Planta (Berl.) 124, 199--205 (1975) 9 by Springer-Verlag 1975
An Effect of Ethylene on the Endoplasmic Reticulum of Expanding Cells of Etiolated Shoots of Pisum sativum L. J. A. S a r g e n t a n d D a p h n e J. Osborne Agricultural Research Council Unit of Developmental Botany, 181A Huntingdon Road, Cambridge CB30DY, U. K. Received 25 February 1975; accepted 24 March 1975
Summary. Within 6 h of supplying ethylene to intact etiolated seedlings of Pisum sativum L. increasingly long profiles of rough and smooth endoplasmie retieulum (ER) appear in sections of epidermal and cortical cells from the hook region. By 24 h some profiles exceed 10 ~m in length, more than 10 times the length of the longest commonly observed in control tissue. These profiles are not artifacts of the different preparations, for similar extended profiles occur in sections of pellets of rough EI~ which have been prepared from subapical internodes of treated seedlings by extraction and separation of the membranes on sucrose gradients. I t is proposed that the changes in rough ER result from a 'stabilisation' of the membranes brought about by a reduced rate of phospholipid turnover in the presence of ethylene. Introduction Shoots of d a r k - g r o w n pea seedlings h a v e been used e x t e n s i v e l y in studies of t h e m e c h a n i s m of a c t i o n of hormones in controlling t h e g r o w t h a n d d e v e l o p m e n t of p l a n t cells a n d tissues. W h e n seedlings are exposed to e t h y l e n e l o n g i t u d i n a l e x p a n s i o n of s u b a p i c a l cells is severely restricted, a response which has been c o r r e l a t e d with changes in t h e c o n t e n t of h y d r o x y p r o l i n e - r i c h proteins in t h e walls (Ridge a n d Osborne, 1971) a n d t h e deposition of new cellulose wall microfibrils o r i e n t a t e d l o n g i t u d i n a l l y r a t h e r t h a n t r a n s v e r s e l y (Sargent et al., 1974). I n a d d i t i o n ethylene induces a t h i c k e n i n g of t h e cell walls in this tissue, increases reaching 2-3 fold b y 4 d a y s (Sargent et al., 1973). E x a m i n a t i o n of m i e r o g r a p h s which i l l u s t r a t e d t h a t p h e n o m e n o n showed clearly t h a t e t h y l e n e t r e a t m e n t resulted also in changes in t h e u l t r a s t r u e t u r e of t h e cytoplasm. I n p a r t i c u l a r t h e o r g a n i z a t i o n of t h e endoplasmie r e t i c u l u m (ER) was clearly modified in subapical tissue (Osborne, R i d g e a n d Sargent, 1972, Fig. 4; S a r g e n t etal., 1973, Fig. 2). F u r t h e r studies which r e v e a l e d t h e e x t e n t to which t h e o r g a n i s a t i o n of t h e m e m b r a n e s y s t e m is altered b y e t h y l e n e are r e p o r t e d here. Materials and Methods Seedlings of Pisum sativum L. cv. Alaska were grown, as previously described, in boxes of sand in darkness at 24 ~C. Six days after sowing, growth of the second internode was complete but the third had only just begun to elongate. Intact seedlings were treated with ethylene by placing them, in their boxes, in glass or pcrspex tanks containing the gas at 100 ~zl/1. Controls were placed in similar tanks to which no ethylene was added and in which ambient ethylene was kept at a low level by the inclusion of a beaker containing a solution of 0.25 M ttg C104 in 2.0 M t t CIO4. All tanks contained KOH pellets to absorb C02.
J.A. Sargent and D. J. Osborne
Examination o / E R in Intact Cells. Best fixation can be obtained in cells in which vacuoles are relatively small. For this reason, much of the electron microscopy involved examination of tissues from the hook region of the shoot apex. One, 3, 6, 12 and 24 h after first treating the seedlings with ethylene, individual plants were removed from the tanks and small pieces of hook tissue excised in a fixative solution consisting of 6% glutarMdehyde and 3% sucrose in 0.1 iV[sodium eacodylate buffer at pit 7.1. The tissue was subsequently post-fixed in 2 % osmium tetroxide, dehydrated, and embedded in Spurr's resin. Thin sections were cut and stained 7 min in a 50% ethanol solution saturated with uranyl acetate followed by 2 rain in Reynold's lead citrate solution. Examination o/Isolated ER. Membrane fractions were prepared from excised internode segments whose limits had been defined by charcoal-lanolin marks placed 10 mm apart on the third internode at the start of treatment, the upper of the two marks being just below the hook (see Fig. 1. Sargent et al., 1974). After 24 h, the marks on control shoots were approximately 30 mm apart and 33 of these segments weighed 2.198 g. The marked regions of ethylenetreated peas were approximately 11 mm long and 33 of these segments together weighed 1.001 g. Each batch of segments were frozen and ground in liquid nitrogen in a cooled mortar. All subsequent steps in the isolation were conducted at 0-2~ The powdered tissue was transferred to a glass homogeniser and gently fragmented in 2 ml of a buffer (ptI 7.5, 20~ consisting of 5• Tris, 2.5• KC1, 5• MgS04, 10-aM dithiothreitol, 0.375 M Sucrose. After making up to 8 ml with buffer, the homogenate was centrifuged at 12000 rpm. in an M.S.E. 18 (Measuring and Scientific Equipment, London) for 15 rain. Aliquots of the supernatent solution were then layered on a discontinuous gradient of sucrose solutions consisting of 0.9 M (1 ml), 1.3 M (2 ml) and 2.0 M (2 ml), and fractionated by centrifugation for 90 min in a Spineo 65 fixed angle rotor at 50000 rpm. l%ibosomespelleted at the bottom of the tube, leaving predominantly smooth and rough membrane fractions at the bottom of the 0.9 M and 1.3 M layers, respectively. These were collected, suspended in buffered glutarMdehyde, and pelleted at 18000 rpm. in an M.S.E. 18 for 30 rain. The compact pellets were prepared for electron microscopy in a manner similar to that described for the hook tissue.
Results E x a m i n a t i o n of sections cut either l o n g i t u d i n a l l y or transversely t h r o u g h b o t h epidermal a n d cortical cells revealed a m a r k e d change in the appearance of profiles of E R following ethylene t r e a t m e n t . Electron micrographs of sections of cortical tissue from control a n d e t h y l e n e - t r e a t e d (24 h) seedlings are shown in Fig. 1. I n the controls, few Et~ profiles exceed 1 Exm in l e n g t h ; most are considerably shorter. I n ethylene, however, the f o r m a t i o n of E R with m a n y profiles up to 10 l~m or more in length can be seen. A l t h o u g h these longer profiles are n o t associated with ribosomes along their whole length t h e y are, to a large extent, ':rough" E R . Also, " s m o o t h " cisternae or vesicles seem more c o m m o n i n the cytoplasm close to the cell wall, a n d crenellations of the p l a s m a l e m m a suggest t h a t these vesicles m a y fuse with the plasma m e m b r a n e . The reorganisation of E R which results from ethylene t r e a t m e n t is n o t confined to cells of the hook; it occurs in subapieal tissue a n d can be d e m o n s t r a t e d in m e m b r a n e preparations isolated from t h i r d internode tissue. E l e c t r o n micrographs of t h i n sections of the m e m b r a n e pellets prepared from fractions t h a t sedimented a t the b o t t o m of the 1.3 M sucrose layer are shown i n Fig. 2. The control pellet (Fig. 2a) is compact a n d composed of n u m e r o u s short profiles of m e m b r a n e most of which s u p p o r t ribosomes. The bulk of the pellet from ethylene-treated plants (Fig. 2b) is composed of similar material b u t embedded in the pellet are sheets of double m e m b r a n e bearing a b u n d a n t ribosomes.
Effect of Ethylene on the ER
Fig. 1 a and b. Electron microgmphs of sections through cortical cells from the hook region of 6 day old etiolated pea plants. (a) Control, (b) Treated with ethylene (100 tzl/1) for 24 h. • 12300
Such long, continuous profiles of rough E R were never f o u n d in a n y of t h e control pellets. The effect of ethylene on E R as i l l u s t r a t e d in Figs. 1 a n d 2 was r e a d i l y o b s e r v e d following a t r e a t m e n t period of 24 h. E l e c t r o n microscopic e x a m i n a t i o n of t h i n sections of h o o k tissue fixed after 12 h t r e a t m e n t also r e v e a l e d a d i s t i n c t a l t e r a t i o n in t h e a p p e a r a n c e of E R profiles, b u t it was impossible to decide b y visual o b s e r v a t i o n alone w h e t h e r the effect was a p p a r e n t a t earlier times. I n order to d e t e r m i n e how soon after t r e a t m e n t a reorganisation of t h e E R could be d e t e c t e d t h e response was q u a n t i f i e d in t h e following way. A large n u m b e r of electron m i e r o g r a p h s were p r e p a r e d from areas selected a t r a n d o m over sections t h r o u g h e p i d e r m a l cells a n d t h r o u g h cortical cells from a region a p p r o x i m a t e l y four cells r e m o v e d from t h e epidermis. This was done for b o t h eontrol a n d t r e a t e d tissue fixed a t 0, 1, 3, 6, 12 a n d 24 h after t r a n s f e r of t h e seedlings to t h e t a n k s . The a r e a of c y t o p l a s m (excluding nuclei a n d m a j o r vacuoles) on each m i e r o g r a p h was t h e n d e t e r m i n e d a n d within each of t h e s e areas t h e length of e v e r y reeognisable profile of EI~ was measured. Since it was impossible to distinguish between c y t o p l a s m i c vesicles a n d EI~ profiles s h o r t e r
J.A. S~rgent and D. J. Osborne
Fig. 2a and b. Electron micrographs of sections through pellets of endoplasmie reticulum prepared from the subapical region of 6 day old etiolated pea plants. (a) Control, (b) Treated with ethylene (100 ~1/1) for 24 h. • 19000
than 0.25 ~m, only lengths of profiles exceeding this value were recorded. The data were bulked for each set of micrographs and the numbers of profiles grouped into classes according to their length. Histograms (Fig. 3) were constructed expressing E R profile length against the product of that length and the number of profiles of that length per unit area of cytoplasm section. Differences in the histogram patterns are clearly evident in both epidermal and cortical cells after 12 h (not presented) and 24 h treatment but an effect is also apparent after only 6 h. The patterns for the 1 and 3 h data did not differ from those for the controls and a pattern intermediate between 6 and 24 h patterns was revealed for the 12 h data.
Effect of Ethylene on the EI~
6 hour s
Z 0'8 X
24 h o u r s
0-4 r~ LU 0
2 E.R. l e n g t h
Fig. 3. The effect of ethylene (100 BI/t) on the length of profiles of endoplasmic reticulum observed in sections of epidermal and cortical cells from the hook region of 6 day old etiolated pea plants treated for 6 or 24 h. Profiles shorter than 0.25 y.m were not recorded
The stimulation of rough E R formation following hormone treatments has been reported in both plant and animal tissues (Jones, 1969a, b; Tara, 1968) and m a y lead to the enhancement of protein synthesis and secretion which frequently results from such treatments. That the metabolism or activity of membrane systems m a y be involved in the ethylene response is suggested by several results. For example, a changed synthesis of total phospholipid occurs when ethylene is supplied to intact pea plants. I t was shown by Irvine and Osborne (1973), that incorporation of [14C]glycerol into phospholipid during 45 rain is reduced by 30-50% by a previous exposure of the plants to 10 F1/1 ethylene for only 2.5 h. Similar reductions were found 2.5 hours after the enhanced ethylene production t h a t occurs in shoots when they are handled or "wounded" in any way. Over a period of 18 hours in 10 B1/1 ethylene there is a small reduction in total phospholipid (as measured by total phosphate in the phospholipid fraction) which does not exceed 10% (Irvine and Osborne, 1975). Therefore, either the synthesis represents a very small part of the total phospholipid content of the cell, or breakdown occurs at a rate which is somewhat in excess of t h a t of synthesis. This indicates that ethylene initiates a reorganisation, rather than a proliferation of the endomembrane system (Palade, Siekewitz and Caro, 1962; Frey-Wyssling, Ldpez-Ss and Miihlethaler, 1964; Morr6, Mollenhauer and Bracker, 1971). The enrichment of the cell wall with protein of an enhanced hydroxyproline content
J.A. Sargent and D. J. Osborne
is one of the effects of ethylene (Ridge and Osborne, 1970, 1971), and this could be correlated with an alteration in the morphology of E R on which wall proteins are synthesised. If this response involves a stimulation of the transport t h r o u g h the Golgi apparatus of protein synthesised on and secreted into the E R , it might be expected t h a t a greater vesiculation of the E R would occur, the vesicles transporting the protein to the dictyosome cisternae. Clearly, however, the overall effect of ethylene is the reverse: instead, the profiles appear longer t h a n those found in control tissue. The sheets of E R induced by ethylene m a y therefore be perforated b y fewer holes t h a n normal, and/or the holes are smaller. Valdovinos et al. (1971, 1972) have described similar ultrastructural changes in abscission zone cells in response to ethylene and Shore and Maclaehlan (1974)listed a shift of cellulose synthetase activity from the Golgi to the smooth E R after auxin treatment. I n view of the biphasic responses of pea stems to auxin, this response could have been p a r t l y a response to auxin-induced ethylene production (Sargent, Ataek and Osborne, 1974). A change in the p a t t e r n of m e m b r a n e turnover in ethylene, with a differential conversion of one class of m e m b r a n e to another m a y be the prime cause of the altered morphology of the E R at 6 hours. If a general slowing of the rate of turnover of membranes leads to a "stabilisation" of the E R a consequent decrease in fragmentation could result in a change in the rate at which membranes are transferred and transformed along the endomembrane pathways. Disturbances in m e m b r a n e metabolism and conformation could be initial steps in modifying the p a t t e r n of cell growth and form t h a t are the result of hormone treatments.
Reterences Frey-Wyssling, A., LSpez-S~ez, J. F., Miihlethaler, K. : Formation and development of the cell plate. J. Ultrastruct. Res. 10, 422432 (1964) Irvine, 1%.F., Osborne, D. J. : The effect of ethylene on [1-1aC] glycerol incorporation into phospholipids of etiolated pea stems. Biochem. J. 186, 1133-1135 (1973) Irvine, R. F., Osborne, D. J. : The effect of ethylene on the synthesis of endoplasmic reticulum in etiolated pea stems. Trans. Biochem. Soe., in Press (1975) Jones, R. L. : Gibberellic acid and the fine structure of barley aleurone cells. I. Changes during the lag-phase of a-amylase synthesis. Planta (Berl.) 87, 119-133 (1969a) Jones, R. L. : Gibberellic acid and the fine structure of barley aleurone cells. II. Changes during synthesis and secretion of u-amylase. Planta (Berl.) 88, 73-86 (1969b) MorrO, D. J., ~ollenhauer, H. H., Bracker, C. E. : Origin and continuity of Golgi apparatus. In: Origin and continuity of cell organelles. Reinert, J., Ursprung, It., eds. Berlin-Heidelberg-New York: Springer t971 Osborne, D. J., l~idge, I., Sargent, J.A.: Ethylene and the growth of plant cells: role of peroxidase and hydroxyproline-rich proteins. In: :Plant growth substances 1970, p. 534-542. Carr, D. J., ed. Berlin-Heidelberg-New York: S13ringer 1972 Palade, G. E., Siekewitz, :P., Caro, C. G. : The exocrine pancreas. CIBA Foundation Symp. 2349 (1962) l~idge, I., Osborne, D. J.: Hydroxyproline and peroxidase in cell walls of Piaum aativum: regulation by ethylene. J. exp. Bot. 21, 843-856 (1970) Ridge, I., Osborne, D. J. : Role of peroxidase when hydroxyproline-rich protein in plant cell walls is increased by ethylene, bTature (Lond.) Hew Biol. 229, 205-208 (1971)
Effect of Ethylene on the ER
Sargent, J. A., Atack, A. V., Osborne, D. J. : Orientation of cell growth in the etiolated pea stem. Effect of ethylene and auxin on cell wall deposition. Planta (Bed.) 109, 185-192 (1973) Sargent, J. A., Atack, A. V., Osborne, D. J. : Auxin and ethylene control of growth in epidermal cells of Pisum sativum: A biphasic response to auxin. Planta (Bed.) 115, 213-225 (1974) Shore, G., Maclaehlan, C. A. : Changes in subeellular location of cellulase synthetases during growth. Plant Physiol. 54, 89 (1974) Tara, J. R. : Hormonal regnJlation of growth and protein synthesis. Iqature (Lond.) 219, 331-337 (1968) Valdovinos, J. G., Jensen, T. E., Sicko, L. M. : Ethylene induced rough endoplasmic reticula in abscission cells. Plant Physiol. 47, 162-163 (1971) Valdovinos, J. G., Jensen, T. E., Sicko, L. M. : Fine structure of abscission Zones. IV. Effect of ethylene on the ultrastructure of abscission cells of tobacco flower pedicels. Planta (Bed.) 102, 324-333 (1972)
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