Constitutive Steroidal Glycoalkaloid Biosynthesis in Tomato is Regulated by the Clade IIIe Basic Helix-Loop-Helix Transcription Factors MYC1 and MYC2

Abstract

One sentence summary: The clade IIIe basic helix-loop-helix transcription factors MYC1 and 22 MYC2 control the constitutive biosynthesis of tomato steroidal glycoalkaloids and might do so 23 independently of jasmonate signaling.24 Footnotes: 25 ABSTRACT 33 Specialized metabolites are produced by plants to fend off biotic enemies. Across the plant kingdom, 34 the biosynthesis of these defense compounds is promoted by jasmonate signaling in which clade IIIe 35 basic helix-loop-helix (bHLH) transcription factors take on a central role. Tomato (Solanum 36 lycopersicum) produces cholesterol-derived steroidal glycoalkaloids (SGAs) that act as 37 phytoanticipins against a broad variety of herbivores and pathogens. The biosynthesis of SGAs 38 from cholesterol occurs constitutively in tomato plants and can be further stimulated by jasmonates. 39 Here, we demonstrate that the two tomato clade IIIe bHLH transcription factors, MYC1 and 40 MYC2, redundantly and specifically control the constitutive biosynthesis of SGAs. Double myc1 41 myc2 loss-of-function tomato hairy roots displayed suppressed constitutive expression of cholesterol 42 and SGA biosynthesis genes, and consequently severely reduced levels of the main tomato SGAs -43 tomatine and dehydrotomatine. In contrast, basal expression of genes involved in canonical 44 jasmonate signaling or in the biosynthesis of highly jasmonate-inducible phenylpropanoid-45 polyamine conjugates was not affected. Furthermore, CRISPR-Cas9(VQR)-mediated genome 46 editing of a specific cis-regulatory element, targeted by MYC1/2, in the promoter of a cholesterol 47 109 TFs ensure constitutive SGA biosynthesis by redundantly controlling basal expression of both 110 cholesterol and SGA pathway genes. Although MYC-regulated specialized metabolism is 111 typically induced following an elevation in intracellular JA-Ile levels, we found that the 112 constitutive production of SGAs only partially relies on COI1-dependent JA signaling. Finally, 113 6 CRISPR-Cas9(VQR) genome editing of an endogenous G-box, which is targeted by MYC1/2, in 114 the promoter of the cholesterol biosynthesis gene STEROL C-5(6) DESATURASE 2 (C5-SD2) 115 leads to decreased constitutive expression of C5-SD2. This further supports the role of the tomato 116 clade IIIe bHLH TFs in the regulation of constitutive SGA biosynthesis. 117 RESULTS 118 256G-box in planta, we decided to target this cis-regulatory element in tomato hairy roots by genome 257 editing using an engineered version of Cas9 that recognizes 5'-NGA-3' as protospacer adjacent 258 motif (PAM) (Kleinstiver et al., 2015). Three independent hairy root lines, denominated g lines, 259 were generated in which the G-box motif was either deleted or disrupted. Line g #1 contains a 33-260 bp deletion removing the G-box, while g #2 and g #3 carry a thymidine insertion and a 2-bp deletion 261 within the G-box, respectively ( Figure 11A). The 2-bp deletion in line g #3 creates an alternative 262 G-box (5'-CACGTT-3'). The transcript level of C5-SD2 was significantly reduced...

Steroidal alkaloids defence metabolism and plant growth are modulated by the joint action of gibberellin and jasmonate signalling

“…glucosinolates in Arabidopsis; Howe et al, 2018), to the best of our knowledge in all green tissues of the tomato plant. In addition, biosynthesis of these defence metabolites is induced in response to JA-mediated activation of MYC1 and MYC2, suggesting their role in both constitutive as well as JA-dependent defence responses (Swinnen et al, 2020). This resembles the proposed mechanism for the regulation of glucosinolate accumulation in Arabidopsis (Schweizer et al, 2013).…”

Integrated view of plant metabolic defense with particular focus on chewing herbivores

“…While biosynthesized in roots, leaf nicotine levels are controlled by transport mechanisms that involve nicotine uptake permease (NUP1) (Hildreth et al, 2011). Several regulators of steroidal glycoalkaloids were identified in tomato, including AP2/ERF transcription factor GAME9 (Cardenas et al, 2016), bHLH type regulators, MYC1 and MYC2 (Swinnen et al, 2020), and MYB12, which is regulated by stress‐responsive sly‐microRNA1916 (Chen et al, 2019b). The aliphatic and indolic glucosinolate biosynthesis in Arabidopsis is dependent on MYB28‐MYB29‐MYB76 and MYB34‐MYB51‐MYB122 genes (clade subgroup 12), respectively (reviewed in Mitreiter and Gigolashvili, 2021).…”

Bitter and sweet make tomato hard to (b)eat

“…This process involves GAME31, a 2-oxoglutarate-dependent dioxygenase (also known as Sl23DOX) that catalyzes the hydroxylation of atomatine, and the recently identified glycosyltransferase GAME5, which produces esculeoside A (Fig. 1;C ardenas et al, 2019;Nakayasu et al, 2020;Szyma nski et al, 2020).…”

“…The regulation of α‐tomatine metabolism involves the GAME9 gene, a member of the APETALA2/Ethylene Response Factor family, also referred to as JRE4 (Cárdenas et al ., 2016; Thagun et al ., 2016). More recently, additional genes involved in the (positive or negative) regulation of α‐tomatine metabolism have been identified (Wang et al ., 2018; Zhao et al ., 2018; Chen et al ., 2019; Swinnen et al ., 2020; see Table 1).…”