Control of anthocyanin and non-flavonoid compounds by anthocyanin-regulating MYB and bHLH transcription factors in Nicotiana benthamiana leaves

Coloration of plant organs such as fruit, leaves and flowers through anthocyanin production is governed by a combination of MYB and bHLH type transcription factors (TFs). In this study we introduced Rosea1 (ROS1, a MYB type) and Delila (DEL, a bHLH type), into Nicotiana benthamiana leaves by agroinfiltration. ROS1 and DEL form a pair of well-characterized TFs from Snapdragon (Antirrhinum majus), which specifically induce anthocyanin accumulation when expressed in tomato fruit. In N. benthamiana, robust induction of a single anthocyanin, delphinidin-3-rutinoside (D3R) was observed after expression of both ROS1 and DEL. Surprisingly in addition to D3R, a range of additional metabolites were also strongly and specifically up-regulated upon expression of ROS1 and DEL. Except for the D3R, these induced compounds were not derived from the flavonoid pathway. Most notable among these are nornicotine conjugates with butanoyl, hexanoyl, and octanoyl hydrophobic moieties, and phenylpropanoid-polyamine conjugates such as caffeoyl putrescine. The defensive properties of the induced molecules were addressed in bioassays using the tobacco specialist lepidopteran insect Manduca sexta. Our study showed that the effect of ROS1 and DEL expression in N. benthamiana leaves extends beyond the flavonoid pathway. Apparently the same transcription factor may regulate different secondary metabolite pathways in different plant species.

Introduction

Higher plants produce a large variety of low-molecular weight secondary compounds, such as phenylpropanoids, terpenoids, and alkaloids (Pichersky and Gang, 2000). Anthocyanins are colorants, that may appear red, purple, or blue depending on their chemical composition and the pH. Anthocyanins are a specific class of flavonoids synthesized via the phenylpropanoid pathway that have been part of the human diet in the form of fruit and berries since ancient times. There is increasing scientific evidence that anthocyanins provide health-promoting benefits (He and Giusti, 2010; Martin et al., 2011).

The patterns of anthocyanin coloration in different plant organs and tissues of higher plants is under the control of specific transcription factors (TFs) of the MYB and basic helix-loop-helix (bHLH) families (Petroni and Tonelli, 2011). MYB TFs that regulate anthocyanin biosynthesis belong to the R2R3-type MYB family (Stracke et al., 2001; Feller et al., 2011). Members of this protein family regulate diverse processes such as the phenylpropanoid pathway, tryptophan biosynthesis, epithelial cell fate identity and plant responses to environmental factors, and they also play a role in mediating hormone actions (Stracke et al., 2001; Broun, 2005; Dubos et al., 2010). R2R3-MYB proteins execute their regulatory functions through physical association with bHLH types of TFs and a WD repeat protein (Ramsay and Glover, 2005). Upon ectopic expression, many of the R2R3-MYB TFs, e.g., Rosea1 (ROS1) from Antirrhinum majus (Schwinn et al., 2006), Arabidopsis MYB75/PAP1 (Kranz et al., 1998) or the maize C1 (Cone et al., 1986), are able to induce anthocyanin production in a variety of divergent mono- and dicot plant species such as tomato, Arabidopsis, petunia and maize (Cone et al., 1986; Spelt et al., 2000; Teng et al., 2005; Butelli et al., 2008). For the activity of ROS1, a bHLH factor, such as Delila (DEL) from A. majus, is often required (Butelli et al., 2008) although in tobacco it has been shown that the expression of ROS1 alone could mediate anthocyanin formation (Orzaez et al., 2009).

There are many examples of TFs that execute the same function in different plant species. It has been described that MYB TFs such as ROS1 and PAP1 can induce accumulation of specific anthocyanins (Tohge et al., 2005; Butelli et al., 2008; Bhargava et al., 2013). Knowledge of the effects of such TFs on metabolites from different pathways has been scarce. It has been described that the overexpression of PAP1 in Arabidopsis leads to accumulation of anthocyanins, and concomitantly to a decrease in secondary cell wall components such as lignin and polysaccharides (Bhargava et al., 2013), and also of procyanidins (Tohge et al., 2005). On the other hand, compounds more closely related to anthocyanins, such as flavonols, have been described to be co-induced with anthocyanins (Butelli et al., 2008). Nicotiana is a plant genus that is known for its broad set of defensive molecules, including phenolic (Gaquerel et al., 2014) and nicotine-derived compounds (Dawson, 1945), and for its ability to produce anthocyanins (Deluc et al., 2006). It is therefore an interesting platform to monitor cross-talk between different metabolite groups upon overexpression of TFs regulating secondary metabolism.

In this work, the effects of expression of ROS1 and DEL from A. majus on Nicotiana benthamiana metabolites was estimated using untargeted LC-MS analysis. In line with their well-characterized role as anthocyanin-specific TFs, ROS1 and DEL overexpression resulted in the accumulation of the anthocyanin delphinidin 3-rutinoside (D3R). Surprisingly, accumulation of phenolamides as well as nornicotine conjugates with butan-, hexan-, and octan-oyl hydrophobic moieties was also observed. These compounds are defence N. benthamiana compounds with an established activity against insect herbivores (Severson et al., 1988; Kaur et al., 2010). Our study has shown that the effect of ROS1 and DEL expression in N. benthamiana therefore extends beyond anthocyanin production. Apparently, the same transcription factor pair can activate different secondary metabolite pathways in different plant species.

Materials and Methods

Materials and Constructs

Standards of delphindin 3-rutinoside (Extrasynthese, Genay, France), chlorogenic acid, nicotine, and tryptophan (Sigma, St Louis, MO, USA) were used in concentrations ranging from 3.12 to 200 μg/ml. cDNAs from ROS1 and DEL were amplified by PCR using genomic DNA of E8:ROS/DEL tomato fruits obtained from (Butelli et al., 2008). PCR products were gel purified and TOPO cloned into the pCR8/GW/TOPO-TA vector (Invitrogen). After sequence verification the ROS1 and DEL fragments were transferred by GATEWAY recombination to pK7WG2¹ to create 35S-ROS1 and 35S-DEL. The plasmids obtained were then introduced into Agrobacterium tumefaciens AGL0 (Lazo et al., 1991). AGLO harboring pBINPLUS (pBIN) plasmid (van Engelen et al., 1995) was used as a negative control.

Agroinfiltration

Nicotiana benthamiana infiltrations were carried out as described previously (van Herpen et al., 2010). Briefly, Agrobacterium strains were grown at 28°C for 24 h in LB medium with antibiotics. Cells were resuspended in MES buffer (10 mM MES; 10 mM MgCl2; 100 mM acetosyringone) to a final OD₆₀₀ of 0.5, followed by incubation for 150 min. Agrobacterium strains (pBIN, 35S-ROS1 and/or 35S-DEL) were mixed in equal volumes. N. benthamiana plants were grown in a greenhouse with 16 h light at 28°C. Strain mixtures were infiltrated into the abaxial side of leaves of four-week-old plants using a 1 mL syringe. The plants were grown under greenhouse conditions before further analysis.

For each reported metabolite, data derive from three independent infiltration experiments, performed on different dates. Within each infiltration experiment, the same Agrobacterium solutions were used for three independent leaves on different N. benthamiana plants. Each leaf was analyzed individually.

Metabolite Analysis

Metabolite analysis was essentially performed as described in De Vos et al. (2007). Leaves were harvested 5 days post infiltration, snapfrozen in liquid nitrogen and ground to a fine powder. Exactly 200 mg (+/–2 mg) of powder was extracted with 800 μL of methanol containing 1% formic acid. Extracts were sonicated for 15 min, centrifuged at 12500×g for 10 min and filtered through 0.45 μm filters (Minisart SRP4, Biotech GmbH, Germany). An Accela High Pressure Liquid Chromatography system with a photodiode array (HPLC-PDA; Thermo) coupled to an LTQ Ion Trap-Orbitrap Fourier Transformed Mass Spectrometer (FTMS; Thermo) hybrid system was used to detect, identify and quantify compounds (van der Hooft et al., 2012a). A LUNA 3 μ C18 (2) 150 × 2.00 mm column (Phenomenex, USA) was used to separate the extracted metabolites, with MQ water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B) as solvents. A linear gradient from 5 to 35% B at a flow rate of 0.19 ml/min was used. The FTMS was set at a mass resolution of 60,000 HWHM and a mass range of m/z 85-1200, using electrospray ionization in positive mode. Identification of detected compounds was based on retention time, accurate masses of both the parent and fragment ions, in combination with any PDA absorbance spectra (recorded at 240–600 nm).

Data analysis was performed in an untargeted manner, essentially as described (De Vos et al., 2007). Visualization of the HPLC-PDA-FTMS data was performed using Xcalibur 2.1 software (Thermo). The MetAlign software package² was used for baseline correction, noise estimation, and mass peak alignment (Lommen, 2009). Threshold for peak detection was set at 10000 ions per scan. Mass peaks originating from the same metabolite, including adducts, fragments and isotopes, were subsequently clustered into so-called reconstructed metabolites, using MSClust software (Tikunov et al., 2012). Zero values for compound intensity, i.e., absent in sample, were replaced by random values between 0 and 1. The resulting relative intensity levels of compounds were then used for further statistical analysis, using Student’s t-test. Metabolites of interest were annotated by querying literature data for metabolites, previously detected in N. benthamiana or other Nicotiana species. In addition, we checked for the presence of compounds that have previously been identified from other plant sources and analyzed at the same LC-MS conditions (Moco et al., 2007; van der Hooft et al., 2012b). Quantification of selected compounds in the leaf extracts was performed using the same HPLC-PDA-Orbitrap FTMS system and conditions, using dilution series of authentic standards.

Insect Assays

Manduca sexta bioassays were carried out as follows. Infiltrated N. benthamiana leaves were collected after 4 days of infiltration. Leaves were placed in Petri dishes with a wet filter paper and their petioles were placed in Eppendorf tubes that contained 1% agar in water. Per infiltration construct four replicates were used, and every replicate included five larvae. Leaves and larvae were incubated in the dark at 25°C. Leaves were replaced on day 3, and on day 5 the weights of individual larvae were recorded.

Results

Phenolic and Non-Phenolic Molecules Regulated by ROS1 and DEL in N. benthamiana

We set out to investigate the effect of ROS1 and DEL in N. bentamiana on metabolite profiles by transient expression using agroinfiltration. Leaves of four-week-old N. benthamiana plants were infiltrated with Agrobacterium suspensions and harvested, unless indicated, 5 days after infiltration. Anthocyanin formation could be observed by occurrence of a purple color in the leaves, but no other phenomena could be observed that would distinguish a ROS1 and DEL infiltrated leaf from a pBIN infiltrated leaf (supplemental Figure S1). Aqueous-methanol extracts of the leaves were subjected to HPLC-PDA-Orbitrap FTMS based global profiling, enabling the detection of a large variety of secondary metabolites, including anthocyanins (De Vos et al., 2007). Infiltration of ROS1 and DEL, induced a single peak with UV/Vis-absorbance at 520 nm, as detected by the photodiode array detector (PDA). This peak displayed a typical anthocyanin absorption spectrum and a monoisotopic mass of [M+H] = 611.158 m/z (Figure 1A, inserted panel). The observed m/z, retention time, absorption spectrum, and MSMS fragmentation pattern (Table 1) corresponded to that of the delphinidin 3-rutinoside (D3R) standard. Surprisingly, however, the LC-MS analysis revealed that, next to D3R, a range of other compounds not absorbing light at 520 nm, were also highly upregulated by ROS1 and DEL expression (Figure 1B). To investigate the reproducibility of induction of these compounds, we performed two new independent infiltration experiments, each in three replicates. Data were processed in an untargeted manner and analyzed for compounds that were significantly altered (students t-test, p < 0.05) in ROS1 and DEL infiltrated plants as compared to the control pBIN plants (Table 1). Most of the induced compounds were already present in the control pBIN plants, and could be annotated by comparison with authentic standards, or by MSMS analysis and comparison to literature data of Nicotiana compounds. Figure 2 illustrates their relative changes in content induced by ROS1 and DEL infiltration.

FIGURE 1

FIGURE 1. (A) HPLC-Photodiode array detection at 520 nm of extracts from pBIN (control) and ROS1&DEL infiltrated Nicotiana benthamiana leaves. Inserted panel shows the detected accurate mass and the UV-Vis absorbance spectrum of the delphinidin-3-rutinoside (D3R) peak. (B) HPLC-MS chromatograms of the samples described in (A). The relative abundance of mass signals is, in both cases, 1e7 at 100%. Indicated peak numbers refer to Table 1.

TABLE 1

TABLE 1. List of significantly changing compounds (students t-test, p < 0.05) in the ROS1&DEL infiltrated leaves as compared to pBIN (empty vector) infiltrated leaves.

FIGURE 2

FIGURE 2. (A) Diagram showing the ratios of the MS signals of significantly changed (p < 0.05) compounds in the ROS1&DEL infiltrated leaves as compared to the empty vector pBIN. (B) Chemical structures of the down-regulated (left) and up-regulated (right) compounds.

As expected, D3R was highly upregulated in the ROS1 and DEL infiltrated plants. In addition, a number of compounds which are derived from the phenylpropanoid pathway but which do not qualify as anthocyanins or flavonoids were detected. Among these was a group of phenolamides, consisting of hydroxycinnamic acids conjugated to polyamines such as p-coumaroyl-, caffeoyl-, and feruloyl-putrescine. On the other hand, down-regulation of two higher molecular weight caffeoyl-polyamine conjugates, i.e., N-(3-aminopropyl)-N-[4-[[(2E)-3-(3,4-dihydroxyphenyl)-1-oxo-2-propen-1-yl]amino]butyl]-3,4-dihydroxybenzenepropan amide, abbreviated as NB13, and N-[3-[[4-[[3-(3,4-dihydroxyphenyl)-1-oxo-2-propen-1-yl]amino]butyl]amino]propyl]-3,4-dihydroxybenzenepropanamide, abbreviated as NB16, were down-regulated (Table 1). Caffeoylquinic acids and tryptophan were found to be upregulated, indicating a general up-regulation of products of the shikimate pathway. Surprisingly, while nicotine levels were found to be down-regulated, a number of nornicotine conjugates carrying butanoyl, hexanoyl, and octanoyl hydrophobic moieties, which are not related to the phenylpropanoid pathway, were significantly (students t-test, p < 0.05) up-regulated.

Thus, ROS1 and DEL expression results in a range of both phenolic and non-phenolic metabolites in N. benthamiana (Figure 2). The mechanism of this induction (by direct gene activation of by indirect effects) remains elusive.

ROS1 is Required for the Induction of New Compounds and DEL Only Potentiates and Enhances ROS1 Activity

Earlier reports indicate that in N. tabacum, which is a close relative of N. benthamiana, ROS1 alone is sufficient for the induction of anthocyanins (Orzaez et al., 2009). We compared the effect of infiltration of combination of 35S-ROS1&35S-DEL against 35S-ROS1 alone and 35S-DEL alone on the global secondary metabolite profile (Figure 3). LC-MS analysis revealed that the anthocyanin D3R was still induced by ROS1, albeit two to four fold less than upon ROS&DEL co-infiltration. Other compounds, such as the putrescine conjugates and the nornicotine conjugates, were much less induced in the ROS1 only infiltration, while the DEL only infiltration hardly induced any changes in biochemical profile. In our interpretation, this indicated that ROS1 is essential for the changes in the metabolite profiles that were observed in N. benthamiana, while DEL is required for robust ROS1 activity. Levels of D3R, nicotine and 5-0-(E)-caffeoylquinic acid were quantified in the LC-MS using a dilution series of purified compounds (Figure 3B). D3R was undetectable in both the empty vector pBIN and DEL infiltrated plants. ROS1 infiltration alone yielded about 0.3 mg/g fresh weight of D3R. Combining ROS1 with DEL increased the D3R level about twofold. In control plants, the phenylpropanoid 5-0-(E)-caffeoylquinic acid was already present in well-detectable amounts. Infiltration of ROS1 alone increased its level significantly (students t-test, p < 0.05), while ROS&DEL co-infiltration showed doubled this effect, to 0.8 mg/g fresh weight. Thus, 5-0-(E)-caffeoyl quinic acid increased by a similar manner as D3R upon ROS1&DEL infiltration, relative to ROS1 alone. This result suggests that a general activation of the phenylpropanoid pathway has occurred, rather than a specific activation of the flavonoid/anthocyanin pathway. Nicotine levels were significantly (students t-test, p < 0.05) down-regulated by fivefold in the combination of ROS1&DEL, as compared to the control pBIN infiltrated leaves. Small nicotine down-regulation was also observed in ROS alone infiltration but that change was not significant.

FIGURE 3

FIGURE 3. (A) Diagram showing the ratios of the MS signals of significantly changed compounds (students t-test, p < 0.05) in the leaves infiltrated with ROS1, DEL, and ROS1&DEL as compared to the empty vector pBIN. (B) Quantity, in milligrams/gram fresh weight (FW), of selected compounds present in (A) using a dilution series of commercially available standards. Stars indicate a significant change in the particular compound (Student’s t-test, n = 3; *p < 0.05; **p < 0.01) as compared to pBIN.

Defensive Functions of the Metabolites Induced by ROS1 and DEL

Both nornicotine conjugates and phenolamines have been described as plant defensive molecules against insect herbivores (Zador and Jones, 1986; Severson et al., 1988; Laue et al., 2000; Gaquerel et al., 2014). In addition there is a growing evidence that anthocyanin by itself might have a role in the plant defense against herbivores (Lev-Yadun and Gould, 2009; Markwick et al., 2013). We investigated if plant leaves ectopically expressing ROS1 and DEL exert altered resistance against the tobacco hornworm, M. sexta. M. sexta is a solanaceous specialist adapted to nicotine in tobacco, but not to phenolamines and octanoyl nornicotine (Severson et al., 1988; Kaur et al., 2010). We have fed M. sexta first instar larvae for 5 days with leaves infiltrated with ROS1 and DEL, both alone and in combination, and have compared them to pBIN controls. In Figure 4, the average larval weights after 5 days of feeding are shown. M. sexta larvae fed on pBIN or DEL infiltrated leaves did not differ significantly in weight. In contrast, a significant weight reduction (t-test, p < 0.05) was observed when larvae were fed on leaves expressing ROS1 or ROS1 and DEL.

FIGURE 4

FIGURE 4. Average larval weight of 20 larvae after 5 days of feeding on leaves infiltrated with the following constructs: pBIN, ROS1, DEL, and ROS1&DEL. Star indicates a significant change (Student’s t-test, n = 20, p < 0.05) of the larval weight as compared to larvae fed on the empty vector pBIN treated leaves.

These results indicate that anthocyanin-regulating ROS1 and DEL TFs act as defensive anti-herbivore TFs in N. benthamiana. The available data do not allow to distinguish the roles of the different compound classes in the anti-herbivore effect.

Discussion

In this study we report that ectopic expression of two anthocyanin-specific TFs from A. majus (ROS1 and DEL) in N. benthamiana induces, besides a single anthocyanin D3R, a range of non-flavonoid, defensive, anti-herbivore molecules. In contrast, in tomato and in A. majus, ROS1 and DEL appear to specifically induce anthocyanins and related flavonoids, while no other molecules of different chemical classes have been reported (Goodrich et al., 1992; Schwinn et al., 2006; Butelli et al., 2008). Most literature describing overexpression of related anthocyanin-regulating MYB TFs study only report changes in anthocyanin levels, while changes in other metabolites were often not studied (Aharoni et al., 2001; Kobayashi et al., 2002; Mathews et al., 2003; Vimolmangkang et al., 2013). Arabidopsis overexpressing MYB75/PAP1 showed reductions in cell-wall components (Bhargava et al., 2013), while an untargeted metabolome analysis showed predominant induction of anthocyanins and flavonols (Tohge et al., 2005).

The type of anthocyanins induced by ROS1 and DEL is much different in N. benthamiana leaves as compared to tomato fruit. In contrast to the single D3R in N. benthamiana, in tomato six different, glycosylated, methylated, and acylated forms of anthocyanin have been detected upon ROS1 and DEL expression (Butelli et al., 2008). This difference in anthocyanin types could be caused by a more limited anthocyanin modification potential in N. benthamiana, as compared to tomato. Indeed ectopic expression in N. benthamiana of MYB75/PAP1 (Hugueney et al., 2009) also resulted in D3R as the major anthocyanin accumulating in the leaves. Apparently, N. benthamiana, in contrast to tomato, does not display activity of anthocyanin methyltransferases, anthocyanin 5-glucosyltransferases and anthocyanin acyltransferases, required for specific anthocyanin modifications. Whether genes with these specific functions are absent from the N. benthamiana genome, or are not subject to regulation by these TFs is not clear.

While ROS1 is essential to induce accumulation of different compounds in N. benthamiana, our study suggests that DEL is only required to potentiate the functions of ROS1. It is likely that N. benthamiana produces an endogenous DEL-type of bHLH transcription factor, which can support activity of ROS1.

One of the major compound classes induced by ROS1 and DEL in N. benthamiana is the phenylpropanoid-polyamines. Studies in N. attenuata have revealed important functions of these compounds as plant defense factors protecting against insect herbivores (Gaquerel et al., 2014). Their biosynthesis is under control of NaMYB8 (Kaur et al., 2010). This TF is from the same R2R3-MYB family as ROS1, and seems to overlap to some extent in the secondary metabolism pathways it regulates. Similar to ROS1 in N. benthamiana, NaMYB8 in N. attenuata induces multiple phenylpropanoid-polyamine conjugates. While ROS1 induces formation of the anthocyanin D3R, NaMYB8 in N. attenuata induces formation of the flavonol quercetin rutinoside (Kaur et al., 2010). Interestingly, three hydroxycinnamoyl-coenzyme A: polyamine transferases from the BAHD family have been identified in N. attenuata, and they are strongly upregulated by NaMYB8 (Onkokesung et al., 2012).

Our study suggests a TF-regulated production of octanoyl-nornicotine and similar conjugates in plants. The insecticidal properties of these compounds (Severson et al., 1988) warrant an investigation into their biosynthesis and regulation. These molecules are not known to be regulated by NaMYB8 in N. attenuata (Kaur et al., 2010; Onkokesung et al., 2012). The biosynthesis of these nornicotine conjugates has been hypothesized to be directly linked to the pool of nicotine (Zador and Jones, 1986). Indeed we observed that increasing octanoyl-nornicotine and similar derivatives coincided with a reduction of the total unconjugated nicotine levels (Figures 2 and 3). Nicotine is synthesized in the roots of multiple Nicotiana species. Transport to the aerial part of the plant is followed by its demethylation to nornicotine (Dawson, 1945). Subsequently, a yet unknown acyltransferase should transfer them to small fatty acid CoA conjugates. Acyl-nornicotine conjugates are normally present in the trichome exudate produced in the epidermis of the aerial parts of different Nicotiana species (Zador and Jones, 1986; Severson et al., 1988; Laue et al., 2000). Future experiments using ROS1 and DEL activation and gene expression studies may help to identify genes involved in the biosynthetic pathway toward acyl-nornicotine conjugates. It will be particularly interesting to identify the acyltransferase enzyme responsible for conjugation of nornicotine to acyl groups, and to compare its activity to both the N. attenuata acyltransferases involved in production of phenolic polyamine conjugates (Onkokesung et al., 2012) and putative tomato enzymes induced by ROS1 and DEL involved in anthocyanin acylation (Butelli et al., 2008).

In summary, the regulation of secondary metabolites by ROS1 and DEL in N. benthamiana extends beyond flavonoid production. ROS1 and DEL activate only partly understood pathways leading to the accumulation of phenolamines and nornicotine-conjugates. Therefore ROS1 and DEL have emerged as potentially valuable tools to investigate multiple secondary metabolite branches in different species.

Author Contributions

Nikolay S. Outchkourov designed de study, performed experiments, and wrote the manuscript. Carlos A. Carollo and Ric C. H. de Vos analyzed the LC-MS data. Victoria Gomez-Roldan generated the 35S-ROS1 and 35S-DEL constructs. Dirk Bosch, Ric C. H. de Vos, and Robert D. Hall facilitated the study and provided analysis and discussions. Jules Beekwilder provided discussions, data analysis, interpretations, and edited the manuscript.

Conflict of Interest Statement

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.

Acknowledgments

The authors wish to thank to Bert Schipper and Harrie Jonker for the LC-MS measurements. M. sexta eggs were a kind gift from Emma van der Woude, Department of Entomology Wageningen University, The Netherlands. This research was supported by the “Platform Green Synthetic Biology” program³ funded by the Netherlands Genomics Initiative. Jules Beekwilder and Robert D. Hall acknowledge support by the EU 7th Framework ATHENA project (FP7-KBBE-2009-3-245121-ATHENA). Robert D. Hall, Ric C. H. de Vos, and Victoria Gomez-Roldan acknowledge support from the Netherlands Metabolomics Centre and the Netherlands Consortium for Systems Biology, both financed under the auspices of the Netherlands Genomics Initiative. The authors thank Prof. Cathie Martin for the E8:ROS/DEL tomato seeds (Butelli et al., 2008).

Supplementary Material

The Supplementary Material for this article can be found online at: http://www.frontiersin.org/journal/10.3389/fpls.2014.00519/abstract

FIGURE S1. Picture of plants five days post infiltration. On the left plant two leaves infiltrated with pBIN are indicated, serving as a control. On the right plant two leaves infiltrated with ROS1&DEL have been indicated. Note the darker coloration of the ROS1&DEL infiltrated leaves.

Footnotes

1. ^ http://gateway.psb.ugent.be
2. ^ www.metAlign.nl
3. ^ http://www.pgsb.nl/

References

Aharoni, A., De Vos, C. H., Wein, M., Sun, Z., Greco, R., Kroon, A.,et al. (2001). The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco. Plant J. 28, 319–332. doi: 10.1046/j.1365-313X.2001.01154.x

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Ben-Hayyim, G., Damon, J. P., Martin-Tanguy, J., and Tepfer, D. (1994). Changing root system architecture through inhibition of putrescine and feruloyl putrescine accumulation. FEBS Lett. 342, 145–148. doi: 10.1016/0014-5793(94)80489-3

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Bhargava, A., Ahad, A., Wang, S., Mansfield, S. D., Haughn, G. W., Douglas, C. J.,et al. (2013). The interacting MYB75 and KNAT7 transcription factors modulate secondary cell wall deposition both in stems and seed coat in Arabidopsis. Planta 237, 1199–1211. doi: 10.1007/s00425-012-1821-9

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Bolt, A. J. N. (1972). 1′-Hexanoylnornicotine and 1′-Octanoylnornicotine from Tobacco. Phytochemistry 11, 2341–2343. doi: 10.1016/S0031-9422(00)88406-X

CrossRef Full Text | Google Scholar

Broun, P. (2005). Transcriptional control of flavonoid biosynthesis: a complex network of conserved regulators involved in multiple aspects of differentiation in Arabidopsis. Curr. Opin. Plant Biol. 8, 272–279. doi: 10.1016/j.pbi.2005.03.006

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Butelli, E., Titta, L., Giorgio, M., Mock, H. P., Matros, A., Peterek, S.,et al. (2008). Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. Nat. Biotechnol. 26, 1301–1308. doi: 10.1038/nbt.1506

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Cone, K. C., Burr, F. A., and Burr, B. (1986). Molecular analysis of the maize anthocyanin regulatory locus C1. Proc. Natl. Acad Sci. U.S.A. 83, 9631–9635. doi: 10.1073/pnas.83.24.9631

CrossRef Full Text | Google Scholar

Dawson, R. F. (1945). An experimental analysis of alkaloid production in Nicotiana – the origin of Nornicotine. Am. J. Bot. 32, 416–423. doi: 10.2307/2437360

CrossRef Full Text | Google Scholar

Deluc, L., Barrieu, F., Marchive, C., Lauvergeat, V., Decendit, A., Richard, T.,et al. (2006). Characterization of a grapevine R2R3-MYB transcription factor that regulates the phenylpropanoid pathway. Plant Physiol. 140, 499–511. doi: 10.1104/pp.105.067231

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

De Vos, R., Moco, C. S., Lommen, A., Keurentjes, J. J., Bino, R. J., and Hall, R. D. (2007). Untargeted large-scale plant metabolomics using liquid chromatography coupled to mass spectrometry. Nat. Protoc. 2, 778–791. doi: 10.1038/nprot.2007.95

CrossRef Full Text | Google Scholar

Dubos, C., Stracke, R., Grotewold, E., Weisshaar, B., Martin, C., and Lepiniec, L. (2010). MYB transcription factors in Arabidopsis. Trends Plant Sci. 15, 573–581. doi: 10.1016/j.tplants.2010.06.005

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Feller, A., Machemer, K., Braun, E. L., and Grotewold, E. (2011). Evolutionary and comparative analysis of MYB and bHLH plant transcription factors. Plant J. 66, 94–116. doi: 10.1111/j.1365-313X.2010.04459.x

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Gaquerel, E., Gulati, J., and Baldwin, I. T. (2014). Revealing insect herbivory-induced phenolamide metabolism: from single genes to metabolic network plasticity analysis. Plant J. 79, 679–692. doi: 10.1111/tpj.12503

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Gaquerel, E., Kotkar, H., Onkokesung, N., Galis, I., and Baldwin, I. T. (2013). Silencing an N-acyltransferase-like involved in lignin biosynthesis in Nicotiana attenuata dramatically alters herbivory-induced phenolamide metabolism. PLoS ONE 8:e62336. doi: 10.1371/journal.pone.0062336

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Goodrich, J., Carpenter, R., and Coen, E. S. (1992). A common gene regulates pigmentation pattern in diverse plant species. Cell 68, 955–964. doi: 10.1016/0092-8674(92)90038-E

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

He, J. A., and Giusti, M. M. (2010). Anthocyanins: natural colorants with health-promoting properties. Ann. Rev. Food Sci. Technol. 1, 163–187. doi: 10.1146/annurev.food.080708.100754

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Hugueney, P., Provenzano, S., Verries, C., Ferrandino, A., Meudec, E., Batelli, G.,et al. (2009). A novel cation-dependent O-methyltransferase involved in anthocyanin methylation in grapevine. Plant Physiol. 150, 2057–2070. doi: 10.1104/pp.109.140376

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Kaur, H., Heinzel, N., Schottner, M., Baldwin, I. T., and Galis, I. (2010). R2R3-NaMYB8 regulates the accumulation of phenylpropanoid-polyamine conjugates, which are essential for local and systemic defense against insect herbivores in Nicotiana attenuata. Plant Physiol. 152, 1731–1747. doi: 10.1104/pp.109.151738

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Kobayashi, S., Ishimaru, M., Hiraoka, K., and Honda, C. (2002). Myb-related genes of the Kyoho grape (Vitis labruscana) regulate anthocyanin biosynthesis. Planta 215, 924–933. doi: 10.1007/s00425-002-0830-5

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Kranz, H. D., Denekamp, M., Greco, R., Jin, H., Leyva, A., Meissner, R. C.,et al. (1998). Towards functional characterisation of the members of the R2R3-MYB gene family from Arabidopsis thaliana. Plant J. 16, 263–276. doi: 10.1046/j.1365-313x.1998.00278.x

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Laue, G., Preston, C. A., and Baldwin, I. T. (2000). Fast track to the trichome: induction of N-acyl nornicotines precedes nicotine induction in Nicotiana repanda. Planta 210, 510–514. doi: 10.1007/s004250050038

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Lazo, G. R., Stein, P. A., and Ludwig, R. A. (1991). A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Biotechnology 9, 963–967. doi: 10.1038/nbt1091-963

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Lev-Yadun, S., and Gould, K. S. (2009). “Role of anthocyanins in plant defence,” in: Anthocyanins, Biosynthesis, Function and Applications, eds K. Gould, K. M. Davies, and C. Weinfield (New York: Springer).

Google Scholar

Lommen, A. (2009). MetAlign: interface-driven, versatile metabolomics tool for hyphenated full-scan mass spectrometry data preprocessing. Anal. Chem. 81, 3079–3086. doi: 10.1021/ac900036d

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Markwick, N. P., Poulton, J., Espley, R. V., Rowan, D. D., McGhie, T. K., Wadasinghe, G.,et al. (2013). Red-foliaged apples affect the establishment, growth, and development of the light brown apple moth, Epiphyas postvittana. Entomol. Exp. Appl. 146, 261–275. doi: 10.1111/eea.12024

CrossRef Full Text | Google Scholar

Martin, C., Butelli, E., Petroni, K., and Tonelli, C. (2011). How can research on plants contribute to promoting human health? Plant Cell 23, 1685–1699. doi: 10.1105/tpc.111.083279

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Mathews, H., Clendennen, S. K., Caldwell, C. G., Liu, X. L., Connors, K., Matheis, N.,et al. (2003). Activation tagging in tomato identifies a transcriptional regulator of anthocyanin biosynthesis, modification, and transport. Plant Cell 15, 1689–1703. doi: 10.1105/tpc.012963

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Matsushita, H., Tsujino, Y., Yoshida, D., Saito, A., Kisaki, T., Kato, K.,et al. (1979). New minor alkaloids in flue-cured tobacco leaf (Nicotiana tabacum Cv by-260-9). Agric. Biol. Chem. 43, 193–194. doi: 10.1271/bbb1961.43.193

CrossRef Full Text | Google Scholar

Miyano, M., Yasumatsu, N., Matsushita, H., and Nishida, K. (1981). 1′-(6-Hydroxyoctanoyl)Nornicotine and 1′-(7-Hydroxyoctanoyl)Nornicotine, 2 new alkaloids from Japanese domestic tobacco. Agric. Biol. Chem. 45, 1029–1032. doi: 10.1271/bbb1961.45.1029

CrossRef Full Text | Google Scholar

Moco, S., Capanoglu, E., Tikunov, Y., Bino, R. J., Boyacioglu, D., Hall, R. D.,et al. (2007). Tissue specialization at the metabolite level is perceived during the development of tomato fruit. J. Exp. Bot. 58, 4131–4146. doi: 10.1093/jxb/erm271

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Onkokesung, N., Gaquerel, E., Kotkar, H., Kaur, H., Baldwin, I. T., and Galis, I. (2012). MYB8 controls inducible phenolamide levels by activating three novel hydroxycinnamoyl-coenzyme A: polyamine transferases in Nicotiana attenuata. Plant Physiol. 158, 389–407. doi: 10.1104/pp.111.187229

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Orzaez, D., Medina, A., Torre, S., Fernandez-Moreno, J. P., Rambla, J. L., Fernandez-Del-Carmen, A.,et al. (2009). A visual reporter system for virus-induced gene silencing in tomato fruit based on anthocyanin accumulation. Plant Physiol. 150, 1122–1134. doi: 10.1104/pp.109.139006

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Osawa, Y., Tochigi, B., Tochigi, M., Ohnishi, S., Watanabe, Y., Bullion, K.,et al. (1990). Aromatase inhibitors in cigarette smoke, tobacco leaves and other plants. J. Enzyme Inhib. 4, 187–200. doi: 10.3109/14756369009040741

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Petroni, K., and Tonelli, C. (2011). Recent advances on the regulation of anthocyanin synthesis in reproductive organs. Plant Sci. 181, 219–229. doi: 10.1016/j.plantsci.2011.05.009

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Pichersky, E., and Gang, D. R. (2000). Genetics and biochemistry of secondary metabolites in plants: an evolutionary perspective. Trends Plant Sci. 5, 439–445. doi: 10.1016/S1360-1385(00)01741-6

CrossRef Full Text | Google Scholar

Ramsay, N. A., and Glover, B. J. (2005). MYB-bHLH-WD40 protein complex and the evolution of cellular diversity. Trends Plant Sci. 10, 63–70. doi: 10.1016/j.tplants.2004.12.011

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Schwinn, K., Venail, J., Shang, Y., Mackay, S., Alm, V., Butelli, E.,et al. (2006). A small family of MYB-regulatory genes controls floral pigmentation intensity and patterning in the genus Antirrhinum. Plant Cell 18, 831–851. doi: 10.1105/tpc.105.039255

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Severson, R. F., Huesing, J. E., Jones, D., Arrendale, R. F., and Sisson, V. A. (1988). Identification of tobacco hornworm antibiosis factor from cuticulae of repandae section of Nicotiana species. J. Chem. Ecol. 14, 1485–1494. doi: 10.1007/BF01012420

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Snook, M. E., Costello, C. E., Arrendale, R. F., Sisson, V. A., and Chortyk, O. T. (1988). Isolation and identification of polyphenols in the flowers of the Nicotiana species. Bull. Liaison - Groupe Polyphenols 14, 42–45.

Pubmed Abstract | Pubmed Full Text | Google Scholar

Spelt, C., Quattrocchio, F., Mol, J. N., and Koes, R. (2000). Anthocyanin1 of petunia encodes a basic helix-loop-helix protein that directly activates transcription of structural anthocyanin genes. Plant Cell 12, 1619–1632. doi: 10.1105/tpc.12.9.1619

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Stracke, R., Werber, M., and Weisshaar, B. (2001). The R2R3-MYB gene family in Arabidopsis thaliana. Curr. Opin. Plant Biol. 4, 447–456. doi: 10.1016/S1369-5266(00)00199-0

CrossRef Full Text | Google Scholar

Teng, S., Keurentjes, J., Bentsink, L., Koornneef, M., and Smeekens, S. (2005). Sucrose-specific induction of anthocyanin biosynthesis in Arabidopsis requires the MYB75/PAP1 gene. Plant Physiol. 139, 1840–1852. doi: 10.1104/pp.105.066688

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Tikunov, Y. M., Laptenok, S., Hall, R. D., Bovy, A., and de Vos, R. C. (2012). MSClust: a tool for unsupervised mass spectra extraction of chromatography-mass spectrometry ion-wise aligned data. Metabolomics 8, 714–718. doi: 10.1007/s11306-011-0368-2

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Tohge, T., Nishiyama, Y., Hirai, M. Y., Yano, M., Nakajima, J., Awazuhara, M.,et al. (2005). Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis plants over-expressing an MYB transcription factor. Plant J. 42, 218–235. doi: 10.1111/j.1365-313X.2005.02371.x

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

van der Hooft, J. J. J., Akermi, M., Unlu, F. Y., Mihaleva, V., Roldan, V. G., Bino, R.J.,et al. (2012a). Structural annotation and elucidation of conjugated phenolic compounds in black, green, and white tea extracts. J. Agric. Food Chem. 60, 8841–8850. doi: 10.1021/jf300297y

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

van der Hooft, J. J. J., Vervoort, J., Bino, R. J., and de Vos, R. C. H. (2012b). Spectral trees as a robust annotation tool in LC-MS based metabolomics. Metabolomics 8, 691–703. doi: 10.1007/s11306-011-0363-7

CrossRef Full Text | Google Scholar

van Engelen, F. A., Molthoff, J. W., Conner, A. J., Nap, J. P., Pereira, A., and Stiekema, W. J. (1995). pBINPLUS: an improved plant transformation vector based on pBIN19. Transgenic Res. 4, 288–290. doi: 10.1007/BF01969123

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

van Herpen, T. W., Cankar, K., Nogueira, M., Bosch, D., Bouwmeester, H. J., and Beekwilder, J. (2010). Nicotiana benthamiana as a production platform for artemisinin precursors. PLoS ONE 5:e14222. doi: 10.1371/journal.pone.0014222

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Vimolmangkang, S., Han, Y. P., Wei, G. C., and Korban, S. S., (2013). An apple MYB transcription factor, MdMYB3, is involved in regulation of anthocyanin biosynthesis and flower development. BMC Plant Biol. 13:176. doi: 10.1186/1471-2229-13-176

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar

Zador, E., and Jones, D. (1986). The biosynthesis of a novel nicotine alkaloid in the trichomes of Nicotiana stocktonii. Plant Physiol. 82, 479–484. doi: 10.1104/pp.82.2.479

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text | Google Scholar