N-acyl-homoserine lactones (AHLs) belong to a class of bacterial quorum-sensing signals important for bacterial cell-to-cell communication. We evaluated Arabidopsis thaliana growth responses to a variety of AHLs ranging from 4 to 14 carbons in length, focusing on alterations in post-embryonic root development as a way to determine the biological activity of these signals. The compounds affected primary root growth, lateral root formation and root hair development, and in particular, N-decanoyl-HL (C10-HL) was found to be the most active AHL in altering root system architecture. Developmental changes elicited by C10-HL were related to altered expression of cell division and differentiation marker lines pPRZ1:uidA, CycB1:uidA and pAtEXP7:uidA in Arabidopsis roots. Although the effects of C10-HL were similar to those produced by auxins in modulating root system architecture, the primary and lateral root response to this compound was found to be independent of auxin signalling. Furthermore, we show that mutant and overexpressor lines for an Arabidopsis fatty acid amide hydrolase gene (AtFAAH) sustained altered growth response to C10-HL. All together, our results suggest that AHLs alter root development in Arabidopsis and that plants posses the enzymatic machinery to metabolize these compounds.
Many bacterial species use small molecule signalling to communicate with each other and to coordinate their growth activities – a process commonly referred to as quorum sensing (QS) (Taga & Bassler 2003; Reading & Sperandio 2006). Diverse gram-negative bacteria produce N-acyl-homoserine lactones (AHLs); these compounds contain a conserved homoserine lactone (HL) ring and an amide (N)-linked acyl side chain. The acyl groups of naturally occurring AHLs range from 4 to 18 carbons in length; they can be saturated or unsaturated and with or without a C-3 substituent (Waters & Bassler 2005; Camilli & Bassler 2006). These chemical signals are produced by specific enzymes, and they are detected by specific receptors (Pearson et al. 1994; Parsek et al. 1999). Moreover, the specific activity of the different compounds can be determined by the lactone ring, the amide group and the fatty acid chain length (Zhu et al. 1998; Vannini et al. 2002; Raffa et al. 2004).
The presence of AHL-producing bacteria in the rhizosphere of tomato induced the salicylic acid and ethylene-dependent defence response, which plays an important role in the activation of systemic resistance in plants and conferred resistance to the fungal pathogen Alternaria alternata (Schuhegger et al. 2006).
In addition, certain Rhizobium mutants that fail to produce or sense AHLs were unable to nodulate legume plants, suggesting that AHLs might also participate in beneficial plant–bacteria interactions (Rosemeyer et al. 1998; Daniels et al. 2002; Zheng et al. 2006). Mathesius et al. (2003) showed that Medicago truncatula plants are able to sense AHLs and that low concentrations of these compounds elicit major changes in protein expression. The possibility is open that plants could sense AHLs to respond to bacterial populations.
Plants produce substances that mimic AHLs. At least 10 chromatographically separable active compounds can be detected in root exudates of M. truncatula (Gao et al. 2003). These plant compounds can affect QS responses in bacteria, indicating that plants produce compounds that appear to be AHL signal mimics (Teplitski, Robinson & Bauer 2000; Gao et al. 2003). None of these compounds has yet been chemically identified, so their structural similarities to bacterial AHLs are unknown.
Plants produce compounds with structural similarity to AHLs, including N-acyl ethanolamines (NAEs) and alkamides. These compounds have been considered a novel class of plant signals because of their wide distribution in plants and their potent biological activities (Chapman 2004; López-Bucio et al. 2006; Morquecho-Contreras & López-Bucio 2007). A role of NAEs in plant development has been inferred from the work by Blancaflor, Huo and Chapman (2003), who showed that application of micromolar concentrations of N-lauroylethanolamide (NAE12:0) to Arabidopsis seedlings inhibited primary root growth and stimulated lateral root development. Similarly, alkamides isolated from plants have been found to alter root and shoot system architecture in Arabidopsis by affecting cell division and differentiation processes. Morphogenetic responses affected by alkamides included primary root growth, lateral root formation, root hair formation and growth, adventitious root formation, and leaf formation (Ramírez-Chávez et al. 2004; López-Bucio et al. 2007; Campos-Cuevas et al. 2008).
The similarity in chemical structures of N-decanoyl homoserine lactone (C10-AHL, a bacterial QS compound) with NAE10:0 and N-isobutyl decanamide (an alkamide) (Fig. 1) provides us with the basis to hypothesize that AHLs could alter plant development. AHLs are composed of an acyl chain linked to an amide, being the most important difference the presence of a homoserine lactone ring in the side chain in AHLs, which is absent in NAEs and alkamides. Perhaps more compelling is the fact that AHLs are widely distributed in bacteria that associate with plants (Cha et al. 1998; Elasri et al. 2001; Khmel et al. 2002; D’Angelo-Picard et al. 2005), and plants produce compounds that affect the bacterial QS (Gao et al. 2003; Teplitski et al. 2004).
To test the hypothesis that AHLs alter plant development and to explore in more detail the cellular basis of plant responses to these compounds, we evaluated the root developmental responses of Arabidopsis to exogenous applications of AHLs with acyl chain length from 4 to 14 carbons. Our results provide cellular, physiological and genetic evidence that AHLs modulate root system architecture and that plants posses the enzymatic machinery to metabolize these compounds.
MATERIALS AND METHODS
Plant material and growth conditions
Arabidopsis (Col-0 ecotype) and the transgenic lines pPRZ1:uidA (Sieberer et al. 2003), CyCB1:uidA (Colón-Carmona et al. 1999), pAtEXP7:uidA (Cho & Cosgrove 2002), DR5:uidA (Ulmasov et al. 1997), AtFAAH overexpressor OE7A and mutant KO118043 (Wang et al. 2006) lines, aux1-7 (Pickett, Wilson & Estelle 1990), doc1 (Li, Altschmied & Chory 1994) and axr2 (Timpte, Wilson & Estelle 1994) were used for all experiments. Seeds were surface sterilized with 95% (v/v) ethanol for 5 min and 20% (v/v) bleach for 7 min. After five washes with sterile distilled water, seeds were germinated and grown on agar plates containing 0.2× MS medium (Murashige & Skoog 1962). MS medium (Murashige and Skoog basal salts mixture, Cat. M5524) was purchased from Sigma (St. Louis, MO, USA). The suggested formulation is 4.3 g L−1 of salts for a 1× concentration of medium; we used 0.9 g L−1, which we consider and refer to as MS 0.2×. This medium lacks amino acids and vitamins. N-acyl-homoserine lactones were purchased from Sigma. Ethanol-dissolved compounds were added to cooled (50 °C) molten medium and poured into plates. In control seedlings, we added the solvent in equal amounts as present in the greatest concentration of AHL tested. Phytagar (micropropagation grade) was purchased from Phytotechnology (Shawnee Mission, KS, USA). Plants were placed in a plant growth chamber (Percival Scientific AR-95L) with a photoperiod of 16 h of light, 8 h of darkness, light intensity of 100 µmol m2 s−1 and temperature of 22 °C.
Analysis of growth
Growth of primary roots was registered using a rule. Lateral root number and lateral root primordia (LRP) were determined by counting the lateral roots or LRP present in the primary root, from the tip to the root/stem transition. Lateral root and primordia densities were determined by dividing the lateral root number by the primary root length and expressed as LRP cm−1. Trichoblasts and root hairs were measured in a 500 µm region from the primary root tip. The average length of root hairs was determined upon measuring 10 hairs, taking as a reference the root protoxilematic plane to locate the radical hair base in the epidermic cell. Fresh weight of plants was determined with an Ohaus analytical balance (Ohaus Corporation, Pine Brook, NJ, USA) with a 0.0001 g precision value. For all experiments, the overall data were statistically analysed in the SPSS 10 program (SPSS, Chicago, IL, USA). Univariate and multivariate analyses with a Tukey's post hoc test were used for testing differences in growth and root developmental responses in wild type (WT). Different letters are used to indicate means that differ significantly (P < 0.05).
Transgenic plants that express the uidA reporter gene (Jefferson, Kavanagh & Bevan 1987) were stained in 0.1% X-Gluc (5-bromo-4-chlorium-3-indolyl, β-D-glucuronide) in phosphate buffer (NaH2PO4 and Na2HPO4, 0.1 m, pH 7) with 2 mm potassium ferrocyanide and 2 mm potassium ferricyanide, for 12 h at 37 °C. Plants were cleared and fixed with 0.24 N HCl in 20% methanol (v/v) and incubated for 60 min at 62 °C. The solution was substituted for 7% NaOH (w/v) in 60% ethanol (v/v) for 20 min at room temperature. Plants were dehydrated with ethanol treatments at 40, 20 and 10% (v/v) for a 24 h period each, and fixed in 50% glycerol (v/v). The processed roots were included in glass slips and sealed with commercial nail varnish.
The Arabidopsis thaliana root system was analysed with a stereoscopic microscope (Leica MZ6, Leica Microsystems, Wetzlar, Germany). Total lateral roots were counted at 30× magnification. The primordial phases of primary and lateral roots were analysed in semi-permanent preparations of cleared roots using a composed microscope (Axiostar Zeiss Plus, Carl Zeiss, Göttingen, Germany) at 100× or 400× magnifications. Images were captured with a Sony Cyber-shot DSC-S75 digital camera (Sony Electronics Inc., Oradell, NJ, USA) adapted to the microscope and processed with the Zeiss Axio Vision 4AC software (Carl Zeiss).
AHLs modify Arabidopsis root system architecture
AHLs are a diverse class of hormones used by gram-negative bacteria for cell-to-cell communication. Thus, the hypothesis that plants could sense AHLs was investigated by testing the Arabidopsis root developmental responses to these compounds. Three main processes are involved in determining root system architecture: (1) primary root growth; (2) lateral root formation; and (3) root hair development, thus contributing to the total absorptive capacity for water and nutrients. We evaluated the effect of seven AHLs with different chain lengths (Fig. 2a–g) on primary root growth.
To determine the effect of AHLs on growth, Arabidopsis plants were germinated and grown on 0.2× MS-agar media supplemented with AHL concentrations from 12 to 192 µm and primary root length recorded 10 d after germination (d.a.g.). We found that the effects of each AHL were dependent on the structure and the concentration of the compound. Small-chain (C4 and C6) AHLs did not significantly affect primary root growth (Fig. 3a). On the contrary, medium-chain AHLs showed dose-dependent inhibitory effects on primary root growth, for which C10-HL showed the greatest biological activity (Fig. 3a). C10-HL concentration of 48 µm caused an 80% reduction in root primary length. Interestingly, the decrease of primary root length by AHL treatment was accompanied by a shift in root system architecture from a taproot architecture to a more branched exploratory system (Fig. 3b–q). Similar effects were observed for C8, C12 and C14-AHL, albeit at increased concentrations of the compounds (Fig. 3b–q). These results indicate that the length of the acyl chain appears to be important for AHL activity to alter root system architecture in Arabidopsis.
C10-HL promotes lateral root development in Arabidopsis seedlings
The effect of AHLs promoting lateral root formation could be due to a stimulation of the emergence of pre-existing LRP or to de novo formation of additional LRP. To establish the developmental basis for the effect of C10-AHL on lateral root formation, LRP originating in the primary root were quantified at 7 d.a.g. We used pPRZ1:GUS seedlings to visualize LRP in primary roots. pPRZ1:GUS seedlings were grown for 6 d in MS 0.2× medium supplied with 12 to 192 µm concentrations of C10-HL, and then stained for β-glucuronidase (GUS) activity and cleared to enable LRP at early stages of development to be visualized and counted. Each LRP was classified according to its stage of development as reported by Zhang et al. (1999), who consider the following stages: stage A, up to three cell layers; stage B, unemerged, of more than three cell layers; stage C, LR emerged of less than 0.5 mm in length; stage D, lateral root larger than 0.5 mm.
The LRP density (number of LRP cm−1) revealed that plants treated with 48 µm or greater concentration of C10-HL formed a significantly higher number of LRP. A 192 µm concentration of this compound elicited a fivefold increase in LRP density when compared with solvent-treated control seedlings (Fig. 4a). The stage distribution of LRP was also affected by C10-HL. Treatments from 24 to 192 µm C10-HL increased the number of LRP in stage D compared with the control (Fig. 4b). The observations that C10-HL treatments increased LRP density and the transition of LRP from early developmental stages (stages A, B and C) to an advanced developmental stage (stage D) suggest that C10-HL can promote root branching by inducing LRP initiation and further growth of LRP.
AHLs alter root hair development
Root hairs are root epidermal cells that participate in nutrient and water uptake. To analyse whether AHLs could alter root hair development, we performed experiments in which Arabidopsis WT (Col-0) seedlings were germinated and grown on the surface of agar plates containing different concentrations of five AHLs from 3-oxo-C6-HL to C14-HL.
Root hair parameters were analysed 9 d.a.g. on primary roots of solvent-treated or AHL-treated seedlings. Figure 5 shows representative photographs of root hairs formed in the different concentrations of AHLs. Solvent-treated control seedlings showed the cellular typical organization of the primary root tip, with root hairs forming at a distance from the root tip (Fig. 5a). Small-chain (C6 and C8) AHLs did not modify root hair development (Fig. 5b–j), while C10 and C12-HL altered root hair development in a dose-dependent way. In particular, C10-HL at concentrations from 12 to 48 µm promoted root hair formation close to the root tip (Fig. 5k–n), whereas a greater concentration was found to stimulate lateral root formation at the root tip region (Fig. 5o). C12-HL stimulated root hair formation and growth at concentrations from 48 to 96 µm (Fig. 5p–t). In the greatest concentration tested, root hair formation close to the root tip could be observed (Fig. 5t). C14-HL effects on root hair development were similar to those observed for small-chain AHLs (Fig. 5u–y). C10 and C12-HL showed the clearest promoting effects on root hair development, indicating the importance of acyl chain length for biological activity of AHLs.
To investigate more deeply the effects of C10-HL on root hair density, we measured the trichoblast length and root hair length on 6-day-old Arabidopsis seedlings subjected to different concentrations of this compound. Trichoblasts are the hair formation root epidermal cells that form cell files along the root surface. We found a dose-dependent decrease in trichoblast length in response to C10-HL treatment (Fig. 6a), while root hair length significantly increased at 12 µm C10-HL treatment (Fig. 6b). The most stimulating effects of C10-HL on root hair growth were observed in concentrations of 24 µm or greater, in which a roughly twofold increase in root hair length was recorded in roots of C10-HL-treated plants when compared with roots of plants treated with solvent only (Fig. 6b). These results suggest that medium-chain AHLs alter root hair development and that C10-HL increases root hair density and root hair growth.
C10-HL affects cell division and differentiation in Arabidopsis roots
The post-embryonic root developmental effects of medium-chain AHLs in Arabidopsis seedlings suggested that these compounds could play an important role in cell division. Next, we used C10-HL, the most active AHL among those investigated (Fig. 3), to study the effects of this compound on cell division and differentiation gene expression markers.
To investigate the pattern of cell division in response to C10-HL, we analysed the expression of pPRZ1:uidA, which marks only active meristems (Sieberer et al. 2003) and CyCB1:uidA, which is expressed only in cells in the G2/M phase of the cell cycle and is a marker of mitotic activity (Colón-Carmona et al. 1999). In addition, to evaluate the effects of C10-HL on cell differentiation, we used pAtEXP7:uidA, the expression of which strongly correlates with initiation of root hair development (Cho & Cosgrove 2002). Strong primary root growth inhibition under 48 µm or greater concentrations of C10-HL correlated with the loss of GUS expression in the primary root meristem of pPRZ1:uidA transgenic seedlings (Fig. 7a–f, arrows), and with the lack of cell divisions in the roots of CycB1:uidA plants (Fig. 7g–l). These effects were accompanied by an accelerated cell differentiation program revealed by pAtEXP7:uidA expression and root hair formation closer to the root tip at concentrations from 24 to 48 µm of C10-HL, and by lateral root formation at greater concentrations of this compound (Fig. 7m–r). We also measured the length of pPRZ1:uidA expression zone. It was observed that in solvent-treated control seedlings, the meristem comprised around 350 µm. Interestingly, C10-HL treatments showed a dose-dependent inhibition in the size of the meristem, in which concentrations greater than 48 µm dramatically decreased the pPRZ1:uidA GUS expression region (Fig. 8). These results indicate that C10-HL inhibits primary root growth by affecting cell division in the meristem.
The effect of C10-HL on root development is independent of auxin signalling
The impact of C10-HL on root hair development is similar to the effect of auxin when exogenously applied to Arabidopsis seedlings. Also, roots are shorter, root hairs develop closer to the root tip and lateral root formation is induced. To investigate whether C10-HL might or rather does not function via auxin-regulated processes, we conducted analyses of the expression of the GUS reporter gene in Arabidopsis transgenic seedlings harbouring the DR5:uidA gene construct. This reporter line has been useful in studying auxin-regulated gene expression in Arabidopsis (Ulmasov et al. 1997). Figure 9 shows histochemical staining for roots of transgenic DR5:uidA seedlings that were grown for 10 d in medium supplied with the solvent, indole-3-acetic acid (IAA) or C10-HL. In solvent-treated control seedlings, DR5:uidA expression is located primarily in the columella and quiescent centre at the root tip region (Fig. 9a). DR5:uidA seedlings grown in a concentration of 1 µm IAA showed GUS activity throughout the primary root (Fig. 9b). In contrast, the pattern of GUS expression in DR5:uidA seedlings treated up to 192 µm C10-HL remained similar (Fig. 9c,d) or decreased (Fig. 9e–g, arrows) in the primary root tip. The results from DR5:uidA reporter expression analyses suggest that C10-HL did not induce auxin-responsive gene expression in Arabidopsis seedlings.
Several auxin-related mutants have been identified using screens for resistance to growth inhibitory amounts of auxin. To determine if C10-HL operates in a genetically defined auxin pathway, WT Arabidopsis seedlings (Col-0) and the auxin-related mutants aux1-7, axr2 and doc1 were evaluated in primary root growth response assays to IAA or C10-HL. Firstly, to confirm auxin resistance of these mutant lines, homozygous aux1-7, axr2 and doc1 mutant seedlings were screened for resistance to IAA based on primary root growth. In these experiments, aux1-7 and axr2 were resistant to the inhibition of primary root elongation by IAA when compared with WT seedlings (Supporting Information Fig. S1). These mutants also failed to form abundant root hairs at the root tip region in response to increasing IAA concentration in the medium, a phenotype associated with increased auxin resistance (Supporting Information Fig. S2).
Next we tested the effects of 48 µm C10-HL on primary root growth and lateral root density (fully emerged lateral roots per centimetre of primary root) of the aux1-7, doc1, and axr2 auxin-related mutants. As shown in Fig. 10, C10-HL treatment with 48 µm caused a 90% inhibition in primary root growth in WT plants of the Col-0 ecotype (Fig. 10a). In media supplied with the solvent, doc1 mutant seedlings showed a significantly reduced growth of the primary root, while aux1-7 and axr2 showed a modest yet not statistically significant increase in primary root growth. Despite this difference in primary root growth, when aux1-7, doc1 and axr2 seedlings were grown in medium supplied with 48 µm C10-HL, an inhibition in primary root growth similar to that of WT plants could be observed (Fig. 10a). Our analysis of lateral root density in WT and mutant seedlings showed that lateral root density increases in WT, aux1-7 and axr2 to a similar extent (Fig. 10b). Even doc1 seedlings, which failed to form lateral roots on control medium, were found to activate lateral root formation in response to C10-HL (Fig. 10b). Because C10-HL failed to induce auxin-regulated gene expression and aux1-7, doc1 and axr2 auxin-related mutants showed WT root developmental changes to C10-HL, we conclude that auxin may not be involved in Arabidopsis responses to C10-HL.
C10-HL did not rescue the root hair defective phenotype of the auxin-related rhd6 Arabidopsis mutant
Auxin-related mutations have been found to alter root hair development (Parker et al. 2000). Of particular interest is the rhd6 mutant, which is defective on root hair initiation and has been previously shown to be rescued by auxin (Masucci & Schiefelbein 1994). We used the rhd6 mutant as a tool to probe further the mechanism of C10-HL action. We compared the root hair response of Arabidopsis WT seedlings and rhd6 mutants with IAA and C10-HL at 7 d.a.g. As shown in Fig. 11, treatments with 1 µm IAA stimulated root hair elongation and increased root hair formation at the primary root tip region in Arabidopsis WT seedlings (Fig. 11a,b). A similar effect was observed with 24 µm C10-HL (Fig. 11c), while a 48 µm concentration of this compound elicited lateral root formation (Fig. 11d). rhd6 mutant seedlings grown in medium without auxin were completely devoid of root hairs (Fig. 11e). As previously reported (Masucci & Schiefelbein 1994), IAA was found to rescue the rhd6 root hair defective phenotype (Fig. 11f). The root hairs produced under IAA treatment exhibited normal growth and morphology, whereas treatment with 24 or 48 µm C10-HL failed to induce root hairs in rhd6 (Fig. 11g,h). These results imply that the application of C10-HL cannot suppress the root hair formation defects of rhd6, indicating an auxin-independent mechanism of action of this compound in the plant.
Manipulation of Arabidopsis fatty acid amide hydrolase modifies sensitivity to C10-HL
Recently, a fatty acid amide hydrolase (FAAH) enzyme that rapidly hydrolyses NAEs, molecules structurally related to AHLs and alkamides (for the structure of NAE10:0, see Fig. 1), has been cloned from Arabidopsis (Wang et al. 2006). To investigate whether AtFAAH could play a role on in vivo metabolism of AHLs, we analysed the growth of AtFAAH mutant and overexpressor seedlings from lines previously reported by Wang et al. (2006), in response to C10-HL treatments. The relationship between NAEs and AHLs was revealed with AtFAAH overexpressor seedlings (OE2A), which were more tolerant to C10-HL primary root growth arrest, and AtFAAH mutants (faah; KO118043), which were hypersensitive to C10-HL (Fig. 12a). The differential sensitivity of FAAH overexpressors and mutants to C10-HL could be confirmed in bioassays comparing general plant development. It could be observed that seedlings of AtFAAH overexpressors displayed less sensitivity towards exogenous C10-HL in terms of shoot and root growth, and faah mutants were more sensitive than WT seedlings (Fig. 12b–j). These results suggest that manipulation of AtFAAH alters plant responses to a highly active AHL.
AHLs modify Arabidopsis root system architecture
In this work, we show that AHLs, a class of bacteria-produced amino compound-containing lipids and structurally related to NAEs and alkamides (Figs 1 & 2), modify post-embryonic root development. AHLs are used by gram-negative bacteria to monitor population density, a term commonly referred to as quorum sensing (QS). QS was first described in Vibrio fischeri, a bacterial symbiotic species that provides its marine eukaryotic hosts with light. This bacterium produces AHL compounds that were shown to be freely diffusible across the bacterial membranes and accumulate in the cell environment. For bacteria, these AHLs act as cell density cues.
Medium (C8-C14)-chained AHL compounds showed a dose-dependent effect on root architecture, altering primary root growth, lateral root formation and root hair development (Figs 3–6). Particularly, C10-HL showed the greatest activity in inhibiting root growth and stimulating lateral root and root hair development (Figs 3–6). These results indicate that AHLs can alter root morphogenesis and provides a mechanism to explain the beneficial effects of certain AHL-producing rhizobacteria to promote plant growth, perhaps by modulating root development (Venturi 2006).
Several strains of Pseudomonas have been studied for their ability to colonize plant-related niches, such as the rhizosphere (e.g. Pseudomonas aeruginosa, Pseudomonas fluorescens and Pseudomonas putida), where they can act as plant growth-promoting rhizobacteria by antagonizing plant-deleterious microorganisms and through the production of traits that directly influence plant disease resistance and growth (Venturi 2006). The rhizospheric P. putida plant beneficial strains WCS358 and IsoF produce 3-oxo-C12-AHL, whereas in the rhizosphere-colonizing biocontrol P. fluorescens strain F113, the production of three AHL molecules has been reported, including C10-HL (Laue et al. 2000; Venturi 2006). Interestingly, C10-HL and C12-HL seem to be also produced in the nitrogen-fixing bacterial symbiont Sinorhizobium meliloti (Marketon et al. 2002; Teplitski et al. 2003). This information, along with the effects of C8-to-C12-HL on root development, opens new possibilities to identify plant growth-promoting bacterial strains based on AHL production.
Our results with cell division markers pPRZ1:uidA and CycB1:uidA show that primary root growth inhibition by C10-HL occurs by the alteration of cell division in the primary root meristem, probably during the G2-M phase transition of the cell cycle or at an earlier phase (Figs 7 & 8). These effects were accompanied by differentiation processes, including the formation of root hairs close to the root tip (Fig. 5). Rhizobacteria multiply to high densities on plant root surfaces where root exudates and root cell lysates provide nutrient sources. Sometimes, they exceed 100 times those densities found in the bulk soil (Campbell & Greaves 1990). The possibility is open that both the identity and the quantity of AHLs produced by rhizobacteria could modulate cell division in the meristem. Alterations in lateral root formation and root hair proliferation may provide a greater root surface area for bacterial colonization. In turn, increased absorptive surface by branched roots may increase water and nutrient uptake capacity of plants. We speculate that production of AHL compounds by plant-associated bacteria might benefit plant hosts by initiating or reinforcing symbiotic behaviours with bacterial partners.
Although the effects of C10-HL on root development are similar to those of auxins, our results show that the classic auxin-signalling pathway might be not involved in the root architectural responses of Arabidopsis seedlings to C10-HL. This hypothesis is mainly based on three lines of evidence: expression studies of the DR5:GUS marker (Fig. 9), the WT responses of the aux1-7 and axr2 auxin-related mutants to treatments with C10-HL (Fig. 10), and the failure of this compound to elicit normal root hair formation in the rhd6 Arabidopsis mutant (Fig. 11), indicating that the root developmental effects induced by C10-HL are unlikely mediated by the known auxin-signalling pathway.
Arabidopsis fatty acid amide hydrolase manipulation modifies sensitivity to C10-HL
Our results provide evidence that AHLs are signals with activity in plants. In bacteria, QS can be interfered by targeting the signal itself for destruction, preventing it from accumulating. Two different bacterial enzymes have been reported to have AHL-degrading activity: an AHL lactonase initially found in Bacillus sp. and an AHL acylase reported in several bacterial species (reviewed by Roche et al. 2004). Recently, a fatty acid amide hydrolase, which hydrolyses NAEs was identified in A. thaliana and their levels manipulated by mutational and transgenic means (Wang et al. 2006). The availability of AtFAAH knockouts and overexpressor lines provided us with the basis to test the possibility that such AtFAAH activity may already act in AHL degradation. Our findings that manipulation of fatty acid amide hydrolase activity in Arabidopsis modifies sensitivity to C10-HL (Fig. 12), suggest a mechanism by which plants can metabolize bacterial AHLs. This result suggests that plants posses the enzymatic machinery to degrade these compounds.
For bacterial QS, the roles that AHL-degrading enzymes play in their natural ecological niche remain far from clear (Roche et al. 2004). Both for NAEs and AHLs, the signal decay may have a function in the regulation of plant developmental processes or in plant recognition of bacterial symbionts or pathogens.
In animals, AHLs can act as virulence agents. In plant pathogenic bacteria, mutations affecting production of AHLs affect virulence (Taguchi et al. 2006; Hussain et al. 2008). Although the ultimate cause of this reduced virulence could be lack of QS in bacteria, our results indicate that in plants the role played by AHLs would be rather different. An important alternative to combat pathogens of plants is the alteration of virulence factors by interfering with QS. In this regard, the aiiA gene from Bacillus sp. encoding for an AHL lactonase has been cloned into tobacco and potato and found to confer resistance to infection by Erwinia carotovora, inhibiting the production of virulence determinants regulated by QS and allowing the host defences to effectively combat the infection (Dong et al. 2001). Future work is required in order to determine whether AtFAAH plays a role on QS signal modulation in addition to its suggested role in plant morphogenesis (Wang et al. 2006).
AHLs: novel signals for plant–bacteria communication?
In recent years, evidence has accumulated that AHL molecules affect plant physiological responses. Mathesius et al. (2003) showed that M. truncatula roots respond to AHL exposure by changes in expression of over 150 genes, depending on AHL structure, concentration and time of exposure. AHL-mimicking compounds and AHL inhibitory activities were reported in exudates from pea seedlings (Gao et al. 2003; Teplitski et al. 2004). On the other hand, previous research showed that plants produce a wide range of alkamides and NAEs, which are active in modulating plant development (Chapman 2004; López-Bucio et al. 2006). This information provides a scenario in which small lipid amides could play a role in bacteria–plant interactions.
In Vibrio harveyi, an AHL called autoinducer-1 (AI-1) acts as a species-specific QS signal, which activates a two-component signalling system (Cao & Meighen 1989; Freeman & Bassler 1999;Timmen, Bassler & Jung 2006). Plants possess two-component signalling systems underlying the regulation of growth and development in response to cytokinins and ethylene (Mizuno 2005). In this regard, our recent research has revealed an important interaction between alkamides and cytokinin signalling. In such work, proliferative growth activity elicited by N-isobutyl decanamide on callus formation in leaves and lateral root formation in roots was decreased or was even absent in Arabidopsis mutants lacking one, two or three of the putative cytokinin receptors CRE1, AHK2 and AHK3 (López-Bucio et al. 2007). The triple cytokinin receptor mutant cre1-12/ahk2-2/ahk3-3 was particularly insensitive to high alkamide concentrations in terms of developmental alterations, indicating that N-isobutyl decanamide requires, at least in part, a functional cytokinin-signalling pathway to control meristematic activity and differentiation processes. Whether small lipid amides act as ligands of cytokinin receptors remains to be determined; however, the possibility is open that AHLs, NAEs and alkamides could regulate plant development by modulating two-component signalling systems.
We gratefully acknowledge Elison B. Blancaflor, Peter Doerner, Hyung Taeg Cho, Christian Luschnig, Tom Guilfoyle, Bonnie Bartel and John W. Schiefelbein for kindly providing us with seeds of Arabidopsis transgenic and mutant lines. This work was supported by grants from the Consejo Nacional de Ciencia y Tecnología (CONACYT, Mexico, grant no. 43978), Consejo Estatal de Ciencia y Tecnología (COECYT, Mexico, grant no. CB0702110-0) and Consejo de la Investigación Científica (UMSNH, México, grant no. CIC 2.26).