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

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<ul><li><p>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 23:52</p><p>Your use of the JSTOR archive indicates your acceptance of the Terms &amp; Conditions of Use, available at .http://www.jstor.org/page/info/about/policies/terms.jsp</p><p> .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.</p><p> .</p><p>Wiley and New Phytologist Trust are collaborating with JSTOR to digitize, preserve and extend access to NewPhytologist.</p><p>http://www.jstor.org </p><p>This content downloaded from 185.44.78.129 on Tue, 17 Jun 2014 23:52:57 PMAll use subject to JSTOR Terms and Conditions</p><p>http://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.jsp</p></li><li><p>New Phytol. (1978) 80,557-565. </p><p>HORMONES IN PLANTS BEARING NITROGEN-FIXING ROOT NODULES: THE NODULE AS A SOURCE OF CYTOKININS IN </p><p>ALNUS GL UTINOSA (L.) GAERTN. </p><p>By C. T. WHEELER and I. E. HENSON* </p><p>Department of Botany, University of Glasgow, Glasgow G12 8QQ, Scotland </p><p>(Received 29 November 1977) </p><p>SUMMARY </p><p>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. </p><p>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. </p><p>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. </p><p>INTRODUCTION </p><p>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). </p><p>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 </p><p>* Present address: Plant Breeding Institute, Maris Lane, Trumpington, Cambridge CB2 2LQ. OO28-646X/78/0500-0557$02.00 ?1978 Blackwell Scientiflc Publications </p><p>557 </p><p>This content downloaded from 185.44.78.129 on Tue, 17 Jun 2014 23:52:57 PMAll use subject to JSTOR Terms and Conditions</p><p>http://www.jstor.org/page/info/about/policies/terms.jsp</p></li><li><p>558 C. T. WHEELER and I. E. HENSON </p><p>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. </p><p>MATERIALS AND METHODS Plant material </p><p>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. </p><p>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. </p><p>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. </p><p>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. </p><p>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. </p><p>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. </p><p>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 </p><p>This content downloaded from 185.44.78.129 on Tue, 17 Jun 2014 23:52:57 PMAll use subject to JSTOR Terms and Conditions</p><p>http://www.jstor.org/page/info/about/policies/terms.jsp</p></li><li><p>Cytokinins in alder nodules 559 </p><p>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. </p><p>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). </p><p>Determination of total nitrogen, chlorophyll and protein contents, leaf area and root tip numbers </p><p>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. </p><p>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. </p><p>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. </p><p>RESULTS </p><p>Comparison of nodulated and non-nodulated plants </p><p>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). </p><p>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. </p><p>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 </p><p>This content downloaded from 185.44.78.129 on Tue, 17 Jun 2014 23:52:57 PMAll use subject to JSTOR Terms and Conditions</p><p>http://www.jstor.org/page/info/about/policies/terms.jsp</p></li><li><p>560 C. T. WHEELER and I. E. HENSON </p><p>Table 1. Comparison of non-nodulated and nodulated plants of Alnus glu tinosa. Experiment 1 (1975) </p><p>(a) Plant growth and nitrogen content </p><p>Total N Fresh wt (g per plant)* (mg g-' dry wt) </p><p>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 </p><p>Total 26.03 (100.0) 18.07 (100.0) </p><p>*Means of sixteen plants. Data in parentheses are percentages of total fresh weight. </p><p>(b) Leaf chlorophyll, leaf area/weight ratios and numbers of root apices </p><p>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 </p><p>No nodules 0.89 72.0 375 4410 Nodulated 1.06 68.5 464 2315 </p><p>(c) Cytokinin activity in Fraction 1. Data are means of two separate extractions and bioassays </p><p>,ug kinetin equivalents ng kinetin equivalents per kg fresh wt per plant* </p><p>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) </p><p>Total 5057 (100.0) 2536 (100.0) </p><p>*Data in parentheses are percentages of total cytokinin content. </p><p>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). </p><p>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. </p><p>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 </p><p>This content downloaded from 185.44.78.129 on Tue, 17 Jun 2014 23:52:57 PMAll use subject to JSTOR Terms and Conditions</p><p>http://www.jstor.org/page/info/about/policies...</p></li></ul>