Short title: Seed-specific saponin biosynthesis in Medicago One-sentence summary: The discovery of a seed-specific regulator of hemolytic saponin biosynthesis in Medicago truncatula led to the identification of the missing P450 of the hemolytic saponin biosynthesis branch.

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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...