Phytochelatins (PCs) are thiol-rich oligopeptides that facilitate the sequestration in the vacuo-lysosomal compartments of several metal(loid)s (Cobbett, 2000; Mendoza-Cózatl et al., 2011; Rea, 2012). PCs are post-translationally synthesized by the enzyme PCS, a γ-glutamylcysteine dipeptidyl (trans)peptidase (EC 2.3.2.15) (Grill et al., 1989; Rauser, 1990; Zenk, 1996; Clemens et al., 1999; Ha et al., 1999; Vatamaniuk et al., 1999, 2000, 2004; Romanyuk et al., 2006; Rea, 2012). In Arabidopsis thaliana the transpeptidation reaction is performed by the AtPCS1 enzyme, characterized for the first time by Ha et al. (1999); indeed, the importance of AtPCS1 in metal detoxification was demonstrated by the A. thaliana cad1-3 mutant, which lacks a functional PCS1, thus being PC-deficient and Cd-hypersensitive (Howden et al., 1995). Likewise, the PCS-deficient mutants of fission yeast Schizosaccharomyces pombe and of the nematode Caenorhabditis elegans are hypersensitive to heavy metals (Ha et al., 1999; Vatamaniuk et al., 2001). Thus, the PC synthesis via PCS activation is essential for metal(loid) detoxification in several organisms, including basal land plants (Degola et al., 2014; Petraglia et al., 2014).

Interestingly, Clay et al. (2009) found a close correlation between AtPCS1 activity and indole glucosinolate biosynthesis. This class of secondary metabolites is constitutively synthesized in plants, predominantly in the Brassicaceae family (Fahey et al., 2001), and include: (1) aliphatic glucosinolates, derived primarily from methionine; (2) indole glucosinolates, derived from tryptophan; and (3) aromatic glucosinolates, from phenylalanine or tyrosine (for a review, see Halkier and Gershenzon, 2006). Glucosinolates are stored in the vacuole, and when the cell is damaged (e.g., by herbivores), they are released from this compartment and hydrolyzed by the endogenous enzyme myrosinase (β-thioglucosidase). The products of the glucosinolate-myrosinase system are biologically active molecules, which include isothiocyanates, nitriles, thiocyanates, oxazolidine-2-thiones, and epithionitriles, with multiple physiological functions as defense against herbivors, parasites and pathogens, and cell signaling (Wittstock and Halkier, 2002; Kim et al., 2008; Wittstock and Burow, 2010; Burow and Halkier, 2017).

In addition, in response to a pathogen or herbivore attack, plants can deposit callose [β(1,3)-glucan] into cell walls, that is a defense mechanism interpretable in terms of “Microbe-Triggered Immunity” (MTI) or, in sporadic cases, of “Herbivore-Triggered Immunity” (HTI) (Thordal-Christensen, 2003; Nürnberger and Lipka, 2005).

Unlike wild-type, the cad1-3 mutant of A. thaliana, which lacks a functional AtPCS1 enzyme, is impaired in callose deposition after Flg22 treatments (Flg22 is a synthetic polypeptide that represents one of the highly conserved epitopes of the bacterial flagellin) (Clay et al., 2009). The cad1-3 mutant also accumulates a callose precursor, the indole glucosinolate 4MOI3M (sin. 4-methoxy-I3G; Clay et al., 2009). Likewise, other mutants from the glucosinolate synthesis pathway, including pen2-1, pen2-2, pen3-1, etc., show an accumulation of 4MOI3M and defects in the callose response after Flg22 treatment (Clay et al., 2009; Luna et al., 2011). Transcriptional data of PEN2, PEN3, and PCS1 genes suggest their mutual interaction aimed at the hydrolysis of 4MOI3M, with consequent callose deposition (Clay et al., 2009). Concerning this, PEN2 acts as a putative myrosinase enzyme (a type of β-thio-glucoside glycohydrolase), which hydrolyzes indole glucosinolates in planta (Lipka et al., 2005; Bednarek et al., 2009), and represents an important component in oomycete defense response (Schlaeppi and Mauch, 2010). PEN3 is an ABC transporter located on the plasma membrane, able to export Cd and IBA from the cell (Kim et al., 2007; Strader and Bartel, 2009). Its involvement in the movement of toxic metabolites toward pathogen penetration sites has been also hypothesized (Stein et al., 2006; Bednarek et al., 2009), since a higher accumulation of PEN3 has been found under fungal appressoria during infection (Stein et al., 2006).

Thus, considering that the cad1-3 mutant is defective in callose deposition (Clay et al., 2009), AtPCS1 may possibly play a relevant role in the innate immune response, particularly as far as MTI is concerned. In line with this, the cad1-3 mutant has been demonstrated to be more susceptible to the oomycete Phytophthora infestans compared to the wild-type (Kühnlenz et al., 2015).

All this assumed, it should also be considered that the phenylpropanoid pathway is a starting point for the production of a specialized number of metabolites such as lignins, sinapates, and flavonoids; and that some investigations have shown the existence of a possible crosstalk between glucosinolate and phenylpropanoid metabolic pathways (Hemm et al., 2003; Kliebenstein et al., 2005; Kim et al., 2015). For example, the incorporation of the sinapoyl moiety might occur through a sinapoyl-CoA co-substrate leading to the biosynthesis of sinapoylated glucosinolates (Sønderby et al., 2010). Besides, Kim et al. (2015) showed that ref5-1 mutants (A. thaliana reduced epidermal fluorescence5), which are defective in the accumulation of soluble phenylpropanoids, had a missense mutation in CYP83B1, a key enzyme in indole glucosinolate biosynthesis.

Consequently, the lack of PCS1 (as occurs in the cad1-3 mutant) might alter both the glucosinolate and, the phenylpropanoid pathway, the latter having potential repercussions on metabolism of monolignols, the degree of lignification, and the biosynthesis of other phenolic compounds, i.e., flavonoids. In fact, Cd treatment upregulated the transcript levels of genes encoding monolignol biosynthetic enzymes in both roots and shoots (Herbette et al., 2006), suggesting subsequent increases in lignin biosynthesis. Interestingly, some flavonoids can act as negative regulators of auxin movement in vivo (Murphy et al., 2000; Brown et al., 2001; Peer et al., 2001; Buer and Muday, 2004; Buer et al., 2013; Kuhn et al., 2016), and therefore variations in their levels might affect auxin content and distribution in planta.

To revisit the role of PCS1 in plants and to elucidate its function in the secondary metabolism, we compared here the metabolome of A. thaliana wild-type and PCS1-deficient cad1-3 mutant. Indeed, we reported data showing that – in addition to the glucosinolate pathway – AtPCS1 plays a role in regulating the phenylpropanoid pathway. In the glucosinolate pathway, AtPCS1 influences callose deposition, with a consequent increased resistance to high Cd concentrations and to the hemibiotrophic bacterial pathogen Pseudomonas syringae. In the phenylpropanoid pathway, AtPCS1 modulates the levels of hydroxycinnamic acids, lignin and flavonols, with consequent variations in auxin content, both in shoots and in roots.

Overall, our results are consistent with the role of the AtPCS enzyme in the accumulation of indole and aliphatic glucosinolates (Schlaeppi et al., 2008). To this end, in agreement with Clay et al. (2009), we found an increased level of 4MOI3M in shoots of cad1-3 plants grown under control conditions. 4MOI3M has deterrent properties, and its breakdown products contribute to the defense against pathogens and herbivores (Halkier and Gershenzon, 2006; Kim and Jander, 2007; Bednarek et al., 2009). Our results revealed an increase in 4MOI3M in cad1-3 plants also after Cd exposure, by a factor of about 1.5 and 4 in shoots and roots, respectively. These data thus highlight that the 4MOI3M glucosinolate plays a key role in the activation of common response pathways toward Cd toxicity and biotic stress. We found an accumulation of 4MSOB, 7MSOH, and 8MSOO in shoots of cad1-3 plants grown in control conditions, as well as in roots of this mutant after Cd treatment, presumably as a consequence of the 4MOI3M relatively high accumulation. Likewise, according to Kim and Jander (2007), the accumulation of 4MOI3M after aphid infestation of A. thaliana leaves significantly stimulated the synthesis of some aliphatic glucosinolates.

To test if the alteration of the glucosinolate pathway in cad1-3 affects plant response to bacterial pathogens, other than to the oomycete Phytophthora infestans (Kühnlenz et al., 2015), we compared the sensitivity of wild-type and cad1-3 plants to the hemibiotrophic bacterium P. syringae DC3000. Callose is one of most important mechanism of defense responses to pathogens, and the cad1-3 mutant shows defects in callose deposition after Flg22 treatment. As expected, our cad1-3 plants were more susceptible to PsDC3000 than wild-type. A similar trend for the same pathogen was found for the pen2-1 mutant which accumulates 4MOI3M and is impaired in callose deposition. We used as a control the pad4 mutant, which is more sensitive to pathogen as well (Xing and Chen, 2006). Therefore, the catabolic products of 4MOI3M are components of the defense response toward biotic stress in A. thaliana. We found that this response mechanism involves AtPCS1.

The synthesis of callose is also activated under exposure to toxic heavy metals (Ueki and Citovsky, 2002, 2005), but callose production after Cd treatment has never been experimentally validated. We noticed that Cd exposure induces callose deposition only in wild-type plants, but this event was shown to be not as prominent as following Flg22 treatment. We also found that Cd treatment did not stimulate callose deposition in cad1-3 and pen2-1 mutants. The lack of stimulation could be due to the remarkable accumulation of 4MOI3M in cad1-3 plants, also as a response to metal stress. These results highlight that AtPCS1 protects plants against heavy metal toxicity not only by producing PCs, but also by contributing to callose deposition. The proposed pathway may involve the conjugation of isothiocyanates (which are the products of PEN2-catalyzed glucosinolate hydrolysis) with GSH, and the subsequent glycine cleavage by AtPCS1 (Bednarek et al., 2009; Kühnlenz et al., 2015). In addition, our data suggest the importance of callose deposition in protecting plants particularly against high concentrations of Cd, since the callose-defective mutant pen2-1 showed an increased sensitivity to 50 and 75 μM Cd, compared with wild-type plants.

Interestingly, our metabolomic study also showed an upregulation of the phenylpropanoid pathway in cad1-3 plants. Specifically, accumulation of sinapic acid hexoside was found in shoots of cad1-3 plants, both under control conditions and exposed to Cd. Sinapic acid is involved in lignin biosynthesis (Lim et al., 2001). In fact, in line with our findings, exposure to Cd upregulated the transcript levels of genes encoding proteins involved in monolignol synthesis (Herbette et al., 2006), suggesting a possible consequent increase in lignin biosynthesis as well. In general, an increased lignification can be interpreted as an avoidance mechanism aimed at reducing the metal entry inside the cells, the inner tissues, the xylem flow, etc. The increase in the cell wall thickness can in fact lead to a substantial extension of the surfaces potentially useful for the immobilization of toxic metals (Van Belleghem et al., 2007; Sanità di Toppi et al., 2012). Since cad1-3 plants are not able to produce PCs and are impaired in callose deposition, the increased levels sinapic acid hexoside could result in an augmented lignin biosynthesis. This evidence was supported by the increased resistance of the cad1-3 stems to stretch, indicative of rigidity increments of cell walls, and also by the increase in lignin staining. Thus, an augmented lignification can represent a further mechanism for counteracting Cd toxicity in cad1-3 plants, which lack a major PC-dependent, and in part an auxiliary callose-dependent, heavy metal protective systems.

In our experiments, besides an increased level of sinapic acid hexoside in the shoots, Cd stress also raised the levels of a ferulic acid derivative in the roots of cad1-3 plants vs. wild-type. Intriguingly, such increase could be one explanation for the strong decrease in primary root length observed in the former. In fact, the formation of diferulic linkages typically occurs at various stages during cell wall formation (Passardi et al., 2004). Changes in the accumulation of cell wall-bound phenolics, such as ferulic acid derivatives, are typically reported under abiotic stress conditions (i.e., drought and salt stress) and are involved in the progressive inhibition of root growth (Fan et al., 2006; Neves et al., 2010). As a consequence, the reduction in root growth possibly mediated by the ferulic acid derivative accumulation may facilitate the root response to toxic metal exposure.

Although glucosinolates and phenylpropanoids are synthesized through different biosynthetic pathways and have distinct functions, some evidence indicates a link between these two pathways (Hemm et al., 2003; Kim et al., 2015). In fact, genetic and biochemical investigations suggested that the crosstalk between the indole glucosinolate pathway and the early steps of phenylpropanoid biosynthesis could be regulated by the levels of IAOx or by a subsequent metabolite (Kim et al., 2015). On this basis, we may surmise that also in our experiments the increased content in sinapic acid hexoside in the shoots might be related to a possible decrease in the IAOx content (not measured). In addition, it may have taken place a carbon re-routing toward phenylpropanoid biosynthesis caused by lack of PCS, which might have rendered plants in a pseudo-state of stress responsiveness even in the absence of Cd.

We also found increased levels of other metabolites from the phenylpropanoid pathway, particularly some flavonols. In fact, two glycosylated forms of kaempferol and quercetin were detected at higher relative levels in cad1-3 shoots, compared with wild-type ones. These flavonols regulate auxin retention and transport in vivo (Murphy et al., 2000; Brown et al., 2001; Peer et al., 2001; Buer and Muday, 2004; Buer et al., 2013). Experiments using an allelic series of tt4 mutants, with defects in the gene encoding chalcone synthase and characterized by the absence of flavonoids, have revealed that these phenolic compounds likely alter auxin transport in A. thaliana (Brown et al., 2001). Indeed, flavonols act as regulators of cellular auxin efflux and consequent auxin polar transport (Murphy et al., 2000; Buer and Muday, 2004; Peer and Murphy, 2007; Rusak et al., 2010).

To analyze whether the higher flavonol levels found in cad1-3 plants affect auxin levels in shoots and roots, we measured the concentrations of auxins (IAA, IBA, ICA, and MeIAA) in wild-type and cad1-3 plants, grown under control conditions. Interestingly, the cad1-3 mutant accumulated lower levels of total auxins in shoots and roots compared with wild-type plants. The abundance of kaempferol and quercetin derivatives found in cad1-3 shoots is in fact well correlated with the lower auxin level of this mutant, both in shoots and in roots. In agreement with our findings, kaempferol 3-O-rhamnoside-7-O-rhamnoside has been recently identified as an endogenous polar auxin transport inhibitor in A. thaliana shoots in a dose-dependent manner (Yin et al., 2014). Also, the formation of kaempferol 3-O-rhamnoside-7-O-rhamnoside is subordinated to a specific 3-O-rhamnosyltransferase, which is relatively abundant at the A. thaliana shoot apex, but almost absent in the roots (Jones et al., 2003). In addition, Kuhn et al. (2016) indicated that flavonols, and in particular 7-rhamnosylated ones, influence auxin transport and turnover, thus modifying the overall auxin levels in planta. Indeed, flavonoids can also accumulate in target-tissues far from the synthesis site, indicating that intermediates in their pathway can move long distances (Buer et al., 2007, 2008). Flavonoids have also been demonstrated to effectively modulate auxin signaling, not only by regulating auxin gradient (Grunewald et al., 2012) and flux (Michniewicz et al., 2007), but by buffering the formation of reactive oxygen species (ROS) (Peer et al., 2013) and ROS-dependent auxin oxidation (Peer et al., 2013). In our experiments, the flavonols might have regulated the auxin content at different levels, both modulating intra- and cell-to-cell auxin movements (Adamowski and Friml, 2015) and reducing auxin oxidation (Peer et al., 2013).

Not least, some intermediate steps in indole glucosinolate biosynthesis are shared not only with the biosynthesis of camalexin, which was found to be less accumulated in cad1-3 plants vs. wild-type after pathogen infection (Su et al., 2011), but also with ICA biosynthesis (Böttcher et al., 2014), which plays also an important role in plant–pathogen defense response (Böttcher et al., 2009, 2014). The significantly lower concentration of ICA in cad1-3 plants compared with wild-type may be directly related to lack of AtPCS1, because ICA is synthesized in the glucosinolate pathway through A. thaliana aldehyde oxidase1 (AAO1) and Cytochrome P450 (CYP)71B6, starting from IAN (Böttcher et al., 2014). Since AAO1 and CYP71B6 are transcriptionally coexpressed with camalexin biosynthetic genes, the role of ICA has been recently revised, not simply as an IAA catabolite, but also as a metabolite involved in induced chemical defense against biotic agents (Böttcher et al., 2014).

It has been shown that camalexin is derived from IAOx, which is synthesized from tryptophane by the cytochrome P450 enzymes CYP79B2 and CYP79B3 (Glawischnig et al., 2004). IAOx is also an intermediate in the biosynthesis of indole glucosinolates and a precursor for IAA. This makes IAOx a possible key point in the branching of different metabolic pathways. Other evidence has shown that IAOx is a precursor of IAA, and because of defects in CYP83B1 activity, sur2 mutants have phenotypes that are presumed to be associated with auxin overaccumulation (Delarue et al., 1998; Barlier et al., 2000; Bak et al., 2001). The low internal concentration of IAA derivatives we found in shoots of cad1-3 mutants might also be related to the accumulation in indole glucosinolate (such as 4MOI3M), caused by a lack of PCS1 which hydrolyses this compound. Lastly, since tryptophan is the precursor for de novo biosynthesis of both IAA and indole-glucosinolates, competition for the same substrate may have concurred to cause a decrease in IAA derivatives concomitantly to an increase in 4MOI3M in the shoots of cad1-3 mutants, even in the absence of Cd.

This work highlights some novel aspects of AtPCS1 functions in A. thaliana. First, our study reveals the role of AtPCS1 in Cd and bacterial pathogenic resistance fully enabling callose deposition. Second, the performed analyses confirm the role of AtPCS1 in managing the glucosinolate pathway, and support the AtPCS1 involvement in the regulation of the phenylpropanoid pathway, with particular reference to the degree of stem lignification. Third, the loss of AtPCS1 suggests possible effects on the modulation of auxin content and distribution in planta.

MD set up the experimental plans, together with OV and LS, and performed most of the experiments. SP and EB contributed to carry out the experiments on bacterial growth assays. FG and MC executed the metabolite extraction and HPLC-ESI-MS analyses. MRC performed the measurements on lignification. AS, CB, and AA performed the auxin quali- quantification. LS and OV conceptualized the overall structure of the experimental work and critically edited it. LS and CB revised the text based on feedback from all the coauthors.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.