Enhancement of Root Growth and Alteration in Cytokinin Distribution in FarnesoI-Treated Barley Seedlings

KEITH WARDLE and KEITH C. SHORT

Department of Biology, North East London Polytechnic, Romford Road, London E 15 4 LZ, UK

Received November 19, 1980 . Accepted December 29, 1980

Summary

Barley, treated at root emergence with 10 mg I-I farnesol exhibited, within a three day period, enhanced root growth compared to untreated plants. Extraction of free cytokinins revealed a high activity in the shoots of untreated plants whereas in the farnesol-treatments such activity was entirely confined to the roots. Farnesol did not exhibit cytokinin activity in the soybean callus bioassay. The results suggest that farnesol may inhibit the basipetal transport of cytokinins from the rOOt.

Key w ords: Hordeum vulgare, root growth, cytokinin, lamesol.

Introduction

Farnesol pyrophosphate is an intermediary meuabolite which has a widespread occurrence in plants, animals and insects. In plants a possible role for farnesol is becoming established in conditions of stress as it has been demonstrated that: farnesol accumulates in the leaves of waterstressed Sorghum (Ogunkanmi et aI., 1974) and maize (Wilson and Davies, 1977) . Furthermore, farnesol causes stomatal closure in epidermal strips of Commelina communis (Wellburn et ai., 1974) and in whole plants of Sorghum without permanent damage to stomatal functioning (Fenton et aI., 1977). Attempts to explain the antitranspirant activity of farnesol also indica,tes an effect on membrane permeability especially of ,the chloroplasts (Fenton et ai., 1976; Milborrow, 1979; Will mer et ai. , 1978). Enhanced growth of tissues has also been reported following farnesol application in excised pea internodes (Stowe and Hudson, 1969) and root growth in tissue cultures (Wardle and Simpkins, 1980).

Rapid responses to farnesol, e.g. stomatal closure, are presumably due to ~ direct action, however, the central position of farnesol in several metabolic pathways allows for a potentially large number of metabolites to be implicated in ~ny longer term effects. The objective of this study was to investigate the cause of farnesol-enhanced root growth in Hordeum vulgare.

Materials and Methods

Famesol : Farnesol (mixed isomers, Sigma) was prepared by vigorous shaking In water containing a few drops of teepol and was used at 10 mg I-I.

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184 KEITH WARDLE and KEITH C. SHORT

Barley and growth conditions: Barley seeds were soaked in water for 2 h, drained and kept in a moist atmosphere. This procedure was repeated 24 h later, and by 72 h root emer­gence had occurred. The germinating seeds were grown in 9 cm diameter Petri dishes on 2 filter papers (Whatman * 1) with 5 ml water or farnesol solution and incubated in the dark at 25 ± 2 DC for 3 days. Each Petri dish contained five plants.

Extraction of free cytokinins: Barley plants cv. Printa (500 per treatment) were divided into roots and shoots prior to homogenisation in a chilled mortar with 80 Ofo (v/ v) ethanol. The homogenate was extracted three times in 80 Ofo ethanol at -20 DC for 24 h. The com­bined 80 % ethanol extracts were pooled, concentrated in vacuo (40 DC), and the aqueous phase adjusted to pH 8.0. Free cytokinins were extracted from the aqueous fraction with butan-I-ol (3 times) which was pooled and concentrated in vacuo (40 DC). The concentra­ted butan-I-ol phase was applied as a streak onto TLC plates (20 X 20 em) coated with Kieselgel 60 GFm (merck) and developed in chloroform-acetone-methanol (1: 1 : 1, v/v/ v). Following development each chomatogram was divided into 10 equal R f zones and eluted twice with 80 Ofo ethanol.

Bioassay 0/ cytokinins : Each eluate was further concentrated in vacuo (40 DC) prior to being incorporated into SCF medium (Fosket and Torrey, 1969). The medium (50 ml) was dispensed into 2 Petri dishes after autoclaving and innoculated with 5 pieces of cytokinin­dependent soybean (Glycine max L. Merril. cv. Biloxi) callus. The cultures were maintained at 25 ± 2 DC under a 16 h photoperiod. After 21 days the fresh weight of each callus was determined.

Results and Discussion

Previous studies have shewn that farnesel can enhance reet growth of excised cultured embryos of barley (Wardle and Simpkins, 1980). In this study, the results in Table 1 demonstrate that this effect is very pronounced during the early stages of root elongation following germin<l!tion. Differences existed between cultivars not only in the untreated samples but also in the extent to which faroesol stimulated elongation. Although some variation was experienced in replicate experiments performed on different days, all cultivars did exhibit enhanced root elongation in the presence of farnesol in comparison to untrea'ted samples. Pronounced differences are known to occur between barley cultivars in response to a given treatment. Singh et al., (1972) demonstrated variations in the level of free proline accumulated under water-stress. With all barley cu1tivars tested in this study it was noted that the farnesol treatment significantly reduced the number of root hairs but this effect was not determined quantitatively.

Interactions of farnesol with auxins and cytokinins on barley root growth were examined and it was found that both hormones at high concentrations (1 and 10 mg I-i) were inhibitory to root growth. However, root growth was greater in hormone

treatments supplemented with farnesol than in those lacking this compound (unpublished). From these studies it was found that .a significant growth-promotive effect occurred in the presence of farnesol and adenine sulphate. Van Staden (1979) has recently demonstrated 'that a cytokinin-dependent soya bean callus can grow (and therefore synthesise cytokinins) in the presence of adenine and mevalonic acid.

z. P/lanzenphysiol. Bd. 102. s. 183-188. 1981.

Root growth and cytokinin distribution in farnesol-treated barley 185

Table I: The effect of farnesol on root elongation (em) of barley (treated at root emergence) after an incubation period of 3 days (± s. e. mean).

Cultivar Untreated Farnesol Farnesol as % of (water) (10 mg I') untreated

Printa 14.3 ± 0.9 23.5 ± 1.6 164

Sonja 17.3 ± 1.3 24.1 ± 1.6 139

Keg 18.7±1.1 25.6 ± 1.6 137

Porth as 18.9 ± 0.9 26.6 ± 1.2 141

Marris Otter 21.0 ± 1.4 31.1±1.7 148

Proctor 21.6±1.2 26.6 ± 1.5 123

Athas 22.6 ± 2.1 26.1±1.6 115

Wing 23.0 ± 1.4 29.8 ± 2.0 130

Since mevalonic acid is also a precursor of farnesol the possibility was considered that barley roots could utilise farnesol in the endogenous production of cytokinins.

Farnesol and adenine sulphate, separately and in combination, were incorporated into SCF medium and tested for cytokinin activity using the soybean callus bioassay. Adenine sulphate induced some tissue growth in soybean callus whereas farnesol did not (Table 2). Furthermore, farnesol in combination with adenine sulphate reduced the growth of the callus (Table 2). It was therefore concluded that farnesol did not directly exhibit any cytokinin activity.

Additional support for this view comes from ,the work of Biddin&ton et aI., (1980). These workers found that cytokinins stimulated the germination of celery seeds. In our studies celery seeds failed to germinate wi,th water and in the presence of 10 mg 1-1 farnesol germination was not increased, suggesting that farnesol does not directly exhibit cytokinin activity.

If the assumption is made that farnesol-induced root elongation is mediated via a cytokinin response then two possibilities exist: (a) that a metabolite of farnesol is converted to or possessed cytokinin activity, or (b) farnesol itself in some manner altered the production and/or distribution of endogenous cytokinins.

Table 2: Effect of kinetin, farnesol, adenine sulphate, and farnesol plus adenine sulphate on the growth of soybean callus.

Supplement to SCF medium

None (basal medium) Kinetin (100 fLg 1-1) Farnesol (10 mg I') Adenine sulphate (10 mg [.') Farneso[ (10 mg ['') + Adenine sulphate (10 mg [")

, All after 21 days (± s.e. mean).

Fresh weight (g)'

0.0681 ± 0.0037 1.4056 ± 0.0477 0.1752 ± 0.0113 0.8007 ± 0.0291

0.5471 ± 0.1441

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186 KEITH WARDLE and KEITH C. SHORT

Van Staden (1979), using radioactive adenine and mevalonic acid, demonstrated th<l!t soybean cells can synthesise sufficient cytokinin for growth, but was unable to show such a pathway unequivically because of the involvement of the two compounds in many diverse pathways. For these reasons labelling experiments were not attempted in this study and so changes in endogenous cytokinins were investigated.

Analysis of barley plants which were not exposed to farnesol revealed that cytokinin activity was located only in the shoots and little activity was present in roots (Fig. 1). However, in the farnesol-treated plants the cytokinin activity was totally confined to the roots (Fig. 1). Since cytokinins are known to be produced in the root apex (Short and Torrey, 1972) it is possible that farnesol, by an unidentified mechanism prevents the export of c}"tokinins from the root to the shoot system. The maintenance of cytokinins in the roots in therefore likely to enhance cell-proliferative activity and result in a stimulation of root growth. Further

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Fig. 1: Cytokinin activity determined by the soybean callus bioassay from R f zones of the butan-I-ol phase developed in chloroform-acetone-methanol (1 : 1 : 1, v/v/v) from extracts of (a) untreated roots, (b) farnesol-treated roots, (c) untreated shoots, and (d) famesol-trea­ted shoots.

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Root growth and cytOkinin distribution in farnesol-treated barley 187

evidence is, however, required to confirm this as the mode of action of farnesol on barley roots.

Another possible explanation for the observed root-growth effect is that farnesol may redirect the transport of cytokinins which are present in the seed, towards the root. Previous work by Wardle and Simpkins (1980) and the adenine sulphate effect reported here would appear to discount this possibility.

Farnesol has the potential use as an antitranspirant because of its ability to induce stomatal closure. However, an effect on cytokinin distribution may limit such use since cytokinins are involved in the delay of leaf senescence (Dumbroff and Walker, 1979). In addition, farnesol-induced damage of chloroplast membranes (Fenton et al., 1976) would constitute a further limitation on its use as an antitranspirant.

In conclusion, farnesol stimulates root growth in barley and it appears that this is due to the prevention of transport of cytokinins from the root to the shoot since farnesol itself does not exhibit cytokinin activity.

Acknowledgements

Thanks are expressed to MuntOn and Fison Ltd., (StOwmarket, Suffolk) for the gift of the barley cultivars.

References

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