Some Ultrastructural and Enzymatic Effects of Water Stress in Cotton (Gossypium Hirsutum L.) Leaves (Acid Phosphatase/Acid Lipase/Alkaline Lipase)

Proc. Nat. Acad. Sci. USA Vol. 71, No. 8, pp. 3243-3247, August 1974

Some Ultrastructural and Enzymatic Effects of Water Stress in Cotton (Gossypium hirsutum L.) Leaves (acid phosphatase/acid lipase/alkaline lipase)

JORGE VIEIRA DA SILVA*, AUBREY W. NAYLOR, AND PAUL J. KRAMER Department of Botany, Duke University, Durham, North Carolina 27706 Contributed by Paul J. Kramer, May 30, 1974

ABSTRACT Water stress induced by floating discs cut boxylation of glycine occurs after lipase treatment of mito- from cotton leaves (Gossypium hirsutum L. cultivar chondria Stoneville) on a polyethylene glycol solution (water poten- (23). tial, -10 bars) was associated with marked alteration of Results, thus far obtained by indirect means, support the ultrastructural organization of both chloroplasts and hypothesis that water stress in drought sensitive species leads mitochondria. Ultrastructural organization of chloro- to hydrolytic activity that degrades not only storage products plasts was sometimes almost completely destroyed; per- but the structural framework of organelles such as ribosomes, oxisomes seemed not to be affected; and chloroplast ribosomes disappeared. Also accompanying water stress chloroplasts, and mitochondria. Ultrastructural and micro- was a sharp increase in activity of acid phosphatase chemical evidence is reported here that such deterioration [orthoplhosphoric-monoester phosplhohydrolase (acid opti- occurs in cotton (Gossypium hirsutum L. cv. Stoneville) during mum), EC 3.1.3.2], and acid and alkaline lipase [glycerol water stress. ester hydrolase EC 3.1.1.3] within chloroplasts. Only acid lipase activity was detected inside mitochondria of stressed MATERIALS AND METHODS discs. These alterations in cell organization and enzy- mology may account for at least part of the previously Cotton plants (Gossypium hirsutum L. cultivar Stoneville) reported effects of water stress on the CO2 compensation were grown in the greenhouse under natural light during the point, photochemical reactions, and photorespiration. summer of 1972 and in the Duke University unit of the Southeastern Plant Environmental Laboratories. The third Water stress in some drought intolerant seed plants is accom- leaf from the top of the main axis was sampled at 9 p.m. when panied by increased activity of several hydrolytic enzymes the plants were 2 months old. While avoiding main veins, [see tabulation by Todd (1) and (2-7)], by change in their discs of 1.2 cm in diameter were cut from the leaves. The discs intracellular compartmentation (3), and even by loss of part were floated, abaxial surface up, for 20 hr on a polyethylene of the protein making machinery (8, 9). One of the hydrolytic glycol 1540 solution adjusted to -10 bars at 200 under dim enzymes affected, acid phosphatase, appears to be concen- light (800 lux). The relative advantages of using polyethylene trated in the chloroplast. Water stress is accompanied not glycol over mannitol have been discussed by Mfichel (24) and only by increased acid phosphatase activity (5, 6) but also by Lawlor (25). Controls were cut from the same tissue and release of this enzyme from the organelle. Chloroplasts from floated in a similar way on distilled water. drought intolerant cotton plants have been shown to release After the stress treatment, 0.5 X 8-mm strips were sliced not only ribonucleic acid into the cytoplasm but ribosomes as from the discs (avoiding the border) and fixed in 4.2% well (10). In contrast, drought resistant cotton species have a glutaraldehyde in 0.1 M cacodylate buffer pH 7.2 for 2 hr relatively stable compartmentation of their enzymes in- at 40 in the absence of polyethylene glycol 1540. After washing cluding acid phosphatase (6); this is perhaps clue to membrane in buffer, the strips to be tested for enzymatic activity were stability in these taxa. incubaeed 8 hr at room temperature in the following solutions: The effects of water stress on photosynthesis (3, 11-13) (a) For acid phosphatase [EC 3.1.3.2] activity the medium appear to be duplicatable by the action of lipase (14-16), of Gomori (26) was used in 0.1 M acetate buffer at pH 5.3. A addition of certain unsaturated fatty acids (17-20), and by control with lead nitrate (0.003 AM) was done. aging as shown in isolated plastids (14, 17, 18, 21). Em- (b) For acid aud alkaline lipase (esterase) [EC 3.1.1.3] ac- pirically, the addition of inorganic phosphate inhibits CO2 up- tivity, the incubating medium (27, 28) contained 0.2% Tween take and changes the CO2 compensation point (22). In vivo, a 60, a substance consisting of a complex mixture of polyoxy- rise in phosphatase activity could lead to an accumulation of ethylene ethers of mixed partial stearic esters of sorbitol an- inorganic phosphate. Photorespiration is decreased by desicca- hydrides, which is known not to have any swelling effects on tion. This particular stress effect could be due to a reduction chloroplasts. Acid lipase activity was detected by using a in the production of glycolate. Enhanced lipase activity could modified (21) Gomori lead precipitation technique in which also lead to reduced glycine decarboxylation. This specula- an 0.1 M acetate buffer at pH 5.3 without sodium taurocho- tion is supported by the finding that inhibition of the decar- late was used. Alkaline lipase activity was detected by incubating in a 0.1 M cacodylate buffer at pH 7.2 in the presence * Present address: Universite de Paris VII, Laboratoire d'Pcol- of 0.25% sodium taurocholate. ogie G6n6rale et Appliqulle, 2, Place Jussieti-Tour 54-64, 3 eme (c) A stressed control was run using the same conditions but tage, Paris 75005 France. without any cytochemical tests. 3243 3244 Botany: Da Silva et al. Proc. Nat. Acad. Sci. USA 71 (1974)

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After fixation, the strips were washed in 0.1 M cacodylate the lead deposits seen in Figs. 1, 3, and 5 did indeed result buffer at pH 7.2, post-fixed in unbuffered aqueous 2% from reactions traceable to enzyme activity. osmium tetroxide (w/v) for 2 hr, rinsed repeatedly in water, A great deal of acid lipase activity can be observed (Fig. 3) and dehydrated with ethanol. The dehydrated samples were between the chloroplast lamellae in stressed cells when com- embedded in Spurr's (29) epoxy resin, cut with a Dii Pont pared to the control. Mitochondria in the stressed cells show diamond knife, and post-stained with aqueous uranyl acetate. an even more intense acid lipase activity than does the chloro- Lead was avoided when enzymatic activity was being tested. plast with lipid droplets outside. Peroxisomes, in contrast, Examination of the sections was done with the aid of a seem to have very little lipase activity. Alkaline lipase seems Siemens Elmiscop 101. to be limited to the chloroplast (Fig. 5) with almost no activity observed within the mitochondria. RESULTS AND DISCUSSION The localization of acid and alkaline lipase inside the chloroplast and acid lipase in mitochondria together with their ap- Tissues from discs floated on distilled water (control) for 20 parent activation by water stress, points to the potential role hr showed no evidence of ultrastructural changes resulting these enzymes may play in membrane degradation. Accom- from wounding or incubation. Water stress, however, resulted panying such actions would be obvious microstructural in various degrees of degeneration of organelles and mem- changes such as have been observed here and in other water branes within the same period. Chloroplast ribosomes were stressed plants (31, 32, 2). apparently among the first structures to disappear. Chloro- The peroxisomes did not appear to harbour significant plast thylakoid structure was profoundly affected. In many amounts of any one of the hydrolytic enzymes studied and instances the characteristic fine structural organization was their form seemed unaffected by the stress treatment. lost. This is made evident by a comparison of stressed tissues (Figs. 1, 3, and 5) and unstressed controls (Figs. 2, 4, and 6). CONCLUSION Cristae in mitochondria of water stressed samples were gen- Water stress treatment of Stoneville cotton leaf discs was ac- erally less distinct than were those of the controls (Figs. 5 and companied by loss of internal structural organization of both 6). Peroxisomes were less affected than either chloroplasts or chloroplasts and mitochondria. Ribosomes disappeared from mitochondria (Fig. 5). Single membrane bodies found in the the chloroplasts and thylakoid membranes lost their charac- chloroplasts appeared not to be much affected by the stress teristic staining properties. This was paralleled by a detectable treatment (Fig. 3). increase in enzymatic activity as measured by histochemical The decrease in matrix density and prominence of mito- techniques. Acid phosphatase and both acid and alkaline chondrial cristae with dehydration have already been reported lipase activity increased in the stressed chloroplasts. M\ean- in maize roots (30). Similar effects on the ultrastructure of while, acid lipase activity, alone among the three enzymes chloroplasts and mitochondria have been observed in Atriplex studied, was found in mitochondria from stressed tissue. (31) and cotton (32). Accompanying these structural changes Several inferences can be made with respect to the effect has been extensive swelling of the organelles. Ultimately, of water stress on photosynthesis and respiration in Stoneville disintegration might be expected. Indeed, a reduction in cotton and perhaps other drought sensitive species. Release of number o F chloroplasts in water stressed wheat has been noted inorganic phosphate, detected here by the Gomori (26) reac- (33). tion has been confirmed by direct analysis of stressed tissues The ultrastructural changes seem to be accompanied by (Nguyen Duc, unpublished results). Others have observed modifications of enzymatic activity within the chloroplast and the inhibitory effect of reduction in the water content of mitochondria. In Fig. 1 the dark spots inside the chloroplast chloroplasts on photosynthetic phosphorylation (3, 34). Inor- localize the activity of acid phosphatase, while no such spots ganic phosphate loss corresponds to the reversible inhibition could be found either in the water control (Fig. 2), in the lead of photophosphorylation and CO2 reductive activity observed stained control or in the control without evtochemical tests at the beginning of a water stress treatment (3, 35). (data not shown). This confirms the earlier observation (7) Inhibition of C02 absorption by inorganic phosphate in made on density gradient separated organelles, that showed isolated chloroplasts has previously been observed (36). Un- phosl)hatase activity within chloroplasts of stressed plants. der natural soil drought conditions, water stress in several Apparently phosphatase activity is latent in the nonstressed species of cotton is accompanied by differences in dry matter plant. With the onset of water stress, most of the acid phos- accumulation and acid phosphatase activity (37). The rela- phatase leaks out of chloroplasts (7). Therefore, the activity tionship appears to be inverse. The more drought resistant the observed in Fig. 1 is probably only a fraction of the total. It is species, the lower the specific activity of the enzyme (6, 37). difficult to judge the cytoplasmic activity, if any, from Fig. 1 Enhanced activity of lipase in water stressed leaf tissue can since the boundary of the chloroplast is not clear. account for a significant part of the long lasting effect of water The droughted control without cytochemical tests showed stress on the chloroplast. This is true because 22% of the clearly that no electron dense sl)ots resulted from the poly- plastid's makeup is lipid (38) and membranes are known to ethylene glycol treatment. It may, therefore, be Inferred that be rich in phospholipids (39). Loss of membrane integrity

FIGS. 1-4 (on preceding page). Ch, chloroplast; i3f, mitochondrion; P, peroxisome; R, ribosomes; Srnb, single membrane bodies (inside the chloroplasts); S, starch; Ph, phosphatase induced lead deposits. (1) Acid phosphatase (Gomori) activity inside the chloroplast of a stressed leaf disc. A decrease in structure and loss of definition can be observed. (2) Chloroplast and mitochondrion from disc floated on distilled water and incubated in Gomori's medium. Little or no phosphatase activity is evident. Good structural definition can be seen. (.3) Acid lipase in a stressed leaf disc appears very strong in the mitochondria and between the lamellae in the chloroplast. Lipid droplets are visible outside the chloroplast. (4) Water control for (3). Acid lipase activity extremely feeble in the chloroplast and mitochondrion. 3246 Botany: Da Silva et al. Proc. Nat. Acad. Sci. USA 71 (1974)

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FIGS. .5 and 6. Symbols are same as in Figs. 1-4. (5) Alkaline lipase activity of a stressed leaf disc. Very feeble activity is seen in the mitochondrion, but strong activity is visible between the chloroplast lamellae. (6) Water control of Fig. 5 tested for alkaline lipase activity. Almost none was present. Proc. Nat. Acad. Sci. USA 71 (1974) Subcellular Effects of Water Stress 3247

probably accounts for the known effect of desiccation* on 14. -Butler, W. L. & Okayama, S. (1971) Biochim. Biophys. the C02 compensation point (22). Depression of photochemical Acta 245, 237-239. 15. Mantai, K. E. (1970) Plant Physiol. 45, 563-566. reactions is known to occur in the presence of free fatty acids 16. Okayama, S. (1964) Plant Cell Physiol. 5, 145-156. (3, 11-14, 19, 20). Specifically, fatty acids interfere with the 17. Bamberger, E. S. & Park, It. S. (1966) Plant Physiol. 41, Hill reaction (19, 34) and photoreduction of the C-550 com- 1591-1600. ponent of photosystem II (14). 18. Constantopoulos, G. & Kenyon, C. N. (1968) Plant Physiol. 43, 531-536. The observed increase in acid lipase activity of mitochondria 19. Krogmann, D. W. & Jagendorf, A. T. (1959) Arch. Bio- from water stressed leaves may account for the altered swelling chem. Biophys. 80, 421-430. and ion transport characteristics that Miller et al. (40) noted. 20. McCarty, R. E. & Jagendorf, A. T. (1965) Plant Physiol. 40, The uncoupling of oxidative phosphorylation found to occur 725-735. in mitochondria from wilting tissue (41) may also be hy- 21. Siegenthaler, P. A. (1972) Biochim. Biophys. Acta 275, 182- 191. pothesized to result from membrane disruption as well as from 22. Nguyen Due, A. T. & Vieira da Silva, J. (1971) C. R. BI. phosphatase activity. Furthermore, it is conceivable that the Acad. Sci. 273, 1291-1294. decrease in photorespiration noted in stressed plants (22, 42) 23. Kisaki, T., Ayako, I. & Tolbert, N. E. (1971) Plant Cell results from lipase attack on membranes. Physiol. 12, 267-273. 24. Michel, B. E. (1970) Plant Physiol. 45, 507-509. The preliminary ultrastructural and cytochemical evidence 25. Lawlor, D. W. (1970) New Phytol. 69, 501-513. provided here is thought to help in understanding the effect 26. Gomori, G. (1952) Histochemistry. Prinzciples and Practice of water stress on photosynthesis and respiration at the en- (University of Chicago Press, Chicago). zymatic level. Ultrastructural changes accompanying re- 27. Murata, F., Yokota, S. & Nagata, T. (1968) Histochemie 13, covery from water stress still require investigation. Further- 215-222. 28. Nagata, T. & Murata, F. (1972) Histochemie 29, 8-15. more, ultrastructural differences in drought resistant and 29. Spurr, A. R. (1969) J. Ultrastruct. Res. 26, 31-43. sensitive species subjected to desiccation also must be de- 30. Nir, I., Klein, S. & Poljakoff-Miayber (1969) Aust. J. Biol. termined. Sci. 22, 17-33. 31. Blumenthal-Goldschmidt, S. & Poljakoff-Mayber, J. (1968) This work was supported by a National Science Foundation Aust. J. Bot. 16, 469-478. Grant GB-30367X to P. J. Kramer. The Phytotron facilities used 32. Gausman, H. W., Baur, P. S., Jr., Porterfield, MA. P. & were supported by NSF Grant GB-28950 to H. Hellmers. We are Cardenas, C. (1972) Agron?. J. 64, 133-136. grateful to and wish to thank AIiss Rose Broome and Air. AMark 33. Todd, G. W. & Basler, E. (1965) Phyton (Buenos Aires) 22, Norris for their help with the electron microscopy. 79-85. 34. Santarius, K. A. & Heber, U. (1967) Planta 73, 109-137. 1. Todd, G. W. (1972) in Water Deficits and Plant Growth, ed. 35. Pham Thi, A. T. (1972) Conatribution a l'tude de l'actiona de la Kozlowski, T. T. (Academic Press, New York), Vol. 3, pp. secheresse sur la photosynthese et la respiration du cotonnier 177-216. (Gossypium hirsutum L.), Thesis doctorate 3d cycle Uni- 2. Nir, I. & PoIjakoff-Mayber, A. (1966) Isr. J. Bot. 15, 12-16. versity of Paris-south. 3. Nir, I. & PoIjakoff-Mayber, A. (1967) Nature 213, 418-419. 36. Champigny, M.-L. & Miginiac-M\Iaslow, M. (1971) Biochim. 4. Vieira da Silva, J. (1968) C. R. H. Acad. Sci. 266, 2412-2413. Biophys. Acta 234, 335-343. 5. Vieira da Silva, J. (1968) C. R. H. Acad. Sci. 267, 729-732. 37. Vieira da Silva, J. (1970) UNESCO, Symposium on Plant 6. Vieira da Silva, J. (1969) Z. Pfianzenphysiol. 60, 385-387. Responses to Climatic Factors, Uppsala, 15-20 September, 7. Vieira da Silva, J. (1970) Physiol. Veg. 8, 413-447. pp. 213-220. 8. Chen, D., Sarid, S. & Katchalski, E. (1968) Proc. Nat. 38. Kirk, J. T. 0. & Tilney-Bassett, R. A. E. (1967) in The Acad. Sci. USA 61, 1378-1383. Plastids (W. H. Freeman Co., San Francisco), pp. 10-13. 9. Hsiao, T. C. (1970) Plant Physiol. 46, 281-285. 39. Harrison, J. S. & Trevelyan, W. E. (1963) Nature 200, 10. Marin, B. & Vieira da Silva, J. (1972) Physiol. Plant. 27, 1189-1190. 150-155. 40. Miller, R. J., Bell, D. T. & Koeppe, D. E. (1971) Plant 11. Boyer, J. S. & Bowen, B. L. (1970) Plant Physiol. 45, 612- Physiol. 48, 229-231. 615. 41. Zholkevich, V. N. & Rogacheva, A. Y. (1968) Fiziol. Rast 12. Fry, K. E. (1970) Plant Physiol. 45, 465-469. 15, 537-545 (English Summary, 1969) Biol. Abstr. 50, 89033. 13. Vieira da Silva, J. & Veltkamp, J. (1970) C. R. H. Acad. Sci. 42. Nguyen Duc, A. T. & Vieira da Silva, J. (1972) C. R. H. 271, 1376-1379. Acad. Sci. 274, 3234-3237.