Effects of Phenolic Compounds on Agrobacterium Vir Genes and Gene

Plant Science 162 (2002) 733Á/743 www.elsevier.com/locate/plantsci

Effects of phenolic compounds on Agrobacterium vir genes and gene

transfer induction*/a plausible molecular mechanism of phenol binding protein activation

Philippe Joubert a, Daniel Beaupe`re b, Philippe Lelie`vre b, Anne Wadouachi b, Rajbir S. Sangwan a,*, Brigitte S. Sangwan-Norreel a

a UPRES EA 2084, Laboratoire Androgene`se et Biotechnologie, Faculte des Sciences, Universite´ de Picardie Jules Verne, 33 rue Saint-Leu, 80039 Amiens Cedex, France b Laboratoire de Chimie Organique, Universite´ de Picardie Jules Verne, 33 rue Saint-Leu, 80039 Amiens Cedex, France

Received 31 October 2001; received in revised form 8 January 2002; accepted 8 January 2002

Abstract

We have studied the effects of nine new phenolic compounds: 4-(4-hydroxy-3-methoxyphenyl)-but-3-en-2-one 4, 4-(4-hydroxy-3- methoxyphenyl)-but-3-en-2-one 5, 4-hydroxy-3,5-dimethoxycinnamyl alcohol or sinapyl alcohol 6, 4-hydroxy-3,5-dimethoxy-N,N- dimethylcinnamamide 7, N-ethyl-4-hydroxy-3,5-dimethoxycinnamamide 8, 1-(4-hydroxy-3,5-dimethoxyphenyl)-3-(4-hydroxy-3,5- dimethoxyphenyl)-prop-2-en-1-one 11, 1-(4-hydroxyphenyl)-3-(4-hydroxyphenyl)-prop-2-en-1-one 12, 4-(4-hydroxy-3,5-dimethoxyphenyl)-butan-2-one 13, and 1-(4-hydroxy-3,5-dimethoxyphenyl)-3-(4-hydroxyphenyl)-propan-1-one 14,onAgrobacterium virulence gene induction, on Agrobacterium-mediated gene transfer, and on both transient and stable transformation rates on Petunia and tobacco. We confirmed that virulence induction and transformation rates are increased by the use of phenolic vir inducers bearing an unsaturated lateral chain. However, we found that the presence of at least one ortho-methoxy group in the phenolic compounds is required to increase the vir gene induction. Moreover, we synthesized phenolic compounds 13 and 14 with a saturated lateral chain from the corresponding compounds 4-(4-hydroxy-3,5-dimethoxyphenyl)-but-3-en-2-one 2 and 1-(4-hydroxy- 3,5-dimethoxyphenyl)-3-(4-hydroxyphenyl)-prop-2-en-1-one 9, in order to stop conjugation between hydroxyl and carbonyl groups in these molecules. These compounds showed a lower efficiency of both vir induction and gene transfer. Furthermore, we also synthesized two amides derivatives 7 and 8 from sinapinic acid. The nature of alkyl groups on nitrogen is essential to the vir induction and gene transfer, and we observed that N-dimethylamide compound 7 is less active than N-ethyl compound 8. This may be due to the difference of the electron-donating effect between methyl and ethyl groups. We present a model for the molecular mechanism of VirA activation by phenols. # 2002 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Petunia (Petunia hybrida); Tobacco (Nicotiana tabacum); Agrobacterium tumefaciens; Vir gene induction; Transformation induction; VirA; Phenolic compounds

1. Introduction chv */chromosomal virulence*/genes are involved in the recognition and immobilization of the bacteria on Among the different techniques used for the intro- the epidermal plant cell surface [5]. The vir genes are duction of foreign genes in plants, Agrobacterium located on an extra-chromosomal DNA replicon, the tumefaciens-mediated transfer remains the most popular Tumour-inducing plasmid (Ti). The induction of vir and a powerful one [1Á/4]. It is accepted that Agrobac- genes leads to the transfer of a part of the Ti plasmid: terium-mediated transfer requires the activation of two the transferred-DNA (T-DNA). Comprehensivereviews Agrobacterium gene families: chv and vir genes. The on the DNA transfer process from the bacteria to the plant cells are available [4,6,7]. The vir genes are specifically induced by phenolic * Corresponding author. Tel.: 33-3-22-82-76-49; fax: 33-3-22- 82-76-12. compounds. First, intermediates of lignin synthesis or E-mail address: [email protected] (R.S. Sangwan). phenolic precursors, such as acetosyringone 1 (AS)and

0168-9452/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 0 2 ) 0 0 0 1 2 - 2 734 P. Joubert et al. / Plant Science 162 (2002) 733Á/743 hydroxy-AS [8,9], are chemoattractants at very low mechanism for possible molecular interactions between concentrations, but become vir inducers at high con- phenolics and VirA complex protein site. centrations [10Á/13]. A variety of other phenols have been described and their vir gene induction abilities investigated [14Á/16]. Furthermore, other families of 2. Material and methods phenolic compound such as hydroxycinnamides are known to act as vir gene inducers [17]. It has also 2.1. Chemical structures, synthesis and description been reported that monosaccharides such as D-glucose, D-galactose and D-xylose, which are ‘long distance’ The structures and details of different chemical attractors of Agrobacteria, work in synergy with phe- functions used are reported on Fig. 1 and Table 1. nolic compounds in order to increase vir gene induction Simple phenolic compounds 1, 3 and 6 were purchased [18,19] and enlarge the recognition profiles of sensitive from Aldrich (Strasbourg, France). Typical experimen- Agrobacteria strains to phenolic compounds [20]. tal procedures for synthesis of all other derivatives. Furthermore, opines [21] and flavonoid compounds [22] may be involved in vir gene induction. It was demonstrated [23] that if the two basic elements 2.1.1. General procedure for synthesis of benzalacetones required for phenol-mediated induction of virulence (2, 4, 5) and chalcones (9Á/12) gene expression, VirA and VirG are encoded on the Ti An ether solution of borontrifluoride (1 eq.) and plasmid, they are dependent on the chromosomal back- ketone (R/CO/CH3: 2 eq.) were added successively dropwise to a solution of benzaldehyde derivative ground for even the very first stage for signal perception. 3 In the vir gene induction process, the first stage is the (HO,R1,R2-PhCHO: 8.2/10 mol) in CH2Cl2 (10 recognition of phenols by a transmembrane VirA ml). The resulting solution was stirred for 24 h at room temperature, then neutralized with NaOAc and protein encoded by the virA gene [24Á/26]. The recognition and autophosphorylation of VirA [27] leads to the extracted with CH2Cl2. The combined extracts were phosphorylation of the VirG cytoplasmic protein [28] washed with water, dried over Na2SO4 and filtered. The which, in turn, induces the expression of other vir genes. organic layers were concentrated under reduced pressure Moreover, a direct molecular mechanism which may and the crude product purificated by column chromato- explain the action of AS on VirA was put forward graphy on silica gel (n-hexane-etOAc). a,b-ethylenic [16,29,30]. It was suggested that an acidic VirA residue ketones were obtained in 60Á/75% yield. The same may protonate the carbonyl group of phenolic com- aldolisation procedure was applied to acetophenone pounds. The electron resonance delocalization leads to derivative (HO,R1,R2 /PhCOCH3) for synthesis of a,b- an electron transfer from the phenol to the acidic ethylenic ketone 9. residue. Then the phenol can protonate a VirA basic residue, leading to the autophosphorylation of VirA and 2.1.2. General procedure for ketones 13 and 14 then to the phosphorylation of VirG. They were obtained by the hydrogenation of a,b- It was suggested [31] that the VirA protein may not be ethylenic ketones 2 and 9 [34]. In this procedure, a directly involved in the bonding of phenolic compounds. toluene solution of unsaturated compound 2 and 9 (0.25 1 1 Indeed, two chromosome-encoded proteins (p10 and mol l ) and 1-phenylethanol (0.25 mol l ) and hy- p21) were discovered which could act as receptors of dridotetrakis (tris tri-phenylphosphine) rhodium I [35] 2 phenols and mediate interaction between phenolics and (RhH(PPh3)4: 4.10 eq.) was heated at 50 8C for 2 h. VirA. Turk et al. [32] demonstrated that the periplasmic After solvent evaporation, the purification by chroma- domain of VirA does not react with AS, whereas its tography on silica gel led to products 13 and 14 in 92 cytoplasmic domain (aa283Á/aa304) is able to sense the and 96% yield respectively. signal whenever AS is present in the medium. Recently, Dye´ and Delmotte [33] discovered another Agrobacter- 2.1.3. General procedure for amides 7 and 8 ium-phenol binding protein, Pbp39, but whether this N,N?-dicyclohexylcarbodiimide (1.09 eq.) was added new protein is a vir gene inducer has not yet been to a suspension of 4-hydroxy-3,5-dimethoxycinnamic determined. acid in dry CH2Cl2 under N2 atmosphere. The resulting In order to understand the molecular interactions suspension was stirred for 30 min at room temperature. between phenolic compounds and the VirA complex Then a solution of amine (dimethylamine0/7 or protein or any other receptor proteins such as p10 and ethylamine0/8) 2M in THF (1.2 eq.) was added drop- p21, we havedeveloped new phenolic compounds and wise. The mixture was stirred at room temperature for tested their effects on vir induction. Furthermore, these 12 h. The dicyclohexylurea (DCU) was filtered off, new phenols were tested on tobacco and Petunia washed with CH2Cl2 and the filtrate was concentrated transformation and, as the results compared favourably under reduced pressure. The purification of the residue with their vir induction, this has enabled us to suggest a by flash chromatography on silica gel (EtOAc, EtOAc/ P. Joubert et al. / Plant Science 162 (2002) 733Á/743 735

Fig. 1. General structures of tested phenolic compounds, which were AS, benzalacetones and chalcones derived. Arrows indicate ethylenic bonds, which were hydrogenated on the 13 and 14 compounds.

EtOH 14:1) led to compounds 7 or 8 as yellow solids in v) sucrose and 0.8% Difco agar, pH adjusted to 5.6 80 and 82% yield respectively. before autoclaving (120 8C for 20 min). The cultures were maintained in a culture room at 27 8C under a 2 1 2.1.4. Physical data light regime of fluorescent light (:/32 E m s )16h The physical characteristics of all known compounds a day. were in perfect accordance with the literature values. All unknown compounds were characterised by elementary 2.3. Bacterial strains analysis and NMR Spectroscopy. A. tumefaciens A348 pSM219 virH::lacZ (pinF::lacZ) 2.2. Plant material was used to evaluate vir gene induction. It was kindly supplied by B. Hohn (FMI, Basel, Switzerland). A. Leaves of Petunia hybrida and Nicotiana tabacum tumefaciens EHA105 (pG3.3) carrying a binary vector were cultured on a basal medium (MS) that contained with NPTII and GUS genes was kindly provided by S. salts and vitamins of Murashige and Skoog [36], 3% (w/ Hamon-Poirier (CNRS Gif/Yvette, France). Bacteria

Table 1 Description of various phenolic compounds tested

No. R1 R2 R3 Usual name

1 OCH3 OCH3 COCH3 Acetosyringone (AS) Benzalacetones derived

2 OCH3 OCH3 COCH3 Á/ 3 OCH3 OCH3 CO2H Sinapinic acid 4 OCH3 H COCH3 Á/ 5 H H COCH3 Á/ 6 OCH3 OCH3 CH2OH Sinapyl alcohol 7 OCH3 OCH3 CON(CH3)2 Á/ 8 OCH3 OCH3 CONHCH2CH3 Á/ Chalcones derived

No. R1 R2 R1? R2?

9 H H OCH3 OCH3 10 OCH3 OCH3 HH 11 OCH3 OCH3 OCH3 OCH3 12 HHHH Reduced compounds 13 Same 10 with reduced ethylenic bond 14 Same 12 with reduced ethylenic bond

Showing the substitutions at R1,R2,R1?,R2? and R3. Compounds 1, 2, 3, 9 and 10: Joubert et al. [40], and this work. Compounds 4, 5, 6, 7, 8, 11, 12, 13 and 14: this work. 736 P. Joubert et al. / Plant Science 162 (2002) 733Á/743 colonies were picked from selection plates (Luria Broth e´ales et des Fourrages (Paris, France). In each case, we medium/carbenicillin 100 mg/l (A348) or kanamycin proceeded to a variance analysis. This test gaveus 100 mg/l (EHA105)) and grown overnight in Luria probable differences between phenolic treatments. Ad-

Broth liquid medium [37] with the appropriate anti- ditional investigations with a NewmanÁ/Kuels test biotics added, at 28 8C, in a rotary shaker (210 rpm). classified these treatments in decreasing order with a 95% safety margin. 2.4. Determination of vir gene expression

To test vir gene induction, an overnight colony of 3. Results Agrobacterium A348 (pSM219) virH::lacZ was centri- fuged and suspended in 15 ml of mannitol-phosphate 3.1. Action of benzalacetones and chalcones on vir gene buffer (0.6 M mannitol 0.01 M NaPi, pH 5.2). A 100 ml expression and transformation rates aliquot of this bacterial suspension was added to a 900 ml MSPS liquid medium (MS medium supplemented Benzalacetone derivatives 2, 4 and 5, which differ by with 62.5 mM NaPi and 3% sucrose, pH 5.25), to which the number of methoxy groups, have been tested (Table the phenolic compound to be tested was also added, and 1). As reported in our previous work [40], we noted (Fig. grown for 18 h at 28 8C. The expression of vir genes 2) that a compound without methoxy groups, benzala- was monitored through the measurement of b-galacto- cetone 5, does not induce vir functions, whereas the sidase activity of the fusion gene. Bacterial cells were presence of one of them, in benzalacetone 4, leads to less harvested and b-gal activity [37] determined as described active(2/5, 3/2) vir inducers. Similarly, the chalcone by Sambrook et al. [38]. derivative 12, lacking methoxy group as compared to the structure of compound 11, does not induce vir genes, 2.5. Inoculation of explants whereas 11 had an effect on the vir induction similar to AS effect. Young leaves were collected from 2 months-old However, the tetra-methoxylated compound 11 tobacco and petunia plants. Leaf pieces (1 cm2) were showed a significant decrease of vir induction ability dipped into a suspension EHA105 strain (1 ml overnight compared to its analogue derivatives 9 and 10.We culture of bacteria/9 ml of MS medium) for 20 min, suggest that the presence of four methoxy groups blotted dry on sterile paper, and incubated on MS basal increases the molecular bulk of the phenolic molecule medium with the appropriate phenolic compound for 3 and consequently reduce its affinity to the VirA receptor days. Cocultured explants were washed in liquid MS site. medium in the presence of 500 mg/l cefotaxim and In all cases, we noted that activity of 2, 9 and 10 on vir blotted dry on sterile paper. They were then plated on induction was very high and greater than AS. From the MS medium containing 300 mg/l cefotaxim, 250 mg/l hypothesis of Hess et al. [16], the conjugated structures kanamycin and: (i) 4.44 mM BAP/0.57 mM IAA for led to a delocalization of electrons by mesomeric effect tobacco plants; and (ii) 4.44 mM BAP/2.68 mM NAA on the whole molecule (Fig. 3). In comparison with AS, for petunia plants, for direct selection and regeneration the additional double bond on R3 alkyl chain should of transformed callus and buds. contribute to a better stabilisation of the mesomeric form, thereby allowing the phenolic proton to leave 2.6. Transient and stable transformation results more easily and then activate phenol binding protein before speeding up the VirA. Transient transformation was assessed 3 days after Following tests of these compounds as vir inducers in coculture. Leaf explants were ground and extracts used Agrobacterium, we tested them for tobacco transforma- to measure b-glucuronidase activity, as described by tion efficiency. Most compounds displayed positive Jefferson [39]. Four or five weeks after coculture, effects on transformation results (Fig. 4). Phenolics 2, regenerated transformed (kanamycin resistant) callus 9 and 1 (AS) appeared to be the strongest vir inducers and buds of tobacco and petunia, respectively, were with the production of twice as many transformed callus counted. The average number of regenerates per explant than in control experiment (control: leaf explants was then established. Forty five explants were cocul- cocultured without any phenolic) while compound 3, tured per series and all experiments repeated three times. of a limited vir induction ability, did not enhance the transformation rate. These results confirm the impor- 2.7. Statistical studies tance of these compounds and their effects, on the virulence of Agrobacterium, and hence on T-DNA All experiments were controlled through statistical transfer. analysis carried out with the STAT-ITCF computer We also checked the GUS gene expression by program provided by the Institut Technique des Ce´r- quantification of b-glucuronidase in order to evaluate P. Joubert et al. / Plant Science 162 (2002) 733Á/743 737

Fig. 2. Action of phenolics on virH gene. All structures were tested at a concentration of 100 mM. Values are average (n9)9s.e. Probability showing differences between media was equal to 1.00 and NewmanÁ/Kuels test classiﬁed medium effects in ﬁve classes noted A to E.

the relationship between vir gene induction, and transient and stable gene transfer. This experiment was aimed at linking vir induction results (Fig. 2) to stable transformation occurrences (Fig. 4) generated by the same molecules. Fig. 5 shows that the most efficient vir inducers molecules (2, 9, 1 (AS), 10 and 11) also result in a high gene transfer frequency in Petunia and tobacco. The gradient of gene transfer and stable transformation seems to follow vir gene induction. Nevertheless, we noted the particularly high level of gene transfer induced Fig. 3. Induction model of phenol binding protein site (VirA) with a by compound 9 on Petunia. Conversely, we tested a non electronic transfer from basic to acidic residue (proposed by Hess et al. vir-inducing compound in order to find out whether it [16]). may also induce a low frequency of gene transfer on Petunia and tobacco. The molecule 5 tested showed a

Fig. 4. Inﬂuence of phenolics on tobacco transformation. The number of callus regenerated per leave explant after 3 weeks’ culture on selection medium is represented. Explants had been cocultured with bacteria for 3 days in the presence of 100 mM tested phenolic compound. The control was a cocultured medium without phenolic. Values are average (n6)9s.e. Probability showing differences between media was 0.95 and NewmanÁ/ Kuels test classiﬁed the phenolic effect in two homogeneous groups noted A and B. 738 P. Joubert et al. / Plant Science 162 (2002) 733Á/743

Fig. 5. Action of phenolic compounds on Agrobacterium-mediated gene transfer. Transient expression of GUS gene in leaf explants of Petunia and tobacco after 3 days of A. tumefaciens cocultivation, in the presence of 100 mM of the tested phenolic compounds. The control represents GUS activity in non-cocultivated explants; the control is GUS activity in explants cocultivated without any phenolic. GUS activities are measured kinetic (nm) apparition of fluorescent 4-MU substrate per minute and per gram of fresh weight. Values are average (n3)9s.e. Probability showing differences between media was equal to 1.00 and NewmanÁ/Kuels test classified the phenolic effect in six homogeneous Petunia groups and five Tobacco groups noted A, B, C, D, E and F. total inability to induce gene transfer and, furthermore, to give products 13 and 14. According to the above a low inhibition of this transfer, a feature presumably mentioned hypothesis, the electron transfer between the related to the general toxicity of phenols. carbonyl and phenolic groups of molecules 13 and 14 was not possible. The acidity of the hydroxyl group was 3.2. Action of hydrogenated benzalacetones and only enhanced by attractive effect of the phenyl group chalcones on vir gene and transformation induction (Fig. 6). As regards vir gene induction, compounds 13 and 14 showed a limited efficiency compared to In relation to the hypothesis of Hess et al. [16], we compounds 2 and 9, respectively (Fig. 7). Such reduced synthesized molecules 13 and 14 and tested them on vir activities accounted for 1/2 and 1/3 of that of the gene induction. The double bonds of compounds 2 and original products, but remained much higher than the 9 were treated under catalytic hydrogenation conditions activities observed in the control experiment.

Fig. 6. Example of electronic delocalization which can affect the aromatic cycle of phenolic compounds and explains the vir activity of compound 13. P. Joubert et al. / Plant Science 162 (2002) 733Á/743 739

Fig. 7. Action of hydrogenated compounds*/13 and 14*/on virH gene induction. Compounds were tested at 100 mM. Values are average (n9)9s.e. Probability showing differences between media was equal to 1.00 and NewmanÁ/Kuels test classiﬁed medium effects in ﬁve classes noted AtoE.

We tested these four molecules 2, 13, 9 and 14 in the From both the molecular mechanism suggested by presence of Agrobacteria throughout cocultivation with Hess et al. [16] and the above results, we believe that the explants of Tobacco and Petunia. After a few weeks on following hypothesis is well-founded. On the one hand, selection medium, we calculated that explants coculti- a non-conjugated lateral chain R3 might explain the vated with 13 and 14 showed a significant decrease in the relative decrease in the vir and transformation induction regenerated callus number. Therefore, compound 14 did (Fig. 7 and Fig. 8). On the other hand, the hydrogena- not enhance the transformation induction. However, tion of this chain changes the initial planar structure of explants cocultivated with 13 maintained a higher level the molecule and induces, in comparison with 2 and 9, a of transformation than the control. new freedom of movement for R3. Therefore, the

Fig. 8. Inﬂuence of phenolics 13 and 14 on Petunia and Tobacco transformation. The number of callus regenerated per leaf explant after 3 weeks’ culture on selection medium for Tobacco and 4 weeks for Petunia is represented. Explants had been cocultured with bacteria for 3 days in the presence of 100 mM tested phenolic compound. The control was a cocultured medium without phenolic. Values are average (n6)9s.e. Probability showing differences between media was 0.95 and NewmanÁ/Kuels test classiﬁed phenolic effect in three homogeneous groups noted A, B and C. 740 P. Joubert et al. / Plant Science 162 (2002) 733Á/743

Fig. 9. Action of nitrogenated compounds*/7 and 8*/on virH gene induction, compared to the corresponding benzalacetones. Compounds were tested at 100 mM. Values are average (n6)9s.e. Probability showing differences between media was equal to 1.00 and NewmanÁ/Kuels test classiﬁed medium effects in ﬁve classes noted A, B, C, D and E. probability of simultaneous recognition of both the The spatial size of the dimethyl groups on nitrogen (7)is chemical phenol and carbonyl functions in the VirA site higher than in the ethyl group (8). This may explain why decreased. it is so difficult for the carbonyl group to reach the VirA acidic receptor site. We noted that compound 6 has a higher vir action 3.3. Action of amino compounds on vir and while its R chain has an alcohol function and not a transformation induction 3 carbonyl function. This molecule could be oxidized by the oxygen in the medium and transformed into It was reported by Dye´ et al. [41,42] that different aldehyde, then acid, which are reliable vir inductors. syringamides are new vir inducers. These compounds appeared to have2Á/20 times stronger action than the syringic acid, depending on the Agrobacterium lacZ fusion strains. We decided to test amides of the sinapinic 4. Discussion acid 3 as vir inducers. So, we synthesized two amide derivatives of 3: the 4-hydroxy-3,5-dimethoxy-N,N- Hess et al. [16] first suggested a hypothetical direct dimethylcinnamamide 7 and the N-ethyl-4-hydroxy- mechanism of VirA activation, when the phenolic 3,5-dimethoxycinnamamide 8. molecule is placed in the receptor protein site VirA. Fig. 9 shows the vir induction of these two products (7 Another hypothetical mechanism implies mediated-pro- and 8) compared with the analogous cinnamic deriva- tein such as p10 and p21 or even the Pbp39 [31,33], tives: 2, 3 (sinapinic acid) and 6 (sinapyl alcohol) and which leads to an indirect mechanism of VirA activa- with the two controls (negative control without any tion. Then, transfer of phenolic proton to the protein phenolic, and positive control with AS in the same basic residue and transfer to the phenol carbonyl group concentration as all other phenolics, 100 mM). In both from the protein acidic residue proton, are possible cases the induction ratios of 3 (sinapinic acid) and 6 through the conjugation of the molecules. (sinapyl alcohol) compared with 2 were equal to 0.6. Here we confirmed that the induction of the Agro- Compound 8 had the same effect as compound 3 (90% bacterium vir genes was essential for an effective gene of it). Compound 7 showed a lower vir induction activity: 43% that of compound 3 and 26% that of Table 2 compound 2. The activity of the two amides did not Comparison of phenolic pKa for some of the tested molecules on reach that of 3 as expected, and as reported by Dye´ et al. Agrobacteria virulence with their vir induction rates [41] for the ethyl amide of syringic acid. However, we Phenolic compounds 31(AS)10 2 noted that the replacement of the two methyl groups of Phenol pKa 7.91 8.04 9.81 9.94 the nitrogen atom (7) by only one ethyl chain (8) vir induction induced an increase in the vir gene induction (8/7/2). P. Joubert et al. / Plant Science 162 (2002) 733Á/743 741

Table 3 Comparison of inductor and mesomeric donor effects of R radicals (Fig. 5) for some of the tested molecules on Agrobacteria virulence, which all have ethylenic bonds on phenol para with their vir induction rates [43]

R radicals CH3 (C6H4)OH OH NHEt N(Me)2 Donor effects Compounds 2 10 3 8 7 Vir induction ()

transfer to the plants. Previously, we had shown that vir case the acidity of the acid group was hidden by the active molecules must have two methoxy groups in esterification. ortho position of phenolic function and one carbonyl From the comparison of pKa of phenol derivatives group on the R3 chain [40]. with the vir induction of some molecules (i), comparing The presence of strong active vir gene phenolic the electron-donating effects with the vir induction (ii), molecules such as AS 1, benzalacetone 2 and chalcones and seeing that there is no possible conjugation between 9, 10 and 11, increased the gene transfer in Tobacco and hydroxyl and carbonyl groups in compound 13 (iii), we believe that the model of phenol binding protein Petunia, and transformation rates 2Á/3 times higher than the control. Therefore, we confirmed that phenolic molecular activation proposed by Hess et al. [16] is molecules are essential in the T-DNA transfer process. highly significant. However, the physical distance be- The presence of the double bonds in the phenolic tween the phenol and carbonyl functions accounts for compounds enhanced the molecular conjugation and 20% with the two active compounds AS 1 and 2 (5.9 and ˚ enable the electronic transfer from phenol to carbonyl 7.5 A respectively). In this case, we cannot easily imagine that there should be only one phenol receptor and finally activated the protein receptor (Fig. 3). type. Recently an A. tumefaciens protein surface*/ According to the above hypothesis of Hess et al. [16], Pbp39*/which binds phenols was discovered [33]. We it is suggested that leaving-ability of the phenolic proton also know that the two proteins p10 and p21 have should be correlated with the vir induction power of similar properties. Moreover, phenols of flavones class, molecules. In order to find out if the ability of ionisation such as kaempferol [22] showed good vir induction. of phenol function was the only induction factor on the These data suggest that VirA may be composed as a VirA site, we calculated various pKa of some tested protein complex made of several phenol receptors, each molecules and compared them to their vir induction of them specialized with one phenol class indirectly power. The values were predicted according to the inducing VirA. method described by Perrin et al. [43]. We check this Finally, we report here that the three compounds with method experimentally by measurement and calculation the most important conjugation fields (2, 9, and 10), also pKa of AS and 2. Tables 2 and 3 shows the pKa and vir have the higher vir induction effect. Their great stability induction of these molecules. The less active compounds could result in an easier electron delocalization on the had the lowest pKa, and the pKa of molecules increased whole molecule, and therefore an easier (more efficient) along with the vir induction power. Thus, it seems that VirA activation. effective induction of phenol binding (VirA) protein decreases with the phenol dissociation. From the above suggested model (Fig. 3), a R radical Acknowledgements can effect the induction power of phenols. This R radical might actively have the donor electronic effect, antago- We thank B. Hohn and A. de Laat for generous gifts nist to the electronic flow from the phenol donor of A. tumefaciens strains; Y. Dessaux and S. Millam for residue. Thus, we compared donor inductor and meso- critical reading and improving the English style of the meric effects of some R radicals with the vir induction manuscript. powers of the corresponding molecules (Tables 2 and 3). For example, we noted that the compound 2, with a high vir induction, has a weak electron-donating group (/ References CH3) whereas the amide 7 with a high electron-donating [1] P.J.J. Hooykaas, R.A. Schilperoort, Agrobacterium and plant effect (/N(CH3)2) has a low vir activity. Moreover, the literature showed [14] that three methyl esters of ferulic, genetic engineering, Plant Mol. Biol. 19 (1992) 15Á/38. [2] P.J.J. Hooykaas, A.G.M. Beijersbergen, The virulence system of sinapinic and syringic acids have a far higher action on Agrobacterium tumefaciens, Annu. Rev. Phytopathol. 32 (1994) vir genes than the corresponding free acids, but in this 157Á/179. 742 P. Joubert et al. / Plant Science 162 (2002) 733Á/743

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