School of Plant Biology, University College of North Wales, Bangor. (Received 22 October 1972) SUMMARY

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1 New Phytol. (1973) 72, THE EFFECT OF ROOT PRUNING AND 6-BENZYL- AMINOPURINE ON THE CHLOROPHYLL CONTENT, ^'^CO^ FIXATION AND THE SHOOT/ROOT RATIO IN SEEDLINGS OF PISUM SATIVUM L. BY C. R. MCDAVID*, G. R. SAGAR AND C. MARSHALL School of Plant Biology, University College of North Wales, Bangor (Received 22 October 1972) SUMMARY Pruning the roots of light-grown pea seedlings every 4 days accelerated senescence of older leaves and reduced their rate of fixation of '*C02. Application of 6-benzylaminopurine to the shoot largely compensated for root removal and increased the shoot/root ratio of both pruned and intact plants. It is suggested that the supply of cytokinins from root to shoot may affect leaves by delaying senescence and maintaining tlieir capacity for photosynthesis; it may also alter the distribution of dry matter within the plant by influencing the proportion of assimilates and other metabolites retained by the shoot. INTRODUCTION Changes in the shoot/root ratio in response to changes in the environment have often been explained in terms of competition between shoot and root for mineral salts, water and photosynthates. lt is becoming increasingly clear, however, that shoot/root interactions involve much more complex control systems in which growth substances are important. Both auxins and gibberellins arc translocated from shoot to root and gibberellins and cytokinins of root origin move into the shoot (Phillips, 1971). Went (1938, 1943) first suggested that the root exerted some hormonal control over shoot growth, and later Chibnall (1954) proposed that protein metabolism in leaves was maintained by metabolites synthesized in the root. The ability of cytokinins to retard chlorophyll and protein degradation in detached leaves (Richmond and Lang, 1957; Mothes, 1964) and in the intact plant (Fletcher, 1969; Adedipc and Fletcher, 1971) and the reports of cytokinin-like activity in the xylem exudatc of many species suggest that the root system may indeed play an important role in infiucncing shoot metabolism. Thus it follows that changing environmental conditions could control the shoot/root ratio by affecting the level of cytokinins supplied to the shoot. Some evidence for this view has been provided by Shah and Loomis (1965) who demonstrated that cytokinins retarded senescence caused by water deficit in sugar beet leaves, and by Vaadia and Itai (1968) who showed that cytokinin activity in root exudates decreased sharply in plants subjected to osmotic and saline stress. Direct application of kinetin to plants has also been shown to influence the shoot/root ratio (Wittwer and Dedolph, 1963). * Present address: Department of Biological Sciences, Universilv of the West Indies St Au.nistine Trinidad. ', i,.

2 466 C. R. MCDAVID, G. R. SAGAR AND C. MARSHALL In tbe experiments reported bere tbe effects of root pruning and application of a cytokinin on some aspects of sboot growth and on the sboot/root ratio of pea seedlings were investigated in an attempt to evaluate tbe role of cytokinins of root origin in tbe correlation between sboot and root growtb. (a) 80 r Cb) Day5 after initial treatment" (c) 80- n r-. (d) l S I 234 I I Harvest Harvest Leaf I Leaf 4 Fig. I. (a-d) The effect of root pruning on the growth of dwarf pea seedlings, (a) and (b) Rate of increase in root and shoot dry weight respectively; (c) chlorophyll content of discs from leaf I and leaf 4 (at the fourth harvest leaf 2 was taken instead of leaf i); (d) radioactivity fixed per unit area of leaf 3. (a) and (b), Control; O, pruned, (e) and (d) Open columns, control; hatched columns, pruned. Vertical lines show three times standard error of the means. MATERIALS AND METHODS Seedlings of dwarf pea [Pisum sativum L. cv. Kelvedon Wonder) were grown in water culture in a growtb room at 20 C with a 14-hour photoperiod and a light intensity of 12.5 klux. A standard Long Ashton nutrient solution was used which was continuously aerated and changed every fourth day. In Expt I when the seedlings were 15 days old, the cotyledons and all lateral roots (which comprised about 75% of the root system at this time) were removed. Control plants were left intact. New lateral roots were removed every fourth day and the weight

3 Shoot/root ratio in peas 467 of root removed was recorded. In order to minimize wilting the humidity was kept high by flooding the surface of the bench and by running a humidifier continuously. In addition, after each pruning treatment, the plants were sprayed with distilled water. Six control and six treated plants were harvested 3, 7, 12 and 16 days after treatment. The third leaf was removed from each plant and batches of six leaves (three from each treatment) were placed on a platform of wire mesh in a plastic tray lined by moist filter paper, covered by clear polythene, and exposed for 5 minutes to 8 /ici '*C02, released from Na2''''CO3 by the addition of excess N HCl. Eight 7-mm-diameter leaf discs were taken immediately afterwards to determine the amount of radioactivity fixed. Four leaf discs were also taken from the first and fourth leaves of each plant for chlorophyll estimation. The dry weights of the shoot and root were also determined. In Expt 2 the cotyledons were again removed at 15 days and the following treatments carried out: (i) control root system left intact; (2) root system left intact but the shoot dipped in a 20 ppm solution of 6-benzylaminopurine (BAP) containing 0.025% agral 90 as wetting agent; (3) all lateral roots removed; (4) all lateral roots removed and shoots treated with BAP as above. There were twelve plants per treatment and the shoots of plants not treated with BAP were dipped into distilled water containing the same concentration of wetting agent. The pruning and BAP treatments were repeated every fourth day. Ten days after treatment the second leaf was removed from the plants and batches of twelve (two from each treatment) were exposed to 12 /ici '^COj for 5 minutes as in Expt i. From each of the two terminal leaflets a i ^-mm-diameter leaf disc was taken, one prior to exposure to ''^COj for chlorophyll estimation and the other immediately after exposure to measure the fixation of "^CO^. The radioaetivity in the leaf discs was determined by liquid scintillation counting using the in-vial wet combustion technique (Shimshi, 1969). The scintillant used was a mixture of two parts NE 233 and one part of Triton X-ioo detergent and an average counting efficiency of 61^-70% was obtained using a Tracerlab two-channel counting system with an automatic external standard. Chlorophyll was measured spectrophotometrically in acetone at 645 and 663 nm and the values in /;g determined using a nomogram (Kirk, 1968). The dry weight of shoot and root were also determined. RESULTS Experiment i Results are shown in Fig. i. The rate of dry weight increase of the root system (including the weight of root removed in the pruning treatment) although initially lower in pruned than in intact plants was similar in the two treatments at the third and fourth harvests. The rate of increase in dry weight of the shoot of pruned plants, however, was reduced to about 30% of that of intact plants by the end of the experiment. The chlorophyll content of the first leaf of intact plants declined as the plants aged but the decline was greatly accelerated in pruned plants so that by the third harvest the chlorophyll content of the first leaf of pruned plants was half that of intact plants. At the final harvest the first leaf of pruned plants was too withered to be sampled and leaf discs were taken instead from the second leaf of intact and pruned plants. The latter contained 40% less chlorophyll than the equivalent leaf of control plants. In contrast the chlorophyll content of the fourth leaf remained constant for the duration of the experiment and did not differ between the two treatments.

4 468 C. R. MCDAVID, G. R. SAGAR AND C. MARSHALL The amount of '*CO2 fixed per unit area of the third leaf was consistently smaller in pruned plants. In intact plants fixation rose to a maximum at the third harvest and then declined, whereas in pruned plants there was a consistent decline so that at the final harvest fixation was reduced to a third of that of intact plants (P = o.oi). 400 (a) 8 (b) I Treatment Total Stioot 2 34 Root I 2 3 Treatment i60r (c) 80r (d) ro O X c Treatment Fig. 2. (a-d) The effect of root pruning and application of BAP to the shoot on the growth of dwarf pea seedlings, (a) Dry weight; (b) shoot/root ratio; (c) chlorophyll content; (d) radioactivity fixed per leaf disc. Vertical lines show three times standard error of the means. Experiment 2 The results of this experiment are shown in Fig. 2. Application of BAP resulted in a temporary release of apical dominance in both pruned and intact plants. Growth in length of the primary root and the production and growth of new lateral roots was much less in BAP-treated plants, an effect reflected in the dry weight of the root system at the end of the experiment. This was 42% smaller in BAP treated intact plants than in control (P = 0.01) and 46% smaller in pruned plants with BAP than in those without (P = o.oi). The BAP treatment changed shoot weights relatively little and consequently the shoot/ root ratio in BAP-treated plants was almost double tbat of the appropriate controls (both

5 Shoot/root ratio in peas 469 significantly different at P = 0.01). However, the shoot/root ratio was artificially high in pruned plants because the weight of root removed was not included in the root dry weight. Loss of chlorophyll from the second leaf was again greatly accelerated by root pruning and the chlorophyll content per unit area was 30% lower in pruned than in intact plants. Application of BAP prevented the loss of chlorophyll associated with the pruning treatment and stimulated a 10% increase in chlorophyll content in intact plants [P = o.oi). Fixation of '"^COi per unit area of the second leaf was reduced in root-pruned plants without BAP to about 50% of that of control plants (P = o.oi), whereas BAP application increased fixation in pruned plants by about 67% (P = o.oi). DISCUSSION Although some wilting of pruned plants occurred immediately after the lateral roots were initially removed, the plants quickly recovered from this and thereafter showed little evidence of water deficit. It is unlikely therefore that the effects of root pruning resulted from an inadequate water supply though it is possible that a retardation in the supply of mineral salts to the shoot may have contributed to the differential response of shoot and root growth to root pruning (Fig. i). Indeed reduction in the supply of minerals was the explanation put forward by Humphries (1958) to explain similar results. The rapid loss of chlorophyll from the older leaves of pruned plants and the ability of BAP to retard such loss and also to largely prevent the decline in fixation of '^COj per unit leaf area accords with the view that a cytokinin regulates protein metabolism in leaves and that the amount of this factor which is supplied to the shoot is regulated by the size of the root system, at least in some species (Buttrose and Mullins, 1968). The presence of natural cytokinins in seedling roots of pea has been demonstrated (Short and Torrey, 1972) and the reduction in shoot growth reported in Expt i as a result of root pruning may have been mediated through a reduction in the supply of cytokinin from root to shoot. Similarly it can be argued that the reduction infixationof ' ^COj in pruned plants may be associated with reduced amounts of cytokinin passing from the root to the shoot with a consequential reduction in chlorophyll and carboxylating enzyme activity (Wareing, Khalifa and Trcharnc, 1968). The increases in shoot/root ratio caused by BAP treatment suggest that this growth regulator affects the distribution of dry weight within the plant. A stimulatory effect of kinetin on shoot growth was reported by Miller (1956). In contrast, however, Wittwer and Dedolph (1963) found that application of kinetin to roots greatly reduced the shoot/ root ratio. The present results and those of Wittwer and Dedolph (1963) are consistent with the suggestion that cytokinins act as mobilizing agents, both directing the movement of nutrients and metabolites to areas of the plant treated with the growth substance as well as preventing the movement of metabolites out of areas of leaves rich in kinetin (Mothes, 1964; Quinlan and Weaver, 1969; Shindy and Weaver, 1970). Furthermore, although endogenous cytokinins are mobile within the plant, the evidence in the literature suggests that the movement of exogenously applied cytokinins, including BAP, is at best very restricted (Thimann, 1963; Sachs and Thimann, 1964). It seems possible therefore that the reduction of root growth in the present experiments was not a direct effect of BAP, but rather the result of a reduction in the supply of assimilates to the roots from the shoots which were treated with BAP. This view is supported by the results of Wittwer and Dedolph (1963) where direct application of kinetin to roots enhanced root growth.

6 470 C. R. MCDAVID, G. R. SAGAR AND C. MARSHALL Thus the proportion of current assimilates and other metabolites retained by the shoot may be dependent on the amount of cytokinin or other hormones supplied from the roots. A reduction in this supply following root pruning or adverse conditions for root growth, e.g. drought and salinity (Vaadia and Itai, 1968) may reduce the sink capacity of the shoot and this would account for the reduced shoot/root ratios under these conditions. ACKNOWLEDGMENTS This work was carried out while one of us (C.R.M.) held a Commonwealth Scholarship and was submitted as partial requirement for a Doctor of Philosophy degree of tbe University of Wales. REFFRFNCES AuiiDiPK, N. O. & FLiiTCHUH, R. A. (1971). Retardation of leaf senescence by benzyladenine in bean plants is not dependent on mobilisation. Can. J. Bot., 49, 59. BuTTROsi;, M. S. & MuLLiNS, M. G. (1968). Proportional reduction in shoot growth of grapevines with root systems maintained at constant relative volumes by repeated pruning. Aiist. J. hid. Sci., 21, CHIBNALL, A. C. (1954). Protein metabolism in rooted runner-bean leaves. New PhytoL, 53, 31. FLETCHEU, R. A. (1969). Retardation of leaf senescence by benzyladenine in intact bean plants. Planta, 89, I. HUMPHRIES, E. G. (1958). Effect of removal of a part of the root system on subsequent growth of root and shoot. Ann. Bol., N.s. 22, 251. KIRK, ]. T. O. (1968). Studies on the dependence of chlorophyll synthesis on protein synthesis in Englena gracilis, together with a nomogram for determination of chlorophyll concentration. Planta, 78, 200. MILLER, C. O. (1956). Similarity of some kinetin and red light effects. PI. PhysioL, Lancaster, 31, 318. MoTHES, K. (1964). The role of kinetin in plant regulation. In: Rej>ulateurs Natnreh de la Croissance Veifetale (Collogue International, Gif-sur-Yvette, 1963), pp C.N.R.S., Paris. PHILLIPS, I. D. J. (1971). Introduction to the Biochemistry and Physiology of Plant Groivth Hormones. McGraw-Hill, New York. QuiNLAN, J. D. & WEAVER, R. J. (1969). Influence of henzyladenine, leaf darkening and ringing on movement of ' 'C-labelled assimilates into expanded leaves of Vitis vinifera L. PL PhysioL, Lancaster, 44> RICHMOND, A. & LANG, A. (1957). Effect of kinetin on protein content and survival of detached Xanthiiim leaves. Science, N.Y., 125, 650. SACHS, T. & THIMANN, K. V. (1964). Release of lateral buds from apical dominance. Nature, Lond., 201, 93y. SHAH, C. B. & LOOMIS, R. S. (1965). Rihonucleic acid and protein metabolism in sugar beet during drought. Physiologia PI., 18, 240. SHORT, K. C. & TORREY, J. G. (1972). Cytokinins in seedling roots of pea. PI. PhysioL, Lancaster, 49, 155. SHIMSHI, D. (1969). A rapid field method for measuring photosynthesis with labelled carbon dioxide. J. exp. Hot.. 20, 381. SHINDY, W. W. & WEAVER, R. J. (1970). Export of photosynthate affected when leaves are pretreated with growth suhstances. Nature, Lond., 227, 301. THIMANN, K. {1963). Plant growth substances; past, present and future. A. Rev. PL PhysioL, 14, i. VAADIA, Y. & ITAI, C. (1968). Interrelationships of growth with reference to the distribution of growth substances. In: Root Growth (Ed. by W. J. Whittington), pp Butterworths, London. WAREING, P. F., KHALIFA, M. M. & TREHARNE, K. ]. (1968). Rate limiting processes in photosynthesis at saturating light intensities. Nature, Lond., 220, WENT, F. W. (1938). Specific factors other than auxin affecting growth and root formation. PL PhysioL, Lancaster, 13, 55. WENT, F. W. (1943). Effect of the root system on tomato stem growth. PL PhysioL, Lancaster, 18, 51. WlTTWER, S. H. & DEDOLPH, R. R. (1963). Some effects of kinetin on the growth and flowering of intact green plants. Am.J. Bot., 50, 330.

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