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Ethylene-dependent ⁄ethylene-independent ABAregulation of tomato plants colonized by arbuscularmycorrhiza fungi

Jose Angel Martın-Rodrıguez1, Rafael Leon-Morcillo1, Horst Vierheilig1, Juan Antonio Ocampo1, Jutta Ludwig-

Muller2 and Jose Manuel Garcıa-Garrido1

1Department of Soil Microbiology and Symbiotic Systems, Estacion Experimental del Zaidın (EEZ), CSIC, Calle Profesor Albareda no1, 18008 Granada,

Spain, 2Institut fur Botanik, Technische Universitat Dresden, Zellescher Weg 20b, 01062 Dresden, Germany

Author for correspondence:Jose Manuel Garcıa Garrido

Tel: +34 958 18 16 00 (ext. 145 ⁄ 302)Email: [email protected]

Received: 7 October 2010

Accepted: 22 November 2010

New Phytologist (2011) 190: 193–205doi: 10.1111/j.1469-8137.2010.03610.x

Key words: abscisic acid, arbuscularmycorrhiza, ethylene, tomato (Solanum

lycopersicum).

Summary

• We investigated the relationship between ABA and ethylene regulating the

formation of the arbuscular mycorrhiza (AM) symbiosis in tomato (Solanum

lycopersicum) plants and tried to define the specific roles played by each of these

phytohormones in the mycorrhization process.

• We analysed the impact of ABA biosynthesis inhibition on mycorrhization by

Glomus intraradices in transgenic tomato plants with an altered ethylene pathway.

We also studied the effects on mycorrhization in sitiens plants treated with the

aminoethoxyvinyl glycine hydrochloride (AVG) ethylene biosynthesis inhibitor and

supplemented with ABA. In addition, the expression of plant and fungal genes

involved in the mycorrhization process was studied.

• ABA biosynthesis inhibition qualitatively altered the parameters of mycorrhiza-

tion in accordance with the plant’s ethylene perception and ethylene biosynthesis

abilities. Inhibition of ABA biosynthesis in wild-type plants negatively affected all

the mycorrhization parameters studied, while tomato mutants impaired in ethyl-

ene synthesis only showed a reduced arbuscular abundance in mycorrhizal roots.

Inhibition of ethylene synthesis in ABA-deficient sitiens plants increased the inten-

sity of mycorrhiza development, while ABA application rescued arbuscule

abundance in the root’s mycorrhizal zones.

• The results of our study show an antagonistic interaction between ABA and eth-

ylene, and different roles of each of the two hormones during AM formation. This

suggests that a dual ethylene-dependent ⁄ ethylene-independent mechanism is

involved in ABA regulation of AM formation.

Introduction

The key to understanding the phenomenon of plant ⁄ funguscompatibility in arbuscular mycorrhiza (AM) interaction isto analyse the recognition mechanisms and moleculesinvolved in this interaction. In this regard, there have beensignificant and relevant discoveries in relation to the signalsassociated with the development of AM symbiosis. In thisrespect, a group of exudate compounds (strigolactones) fromthe plant root has recently been identified and characterizedas hyphal branching inducers of AM fungi (Akiyama et al.,2005). Moreover, a symbiotic plant signal-transduction cas-cade is also activated in plant roots after both rhizobia and

AM fungi are recognized by the plant root, and some genesand gene products have been identified as necessary for fun-gal penetration and arbuscule formation in roots (Parniske,2004, 2008).

Despite recent progress made in signalling events, knowl-edge of the processes required for AM formation is still verylimited, particularly in relation to the biochemical andmorphogenetic events that take place after the fungus entersthe plant root. The establishment of an AM symbiotic inter-action is a successful strategy to improve the nutritionalstatus of both plant and fungus. Fungal penetration andestablishment in the host roots involve a complex sequence ofevents and intracellular modifications (Bonfante-Fasolo &

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Perotto, 1992; Genre et al., 2008; Genre & Bonfante,2010), leading to an interchange of mineral nutrient uptakeby the fungus in exchange for plant carbohydrates.Numerous studies have supported the hypothesis that a finebalance between hormones and nutrient availability (phos-phorus, carbon and nitrogen) is probably important for theregulation of mycorrhizal formation and functioning(Ludwig-Muller, 2000). More recently, mycorrhizationexperiments on transgenic and mutant plants, altered in theirhormonal biosynthesis and ⁄ or perception, have shown thatplant hormones play an important role in the establishmentof functional AM symbiosis (Isayenkov et al., 2005; Herrera-Medina et al., 2007). Additionally, the use of DNA arraymethodologies has advanced our knowledge of certain planthormonal pathways, such as gibberellins and ABA, activatedduring mycorrhization (Fiorilli et al., 2009; Garcıa-Garridoet al., 2010). Thus, variations in the mycorrhization charac-teristics of wild-type and ABA-deficient tomato roots areaccompanied by specific transcriptomic alterations associatedwith differences in mycorrhization status according to theABA content in the roots (Garcıa-Garrido et al., 2010).

Analysis of AM colonization in the sitiens ABA-deficienttomato mutant has shown that ABA is necessary in order tocomplete the arbuscule formation process and its functional-ity and to promote sustained colonization of the plant root(Herrera-Medina et al., 2007). Comparative analysis of twoABA-deficient tomato mutants showed both quantitativeand qualitative differences in the pattern of AM colonization.The highly limited fungal colonization of sitiens mutantsin terms of mycorrhizal intensity and arbuscule formationclosely correlated with their incapacity in ABA biosynthesis.Notabilis plants, whose ABA deficiency in roots was lessaffected, showed lower mycorrhizal intensity only at the endof the mycorrhization process (Martın-Rodrıguez et al., 2010).In addition, ABA deficiency induced ethylene production,and it has been suggested that one of the mechanisms used byABA to determine susceptibility to fungal infection is throughnegative modulation of the ethylene pathway (Herrera-Medina et al., 2007; Martın-Rodrıguez et al., 2010).

The ABA signalling pathway interacts antagonisticallywith the ethylene signalling pathway and vice versa, in orderto modulate plant development (Beaudoin et al., 2000;Ghassemian et al., 2000) and plant disease resistance(Anderson et al., 2004). However, no specific role has beendemonstrated for ethylene during AM symbiosis, althoughrecent results suggest its participation in the regulation of theAM (Penmetsa et al., 2008; Riedel et al., 2008; Zsogonet al., 2008), and our understanding of how the ABA path-way interacts with ethylene pathways during AM formationis very limited. The objective of this study was to analyse therole played by the relationship between ABA and ethylene inregulating the formation of AM symbiosis in tomato plants.Using genetic tools, such as plant mutants defective inhormone production or perception, in combination with

histochemical and molecular biology techniques for theanalysis of AM formation, we studied the mechanism usedby ABA to determine susceptibility to AM fungal root colo-nization. Our results suggest that the reduction in arbuscularabundance in the mycorrhizal roots of sitiens mutants isdirectly associated with their ABA biosynthesis deficiency.The accumulation of ethylene in the roots of these mutantplants as a result of their ABA deficiency mainly affectsmycorrhizal intensity. A dual ethylene-dependent ⁄ ethylene-independent mechanism has been suggested to explain ABAregulation of the AM formation. As ABA is necessary forarbuscule formation, ABA deficiency has a direct negativeeffect on the percentage of arbuscules in mycorrhizal roots.ABA deficiency enhances ethylene content, thus acting as anegative regulator of mycorrhizal intensity.

Materials and Methods

Plant growth and AM inoculation

Seeds of Solanum lycopersicum L. (Mill.) wild-type RheinlandsRuhm (Accession LA0535) and ABA-deficient mutant(Taylor et al., 1988) sitiens (accession LA0575) were obtainedfrom the Tomato Genetics Resource Centre (TGRC) of theUniversity of California. Transgenic tomato seed lines over-expressing the Never Ripe (NR) ethylene receptor (NRO plants)(Ciardi et al., 2000), LeETR6 ethylene receptor antisenselines (ETR6-AS plants) (Kevany et al., 2007), ethylenetransgenic tomato lines expressing a bacterial 1-amino-cyclopropane-1-carboxylate (ACC) deaminase (ACCD-expressing plants) (Klee et al., 1991) and their isogenicwild-type cvs Flora-Dade and UC82B were kindly providedby Dr H. Klee (University of Florida).

Tomato seed sterilization, AM fungi inoculation andplant growth were carried out as previously described byHerrera-Medina et al. (2007). Plant growth and treatmentswere carried out in a growth chamber (16 : 8 h, 25 : 19�C,day : night cycle; relative humidity 50%). Inoculation withGlomus intraradices (DAOM 197198), reclassified as Glomusirregulare according to Stockinger et al. (2009), was carriedout in 200 ml pots. Each seedling was grown in a separatepot and inoculated with a piece of monoxenic culture in aGel-Gro (ICN Biochemicals, Aurora, OH, USA) mediumcontaining 50 G. intraradices spores and infected carrotroots. The monoxenic culture (G. intraradices and carrotroots) was produced according to the method described byChabot et al. (1992). In the noninoculated treatment, theplants were applied with a piece of Gel-Gro medium con-taining only uninfected carrot roots.

One week after planting in pots and weekly thereafter,20 ml of a modified Long Ashton nutrient solution con-taining 25% of the P concentration (Hewitt, 1966) wasadded to prevent mycorrhizal inhibition as a result of excessof phosphorous. Plants were harvested 50 d after inoculation,

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and the root system was washed and rinsed several timeswith sterilized distilled water. The root system was weighedand used for the different measurements according to thenature of the experiments. In each experiment, five inde-pendent plants were analysed per treatment. Sitiens plantswere sprayed daily with water to prevent wilting.

Estimation of root colonization

The nonvital trypan blue histochemical staining procedurewas used according to the Phillips & Hayman (1970)method. Stained roots were observed with a light micro-scope, and the intensity of root cortex colonization by AMfungus was determined as described by Trouvelot et al.(1986) using the MYCOCALC software (http://www.dijon.inra.fr/mychintec/Mycocalc-prg/download.html). The para-meters measured were frequency of colonization (%F),intensity of colonization (%M) and arbuscular abundance(%A) in the whole root as well as intensity of colonization(%m) and arbuscular abundance (%a) in mycorrhizal rootfragments. At least five microscope slides were analysed perbiological replicate, and each slide contains 30 root piecesof 1 cm.

RNA extractions and gene expression

For the reverse transcription polymerase chain reaction (RT-PCR) experiments, total RNA was isolated from 1 g ofpooled material (biological replicate) that contained repre-sentative portions of roots from at least five different plants.Total RNA was isolated from the roots stored at )80�Cusing the RNeasy Plant Mini Kit (Qiagen, Valencia,California) following the manufacturer’s instructions.cDNAs were obtained from 1 lg of total DNase-treatedRNA in a 20 ll reaction volume containing 20 U of avianmyeloblastosis virus (AMV) reverse transcriptase (Roche,Mannheim, Germany), 400 ng of random hexamer primers,1 mM each deoxynucleoside triphosphate (dNTP), 50 U ofRNase inhibitor and 1· reverse transcription buffer. After

reverse transcription, 80 ll of MilliQ water was added toobtain a final volume of 100 ll for each cDNA solution.

Quantitative RT-PCR (qRT-PCR) was carried out tomeasure the transcript abundances of elongation factor 1a(GinEF) and glutamine synthase (GinGS) G. intraradicesgenes and tomato ethylene receptor genes (LeETR3, LeETR4,LeETR6) in mycorrhizal roots. All primer names and corre-sponding sequences are listed in Table 1. qRT-PCR wascarried out with an iCycler apparatus (Bio-Rad). Each 20 llPCR reaction contained 1 ll of diluted cDNA (1 : 10),10 ll 2· SYBR Green Supermix (Bio-Rad), and 200 nM ofeach primer using a 96-well plate. The PCR programme con-sisted of a 3 min incubation at 95�C, followed by 35 cyclesof 30 s at 95�C, 30 s at 58–63�C, and 30 s at 72�C. Thespecificity of the PCR amplification procedure was checkedwith a melting curve after the final cycle of the PCR (70 stepsof 30 s from 60 to 95�C with a heating rate of 0.5�C). qPCRexperiments were carried out from three biological replicatesand the threshold cycle (CT) was determined in triplicate.The relative levels of transcription were calculated by usingthe 2)DDCt method (Livak & Schmittgen, 2001). CT valuesof all genes were normalized to the CT value of the LeEF-1a(X14449) housekeeping gene.

Chemical treatments

The tomato plants were treated in soil with ABA (Sigma),sodium tungstate (Na2WO4) (Panreac, Barcelona, Spain),and aminoethoxyvinyl glycine hydrochloride (AVG)(Sigma-Aldrich, Steinheim, Germany). Sodium tungstate isa potent inhibitor of molybdo-enzymes in plants such asABA aldehyde oxidase (Milborrow, 2001) and AVGstrongly inhibits the conversion of methionine to ACC inthe ethylene biosynthetic pathway (Adams & Yang, 1979).The solutions were prepared by dilution from a stock.Twenty millilitres of the corresponding diluted solution wereapplied twice a week to each 200 ml pot containing onetomato plant. The first application started 1 wk after AMfungal inoculation. Stock solutions contained 1 mM ABA in

Table 1 Primers used in this study for quantitative reverse transcription polymerase chain reaction (qRT-PCR) experiments

Primer name Organism Target gene Primer sequence (5¢–3¢) Reference

GinGS-F Glomus intraradices GinGS (5¢-CCTCAAGGTCCCTATTATTGTTCTG-3¢) Gomez et al.(2009)GinGS-R (5¢-ACGATAATGAGCTTCCACAACGT-3¢)GinEF-F G. intraradices GinEF (5¢-GCTATTTTGATCATTGCCGCC-3¢) Benabdellah et al. (2009)GinEF-R (5¢-TCATTAAAACGTTCTTCCGACC-3¢)EF-1aF Solanum lycopersicum LeEF-1a (5¢-GGTGGCGAGCATGATTTTGA-3¢) Garcıa-Garrido et al. (2010)EF-1aR (5¢-CGAGCCAACCATGGAAAACAA-3¢)ETR3F S. lycopersicum LeETR3 (NR) (5¢-AGGGAACCACTGTCACGTTTG-3¢) Kevany et al. (2008)ETR3R (5¢-CTCTGGGAGGCATAGGTAGCA-3¢)ETR4F S. lycopersicum LeETR4 (5¢-GGTAATCCCAAATCCAGAAGGTTT-3¢) Kevany et al. (2008)ETR4R (5¢-CAATTGATGGCCGCAGTTG-3¢)ETR4F S. lycopersicum LeETR6 (5¢-CAATTGATGGCCGCAGTTG-3¢) Kevany et al. (2008)ETR4R (5¢-GGATGTGGATATGTGGGATTAGAAG-3¢)

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1% ethanol, 25 lM AVG and 10 mM Na2WO4 in water.Control treatments used 0.1% ethanol solution. The finalABA, AVG and sodium tungstate concentrations used werein the range of concentrations used in previous studies(Hansen & Grossmann, 2000; Audenaert et al., 2002;Martın-Rodrıguez et al., 2010).

Ethylene quantification

The ethylene content in roots was measured by placingexcised root systems in a 16 ml test tube sealed with a rub-ber stopper and incubated for 1 h at room temperature. Theaccumulation of ethylene in each tube was determined fromthree different samples of 1 ml taken from the tube using asyringe. Measurements were carried out in a gas chromato-graph (Hewlett Packard 5890, Palo Alto, California) fittedwith a flame ionization detector, using commercial ethyleneas standard for identification and quantification purposes.The ethylene concentration was expressed as g–1 root FW.

ABA quantification

Plant harvest and root processing for ABA quantification intomato roots were carried out according to Martın-Rodrıguez et al. (2010). ABA quantification was repeatedthree times with different pooled root material. Freeze-driedroots (equivalent to minimum 100 mg FW of root material)were extracted with a mixture of isopropanol and acetic acid(95 : 5, v ⁄ v). To each sample was added 100 ng (d6)-ABA(Plant Biotechnology Institute, National Research Councilof Canada, Saskatoon, Canada). Sample preparation was per-formed according to Meixner et al. (2005); the samples wereincubated, continuously shaken for 2 h at 4�C, and centri-fuged for 10 min at 10 000 g; the supernatant was removedand evaporated to dryness under a stream of N2. The residuewas resuspended in methanol, centrifuged again for 10 minat 10 000 g, and the supernatant was removed and placed ina glass vial. The methanol was evaporated under a stream ofN2 and the sample was resuspended in 100 ll ethyl acetate.Methylation was carried out according to the method describedby Cohen (1984) using freshly prepared diazomethane.

Gas chromatography mass spectrometry analysis was carriedout using a Varian Saturn 2100 ion-trap mass spectrometerwith electron impact ionization at 70 eV connected to aVarian CP-3900 gas chromatograph equipped with a CP-8400 autosampler (Varian, Walnut Creek, CA, USA). Forthe analysis, 2 ll of the methylated sample dissolved in 30 llethyl acetate was injected in splitless mode (splitter opening1 : 100 after 1 min) onto a Phenomenex (Aschaffenburg,Germany) ZB-5 column (30 m · 0.25 mm · 0.25 lm) usingHe carrier gas at 1 ml min)1. Injector temperature was250�C, and the temperature programme was 60�C for 1 minfollowed by an increase of 25�C min)1 to 180�C, 5�Cmin)1 to 250�C, 25�C min)1 to 280�C, and then 5 min iso-

thermically at 280�C. The methyl ester of ABA eluted at13.5 min under these conditions. For greater sensitivity, thelSIS mode (Varian Manual; Wells & Huston, 1995) was used.

The endogenous ABA concentration was calculated onthe basis of isotope dilution principles using the ions fromthe methylated substance at m ⁄ z = 190 ⁄ 194 (ions derivedfrom endogenous and d6-ABA concentrations; Walker-Simmons et al., 2000).

Statistical analysis

The data were subjected to one- or two-way ANOVA. Themean values for five replicate samples were compared usingDuncan’s multiple range test (P = 0.05).

Results

Plant ethylene sensitivity mediates the degree offungal colonization after ABA biosynthesis inhibition

Previous results have shown that sodium tungstate appli-cation to roots reduced mycorrhization and had a positiveeffect on ethylene production and a negative impact onABA biosynthesis in tomato plants (Martın-Rodrıguez et al.,2010). In order to determine whether the negative effectof tungstate applications on mycorrhization is always causedby ethylene pathway activation, we studied the impact ofsodium tungstate applications on different mutant tomatoplants with varying degrees of ethylene sensitivity. Theseexperiments show that all the mycorrhization parametersmeasured, except colonization frequency, were negativelyaffected in wild-type Flora-Dade plant roots by the applica-tion of sodium tungstate (Fig. 1a). This effect was limited inwild-type plants to between 16% in %M and 40% in %A,and no negative effect on mycorrhizal parameters wasobserved when the application of ABA was combined withtungstate. Plants with reduced ethylene perception, and thuslower sensitivity to ethylene (NRO), were unaffected bytungstate applications in terms of mycorrhizal frequency andcolonization intensity, and, as with wild-type plants, a 35%reduction was detected in the %a parameter (Fig. 1b). Asthe tungstate application did not affect the %M, the reduc-tion in %A was less pronounced (c. 20%) in NRO plants.Nevertheless, in the presence of tungstate, the plants withincreased sensitivity to ethylene (ETR6-AS) recorded a sharplyreduced mycorrhization parameters (Fig. 1c). In ETR6-ASplants, %M and %m decreased by 60%, while %a para-meters fell by c. 40%. The latter reduction also occurred inwild-type and NRO plants. The sharp decrease in %M and%a parameters with respect to ETR6-AS tungstate-treatedplants reduced their %A by 75% (Fig. 1c). When ABA wasapplied together with tungstate to ETR6-AS plants, %Mand %m parameters reached only 80% of their former valuesin control treatments (Fig. 1c).

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Our results reveal that the reduction in root colonizationby tungstate application (%M) was highly dependent onthe plant genotype (two-way ANOVA, F2,45 = 27.44, P <0.0001). By contrast, the reduction in arbuscular abundance

in the mycorrhizal root zones (%a) was mainly the result oftungstate application (two-way ANOVA, F2,27 = 174.07,P < 0.0001), regardless of the plant’s ethylene sensitivity.Nevertheless, there is a significant interaction between plantgenotype and tungstate ⁄ ABA application factors for the twomycorrhizal parameters, %M (two-way ANOVA, F4,45 =4.21, P = 0.005) and %a (two-way ANOVA, F4,27 = 6.13,P = 0.001), demonstrating that these factors interact.

Fungal colonization was also quantified by qRT-PCR atthe molecular level to verify the microscopic measurementof mycorrhization in tungstate, tungstate plus ABA-treatedand nontreated plants with varying degrees of ethylene sen-sitivity. We quantified the accumulation of mRNA for theG. intraradices elongation factor 1-alpha gene (GinEF)(GenBank accession no. DQ2826112) in tomato roots inorder to measure the rate of fungal colonization. The pres-ence of arbuscules in the whole mycorrhizal root wasquantified at the molecular level through transcript detec-tion in the G. intraradices glutamine synthase gene(GinGS), which has been described elsewhere as arbuscule-expressed (Gomez et al., 2009). The data in Fig. 1(d) showthe M values for gene induction. M value represents thelog2 ratio of gene expression in treated plants with respectto gene expression for nontreated plants. A more significantchange in GinEF expression was observed in relation totungstate-treated ETR6-AS plants, where M values werefound to be )2 (four times down-regulated) (Fig. 1d). TheM values for GinEF were )0.9 (almost twice down-regu-lated) and )0.4 (< 1.5 times down-regulated) in tungstatewild-type and NRO-treated plants, respectively. GinGSexpression decreased threefold in wild-type and nearlyeightfold in ETR6-AS mycorrhizal roots treated with tung-state (M values of nearly )1.5 and )2.7, respectively)(Fig. 1d). The application of tungstate affected GinGS geneexpression in NRO plants to a lesser degree than in the other

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Fig. 1 Effect of sodium tungstate application on mycorrhizationparameters (a, b, c) and expression of Glomus intraradices GinGS andGinEF genes (d) in plants with different ethylene sensitivities. After1 wk of transplanting and inoculation with G. intraradices, wild-typeFlora-Dade, ethylene-insensitive NRO and ethylene-hypersensitiveETR6-AS plants were treated with sodium tungstate alone (1.5 mM,black bars, a–c) and in combination with ABA (75 lM, grey bars, a–c). White bars, control (a–c). In (d) white bars, treated with sodiumtungstate; black bars, sodium tungstate + ABA. Solutions wereapplied to soil twice per wk and mycorrhization was measured 50 dafter inoculation. Mycorrhization parameters of frequency (%F),intensity (%M) and arbuscular abundance in the whole root (%A),and intensity (%m) and arbuscular abundance (%a) in mycorrhizalzones of the root were determined using MYCOCALC software.Quantitative reverse transcription polymerase chain reaction datarepresent the M value (log2 ratio). The value of M is 0 if there is nochange, and +1 or )1 if there is a twofold induction or reduction withrespect to the expression of each gene under nontreatmentconditions. Values are means ± SE. For each mycorrhizationparameter, bars with similar letters are not significantly different(P = 0.05) according to Duncan’s multiple range test.






plant varieties, while the application of ABA in combinationwith tungstate rescued both GinEF and GinGS expressionin all plants (Fig. 1d).

These results therefore show that genetic manipulation inthe ethylene receptors affects the mycorrhization processafter tungstate application differently according to ethylenesensitivity, and clearly illustrate that the effect of the reduc-tion in mycorrhizal root colonization as a result of tungstateapplication is mediated by the ethylene pathway.

We also performed LeETR3 (NR) and LeETR6 geneexpression analysis in order to confirm the activation of theethylene pathway during AM formation. qRT-PCR experi-ments with specific primers on LeETR3 and LeETR6 genefamilies were carried out during mycorrhization on wild-typeand ABA-deficient sitiens tomato plant roots, which are inher-ently capable of increasing ethylene. An induction of bothethylene receptor family genes during mycorrhization of wild-type plant roots was clearly observed (Fig. 2). In sitiens plants,both mycorrhizal and nonmycorrhizal root plants showed up-regulation of ETR6 and ETR3 tomato ethylene receptor genescomparing with wild-type nonmycorrhizal plants (Fig. 2).

Discrete ethylene and ABA roles during AM formation

To determine the ethylene-independent effect of ABA-regulated AM formation in tomato plants, we studied theeffect of sodium tungstate application on ABA concen-trations, ethylene production and mycorrhizal formation inACCD-expressing transgenic tomato plants. This trans-genic line expresses a bacterial ACC deaminase (Klee et al.,1991), which is involved in irreversible degradation of theethylene precursor ACC. Consequently, these plants exhibiteda reduction in ethylene content as a result of ethylene biosynthesis

impairment. This transgenic line has ABA concentrationssimilar to those of the wild-type, while the decrease in ABAcontent as a result of tungstate application was similar inboth wild-type and ACCD-expressing plants (Fig. 3a).There was no significant interaction between genotype andsodium tungstate ⁄ ABA application factors using two-wayANOVA (F2,12 = 1.77, P = 0.212), indicating that the ABAcontent in roots is only dependent on the tungstate ⁄ ABAapplication factor. The concentration of ethylene in ACCD-expressing plant roots was always below that for wild-typeplants (Fig. 3b). In the case of ethylene content, the inter-action between genotype and sodium tungstate ⁄ ABAapplication factors was significant, demonstrating that theeffect on ethylene content of tungstate application wasdependent on plant ethylene synthesis capacity (two-wayANOVA, F2,12 = 18, P = 0.0002) (Fig. 3b). This indicatesthat this transgenic tomato line is an ideal tool for separatelyanalysing the effects of ABA and ethylene.

The application of sodium tungstate to wild-type UC82Bplants negatively affected all the parameters of mycorrhiza-tion measured in the experiment, such as mycorrhizalfrequency and intensity and arbuscular abundance (Fig. 3c).Nevertheless, in ACCD-expressing plants, only arbuscularabundance (%a) was negatively affected following tungstateapplication (Fig. 3d). In the case of %M there was a signifi-cant interaction between genotype and sodium tungstate ⁄ABA application factors (two-way ANOVA, F2,18 = 3.88,P = 0.05), demonstrating that the application of tungstateselectively altered the %M parameter according to the plantphenotype studied.

The application of ABA combined with sodium tungstaterescued all the mycorrhization parameters in both wild-typeUC82B and ACCD-expressing plants (Fig. 3c,d). Even thevalues for mycorrhizal intensity (%M and %m) were signifi-cantly higher in ACCD-expressing plants treated withtungstate plus ABA. After tungstate application, the myco-rrhization parameters for wild-type UC82B plants werelower, while ABA content decreased and ethylene productionin roots increased (Fig. 3a,b). The decrease of almost 40% inABA content following tungstate application was similarfor both wild-type UC82B and ACCD-expressing plants(Fig. 3a). The concentration of ethylene in ACCD-express-ing plants remained below that recorded for UC82Bnontreated plants as well as those treated with sodium tung-state (Fig. 3b). For the %a parameter there was no significantinteraction between genotype and sodium tungstate ⁄ABA appli-cation factors (two-way ANOVA, F2,18 = 0.18, P = 0.836),indicating that the reduction in %a observed in these plants wasmainly caused by the reduction in ABA biosynthesis caused bytungstate application (two-way ANOVA, F2,18 = 16.32, P =0.0001). The decrease in %M and %a for tungstate-treatedUC82B plants reduced %A in these plants almost 2.2-fold.In ACCD-expressing plants, the application of tungstateonly affected the percentage of arbuscules (%a) in the myco-

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Fig. 2 Expression of LeETR6 and LeETR3 ethylene receptor genes inroots of wild-type Rheinlands Ruhm (Rhe) and ABA-deficient sitiens

(Sit, white bars) tomato plants colonized (I, black bars) andnoncolonized (NI, grey bars) by Glomus intraradices. Geneexpression was measured by quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR) 50 d after inoculation. qRT-PCR data represent the M value (log2 ratio). The value of M is 0 ifthere is no change, and +1 or )1 if there is a twofold induction orreduction with respect to the expression of each gene in wild-typeplants under noncolonized conditions. Values are means ± SE. Barswith similar letters are not significantly different (P = 0.05)according to Duncan’s multiple range test.

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rrhizal zones of the root and did not affect mycorrhizal intensity(%M). Consequently, the %A parameter for tungstate-treatedACCD-expressing plants (calculated from %a and %M data) fellonly 1.4-fold (Fig. 3d).

Fungal colonization was also quantified by qRT-PCR atthe molecular level to verify the microscopic measurementsof mycorrhization in tungstate, tungstate plus ABA-treatedand nontreated wild-type UC82B and ACCD-expressingtransgenic plants. The data shown in Fig. 4 represent the Mvalue for gene induction. M value represents the log2 ratio ofgene expression in treated plants with respect to gene expres-sion for nontreated plants in each genotype. No significantchange in GinEF expression was observed for ACCD-expressing plants, as the M values for tungstate and tungstateplus ABA treatments were found to be between 0 and )1 (lessthan twice down-regulation). Nevertheless, GinEF expres-sion decreased in wild-type UC82B mycorrhizal rootstreated with tungstate (M value of nearly )4, or 16 timesdown-regulation), and the application of ABA in combina-tion with tungstate rescued GinEF expression in these plants(the M value was up from )4 to )1). The data for GinGSgene expression, reflecting the presence of fungal activity inarbuscule cells, show that tungstate treatment negativelyaffects GinGS gene expression in both types of plants. Theapplication of tungstate diminished GinGS expression almosteight- and fourfold in wild-type and ACCD-expressing trans-

genic plants, respectively (M values of )2.7 and )1.8 forwild-type and ACCD-expressing plants, respectively). Therewas no negative impact on GinGS gene expression when theapplication of ABA was combined with tungstate (Fig. 4).

LeETR4 and LeETR6 gene expression analysis was carriedout to determine the differing responses at the ethylene sig-nalling pathway activation level in UC82B wild-type andACCD-expressing plants after tunsgstate applications.LeETR4 and LeETR6 ethylene receptor transcript accumu-lation was clearly induced in wild-type plant roots whentungstate was applied on its own, while the application oftungstate combined with ABA only had a slightly positiveeffect on transcript accumulation of LeETR4 (Fig. 5). InACCD-expressing plants, the LeETR4-6 ethylene receptortranscript accumulation was unaffected by tungstate appliedeither alone or in combination with ABA (Fig. 5). LeETR4expression levels were greater than and equal to levels foundfor LeETR6 in wild-type and ACCD-expressing plants,respectively (Fig. 5).

The dual ABA deficiency ⁄ ethylene enhancementmechanism causing mycorrhiza impairment in ABA-deficient mutant sitiens plants

To confirm that the reduced percentage of arbuscules recordedin mycorrhizal sitiens plants and wild-type mycorrhizal roots

ABA content

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(c) Mycorrhization parameters(UC82B wild-type plants)

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Fig. 3 Effect of sodium tungstate application on ABA content (UC82B, open bars; 1-aminocyclopropane-1-carboxylate deaminase (ACCD)-expressing, black bars, (a) and ethylene accumulation (ET) in roots (UC82B, open bars; ACCD-expressing, black bars, (b) and mycorrhizationparameters (control, open bars; sodium tungstate, black bars; sodium tungstate + ABA, grey bars, (c, d) of plants with different ethylenesynthesis capacities. After 1 wk of transplanting and inoculation with Glomus intraradices, wild-type UC82B and the ethylene transgenic tomatolines expressing ACCD, tomato plants were treated with sodium tungstate alone (1.5 mM) and in combination with ABA (75 lM). Solutionswere applied to soil twice per wk and the mycorrhization parameters, ABA content and ethylene accumulation were measured 50 d afterinoculation. Mycorrhization parameters of frequency (%F), intensity (%M) and arbuscular abundance in the whole root (%A), and intensity(%m) and arbuscular abundance (%a) in mycorrhizal zones of the root were determined using MYCOCALC software. Values are means ± SE.For each mycorrhization parameter, bars with similar letters are not significantly different (P = 0.05) according to Duncan’s multiple range test.






treated with tungstate was specifically caused by ABA sup-pression rather than ethylene enhancement, we performedexperiments where ethylene synthesis was blocked in sitiensplants by the application of AVG. AVG and ABA wereapplied alone and together to sitiens-planted soil, and data onfrequency, intensity and arbuscule abundance in mycorrhizalroots were collected. No significant variations in the valuesfor mycorrhization frequency were observed in sitiens plantsafter ABA treatment as compared with nontreated sitiensplants (Fig. 6). A slight increase in mycorrhization frequencywas observed after AVG and AVG + ABA treatments. Thisincrease in frequency was sufficient to reach the valuesrecorded for the wild-type plants (Fig. 6). Nevertheless, theintensity of mycorrhiza development noticeably increased inthe plants treated with AVG (alone or in combination withABA) (Fig. 6). The percentage of arbuscule abundance (%a)in the colonized parts of the roots was rescued to the valuesrecorded by wild-type Rheinlands Ruhm plants only whenABA was present (Fig. 6). The single application of ABA orAVG did not rescue the percentage of arbuscules in wholesitiens roots, while only the combined application of bothcompounds had a positive effect on the recovery of this para-meter in mycorrhizal sitiens roots (Fig. 6).

Aminoethoxyvinyl glycine hydrochloride, applied aloneor in combination with ABA, reduced ethylene productionin sitiens plants to the amounts recorded by wild-typeplants, while the application of ABA alone was not effectivein significantly decreasing ethylene concentrations in sitiensplants (Fig. 7).

GinEF GinGS

b,c

d,e

b

a

e

ee

c,d

–5

–4

–3

–2

–1

0

1

UC82B wild-type

ACCD-expressing

UC82B wild-type

ACCD-expressing

M v

alu

e

Fig. 4 Quantitative reverse transcription polymerase chain reaction(qRT-PCR) analysis of the expression of Glomus intraradices GinGSand GinEF genes in root plants with different ethylene synthesiscapacities during arbuscular mycorrhiza (AM) formation. After 1 wkof transplanting and inoculation with G. intraradices, wild-typeUC82B and the ethylene transgenic tomato lines expressing1-aminocyclopropane-1-carboxylate deaminase (ACCD), tomatoplants were treated with sodium tungstate alone (1.5 mM, openbars) and in combination with ABA (75 lM, closed bars). Solutionswere applied to soil twice per wk and data for qRT-PCR weremeasured in 50-d-old roots after inoculation. qRT-PCR data representthe M value (log2 ratio). The value of M is 0 if there is no change, and+1 or )1 if there is a twofold induction or reduction with respect tothe expression of each gene under nontreatment conditions. Valuesare means ± SE. Bars with similar letters are not significantly different(P = 0.05) according to Duncan’s multiple range test.

LeETR4 LeETR6

d

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UC82B wild-type

ACCD-expressing

UC82B wild-type

ACCD-expressing

M v

alu

e

Fig. 5 Quantitative reverse transcription polymerase chain reaction(qRT-PCR) analysis of the expression of LeETR4 and LeETR6 ethylenereceptor genes in root plants with different ethylene synthesiscapacities during arbuscular mycorrhiza (AM) formation. After 1 wkof transplanting and inoculation with Glomus intraradices, wild-typeUC82B and the ethylene transgenic tomato lines expressing1-aminocyclopropane-1-carboxylate deaminase (ACCD), tomatoplants were treated with sodium tungstate alone (1,5 mM, open bars)and in combination with ABA (75 lM, closed bars). Solutions wereapplied to soil twice per wk and data for qRT-PCR were obtainedfrom 50-d-old roots after inoculation. qRT-PCR data represent theM value (log2 ratio). The value of M is 0 if there is no change, and +1or )1 if there is a twofold induction or reduction with respect to theexpression of each gene under nontreatment conditions. Values aremeans ± SE. Bars with similar letters are not significantly different(P = 0.05) according to Duncan’s multiple range test.

Mycorrhization parameters

c

b

c

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a

a

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ab

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a

a

b

b

b

b

0102030405060708090

100

%F %M %a %A

%

Rhe 0Sit 0Sit AVGSit ABASit AVG + ABA

Fig. 6 Effect of ABA and aminoethoxyvinyl glycine hydrochloride(AVG) application on mycorrhization parameters of ABA-deficientsitiens tomato plants. After 1 wk of transplanting and inoculationwith Glomus intraradices, wild-type (Rhe 0) and a set of sitiens (Sit0) tomato plants were treated with 0.1% ethanol solution, andthree sets of sitiens plants were treated with AVG alone and incombination with ABA (Sit AVG; Sit ABA; Sit AVG + ABA). AVG(50 lM) and ABA (75 lM) solutions were applied to soil twice perwk. Mycorrhization parameters of frequency (%F), intensity (%M),arbuscular abundance in mycorrhizal zones of the root (%a), andarbuscular abundance in whole root (%A) were determined 50 dafter inoculation using MYCOCALC software. Values aremeans ± SE. For each mycorrhization parameter bars with similarletters do not significantly differ (P = 0.05) according to Duncan’smultiple range test.

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Discussion

As in previous studies on plant growth and development,ABA-deficient mutants were used in our study to investigatethe role of ABA during AM formation. Recent research hasshown that ABA-deficient sitiens plants are less susceptible toG. intraradices infection than wild-type plants and that ABAis involved in arbuscule formation and its functionality topromote sustainable colonization of the plant root during theestablishment of AM (Herrera-Medina et al., 2007). Anadditional role for ABA in the establishment of AM symbiosiscannot therefore be ruled out, as the reduction in AM coloni-zation observed in sitiens may also be caused by a reduction instrigolactone production by this mutant (Lopez-Raez et al.,2010). Interestingly, the exogenous application of ABA tositiens did not affect strigolactone production (Lopez-Raezet al., 2010). The failure to rescue sitiens strigolactonephenotype with exogenous ABA applications is in line withthe reported failure to complement sitiens AM-colonizationphenotype by exogenous ABA application (Aroca et al.,2008). The application of ABA to sitiens plants normalizedgrowth and led to an almost complete recovery in the freq-uency and intensity of alkaline phosphatase activity.Nevertheless, exogenous ABA applications were unable torestore the frequency and intensity of mycorrhization(Herrera-Medina et al., 2007).

There was some evidence that the role played by ABA inAM formation could, at least in part, be attributable toantagonistic interaction with ethylene (Herrera-Medinaet al., 2007; Martın-Rodrıguez et al., 2010). We thereforeinvestigated the relationship between ABA and ethylenewith respect to the regulation of the formation of AM

symbiosis in tomato plants and attempted to define thespecific roles of these different phytohormones during themycorrhization process. Previous studies have shown acorrelation between ABA root content and limitations inAM fungal colonization with respect to the ABA-deficientnotabilis and sitiens mutants in which the root ethylenecontent increased compared with their respective wild-typeplants (Martın-Rodrıguez et al., 2010). This suggests thatABA plays a role in symbiosis functionality regardless of theethylene content in roots. In addition, sodium tungstateapplication to roots caused a reduction in mycorrhizationand had a positive effect on ethylene production and anegative impact on ABA biosynthesis in tomato plants(Martın-Rodrıguez et al., 2010). It is therefore necessary todetermine whether the parameters indicating the negativeimpact of mycorrhization caused by ABA deficiency wereaffected by the decrease in ABA or by the ethylene accu-mulation associated with ABA deficiency.

To answer this question, we used two alternative strate-gies: analysis of the effects on the pattern of mycorrhizationas a result of the inhibition of ABA biosynthesis in trans-genic tomato plants with an altered ethylene pathway; andcomparison of the effects on mycorrhization between sitiensplants treated with the AVG ethylene biosynthesis inhibitoralone and those combined with ABA applications. We usedtwo different kinds of transgenic tomato lines with alteredethylene pathways. The NRO and ETR6-AS lines corre-spond to plants whose ethylene perception was affected byalterations in the expression of ethylene receptors (Ciardiet al., 2000; Kevany et al., 2007). The ACCD-expressingtransgenic plant line was made up of plants with ethylenesynthesis impairment (Klee et al., 1991).

Our results reveal that the reduction in root colonizationby tungstate application correlates with the increased sensi-tivity of plants to ethylene. The NRO plants with higherinsensitivity to ethylene as a result of overexpression ofthe NR receptor (Ciardi et al., 2000) were unaffected bytungstate application with respect to the percentage of myco-rrhizal intensity of colonization. This result was wellcorrelated with the finding that the expression of GinEF genewas unchanged in NRO mycorrhizal root after tungstateapplication. NRO transgenic plants overexpress the NRtomato ethylene receptor, which reduces ethylene sensitivityin seedlings and mature plants, indicating that this receptor isa negative regulator of ethylene response (Ciardi et al.,2000). Conversely, ETR6-AS plants, with increased sensitiv-ity to ethylene as a result of ETR6 mRNA inactivation byantisense overexpression (Kevany et al., 2007), were moreaffected in the parameters of intensity of mycorrhization(%M, %m), and decrease in GinEF expression when tung-state was applied either alone or in combination with ABA.The reduction in LeETR6 mRNA levels in ETR6-AS linesproduced early-ripening phenotypes, which are consistentwith a constitutive ethylene response (Kevany et al., 2007).

Ethylene content

a,b

b,ca,b

c

a

0

0.05

0.1

0.15

0.2

0.25

Rhe 0 Sit 0 Sit AVG Sit AVG +ABA

Sit ABA

Treatments

ng

g–1

h–1

Fig. 7 Effect of ABA and aminoethoxyvinyl glycine hydrochloride(AVG) application on ethylene content in roots of ABA-deficientsitiens tomato plants colonized by Glomus intraradices. After 1 wkof transplanting and inoculation with G. intraradices, wild-type (Rhe0) and a set of sitiens (Sit 0) tomato plants were treated with 0.1%ethanol solution and three sets of sitiens plants were treated withAVG alone and in combination with ABA (Sit AVG; Sit ABA; SitAVG + ABA). AVG (50 lM) and ABA (75 lM) solutions wereapplied to soil twice per wk, and ethylene content was determined50 d after inoculation. Values are means ± SE. Bars with similarletters do not significantly differ (P = 0.05) according to Duncan’smultiple range test.






Our results clearly show that the impact of this reduction onthe intensity of mycorrhizal root colonization caused bytungstate application is mediated by the ethylene pathway.This result is in line with previous data where epinastic (epi)and Never ripe (Nr) mutants, ethylene overproducer and lowsensitivity, respectively, have the intraradical colonization byGlomus clarum highly inhibited, as compared with the con-trol Micro-Tom (Zsogon et al., 2008). Furthermore, qRT-PCR analysis of LeETR6 and LeETR3 ethylene receptorgenes indicates that their expression was enhanced in wild-type Rheinlands Ruhm as a result of mycorrhization, suggest-ing the role of ethylene during AM formation. In sitiensroots, LeETR6 and LeETR3 ethylene receptor gene expressionwas constitutively increased regardless of mycorrhization, asthese plants are inherently capable of increasing ethylene.

By contrast, the presence of tungstate reduced arbuscularabundance in the mycorrhizal root zones (%a) regardless ofthe plant’s ethylene sensitivity. In this sense, a reduction inGinGS gene expression, a marker for the presence of arbus-cules in roots, was observed in all plant varieties treatedwith tungstate. The data of GinGS expression were bettercorrelated with the parameter of abundance of arbuscules inwhole mycorrhizal root (%A) since both represent valuesfrom the whole root system. Unlike the process observedfor the intensity of colonization in roots, these results clearlyshow that the impact of this reduction on the arbusculeabundance in the mycorrhizal zones of the roots (%a) as aresult of tungstate application is not mediated by theethylene pathway.

1-Aminocyclopropane-1-carboxylate deaminase-express-ing transgenic plants (ACCD plants) are subject to ethylenesynthesis impairment as a result of the catabolism of itsimmediate precursor, ACC, by the action of the bacterialACC deaminase cloned and introduced into tomato plants(Klee et al., 1991). We used this transgenic tomato plantline to separately analyse the effect of both ABA and ethyl-ene. ACCD-expressing plants, which have less ethylenecontent in their roots, showed no alteration in mycorrhiza-tion capacity. These plants also responded to tungstateapplication in a similar way to the UC82B wild-type,recording a reduction of nearly 40% in ABA content inroots. The decrease in ethylene synthesis in transgenicACCD-expressing plants did not cause any apparent vegeta-tive phenotypic abnormalities, and only some delays in fruitripening have been observed (Klee et al., 1991). Subtle dif-ferences in response to the environmental effects ontransgenic plants cannot be ruled out (Klee et al., 1991).

In our experiments, the application of tungstate selectivelyaltered the parameters of mycorrhization according to theplant phenotype studied. In ACCD-expressing plants, onlythe percentage of arbuscules (%a) in mycorrhizal roots wasaffected, while in wild-type UC82B plants, the application oftungstate affected all the mycorrhization parameters. Thehistochemical data correlated closely with the transcript accu-

mulation results for GinGS and GinEF. The pattern oftranscript accumulation for GinGS, a fungal gene expressedin arbuscular cells (Gomez et al., 2009) paralleled the dataon arbuscule abundance (%a, %A) in mycorrhizal roots. Aswith these parameters, GinGS gene expression was down-regulated in both wild-type and ACCD-expressing plantsafter tungstate application. On the other hand, the expressionpattern of GinEF was closely correlated with the data ofmycorrhizal intensity (%M) and its expression was down-regulated only in wild-type plants after tungstate application.

The reduction in mycorrhization parameters for UC82Bplants was associated with the effects of lower ABA contentand higher ethylene production in roots, whereas the reduc-tion in %a observed in ACCD-expressing plants wasmainly caused by the decrease in ABA biosynthesis. In con-trast to wild-type UC82B, no changes in the expression ofthe ETR4 or ETR6 tomato ethylene receptors were reportedfollowing tungstate application in ACCD-expressing plants.These genes are ethylene-inducible (Kevany et al., 2007),and the absence of up-regulation confirms that the ethyleneresponse in ACCD-expressing plants after tungstate appli-cation was ineffective.

These results indicate that ABA positively affects arbus-cule formation (measured as %a), while ethylene mainlyregulated the ratio of colonization (measured as mycorrhizalintensity). To confirm this hypothesis, we determined theeffect of ABA and an ethylene synthesis inhibitor on the res-cue of mycorrhiza parameters in sitiens plants. We clearlydemonstrated here that mycorrhiza rescue in sitiens plantsvaried depending on the compound applied. The data showa significant increase in the intensity of mycorrhiza develop-ment associated with the inhibition of ethylene biosynthesisresulting from AVG treatment (alone or in combinationwith ABA) as well as a rescue of arbuscule abundance insitiens mycorrhizal roots when ABA is present.

In previous work, the higher rate of recovery for fungalalkaline phosphatase activity in the sitiens mutant occurredafter ABA applications but not with silver thiosulphate appli-cations, which blocked ethylene perception, suggesting thatABA may have an additional role in arbuscular functionalityapart from its function as an inhibitor of ethylene production(Herrera-Medina et al., 2007).The results shown in thisstudy are in line with previous data, which confirmed thatABA applications restored fungal alkaline phosphatase activ-ity (mostly associated with arbuscules) in sitiens roots, thoughnot fungal spread, as measured by trypan blue staining(Herrera-Medina et al., 2007). In a previous study, therecovery rate reached 25% in %F, while only a slight increasein %a was observed in sitiens mycorrhizal roots after ABAapplication (Herrera-Medina et al., 2007). However, in ourexperiments, we found that the exogenous ABA applicationswere unable to restore mycorrhization frequency or fully res-cue arbuscule abundance. Although similar plant and fungallines were used, this discrepancy could be caused by differing

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colonization dynamics in both experiments, as, comparedwith previous studies, the %F and %a values recorded in ourstudy for nontreated sitiens plants were > three times higherfor plants with approximately the same harvest time. In a my-corrhization experiment carried out in parallel with thatshown in Fig. 6, we used G. mosseae as a fungal inoculum,and observed a similar increase in the intensity of mycorrhizadevelopment associated with the inhibition of ethylene bio-synthesis caused by AVG treatment. We also noted thatarbuscular abundance in sitiens mycorrhizal roots was rescuedwhen ABA was present (Supporting Information, Fig. S1).It is therefore clear that, in zones with sitiens mycorrhizalroots colonized by the fungus, the exogenously applied ABArestored arbuscular formation and functioning capacity, as mea-sured by fungal alkaline phosphatase activity (Herrera-Medinaet al., 2007) or in terms of arbuscular abundance rescue insitiens mycorrhizal roots as shown in the current study.

The values for ethylene production in roots demonstratethat AVG, applied alone and in combination with ABA,reduced ethylene production in sitiens plants to wild-typeplant amounts, whereas the application of ABA alone wasnot sufficiently effective in significantly decreasing theamount of ethylene in sitiens plants. Interestingly, whenABA was exogenously applied to sitiens plants, a completerescue of arbuscular abundance was observed, while theapplication of AVG did not produce a total recovery inmycorrhization intensity. The observed differential responseto similar ethylene concentrations in wild-type and sitiensplants could be a result of their different ethylene sensitivi-ties. The effect of the reduction in ethylene concentrationsas a result of exogenous AVG application to the mutantplant may not have been sufficient to restore all physiologicaldeficiencies caused by the mutation affecting mycorrhizalcolonization.

This study shows that a dual ethylene-dependent ⁄ ethyl-ene-independent mechanism is associated with ABAregulation of the AM formation (Fig. 8). ABA is necessaryfor arbuscule formation, indicating that ABA deficiency hasa direct negative effect on the percentage of arbuscules inmycorrhizal roots. ABA deficiency enhances ethylene con-tent, which functions as a negative regulator of mycorrhizalintensity. This is in line with previous data, where the exog-enous application of ethylene restricted the spread of theAM fungus along the length of the root’s axis, although itsmovement towards the inner cortex, where arbuscules areformed, did not appear to be impeded (Geil et al., 2001).Furthermore, ethylene has been suggested as a negative reg-ulator in early infection events such as infection threadformation or elongation and cortical cell activation duringnodulation in legumes (Sugawara et al., 2006), and earlyphases of the symbiotic interaction of Medicago truncatulawith mycorrhizal fungi (Penmetsa et al., 2008).

In this study, the application of ABA to mycorrhizalsitiens roots led to a recovery in arbuscule formation capac-

ity. Further research is required to clarify the role of ABA inarbuscule formation and to identify putative crosstalkbetween plant hormones in this process.

Acknowledgements

We wish to thank the Tomato Genetics Resource Centre(TGRC) of the University of California and Dr H. Klee atthe University of Florida for providing tomato seeds. Wewould also like to thank Isabel Tamayo and Nuria Molinerofor their help with the plant experiments. Financial supportfor this study was provided by grants from the ComisionInterministerial de Ciencia y Tecnologıa (CICYT) andFondos Europeos de Desarrollo Regional (FEDER) throughthe Ministerio de Ciencia e Innovacion, Spain (AGL2005-

Arbuscule formation and functioning

?

ABA

Ethylene

AVG

Sodium tungstatesitiens plants

Strigolactones?

Fungal spreadin the root

Hyphal branchingand apresoria formation

?

ABA

Ethylene

Fig. 8 Proposed model of the regulation of arbuscularmycorrhization in tomato roots by ABA ⁄ ethylene interaction. A dualethylene-dependent ⁄ ethylene-independent mechanism issuggested for ABA regulation of arbuscular mycorrhiza (AM)formation. ABA is necessary for the process of arbuscule formationand functioning, and an additional role played by ABA in theestablishment of AM symbiosis mediated by strigolactoneproduction cannot be ruled out (dashed arrow). In addition, ABAinteracts antagonistically with ethylene, which negatively regulatesthe ratio of fungal spread in the root. ABA deficiency as a result ofsodium tungstate application or mutation in sitiens plants reducesarbuscular abundance and increases ethylene concentrations,causing a decrease in the rate of colonization. Inhibition of ethylenesynthesis by aminoethoxyvinyl glycine hydrochloride (AVG) inABA-deficient sitiens plants specifically increases the intensity ofmycorrhiza development.






0639; AGL2008-00742) as well as the Junta de Andalucıa(Research Group BIO 260). J.A.M.R. and R.L.M. were sup-ported by fellowships from the FPU-MICINN and JAE-preCSIC programmes, respectively.

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Supporting Information

Additional Supporting Information may be found in theonline version of this article.

Fig. S1 Effect of ABA and AVG application on mycorrhiza-tion of ABA-deficient sitiens tomato plants.

Please note: Wiley-Blackwell are not responsible for thecontent or functionality of any supporting informationsupplied by the authors. Any queries (other than missingmaterial) should be directed to the New Phytologist CentralOffice.






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