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 23:52

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  • New Phytol. (1978) 80,557-565.



    By C. T. WHEELER and I. E. HENSON*

    Department of Botany, University of Glasgow, Glasgow G12 8QQ, Scotland

    (Received 29 November 1977)


    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.


    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


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  • 558 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

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  • Cytokinins 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.