Antoine Champagne

NIR Determination of Major Constituents in Tropical Root and Tuber Crop Flours

Tropical root and tuber crops (cassava, sweet potato, taro, and yam) are staples in developing countries where rapid urbanization is strengthening the demand for flour based foods. Quality control techniques are still under development, and when available, laboratory analyses are too expensive. The objectives of this study were to calibrate Near-infrared spectroscopy (NIRS) for routine analysis of flours and to test its reliability to determine their major constituents. Flours prepared from 472 accessions (traditional varieties and breeding lines) were analyzed for their starch, total sugars, cellulose, total nitrogen, and ash (total minerals) contents. The near-infrared (350-2500 nm) spectra of all samples were measured. Calibration equations with cross and independent validation for all analytical characteristics were computed using the partial least squares method. Models were developed separately for each of the four crop species and by combining data from all spp. to predict values within each of them. The quality of prediction was evaluated on a test set of 94 accessions (20%) by standard error of prediction (SEP) and r2 parameters between the measured and the predicted values from cross-validation. Starch, sugar, and total nitrogen content could be predicted, respectively, with 87%, 86%, and 93% confidence, whereas ash (minerals) could be predicted with 71%, and cellulose was not predictable (r2=0.31). The statistical parameters obtained for starch, sugars, and total nitrogen are of special interest for flour quality control. These constituents are quantitatively the most important in the chemical composition of flours, and starch content is negatively correlated with sugars and total nitrogen. NIRS is a low cost technique well adapted to the conditions in developing countries and can be used for the high-throughput screening of a great number of samples. Possible applications are discussed.

Proteomics of nonmodel plant species

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.

In-depth proteome mining of cultured Catharanthus roseus cells identifies candidate proteins involved in the synthesis and transport of secondary metabolites

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.

Comparative Proteomics of Short and Tall Glandular Trichomes of Nicotiana tabacum Reveals Differential Metabolic Activities.

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.

Proteomics of terpenoid biosynthesis and secretion in trichomes of higher plant species.

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.

Chemotype profiling to guide breeders and explore traditional selection of tropical root crops in Vanuatu, South Pacific

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.

Biofortification of taro (Colocasia esculenta) through breeding for increased contents in carotenoids and anthocyanins

Abstract 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 color, 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. Total sugars, cellulose and mineral contents presented moderate increases in hybrids. Carotenoids and anthocyanins contents were not correlated with corm flesh colors, most likely in relation with the wide range of the observed variability. Anthocyanin contents could not be increased in the selected hybrids, while total carotenoid contents were increased by more than fourfolds. The results of this study indicate that carotenoid contents can be rapidly improved by selecting plants of good agronomic performance and corm shape with increased density of yellow and orange colors. Potential applications to taro breeding programs are discussed.

A comprehensive proteome map of glandular trichomes of hop (Humulus lupulus L.) female cones: Identification of biosynthetic pathways of the major terpenoid-related compounds and possible transport proteins

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 1015 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.

Toward a better understanding of the mechanisms of symbiosis: a comprehensive proteome map of a nascent insect symbiont

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 deploy 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.