The question of plant hormone binding sites

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cult to see how multiple mutations giving 3 specific changes in activity (such as the altered ratios of affinities in Lamb pro- 4 tein, example (1)) may be predicted a 5 priori for in vitro modifications. More speculatively, affinity selections 6 can also be hoped to result in changes in surface composition and protein reloca- 7 tion, as suggested in Sections (3) and (4). Since the experimental strategies 8 for such selections have been estab- 9 lished, the feasibility of these enrichments can be tested in the near future. 10

Acknowledgements Financial support for this project was supplied through the Australian Research Grants Committee.

11 12

13 14

References

1 Lowe, C. R. (1979) in Laboratory Techniques in Biochemistry and Molecular Biology (Work, T. S. and Work, E., eds), Vol. 7, pp. 267-522, 15 North-Holland 2 Platsoucas, C. D. and Catsimpoolas, N. (1980) in Methods of Cell Separation (Catsimpoolas,

N., ed.), Vol. 3, pp. 157-200, Plenum Press Briles, E. G. (1982) Int. Rev. Cytol. 75, !01-165 Ferenci, T. and Lee, K.-S. (1982) J. Mok Biok 160, 431-444 Ferenci, T. (1980) Eur. J. Biochem. 108, 631-636 Kamio, Y. and Nikaido, H. (1976) Biochemistry. US 15, 2561-2569 Ferenci, T., Sehwentorat, M., UIIrich, S. and Vilmart, J. (1980) J. Bacteriol. 142, 521-526 Ferenei, T. (1983) Appl. Env. Microbiol. 45, 384-388 Ferenci, T. and Boos, W. (1980) J. Supramol. Struct. 13, 101-116 Luckey, M. and Nikaido, H. (1980) Proc. Natl Acad. Sci. USA 77, 16%171 Ferenci, T. and Lee, K.-S. (1983)J. Bacteriol. 154, 984-987 D6barbouill~, M., Shuman, H. A., Silhavy, T. J. and Schwartz, M. (1978) J. Mol. Biol. 124, 35%371 Chapon, C. (1982) J. Bacteriol. 150, 722-729 Pless, D. D., Lee, Y. C., Roseman, S. and Schmaar, R. L. (1983) J. Biol. Chem. 258, 2340-2349 Ward, J. B. and Berkeley, R. C. W. (1980) in Microbial Adhesion to Surfaces (Berkeley, R. C. W., Lynch, J. M., Melling, J., Rutter, P. R. and Vincent, B., eds), pp. 47-66, Ellis

Horwood Ltd 16 Hall, A. N., Hogg, S. D. and Phillips, G. O. (1978) J. Appl. Bacteriol. 44, 215-233 17 Hall, A. N. and Jafri, S. S. A. (1980) J. Gen. Microbiol. 117, 263-265 18 Rosenberg, M. and Rosenberg, E. (1981) J. Bacteriol. 148, 51-57 19 Rosenberg, M., Bayer, E. A., Delarea, J. and Rosenberg, E. (1982) Appl. Env. Microbiol. 44, 92%937 20 Hermansson, M., Kjelleberg, S., Korhonen, T. K. and Stcnstr6m, T.-A. (1982) Arch. Microbiol. 131,308-312 21 Michaelis, S. and Beckwith, J. (1982) Annu. Rev. Microbiol. 36, 435-465 22 Chihata, I. and Tosa, T. (1980) Annu. Rev. Biophys. Bioeng. 10, 197-216 23 Silhavy, T. J., Shuman, H. A., Beckwith, J. and Schwartz, M. (1977) Proc. NadAcad. Sci. USA 74, 5411-5415 24 Weinstock, G. M., ap Rhys, C., Berman, M. L., Hampar, B., Jackson, D., Silhavy, T. J., Weisemann, J. and Zweig, M. 0983) Proc. Natl Acad. Sci. USA 80, 4432-4436 25 Bayer, E. A., Rosenberg, E. and Gutnick, D. (1981) J. Gen. Microbiol. 127, 295-3(D 26 Gabay, J. and Schwartz, M. (1982) J. Biol. Chem. 257, 6627~630 27 Ulmer, K. M. (1983) Science 219, 666-671

Letters to the Editor The question of plant hormone binding sites SIR: Reading the discussion forum on plant development in the October edition of T I B S ~, I was reminded of an article by E. W. Sinnott in which he used the analogy of coins and coin-operated machines to hormones and cells respectively2. Each 'coin' can affect only some of the total range of 'machines' and each 'machine' can respond to only some of the total range of 'coins', depending on the internal design of the machine. I find this analogy very helpful in clarifying the difference between placing the major emphasis in the control of plant development on the 'substance' (i.e. the hormone) or the 'system' (i.e. the tissue sensitivity). The occurrence of specific 'sensitive' target tissues for mammalian hormones is a well-established concept, and the primary biochemical responses to hormone-receptor binding are quite well understood 3'4. However, although binding sites have been identified for all the major phytohormones, in no case is the primary biochemical response to binding known; it is, therefore, not yet proven that such binding sites are true receptors which thus might be involved in the control of tissue sensitivity5. Trewavas ~ suggests that hormones act by modifying PM structure or function (this process possibly being mediated by

a receptor) and that similar modifications can occur in response to other stimuli. This hypothesis is quite plausible, and has tentative support in the suggestion that the major auxin binding site of maize coleoptiles (localized on the endoplasmic reticulum by density gradient centrifugation) may act by pumping protons into vesicles which are subsequently released to the cell exterior 6 thus causing 'acid-growth '7. The fungal toxin fusicoccin can cause a similar morphogenetic response by stimulation of proton efflux due to a plasma-membrane ATPaseS; it appears, therefore, that two distinct compounds can cause the same response but by different mechanisms. However, the existence of three membrane-bound binding sites 9 and at least one cytoplasmic bindingsite l° for auxin in maize coleotiles will make their putative roles in the control of tissue morphogenesis and hormone sensitivity much more difficult to unravel. Work in our laboratories has shown that there is little difference in the affinity constants and concentrations (pmol/ g.f.wt) of ethylene binding sites isolated from leaves, stem and callus of tobacco (Nicotiana tabacum or from cotyledons or callus of bean (Phaseolus vulgaris), suggesting little difference exists in the sensitivity of these tissues to ethylene tl.

However, until the physiological role of ethylene in these tissues is known, it would be premature to draw any firm conclusions from this data. An alternative approach would be to ask what factors can regulate tissue sensitivity, by regulating the concentration of hormone receptors; are such differences due to cell interactions or similar phenomena 12, or can receptor concentration be influenced by the hormone itself, as is the case in many mammalian systemsl3? Work in our laboratories has indicated that the concentration of N A A binding sites in tobacco callus increases 10-fold with a 1 000-fold increase in the N A A concentration of the culture media, and Trewavas himself has found that application of 2-4D to cultured explants of jerusalem artichoke tuber tissue caused the induction of 2-4D binding activity~4. Similarly, high-affinity I A A binding sites have been induced in A r e n a sativa roots by pre-incubation with IAA, and this process could be prevented by the presence of cycloheximide. Thus it appears that auxins enhance the synthesis/ activity of their own binding-sites, as do the mammalian hormones angiotensin and prolactin ~. In a different vein, an article has appeared reporting that a very marked variation occurs in the concentration of cytoplasmic auxin binding sites through

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T I B S - February 1984

a culture passage, possibly due to a wound response I~L To conclude, if it is accepted that the concentration of hormone binding sites is a reasonable criterion for assessing tissue sensitivity, the sparse literature available indicates that such sensitivity may be influenced by certain environmental factors and also by the actual hormone concentration itself; further research along these lines may well prove very informative.

References 1 Trewavas, A. J. and Cleland, R. E. (1983) Trends Biochem. Sci. 8, 354-357 2 Sinnott, E. W. (1946) Amer. Nat. 80, 497-505

3 Cuatrecasas, P. and Greaves, M. F. Receptors and Recognition (all volumes), Chapman and Hall 4 Cuatrecasas, P., Hollenberg, M. D., Chang, K.-J. and Bennett, V. (1977) Recent Adv. Cell Res. 31, 37-62 5 Dodds, J. H. and Hall, M. A. (1980) Sci. Prog. (Oxf.) 66, 513--535 6 Ray, P. M. (1977) Plant Physiol. 59, 594-599 7 Rayle, D. L. and Cleland, R. E. (1970) Plant Physiol. 46, 250-253 8 Marre, E. (1979) Annu. Rev. Plant Physiol. 30, 273-288 9 Dohrmann, U., Hertel, R. and Kowalik, H. (1978) Planta 140, 97-104 10 Murphy, G. J. P. (1980) Plant Sci. Lett. 19, 157-168 11 Dodds, J. H., Bengochea, T. and Starting, R. J. (1982) Proc. 5th Int. Con. Plant Tissue and Ceil Cult. (Fujiwara, A., ed.), pp. 5%58,

A third dimension in the control of plant development SIR: The title of your October 1983 Discussion Forum, 'Is plant development regulated by changes in the concentration of growth substances or by changes in sensitivity to growth substances?' seems to rule out any consideration of the possibility that some developmental responses might be dependent on neither changes in hormone concentration n o r changes in hormone sensitivity of the tissue. This type of hormone-independent response is clearly recognized by Trewavas and there is growing evidence of the importance of such phenomena, particularly in cases where the plant responds rapidly (< 300 s) to environmental stimuli. We would also consider shoot tropistic responses to be included in this category. The results of our recent studies ~.2.3 and those of others 4"5 on the rapid growth rate changes bringing about tropistic curvatures in shoots, suggest that these changes in growth rate are not caused by changes in the concentration of hormones. We would not accept Cleland's view that changes in the concentration of auxin can explain such growth rate changes - in our opinion the changes found are too small, they have not been shown to occur rapidly enough and the hormone changes do not correlate well with the complex growth patterns causing curvature. However, poor correlations have not in the past disturbed plant hormonologists as there is always the excuse, noted by Cleland, that changes in some particular fraction of the total hormone might correlate well. Unfortunately, Trewavas may have offered yet another straw for plant hormonologists to clutch at, as they can now evoke ~mewhat nebulous sensitivity arguments after analyses of even the smallest hormone compartment have

failed to reveal appropriate changes. After 50 years of analysing plant hormones, admittedly usually imperfectly, it is now accepted that changes in plant hormone levels do not provide an adequate explanation of many developmental changes. By all means let us consider whether hormone sensitivity might not provide us with a better explanation of some responses but let us also reconsider whether hormonal control need be the only way in which plant cells regulate basic processes.

References 1 Firn, R. D. and Digby, J. (1980) Annu. Rev.

Jap. Ass. Plant Tissue Culture, Tokyo 12 Graham, C. F. and Wareing, P. F. (1976) The Developmental Biology of Plants and Animals, Blackwell Scientific Publishers 13 Lefkowitz, R. J., ed. (1982) Receptors and Recognition, Series B, Vol. 13, Chapman and Hall 14 Trewavas, A. J. (1980) Phytochemistry 19, 1303-1308 15 Bhattacharyya, K. and Biswas, B. B. (1982) Phytochemistry 21, 1207-1211 16 Ostroom, H., Kulescha, Z., van Vliet, Th. B. and Libbenga, K. R. (1980) Planta 149, 44-47 ROBERT J. STARLING

Department of Plant Biology, University of Birmingham, PO Box 363, Birmingham B15 2T-F, UK.

Plant Physiol. 31, 131-148 2 Franssen, J. M., Cooke, S. A., Digby, J. and Fire, R . D . (t981) Z. Pflanzenphysiol. 103, 207-216 3 Digby, J., Firn, R. D. and Carrington, S. C. M. (1982) in Plant Growth Substances 1982' (Wareing, P. F., ed.), pp. 519-528, Academic Press 4 Hart, J. W., Gordon, D. C. and MacDonald, I. R. (1982) Plant Cell Envir. 5, 361-366 5 MacDonald, I. R., Hart, J. W. and Gordon, D. C. (1983) Plant Cell and Envir. 6, 401-406 RICHARD FIRN a n d JOI~IN DIGBY

Department of Biology, University of York, York YO1 5DD, UK.

Antizymes: inhibitor proteins or regulatory subunits? SIR: It has been recognized for over half a century that an important factor influencing the activity of many enzymes in vivo is their association with other proteins. As early as 1930, Haldane ~ described 'antienzymes' as a class of proteins encompassing both immunoglobulins and other types of inhibitory proteins; since that time, the number and types of such known interactions have grown impressively. Recently, the discovery of different regulatory proteins that inhibit a specific target enzyme has been accompanied by changing terminology that accentuates the uniqueness of each regulatory protein, while obscuring its general membership in the class of regulatory subunits. It has become apparent that the use of regulatory subunits to govern the function of structural proteins, transport proteins, and enzymes is widespread. From the extant characterized examples, some generalizations can be made:

(1) The association of a protein with its regulatory subunit affects the affinity and/or specificity of ligand binding either by alteration in tertiary structure or by steric impedance resulting from the aggregation itself. (2) In many cases, aggregation of a protein with its regulatory subunit is mediated by a small molecule effector(s) since most regulatory subunits are themselves controlled either by chemical modification or by small metabolites. A considerable spectrum is apparent in the types of regulatory, subunits, examples of which are given in Table I. The list of proteins is in no way comprehensive, but was chosen to illustrate diversity. At one extreme is aspartate carbamoyltransferase, where the regulatory subunit could be viewed as a genetically independently produced allosteric site. At the other extreme is the complex of two enzymes, ornithine