Contents lists available at ScienceDirect Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy Research article Metabolomics-guided investigations of unintended effects of the expression of the hydroxycinnamoyl quinate hydroxycinnamoyltransferase (hqt1) gene from Cynara cardunculus var. scolymus in Nicotiana tabacum cell cultures S.P. Mudau a , P.A. Steenkamp a , L.A. Piater a , M. De Palma b , M. Tucci b , N.E. Madala a,∗ , I.A. Dubery a,∗∗ a Department of Biochemistry, University of Johannesburg, P.O. Box 524, Auckland Park, 2006, South Africa b CNR – Istituto di Bioscienze e BioRisorse via Università 133, 80055, Portici, Naples, Italy ARTICLE INFO Keywords: Cell culture Chlorogenic acids Benzoic acid Hqt gene Hydroxycinnamoyltransferase Metabolomics profiling Phenylpropanoid pathway ABSTRACT Chlorogenic acids (CGAs) are phenolic compounds biosynthesized in the phenylpropanoid pathway, with hy- droxycinnamoyl quinate hydroxycinnamoyltransferase (HQT) as the key enzyme. Variation of CGAs has been noted in different plants, with globe artichoke (Cynara cardunculus var. scolymus L.) producing high amounts and a diverse spectrum of CGAs in its leaves. In the current study, the effect of overexpression of the hqt1 transgene from globe artichoke in tobacco was evaluated at the metabolome level. Here, metabolomic approaches based on ultra-high performance liquid chromatography coupled to mass spectrometry, together with chemometric models such as principal component analysis and orthogonal partial least square discriminant analysis, were employed to evaluate altered metabolic changes due to hqt1 overexpression. CGA profiles (caffeoylquinic acids: 3-CQA, 4-CQA and 5-CQA; p-coumaroylquinic acids: 4-pCoQA and 5-pCoQA; and 4,5-di-caffeoylquinic acid) of transgenic tobacco cell cultures were detected at lower concentrations than in the wild type. Interestingly, the cells were found to rather accumulate, as an unintended effect, abscisic acid - and benzoic acid derivatives. The results suggest that insertion of hqt1 in tobacco, and overexpression in undifferentiated cells, led to rechannelling of the phenylpropanoid pathway to accumulate benzoic acids. These findings proved to be contrary to the results shown elsewhere in leaf tissues, thus indicating differential metabolic control and regulation in the un- differentiated cell culture system. 1. Introduction Phenolic acids originating from L-phenylalanine (phenylpropa- noids) are members of a widely distributed group of secondary meta- bolites, and contain an aromatic ring with hydroxyl functional group(s) (Le Roy et al., 2016). These compounds include two main classes that can be distinguished based on the structures: benzoic acid (BA) deri- vatives (e.g. hydroxybenzoic acids, HBAs) and cinnamic acid derivatives (e.g. hydroxycinnamic acids, HCAs) (Khadem and Marles, 2010). Chlorogenic acids (CGAs) are ester compounds formed between trans- hydroxycinnamic acids and (-)-quinic acids [1L-1(OH), 3,4/5-tetra- hydroxycyclohexane carboxylic acid] (Clifford et al., 2005; Jaiswal and Kuhnert, 2011; Jaiswal et al., 2014). The most studied CGAs are esters of quinic acids with coumaric-, caffeic- and ferulic acids, resulting in p- coumaroylquinic acids (p-CoQA), caffeoylquinic acids (CQA) and fer- uloylquinic acids (FQA) respectively (Jaiswal et al., 2014). In addition, CGAs occur in various structural forms due to multi-acylation on quinic acid that brings about different structural hierarchies namely mono- acyl, di-acyl, tri-acyl and, rarely, tetra-acyl derivatives (Jaiswal and Kuhnert, 2011). The CGA content varies from one plant to another. For instance, tri-CQA have been reported to have a limited presence in Lactuca sativa var. Crispa L. (Tamura et al., 2006), whilst artichoke has been reported to contain all mono-acyl and di-acyl CGAs (Sonnante et al., 2010). CGAs are naturally-occurring plant defence metabolites that have been identified as resistance bio-markers, herbivore feeding retardants (Jansen et al., 2008) and are also induced when plants undergo bac- terial and fungal infection (Marques and Farah, 2009). Previously we proposed a role for the interconversion of the phytoalexin - and phy- toanticipin - roles of CGAs through storage and conjugation (Mhlongo et al., 2014). High contents of CGAs have been reported in plants such as pears, apples, arnica, artichoke, coffee beans, tobacco, burdock, https://doi.org/10.1016/j.plaphy.2018.04.005 Received 20 February 2018; Received in revised form 3 April 2018; Accepted 3 April 2018 ∗ Corresponding author. ∗∗ Corresponding author. E-mail addresses: emadala@uj.ac.za (N.E. Madala), idubery@uj.ac.za (I.A. Dubery). Plant Physiology and Biochemistry 127 (2018) 287–298 Available online 05 April 2018 0981-9428/ © 2018 Elsevier Masson SAS. All rights reserved. T