Integrated genetic and metabolic characterization of Latin American cassava (Manihot esculenta) germplasm.

Cassava (Manihot esculenta Crantz) is an important staple crop for food security in Africa and South America. The present study describes an integrated genomic and metabolomic approach to the characterization of Latin American cassava germplasm. Classification based on genotyping correlated with the leaf metabolome and indicated a key finding of adaption to specific eco-geographical environments. In contrast, the root metabolome did not relate to genotypic clustering, suggesting the different spatial regulation of this tissue's metabolome. The data were used to generate pan-metabolomes for specific tissues, and the inclusion of phenotypic data enabled the identification of metabolic sectors underlying traits of interest. For example, tolerance to whiteflies (Aleurotrachelus socialis) was not linked directly to cyanide content but to cell wall-related phenylpropanoid or apocarotenoid content. Collectively, these data advance the community resources and provide valuable insight into new candidate parental breeding materials with traits of interest directly related to combating food security.

Divergent contribution of the MVA and MEP pathways to the formation of polyprenols and dolichols in Arabidopsis.

Isoprenoids, including dolichols (Dols) and polyprenols (Prens), are ubiquitous components of eukaryotic cells. In plant cells, there are two pathways that produce precursors utilized for isoprenoid biosynthesis: the mevalonate (MVA) pathway and the methylerythritol phosphate (MEP) pathway. In this work, the contribution of these two pathways to the biosynthesis of Prens and Dols was addressed using an in planta experimental model. Treatment of plants with pathway-specific inhibitors and analysis of the effects of various light conditions indicated distinct biosynthetic origin of Prens and Dols. Feeding with deuteriated, pathway-specific precursors revealed that Dols, present in leaves and roots, were derived from both MEP and MVA pathways and their relative contributions were modulated in response to precursor availability. In contrast, Prens, present in leaves, were almost exclusively synthesized via the MEP pathway. Furthermore, results obtained using a newly introduced here 'competitive' labeling method, designed so as to neutralize the imbalance of metabolic flow resulting from feeding with a single pathway-specific precursor, suggest that under these experimental conditions one fraction of Prens and Dols is synthesized solely from endogenous precursors (deoxyxylulose or mevalonate), while the other fraction is synthesized concomitantly from endogenous and exogenous precursors. Additionally, this report describes a novel methodology for quantitative separation of 2H and 13C distributions observed for isotopologues of metabolically labeled isoprenoids. Collectively, these in planta results show that Dol biosynthesis, which uses both pathways, is significantly modulated depending on pathway productivity, while Prens are consistently derived from the MEP pathway.

Integrative transcriptomics reveals association of abscisic acid and lignin pathways with cassava whitefly resistance.

BACKGROUND: Whiteflies are a global threat to crop yields, including the African subsistence crop cassava (Manihot esculenta). Outbreaks of superabundant whitefly populations throughout Eastern and Central Africa in recent years have dramatically increased the pressures of whitefly feeding and virus transmission on cassava. Whitefly-transmitted viral diseases threaten the food security of hundreds of millions of African farmers, highlighting the need for developing and deploying whitefly-resistant cassava. However, plant resistance to whiteflies remains largely poorly characterized at the genetic and molecular levels. Knowledge of cassava-defense programs also remains incomplete, limiting characterization of whitefly-resistance mechanisms. To better understand the genetic basis of whitefly resistance in cassava, we define the defense hormone- and Aleurotrachelus socialis (whitefly)-responsive transcriptome of whitefly-susceptible (COL2246) and whitefly-resistant (ECU72) cassava using RNA-seq. For broader comparison, hormone-responsive transcriptomes of Arabidopsis thaliana were also generated. RESULTS: Whitefly infestation, salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and abscisic acid (ABA) transcriptome responses of ECU72 and COL2246 were defined and analyzed. Strikingly, SA responses were largely reciprocal between the two cassava genotypes and we suggest candidate regulators. While susceptibility was associated with SA in COL2246, resistance to whitefly in ECU72 was associated with ABA, with SA-ABA antagonism observed. This was evidenced by expression of genes within the SA and ABA pathways and hormone levels during A. socialis infestation. Gene-enrichment analyses of whitefly- and hormone-responsive genes suggest the importance of fast-acting cell wall defenses (e.g., elicitor recognition, lignin biosynthesis) during early infestation stages in whitefly-resistant ECU72. A surge of ineffective immune and SA responses characterized the whitefly-susceptible COL2246's response to late-stage nymphs. Lastly, in comparison with the model plant Arabidopsis, cassava's hormone-responsive genes showed striking divergence in expression. CONCLUSIONS: This study provides the first characterization of cassava's global transcriptome responses to whitefly infestation and defense hormone treatment. Our analyses of ECU72 and COL2246 uncovered possible whitefly resistance/susceptibility mechanisms in cassava. Comparative analysis of cassava and Arabidopsis demonstrated that defense programs in Arabidopsis may not always mirror those in crop species. More broadly, our hormone-responsive transcriptomes will also provide a baseline for the cassava community to better understand global responses to other yield-limiting pests/pathogens.

Multilevel interactions between native and ectopic isoprenoid pathways affect global metabolism in rice.

Isoprenoids are natural products derived from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In plants, these precursors are synthesized via the cytosolic mevalonate (MVA) and plastidial methylerythritol phosphate (MEP) pathways. The regulation of these pathways must therefore be understood in detail to develop effective strategies for isoprenoid metabolic engineering. We hypothesized that the strict regulation of the native MVA pathway could be circumvented by expressing an ectopic plastidial MVA pathway that increases the accumulation of IPP and DMAPP in plastids. We therefore introduced genes encoding the plastid-targeted enzymes HMGS, tHMGR, MK, PMK and MVD and the nuclear-targeted transcription factor WR1 into rice and evaluated the impact of their endosperm-specific expression on (1) endogenous metabolism at the transcriptomic and metabolomic levels, (2) the synthesis of phytohormones, carbohydrates and fatty acids, and (3) the macroscopic phenotype including seed morphology. We found that the ectopic plastidial MVA pathway enhanced the expression of endogenous cytosolic MVA pathway genes while suppressing the native plastidial MEP pathway, increasing the production of certain sterols and tocopherols. Plants carrying the ectopic MVA pathway only survived if WR1 was also expressed to replenish the plastid acetyl-CoA pool. The transgenic plants produced higher levels of fatty acids, abscisic acid, gibberellins and lutein, reflecting crosstalk between phytohormones and secondary metabolism.

Metabolite database for root, tuber, and banana crops to facilitate modern breeding in understudied crops.

Roots, tubers, and bananas (RTB) are vital staples for food security in the world's poorest nations. A major constraint to current RTB breeding programmes is limited knowledge on the available diversity due to lack of efficient germplasm characterization and structure. In recent years large-scale efforts have begun to elucidate the genetic and phenotypic diversity of germplasm collections and populations and, yet, biochemical measurements have often been overlooked despite metabolite composition being directly associated with agronomic and consumer traits. Here we present a compound database and concentration range for metabolites detected in the major RTB crops: banana (Musa spp.), cassava (Manihot esculenta), potato (Solanum tuberosum), sweet potato (Ipomoea batatas), and yam (Dioscorea spp.), following metabolomics-based diversity screening of global collections held within the CGIAR institutes. The dataset including 711 chemical features provides a valuable resource regarding the comparative biochemical composition of each RTB crop and highlights the potential diversity available for incorporation into crop improvement programmes. Particularly, the tropical crops cassava, sweet potato and banana displayed more complex compositional metabolite profiles with representations of up to 22 chemical classes (unknowns excluded) than that of potato, for which only metabolites from 10 chemical classes were detected. Additionally, over 20% of biochemical signatures remained unidentified for every crop analyzed. Integration of metabolomics with the on-going genomic and phenotypic studies will enhance 'omics-wide associations of molecular signatures with agronomic and consumer traits via easily quantifiable biochemical markers to aid gene discovery and functional characterization.

A transcriptomic, metabolomic and cellular approach to the physiological adaptation of tomato fruit to high temperature.

High temperatures can negatively influence plant growth and development. Besides yield, the effects of heat stress on fruit quality traits remain poorly characterised. In tomato, insights into how fruits regulate cellular metabolism in response to heat stress could contribute to the development of heat-tolerant varieties, without detrimental effects on quality. In the present study, the changes occurring in wild type tomato fruits after exposure to transient heat stress have been elucidated at the transcriptome, cellular and metabolite level. An impact on fruit quality was evident as nutritional attributes changed in response to heat stress. Fruit carotenogenesis was affected, predominantly at the stage of phytoene formation, although altered desaturation/isomerisation arose during the transient exposure to high temperatures. Plastidial isoprenoid compounds showed subtle alterations in their distribution within chromoplast sub-compartments. Metabolite profiling suggests limited effects on primary/intermediary metabolism but lipid remodelling was evident. The heat-induced molecular signatures included the accumulation of sucrose and triacylglycerols, and a decrease in the degree of membrane lipid unsaturation, which influenced the volatile profile. Collectively, these data provide valuable insights into the underlying biochemical and molecular adaptation of fruit to heat stress and will impact on our ability to develop future climate resilient tomato varieties.

Nitrogen inputs influence vegetative metabolism in maize engineered with a seed-specific carotenoid pathway.

KEY MESSAGE: Metabolomic profiling of a maize line engineered with an endosperm-specific carotenogenic pathway revealed unexpected metabolic readjustments of primary metabolism in leaves and roots. High-carotenoid (HC) maize was engineered to accumulate high levels of carotenoids in the endosperm. The metabolic interventions influenced the flux through non-target pathways in tissues that were not affected by the targeted intervention. HC maize at the vegetative stage also showed a reduced susceptibility to insect feeding. It is unknown, however, whether the metabolic history of the embryo has any impact on the metabolite composition in vegetative tissues. We, therefore, compared HC maize and its isogenic counterpart (M37W) to test the hypothesis that boosting the carotenoid content in the endosperm triggers compensatory effects in core metabolism in vegetative tissues. Specifically, we investigated whether the metabolite composition of leaves and roots at the V6 stage differs between HC and M37W, and whether N inputs further alter the core metabolism of HC compared to M37W. We found an increase in the abundance of organic acids from the tricarboxylic acid (TCA) cycle in HC even under restricted N conditions. In contrast, low levels of carotenoids and chlorophyll were measured regardless of N levels. Sugars were also significantly depleted in HC under low N. We propose a model explaining the observed genotype-dependent and input-dependent effects, in which organic acids derived from the TCA cycle accumulate during vegetative growth and contribute to the increased demand for pyruvate and/or acetyl-CoA in the endosperm and embryo. This response may in part reflect the transgenerational priming of vegetative tissues in the embryo induced by the increased demand for metabolic precursors during seed development in the previous generation.

Genome-wide analyses of cassava Pathogenesis-related (PR) gene families reveal core transcriptome responses to whitefly infestation, salicylic acid and jasmonic acid.

BACKGROUND: Whiteflies are a threat to cassava (Manihot esculenta), an important staple food in many tropical/subtropical regions. Understanding the molecular mechanisms regulating cassava's responses against this pest is crucial for developing control strategies. Pathogenesis-related (PR) protein families are an integral part of plant immunity. With the availability of whole genome sequences, the annotation and expression programs of the full complement of PR genes in an organism can now be achieved. An understanding of the responses of the entire complement of PR genes during biotic stress and to the defense hormones, salicylic acid (SA) and jasmonic acid (JA), is lacking. Here, we analyze the responses of cassava PR genes to whiteflies, SA, JA, and other biotic aggressors. RESULTS: The cassava genome possesses 14 of the 17 plant PR families, with a total of 447 PR genes. A cassava PR gene nomenclature is proposed. Phylogenetic relatedness of cassava PR proteins to each other and to homologs in poplar, rice and Arabidopsis identified cassava-specific PR gene family expansions. The temporal programs of PR gene expression in response to the whitefly (Aleurotrachelus socialis) in four whitefly-susceptible cassava genotypes showed that 167 of the 447 PR genes were regulated after whitefly infestation. While the timing of PR gene expression varied, over 37% of whitefly-regulated PR genes were downregulated in all four genotypes. Notably, whitefly-responsive PR genes were largely coordinately regulated by SA and JA. The analysis of cassava PR gene expression in response to five other biotic stresses revealed a strong positive correlation between whitefly and Xanthomonas axonopodis and Cassava Brown Streak Virus responses and negative correlations between whitefly and Cassava Mosaic Virus responses. Finally, certain associations between PR genes in cassava expansions and response to biotic stresses were observed among PR families. CONCLUSIONS: This study represents the first genome-wide characterization of PR genes in cassava. PR gene responses to six biotic stresses and to SA and JA are demonstrably different to other angiosperms. We propose that our approach could be applied in other species to fully understand PR gene regulation by pathogens, pests and the canonical defense hormones SA and JA.

Characterization of CRISPR Mutants Targeting Genes Modulating Pectin Degradation in Ripening Tomato.

Tomato (Solanum lycopersicum) is a globally important crop with an economic value in the tens of billions of dollars, and a significant supplier of essential vitamins, minerals, and phytochemicals in the human diet. Shelf life is a key quality trait related to alterations in cuticle properties and remodeling of the fruit cell walls. Studies with transgenic tomato plants undertaken over the last 20 years have indicated that a range of pectin-degrading enzymes are involved in cell wall remodeling. These studies usually involved silencing of only a single gene and it has proved difficult to compare the effects of silencing these genes across the different experimental systems. Here we report the generation of CRISPR-based mutants in the ripening-related genes encoding the pectin-degrading enzymes pectate lyase (PL), polygalacturonase 2a (PG2a), and ß-galactanase (TBG4). Comparison of the physiochemical properties of the fruits from a range of PL, PG2a, and TBG4 CRISPR lines demonstrated that only mutations in PL resulted in firmer fruits, although mutations in PG2a and TBG4 influenced fruit color and weight. Pectin localization, distribution, and solubility in the pericarp cells of the CRISPR mutant fruits were investigated using the monoclonal antibody probes LM19 to deesterified homogalacturonan, INRA-RU1 to rhamnogalacturonan I, LM5 to ß-1,4-galactan, and LM6 to arabinan epitopes, respectively. The data indicate that PL, PG2a, and TBG4 act on separate cell wall domains and the importance of cellulose microfibril-associated pectin is reflected in its increased occurrence in the different mutant lines.

A metabolomics characterisation of natural variation in the resistance of cassava to whitefly.

BACKGROUND: Cassava whitefly outbreaks were initially reported in East and Central Africa cassava (Manihot esculenta Crantz) growing regions in the 1990's and have now spread to other geographical locations, becoming a global pest severely affecting farmers and smallholder income. Whiteflies impact plant yield via feeding and vectoring cassava mosaic and brown streak viruses, making roots unsuitable for food or trading. Deployment of virus resistant varieties has had little impact on whitefly populations and therefore development of whitefly resistant varieties is also necessary as part of integrated pest management strategies. Suitable sources of whitefly resistance exist in germplasm collections that require further characterization to facilitate and assist breeding programs. RESULTS: In the present work, a hierarchical metabolomics approach has been employed to investigate the underlying biochemical mechanisms associated with whitefly resistance by comparing two naturally occurring accessions of cassava, one susceptible and one resistant to whitefly. Quantitative differences between genotypes detected at pre-infestation stages were consistently observed at each time point throughout the course of the whitefly infestation. This prevalent differential feature suggests that inherent genotypic differences override the response induced by the presence of whitefly and that they are directly linked with the phenotype observed. The most significant quantitative changes relating to whitefly susceptibility were linked to the phenylpropanoid super-pathway and its linked sub-pathways: monolignol, flavonoid and lignan biosynthesis. These findings suggest that the lignification process in the susceptible variety is less active, as the susceptible accession deposits less lignin and accumulates monolignol intermediates and derivatives thereof, differences that are maintained during the time-course of the infestation. CONCLUSIONS: Resistance mechanism associated to the cassava whitefly-resistant accession ECU72 is an antixenosis strategy based on reinforcement of cell walls. Both resistant and susceptible accessions respond differently to whitefly attack at biochemical level, but the inherent metabolic differences are directly linked to the resistance phenotype rather than an induced response in the plant.

The Formation and Sequestration of Nonendogenous Ketocarotenoids in Transgenic Nicotiana glauca.

Ketolated and hydroxylated carotenoids are high-value compounds with industrial, food, and feed applications. Chemical synthesis is currently the production method of choice for these compounds, with no amenable plant sources readily available. In this study, the 4,4' ß-oxygenase (crtW) and 3,3' ß-hydroxylase (crtZ) genes from Brevundimonas sp. SD-212 were expressed under constitutive transcriptional control in Nicotiana glauca, which has an emerging potential as a biofuel and biorefining feedstock. The transgenic lines produced significant levels of nonendogenous carotenoids in all tissues. In leaf and flower, the carotenoids (∼0.5% dry weight) included 0.3% and 0.48%, respectively, of nonendogenous ketolated and hydroxylated carotenoids. These were 4-ketolutein, echinenone (and its 3-hydroxy derivatives), canthaxanthin, phoenicoxanthin, 4-ketozeaxanthin, and astaxanthin. Stable, homozygous genotypes expressing both transgenes inherited the chemotype. Subcellular fractionation of vegetative tissues and microscopic analysis revealed the presence of ketocarotenoids in thylakoid membranes, not predominantly in the photosynthetic complexes but in plastoglobules. Despite ketocarotenoid production and changes in cellular ultrastructure, intermediary metabolite levels were not dramatically affected. The study illustrates the utility of Brevundimonas sp. SD-212 CRTZ and CRTW to produce ketocarotenoids in a plant species that is being evaluated as a biorefining feedstock, the adaptation of the plastid to sequester nonendogenous carotenoids, and the robustness of plant metabolism to these changes.

Metabolic engineering of astaxanthin biosynthesis in maize endosperm and characterization of a prototype high oil hybrid.

Maize was genetically engineered for the biosynthesis of the high value carotenoid astaxanthin in the kernel endosperm. Introduction of a ß-carotene hydroxylase and a ß-carotene ketolase into a white maize genetic background extended the carotenoid pathway to astaxanthin. Simultaneously, phytoene synthase, the controlling enzyme of carotenogenesis, was over-expressed for enhanced carotenoid production and lycopene ε-cyclase was knocked-down to direct more precursors into the ß-branch of the extended ketocarotenoid pathway which ends with astaxanthin. This astaxanthin-accumulating transgenic line was crossed into a high oil- maize genotype in order to increase the storage capacity for lipophilic astaxanthin. The high oil astaxanthin hybrid was compared to its astaxanthin producing parent. We report an in depth metabolomic and proteomic analysis which revealed major up- or down- regulation of genes involved in primary metabolism. Specifically, amino acid biosynthesis and the citric acid cycle which compete with the synthesis or utilization of pyruvate and glyceraldehyde 3-phosphate, the precursors for carotenogenesis, were down-regulated. Nevertheless, principal component analysis demonstrated that this compositional change is within the range of the two wild type parents used to generate the high oil producing astaxanthin hybrid.

Annotation and functional assignment of the genes for the C30 carotenoid pathways from the genomes of two bacteria: Bacillus indicus and Bacillus firmus.

Bacillus indicus and Bacillus firmus synthesize C30 carotenoids via farnesyl pyrophosphate, forming apophytoene as the first committed step in the pathway. The products of the pathways were methyl 4'-[6-O-acyl-glycosyl)oxy]-4,4'-diapolycopen-4-oic acid and 4,4'-diapolycopen-4,4'-dioic acid with putative glycosyl esters. The genomes of both bacteria were sequenced, and the genes for their early terpenoid and specific carotenoid pathways annotated. All genes for a functional 1-deoxy-d-xylulose 5-phosphate synthase pathway were identified in both species, whereas genes of the mevalonate pathway were absent. The genes for specific carotenoid synthesis and conversion were found on gene clusters which were organized differently in the two species. The genes involved in the formation of the carotenoid cores were assigned by functional complementation in Escherichia coli. This bacterium was co-transformed with a plasmid mediating the formation of the putative substrate and a second plasmid with the gene of interest. Carotenoid products in the transformants were determined by HPLC. Using this approach, we identified the genes for a 4,4'-diapophytoene synthase (crtM), 4,4'-diapophytoene desaturase (crtNa), 4,4'-diapolycopene ketolase (crtNb) and 4,4'-diapolycopene aldehyde oxidase (crtNc). The three crtN genes were closely related and belonged to the crtI gene family with a similar reaction mechanism of their enzyme products. Additional genes encoding glycosyltransferases and acyltransferases for the modification of the carotenoid skeleton of the diapolycopenoic acids were identified by comparison with the corresponding genes from other bacteria.

Combined transcript, proteome, and metabolite analysis of transgenic maize seeds engineered for enhanced carotenoid synthesis reveals pleotropic effects in core metabolism.

The aim of this study was to assess whether endosperm-specific carotenoid biosynthesis influenced core metabolic processes in maize embryo and endosperm and how global seed metabolism adapted to this expanded biosynthetic capacity. Although enhancement of carotenoid biosynthesis was targeted to the endosperm of maize kernels, a concurrent up-regulation of sterol and fatty acid biosynthesis in the embryo was measured. Targeted terpenoid analysis, and non-targeted metabolomic, proteomic, and transcriptomic profiling revealed changes especially in carbohydrate metabolism in the transgenic line. In-depth analysis of the data, including changes of metabolite pools and increased enzyme and transcript concentrations, gave a first insight into the metabolic variation precipitated by the higher up-stream metabolite demand by the extended biosynthesis capacities for terpenoids and fatty acids. An integrative model is put forward to explain the metabolic regulation for the increased provision of terpenoid and fatty acid precursors, particularly glyceraldehyde 3-phosphate and pyruvate or acetyl-CoA from imported fructose and glucose. The model was supported by higher activities of fructokinase, glucose 6-phosphate isomerase, and fructose 1,6-bisphosphate aldolase indicating a higher flux through the glycolytic pathway. Although pyruvate and acetyl-CoA utilization was higher in the engineered line, pyruvate kinase activity was lower. A sufficient provision of both metabolites may be supported by a by-pass in a reaction sequence involving phosphoenolpyruvate carboxylase, malate dehydrogenase, and malic enzyme.

Functional characterization of long-chain prenyl diphosphate synthases from tomato.

The electron transfer molecules plastoquinone and ubiquinone are formed by the condensation of aromatic head groups with long-chain prenyl diphosphates. In the present paper we report the cloning and characterization of two genes from tomato (Solanum lycopersicum) responsible for the production of solanesyl and decaprenyl diphosphates. SlSPS (S. lycopersicum solanesyl diphosphate synthase) is targeted to the plastid and both solanesol and plastoquinone are associated with thylakoid membranes. A second gene [SlDPS (S. lycopersicum solanesyl decaprenyl diphosphate synthase)], encodes a long-chain prenyl diphosphate synthase with a different subcellular localization from SlSPS and can utilize geranyl, farnesyl or geranylgeranyl diphosphates in the synthesis of C45 and C50 prenyl diphosphates. When expressed in Escherichia coli, SlSPS and SlDPS extend the prenyl chain length of the endogenous ubiquinone to nine and ten isoprene units respectively. In planta, constitutive overexpression of SlSPS elevated the plastoquinone content of immature tobacco leaves. Virus-induced gene silencing showed that SlSPS is necessary for normal chloroplast structure and function. Plants silenced for SlSPS were photobleached and accumulated phytoene, whereas silencing SlDPS did not affect leaf appearance, but impacted on primary metabolism. The two genes were not able to complement silencing of each other. These findings indicate a requirement for two long-chain prenyl diphosphate synthases in the tomato.

Changing biosynthesis of terpenoid percursors in rice through synthetic biology.

Many highly valued chemicals in the pharmaceutical, biotechnological, cosmetic, and biomedical industries belong to the terpenoid family. Biosynthesis of these chemicals relies on polymerization of Isopentenyl di-phosphate (IPP) and/or dimethylallyl diphosphate (DMAPP) monomers, which plants synthesize using two alternative pathways: a cytosolic mevalonic acid (MVA) pathway and a plastidic methyleritritol-4-phosphate (MEP) pathway. As such, developing plants for use as a platform to use IPP/DMAPP and produce high value terpenoids is an important biotechnological goal. Still, IPP/DMAPP are the precursors to many plant developmental hormones. This creates severe challenges in redirecting IPP/DMAPP towards production of non-cognate plant metabolites. A potential solution to this problem is increasing the IPP/DMAPP production flux in planta. Here, we aimed at discovering, understanding, and predicting the effects of increasing IPP/DMAPP production in plants through modelling. We used synthetic biology to create rice lines containing an additional ectopic MVA biosynthetic pathway for producing IPP/DMAPP. The rice lines express three alternative versions of the additional MVA pathway in the plastid, in addition to the normal endogenous pathways. We collected data for changes in macroscopic and molecular phenotypes, gene expression, isoprenoid content, and hormone abundance in those lines. To integrate the molecular and macroscopic data and develop a more in depth understanding of the effects of engineering the exogenous pathway in the mutant rice lines, we developed and analyzed data-centric, line-specific, multilevel mathematical models. These models connect the effects of variations in hormones and gene expression to changes in macroscopic plant phenotype and metabolite concentrations within the MVA and MEP pathways of WT and mutant rice lines. Our models allow us to predict how an exogenous IPP/DMAPP biosynthetic pathway affects the flux of terpenoid precursors. We also quantify the long-term effect of plant hormones on the dynamic behavior of IPP/DMAPP biosynthetic pathways in seeds, and predict plant characteristics, such as plant height, leaf size, and chlorophyll content from molecular data. In addition, our models are a tool that can be used in the future to help in prioritizing re-engineering strategies for the exogenous pathway in order to achieve specific metabolic goals.

Identification and the developmental formation of carotenoid pigments in the yellow/orange Bacillus spore-formers.

Spore-forming Bacillus species capable of synthesising carotenoid pigments have recently been isolated. To date the detailed characterisation of these carotenoids and their formation has not been described. In the present article biochemical analysis on the carotenoids responsible for the yellow/orange pigmentation present in Bacilli has been carried out and the identity of the carotenoids present was elucidated. Chromatographic, UV/Vis and Mass Spectral (MS) data have revealed the exclusive presence of a C(30) carotenoid biosynthetic pathway in Bacillus species. Apophytoene was detected representing the first genuine carotenoid formed by this pathway. Cultivation in the presence of diphenylamine (DPA), a known inhibitor of pathway desaturation resulted in the accumulation of apophytoene along with other intermediates of desaturation (e.g. apophytofluene and apo-ζ-carotene). The most abundant carotenoids present in the Bacillus species were oxygenated derivatives of apolycopene, which have either undergone glycosylation and/or esterification. The presence of fatty acid moieties (C(9) to C(15)) attached to the sugar residue via an ester linkage was revealed by saponification and MS/MS analysis. In source fragmentation showed the presence of a hexose sugar associated with apolycopene derivatives. The most abundant apocarotenoids determined were glycosyl-apolycopene and glycosyl-4'-methyl-apolycopenoate esters. Analysis of these carotenoids over the developmental formation of spores revealed that 5-glycosyl-4'-methyl-apolycopenoate was related to sporulation. Potential biosynthetic pathways for the formation of these apocarotenoids in vegetative cells and spores have been reconstructed from intermediates and end-products were elucidated.

The biosynthetic pathway to a novel derivative of 4,4'-diapolycopene-4,4'-oate in a red strain of Sporosarcina aquimarina.

In a red bacterial strain SF238 belonging to Sporosarcina aquimarina, a C(30) carotenoid biosynthetic pathway was identified. It has been reconstructed by analysis of intermediates that accumulate in two different pigment mutants. It starts with the synthesis of 4,4'-diapophytoene and proceeds with its desaturation to 4,4'-diapolycopene, which is then oxidized to 4,4'-diapolycopene-4,4'-dioate. Using a combination of HPLC-PDA and LC-MS/MS analyses, the final product of this pathway was identified as acetyl-4,4'-diapolycopene-4,4'-dioate. This is a novel carotenoid not reported in any organisms to date. It could be demonstrated that this carotenoid has excellent antioxidative properties to protect from photosensitized peroxidation reactions like other related 4,4'-diapolycopene-4,4'-dioate derivatives.

Isolation and characterization of sub-plastidial fractions from carotenoid rich fruits.

Carotenoid biosynthesis and sequestration in higher plants occurs in the plastid organelle. Among diverse germplasm collections displaying natural variation for carotenoids and outputs from metabolic engineering experiments it has become clear that plastid type and numbers can have important implications on the quantitative composition of carotenoids accumulating. Therefore, it is important to characterize these organelles to fully evaluate the potential of the germplasm to enhance carotenoids and create nutrient dense fruits and vegetables. In this article the procedures used to isolate sub-plastidial structures from carotenoid-rich Solanaceae fruits (tomato and Capsicum) are described.

Metabolomic approaches for the characterization of carotenoid metabolic engineering in planta.

Carotenoid biosynthesis has now been subjected to metabolic engineering for over two decades. The outputs clearly show that carotenoid formation is an integral component of metabolism. Perturbations can affect intermediary metabolism and other isoprenoids. The advances in omic technologies have enabled the quantitative assessment of changes in the transcriptome, proteome and metabolome in response to altered carotenoid biosynthesis. In the present article, the approaches and procedures relating to the capture of the metabolome in response to modulation of the carotenoid biosynthetic pathway are described. These data will contribute to the fundamental understanding of metabolic biology, underpinning future rationale design of New Plant Breeding Techniques (NPBTs) and associated regulatory affairs.