2.1. Effects of Short Daily UV Treatment of the Phyllosphere on the Leaf and Root Metabolism of Tomato Plants
The UHPLCESI/QTOF-MS analysis, coupled with a comprehensive database for primary and secondary metabolites identification (PlantCyc), allowed for the detection of about 3000 metabolites in leaves and 2800 metabolites in roots. The whole list of metabolites detected is provided as
supplementary material, together with abundances and composite mass spectra (
Table S2). Given the broad chemical diversity in the metabolome, several multivariate statistical analyses were performed to allow a better understanding of the modifications caused by the UV treatment.
The output of the unsupervised fold-change-based hierarchical clustering (HCA) performed on the foliar metabolomic profile (
Figure 1) showed a separation between UV and CTR samples, with the main clustering between UV-rec and the other groups. However, in the case of roots, the output of the HCA (
Figure 1) showed the main clustering between CTR-rec and the other samples, with UV groups being clustered together irrespective of the time point considered. This preliminary analysis highlighted a different behavior of the two organs’ metabolism.
Then, an OPLS-DA supervised model (
Figure 2a,b for leaves,
Figure 2c,d for roots) was used to enhance the interpretation of the variables that maximize the differences among sample groups for each investigated organ.
Figure 2a,c clearly empathized the difference existing between CTR and UV samples (independently from the time point considered) in both leaves and roots. Consequently, Variable Importance for Prediction (VIP) analysis was carried out to show which metabolites weighed the most in the loading plot of these supervised models.
Table 1 lists the metabolic markers in leaves, with 122 compounds in total, related to
Figure 2a, with a VIP score higher than 1.1.
Univariate statistical analysis was performed to identify the VIP markers displaying a significant fold-change (FC) between UV and CTR foliar samples. About half of the compounds listed displayed a significant p value. Some compounds such as 2-oxoadipate, oxalosuccinate, scopolamine, 9-[6(S),9-diamino-5,6,7,8,9-pentadeoxy-β-D-ribo-nonafuranosyl]-9H-purin-6-amine, 3-hydroxy-9-apo-δ-caroten-9-one, and tricoumaroyl spermidine showed a remarkable and statistically significant enhanced FC in respect to CTR samples. On the contrary, N-P-tosyl-L-phenylalanyl chloromethyl ketone, 1-[18-hydroxyoeoyl]-2-[18-hydroxy-linoleoyl]-sn-glycerol, (22R,23R)-28-homocastasterone, and cyclo-dopa 5-O-glucoside reported a marked and significantly reduced FC in respect to CTR samples.
Roots VIP metabolic markers related to the OPLS-DA plot are shown in
Figure 2c and listed in
Table 2.
As for leaves, univariate statistical analysis led to identifying the compounds with a significant FC in respect to CTR root samples. Among 169 markers, 62 compounds displayed a significant FC. In particular, 10-deacetyl-2-debenzoylbaccatin III, a pentose, N-methylanthranilate, L-α-(methylenecyclopropyl)-glycine, 5,10-methylenetetrahydropteroyl mono-L-glutamate, all-trans-hexaprenyl diphosphate, 4-(β-D-glucosyloxy) benzoate, L-nicotianamine, dTMP, and N-(4-aminobenzoyl)-L-glutamate showed a remarkably enhanced or reduced (cycloheptadienyl/sinapate) FC in respect to CTR samples.
Finally, among the hundreds of VIP markers resulting from the OPLSDA plots, only eight markers were found to be in common between leaves and roots, and these were compounds related to the secondary metabolism such as O-sinapoylglucarolactone, cyanidin 3-O-β-D-p-coumaroylglucoside/cyanidin 3-(p-coumaroyl) glucoside/pelargonidin 3-O-β-D-caffeoylglucoside, scopolamine, aminoacids such as cyclogutamate and N-methylanthranilate, and 5-[[4-methoxy-3-(phenylmethoxy)phenyl]methyl]-2,4-pyrimidinediamine.
The OPLS-DA in
Figure 2b,d shows the effects of UV and time factors in leaves and roots, respectively. A good separation among the different groups is visible in both organs; however, in leaves, the treatment played the main role for the distribution along the first vector, with UV samples placed in the negative half of the plot and CTR samples in the positive half, while the sampling time did not have a great effect. Surprisingly roots exhibited not only the effect of the UV treatment, with UV samples and CTR samples in the positive and negative half of the score plot considering the second vector, but also a time effect, with a good separation of the samples along the first vector according to this factor.
2.2. Outputs of the Volcano Analysis on the Metabolome
Differential compounds, with individual
p values and fold-change resulting from the volcano plot, are fully provided as
supplementary material (Table S3). To visualise how the metabolic pathways were modulated by short daily UV treatments, the differential compounds resulting from the volcano analysis were interpreted by the Omics Viewer Dashboard of the PlantCyc Pathway Tool software.
Figure 3 shows several metabolic pathways in leaves and roots, with the main focus on biosynthesis and degradation processes.
Leaves of UV-11d plants displayed an increased fold-change in the amino acid and hormone biosynthesis (+28 and +23 log
2FC, respectively) in respect to the relative CTR, while the fatty acid/lipid and secondary metabolites showed a decrease in respect to CTR leaves on the same day (−24 and −100 log
2FC, respectively,
Figure 3). Especially the latter biosynthetic process was still reduced after the recovery, while fatty acid/lipid biosynthesis increased in respect to CTR (+47 log
2FC). Fatty acids, as components of the cellular membrane, also play a role in stress signalling responses leading to the alteration in the fluidity of the membrane. As an example, the release of unsaturated fatty acids is able to activate the defensive genes in tomato leaves under herbivore wounding [
25]. Yang and collaborators [
26], studying the effects of high UV-B irradiation (120.8 μW cm
−2 for 5 h followed by incubation with dark) on
Clematis terniflora, found an accumulation of linolenic and linoleic acids. In our experiment, the fluctuation in the FA/lipid synthesis reported in UV-treated leaves and the increase in their biosynthesis after 3 days of recovery suggest a remodelling of the membrane in respect to untreated plants and once the treatment was removed. Specifically, the lipids assigned to the biosynthetic process comprised, among others, two galactolipids, one phospholipid, three sterols, and three polyunsaturated fatty acids. Among these latter, (12Z,15Z)-9,10-epoxyoctadeca-12,15-dienoate and (9Z,12Z)-15,16-dihydroxyoctadeca-9,12-dienoate, which are involved in the production of hydroxy fatty acids from α-linolenic acid, were oppositely affected by the UV-B exposure, being increased or decreased, respectively. Since some hydroxylated fatty acids act as antifungal agents [
27], the influence of UV-B radiation on this pathway may lead to changes in plant–pathogen interactions.
Two of the three sterols identified as differential compounds according to volcano analysis (4α-formyl-4β-methyl-5α-cholesta-8,24-dien-3β-ol and 4α-carboxy-5α-cholesta-8,24-dien-3β-ol) markedly accumulated in UV-B-treated leaves after three days of recovery, similarly to the triterpene precursor, presqualene diphosphate (
Table S3). The two sterols identified in the present research are cholesterol precursors. Differently from mammals, who synthesize cholesterol, plants produce a complex mix of sterols, among which sitosterol, stigmasterol, and campesterol are the most representative compounds, though, in some plants, as in the Solanaceae family, cholesterol is present in significant amounts [
28]. Sterols are intrinsic components of the cell membrane, therefore their increase after the end of UV-B treatment may represent a mechanism of repairing possible damage induced by UV-B radiation to restore membrane fluidity and permeability. A slightly increased lipid peroxidation was indeed observed in UV-B-irradiated Micro-Tom leaves on day 11, while, at the end of the recovery period, this parameter was similar between control and treated leaves [
29]. It is well known that flavonoids can protect against lipid peroxidation caused by stressing conditions such as photoinhibition [
30,
31], and different studies have indicated that UV-B can increase lipid peroxidation levels, usually linked with a decrease in chlorophylls concentration and photosynthesis inhibition [
32]. In particular, Liu et al. [
33] found a significant correlation between anthocyanidins and lipid peroxidation, suggesting that the content of those antioxidant compounds may be linked to the extent of lipid oxidation. In this study, the FC of anthocyanin in leaves was slightly decreased in respect to CTR plants (paragraph 2.4), which is in line with the quantification reported in our recent paper [
34] after 11 days. One could assume that flavonoids might have been consumed in reactions aimed to interrupt the oxidative process occurring at the expense of cellular components such as membranes. However, as we reported in our previous papers [
29,
34], the UV-B treatment applied in this study did not cause severe damages to the photosynthetic apparatus, as indicated by the higher total chlorophyll concentration detected after 11 days in treated plants, and no differences in respect to CTR, considering the PSII efficiency, were detected. Moreover, as showed in [
34], we found a reduced expression of a gene related to anthocyanin biosynthesis on previous days, suggesting a likely reduced biosynthesis of these compounds in the leaves of treated plants.
An increase in sterol (sitosterol and stigmasterol) accumulation is reported in grapevine leaves following a ‘‘field-simulating’’ dose of UV-B radiation (4.75 kJ m
−2 per day) administered at low intensity (16 h at 8.25 μW cm
−2) and, less intensely, also at high intensity (4 h at 33 μW cm
−2). This latter treatment induced an increase in antioxidant compounds, such as mono- and diterpenes and tocopherol and phytol [
35]. In our study, the accumulation of some monoterpenes, diterpenes, and sesquiterpenes, among which were some phytoalexins, was stimulated in UV-exposed tomato leaves, indicating the activation of both MEP and mevalonate biosynthetic pathways. Such a response was generally more pronounced after 11 days of treatment (
Table S3). Beside changes in sterol levels, in our research, UV-B treated leaves underwent a decrease in 1-18:3-2-16:2-monogalactosyldiacylglycerol and an increase in the amount of 1-16:0-2-18:3-digalactosyldiacylglycerol, particularly after 3 days of recovery, indicating that UV-B radiation modifies the lipidic composition of membranes.
After 3 days of recovery, hormone degradation was strongly increased (+43 log
2FC), suggesting the necessity of modulating the hormone homeostasis. Interestingly, in UV-11d leaves, the reduced secondary metabolites synthesis was parallel to a lower degradation process (
Figure 3). A more detailed representation of the secondary metabolites pathway, which is the typical process involved in the UV response and acclimation, is shown in
Figure S1, where, in particular, the phenylpropanoid biosynthetic and degradation pathways demonstrated a strong reduction in treated leaves.
Roots displayed a different behaviour from leaves, in particular, in the case of the secondary metabolism biosynthesis, which appeared to be enhanced in UV-11d in respect to its CTR (+191 log
2FC,
Figure 3). After examining the secondary metabolites in detail, pathway biosynthesis led us to identify an increase in terpenoid (+60 log
2FC) and phenylpropanoid derivative (+16 log
2FC) biosynthesis (
Figure S1) in irradiated plants with respect to CTR at the 11-day treatment. However, phenylpropanoid biosynthesis was greatly reduced after the recovery (−47 log
2FC). Degradative reactions of the secondary metabolites (
Figure 3) showed a reduction in the fold-change after the recovery in respect to CTR roots (−13 log
2FC). Concerning the carbohydrate metabolism, degradative processes were strongly enhanced on both day 11 of treatment and after the recovery (about +13 log
2FC), with respect to roots of control plants, likely suggesting a mobilization of storage forms. The degradation of root carbohydrates might have been the source of precursors needed for the accumulation of secondary metabolites in this organ such as the flavonoid class, which will be discussed later in this article.
As observed in leaves, two monogalactosyldiacylglycerols showed an opposite behaviour following UV-B treatment also in roots. This finding, together with the decreased 4a-carboxy-5a-cholesta-7,24-dien-3b-ol root content detected in the roots of UV-B-treated plants, in accordance with the report of decreased cholesterol and accumulation of two steryl esters in roots of
Withania somnifera plants irradiated with supplemental UV-B (3.6 kJ m
−2 day
−1 above ambient level [
36], suggests a rearrangement of membrane lipid composition due to UV-B irradiation also in the non-irradiated organ.
Based on the unexpected modulation of the biochemical processes particularly related to the phenylpropanoid pathway, phenolic compounds were profiled in tomato leaves and roots, since it is highly recognised that phenolic compounds are involved in many plant processes and play a fundamental role in the acclimation towards UV, especially UV-B radiation.
2.3. Effects of Short Daily UV Treatment on the Phenolic Profile of Tomato Leaves and Roots
Since the UHPLC-ESI/QTOF-MS system coupled with a comprehensive phenolic database (Phenol-Explorer) allowed for the detection and identification of hundreds of compounds (232 and 183 compounds in leaves and roots, respectively), an OPLS-DA model was built for both organs, and for the metabolomics, to extrapolate the UV-induced phenolic modifications.
In leaves, the OPLS-DA analysis (
Figure 4a) revealed a clear distinction between CTR and UV-treated groups, indicating that, regardless of the sampling time, the irradiation did result in effectively modulating the phenolic pattern.
Variable Importance for Prediction (VIP) analysis was carried out to show which phenolic compounds weighed the most in the loading plot of the OPLS-DA model reported in
Figure 4a.
Table 3 shows the 33 phenolic markers with a VIP score higher than 1.1, classified according to their class and subclass.
The most abundant class was the flavonoid one (15 compounds in total) followed by the phenolic acids (12), while anthocyanins (5), flavonols (5), and hydroxycinnamic acids (10) were the most represented subclasses. At the same time, to understand which of the VIP markers displayed a significant fold-change between UV and CTR foliar samples, a univariate statistical analysis was performed and highlighted three compounds with a significant p value. These are rosmarinic acid (−4 log2FC), caffeoyl aspartic acid (+0.52 log2FC), and cirsimaritin (+0.72 log2FC). Besides the UV protection of phenolic acids and flavonoids, they also play an important role as antifungal and antimicrobial compounds.
Furthermore, both sampling time and UV treatment played a role in the group separation along with the vectors (
Figure 4b). Indeed, the first vector provided a clear separation of all groups, with CTR-11d being the only one located in the positive half of the plot, while, according to the second vector, only UV-11d was positioned in the positive half. However, upon obtaining a general overview of the OPLS-DA output, control, and UV-treated samples, they are shown to be well clustered within each group and well separated from the others, indicating good homogeneity among the replicates and a highly different phenolic profile according to both the treatment and the sampling time.
The OPLS-DA analysis on root tissue revealed a clear distinction between control and UV-treated plants (
Figure 4c). This result suggests that the UV irradiation of the plant’s aerial part did indirectly alter the phenolic profile also within the below-ground tissues. The details of the VIP analysis performed on root tissue data are indicated in
Table 4, where 25 phenolics with a VIP score higher than 1.1 are classified according to their class and subclass.
The most abundant class was the flavonoid one (12 compounds in total) followed by the phenolic acids (7), while anthocyanins (6) and hydroxycinnamic acids (5) represent the large subclasses. As for leaves, a univariate statistical analysis of the FC was performed, and p-Coumaroyl glycolic acid, a hydroxycinnamic acid, was the only compound with a significant
p value. This phenolic acid was significantly reduced by UV treatment (−10 log
2FC) and, as reported by Kadam et al. [
37], it is known for its antioxidant and anti-inflammatory properties.
Moreover, considering both time and UV treatment factors (
Figure 4d), a good separation among all groups occurred within the score plot. In detail, the first vector highlights the effect of the sampling time factor, since both CTR-11d and UV-11d plants were overlapped and placed in the negative half of the plot, while CTR-rec and UV-rec were in the positive half, although well distinct from each other. According to the second vector, the UV-rec group resulted in being the only one located in the positive half of the plot, distant from all the other groups.
2.4. Fold-Change Analysis on the Comprehensive Dataset of Leaves and Roots Phenolic Compounds
To investigate the accumulation of the most UV-responsive phenolic classes and subclasses in detail, a fold change (FC) analysis of UV in respect to controls was carried out on the base of the whole phenolics dataset, considering both the sampling times (11 days of UV exposure and 3 days after the end of the treatment) and both tissues (leaves and roots) (
Figure 5). A detailed list of the phenolic molecules and their fold change are listed in
Tables S4 and S5.
A general decrease in the phenolic class abundance was detected in the UV-11d leaves, especially for phenolic acids, which showed a −0.57 log
2FC in respect to control leaves (
Figure 5a). After the 3 days of recovery, most classes were still down accumulated in UV-rec, with flavonoids and phenolic acids showing a −0.13 and −0.18 log
2FC and lignans being the most affected phenolic subclass, with −0.68 log
2FC in respect to control (
Figure 5a). UV-induced responses typically trigger the accumulation of phenylpropanoids due to their ROS scavenging and screening activity; although, recently, several studies have showed the complexity of the phenolic response to this wavelength, depending on the exposure time, plant species, and phenolic class considered [
38]. However, after 11 days of treatments, Micro-Tom tomato plants have already acclimated to the supplemental UV irradiation, as showed in our previous study [
29], where many physiological parameters were checked to ensure that the UV dose applied did not induce stress-related responses. Indeed, we detected a reduced content of total phenols on day 11 of UV (according to the Folin–Ciocalteau method), and no differences were found in terms of flavonoid concentration. In contrast, a slightly increased phenols and flavonoids content was detected in previous days. According to these data, we speculate that the well-known and widely described induction of phenolics biosynthesis could be an early response under UV-B conditions, with fluctuations depending on the antioxidant status of the plant. After the plant acclimated to low UV-B doses and the morphogenic responses took place, e.g., after an increase in leaf thickness, lignification, trichome density [
39,
40,
41], it is likely that the plant energy and resources are re-addressed mainly to primary rather than secondary metabolism, so that physiological growth and development can be restored. Indeed, as reported by Coffey et al. [
42], the inhibiting effect of UV-B radiation on the leaf growth of
A. thaliana, grown in outdoor conditions for 7 months, ten days a month, was detected only under summer intensities, and it was not related to the activation of the UVR8-signalling pathway. The authors also found no changes in the concentration of UV-absorbing pigments; however, nothing was reported about the composition of the pool. Robson et al. [
16] also argued that the UV effects on leaf growth are complex and that, once the plants acclimated to the UV stimulus, normal development can be restored, leading to compensatory effects on the enlargement of the leaf area. According to our hypothesis of a UV-triggered modification in the morphology of the leaves, the reduced levels of lignans detected in UV-rec leaves suggest that these compounds were cross-linked for lignin biosynthesis [
43].
Very few studies have focused on UV-B perception in roots [
15,
44], and most of them have applied high UV-B intensities, which likely induced the onset of stress responses overlapping the activation of the UVR8 pathway [
45,
46]. To the best of our knowledge, no previous works have investigated the changes in the phenolic profile of roots under short daily UV conditions. The fold-change analysis on the root phenolic abundance dataset (
Figure 5b) revealed a different behaviour between the two plant organs. The roots of treated plants showed an increase in flavonoids (+0.35 log
2FC) and lignans (+0.26 log
2FC) in the UV-11d group. After the recovery, an opposite behaviour was shown by lignans (−0.27 log
2FC) and phenolic acids (−0.60-fold) whose concentrations were both reduced, while flavonoids were still increased (+0.64-fold).
Consequently, to better understand how the treatment influenced the flavonoids (
Figure 5c,d) and phenolic acids (
Figure 5e,f), the fold change analysis was also carried out at subclass levels in both organs.
Flavonoids in UV-11d leaves showed an increased log2FC of dihydrochalcones, flavanols, flavanones, and flavones (+0.25, +0.91, +1.47, +0.93, respectively), while anthocyanins, dihydroflavonols, and isoflavonoids were reduced (−0.16, −0.81 and −0.43, respectively) compared to the correspondent control. After the 3-day recovery, leaves of treated plants still exhibited an increase in flavanols and flavones (+0.44 and +0.22 log2FC, respectively) and a persistent decrease in anthocyanins and dihydroflavonols (−0.13 and −0.68, respectively). Conversely, flavanones (−0.22 log2FC), flavonols (−0.30), and isoflavonoids (+1.45) demonstrated an opposite behaviour in respect to what was observed after 11 days. In root tissue, all the flavonoid subclasses generally increased their abundance at both sampling times, with flavones showing the highest increase (+0.74 and +1.52 log2FC in UV-11d and UV-rec, respectively). Only isoflavonoids were reduced after the recovery period, with a −1.69-fold decrease in respect to controls.
Besides their function as antioxidants, flavonoids can modulate the phytohormone signalling as well. Polar auxin (IAA) transport was increased in studies with
Arabidopsis mutant, with reduced levels of flavonoids aglycones [
47,
48,
49]. In our study flavonols and flavones, which have been demonstrated to be able to inhibit polar IAA transport and signalling [
50,
51], were found to be enhanced in UV-11d leaves, likely causing a reduced basipetal movement of this hormone towards roots. Indeed, a reduced level of IAA concentrations was detected in the roots of UV-treated plants after 3 days of recovery [
29], although a reduced biosynthesis in this organ could be another factor affecting its amount. Although less concentrated in respect to the aerial part of the plant, root flavonoids have significant roles in the regulation of root growth, within the nitrogen cycle, and among allelopathic interactions [
52]. Flavonoids can exert signalling actions in lower concentrations than those necessary for ROS scavenging and UV absorption [
50,
53]. The increased levels of flavonols and flavones detected in roots of UV-11d may play a role in creating an IAA gradient at cellular and tissue levels, thus regulating root growth. However, since the flavonoid aglycones are located in tissue involved in auxin translocation, it is reasonable to consider that 8 flavonol glycosides out of 11 flavonols were found, and only 1 flavone glycoside out of 6 flavones in leaves, while, in roots, there were 6 flavonol glycosides out of 9 flavonols and 1 flavone glycoside out of 5 flavones. Further investigations are needed to verify the link between the IAA metabolism and flavonoids under short daily UV treatments, considering both leaves and roots, to gain a complete understanding of this phenomenon. The presence of flavonoids has been reported in the root exudates released into the rhizosphere, where these compounds act as regulators of nutrient availability, thanks to their reducing potential and chelating ability and promotion of root-rhizobia symbiosis [
54]. Though we did not study the flavonoid content and composition of the root exudated, the increased flavonoid content (FC) in the roots of treated plants, detected in our experiment, suggests a potential benefit for irradiated plants in minerals uptake.
Concerning the phenolic acids, a general decrease in hydroxybenzoic (HB) and hydroxycinnamic (HC) acids occurred in leaves at both UV-11d and UV-rec. Notably, the strongest decrease was detected at 11 days for HC (−0.60 log
2FC) and after the 3-day recovery period for HB (−1.43-fold). In a study on the effects of UV-modulated resistance to herbivory in tomato plants (0.34 kJ m
−2 day
−1 UV dose), it was found that chlorogenic acid and rutin, the two major leaf phenolic compounds in this species, but generally the whole leaf metabolome, did not change after 14-day UV exposure [
55]. This might suggest that the secondary metabolism could have been affected at earlier days of the UV treatment. HC and HB in roots displayed an opposite trend, with −1.29 and +0.61 log
2FC for HB and HC at 11 days, respectively, and +0.87 and −0.48 log
2FC for HB and HC after the 3-day recovery period. As already mentioned, phenolic acids are involved in plant growth, antioxidant defence, and plant/microbe interaction [
56,
57]. Indeed, these compounds can be secreted by roots and act as allelochemicals by suppressing the proliferation of pathogens [
58].