NMR-based Metabolomics to Study the Cold-acclimation Strategy of Two Miscanthus Genotypes.

INTRODUCTION: Abiotic stress is a major cause of yield loss in plant culture. Miscanthus, a perennial C4 grass, is now considered a major source of renewable energy, especially for biofuel production. During the first year of planting in Northern Europe, Miscanthus was exposed to frost temperature, which generated high mortality in young plants and large loss of yield. One strategy to avoid such loss is to apply cold-acclimation, which confers on plants a better resistance to low temperature. OBJECTIVES: The aim of this study is to describe the effect of a cold-acclimation period on the metabolome of two Miscanthus genotypes that vary in their frost sensitivity at the juvenile stage. Miscanthus × giganteus (GIG) is particularly sensitive to frost, whereas Miscanthus sinensis August Feder (AUG) is tolerant. MATERIALS AND METHODS: Polar metabolite extraction was performed on Miscanthus, grown in non-acclimated or cold-acclimated conditions. Extracts were analysed by 1 H-NMR followed by multivariate statistical analysis. Discriminant metabolites were identified. RESULTS: More than 40 metabolites were identified in the two Miscanthus genotypes. GIG and AUG showed a different metabolic background before cold treatment, probably related to their genetic background. After cold-acclimation, GIG and AUG metabolomes remained different. The tolerant genotype showed notably higher levels of accumulation in proline, sucrose and maltose when subjected to cold. CONCLUSION: These two genotypes seem to have a different adaptation strategy in cold conditions. The studied change in the metabolome concerns different types of molecules related to the cold-tolerant behaviour of Miscanthus. Copyright © 2016 John Wiley & Sons, Ltd.

Cell Wall Metabolism in Response to Abiotic Stress.

This review focuses on the responses of the plant cell wall to several abiotic stresses including drought, flooding, heat, cold, salt, heavy metals, light, and air pollutants. The effects of stress on cell wall metabolism are discussed at the physiological (morphogenic), transcriptomic, proteomic and biochemical levels. The analysis of a large set of data shows that the plant response is highly complex. The overall effects of most abiotic stress are often dependent on the plant species, the genotype, the age of the plant, the timing of the stress application, and the intensity of this stress. This shows the difficulty of identifying a common pattern of stress response in cell wall architecture that could enable adaptation and/or resistance to abiotic stress. However, in most cases, two main mechanisms can be highlighted: (i) an increased level in xyloglucan endotransglucosylase/hydrolase (XTH) and expansin proteins, associated with an increase in the degree of rhamnogalacturonan I branching that maintains cell wall plasticity and (ii) an increased cell wall thickening by reinforcement of the secondary wall with hemicellulose and lignin deposition. Taken together, these results show the need to undertake large-scale analyses, using multidisciplinary approaches, to unravel the consequences of stress on the cell wall. This will help identify the key components that could be targeted to improve biomass production under stress conditions.

Structural alteration of cell wall pectins accompanies pea development in response to cold.

Pea (Pisum sativum) cell wall metabolism in response to chilling was investigated in a frost-sensitive genotype 'Terese' and a frost-tolerant genotype 'Champagne'. Cell walls isolated from stipules of cold acclimated and non-acclimated plants showed that cold temperatures induce changes in polymers containing xylose, arabinose, galactose and galacturonic acid residues. In the tolerant cultivar Champagne, acclimation is accompanied by increases in homogalacturonan, xylogalacturonan and highly branched Rhamnogalacturonan I with branched and unbranched (1→5)-α-arabinans and (1→4)-ß-galactans. In contrast, the sensitive cultivar Terese accumulates substantial amounts of (1→4)-ß-xylans and glucuronoxylan, but not the pectins. Greater JIM7 labeling was observed in Champagne compared to Terese, indicating that cold acclimation also induces an increase in the degree of methylesterification of pectins. Significant decrease in polygalacturonase activities in both genotypes were observed at the end of cold acclimation. These data indicate a role for esterified pectins in cold tolerance. The possible functions for pectins and their associated arabinans and galactans in cold acclimation are discussed.

Cell wall compositional modifications of Miscanthus ecotypes in response to cold acclimation.

Miscanthus, a potential energy crop grass, can be damaged by late frost when shoots emerge too early in the spring and during the first winter after planting. The effects of cold acclimation on cell wall composition were investigated in a frost-sensitive clone of Miscanthus x giganteus compared to frost-tolerant clone, Miscanthus sinensis August Feder, and an intermediate frost-tolerant clone, M. sinensis Goliath. Cellulose and lignin contents were higher in M. x giganteus than in the M. sinensis genotypes. In ambient temperature controls, each clone displayed different glucuronoarabinoxylan (GAX) contents and degree of arabinose substitution on the xylan backbone. During cold acclimation, an increase in (1→3),(1→4)-ß-D-glucan content was observed in all genotypes. Uronic acid level increased in the frost sensitive genotype but decreased in the frost tolerant genotypes in response to cold. In all clones, major changes in cell wall composition were observed with modifications in phenylalanine ammonia-lyase (PAL) and cinnamyl alcohol dehydrogenase (CAD) activities in both non- and cold-acclimated experiments. A large increase in CAD activity under cold stress was displayed in each clone, but it was largest in the frost-tolerant clone, M. sinensis August Feder. The marked increase in PAL activity observed in the frost-tolerant clones under cold acclimation, suggests a reorientation of the products towards the phenylpropanoid pathway or aromatic synthesis. How changes in cell wall physical properties can impact frost tolerance is discussed.

The transcription factor BELLRINGER modulates phyllotaxis by regulating the expression of a pectin methylesterase in Arabidopsis.

Plant leaves and flowers are positioned along the stem in a regular pattern. This pattern, which is referred to as phyllotaxis, is generated through the precise emergence of lateral organs and is controlled by gradients of the plant hormone auxin. This pattern is actively maintained during stem growth through controlled cell proliferation and elongation. The formation of new organs is known to depend on changes in cell wall chemistry, in particular the demethylesterification of homogalacturonans, one of the main pectic components. Here we report a dual function for the homeodomain transcription factor BELLRINGER (BLR) in the establishment and maintenance of the phyllotactic pattern in Arabidopsis. BLR is required for the establishment of normal phyllotaxis through the exclusion of pectin methylesterase PME5 expression from the meristem dome and for the maintenance of phyllotaxis through the activation of PME5 in the elongating stem. These results provide new insights into the role of pectin demethylesterification in organ initiation and cell elongation and identify an important component of the regulation mechanism involved.

Identification of pectin methylesterase 3 as a basic pectin methylesterase isoform involved in adventitious rooting in Arabidopsis thaliana.

• Here, we focused on the biochemical characterization of the Arabidopsis thaliana pectin methylesterase 3 gene (AtPME3; At3g14310) and its role in plant development. • A combination of biochemical, gene expression, Fourier transform-infrared (FT-IR) microspectroscopy and reverse genetics approaches were used. • We showed that AtPME3 is ubiquitously expressed in A. thaliana, particularly in vascular tissues. In cell wall-enriched fractions, only the mature part of the protein was identified, suggesting that it is processed before targeting the cell wall. In all the organs tested, PME activity was reduced in the atpme3-1 mutant compared with the wild type. This was related to the disappearance of an activity band corresponding to a pI of 9.6 revealed by a zymogram. Analysis of the cell wall composition showed that the degree of methylesterification (DM) of galacturonic acids was affected in the atpme3-1 mutant. A change in the number of adventitious roots was found in the mutant, which correlated with the expression of the gene in adventitious root primordia. • Our results enable the characterization of AtPME3 as a major basic PME isoform in A. thaliana and highlight its role in adventitious rooting.

Major changes in the cell wall during silique development in Arabidopsis thaliana.

Fruit development is a highly complex process, which involves major changes in plant metabolism leading to cell growth and differentiation. Changes in cell wall composition and structure play a major role in modulating cell growth. We investigated the changes in cell wall composition and the activities of associated enzymes during the dry fruit development of the model plant Arabidopsis thaliana. Silique development is characterized by several specific phases leading to fruit dehiscence and seed dispersal. We showed that early phases of silique growth were characterized by specific changes in non-cellulosic sugar content (rhamnose, arabinose, xylose, galactose and galacturonic acid). Xyloglucan oligosaccharide mass profiling further showed a strong increase in O-acetylated xyloglucans over the course of silique development, which could suggest a decreased capacity of xyloglucans to be associated with each other or to cellulose. The degree of methylesterification, mediated by the activity of pectin methylesterases (PMEs), decreased over the course of silique growth and dehiscence. The major changes in cell wall composition revealed by our analysis suggest that it could be major determinants in modulating cell wall rheology leading to growth or growth arrest.

Comprehensive expression profiling of the pectin methylesterase gene family during silique development in Arabidopsis thaliana.

Pectin methylesterases (PME, EC. 3.1.1.11) are enzymes that demethylesterify plant cell wall pectins in muro. In Arabidopsis thaliana, putative PME proteins are thought to be encoded by a 66-member gene family. This study used real-time RT-PCR to gain an overview of the expression of the entire family at eight silique developmental stages, in flower buds and in vegetative tissue in the Arabidopsis. Only 15% of the PMEs were not expressed at any of the developmental stages studied. Among expressed PMEs, expression data could be clustered into five distinct groups: 19 PMEs highly or uniquely expressed in floral buds, 4 PMEs uniquely expressed at mid-silique developmental stages, 16 PMEs highly or uniquely expressed in silique at late developmental stages, 16 PMEs mostly ubiquitously expressed, and 1 PME with a specific expression pattern, i.e. not expressed during early silique development. Comparison of expression and phylogenetic profiles showed that, within phylogenetic group 2, all but one PME belong to the floral bud expression group. Similar results were shown for a subset of one of the phylogenetic group, which differed from others by containing most of the PMEs that do not possess any PRO part next to their catalytic part. Expression data were confirmed by two promoter:GUS transgenic plant analysis revealing a PME expressed in pollen and one in young seeds. Our results highlight the high diversity of PME expression profiles. They are discussed with regard to the role of PMEs in fruit development and cell growth.

High accumulation of dehydrodiconiferyl alcohol-4-beta-D: -glucoside in free and immobilized Linum usitatissimum cell cultures.

As flaxseed mainly accumulates lignans (secoisolariciresinol diglucoside and matairesinol), these compounds were barely or not detected in plant cell suspensions initiated from Linum usitatissimum. In contrast, these cell suspensions were shown to accumulate substantial amounts of a neolignan identified as dehydrodiconiferyl alcohol-4-beta-D: -glucoside (DCG) (up to 47.7 mg g(-1) DW). The formation of this pharmacologically active compound was evaluated as a function of cell growth and in relation to phytohormone balance of the culture media. After establishment of efficient culture conditions, production of DCG was investigated in immobilized plant cell suspensions initiated from plantlet roots of L. usitatissimum. The results indicate that immobilization enhances the DCG production up to 60.0 mg g(-1) DW but depresses the cell growth resulting in no improvement of the total DCG yield. Nevertheless, with immobilized cell suspensions, a release of DCG into the medium is observed allowing an easier recovery.

Stereoselectivity of the demethylation of nicotine piperidine homologues by Nicotiana plumbaginifolia cell suspension cultures.

The metabolism of (R,S)-N-methylanabasine and (R,S)-N-methylanatabine has been studied in a cell suspension culture of Nicotiana plumbaginifolia. Both substrates are effectively demethylated, anabasine or anatabine, respectively, accumulating in the medium. Similarly, there is strong stereoselectivity for the (R)-isomers of both substrates. The kinetics of metabolism of (R,S)-N-methylanabasine differ significantly from those of nicotine in that no further degradation of the initial demethylation product occurs. (R,S)-N-Methylanatabine, however, shows kinetics closer to those of nicotine, with loss of alkaloid from the system. Further more, (R,S)-N-methylanabasine does not diminish (S)-nicotine demethylation, indicating a lack of competition. However, the metabolism of (S)-nicotine is affected by the presence of (R,S)-N-methylanabasine. Hence, the demethylation of the piperidine homologues of nicotine is seen to be similar but not identical to that of the pyridine analogues. The implications of these different metabolic profiles in relation to the demethylation activity are discussed.

Variation in the accumulation levels of N,N-dimethyltryptamine in micropropagated trees and in in vitro cultures of Mimosa tenuiflora.

The present article reports the accumulation of N,N-dimethyltryptamine and its metabolic precursors (tryptophan, tryptamine) in different organs of micropropagated Mimosa tenuiflora trees (leaves, flowers and bark) subjected to seasonal variations (January and June), as well as in in vitro cultures (plantlets and calluses) of this plant species. The accumulation of all the tested compounds varied according to the organ, the month of collection, and age of the plant material. In all cases, the neurotoxic compound N,N-dimethyltryptamine (DMT) was detected with the lowest concentration 0.01% dry weight (DW) in flowers, and the highest 0.33% DW in bark. For the in vitro cultures, DMT was present in high yields in plantlets (0.1-0.2% DW), while in calluses this compound was initially detected but its concentration decreased significantly in the subsequent subcultures.