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  • Flavan-Containing Cells Delimit Frankia-Infected Compartments in Casuarina glauca Nodules1

    Laurent Laplaze, Hassen Gherbi, Thierry Frutz, Katharina Pawlowski, Claudine Franche, Jean-Jacques Macheix, Florence Auguy, Didier Bogusz, and Emile Duhoux*

    Physiologie Cellulaire et Moléculaire des Arbres, GeneTrop Institut de Recherche pour le Développement, 911 Avenue Agropolis, 34032 Montpellier cedex 1, France (L.L., H.G., T.F., C.F., F.A., D.B., E.D.); Department of

    Molecular Biology, Agricultural University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands (K.P.); Biochemie der Pflanze, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Untere Karspüle 2, 37073

    Göttingen, Germany (K.P.); and Laboratoire de Biotechnologie et Physiologie Végétale Appliquée, Université Montpellier 2, Place Eugène Bataillon, 34095 Montpellier cedex 5, France (J.-J.M.)

    We investigated the involvement of polyphenols in the Casuarina glauca-Frankia symbiosis. Histological analysis revealed a cell- specific accumulation of phenolics in C. glauca nodule lobes, cre- ating a compartmentation in the cortex. Histochemical and bio- chemical analyses indicated that these phenolic compounds belong to the flavan class of flavonoids. We show that the same compounds were synthesized in nodules and uninfected roots. However, the amount of each flavan was dramatically increased in nodules com- pared with uninfected roots. The use of in situ hybridization estab- lished that chalcone synthase transcripts accumulate in flavan- containing cells at the apex of the nodule lobe. Our findings are discussed in view of the possible role of flavans in plant-microbe interactions.

    Flavonoids are secondary metabolites derived from the phenylpropanoid pathway. They are involved in various biological processes, including flower pigmentation (an- thocyanins) and protection against UV irradiation (Shirley, 1996). Flavonoids play key roles at different levels of plant- microbe interactions (Dixon and Paiva, 1995; Shirley, 1996). In legumes the accumulation of flavonoid compounds, identified as phytoalexins, occurs in response to pathogen attack. These compounds have been shown to prevent the spread of the pathogen (Dixon and Paiva, 1995). Particular flavonoids have also been implicated in the establishment of pathogenic and symbiotic plant-microbe interactions, in particular, in the Rhizobium-legume symbiosis (Peters and Verma, 1990). Specific flavonoids released from the roots of legumes interact with the NodD protein of Rhizobium to activate transcription of other nod genes responsible for the synthesis of Nod factors (Dénarié et al., 1996; Long, 1996). Aside from their role as signal molecules in root exudates, flavonoids might also be involved in the morphogenesis of legume nodules. Since specific flavonoids can inhibit polar auxin transport (Jacobs and Rubery, 1988), they were pro- posed to change the phytohormone balance during nodule

    initiation (Hirsch et al., 1989; Yang et al., 1992; Mathesius et al., 1998). Moreover, Charrier et al. (1998) suggested a role of flavonoids in modulating nutrient exchanges and enzy- matic activities in legume nodules. Furthermore, the accu- mulation of flavonoids in some ineffective symbioses have been related to a host defense response elicited by rhizobia, which could be part of the regulation of nodulation (Gross- kopf et al., 1993).

    The flavonoid biosynthetic pathway has been extensively studied in legumes (see e.g. Paiva et al., 1991; Maxwell et al., 1993; McKahn and Hirsch, 1994; Charrier et al., 1995; Djordjevic et al., 1997). Chalcone synthase (CHS), which catalyzes the first step of flavonoid biosynthesis, is a key enzyme in this pathway. It condenses three molecules of malonyl CoA with 4-coumaroyl CoA to produce chalcones (Martin, 1993). CHS genes have been cloned from many plants and are often encoded by small gene families (Mar- tin, 1993). Several authors have reported that CHS is a key regulatory enzyme of flavonoid biosynthesis (Hahlbrock and Scheel, 1989) and that the study of CHS gene expres- sion is a good molecular marker of flavonoid production. CHS gene expression is subject to complex developmental and environmental regulation (Hahlbrook and Scheel, 1989; Dixon and Paiva, 1995). Its expression is induced by nitrogen deprivation in alfalfa (Coronado et al., 1995) and upon inoculation by rhizobia in soybean and vetch (Es- tabrook and Sengupta-Gopalan, 1991; Recourt et al., 1992). In pea the highest level of chs expression was found in uninfected apical parts of the nodule, suggesting a devel- opmental function of flavonoids (Yang et al., 1992).

    Whereas rhizobia interact with legumes, Frankia strains establish root nodule symbioses with eight different angio- sperm families collectively called actinorhizal plants (Ben- son and Silvester, 1993). The mode of infection depends on the host plant. Two modes of infection of actinorhizal plants have been described: intercellular and intracellular (Berry and Sunell, 1990; Franche et al., 1998). Intracellular infection (e.g. of Casuarina glauca) starts with root hair curling induced by an unknown Frankia signal. Bacteria penetrate a curled root hair and grow intracellularly. Root hair infection induces limited cell divisions in the cortex, giving rise to the so-called prenodule. Frankia infects some

    1 This research was supported by the Institut de Recherche pour le Développement.

    * Corresponding author; e-mail [email protected]; fax 33– 4 – 67– 63– 82– 65.

    Plant Physiology, September 1999, Vol. 121, pp. 113–122, www.plantphysiol.org © 1999 American Society of Plant Physiologists


  • of the prenodule cells. Concomitantly with prenodule for- mation, cell divisions occur in the pericycle opposite to a protoxylem pole, leading to the formation of a nodule primordium. Its cells become infected by bacterial hyphae coming from the prenodule. The involvement of flavonoids in actinorhizal symbioses is poorly understood. Because of the similarities of the infection process between some acti- norhizal plants and legumes, flavonoids were proposed to act as plant signals activating the production of a Frankia root hair-deforming factor (Prin and Rougier, 1987; Van Ghelue et al., 1997).

    As part of our investigation into plant responses to Frankia infection, we characterized the major polyphenol compounds and monitored chs gene expression in C. glauca nodules. We report a cell-specific accumulation of phenolic compounds in cell layers delimiting Frankia-infected areas of the nodule cortex. Histochemical and biochemical anal- yses indicated that compounds found in these cell strands belong to the flavan group of flavonoids. We also report that high levels of CHS transcripts were found in the uninfected tannin-containing cells at the apex of the nodule lobes. Finally, we discuss the possible functions of flavans in the C. glauca-Frankia interaction.


    Plant Material

    Casuarina glauca plants were grown in a greenhouse and inoculated with Frankia as previously described (Gherbi et al., 1997). Mature nodules (three to six lobes) from 5- to 6-month-old plants were harvested 3 to 4 weeks after in- oculation and used for the experiments.


    Plant material was fixed and embedded in paraffin as described previously (Gherbi et al., 1997). Sections (7 mm) were cut with a microtome (Jun GRM 2055, Leica Micro- systems, Wetzlar, Germany). After staining, sections were mounted in the staining reagent or in glycerine plus water (15%, v/v) and examined with a light microscope (model DMR13, Leica). Two filter sets were used: a UV filter set with a 340- to 380-nm excitation and a 425-nm barrier filter, and a blue filter set with a 450- to 490-nm excitation filter and a 515-nm barrier filter. For the structural study, sec- tions were stained with 0.025% (w/v) toluidine blue.

    Flavonoid compounds were detected with Neu’s reagent (Neu, 1956). Sections were immersed in 1% (w/v) 2-aminoethyl-diphenyl borinate (Fluka, Milwaukee, WI) in absolute methanol for 2 to 5 min, mounted in glycerine water, and observed with epifluorescence. The results were confirmed with Wilson’s reagent (Hariri et al., 1991).

    A reagent of vanillin-HCl (Sarkar and Howarth, 1976) was employed for analysis of catechins and condensed tannins. Sections were immersed in 10% (w/v) vanillin in 1 volume of absolute ethanol mixed with 1 volume of con- centrated HCl, mounted in this reagent, and observed with a light microscope. Results were confirmed using 4-dimethyl-aminocinnamaldehyde staining (Feucht and

    Treutter, 1990) on handmade thick sections. Hydrolyzable tannins were detected according to the method of Schnei- der (1977).

    Extraction and Analysis of Soluble Phenolics

    Plant material was ground in cold ethanol:water (4:1) containing 2% (w/v) K2SO3. The extract was agitated for 15 min at 4°C and vacuum filtered. This step was repeated three times. After ethanol evaporation, the aqueous phase was extracted four times with EtOAc containing 20% (w/v) (NH4)2SO4 and 2% (w/v) metaphosphoric acid. The or- ganic phase was then reduced to dryness in vacuo and the residue was dissolved in MeOH. Root and nodule extractions were repeated two times using different tissue samples.

    Methanolic extracts were analyzed by HPLC. The liquid chromatograph (model 600E, Waters, Milford, MA) was equipped with a 5-mm C18 column (250 3 5 mm; Spheri- sorb, Machery and Nagel, Düren, Germany). The following linear gradient elution system was applied at a flow rate of 1 mL min21: within 13 min from 7% to 15% solvent B (acetonitrile) in solvent A (water), within 17 min at 15% B, within 5 min from 15% to 19% B, within 15 min at 19% B, within 5 min from 19%