Protoplasma (1986) I35:67-70

�9 by Springer-Verlag 1986

Fixation of a Vacuole-Associated Network of Channels in Protein-Storing Pea Cotyledon Cells

STUART CRAIG*

CSIRO, Division of Plant Industry, Canberra ACT

Received October 16, 1985 Accepted July I0, 1986

Summary

In cells of developing pea cotyledons fixed at ambient temperature, electron-dense cisternae often encircle vacuoles and occasionally are continuous with vacuolar protein deposits. In cotyledons chilled prior to fixation, the cisternae are present as an anastomosing network of channels that make multiple contacts with vacuolar protein. It is suggested that during fixation at ambient temperature, many of the contacts with vacuoles are lost and that much of the complexity of the network is iost, giving way to a system of largely unbranched cisternae. The network may be a mobile phase involved m reserve protein accumulation and concentration within the vacuole,

K(~l:word, s: Pea seed: Protein deposits: Fixation; ChiiIing.

I. Introduction

Developing seeds synthesize and store large amounts of storage or reserve proteins. Much is known of this process, particularly in legume seeds (for reviews, see CHRISPEF.LS 1984, 1985, HJ~6~NS 1984). By a range of techniques, it has been established that storage proteins are synthesized on endoplasmic reticulum-bound poly- somes, moved into the ER lumen, processed within the Golgi complex and stored within the vacuolar system. In protein secreting systems, the ER, Golgi and vacuolar compartments are generally considered as more or less discrete entities, and little specific in- formation is available on how the proteins pass from one compartment to the next. Ultrastructural studies have provided some information on this pathway.

* Correspondence and Reprints: CSIRO, Division of Plant Industry, P.(). Box 1600, Canberra ACT2601. Australia.

Depending on the tissue examined, ER has been seen to be either continuous with Golgi membranes or not connected (HAND and OLIVER 1981, JUNIPER etal. 1982). In the latter case, there are often small vesicle-like structures, the so-called transitional vesicles, between the cis or forming face of the Golgi, and an adjacent ER cisterna, and these are presumed to be mobile carriers between the two membrane systems. Similarly, vesicles, often with dense contents, may be present towards the trans or mature face of the Golgi and these are the presumed vehicle by which the proteins are moved to the storage vacuoles. Electron micrographs provide static images of what in most cases are dynamic systems. Unfortunately, trans- itional vesicles and Golgi vesicles do not possess "arrows" indicating in which direction, if any, they are moving. Our preconceived ideas generally determine this, just as they do for what constitutes "good" fine structural preservation (MERSEY and McCuLLv [978). The present report addresses the question of storage protein accumulation into vacuoles in developing pea cotyledon cells. Storage protein synthesis in pea seeds follows the general pattern outlined above, and using immunogold techniques, we showed globulin (vicilin) storage protein associated with ER, Golgi complexes, vacuoles and protein bodies (CRAI~ and GOODCH~LD 1984a, b, CRAIC~ and MILLER 1984). In peas, an additional step or component in the pathway is apparent. Electron-dense cisternae of variable morphology and containing storage protein were frequently observed, but only occasionally were

68 STUART CRAIO: Fixation of a Vacuole-Associated Network

they continuous with vacuolar protein deposits (CRMG and GOODCmLD 1984 a). Evidence is presented here that these electron-dense cisternae are part of an anastomos-

ing network continuous with the vacuoles, and that the

forms depicted earlier (CaAIG and GOODCHmD 1984 a) may be an artefact resulting from the action of fixative on mobile cytoplasm.

2. Materials and Methods

Pisum sativum L. PI/G 086, a line ofcv. Greenfeast was grown in the CSIRO phytotron under controlled conditions as described earlier (M1LLERD and SPENCER 1974), except for growth temperature (20 ~ and daylength (16 hours). Pods were harvested at 20-22 DAF (days after flowering), and both pods and isolated cotyledons were wrapped in aluminium foil and placed on ice for 2-4hours. Seeds were removed from the cold pods, and the cotyledons dissected into ice-cold 3% glutaraldehyde in 25 mM Na-phosphate, pH 7.1, containing 200raM sucrose. Fixation was for 1.5hours on ice, followed after buffer washing by postfixation in l-2% OsO 4 in the same buffer for a further 1.5 hours. Samples were brought to 22 ~ in buffer and dehydrated through ethanol and embedded in L R White resin (CRAIG and MILLER 1984). Sections were cut, stained with uranium and lead salts, and examined at 60 kV in a JEOL JEM 100 S electron microscope. On-grid immunogold labeling was carried out as described previously (CRAIG and MILLER 1984).

3. Results and Discussion

Parenchyma cells of pea cotyledons are up to 100 jam in diameter (CRAIG et al. 1979), and thus may be expected to possess some mechanism for mixing the cytoplasm.

In protoplasts of these cells, cytoplasmic streaming takes place (GooDct~ILD and CP, Am 1982). Assuming that streaming is not an artefact of protoplast pro- duction and that it occurs in intact cotyledons, then fixation of these large, vacuolate cells is likely to be difficult. I f forces are exerted to move organelles through the cytoplasm, then it seems likely that in the presence of cross-linking agents like glutaraldehyde, additional stresses will be established and that these

could break continuities between, for example, ER and vacuoles or between ER and Golgi membranes. To address this problem in pea cotyledon cells, tissue was chilled to stop cytoplasmic streaming before fixa- tion. The most obvious change in chilled tissue was the presence of an anastomosing network of electron-dense material that made frequent contacts with vacuolar protein deposits (Fig. 1). This network was most devel- oped in cotyledons from intact pods chilled for 4 hours before fixation. Although reconstructions from serial sections were not produced, from examination of many separate areas

such as those in Figs. 1 and 3, it appears that the anastomosing network consists of a system of cisternae that extend from the vacuole into the cytoplasm. The cisternae repeatedly branch and fuse, and are frequent- ly continuous with small vacuoles or vesicles (Fig. 3, *).

This is in contrast to the situation in tissue fixed at ambient temperature where the cisternae rarely branch or fuse, and where they make infrequent contact with vacuolar protein. Small dilations or vesicles are, how- ever, common (Fig. 2). In chilled tissue, the anastomosing network was present to the exclusion of cisternae in the forms seen in Fig. 2

and in Figs. 7 11 of CaA~G and GOODCmLD (1984a). Furthermore, the change in appearance from simple cisternae to anastomosing network is reversible, as in chilled tissue warmed to ambient (22 ~ for as little as

0.5 hours before fixation, the network was absent (not illustrated), and cisternae similar to those in Fig. 2 were

present. This is consistent with, but does not prove, that the changes are not an artefact of the chilling treatment. This contention is supported by the observation (LANGENBERG 1979) that crystalline viral inclusions stabilized in leaf tissue by chilling, were not an artefact of the chilling protocol.

Most of the network contains electron-dense material to which antibodies specific for the storage protein vicilin bind (Fig. 3), confirming the role of this system of channels in storage protein accumulation. The vicilin-containing material is clearly continuous from the cisternae into the vacuolar deposits. In tissue fixed at ambient temperature, and in vacuoles in chilled tissue that have no associated anastomosing network, the vacuolar deposits invariably project into the vacuole lumen from the tonoplast (Fig. 1, upper vacuole). Where the anas tomos ing network is con- tinuous with the vacuole, protein deposits are irregular in shape and often extend out from where a "projected" smooth tonoplast would lie (Fig. 1, lower vacuole and Fig. 3, PD). Thus it appears that the vacuolar protein

deposits are pliable and not immobile, gelled masses. This was also evident in our earlier study of the effects of different fixation protocols on the appearance of vacuolar protein (GoODCHILD and CRAIG 1982). Further interpretation of the interaction between the network and vacuole is somewhat speculative as one cannot identify the delimiting membrane where it has adhering electon-dense protein. Tonoplast can be seen where there is no adhering protein material (Figs. 1 and 3); similarly, bounding membrane can be seen on the cisternal network where there is a discontinuity in the contents and where the membrane orientation within

STUARI CRAIG: Fixation of a Vacuole-Associated Network 69

Fig. 1. Electron micrograph of anastomosing network ( �9 ) associated with vacuolar (V) protein deposits (PD) in tissue chilled for 4 hours prior to fixation. Open arrows indicate where limiting membrane can be identified on the cisternal network. 22 DAF

Fig. 2. Electron micrograph of electron-dense cisterna ( �9 ) present in tissue fixed at ambient temperature. Section was labeled for vicilin with affinity purified antibodies, followed by protein A-gold. FD protein deposit, V vacuole, 22 DAF

l:ig. 3. Section from the specimen in Fig. i following immunogold labeling to detect vicilin, showing the involvement of the anastomosing network ( � 9 in protein accumulation. The network is frequently in contact with small vacuoles or vesicles (*). 22 DAF

70 STUART CRAIG: Fixation of a Vacuole-Associated Network

the section is favorable (Fig. 1, open arrows). Thus, membrane is presumed to be continuous from the tonoplast to the cisternal network. The appearance of the anastomosing network de- scribed here in cotyledon cells bears some similarities to the mobile, pleomorphic canalicular system described in plant hair cells (O'BRIEN etal. 1973, MERSEY and McCULLV 1978) that was closely associated with cytoplasmic streaming, but which was lost during fixation and gave rise to an artefactual set of vesicles. Unfortunately cotyledon cells are not well suited to observation in vivo; their large size and high starch content together have prevented observation of the anastomosing network in vivo in hand sections. It is proposed that a) in developing pea cotyledons there is a network of cisternae forming a mobile channel system extending from the vacuolar protein deposits into the cytoplasm, and b) that during fixation most of the connections with vacuoles are severed and the cisternae fragment. The function of these cisternae is unclear, although one can speculate a role in storage protein accumulation. The surface area of the network must be considerable and could therefore increase the potential membrane area with which Golgi vesicles could fuse, discharging their contents. Associations between either the cister- nae and Golgi complexes, or between the ER and the cisternae were not observed. The mechanism and site at which storage protein becomes electron-dense, and presumably concentrated, has not been addressed in detail, although the Golgi complex could be proposed as this is where electron-dense storage protein can first be detected in the ER-Golgi-vacuole pathway. If con- centration also takes place within the vacuole, then removal of water must occur. The necessarily high surface to volume ratio of the cisternal network may facilitate removal of water and thus concentration of the protein. The dramatic changes described here in a vacuole- associated network of cisternae is another example of the inability of chemical fixation to stabilize certain dynamic intracellular structures. Freezing of unfixed cotyledon tissue has not been possible to date, but recent progress in the use of high pressure freezing (CRA~C and STAEI~ELIN 1986), may enable further clarification of these structures.

Acknowledgements

CELIA MILLER provided excellent technical assistance.

References

CHRISPEELS MJ (1984) Biosynthesis, processing and transport of storage proteins and lectins in cotyledons of developing legume seeds. Philos Trans R Soc Lond [Biol] B 304:309-322

- - (1985) The role of the Golgi apparatus in the transport and post- translational modification of vacuolar (protein body) proteins. In: MIELIN B (ed) Oxford surveys of plant molecular and cell biology, vol 2. Oxford University Press, Oxford, pp 43-68

CRAIG S, GOODCHILD D J, HARDHAM AR (1979) Structural aspects of protein accumulation in developing pea eotyledons. 1. Quali- tative and quantitative changes in parenchyma cell vacuoles. Aust J Plant Physiol 6:81-98

- - - - (1984a) Periodate-acid treatment of sections permits on-grid localisation of pea seed vicilin in ER and Golgi. Protoplasma 122: 35-44

- - - - (1984b) Golgi-mediated vicilin accumulation in pea cotyledon cells is redirected by monensin and nigericin. Proto- plasma 122:91-97

--MILLER C (1984) L R White resin and improved on-grid immunogold detection of vicilin, a pea seed storage protein. Cell Biol Int Rep 8:879-886

- - STAEHELIN LA (1986) Application of high pressure freezing and freeze fracture to study dynamic events in plant cells. In: BAILEY WG (ed) Proceedings. E.M. Society of America, vol 44. San Francisco Press, San Francisco 99:234-235

GOODCHILD DJ, CRAIG S (1982) Structural aspects of protein accumulation in developing pea cotyledons. IV. Effects of preparative procedures on structural integrity. Aust J Plant Physiol 9:689-704

HAND AR, OLIVER C (198 l) The Golgi apparatus: protein transport and packaging in secretory cells. Methods Cell Biol 23:137-153

HIGGINS TJV (1984) Synthesis and regulation of major proteins in seeds. Ann Rev Plant Physiol 35:191-221

JUNIPER BE, HAWES CR, HORNE JC (1982) The relationships between the dictyosomes and the forms of endoplasmic reticulum in plant cells with different export programs. Bot Gaz 143: 135- 145

LANGENBERG WG (1979) Chilling of tissue before glutaraldehyde fixation preserves fragile inclusions of several plant viruses. J Ultrastruct Res 66: 120-13l

MEP~SEY B, McCuLLY ME (1978) Monitoring the course of fixation of plant cells. J Microsc 114:49-76

MILLERD A, SPENCER D (1974) Changes in the RNA synthesising activity and template activity in nuclei from cotyledons of developing seeds. Aust J Plant Physiol 1:331-341

O'BRIEN TP, Kuo J, MCCULLY ME, ZEE SY (1973) Coagulant and non-coagulant fixation of plant cells. Aust J Biol Sci 26: 1231- 1250