Exploring the potential role of four Rhizophagus irregularis nuclear effectors: opportunities and technical limitations.

Arbuscular mycorrhizal fungi (AMF) are obligate symbionts that interact with the roots of most land plants. The genome of the AMF model species Rhizophagus irregularis contains hundreds of predicted small effector proteins that are secreted extracellularly but also into the plant cells to suppress plant immunity and modify plant physiology to establish a niche for growth. Here, we investigated the role of four nuclear-localized putative effectors, i.e., GLOIN707, GLOIN781, GLOIN261, and RiSP749, in mycorrhization and plant growth. We initially intended to execute the functional studies in Solanum lycopersicum, a host plant of economic interest not previously used for AMF effector biology, but extended our studies to the model host Medicago truncatula as well as the non-host Arabidopsis thaliana because of the technical advantages of working with these models. Furthermore, for three effectors, the implementation of reverse genetic tools, yeast two-hybrid screening and whole-genome transcriptome analysis revealed potential host plant nuclear targets and the downstream triggered transcriptional responses. We identified and validated a host protein interactors participating in mycorrhization in the host.S. lycopersicum and demonstrated by transcriptomics the effectors possible involvement in different molecular processes, i.e., the regulation of DNA replication, methylglyoxal detoxification, and RNA splicing. We conclude that R. irregularis nuclear-localized effector proteins may act on different pathways to modulate symbiosis and plant physiology and discuss the pros and cons of the tools used.

A tomato B-box protein regulates plant development and fruit quality through the interaction with PIF4, HY5 and RIN transcription factors.

During the last decade, the knowledge about BBX proteins has abruptly increased. Genome-wide studies identified BBX gene family in several ornamental, industry and food crops; however, the reports regarding the role of these genes as regulators of agronomically important traits are scarce. Here, by phenotyping a knockout mutant, we performed a comprehensive functional characterization of the tomato locus Solyc12g089240, hereafter called SlBBX20. The data revealed the encoded protein as a positive regulator of light signaling affecting several physiological processes during plant lifespan. By the inhibition of PHYTOCHROME INTERACTING FACTOR 4 (SlPIF4)-auxin crosstalk, SlBBX20 regulates photomorphogenesis. Later, it controls the balance between cell division and expansion to guarantee the correct vegetative and reproductive development. In fruits, SlBBX20 is transcriptionally induced by the master transcription factor RIPENING INHIBITOR (SlRIN) and, together with ELONGATED HYPOCOTYL 5 (SlHY5), upregulates flavonoids biosynthetic genes. Finally, SlBBX20 promotes the accumulation of steroidal glycoalkaloids and attenuates Botrytis cinerea infection. This work clearly demonstrates that BBX proteins are multilayer regulators of plant physiology, not only because they affect multiple processes along plant development but also regulate other genes at the transcriptional and post-translational levels.

Identification and characterization of CYP71 subclade cytochrome P450 enzymes involved in the biosynthesis of bitterness compounds in Cichorium intybus.

Industrial chicory (Cichorium intybus var. sativum) and witloof (C. intybus var. foliosum) are crops with an important economic value, mainly cultivated for inulin production and as a leafy vegetable, respectively. Both crops are rich in nutritionally relevant specialized metabolites with beneficial effects for human health. However, their bitter taste, caused by the sesquiterpene lactones (SLs) produced in leaves and taproot, limits wider applications in the food industry. Changing the bitterness would thus create new opportunities with a great economic impact. Known genes encoding enzymes involved in the SL biosynthetic pathway are GERMACRENE A SYNTHASE (GAS), GERMACRENE A OXIDASE (GAO), COSTUNOLIDE SYNTHASE (COS) and KAUNIOLIDE SYNTHASE (KLS). In this study, we integrated genome and transcriptome mining to further unravel SL biosynthesis. We found that C. intybus SL biosynthesis is controlled by the phytohormone methyl jasmonate (MeJA). Gene family annotation and MeJA inducibility enabled the pinpointing of candidate genes related with the SL biosynthetic pathway. We specifically focused on members of subclade CYP71 of the cytochrome P450 family. We verified the biochemical activity of 14 C. intybus CYP71 enzymes transiently produced in Nicotiana benthamiana and identified several functional paralogs for each of the GAO, COS and KLS genes, pointing to redundancy in and robustness of the SL biosynthetic pathway. Gene functionality was further analyzed using CRISPR/Cas9 genome editing in C. intybus. Metabolite profiling of mutant C. intybus lines demonstrated a successful reduction in SL metabolite production. Together, this study increases our insights into the C. intybus SL biosynthetic pathway and paves the way for the engineering of C. intybus bitterness.

Cataloguing Protein Complexes In Planta Using TurboID-Catalyzed Proximity Labeling.

Mapping protein-protein interactions is crucial to understand protein function. Recent advances in proximity-dependent biotinylation (BioID) coupled to mass spectrometry (MS) allow the characterization of protein complexes in diverse plant models. Here, we describe the use of BioID in hairy root cultures of tomato and provide detailed information on how to analyze the data obtained by MS.

Structural and functional diversity in plant specialized metabolism signals and products: The case of oxylipins and triterpenes.

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.

A redundant transcription factor network steers spatiotemporal Arabidopsis triterpene synthesis.

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.

Engineering the plant metabolic system by exploiting metabolic regulation.

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.

The Non-JAZ TIFY Protein TIFY8 of Arabidopsis thaliana Interacts with the HD-ZIP III Transcription Factor REVOLUTA and Regulates Leaf Senescence.

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.

The saponin bomb: a nucleolar-localized ß-glucosidase hydrolyzes triterpene saponins in Medicago truncatula.

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 ß-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 ß-glucosidase-dependent defense systems for glucosinolates and cyanogenic glucosides.

Rhizogenic Agrobacterium protein RolB interacts with the TOPLESS repressor proteins to reprogram plant immunity and development.

Rhizogenic Agrobacterium strains comprise biotrophic pathogens that cause hairy root disease (HRD) on hydroponically grown Solanaceae and Cucurbitaceae crops, besides being widely explored agents for the creation of hairy root cultures for the sustainable production of plant-specialized metabolites. Hairy root formation is mediated through the expression of genes encoded on the T-DNA of the root-inducing (Ri) plasmid, of which several, including root oncogenic locus B (rolB), play a major role in hairy root development. Despite decades of research, the exact molecular function of the proteins encoded by the rol genes remains enigmatic. Here, by means of TurboID-mediated proximity labeling in tomato (Solanum lycopersicum) hairy roots, we identified the repressor proteins TOPLESS (TPL) and Novel Interactor of JAZ (NINJA) as direct interactors of RolB. Although these interactions allow RolB to act as a transcriptional repressor, our data hint at another in planta function of the RolB oncoprotein. Hence, by a series of plant bioassays, transcriptomic and DNA-binding site enrichment analyses, we conclude that RolB can mitigate the TPL functioning so that it leads to a specific and partial reprogramming of phytohormone signaling, immunity, growth, and developmental processes. Our data support a model in which RolB manipulates host transcription, at least in part, through interaction with TPL, to facilitate hairy root development. Thereby, we provide important mechanistic insights into this renowned oncoprotein in HRD.

Clustered regularly interspaced short palindromic repeats tools for plant metabolic engineering: achievements and perspectives.

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.

Identification of Plant Protein-Metabolite Interactions by Limited Proteolysis-Coupled Mass Spectrometry (LiP-MS).

The interactions between metabolites and proteins constitute crucial events in cell signaling and metabolism. In recent years, large-scale proteomics techniques have emerged to identify and characterize protein-metabolite interactions. However, their implementation in plants is generally lagging behind, preventing a complete understanding of the regulatory mechanisms governing plant physiology. Recently, a novel approach to identify metabolite-binding proteins, namely, limited proteolysis-coupled mass spectrometry (LiP-MS), was developed originally for microbial proteomes. Here, we present an adapted and accessible version of the LiP-MS protocol for use in plants. Plant proteomes are extracted and incubated with the metabolite of interest or control treatment, followed by a limited digestion by a nonspecific/promiscuous protease. Subsequently, a conventional shotgun proteomics sample preparation is performed including a complete digestion with the sequence-specific protease trypsin. Finally, label-free proteomics analysis is applied to identify structure-dependent proteolytic patterns corresponding to protein targets of the specific metabolite and their binding sites. Given its amenability to relatively high throughput, the LiP-MS approach may open a potent avenue for the discovery of novel regulatory mechanisms in plant species.

Corrigendum: Interference between ER stress-related bZIP-type and jasmonate-inducible bHLH-type transcription factors in the regulation of triterpene saponin biosynthesis in Medicago truncatula.

[This corrects the article DOI: 10.3389/fpls.2022.903793.].

Interference between ER stress-related bZIP-type and jasmonate-inducible bHLH-type transcription factors in the regulation of triterpene saponin biosynthesis in Medicago truncatula.

Triterpene saponins (TS) are a structurally diverse group of metabolites that are widely distributed in plants. They primarily serve as defense compounds and their production is often triggered by biotic stresses through signaling cascades that are modulated by phytohormones such as the jasmonates (JA). Two JA-modulated basic helix-loop-helix (bHLH) transcription factors (TFs), triterpene saponin biosynthesis activating regulator 1 (TSAR1) and TSAR2, have previously been identified as direct activators of TS biosynthesis in the model legume Medicago truncatula. Here, we report on the involvement of the core endoplasmic reticulum (ER) stress-related basic leucine zipper (bZIP) TFs bZIP17 and bZIP60 in the regulation of TS biosynthesis. Expression and processing of M. truncatula bZIP17 and bZIP60 proteins were altered in roots with perturbed TS biosynthesis or treated with JA. Accordingly, such roots displayed an altered ER network structure. M. truncatula bZIP17 and bZIP60 proteins were shown to localize in the nucleus and appeared to be capable of interfering with the TSAR-mediated transactivation of TS biosynthesis genes. Furthermore, interference between ER stress-related bZIP and JA-modulated bHLH TFs in the regulation of JA-dependent terpene biosynthetic pathways may be widespread in the plant kingdom, as we demonstrate that it also occurs in the regulation of monoterpene indole alkaloid biosynthesis in the medicinal plant Catharanthus roseus.

The basic helix-loop-helix transcription factors MYC1 and MYC2 have a dual role in the regulation of constitutive and stress-inducible specialized metabolism in tomato.

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.

Regulation of jasmonate signaling by reversible acetylation of TOPLESS in Arabidopsis.

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.

Engineering a highly sensitive biosensor for abscisic acid in mammalian cells.

Abscisic acid (ABA) is a signalling 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 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.

Transient Gene Expression in Catharanthus roseus Flower Petals Using Agroinfiltration.

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.

Touch signaling and thigmomorphogenesis are regulated by complementary CAMTA3- and JA-dependent pathways.

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 TCH2 and TCH4. In agreement, camta3 mutants show a near complete loss of thigmomorphogenesis and touch-induced delay of flowering. In conclusion, we have now identified key regulators of two independent touch-signaling pathways.

Overview of Witloof Chicory (Cichorium intybus L.) Discolorations and Their Underlying Physiological and Biochemical Causes.

Many fruits and vegetables suffer from unwanted discolorations that reduce product quality, leading to substantial losses along the supply chain. Witloof chicory (Cichorium intybus L. var. foliosum), a specialty crop characterized by its unique bitter taste and crunchiness, is particularly sensitive to various types of red and brown discolorations. The etiolated vegetable suffers from three predominant color disorders, i.e., core browning, internal leaf reddening, and leaf edge browning. Additionally, several less frequently observed color disorders such as hollow pith, external red, and point noir can also negatively affect crop quality. In this article, we bring together fragmented literature and present a comprehensive overview of the different discoloration types in chicory, and discuss their potential underlying physiological causes, including laticifer rupture, calcium deficiency, and a disturbed water distribution. We also describe the role of environmental cues that influence discoloration incidence, including cultivation and postharvest storage conditions such as forcing and storage temperature, root ripeness and the duration of the forcing process. Finally, we zoom in on the underlying biochemical pathways that govern color disorders in witloof chicory, with a strong emphasis on polyphenol oxidase.