Biochem. Physiol. Pflanzen 179, 359-368 (1984)

Studies on Root Nodules of Leguminous Trees:1. Seasonal Variation of Plant Hormones and IAA Metabolism with

Reference to Nitrogen Fixation in Pterocarpus mursupium ROXB.

T. K. DANGAR and P. S. BASU

Department of Botany, University of Burdwan, India

Kea Term Index: root nodules, nitrogenase, endogenous phytohormones, indole acetic acid,gibberellic acid, abscisic acid, cytokinin; Pterocarpus rnursupium, Rhizobium spec.

Summary

Higher amounts of plant growth substances such as, indole acetic acid (IAA), cytokinin-likesubstances (CK), gibberellic acid-like substances (GA), abscisic acid-like substance (ABA) were ob­tained from mature root nodules of the leguminous tree, Pterocarpus mursupiurn as compared withyoung or old ones. Higher levels of nitrogenase (N2-ase) activity together with IAA metabolism asevaluated by levels of indole acetic acid oxidase (IAA oxidase), methyleneoxindole reductase (MeOxreductase), phenol, peroxidase, and polyphenol oxidase in mature nodules indicated higher metabolicefficiency of mature noduJes than in young or old ones. In mature nodules IAA and CK togetherwith N2-ase were highest in the rainy season, whereas, GA and ABA were highest in winter. N2-asealso was lowest in winter. Thus, N2-ase may have positive relations with IAA and CK and negativerelations with GA and ABA.

Metabolism of IAA, was found to change inversely with the changes of IAA oxidase activitywhich was again regulated by changes of phenol levels. The phenol level, in turn, was regulated bythe activities of peroxidase and polyphenoloxidase in the nodules.

Tryptophan (Trp) level showed a positive relation with the IAA level in the nodules.In spite of the variation in content of the hormones with seasons, the size of the nodules was

fixed at a definite age in all the seasons indicating that hormone contents were not solely relatedto the development of nodules.

The roots of the plant did not show any N2-ase activity and always had lower levels of hormones.The IAA metabolic rate was much more pronounced in roots than in nodules which may be the causeof the lower IAA level in them.

Introduction

Information on the presence of extractable plant growth substances and their meta­bolism in root nodules and roots of leguminous trees is almost lacking. SINHA and BASU

(1981) reported IAA content and its metabolism in the leguminous tree, Pongamiapinnata. The other studies on IAA content by several workers were restricted to actino­mycete root nodules of some non-leguminous trees such as, Myrica and Casuarina(SILVER et al. 1966) or Alnus (DULLAART 1970; WHEELER et al. 1979). The seasonal

Abbreviations: ABA, abscisic acid; GA, gibberellin; CK, cytokinin; IAA, indoleacetic acid;N2-ase, nitrogenase; MeOx, methylene oxindole; TLC, thin layer chromatography

360 T. K. DANGAR and P. S. BAsu

changes of CK and GA were studied in the non-leguminous actinomycete-root nodulesof Alnus (HENSON and WHEELER 1977 a, 1977b; WHEELER et al. 1979).

The purpose of this study is to gather some new information on the presence andmetabolism of plant hormones in the rhizobial root nodules of a leguminous tree P.mursupium and to establish the relations of N2-ase with the hormone contents andmetabolism in nodules. The results on hormone contents and nitrogen fixation wouldelucidate the nodule-plant relation for better growth and development of the plant.

Materials and Methods

Fresh nodules and roots of P. mursupium Roxb. were used for the study. The nodules werecatagorised into young, mature and old on the basis of age and size. The soil moisture of the experi­mental plot was maintained to 22 ± 3 % by regular watering and loosening the soil throughout theyear to provide similar soil moisture and pressure to the nodules during development. The variationin the seasonal conditions did not alter the morphological development of the nodules. In all theseasons the nodules reached an average size within 15 d after which their size and morphology didnot change greatly. The size of nodules provided to group them easily into young and postyoungstage. Nitrogenase activity was then considered to distinguish old nodules from mature ones, as theN2-ase activity declines in old nodules. Nitrogenase activity remained almost same from 20-40 dof age, after which it declined. The nodules after 40 d were considered as old. Young nodules were5-15 d old and 2-4 mm in diameter, mature nodules were 20-40 d old and 6-16 mm in diameterand old nodules were 45-50 d old and 18-20 mm in diameter. IAA and cytokinin (CK) wereassessed in rainy season (June-August) but GA and ABA were determined in winter (November­February) from young, mature and old nodules. For seasonal studies only the mature nodules wereconsidered.

IAA was extracted and identified by comparing the Rr (0.85) with authentic IAA on thin layerchromatography (TLC) plates, as well as bioassay, and assessed colorimetrically, following SINHAand BAsu (1981). The colour was extracted in chloroform before assay.

GA was extracted according to OBATA-SASAMATO and SUZUKI (1979) but using H2S04 insteadof HaP04 for pH adjustment. The extract was purified by thin layer chromatography (TLC) on silicagel G developed with isopropanol: ammonia: water (10: 1: 1, by volume). The gel layer of 0.8:-0.9Rr was scraped off comparing the UV flouresccnt spot (0.84 Rr) with authentic GAa and eluated inmethanol-ethylacetate (1: 1, by volume) mixture. Eluate was dried and bioassayed according toFRANI\LAND and WAREING (1960) with letuce hypocotyl and estimated as GAa equivalents comparingthe hypocotyl growth with that of authentic GAa. The lower limit of detection by this method was1.5 ng/m!.

The ABA was extracted following RUDICH et al. (1972). The extract was purified by TLC onsilica gel G, developed with propanol: n-butanol: ammonia: water (6: 2: 1: 1, by volume). The0.75-0.8 Rr zone of the gel layer was scraped off comparing the UV flourescent spot (0.77 Rr) withauthentic ABA and eluated in methanol, dried, taken in water solution and tested by bio-assay withwheat coleoptile negetive elongation (WRIGHT and HIRON 1972) specific for ABA. Extract fromthat zone was estimated as ABA equivalents by UV absorption at 245 nm following GLENN et a!.(1972).

Cytokinin (CK)-like substances were extracted following OBATA-SASAMATO and SUZUKI (1979)and purified by TLC on silica gel G developed with chloroform: methanol (4: 1, by volume) accordingto SYONO et a!. (1976). The 0.8-D.9 Rr zone of the chromatogram was scraped off comparing theUV fluorescent spot (0.85 Rr) with authentic kinetin, eluated, taken in water solution, and tested bychlorophyll synthesis in cucumber (Cucumis sativus cv. long green) cotyledons following FLETCHERand MCClJLLAH (1971). CK was determined as kinetin equivalents by the UV absorption at 268 nmaccording to GLENN et a!. (1972).

Phytohormones and Root Nodules of Pterocarpus 361

Total phenol was extracted and assessed colorimetrically as ferulic acid equivalents accordingto BRAY and THORPE (1954).

Tryp was extracted following NITSCH (1955) and assessed colorimetrically following HASSAN(1975).IAA oxidase was extracted in K"HP04 buffer, pH 5.3 and saturated with solid (NH4)aS04 upto

95 %, centrifuged, the residue dissolved and dialysed at 3°C for 12 h in the same buffer with 3changes. The dialysed extract was used as the enzyme source. The enzyme activity was measuredaccording to SINHA and BAsu (1981).

Peroxidase and polyphenol oxidase were extracted and assayed following KAR and MISHRA(1976).

Na-ase activity was measured in terms of reduction of acetylene to ethylene and the latter wasdetermined colorimetrically, following the method of LA RUE and KURZ (1973), according to themthe method was as efficient as GLC.

Enzyme proteins were estimated according to LOWRY et al. (1951) after precipitating the proteinby 50 % trichloroacetic acid and dissolving the precipitate with 1 N NaOH.

Results

Both IAA and GA contents were higher in mature nodules than in young or oldones (Fig. IA). But CK content was almost the same in young and mature nodules anddeclined sharply to 1/4th in old nodules (Fig. 1A). There was little difference in ABAcontent of the nodules with age and size. Tryp and phenol contents (Fig. 1B) alsofollowed a similar trend as IAA or GA within the three categories of nodules.

N2-ase activity was about three times higher in mature nodules than the others(Fig.2A). Peroxidase activity (Fig. 2A) gradually increased with age upto maturity

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24 Biochem. Physiol. Pflanzen, Bd.179

362 T. K. DANGAR and P. S. BASU

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Phytohormones and Root Nodules of Pterocarpus

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Fig. 4. Seasonal variation in activity of fAA-oxidase (circle) and MeOx reductase (triangle) in maturenodule (open symbol) and root (closed symbol).Other conditions are same as in Fig. 3.

and then remained unchanged. But polyphenol oxidase activity gradually increasedabout twice from young to mature nodules, then declined in old nodules (Fig. 2A). IAAoxidase and MeOx reductase (Fig. 2B) activities also followed the similar trend of changesas N2-ase. Crude IAA-oxidase extract showed very low activity (data not shown).

Fig. 3 shows the seasonal changes of IAA and CK content in mature nodules. IAAcontent was at a maximum in September i.e. just after the rainy season, declined toabout 1/4th in February, i.e. in winter, and then gradually increased again. Similarly,CK content was highest in the rainy season (July) and declined in winter (January).The roots of the plant contained very low amounts of hormones and showed nosignificant changes throughout the year.

Unlike IAA or CK; GA and ABA contents in nodules were highest in the winter andwere very low in the rainy season (Fig. 3), but in roots the levels were very much lowerand remained the same throughout the year.

IAA oxidase and MeOx reductase were higher in roots than those of nodules through­out the year, except after the winter when MeOx reductase was lower in roots thannodules (Fig. 4). In the nodules these enzymes of the IAA degradation pathway werelower during the rainy season and higher in the winter months (Fig. 4).

The tryp and free phenol contents (Fig. 5) in nodules were always higher than inthe roots throughout the year and followed similar patterns of changes as the IAAcontent. They reached a maximum in the rainy season and declined in winter. Phenolcontent (Fig. 5) in roots also followed similar changes as in the nodules but tryp contentof roots was less responsive to the seasonal changes.24*

364

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T. K. DANGAR and P. S. BASU

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Fig. 5. Seasonal variation in phenol (circle) and'tryp (triangle) content in mature nodule (open symbol)and root (closed symbol).Other conditions are same as in Fig. 3.

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Fig. 6. Seasonal variation in activity of peroxidase (circle), N 2-ase (square) and polyphenol oxidase(triangle) of mature nodule (open symbol) and root (closed symbol).Other conditions are same as in Fig. 3.

Phytohormones and Root Nodules of Plerocarpus 365

Levels of both peroxidase and polyphenol oxidase were higher in roots than in no­dules and the peroxidase of roots showed higher activity. The enzymes were less activeduring the rainy season and the activity increased to reach the maximum in the follow­ing May (Fig. 6).

As evident from N2-ase activity (Fig. 6), the nodules were able to fix more nitrogenduring the rainy season (August) and were about twenty times more active than in thewinter (January). The N2-ase activity remained almost the same from January toMay. The roots showed no N2-ase activity.

Discussion

The mature nodules contained more IAA than young or old ones (Fig. 1A) and hadmore IAA oxidase activity (Fig. 2B). The presence of a higher level of IAA oxidasewas expected to lower the IAA level, but this was not observed. High tryp content(Fig. 1 B), the precursor of IAA, in mature nodules probably maintained the higherIAA level through synthesis. Besides, high free phenol content also inhibited the IAAoxidase activity as the enzyme activity was observed only after removal of the phenolsby (NH4hS04 saturation and dialysis. The higher phenol content was observed (Fig. 1B)when peroxidase and polyphenol oxidase (Fig. 2B) were high which would be due tohigher rate of synthesis of phenol by phenylalanine ammonia lyase (VENCE 1978). Thelower oxidation rate of IAA leads to less production of MeOx, hence there was nodifference in MeOx reductase until maturity of the nodules (Fig. 2B).

The mature nodules also contained more GA than young or old ones (Fig. 1A) whichcould be due to its participation in regulation of growth and development of the plant,as suggested by LIBBENGA and BOGERS (1974). Participation of CK during noduledevelopment was indicated by the same level of CK in both young and mature nodules.The lower level of all the hormones and related enzymes in the old nodules was dueto cessation of growth and metabolism in them.

Being a degrading phase plant growth regulator, ABA has a much lower level inthe nodules (Fig.1A), having a tendency to increase with age. The highest N2-aseactivity in mature nodules (Fig. 2A) showed the active participation of Rhizobium inthe symbiosis, which might be related to other hormones.

Seasonal changes of IAA (Fig. 3) and N2-ase (Fig. 6) in mature nodules showed thesame patterns of changes and are expected to be related with each other. But DULLAART(1970) failed to show any variation of IAA in the actinomycete nodules of Alder.

The observed inverse relation of IAA with IAA oxidase and MeOx reductase (Fig. 4)during seasonal changes maintained a threshold level of IAA in the nodules. The freephenol (Fig. 5) also played a role in regulating the activity of IAA oxidase. As peroxidaseand polyphenol oxidase regulate the level of the phenols, an inverse seasonal variationof the phenols with these enzymes was observed (Fig. 6). The IAA content of the no­dules was regulated not only by IAA oxidase, MeOx reductase, phenols, peroxidase,and polyphenol oxidase, but tryp content also had a regulatory role, as evidenced bya similar seasonal variation with IAA, ensuring its biosynthesis (Fig. 5).

366 T. K. DANGAR and P. S. BASU

In the roots the IAA level was maintained at a low level throughout the year (Fig. 3).This was maintained by high IAA oxidase and MeOx reductase (Fig. 4), more activeperoxidase and polyphenol oxidase (Fig. 6), and low phenol content (Fig. 5). The lowtryp content (Fig. 5) and a high degradation rate of IAA maintained a low level of IAAin roots throughout the year. Absence of N2-ase activity and very low level of IAA inroots (Fig. 3) strengthen the view that IAA level may have a relation to N2-ase activity.

Like IAA, CK (Fig. 3) content also showed a similar seasonal variation with N2-ase(Fig. 6). A similar relation was observed by WHEELER et al. (1979) in actinomycetenodules of Alnus glutinosa. But seasonal variation of GA (Fig. 3) has a negative rela­tion to N2-ase activity. Such a relation was also observed in actinomycete nodules(WHEELER et al. 1979). Seasonal variation of ABA (Fig. 3) showed an inverse relationto N2-ase like that of GA.

In roots seasonal changes in content of GA (Fig. 3) or CK (Fig. 3) was not observedand the level was very low. Roots had no N2-ase activity. It would not be unreasonableto think that these hormones may also have a relation to N2-ase activity.

P. mursupium flowers in October, just after the rainy season. The plant maintainsa higher metabolic status at that time, and the demand of nitrogen also increases. TheN2-ase activity and levels of IAA and CK are also increased in the nodules, more orless at the same period, but GA and ABA levels were low. After flowering, when theplant returns to a less active state of metabolism, the N2-ase, IAA, and CK return to alow level (in winter). This indicates a close relation of the growth and development ofthe plant to the hormone metabolism of the nodules.

Formation and growth of the nodules are regulated by their hormone content(LIBBENGA and BOGERS 1974). The size of the nodules was definite at a definite age,though the hormone content at that age varied throughout the year. So the hormonesare not solely responsible for size of the nodules.

The Rhizobium from P. mursupium can produce IAA in culture (data not shown).Besides, there also are reports that Rhizobium can produce IAA and GA (LIBBENGA ;tndBOGERS 1974). Rhizobium sp. from other sources are known to produce IAA (SINHA andBAsu 1981) and CK (PHILLIPS and TORREY 1970) in culture. So, nodular hormonesmight be, at least partly, metabolised by the bacteria.

The IAA produced in the nodule was at least partly metabolised in it as evidencedby the presence of IAA-metabolic enzymes, besides, IAA was observed to be transportedfrom the actinomycete nodules of Alnus to other parts of the plant (WHEELER et al.1979). In P. mursupium IAA content in the mature nodules was about 50 times higherthan in Alnus and is also expected to be transported to other plant parts. WHEELERet al. (1979) showed a relation of nodular CK or GA with bud development and leafshedding in Alnus. A similar relation could also be expected in Rhizobium nodules ofP. mursupium which starts leaf shedding in late winter.

It may be concluded from the above observations that the root nodules are notonly the source of nitrogen to the plant but also the source of hormones which mayhave a regulatory role on growth and development of the plant.

Phytohormones and Root Nodules of Pterocarpus

Acknowledgement

The authors wish to acknowledge CSIR, India, for financial assistance.

References

367

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368 T. K. DAN GAR and P. S. BAsu, Phytohormones and Root Nodules of Pterocarpus

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Received April 27, 1983; revised form accepted December 16, 1983

Author's address: Dr. P. S. BAsu, Department of Botany, University of Burdwan, Golapbag,Burdwan 713104, West Bengal, India.