Hormones in Plants Bearing Nitrogen-Fixing Root Nodules: The Nodule as a Source of Cytokinins in Alnus glutinosa (L.) Gaertn

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Hormones in Plants Bearing Nitrogen-Fixing Root Nodules: The Nodule as a Source ofCytokinins in Alnus glutinosa (L.) GaertnAuthor(s): C. T. Wheeler and I. E. HensonSource: New Phytologist, Vol. 80, No. 3 (May, 1978), pp. 557-565Published by: Wiley on behalf of the New Phytologist TrustStable URL: http://www.jstor.org/stable/2431213 .Accessed: 17/06/2014 23:52Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at .http://www.jstor.org/page/info/about/policies/terms.jsp .JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range ofcontent in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new formsof scholarship. For more information about JSTOR, please contact support@jstor.org. .Wiley and New Phytologist Trust are collaborating with JSTOR to digitize, preserve and extend access to NewPhytologist.http://www.jstor.org This content downloaded from 185.44.78.129 on Tue, 17 Jun 2014 23:52:57 PMAll use subject to JSTOR Terms and Conditionshttp://www.jstor.org/action/showPublisher?publisherCode=blackhttp://www.jstor.org/action/showPublisher?publisherCode=npthttp://www.jstor.org/stable/2431213?origin=JSTOR-pdfhttp://www.jstor.org/page/info/about/policies/terms.jsphttp://www.jstor.org/page/info/about/policies/terms.jspNew Phytol. (1978) 80,557-565. HORMONES IN PLANTS BEARING NITROGEN-FIXING ROOT NODULES: THE NODULE AS A SOURCE OF CYTOKININS IN ALNUS GL UTINOSA (L.) GAERTN. By C. T. WHEELER and I. E. HENSON* Department of Botany, University of Glasgow, Glasgow G12 8QQ, Scotland (Received 29 November 1977) SUMMARY Levels of endogenous cytokinins in various parts of nodulated and non-nodulated alder plants were estimated using the soybean callus bioassay. In three separate experiments, differences in levels of total plant cytokinin activity were attributed primarily to growth differences between plants, other than nodulation. The higher percentage of total plant cyto- kinin activity found in the leaves of non-nodulated plants may be related at least in part to the better development of the root system of these plants compared to those with nodules. In other experiments growth differences were minimized by comparing intact plants with plants which were either denodulated or root-pruned or both. All three surgical treat- ments had similar effects, reducing levels of cytokinins in the stems, while leaf cytokinin levels were not affected greatly. The relative stability of leaf cytokinin levels was also evident following leaf detachment. Factors responsible for the maintenance of cytokinin levels in the leaf are discussed. The results suggest that nodules are unlikely to make a major contribution to shoot cytokinin content, although export of endogenous cytokinins from the nodules cannot be excluded entirely. INTRODUCTION Experimental support for the suggestion (Rodriguez-Barrueco, 1968; Gibson, 1974; Becking, 1975; Gladstones, Loneragan and Goodchild, 1977), that nodules may export cytokinins to the rest of the plant, thus supplementing the roots as a source of hormone (Kende, 1971; Skene, 1975), has been obtained previously by radiotracer studies with [8-14C] -zeatin. Following application by micropipette to the root nodules of Alnus glutinosa (L.) Gaertn., radioactivity from [8-14C] -zeatin was found in all parts of the plant within 24 h (Henson and Wheeler, 1977b). A significant portion of the n-butanol-soluble radioactivity in stems and leaves was associated with zeatin, while zeatin riboside was a prominent metabolite of this cytokinin. Movement to the shoot was not interrupted by stem-ringing, suggesting that transport was mainly in the xylem (I. E. Henson, unpublished). Hence, the transport of nodule cytokinins to other plant parts may occur. Nevertheless, the transport of exogenous cytokinin, applied in relatively large doses by micropipette, may not reflect the actual movement of endogenous compounds. As alternative approaches we have attempted to assess the extent to which nodules may affect levels of cytokinin activity in other plant parts, firstly by means of direct comparisons between nodulated and * Present address: Plant Breeding Institute, Maris Lane, Trumpington, Cambridge CB2 2LQ. OO28-646X/78/0500-0557$02.00 ?1978 Blackwell Scientiflc Publications 557 This content downloaded from 185.44.78.129 on Tue, 17 Jun 2014 23:52:57 PMAll use subject to JSTOR Terms and Conditionshttp://www.jstor.org/page/info/about/policies/terms.jsp558 C. T. WHEELER and I. E. HENSON non-nodulated plants supplied with combined nitrogen, and secondly, by assessment of the effects of denodulation and root pruning on cytokinin levels. The limitations of these approaches are discussed further below. MATERIALS AND METHODS Plant material Plants of Alnus glutinosa were raised in a heated glasshouse from seed collected locally. Nodulated plants were obtained by inoculation of seedling roots with a crushed nodule suspension. The plants were grown in water culture in half-strength Crone's solution (nitrogen- free formula), supplemented where necessary with nitrogenous salts as described below. In Experiment 1 (1975), the nutrient solution was supplemented with 15 mg, and later, before inoculation at 10 weeks after sowing, with 30 mg N per 1 as ammonium nitrate. The amount of N supplied to the inoculated plants was then reduced progressively and removed altogether 7 weeks before harvest. Uninoculated plants continued to receive combined N, the amount being increased progressively to 100 mg per 1. Culture solutions were changed regularly (every 10-14 days during the period of most rapid growth) and the pH adjusted to pH 4.5-4.8 as necessary. A check in the growth of the nodulated plants occurred 14-15 weeks after sowing due to failure to adjust the culture solution pH at this time. The plants were harvested 6 months after sowing in early September. In Experiment 2 (1976), seedlings were supplied initially with 7.5 mg N per l as ammonium sulphate until inoculation 6 weeks after sowing. Nitrogen was withheld from inoculated plants 4 weeks after inoculation. Uninoculated plants continued to be supplied with com- bined N, which was increased progressively to 50 mg per 1. The culture solutions were main- tained as described above and plants harvested 6 months after sowing in late July. In Experiment 3 (1977), seedlings were supplied with 10 mg N per 1 as ammonium sul- phate until inoculation 8 weeks after sowing. Nitrogen was withheld from inoculated plants 3 weeks after inoculation, but was restored after 7 days because of poor plant growth. Nitrogen was withheld completely from these plants 9 weeks after inoculation, when good nodulation was observed. Uninoculated plants continued to be supplied with combined N, which was increased progressively to 50 mg N per 1. Culture solutions were maintained as described above and plants harvested 7 months after sowing in late July. In experiments to study the effects of root pruning and denodulation, all plants were inoculated and combined nitrogen was withheld 4 weeks after inoculation. Seven days before harvest (6 months after sowing) the plants were divided into four comparable groups for denodulation and root pruning. All nodules visible to the eye were removed during denodulation while the lower half to two thirds of the root system (equivalent to about 25% of total root fresh weight) was removed by pruning. Combined nitrogen was then sup- plied to denodulated plants until the experiment was harvested. Conservation of cytokinin activity in detached leaves was studied using mature, expanded leaves which were supplied with nutrient solution (quarter strength Crone's salts plus 50 mg 11 N as ammonium nitrate) through the petiole base for 1-3 days before extraction. Detached leaves and plants from which 'attached', control leaves were to be taken, were maintained in a controlled environment room with 20 h photoperiod, 190C day and 150C night tempera- tures. Extraction, partial purification, chromatography and bioassay of cytokinins Plant material was extracted three times in methanol:water (4:1, v/v) using 10 ml/g This content downloaded from 185.44.78.129 on Tue, 17 Jun 2014 23:52:57 PMAll use subject to JSTOR Terms and Conditionshttp://www.jstor.org/page/info/about/policies/terms.jspCytokinins in alder nodules 559 fresh weight, and the methanol extract was partially purified as described by Henson and Wheeler (1976) to yield two fractions with cytokinin activity. Fraction I contains cytokinin free bases and their glycosides and was found, as in previous work (Henson and Wheeler, 1976; 1977a), to contain the majority of the cytokinin activity. Results are presented for this fraction only, as activity in the other fraction (expected to contain ribosides, derived from ribotides by enzymic hydrolysis) was comparatively minor. Purified fractions were chromatographed on Whatman 3MM paper, developed in iso- propanol: ammonia: water (10: 1: 1 v/v) and subsequently tested for cytokinin activity using the soybean callus bioassay (Miller, 1968). For further details see Henson and Wheeler (1976, 1977a). Determination of total nitrogen, chlorophyll and protein contents, leaf area and root tip numbers Total nitrogen of oven-dried (900C for 24 h) material was determined by a mico-Kjeldahl procedure. The chlorophyll content of 80% acetone extracts of leaves was determined according to Arnon (1949) and leaf areas by comparison of the weights of cut-outs of Xerox prints of detached leaves with weight/area calibration curves. Protein levels were measured by the method of Lowry et al. (1951) after extraction of leaves in 10% trichloroacetic acid, removal of chlorophyll and lipids by successive extraction of the TCA-insoluble residue with ethanol (twice) and ether: ethanol: chloroform (2: 1: 1, v/v, twice) and resolubilization of the residue in 1 N NaOH (900C, 15 min). Bovine serum albumin was used as standard. Root tip numbers were assessed by visual counts of at least three weighed root samples (0.1-0.2 g fresh weight) per plant. Five plants were assessed for each treatment. RESULTS Comparison of nodulated and non-nodulated plants Experiment 1. Despite a check in the growth of nodulated plants (see Methods) the concentration of total nitrogen in the two treatments was not greatly different except in the leaves, where there was a slightly higher level in nodulated plants (Table la). Differences in the concentration of chlorophyll in the leaves of the two sets of plants and in the ratio of leaf area/weight were also small (Table lb). The large differences in root fresh weight per plant were reflected in the greater number of root apices of non-nodulated plants, al- though the frequency of occurrence of root apices (number per g fresh weight) was actually greater in the nodulated plants (Table lb). Non-nodulated plants contained higher levels of cytokinin activity, both on a fresh weight basis and per plant, than nodulated plants (Table lc). Total cytokinin levels of nodulated plants were only about 50% of non-nodulated plants. The highest percentage of total plant cytokinin activity was contained in the root system (roots alone or roots + nodules) and was almost equal in the two sets of plants. The stems of nodulated plants contained a higher percentage of total plant cytokinin than those of non-nodulated plants while the reverse was true of the leaves. Experiment 2. The reduced root development of nodulated plants was the main growth difference between the two sets of plants in this experiment (Table 2a). As in Experiment 1, the frequency of occurrence of root apices was higher in nodulated plants but the larger This content downloaded from 185.44.78.129 on Tue, 17 Jun 2014 23:52:57 PMAll use subject to JSTOR Terms and Conditionshttp://www.jstor.org/page/info/about/policies/terms.jsp560 C. T. WHEELER and I. E. HENSON Table 1. Comparison of non-nodulated and nodulated plants of Alnus glu tinosa. Experiment 1 (1975) (a) Plant growth and nitrogen content Total N Fresh wt (g per plant)* (mg g-' dry wt) Plant part No nodules Nodulated No nodules Nodulated Leaves 7.89 (30.3) 7.50 (41.5) 18.10 21.60 Stems 6.3 8 (24.5) 4.93 (27.4) 9.05 8.90 Roots 11.76 (45.2) 4.99 (27.6) 15.15 15.08 Nodules - - 0.65 (3.5) - 30.30 Total 26.03 (100.0) 18.07 (100.0) *Means of sixteen plants. Data in parentheses are percentages of total fresh weight. (b) Leaf chlorophyll, leaf area/weight ratios and numbers of root apices Leaf chlorophyll Leaf area/wt Number of root apices (mg g-1 (cm2 g-1 g- Iroot Per fresh wt) fresh wt) fresh wt plant No nodules 0.89 72.0 375 4410 Nodulated 1.06 68.5 464 2315 (c) Cytokinin activity in Fraction 1. Data are means of two separate extractions and bioassays ,ug kinetin equivalents ng kinetin equivalents per kg fresh wt per plant* Plant part No nodules Nodulated No nodules Nodulated Leaves 165 59 1301 (25.7) 445 (17.5) Stems 211 194 1347 (26.6) 953 (37.6) Roots 204 155 2409 (47.6) 774 (30.5) Nodules - 560 - - 364 (14.4) Total 5057 (100.0) 2536 (100.0) *Data in parentheses are percentages of total cytokinin content. root fresh weight of non-nodulated plants ensured that these had more apices per plant (Table 2b). Levels of total nitrogen and leaf chlorophyll were slightly higher in nodulated plants as were leaf area/weight ratios (Table 2a,b). By contrast to Experiment 1, higher levels of cytokinin activity were found in nodulated plants, with the roots and stems showing the largest differences (Table 2c). On a per plant basis, cytokinin levels of nodulated plants were more than twice those of non-nodulated plants, despite the slightly larger total fresh weight of the latter. About 50% of the total cytokinin activity of nodulated plants was concentrated in the root system (root + nodules) with the remaining 50% distributed almost equally between leaves and stems. In non-nodulated plants, the leaves contained about 50% of the total activity with the remainder distributed almost equally between roots and stems. Experiment 3. The stem fresh weight of plants in this experiment was somewhat higher than that of the plants in Experiment 2 but leaf fresh weight was rather less (Table 3a). Root fresh weight comprised a similar percentage of total plant fresh weight as in experiment 2 but the numbers of root apices per plant was lower than in experiment 2 by about 45% in both treatments (Table 3b). However, the occurrence of a higher frequency of root apices in nodulated plants and a larger number of root apices per non-nodulated plant was in accord with ithe results obtained for plants in Experiments 1 and 2. The levels of total nitrogen This content downloaded from 185.44.78.129 on Tue, 17 Jun 2014 23:52:57 PMAll use subject to JSTOR Terms and Conditionshttp://www.jstor.org/page/info/about/policies/terms.jspCytokinins in alder nodules 561 Table 2. Comparison of non-nodulated and nodulated plants of Alnus glutinosa. Experiment 2 (1976) (a) Plant growth and nitrogen content Total N Fresh wt (g per plant)* (mg g-1 dry wt) Plant part No nodules Nodulated No nodules nodulated Leaves 4.50 (39.7) 4.22 (44.5) 17.1 21.3 Stems 2.33 (20.6) 2.22 (23.4) 6.8 10.5 Roots 4.50 (39.7) 2.39 (25.2) 11.5 13.5 Nodules - - 0.65 (6.9) - 29.6 Total 11.33 (100.0) 9.48 (100.0) *Means of eighteen plants. Data in parentheses are percentages of total plant weight. (b) Leaf chlorophyll, leaf area/weight ratios and numbers of root apices Leaf chlorophyll Leaf area/wt Number of root apices (mg g-1 (cm2 g- g1- root Per fresh wt) fresh wt) fresh wt plant No nodules 0.64 65.6 571 2569 Nodulated 0.87 71.3 924 2208 (c) Cytokinin activity in Fraction 1. Data are means of three separate extractions and bioassays ,g kinetin equivalents ng kinetin equivalents per kg fresh wt per plant* Plant part No nodules Nodulated No nodules Nodulated Leaves 42.4 59.3 196 (52.0) 254 (27.3) Stems 36.2 91.3 84 (22.3) 209 (22.5) Roots 21.4 136.6 97 (25.7) 321 (34.5) Nodules - 223.8 - - 146 (15.7) Total 377 (100.0) 930 (100.0) *Data in parentheses are percentages of total cytokinin content. in nodulated plants were higher than in non-nodulated plants (Table 3a) but leaf chlorophyll contents and leaf area/weight ratios were similar for both sets of plants (Table 3b). That the cytokinin content of non-nodulated plants was somewhat higher than nodulated plants, was attributable partly to their greater size and partly to higher cytokinin levels in the leaves of these plants. Effects of root pruning and denodulation on cytokinin levels In order to minimize the effects of growth differences between sets of nodulated and non-nodulated plants, the use of denodulation techniques was explored as a means of in- vestigating contributions made by the nodules to the shoot. As roots are probably a major source of cytokinins for the shoot, the effects of denodulation were compared with those of root pruning. Bioassay of extracts of treated plants showed little effect of either denodulation or root pruning, or a combination of both treatments, on leaf cytokinin levels (Table 4), although it should be noted that in all plants, including untreated controls, leaf cytokinin levels were lower than in other experiments (Tables 1-3). In stems, cytokinin levels in both denodulated and root-pruned plants were about 45% lower than controls although no greater reduction in levels was obtained when the two treatments were combined. Separate assays of root nodule extracts showed the cytokinin content of the nodules to be unaffected by root pruning. This content downloaded from 185.44.78.129 on Tue, 17 Jun 2014 23:52:57 PMAll use subject to JSTOR Terms and Conditionshttp://www.jstor.org/page/info/about/policies/terms.jsp562 C. T. WHEELER and I. E. HENSON Table 3. Comparison of non-nodulated and nodulated plants of Alnus glutinosa. Experiment 3 (1977) (a) Plant growth and nitrogen con tent Total N Fresh wt (g per plant)* (mg g'- dry wt) Plant part No nodules Nodulated No nodules Nodulated Leaves 4.17 (35.2) 3.45 (38.9) 18.4 25.0 Stems 3.45 (29.2) 2.46 (27.7) 6.7 8.8 Roots 4.21 (35.6) 2.32 (26.2) 12.1 l 20.1 Nodules 0.64 (7.2) - Total 11.83 (100.0) 8.87 (100.0) *Means of twenty-eight plants. Data in parentheses are percentages of total fresh weight. (b) Leaf chlorophyll, leaf area/weight ratios and numbers of root apices Leaf chlorophyll Leaf area/wt Number of root apices (mg g-1 (cm2 g-I g-1 root Per fresh wt) fresh wt) fresh wt plant No nodules 1.18 70.1 337 1419 Nodulated 1.14 69.1 537 1245 (c) Cytokinin activity in Fraction 1. Data are means of five replicate bioassays Atg kinetin equivalents ng kinetin equivalents per kg fresh wt per plant* Plant part No nodules Nodulated No nodules Nodulated Leaves 40.0 27.5 167 (52) 95 (32) Stems 11.0 22.1 38 (12) 54 (18) Roots 27.3 42.9 115 (36) 100 (35) Nodules - N.D. - - N.D. (15) Total 320 (100) 249 (85) *Data in parentheses are percentages of total cytokinin content. The cytokinin content of the nodules was not determined (N.D.) and percentage contents of nodulated plants have been calculated assuming that the nodules contain 15% of total plant activity (average of percentage content of nodules in Experi- ments 1 and 2.). Table 4. Effect of denodulation and root pruning on the cytokinin content of stems and leaves ofAlnus glutinosa Treatment ,ug kinetin equivalents per kg fresh wt Leaves Stems Control 18.1 (100.0) 52.7 (100.0) Nodules removed 15.7 (86.7) 29.0 (55.0) Roots pruned 20.9 (115.4) 28.9 (54.8) Nodules removed and roots pruned 23.3 (128.7) 28.0 (53.1) Data are means of two separate extractions and bioassays. Values in parenthesis are percentages of controls. Effects of detachment on leaf cytokinin levels The lack of effect of root surgical treatments on leaf cytokinin levels may have been a consequence either of an additional import of cytokinins stored in the stem, of a slow rate of utilization, or of formation within the leaf. Experiments were conducted with detached leaves to investigate these possibilities. In an initial experiment no significant differences were found between the levels of cyto- kinin activity in attached (control) leaves and leaves detached 24 h prior to extraction. After This content downloaded from 185.44.78.129 on Tue, 17 Jun 2014 23:52:57 PMAll use subject to JSTOR Terms and Conditionshttp://www.jstor.org/page/info/about/policies/terms.jspCytokinins in alder nodules 563 detachment for three days however, a small difference was evident (Table 5) with the levels of cytokinin in detached leaves being reduced by 17% (Peak I at RF 0.3) and 30% (Peak II at RF 0.6) below the controls. Leaf chlorophyll and protein levels both declined substantially during this period, to 62% and 53% respectively of control levels. Table 5. Effects of detachment on cytokinin activity, chlorophyll, and protein levels in leaves of Alntts glu tinosa Treatment Cytokinin activity* Chlorophyll Protein (,ig kinetin equivalents kg-' fresh w t) (mg g-1 dry wt) (mg g-l dry wt) Peak I Peak II Total Attached 11.5 13.1 24.6 13.3 143.8 Three days detached 9.6 9.2 18.8 8.3 77.1 *Means of two experiments. DISCUSSION Some of the difficulties associated with experiments in which a comparison of physiological or metabolic data obtained from nodulated or non-nodulated plants is attempted are illus- trated by the results of Experiments 1-3. Although differences in shoot growth of nodulated and non-nodulated plants were minimized by control of the supply of combined nitrogen, the reduced root fresh weight of nodulated plants, compared with non-nodulated plants, dic- tates that considerable caution must be exercised when attempting to correlate nodulation with the cytokinin content. Differences in the conditions of plant growth in each experi- ment presumably were mainly responsible for variations in the levels and in the distribution of cytokinin between different plant parts; e.g. although in Experiment 2 the total cytokinin content of nodulated plants was much greater than that of non-nodulated plants, the reverse was true in Experiments 1 and 3. The variability in the relative amounts and distribution of cytokinins on separate occasions suggests that nodules had little or no direct effect on the cytokinin content of other plant parts compared to the effects of other changes resulting from interactions between the plant and its environment. The only consistent difference in the cytokinin content of the various parts of nodulated and non-nodulated plants was the higher percentage of the total plant cytokinins in the leaves of the latter plants (Tables lc-3c). This again would suggest that, despite the high cytokinin content of the nodules (Tables lc, 2c; Henson and Wheeler, 1977a), translocation of cyto- kinins from the nodule to the shoot either does not occur or is of only minor significance in relation to other sources of supply and other factors in determining cytokinin levels in other plant parts. The roots have been suggested as a major source of cytokinins for the shoot (Kende, 1971; Skene, 1975) so that the larger percentage of total plant cytokinin activity in the leaves of non-nodulated alders may reflect particularly the greater root growth and greater number of root tips of non-nodulated plants. Surgical treatments were used as an alternative method of investigating relationships between the presence of nodules and plant cytokinin levels. Although the developmental differences which arise between nodulated and non-nodulated plants during prolonged growth can be minimized by this technique, these particular experimental difficulties may be replaced by artefacts attributable to wounding effects or to the replacement of the nodule as the N source by a supply of combined N in the culture medium. In the experiments con- ducted here (Table 4), the similarity of the effects upon shoot cytokinin levels of nodule This content downloaded from 185.44.78.129 on Tue, 17 Jun 2014 23:52:57 PMAll use subject to JSTOR Terms and Conditionshttp://www.jstor.org/page/info/about/policies/terms.jsp564 C. T. WHEELER and I. E. HENSON excision, and of root pruning, and of both of these treatments combined, do suggest that wounding itself may be an important factor limiting translocation of cytokinins from the root system to the shoot in alder. Although leaf cytokinin levels were affected little by the various surgical treatments such treatments did reduce levels in stems, implying either considerable turnover, or movement of cytokinins out of the stem. It is possible therefore, that stabilization of leaf cytokinin levels during partial interruption of supplies from the root might have been achieved by drawing on 'pools' of cytokinins in the stem. That this is not the sole mechanism for maintenance of leaf cytokinin levels was suggested by the absence, following leaf detachment, of any rapid decline in cytokinin levels such as that observed, for example, in leaves of Xanthium stru- marium (Henson and Wareing, 1977). It is thus probable that stabilization of leaf cytokinin levels in alder is achieved partly by mechanisms within the leaf. For example, the cytokinins may undergo only a slow rate of turnover or they may be renewed in situ by biosynthesis or release from 'bound' forms. It is not possible at present to determine which of these alternatives is operative. Although radioactive zeatin is metabolized rapidly when fed to de- tached alder leaves (Henson, 1978) rates of metabolism of this cytokinin following its exogenous application may not reflect that of the endogenous cytokinins, due to differences in the concentration, localization and nature of these compounds in the tissue. Reports of increases in cytokinin activity in detached leaves and cotyledons (Hewett and Wareing, 1973; Uheda and Kuraishi, 1977) provide some support also for the occurrence of cytokinin synthesis within the leaf. The results presented here do not support the suggestion that alder nodules are a sub- stantial source of cytokinins for other plant parts. Translocation of radioactive cytokinin, applied to nodules by micropipette (Henson and Wheeler, 1977b), may occur by pathways not normally available to the endogenous nodule hormones. The possibility remains, however that a relatively minor local movement of nodule cytokinins may occur which would be too small to demonstrate by the comparative approaches adopted here but which could still have a significant effect upon physiological and developmental events in adjacent root tissues. ACKNOWLEDGMENT We are grateful to the Science Research Council for financial support (grant no. B/RG/7 1340). REFERENCES ARNON, D. I. (1949). Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1. BECKING, J. H. (1975). Root nodules in non-legumes. In: The Development and Function of Roots. Third Cabot Symp. (Ed. by J. G. Torrey and D. T. Clarkson) pp. 507-566. Academic Press, New York. GIBSON, A. H. (1974). The control of dinitrogen assimilation by nodulated legumes. In: Mechanisms of Regulation of Plant Growth (Ed. by R. L. Bieleski, A. R. Ferguson and M. M. Cresswell) pp. 13-22. Bull. Roy. Soc. N.Z. Wellington, 12. GLADSTONES, J. S., LONERAGAN, J. F. & GOODCHILD, N. A. (1977). Field responses to cobalt and molybdenum by different legume species, with inferences on the role of cobalt in legume growth. Aust. J Agric. Res. 28, 619. HENSON, I. E. (1978). Types, formation and metabolism of cytokinins in leaves of Alnus glutinosa (L.) Gaertn. J. exp. Bot. 29, (in press). HENSON, I. E. & WAREING, P. F. (1977). Cytokinins in Xanthium strumarium L.: the metabolism of cytokinins in detached leaves and buds in relation to photoperiod. New Phytol. 78, 27. This content downloaded from 185.44.78.129 on Tue, 17 Jun 2014 23:52:57 PMAll use subject to JSTOR Terms and Conditionshttp://www.jstor.org/page/info/about/policies/terms.jspCytokinins in alder nodules 565 HENSON, I. E. & WHEELER, C. T. (1976). Hormones in plants bearing nitrogen-fixing root nodules: The distribution of cytokinins in Vicia faba L. New Phytol. 76, 433. HENSON, I. E. & WHEELER, C. T. (1977a). Hormones in plants bearing nitrogen-fixing root nodules: Distribution and seasonal changes in levels of cytokinins in Alnus glutinosa (L.) Gaertn. J exp. Bot. 28, 205. HENSON, I. E. & WHEELER, C. T. (1977b). Hormones in plants bearing nitrogen-fixing root nodules: Cytokinin transport from the root nodules of Alnusglutinosa (L.) Gaertn.J. exp. Bot. 28, 1099. HEWETT, E. W. & WAREING, P. F. (1973). Cytokinins in Populus x robusta (Schneid.): Light effects on endogenous levels. Plan ta, 114, 119. KENDE, H. (1971). The cytokinins. Int. rev. Cytol. 31, 301. LOWRY, D. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265. MILLER, C. 0. (1968). Naturally occurring cytokinins. In: The Biochemistry and Physiology of Plant Growth Substances. (Ed. by F. Wightman and G. Setterfield) pp. 33-45. Runge Press, Ottawa. RODRIGUEZ-BARRUECO, C. (1968). In: VII. Simposio Internazionale di Agrochimica. La sintesi biologica delle proteine, pp. 354-360. SKENE, K. G. M. (1975). Cytokinin production by roots as a factor in the control of plant growth. In: The Development and Function of Roots. Third Cabot Symp. (Ed. by J. G. Torrey and D. T. Clarkson) pp. 365-396. Academic Press, New York. UHEDA, E. & KURAISHI, S. (1977). Increase of cytokinin activity in detached etiolated cotyledons of squash after illumination. Plant and Cell Physiol. 18, 481. This content downloaded from 185.44.78.129 on Tue, 17 Jun 2014 23:52:57 PMAll use subject to JSTOR Terms and Conditionshttp://www.jstor.org/page/info/about/policies/terms.jspArticle Contentsp. 557p. 558p. 559p. 560p. 561p. 562p. 563p. 564p. 565Issue Table of ContentsNew Phytologist, Vol. 80, No. 3 (May, 1978), pp. i-ii+489-647+i-xivVolume Information [pp. ]Front Matter [pp. ]A Suggested Cycle of Plastid Developmental Interrelationships [pp. 489-502]Cell Wall Structure and Elongation Growth in Zea mays Coleoptile Tissue [pp. 503-516]Cell Differentiation in Embryoids and Plantlets of Celery Tissue Cultures [pp. 517-521]Plant and Leaf Resistance to Gaseous Air Pollution Stress [pp. 523-534]Response to Soil Moisture Supply in Three Leguminous Species. I. Growth, Reproduction and Mortality [pp. 535-545]Response to Soil Moisture Supply in Three Leguminous Species. II. Rate of N2(C2H2)-Fixation [pp. 547-555]Hormones in Plants Bearing Nitrogen-Fixing Root Nodules: The Nodule as a Source of Cytokinins in Alnus glutinosa (L.) Gaertn [pp. 557-565]Interaction between a Va Mycorrhiza and Azotobacter and their Effects on Rhizosphere Microflora and Plant Growth [pp. 567-573]Phosphorus Concentrations in Plants Responsible for Inhibition of Mycorrhizal Infection [pp. 575-578]A Scanning Electron Microscopy Study of 'Vesicular Bodies' in Mycorrhizal Roots of Pinus mugo (Turra) [pp. 579-582]The Azolla-Anabaena azollae Relationship. VI. Morphological Aspects of the Association [pp. 583-593]Uptake of Fuberidazole and Thiabend Azole by Penicillium digitatum, cunninghamella echinulata and Potato Slices [pp. 595-605]Root Exudation in Cowpea and Sorghum and the Effect on Spore Germination and Growth of Some Soil Fusaria [pp. 607-612]Salt Tolerance in Pasture Grasses [pp. 613-622]The Measurement of Tolerance to Edaphic Factors by Means of Root Growth [pp. 623-633]Ecophysiological Aspects of Zinc Tolerance in Silene maritima With [pp. 635-642]ReviewsReview: untitled [pp. 643]Review: untitled [pp. 644]Review: untitled [pp. 644-645]Review: untitled [pp. 645]Review: untitled [pp. 645-646]Review: untitled [pp. 646-647]Back Matter [pp. ]

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