Growth conditions trigger genotype-specific metabolic responses that affect the nutritional quality of kale cultivars.

Kales (Brassica oleracea convar acephala) are fast-growing, nutritious leafy vegetables ideal for year-round indoor farming. However, selection of best cultivars for growth under artificial lighting necessitates a deeper understanding of leaf metabolism in different kale types. Here we examined a curly leaved cultivar Half Tall and a lacinato type cultivar Black Magic under moderate growth light (130 µmol photons m-1s-1/22°C) and high light (800 µmol photons m-1s-1/26°C) conditions. These conditions induced genotype-dependent differences in nutritionally important metabolites, especially anthocyanins and glucosinolates (GSLs), in the kale cultivars. In the pale green Half Tall, growth under high light conditions did not induce changes in either pigmentation or total GSL content. In contrast, the purple pigmentation of Black Magic intensified due to increased anthocyanin accumulation. Black Magic showed reduced amounts of indole GSLs and increased amounts of aliphatic GSLs under high light conditions, with notable cultivar-specific adjustments in individual GSL species. Correlation analysis of metabolite profiles suggested cultivar-specific metabolic interplay between serine biosynthesis and the production of indole GSLs. RNA sequencing identified candidate genes encoding metabolic enzymes and regulatory components behind anthocyanin and GSL biosynthesis. These findings improve the understanding of leaf metabolism and its effects on the nutritional quality of kale cultivars.

Cryptococcus neoformans: plant-microbe interactions and ecology.

While the opportunistic human pathogens Cryptococcus neoformans and Cryptococcus gattii are often isolated from plants and plant-related material, evidence suggests that these Cryptococcus species do not directly infect plants. Studies find that plants are important for Cryptococcus mating and dispersal. However, these studies have not provided enough detail about how plants and these fungi interact, especially in ways that could show the fungi are capable of causing disease. This review synthesizes recent findings from studies utilizing different plant models associated with the ecology of C. neoformans and C. gattii. Unanswered questions about their environmental role are highlighted. Overall, current research indicates that Cryptococcus utilizes plants as a substrate rather than harming them, arguing against Cryptococcus as a genuine plant pathogen. We hypothesize that plants represent reservoirs that aid dispersal, not hosts vulnerable to infection.

Heterologous expression of PtAAS1 reveals the metabolic potential of the common plant metabolite phenylacetaldehyde for auxin synthesis in planta.

Aromatic aldehydes and amines are common plant metabolites involved in several specialized metabolite biosynthesis pathways. Recently, we showed that the aromatic aldehyde synthase PtAAS1 and the aromatic amino acid decarboxylase PtAADC1 contribute to the herbivory-induced formation of volatile 2-phenylethanol and its glucoside 2-phenylethyl-ß-D-glucopyranoside in Populus trichocarpa. To unravel alternative metabolic fates of phenylacetaldehyde and 2-phenylethylamine beyond alcohol and alcohol glucoside formation, we heterologously expressed PtAAS1 and PtAADC1 in Nicotiana benthamiana and analyzed plant extracts using untargeted LC-qTOF-MS and targeted LC-MS/MS analysis. While the metabolomes of PtAADC1-expressing plants did not significantly differ from those of control plants, expression of PtAAS1 resulted in the accumulation of phenylacetic acid (PAA) and PAA-amino acid conjugates, identified as PAA-aspartate and PAA-glutamate. Herbivory-damaged poplar leaves revealed significantly induced accumulation of PAA-Asp, while levels of PAA remained unaltered upon herbivory. Transcriptome analysis showed that members of auxin-amido synthetase GH3 genes involved in the conjugation of auxins with amino acids were significantly upregulated upon herbivory in P. trichocarpa leaves. Overall, our data indicates that phenylacetaldehyde generated by poplar PtAAS1 serves as a hub metabolite linking the biosynthesis of volatile, non-volatile herbivory-induced specialized metabolites, and phytohormones, suggesting that plant growth and defense can be balanced on a metabolic level.

Glucosinolate Catabolism Maintains Glucosinolate Profiles and Transport in Sulfur-Starved Arabidopsis.

Glucosinolates (GSLs) are sulfur (S)-rich specialized metabolites present in Brassicales order plants. Our previous study found that GSL can function as a S source in Arabidopsis seedlings via its catabolism catalyzed by two ß-glucosidases (BGLUs), BGLU28 and BGLU30. However, as GSL profiles in plants vary among growth stages and organs, the potential contribution of BGLU28/30-dependent GSL catabolism at the reproductive growth stage needs verification. Thus, in this study, we assessed growth, metabolic and transcriptional phenotypes of mature bglu28/30 double mutants grown under different S conditions. Our results showed that compared to wild-type plants grown under -S, mature bglu28/30 mutants displayed impaired growth and accumulated increased levels of GSL in their reproductive organs and rosette leaves of before-bolting plants. In contrast, the levels of primary S-containing metabolites, glutathione and cysteine decreased in their mature seeds. Furthermore, the transport of GSL from rosette leaves to the reproductive organs was stimulated in the bglu28/30 mutants under -S. Transcriptome analysis revealed that genes related to other biological processes, such as ethylene response, defense response and plant response to heat, responded differentially to -S in the bglu28/30 mutants. Altogether, these findings broadened our understanding of the roles of BGLU28/30-dependent GSL catabolism in plant adaptation to nutrient stress.

Phytoalexins of the crucifer Barbarea vulgaris: Structural profile and correlation with glucosinolate turnover.

Phytoalexins are antimicrobial plant metabolites elicited by microbial attack or abiotic stress. We investigated phytoalexin profiles after foliar abiotic elicitation in the crucifer Barbarea vulgaris and interactions with the glucosinolate-myrosinase system. The treatment for abiotic elicitation was a foliar spray with CuCl2 solution, a usual eliciting agent, and three independent experiments were carried out. Two genotypes of B. vulgaris (G-type and P-type) accumulated the same three major phytoalexins in rosette leaves after treatment: phenyl-containing nasturlexin D and indole-containing cyclonasturlexin and cyclobrassinin. Phytoalexin levels were investigated daily by UHPLC-QToF MS and tended to differ among plant types and individual phytoalexins. In roots, phytoalexins were low or not detected. In treated leaves, typical total phytoalexin levels were in the range 1-10 nmol/g fresh wt. during three days after treatment while typical total glucosinolate (GSL) levels were three orders of magnitude higher. Levels of some minor GSLs responded to the treatment: phenethylGSL (PE) and 4-substituted indole GSLs. Levels of PE, a suggested nasturlexin D precursor, were lower in treated plants than controls. Another suggested precursor GSL, 3-hydroxyPE, was not detected, suggesting PE hydrolysis to be a key biosynthetic step. Levels of 4-substituted indole GSLs differed markedly between treated and control plants in most experiments, but not in a consistent way. The dominant GSLs, glucobarbarins, are not believed to be phytoalexin precursors. We observed statistically significant linear correlations between total major phytoalexins and the glucobarbarin products barbarin and resedine, suggesting that GSL turnover for phytoalexin biosynthesis was unspecific. In contrast, we did not find correlations between total major phytoalexins and raphanusamic acid or total glucobarbarins and barbarin. In conclusion, two groups of phytoalexins were detected in B. vulgaris, apparently derived from the GSLs PE and indol-3-ylmethylGSL. Phytoalexin biosynthesis was accompanied by depletion of the precursor PE and by turnover of major non-precursor GSLs to resedine. This work paves the way for identifying and characterizing genes and enzymes in the biosyntheses of phytoalexins and resedine.

Polygenic pathogen networks influence transcriptional plasticity in the Arabidopsis-Botrytis pathosystem.

Bidirectional flow of information shapes the outcome of the host-pathogen interactions and depends on the genetics of each organism. Recent work has begun to use co-transcriptomic studies to shed light on this bidirectional flow, but it is unclear how plastic the co-transcriptome is in response to genetic variation in both the host and pathogen. To study co-transcriptome plasticity, we conducted transcriptomics using natural genetic variation in the pathogen, Botrytis cinerea, and large-effect genetic variation abolishing defense signaling pathways within the host, Arabidopsis thaliana. We show that genetic variation in the pathogen has a greater influence on the co-transcriptome than mutations that abolish defense signaling pathways in the host. Genome-wide association mapping using the pathogens' genetic variation and both organisms' transcriptomes allowed an assessment of how the pathogen modulates plasticity in response to the host. This showed that the differences in both organism's responses were linked to trans-expression quantitative trait loci (eQTL) hotspots within the pathogen's genome. These hotspots control gene sets in either the host or pathogen and show differential allele sensitivity to the host's genetic variation rather than qualitative host specificity. Interestingly, nearly all the trans-eQTL hotspots were unique to the host or pathogen transcriptomes. In this system of differential plasticity, the pathogen mediates the shift in the co-transcriptome more than the host.

Cytosolic phosphofructokinases are important for sugar homeostasis in leaves of Arabidopsis thaliana.

BACKGROUND AND AIMS: ATP-dependent phosphofructokinases (PFKs) catalyse phosphorylation of the carbon-1 position of fructose-6-phosphate, to form fructose-1,6-bisphosphate. In the cytosol, this is considered a key step in channelling carbon into glycolysis. Arabidopsis thaliana has seven genes encoding PFK isoforms, two chloroplastic and five cytosolic. This study focuses on the four major cytosolic isoforms of PFK in vegetative tissues of A. thaliana. METHODS: We isolated homozygous knockout individual mutants (pfk1, pfk3, pfk6 and pfk7) and two double mutants (pfk1/7 and pfk3/6), and characterized their growth and metabolic phenotypes. KEY RESULTS: In contrast to single mutants and the double mutant pfk3/6 for the hypoxia-responsive isoforms, the double mutant pfk1/7 had reduced PFK activity and showed a clear visual and metabolic phenotype with reduced shoot growth, early flowering and elevated hexose levels. This mutant also has an altered ratio of short/long aliphatic glucosinolates and an altered root-shoot distribution. Surprisingly, this mutant does not show any major changes in short-term carbon flux and in levels of hexose-phosphates. CONCLUSIONS: We conclude that the two isoforms PFK1 and PFK7 are important for sugar homeostasis in leaf metabolism and apparently in source-sink relationships in A. thaliana, while PFK3 and PFK6 only play a minor role under normal growth conditions.

Evolution of A bHLH Interaction Motif.

Intrinsically disordered proteins and regions with their associated short linear motifs play key roles in transcriptional regulation. The disordered MYC-interaction motif (MIM) mediates interactions between MYC and MYB transcription factors in Arabidopsis thaliana that are critical for constitutive and induced glucosinolate (GLS) biosynthesis. GLSs comprise a class of plant defense compounds that evolved in the ancestor of the Brassicales order. We used a diverse set of search strategies to discover additional occurrences of the MIM in other proteins and in other organisms and evaluate the findings by means of structural predictions, interaction assays, and biophysical experiments. Our search revealed numerous MIM instances spread throughout the angiosperm lineage. Experiments verify that several of the newly discovered MIM-containing proteins interact with MYC TFs. Only hits found within the same transcription factor family and having similar characteristics could be validated, indicating that structural predictions and sequence similarity are good indicators of whether the presence of a MIM mediates interaction. The experimentally validated MIMs are found in organisms outside the Brassicales order, showing that MIM function is broader than regulating GLS biosynthesis.

Arabidopsis thaliana transcription factors MYB28 and MYB29 shape ammonium stress responses by regulating Fe homeostasis.

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.

Induction and Priming of Plant Defense by Root-Associated Insect-Pathogenic Fungi.

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.

Root-type ferredoxin-NADP+ oxidoreductase isoforms in Arabidopsis thaliana: Expression patterns, location and stress responses.

In Arabidopsis, two leaf-type ferredoxin-NADP+ oxidoreductase (LFNR) isoforms function in photosynthetic electron flow in reduction of NADP+ , while two root-type FNR (RFNR) isoforms catalyse reduction of ferredoxin in non-photosynthetic plastids. As the key to understanding, the function of RFNRs might lie in their spatial and temporal distribution in different plant tissues and cell types, we examined expression of RFNR1 and RFNR2 genes using ß-glucuronidase (GUS) reporter lines and investigated accumulation of distinct RFNR isoforms using a GFP approach and Western blotting upon various stresses. We show that while RFNR1 promoter is active in leaf veins, root tips and in the stele of roots, RFNR2 promoter activity is present in leaf tips and root stele, epidermis and cortex. RFNR1 protein accumulates as a soluble protein within the plastids of root stele cells, while RFNR2 is mainly present in the outer root layers. Ozone treatment of plants enhanced accumulation of RFNR1, whereas low temperature treatment specifically affected RFNR2 accumulation in roots. We further discuss the physiological roles of RFNR1 and RFNR2 based on characterization of rfnr1 and rfnr2 knock-out plants and show that although the function of these proteins is partly redundant, the RFNR proteins are essential for plant development and survival.

Heterologous microProtein expression identifies LITTLE NINJA, a dominant regulator of jasmonic acid signaling.

MicroProteins are small, often single-domain proteins that are sequence-related to larger, often multidomain proteins. Here, we used a combination of comparative genomics and heterologous synthetic misexpression to isolate functional cereal microProtein regulators. Our approach identified LITTLE NINJA (LNJ), a microProtein that acts as a modulator of jasmonic acid (JA) signaling. Ectopic expression of LNJ in Arabidopsis resulted in stunted plants that resembled the decuple JAZ (jazD) mutant. In fact, comparing the transcriptomes of transgenic LNJ overexpressor plants and jazD revealed a large overlap of deregulated genes, suggesting that ectopic LNJ expression altered JA signaling. Transgenic Brachypodium plants with elevated LNJ expression levels showed deregulation of JA signaling as well and displayed reduced growth and enhanced production of side shoots (tiller). This tillering effect was transferable between grass species, and overexpression of LNJ in barley and rice caused similar traits. We used a clustered regularly interspaced short palindromic repeats (CRISPR) approach and created a LNJ-like protein in Arabidopsis by deleting parts of the coding sentence of the AFP2 gene that encodes a NINJA-domain protein. These afp2-crispr mutants were also stunted in size and resembled jazD Thus, similar genome-engineering approaches can be exploited as a future tool to create LNJ proteins and produce cereals with altered architectures.

Insights into the community structure and lifestyle of the fungal root endophytes of tomato by combining amplicon sequencing and isolation approaches with phytohormone profiling.

Little is known about the influence of host genotype and phytohormones on the composition of fungal endophytic communities. We investigated the influence of host genotype and phytohormones on the structure of the fungal endophytic communities of tomato roots by amplicon sequencing of the ITS1 region and combined this approach with isolation and functional characterization of the isolates. A significant effect of the host genotype on the dominant fungal species was found by comparing the cultivars Castlemart and UC82B and, surprisingly, root pathogens were among the most abundant taxa. In contrast, smaller changes in the relative abundance of the dominant species were found in mutants impaired in jasmonic acid biosynthesis (def1) and ethylene biosynthesis (8338) compared to the respective wild types. However, def1 showed significantly higher species richness compared to the wild type. Analysis of the phytohormone profiles of these genotypes indicates that changes in the phytohormone balance may contribute to this difference in species richness. Assessing the lifestyle of isolated fungi on tomato seedlings revealed the presence of both beneficial endophytes and latent pathogens in roots of asymptomatic plants, suggesting that the interactions between members of the microbiome maintain the equilibrium in the community preventing pathogens from causing disease.

Diverse Allyl Glucosinolate Catabolites Independently Influence Root Growth and Development.

Glucosinolates (GSLs) are sulfur-containing defense metabolites produced in the Brassicales, including the model plant Arabidopsis (Arabidopsis thaliana). Previous work suggests that specific GSLs may function as signals to provide direct feedback regulation within the plant to calibrate defense and growth. These GSLs include allyl-GSL, a defense metabolite that is one of the most widespread GSLs in Brassicaceae and has also been associated with growth inhibition. Here we show that at least three separate potential catabolic products of allyl-GSL or closely related compounds affect growth and development by altering different mechanisms influencing plant development. Two of the catabolites, raphanusamic acid and 3-butenoic acid, differentially affect processes downstream of the auxin signaling cascade. Another catabolite, acrylic acid, affects meristem development by influencing the progression of the cell cycle. These independent signaling events propagated by the different catabolites enable the plant to execute a specific response that is optimal to any given environment.

IDDomainSpotter: Compositional bias reveals domains in long disordered protein regions-Insights from transcription factors.

Protein domains constitute regions of distinct structural properties and molecular functions that are retained when removed from the rest of the protein. However, due to the lack of tertiary structure, the identification of domains has been largely neglected for long (>50 residues) intrinsically disordered regions. Here we present a sequence-based approach to assess and visualize domain organization in long intrinsically disordered regions based on compositional sequence biases. An online tool to find putative intrinsically disordered domains (IDDomainSpotter) in any protein sequence or sequence alignment using any particular sequence trait is available at http://www.bio.ku.dk/sbinlab/IDDomainSpotter. Using this tool, we have identified a putative domain enriched in hydrophilic and disorder-promoting residues (Pro, Ser, and Thr) and depleted in positive charges (Arg and Lys) bordering the folded DNA-binding domains of several transcription factors (p53, GCR, NAC46, MYB28, and MYB29). This domain, from two different MYB transcription factors, was characterized biophysically to determine its properties. Our analyses show the domain to be extended, dynamic and highly disordered. It connects the DNA-binding domain to other disordered domains and is present and conserved in several transcription factors from different families and domains of life. This example illustrates the potential of IDDomainSpotter to predict, from sequence alone, putative domains of functional interest in otherwise uncharacterized disordered proteins.

PROTEIN PHOSPHATASE 2A-B'γ Controls Botrytis cinerea Resistance and Developmental Leaf Senescence.

Plants optimize their growth and survival through highly integrated regulatory networks that coordinate defensive measures and developmental transitions in response to environmental cues. Protein phosphatase 2A (PP2A) is a key signaling component that controls stress reactions and growth at different stages of plant development, and the PP2A regulatory subunit PP2A-B'γ is required for negative regulation of pathogenesis responses and for maintenance of cell homeostasis in short-day conditions. Here, we report molecular mechanisms by which PP2A-B'γ regulates Botrytis cinerea resistance and leaf senescence in Arabidopsis (Arabidopsis thaliana). We extend the molecular functionality of PP2A-B'γ to a protein kinase-phosphatase interaction with the defense-associated calcium-dependent protein kinase CPK1 and present indications this interaction may function to control CPK1 activity. In presenescent leaf tissues, PP2A-B'γ is also required to negatively control the expression of salicylic acid-related defense genes, which have recently proven vital in plant resistance to necrotrophic fungal pathogens. In addition, we find the premature leaf yellowing of pp2a-b'γ depends on salicylic acid biosynthesis via SALICYLIC ACID INDUCTION DEFICIENT2 and bears the hallmarks of developmental leaf senescence. We propose PP2A-B'γ age-dependently controls salicylic acid-related signaling in plant immunity and developmental leaf senescence.

Differential partitioning of thiols and glucosinolates between shoot and root in Chinese cabbage upon excess zinc exposure.

Zinc (Zn) is one of the important elements of plant growth, however, at elevated level it is toxic. Exposure of Chinese cabbage to elevated Zn2+ concentrations (5 and 10 µM ZnCl2) resulted in enhancement of total sulfur and organic sulfur concentration. Transcript level of APS reductase (APR) as a key enzyme in biosynthesis of primary sulfur compounds (cysteine and thiols), was up-regulated in both shoot and root upon exposure to elevated Zn2+, which was accompanied by an increase in the concentration of cysteine in both tissues. In contrast, the concentration of thiols increased only in the root by 5.5 and 15-fold at 5 and 10 µM Zn2+, respectively, which was in accompanied by an upregulation of ATP sulfurylase, an enzyme responsible for activation of sulfate. An elevated content of glucosinolates, mostly indolic glucosinolates, only in the shoot of plants exposed to excess level of Zn2+ coincided with an increase in gene expression of key biosynthetic enzymes and regulators (CYP79B3, CYP83B1, MYB34). Thus distinct acuumulation patterns of sulfur containing compounds in root and shoot of Chinese cabbage may be a strategy for Chinese cabbage to combat with exposure to excess Zn.

Glucosinolate structural diversity, identification, chemical synthesis and metabolism in plants.

The glucosinolates (GSLs) is a well-defined group of plant metabolites characterized by having an S-ß-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.

Metabolic engineering of Synechocystis sp. PCC 6803 for the production of aromatic amino acids and derived phenylpropanoids.

In light of the climate change challenge, the advantageous trait of using solar energy and carbon dioxide to produce organic molecules has granted cyanobacteria deserved interest as hosts for metabolic engineering. Importantly, these organisms do not directly compete with agricultural resources. Aromatic amino acids and derived phenylpropanoids are of high importance because they are used by the pharmaceutical, food, cosmetic, and agricultural industries as precursors of active ingredients. Amino acids are traditionally produced by extraction from protein hydrolysates, chemical synthesis or fermentation pathways using heterotrophic microorganisms. In this work we demonstrate for the first time the efficient overproduction of phenylalanine and tyrosine from CO2 in a Synechocystis sp. PCC 6803 strain heterologously expressing the feedback-inhibition-resistant AroG and TyrA enzymes from E. coli. Production titers reached 904 ±â€¯53 mg/gDW (580 ±â€¯34 mg/L) of phenylalanine and 64 ±â€¯3.7 mg/gDW (41 ±â€¯2.3 mg/L) of tyrosine after 10 days of photoautotrophic growth. We estimate that the production of the two amino acids corresponds to 56% of the total fixed carbon. Phenylalanine and tyrosine are the precursors for phenylpropanoids, thus, we tested the functionality of several phenylpropanoid biosynthetic enzymes in the generated cyanobacterium strains and successfully achieved the production of 470 ±â€¯70 mg/gDW (207 mg/L) of p-coumaric acid, 267 ±â€¯31 mg/gDW (114 mg/L) of cinnamic acid and 47.4 ±â€¯13.9 mg/gDW (12.6 mg/L) of caffeic acid after 6 days of photoautotrophic growth. All compounds were secreted to the growth medium. Our work enlarges the repertoire and yield of heterologous chemicals produced by Synechocystis and contributes to extend the molecular knowledge about this cyanobacterium.

Coordination of Glucosinolate Biosynthesis and Turnover Under Different Nutrient Conditions.

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 Arabidopsis thaliana 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 in planta 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.