GAME9 regulates the biosynthesis of steroidal alkaloids and upstream isoprenoids in the plant mevalonate pathway

Steroidal glycoalkaloids (SGAs) are cholesterol-derived molecules produced by solanaceous species. They contribute to pathogen defence but are toxic to humans and considered as anti-nutritional compounds. Here we show that GLYCOALKALOID METABOLISM 9 (GAME9), an APETALA2/Ethylene Response Factor, related to regulators of alkaloid production in tobacco and Catharanthus roseus, controls SGA biosynthesis. GAME9 knockdown and overexpression in tomato and potato alters expression of SGAs and upstream mevalonate pathway genes including the cholesterol biosynthesis gene STEROL SIDE CHAIN REDUCTASE 2 (SSR2). Levels of SGAs, C24-alkylsterols and the upstream mevalonate and cholesterol pathways intermediates are modified in these plants. Delta(7)-STEROL-C5(6)-DESATURASE (C5-SD) in the hitherto unresolved cholesterol pathway is a direct target of GAME9. Transactivation and promoter-binding assays show that GAME9 exerts its activity either directly or cooperatively with the SlMYC2 transcription factor as in the case of the C5-SD gene promoter. Our findings provide insight into the regulation of SGA biosynthesis and means for manipulating these metabolites in crops

Altered Levels of Aroma and Volatiles by Metabolic Engineering of Shikimate Pathway Genes in Tomato Fruits

The tomato (Solanum lycopersicum) fruit is an excellent source of antioxidants, dietary fibers, minerals and vitamins and therefore has been referred to as a “functional food”. Ripe tomato fruits produce a large number of specialized metabolites including volatile organic compounds. These volatiles serve as key components of the tomato fruit flavor, participate in plant pathogen and herbivore defense, and are used to attract seed dispersers. A major class of specialized metabolites is derived from the shikimate pathway followed by aromatic amino acid biosynthesis of phenylalanine, tyrosine and tryptophan. We attempted to modify tomato fruit flavor by overexpressing key regulatory genes in the shikimate pathway. Bacterial genes encoding feedback-insensitive variants of 3-Deoxy-D-Arabino-Heptulosonate 7-Phosphate Synthase (DAHPS; AroG209-9) and bi-functional Chorismate Mutase/Prephenate Dehydratase (CM/PDT; PheA12) were expressed under the control of a fruit-specific promoter. We crossed these transgenes to generate tomato plants expressing both the AroG209 and PheA12 genes. Overexpression of the AroG209-9 gene had a dramatic effect on the overall metabolic profile of the fruit, including enhanced levels of multiple volatile and non-volatile metabolites. In contrast, the PheA12 overexpression line exhibited minor metabolic effects compared to the wild type fruit. Co-expression of both the AroG209-9 and PheA12 genes in tomato resulted overall in a similar metabolic effect to that of expressing only the AroG209-9 gene. However, the aroma ranking attributes of the tomato fruits from PheA12//AroG209-9 were unique and different from those of the lines expressing a single gene, suggesting a contribution of the PheA12 gene to the overall metabolic profile. We suggest that expression of bacterial genes encoding feedback-insensitive enzymes of the shikimate pathway in tomato fruits provides a useful metabolic engineering tool for the modification of fruits aroma and the generation of new combinations of tomato flavors

The WEIZMASS spectral library for high-confidence metabolite identification

Annotation of metabolites is an essential, yet problematic, aspect of mass spectrometry (MS)-based metabolomics assays. The current repertoire of definitive annotations of metabolite spectra in public MS databases is limited and suffers from lack of chemical and taxonomic diversity. Furthermore, the heterogeneity of the data prevents the development of universally applicable metabolite annotation tools. Here we present a combined experimental and computational platform to advance this key issue in metabolomics. WEIZMASS is a unique reference metabolite spectral library developed from high-resolution MS data acquired from a structurally diverse set of 3,540 plant metabolites. We also present MatchWeiz, a multi-module strategy using a probabilistic approach to match library and experimental data. This strategy allows efficient and high-confidence identification of dozens of metabolites in model and exotic plants, including metabolites not previously reported in plants or found in few plant species to date.</p

DataSheet1_Metabolic diversity in a collection of wild and cultivated Brassica rapa subspecies.zip

Brassica rapa (B. rapa) and its subspecies contain many bioactive metabolites that are important for plant defense and human health. This study aimed at investigating the metabolite composition and variation among a large collection of B. rapa genotypes, including subspecies and their accessions. Metabolite profiling of leaves of 102 B. rapa genotypes was performed using ultra-performance liquid chromatography coupled with a photodiode array detector and quadrupole time-of-flight mass spectrometry (UPLC-PDA-QTOF-MS/MS). In total, 346 metabolites belonging to different chemical classes were tentatively identified; 36 out of them were assigned with high confidence using authentic standards and 184 were those reported in B. rapa leaves for the first time. The accumulation and variation of metabolites among genotypes were characterized and compared to their phylogenetic distance. We found 47 metabolites, mostly representing anthocyanins, flavonols, and hydroxycinnamic acid derivatives that displayed a significant correlation to the phylogenetic relatedness and determined four major phylometabolic branches; 1) Chinese cabbage, 2) yellow sarson and rapid cycling, 3) the mizuna-komatsuna-turnip-caitai; and 4) a mixed cluster. These metabolites denote the selective pressure on the metabolic network during B. rapa breeding. We present a unique study that combines metabolite profiling data with phylogenetic analysis in a large collection of B. rapa subspecies. We showed how selective breeding utilizes the biochemical potential of wild B. rapa leading to highly diverse metabolic phenotypes. Our work provides the basis for further studies on B. rapa metabolism and nutritional traits improvement.</p

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

[EN] 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: Delta y. 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 Delta y 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.We are grateful to Prof. Daisuke Shibata and Prof. Tohru Ariizumi for providing the pink fruit mutant lines. We thank Justin Lashbrooke for his collaboration in establishing optimal conditions in order to obtain the RNA-Seq cDNA libraries in tomato fruit. We thank Shelly Hen-Avivi for growing the mutant and wild plant types. We are also grateful to Dr. Kristina Gruden for providing us with the tomato mapping file to identify the genes involved in primary metabolism by using the online GoMapMan tool. We thank the Bioinformatics Core Service at the IBMCP for the support provided. Our special thanks to Joss Rose for his comments and careful editing of the final version of the manuscript.Fernández Moreno, JP.; Tzfadia, O.; Forment Millet, JJ.; Presa Castro, S.; Rogachev, I.; Meir, S.; Orzáez Calatayud, DV.... (2016). Characterization of a New Pink-Fruited Tomato Mutant Results in the Identification of a Null Allele of the SlMYB12 Transcription Factor. PLANT PHYSIOLOGY. 171(3):1821-1836. doi:10.1104/pp.16.00282S18211836171

A BAHD-type acyltransferase concludes the biosynthetic pathway of non-bitter glycoalkaloids in ripe tomato fruit

Abstract Tomato is the highest value fruit and vegetable crop worldwide, yet produces α-tomatine, a renowned toxic and bitter-tasting anti-nutritional steroidal glycoalkaloid (SGA) involved in plant defense. A suite of modifications during tomato fruit maturation and ripening converts α-tomatine to the non-bitter and less toxic Esculeoside A. This important metabolic shift prevents bitterness and toxicity in ripe tomato fruit. While the enzymes catalyzing glycosylation and hydroxylation reactions in the Esculeoside A pathway have been resolved, the proposed acetylating step remains, to date, elusive. Here, we discovered that GAME36 (GLYCOALKALOID METABOLISM36), a BAHD-type acyltransferase catalyzes SGA-acetylation in cultivated and wild tomatoes. This finding completes the elucidation of the core Esculeoside A biosynthetic pathway in ripe tomato, allowing reconstitution of Esculeoside A production in heterologous microbial and plant hosts. The involvement of GAME36 in bitter SGA detoxification pathway points to a key role in the evolution of sweet-tasting tomato as well as in the domestication and breeding of modern cultivated tomato fruit

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

Uncovering Pathways Highly Correlated to NUE through a Combined Metabolomics and Transcriptomics Approach in Eggplant

Nitrogen (N) fertilization is one of the main inputs to increase crop yield and food production. However, crops utilize only 30&ndash;40% of N applied; the remainder is leached into the soil, causing environmental and health damage. In this scenario, the improvement of nitrogen-use efficiency (NUE) will be an essential strategy for sustainable agriculture. Here, we compared two pairs of NUE-contrasting eggplant (Solanum melongena L.) genotypes, employing GC-MS and UPLC-qTOF-MS-based technologies to determine the differential profiles of primary and secondary metabolites in root and shoot tissues, under N starvation as well as at short- and long-term N-limiting resupply. Firstly, differences in the primary metabolism pathways of shoots related to alanine, aspartate and glutamate; starch, sucrose and glycine; serine and threonine; and in secondary metabolites biosynthesis were detected. An integrated analysis between differentially accumulated metabolites and expressed transcripts highlighted a key role of glycine accumulation and the related glyA transcript in the N-use-efficient genotypes to cope with N-limiting stress. Interestingly, a correlation between both sucrose synthase (SUS)- and fructokinase (scrK)-transcript abundances, as well as D-glucose and D-fructose accumulation, appeared useful to distinguish the N-use-efficient genotypes. Furthermore, increased levels of L-aspartate and L-asparagine in the N-use-efficient genotypes at short-term low-N exposure were detected. Granule-bound starch synthase (WAXY) and endoglucanase (E3.2.1.4) downregulation at long-term N stress was observed. Therefore, genes and metabolites related to these pathways could be exploited to improve NUE in eggplant