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Plants produce a large variety of highly functionalized terpenoids. Functional groups such as partially unsaturated rings and carboxyl groups provide handles to use these compounds as feedstock for biobased commodity chemicals. For instance, methylperillate, a monoterpenoid found in Salvia dorisiana, may be used for this purpose, as it carries both an unsaturated ring and a methylated carboxyl group. The biosynthetic pathway of methylperillate in plants is still unclear. In this work, we identified glandular trichomes from S. dorisiana as the location of biosynthesis and storage of methylperillate. mRNA from purified trichomes was used to identify four genes that can encode the pathway from geranyl diphosphate towards methylperillate. This pathway includes a (-)-limonene synthase (SdLS), a limonene 7-hydroxylase (SdL7H, CYP71A76), and a perillyl alcohol dehydrogenase (SdPOHDH). We also identified a terpene acid methyltransferase, perillic acid O-methyltransferase (SdPAOMT), with homology to salicylic acid OMTs. Transient expression in Nicotiana benthamiana of these four genes, in combination with a geranyl diphosphate synthase to boost precursor formation, resulted in production of methylperillate. This demonstrates the potential of these enzymes for metabolic engineering of a feedstock for biobased commodity chemicals.

Gene expression in different tissues of Salvia dorisiana, Gene expression in different tissues of Salvia dorisiana

RNAseq of Salvia dorisiana plant parts Overall design: RNAseq of Salvia dorisiana plant parts, RNAseq of Salvia dorisiana plant parts Overall design: RNAseq of Salvia dorisiana plant parts

Female flowers of hop (Humulus lupulus) are an essential source of terpenoid-related compounds, which are mainly used as flavoring in the beer brewing process. The compounds involved are bitter acids, terpenophenolics, as well as mono- and sesquiterpenoids. In this work, we analyzed the proteome of purified glandular trichomes (lupulin glands) from female flowers, which produce and accumulate these compounds. An extensive 2D-LC-MS/MS analysis identified 1,015 proteins. Of these, most correspond to housekeeping and primary metabolism-related proteins, albeit predominantly including amino acid and lipid metabolism, which feeds the specialized (secondary) metabolism. Indeed, 75 proteins belong to the specialized metabolism. No less than 40 enzymes are involved in the synthesis of terpenoid-derived compounds and 21 are predicted transporters, some of which might be involved in the transport of specialized metabolites. We discuss the possible routes involved in the intra- and intercellular translocation of terpenoids and their precursors. This comprehensive proteomic map of the glandular trichomes of hop female flowers represents a valuable resource to improve our knowledge on the function of glandular trichomes. This article is protected by copyright. All rights reserved.

Symbiotic bacteria are common in insects and can affect various aspects of their hosts’ biology. Although the effects of insect symbionts have been clarified for various insect symbiosis models, due to the difficulty of cultivating them in vitro, there is still limited knowledge available on the molecular features that drive symbiosis. Serratia symbiotica is one of the most common symbionts found in aphids. The recent findings of free-living strains that are considered as nascent partners of aphids provide the opportunity to examine the molecular mechanisms that a symbiont can deployed at the early stages of the symbiosis (i.e., symbiotic factors). In this work, a proteomic approach was used to establish a comprehensive proteome map of the free-living S. symbiotica strain CWBI-2.3T. Most of the 720 proteins identified are related to housekeeping or primary metabolism. Of these, 76 were identified as candidate proteins possibly promoting host colonization. Our results provide strong evidence that S. symbiotica CWBI-2.3T is well-armed for invading insect host tissues, and suggest that certain molecular features usually harbored by pathogenic bacteria are no longer present. This comprehensive proteome map provides a series of candidate genes for further studies to understand the molecular cross-talk between insects and symbiotic bacteria.

Among the specialized (secondary) plant metabolites, terpenoids represent the most diverse family and are often involved in the defense against pathogens and herbivores. Terpenoids can be produced both constitutively and in response to the environment. At the front line of this defense strategy are the glandular trichomes, which are organs dedicated primarily to the production of specialized metabolites. Mass spectrometry-based proteomics is a powerful tool, which is very useful to investigate enzymes involved in metabolic pathways, such as the synthesis and secretion of terpenoids in glandular trichomes. Here we review the strategies used to investigate the specific roles of these particular organs from non-model plant species, mainly belonging to the Lamiaceae, Solanaceae, and Cannabaceae families. We discuss how proteomics helps to accurately pinpoint candidate proteins to be functionally characterized, and how technological progresses create opportunities for studying low-abundance proteins, such as the ones related to the synthesis and transport of specialized metabolites. This article is part of a Special Issue entitled: Plant Proteomics - a bridge between fundamental processes and crop production, edited by Dr. Hans-Peter Mock.

Leaf glandular trichomes (epidermal hairs) actively synthesize secondary metabolites, many of which are the frontline of plant defense. In Nicotiana tabacum, tall and short glandular trichomes have been identified. While the former have been extensively studied and match the classic picture of trichome function, the short trichomes have remained relatively uncharacterized. We have set up a procedure based on centrifugation on Percoll density gradients to obtain separate tall and short trichome fractions purified to >85%. We then investigated the proteome of both trichome types combining 2D-LC fractionation of tryptic peptides and quantification of a set of 461 protein groups using isobaric tags for relative and absolute quantitation. Almost the entire pathway leading to the synthesis of diterpenes was identified in the tall trichomes. Indications for their key roles in the synthesis of cuticular compounds were also found. Concerning the short glandular trichomes, ribosomal proteins and enzymes such phosphoenolpyruvate carboxykinase and polyphenol oxidase were more abundant than in the tall glandular trichomes. These results are discussed in the frame of several hypotheses regarding the respective roles of short and long glandular trichomes.

Peltate glandular trichomes from Mentha spicata were purified on a Percoll gradient and soluble and membrane proteins were trypsinized and the peptides were separated by nano-LC fractionation and analyzed by MALDI-MS/MS. The vast majority of the 1666 proteins identified were housekeeping proteins or involved in the primary metabolism. However, 57 were predicted to be involved in the secondary metabolism. Of these, 21 were involved in the synthesis of phenylpropanoids and phenolics and 32 in terpenoid synthesis. Of the 14 membrane transporters identified, the 11 ATP-binding cassette transporters provide good material for assessing whether active transport is required for the transfer of monoterpenoid intermediates between cellular compartments and for the secretion of the final products into the subcuticular storage cavity. In conclusion, this proteome analysis of M. spicata peltate trichomes has identified several candidate proteins that might be involved in terpenoid synthesis and transport. The data have been deposited to the ProteomeXchange with identifier PXD000352 (http://proteomecentral.proteomexchange.org/dataset/PXD000352).

Until recently, large scale proteomic investigations in the plant field have only been possible for a few model species for which the whole genome sequence had been fully determined. In contrast, for many other species with a strong economic interest as sources of human food and animal feed, as well as industrial and pharmacological molecules, little was known about their genome sequence and identifying the proteome in these species was still considered challenging. However, progress has been made as a result of several recent advances in proteomics tools, e.g. in MS technology and data search programs, and the increasing availability of genomic and cDNA sequences from various species. Moreover, next-generation sequencing technologies now make it possible to rapidly determine, at a reasonable cost, the genome or RNA sequence of species not currently considered as models, thus considerably expanding the plant sequence databases. This review will show how these advances make it possible to identify a large set of proteins, even for species for which few sequences are currently available.

Madagascar periwinkle (Catharanthus roseus) is the major source of terpenoid indole alkaloids, such as vinblastine or vincristine, used as natural drugs against various cancers. In this study, we have extensively analyzed the proteome of cultured C. roseus cells. Comparison of the proteomes of two independent cell lines with different terpenoid indole alkaloid metabolism by 2D-DIGE revealed 358 proteins that differed quantitatively by at least a twofold average ratio. Of these, 172 were identified by MS; most corresponded to housekeeping proteins. Less abundant proteins were identified by LC separation of tryptic peptides of proteins from one of the lines. We identified 1663 proteins, most of which are housekeeping proteins or involved in primary metabolism. However, 63 enzymes potentially involved in secondary metabolism were also identified, of which 22 are involved in terpenoid indole alkaloid biosynthesis and 16 are predicted transporters putatively involved in secondary metabolite transport. About 30% of the proteins identified have an unclear or unknown function, indicating important gaps in knowledge of plant metabolism. This study is an important step toward elucidating the proteome of C. roseus, which is critical for a better understanding of how this plant synthesizes terpenoid indole alkaloids.

Biofortification of taro (Colocasia esculenta) has never been studied. The aim of the present study is to compare the chemical compositions and individual constituent contents for major compounds (starch, sugars, cellulose, proteins, minerals), carotenoids and anthocyanins between parents and hybrids selected first for their agronomic performance and second for their corm characteristics (flesh colour, quality and taste). For major compounds, 45 selected hybrids were compared to 66 cultivars and for carotenoids and anthocyanins, 34 selected hybrids were compared to 79 cultivars. All plants were planted the same day within the same plot and harvested together to avoid environmental factors effects. Total sugars, cellulose and minerals contents presented moderate increases in hybrids. Carotenoids and anthocyanins contents were correlated with corm flesh colours. Anthocyanins contents could not be increased in the selected hybrids. However, total carotenoids contents were increased by more than four folds in the hybrids. The results of this study indicate that carotenoids contents can be rapidly improved by selecting plants of good agronomic performance and corm shape with increased density of yellow and orange colours. Potential applications to taro breeding programmes are discussed.

Over-expression and co-silencing of NtPDR1 in Nicotiana tabacum hairy roots.

Mining the proteome of Catharanthus roseus cell lines. SmartCell research meeting.

Tropical root and tuber crops are staple foods in developing countries where they are the main source of carbohydrates and a potential source of secondary metabolites such as carotenoids. Enhancing carotenoid content has considerable potential for improving human nutrition in these areas. Except for cassava and sweet potato, carotenoid content has not been evaluated in large germplasm collections. We describe a simple HPLC-DAD-based protocol for the reliable characterization of carotenoids in root crops. Less than 5% variation in carotenoid contentwas found among samples collected from tubers of the same plant or from clones of the same genotype. Carotenoids were evaluated in 153 accessions representing 10 different species. Each species had a specific profile of carotenoids, and accessions of the same species differed greatly in their individual content. The database assembled during this study represents a useful tool for nutritionists and breeders working on biofortification of root crops with carotenoids.

Agronomie selection generates and maintains agrobiodiversity which can be regarded as an essential resource. To describe phenotypic diversity, genotypes as well as their chemical expression, chemotypes, have been widely used. Tropical root and tuber crops are vegetatively propagated and their genetic bases are often narrow. Therefore, unveiling the impressive phenotypic diversity is still rather difficult through genotyping alone, whereas chemotype studies are very informative. Tropical root crops are staples and represent a good source of compounds related to health benefits and used in food and pharmaceutical indu stries. Those crops have keyroles to play in food security for developing countries and so plant breeding cannot be neglected. Biofortification is effective and does not imply change of dietary behaviours. Analysing, computing and compiling data bases for chernical data on diversity of neglected crops are still needed. Biofortification involving mass selection and profiling of numerous accessions is , however, time-consuming. Thus, efficient protocols and tools facilitating this process are required. Chemotypic variability within Vanuatu germplasm was characterised through core samples from different species and relationships between primary compounds, secondary metabolites and local preferences have been studied. Useful correlations were shown, thus clarifying traditional selection process. This process has permitted the definition of ideotypes that will be interesting for the management of breeding programmes. At least for the two most important species in Vanuatu - taro Colocasia esculenta and the greater yam Dioscorea alata - our work is the fust broad screening of germplasm. The study of carotenoid and anthocyanin content indicates that sorne cultivars show potential for commercial exploitation. With the aim of improving staple foods by plant breeding, we identified accessions to be selected as parents for future crosses.

The objectives of the present study were to characterize good-quality cultivars, identify relationships between local eating preferences and primary compound content, and reveal biofortification potential in tropical root crop species aroids, yams, cassava, and sweet potato. A core sample of about 500 cultivars was assembled to represent the widest agro-morphological diversity. Very high coefficients of variation were found within species for proteins, sugars, cellulose, and mineral contents, whereas starch exhibited the lowest variation. Starch content was negatively correlated with other primary compound contents. For the national dish in Vanuatu, consumers prefer cultivars with high starch content. In contrast, preferences for daily consumption of boiled or roasted tubers are linked to average starch content, indicating great potential for improving primary compounds. Interestingly, relationships between flesh color and requirements for the traditional dish were revealed, suggesting opportunities for biofortification. The data produced will assist breeders in adopting appropriate biofortification strategies.

The objectives of the present study were to characterize good-quality cultivars, identify relationships between local eating preferences and primary compound content, and reveal biofortification potential in tropical root crop species aroids, yams, cassava, and sweet potato. A core sample of about 500 cultivars was assembled to represent the widest agro-morphological diversity. Very high coefficients of variation were found within species for proteins, sugars, cellulose, and mineral contents, whereas starch exhibited the lowest variation. Starch content was negatively correlated with other primary compound contents. For the national dish in Vanuatu, consumers prefer cultivars with high starch content. In contrast, preferences for daily consumption of boiled or roasted tubers are linked to average starch content, indicating great potential for improving primary compounds. Interestingly, relationships between flesh color and requirements for the traditional dish were revealed, suggesting opportunities for biofortification. The data produced will assist breeders in adopting appropriate biofortification strategies.

In Vanuatu, Melanesia, root crops (aroids, cassava, sweet potato and yams) are staples. Several methods exist and are employed to process and cook these foods such as boiling, roasting or baking on hot stones. In Vanuatu, unlike other Melanesian and Pacific countries, these starchy foods are processed into the traditional dish called laplap, which is a pudding-like dish prepared from hand-grated fruits, corms, roots or tubers. The raw paste is wrapped in Heliconia indica leaves and slowly steamed in a ground-oven. Producers and consumers alike are unanimous to state that some cultivars are suitable for laplap while others are not. It is still unknown what the chemical characteristics of so-called “good” varieties are and how chemotypes are traditionally selected.

Ce travail fait partie du projet « préservation et utilisation de l’agrobiodiversité de plantes à racines et tubercules au Vanouatou », et a donc pour objectif la sélection variétale. Puisque l’amélioration de plantes à multiplication végétative passe par le choix judicieux de parents ainsi que par le criblage de grands nombres au niveau des descendances, une caractérisation physico-chimique précise est nécessaire. Un échantillonnage représentant les principales plantes à racines et tubercules consommées au Vanouatou (Mélanésie), a donc été choisi pour constituer un large spectre de plantes appartenant à quatre familles botaniques différentes. Les isoprénoïdes ont été extraits à l’acétone à partir de 163 échantillons lyophilisés, et les profils réalisés par CLHP. Les teneurs et compositions ont été déterminées à l’aide d’un détecteur à barrette de diodes et de standards choisis dans la voie de biosynthèse des caroténoïdes. Ceci permet d’établir des profils métaboliques pour étudier, chez ces plantes, la variabilité au niveau interspécifique.

Many developing countries have found great difficulty in sustaining the conservation and genetic improvement of root crops. Conservation is fraught with difficulty: ex situ collections are expensive to maintain and methods for on-farm conservation have not been studied. This project is testing the hypothesis that the geographical distribution of allelic diversity can represent an efficient and practical alternative to present conservation and use activities.