Fatty alcohols, a minor component of the tree tobacco surface wax, are associated with defence against caterpillar herbivory.

Despite decades of research resulting in a comprehensive understanding of epicuticular wax metabolism, the function of these almost ubiquitous metabolites in plant-herbivore interactions remains unresolved. In this study, we examined the effects of CRISPR-induced knockout mutations in four Nicotiana glauca (tree tobacco) wax metabolism genes. These mutations cause a wide range of changes in epicuticular wax composition, leading to altered interactions with insects and snails. Three interaction classes were examined: chewing herbivory by seven caterpillars and one snail species, phloem feeding by Myzus persicae (green peach aphid) and oviposition by Bemisia tabaci (whitefly). Although total wax load and alkane abundance did not affect caterpillar growth, a correlation across species, showed that fatty alcohols, a minor component of N. glauca surface waxes, negatively affected the growth of both a generalist caterpillar (Spodoptera littoralis) and a tobacco-feeding specialist (Manduca sexta). This negative correlation was overshadowed by the stronger effect of anabasine, a nicotine isomer, and was apparent when fatty alcohols were added to an artificial lepidopteran diet. By contrast, snails fed more on waxy leaves. Aphid reproduction and feeding activity were unaffected by wax composition but were potentially affected by altered cutin composition. Wax crystal morphology could explain the preference of B. tabaci to lay eggs on waxy wild-type plants relative to both alkane and fatty alcohol-deficient mutants. Together, our results suggest that the varied responses among herbivore classes and species are likely to be a consequence of the co-evolution that shaped the specific effects of different surface wax components in plant-herbivore interactions.

Rhizosphere microbiome mediates systemic root metabolite exudation by root-to-root signaling.

Microbial communities associated with roots confer specific functions to their hosts, thereby modulating plant growth, health, and productivity. Yet, seminal questions remain largely unaddressed including whether and how the rhizosphere microbiome modulates root metabolism and exudation and, consequently, how plants fine tune this complex belowground web of interactions. Here we show that, through a process termed systemically induced root exudation of metabolites (SIREM), different microbial communities induce specific systemic changes in tomato root exudation. For instance, systemic exudation of acylsugars secondary metabolites is triggered by local colonization of bacteria affiliated with the genus Bacillus Moreover, both leaf and systemic root metabolomes and transcriptomes change according to the rhizosphere microbial community structure. Analysis of the systemic root metabolome points to glycosylated azelaic acid as a potential microbiome-induced signaling molecule that is subsequently exuded as free azelaic acid. Our results demonstrate that rhizosphere microbiome assembly drives the SIREM process at the molecular and chemical levels. It highlights a thus-far unexplored long-distance signaling phenomenon that may regulate soil conditioning.

2-oxoglutarate-dependent dioxygenases drive expansion of steroidal alkaloid structural diversity in the genus Solanum.

Solanum steroidal glycoalkaloids (SGAs) are renowned defence metabolites exhibiting spectacular structural diversity. Genes and enzymes generating the SGA precursor pathway, SGA scaffold and glycosylated forms have been largely identified. Yet, the majority of downstream metabolic steps creating the vast repertoire of SGAs remain untapped. Here, we discovered that members of the 2-OXOGLUTARATE-DEPENDENT DIOXYGENASE (2-ODD) family play a prominent role in SGA metabolism, carrying out three distinct backbone-modifying oxidative steps in addition to the three formerly reported pathway reactions. The GLYCOALKALOID METABOLISM34 (GAME34) enzyme catalyses the conversion of core SGAs to habrochaitosides in wild tomato S. habrochaites. Cultivated tomato plants overexpressing GAME34 ectopically accumulate habrochaitosides. These habrochaitoside enriched plants extracts potently inhibit Puccinia spp. spore germination, a significant Solanaceae crops fungal pathogen. Another 2-ODD enzyme, GAME33, acts as a desaturase (via hydroxylation and E/F ring rearrangement) forming unique, yet unreported SGAs. Conversion of bitter α-tomatine to ripe fruit, nonbitter SGAs (e.g. esculeoside A) requires two hydroxylations; while the known GAME31 2-ODD enzyme catalyses hydroxytomatine formation, we find that GAME40 catalyses the penultimate step in the pathway and generates acetoxy-hydroxytomatine towards esculeosides accumulation. Our results highlight the significant contribution of 2-ODD enzymes to the remarkable structural diversity found in plant steroidal specialized metabolism.

Steroidal alkaloids defence metabolism and plant growth are modulated by the joint action of gibberellin and jasmonate signalling.

Steroidal glycoalkaloids (SGAs) are protective metabolites constitutively produced by Solanaceae species. Genes and enzymes generating the vast structural diversity of SGAs have been largely identified. Yet, mechanisms of hormone pathways coordinating defence (jasmonate; JA) and growth (gibberellin; GA) controlling SGAs metabolism remain unclear. We used tomato to decipher the hormonal regulation of SGAs metabolism during growth vs defence tradeoff. This was performed by genetic and biochemical characterisation of different JA and GA pathways components, coupled with in vitro experiments to elucidate the crosstalk between these hormone pathways mediating SGAs metabolism. We discovered that reduced active JA results in decreased SGA production, while low levels of GA or its receptor led to elevated SGA accumulation. We showed that MYC1 and MYC2 transcription factors mediate the JA/GA crosstalk by transcriptional activation of SGA biosynthesis and GA catabolism genes. Furthermore, MYC1 and MYC2 transcriptionally regulate the GA signalling suppressor DELLA that by itself interferes in JA-mediated SGA control by modulating MYC activity through protein-protein interaction. Chemical and fungal pathogen treatments reinforced the concept of JA/GA crosstalk during SGA metabolism. These findings revealed the mechanism of JA/GA interplay in SGA biosynthesis to balance the cost of chemical defence with growth.

Short-chain dehydrogenase/reductase governs steroidal specialized metabolites structural diversity and toxicity in the genus Solanum.

Thousands of specialized, steroidal metabolites are found in a wide spectrum of plants. These include the steroidal glycoalkaloids (SGAs), produced primarily by most species of the genus Solanum, and metabolites belonging to the steroidal saponins class that are widespread throughout the plant kingdom. SGAs play a protective role in plants and have potent activity in mammals, including antinutritional effects in humans. The presence or absence of the double bond at the C-5,6 position (unsaturated and saturated, respectively) creates vast structural diversity within this metabolite class and determines the degree of SGA toxicity. For many years, the elimination of the double bond from unsaturated SGAs was presumed to occur through a single hydrogenation step. In contrast to this prior assumption, here, we show that the tomato GLYCOALKALOID METABOLISM25 (GAME25), a short-chain dehydrogenase/reductase, catalyzes the first of three prospective reactions required to reduce the C-5,6 double bond in dehydrotomatidine to form tomatidine. The recombinant GAME25 enzyme displayed 3ß-hydroxysteroid dehydrogenase/Δ5,4 isomerase activity not only on diverse steroidal alkaloid aglycone substrates but also on steroidal saponin aglycones. Notably, GAME25 down-regulation rerouted the entire tomato SGA repertoire toward the dehydro-SGAs branch rather than forming the typically abundant saturated α-tomatine derivatives. Overexpressing the tomato GAME25 in the tomato plant resulted in significant accumulation of α-tomatine in ripe fruit, while heterologous expression in cultivated eggplant generated saturated SGAs and atypical saturated steroidal saponin glycosides. This study demonstrates how a single scaffold modification of steroidal metabolites in plants results in extensive structural diversity and modulation of product toxicity.

A Multilevel Study of Melon Fruit Reticulation Provides Insight into Skin Ligno-Suberization Hallmarks.

The skin of fleshy fruit is typically covered by a thick cuticle. Some fruit species develop different forms of layers directly above their skin. Reticulation, for example, is a specialized suberin-based coating that ornaments some commercially important melon (Cucumis melo) fruit and is an important quality trait. Despite its importance, the structural, molecular, and biochemical features associated with reticulation are not fully understood. Here, we performed a multilevel investigation of structural attributes, chemical composition, and gene expression profiles on a set of reticulated and smooth skin melons. High-resolution microscopy, surface profiling, and histochemical staining assays show that reticulation comprises cells with heavily suberized walls accumulating large amounts of typical suberin monomers, as well as lignified cells localized underneath the specialized suberized cell layer. Reticulated skin was characterized by induced expression of biosynthetic genes acting in the core phenylpropanoid, suberin, lignin, and lignan pathways. Transcripts of genes associated with lipid polymer assembly, cell wall organization, and loosening were highly enriched in reticulated skin tissue. These signatures were exclusive to reticulated structures and absent in both the smooth surfaces observed in between reticulated regions and in the skin of smooth fruit. Our data provide important insights into the molecular and metabolic bases of reticulation and its tight association with skin ligno-suberization during melon fruit development. Moreover, these insights are likely to contribute to melon breeding programs aimed at improving postharvest qualities associated with fleshy fruit surface layers.

Tissue-specific organic acid metabolism in reproductive and non-reproductive parts of the fig fruit is partially induced by pollination.

Organic acids are important components of overall fruit quality through flavor, taste, nutritional and medicinal values. Pollinated fig (Ficus carica L.) fruit quality is enhanced by increased acidity. We quantified the major organic acids and characterized the expression pattern of organic acid metabolic pathway-related genes in the reproductive part - inflorescence and non-reproductive part - receptacle of parthenocarpic and pollinated fig fruit during ripening. Essentially, pollinated fruit contains seeds in the inflorescence, as opposed to no seeds in the parthenocarpic inflorescence. The major organic acids - citrate and malate - were found in relatively high quantities in the inflorescence compared to the receptacle of both parthenocarpic and pollinated fig fruit. Notably, pollination increased citric acid content significantly in both inflorescence and receptacle. Genes related to the phosphoenolpyruvate carboxylase (PEPC) cycle, tricarboxylic acid cycle, citrate catabolism and glyoxylate cycle were identified in fig fruit. Expression levels of most of these genes were higher in inflorescences than in receptacles. In particular, FcPEPC and FcFUM (encoding fumarase) had significantly higher expression in the inflorescence of pollinated fruit. Most importantly, expression of the glyoxylate cycle genes FcMLS and FcICL (encoding malate synthase and isocitrate lyase, respectively) was induced to strikingly high levels in the inflorescence by pollination, and their expression level was highly positively correlated with the contents of all organic acids. Therefore, the glyoxylate cycle may be responsible for altering the accumulation of organic acids to upgrade the fruit taste during ripening, especially in the pollinated, seeded inflorescence.

Indole Derivatives Maintain the Status Quo Between Beneficial Biofilms and Their Plant Hosts.

Biofilms formed by bacteria on plant roots play an important role in maintaining an optimal rhizosphere environment that supports plant growth and fitness. Bacillus subtilis is a potent plant growth promoter, forming biofilms that play a key role in protecting the host from fungal and bacterial infections. In this work, we demonstrate that the development of B. subtilis biofilms is antagonized by specific indole derivatives that accumulate during symbiotic interactions with plant hosts. Indole derivatives are more potent signals when the plant polysaccharide xylan serves as a carbon source, a mechanism to sustain beneficial biofilms at a biomass that can be supported by the plant. Moreover, B. subtilis biofilms formed by mutants resistant to indole derivatives become deleterious to the plants due to their capacity to consume and recycle plant polysaccharides. These results demonstrate how a dynamic metabolite-based dialogue can promote homeostasis between plant hosts and their beneficial biofilm communities.

AUXIN RESPONSE FACTOR 2 Intersects Hormonal Signals in the Regulation of Tomato Fruit Ripening.

The involvement of ethylene in fruit ripening is well documented, though knowledge regarding the crosstalk between ethylene and other hormones in ripening is lacking. We discovered that AUXIN RESPONSE FACTOR 2A (ARF2A), a recognized auxin signaling component, functions in the control of ripening. ARF2A expression is ripening regulated and reduced in the rin, nor and nr ripening mutants. It is also responsive to exogenous application of ethylene, auxin and abscisic acid (ABA). Over-expressing ARF2A in tomato resulted in blotchy ripening in which certain fruit regions turn red and possess accelerated ripening. ARF2A over-expressing fruit displayed early ethylene emission and ethylene signaling inhibition delayed their ripening phenotype, suggesting ethylene dependency. Both green and red fruit regions showed the induction of ethylene signaling components and master regulators of ripening. Comprehensive hormone profiling revealed that altered ARF2A expression in fruit significantly modified abscisates, cytokinins and salicylic acid while gibberellic acid and auxin metabolites were unaffected. Silencing of ARF2A further validated these observations as reducing ARF2A expression let to retarded fruit ripening, parthenocarpy and a disturbed hormonal profile. Finally, we show that ARF2A both homodimerizes and interacts with the ABA STRESS RIPENING (ASR1) protein, suggesting that ASR1 might be linking ABA and ethylene-dependent ripening. These results revealed that ARF2A interconnects signals of ethylene and additional hormones to co-ordinate the capacity of fruit tissue to initiate the complex ripening process.

Dynamic metabolic reprogramming of steroidal glycol-alkaloid and phenylpropanoid biosynthesis may impart early blight resistance in wild tomato (Solanum arcanum Peralta).

KEY MESSAGE: Exploration with high throughput leaf metabolomics along with functional genomics in wild tomato unreveal potential role of steroidal glyco-alkaloids and phenylpropanoids during early blight resistance. Alternaria solani severely affects tomato (Solanum lycopersicum L.) yield causing early blight (EB) disease in tropical environment. Wild relative, Solanum arcanum Peralta could be a potential source of EB resistance; however, its underlying molecular mechanism largely remains unexplored. Hence, non-targeted metabolomics was applied on resistant and susceptible S. arcanum accessions upon A. solani inoculation to unravel metabolic dynamics during different stages of disease progression. Total 2047 potential metabolite peaks (mass signals) were detected of which 681 and 684 metabolites revealed significant modulation and clear differentiation in resistant and susceptible accessions, respectively. Majority of the EB-triggered metabolic changes were active from steroidal glycol-alkaloid (SGA), lignin and flavonoid biosynthetic pathways. Further, biochemical and gene expression analyses of key enzymes from these pathways positively correlated with phenotypic variation in the S. arcanum accessions indicating their potential role in EB. Additionally, transcription factors regulating lignin biosynthesis were also up-regulated in resistant plants and electrophoretic mobility shift assay revealed sequence-specific binding of rSaWRKY1 with MYB20 promoter. Moreover, transcript accumulation of key genes from phenylpropanoid and SGA pathways along with WRKY and MYB in WRKY1 transgenic tomato lines supported above findings. Overall, this study highlights vital roles of SGAs as phytoalexins and phenylpropanoids along with lignin accumulation unrevealing possible mechanistic basis of EB resistance in wild tomato.

Characterization of a New Pink-Fruited Tomato Mutant Results in the Identification of a Null Allele of the SlMYB12 Transcription Factor.

The identification and characterization of new tomato (Solanum lycopersicum) mutants affected in fruit pigmentation and nutritional content can provide valuable insights into the underlying biology, as well as a source of new alleles for breeding programs. To date, all characterized pink-pigmented tomato fruit mutants appear to result from low SlMYB12 transcript levels in the fruit skin. Two new mutant lines displaying a pink fruit phenotype (pf1 and pf2) were characterized in this study. In the pf mutants, SlMYB12 transcripts accumulated to wild-type levels but exhibited the same truncation, which resulted in the absence of the essential MYB activation domain coding region. Allelism and complementation tests revealed that both pf mutants were allelic to the y locus and showed the same recessive null allele in homozygosis: Δy A set of molecular and metabolic effects, reminiscent of those observed in the Arabidopsis (Arabidopsis thaliana) myb11 myb12 myb111 triple mutant, were found in the tomato Δy mutants. To our knowledge, these have not been described previously, and our data support the idea of their being null mutants, in contrast to previously described transcriptional hypomorphic pink fruit lines. We detected a reduction in the expression of several flavonol glycosides and some associated glycosyl transferases. Transcriptome analysis further revealed that the effects of the pf mutations extended beyond the flavonoid pathway into the interface between primary and secondary metabolism. Finally, screening for Myb-binding sites in the candidate gene promoter sequences revealed that 141 of the 152 co-down-regulated genes may be direct targets of SlMYB12 regulation.

The polyketide synthase OsPKS2 is essential for pollen exine and Ubisch body patterning in rice.

Lipid and phenolic metabolism are important for pollen exine formation. In Arabidopsis, polyketide synthases (PKSs) are essential for both sporopollenin biosynthesis and exine formation. Here, we characterized the role of a polyketide synthase (OsPKS2) in male reproduction of rice (Oryza sativa). Recombinant OsPKS2 catalyzed the condensation of fatty acyl-CoA with malonyl-CoA to generate triketide and tetraketide α-pyrones, the main components of pollen exine. Indeed, the ospks2 mutant had defective exine patterning and was male sterile. However, the mutant showed no significant reduction in sporopollenin accumulation. Compared with the WT (wild type), ospks2 displayed unconfined and amorphous tectum and nexine layers in the exine, and less organized Ubisch bodies. Like the pksb/lap5 mutant of the Arabidopsis ortholog, ospks2 showed broad alterations in the profiles of anther-related phenolic compounds. However, unlike pksb/lap5, in which most detected phenolics were substantially decreased, ospks2 accumulated higher levels of phenolics. Based on these results and our observation that OsPKS2 is unable to fully restore the exine defects in the pksb/lap5, we propose that PKS proteins have functionally diversified during evolution. Collectively, our results suggest that PKSs represent a conserved and diversified biochemical pathway for anther and pollen development in higher plants.

Differential effects of ambient or diminished CO2 and O2 levels on thylakoid membrane structure in light-stressed plants.

Over-reduction of the photosynthetic electron transport chain may severely damage the photosynthetic apparatus as well as other constituents of the chloroplast and the cell. Here, we exposed Arabidopsis leaves to saturating light either under normal atmospheric conditions or under CO2--and O2 -limiting conditions, which greatly increase excitation and electron pressures by draining terminal electron acceptors. The two treatments were found to have very different, often opposing, effects on the structure of the thylakoid membranes, including the width of the granal lumenal compartment. Modulation of the latter is proposed to be related to movements of ions across the thylakoid membrane, which alter the relative osmolarity of the lumen and stroma and affect the partitioning of the proton motive force into its electrical and osmotic components. The resulting changes in thylakoid organization and lumenal width should facilitate the repair of photodamaged photosystem II complexes in response to light stress under ambient conditions, but are expected to inhibit the repair cycle when the light stress occurs concurrently with CO2 and O2 depletion. Under the latter conditions, the changes in thylakoid structure are predicted to complement other processes that restrict the flow of electrons into the high-potential chain, thus moderating the production of deleterious reactive oxygen species at photosystem I.

Altered metabolite accumulation in tomato fruits by coexpressing a feedback-insensitive AroG and the PhODO1 MYB-type transcription factor.

Targeted manipulation of phenylalanine (Phe) synthesis is a potentially powerful strategy to boost biologically and economically important metabolites, including phenylpropanoids, aromatic volatiles and other specialized plant metabolites. Here, we use two transgenes to significantly increase the levels of aromatic amino acids, tomato flavour-associated volatiles and antioxidant phenylpropanoids. Overexpression of the petunia MYB transcript factor, ODORANT1 (ODO1), combined with expression of a feedback-insensitive E. coli 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (AroG), altered the levels of multiple primary and secondary metabolites in tomato fruit, boosting levels of multiple secondary metabolites. Our results indicate that coexpression of AroG and ODO1 has a dual effect on Phe and related biosynthetic pathways: (i) positively impacting tyrosine (Tyr) and antioxidant related metabolites, including ones derived from coumaric acid and ferulic acid; (ii) negatively impacting other downstream secondary metabolites of the Phe pathway, including kaempferol-, naringenin- and quercetin-derived metabolites, as well as aromatic volatiles. The metabolite profiles were distinct from those obtained with either single transgene. In addition to providing fruits that are increased in flavour and nutritional chemicals, coexpression of the two genes provides insights into regulation of branches of phenylpropanoid metabolic pathways.

Orchestration of thiamin biosynthesis and central metabolism by combined action of the thiamin pyrophosphate riboswitch and the circadian clock in Arabidopsis.

Riboswitches are natural RNA elements that posttranscriptionally regulate gene expression by binding small molecules and thereby autonomously control intracellular levels of these metabolites. Although riboswitch-based mechanisms have been examined extensively, the integration of their activity with global physiology and metabolism has been largely overlooked. Here, we explored the regulation of thiamin biosynthesis and the consequences of thiamin pyrophosphate riboswitch deficiency on metabolism in Arabidopsis thaliana. Our results show that thiamin biosynthesis is largely regulated by the circadian clock via the activity of the THIAMIN C SYNTHASE (THIC) promoter, while the riboswitch located at the 3' untranslated region of this gene controls overall thiamin biosynthesis. Surprisingly, the results also indicate that the rate of thiamin biosynthesis directs the activity of thiamin-requiring enzymes and consecutively determines the rate of carbohydrate oxidation via the tricarboxylic acid cycle and pentose-phosphate pathway. Our model suggests that in Arabidopsis, the THIC promoter and the thiamin-pyrophosphate riboswitch act simultaneously to tightly regulate thiamin biosynthesis in a circadian manner and consequently sense and control vital points of core cellular metabolism.

Reconstruction of Arabidopsis metabolic network models accounting for subcellular compartmentalization and tissue-specificity.

Plant metabolic engineering is commonly used in the production of functional foods and quality trait improvement. However, to date, computational model-based approaches have only been scarcely used in this important endeavor, in marked contrast to their prominent success in microbial metabolic engineering. In this study we present a computational pipeline for the reconstruction of fully compartmentalized tissue-specific models of Arabidopsis thaliana on a genome scale. This reconstruction involves automatic extraction of known biochemical reactions in Arabidopsis for both primary and secondary metabolism, automatic gap-filling, and the implementation of methods for determining subcellular localization and tissue assignment of enzymes. The reconstructed tissue models are amenable for constraint-based modeling analysis, and significantly extend upon previous model reconstructions. A set of computational validations (i.e., cross-validation tests, simulations of known metabolic functionalities) and experimental validations (comparison with experimental metabolomics datasets under various compartments and tissues) strongly testify to the predictive ability of the models. The utility of the derived models was demonstrated in the prediction of measured fluxes in metabolically engineered seed strains and the design of genetic manipulations that are expected to increase vitamin E content, a significant nutrient for human health. Overall, the reconstructed tissue models are expected to lay down the foundations for computational-based rational design of plant metabolic engineering. The reconstructed compartmentalized Arabidopsis tissue models are MIRIAM-compliant and are available upon request.

GLYCOALKALOID METABOLISM1 is required for steroidal alkaloid glycosylation and prevention of phytotoxicity in tomato.

Steroidal alkaloids (SAs) are triterpene-derived specialized metabolites found in members of the Solanaceae family that provide plants with a chemical barrier against a broad range of pathogens. Their biosynthesis involves the action of glycosyltransferases to form steroidal glycoalkaloids (SGAs). To elucidate the metabolism of SGAs in the Solanaceae family, we examined the tomato (Solanum lycopersicum) GLYCOALKALOID METABOLISM1 (GAME1) gene. Our findings imply that GAME1 is a galactosyltransferase, largely performing glycosylation of the aglycone tomatidine, resulting in SGA production in green tissues. Downregulation of GAME1 resulted in an almost 50% reduction in α-tomatine levels (the major SGA in tomato) and a large increase in its precursors (i.e., tomatidenol and tomatidine). Surprisingly, GAME1-silenced plants displayed growth retardation and severe morphological phenotypes that we suggest occur as a result of altered membrane sterol levels caused by the accumulation of the aglycone tomatidine. Together, these findings highlight the role of GAME1 in the glycosylation of SAs and in reducing the toxicity of SA metabolites to the plant cell.

A NAC triad modulates plant immunity by negatively regulating N-hydroxy pipecolic acid biosynthesis.

N-hydroxy pipecolic acid (NHP) plays an important role in plant immunity. In contrast to its biosynthesis, our current knowledge with respect to the transcriptional regulation of the NHP pathway is limited. This study commences with the engineering of Arabidopsis plants that constitutively produce high NHP levels and display enhanced immunity. Label-free proteomics reveals a NAC-type transcription factor (NAC90) that is strongly induced in these plants. We find that NAC90 is a target gene of SAR DEFICIENT 1 (SARD1) and induced by pathogen, salicylic acid (SA), and NHP. NAC90 knockout mutants exhibit constitutive immune activation, earlier senescence, higher levels of NHP and SA, as well as increased expression of NHP and SA biosynthetic genes. In contrast, NAC90 overexpression lines are compromised in disease resistance and accumulated reduced levels of NHP and SA. NAC90 could interact with NAC61 and NAC36 which are also induced by pathogen, SA, and NHP. We next discover that this protein triad directly represses expression of the NHP and SA biosynthetic genes AGD2-LIKE DEFENSE RESPONSE PROTEIN 1 (ALD1), FLAVIN MONOOXYGENASE 1 (FMO1), and ISOCHORISMATE SYNTHASE 1 (ICS1). Constitutive immune response in nac90 is abolished once blocking NHP biosynthesis in the fmo1 background, signifying that NAC90 negative regulation of immunity is mediated via NHP biosynthesis. Our findings expand the currently documented NHP regulatory network suggesting a model that together with NHP glycosylation, NAC repressors take part in a 'gas-and-brake' transcriptional mechanism to control NHP production and the plant growth and defense trade-off.

The biosynthetic pathway of the hallucinogen mescaline and its heterologous reconstruction.

Mescaline, among the earliest identified natural hallucinogens, holds great potential in psychotherapy treatment. Nonetheless, despite the existence of a postulated biosynthetic pathway for more than half a century, the specific enzymes involved in this process are yet to be identified. In this study, we investigated the cactus Lophophora williamsii (Peyote), the largest known natural producer of the phenethylamine mescaline. We employed a multi-faceted approach, combining de novo whole-genome and transcriptome sequencing with comprehensive chemical profiling, enzymatic assays, molecular modeling, and pathway engineering for pathway elucidation. We identified four groups of enzymes responsible for the six catalytic steps in the mescaline biosynthetic pathway, and an N-methyltransferase enzyme that N-methylates all phenethylamine intermediates, likely modulating mescaline levels in Peyote. Finally, we reconstructed the mescaline biosynthetic pathway in both Nicotiana benthamiana plants and yeast cells, providing novel insights into several challenges hindering complete heterologous mescaline production. Taken together, our study opens up avenues for exploration of sustainable production approaches and responsible utilization of mescaline, safeguarding this valuable natural resource for future generations.