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Hormones in Plants Bearing Nitrogen-Fixing Root Nodules: Gibberellin-Like Substances in Alnus glutinosa (L.) Gaertn. Author(s): I. E. Henson and C. T. Wheeler Source: New Phytologist, Vol. 78, No. 2 (Mar., 1977), pp. 373-381 Published by: Wiley on behalf of the New Phytologist Trust Stable URL: http://www.jstor.org/stable/2433362 . Accessed: 18/06/2014 22:25 Your 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 of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact [email protected]. . Wiley and New Phytologist Trust are collaborating with JSTOR to digitize, preserve and extend access to New Phytologist. http://www.jstor.org This content downloaded from 195.78.109.119 on Wed, 18 Jun 2014 22:25:56 PM All use subject to JSTOR Terms and Conditions

Hormones in Plants Bearing Nitrogen-Fixing Root Nodules: Gibberellin-Like Substances in Alnus glutinosa (L.) Gaertn

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Hormones in Plants Bearing Nitrogen-Fixing Root Nodules: Gibberellin-Like Substances inAlnus glutinosa (L.) Gaertn.Author(s): I. E. Henson and C. T. WheelerSource: New Phytologist, Vol. 78, No. 2 (Mar., 1977), pp. 373-381Published by: Wiley on behalf of the New Phytologist TrustStable URL: http://www.jstor.org/stable/2433362 .

Accessed: 18/06/2014 22:25

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.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 [email protected].

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New Phytol. (1977) 78, 373-381.

HORMONES IN PLANTS BEARING NITROGEN-FIXING ROOT NODULES: GIBBERELLIN-LIKE SUBSTANCES IN

ALNUS GL UTINOSA (L.) GAERTN.

BY I. E. HENSON and C. T. WHEELER

Department of Botany, University of Glasgow

(Received 28 July 19 76)

SUMMARY

The content of gibberellin-like (GA-like) substances in various parts of young alder (Alnus glutinosa (L.) Gaertn.) plants was estimated by means of the lettuce hypocotyl bioassay. The highest levels of GA-like activity were found in the root nodules, of dormant plants, plants emerging from dormancy, and plants in full leaf.

No major differences were found in the roots and leaves of nodulated as opposed to non- nodulated plants, although the stems of nodulated plants did contain lower levels of GA-like activity.

The detection of seasonal changes in GA-like activity in root nodules from mature trees was dependent upon the method of chromatography employed. One major peak of biolo- gical activity was detected following paper chromatography and no large changes were de- tected in the level of this component. In contrast, three zones of GA-like activity could be detected after thin layer chromatography (TLC) and there were marked seasonal changes in activity in these three zones. This can be attributed either to changes in levels of the GA-like substances themselves and/or to variations in the amounts of impurities interfering with the response of the bioassay.

A number of peaks of GA-like activity were detected by lettuce and rice bioassays follow- ing analysis of a large-scale extract on a preparative high performance liquid chromatograph (HPLC). However, both the total number and the identity of the substances responsible for this activity, remain to be determined.

INTRODUCTION

The presence of GA-like substances in nitrogen-fixing root nodules has received relatively little attention. Radley (1961), using a dwarf pea assay, demonstrated that nodules of Pisum sativum and Phaseolus vulgaris contained easily detectable levels of GA-like substances whereas the normal roots did not. Dullaart and Duba (1970) found similarly, that nodules of Lupinus luteus had much higher levels of GA-like activity than did the roots. In view of the stimulatory effect of GA3 on auxin production from tryptophan by cell-free nodule extracts, these latter workers speculated that the presence of large amounts of GAs in the nodules of L. luteus might be responsible for the high levels of nodule auxin (Dullaart, 1967).

The nodules of Alnus glutinosa (L.) Gaertn., a non-leguminous nitrogen-fixing species, have been shown, in common with nodules of several legumes, to contain high auxin levels (Dullaart, 1970). The GA content of this plant does not, however, appear to have been in- vestigated previously. We report here results of some preliminary studies concerning the presence of GA-like activity in A. glutinosa.

373

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374 I. E. HENSON and C. T. WHEELER

MATERIALS AND METHODS

Plant material Plants of Alnus glutinosa were raised in a glasshouse from seed collected locally.

Nodulated plants were obtained by treatment of the roots, at the two-leaf stage, with a crushed nodule preparation. The plants were grown mainly in pots in 'Peralite' (British Gypsum Co.) as described previously (Wheeler, 1969), and watered with half-strength Crones solution (nitrogen-free formulae) at intervals.

In experiments to compare the hormone content of nodulated and non-nodulated plants, seedlings were grown in half-strength Crones solution, supplemented with ammonium nitrate (15 mg and later 30 mg N I-) for ten weeks, when half the plants were inoculated with crushed nodule inoculum. Combined nitrogen supplied to the inoculated plants was reduced to 10 mg and then 5 mg N I1- and removed entirely 7 weeks prior to harvest. Uninoculated plants continued to receive combined N, finally at a concentration of 100 mg N I-1.

For the seasonal study, nodules were obtained from mature fruiting trees 12-20 years of age growing at Milngavie near Glasgow. Samples were collected from several trees at any one harvest date and bulked prior to extraction.

Extraction of plant material Plant material was extracted directly after harvesting without prior storage. Tissues

were homogenized in methanol: water (4: 1 v/v) using 10 ml g-' fresh weight, and the extracts stirred for several hours at + 10C. The extracts were filtered and the residues re- extracted twice more. The filtrates were combined and reduced to aqueous in vacuo at 300C.

Partial purification of the extracts Each extract was adjusted to pH 3.5 and centrifuged for 30 min at 20,000 g. The

supernatant was then passed through a column of the cation-exchange resin Zerolit 225 (SRC 14, 52-100 mesh, NH4+ form) using 2.0 ml resin g-1 initial fresh weight. Anions were washed from the column with distilled water (10 column volumes). The water wash was reduced to 100 ml and partitioned at pH 2.5 four times against equal volumes of redistilled ethyl acetate. The combined organic phases were reduced to dryness, the residue redissolved in a small volume of 0.1 M K2HPO4, and applied to a column (1.9 X 15.0 cm for 100 g fresh weight sample) of insoluble polyvinylpyrrolidone (PVP) (Glen et al., 1972), previously washed with two column volumes of 0.1 M K2HPO4. The PVP column was eluted with 2.5 column volumes of the same buffer, the eluate adjusted to pH 2.5 and partitioned four times against ethyl acetate. The combined ethyl acetate phases were dried over anhydrous Na2SO4, filtered and reduced to dryness in vacuo.

The partially purified residue was next redissolved in about 1 ml of 20o aqueous acetone and applied to a column (1.9 X 24.0 cm for 100 g fresh weight sample) of charcoal: celite 535 (1.2 w/w) (MacMillan and Pryce, 1968) which was eluted in sequence with 20o (1.7 column volumes), 80o (4.2 column volumes) and 100% (1.7 column volumes) acetone. The 80%po and 100% acetone fractions were combined and reduced to dryness.

Prior to analysis of a large scale extract by high performance liquid chromatography a further purification step was introduced. Following the charcoal: celite column, the extract was further purified by gel permeation chromatography (GPC) as described by Reeve and

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Hormones in alder roots 375

Crozier (1976). Fractions corresponding to the elution volume of free acidic GAs were collected and bulked.

Chromatography Extracts, dissolved in methanol: water (4: 1 v/v), were strip-loaded on to paper or

thin layer chromatograms. Paper chromatograms (Whatman 1MM) were developed in iso- propanol ammonia: water (10: 1: 1 v/v) to about 30 cm and dried in a cool air stream prior to bioassay.

Thin layer chromatography (TLC) was conducted using 20 X 20 cm precoated plates of silica gel G (Camlab) 0.25 cm thick. The plates were washed exhaustively prior to use in the developing solvent, chloroform : ethyl actate : acetic acid (60: 40: 5 v/v). After developing to 15 cm, the plates were dried in a cool air stream, and divided into 1.0 cm zones, and each zone eluted three times with 5 ml methanol: water (4: 1 v/v). The eluates were evaporated directly in the 4.5-cm diameter Petri dishes used for bioassay. Marker spots of GA3 were located after TLC by spraying with 5% sulphuric acid in ethanol, drying at 800C, and viewing under a u.v. lamp.

A large-scale extract (2 kg) of nodules, further purified by GPC, was injected on to pre- parative high performance liquid chromatography (HPLC) column (Reeve et al., 1976). The 10 X 450 mm column comprised a 0.5 M formic acid stationary phase on a Partisil 20 support and was eluted with a 0-1OO%o ethyl acetate-hexane gradient. Two hundred fractions each of 3 ml were collected. Aliquots of two adjacent fractions were bulked for bioassay. Samples of high specific activity [3H]-GA1, [3H]-GA4, [3H]-GA9 and [3H]-GA20 were used as internal markers and located by counting 1% aliquots of each fraction (dissolved in 1.0 ml methanol to which was added toluene containing 4 g PPO and 0.2 g POPOP l-l) in a liquid scintillation spectrometer (Tracerlab Coumatic 200). Although readily detected by radio- assay the [3H]-GAs were not present in sufficient amounts to be detected by bioassay.

Bioassays The lettuce hypocotyl bioassay (Frankland and Wareing, 1960), as modified by Loveys

(1970), was used routinely to detect GA-like substances. Seeds were germinated in darkness for 18 h at 260 C. Ten seeds were then transferred to each 4.5-cm Petri dish containing the test solutions (1.0 ml). Hypocotyl lengths were measured after 3 days further growth under high intensity fluorescent light at 260C. The cultivar Arctic was used.

A rice seedling assay, using the cultivar Tan-ginbozu, was also employed. The method was similar to that of Ogawa (1963). Seeds were soaked in distilled water for 48 h, and trans- ferred to moist filter paper for a further 24 h before being placed in the test solutions (0.5 ml). The length of the second leaf sheath was measured after 7 days at 280C.

RESULTS

Levels of GA-like substances in various plant parts Initially, a comparison was made of the levels of GA-like activity in nodules and roots

of actively growing plants bearing fully expanded leaves, harvested in December, 1974 when 6 months old and maintained in a non-dormant state in a heated glasshouse with supple- mentary illumination to give a 16 h photoperiod. The results are shown in Fig. 1 together

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376 I. E. HENSON and C. T. WHEELER

with those of later extractions of 1 1-month old dormant plants harvested in February, 1975 after overwintering in a cold glasshouse with natural illumination. In both cases the levels of activity in the nodules were more than six times those in equivalent amounts of root tissue.

A more comprehensive analysis was made of 10-13-month old plants from the cold glass- house, emerging from dormancy in the spring of 1975. All parts of the plants were extracted. As shown in Table 1, GA-like activity was in all cases greatest in the nodules, and in these, tended to fall slightly as the plants broke dormancy. Although the nodules constituted only a small part of the total plant fresh weight (c. 4-5%), they nevertheless contributed signi- ficantly to the total GA-like substances in the plant.

Lg GA3 JLg GA3

- 5x 10-2 6 ( a ) ( b) -5X102

^S~~~~~~ 3XO- [ I, -xIO-',G3 E 3 _ -

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Fig. 1 Fig. 2

Fig. 1. Lettuce hypocotyl bioassay of extracts of nodules (a, b) and roots (c, d) of dormant (a, c) and non-dormant (b, d) alder plants. Extracts (10 g fresh weight equivalents) were chromatographed on 1 MM paper in iso-propanol: 0.88 sg ammonia: water (10: 1: 1 v/v). Shaded areas represent significant promotions above control (P= 0.01). Total levels of GA-like substances (Mlg GA3 equivalents per kg fresh weight) were: (a) 3.1; (b) 4.9; (c) 0.5 and (d) 0.5. Fig. 2. Lettuce hypocotyl bioassay of extract of nodules collected on 10 October, 1974 from mature trees. 24 g fresh weight equivalent was chromatographed by TLC on silica gel in chloroform: ethyl acetate: acetic acid (60 : 40; 5 v/v). Shaded areas represent growth promotions significantly greater than controls (P = 0.01). Individual peaks of activity are labelled I, II and III. The position of co-chromatographed GA3 standard is indicated by horizontal bar.

Comparison of nodulated and non-nodulated plants Table 2 shows the levels of GA-ike activity in various parts of nodulated and non-

nodulated plants grown in solution culture and harvested in early September, 6 months after sowing. The distribution of fresh weight in the two groups of plants was not identical, the main difference being that the non-nodulated group possessed larger root systems, nodule formation apparently serving to inhibit root development. However, comparable parts of the two sets of plants did not differ greatly either in their nitrogen (determined by the Kjeldahl method), or leaf chlorophyll contents.

The nodules were once again found to have higher GA-like activity than other plant parts, contributing almost 1r0% to the total GA-like content, although comprising only 3.5% of the total fresh weight. In these plants the roots had higher GA-like activity than previously found, the levels being highest in the nodulated plants on a fresh weight basis, although,

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Hormones in alder roots 377

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378 I. E. HENSON and C. T. WHEELER

due to the differences in root growth, the total activity per plant of the root systems was similar in both cases. Levels of GA-like substances in the leaves were the same in both treat- ments. Only in the stems was there a substantial difference in GA content, being greater in non-nodulated plants.

Seasonal changes in GA-like substances in nodules When extracts of nodules, obtained from mature trees in the field, were subjected to

paper chromatography, only slight seasonal variations were recorded in GA-like activity (Table 3). The active constituent of the nodules always resolved into one major peak on the chromatograms (R F = c. 0.45). Levels were consistently higher than those detected in nodules of young pot-grown plants.

When aliquots of the same extracts were tested after TLC, up to three distinct peaks of activity could be identified, designated peak I (at c. RF 0.05), peak II (c. RF 0.4) and peak III (c. RF 0.85) (Fig. 2). Peak III, when detected, was generally present only in trace amounts. The total activity was always lower following TLC than with paper chromatography, and furthermore, there was a pronounced seasonal change, both in total activity and in the activity of individual peaks (Fig. 3). Levels of activity, particularly of peak II, fell substantially as the trees emerged from dormancy, remained low throughout the growing season and only increased again near leaf fall. Levels of peak I were less affected than peak II, and although

Table 2. A comparison of non-nodulated and nodulated plants of Alnus gluti- nosa: gibberellin-like activity as estimated by the lettuce hypocotyl bioassay, following paper chromatography of extracts. Data are means of two separate

extractions and bioassays

Plant part ,g GA3 equivalents per kg fresh weight ng GA3 equivalents per plant

No nodules Nodulated No nodules Nodulated

Leaves 2.1 2.1 17.0 16.0 Stems 2.1 0.9 13.0 4.0 Roots 0.8 1.6 9.0 8.0 Nodules - 4.6 - 3.0

Total 39.0 31.0

Table 3. Seasonal levels of gibberellin-like activity in nodules of Alnus glutinosa estimated by the lettuce hypocotyl bioassay following paper

chromatography of extracts

Date of collection Growth stage ,g GA3 equivalent per kg fresh weight

1974 10 Oct Leaves mature 8.5

1975 9 Jan Dormancy 7.4 1 April Dormancy 6.1 24 April Bud break 8.3 20 May Leaves half expanded 8.6 2 July Leaves fully expanded 7.2 12 Aug Leaves fully expanded 6.6 30 Sept Leaves mature 7.8

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Hormones in alder roots 379

they did not actually increase as peak II declined, there was a steady change in the relative contribution made by the two peaks to the total activity.

Analysis of GA-like substances in nodules by HPLC In order to attempt to clarify further the nature of the GA-like substances present in

the nodules, an extract was prepared from 2 kg of tissue and applied to the HPLC column. Bioassay of aliquots of the column eluate revealed the presence of several peaks active in the

(a) (b) (c) (d) (e) (a)

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Oct. Nov. Dec. Jan. Feb. March April May June July Aug. Sept. 1974 1975

Date of collection

Fig. 3. Seasonal changes in levels of GA-like substances in nodules as detected following TLC. (s) Peak I; (o) Peak II; (o) Peak III. Vertical arrows indicate stages in the growth cycle: (a) leaves mature, senescing. (b) plants dormant (c) bud break (> 50%) (d) leaves expanding (e) leaves expanded.

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F:ig. 4. Analysis of GA-like substances in nodules by HPLC. A sample derived from 2 kg of nodules was co-injected with high specific activity 3[H]-GAs and aliquots assayed for bio- logical and radio-activity. (a) Lettuce hypocotyl bioassay (1/10th aliquots). (b) Rice seed- ling bioassay (1/Sth aliquots). (c) Radioactivity (1/1OOth aliquots). Biologically active peaks are numbered 1-6.

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380 I. E. HENSON and C. T. WHEELER

lettuce and rice assays (Fig. 4). Peak 3 and peak 4 co-eluted with GA4 and GA20 respectively and hence could contain either these GAs or their double bond isomers, GA7 and GA5. These two peaks, as well as peak 2, were found to migrate to an RF on TLC similar to that of peak II; therefore these peaks could all contribute to the TLC peak II. Peak I on HPLC ran close to GA9 and hence was a likely candidate for TLC peak III, but proved, however, to have a distinctly different RF (0.55) to III when re-run on TLC.

DISCUSSION

The levels of GA-like activity, as measured by the lettuce hypocotyl bioassay, were found to be higher in the root nodules than in other parts of young alder plants, irrespective of whether the plants were dormant or actively growing. The lower levels of activity in normal root tissue of Alnus glutinosa, from both nodulated and non-nodulated plants, parallels the situation reported for leguminous speciesby Radley (1961) and by Dullaart and Duba (1970). However, the levels of activity in alder nodules were much lower than those reported for legume nodules by the above authors.

The occurrence of high hormone levels in nitrogen-fixing root nodules has frequently led to speculation as to whether these hormones are translocated to other plant parts, the changes in hormone levels so produced being responsible for some of the special physio- logical features associated with nodulated plants (Rodriguez-Barrueco, 1968; Gibson, 1974; Becking, 1975). No one experiment is likely to answer this important question; the approach adopted here was to compare levels of GA-like activity in both nodulated and non-nodulated plants, the latter supplied with combined nitrogen. One problem with this experiment is that differences between the two sets of plants may be due solely to the different modes of nutri- tion rather than to the presence or otherwise of nodules. However, the absence of any large differences in the GA content of the two sets of plants suggests that the nodule GAs do not contribute greatly to the GA pools in other plant parts. The significance of decreased acti- vity in the stems of nodulated plants, suggested by the bioassays, is unclear; in view of the possibility that exchange of GAs may occur between roots and shoots (Crozier and Reid, 1971) this may be related to the reduced root growth of these plants.

Levels of GA-like activity were high both in the nodules of young dormant plants, inactive in nitrogen fixation, and in the nodules of young plants in full leaf, dependent on their no- dules for a supply of fixed nitrogen. Although large seasonal changes in total activity in the nodules of mature trees in the field were not detected following bioassay of paper chromato- grams of extracts (Table 3), marked changes were recorded in both the levels of individual peaks and in total activity, following TLC of aliquots of these extracts. Total activity was greatest during dormancy and declined with the onset of shoot growth in the spring, the major quantitative change occurring in peak II.

The discrepancy in the results obtained with the two chromatographic systems may best be explained by postulating changes in the levels of the individual GAs or changes in the levels of inhibitory substances co-chromatographing with the active peaks on TLC, or, more likely, a combination of these factors. It is not known at present which of the two patterns of seasonal variation in GA-like activity suggested by the two methods of chromatography most closely reflect the actual changes occurring in the nodules. It is evident that a more precise understanding of the nature of the GA-like substances is required prior to undertak- ing further work on their physiological role. High performance liquid chromatography would appear to be a useful tool for analysis of GAs and a preliminary examination of a nodule

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Hormones in alder roots 381

extract using this method indicated the presence of a number of compounds with GA-like activity. Further investigation into the nature of these compounds and their levels in the nodule during the various phases of plant growth is called for.

ACKNOWLEDGMENTS

We wish to thank Dr A. Crozier for use of chromatographic facilities, for generously pro- viding samples of [3H]-gibberellins and lettuce and rice seed and for contructive suggestions on the preparation of the manuscript. We are especially grateful to Mr D.R. Reeve for much help with column chromatography and to Mrs M. E. McLaughlin for assistance with the cul- ture of the alder plants.

The work was supported with a grant (No. B/RG/71340) from the Science Research Council.

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