# Source paper archive - Source title: CrJAMs are transcriptional repressors of the monoterpenoid indole alkaloid pathway in Catharanthus roseus - Author(s): Breselge, S., Traverse, K. K. F., cole-osborn, l. F., Sathitloetsakun, S., Duerr, K., Wang, J., Childs, K., Cram, E. J., Lee-Parsons, C. W. T. - Source landing URL: https://www.biorxiv.org/content/10.64898/2026.11.02.763677v1 - PDF URL: https://www.biorxiv.org/content/10.64898/2026.11.02.763677v1.full.pdf - PDF SHA-256: ce5825af9c45110a22fb1086d0b09b5b842c989c5369665c1bd22023053e2d10 - Converter: pymupdf4llm 0.0.27 - Limits: Automated PDF text extraction; reading order, equations, tables and formatting may be incomplete or incorrect. Images are omitted, and no OCR was performed. Markup and links are neutralized for safe downloading. Consult the original PDF for the authoritative version. --- [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 CrJAMs are transcriptional repressors of the monoterpenoid indole alkaloid pathway in 2 _Catharanthus roseus_ 3 4 **Author names & affiliations:** Samuel Breselge [1], Krystyna K. F. Traverse [2], Lauren F. Cole5 Osborn [2,3], Suphinya Sathitloetsakun [4], Kristina Dürr [4,5], Jie Wang [6], Kevin L. Childs [6], Erin J. 6 Cram [1], Carolyn W.T. Lee-Parsons [2,3,4] 7 8 9 10 11 SB, KKFT, LFCO are shared first authors 12 1 Northeastern University, Department of Biology, Boston, MA, USA 02115 13 2 Northeastern University, Department of Chemical Engineering, Boston, MA 14 3 Northeastern University, Department of Bioengineering, Boston, MA 15 4 Northeastern University, Department of Chemistry and Chemical Biology, Boston, MA 16 5 Leibniz University, Department of Life Science, Hanover, Germany 17 6 Michigan State University, Department of Plant Biology, East Lansing, MI, USA 18 19 **Corresponding author:** Carolyn Lee-Parsons, ca.lee@northeastern.edu 20 21 22 23 24 25 1 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 26 **Abstract (200 word limit)** 27 _Catharanthus roseus_ produces monoterpenoid indole alkaloids (MIAs) in response to the 28 defense hormone jasmonate (JA). In _Arabidopsis_, JA regulates defense processes through MYC2 29 and JA-Associated MYC2a-like transcription factors (JAMs). Here we characterize two previously 30 identified _C. roseus_ JAMs, CrJAM2 and CrJAM3, as regulators of MIA biosynthesis. 31 Functional screens using viral-induced gene silencing and transient promoter activity 32 assays showed that CrJAMs repress the MIA pathway; silencing both _CrJAM2_ and _CrJAM3_ 33 increased expression of MIA pathway genes and MIA levels while their individual overexpression 34 repressed promoter activity. To assess their activity broadly across MIA regulatory and 35 biosynthetic genes, we generated _C. roseus_ hairy root cultures either constitutively overexpressing 36 individual _CrJAMs_ or _CRISPR-Cas9_ creating mutations in multiple _CrJAMs_, and profiled their 37 expression by RNA-Seq. _CrJAM2_ overexpression decreased expression of most MIA biosynthetic 38 genes relative to the _GUS_ control, while double mutant lines showed increased basal expression 39 of these genes relative to non-targeting controls. Notably, methyl JA treatment perturbs this 40 equilibrium, reversing these trends at 24 hrs in both _CrJAM_ overexpression and mutant lines 41 relative to basal conditions. 42 In summary, CrJAMs broadly repress MIA regulatory and biosynthetic gene expression 43 and together with CrJAZ and CrMYC2a regulates MIA biosynthesis. 44 45 **Key words (5-10):** JA-Associated MYC2a-like (JAMs), bHLH IIId transcription factors, 46 jasmonate-responsive, _Catharanthus roseus_, CRISPR-Cas9, hairy roots 47 48 **Summary sentence (300 characters with spaces limit):** We characterize two previously 49 identified _Catharanthus roseus_ JAMs; CrJAM2 and CrJAM3 regulate overlapping and unique 50 genes and broadly repress the expression of monoterpenoid indole alkaloid regulatory and 51 biosynthetic genes. 52 2 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 53 **1.** **Introduction** 54 _Catharanthus roseus_ produces over 130 different alkaloids (Van der Heijden et al., 2004), 55 including the valuable chemotherapeutics, vinblastine and vincristine (Noble et al., 1958; Aslam 56 et al., 2010; Škubník et al., 2021). Vinblastine and vincristine are produced from the condensation 57 of precursors from the monoterpenoid indole alkaloid (MIA) pathway; this defense-associated 58 pathway is induced by jasmonates (JAs; Aerts et al., 1994), hormones that act in various 59 developmental and stress responses (Wasternack and Strnad, 2018). 60 Many molecular components of the JA signaling pathway are conserved between the model 61 organism _Arabidopsis thaliana_ and other plant species (Thaler et al., 2012). For instance, _A._ 62 _thaliana_ and _C. roseus_ share the early steps of JA perception and regulation, e.g. the role of JAZ 63 (Jasmonate ZIM‐domain) and MYC2 (Zhang et al., 2011; Patra et al., 2018). In addition, a class 64 of JA-induced basic helix-loop-helix (bHLH) repressors in _A thaliana,_ known as JAMs 65 (Jasmonate-associated MYC2-like transcription factors), were identified in _C. roseus_ (Patra et al., 66 2018; Schweizer et al., 2018) and are characterized in this paper. 67 MYCs and JAMs are members of the bHLH transcription factor (TFs) family which play 68 a key role in mediating JA responses. In _A. thaliana_, bHLH subgroup IIIe contains key activators 69 of JA-responses, _AtMYC2_, _AtMYC3_, _AtMYC4_, and _AtMYC5_, while subgroup IIId contains 70 repressors of JA-responses _, AtbHLH17_ ( _AtJAM1_ ), _AtbHLH13_ ( _AtJAM2_ ), _AtbHLH03_ ( _AtJAM3_ ), 71 and _AtbHLH14_ (Heim et al., 2003). In the absence of JAs, JAZ proteins bind and inhibit JA 72 responsive TFs like MYCs and JAMs (see top tier of Figure 1; reviewed in Chini et al., 2016; Li 73 et al., 2024). In the presence of JAs, the SCF [COI1] -JAZ (Skp/Cullin/F-box complex with 74 CORONATINE INSENSITIVE1 and JAZ) coreceptor complex forms, leading to the 75 ubiquitination and degradation of JAZ proteins (Thines et al., 2007). The degradation of JAZ 3 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 76 releases MYCs and JAMs. Released MYC2 binds to G-boxes (CACGTG and its variants) and 77 activates JA-responsive gene expression (Lorenz et al., 2004; Dombrecht et al., 2007; Fonseca et 78 al., 2014); MYC2 also activates the expression of _JAZ_ and _JAM_ (Chini et al., 2007; Sasaki 79 Sekimoto et al., 2013), regenerating JAZ. JAMs also bind to G-boxes and its variants but lack an 80 activator domain; therefore, JAM proteins compete with MYC2 and attenuate JA-responses 81 (Fonseca et al., 2014). For instance, AtMYCs activate while AtJAMs repress the expression of the 82 senescence-associated protein _SAG29_, fine-tuning JA-induced leaf senescence (Qi et al., 2015). 83 The competition of JAMs and MYCs for the same binding site appears to fine-tune defense and 84 developmental responses. 85 In _C. roseus_, additional components of the JA-responsive network regulating MIA 86 biosynthesis have been identified (Figure 1). As in _A. thaliana_, CrJAZ proteins interact and repress 87 CrMYC2a (Patra et al., 2018; Schweizer et al., 2018). Upon CrJAZ degradation, CrMYC2a 88 (particularly the derepressed CrMYC2a [D126N] ) activates MIA pathway gene expression and MIA 89 accumulation (Zhang et al., 2011; Schweizer et al., 2018) by inducing the expression of key MIA 90 pathway activators like CrBIS (bHLH Iridoid Synthesis) and CrORCA (Octadecanoid-Responsive 91 Catharanthus AP2-domain) (see second tier of Figure 1; Van Der Fits et al., 2000; Zhang et al., 92 2011; Van Moerkercke et al., 2015, 2016; Paul et al., 2017; Schweizer et al., 2018; Colinas et al., 93 2021); CrMYC2a, CrBIS, and CrORCA then directly induce the expression of the MIA pathway 94 genes (see third tier of Figure 1; Zhang et al., 2011; Schweizer et al., 2018). The proposed 95 regulation of _CrBIS_ and of the genes in the seco-iridoid pathway by CrMYC2a differ between 96 Patra et al (2018) and Schweizer et al (2018), potentially due to the different tissue context studied. 97 In addition to CrJAZs, other JA-induced repressors of MIA biosynthesis have been characterized 98 (not shown in Figure 1), such as CrZCTs (zinc finger _Catharanthus_ Transcription Factors) and 4 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 99 CrGBFs (G-box Binding Factors) (Sibéril et al., 2001; Pauw et al., 2004; Schweizer et al., 2018). 100 Of these repressors, CrZCTs have been studied most extensively (Pauw et al., 2004; Peebles et al., 101 2009; Goklany et al., 2013; Li et al., 2013; Rizvi et al., 2016; Traverse et al., 2024). 102 Here we characterize the JA-induced repressors, JAMs, in _C. roseus_ . We hypothesize that 103 CrJAMs attenuate JA-responses similarly as in _A. thaliana_ . Using the _A. thaliana_ JAM amino acid 104 sequences, we identified only the previously reported three bHLH IIId subgroup members: 105 CrJAM2, CrJAM3 (Schweizer et al., 2018), and Repressor of MYC2 Targets 1 (RMT1; Patra et 106 al., 2018). Overexpression of _CrRMT1_ represses CrMYC2 targets ( _CrMYC2_, _CrORCA3_, and 107 _CrSTR1_ ) and reduces MIAs (catharanthine, tabersonine, and ajmalicine) in hairy roots (Patra et 108 al., 2018). 109 Here we focus on characterizing _CrJAM2_ and _CrJAM3_ . In an initial screen, virus-induced 110 gene silencing (VIGS) of _CrJAM2_ and _CrJAM3_ together increased the expression of several MIA 111 activators and pathway genes and increased specific MIAs. In addition, we demonstrated that 112 CrJAM2 and CrJAM3 significantly repress the transcriptional promoter activity of several MIA 113 activators and pathway genes in transiently transformed _C. roseus_ seedlings. To evaluate the 114 regulation of the entire MIA biosynthetic pathway, we developed hairy root cultures that 115 overexpress either the individual _CrJAMs_ or _CRISPR-Cas9_ to create double or triple mutants and 116 conducted a global RNA-Seq. These results show that JAMs broadly act as transcriptional 117 repressors of the MIA regulatory and biosynthetic genes in _C. roseus_ . 118 **2.** **Methods** 119 **Accession numbers** 120 _A. thaliana_ _bHLH_ _IIId_ genes: _AtbHLH03/AtJAM3_ ( _Arabidopsis_ Genome Initiative number: 121 AT4G16430), _AtbHLH13/AtJAM2_ (AT1G01260), _AtbHLH14_ (AT4G00870), _AtbHLH17/AtJAM1_ 5 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 122 (AT2G46510). _C. roseus_ _bHLH_ _IIId_ genes: _CrJAM2_ (GenBank: MG676662), _CrJAM3_ (GenBank: 123 MG676663), _CrRMT1_ (GenBank: KY851107). _C. roseus_ MIA pathway enzymes and TFs used to 124 identify their promoters and 5’UTRs: _CrBIS1_ (GenBank: KM409646), _CrBIS2_ (GenBank: 125 KM409645), _CrG8O_ (also referred to as _CrG10H_, GenBank: AJ251269), _CrGO_ (GenBank: 126 MF770508), _CrLAMT_ (GenBank: EU057974), _CrMYC2a_ (GenBank: AF283507), _CrORCA3_ 127 (GenBank: AJ251249), _CrSTR1_ (GenBank: Y10182), _CrT16H1_ (GenBank: FJ647194), _CrT16H2_ 128 (GenBank: JF742645), _CrT19H_ (GenBank: HQ901597), _CrZCT1_ (GenBank: AJ632082). 129 **Identification of** _**C. roseus**_ **bHLH proteins** 130 To identify bHLH proteins in _C. roseus_, the bHLH domains of the following proteins were 131 used in a BlastP search against the _C. roseus_ genome version 3 (Li et al., 2023): CRO_07G000280 132 (CrMYC2a), CRO_08G020500 (CrBIS1), CRO_07G004820 (CrMYC1), CRO_04G030430 (a 133 bHLH DNA-binding superfamily protein), CRO_05G017550 (a bHLH DNA-binding superfamily 134 protein), AT1G18400.1 (AtBEE1), AT1G68240.1, AT2G46510.1 (AtJAM1), AT4G00480.1 135 (AtMYC1), AT1G32640.1 (AtMYC2), and AT1G09530.1 (AtPIF3). Hits with E-values ≤0.05 136 were included for further consideration. For each hit, the longest isoform sequence was used to 137 generate a list of candidate proteins. Proteins were scanned using the Pfam-A v37.3 database and 138 the HMMER3 package version 3.4 with domain specific E-value cutoffs in CLC Main Workbench 139 8.1.3 (QIAGEN). Hits were further analyzed for the presence of bHLH domains using several 140 databases through InterPro (Mitchell et al., 2019) and the domain annotated using PROSITE 141 (Sigrist et al., 2013) and aligned to identify the 5-9-13 motif (Heim et al., 2003; Toledo-Ortiz et 142 al., 2003). 6 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 143 **Identification of** _**C. roseus**_ **bHLH IIId proteins** 144 Amino acid sequences of the four _A. thaliana_ bHLH IIId proteins (AtbHLH17, AtbHLH13, 145 AtbHLH3, and AtbHLH14) were used in a BlastP search with the _C. roseus_ genome v3 (Li et al., 146 2023) in CLC Main Workbench. The best hits were used for a NCBI Blast search to identify if the 147 sequences were reported previously. 148 **Construction of phylogenetic trees** 149 Amino acid sequences were aligned in CLC Main Workbench (gap open cost = 10; gap 150 extension cost = 1; end gap cost = as any other; alignment mode = very accurate). Identified 151 domains were aligned as described above and manually curated. The amino acid alignment was 152 used to create a maximum likelihood phylogenetic tree (Neighbor Joining; WAG; 100 replicates 153 for full-length proteins and 1000 replicates for domains) in CLC Main Workbench. 154 **Hierarchical clustering of** _**C. roseus**_ **RNA-Seq data** 155 RNA-Seq reads were downloaded from NCBI-SRA databases (BioProject: 156 PRJNA252611) and processed to obtain sequencing reads in FASTQ format. Reads were 157 processed to trim the sequencing adapter sequences and read positions with low sequencing quality 158 using Trimmomatics version 0.36 (Bolger et al., 2014). Clean reads were aligned to the _C. roseus_ 159 reference genome v2 using STAR version 2.5.2b (Dobin et al., 2013). Aligned sequences were 160 counted using the featureCount version 1.22 package (Liao et al., 2014) in R to get number of 161 reads aligned to a gene feature. Read counts data were normalized to get gene expression values 162 in FPKM (fragments per kilo base million). Heatmap was generated using gplots package (Warnes 163 et al., 2009) in R. 164 **Cloning of VIGS vectors** 7 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 165 Fragments (400 bp) targeting _CrJAM2_ or _CrJAM3_ were designed using the Sol Genomics 166 Network VIGS tool (Fernandez-Pozo et al., 2015) (n-mer = 23, mismatches = 0), which minimized 167 off-targets in the _C. roseus_ version 2 transcriptome (Franke et al., 2019). Fragments were amplified 168 from previously cloned plasmids using Phusion High-Fidelity DNA Polymerase (New England 169 Biolabs) with primers listed in Table S 3. Fragments were inserted, either individually or together, 170 into pTRV2-GG (Addgene plasmid #105349) using modular cloning and confirmed with Sanger 171 Sequencing. Final plasmids were confirmed with a restriction enzyme digest and then 172 electroporated into _Agrobacterium tumefaciens_ GV3101 (pMP90). Plasmids targeting _CHLH_ and 173 _GFP_ for silencing were cloned previously (Cole-Osborn et al., 2024a). 174 **Virus-induced gene silencing (VIGS)** 175 _C. roseus_ Little Bright Eye (NESeeds) were sterilized, germinated, and finally cultivated 176 in soil under the 16 hr light / 8 hr dark photoperiod until two true leaves appeared as described 177 previously (Cole-Osborn et al., 2024a). Once seedlings had two true leaves, they were infected 178 with _A. tumefaciens_ according to the pinch-wounding method (Liscombe and O’Connor, 2011). 179 A single colony of _A. tumefaciens_ GV3101 (pMP90) harboring pTRV1 or pTRV2 was used 180 to inoculate LB with the appropriate antibiotics, cultivated, and prepared as described in Cole 181 Osborn et al (2024a). _A. tumefaciens_ strains containing pTRV1 and pTRV2 plasmids were then 182 combined in a 1:1 ratio (OD 600 of each strain = 2-4). Modified tweezers were dipped into the _A._ 183 _tumefaciens_ solution and the plant was pinched three times in the highest internode beneath the 184 shoot apical meristem. 185 After infection, plants were cultivated in the dark for two days before being placed back 186 into a 16 hour light / 8 hour dark photoperiod as described previously (Cole-Osborn et al., 2024a). 187 Plants were grown until two pairs of leaves emerged after silencing and the _CHLH_ -silenced plant 8 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 188 exhibited yellow leaves. At this point, the two youngest leaf pairs were harvested for RNA and 189 alkaloid extraction. 190 **RNA extraction** 191 Briefly, RNA was extracted from liquid nitrogen flash-frozen leaves or hairy root tissue; 192 tissue was crushed by shaking in the Mini-BeadBeater-16, extracted with RNAzol-RT (Molecular 193 Research Center) and the Direct-zol RNA Miniprep Plus Kit (Zymo Research) with on-column 194 DNAse treatment to remove genomic DNA as described previously (Cole-Osborn et al., 2024a). 195 For RNA-Seq experiments, an additional DNAse treatment was performed using the TURBO 196 DNA-freeTM kit (Invitrogen), according to manufacturer’s instructions. RNA integrity was 197 assessed using agarose gel electrophoresis, and concentration and purity were quantified with a 198 NanoDrop (ND-1000 Spectrophotometer; ThermoScientific). 199 **Monitoring gene expression** 200 cDNA was synthesized from extracted RNA using either the SuperScript II First-Strand 201 Synthesis System (Invitrogen) or the LunaScript RT SuperMix Kit (New England Biolabs) with 202 up to 2.5 µg of RNA, according to manufacturer’s instructions. cDNA was diluted 1:4, and 1 µL 203 was used in a 10 µL reaction with SYBR Green ROX qPCR Master Mix (Qiagen or ABClonal) 204 and 300 nM primers. Amplification efficiency for each primer set was confirmed using Ct values 205 over a range of cDNA dilutions and was 100% ± 10% for each gene monitored. If primers did not 206 show 100% efficiency, the concentrations of the forward and reverse primers were varied until 207 100% efficiency was achieved. Optimal concentrations of primers that differed from the standard 208 300 nM for both forward and reverse primers were as follows: JAM2q_set3F = 600 nM; 209 JAM2q_set3R = 600 nM; JAM3q_set3F = 600 nM; JAM3q_set3R = 600 nM; Orca3-forward = 9 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 210 300 nM; Orca3-reverse = 600 nM. Specificity of each primer set was confirmed by gel 211 electrophoresis and sequencing. 212 Reactions were run on the MX3000P (Agilent) or CFX96 (Bio-Rad) qPCR instrument 213 using the thermocycler protocol as described previously (Cole-Osborn et al., 2024a). Transcript 214 levels were normalized to the housekeeping gene, SAND (Pollier et al., 2014), and fold changes 215 relative to the negative control condition were calculated according to the 2-∆∆Ct method (Livak 216 and Schmittgen, 2001). SAND Ct values in no reverse-transcriptase controls were confirmed to be 217 at least 5 Ct values above the respective experimental sample, indicating minimal genomic DNA 218 contamination (Svec et al., 2015). 219 **Alkaloid extraction from leaf tissue** 220 At the time of harvest, individual leaves were weighed to determine their fresh weight, 221 flash-frozen in liquid nitrogen, crushed using a Mini-BeadBeater-16 (Biospec), and extracted in 222 methanol, as described previously (Cole-Osborn et al., 2024a). Ajmaline (50 ng) per mg tissue 223 served as an internal standard prior to extraction. After extraction, concentration, and 224 centrifugation steps, the final extract was diluted 1:50 in 50% methanol/50% water. 225 **Quantification of alkaloid content in leaf tissue with HPLC-MS/MS** 226 Alkaloid quantification was performed at the Mass Spectrometry Facility at Northeastern 227 University, as described previously (Cole-Osborn et al., 2024a). Briefly, alkaloids were separated 228 using the Thermo Scientific [TM] Vanquish HPLC system with a Phenomenex Luna Omega LC 229 column and detected on a Tandem HRMS orbitrap mass analyzer (Thermo Scientific Exploris 240) 230 coupled to an electrospray ionization (H-ESI) source in the positive mode. Quantitative analysis 231 was performed in the full scan MS with data-dependent tandem mass spectrometry. For 10 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 232 normalization, peak areas of each compound were divided by the peak area of ajmaline in each 233 sample. 234 **Vector construction** 235 All vectors (except pSB310-312) were constructed using the Golden Gate system, MoClo 236 (Weber et al., 2011). Vectors and parts were used from Weber et al., 2011, Engler et al., 2014, and 237 Mortensen et al., 2019. All sequences from _C. roseus_ (Little Bright Eye) gDNA or cDNA were 238 amplified with Phusion High-Fidelity DNA Polymerase. All new promoters and coding sequences 239 were confirmed by Sanger Sequencing. Correct assembly of transcriptional units (L1 vectors) and 240 expression vectors (L2 vectors) were confirmed by partial sequencing, colony PCR, and/or 241 restriction digest. All final vectors were assembled in the pSB90 vector backbone (Mortensen et 242 al., 2019) containing a constitutively active _VirG_ gene ( _VirGN54D_ ) for increased _Agrobacterium_ 243 virulence (Pazour et al., 1992) and _KanR_ as bacterial selection marker. Vector cartoons for the 244 final vectors are given in Figure S 6. 245 **Cloning of** _**CrJAMs**_ **and MIA pathway promoters** 246 _CrJAM2_ and _CrJAM3_ coding sequences were amplified as fragments to remove and 247 replace type IIS restriction enzyme recognition sites ( _BpiI, Eco31I, Esp3I_ ) with synonymous 248 codons by mutating an A to T or a T to A within the recognition site. Fragments were cloned into 249 pAGM1311 (Weber et al., 2011) before combining in pICH41308 (Weber et al., 2011). 250 Oligonucleotides used in cloning are given in Table S 3 and coding sequences of _CrJAM2_ and 251 _CrJAM3_ are given in Table S 6. 252 The promoters and 5’UTRs of several MIA pathway transcription factors and pathway 253 enzymes (including the MEP-secoiridoid pathway) were used to test if CrJAM2 and CrJAM3 can 254 repress their activity. Promoter and 5’UTR sequences of the genes of interest were identified with 11 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 255 Blast using the coding sequences (see section ‘Accession numbers’) against the _C. roseus_ genome 256 v2 (Franke et al., 2019; v3 was not available when constructs were cloned) in CLC Main 257 Workbench. Up to 2 kb upstream of the translational start site was amplified from _C. roseus_ Little 258 Bright Eye gDNA and cloned into pICH41295 (Weber et al., 2011). _BpiI_ or _Eco31I_ sites were 259 mutated as described above. In the process no E-boxes (CANNTG) were created or removed. 260 Primers can be found in Table S 3 and promoter sequences are given in Table S 6. 261 There are two different gene locations with 100% exon identity for _CrGO_ in the genome 262 of the _C. roseus_ Sunstorm Apricot variety (cro_v2_scaffold_43, Franke et al _._, 2019) but their 5’ 263 sequences differ. The 5’ region from 324.327 to 325.969 in cro_v2_scaffold_43 was chosen to 264 represent the _GO_ promoter. The amplified _CrT16H2_ 5’ sequence from _C. roseus_ Little Bright Eye 265 variety gDNA contained a 653 bp deletion compared to the expected sequence in the genome of 266 the _C. roseus_ Sunstorm Apricot variety (Franke et al., 2019). 267 **Promoter activity assays in** _**C. roseus**_ **seedlings** 268 To test their ability to repress the above-mentioned _C. roseus_ promoters, _CrJAM2_ and 269 _CrJAM3_ were transiently overexpressed in _C. roseus_ seedlings using the efficient _Agrobacterium_ 270 mediated seedling infiltration (EASI) method (Mortensen et al., 2019, 2022). Briefly, 10-day old 271 _C. roseus_ seedlings were vacuum-infiltrated with two strains of _A. tumefaciens_ GV3101(pMP90). 272 The first strain contained the effector (at OD 600 : 0.34), either _CaMV2x35S::GUS_ (pSB161, serving 273 as the control), _CaMV2x35S::CrJAM2_ (pSB270), or _CaMV2x35S::CrJAM3_ (pSB271). The second 274 strain contained the promoter plus 5’UTR of MIA TF or enzyme driving the _FLUC_ reporter (at 275 OD 600 : 0.06) and the constitutive _AtNos-_ driven _RLUC_ reporter for normalizing transformation 276 efficiency _._ Cartoons of vectors are given in Figure S 6. 12 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 277 Transformed seedlings were harvested 3 days post-infection and luciferase assays 278 performed using the Luc-Pair [TM] Duo-Luciferase HT Assay Kit (Genecopeia) as described in 279 Mortensen et al. (2019). For the promoter of the gene of interest, the relative transcriptional 280 promoter activity was calculated by dividing the ratio of FLUC (firefly luciferase) to RLUC 281 ( _Renilla_ luciferase) luminescence of the effector condition ( _JAM2_ or _JAM3_ overexpression) by the 282 ratio of FLUC to RLUC luminescence of the _GUS_ control. The experiment was carried out in three 283 independent assays with 10 biological replicates per assay and each biological replicate or data 284 point consisting of 2 seedlings (Figure 4, Figure S 7). 285 **Guide design and cloning for CRISPR-Cas9 deletion of** _**CrJAM2**_ **,** _**CrJAM3**_ **, and** _**CrRMT1**_ 286 Guide sequences targeting genomic regions of _CrJAM2_, _CrJAM3_, and _CrRMT1_ were 287 designed using Benchling.com and on-target scores were predicted (Doench et al., 2016). Guides 288 with high on-target scores (>50) and low chances of off-targets (16/20 or less identity to closest 289 other Blast hit in _C. roseus_ genome v2) were chosen (Table S 5). Guides were cloned into the 290 improved pDGE vector system. The pDGE vectors contain improvements in the sgRNA scaffolds 291 and in the _Cas9_ sequence and their demonstrated improvements are described in (Grützner et al., 292 2021). 293 **Development and maintenance of stable transgenic hairy roots** 294 Transgenic hairy roots were developed and maintained as described previously (Rizvi et 295 al., 2015; Traverse et al., 2022). For the overexpression lines _,_ the _Agrobacterium_ _rhizogenes_ 296 R1000 contained one of the following constructs: _CaMV2x35S::CrJAM2_ (pSB297), 297 _CaMV2x35S::CrJAM3_ (pSB298), or _CaMV2x35S::GUS_ (control line, pSB299, Figure S 6). For 298 deletion lines _, A. rhizogenes_ R1000 contained one of the following constructs: CRISPR-Cas9 13 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 299 vectors with sgRNAs targeting 1) _CrJAM2_ and _CrJAM3_ (pSB311), 2) _CrJAM2_, _CrJAM3,_ and 300 _CrRMT1_ (pSB312), or 3) the control without sgRNAs (pSB310, Figure S 6). 301 **Confirmation of transgenic hairy root lines** 302 Hairy root tissue was harvested and extracted for genomic DNA (GeneJET Plant Genomic 303 DNA Purification Mini Kit; Thermo Scientific) and genes confirmed by PCR as described 304 previously (Rizvi et al., 2015; Traverse et al., 2022). In addition, the presence of _GUS_ (control 305 line, pSB299), _CrJAM2_ (pSB297), or _CrJAM3_ (pSB298) in the overexpression lines was 306 confirmed using primers shown in Table S 3. 307 For the deletion lines, the amplification of the sgRNA region or the Cas9 and promoter 308 region in the control lines lacking guides was also confirmed using primers shown in Table S 4; to 309 evaluate the edits, the genomic regions of _CrJAM2_, _CrJAM3_, and _CrRMT1_ from the different hairy 310 root lines were amplified from gDNA using primers in Table S 4 and gel-purified PCR products 311 were sent for Sanger sequencing. PCR products with different mutations were cloned into 312 pAGM1311 (Weber et al., 2011) and individual clones were sent for sequencing to distinguish 313 different allelic mutations. 314 **Illumina RNA-Seq** 315 Total RNA samples were submitted to the MSU Genomics Core for Illumina sequencing. 316 Samples were checked for RNA integrity using an Agilent 2100 Bioanalyzer, and all samples had 317 RIN scores better than 7.0. Illumina TruSeq Stranded mRNA libraries were prepared following 318 the manufacturer's protocol. Libraries were checked using Qubit dsDNA High Sensitivity and 319 Agilent 4200 TapeStation High Sensitivity DNA1000 assays. The libraries were combined on an 320 equimolar basis into multiple pools, which were quantified using a Kapa Biosystems Illumina 321 Library Quantification qPCR kit. Library pools were each sequenced on one lane of an Illumina 14 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 322 HiSeq 4000 flow cell using single end 50 bp chemistry. Demultiplexed FastQ files were prepared 323 for each RNA sample. A minimum of 30 million reads was generated for each sample. FastQ files 324 have been deposited in NCBI Sequence Read Archive (BioProject: PRJNA1001907). 325 **Analysis of differentially expressed genes in transgenic hairy root lines** 326 Reads were prepared, aligned to the _C. roseus_ genome, and differentially expressed genes 327 were identified. All reads were trimmed of adapters and low-quality bases with Trimmomatic 328 version 0.36 (Bolger et al., 2014). The _C. roseus_ v2 genome and annotation files were downloaded 329 from Dryad (https://doi.org/10.5061/dryad.08vv50n; Jakob et al 2018), and cleaned RNA-seq 330 reads were aligned to the genome assembly using STAR version 2.5.2b (Dobin et al 2013). Only 331 uniquely mapping reads were retained. The v2 _C. roseus_ gene annotation was supplemented with 332 missing ZCT1 and ZCT4 gene models. The featureCounts function from the Rsubread version 3 333 R package was used to assign read counts to all genes (Liao et al 2013). Read counts were 334 normalized using the TMM function from the edgeR package (Robinson et al 2010), and genes 335 with fewer than 5 counts in five or more samples were discarded. Count data were transformed 336 with the voom function from the limma R package (Law et al 2014). A linear model (genotype + 337 time + genotype:time) was used to fit the data with the lmfit function from limma, and genes with 338 _p_ -values less than 0.01 were identified as differentially expressed. Significantly differentially 339 expressed genes were then filtered for those genes with log 2 -fold change greater than 2. 340 **Statistical analysis of RNA-Seq data** 341 Fold change in gene expression of the two highest _CrJAM2_ and two highest _CrJAM3_ 342 overexpressing lines were calculated from their individual RPKM divided by the average RPKM 343 of two _GUS_ -overexpressing control lines for each time point and then transformed with log 2 . Fold 344 change in gene expression of two individual _jam2jam3_ and two individual _jam2jam3rmt1_ -mutant 15 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 345 lines were calculated from their individual RPKM divided by the average RPKM of four no-guide 346 control lines for each time point and then transformed with log 2 . Fold changes were unable to be 347 calculated for genes that were not expressed in the control condition. Heatmaps were constructed 348 using GraphPad Prism. 349 **Results** 350 _**C. roseus**_ **genome encodes for three bHLH IIId JAM-like transcription factors** 351 Shown in Figure S 1, we identified 105 bHLH proteins and three bHLH IIId TFs in the _C._ 352 _roseus_ genome version 3 (Li et al., 2023) as compared to 164 bHLH proteins and four bHLH IIId 353 TFs in _A. thaliana_ (Bailey et al., 2003; Heim et al., 2003; Hao et al., 2021; Qian et al., 2021). 354 Together, the phylogenetic tree of all bHLH TFs from _C. roseus_ (Figure S 1) and the BlastP results 355 (Table S 1) suggest that the bHLH IIId subgroup consists of just three TFs in _C. roseus,_ which 356 were previously identified by Patra et al. (2018) and Schweizer et al. (2018): CrJAM2, CrJAM3, 357 and CrRMT1. 358 We compared the bHLH domain of CrJAMs and CrMYC2a for their DNA recognition 359 sequences (Figure 2). The characteristic bHLH domain consists of a N-terminal conserved 360 sequence of basic amino acids, followed by a conserved amphipathic a-Helix, a Loop of variable 361 length (up to 14 amino acids and average of 6 amino acids), and a conserved second amphipathic 362 a-Helix (Heim et al., 2003). The first a-helix domain, specifically the amino acid positions 5-9 363 13, influences the recognition of target DNA sequences by these TFs (Heim et al., 2003; Toledo 364 Ortiz et al., 2003); CrJAMs, CrMYCs, and CrBIS all contain the H-E-R amino acids in the 5-9-13 365 motif within the first a-helix domain (Figure S 1). Therefore, they are expected to bind to the same 366 DNA sequence (G-box: CACGTG; Heim et al., 2003). 16 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 367 The domain responsible for the repressive activity of JAMs has not been clearly identified 368 in the literature. In _Arabidopsis_, the homologs AtJAM2 and AtJAM3 lack an activation domain 369 found in AtMYC2 and therefore are assumed to be passive repressors, competing for the same 370 binding site with AtMYC2 (Fonseca et al., 2014). In contrast, Huang et al (2018) showed that the 371 conserved N-terminal region of AtJAM2 is necessary for repression while the C-terminal region, 372 which includes the bHLH domain, is insufficient for repression. Therefore, functional assays are 373 needed to confirm that the candidate TF is a repressor. 374 The three bHLH IIId TFs cluster closely with the bHLH IIIe TFs, CrMYC2a and CrMYC2c 375 (Figure S 1). CrRMT1 (Patra et al., 2018) and CrMYC2b (Schweizer et al., 2018) share ~98% 376 nucleotide (1311/1332 bp) and amino acid (434/443 aa) identity and the genes map to the same 377 locus in the _C. roseus_ v3 genome, suggesting that CrRMT1 and CrMYC2b refer to the same gene. 378 The reported differences in the gene sequences may be due to single nucleotide polymorphisms 379 between different _C. roseus_ varieties. Of these three bHLH IIId TFs in _C. roseus,_ CrRMT1 was 380 previously characterized and shown to repress CrMYC2 targets (Patra et al., 2018). CrJAM2 (1812 381 bp) and CrJAM3 (1473 bp) were only tested previously for their ability to regulate and repress the 382 _CrBIS1_ promoter (Schweizer et al., 2018). Since CrRMT1 has been studied in detail (Patra et al., 383 2018), we mainly focused on characterizing CrJAM2 and CrJAM3 in this paper. 384 Previously published transcriptome data supported the JA-responsiveness of _CrJAM2_ and 385 _CrJAM3,_ particularly in cell suspensions and/or sterile seedlings (Góngora-Castillo et al., 2012) 386 (Figure S 2). Co-expression analysis of _CrJAM2_ and _CrJAM3_ with known MIA pathway enzymes 387 and TFs showed that _CrJAM3_ and _CrMYC2a_ shared the most similar expression, while _CrJAM2_ 388 and _CrRMT1_ shared the most similar expression (Figure S 3). Since a repression domain in JAMs 389 has not been clearly identified, functional assays are necessary to clearly distinguish between the 17 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 390 MYC2 and JAM proteins, which act as activators and repressors, respectively. CrMYC2a activity 391 was previously characterized by Zhang et al. (2011) and Schweizer et al. (2018). 392 **Silencing** _**CrJAM2**_ **and** _**CrJAM3**_ **generally increased MIA pathway gene expression and** 393 **MIA production** 394 We silenced _CrJAM2_ and _CrJAM3_ in _C. roseus_ leaves using virus-induced gene silencing 395 (VIGS) and analyzed its effect on gene expression and MIA production. To ensure sufficient 396 silencing of both _CrJAM2_ and _CrJAM3_, we first tested the efficacy of silencing two genes at once 397 and the order of the silencing fragments on overall silencing. Targeting both _CrJAM2_ and _CrJAM3_ 398 in the same construct was just as efficient as targeting either _CrJAM2_ or _CrJAM3_ alone and the 399 order of the silencing fragment when targeting both _CrJAM_ genes also did not matter; all silencing 400 constructs led to ~50% silencing of the targeted gene(s) (Figure S 4). 401 Using the _JAM3/2_ construct, significant silencing of _CrJAM2_ and _CrJAM3_ (by 36 and 62%, 402 respectively) was confirmed in the second leaf pair to emerge after infection (immature leaf, Figure 403 3A), but not in the first leaf pair (mature leaf, not shown); thus, we only proceeded with analyzing 404 the gene expression and the MIA content of the immature leaves. 405 We observed ~2-fold or higher increases in _BIS1, BIS2, TDC,_ and _T16H1_ when _CrJAM2_ 406 and _CrJAM3_ were both silenced (Figure 3B), supporting the role of CrJAMs as repressors of the 407 MIA pathway. We could not monitor _ORCA3_ as its expression was too low and variable to 408 accurately quantify. We also observed non-significant increases in specific MIAs when both 409 _CrJAM2_ and _CrJAM3_ were silenced in the immature leaves (33% increase in vindoline, 86% 410 increase in ajmalicine, 51% increase in serpentine, no changes in catharanthine, Figure 3C). Given 411 the observed increase in vindoline, we checked the expression of the other vindoline pathway 412 genes. Several vindoline pathway genes increased by 40 - 60% while two genes decreased with 413 _JAM3/2_ silencing (Figure S 5). 18 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 414 The transient nature of VIGS yields partial silencing, leading to non-significant changes in 415 gene expression and metabolite levels. However, our findings are consistent with the potential role 416 of CrJAMs as repressors of MIA biosynthesis. 417 **CrJAM2 and CrJAM3 repressed the transcriptional promoter activity of several MIA** 418 **pathway TFs and genes in transiently transformed** _**C. roseus**_ **seedlings** 419 In validating CrJAM2 and CrJAM3 as repressors of the MIA pathway, we cloned five 420 promoters from MIA pathway regulators ( _MYC2a_, _BIS1_, _BIS2_, _ORCA3_, and _ZCT1_ ) and seven 421 promoters from MIA pathway enzymes ( _G8O_ or _G10H_, _LAMT_, _STR1_, _GO_, _T16H1_, _T16H2,_ and 422 the root-specific _T19H_ ) to drive the expression of the _FLUC_ reporter gene (Table S 2; Table S 6; 423 Figure S 6, Figure S 7). Either _CrJAM2_ or _CrJAM3_ was transiently co-expressed with these 424 promoter-reporter constructs in _C. roseus_ seedlings using the Efficient _Agrobacterium-_ mediated 425 Seedling Infiltration (EASI) method (Mortensen et al., 2019, 2022; Cole-Osborn et al., 2024b). If 426 CrJAM repressed these promoters (directly or indirectly), luminescence would decrease relative 427 to that of the overexpressed negative effector control, _GUS_ . 428 The overexpression of either _CrJAM2_ or _CrJAM3_ significantly reduced the promoter 429 activity of activators ( _pBIS1, pBIS2,_ and _pORCA3_ ) and MIA pathway enzymes ( _pG8O_, _pGO_, and 430 _pT16H1_ ) (Figure 4A). These results complement the known regulation of the genes by 431 MYC2a [D126N], BIS1/2, and ORCA3 (Figure 4b; Schweizer et al., 2018; Traverse et al., 2024). Since 432 CrJAM, CrMYC2a, and CrBIS all bind to the same DNA sequence, promoters activated by 433 CrMYC2a or CrMYC2a [D126N] ( _pBIS1, pBIS2,_ _pORCA3, pZCT1, pG8O, pLAMT, pSTR1, pGO,_ and 434 _pT19H_ ) and by CrBIS ( _pBIS2,_ _pORCA3,_ and _pG8O_ ) would also be expected to be repressed by 435 CrJAMs; our results differed in that we did not observe a significant repression of _pZCT1, pLAMT,_ 436 _pSTR1,_ and _pT19H_, observed repression of _pT16H1_, and observed unexpected activation of 437 _pT16H2_ . These differences might be attributed to the evaluation methods used (promoter driving 19 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 438 reporter versus quantitative gene expression) and the tissue contexts used (seedlings versus 439 flowers), resulting in difference in basal expression levels and activator levels that compete with 440 the CrJAM2 and CrJAM3. 441 Our transient assay showed that CrJAM2 and CrJAM3 significantly repressed the promoter 442 activity of key activator TFs ( _BIS1, BIS2,_ and _ORCA3_ ) and several MIA pathway enzymes ( _G8O_, 443 _GO_, and _T16H1_ ), supporting their potential role as repressors of the MIA pathway. 444 **Overexpression of** _**CrJAM2**_ **or** _**CrJAM3**_ **in hairy root lines repressed MIA pathway gene** 445 **expression** 446 To evaluate the regulation of the entire MIA biosynthetic pathway, we generated hairy root 447 lines that constitutively overexpressed either _GUS_ (negative effector control), _CrJAM2_, or 448 _CrJAM3_ under the control of the strong _CaMV 2x35S_ promoter and monitored the effect of their 449 overexpression by RNA-Seq. Multiple independent lines were evaluated. 450 _CrJAM2_ and _CrJAM3_ overexpression lines resulted in up to a 23-fold and 17-fold increase 451 of _CrJAM2_ and _CrJAM3_ expression, respectively (Figure S 8). Interestingly, the constitutive 452 overexpression of _CrJAM2_ led to an average reduction of _CrJAM3_ expression by 27% while the 453 overexpression of _CrJAM3_ led to an average reduction of _CrJAM2_ expression by 38%. Since 454 MYC2a activates _JAM_ expression and since MYC2a and JAMs bind to the same promoter target, 455 the overexpression of one _JAM_ would be expected to repress the expression of the other _JAM_ . 456 Feedback regulation of MIA TFs (like _CrJAMs_ ) was also observed with the overexpression of 457 _CrMYC2a_ (Patra et al., 2018), _CrBIS_ (Schweizer et al., 2018), or _CrORCA_ (Schweizer et al., 2018). 458 Next, the gene expression profile of the two strongest expressing _CrJAM2_ 459 (rSB02_297_07_2, rSB02_297_09_1) and the two strongest expressing _CrJAM3_ 460 (rSB02_298_32_1, rSB02_298_33_1) hairy root lines from Figure S 8 were assessed and are 461 presented as a heat map in Figure 5. Root lines were elicited with methyl JA (250 µM MJ) and 20 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 462 sampled at basal conditions (0 hr), 0.5 hr, and 24 hrs. The 0.5 hr time point after MJ addition 463 monitors expression resulting from pre-existing TFs like MYC2a and JAMs released upon the 464 degradation of the JAZ repressor, while the 24 hr time point after MJ addition monitors the 465 expression from newly transcribed TFs (Goklany et al., 2013; Rizvi et al., 2016). 466 In general, the constitutive overexpression of _CrJAM2_ repressed the expression of most 467 MIA pathway genes relative to the negative control lines at either 0 or 0.5 hr; specifically, upstream 468 MIA transporter and pathway genes ( _NPF2.4_, _TDC, SGD, HYS_ ) _,_ downstream MIA pathway genes 469 ( _CS/HL1, VNS_ ), and the root-associated pathway genes ( _V19H, MAT, TEX2, T19H, TAT_ ) were 470 strongly repressed by >75% in both _CrJAM2_ overexpression lines relative to the negative control 471 lines (Figure 5C). The constitutive overexpression of _CrJAM3_ only strongly repressed the 472 expression of a few MIA pathway genes at either 0 or 0.5 hr compared with the overexpression of 473 _CrJAM2_ ; specifically, only _NPF2.4_ and _SGD_ were repressed by at least 75% in both _CrJAM3_ 474 lines _._ In contrast to the _CrJAM2_ lines, the _CrJAM3_ lines showed increased rather than decreased 475 expression of genes encoding the root-associated pathway ( _HL3, V19H, MAT_ ), by at least 2-fold 476 relative to the negative control at either 0 or 0.5 hr, suggesting that _CrJAM2_ may specifically 477 repress the expression of the root-associated pathway. The transcript levels of most aerial 478 associated vindoline pathway genes were either too low to measure or very low relative to the 479 other genes (not shown). 480 However, after 24 hrs, MJ resulted in higher MIA pathway gene expression in the _CrJAM_ 481 overexpressing lines (three of the four lines, except for rSB02_298_33_1) than the negative control 482 lines; specifically, MIA pathway genes ( _LAMT, SS, VNS_ ) increased by greater than 2-fold relative 483 to the negative control lines; _CrJAM3_ overexpressing line (rSB_02_298_32_1) showed the 484 strongest increases in the expression of genes in serpentine and catharanthine biosynthesis ( _SS_, 21 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 485 _HL1/CS_ by >4-fold) and the root-associated pathways ( _HL3, V19H, MAT_ by >8-fold) compared to 486 negative controls. Only _NPF2.4_ and _SGD_ remained repressed (by >50%) 24 hrs after MJ addition 487 in these three _CrJAM_ lines. 488 The constitutive overexpression of _CrJAM2_ (and _CrJAM3_ to a lesser extent) in hairy root 489 cultures repressed the expression of most MIA pathway genes at either 0 or 0.5 hr after MJ 490 addition, consistent with the results from the promoter transactivation assay in seedlings; however, 491 this repression in hairy root cultures was released 24 hrs after MJ addition, resulting in higher 492 expression of MIA pathway genes compared to the MJ-treated negative control lines. In a 493 subsequent section, we present how the level of activators to repressors correlates with the released 494 repression of the MIA pathway. 495 **Deletion** **of** _**CrJAMs**_ **in hairy root lines increased MIA pathway gene expression** 496 We also generated hairy root lines with multiple _CrJAM_ deletions and characterized their 497 effect on MIA gene expression through RNA-Seq. We used an optimized CRISPR-Cas9 construct 498 containing 13 introns and an array of eight guide RNAs to successfully introduce mutations to 499 multiple _CrJAMs_ (Grützner et al., 2021). Double and triple mutant lines were generated targeting 500 the following combination of _CrJAMs_ : 1) _CrJAM2_ and _CrJAM3_ (using pSB311, Figure S 6) or 2) 501 _CrJAM2_, _CrJAM3_, and _CrRMT1_ (using pSB312, Figure S 6); the negative control lines 502 overexpressed _Cas9_ with no guide RNAs _._ 503 CRISPR-Cas mediated genome editing is highly specific, nevertheless the occurrence of 504 off-target mutations is possible (Doench et al., 2016). Ordon et al. (2017) did not observe any 505 negative effects from off-targets in transgenic _Nicotiana benthamiana_ and _A. thaliana_ lines 506 generated with the original pDGE vector system (used to construct pSB311 and pSB312). The 507 presence of 2 or 3 mismatches between guide and target reduced the occurrence of indels in human 22 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 508 cells (Slaymaker et al., 2016). Therefore, we minimized off-target mutations by choosing guides 509 with greater than 4 or 5 mismatches (i.e. a maximum identity of 15/20 or 16/20 bp) to the closest 510 off-targets (Table S 5). 511 Four double mutant lines and four triple mutant lines were characterized in detail for 512 mutations in _CrJAM2_, _CrJAM3_, and _CrRMT1_ (Figure S 9 – S 13). CRISPR-Cas9 consistently 513 introduced mutations at each guide RNA location in the double and triple mutant lines (Figure S 514 9 and S 10) but did not introduce mutations in the target sites of the negative control line expressing 515 _Cas9_ with no guide RNAs ( _CrJAMs_ were sequenced in two control lines). These mutations ranged 516 from small indels to large deletions (>1000 bp) between different sgRNA target sites, as well as 517 the integration of reverse complementary sequences between target sites (Figure S 10). 518 Surprisingly, many lines appeared to have homozygous mutations (7 homozygous out of 20 519 potentially different alleles). Only one chimeric line with three different mutations was observed 520 (CrJAM3 of rSB02_311_12_01, Figure S 9), suggesting that the mutations might occur early on 521 during hairy root formation. 522 While the mutations of all four double mutant and four triple mutant lines were sequenced 523 and characterized (Figure S 11 – S 13), only two double mutant and two triple mutant lines were 524 further investigated for gene expression profiling. The functionality of the CrJAMs in these lines 525 is summarized in Table 1. The functionality of a specific CrJAM was predicted based on the 526 presence of the complete bHLH domain. If the bHLH domain was truncated, this CrJAM was 527 considered non-functional since both its DNA-binding and its bHLH-interacting domains were 528 disrupted. If mutations occurred after the H-E-R motif, this CrJAM was considered potentially 529 functional since DNA-binding might still be possible although its bHLH-interacting domain would 530 be missing. Both CrJAM2 and CrJAM3 were non-functional for one double mutant 23 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 531 (rSB02_311_08_1 or _jam2_ _[AB]_ _jam3_ _[AB]_ ) while one allele of CrJAM2 was potentially functional for 532 the other double mutant line (rSB02_311_12_1 or _jam2_ _[A?]_ _jam3_ _[ABC]_ ). Similarly, all three CrJAMs 533 were non-functional for one triple mutant (rSB02_312_26_1 or _jam2_ _[AB]_ _jam3_ _[AB]_ _rmt1_ _[AB]_ ) while one 534 allele of CrJAM3 was potentially functional for the other triple mutant line (rSB02_312_03_2 or 535 _jam2_ _[AB]_ _jam3_ _[A?]_ _rmt1_ _[AB]_ ). 536 These CRISPR-mutated hairy root lines were elicited with MJ (250 µM) and sampled for 537 RNA-Seq at basal conditions (0 hr), 0.5 hr, and 24 hrs . The expression of most MIA pathway genes 538 was either unaffected or slightly increased in both double mutant lines compared to the control 539 lines at 0 and 0.5 hr (~2-fold or less; Figure 5E); however, the expression of MIA pathway genes 540 was not consistent between the two triple mutant lines. One line (rSB02_312_03_2 or 541 _jam2_ _[AB]_ _jam3_ _[A?]_ _rmt1_ _[AB]_ ) displayed increased expression of most MIA pathway genes while the other 542 line (rSB02_312_26_1 or _jam2_ _[AB]_ _jam3_ _[AB]_ _rmt1_ _[AB]_ ) showed increased expression of only a few genes. 543 Key steps that were strongly repressed in the _CrJAM2_ overexpressing lines (like _NPF2.4, TDC,_ 544 _SGD, HYS_, _VNS_ ) and in the root-associated pathway ( _HL3, TEX2, T19H, TAT_ ) were no longer 545 repressed in the _CrJAM_ double mutant lines at 0 or 0.5 hr; the similar expression profile observed 546 in both double mutant lines suggests that both alleles of _CrJAM2_ in rSB02_311_12_1 were non 547 functional (behaved like _jam2_ _[AB]_ _jam3_ _[AB]_ ). Only the expression of _CS/HL1_ remained strongly 548 repressed in both double mutant lines (so strongly repressed in rSB02_311_12_1 that the 549 transcripts could not be quantified). The transcript levels of most aerial-associated vindoline 550 pathway genes were either too low to measure or very low relative to the other genes (not shown). 551 However, after 24 hrs, MJ resulted in either no change or lower expression of most MIA 552 pathway genes in the double mutant lines than the negative control lines; specifically, downstream 553 MIA pathway genes ( _SS, V19H, MAT_ ) were strongly decreased by greater than 75% relative to the 24 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 554 negative control lines. The expression of MIA pathway genes at 24 hrs with the addition of MJ 555 was not consistent between the two triple mutant lines: one triple mutant (rSB02_312_03_2) 556 showed >2-fold increases in the expression of MIA pathway genes (10 out of 39 genes) while the 557 other triple mutant (rSB02_312_26_1) showed >50% decreases in the expression of MIA pathway 558 genes (8 out of 39 genes) relative to the control _._ 559 Overall, CRISPR-Cas9 editing was highly effective in _C. roseus_ hairy root cultures with 560 mutations at 100% of the guide RNA target sites. Double mutant lines showed slightly increased 561 expression of the MIA pathway genes compared to the control lines while only one triple mutant 562 line exhibited strong activation of MIA gene expression at 0 or 0.5 hr. However, at 24 hrs after MJ 563 addition, the double mutant lines showed decreased expression of MIA pathway genes compared 564 to the MJ-treated negative control lines. Next, we present how the level of activators to repressors 565 correlates with this reversal in MIA pathway expression. 566 **Overexpression of** _**CrJAMs**_ **repressed, while loss of** _**CrJAMs**_ **activated, the basal expression** 567 **of the transcriptional regulators of MIA biosynthesis** 568 The overexpression or mutation of _CrJAMs_ impacted the expression of transcriptional 569 regulators similarly to that of the MIA pathway genes, as shown in Figure 6. As with the expression 570 of MIA pathway genes, the constitutive overexpression of _CrJAM2_, and _CrJAM3_ to a lesser extent, 571 repressed the expression of both activators and repressors at either 0 or 0.5 hr (Figure 6); strongly 572 repressed genes in the _CrJAM2_ lines (by greater than 75% decrease compared to the _GUS_ lines) 573 included the activators _ORCA2_, _ORCA3_, _ORCA5_, and _ORCA6_, and the repressors _JAZ1_ and _JAZ2_ ; 574 _BIS2_ and _BIS3_ were also repressed (by less than 75%). Since MYC2a activates the expression of 575 _BIS_, _ORCA_, and _JAZ_ (see regulatory network in Figure 1), the constitutive overexpression of _JAMs_ 576 would be expected to outcompete MYC2a for promoter binding, resulting in repression of _BIS,_ 577 _ORCA, JAZ,_ and MIA pathway genes as observed. 25 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 578 By 24 hr after MJ addition, the repression of most MIA activators in _CrJAM_ 579 overexpressing lines was released; activators such as _MYC2a_, _BIS3_, and _ORCA4_ were now induced 580 relative to the negative control, while repressors such as _JAZ_ and _ZCT1_ remained repressed (Figure 581 6A). The increased expression of activators relative to repressors could explain the induction of 582 MIA pathway genes at 24 hr in three of the four _CrJAM_ -overexpressing lines. 583 Conversely, the double and triple mutations of _CrJAMs_ resulted in higher expression of 584 both activators ( _BISs_ and _ORCAs_ ) and repressors ( _JAZs_ ) relative to the negative control at either 0 585 or 0.5 hr (Figure 6B) as expected. However, at 24 hr after MJ addition, the expression of the 586 activators in the double mutant lines became repressed while the repressors remained induced 587 relative to the negative control lines; the increased expression of repressors relative to activators 588 could explain the inhibition of the MIA pathway genes at 24 hrs (Figure 5E). Unlike the double 589 mutant, the activators and repressors in the triple mutant lines remained slightly induced relative 590 to the negative control 24 hrs after MJ treatment. 591 **Discussion** 592 In this study, we investigated the hypothesis that CrJAMs repress MIA biosynthesis. To 593 answer this question, we overexpressed or silenced _CrJAM2_ and _CrJAM3_ using four 594 complementary methods: transient silencing in leaves (VIGS), transient overexpression in 595 seedlings (EASI), stable overexpression in hairy root cultures, and stable CRISPR-mediated 596 knockouts in hairy root cultures. Each method explored the effect of CrJAMs in different contexts 597 (tissue types, overexpression versus silencing, and application of exogenous JA) on the MIA 598 pathway at different layers of regulation (promoter activation, transcript levels, and alkaloid 599 levels). Despite major differences between the four methods, we consistently observed evidence 600 of CrJAMs acting as repressors of the MIA biosynthetic pathway. We integrate this new evidence, 26 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 601 the role of JAMs, into an updated model illustrating how the transcription factor triad of JAZ, 602 MYC2a, and JAM regulates MIA biosynthesis in _C. roseus_ (Figure 1). 603 Evidence for regulation by this transcriptional triad comes from both _A. thaliana_ and _C._ 604 _roseus_ in the literature or in this study, suggesting a conserved pathway modulating the JA 605 mediated defense response. The triad consists of multiple feedforward and feedback loops that 606 work together to create a time-constrained pulse of JA-activated gene expression. First, JAZ binds 607 and holds MYC2 and JAMs captive in Arabidopsis (Chini et al., 2007; Sasaki-Sekimoto et al., 608 2014) and _C. roseus_ (Schweizer et al., 2018; only CrRMT1 was studied in Patra et al. 2018). In 609 the presence of JA-Ile, JAZ is degraded and MYC2 and JAMs are released. In _C. roseus,_ MYC2a 610 then activates a feedforward cascade (Figure 1), increasing the expression of _ORCA_ and _BIS_ 611 activators and the expression of MIA pathway enzymes (Zhang et al., 2011; Schweizer et al., 612 2018). ORCA and BIS further activate expression of MIA pathway genes (Van der Fits and 613 Memelink, 2000; Van Moerkercke et al., 2015; Van Moerkercke et al., 2016; Schweizer et al., 614 2018). 615 JAM proteins are simultaneously released from interaction with JAZ repressors (Sasaki 616 Sekimoto et al., 2013; Song et al., 2013) and compete with MYC2 in binding to the same promoter 617 elements in _A. thaliana_ (Fonseca et al., 2014) and _C. roseus_ . For instance, in _C. roseus_, Zhang et 618 al. (2011) and Patra et al. (2018) showed that MYC2a and CrRMT1 activate and repress the 619 _ORCA3_ promoter, respectively, by binding to the same T/G-boxes. In addition to regulation of 620 _ORCA3_, we consistently observed that CrJAM2 and CrJAM3 repressed the expression of the _BIS_ 621 and _ORCA_ family (Figure 1, line 2 and line 4), as follows: VIGS of _CrJAM2_ and _CrJAM3_ together 622 led to >2-fold increases in _BIS1_ and _BIS2_ (Figure 3B); overexpression of _CrJAM2_ or _CrJAM3_ in 623 seedlings significantly reduced the promoter activity of _BIS1_, _BIS2_, and _ORCA3_ (Figure 4); 27 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 624 overexpression of _CrJAM2_ or _CrJAM3_ in hairy roots decreased the basal transcript levels of 625 _ORCA2_, _ORCA3_, _ORCA5_, and _ORCA6_ by over 75%, and of _BIS2, BIS3_, and _ORCA4_ more 626 moderately (Figure 6A). Finally, both the _jam_ double and triple mutant lines exhibited increased 627 expression of multiple _BIS_ and _ORCA_ genes at either 0 or 0.5 hrs after MeJA addition. These 628 results are consistent with Schweizer et al. (2018), who observed repression of _pBIS1_ with transient 629 overexpression of _CrJAM2_ or _CrJAM3_, and Patra et al. (2018), who showed repression of _pORCA3_ 630 with transient overexpression of _CrRMT1_ . Overall, CrJAM2, CrJAM3, and CrRMT1 have 631 overlapping roles in repressing the expression of multiple _BIS_ and _ORCA_ activators. 632 Like its repression of _BIS_ and _ORCA_ activators, JAMs repress the expression of the MEP 633 secoiridoid and MIA pathway. We observed that the overexpression of either _CrJAM2_ or _CrJAM3_ 634 broadly repressed genes in these pathways. For example, transient expression of either _CrJAM2_ or 635 _CrJAM3_ significantly reduced promoter activity of MEP-secoiridoid ( _pG8O_ ) and MIA pathway 636 ( _pGO_ ), genes previously shown to be regulated by the derepressed CrMYC2a [D126N ] in _C. roseus_ 637 flowers (Schweizer et al., 2018) _._ However, we did not observe an effect on _pZCT1_, _pLAMT_, 638 _pSTR1_, and _pT19H_ genes that were previously shown to be activated by CrMYC2a [D126N ] 639 (Schweizer et al., 2018; Traverse et al., 2024). In addition, we observed that CrJAMs repressed 640 _pT16H1_ but activated _pT16H2_, which were not shown to be regulated by CrMYC2a [D126N] 641 (Schweizer et al. 2018) _._ These differences between our results, Patra et al. (2018), and Schweizer 642 et al. (2018) might be attributed to the distinct role of the individual CrJAMs and to the evaluation 643 methods and tissue context used. 644 Furthermore, RNAseq profiling of hairy root lines showed the broad extent of regulation 645 by CrJAMs. To our knowledge, this is the first evidence of the broad regulation of the MIA 646 pathway by CrJAMs. For instance, we discovered that CrJAM2 repress genes in the upstream MIA 28 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 647 pathway (Figure 1, line 3: _NPF2.4_, _TDC, SGD, HYS_ ), downstream MIA pathway genes (line 3: 648 _CS/HL1, VNS_ ), and the root-associated pathway ( _V19H, MAT, TEX2, T19H, TAT_ ) by greater than 649 75% compared to the control lines (Figure 5) and the expression of the MEP-secoiridoid pathway 650 genes by less than 75% (Figure 1, line 5). 651 Conversely, the _jam_ double mutant lines (and one triple mutant line: rSB02_312_3_02 or 652 _jam2_ _[AB]_ _jam3_ _[A?]_ _rmt_ _[AB]_ ) exhibited an overall increase in the expression of the MIA regulatory genes 653 and nearly all MIA pathway genes at either 0 or 0.5 hr (Figure 5, Figure 6); importantly, the 654 increase in the basal gene expression relative to no guide control lines (at 0 hr) occurred in the 655 absence of MJ addition. In these lines, _NPF2.4, TDC, SGD, HYS, VNS,_ and most root-associated 656 pathway genes ( _HL3, TEX2, T19H,_ and _TAT_ ) were either no longer or less strongly repressed as 657 compared to the _CrJAM2_ lines. CrJAM2 appears to specifically repress the root-associated 658 pathway ( _HL3, V19H, MAT, TEX2, T19H, TAT_ ) since the expression of most of these genes 659 increased in the _CrJAM3_ overexpression and both the double and triple mutant lines. Only the 660 expression of _CS/HL1_ remained strongly repressed in both double mutant lines, suggesting that 661 the wildtype CrRMT1 is repressing _CS/HL1_ . Our results indicate that the individual CrJAMs 662 repress overlapping as well as different genes in the MIA pathway. 663 Consistent with the effect on promoter activity and transcript level, we observed 33 – 86% 664 increases in MIA alkaloids in _CrJAM2_ and _CrJAM3_ silenced leaves (Figure 3C). Patra et al. (2018) 665 showed that the overexpression of _CrRMT1_ in _C. roseus_ hairy root cultures decreased ajmalicine, 666 catharanthine, and tabersonine production. Unfortunately, our hairy root lines with either the 667 overexpression or deletion of _CrJAMs_ showed poor viability with prolonged subcultures and 668 subsequently died before alkaloid profiling could be completed. The constitutive overexpression 669 of the _CrJAM_ repressors or the _Cas9_ endonuclease may have caused toxicity during prolonged 29 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 670 culturing (Zhang et al., 2019; Gutierrez-Valdes et al., 2020). Future studies could employ an 671 inducible expression system to improve root culture viability. 672 In parallel with the feedforward induction of activators, MYC2 initiates a negative 673 feedback loop by activating both _JAZ_ and _JAM_ expression, previously shown in both Arabidopsis 674 and _C. roseus_ (Chini et al., 2007; Sasaki-Sekimoto et al., 2013, Patra et al. 2018). In addition, we 675 observed that CrJAMs regulate _CrJAZ_ expression in our stable hairy root cultures. When _CrJAM2_ 676 was constitutively overexpressed in _C. roseus_ hairy roots (and _CrJAM3_ a lesser extent), _CrJAZ1_ 677 and _CrJAZ2_ expression was repressed compared to the control, at all timepoints after MeJA 678 addition. Similarly, in the CRISPR-mutated lines, _CrJAZ1_ and _CrJAZ2_ expression was elevated 679 compared to the control, at all timepoints after MeJA addition. This finding is consistent with the 680 prior observation that CrMYC2a regulates _CrJAZ_ expression (Patra et al., 2018), and since 681 CrJAMs directly compete with CrMYC2a targets, CrJAMs repress _CrJAZ_ expression. However, 682 CrJAMs do not likely regulate _CrMYC2a_ expression since the _CrMYC2a_ promoter lacks G-box 683 and its variants; in Arabidopsis, JAM and MYC2 proteins also do not interact with each other 684 (Nakata et al., 2013). This is consistent with the findings of Schweizer et al. (2018) who found that 685 endogenous expression of _MYC2a_ did not change with overexpression of _CrMYC2a_ _[D126N]_ in _C._ 686 _roseus_ flowers. This negative regulation of _JAZ_ expression by CrJAMs may be responsible for the 687 reversal of gene expression trends we observed 24 hours after JA addition. Repression of _CrJAZ1_ 688 and _CrJAZ2_ by CrJAM2 and CrJAM3 adds further nuance to the JA-mediated regulatory triad 689 involved in MIA biosynthesis regulation (Figure 1, line 1). 30 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 690 **Conclusion** 691 In summary, we investigated CrJAMs as repressors of MIA biosynthesis. We characterized 692 the recently identified CrJAM2 and CrJAM3 through complementary methods involving distinct 693 tissue types, overexpression versus silencing, and application of the MIA pathway elicitor, JA. 694 Based on this collective evidence, we identified CrJAMs as repressors of the MIA regulatory and 695 MIA biosynthetic pathway. CrMYC2a and CrJAMs appear to modulate the extent of JA-mediated 696 expression of MIA biosynthesis. _CrJAM2_ appears to specifically repress the root-associated 697 pathway ( _HL3, V19H, MAT, TEX2, T19H, TAT_ ) while CrRMT1 appears to repress _CS/HL1_ 698 expression. Our results suggest that CrJAM2 and CrJAM3, along with the previously characterized 699 CrRMT1, share redundant function, contributing to the robust regulation of JA-inducible MIA 700 biosynthesis in _C. roseus_ . 701 31 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 702 **Glossary** 703 VIGS – Viral Induced Gene Silencing 704 EASI – Efficient Agro-mediated Seedling Infiltration 705 JAZ – Jasmonate ZIM-domain protein 706 JA – Jasmonic Acid 707 MJ – Methyl Jasmonate 708 bHLH – Basic Helix-Loop-helix 709 710 **Author contributions: CRediT** 711 Samuel Breselge: Writing – original draft, review & editing, Investigation, Conceptualization. 712 Krystyna K. F. Traverse: Writing – original draft, review & editing, Investigation. Lauren F. Cole 713 Osborn: Writing – original draft, review & editing, Investigation. Suphinya Sathitloetsakun: 714 Investigation; Kristina Erwardt: Investigation; Jie Wang: Investigation; Kevin L. Childs: Writing 715 – review & editing, Supervision, Investigation. Erin J. Cram: Writing – review & editing, 716 Supervision, Funding acquisition. Carolyn W.T. Lee-Parsons: Writing – original draft, review & 717 editing, Supervision, Project administration, Funding acquisition, Conceptualization. 718 719 **Acknowledgements** 720 The authors would like to thank Dr. Johannes Stuttmann and Dr. Sylvestre Marillonnet for 721 generously providing the pDGE vector. 722 723 724 32 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 725 **Funding** 726 This work was supported by the National Science Foundation (NSF) MCB Award #2031237 to 727 CLP and EC. 728 33 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 729 **References** 730 Aerts, R. J., Gisi, D., Carolis, E. De, Luca, V. De, and Baumann, T. W. (1994). Methyl jasmonate 731 vapor increases the develpmentally controlled synthesis of alkaloids in Catharanthus and 732 Cinchona seedlings. _Plant Cell_ 5, 635–643. doi: 10.1111/j.1365-313X.1994.00635.x 733 Aslam, J., Khan, S. H., Siddiqui, Z. H., Fatima, Z., Maqsood, M., Bhat, M. A., et al. (2010). 734 Catharanthus roseus (L.) G. Don. An Important Drug: It’s Applications and Production. _Int._ 735 _J. Compr. Pharm._ 1, 1–16. 736 Bailey, P. C., Martin, C., Toledo-Ortiz, G., Quail, P. H., Huq, E., Heim, M. A., et al. (2003). Update 737 on the Basic Helix-Loop-Helix Transcription Factor Gene Family in Arabidopsis thaliana. 738 _Plant Cell Online_ 15, 2497–2502. doi: 10.1105/tpc.151140 739 Bernonville, T. D., Carqueijeiro, I., Lanoue, A., Lafontaine, F., Sánchez Bel, P., Liesecke, F., et 740 al. (2017). Folivory elicits a strong defense reaction in Catharanthus roseus: Metabolomic 741 and transcriptomic analyses reveal distinct local and systemic responses. _Sci. Rep._ 7, 1–14. 742 doi: 10.1038/srep40453 743 Bolger, A. M., Lohse, M., and Usadel, B. (2014). Trimmomatic: A flexible trimmer for Illumina 744 sequence data. _Bioinformatics_ 30, 2114–2120. doi: 10.1093/bioinformatics/btu170 745 Chini, A., Fonseca, S., Fernández, G., Adie, B., Chico, J. M., Lorenzo, O., et al. (2007). The JAZ 746 family of repressors is the missing link in jasmonate signalling. _Nature_ 448, 666–671. doi: 747 10.1038/nature06006 748 Chini, A., Gimenez-Ibanez, S., Goossens, A., and Solano, R. (2016). Redundancy and specificity 749 in jasmonate signalling. _Curr. Opin. Plant Biol._ 33, 147–156. doi: 10.1016/j.pbi.2016.07.005 750 Cole-Osborn, L. F., McCallan, S. A., Prifti, O., Abu, R., Sjoelund, V., and Lee-Parsons, C. W. T. 751 (2024a). The role of the Golden2-like (GLK) transcription factor in regulating terpenoid 752 indole alkaloid biosynthesis in Catharanthus roseus. _Plant Cell Rep._ 43, 1–20. doi: 753 10.1007/s00299-024-03208-9 754 Cole-Osborn, L. F., Meehan, E., and Lee-Parsons, C. W. T. (2024b). Critical parameters for robust 755 Agrobacterium-mediated transient transformation and quantitative promoter assays in 756 Catharanthus roseus seedlings. _Plant Direct_ 8, 1–14. doi: 10.1002/pld3.596 757 Colinas, M., Pollier, J., Vaneechoutte, D., Malat, D. G., Schweizer, F., De Milde, L., et al. (2021). 758 Subfunctionalization of Paralog Transcription Factors Contributes to Regulation of Alkaloid 759 Pathway Branch Choice in Catharanthus roseus. _Front. Plant Sci._ 12. doi: 760 10.3389/fpls.2021.687406 761 Dobin, A., Davis, C. A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., et al. (2013). STAR: 762 Ultrafast universal RNA-seq aligner. _Bioinformatics_ 29, 15–21. doi: 763 10.1093/bioinformatics/bts635 764 Doench, J. G., Fusi, N., Sullender, M., Hegde, M., Vaimberg, E. W., Donovan, K. F., et al. (2016). 765 Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR766 Cas9. _Nat. Biotechnol._ 34, 184–191. doi: 10.1038/nbt.3437 767 Dombrecht, B., Gang, P. X., Sprague, S. J., Kirkegaard, J. A., Ross, J. J., Reid, J. B., et al. (2007). 768 MYC2 differentially modulates diverse jasmonate-dependent functions in Arabidopsis. _Plant_ 769 _Cell_ 19, 2225–2245. doi: 10.1105/tpc.106.048017 770 Engler, C., Youles, M., Gruetzner, R., Ehnert, T.-M., Werner, S., Jones, J. D. G., et al. (2014). A 771 Golden Gate Modular Cloning Toolbox for Plants. _ACS Synth. Biol._ 3, 839–843. doi: 772 10.1021/sb4001504 773 Fernandez-Pozo, N., Rosli, H. G., Martin, G. B., and Mueller, L. A. (2015). The SGN VIGS Tool: 34 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 774 User-Friendly Software to Design Virus-Induced Gene Silencing (VIGS) Constructs for 775 Functional Genomics. _Mol. Plant_ 8, 486–488. doi: 10.1016/j.molp.2014.11.024 776 Fonseca, S., Fernández-Calvo, P., Fernández, G. M., Díez-Díaz, M., Gimenez-Ibanez, S., López777 Vidriero, I., et al. (2014). bHLH003, bHLH013 and bHLH017 are new targets of JAZ 778 repressors negatively regulating JA responses. _PLoS_ _One_ 9, 1–12. doi: 779 10.1371/journal.pone.0086182 780 Franke, J., Kim, J., Hamilton, J. P., Zhao, D., Pham, G. M., Wiegert-Rininger, K., et al. (2018). 781 Data from: Gene discovery in Gelsemium highlights conserved gene clusters in monoterpene 782 indole alkaloid biosynthesis. Available at: 783 https://datadryad.org/stash/dataset/doi:10.5061/dryad.08vv50n 784 Franke, J., Kim, J., Hamilton, J. P., Zhao, D., Pham, G. M., Wiegert-Rininger, K., et al. (2019). 785 Gene Discovery in Gelsemium Highlights Conserved Gene Clusters in Monoterpene Indole 786 Alkaloid Biosynthesis. _Chembiochem_ 20, 83–87. doi: 10.1002/cbic.201800592 787 Goklany, S., Rizvi, N. F., Loring, R. H., Cram, E. J., and Lee-Parsons, C. W. T. (2013). Jasmonate788 dependent alkaloid biosynthesis in Catharanthus Roseus hairy root cultures is correlated with 789 the relative expression of Orca and Zct transcription factors. _Biotechnol. Prog._ 29, 1367– 790 1376. doi: 10.1002/btpr.1801 791 Góngora-Castillo, E., Childs, K. L., Fedewa, G., Hamilton, J. P., Liscombe, D. K., Magallanes792 Lundback, M., et al. (2012). Development of Transcriptomic Resources for Interrogating the 793 Biosynthesis of Monoterpene Indole Alkaloids in Medicinal Plant Species. _PLoS One_ 7. doi: 794 10.1371/journal.pone.0052506 795 Grützner, R., Martin, P., Horn, C., Mortensen, S., Cram, E. J., Lee-Parsons, C. W. T., et al. (2021). 796 High-efficiency genome editing in plants mediated by a Cas9 gene containing multiple 797 introns. _Plant Commun._ 2, 1–15. doi: 10.1016/j.xplc.2020.100135 798 Gutierrez-Valdes, N., Häkkinen, S. T., Lemasson, C., Guillet, M., Oksman-Caldentey, K. M., 799 Ritala, A., et al. (2020). Hairy Root Cultures—A Versatile Tool With Multiple Applications. 800 _Front. Plant Sci._ 11. doi: 10.3389/fpls.2020.00033 801 Haas, J. H., Moore, L. W., Ream, W., and Manulis, S. (1995). Universal PCR primers for detection 802 of phytopathogenic Agrobacterium strains. _Appl. Environ. Microbiol._ 61, 2879–2884. doi: 803 10.1128/aem.61.8.2879-2884.1995 804 Hao, Y., Zong, X., Ren, P., Qian, Y., and Fu, A. (2021). Basic helix‐loop‐helix (Bhlh) transcription 805 factors regulate a wide range of functions in arabidopsis. _Int. J. Mol. Sci._ 22. doi: 806 10.3390/ijms22137152 807 Heim, M. A., Jakoby, M., Werber, M., Martin, C., Weisshaar, B., and Bailey, P. C. (2003). The 808 basic helix-loop-helix transcription factor family in plants: A genome-wide study of protein 809 structure and functional diversity. _Mol._ _Biol._ _Evol._ 20, 735–747. doi: 810 10.1093/molbev/msg088 811 Huang, H., Gao, H., Liu, B., Fan, M., Wang, J., Wang, C., et al. (2018). bHLH13 Regulates 812 Jasmonate-Mediated Defense Responses and Growth. _Evol. Bioinforma._ 14, 1–8. doi: 813 10.1177/1176934318790265 814 Li, C., Wood, J. C., Hamilton, J. P., Rodriguez Lopez, C. E., Payne, R. M. E., Serna Guerrero, D. 815 A., et al. (2023). Data for: Single-cell multi-omics in the medicinal plant Catharanthus roseus 816 [Dataset]. doi: doi.org/10.5061/dryad.d2547d851 817 Li, C. Y., Leopold, A. L., Sander, G. W., Shanks, J. V., Zhao, L., and Gibson, S. I. (2013). The 818 ORCA2 transcription factor plays a key role in regulation of the terpenoid indole alkaloid 819 pathway. _BMC Plant Biol._ 13, 1–17. doi: 10.1186/1471-2229-13-155 35 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 820 Li, Z., Huang, Y., Shen, Z., Wu, M., Huang, M., Hong, S. B., et al. (2024). _Advances in functional_ 821 _studies of plant MYC transcription factors_ . Springer Berlin Heidelberg. doi: 10.1007/s00122822 024-04697-8 823 Liao, Y., Smyth, G. K., and Shi, W. (2014). FeatureCounts: An efficient general purpose program 824 for assigning sequence reads to genomic features. _Bioinformatics_ 30, 923–930. doi: 825 10.1093/bioinformatics/btt656 826 Liscombe, D. K., and O’Connor, S. E. (2011). A virus-induced gene silencing approach to 827 understanding alkaloid metabolism in Catharanthus roseus. _Phytochemistry_ 72, 1969–1977. 828 doi: 10.1016/j.phytochem.2011.07.001 829 Livak, K. J., and Schmittgen, T. D. (2001). Analysis of relative gene expression data using real830 time quantitative PCR and the 2-ΔΔCT method. _Methods_ 25, 402–408. doi: 831 10.1006/meth.2001.1262 832 Lorenz, O., Chico, J. M., Sánchez-Serran, J. J., and Solano, R. (2004). JASMONATE833 INSENSITIVE1 Encodes a MYC Transcription Factor Essential to Discriminate between 834 Different Jasmonate-Regulated Defense Responses in Arabidopsis. _Plant Cell_ 16, 1938– 835 1950. doi: 10.1105/tpc.022319.with 836 Mitchell, A. L., Attwood, T. K., Babbitt, P. C., Blum, M., Bork, P., Bridge, A., et al. (2019). 837 InterPro in 2019: Improving coverage, classification and access to protein sequence 838 annotations. _Nucleic Acids Res._ 47, D351–D360. doi: 10.1093/nar/gky1100 839 Mortensen, S., Bernal-Franco, D., Cole, L. F., Sathitloetsakun, S., Cram, E. J., and Lee-Parsons, 840 C. W. T. (2019). Easi transformation: An efficient transient expression method for analyzing 841 gene function in Catharanthus roseus seedlings. _Front. Plant Sci._ 10, 1–17. doi: 842 10.3389/fpls.2019.00755 843 Mortensen, S., Cole, L. F., Bernal-Franco, D., Sathitloetsakun, S., Cram, E. J., and Lee-Parsons, 844 C. W. T. (2022). “EASI Transformation Protocol: An Agrobacterium-Mediated Transient 845 Transformation Protocol for Catharanthus roseus Seedlings,” in _Methods in Molecular_ 846 _Biology_, eds. V. Courdevault and S. Besseau (Springer Nature), 249–262. Available at: 847 https://link.springer.com/protocol/10.1007/978-1-0716-2349-7_18 (Accessed May 11, 848 2023). 849 Nakata, M., Mitsuda, N., Herde, M., Koo, A. J. K., Moreno, J. E., Suzuki, K., et al. (2013). A 850 bHLH-type transcription factor, ABA-INDUCIBLE BHLH-TYPE TRANSCRIPTION 851 FACTOR/JA-ASSOCIATED MYC2-LIKE1, acts as a repressor to negatively regulate 852 jasmonate signaling in Arabidopsis. _Plant Cell_ 25, 1641–1656. doi: 10.1105/tpc.113.111112 853 Noble, R. L., Beer, C. T., and Cutts, J. H. (1958). Role of chance observations in chemotherapy: 854 Vinca rosea. _Ann. N. Y. Acad. Sci._ 76, 882–894. doi: 10.1111/j.1749-6632.1958.tb54906.x 855 Ordon, J., Gantner, J., Kemna, J., Schwalgun, L., Reschke, M., Streubel, J., et al. (2017). 856 Generation of chromosomal deletions in dicotyledonous plants employing a user-friendly 857 genome editing toolkit. _Plant J._ 89, 155–168. doi: 10.1111/tpj.13319 858 Patra, B., Pattanaik, S., Schluttenhofer, C., and Yuan, L. (2018). A network of jasmonate859 responsive bHLH factors modulate monoterpenoid indole alkaloid biosynthesis in 860 Catharanthus roseus. _New Phytol._ 217, 1566–1581. doi: 10.1111/nph.14910 861 Paul, P., Singh, S. K., Patra, B., Sui, X., Pattanaik, S., and Yuan, L. (2017). A differentially 862 regulated AP2/ERF transcription factor gene cluster acts downstream of a MAP kinase 863 cascade to modulate terpenoid indole alkaloid biosynthesis in Catharanthus roseus. _New_ 864 _Phytol._ 213, 1107–1123. doi: 10.1111/nph.14252 865 Pauw, B., Hilliou, F. A. O., Martin, V. S., Chatel, G., De Wolf, C. J. F., Champion, A., et al. 36 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 866 (2004). Zinc finger proteins act as transcriptional repressors of alkaloid biosynthesis genes in 867 Catharanthus roseus. _J. Biol. Chem._ 279, 52940–52948. doi: 10.1074/jbc.M404391200 868 Pazour, G. J., Ta, C. N., and Das, A. (1992). Constitutive mutations of Agrobacterium tumefaciens 869 transcriptional activator virG. _J. Bacteriol._ 174, 4169–4174. doi: 10.1128/jb.174.12.4169870 4174.1992 871 Peebles, C. A. M., Hughes, E. H., Shanks, J. V., and San, K. Y. (2009). Transcriptional response 872 of the terpenoid indole alkaloid pathway to the overexpression of ORCA3 along with 873 jasmonic acid elicitation of Catharanthus roseus hairy roots over time. _Metab. Eng._ 11, 76– 874 86. doi: 10.1016/j.ymben.2008.09.002 875 Pollier, J., Vanden Bossche, R., Rischer, H., and Goossens, A. (2014). Selection and validation of 876 reference genes for transcript normalization in gene expression studies in Catharanthus 877 roseus. _Plant Physiol. Biochem._ 83, 20–25. doi: 10.1016/j.plaphy.2014.07.004 878 Qi, T., Wang, J., Huang, H., Liu, B., Gao, H., Liu, Y., et al. (2015). Regulation of Jasmonate879 Induced Leaf Senescence by Antagonism between bHLH Subgroup IIIe and IIId Factors in 880 Arabidopsis. _Plant Cell_ 27, 1634–1649. doi: 10.1105/tpc.15.00110 881 Qian, Y., Zhang, T., Yu, Y., Gou, L., Yang, J., Xu, J., et al. (2021). Regulatory Mechanisms of 882 bHLH Transcription Factors in Plant Adaptive Responses to Various Abiotic Stresses. _Front._ 883 _Plant Sci._ 12. doi: 10.3389/fpls.2021.677611 884 Rizvi, N. F., Cornejo, M., Stein, K., Weaver, J., Cram, E. J., and Lee-Parsons, C. W. T. (2015). 885 An efficient transformation method for estrogen-inducible transgene expression in 886 Catharanthus roseus hairy roots. _Plant Cell. Tissue Organ Cult._ 120, 475–487. doi: 887 10.1007/s11240-014-0614-1 888 Rizvi, N. F., Weaver, J. D., Cram, E. J., and Lee-Parsons, C. W. T. (2016). Silencing the 889 Transcriptional Repressor, ZCT1, Illustrates the Tight Regulation of Terpenoid Indole 890 Alkaloid Biosynthesis in Catharanthus roseus Hairy Roots. _PLoS One_ 11, e0159712. doi: 891 10.1371/journal.pone.0159712 892 Sasaki-sekimoto, Y., Jikumaru, Y., Obayashi, T., Saito, H., Masuda, S., Kamiya, Y., et al. (2013). 893 Basic Helix-Loop-Helix Transcription Factors JAM2, and JAM3 Are Negative Regulators 894 of Jasmonate. 163, 291–304. doi: 10.1104/pp.113.220129 895 Sasaki-Sekimoto, Y., Jikumaru, Y., Obayashi, T., Saito, H., Masuda, S., Kamiya, Y., et al. (2013). 896 Basic helix-loop-helix transcription factors JASMONATE-ASSOCIATED MYC2-LIKE1 897 (JAM1), JAM2, and JAM3 are negative regulators of jasmonate responses in Arabidopsis. 898 _Plant Physiol._ 163, 291–304. doi: 10.1104/pp.113.220129 899 Schweizer, F., Colinas, M., Pollier, J., Van Moerkercke, A., Vanden Bossche, R., de Clercq, R., et 900 al. (2018). An engineered combinatorial module of transcription factors boosts production of 901 monoterpenoid indole alkaloids in Catharanthus roseus. _Metab. Eng._ 48, 150–162. doi: 902 10.1016/j.ymben.2018.05.016 903 Sibéril, Y., Benhamron, S., Memelink, J., Giglioli-Guivarc&apos;h, N., Thiersault, M., Boisson, 904 B., et al. (2001). Catharanthus roseus G-box binding factors 1 and 2 act as repressors of 905 strictosidine synthase gene expression in cell cultures. _Plant Mol. Biol._ 45, 477–488. doi: 906 10.1023/A:1010650906695 907 Sigrist, C. J. A., De Castro, E., Cerutti, L., Cuche, B. A., Hulo, N., Bridge, A., et al. (2013). New 908 and continuing developments at PROSITE. _Nucleic Acids Res._ 41, 344–347. doi: 909 10.1093/nar/gks1067 910 Škubník, J., Pavlíčková, V. S., Ruml, T., and Rimpelová, S. (2021). Vincristine in combination 911 therapy of cancer: Emerging trends in clinics. _Biology_ _(Basel)._ 10. doi: 37 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 912 10.3390/biology10090849 913 Slaymaker, I. M., Gao, L., Zetsche, B., Scott, D. A., Yan, W. X., and Zhang, F. (2016). Rationally 914 engineered Cas9 nucleases with improved specificity. _Science (80-. )._ 351, 84–88. doi: 915 10.1126/science.aad5227 916 Song, S., Qi, T., Fan, M., Zhang, X., Gao, H., Huang, H., et al. (2013). The bHLH Subgroup IIId 917 Factors Negatively Regulate Jasmonate-Mediated Plant Defense and Development. _PLoS_ 918 _Genet._ 9, 1–19. doi: 10.1371/journal.pgen.1003653 919 Suttipanta, N., Pattanaik, S., Kulshrestha, M., Patra, B., Singh, S. K., and Yuan, L. (2011). The 920 transcription factor CrWRKY1 positively regulates the terpenoid indole alkaloid biosynthesis 921 in Catharanthus roseus. _Plant Physiol._ 157, 2081–2093. doi: 10.1104/pp.111.181834 922 Svec, D., Tichopad, A., Novosadova, V., Pfaffl, M. W., and Kubista, M. (2015). How good is a 923 PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency 924 assessments. _Biomol. Detect. Quantif._ 3, 9–16. doi: 10.1016/j.bdq.2015.01.005 925 Thaler, J. S., Humphrey, P. T., and Whiteman, N. K. (2012). Evolution of jasmonate and salicylate 926 signal crosstalk. _Trends Plant Sci._ 17, 260–270. doi: 10.1016/j.tplants.2012.02.010 927 Thines, B., Katsir, L., Melotto, M., Niu, Y., Mandaokar, A., Liu, G., et al. (2007). JAZ repressor 928 proteins are targets of the SCFCOI1 complex during jasmonate signalling. _Nature_ 448, 661– 929 665. doi: 10.1038/nature05960 930 Toledo-Ortiz, G., Huq, E., and Quail, P. H. (2003). The Arabidopsis basic/helix-loop-helix 931 transcription factor family. _Plant Cell_ 15, 1749–1770. doi: 10.1105/tpc.013839 932 Traverse, K. K. F., Breselge, S., Trautman, J. G., Dee, A., Wang, J., Childs, K. L., et al. (2024). 933 Characterization of the ZCTs, a subgroup of Cys2-His2 zinc finger transcription factors 934 regulating alkaloid biosynthesis in Catharanthus roseus. _Plant Cell Rep._ 43, 1–27. doi: 935 10.1007/s00299-024-03295-8 936 Traverse, K. K. F., Mortensen, S., Trautman, J. G., Danison, H., Rizvi, N. F., and Lee-Parsons, C. 937 W. T. (2022). “Generation of Stable Catharanthus roseus Hairy Root Lines with 938 Agrobacterium rhizogenes,” in _Methods in Molecular Biology_, ed. A. G. Fett-Neto (Springer), 939 129–144. doi: 10.1007/978-1-0716-2185-1_11 940 Van Der Fits, L., Memelink, J., Fits, L., and Memelink, J. (2000). ORCA3, a jasmonate-responsive 941 transcriptional regulator of plant primary and secondary metabolism. _Science (80-. )._ 289, 942 295–297. doi: 10.1126/science.289.5477.295 943 Van der Heijden, R., Jacobs, D., Snoeijer, W., Hallard, D., and Verpoorte, R. (2004). The 944 Catharanthus Alkaloids: Pharamacognosy and Biotechnology. _Curr Med Chem_ 11, 607–628. 945 Van Moerkercke, A., Steensma, P., Gariboldi, I., Espoz, J., Purnama, P. C., Schweizer, F., et al. 946 (2016). The basic helix-loop-helix transcription factor BIS2 is essential for monoterpenoid 947 indole alkaloid production in the medicinal plant Catharanthus roseus. _Plant J._ 88, 3–12. doi: 948 10.1111/tpj.13230 949 Van Moerkercke, A., Steensma, P., Schweizer, F., Pollier, J., Gariboldi, I., Payne, R., et al. (2015). 950 The bHLH transcription factor BIS1 controls the iridoid branch of the monoterpenoid indole 951 alkaloid pathway in Catharanthus roseus. _Proc. Natl. Acad. Sci. U. S. A._ 112, 8130–8135. doi: 952 10.1073/pnas.1504951112 953 Warnes, G. R., Bolker, B., Bonebakker, L., Gentleman, R., Liaw, W. H. A., Lumley, T., et al. 954 (2009). gplots: Various R Programming Tools for Plotting Data. _R Packag. version_ . 955 Wasternack, C., and Strnad, M. (2018). Jasmonates: News on Occurrence, Biosynthesis, 956 Metabolism and Action of an Ancient Group of Signaling Compounds. _Int. J. Mol. Sci._ 19, 957 2539. doi: 10.3390/ijms19092539 38 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 958 Weber, E., Engler, C., Gruetzner, R., Werner, S., and Marillonnet, S. (2011). A Modular Cloning 959 System for Standardized Assembly of Multigene Constructs. _PLoS One_ 6, e16765. doi: 960 10.1371/journal.pone.0016765 961 Zhang, H., Hedhili, S., Montiel, G., Zhang, Y., Chatel, G., Pré, M., et al. (2011). The basic helix962 loop-helix transcription factor CrMYC2 controls the jasmonate-responsive expression of the 963 ORCA genes that regulate alkaloid biosynthesis in Catharanthus roseus. _Plant J._ 67, 61–71. 964 doi: 10.1111/j.1365-313X.2011.04575.x 965 Zhang, Y., Malzahn, A. A., Sretenovic, S., and Qi, Y. (2019). The emerging and uncultivated 966 potential of CRISPR technology in plant science. _Nat. Plants_ 5, 778–794. doi: 967 10.1038/s41477-019-0461-5 968 39 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 CrJAMs are transcriptional repressors of the monoterpenoid indole alkaloid pathway in 2 _Catharanthus roseus_ 3 4 **Author names & affiliations:** Samuel Breselge [1], Krystyna K. F. Traverse [2], Lauren F. Cole5 Osborn [2,3], Suphinya Sathitloetsakun [4], Kristina Dürr [4,5], Jie Wang [6], Kevin L. Childs [6], Erin J. 6 Cram [1], Carolyn W.T. Lee-Parsons [2,3,4] 7 8 9 10 11 SB, KKFT, LFCO are shared first authors 12 1 Northeastern University, Department of Biology, Boston, MA, USA 02115 13 2 Northeastern University, Department of Chemical Engineering, Boston, MA 14 3 Northeastern University, Department of Bioengineering, Boston, MA 15 4 Northeastern University, Department of Chemistry and Chemical Biology, Boston, MA 16 5 Leibniz University, Department of Life Science, Hanover, Germany 17 6 Michigan State University, Department of Plant Biology, East Lansing, MI, USA 18 19 **Corresponding author:** Carolyn Lee-Parsons, ca.lee@northeastern.edu 20 21 22 23 24 25 1 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 26 **Figures** 27 28 **Figure 1.** Model for the jasmonate-responsive regulation of MIA biosynthesis by JAZ, MYC2a, 29 ORCA, and BIS previously proposed in the literature, overlaid with regulation by JAM from this 30 paper 31 The regulation of MIA biosynthesis by MYC2a, ORCA, and BIS in this figure was previously 32 published in Schweizer et al., 2018. The regulation of MIA biosynthesis by JAMs is investigated 33 and presented in this paper and overlaid on the figure adapted from Schweizer et al., 2018. 34 Activation is indicated by green lines and green arrowheads whereas inhibition is indicated by red 35 lines and red flat-head arrow. Solid lines indicate confirmed protein-protein interactions whereas 2 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 36 dashed lines indicate either confirmed protein-DNA interactions or regulation based on 37 overexpression of JAMs or MYC2a. JA-Ile: jasmonate-isoleucine conjugate; GPP: geranyl 38 pyrophosphate. Please refer to the Introduction and the Discussion Sections for detailed 39 descriptions of the regulatory relationships; the grey numbers indicate relationships inferred from 40 the experimental results and summarized in the Discussion. 41 3 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 42 43 **Figure 2.** Protein alignment of JAMs from _A. thaliana_ and _C. roseus_ . 44 Protein sequences of JAMs from _A. thaliana_ and _C. roseus_ were downloaded from the NCBI 45 database: AtJAM1 (AT2G46510), CrRMT1(KY851107), AtJAM2 (AT1G01260), CrJAM2 46 (MG676662), AtJAM3 (AT4G16430), CrJAM3 (MG676663), AtbHLH14 (AT4G00870), 47 CrMYC2a (CRO_07G000280). Protein sequences were aligned in CLC Main Workbench (Gap 48 open cost = 10.0, Gap extension cost = 1.0, End gap cost = as any other) and shaded for 4 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 49 homologous regions using Boxshade. Black shading indicates residues that are identical to the 50 consensus while grey shading indicates residues that are similar to the consensus. The bHLH 51 region (as predicted by InterPro) is highlighted in yellow and the 5-9-13 motif (amino acids H-E 52 R) is indicated above the alignment. 53 54 5 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 55 56 **Figure 3.** Virus-induced gene silencing of _CrJAM2_ and _CrJAM3_ in _C. roseus_ leaves (indicated 57 as _JAM3/2_ ) led to increases in MIA gene expression and alkaloid accumulation. 58 **(A)** _CrJAM2_ and _CrJAM3_ were significantly silenced in _JAM3/2_ -silenced plants. **(B)** Silencing 59 _CrJAM2_ and _CrJAM3_ led to non-significant increases in the gene expression of most MIA TFs 60 and MIA pathway genes. Relative gene expression was measured with qPCR and calculated using 61 the 2 [-∆∆Ct] method (Livak and Schmittgen, 2001) relative to the control condition ( _GFP_ -silenced 62 plants) and normalized relative to the housekeeping gene, _SAND_ (Pollier et al., 2014). * q < 0.05 63 according to an unpaired two-tailed t-test on ∆∆Ct values with a false discovery rate of 5% (two 64 stage linear step-up procedure of Benjamini, Krieger and Yekutieli, with 17 genes); vindoline 65 pathway genes are shown in Figure S 5. **(C)** Alkaloid accumulation (vindoline, catharanthine, 6 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 66 ajmalicine, and serpentine) increased in _JAM3/2_ -silenced plants. Metabolite levels were quantified 67 from the peak area of the extracted ion chromatogram using HPLC-MS/MS divided by the peak 68 area of the internal standard, ajmaline. RNA and alkaloids were extracted from the second leaf pair 69 to emerge after infection for each plant (n = 5). No statistical significance in alkaloid levels was 70 found according to an unpaired two-tailed t-test on normalized peak areas with a false discovery 71 rate of 5% (two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, with 4 72 metabolites). Boxes represent the 25th and 75th percentile with a line marking the median. 73 Whiskers extend to the minimum and maximum. 74 7 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 75 **A.** 76 77 **B.** 78 |Col1|Promoter|Activatedby|Col4|Col5|Relative Promoter<br>Activity|Col7| |---|---|---|---|---|---|---| ||Promoter|MYC2a|BIS1/2|ORCA3|_JAM2_OE|_JAM3_OE| ||_pMYC2a_||||1.01|1.13| ||_pBIS1_||||0.66|0.94| ||_pBIS2_||||0.62|0.67| ||_pORCA3_||||0.68|0.63| ||_pZCT1_||||1.15|1.17| |||||||| ||_pG8O_||||0.58|0.97| ||_pLAMT_||||1.09|1.17| ||_pSTR1_||||1.15|1.30| ||_pGO_||||0.74|0.58| ||_pT16H1_||||0.25|0.47| ||_pT16H2_||||1.17|**^**1.65| |Root<br>specific|_pT19H_||||0.95|0.87| 8 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 79 **Figure 4.** The overexpression of either _CrJAM2_ or _CrJAM3_ represses _C. roseus_ promoters in 80 transiently transformed _C. roseus_ seedlings 81 **(A)** _C. roseus_ seedlings were vacuum-infiltrated with a combination of two strains of _A._ 82 _tumefaciens_ (total OD 600 = 0.4): 1) strain containing the promoter and its 5’UTR driving the 83 _FLUC-I_ reporter (Figure S 6; OD 600 = 0.06), and 2) strain containing a _CaMV2x35S_ driven effector 84 for transactivation (either _GUS_ as the negative effector control – pSB161 (Addgene ID #123197), 85 _CrJAM2_ – pSB270, or _CrJAM3_ – pSB271; OD 600 = 0.34). Samples were harvested three days post 86 infection. The relative promoter activity was the ratio of FLUC to RLUC luminescence for each 87 sample normalized to the average FLUC to RLUC luminescence of the _GUS_ overexpression 88 control (set to 1). The experiment was carried out with ten replicates per condition and three 89 independent experiments were performed (n = 30 replicates per condition). The black horizontal 90 line within the boxes shows the median, and the ends of the boxes show the 25 [th] and 75 [th] percentile. 91 The data was log-normal transformed to obtain normally distributed data. Significant differences 92 compared to the _GUS_ control were determined using Dunnett’s method on log-normal transformed 93 data. P-values <0.05 are indicated with one star (*), <0.01 are indicated with two stars (**), and 94 <0.001 are indicated with three stars (***), and <0.0001 are indicated with four stars (****). **(B)** 95 Statistical analysis was performed with the Dunnett’s method on log normal transformed data. 96 Significant promoter activity changes (P-values <0.05) are highlighted in red (^ indicates 97 activation rather than repression). Included in the table are genes known to be activated by 98 MYC2a [D126N], BIS1/2, ORCA3 based on findings reported in Schweizer et al. (2018) and Traverse 99 et al., (2024). 100 9 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 101 10 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 102 103 104 **Figure 5.** Gene expression profile of _CrJAM_ overexpressing and CRISPR-mutated lines. 11 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 105 **(A)** The _C. roseus_ MIA biosynthetic pathway is color-coded to correspond to the same pathway 106 genes in the heat map shown in part C and E; regulation of genes by MYC2a, BIS1/BIS2, and 107 ORCA3 (Schweizer et al., 2018) is indicated with arrows (yellow, blue, and green, respectively). 108 **(B)** This heat map shows the transcript abundance of MIA pathway in two _GUS_ overexpressing 109 hairy root lines as average reads per kilobase of transcript per million (RKPM) mapped reads at 0 110 hours after methyl jasmonate elicitation. **(C)** This heat map show changes in gene expression at 0, 111 0.5, and 24 hours after methyl jasmonate elicitation in transgenic hairy root lines overexpressing 112 either _CrJAM2_ or _CrJAM3_, normalized to _GUS_ overexpression control lines. **(D)** This heat map 113 shows the transcript abundance of MIA pathway in four hairy root lines overexpressing _Cas9_ with 114 no guides as average reads per kilobase of transcript per million (RKPM) mapped reads at 0 hours 115 after methyl jasmonate elicitation. **(E)** This heat map show changes in gene expression at 0, 0.5, 116 and 24 hours after methyl jasmonate elicitation in transgenic hairy root lines mutated using 117 CRISPR-Cas9, i.e _. jam2jam3_ or _jam2jam3rmt1_, normalized to a no guide control line. The 118 functionality of the CrJAMs in these mutant lines are summarized in Table 1. 119 120 121 12 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 122 13 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 123 **Figure 6.** Gene expression profile of activators and repressors in CrJAM overexpressing and 124 CRISPR-mutated lines. 125 Heat maps show RNA-Seq data shifts in transcriptional activators and repressors at 0, 0.5, and 24 126 hours after methyl jasmonate elicitation in **(A)** transgenic hairy root lines overexpressing either 127 _CrJAM2_ or _CrJAM3_, normalized to _GUS_ overexpression control lines or **(B)** transgenic hairy root 128 lines where _CrJAMs_ were mutated using CRISPR-Cas9, i.e. _jam2jam3_ or _jam2jam3rmt1_, 129 normalized to a no guide control line. See Figure 1 for the regulatory model and Figure 5 A for the 130 biosynthetic pathway. 131 14 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.02.763677; this version posted November 3, 2026. The copyright holder for this preprint](https://doi.org/10.64898/2026.11.02.763677) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 132 **Table 1.** Summary of the functionality of the CrJAMs in the double and triple mutant lines 133 134 135 136 137 138 The number within the ( ) is the location of the amino acid based on the number bar above the amino acids in Figure S11, S12, S13. 1 See Figure S11 for the amino acid alignment of the CrJAM2 in the CRISPR mutant lines 2 See Figure S12 for the amino acid alignment of the CrJAM3 in the CRISPR mutant lines 3 See Figure S13 for the amino acid alignment of the CrRMT1 in the CRISPR mutant lines 15