Effects of the exposure of roots of Alnusglutinosa to light on flavonoids and nodulation1
M. Hughes, C. Donnelly, A. Crozier, and C.T. Wheeler
Abstract: Exposure of the roots of Alnus glutinosa (L.) Gaertn. to white light stimulated within 5 days a substantialincrease in the content of quercetin and kaempferol, two major flavonols in acid hydrolysates of both roots and rootexudates. Both compounds were detected also in hydrolysates of root extracts of Myrica gale and Casuarinaequisetifolia, the latter a species not nodulated by Frankia strains effective on Alnus. A 7-fold reduction in nodulationof seedlings 12 days after inoculation with Frankia preincubated for 24 h with kaempferol supported the possibilitythat flavonols might be involved in the regulation of nodulation. Nodulation of seedling roots that were inoculated withFrankia and then exposed to light was inhibited by 50% after 11 days compared with seedlings with darkened roots.This effect of light treatment was preceded by a 30-fold increase in quercetin and kaempferol. The inhibitory effects onnodulation of preincubation of Frankia with kaempferol persisted for 18 days after inoculation, but there was nosignificant effect on nodulation after prolonging exposure of the root system to light for 40 days. The data supportindirectly the suggestion that the balance between stimulatory and inhibitory flavonoids in roots and root exudates maycontribute to the regulation of nodulation of actinorhizal plants.
Key words: Alnus glutinosa, flavonoids, Frankia, kaempferol, nodulation, roots.
Rsum : Lexposition la lumire blanche pendant 5 jours des racines de lAlnus glutinosa (L.) Gaertn. stimule uneaugmentation substantielle de la teneur en querctine et en kampfrol, deux flavonols majeurs quon retrouve dans leshydrolysats des racines ainsi que des exsudats. On retrouve galement ces deux composs dans les hydrolysatsdextraits racinaires du Myrica gale et du Casuarina equisetifolia, cette dernire espce de formant pas de nodules avecles souches de Frankia actives sur Alnus. Une rduction de sept fois dans la nodulation de plantules ges de 12 joursaprs inoculation avec du Frankia, pr-incub pendant 24 h dans du kampfrol, supporte la possibilit que desflavonols pourraient tre impliqus dans la rgulation de la nodulation. La nodulation de racines de plantules, inoculesavec du Frankia et exposes par la suite la lumire, a t inhibe 50% aprs 11 jours, comparativement desracines maintenues lobscurit. Cet effet du traitement la lumire est prcd dune augmention de 30 fois de laquerctine et du kamfrol. Les effets inhibiteurs sur la nodulation, dune pr-incubation des Frankia avec le kampfrol,persistent pendant 18 jours aprs linoculation, mais il ny a pas deffet significatif sur la nodulation lorsquonprolonge lexposition la lumire du systme racinaire pendant 40 jours. Les donnes supportent indirectement lasuggestion que la balance entre les flavonodes stimulateurs et inhibiteurs, dans les racines et les exsudats racinaires,pourrait contribuer la rgulation de la nodulation chez les plantes actinorhiziennes.
Mots cls : Alnus glutinosa, flavonodes, Frankia, kampfrol, nodulation, racines.
[Traduit par la Rdaction] Hughes et al. 1315
Flavonoids are involved in the initiation of the legumerhizobium symbiosis through their action as specific reg-ulators of the expression of bacterial nodulation genes(Redmond et al. 1986). The development of a nod gene re-porter system to screen potential regulatory compounds ex-creted by legume roots was of major importance for the
success of these studies. In actinorhizal symbioses, Frankiagenes with similar functions to the nod genes of rhizobiumhave not yet been identified with certainty and consequentlysuitable reporter gene systems have not been developed.However, there has been much interest in the possibility thatflavonoids, or other phenolics excreted by seedling roots,may have regulatory roles similar to that in legumes. Thispossibility was strengthened by observations that nodulationof Alnus rubra Bong. is enhanced or inhibited by compo-nents of seed wash, tentatively identified as flavonones andisoflavones (Benoit and Berry 1997). In the absence of suit-able molecular methods to screen for nodulation gene regu-lators, an alternative approach to investigate the involvementof flavonoids in nodulation is described here.
Flavonoid biosynthesis genes are transcriptionally acti-vated by light in the plant, where flavonoids may provideprotection against ultraviolet light (Schmelzer et al. 1988;Stapleton 1992; Kubasek et al. 1992). Exposure of root sys-tems to light can stimulate the synthesis of flavonoids, and
Can. J. Bot. 77: 13111315 (1999) 1999 NRC Canada
Received June 25, 1998.
M. Hughes, C. Donnelly, A Crozier, and C.T. Wheeler.2
Plant Science Group, Division of Biochemistry & MolecularBiology, Glasgow University, Glasgow G12 8QQ, UnitedKingdom.
1This paper was presented at the 11th InternationalConference on Frankia and Actinorhizal Plants, June 711,1998, University of Illinois at UrbanaChampaign.
2Author to whom all correspondence should be addressed.e-mail: C.Wheeler@bio.gla.ac.uk
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accumulation of anthocyanins in both roots and nodules ofalders can be observed soon after exposure to light. In peas,suppression of nodulation by exposure of roots to light wasascribed to increased ethylene production (Lee and LaRue1992). The possibility that inhibition of nodulation could bedue to other causal or contributory events, such as changesin flavonoid biosynthesis, was not considered.
The aim of the present study was to investigate whethernodulation of Alnus glutinosa (L.) Gaertn. is inhibited bylight and whether such inhibition could be related to changesin flavonoid biosynthesis. The occurrence of flavonols in se-cretions of seedling roots has been determined and the re-sults of preliminary experiments to examine the effect onnodulation of preincubation of Frankia with flavonols aredescribed.
Germination and growth of seedlingsSeed of A. glutinosa was sown in presterilized seed trays con-
taining a 1:1 mixture of medium- and fine-grade Perlite moistenedwith water containing 0.25 g Crones N-free salts and 0.2 mLHoaglands AZ nutrient solution per litre (Hooker and Wheeler1987). The trays were placed in a growth room under Osram warmwhite fluorescent tubes (irradiance: 120 mmolm2s1, photoperiod16 h, 23C light : 20C dark). Seedlings with well-developed firsttrue leaves (68 weeks old) were transferred to ceramic pots con-taining 0.5 g Crones N-free salts, 0.5 mL AZ nutrients solution,and 4 drops of Liquinure (Fisons Ltd., U.K.) per 2 L of water. Theroots were kept in darkness.
For light treatment for anthocyanin analysis, plants were trans-ferred after 68 weeks to 2-L clear glass jars with fresh culturemedium and grown for 3 weeks prior to harvest.
Growth of sterile seedlings and collection of rootexudate
Alnus glutinosa seeds were sterilized by agitating in sterile dis-tilled water + 5 drops Tween 80 per litre for 5 min, soaking in96% propan-2-ol for 1 min, and then agitating in 10% bleach for10 min. Seeds were then rinsed three times with 5% hydrogenperoxide, followed by five rinses with sterile distilled water. Thetreated seeds were plated on 9 cm diameter Petri dishes containing0.8% water agar, 15 seeds per dish, and incubated in the dark at25C for 34 days. Germinating seeds were then transferred to thegrowth room (conditions as above). When roots were 1 cm inlength, seedlings were transferred to sterile cellulose fibre rods(Sorbarod, Ilacon Ltd., Kent, U.K.) contained in 28-mL glass vi-als and moistened with 5 mL filtered Crones + Hoaglands AZnutrients and grown on for a further 8 weeks.
Seedlings were carefully removed from the Sorbarods, whichwere then squeezed and washed with distilled water. Liquid con-taining root exudate from 20 vials was combined and concentratedby lyophilisation.
HPLC analysis of root exudates (Crozier et al. 1997)Hydrolyzed root exudates were analysed using a Shimadzu
(Kyoto, Japan) LC-10A series liquid chromatograph consisting ofan SCL-10A VP system controller, two LC-10AT VP pumps, anSIL-10AD VP autoinjector with sample cooler, a CTO-10A VPcolumn oven, an SPD-10A VP UV-VIS detector, and an RF-10AXL fluorimeter linked to a Reeve Analytical (Glasgow, U.K.) 2700data handling system. Reverse phase separations were carried outat 40C using a 150 3.0 mm i.d. 4-mm Genesis C18 cartridge col-umn fitted with a Genesis guard cartridge (Jones Chromatography,
Mid-Glamorgan, U.K.). The mobile phase was a 20-min, 2040%gradient of acetonitrile in water adjusted to pH 2.5 with trifluoro-acetic acid pumped at a flow rate of 0.5 mLmin1. Column eluentwas first directed to the UV absorbance monitor operating at365 nm. Postcolumn derivatization was achieved by the addition ofmethanolic aluminium nitrate containing 7.5% glacial acetic acid(Hollman et al. 1996), pumped at 0.5 mLmin1 by a Reeve analyt-ical model 9802 pump. The mixture was directed to a RF-10Afluorimeter, and fluorescent flavonol complexes were detected atexcitation 420 nm and emission 495 nm. The limits of detectionwere 5 ng with UV and 0.1 ng with fluorescence. Flavonoid stan-dards for calibration and co-chromatography were purchased fromSigma Chemicals, Poole, U.K.
Extraction and acid hydrolysis of flavonoidsTissues were frozen in liquid nitrogen and lyophilised. Samples
were ground to a fine powder in liquid nitrogen and stored at70C. Dried sample (15 mg) was hydrolyzed in a 5-mLReactiVial (Pierce, Rockford, U.S.A.) in 2 mL of 1.2 M hydrochlo-ric acid in 50% methanol, containing 25 mM sodium diethyl di-thiocarbamate as an antioxidant and 100 ng morin as internalstandard, at 90C in a ReactiTherm heatingstirring module. Ali-quots of unhydrolyzed samples were removed prior to heating.Samples were centrifuged at 13 000 rpm in a Sanyo Micro-Centaurcentrifuge before HPLC, as above. Root exudates, 300 L, werehydrolyzed similarly in 2 mL 1.2 M hydrochloric acid in 50%methanol and 25 mM sodium diethyl dithiocarbamate but withoutmorin.
Exposure of seedling roots to white light andinoculation with Frankia
Seedlings (8 weeks old) were suspended in UV transparent cel-lulose acetate film covering the top of 250-mL glass beakers withtheir roots partially in filtered Crones solution. Dark seedlingswere grown with their roots suspended through black plastic topscovering filtered Crones solution in opaque jars. Frankia strainUGL010710 was cultured in propionate medium as described pre-viously (Hooker and Wheeler 1987), with or without kaempferol.Kaempferol was added by filter sterilization to 100-mL cultures toa final concentration of 106 M. Cultures with kaempferol were in-cubated in the dark at 24C for 24 h. All cultures were washed se-quentially with water on harvest, with centrifugation at 1200 gbetween each wash. The final pellet (2.5 mL) was suspended in50 mL water and gently homogenized. Seedlings were inoculatedwith 0.5 mL of this suspension. Seedlings were maintained in thegrowth room, lit by Osram warm white fluorescent tubes with 16-hphotoperiod, as above.
Flavonoids of roots of alder seedlings
AnthocyaninsThe roots of seedlings grown on Sorbarods exposed to
light showed high pigmentation, characteristic of anthocy-anins. Indication of the identity of the major anthocyaninswas obtained by co-chromatography of tissue extracts (15%acetic acid in 85% methanol) by (i) two-dimensional chro-matography on Polygram Cel 300 (Machery-Nagel) utilisingn-butanol acetic acid water (4:1:5) and (ii) HPLC (30C,absorbance detector 535 nm) on a 150 4.6 mm i.d. Waters5-m symmetry C18 reverse phase column eluted with a 5-min linear gradient of 020% methanol in water (pH 1.5with trifluoracetic acid), followed by a 35-min gradient of2055% methanol. The major compounds detected in pre-
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hydrolyzed or hydrolyzed (1 M HCl in methanol for 2 hat 90C) extracts were indicative of cyanidin-3-glucoside,cyanidin-3-galactoside, and delphinidin as components ofleaf, stem, root, and nodule extracts.
FlavonolsThe occurrence of quercetin and kaempferol in hydro-
lyzed alder root extract was indicated by co-chromatographywith authentic standards, utilising both absorbance and fluo-rescence detection. The latter technique is highly specific forthe detection of flavonols (Hollman et al. 1996) and thepresence of other peaks suggests that extracts contained sev-eral unidentified flavonols (Fig. 1). There were no signifi-cant differences in the chromatograms of root extracts fromnodulated and non-nodulated plants. Comparison of chro-matographs with those from hydrolyzed root extracts of nod-ulated, 1-year-old Myrica gale and Casuarina equisetifoliashowed that quercetin and kaempferol were present in allthree species but that several peaks, indicated with arrows,were unique to a particular species.
Flavonoids of seedling root exudatesAnalysis by HPLC of hydrolyzed root exudates showed
several peaks (Fig. 2), with the two main peaks co-chro-matographing with quercetin and kaempferol. The averagecontent of flavonols in root exudate from one seedling calcu-lated from peak areas and calibration curves for authenticstandards, was 1.3 ng for quercetin and 0.12 ng for kaemp-ferol. No free or conjugated anthocyanins were detected inroot exudates.
Effect on nodulation of preincubation of Frankia withkaempferol
Preincubation of Frankia with kaempferol for 24 h priorto inoculation reduced nodulation of seedlings 7-fold 12 daysafter inoculation (Table 1). The inhibitory effects on nod-ulation of preincubation of Frankia with kaempferol stillpersisted after 18 days, when the number of nodules on rootsof kaempferol-treated seedlings was almost 4 times less thanthat of the controls.
Effects of light on the flavonol content and nodulationof alder seedling roots
Exposure of seedling roots to white light during the 16-hphotoperiod stimulated after 5 days a 46- and 4-fold increasein free quercetin and kaempferol, respectively. Quercetinconjugate was not detected but kaempferol conjugates in-creased 70-fold (Table 2). Seedling roots, inoculated withFrankia at the time of exposure to light, showed a 50% re-duction in nodule number after 11 days but there was no sig-
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Hughes et al. 1313
Fig. 2. Chromatography by reversed phase HPLC of acidhydrolyzed extract of root exudate of Alnus glutinosa (Q,quercetin; K, kaempferol).
Fig. 1. Chromatography by reversed phase HPLC of(A) standard flavonols (M, myricetin; Q, quercetin; K,kaempferol), (B) hydrolyzed extract of roots of Casuarinaequisetifolia, (C) hydrolyzed extract of roots of Myrica gale, and(D) hydrolyzed extract of roots of Alnus glutinosa. Arrowsindicate some p...