THE ROLE OF LEAVES IN AUXIN AND BORON-DEPENDENT ROOTING OF STEM CUTTINGS OF PHASEOLUS AUREUS ROXB.

Transcription

1 jv«w Phytol. (1980) 84, THE ROLE OF LEAVES IN AUXIN AND BORON-DEPENDENT ROOTING OF STEM CUTTINGS OF PHASEOLUS AUREUS ROXB. BY W. MIDDLETON, B. C. JARVIS AND A. BOOTH Department of Botany, The University of Sheffield, Sheffield SIO 2TN, U.K. (Accepted 22 August 1979) SUMMARY The promotion of rooting of cuttings of Phaseolus aureus by indolebutyric acid () occurs only when leaves are present. may have some direct action on root initiation in the hypocotyl but its main action is in the leaves where it leads to an early increase m free sugars A later increase in sugars in the hypocotyl is largely dependent on exogenous boron which is essential for root development in cuttings of light-grown material. also stimulates and boron promotes the accumulation of ^*C in hypocotyls when [i*c]sucrose is applied to the leaves. These events occur before roots develop on the hypocotyl. Illumination of the leaves of cuttings during the rooting treatment ( for 1 day, followed by boron for 6 days) is beneficial for root development. A progressive loss of response to and boron by cuttings aged in water prior to the rooting treatment is greater in cuttings with leaves in the light during the period of ageing than with leaves in darkness. Possible roles of leaves, auxm and boron in rooting are discussed. INTRODUCTION Cuttings taken from seedlings of Phaseolus aureus Roxb. grown in the light often fail to produce visible roots or even primordia when placed in deionized water. When cuttings are placed in an auxin solution, mitoses occur in the phloem parenchyma at the sites at which root primordia are known to develop (Middleton, 1977) However, primordia appear and roots emerge from the cuttings readily when they are treated subsequently with a solution containing boron. Therefore, it appears that auxin stimulates the initial events of root development but that root growth, even from its earliest stages, is dependent upon an adequate supply of boron (Middleton, Jarvis and Booth, 1978b).,.,., c The existence of endogenous factors apart from auxins which stimulate rooting ot cuttings has been suggested by many workers following the initial report by Bouillene and Went (1933). Such factors seemingly arise in the leaves, buds and cotyledons and include carbohydrates, nitrogenous compounds (van Overbeek, Gordon and Gregory 1946) and a variety of auxin synergists (Gorter, 1958). Treatments which promote rooting may increase the availability of carbohydrates at the site of root development. For example, auxin leads to the mobilization of carbohydrates from the leaves to the hypocotyls of P. vulgaris (Stuart, 1939). More recent work suggests that auxin may act directly in the control of such movement (Booth et al, 1962; Patrick and Wareing, 1973). Altman and Wareing (1975) propose an mdirect effect on carbohydrate accumulation owing to an increased ' sink' resulting from auxm-induced rooting in addition to a direct effect of auxin on carbohydrate movement. Direct control of carbohydrate movement by boron was postulated by Gauch and Dugger X/80/ $02.00/ The New Phytologist

2 252 W. MIDDLETON, B. C. JARVIS AND A. BOOTH (1953). It was proposed that an ionizable boron-sucrose complex facilitates movement of boron through cell membranes. Despite evidence favouring regulatory roles for auxin and boron in carbohydrate translocation and root development, much of the previous work has involved the use of cuttings with established roots acting as sinks when the basal accumulation of carbohydrates could be a consequence and not a cause of rooting. Further complexities arise owing to the implication of auxins in the control of boron levels in tissues (Brunstetter et al., 1948) and because boron might regulate the metabolism, transport or action of auxin (Robertson and Loughman, 1974). Since compounds which influence rooting appear to arise in the leaves, the present report concerns the influence of leaves on root initiation and growth. The effects of auxin and boron on carbohydrate levels in different parts of the cutting and on the translocation of [^*C] sucrose from leaves to the base of the hypocotyl where roots develop has been ascertained. The influence of light has also been studied both prior to, and during the period of, root development. MATERIALS AND METHODS Preparation and treatment of cuttings Details of growth conditions, preparation of cuttings from light-grown seedlings of P. aureus and chemical treatments were as described by Middleton et al. (1978a). Indolebutyric acid () was applied for the initial 24 h of the rooting treatment at a concentration of 10"* mol \-^ in 2 ml 1-^ ethanol, and ethanol (2 ml \-^) was used as the control. Cuttings were then transferred to deionized water or 10 mg 1~^ boric acid solution containing 50 mg T^ calcium chloride for a further 6 d when root numbers and lengths were determined. In one experiment, cuttings were aged in deionized water prior to the standard rooting treatment. The leaves of these cuttings were covered with loose-fitting clear or opaque polythene envelopes during ageing and rooting treatments. Carbohydrate studies For each treatment, at a given sampling time, the basal 1-5 cm of the hypocotyl, the basal 1-5 cm of the epicotyl and the leaves from 16 cuttings were extracted after immersion in liquid nitrogen by homogenization with three changes of 800 ml h^ aqueous ethanol. Trimethylsilyl derivates were prepared according to Sweeley et al. (1963) and the carbohydrates analysed quantitatively by gas-liquid chromatography (HoUigan and Drew, 1971) with calibration curves based on peak areas. Values for glucose, fructose and sucrose were combined and are expressed as total free sugars. Afyo-inositol and another inositol were also present but did not vary in levels. Ethyly?-glucoside synthesized in response to the presence of ethanol showed changes in level only in the first 24 h, as previously described (Middleton et al.., 1978a). In a separate experiment, pcjsucrose (0-2/fCi, 610 mci mmol^^, in 5 fa of 0-2 ml l"'^ Tween 80) was applied to the upper surface of one leaf on each cutting 10 h from the end of or boron treatments. The solution was placed in lanolin rings (5 mm diam.) at the proximal ends of the leaves, the areas of which were rewetted hourly with 5 [A of Tween 80 solution. Fight cuttings were used per treatment. The basal 1-5 cm of the hypocotyls were extracted in 800 ml l^^ aqueous ethanol, the extracts dried in vacuo and the residue partitioned between chloroform and water (1:1)-

3 Rooting of stem cuttings 253 Chlorophyll was removed in the chloroform and the aqueous fractions were assayed for radioactivity using a liquid scintillation spectrometer and the scintillation fluid of Turner (1968). Quench correction was applied by the channels ratio method. RESULTS In water-treated controls, seven roots developed at the base of the cutting whereas, in, 50 roots of uniform length distributed along the whole of the hypocotyl were formed (Table 1). Only when both primary leaves were excised was there a significant reduction in the number of roots formed. Mean root lengths fell into two categories, 6 to 8 and 2 to 3 mm, correlated with the presence or absence of leaves. promoted Table 1. The effect of excision of parts of cuttings 0/Phaseolus aureus on root development Treatment Day 1 Part excised Number of roots per cutting Mean root length (mm) None (control) Apical bud One leaf Both leaves Both leaves + bud None (control) Apical bud One leaf Both leaves Both leaves + bud * 21* » 8-4* * 2-8* * 22* Cuttings standing for the first day in deionized water or solution (both containing 2 ml I~^ ethanol) were transferred to a solution containing boron for days 2 to 7. * Indicates a significant difference from the appropriate controls at the 0-1 % level. root number irrespective of the presence or absence of leaves but it did not affect mean root length. When leaves were present, treatments with water or resulted in comparable values for mean root length, yet gave a sevenfold increase in the number of roots formed. Without leaves, only two short roots developed after pretreatment in water. In, similar short roots were regenerated but there was a fourfold increase in the number formed. Thus, although enhanced total root growth in cuttings with and without leaves, its effect was much greater in their presence. Since sugar may be a limiting factor for both root initiation and growth, the effects of on total free sugar levels have been compared in leaves, epicotyls and hypocotyls of cuttings treated for one day in or in water. As the development of roots in light-grown cuttings is dependent on the presence of boron in the rooting solution (Middleton et al., 1978b), cuttings from both and water first-day treatments were subsequently transferred to water and to a solution containing boron. Treatment with led to a marked and immediate increase in sugars in the leaves and, to a lesser extent, in the epicotyls (Fig. 1). Subsequently, sugar levels in the leaves and epicotyl declined and there were concomitant increases in sugars in the hypocotyl, the site of root development. A substantial increase in sugars in the hypocotyl occurred only when boron was present. Samples from cuttings in water showed almost no changes m sugar levels in the first day. Sugars increased in the leaves in the second and third

4 254 W. MIDDLETON, B. C. JARVIS AND A. BOOTH days. Again, sugar levels increased in the epicotyl and hypocotyl only when boron was present. Thus, the two treatments which led to the largest increases in sugars in the hypocotyl were those which resulted in root development. However, there was not a close 600 S I ''00 ^ 200' 0 ^ 400 (b) O -o 600 C) I * 400 I 200'.Q... 0 I I 2 3 Days Fig. 1. The effects of and boron on total sugar levels in the (a) leaf, (b) epicotyl and (c) hypocotyl of cuttings of Phaseolus aureus. Cuttings standing for the first day in water (O) or in ( ) (both containing 2 ml 1~^ ethanol) ( ) were transferred to solution containing boron ( ) or to deionized water ( ) for the second and third days.

5 Rooting of stem cuttings 255 correspondence between sugar levels and the numbers of roots formed in these treatments (seven with water followed by boron, 50 with followed by boron). Cuttings transferred from to water showed some increase in sugars in the hypocotyl but did not develop roots. Sugar analyses give no indication of the rate of utilization of sugars in respiration and growth because continued photosynthesis in the leaves or the hydrolysis of reserves will supply translocates to the rooting region. Measurements of ^*C activity in hypocotyls following foliar application of [^*C]sucrose to leaves showed that translocation to the hypocotyl did take place in the first 24 h and that the amount of ^*C transported was almost doubled with (Table 2), notwithstanding the larger pool of free sugar in the leaves in this treatment and the absence of root 'sinks'. Also, much greater ^*C activity was recovered from the hypocotyls of -treated cuttings transferred to a solution containing boron as compared to water. Table 2. ^*C activity in the basal halves of hypocotyls of cuttings of Phaseolus aureus following foliar application of \^^C']siu:rose 10 h before the end of the given treatment Treatment Radioactivity Day 1 Day 2 Boron D min-i»/ 0 of applied If the action of leaves in supporting -promoted rooting is mediated by products of carbon assimilation, then rooting should vary according to the amount of light received by the leaves. A simple experiment was designed in which the primary leaves were held in loose-fitting clear or opaque polythene envelopes. The remaining parts of the cuttings were in the light. Cuttings with envelopes were stood in water for up to 4 days in order to increase the differences between these treatments. The cuttings were then subjected to the standard rooting treatment of for 1 day followed by boron solution for 6 days. Illumination of the leaves of fresh cuttings w^as markedly beneficial to root initiation since some 40 % more roots were formed than on cuttings with darkened leaves (Fig. 2). Ageing prior to rooting treatment resulted in a rapid and progressive loss of rooting which was accelerated by illumination during ageing. In contrast, illumination during the subsequent rooting period promoted rooting although this effect was lost in cuttings aged for more than 2 days with leaves in the light. Ageing in the light led to reduced mean root length but had no effect in the dark. The total length of roots per cutting decreased with ageing in light and dark. Total length of roots is influenced by root number rather than mean length because of the large differences between the number of roots initiated under the different conditions. Light during rooting was beneficial to root growth except where cuttings were aged for more than 2 days in the light. The basis for the ageing effects could be leaf senescence in the sense of proteolysis and chlorophyll degradation. It would be necessary to suppose that such senescence

6 256 W. MIDDLETON, B. C. JARVIS AND A. BOOTH c o o E E ^ CT C 100 o 50 D O 10 ±J Period of aaeina (davs) Fig. 2 The effects of ageing cuttings of Phaseolus aureus for 0 to 4 days in water prior to a root-inducing treatment ( for 1 day, followed by boron for 6 days) on root initiation and growth. Cuttings were aged with their leaves in the light (O and #) or in the dark (D and ). During tbe root-inducing treatment they were maintained with their leaves in the light ( ) or in the dark ( ). Lines are drawn through fitted values derived from logarithmically transformed raw data analysed according to Hunt and Parsons (1974). Vertical bars denote 95 % confidence limits.

7 Rooting of stem cuttings 257 is accelerated in the light. However, senescence was not apparent visually within the period of ageing in either light or dark. In cuttings aged for 3 days, illuminated leaves had 770 fig of sugar per leaf and darkened leaves had 345 /tg so that the diminished rooting of cuttings aged in the light clearly does not result from deficiency of sugars in the leaves. DISCUSSION It is convenient to consider root initiation and root growth as distinct processes and to regard auxin as the root initiator with boron being required to maintain root growth (Hemberg, 1951; Middleton et ah, 1978b). However, conclusions about the factors limiting root development require assumptions to be made about endogenous substances in addition to those given in treatments. Boron alone does support the development of a small number of roots, and, generally, it is assumed that the auxin requirement for initiation is met by endogenous auxin. In P. aureus, this would be derived from the leaves. alone results in no root development under the experimental conditions used here and this is believed to result from a lack of boron for ^owth. A greatly increased number of roots results when is followed by the supply of boron. The excision experiment has confirmed the well-established role of leaves as sources of substances required for the development of adventitious roots on cuttings (see, for example, Altman and Wareing, 1975). Such substances must be transported basipetally and accumulate at the base of the cutting. At the time cuttings were prepared, sufficient rooting substances were present in epicotyl and hypocotyl to allow approximately two roots to develop (water treatments, without leaves). The additional five roots formed when leaves are present must result from the movement of rooting substances out of leaves during the rooting period. Rooting may be limited by endogeous auxin in water-treated cuttings and hence treatment with would be expected to increase the number of roots formed. In cuttings without leaves, promotes the development of some six roots more than the two formed with water treatment. In cuttings with leaves the additional roots resulting from treatment exceed 40. Thus, although may have a direct altect on rooting by substituting for endogenous auxin, it has an additional action which operates indirectly and chiefly via the leaves. Labelled auxin is readily detectable in leaves within 2 h of supplying [l-^*c]auxin in solution to the hypocotyl (unpublished observation). Sugar analyses indicate that alone promotes an immediate increase in free sugars in the leaves but boron alone does not. Boron leads to increases in sugars in the hypocotyls. An obvious interpretation is that enhances the concentration of sugars in the leaves and that boron facilitaties their transport to the hypocotyl. The higher level of sugar in the hypocotyl, linked with a direct action of, then leads to substantial root initiation. However, during the first day, when root initiation takes place (Middleton, 1977), sugar levels in the hypocotyl have not been enhanced by rootinducing treatments. This suggests that root initiation is not limited by sugars. After 3 days, when developing root primordia will be utilizing sugars, free sugars in the hypocotyl have increased and have increased most in the treatment followed by boron in which the highest number of root primordia are present. Sugars would not be expected to be limiting root growth here and this is borne out by the finding that mean root length in this treatment is identical with that in cuttings transferred from to boron in which far fewer roots are formed.

8 258 W. MIDDLETON, B. C. JARVIS AND A. BOOTH In contrast with the evidence from sugar analyses, does enhance the accumulation of i*c activity in hypocotyls after 24 h, following foliar application of ["C] sucrose. However, sugars may be only one of a number of substances mobilized in response to treatment with and a substance other than sugars may be the limiting factor for root initiation. The ageing experiment reveals a paradoxical situation in which illumination of the leaves of cuttings during treatment with followed by boron promotes rootiag, whereas illumination with the cuttings standing in water decreases the ability of and boron subsequently to promote rooting. A possible key to interpreting these effects is boron. It is likely that incipient boron deficiency occurs shortly after cuttings are made since roots are not produced by cuttings in water but an average of seven develop the presence of boron. Boron readily complexes with cw-hydroxyl configurations. Levels of compounds containing these are likely to be higher in the light since secondary carbon metabolism mediated by phenylalanine ammonia lyase is light-stimulated (Zucker, 1965) and would lead to synthesis of polyphenols capable of complexing with boron (Lee and Aronoff, 1965). In P. aureus, dark-grown seedlings do not develop the phenolic-based pigments which are present in light-grown seedlings and which are indicative of secondary metabolism. Early root development in hypocotyls of dark-grown cuttings is not dependent on an exogenous supply of boron but boron is required for root growth (Middleton et al, 1978b). Thus light brings forward in time a requirement for boron. Consequently, during ageing, cuttings with leaves in the light may be expected to become boron deficient earlier than cuttings with their leaves in darkness. The increased rooting of cuttings with leaves in the light and the reversal of the effects of light during ageing suggest that rooting substances are formed during the period of exposure to followed by boron. Changes in leaf metabolism brought about by boron deficiency in the ageing period may lower the capacity of the leaves to respond to the rooting treatment. The production or mobilization of rooting substances does not proceed at its former level when boron is supplied after ageing. The similarities between deficiency symptoms for boron and auxin were remarked upon by Eaton (1940) and his suggestion that boron functions in auxin synthesis was supported by the experiments of Dyar and Webb (1961). The leaf-formed rooting substances could include endogenous auxin and the loss of response of aged cuttings to rooting treatments might indicate a gradual failure of auxin biosynthesis, brought about by light-accelerated boron deficiency. On this basis, the chief effect of would be to enhance substrate levels in the leaves of fresh cuttings such that the synthesis of endogenous auxin would be promoted. Current work is concerned with investigating such a possibility. ACKNOWLEDGEMENT The S.R.C. studentship awarded to W.M. is gratefully acknowledged. REFERENCES ALTMAN, A. & WAREING, P. F. (1975). The effect of IAA on sugar accumulation and basipetal transport of "C-labelled assimilates in relation to root formation in Phaseolus vulgaris cuttings. Physiowgi x ^ X M o f K;? J., DAViBS, C. R., JONES, H. & WAKEINO, P. F. (1962)^ Effects of indolyl-3- acetic acid on the movement of nutrients within plants. Nature, London, iy4, lyn.

9 Rooting of stem cuttings 259 BouiLLENE, R. and WENT, F. W. (1933). Discussion des resultats obtenus dans les experiences de neoformation radiculaire chez Impatiens balsamina et acalypha. Annales du Jardin botanique Buitenzorg, 43, 155. BRUNSTETTER, B. C, MYERS, A. T., MITCHELL, J. W., STEWART, W. S. & KAUFMAN, M. W. (1948). Mineral composition of bean stems treated with 3-indoleacetic acid. Botanical Gazette, 109, 268. DYAR, J. J. & WEBB, K. L. (1961). A relationship between boron and auxin in "C translocation in bean plants. Plant Physiology, Lancaster, 36, 672. EATON, F. M. (1940). Interrelations in tbe effects of boron and indoleacetic acid in plant growth. Botanical Gazette, 101, 700. GAUCH, H. G. & DUGGER, W. M. JR. (1953). Tbe role of boron in the translocation of sucrose. Plant Physiology, Lancaster, 28, 457. GoRTER, C. J. (1958). Synergism of indole and indole-3-acetic acid in root production of Phaseolus cuttings. Physiologia Plantarum, 11, 1. HEMBERG, T. (1951). Rooting experiments with hypocotyls of Phaseolus vulgaris. L. Physiologia Plantarum, 4, 358. HoLLiGAN, P. M. & DREW, E. A. (1971). Routine analysis by gas-liquid chromatography of soluble carbohydrates in extracts of plant tissues. II. Quantitative analysis of standard carbobydrates, and the separation and estimation of soluble sugars and polyols from a variety of plant tissues. New Phytologist, 70, 271. HUNT, R. & PARSONS, I. T. (1974). A computer program for deriving growth functions in plant growth Analysis. Journal of Applied Ecology, 11, LEE, S. & ARONOFF, S. (1967). Boron in plants: A biochemical role. Science, New York, 158, 798. MIDDLETON, W. (1977). Root development in cuttings oi Phaseolus aureus Roxb. Pb.D. thesis. University of Sheffield. MIDDLETON, W., JARVIS, B. C. & BOOTH, A. (1978a). The effects of ethanol on rooting and carbohydrate metabolism in stem cuttings of Phaseolus aureus Roxb. New Phytologist, 81, 279. MIDDLETON, W., JARVIS, B. C. & BOOTH, A. (1978b). The boron requirement for root development in stem cuttings of Phaseolus aureus Roxb. New Phytologist, 81, 287. OvERBEEK, J. VAN, GORDON, S. A. & GREGORY, L. E. (1946). An analysis of the function of the leaf in the process of root formation in cuttings. American Journal of Botany, 33, 100. PATRICK, J. W. & WAREING, P. F. (1973). Auxin-promoted transport of metabolites in stems of Phaseolus vulgaris L. Journal of Experimental Botany, 24, ROBERTSON, G. A. & LOUGHMAN, B. C. (1974). Response to boron deficiency: a comparison with responses produced by chemical methods of retarding root elongation. New Phytologist, 73, 821. STUART, N. W. (1939). Nitrogen and carbohydrate metabolism of bean cuttings as affected by indoleacetic acid. Botanical Gazette, 100, 298. SwEELEY, C. C, BENTLEY, R., MAKITA, M. & WELLS, W. W. (1963). Gas-liquid chromatography of trimethylsilyl derivatives of sugars and related substances. Journal of the American Chemical Society, 85, TURNER, J. C. (1968). Triton X-100 scintillant for carbon-14 labelled materials. International Journal of Applied Radiation and Isotopes. 19, 557. ZucKER, M. (1965). Induction of phenylalanine deaminase by light and its relation to chlorogenic acid synthesis in potato tuber tissue. Plant Physiology, Lancaster, 40, 779.

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