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1. Characterization of the ZCTs, a subgroup of Cys2-His2 zinc finger transcription factors regulating alkaloid biosynthesis in Catharanthus roseus

   https://doi.org/10.1007/s00299-024-03295-8  

   Traverse, Krystyna_K F; Breselge, Samuel; Trautman, Juliet G; Dee, Amanda; Wang, Jie; Childs, Kevin L; Lee-Parsons, Carolyn_W T (September 2024, Plant Cell Reports)

   Abstract Key MessageTheC. roseus ZCTsare jasmonate-responsive, can be induced by CrMYC2a, and can act as significant regulators of the terpenoid indole alkaloid pathway when highly expressed. AbstractCatharanthus roseusis the sole known producer of the anti-cancer terpenoid indole alkaloids (TIAs), vinblastine and vincristine. While the enzymatic steps of the pathway have been elucidated, an understanding of its regulation is still emerging. The present study characterizes an important subgroup of Cys2-His2 zinc finger transcription factors known asZinc fingerCatharanthusTranscription factors (ZCTs).We identified three new ZCT members (named ZCT4, ZCT5, and ZCT6) that clustered with the putative repressors of the TIA pathway, ZCT1, ZCT2, and ZCT3. We characterized the role of these six ZCTs as potential redundant regulators of the TIA pathway, and their tissue-specific and jasmonate-responsive expression. These ZCTs share high sequence conservation in their two Cys2-His2 zinc finger domains but differ in the spacer length and sequence between these zinc fingers. The transient overexpression ofZCTsin seedlings significantly repressed the promoters of the terpenoid (pLAMT) and condensation branch (pSTR1) of the TIA pathway, consistent with that previously reported for ZCT1, ZCT2, and ZCT3. In addition, ZCTs significantly repressed and indirectly activated several promoters of the vindoline pathway (not previously studied). TheZCTsdiffered in their tissue-specific expression but similarly increased with jasmonate in a dosage-dependent manner (except forZCT5). We showed significant activation of thepZCT1andpZCT3promoters by the de-repressed CrMYC2a, suggesting that the jasmonate-responsive expression of theZCTscan be mediated by CrMYC2a. In summary, theC. roseus ZCTsare jasmonate-responsive, can be induced by CrMYC2a, and can act as significant regulators of the TIA pathway when highly expressed. 

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2. Cell type aware regulatory landscapes governing monoterpene indole alkaloid biosynthesis in the medicinal plant Catharanthus roseus

   https://doi.org/10.1111/nph.20208  

   Li, Chenxin; Colinas, Maite; Wood, Joshua C; Vaillancourt, Brieanne; Hamilton, John P; Jones, Sophia L; Caputi, Lorenzo; O'Connor, Sarah E; Buell, C Robin (January 2025, New Phytologist)

   Summary In plants, the biosynthetic pathways of some specialized metabolites are partitioned into specialized or rare cell types, as exemplified by the monoterpenoid indole alkaloid (MIA) pathway ofCatharanthus roseus(Madagascar Periwinkle), the source of the anticancer compounds vinblastine and vincristine. In the leaf, theC. roseusMIA biosynthetic pathway is partitioned into three cell types with the final known steps of the pathway expressed in the rare cell type termed idioblast. How cell‐type specificity of MIA biosynthesis is achieved is poorly understood.We generated single‐cell multi‐omics data fromC. roseusleaves. Integrating gene expression and chromatin accessibility profiles across single cells, as well as transcription factor (TF)‐binding site profiles, we constructed a cell‐type‐aware gene regulatory network for MIA biosynthesis.We showcased cell‐type‐specific TFs as well as cell‐type‐specificcis‐regulatory elements. Using motif enrichment analysis, co‐expression across cell types, and functional validation approaches, we discovered a novel idioblast‐specific TF (Idioblast MYB1,CrIDM1) that activates expression of late‐stage MIA biosynthetic genes in the idioblast.These analyses not only led to the discovery of the first documented cell‐type‐specific TF that regulates the expression of two idioblast‐specific biosynthetic genes within an idioblast metabolic regulon but also provides insights into cell‐type‐specific metabolic regulation. 

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3. Critical parameters for robust Agrobacterium ‐mediated transient transformation and quantitative promoter assays in Catharanthus roseus seedlings

   https://doi.org/10.1002/pld3.596  

   Cole‐Osborn, Lauren F; Meehan, Emma; Lee‐Parsons, Carolyn_W T (June 2024, Plant Direct)

   Abstract Agrobacterium‐mediated transient expression methods are widely used to study gene function in both model and non‐model plants. Using a dual‐luciferase assay, we quantified the effect ofAgrobacterium‐infiltration parameters on the transient transformation efficiency ofCatharanthus roseusseedlings. We showed that transformation efficiency is highly sensitive to seedling developmental state and a pre‐ and post‐infiltration dark incubation and is less sensitive to theAgrobacteriumgrowth stage. For example, 5 versus 6 days of germination in the dark increased seedling transformation efficiency by seven‐ to eight‐fold while a dark incubation pre‐ and post‐infiltration increased transformation efficiency by five‐ to 13‐fold.Agrobacteriumin exponential compared with stationary phase increased transformation efficiency by two‐fold. Finally, we quantified the variation in ourAgrobacterium‐infiltration method in replicate infiltrations and experiments. Within a given experiment, significant differences of up to 2.6‐fold in raw firefly luciferase (FLUC) and rawRenillaluciferase (RLUC) luminescence occurred in replicate infiltrations. These differences were significantly reduced when FLUC was normalized to RLUC values, highlighting the utility of including a reference reporter to minimize false positives. Including a second experimental replicate further reduced the potential for false positives. This optimization and quantitative validation ofAgrobacteriuminfiltration inC. roseusseedlings will facilitate the study of this important medicinal plant and will expand the application ofAgrobacterium‐mediated transformation methods in other plant species. 

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4. Transcriptomic Analysis Reveals the Mechanism of MtLOX24 in Response to Methyl Jasmonate Stress in Medicago truncatula

   https://doi.org/10.3390/agriculture14071076  

   Xu, Lei; Xu, Yanchao; Lv, Huanhuan; Xu, Yanran; Wen, Jiangqi; Li, Mingna; Kang, Junmei; Liu, Zhipeng; Yang, Qingchuan; Long, Ruicai (July 2024, Agriculture)

   Lipoxygenase (LOX) is associated with responses to plant hormones, environmental stresses, and signaling substances. Methyl jasmonate (MeJA) treatment triggers the production of LOX, polyphenol oxidase, and protease inhibitors in various plants, producing resistance to herbivory. To examine the response of MtLOX24 to MeJA, the phenotypic and physiological changes in Medicago truncatula MtLOX24 overexpression and lox mutant plants were investigated. Additionally, wild-type R108, the MtLOX24-overexpressing line L4, and the mutant lox-1 were utilized as experimental materials to characterize the differentially expressed genes (DEGs) and metabolic pathways in response to MeJA. The results indicate that after treatment with 200 µM of MeJA, the damage in the mutants lox-1 and lox-2 was more serious than in the overexpressing lines L4 and L6, with more significant leaf wilting, yellowing, and oxidative damage in lox-1 and lox-2. Exogenous application of MeJA induced H2O2 production and POD activity but reduced CAT activity in the lox mutants. Transcriptome analysis revealed 10,238 DEGs in six libraries of normal-growing groups (cR108, cL4, and clox1) and MeJA-treated groups (R108, L4, and lox1). GO and KEGG functional enrichment analysis demonstrated that under normal growth conditions, the DEGs between the cL4 vs. cR108 and the clox-1 vs. cR108 groups were primarily enriched in signaling pathways such as plant–pathogen interactions, flavonoid biosynthesis, plant hormone signal transduction, the MAPK signaling pathway, and glutathione metabolism. The DEGs of the R108 vs. cR108 and L4 vs. cL4 groups after MeJA treatment were mainly enriched in glutathione metabolism, phenylpropanoid biosynthesis, the MAPK signaling pathway, circadian rhythm, and α-linolenic acid metabolism. Among them, under normal growth conditions, genes like PTI5, PR1, HSPs, PALs, CAD, CCoAOMT, and CYPs showed significant differences between L4 and the wild type, suggesting that the expression of these genes is impacted by MtLOX24 overexpression. CDPKs, CaMCMLs, IFS, JAZ, and other genes were also significantly different between L4 and the wild type upon MeJA treatment, suggesting that they might be important genes involved in JA signaling. This study provides a reference for the study of the response mechanism of MtLOX24 under MeJA signaling. 

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5. Indole-3-acetic acid (IAA) protects Azospirillum brasilense from indole-induced stress

   https://doi.org/10.1128/aem.02384-24  

   Ganusova, Elena E; Banerjee, Ishita; Seats, Trey; Alexandre, Gladys (April 2025, Applied and Environmental Microbiology)

   Glass, Jennifer B (Ed.)

   ABSTRACT Azospirillum brasilenseis plant-growth promoting rhizobacteria that produces the phytohormone indole-3-acetic acid (IAA) to induce changes in plant root architecture. The major pathway for IAA biosynthesis inA. brasilenseconverts tryptophan into indole-3-pyruvic acid (I3P) and then, through the rate-limiting enzyme, indole-3-pyruvate decarboxylase (IpdC), into IAA. Here, we characterize the potential role for IAA biosynthesis in the physiology of these bacteria by characterizing the expression pattern of theipdCpromoter, analyzing anA. brasilense ipdCmutant using multiple physiological assays and characterizing the effect of I3P, which likely accumulates in the absence ofipdCand affects bacterial physiology. We found that theipdCmutant derivative has a reduced growth rate and an altered physiology, including reduced translation activity as well as a more depolarized membrane potential compared to the parent strain. Similar effects could be recapitulated in the parent strain by exposing these cells to increasing concentrations of I3P, as well as other indole intermediates of IAA biosynthesis. Our results also indicate a protective role for IAA against the harmful effects of indole derivatives, with exogenous IAA restoring the membrane potential of cells exposed to indole derivatives for prolonged periods. These protective effects appeared to restore cell physiology, including in the wheat rhizosphere. Together, our data suggest that the IAA biosynthesis pathway plays a major role inA. brasilensephysiology by maintaining membrane potential homeostasis and regulating translation, likely to mitigate the potential membrane-damaging effects of indoles that accumulate during growth under stressful conditions.IMPORTANCEIAA is widely synthesized in bacteria, particularly in soil and rhizosphere bacteria, where it functions as a phytohormone to modulate plant root architecture. IAA as a secondary metabolite has been shown to serve as a signaling molecule in several bacterial species, but the role of IAA biosynthesis in the physiology of the producing bacterium remains seldom explored. Results obtained here suggest that IAA serves to protectA. brasilensefrom the toxic effect of indoles, including metabolite biosynthetic precursors of IAA, on membrane potential homeostasis. Given the widespread production of IAA in soil bacteria, this protective effect of IAA may be conserved in diverse soil bacteria. 

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