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Proc. Natl. Acad. Sci. USA Vol. 75, No. 2, pp. 852-856, February 1978 Cytochemical studies on GERL, provacuoles, and vacuoles in meristematic cells of Euphorbia (electron microscopy/ localizations//cellular /membrane flow) FRANCIS MARTY Institut de Biologie cellulaire, Unite d'Enseignement et de Recherche Scientifique de Luminy, Universit6 d'Aix-Marseille, 13288 Marseille Cedex 2, France Communicated by Alex B. Notikoff, November 9,1977

ABSTRACT The genesis of vacuoles has been studied M cacodylate buffer containing 0.22 M sucrose and then washed by high-voltage and conventional electron microscopy. Vacuole again with quick changes in graded buffers from pH 7.4 to 5.0 genesis is a lysosomal multistep rocess: (i) GERL (Golgi-asso- ciated from which appar- and finally in a citrate buffer (pH 4.8) to remove calcium salts. ently form) produces provacuoles into which lysosomal The pieces were incubated at 370 freely floating in phos- and probably other materials appear to be concentrated and phatase (EC 3.1.3.2) medium according to Novikoff (8, 9) or packaged; (ii) GERL-derived provacuoles cooperate to drive a in Bell and Barrnett medium for thiolacetic acid esterase (10). programmed cellular autophagy leading to young vacuoles; and Controls consisted of tissues either incubated without the sub- (iii) the young vacuoles swell and fuse together into a few large strate ( (3-glycerophosphate or cytidine 5'-monophos- mature vacuoles which continue to collect the GERL-derived phate (CMP) for acid phosphatase activities and thiolacetic acid provacuoles throughout the of the cell. for the corresponding acid esterase) or in the presence of sodium Although vacuoles are the most conspicuous membrane- fluoride (0.01 M for acid phosphatase activities and 0.1 M for bounded in differentiated plant cells and their bio- thiolacetic acid esterase). After the buffered rinses, subsequent chemical characterization is in progress (1, 2), the question of processing was conventional. their origin is unsettled despite many light and electron mi- Zinc Iodide-Osmium Reaction. After excision, 2-mm root croscopy studies (3-5). This report describes the use of marker tips were immediately fixed by immersion for 6-24 hr in a enzyme activities and electron microscopy, at conventional as mixture of zinc iodide and osmium tetroxide (ZIO) at room well as very high voltage, to investigate the pattern of vacu- temperature in the dark (11). Controls were fixed for the same olation in root meristematic cells. The results emphasize the role length of time in unbuffered 0.4% aqueous OsO4 at pH 5.9 of GERL* in the origin of vacuoles and establish the sequence without zinc iodide. of related lysosomal events. They suggest that the plant va- Sectioning and Microscopy. Thin sections (up to 800 A) cuome is an intracellular digestive system involved in cellular were examined in the Philips 300 electron microscope at 80kv. autophagy and in internal membrane flow. For morphological studies they were stained either with uranyl acetate and citrate or with 1% aqueous per- MATERIAL AND METHODS manganate; sections from cytochemical specimens were ob- served without counterstain. Thick (1-10 jim) sections of tissues . of Euphorbia characias L. harvested treated by selective cytochemical staining were held in 200- locally from wild were sterilized in 10% commercial mesh uncoated sandwich grids and were examined without bleach for 30 min. The seeds were rinsed, soaked for 18 hr. and counterstain at 2.5 MV with the Toulouse 3 MV electron mi- then placed in petri plates on filter paper moistened with croscope (11). deionized water. As soon as the root emerged, the seedlings were selected, transferred to a humid atmosphere, and grown ver- RESULTS tically at room temperature (220) in the dark. When the root Morphological Sequence. The most undifferentiated root reached 10 ± 2 mm, the seedlings were collected for processing cells just adjacent to the quiescent zone were devoid of vacuoles for electron microscopy. or their identifiable precursors. Nearby, in the youngest cells Morphological Studies. Whole seedlings were quickly im- where vacuolation is initiated, the primordial vacuole precursors mersed in an ice-cold fixative containing either 3% glutaral- or provacuoles arose in the immediate vicinity of dictyosomes dehyde/0. 1 M cacodylate buffer, pH 7.4 or 2.5% glutaral- (Fig. 1 A-D). The Golgi stacks consisted of five or six evenly dehyde/2% formaldehyde (prepared from paraformaldehyde spaced elements. Rough endoplasmic reticulum (ER) sheets immediately before use)/0.09 M cacodylate buffer, pH 7.4. approached the adjacent Golgi stack but provacuoles were not After 30 min, 1.5-mm apical root tips were excised while in seen budding from Golgi elements or from ER cisternae. Rather fixative, left immersed for an additional 90 min, and then they appeared to bud from GERL. GERL was at the "trans" processed as usual for observation by electron microscopy. side (12) of the dictyosomes and consisted of a twisted, Enzyme Activities. Preliminary processing included a short smooth-surfaced, polygonal meshwork of anastomosing tubules fixation in gfutaraldehyde or glutaraldehyde/formaldehyde and saccules, the saccules facing the Golgi stacks. The saccular followed by thorough rinses in 0.1 M cacodylatebuffer (pH 7.4). portions of GERL were separated from the trans element of the Root tips were generally cut axially and, in each experiment, Golgi stack by a distance greater than that between elements some tips were sectioned with the Sorvall TC 2 tissue chopper in the stack. Despite numerous small gaps between smooth ER at approximately 50 Am. Specimens were collected in cold 0.1 Abbreviations: CMP, cytidine 5'-monophosphate; ZIO, zinc iodide- The costs of publication of this article were defrayed in part by the osmium tetroxide; ER, endoplasmic reticulum. payment of page charges. This article must therefore be hereby marked * The acronym GERL has been given by Novikoff (6, 7) to the region "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate of smooth ER that is located at the trans aspect of the this fact. and that appears to produce Lysosomes. 852 Downloaded by guest on October 1, 2021 : Marty Proc. Natl. Acad. Sci. USA 75 (1978) 853

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FIG 1. Portions of meristematic cells from the root tip. AV, autophagic vacuole; ER, endoplasmic reticulum; G. Golgi stack; GE, GERL; M, ; PV, provacuole; V, vacuole. (A) A dictyosome in a meristematic cell. (Not incubated; X57,600.) (B) Meristematic cell. (Incubated with f,-glycerophosphate 45 min. X64,000.) (C) Detailed view of a nascent provacuole. (ZIO stain 12 hr; X51,500.) (D) GERL portion in a highly meristematic cell. (Incubated with CMP 60 mmn section 1 ,um thick; X46,000.) (E) Elongated provacuoles in a vacuolating cell. (Not incubated; X31,250.) (F) Provacuolar channels in a cell. (Incubated with CMP 75 min, section 1 ,um thick; X25,000.) and GERL, direct continuities of rough and smooth ER sug- the bars of cages, around portions of the . It was dif- gested that rough ER gives rise to smooth elements of GERL. ficult to appreciate this architecture in ordinary thin sections In thin sections, nascent provacuoles were seen budding from where equatorial sections through the cage show only short or lying adjacent to GERL as if they arose from GERL at circumferentially arranged profiles (Fig. 2A, arrows). Although junctions of the anastomosing tubules. they would generally be erroneously interpreted as vesicles, In slightly more differentiated cells, provacuoles were ZIO-stained thick specimens (Fig. 2B) made it clear that each elongated into tubes 0. I Am in diameter (Fig. 1E). Their tri- short profile actually was a transverse section through one of dimensional aspect could be ascertained from sections 1 to 10 the tubes forming the bars of a cage. Subsequently, the tubes ,um thick studied by high-voltage electron microscopy (Fig. IF). of each sequestering cage merged laterally to entrap the pre- The branched provacuolar tubes formed continuous tracts viously demarcated portion of cytoplasm in a double-mem- throughout the cytoplasm. Because the tubes were cut at diverse brane system containing a continuous exoplasmic space. This angles, the observed profiles in thin sections were those of sequence of events led to autophagy but, initially, the seques- random sections through the sinuous and interconnected tubes tered cytoplasm was morphologically unaltered and might have (Fig. 1E). contained mitochondria, , dictyosomes, ER, or peroxi- In the next stage, the provacuole tubes were wrapped, like somes. A webbed aspect of the forming double-membrane Downloaded by guest on October 1, 2021 854 Cell Biology: Marty Proc. Natl. Acad. Sci. USA 75 (1978)

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FIG. 2. Portions of meristematic cells from the root tip. Abbreviations as in Fig. 1. (A) Thin equatorial section through a.Rsequestration .R cage. (Not incubated; X18,900.) (B) Section 1 Asm thick including part of sequestration cage. (ZIO stain 12 h; X18,000.) (C) Typical.. ring-like.. ,.x@.:autophagic vacuole in a vacuolating cell. (Not incubated; X37,100.) (D) Autophagic vacuole in a root cell. (Incubated for the demonstration of thiolacetic acid esterase activity; X41,200.) .....t: :_>...... :F j system occasionally was encountered (Fig. 2B, arrows). This and D). The provacuoles showed the same selectivity as GERL, is an intermediate step in the evolution from the sequestering and the level of activity appeared to be higher in provacuoles cage to the autophagic vacuole. Random thin sections through mostly when CMP was used as substrate....(Fig...iF). #Sodium . the complete system gave typical ring-like autophagic vacuoles fluoride (0.01 M) totally inhibited the acid phosphatase activity (Fig. 2 C and D). Elsewhere, provacuoles that were not engaged in GERL and provacuoles. Golgi elements and ER sheets were in the sequestrating process also were found. devoid of cytochemically demonstrable acid phosphatase and In more differentiated cells, 500 ,um from the root tip, ribo- thiolacetic acid esterase activities. somes and membrane-bounded organelles of the sequestered Provacuoles demonstrated high acid phosphatase and cytoplasm were undergoing a "compaction" and the typical thiolacetic acid esterase activities at all steps of their evolution young vacuole was formed when segregated structures and the into autophagic vacuoles. Reaction products from both enzyme inner membrane of the autophagic cavity had been completely activities were abundant in the wrapping autophagic cavities degraded (Fig. 3). All the young vacuoles formed more or less as well as in the provacuole tubes from which they derived simultaneously in the same cell fused together and enlarged to (Figs. iF and 2D). Up to this step of vacuolation, the acid hy- give rise to a few large vacuoles. These vacuoles were able to drolases were kept rigorously in check in the sequestrating engulf bits of cytoplasm by focal invagination of their sur- cavities, and the trapped cytoplasm was free of reaction prod- rounding membrane (Fig. 3B, long arrows) and to collect newly ucts (Fig. 2D). However, some time after the sequestered formed provacuoles by fusion of the respective membranes portion of cytoplasm had been completely closed off, the hy- (Fig. 3 inset, short arrows). drolases appeared to be released from the surrounding exo- Marker Enzymes. The youngest cells displayed marked acid plasmic space into the sequestered cytoplasm. The hydrolases phosphatase activity both in GERL and in nascent provacuoles were associated with the cytoplasm as it underwent with either fl-glycerophosphate or CMP as substrate (Fig. 1 B (Fig. 3A) but in the resulting young vacuoles the acid phos- Downloaded by guest on October 1, 2021 Cell Biology: Marty Proc. Natl. Acad. Sci. USA 75 (1978) 855

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FIG. 3. Portions of meristematic cells from the root tip. Abbreviations as in Fig. 1. (A) Autophagic vacuole in a vacuolating root cell. (Incubated with CMP 60 min; X42,100.) (B) Vacuole in which the sequestered cytoplasm is almost degraded. (Not incubated; X20,600.) (Inset) Provacuoles (arrows) merging with the large vacuole. (X8000.)

phatase and thiolacetic acid esterase activities were associated possesses cytochemically demonstrable acid phosphatase ac- chiefly with the exoplasmic surface of the vacuole mem- tivities and appears to produce lysosomes (8). Morphological brane. and cytochemical features in plant material support the concept of GERL and emphasize its role in the origin of vacuoles (Fig. DISCUSSION 4). Although the nascent provacuoles arise as focal blebbings Cytochemical studies in diverse cell types led Novikoff and his of GERL in the immediate vicinity of Golgi trans faces, the collaborators to consider the smooth-surfaced element at the Golgi apparatus itself appears to be bypassed. Whether ones inner or trans aspect of the Golgi apparatus as a distinct or- personal preference is to exclude GERL from the Golgi complex ganelle (GERL) excluded from the Golgi stack. In neurons of (7, 8) or not (13, 14), a relationship among nascent provacuoles, the dorsal root ganglia where it has been best studied, GERL GERL, and the Golgi stack is probably functionally significant

FIG. 4. Stages in the formation of vacuoles. Abbreviations as in Fig. 1. ER, Golgi apparatus, and GERL are extensively drawn in the upper part of the diagram because they are present at any step of vacuome maturation. The formation of vacuoles during cell differentiation is shown from left to right in the lower portion of the drawing. Downloaded by guest on October 1, 2021 856 Cell Biology: Marty Proc. Natl. Acad. Sci. USA 75 (1978) even though the metabolic interchanges that could occur re- priate to think that the conversion from ER membrane to main to be elucidated. vacuole membrane occurs not at the Golgi stack but at GERL As in previous studies of diverse cell types (6, 15), the during provacuole production. In sequential differentiation, cytochemical results with meristematic plant cells suggest that the provacuole membrane is progressively transformed into GERL is a specialized region where acid hydrolases are pack- vacuole membrane during cellular autophagy; in already aged into nascent provacuoles. These provacuoles can tenta- vacuolated cells it acquires the capacity to fuse directly with tively be identified as lysosomes (16). At any period of devel- the vacuole membrane. This inwardly directed vacuome route opment, provacuoles display cytochemically demonstrable appears remarkably symmetric to the outwardly directed hydrolase activities whereas in GERL these activities are exocytic route during which membranes change from ER-like sometimes more difficult to demonstrate. It remains to be es- to plasma membrane-like. The latter provides for secretion of tablished whether this cytochemical difference indicates that polysaccharides during wall growth (13) whereas the former the provacuoles may function by directly packaging acid hy- provides for parallel vacuome growth. drolases and other materials including, for example, peroxidase in common plant cells, polyhydroxyphenolic compounds in I gratefully acknowledge the aid of Mr. Michel Berthoumieux, Dr. tanin cells, and polyterpenic granules in coenocytic latici- Dickie Grossman, and Mrs. Gilberte Monti in the preparation of the fers. final photographs and text. The author is indebted to Drs. Phyllis and The mechanisms controlling the release of acid hydrolases Alex Novikoff, Dr. Roger Buvat, and Dr. Daniel Branton for helpful into the cytoplasm sequestered by the fusion of the elongated discussions. .This work was supported by the Centre National de la provacuoles (cage bars) is unknown. I have speculated (17) that, Recherche Scientifique, Research Grant CNRS LA 179 (France). because each sequestrating cavity is built by the lateral union of cooperative provacuoles, its outer and inner delimiting 1. Matile, P. (1968) Planta 79,181-196. membranes initially must be identical and impermeable to the 2. Leigh, R. A. & Branton, D. (1976) Plant Physiol. 58,656-662. contained lysosomal enzymes. Later on, the inner membrane, 3. Buvat, R. (1971) in Origin and Continuity of Cell Organelles, deprived of the cytoplasmic interchanges required for main- eds. Reinert, J. & Ursprung, H. (Springer-Verlag, Berlin), pp. taining its integrity, may be altered and may thus become 127-157. permeable to the lysosomal hydrolases that subsequently digest 4. Matile, P. (1975) The Lytic Compartment of Plant Cells. Cell other the outer Biology Monographs (Springer-Verlag, New York), Vol. 1. the sequestered structures. On the hand, 5. Ledbetter, M. C. & Porter, K. R. (1970) Introduction to the Fine membrane in close contact with the cytoplasm remains unal- Structure of Plant Cells (Springer-Verlag, Berlin). tered and impermeable to acid hydrolases whose digestive ac- 6. Novikoff, A. B. (1973) in Lysosomes and Storage Diseases. eds. tivities are thus confined within the forming vacuole. This outer Hers, G. & van Hoof, F. (Academic Press, New York), pp. membrane prevents cellular autolysis and becomes the vacuole 1-41. membrane. The sequestration and subsequent digestion of ri- 7. Novikoff, A. B. (1976) Proc. Natl. Acad. Sci. USA 73, 2781- bosomes, rough ER, and possibly other organelles by these au- 2787. tophagic provacuole-derived systems may be required for the 8. Novikoff, P. M., Novikoff, A. B., Quintana, N. & Hauw, J. J. normal bulk turnover of cellular macromolecules when the (1971) J. Cell Biol. 50,859-886. remodelled in for further 9. Boutry, J. M. & Novikoff, A. B. (1975) Proc. Natl. Acad. Sci. USA meristematic cell is being preparation 72,508-512. growth and for the acquisition of new functions. The vacuoles 10. Bell, M. & Barrnett, R. J. (1965) J. Histochem. Cytochem. 13, formed at the end of the autophagic process are obviously 611-628. identified as plant "residual bodies" (16). They are able to fuse 11. Marty, F. (1973) C.R. Hebd. Seances Acad. Sci. 277, 2681- together and behave as osmotic systems. 2684. The fate of GERL-derived provacuoles in mature cells that 12. Ehrenreich, J. H., Bergeron, J. J. M., Siekevitz, P. & Palade, G. are already vacuolated appears to be different from that in the (1973) J. Cell Biol. 59,45-72. immature, unvacuolated cells. Studies to date indicate that, 13. Morre, D. J., Mollenhauer, H. H. & Bracker, C. E. (1971) in Or- instead of participating in a program of cellular autophagy, igin and Continuity of Cell Organelles, eds. Reinert, J. & provacuoles in already vacuolated, actively growing cells are Ursprung, H. (Springer-Verlag, Berlin), pp. 82-126. from GERL to the vac- 14. Whaley, W. G., Dauwalder, M. & Kephart, J. E. (1971) in Origin transferred directly preexisting large and Continuity of Cell Organelles, eds. Reinert, J. & Ursprung, uoles. By merging with the already existing vacuoles, these H. (Springer-Verlag, Berlin), pp. 1-45. GERL-derived provacuoles would account for tonoplast en- 15. Decker, R. S. (1974) J. Cell Biol. 61, 599-612. largement and vacuolar content accretion during cell 16. de Duve, C. & Wattiaux, R. (1966) Annu. Rev. Physiol. 28, growth. 435-492. Vacuolating plant cells may be a good material for the study 17. Marty, F. (1974) Doctorat es-Sciences, University Aix-Marseille of the unsettled question of membrane flow. It may be appro- (France), pp. 126-127. Downloaded by guest on October 1, 2021