation of maturing pine tracheids: The final stage
TISSUE & C E L L I972 4 (3) 525-528 Published by Longman Group Ltd. Printed in Great Britain
TOMASZ J. WODZICKI* and WALTER J. HUMPHREYS-i
C Y T O D I F F E R E N T I A T I O N OF M A T U R I N G PINE T R A C H E I D S " THE FINAL S T A G E ABSTRACT. The natural process of autolysis of protoplasm terminating the maturation of xylem tracheids starts with the formation of cytoplasmic spherules and the breakdown of cytoplasmic bridges which maintain the unity of the intravacuolar lamellate and filamentous reticulum of cytoplasm immersed in vacuolar sap. Released organelles are subsequently digested in the central vacuole. The process is believed to involve vacuolar membrane transformations. Transmission and scanning electron microscopy of cell interiors were employed. Specimens for S.E.M. were freeze-fractured and freeze-dried, or processed by the critical point method.
MATURATION of tracheids in the secondary xylem of conifers is a n ontogenetic stage of cytodifferentiation d u r i n g which the seconda r y wall is deposited (Wilson et al., 1966). It terminates in the complete b r e a k d o w n a n d autolysis of the protoplast. Thereafter the tracheids function in water t r a n s p o r t a n d provide mechanical properties of stem xylem. T h e a m o u n t of cell wall material deposited is related to the d u r a t i o n of the m a t u r a t i o n phase, which varies during the season, cont r i b u t i n g to seasonal changes in w o o d density (Wodzicki, 1971). T h e precise m e c h a n i s m t h a t controls the d u r a t i o n of t r a c h e i d m a t u r a t i o n is not k n o w n . Yet its u n d e r s t a n d i n g is a key p r o b l e m in the regulation of w o o d quality a n d cellulose production. So far it is k n o w n t h a t all i m p o r t a n t organelles such as the nucleus, m i t o c h o n d r i a a n d dictyosomes are present in m a t u r i n g xylem elements until they are suddenly autolysed ( C r o n s h a w a n d Bouck, 1965). D u e to extreme v a c u o l a t i o n a n d rapid degenerative changes the investigation of cytoplasmic u l t r a s t r u c t u r e at the final stage o f m a t u r a t i o n is very difficult, b u t the recognized impor-
t a n c e of such i n f o r m a t i o n for current physiological studies m o t i v a t e d this work. Small cubes of tissue (1 m m :~) c o n t a i n i n g xylem, c a m b i a l zone a n d p h l o e m isolated in mid-July a n d early S e p t e m b e r from an eight-year-old stem of Ph;us echinata Mill. were fixed for two h o u r s at 4 : C in 2'.;, giutaraidehyde buffered to p H 7-2 with 0.1 M s o d i u m cacodylate. F o r t r a n s m i s s i o n electron microscopy the cubes were postfixed for two hours in 2% o s m i u m tetroxide a n d e m b e d d e d in m a r a g l a s ( F r e e m a n and Spurlock, 1962). C u b e s of tissue fixed in either glutaraldehyde alone or post-fixed in osmic acid were stored for m o n t h s in cold 5% sucrose buffered to p H 7.2 with 0'1 M s o d i u m caeodylate. T h e blocks were subsequently washed for one h o u r in several changes of glass-redistilled w a t e r a n d processed in the following different ways: (a) Cubes were positioned in a n a l u m i n u m specimen holder w i t h the radial surface exposed, rapidly frozen and fractured under liquid nitrogen, a n d t h e n freeze-dried in a h i g h v a c u u m (10 6 torr) overnight. (b) O t h e r tissue was dried by A n d e r s o n ' s critical p o i n t m e t h o d (Anderson, 1969). All specimens were coated with a layer of gold 200 400 * Tree Physiology Laboratory, School of Forest thick before being observed in the s c a n n i n g Resources, University of Georgia, and Faculty of electron microscope. Forestry, Agricultural University, Warsaw, Poland. T h e p r o t o p l a s t o f tracheids which enter t Department of Zoology, University of Georgia, the m a t u r a t i o n phase is already differentiated Athens, Georgia 30601. into two i n t e r c o n n e c t e d p o r t i o n s : the layer Manuscript received 20 March 1972. lining the internal surface of the cell wall a n d Revised 4 May 1972. 525
WODZICKI AND HUMPHREYS
526 into the p o r t i o n of the cell interior w h i c h represents the t h i c k infoldings a n d strands of cytoplasm with enclosed organelles surr o u n d e d with vacuolar sap (Fig. 1). T h e lining layer of cytoplasm is covered on its external surface by the p l a s m a l e m m a , whereas the i n t r a v a c u o l a r cytoplasm is enclosed in v a c u o l a r m e m b r a n e s . Specializations of f u n c t i o n of the two p o r t i o n s of cytoplasm m a y be envisaged as, for example, only the lining layer is directly involved in cell wall f o r m a t i o n . As m a t u r a t i o n advances, infoldings a n d strands of i n t r a v a c u o l a r cytoplasm are t r a n s f o r m e d to a fine i n t r a v a c u o l a r reticulum c o m p o s e d of an integrated n e t w o r k of p r o t o p l a s m i c lamellae and filaments. M i t o c h o n d r i a , plastids a n d lipid droplets are frequently observed in these structures as being separated from the v a c u o l a r sap only by a thin layer of cytoplasm a n d the v a c u o l a r m e m b r a n e s (Fig. 2, 4), plastids c o n t a i n i n g starch grains can be distinguished in s c a n n i n g electron m i c r o g r a p h s b y the asymmetric shapes of the organelles (Fig. 3). Transmission electron m i c r o s c o p y confirms this identification. P o r t i o n s of the cytoplasmic septations between regions occupied by
organelles b e c o m e exceedingly thin at the stage preceding destruction o f the p r o t o p l a s m (Fig. 2, 4). T h e p o r t i o n s of intravacuolar strands of cytoplasm with mitochondria, chloroplasts a n d possibly o t h e r organelles r e m a i n more or less spherical, suggesting little change in their structure. These findings are in a g r e e m e n t with the investigations of others ( C r o n s h a w a n d Bouck, 1965) who observed m i t o c h o n d r i a separated f r o m cytoplasm in the v a c u o l a r sap of differentiating xylem elements at the very end of m a t u r a t i o n . Various sizes of cytoplasmic spherules b u d off from these septations at the stage preceding the disi n t e g r a t i o n of the p r o t o p l a s t (Fig. 6, 7). This suggests an i m p o r t a n t change in properties of the vacuolar m e m b r a n e s . W h e n the process advances, cytoplasmic bridges are b r o k e n between p o r t i o n s of cytoplasm enclosing organelles a n d the latter are released into the vacuole (Fig. 5). T h e rod-like a n d filamentous extensions of the i n t r a v a c u o l a r reticulum of cytoplasm (Fig. 8) may represent the sites of such breakdown. T h e nucleus is f o u n d a m o n g the last autolysed c o m p o n e n t s of the protoplast.
Structural transformations of cytoplasm in maturing axial tracheids of pine stem xylem. Micrographs represent radial sections or exposures of the cell interior after a part of the cell wall has been removed. Fig. 1. Tracheid at the early stage of maturation. The cell wail lining layer of cytoplasm is indicated by arrows, and cytoplasmic compartmentation is clearly shown. Specimen sectioned after drying by the critical point method. 1000. Figs. 2 and 3. Advanced stage of maturation. Specimen freeze-fractured and Freezedried. The lamellate intravacuolar reticulum of cytoplasm is seen in Fig, 2. ,2000. Various sizes and shapes of cytoplasmic bodies enclose organelles, and plastids with asymmetric shapes (arrow) contain starch grains as shown enlarged in Fig. 3. " 5000. Fig. 4. Portion of the intravacuolar cytoplasmic reticulum with enclosed organelles at the advanced stage of tracheid maturation as viewed by transmission electron microscopy. The vacuolar membranes are shown at arrows. :, I 1,000. Fig. 5. Final stage of tracheid maturation. Isolated cytoplasmic spherules are found in the central vacuole. The vacuolar membrane of the cytoplasm lining the cell wall disintegrates (arrows). 11,000. Figs. 6 and 7. Cytoplasmic spherules bud offthe surface of the intravacuolar reticulum of the cytoplasm at the stage of maturation preceding disintegration of the protoplast as seen by transmission and scanning electron microscopy respectively. Ceil wall is seen at upper-left corner of Fig. 6. Fig. 6, :<64,000: Fig. 7, \ 5000. Fig. 8. Cytoplasmic bridges of lamellate and filamentous reticulum of intravacuolar cytoplasm.., 8000. Fig. 9. Disorganized cytoplasm at the end of tracheid maturation. ;, 1000. Figs. 7, 8 and 9. Specimens freeze-fractured and freeze-dried.
®
~'
~;~ ~
~,:!i~~'~
WODZICKI AND HUMPHREYS
528 Ultimately the structure of the intravacuolar p o r t i o n o f the cytoplasm is destroyed a n d its r e m n a n t s are separated in the vacuole or it recedes towards the cell wall (Fig. 9). There ate indications t h a t the vacuolar m e m b r a n e of the lining layer of cytoplasm is also d a m a g e d at this stage. T h u s the site of p r o t o p l a s m which is affected primarily to bring a b o u t the t e r m i n a t i o n of tracheid fife m a y be localized at the v a c u o l a r m e m b r a n e s as has been postulated by others ( C r o n s h a w a n d Bouck, 1965). T h e process described a b o v e does n o t agree with the hypothesis ( G a h a n a n d Maple, 1966) suggesting an intracytoplasmic release of hydrolases f r o m lysosome-like particles t h a t w o u l d digest the p r o t o p l a s t of the differentiating xylem elements. W e favor the hypothesis t h a t the digestion of separated cytoplasmic c o m p o n e n t s occurs in the vacuolar sap enriched with hydrolases before the cytoplasmic b r e a k d o w n . The process is obviously different from the p h e n o m e n a of cytosegregation a n d autolysis described by n u m e r o u s a u t h o r s (Matfle, 19691) as being c o n n e c t e d with the n o r m a l t u r n o v e r of organelles, m o b i l i z a t i o n of reserve materials, or as a m e c h a n i s m of cell survival u n d e r stress conditions. The process is c o m p a r a b l e to that recently described for senescing p l a n t cells (Matile a n d W i n k e n b a c h , 1971). T h e t e r m i n a t i o n of t r a c h e i d m a t u r a t i o n seems to
be a two-step process connected with structural changes of the m e m b r a n e s exposed to the v a c u o l a r sap. Firstly, the change brings a b o u t the f o r m a t i o n of cytoplasmic spherules a n d a b r e a k in continuity of the i n t r a v a c u o l a r lamellate a n d filamentous reticulum of cytoplasm. Secondly, a m e m b r a n e t r a n s f o r m a t i o n may allow hydrolytic enzymes of the central vacuole to p e n e t r a t e a n d digest the h i t h e r t o little-damaged organelles. This is conjectural, however, since the causative events in autolysis at this stage are n o t yet u n d e r s t o o d . W h a t e v e r the physiologic m e c h a n i s m is which is responsible for these v a c u o l a r m e m b r a n e transf o r m a t i o n s in m a t u r i n g tracheids, it affects the d u r a t i o n of t h e m a t u r a t i o n p h a s e a n d the time of deposition of cell wall material, a n d therefore the thickness of the cell wall,
Acknowledgements The first a u t h o r t h a n k s D e a n A. M. H e r r i c k of the School of Forest Resources w h i c h m a d e his tenure as a visiting professor at the University of G e o r g i a possible. Special gratitude is due D r C. L. Brown of the Tree Physiology L a b o r a t o r y in which m u c h of this work was done. W e t h a n k Mrs J a n e t J o h n s o n of the central Electron M i c r o s c o p y L a b o r a t o r y for h e r invaluable technical assistance.
References ANDERSON, T. F. 1969. Electron Microscopy of Microorganisms. In Physical Techniques in Biological Research (eds. G. Oster and A. Pollister), Vol. 3, pp. /77 240. CRONSHAW, J. and Bouc•, J. B. 1965. The fine structure of differentiating xylem elements. J. Cell Biol., 24, 415-431. FREEMAN,J. A. and SPURLOCK, B. O. 1962. A new epoxy embedment for electron microscopy. J. Cell Biol., 13, 437-443. GAHAN, P. B. and MAPLE, A. J. 1966. The behaviour of lysosome-like particles during cell differentiation. J. exp. Bol., 17, 151-155. MATILE, PH. 1969. Plant lysosomes. In Lysosomes in Biology and Pathology (eds. J. T. Dingle and H. B. FeII), Vol. 1, pp. 406 430. MATILE, PH. and WINKENBACH, F. 1971. Function of lysosomes and lysosomaI enzymes in the senescing corolla of the morning glory (Ipomoea purpurea). J. exp. Bot., 22, 759-771. WILSON, B. F., WODZICKI, T. J. and ZAHNF:R, R. 1966. Differentiation of cambial derivatives: Proposed terminology. Forest Sci., 12, 438-440. WODZlCK~, T. J. 1971, Mechanism of xylem differentiation in Pintrs sih'estris L. J. exp. Bot., 22, 670--687.
TOMASZ J. WODZICKI* and WALTER J. HUMPHREYS-i
C Y T O D I F F E R E N T I A T I O N OF M A T U R I N G PINE T R A C H E I D S " THE FINAL S T A G E ABSTRACT. The natural process of autolysis of protoplasm terminating the maturation of xylem tracheids starts with the formation of cytoplasmic spherules and the breakdown of cytoplasmic bridges which maintain the unity of the intravacuolar lamellate and filamentous reticulum of cytoplasm immersed in vacuolar sap. Released organelles are subsequently digested in the central vacuole. The process is believed to involve vacuolar membrane transformations. Transmission and scanning electron microscopy of cell interiors were employed. Specimens for S.E.M. were freeze-fractured and freeze-dried, or processed by the critical point method.
MATURATION of tracheids in the secondary xylem of conifers is a n ontogenetic stage of cytodifferentiation d u r i n g which the seconda r y wall is deposited (Wilson et al., 1966). It terminates in the complete b r e a k d o w n a n d autolysis of the protoplast. Thereafter the tracheids function in water t r a n s p o r t a n d provide mechanical properties of stem xylem. T h e a m o u n t of cell wall material deposited is related to the d u r a t i o n of the m a t u r a t i o n phase, which varies during the season, cont r i b u t i n g to seasonal changes in w o o d density (Wodzicki, 1971). T h e precise m e c h a n i s m t h a t controls the d u r a t i o n of t r a c h e i d m a t u r a t i o n is not k n o w n . Yet its u n d e r s t a n d i n g is a key p r o b l e m in the regulation of w o o d quality a n d cellulose production. So far it is k n o w n t h a t all i m p o r t a n t organelles such as the nucleus, m i t o c h o n d r i a a n d dictyosomes are present in m a t u r i n g xylem elements until they are suddenly autolysed ( C r o n s h a w a n d Bouck, 1965). D u e to extreme v a c u o l a t i o n a n d rapid degenerative changes the investigation of cytoplasmic u l t r a s t r u c t u r e at the final stage o f m a t u r a t i o n is very difficult, b u t the recognized impor-
t a n c e of such i n f o r m a t i o n for current physiological studies m o t i v a t e d this work. Small cubes of tissue (1 m m :~) c o n t a i n i n g xylem, c a m b i a l zone a n d p h l o e m isolated in mid-July a n d early S e p t e m b e r from an eight-year-old stem of Ph;us echinata Mill. were fixed for two h o u r s at 4 : C in 2'.;, giutaraidehyde buffered to p H 7-2 with 0.1 M s o d i u m cacodylate. F o r t r a n s m i s s i o n electron microscopy the cubes were postfixed for two hours in 2% o s m i u m tetroxide a n d e m b e d d e d in m a r a g l a s ( F r e e m a n and Spurlock, 1962). C u b e s of tissue fixed in either glutaraldehyde alone or post-fixed in osmic acid were stored for m o n t h s in cold 5% sucrose buffered to p H 7.2 with 0'1 M s o d i u m caeodylate. T h e blocks were subsequently washed for one h o u r in several changes of glass-redistilled w a t e r a n d processed in the following different ways: (a) Cubes were positioned in a n a l u m i n u m specimen holder w i t h the radial surface exposed, rapidly frozen and fractured under liquid nitrogen, a n d t h e n freeze-dried in a h i g h v a c u u m (10 6 torr) overnight. (b) O t h e r tissue was dried by A n d e r s o n ' s critical p o i n t m e t h o d (Anderson, 1969). All specimens were coated with a layer of gold 200 400 * Tree Physiology Laboratory, School of Forest thick before being observed in the s c a n n i n g Resources, University of Georgia, and Faculty of electron microscope. Forestry, Agricultural University, Warsaw, Poland. T h e p r o t o p l a s t o f tracheids which enter t Department of Zoology, University of Georgia, the m a t u r a t i o n phase is already differentiated Athens, Georgia 30601. into two i n t e r c o n n e c t e d p o r t i o n s : the layer Manuscript received 20 March 1972. lining the internal surface of the cell wall a n d Revised 4 May 1972. 525
WODZICKI AND HUMPHREYS
526 into the p o r t i o n of the cell interior w h i c h represents the t h i c k infoldings a n d strands of cytoplasm with enclosed organelles surr o u n d e d with vacuolar sap (Fig. 1). T h e lining layer of cytoplasm is covered on its external surface by the p l a s m a l e m m a , whereas the i n t r a v a c u o l a r cytoplasm is enclosed in v a c u o l a r m e m b r a n e s . Specializations of f u n c t i o n of the two p o r t i o n s of cytoplasm m a y be envisaged as, for example, only the lining layer is directly involved in cell wall f o r m a t i o n . As m a t u r a t i o n advances, infoldings a n d strands of i n t r a v a c u o l a r cytoplasm are t r a n s f o r m e d to a fine i n t r a v a c u o l a r reticulum c o m p o s e d of an integrated n e t w o r k of p r o t o p l a s m i c lamellae and filaments. M i t o c h o n d r i a , plastids a n d lipid droplets are frequently observed in these structures as being separated from the v a c u o l a r sap only by a thin layer of cytoplasm a n d the v a c u o l a r m e m b r a n e s (Fig. 2, 4), plastids c o n t a i n i n g starch grains can be distinguished in s c a n n i n g electron m i c r o g r a p h s b y the asymmetric shapes of the organelles (Fig. 3). Transmission electron m i c r o s c o p y confirms this identification. P o r t i o n s of the cytoplasmic septations between regions occupied by
organelles b e c o m e exceedingly thin at the stage preceding destruction o f the p r o t o p l a s m (Fig. 2, 4). T h e p o r t i o n s of intravacuolar strands of cytoplasm with mitochondria, chloroplasts a n d possibly o t h e r organelles r e m a i n more or less spherical, suggesting little change in their structure. These findings are in a g r e e m e n t with the investigations of others ( C r o n s h a w a n d Bouck, 1965) who observed m i t o c h o n d r i a separated f r o m cytoplasm in the v a c u o l a r sap of differentiating xylem elements at the very end of m a t u r a t i o n . Various sizes of cytoplasmic spherules b u d off from these septations at the stage preceding the disi n t e g r a t i o n of the p r o t o p l a s t (Fig. 6, 7). This suggests an i m p o r t a n t change in properties of the vacuolar m e m b r a n e s . W h e n the process advances, cytoplasmic bridges are b r o k e n between p o r t i o n s of cytoplasm enclosing organelles a n d the latter are released into the vacuole (Fig. 5). T h e rod-like a n d filamentous extensions of the i n t r a v a c u o l a r reticulum of cytoplasm (Fig. 8) may represent the sites of such breakdown. T h e nucleus is f o u n d a m o n g the last autolysed c o m p o n e n t s of the protoplast.
Structural transformations of cytoplasm in maturing axial tracheids of pine stem xylem. Micrographs represent radial sections or exposures of the cell interior after a part of the cell wall has been removed. Fig. 1. Tracheid at the early stage of maturation. The cell wail lining layer of cytoplasm is indicated by arrows, and cytoplasmic compartmentation is clearly shown. Specimen sectioned after drying by the critical point method. 1000. Figs. 2 and 3. Advanced stage of maturation. Specimen freeze-fractured and Freezedried. The lamellate intravacuolar reticulum of cytoplasm is seen in Fig, 2. ,2000. Various sizes and shapes of cytoplasmic bodies enclose organelles, and plastids with asymmetric shapes (arrow) contain starch grains as shown enlarged in Fig. 3. " 5000. Fig. 4. Portion of the intravacuolar cytoplasmic reticulum with enclosed organelles at the advanced stage of tracheid maturation as viewed by transmission electron microscopy. The vacuolar membranes are shown at arrows. :, I 1,000. Fig. 5. Final stage of tracheid maturation. Isolated cytoplasmic spherules are found in the central vacuole. The vacuolar membrane of the cytoplasm lining the cell wall disintegrates (arrows). 11,000. Figs. 6 and 7. Cytoplasmic spherules bud offthe surface of the intravacuolar reticulum of the cytoplasm at the stage of maturation preceding disintegration of the protoplast as seen by transmission and scanning electron microscopy respectively. Ceil wall is seen at upper-left corner of Fig. 6. Fig. 6, :<64,000: Fig. 7, \ 5000. Fig. 8. Cytoplasmic bridges of lamellate and filamentous reticulum of intravacuolar cytoplasm.., 8000. Fig. 9. Disorganized cytoplasm at the end of tracheid maturation. ;, 1000. Figs. 7, 8 and 9. Specimens freeze-fractured and freeze-dried.
®
~'
~;~ ~
~,:!i~~'~
WODZICKI AND HUMPHREYS
528 Ultimately the structure of the intravacuolar p o r t i o n o f the cytoplasm is destroyed a n d its r e m n a n t s are separated in the vacuole or it recedes towards the cell wall (Fig. 9). There ate indications t h a t the vacuolar m e m b r a n e of the lining layer of cytoplasm is also d a m a g e d at this stage. T h u s the site of p r o t o p l a s m which is affected primarily to bring a b o u t the t e r m i n a t i o n of tracheid fife m a y be localized at the v a c u o l a r m e m b r a n e s as has been postulated by others ( C r o n s h a w a n d Bouck, 1965). T h e process described a b o v e does n o t agree with the hypothesis ( G a h a n a n d Maple, 1966) suggesting an intracytoplasmic release of hydrolases f r o m lysosome-like particles t h a t w o u l d digest the p r o t o p l a s t of the differentiating xylem elements. W e favor the hypothesis t h a t the digestion of separated cytoplasmic c o m p o n e n t s occurs in the vacuolar sap enriched with hydrolases before the cytoplasmic b r e a k d o w n . The process is obviously different from the p h e n o m e n a of cytosegregation a n d autolysis described by n u m e r o u s a u t h o r s (Matfle, 19691) as being c o n n e c t e d with the n o r m a l t u r n o v e r of organelles, m o b i l i z a t i o n of reserve materials, or as a m e c h a n i s m of cell survival u n d e r stress conditions. The process is c o m p a r a b l e to that recently described for senescing p l a n t cells (Matile a n d W i n k e n b a c h , 1971). T h e t e r m i n a t i o n of t r a c h e i d m a t u r a t i o n seems to
be a two-step process connected with structural changes of the m e m b r a n e s exposed to the v a c u o l a r sap. Firstly, the change brings a b o u t the f o r m a t i o n of cytoplasmic spherules a n d a b r e a k in continuity of the i n t r a v a c u o l a r lamellate a n d filamentous reticulum of cytoplasm. Secondly, a m e m b r a n e t r a n s f o r m a t i o n may allow hydrolytic enzymes of the central vacuole to p e n e t r a t e a n d digest the h i t h e r t o little-damaged organelles. This is conjectural, however, since the causative events in autolysis at this stage are n o t yet u n d e r s t o o d . W h a t e v e r the physiologic m e c h a n i s m is which is responsible for these v a c u o l a r m e m b r a n e transf o r m a t i o n s in m a t u r i n g tracheids, it affects the d u r a t i o n of t h e m a t u r a t i o n p h a s e a n d the time of deposition of cell wall material, a n d therefore the thickness of the cell wall,
Acknowledgements The first a u t h o r t h a n k s D e a n A. M. H e r r i c k of the School of Forest Resources w h i c h m a d e his tenure as a visiting professor at the University of G e o r g i a possible. Special gratitude is due D r C. L. Brown of the Tree Physiology L a b o r a t o r y in which m u c h of this work was done. W e t h a n k Mrs J a n e t J o h n s o n of the central Electron M i c r o s c o p y L a b o r a t o r y for h e r invaluable technical assistance.
References ANDERSON, T. F. 1969. Electron Microscopy of Microorganisms. In Physical Techniques in Biological Research (eds. G. Oster and A. Pollister), Vol. 3, pp. /77 240. CRONSHAW, J. and Bouc•, J. B. 1965. The fine structure of differentiating xylem elements. J. Cell Biol., 24, 415-431. FREEMAN,J. A. and SPURLOCK, B. O. 1962. A new epoxy embedment for electron microscopy. J. Cell Biol., 13, 437-443. GAHAN, P. B. and MAPLE, A. J. 1966. The behaviour of lysosome-like particles during cell differentiation. J. exp. Bol., 17, 151-155. MATILE, PH. 1969. Plant lysosomes. In Lysosomes in Biology and Pathology (eds. J. T. Dingle and H. B. FeII), Vol. 1, pp. 406 430. MATILE, PH. and WINKENBACH, F. 1971. Function of lysosomes and lysosomaI enzymes in the senescing corolla of the morning glory (Ipomoea purpurea). J. exp. Bot., 22, 759-771. WILSON, B. F., WODZICKI, T. J. and ZAHNF:R, R. 1966. Differentiation of cambial derivatives: Proposed terminology. Forest Sci., 12, 438-440. WODZlCK~, T. J. 1971, Mechanism of xylem differentiation in Pintrs sih'estris L. J. exp. Bot., 22, 670--687.