The MYB transcription factor Emission of Methyl Anthranilate 1 stimulates emission of methyl anthranilate from Medicago truncatula hairy roots.

Plants respond to herbivore or pathogen attacks by activating specific defense programs that include the production of bioactive specialized metabolites to eliminate or deter the attackers. Volatiles play an important role in the interaction of a plant with its environment. Through transcript profiling of jasmonate-elicited Medicago truncatula cells, we identified Emission of Methyl Anthranilate (EMA) 1, a MYB transcription factor that is involved in the emission of the volatile compound methyl anthranilate when expressed in M. truncatula hairy roots, giving them a fruity scent. RNA sequencing (RNA-Seq) analysis of the fragrant roots revealed the upregulation of a methyltransferase that was subsequently characterized to catalyze the O-methylation of anthranilic acid and was hence named M. truncatula anthranilic acid methyl transferase (MtAAMT) 1. Given that direct activation of the MtAAMT1 promoter by EMA1 could not be unambiguously demonstrated, we further probed the RNA-Seq data and identified the repressor protein M. truncatula plant AT-rich sequence and zinc-binding (MtPLATZ) 1. Emission of Methyl Anthranilate 1 binds a tandem repeat of the ACCTAAC motif in the MtPLATZ1 promoter to transactivate gene expression. Overexpression of MtPLATZ1 in transgenic M. truncatula hairy roots led to transcriptional silencing of EMA1, indicating that MtPLATZ1 may be part of a negative feedback loop to control the expression of EMA1. Finally, application of exogenous methyl anthranilate boosted EMA1 and MtAAMT1 expression dramatically, thus also revealing a positive amplification loop. Such positive and negative feedback loops seem to be the norm rather than the exception in the regulation of plant specialized metabolism.

The protein quality control system manages plant defence compound synthesis.

Jasmonates are ubiquitous oxylipin-derived phytohormones that are essential in the regulation of many development, growth and defence processes. Across the plant kingdom, jasmonates act as elicitors of the production of bioactive secondary metabolites that serve in defence against attackers. Knowledge of the conserved jasmonate perception and early signalling machineries is increasing, but the downstream mechanisms that regulate defence metabolism remain largely unknown. Here we show that, in the legume Medicago truncatula, jasmonate recruits the endoplasmic-reticulum-associated degradation (ERAD) quality control system to manage the production of triterpene saponins, widespread bioactive compounds that share a biogenic origin with sterols. An ERAD-type RING membrane-anchor E3 ubiquitin ligase is co-expressed with saponin synthesis enzymes to control the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the rate-limiting enzyme in the supply of the ubiquitous terpene precursor isopentenyl diphosphate. Thus, unrestrained bioactive saponin accumulation is prevented and plant development and integrity secured. This control apparatus is equivalent to the ERAD system that regulates sterol synthesis in yeasts and mammals but that uses distinct E3 ubiquitin ligases, of the HMGR degradation 1 (HRD1) type, to direct destruction of HMGR. Hence, the general principles for the management of sterol and triterpene saponin biosynthesis are conserved across eukaryotes but can be controlled by divergent regulatory cues.

Isolation of protein complexes from the model legume Medicago truncatula by tandem affinity purification in hairy root cultures.

Tandem affinity purification coupled to mass spectrometry (TAP-MS) is one of the most powerful techniques to isolate protein complexes and elucidate protein interaction networks. Here, we describe the development of a TAP-MS strategy for the model legume Medicago truncatula, which is widely studied for its ability to produce valuable natural products and to engage in endosymbiotic interactions. As biological material, transgenic hairy roots, generated through Agrobacterium rhizogenes-mediated transformation of M. truncatula seedlings, were used. As proof of concept, proteins involved in the cell cycle, transcript processing and jasmonate signalling were chosen as bait proteins, resulting in a list of putative interactors, many of which confirm the interologue concept of protein interactions, and which can contribute to biological information about the functioning of these bait proteins in planta. Subsequently, binary protein-protein interactions among baits and preys, and among preys were confirmed by a systematic yeast two-hybrid screen. Together, by establishing a M. truncatula TAP-MS platform, we extended the molecular toolbox of this model species.

Natural product biosynthesis in Medicago species.

The genus Medicago, a member of the legume (Fabaceae) family, comprises 87 species of flowering plants, including the forage crop M. sativa (alfalfa) and the model legume M. truncatula (barrel medic). Medicago species synthesize a variety of bioactive natural products that are used to engage into symbiotic interactions but also serve to deter pathogens and herbivores. For humans, these bioactive natural products often possess promising pharmaceutical properties. In this review, we focus on the two most interesting and well characterized secondary metabolite classes found in Medicago species, the triterpene saponins and the flavonoids, with a detailed overview of their biosynthesis, regulation, and profiling methods. Furthermore, their biological role within the plant as well as their potential utility for human health or other applications is discussed. Finally, we give an overview of the advances made in metabolic engineering in Medicago species and how the development of novel molecular and omics toolkits can influence a better understanding of this genus in terms of specialized metabolism and chemistry. Throughout, we critically analyze the current bottlenecks and speculate on future directions and opportunities for research and exploitation of Medicago metabolism.

A common F-box gene regulates the leucine homeostasis of Medicago truncatula and Arabidopsis thaliana.

The F-box domain is a conserved structural protein motif that most frequently interacts with the SKP1 protein, the core of the SCFs (SKP1-CULLIN-F-box protein ligase) E3 ubiquitin protein ligases. As part of the SCF complexes, the various F-box proteins recruit substrates for degradation through ubiquitination. In this study, we functionally characterized an F-box gene (MtF-box) identified earlier in a population of Tnt1 retrotransposon-tagged mutants of Medicago truncatula and its Arabidopsis thaliana homolog (AtF-box) using gain- and loss-of-function plants. We highlighted the importance of MtF-box in leaf development of M. truncatula. Protein-protein interaction analyses revealed the 2-isopropylmalate synthase (IPMS) protein as a common interactor partner of MtF-box and AtF-box, being a key enzyme in the biosynthesis pathway of the branched-chain amino acid leucine. For further detailed analysis, we focused on AtF-box and its role during the cell division cycle. Based on this work, we suggest a mechanism for the role of the studied F-box gene in regulation of leucine homeostasis, which is important for growth.

Transcriptional machineries in jasmonate-elicited plant secondary metabolism.

Jasmonates (JAs) act as conserved elicitors of plant secondary metabolism. JA perception triggers extensive transcriptional reprogramming leading to the concerted activation of entire metabolic pathways. This observation inspired numerous quests for 'master' regulators capable of enhancing the production of specific sets of valuable plant metabolites. Many transcription factors (TFs), often JA-activated themselves, with a role in the JA-modulated regulation of metabolism were discovered. At the same time, it became clear that metabolic reprogramming is subject to complex control mechanisms integrated in robust cellular networks. In this review, we discuss current knowledge of the effect of JA-modulated TFs in the elicitation of secondary metabolism in the model plant Arabidopsis (Arabidopsis thaliana) and a range of medicinal plant species with structurally divergent secondary metabolites. We draw parallels with the regulation of secondary metabolism in fungi and consider the remaining challenges to map and exploit the transcriptional machineries that drive JA-mediated elicitation of plant secondary metabolism.