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25 Pág., Plant specialized metabolites modulate developmental and ecological functions and comprise many therapeutic and other high-value compounds. However, the mechanisms determining their cell-specific expression remain unknown. Here we describe the transcriptional regulatory network that underlies cell-specific biosynthesis of triterpenes in Arabidopsis thaliana root tips. Expression of thalianol and marneral biosynthesis pathway genes depends on the phytohormone jasmonate and is limited to outer tissues. We show that this is promoted by the activity of redundant bHLH-type transcription factors from two distinct clades and coactivated by homeodomain factors. Conversely, the DOF-type transcription factor DAG1 and other regulators prevent expression of the triterpene pathway genes in inner tissues. We thus show how precise expression of triterpene biosynthesis genes is determined by a robust network of transactivators, coactivators and counteracting repressors., This Article was written in loving memory of A. Van Moerkercke (1979–2021). The authors thank A. Bleys for critically reading the manuscript; D. Gasperini for kindly sharing the ProMYCs:NLS-VENUS reporter lines, and P. Vittorioso for the dag1 mutant, ProDAG1:GUS and DAG1 over-expressing lines; J. R. Wendrich and T. Eekhout for assistance in the launching and analysis of the scRNAseq experiment; and S. Desmet and G. Goeminne from the VIB Metabolomics Core – Ghent for the thalianol profiling. This work was supported by the European Community’s Seventh Framework Program (FP7/2007–2013) under grant agreement 613692-TriForC and H2020 Program under grant agreement 760331-Newcotiana to A.G.; the Special Research Fund from Ghent University to A.G. and A.R. (project BOF18/GOA/013), and M.M. (project BOF20/GOA/012); the Flemish Government (AI Research program) to Y.S.; the Research Foundation Flanders with research project grants to A.G. (G004515N and G008417N) and a postdoctoral fellowship to P.F.-C.; a Swiss National Science Foundation postdoctoral fellowship (P300PA_177831) to M.C.; and a China Scholarship Council PhD scholarship to Y.B. A.O. acknowledges funding support from the John Innes Foundation and the BBSRC Institute Strategic Program Grant ‘Molecules from Nature – Products and Pathways’ (BBS/E/J/000PR9790).

Plants often protect themselves from their own bioactive defense metabolites by storing them in less active forms. Consequently, plants also need systems allowing correct spatiotemporal reactivation of such metabolites, for instance under pathogen or herbivore attack. Via co-expression analysis with public transcriptomes, we determined that the model legume Medicago truncatula has evolved a two-component system composed of a β-glucosidase, denominated G1, and triterpene saponins, which are physically separated from each other in intact cells. G1 expression is root-specific, stress-inducible, and coregulated with that of the genes encoding the triterpene saponin biosynthetic enzymes. However, the G1 protein is stored in the nucleolus and is released and united with its typically vacuolar-stored substrates only upon tissue damage, partly mediated by the surfactant action of the saponins themselves. Subsequently, enzymatic removal of carbohydrate groups from the saponins creates a pool of metabolites with an increased broad-spectrum antimicrobial activity. The evolution of this defense system benefited from both the intrinsic condensation abilities of the enzyme and the bioactivity properties of its substrates. We dub this two-component system the saponin bomb, in analogy with the mustard oil and cyanide bombs, commonly used to describe the renowned β-glucosidase-dependent defense systems for glucosinolates and cyanogenic glucosides.

Plants are the most sophisticated biofactories and sources of food and biofuels present in nature. By engineering plant metabolism, the production of desired compounds can be increased and the nutritional or commercial value of the plant species can be improved. However, this can be challenging because of the complexity of the regulation of multiple genes and the involvement of different protein interactions. To improve metabolic engineering (ME) capabilities, different tools and strategies for rerouting the metabolic pathways have been developed, including genome editing and transcriptional regulation approaches. In addition, cutting-edge technologies have provided new methods for understanding uncharacterized biosynthetic pathways, protein degradation mechanisms, protein-protein interactions, or allosteric feedback, enabling the design of novel ME approaches.

Sterols are produced via complex, multistep biosynthetic pathways involving similar enzymatic conversions in plants, animals and fungi, yielding a variety of sterol metabolites with slightly different chemical properties to exert diverse and specific functions. The role of plant sterols has been studied in the context of cell biological processes, signaling and overall plant development, mainly based on mutants. Due to their essential nature, genetic interference with their function causes pleiotropic developmental defects. An important alternative is to use a pharmacological approach. However, the current toolset for manipulating sterol biosynthesis in plants remains limited. Here, we probed a collection of inhibitors of mammalian cholesterol biosynthesis to identify new inhibitors of plant sterol biosynthesis. We provide evidence that imidazole-type fungicides, bifonazole, clotrimazole and econazole inhibit the obtusifoliol 14α-demethylase CYP51, that is highly conserved among eukaryotes. Surprisingly, we found that the selective estrogen receptor modulator, clomiphene, inhibits sterol biosynthesis, in part by inhibiting the plant-specific cyclopropyl-cycloisomerase CPI1. These results demonstrate that rescreening of the animal sterol biosynthesis pharmacology is an easy approach for identifying novel inhibitors of plant sterol biosynthesis. Such molecules can be used as entry points for the development of plant-specific inhibitors of sterol biosynthesis that can be used in agriculture.

The HD-ZIP III transcription factor REVOLUTA (REV) is involved in early leaf development, as well as in leaf senescence. REV directly binds to the promoters of senescence-associated genes, including the central regulator WRKY53. As this direct regulation appears to be restricted to senescence, we aimed to characterize protein-interaction partners of REV which could mediate this senescence-specificity. The interaction between REV and the TIFY family member TIFY8 was confirmed by yeast two-hybrid assays, as well as by bimolecular fluorescence complementation in planta. This interaction inhibited REV’s function as an activator of WRKY53 expression. Mutation or overexpression of TIFY8 accelerated or delayed senescence, respectively, but did not significantly alter early leaf development. Jasmonic acid (JA) had only a limited effect on TIFY8 expression or function; however, REV appears to be under the control of JA signaling. Accordingly, REV also interacted with many other members of the TIFY family, namely the PEAPODs and several JAZ proteins in the yeast system, which could potentially mediate the JA-response. Therefore, REV appears to be under the control of the TIFY family in two different ways: a JA-independent way through TIFY8, which controls REV function in senescence, and a JA-dependent way through PEAPODs and JAZ proteins.

Metabolic enzymes tend to evolve towards catalytic efficacy, precision and speed. This seems particularly true for ancient and conserved enzymes involved in fundamental cellular processes that are present virtually in every cell and organism and converting and producing relatively limited metabolite numbers. Nevertheless, sessile organisms like plants have an astonishing repertoire of specific (specialized) metabolites that, by numbers and chemical complexity, by far exceed primary metabolites. Most theories agree that early gene duplication, subsequent positive selection and diversifying evolution have allowed relaxed selection of duplicated metabolic genes, thus facilitating the accumulation of mutations that could broaden substrate/product specificity and lower activation barriers and kinetics. Here, we use oxylipins, oxygenated fatty acids of plastidial origin to which the phytohormone jasmonate belongs, and triterpenes, a large group of specialized metabolites whose biosynthesis is often elicited by jasmonates, to showcase the structural and functional diversity of chemical signals and products in plant metabolism.

The plant kingdom represents the biggest source of feedstock, food, and added-value compounds. Engineering plant metabolic pathways to increase the phytochemical production or improve the nutraceutical value of crops is challenging because of the intricate interaction networks that link multiple genes, enzymatic steps, and metabolites, even when pathways are fully elucidated. The development of clustered regularly interspaced short palindromic repeats - CRISPR-associated (CRISPR-Cas) technologies has helped to overcome limitations in metabolic engineering, providing efficient and versatile tools for multigene editing. CRISPR approaches in plants were shown to have a remarkable efficiency in genome editing of different species to improve agronomic and metabolic traits. Here, we give an overview of the different achievements and perspectives of CRISPR technology in plant metabolic engineering.

Pathogens produce effectors to overcome plant immunity, thereby threatening crop yields and global food security. Large-scale interactomic studies have revealed that pathogens from different kingdoms of life target common plant proteins during infection, the so-called effector hubs. These hubs often play central roles in numerous plant processes through their ability to interact with multiple plant proteins. This ability arises partly from the presence of intrinsically disordered domains (IDDs) in their structure. Here, we highlight the role of the TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) and JASMONATE-ZIM DOMAIN (JAZ) transcription regulator families as plant signaling and effector hubs. We consider different evolutionary hypotheses to rationalize the existence of diverse effectors sharing common targets and the possible role of IDDs in this interaction.

Transient transformation methods are frequently used to determine gene function. However, until recently only a few methods have been available in the model medicinal plant Catharanthus roseus. Here, we describe a rapid and highly reproducible protocol for the overexpression of genes of interest by agroinfiltration of C. roseus flower petals. This high throughput method is particularly suitable for screening purposes, for instance, target gene screening of transcription factor candidates, and complements other available methods.

Made available in DSpace on 2021-06-25T10:50:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-04-01 John Innes Foundation Seventh Framework Programme European Regional Development Fund Fonds Wetenschappelijk Onderzoek Plant roots are specialized belowground organs that spatiotemporally shape their development in function of varying soil conditions. This root plasticity relies on intricate molecular networks driven by phytohormones, such as auxin and jasmonate (JA). Loss-of-function of the NOVEL INTERACTOR OF JAZ (NINJA), a core component of the JA signaling pathway, leads to enhanced triterpene biosynthesis, in particular of the thalianol gene cluster, in Arabidopsis thaliana roots. We have investigated the biological role of thalianol and its derivatives by focusing on Thalianol Synthase (THAS) and Thalianol Acyltransferase 2 (THAA2), two thalianol cluster genes that are upregulated in the roots of ninja mutant plants. THAS and THAA2 activity was investigated in yeast, and metabolite and phenotype profiling of thas and thaa2 loss-of-function plants was carried out. THAA2 was shown to be responsible for the acetylation of thalianol and its derivatives, both in yeast and in planta. In addition, THAS and THAA2 activity was shown to modulate root development. Our results indicate that the thalianol pathway is not only controlled by phytohormonal cues, but also may modulate phytohormonal action itself, thereby affecting root development and interaction with the environment. Department of Plant Biotechnology and Bioinformatics Ghent University, Technologiepark 71 VIB Center for Plant Systems Biology, Technologiepark 71 Department of Metabolic Biology John Innes Centre, Norwich Research Park, Colney Lane Laboratory of Molecular Cell Biology KU Leuven, Kasteelpark Arenberg 31 VIB Center for Microbiology, Kasteelpark Arenberg 31 Department of Organic Chemistry Institute of Chemistry São Paulo State University (UNESP) Laboratory of Growth Regulators Institute of Experimental Botany of the Czech Academy of Sciences and Faculty of Science of Palacký University, Šlechtitelů 27 Department of Organic Chemistry Ghent University Department of Forest Genetics and Plant Physiology Umeå Plant Science Centre Swedish University of Agricultural Sciences Lab of Plant Growth Analysis Ghent University Global Campus Department of Organic Chemistry Institute of Chemistry São Paulo State University (UNESP) Seventh Framework Programme: 613692 TriForC European Regional Development Fund: CZ.02.1.01/0.0/0.0/17_048/0007323 Fonds Wetenschappelijk Onderzoek: G004515N Fonds Wetenschappelijk Onderzoek: G008417N

Plants constantly perceive internal and external cues, many of which they need to address to safeguard their proper development and survival. They respond to these cues by selective activation of specific metabolic pathways involving a plethora of molecular players that act and interact in complex networks. In this review, we illustrate and discuss the complexity in the combinatorial control of plant specialized metabolism. We hereby go beyond the intuitive concept of combinatorial control as exerted by modular-acting complexes of transcription factors that govern expression of specialized metabolism genes. To extend this discussion, we also consider all known hierarchical levels of regulation of plant specialized metabolism and their interfaces by referring to reported regulatory concepts from the plant field. Finally, we speculate on possible yet-to-be-discovered regulatory principles of plant specialized metabolism that are inspired by knowledge from other kingdoms of life and areas of biological research.

Plants stand out among eukaryotes due to the large variety of sterols and sterol derivatives that they can produce. These metabolites not only serve as critical determinants of membrane structures, but also act as signaling molecules, as growth-regulating hormones, or as modulators of enzyme activities. Therefore, it is critical to understand the wiring of the biosynthetic pathways by which plants generate these distinct sterols, to allow their manipulation and to dissect their precise physiological roles. Here, we review the complexity and variation of the biosynthetic routes of the most abundant phytosterols and cholesterol in the green lineage and how different enzymes in these pathways are conserved and diverged from humans, yeast, and even bacteria. Many enzymatic steps show a deep evolutionary conservation, while others are executed by completely different enzymes. This has important implications for the use and specificity of available human and yeast sterol biosynthesis inhibitors in plants, and argues for the development of plant-tailored inhibitors of sterol biosynthesis.

The use of proximity-dependent biotin labelling (PDL) approaches coupled with mass spectrometry recently greatly advanced the identification of protein-protein interactions and study of protein complexation. PDL is based on the expression of a promiscuous biotin ligase (PBL), e.g. BirA* or a peroxidase fused to a bait protein of interest. In the presence of biotin as substrate, PBL enables covalent biotin labelling of proteins in the vicinity of the PBL-fused bait in vivo , allowing the subsequent capture and identification of interacting and neighbouring proteins without the need for the protein complex to remain intact during purification. To date, PDL has not been extensively used in plants. Here we present the results of a systematic multi-lab study applying a variety of PDL approaches in several plant systems under various conditions and bait proteins. We show that TurboID is the most promiscuous variant for PDL in plants and establish protocols for its efficient application. We demonstrate the applicability of TurboID in capturing membrane protein interactomes using the Lotus japonicus symbiotically active receptor kinases RLKs NOD FACTOR RECEPTOR 5 (NFR5) and LRR-RLK SYMBIOTIC RECEPTOR-KINASE (SYMRK) as test-cases. Furthermore, we benchmark the efficiency of various PBLs using the octameric endocytic TPLATE complex and compare PDL with one-step AP-MS approaches. Our results indicate that different PDL approaches in plants may differ in signal-to-noise ratio and robustness. We present a straightforward strategy to identify both non-biotinylated as well as biotinylated proteins in plants in a single experimental setup. Finally, we provide initial evidence that this technique has potential to infer structural information of protein complexes. Our methods, tools and adjustable pipelines provide a useful resource for the plant research community.

Plants produce specialized metabolites to protect themselves from biotic enemies. Members of the Solanaceae family accumulate phenylpropanoid-polyamine conjugates (PPCs) in response to attackers while also maintaining a chemical barrier of steroidal glycoalkaloids (SGAs). Across the plant kingdom, biosynthesis of such defense compounds is promoted by jasmonate signaling in which clade IIIe basic helix-loop-helix (bHLH) transcription factors play a central role. By characterizing hairy root mutants obtained through Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated protein 9 (CRISPR-Cas9) genome editing, we show that the tomato clade IIIe bHLH transcription factors, MYC1 and MYC2, redundantly control jasmonate-inducible PPC and SGA production, and are also essential for constitutive SGA biosynthesis. Double myc1 myc2 loss-of-function tomato hairy roots displayed suppressed constitutive expression of SGA biosynthesis genes, and severely reduced levels of the main tomato SGAs α-tomatine and dehydrotomatine. In contrast, basal expression of genes involved in PPC biosynthesis was not affected. CRISPR-Cas9(VQR) genome editing of a specific cis-regulatory element, targeted by MYC1/2, in the promoter of a SGA precursor biosynthesis gene led to decreased constitutive expression of this gene, but did not affect its jasmonate inducibility. Our results demonstrate that clade IIIe bHLH transcriptional regulators have evolved under the control of distinct regulatory cues to specifically steer constitutive and stress-inducible specialized metabolism.

Abscisic acid (ABA) is a signaling molecule conserved in plants, bacteria, fungi and animals. Recently, ABA has gained attention for its pharmacological activities and its potential as a biomarker for the severity of chronic obstructive pulmonary disease (COPD) and glioma. This prompts the development of a reliable, sensitive, rapid, and cost-effective method to quantify ABA levels in mammalian cells and tissues. The previously described ABA biosensor system based on the ABA-dependent interaction between the plant ABA receptor PYL1 and co-receptor ABI1 is not sensitive enough for the low ABA levels seen in mammals. Therefore, we optimized this system by replacing PYL1 with other high-affinity plant PYL proteins. The optimized biosensor system engineered with the PYL8 receptor enabled the quantification of ABA at low concentrations in HEK293T cells.

Plants respond to mechanical stimuli to direct their growth and counteract environmental threats. Mechanical stimulation triggers rapid gene expression changes and affects plant appearance (thigmomorphogenesis) and flowering. Previous studies reported the importance of jasmonic acid (JA) in touch signaling. Here, we used reverse genetics to further characterize the molecular mechanisms underlying touch signaling. We show that Piezo mechanosensitive ion channels have no major role in touch-induced gene expression and thigmomorphogenesis. In contrast, the receptor-like kinase Feronia acts as a strong negative regulator of the JA-dependent branch of touch signaling. Last, we show that calmodulin-binding transcriptional activators CAMTA1/2/3 are key regulators of JA-independent touch signaling. CAMTA1/2/3 cooperate to directly bind the promoters and activate gene expression of JA-independent touch marker genes like

Plants often protect themselves from their own bioactive defense metabolites by storing them in less active forms. Consequently, plants also need systems allowing correct spatiotemporal reactivation of such metabolites, for instance under pathogen attack. Here we show that the model legume Medicago truncatula has evolved a two-component system composed of a β-glucosidase and triterpene saponins, which are physically separated from each other in intact cells. The β-glucosidase, which is stored in the nucleolus and subjected to liquid-liquid phase separation in intact cells, is released and united with its substrates only upon tissue damage, partly mediated by the surfactant action of the saponins themselves. Subsequently, enzymatic removal of carbohydrate groups from the saponins creates a pool of metabolites with an increased broad-spectrum antimicrobial activity. The evolution of this peculiar defense system benefited from both the intrinsic condensation abilities of the enzyme and the bioactivity properties of its substrates.

The plant hormone jasmonate (JA) regulates plant immunity and adaptive growth by orchestrating a genome-wide transcriptional program. Key regulators of JA-responsive gene expression include the master transcription factor MYC2, which is repressed by the conserved Groucho/Tup1-like corepressor TOPLESS (TPL) in the resting state. However, the mechanisms underlying TPL-mediated transcriptional repression of MYC2 activity and hormone-dependent switching between repression and de-repression remain enigmatic. Here, we report the regulation of TPL activity and JA signaling by reversible acetylation of TPL. We found that the histone acetyltransferase GCN5 could mediate TPL acetylation, which enhances its interaction with the NOVEL-INTERACTOR-OF-JAZ (NINJA) adaptor and promotes its recruitment to MYC2 target promoters, facilitating transcriptional repression. Conversely, TPL deacetylation by the histone deacetylase HDA6 weakens TPL-NINJA interaction and inhibits TPL recruitment to MYC2 target promoters, facilitating transcriptional activation. In the resting state, the opposing activities of GCN5 and HDA6 maintain TPL acetylation homeostasis, promoting transcriptional repression activity of TPL. In response to JA elicitation, HDA6 expression is transiently induced, resulted in decreased TPL acetylation and repressor activity, thereby transcriptional activation of MYC2 target genes. Thus, the GCN5-TPL-HDA6 module maintains the homeostasis of acetylated TPL, thereby determining the transcriptional state of JA-responsive genes. Our findings uncovered a mechanism by which the TPL corepressor activity in JA signaling is actively tuned in a rapid and reversible manner.

Many fruits and vegetables suffer from unwanted discolorations that reduce product quality, leading to substantial losses along the supply chain. Witloof chicory (

PHYTOCHROME INTERACTING FACTORS (PIFs) are transcription factors that interact with the photoreceptors phytochromes and integrate multiple signaling pathways related to light, temperature, defense and hormone responses. PIFs have been extensively studied inArabidopsis thaliana, but less is known about their roles in other species. Here, we investigate the role of the two homologs of PIF1 found in tomato (Solanum lycopersicum), namely PIF1a and PIF1b. Analysis of gene expression showed very different patterns, indicating a potential evolutionary divergence in their roles. At the protein level, light regulated the stability of PIF1a, but not PIF1b, further supporting a functional divergence. Phenotypic analyses of CRISPR-Cas9-generated tomato mutants defective in PIF1a or PIF1b or both revealed conserved and newly acquired roles compared to Arabidopsis PIF1. Both PIF1a or PIF1b were found to regulate seed germination, photosynthetic pigment biosynthesis and fruit production. However, only PIF1a-defective mutants showed defects on root hair elongation, flowering time and fruit growth and softening. We did not identify any process altered only in plants lacking PIF1b. Together, these data show that neofunctionalization has taken place in tomato, illustrating the potential of these transcription factors to acquiring new roles in different species.

Pieris rapae and Phyllotreta nemorum are Brassicaceae specialists, but do not feed on Iberis amara spp. that contain cucurbitacins. The cucurbitacins are highly oxygenated triterpenoid, occurring widespread in cucurbitaceous species and in a few other plant families. Using de novo assembled transcriptomics from I. amara, gene co-expression analysis and comparative genomics, we unraveled the evolutionary origin of the insect deterrent cucurbitacins in I. amara. Phylogenetic analysis of five oxidosqualene cyclases and heterologous expression allowed us to identify the first committed enzyme in cucurbitacin biosynthesis in I. amara, cucurbitadienol synthase (IaCPQ). In addition, two species-specific cytochrome P450s (CYP708A16 and CYP708A15) were identified that catalyze the unique C16 and C22 hydroxylation of the cucurbitadienol backbone, enzymatic steps that have not been reported before. Furthermore, the draft genome assembly of I. amara showed that the IaCPQ was localized to the same scaffold together with CYP708A15 but spanning over 100 kb, this contrasts with the highly organized cucurbitacin gene cluster in the cucurbits. These results reveal that cucurbitacin biosynthesis has evolved convergently via different biosynthetic routes in different families rather than through divergence from an ancestral pathway. This study thus provides new insight into the mechanism of recurrent evolution and diversification of a plant defensive chemical.

Medicago truncatula is an established model plant, not only for legume biology, but also for the study of specialized metabolism, which serves to create defense barriers and responses to biotic and abiotic external threats. Triterpene saponins (TS) and flavonoids are the two major classes of specialized metabolites in M. truncatula. Here, we provide a detailed overview of TS biosynthesis and regulation and link this to the spatial and functional organization of TS in M. truncatula. Depending on specific oxidations of the triterpene aglycone backbone, M. truncatula TS can be divided further into two types, the hemolytic and non‐hemolytic TS. TS biosynthesis is positively regulated by jasmonate phytohormones, which is mediated, at least in part, through specific basic helix‐loop‐helix transcription factors. Furthermore, TS production is subjected to post‐translational regulation by protein‐protein interactions or endoplasmic reticulum‐associated degradation of key enzymes of the TS biosynthesis pathway, which allows surveying appropriate TS balances.

Mechanical stimuli, such as wind, rain, and touch affect plant development, growth, pest resistance, and ultimately reproductive success. Using water spray to simulate rain, we demonstrate that jasmonic acid (JA) signaling plays a key role in early gene-expression changes, well before it leads to developmental changes in flowering and plant architecture. The JA-activated transcription factors MYC2/MYC3/MYC4 modulate transiently induced expression of 266 genes, most of which peak within 30 min, and control 52% of genes induced >100-fold. Chromatin immunoprecipitation-sequencing analysis indicates that MYC2 dynamically binds >1,300 promoters and trans -activation assays show that MYC2 activates these promoters. By mining our multiomic datasets, we identified a core MYC2/MYC3/MYC4-dependent “regulon” of 82 genes containing many previously unknown MYC2 targets, including transcription factors bHLH19 and ERF109 . bHLH19 can in turn directly activate the ORA47 promoter, indicating that MYC2/MYC3/MYC4 initiate a hierarchical network of downstream transcription factors. Finally, we also reveal that rapid water spray-induced accumulation of JA and JA-isoleucine is directly controlled by MYC2/MYC3/MYC4 through a positive amplification loop that regulates JA-biosynthesis genes.