International Journal of Molecular Sciences Article Sweet Basil Has Distinct Synthases for Eugenol Biosynthesis in Glandular Trichomes and Roots with Different Regulatory Mechanisms Vaishnavi Amarr Reddy 1,2, Chunhong Li 1,†, Kumar Nadimuthu 1,†, Jessica Gambino Tjhang 1, In-Cheol Jang 1,2 and Sarojam Rajani 1,* 1 Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore 117604, Singapore; [email protected] (V.A.R.); [email protected] (C.L.); [email protected] (K.N.); [email protected] (J.G.T.); [email protected] (I.-C.J.) 2 Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore * Correspondence: [email protected]; Tel.: +65-68727556 † These authors contributed equally to this work. Abstract: Production of a volatile phenylpropene; eugenol in sweet basil is mostly associated with peltate glandular trichomes (PGTs) found aerially. Currently only one eugenol synthase (EGS), ObEGS1 which belongs to PIP family is identified from sweet basil PGTs. Reports of the presence of eugenol in roots led us to analyse other EGSs in roots. We screened for all the PIP family reductase transcripts from the RNA-Seq data. In vivo functional characterization of all the genes in E. coli showed their ability to produce eugenol and were termed as ObEGS2-8. Among all, ObEGS1 displayed highest expression in PGTs and ObEGS4 in roots. Further, eugenol was produced only in the roots of soil-grown plants, but not in roots of aseptically-grown plants. Interestingly, eugenol production could be induced in roots of aseptically-grown plants under elicitation suggesting that eugenol production might occur as a result of environmental cues in roots. The presence of ObEGS4 transcript and protein in aseptically- Citation: Reddy, V.A.; Li, C.; grown plants indicated towards post-translational modifications (PTMs) of ObEGS4. Bioinformatics Nadimuthu, K.; Tjhang, J.G.; Jang, analysis showed possibility of phosphorylation in ObEGS4 which was further confirmed by in vitro I.-C.; Rajani, S. Sweet Basil Has experiment. Our study reveals the presence of multiple eugenol synthases in sweet basil and provides Distinct Synthases for Eugenol new insights into their diversity and tissue specific regulation. Biosynthesis in Glandular Trichomes and Roots with Different Regulatory Mechanisms. Int. J. Mol. Sci. 2021, 22, Keywords: eugenol synthase; phenylpropene; post-translational modifications; phosphorylation; 681. https://doi.org/10.3390/ijms secondary metabolism; sweet basil 22020681 Received: 4 December 2020 Accepted: 9 January 2021 1. Introduction Published: 12 January 2021 Volatile organic compounds (VOCs) produced by plants as part of their secondary metabolism are critical to various biological processes, which includes defence mechanism, Publisher’s Note: MDPI stays neu- protection from ultraviolet irradiation, chemical signalling, plant-plant interactions and tral with regard to jurisdictional clai- plant-environment interactions [1,2]. VOC emissions from plants can be constitutive or ms in published maps and institutio- induced as a response to abiotic or biotic stresses [3–7]. It has been reported that accumu- nal affiliations. lation of VOCs and expression of pathway genes in plants is organ- or tissue- specific or developmental stage specific [8,9]. In recent times, a lot of progress with regards to eluci- dation of pathways leading to the formation of various plant VOCs has been made but Copyright: © 2021 by the authors. Li- information regarding the regulation of these specialized pathway especially under stress censee MDPI, Basel, Switzerland. remains limited. Emerging research has shown that regulations can happen at a transcrip- This article is an open access article tion level, translational level or post-translational level resulting in cell type specific stress distributed under the terms and con- response. Most of the studies on plant VOCs have focussed on emissions from aerial organs, ditions of the Creative Commons At- but recent research shows that root produced VOCs play important and diverse roles in tribution (CC BY) license (https:// the rhizosphere. Root VOCs can affect microbial activity around it [10], alter behaviour of creativecommons.org/licenses/by/ insects [11,12] and mediate belowground plant to plant communications [13]. 4.0/). Int. J. Mol. Sci. 2021, 22, 681. https://doi.org/10.3390/ijms22020681 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 22 diverse roles in the rhizosphere. Root VOCs can affect microbial activity around it [10], alter behaviour of insects [11,12] and mediate belowground plant to plant communica- tions [13]. Ocimum species produce and store a range of volatile phenylpropenes in specialized organs known as peltate glandular trichomes (PGTs), which are found on the aerial parts of the plant. Predominantly produced phenylpropenes in sweet basil varieties (Ocimum basilicum) are eugenol, chavicol and their methylated derivatives. In addition, they also make few terpenoids which includes eucalyptol, linalool and alpha-bergamotene [14,15] (Figure 1A). The first committed step of phenylpropene biosynthesis is catalysed by an Int. J. Mol. Sci. 2021, 22, 681 2 of 22 acyltransferase, belonging to BAHD (Benzyl alcohol O-acetyltransferase, anthocyanin O- hydroxycinnamoyltransferase, N-hydroxycinnamoyl/benzoyltransferase, deacetylvindo- line 4-OcimumO-acetyltransferase)species produce and family store a rangewhich of volatile acetylates phenylpropenes monolignols, in specialized p-coumaryl and co- niferylorgans knownalcohols as peltate to form glandular p-coumaryl trichomes and (PGTs), coniferyl which are acetates found on respectively. the aerial parts These are acted onof theby plant.phenylpropene Predominantly synthases produced phenylpropenes to produce different in sweet basil phenylpropenes varieties (Ocimum (Supplementary basilicum) are eugenol, chavicol and their methylated derivatives. In addition, they also Figuremake few S1). terpenoids These phenylpropene which includes eucalyptol, synthases linalool can and be alpha-bergamotene allylphenol synthases [14,15] (APS), which produce(Figure1A). chavicol/eugenol The first committed stepor ofpropenyl phenylpropenephenol biosynthesis synthases is catalysed (PPS) bywhich an produce p- anol/isoeugenolacyltransferase, belonging [16]. All to BAHDphenylpropene (Benzyl alcohol syntO-acetyltransferase,hases identified anthocyanin are NADPH-dependentO- ar- omatichydroxycinnamoyltransferase, alcohol reductases Nbelonging-hydroxycinnamoyl/benzoyltransferase, to the PIP family, named deacetylvindo- after the first three identi- line 4-O-acetyltransferase) family which acetylates monolignols, p-coumaryl and coniferyl fiedalcohols members, to form ppinoresinol-lariciresinol-coumaryl and coniferyl acetates redu respectively.ctase (PLR) These [17], are isoflavone acted on by reductase (IFR) [18],phenylpropene and phenylcoumaran synthases to produce benzylic different ether phenylpropenes reductase (Supplementary(PCBER) [17]. Figure S1). TheseIn phenylpropene sweet basil synthasesPGTs, eugenol can be allylphenol is produced synthases from (APS), coniferyl which produce acetate chavi- in a reaction cata- col/eugenol or propenylphenol synthases (PPS) which produce p-anol/isoeugenol [16]. All lysedphenylpropene by a phenylpropene synthases identified synthase are NADPH-dependent named eugenol aromatic synthase alcohol 1 (ObEGS1), reductases which was pre- viouslybelonging identified to the PIP family,from namedthe EST after collections the first three constructed identified members, from pinoresinol-basil glands and petunia flowerslariciresinol [14,19]. reductase The (PLR) substrate, [17], isoflavone coniferyl reductase acetat (IFR)e, is [formed18], and phenylcoumaranfrom coniferyl alcohol by the actionbenzylic of ether BAHD reductase family (PCBER) coniferyl [17]. alcohol acetyltransferase (CAAT). The first functionally In sweet basil PGTs, eugenol is produced from coniferyl acetate in a reaction catalysed characterizedby a phenylpropene CAAT synthase was named PhCFAT eugenol from synthase petu 1nia (ObEGS1), [20]. Recently, which was previouslytwo CAATs, ObCAAT1 andidentified ObCAAT2 from the (PhCFAT EST collections homologue) constructed from were basil identified glands and and petunia characterized flowers [14,19]. from sweet basil involvedThe substrate, in eugenol coniferyl acetate, synthesis is formed [21]. from Apart coniferyl from alcohol ObEGS1, by the action EGSs of BAHD havefam- been characterized fromily coniferyl few other alcohol plants acetyltransferase also such (CAAT). as Petunia The first (PhEGS1) functionally characterized[19], Gymnadenia CAAT odoratissima was PhCFAT from petunia [20]. Recently, two CAATs, ObCAAT1 and ObCAAT2 (PhC- (GoEGS1FAT homologue) and GoEGS2) were identified [22], and Clarkia characterized breweri from (CbEGS1 sweet basil and involved CbEGS2) in eugenol [23], rose (RcEGS1) [24],synthesis strawberry [21]. Apart (FaEGS1a from ObEGS1, and EGSsFaEGS1b) have been [25], characterized and carrot from (DcE(I)GS1) few other plants [26]. In the majority ofalso these such plants, as Petunia EGS (PhEGS1) was shown [19], Gymnadenia to act only odoratissima on coniferyl(GoEGS1 acetate and GoEGS2) to form [22 ],eugenol, but in a Clarkia breweri (CbEGS1 and CbEGS2) [23], rose (RcEGS1) [24], strawberry (FaEGS1a and fewFaEGS1b) plants [25 like], and in carrot Larrea (DcE(I)GS1) tridentata [26]. In(LtCES1), the majority EGS of these could