Do peptides control plant growth and development?

correspondence

Do peptides control plant growth and development? The phytohormone auxin plays an important role in plant growth and development. However, there is a nagging concern that its structure appears too simple to explain the wide variety of activities that it is thought to promote. Moreover, there is often a lack of correlation between the concentration of auxin and a tissue response. This has fuelled a lively debate centring on the relationship between phytohormone response and the sensitivity of plant tissues 1. Recent work has suggested that small peptides (ranging in size from 12 to 22 amino acids) may play a role in the cell response to phytohormones (Ref. 2; E. Miklashevichs et al., unpublished). This prompted us to carry out a simple experiment to test whether auxin-induced cell division might be mediated by a factor other than the phytohormone itself. Tobacco protoplasts in culture have an absolute requirement for a distinct auxin : cytokinin ratio for maximal division and growth3; therefore, isolated protoplasts were cultured in medium containing auxin and cytokinin at concentrations that promote optimal rates of cell division 4. Four days later, when the cells were undergoing division, the medium was removed and added to recently isolated protoplasts. These protoplasts had been treated with a nonindole auxin analogue, 2-naphthalene acetic acid (2-NAA), which inhibits auxintriggered protoplast division 5. However, addition of the protoplast supernatant to the 2-NAA-pretreated protoplasts induced division (Fig. 1). The activity triggering division in the supernatant was induced by auxin, but destroyed by heat treatment and by incubation with the protease subtilisin. Other proteases, such as protease K, trypsin or chymotrypsin also destroy activity, whereas the endoprotease Glu-C does not (data not shown). Ultrafiltration of the protoplast supernatant revealed that this activity is retained by a 10-kDa cut-off ultrafiltration membrane (YM 10, Amicon), indicating that the active compound is larger than auxin. Gel filtration on a calibrated Superdex 75 HR column (Pharmacia) demonstrated that the active fraction elutes in the molecular mass range of 2 to 3 kDa. Taken together,

60

50 cO

t

40

t~

o 2

30

£L

20

10

[3 1

2

3

4

5

6

7

8

9

10

Treatment

Fig. 1. Tobacco mesophyll protoplasts were isolated and cultured 4 (1) with 5.5 [I,M naphthalene acetic acid (1-NAA) and 0.9 p~Mkinetin, and (2) with kinetin alone. In treatment 3, the protoplasts were exposed to 50 btM of the anti-auxin 2-NAA for 1 h prior to the addition of 1-NAA. The supernatant from protoplasts cultured in the presence of 1-NAA and kinetin was removed, treated as described below and added to fresh protoplasts pretreated with 2-NAA (4-10). Treatments 4-10: (4) no treatment; (5) heat - 65°C for 10 rain; (6) incubation with the protease subtilisin; (7) filtration through a 10kDa cut-off ultrafiltration membrane (YM 10, Amicon) (residue retained); (8) treatment 7 (filtrate retained); (9) filtration through a Superdex 75 HR 30/10 (Pharmacia) gel filtration column (2.5 kDa fraction retained); (10) treatment 9 (2.5 kDa fraction then incubated with trypsin). Protoplasts were scored visually for cell division four days after isolation. Standard deviations are indicated by error bars. these data suggest that the factor responsible for promoting cell division is a peptide. These results led us to suspect that the effect of auxin in activating protoplast division may not be primary (i.e. acting immediately), but may act by inducing the synthesis of another factor, possibly a peptide. This, in turn, triggers protoplast division and may also define the specificity of auxin action. Further work is required to characterize the activity involved, but the results to date prompt us to question whether the action of auxin ought to be reconsidered. We wonder if the notion of Canny6 about Ashby's law of requisite variety ('A situation can only be controlled by a controller that matches the variety of the situations') may prove to be correct. If this is indeed the case, then plant growth and development might be controlled by small peptide growth factors, in a manner similar to that identified in animals. It will be interesting to test if this is true and whether the same might also apply to the other plant growth substances.

Copyright © 1996 Elsevier Science Ltd. All rights reserved. 1360 - 1385/96/$15.00

Edvins Miklashevichs, Inge Czaja, Horst R6hrig, Jilrgen Schmidt, Michael John, Jeff Schell and Richard Walden Max Planck Institut for ZOchtungsforschung, Carl von Linn~ Weg 10, D-50829 KSIn, Germany

References 1 Davies, P.J. (1995) The plant hormone concept: concentration, sensitivity and transport, in Plant Hormones (Davies, P.J., ed.), pp. 13-38, K]uwer 2 van de Sande, K. et al. (1996) Modification of phytohormoneresponse by a peptide encoded by Enod40 of legumes in a nonlegume, Science 273, 370-373 3 Murashige, T. and Skoog, F. (1962)A revised medium for rapid growth and division and bioassays with tobaccotissue cultures, Physiol. Plant. 15,473-497 4 Walden,R. et al. (1993)Rol genes alter hormone requirements for protoplast growthand modify the expressionofan auxin responsivepromoter, Plant Cell Rep. 12, 551-554 5 RiJhrig, H. et al. Convergent pathways for LCO and auxin signaling in tobaccocells, Proc. Natl. Acad. Sci. U. S. A. (in press) Canny, J. (1985) Ashby's law and the pursuit of plant hormones: a critique of accepted dogmas, using the conceptof variety, Aust. J. Plant Physiol. 12, 1-7 December1996,V01.1,No. 12

41 1