AM symbiosis alters phenolic acid content in tomato roots

Abstract

Arbuscular mycorrhizal (AM) fungi colonize the roots of most plants to establish a mutualistic symbiosis leading to important benefits for plant health. We have recently shown that AM symbiosis alters both transcriptional and hormonal profiles in tomato roots, many of these changes related to plant defense. Here, we analytically demonstrate that the levels of other important defense-related compounds as phenolic acids are also altered in the symbiosis. Both caffeic and chlorogenic acid levels significantly decreased in tomato roots upon mycorrhization, while ferulic acid increased. Moreover, in the case of caffeic acid a differential reduction was observed depending on the colonizing AM fungus. The results confirm that AM associations imply the regulation of plant defense responses, and that the host changes may vary depending on the AM fungus involved. The potential implications of altered phenolic acid levels on plant control over mycorrhizal colonization and in the plant resistance to pathogens is discussed.

Plants have to cope with numerous environmental factors, including abiotic and biotic stresses, to ensure survival, growth and development. Regarding biotic stress, plants have evolved complex and integrated systems to defend themselves against a variety of deleterious organisms. These mechanisms include preformed physical and chemical barriers, but also inducible defenses triggered upon non-self recognition, such as strengthening of the cell wall and activation of several biochemical pathways for the synthesis of defense-related enzymes and diverse secondary metabolites.^1–3 Upon recognition of the invader, the plant alters the levels of some phytohormones—mainly salicylic acid, ethylene and jasmonates—which in turn activate the appropriate defense mechanisms.^4–6 The responses triggered usually include the biosynthesis of compounds with biocidal activity such as alkaloids, terpenoids, flavonoids and phenolic acids.^2,7 Phenolic acids (PAs) are metabolites with a diverse structure characterized by hydroxylated aromatic rings. Despite some constitutive expression, their relative location and concentration may vary in response to cell invasion by microorganisms. PAs can fulfil different functions, as constituents of plant cell walls regulating cell wall plasticity as antimicrobial compounds, and they may act as signaling molecules modulating plant-microbe interactions.^3,7,8 In tomato, the PAs vanillic, caffeic, chlorogenic and ferulic acids have been shown to display biocidal activity and/or associated with resistance to pathogens.⁷ Interestingly, ferulic acid has been also related with the induction of plant resistance by biocontrol agents.⁹

Arbuscular mycorrhizal (AM) symbioses are widespread mutualistic associations established between certain soil fungi and most terrestrial plants.^10,11 In the symbiosis, the AM fungi (AMF) obtain carbohydrates from their host plants and help them to acquire water and mineral nutrients. In addition, they may also increase plant resistance to biotic and abiotic stresses.¹¹ To establish the symbiosis, AMF extensively colonize the root cortex inter and intracellularly, process that requires the modulation of plant defense responses. Accordingly, evidences of both induction and repression of different defense-related responses have been reported.^11–13 Surprisingly, despite the importance of the phenolic compounds in plant defense, very little information is available regarding these metabolites in mycorrhizal systems. We have recently shown that AM symbiosis alters both hormonal and transcriptonal profiles in tomato roots, including changes associated to defense responses, and a regulatory role of jasmonates and other oxylipins in the interaction has been pointed out.¹³ Here, we complement those results with the analysis and quantification in the same mycorrhizal systems of the phenolics caffeic, chlorogenic and ferulic acids by ultra pressure liquid chromatographytandem mass spectrometry (UPLC-MS/MS) in order to get new insights on the involvement of such compounds in the AM symbiosis.

LC-MS/MS Analysis and Quantification of Caffeic and Chlorogenic Acids in Mycorrhizal Roots

To assess whether AM symbiosis alters the levels of phenolic compounds in host roots, non mycorrhizal tomato plants were compared with plants colonized by two different AMF.¹³ Nine weeks after inoculation with the AMF, 41% of the root system was colonized by Glomus intraradices, whereas Glomus mosseae colonized tomato roots to a lesser extent (c. 23%). For the analysis of PAs in mycorrhizal plants, root extracts from non-mycorrhizal control or AMF-colonized plants were obtained and analyzed by UPLC-MS/MS as described before.¹⁴ Interestingly, the levels of chlorogenic acid (caffeoyl quinic acid) (Fig. 1A) were significantly (p < 0.01) lower in roots colonized by both AMF compared to non-mycorrhizal roots. The reduction, about 40%, was similar in both mycorrhizal associations (Fig. 1A). Reduced levels of chlorogenic acid were previously observed in roots of tobacco plants colonized by G. intraradices.¹⁵ However, the authors suggested that this decrease in the steady-state of defense-related compounds in the roots was not a direct consequence of mycorrhization.

Figure 1.

Phenolic content in tomato roots colonized by arbuscular mycorrhizal fungi and chemical structure for different phenolic compounds. Levels of chlorogenic acid (A), caffeic acid (B) and ferulic acid (C) were determined by UPLC-MS/MS in the roots of non-mycorrhizal plants (Nm, dashed bars) and mycorrhizal plants colonized by G. mosseae (Gm, closed bars) or G. intraradices (Gi, open bars). Data points represent the means of five independent replicates ± SE. Data not sharing a letter in common differ significantly (p < 0.01) according to the Fisher's LSD test.

As for chlorogenic acid, a clear decrease in caffeic acid (Fig. 1B) was detected upon mycorrhization by both AMF. However, in this case the reduction on the levels of caffeic acid was different in both interactions (Fig. 1B). The reduction in G. mosseae-colonized roots was about 40% compared with non-mycorrhizal plants, whereas in G. intraradices-colonized roots the reduction was significantly (p < 0.01) higher (c. 75%). Interestingly, G. intraradices showed higher root colonization than G. mosseae, suggesting therefore an inverse correlation between the steady-state levels of caffeic acid and the level of colonization. As far as we know, there is no previous data on the endogenous levels of this compound in mycorrhizal roots. However, Douds and co-workers¹⁶ reported that caffeic acid showed antifungal activity against two different AMF. In addition, treatments of Digitaria sanguinalis plants with caffeic acid decreased mycorrhizal colonization.¹⁷ Thus, an inverse correlation between the levels of some PAs (particularly caffeic acid) and the levels of mycorrhization is likely. In this scenario, we speculate that the host plant use defense mechanisms to control AM fungal colonization and that individual AMF display differential capacity to overcome or regulate plant defenses in order to colonize the host roots. Supporting this hypothesis, we have recently shown a higher induction of defense-related compounds (SA, JA-Ile and 9-LOX oxylipins) in roots colonized by G. mosseae, with a lower colonization capacity, compared to G. intraradices,¹³ suggesting a more exhaustive control of G. mosseae by the plant.

In contrast to the reduction of caffeic and chlorogenic acids, the levels of ferulic acid (Fig. 1C) in mycorrhizal roots colonized by any of the AMF were significantly (p < 0.01) higher than in the non-mycorrhizal control plants (Fig. 1C). Induction of ferulic acid in mycorrhizal roots has been previously reported.¹⁸ Remarkably, ferulic acid may be induced by fungal elicitors and jasmonates.^19,20 Our previous analysis pointed to jasmonates and related compounds as key regulators on the changes associated to the symbiosis in tomato,¹³ and this may be the case also with ferulic acid levels. Increased ferulic acid has been associated to plant resistance to pathogens in resistant cultivars and upon treatment with some biocontrol agents.⁹ Therefore, it is tempting to speculate that elevated ferulic acid levels may contribute to the enhanced resistance of mycorrhizal roots to soil-borne pathogens.

In summary, we show here that in addition to the altered hormonal and transcriptional profiles reported previously,¹³ the levels of the major phenolic acids are also altered in tomato roots upon mycorrhization, confirming the regulation of plant defense mechanisms during the symbiosis. The differences observed in the host response to particular AMFs may account for the different mycorrhization levels achieved in each interaction, and their differential impact on plant resistance to root pathogens.

Addendum to: López-Ráez JA, Verhage A, Fernández I, García JM, Azcón-Aguilar C, Flors V, Pozo MJ. Hormonal and transcriptional profiles highlight common and differential host responses to arbuscular mycorrhizal fungi and the regulation of the oxylipin pathway. J Exp Bot. 2010;61:2589–2601. doi: 10.1093/jxb/erq089.