In complex, constantly changing environments, plants have developed astonishing survival strategies. These elaborated strategies rely on rapid and precise gene regulation mediated by transcription ...factors （TFs）. TFs represent a large fraction of plant genomes and among them, MYBs and basic helix-loop-helix （bHLHs） have unique inherent properties specific to plants. Proteins of these two TF families can act as homo- or heterodimers, associate with proteins from other protein families, or form MYB/bHLH complexes to regulate distinct cellular processes. The ability of MYBs and bHLHs to interact with multiple protein part- ners has evolved to keep up with the increased metabolic complexity of multi-cellular organisms. Associ- ation and disassociation of dynamic TF complexes in response to developmental and environmental cues are controlled through a plethora of regulatory mechanisms specifically modulating TF activity. Regulation of TFs at the protein level is critical for efficient and precise control of their activity, and thus provides the mechanistic basis for a rapid on-and-off switch of TF activity. In this review, examples of post-translational modifications, protein-protein interactions, and subcellular mobilization of TFs are discussed with regard to the relevance of these regulatory mechanisms for the specific activation of MYBs and bHLHs in response to a given environmental stimulus.
The glucosinolates (GSLs) is a well-defined group of plant metabolites characterized by having an S-β-d-glucopyrano unit anomerically connected to an O-sulfated (Z)-thiohydroximate function. After ...enzymatic hydrolysis, the sulfated aglucone can undergo rearrangement to an isothiocyanate, or form a nitrile or other products. The number of GSLs known from plants, satisfactorily characterized by modern spectroscopic methods (NMR and MS) by mid-2018, is 88. In addition, a group of partially characterized structures with highly variable evidence counts for approximately a further 49. This means that the total number of characterized GSLs from plants is somewhere between 88 and 137. The diversity of GSLs in plants is critically reviewed here, resulting in significant discrepancies with previous reviews. In general, the well-characterized GSLs show resemblance to C-skeletons of the amino acids Ala, Val, Leu, Trp, Ile, Phe/Tyr and Met, or to homologs of Ile, Phe/Tyr or Met. Insufficiently characterized, still hypothetic GSLs include straight-chain alkyl GSLs and chain-elongated GSLs derived from Leu. Additional reports (since 2011) of insufficiently characterized GSLs are reviewed. Usually the crucial missing information is correctly interpreted NMR, which is the most effective tool for GSL identification. Hence, modern use of NMR for GSL identification is also reviewed and exemplified. Apart from isolation, GSLs may be obtained by organic synthesis, allowing isotopically labeled GSLs and any kind of side chain. Enzymatic turnover of GSLs in plants depends on a considerable number of enzymes and other protein factors and furthermore depends on GSL structure. Identification of GSLs must be presented transparently and live up to standard requirements in natural product chemistry. Unfortunately, many recent reports fail in these respects, including reports based on chromatography hyphenated to MS. In particular, the possibility of isomers and isobaric structures is frequently ignored. Recent reports are re-evaluated and interpreted as evidence of the existence of “isoGSLs”, i.e. non-GSL isomers of GSLs in plants. For GSL analysis, also with MS-detection, we stress the importance of using authentic standards.
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•List of glucosinolates characterized satisfactorily using NMR and MS data, in total 88.•Selected glucosinolate structures deduced indirectly, in total 47.•Review of glucosinolate nomenclature, chemistry, organic synthesis and plant biochemistry.•Review of glucosinolate identification and guidelines for scientific reporting.
Plants evolved in close contact with a myriad of microorganisms, some of which formed associations with their roots, benefitting from carbohydrates and other plant resources. In exchange, they ...evolved to influence important plant functions, e.g. defense against insect herbivores and other antagonists. Here, we test whether a fungus,
Metarhizium brunneum
, which is mostly known as an insect pathogen, can also associate with plant roots and contribute to above-ground plant defense. Cauliflower (
Brassica oleracea
var. botrytis) seeds were sown together with
M. brunneum
-inoculated rice grains, and the resulting plants subjected to leaf herbivory by the specialist
Plutella xylostella.
Activity of myrosinases, the enzymes activating glucosinolates, was measured before and after herbivory; larval consumption and plant weight at the end of experiments.
Metarhizium brunneum
clearly established in the plant roots, and after herbivory myrosinase activity was substantially higher in
M. brunneum
-treated plants than in controls; before herbivory,
M. brunneum
-treated and control plants did not differ. Leaf consumption was slightly lower in the
M. brunneum
-treated plants whereas total biomass and allocation to above- or below-ground parts was not affected by the
Metarhizium
treatment. Thus,
M. brunneum
associates with roots and primes the plant for a stronger or faster increase in myrosinase activity upon herbivory. Consistent with this, myrosinase function has been suggested to be rate-limiting for induction of the glucosinolate-myrosinase defense system. Our results show that
M. brunneum
, in addition to being an insect pathogen, can associate with plant roots and prime plant defense.
Summary
Although ammonium (NH4+) is a key intermediate of plant nitrogen metabolism, high concentrations of NH4+ in the soil provoke physiological disorders that lead to the development of stress ...symptoms.
Ammonium nutrition was shown to induce the accumulation of glucosinolates (GSLs) in leaves of different Brassicaceae species. To further understand the link between ammonium nutrition and GSLs, we analysed the ammonium stress response of Arabidopsis mutants impaired in GSL metabolic pathway.
We showed that the MYB28 and MYB29 double mutant (myb28myb29), which is almost deprived of aliphatic GSLs, is highly hypersensitive to ammonium nutrition. Moreover, we evidenced that the stress symptoms developed were not a consequence of the lack of aliphatic GSLs. Transcriptomic analysis highlighted the induction of an iron (Fe) deficiency response in myb28myb29 under ammonium nutrition. Consistently, ammonium‐grown myb28myb29 plants showed altered Fe accumulation and homeostasis. Interestingly, we showed overall that growing Arabidopsis with increased Fe availability relieved ammonium stress symptoms and that this was associated with MYB28 and MYB29 expression.
Taken together, our data indicated that the control of Fe homeostasis was crucial for the Arabidopsis response to ammonium nutrition and evidenced that MYB28 and MYB29 play a role in this control.
Dynamically changing environmental conditions promote a complex regulation of plant metabolism and balanced resource investments to development and defense. Plants of the Brassicales order ...constitutively allocate carbon, nitrogen, and sulfur to synthesize glucosinolates as their primary defense metabolites. Previous findings support a model in which steady-state levels of glucosinolates in intact tissues are determined by biosynthesis and turnover through a yet uncharacterized turnover pathway. To investigate glucosinolate turnover in the absence of tissue damage, we quantified exogenously applied allyl glucosinolate and endogenous glucosinolates under different nutrient conditions. Our data shows that, in seedlings of
accession Columbia-0, glucosinolate biosynthesis and turnover are coordinated according to nutrient availability. Whereas exogenous carbon sources had general quantitative effects on glucosinolate accumulation, sulfur or nitrogen limitation resulted in distinct changes in glucosinolate profiles, indicating that these macronutrients provide different regulatory inputs. Raphanusamic acid, a breakdown product that can potentially be formed from all glucosinolate structures appears not to reflect
turnover rates, but instead correlates with increased accumulation of endogenous glucosinolates. Thus, raphanusamic acid could represent a metabolic checkpoint that allows glucosinolate-producing plants to measure the flux through the biosynthetic and/or turnover pathways and thereby to dynamically adjust glucosinolate accumulation in response to internal and external signals.
To optimize fitness a plant should monitor its metabolism to appropriately control growth and defense. Primary metabolism can be measured by the universally conserved TOR (Target of Rapamycin) ...pathway to balance growth and development with the available energy and nutrients. Recent work suggests that plants may measure defense metabolites to potentially provide a strategy ensuring fast reallocation of resources to coordinate plant growth and defense. There is little understanding of mechanisms enabling defense metabolite signaling. To identify mechanisms of defense metabolite signaling, we used glucosinolates, an important class of plant defense metabolites. We report novel signaling properties specific to one distinct glucosinolate, 3-hydroxypropylglucosinolate across plants and fungi. This defense metabolite, or derived compounds, reversibly inhibits root growth and development. 3-hydroxypropylglucosinolate signaling functions via genes in the ancient TOR pathway. If this event is not unique, this raises the possibility that other evolutionarily new plant metabolites may link to ancient signaling pathways.
Glucosinolates constitute the primary defense metabolites in Arabidopsis thaliana (Arabidopsis). Indole and aliphatic glucosinolates, biosynthesized from tryptophan and methionine, respectively, are ...known to serve distinct biological functions. Although all genes in the biosynthetic pathways are identified, and it is known where glucosinolates are stored, it has remained elusive where glucosinolates are produced at the cellular and tissue level. To understand how the spatial organization of the different glucosinolate biosynthetic pathways contributes to their distinct biological functions, we investigated the localization of enzymes of the pathways under constitutive conditions and, for indole glucosinolates, also under induced conditions, by analyzing the spatial distribution of several fluorophore‐tagged enzymes at the whole plant and the cellular level. We show that key steps in the biosynthesis of the different types of glucosinolates are localized in distinct cells in separate as well as overlapping vascular tissues. The presence of glucosinolate biosynthetic enzymes in parenchyma cells of the vasculature may assign new defense‐related functions to these cell types. The knowledge gained in this study is an important prerequisite for understanding the orchestration of chemical defenses from site of synthesis to site of storage and potential (re)mobilization upon attack.
While R2R3 MYB transcription factors are a large gene family of transcription factors within plants, comprehensive functional data in planta are still scarce. A model for studying R2R3 MYB control of ...metabolic networks is the glucosinolates (GLSs), secondary metabolites that control plant resistance against insects and pathogens and carry cancer-preventive properties. Three related members of the R2R3 MYB transcription factor family within Arabidopsis (Arabidopsis thaliana), MYB28, MYB29, and MYB76, are the commonly defined regulators of aliphatic GLS biosynthesis. We utilized new genotypes and systems analysis techniques to test the existing regulatory model in which MYB28 is the dominant regulator, MYB29 plays a minor rheostat role, and MYB76 is largely uninvolved. We unequivocally show that MYB76 is not dependent on MYB28 and MYB29 for induction of aliphatic GLSs and that MYB76 plays a role in determining the spatial distribution of aliphatic GLSs within the leaf, pointing at a potential role of MYB76 in transport regulation. Transcriptional profiling of knockout mutants revealed that GLS metabolite levels are uncoupled from the level of transcript accumulation for aliphatic GLS biosynthetic genes. This uncoupling of chemotypes from biosynthetic transcripts suggests revising our view of the regulation of GLS metabolism from a simple linear transcription factor-promoter model to a more modular system in which transcription factors cause similar chemotypes via nonoverlapping regulatory patterns. Similar regulatory networks might exist in other secondary pathways.
Cytochrome P450s (P450s) are key enzymes in the synthesis of bioactive natural products in plants. Efforts to harness these enzymes for in vitro and whole-cell production of natural products have ...been hampered by difficulties in expressing them heterologously in their active form, and their requirement for NADPH as a source of reducing power. We recently demonstrated targeting and insertion of plant P450s into the photosynthetic membrane and photosynthesis-driven, NADPH-independent P450 catalytic activity mediated by the electron carrier protein ferredoxin. Here, we report the fusion of ferredoxin with P450 CYP79A1 from the model plant Sorghum bicolor, which catalyzes the initial step in the pathway leading to biosynthesis of the cyanogenic glucoside dhurrin. Fusion with ferredoxin allows CYP79A1 to obtain electrons for catalysis by interacting directly with photosystem I. Furthermore, electrons captured by the fused ferredoxin moiety are directed more effectively toward P450 catalytic activity, making the fusion better able to compete with endogenous electron sinks coupled to metabolic pathways. The P450-ferredoxin fusion enzyme obtains reducing power solely from its fused ferredoxin and outperforms unfused CYP79A1 in vivo. This demonstrates greatly enhanced electron transfer from photosystem I to CYP79A1 as a consequence of the fusion. The fusion strategy reported here therefore forms the basis for enhanced partitioning of photosynthetic reducing power toward P450-dependent biosynthesis of important natural products.
The glucosinolate-myrosinase system is an activated chemical defense system found in plants of the Brassicales order. Glucosinolates are stored separately from their hydrolytic enzymes, the ...myrosinases, in plant tissues. Upon tissue damage, e.g. by herbivory, glucosinolates and myrosinases get mixed and glucosinolates are broken down to an array of biologically active compounds of which isothiocyanates are toxic to a wide range of organisms. Specifier proteins occur in some, but not all glucosinolate-containing plants and promote the formation of biologically active non-isothiocyanate products upon myrosinase-catalyzed glucosinolate breakdown.
Based on a phytochemical screening among representatives of the Brassicales order, we selected candidate species for identification of specifier protein cDNAs. We identified ten specifier proteins from a range of species of the Brassicaceae and assigned each of them to one of the three specifier protein types (NSP, nitrile-specifier protein, ESP, epithiospecifier protein, TFP, thiocyanate-forming protein) after heterologous expression in Escherichia coli. Together with nine known specifier proteins and three putative specifier proteins found in databases, we subjected the newly identified specifier proteins to phylogenetic analyses. Specifier proteins formed three major clusters, named AtNSP5-cluster, AtNSP1-cluster, and ESP/TFP cluster. Within the ESP/TFP cluster, specifier proteins grouped according to the Brassicaceae lineage they were identified from. Non-synonymous vs. synonymous substitution rate ratios suggested purifying selection to act on specifier protein genes.
Among specifier proteins, NSPs represent the ancestral activity. The data support a monophyletic origin of ESPs from NSPs. The split between NSPs and ESPs/TFPs happened before the radiation of the core Brassicaceae. Future analyses have to show if TFP activity evolved from ESPs at least twice independently in different Brassicaceae lineages as suggested by the phylogeny. The ability to form non-isothiocyanate products by specifier protein activity may provide plants with a selective advantage. The evolution of specifier proteins in the Brassicaceae demonstrates the plasticity of secondary metabolism within an activated plant defense system.
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