Metabolic engineering of cucurbitacins in Cucurbita pepo hairy roots

In this paper we show that metabolic engineering in Cucurbita pepo hairy roots can be used to both effectively increase and modify cucurbitacins. Cucurbitacins are highly-oxygenated triterpenoids originally described in the Cucurbitaceae family, but have since been found in 15 taxonomically distant plant families. Cucurbitacin B, D, E and I are the most widespread amongst the Cucurbitaceae and they have both important biological and pharmacological activities. In this study C. pepo hairy roots were used as a platform to boost production and alter the structures of the afore mentioned cucurbitacins by metabolic engineering to potentially provide new or more desirable bioactivities. We report that the ability to induce cucurbitacin biosynthesis by basic Helix-Loop-Helix transcription factors is partially conserved within the Cucurbitaceae and therefore can potentially be used as a biotechnological tool to increase cucurbitacins in several genera of this family. Additionally, overexpression of a novel acyltransferase from cucurbitacin producing Iberis amara generates a hitherto undescribed acetylation at the C3-hydroxyl group of the cucurbitadienol backbone. While overexpression of the cytochromes P450 CsCYP88L2 and McCYP88L7 from Cucumis sativus and Momordica charantia (respectively), results in accumulation of new spectral feature as revealed by High resolution liquid chromatography mass spectroscopy analysis; the m/z of the new peak supports it might be a cucurbitacin hydroxylated at the C19 position in C. pepo hairy roots. Finally, this paper is a case study of how hairy roots can be used to metabolically engineer and introduce novel modifications in metabolic pathways that have not been fully elucidated

Metabolomic Characterization of Knockout Mutants in Arabidopsis: Development of a Metabolite Profiling Database for Knockout Mutants in Arabidopsis

Despite recent intensive research efforts in functional genomics, the functions of only a limited number of Arabidopsis (Arabidopsis thaliana) genes have been determined experimentally, and improving gene annotation remains a major challenge in plant science. As metabolite profiling can characterize the metabolomic phenotype of a genetic perturbation in the plant metabolism, it provides clues to the function(s) of genes of interest. We chose 50 Arabidopsis mutants, including a set of characterized and uncharacterized mutants, that resemble wild-type plants. We performed metabolite profiling of the plants using gas chromatography-mass spectrometry. To make the data set available as an efficient public functional genomics tool for hypothesis generation, we developed the Metabolite Profiling Database for Knock-Out Mutants in Arabidopsis (MeKO). It allows the evaluation of whether a mutation affects metabolism during normal plant growth and contains images of mutants, data on differences in metabolite accumulation, and interactive analysis tools. Nonprocessed data, including chromatograms, mass spectra, and experimental metadata, follow the guidelines set by the Metabolomics Standards Initiative and are freely downloadable. Proof-of-concept analysis suggests that MeKO is highly useful for the generation of hypotheses for genes of interest and for improving gene annotation. MeKO is publicly available at http://prime.psc.riken.jp/meko/

American College of Rheumatology Provisional Criteria for Clinically Relevant Improvement in Children and Adolescents With Childhood-Onset Systemic Lupus Erythematosus

10.1002/acr.23834ARTHRITIS CARE & RESEARCH715579-59

Genetic analysis of metabolome-phenotype interactions: from model to crop species

The past decade has seen increased interest from the scientific community, and particularly plant biologists, in integrating metabolic approaches into research aimed at unraveling phenotypic diversity and its underlying genetic variation. Advances in plant metabolomics have enabled large-scale analyses that have identified qualitative and quantitative variation in the metabolic content of various species, and this variation has been linked to genetic factors through genetic-mapping approaches, providing a glimpse of the genetic architecture of the plant metabolome. Parallel analyses of morphological phenotypes and physiological performance characteristics have further enhanced our understanding of the complex molecular mechanisms regulating these quantitative traits. This review aims to illustrate the advantages of including assessments of phenotypic and metabolic diversity in investigations of the genetic basis of complex traits, and the value of this approach in studying agriculturally important crops. We highlight the ground-breaking work on model species and discuss recent achievements in important crop species

Effects of Sulfur Assimilation in Pseudomonas fluorescens SS101 on Growth, Defense, and Metabolome of Different Brassicaceae

Genome-wide analysis of plant-growth-promoting Pseudomonas fluorescens strain SS101 (Pf SS101) followed by site-directed mutagenesis previously suggested that sulfur assimilation may play an important role in growth promotion and induced systemic resistance in Arabidopsis. Here, weinvestigated the effects of sulfur metabolism in Pf SS101 on growth, defense, and shoot metabolomes of Arabidopsis and the Brassica crop, Broccoli. Root tips of seedlings of Arabidopsis and two Broccoli cultivars were treated with Pf SS101 or with a mutant disrupted in the adenylsulfate reductase cysH,a key gene in cysteine and methionine biosynthesis. Phenotyping of plants treated with wild-type Pf SS101 or its cysH mutant revealed that sulfur assimilation in Pf SS101 was associated with enhanced growth of Arabidopsis but with a reduction in shoot biomass of two Broccoli cultivars. Untargetedmetabolomics revealed that cysH-mediated sulfur assimilation in Pf SS101 had significant effects on shoot chemistry of Arabidopsis, in particular on chain elongation of aliphatic glucosinolates (GLSs) and on indole metabolites, including camalexin and the growth hormone indole-3-acetic acid. InBroccoli, Pf SS101 sulfur assimilation significantly upregulated the relative abundance of several shoot metabolites, in particular, indolic GLSs and phenylpropanoids. These metabolome changes in Broccoli plants coincided with Pf SS101-mediated suppression of leaf infections by Xanthomonas campestris.Our study showed the metabolic interconnectedness of plants and their root-associated microbiota

Sucrose and Starch Content Negatively Correlates with PSII Maximum Quantum Efficiency in Tomato (Solanum lycopersicum) Exposed to Abnormal Light/Dark Cycles and Continuous Light"

Light is most important to plants as it fuels photosynthesis and provides clues about the environment. If provided in unnatural long photoperiods, however, it can be harmful and even lethal. Tomato (Solanum lycopersicum), for example, develops mottled chlorosis and necrosis when exposed to continuous light. Understanding the mechanism of these injuries is valuable, as important pathways regulating photosynthesis, such as circadian, retrograde and light signaling pathways are probably involved. Here, we use non-targeted metabolomics and transcriptomics analysis as well as hypothesis-driven experiments with continuous light-tolerant and -sensitive tomato lines to explore the long-standing proposed role of carbohydrate accumulation in this disorder. Analysis of metabolomics and transcriptomics data reveals a clear effect of continuous light on sugar metabolism and photosynthesis. A strong negative correlation between sucrose and starch content with the severity of continuous light-induced damage quantified as the maximum quantum efficiency of PSII (Fv/Fm) was found across several abnormal light/dark cycles, supporting the hypothesis that carbohydrates play an important role in the continuous light-induced injury. We postulate that the continuous light-induced injury in tomato is caused by down-regulation of photosynthesis, showing characteristics of both cytokinin-regulated senescence and light-modulated retrograde signaling. Molecular mechanisms linking carbohydrate accumulation with down-regulation of carbon-fixing enzymes are discussed

Cross-platform comparative analyses of genetic variation in amino acid content in potato tubers

Many of the biochemical pathways for plant amino acid metabolism are known and, at least in model species, most of the genes encoding the biosynthetic enzymes have been identified. How the accumulation of amino acids is regulated is much less well understood and for this genetic analysis can be instrumental. In potato, the nutritional value of the tubers is often determined by the content of essential amino acids such as lysine, tyrosine, methionine and cysteine. Better insight into the genetic determinants underlying the variation in amino acid accumulation in potato could support efforts to improve tuber nutritional quality by breeding. In this study, we used a diploid potato mapping population to explore the genetic basis of amino acid content. Hereto, we compared the use of one non-targeted and two targeted analytical approaches for amino acid analysis, allowing the evaluation of the robustness of amino acid quantification and the number and strength of detected quantitative trait locis (QTLs) across the different analytical platforms. Assessment of the three methodologies revealed a comparable detection of amino acids using non-targeted and targeted approaches. QTL detection across the different analytical platforms was similar, although slight differences in strength and explained variance were observed. The QTL regions were subsequently studied to provide candidate genes for the genetic regulation of amino acid accumulation in potato. Our results are discussed in the context of the detection of amino acid variation and its implications for the identification of QTLs