Priming shares some metabolic features with acquired/induced resistance with the aim of faster and stronger activation of stress-inducible defense reactions upon a subsequent pathogen challenge . Since investigations of priming in plants started with molecular tools, it has improved the understanding of the activation of the plant innate immune system through the identification of defense genes and analysis of signaling pathways leading to SAR and ISR. Many natural and synthetic compounds have been shown to be critical regulators of acquired/induced resistance by means of a priming mechanism, or as direct activators of induced immunity [11, 13, 14]. In addition to the acceleration of the plant’s ability to activate defense responses, the emerging picture is that priming represents an important adaptation or survival mechanism in plants to cope with abiotic and biotic stresses [10, 13, 32]. Although recent results started to unravel the molecular mechanism behind priming, it is still relatively poorly understood. Within this context the present study was undertaken to perform a comparative metabolomics investigation regarding the mode(s) of action and effectiveness of Aza and Hxa as inducers of resistance in N. tabacum cells in support of priming, with the focus on genes and secondary metabolites likely to be involved in host defense. Our findings revealed that the response of cells to treatment with Aza and Hxa is time-dependent, with differential effects on the up- or down-regulation of metabolites as well as altered expression kinetics of selected genes involved in the activation and execution of the plant defense response.
Metabolomics is a qualitative and/ or quantitative approach for the analysis of metabolites under certain physiological states in a biological system [33, 34]. In essence, as the ultimate recipients of biological information flow in cells, the spectrum and level of metabolites not only play a crucial role in the expression of genes and stability of proteins, but also determines the phenotypic properties of the cell or organism . Ultimately, this approach significantly contributes to the understanding of unassigned and unidentified compounds from undefined metabolic pathways [36, 37]. Metabolomics has therefore been used in various recent studies that reported on the physiological processes of plant stress biology, the plant immune system and plant-microbe interactions. The exogenous application of priming agents to plant tissues or cultured cells has shown to be effective models to investigate up- or down-regulation of genes involved in defense-related cellular pathways [38,39,40]. In this way plant bioactive secondary metabolites resulting from these pathways can be rapidly biosynthesized, extracted and analyzed [27, 28, 41].
Secondary metabolites and the defensive roles of the annotated metabolites
Secondary plant metabolites constitute a formidable contributor to the chemical defenses of plants, either as phytoanticipins or phytoalexins . These metabolites include phenylpropanoids, terpenoids, alkaloids and glucosinolates, depending on the particular species [34, 36, 42]. These metabolites play an important role in plant defense systems and environmental adaptation, and their presence fluctuates in response to different environmental stimuli [9, 27, 28]. The knowledge on the accumulation of secondary metabolites in response to infection, has been utilized to study the underlying biochemical responses to stress [9, 27, 40].
The obtained results indicate that the exogenous treatment of tobacco cells with both Aza and Hxa led to the production of mainly HCA compounds (conjugates of caffeic acid and ferulic acid) listed in Table 1. This is in accordance with our previous results [27, 28]. PAL is the key enzyme at the entry point of the phenylpropanoid pathway, leading to the production of cinnamic acid that is further metabolized to various phenolic compounds . Noteworthy, in Fig. 6, there is up-regulation of PAL gene transcripts post 12 h Aza and Hxa elicitation. As a major part of the phytoalexins, phenylpropanoid compounds play a huge role in plant defense against stressors such as hostile environmental conditions [44,45,46]. In addition, biological functions of these compounds include structural support (phenylpropanoid-based polymers) and signaling in plant defense [33, 44]. As such, priming of plants by biotechnological induction of the phenylpropanoid pathway is an effective means of increasing resistance against stress conditions.
HCA-related metabolites have been shown to be widely distributed in plants and compounds such as caffeic acid and ferulic acid naturally occur either in conjugated or non-conjugated forms. Solanum species such as N. tabacum, S. tuberosum and S. lycopersicum are known to produce HCA ester and - amide conjugates. Putrescine, a polyamine, often conjugates to HCAs to give rise to compounds such as caffeoylputrescine and feruloylputrescine [47, 48]. The decarboxylation of tyrosine results in tyramine which is also often conjugated to HCAs upon pathogen infection . In addition, an ester bond can be formed between HCAs and a quinic acid to result in compounds known as chlorogenic acids [9, 27, 28, 45].
In this context, caffeoylputrescine glycoside (biomarker 1) was found to be induced by Hxa whereas the levels of cis-5-caffeoylquinic acid (biomarker 2), feruloylglycoside (biomarker 3), feruloyl-3-methoxytyramine glycoside (biomarker 5), feruloyl-3-methoxytyramine conjugate (biomarker 6) and feruloyl-3-methoxytyramine (biomarker 7) increased after either 6 or 12 h of post-elicitation with both Aza and Hxa (Fig. 4). In comparison, there was an increase of azelaic glycoside (biomarker 4) at 6 h and further at 12 h, and a decrease at 24 h following Aza elicitation. This is indicative that upon uptake and accumulation of Aza, the cells conjugated the molecules with glucose, possibly as a detoxification mechanism or for storage purposes.
Although quantitatively different, the effect of Hxa as a priming agent is qualitatively similar to that of Aza with regard to up-regulation of the early phenylpropanoid pathway, resulting in the biosynthesis of similar derivatives of caffeic acid and ferulic acid. The dynamic responses can be attributed to active metabolism, including synthesis, interconversion and degradation of stored conjugates as a mechanism to rapidly supply demands for HCAs .
Quantitative gene expression analysis
Quantitative expression analyses of twelve genes which included: (i) PAL, HQT and HCT; (ii) EREBP, SAR1-GTPase and THIO; (iii) HSP90, RAR1 and SGT1; (iv) NPR1, PR-1a and Defensin, were performed. The results indicate that the transcripts, associated with the various functional categories discussed below, exhibited different expression kinetics that can be described as early, mid and late responses.
Activation of signal transduction networks after pathogen recognition results in reprogramming of cellular metabolism, which leads to a large change in gene activity. Phenylalanine is synthesized via the shikimate pathway that also leads to the synthesis of quinic acid. PAL is the enzyme involved in the deamination of phenylalanine to trans-cinnamic acid, thus linking primary metabolism to secondary metabolism [43, 50]. The quinic acid pool acts as a reservoir that can be reversibly injected into the main pathway for esterification reactions with HCAs [9, 27]. The production of trans-cinnamic acid (and the HCA derivatives) occurs in response to stress-induced increases in PAL activity, and this represents the first step in the biosynthesis of various phenylpropanoids involved in plant defense: hydroxylated and methoxylated cinnamates, chlorogenic acids, coumarins, flavonoids and lignin precursors.
We investigated the expression of PAL, HCT and HQT. The expression of PAL was significantly up-regulated at 12 h in the cells treated with Aza as well as at 12 and 24 h in the cells treated with Hxa (Fig. 6a). HQT is one of the crucial enzymes for the synthesis of 5-caffeoylquinic acid (a CGA), catalyzing the transesterification reaction of caffeoyl-CoA with quinic acid . CGAs and related derivatives exhibit radical scavenging activity and have been identified as phytoanticipins and resistance biomarkers, as well as non-antimicrobial defense compounds that interferes with infection processes [9, 27, 28, 52]. The expression of HQT was significantly up-regulated at 6 h in the cells treated with Aza and at 12 h in those treated with Hxa (Fig. 6b). HCT functions in an alternative route to CGA synthesis where p-coumaroyl-CoA is first trans-esterified with quinic acid before hydroxylation to yield 5-caffeoylquinic acid and, similarly, plays a critical role in the phenylpropanoid biosynthetic pathway . Our study showed that following Hxa treatment of the cells, the expression of HCT was significantly up-regulated from 6 to 24 h, with maximum expression at 12 h (Fig. 6c). In cells treated with Aza, HCT was significantly up-regulated only at 12 h. Based on the differential expression of the PAL, HQT and HCT genes, we suggest that the priming action of Aza and Hxa involves activation of the early shikimate/phenylpropanoid pathway in support of the chemical defenses associated with disease resistance in plants.
Signal perception and transduction
Priming for enhanced defense against biotic/abiotic stress requires specific cellular signaling components upon treatment with an inducing agent. SAR1-GTPase (a small monomeric GTP-binding protein belonging to the Rho subfamily), associated with plant signaling events, was significantly up-regulated in the tobacco cells treated with Aza from 6 to 12 h as well as cells treated with Hxa from 12 to 24 h. The maximum expression was observed in the cells treated with both Aza and Hxa at 12 h (Fig. 6e). SAR1-GTPase acts as a molecular switch and is involved in intracellular signaling pathways downstream of inducible lectin domain receptor-like kinases, possible receptors for recognition of extracellular pathogen-derived P/MAMPs [54, 55]. As such, treatment with Aza and Hxa triggered the expression of SAR1-GTPase which plays a positive role in plant immunity as seen previously in the tobacco cells treated with isonitrosoacetophenone (INAP) . The results indicate that Aza triggered the expression of SAR1-GTPase earlier than Hxa, but with the latter lasting longer than the Aza effect. However as stated, with both treatments the highest expression level of SAR1-GTPase was observed at 12 h. The results indicate that the mode of action of Aza might be similar to that of Hxa on this key gene.
Transcription factors play an important regulatory function in the onset of priming . EREBP is known to be involved in transcriptional activation and in the cells treated with Aza an up-regulation was observed at 6 h post-elicitation. Up-regulation was also observed for EREBP transcripts in the cells treated with Hxa at 12 and 24 h, with a maximum 10-fold expression at 12 h (Fig. 6d). The results thus indicated that Aza triggered the expression of EREBP earlier than Hxa, but with the latter effect lasting longer and with the highest expression level observed at 12 h. Previous findings revealed that, during plant-pathogen interactions, the rate of ethylene biosynthesis (which is mediated by EREBP) increases rapidly and is linked to the induced transcription of some basic-type PR defense genes . Therefore, EREBP plays an important regulatory role in the onset of priming as also observed previously in N. tabacum cells primed with INAP . Since some defense mechanisms are not directly activated upon induction of the primed state, it can be assumed that the up-regulated expression of EREBP transcripts resulted in the activation of the defense response that is marked by the transcription of the appropriate defense-related genes.
The HSP90, SGT1 and RAR1 proteins form a molecular chaperone complex involved in diverse biological signaling including innate immunity and R gene-mediated disease resistance . This complex coordinately contributes to the stability of the nucleotide-binding leucine-rich repeat (NB-LRR)-containing proteins which are a group of receptors mediating innate immune responses to microbial pathogens. In addition, the HSP90-SGT1-RAR1 complex is crucial in the activation of R proteins and therefore a critical component of the plant immune response . In this study, the expression pattern of HSP90, RAR1 and SGT1 in the cells treated with Hxa showed an up-regulation from 12 to 24 h, with a maximum expression of each gene at 12 h resulting in 4-, 18- and 14-fold increases respectively for each gene (Fig. 6 g,h,i). In the cells treated with Aza, up-regulation was observed in the expression of SGT1 at 6 h, and HSP90 and RAR1 at 12 h. Similar expression patterns showing up-regulation was observed for HSP90 and RAR1 in the cells treated with both priming agents at 12 h. These results indicate the involvement of the HSP90-SGT-RAR1 complex in the responses triggered by Aza and Hxa treatment. Furthermore, the findings also showed that the mode of action of Aza and Hxa is similar on HSP90 and RAR1 at 12 h (Fig. 6 g,h), but that at other time points the effect on these gene transcripts differed.
The treatment of tobacco cells with Aza and Hxa resulted in the induction of the NPR1 gene. NPR1 is an important defense regulator protein that is required for SAR establishment , which is regulated by the endogenous accumulation of the signal molecule SA with NPR1 positioned at the cross-roads of multiple defense pathways . Upon SAR induction a biphasic change in cellular reduction potential occurs, leading in reduction of NPR1 to a monomeric form which accumulates in the nucleus and induces the expression of some PR defense genes via SA pathways [60, 62, 63]. A functional NPR1 gene is required for priming and the NPR1 protein is suggested to be one of the receptors for SA . Previously we reported that NPR1 was significantly induced in N. tabacum cells from 2 to 12 h, with a 10-fold increase at 8 h, following the induction of INAP as priming agent . In this study, the treatment of tobacco cells with Aza led to a significant up-regulation of NPR1 transcripts from 6 to 12 h with a maximum 18-fold increase in expression at 6 h. In comparison, the treatment with Hxa led to a notable up-regulation of NPR1 from 6 to 24 h with a maximum expression of 18-fold increase at 12 h (Fig. 6j). Knowing that the transcripts levels of NPR1 are responsive following SA treatment/accumulation to activate PR-gene expression and SAR, this suggests that the SA signaling pathway is also involved in the tobacco cellular response to Aza and Hxa treatment. Finally, the results showed a similar mode of action of Aza and Hxa to trigger enhanced expression of NPR1, although the maximum expression was observed at different time points.
Thioredoxins are known to function in redox signaling, thereby affecting NPR1 and thus also involved in defense responses. In addition, these enzymes act as chaperones and disulphide isomerases in protein folding of newly synthesized proteins . Here, up-regulation was observed for Thioredoxin at 6 h in cells treated with Aza, and at 12 and 24 h in the cells treated with Hxa, indicating differential responsiveness to the two priming agents (Fig. 6f).
Two important gene transcripts found to be induced by Aza and Hxa are PR-1a and Defensin, the accumulation of which results upon treatment with resistance-inducing agents . PR-1 proteins have routinely been used as molecular markers of SA-dependent SAR, and contribute to increase pathogen resistance by directly exerting harmful effects to microbial invaders during SAR . In contrast, Defensin, that exhibits antifungal and antibacterial activity , is controlled by JA/ET-dependent pathways and is regarded as a marker for JA signaling [62, 65]. We have previously reported that PR-1a and PR-1b transcripts were induced during SAR establishment in tobacco cells following the priming action of INAP which confers resistance to P. syringae pv. tabaci . Here, our findings also revealed that there was a significant up-regulation in the expression of PR-1a at 6 h and a highly significant up-regulation at 24 h in the cells treated with Aza. In contrast, the cells treated with Hxa showed only a slight / non-significant increase of the expression of PR-1a at all time-points (Fig. 6k). In addition, the qPCR data showed that the Defensin transcripts were significantly up-regulated at 6 and 12 h, with a maximum 14-fold increase in expression in cells treated with Hxa (Fig. 6l). In contrast to the Hxa action, the cells treated with Aza displayed no differential expression of Defensin from 6 to 12 h, but rather a significant down-regulation at 24 h. The differential changes in the transcript levels of PR-1a and Defensin indicate that the corresponding genes were responsively dissimilar to the treatments, with PR1a responding to Aza and Defensin responding to Hxa, thus implying differential actions of SA and JA/ET in the action mechanisms of the two inducers.