Exploration of the Diversity of Vicine and Convicine Derivatives in Faba Bean (Vicia faba L.) Cultivars: Insights from LC-MS/MS Spectra.

While numerous Fabaceae seeds are a good nutritional source of high-quality protein, the use of some species is hampered by toxic effects caused by exposure to metabolites that accumulate in the seeds. One such species is the faba or broad bean (Vicia faba L.), which accumulates vicine and convicine. These two glycoalkaloids cause favism, the breakdown of red blood cells in persons with a glucose-6-phosphate dehydrogenase deficiency. Because this is the most common enzyme deficiency worldwide, faba bean breeding efforts have focused on developing cultivars with low levels of these alkaloids. Consequently, quantification methods have been developed; however, they quantify vicine and convicine only and not the derivatives of these compounds that potentially generate the same bio-active molecules. Based on the recognition of previously unknown (con)vicine-containing compounds, we screened the fragmentation spectra of LC-MS/MS data from five faba bean cultivars using the characteristic fragments generated by (con)vicine. This resulted in the recognition of more than a hundred derivatives, of which 89 were tentatively identified. (Con)vicine was mainly derivatized through the addition of sugars, hydroxycinnamic acids, and dicarboxylic acids, with a group of compounds composed of two (con)vicine residues linked by dicarboxyl fatty acids. In general, the abundance profiles of the different derivatives in the five cultivars mimicked that of vicine and convicine, but some showed a derivative-specific profile. The description of the (con)vicine diversity will impact the interpretation of future studies on the biosynthesis of (con)vicine, and the content in potentially bio-active alkaloids in faba beans may be higher than that represented by the quantification of vicine and convicine alone.

Integrated transcriptomics and proteomics analysis reveals muscle metabolism effects of dietary Ulva lactuca and ulvan lyase supplementation in weaned piglets.

Seaweeds, including the green Ulva lactuca, can potentially reduce competition between feed, food, and fuel. They can also contribute to the improved development of weaned piglets. However, their indigestible polysaccharides of the cell wall pose a challenge. This can be addressed through carbohydrase supplementation, such as the recombinant ulvan lyase. The objective of our study was to assess the muscle metabolism of weaned piglets fed with 7% U. lactuca and 0.01% ulvan lyase supplementation, using an integrated transcriptomics (RNA-seq) and proteomics (LC-MS) approach. Feeding piglets with seaweed and enzyme supplementation resulted in reduced macronutrient availability, leading to protein degradation through the proteasome (PSMD2), with resulting amino acids being utilized as an energy source (GOT2, IDH3B). Moreover, mineral element accumulation may have contributed to increased oxidative stress, evident from elevated levels of antioxidant proteins like catalase, as a response to maintaining tissue homeostasis. The upregulation of the gene AQP7, associated with the osmotic stress response, further supports these findings. Consequently, an increase in chaperone activity, including HSP90, was required to repair damaged proteins. Our results suggest that enzymatic supplementation may exacerbate the effects observed from feeding U. lactuca alone, potentially due to side effects of cell wall degradation during digestion.

Impact of Heavy Metals on Cold Acclimation of Salix viminalis Roots.

In nature, plants are exposed to a range of climatic conditions. Those negatively impacting plant growth and survival are called abiotic stresses. Although abiotic stresses have been extensively studied separately, little is known about their interactions. Here, we investigate the impact of long-term mild metal exposure on the cold acclimation of Salix viminalis roots using physiological, transcriptomic, and proteomic approaches. We found that, while metal exposure significantly affected plant morphology and physiology, it did not impede cold acclimation. Cold acclimation alone increased glutathione content and glutathione reductase activity. It also resulted in the increase in transcripts and proteins belonging to the heat-shock proteins and related to the energy metabolism. Exposure to metals decreased antioxidant capacity but increased catalase and superoxide dismutase activity. It also resulted in the overexpression of transcripts and proteins related to metal homeostasis, protein folding, and the antioxidant machinery. The simultaneous exposure to both stressors resulted in effects that were not the simple addition of the effects of both stressors taken separately. At the antioxidant level, the response to both stressors was like the response to metals alone. While this should have led to a reduction of frost tolerance, this was not observed. The impact of the simultaneous exposure to metals and cold acclimation on the transcriptome was unique, while at the proteomic level the cold acclimation component seemed to be dominant. Some genes and proteins displayed positive interaction patterns. These genes and proteins were related to the mitigation and reparation of oxidative damage, sugar catabolism, and the production of lignans, trehalose, and raffinose. Interestingly, none of these genes and proteins belonged to the traditional ROS homeostasis system. These results highlight the importance of the under-studied role of lignans and the ROS damage repair and removal system in plants simultaneously exposed to multiple stressors.

The impact of dietary Laminaria digitata and alginate lyase supplementation on the weaned piglet liver: A comprehensive proteomics and metabolomics approach.

The brown seaweed Laminaria digitata, a novel feedstuff for weaned piglets, has potentially beneficial prebiotic properties. However, its recalcitrant cell wall challenges digestion in monogastrics. Alginate lyase is a promising supplement to mitigate this issue. This study's aim was to investigate the impact of incorporating 10% dietary Laminaria digitata, supplemented with alginate lyase, on the hepatic proteome and metabolome of weaned piglets. These diets introduced minor variations to the metabolome and caused significant shifts in the proteome. Dietary seaweed provided a rich source of n-3 PUFAs that could signal hepatic fatty acid oxidation (FABP, ACADSB and ALDH1B1). This may have affected the oxidative stability of the tissue, requiring an elevated abundance of GST for regulation. The presence of reactive oxygen species likely inflicted protein damage, triggering increased proteolytic activity (LAPTM4B and PSMD4). Alginate lyase supplementation augmented the number of differentially abundant proteins, which included GBE1 and LDHC, contributing to maintain circulating glucose levels by mobilizing glycogen stores and branched-chain amino acids. The enzymatic supplementation with alginate lyase amplified the effects of the seaweed-only diet. An additional filter was employed to test the effect of missing values on the proteomics analysis, which is discussed from a technical perspective. SIGNIFICANCE: Brown seaweeds such as Laminaria digitata have prebiotic and immune-modulatory components, such as laminarin, that can improve weaned piglet health. However, they have recalcitrant cell wall polysaccharides, such as alginate, that can elicit antinutritional effects on the monogastric digestive system. The aim of this study was to evaluate the effect of a high level of dietary L. digitata and alginate lyase supplementation on the hepatic metabolism of weaned piglets, using high throughput Omics approaches.

Enhanced ileum function in weaned piglets via Laminaria digitata and alginate lyase dietary inclusion: A combined proteomics and metabolomics analysis.

Laminaria digitata, a brown seaweed with prebiotic properties, can potentially enhance the resilience of weaned piglets to nutritional distress. However, their cell wall polysaccharides elude digestion by monogastric animals' endogenous enzymes. In vitro studies suggest alginate lyase's ability to degrade such polysaccharides. This study aimed to assess the impact of a 10% dietary inclusion of L. digitata and alginate lyase supplementation on the ileum proteome and metabolome, adopting a hypothesis-generating approach. Findings indicated that control piglets escalated glucose usage as an enteric energy source, as evidenced by the increased abundance of PKLR and PCK2 proteins and decreased tissue glucose concentration. Additionally, the inclusion of seaweed fostered a rise in proteins linked to enhanced enterocyte structural integrity (ACTBL2, CRMP1, FLII, EML2 and MYLK), elevated peptidase activity (NAALADL1 and CAPNS1), and heightened anti-inflammatory activity (C3), underscoring improved intestinal function. In addition, seaweed-fed piglets showed a reduced abundance of proteins related to apoptosis (ERN2) and proteolysis (DPP4). Alginate lyase supplementation appeared to amplify the initial effects of seaweed-only feeding, by boosting the number of differential proteins within the same pathways. This amplification is potentially due to increased intracellular nutrient availability, making a compelling case for further exploration of this dietary approach. SIGNIFICANCE: Pig production used to rely heavily on antibiotics and zinc oxide to deal with post-weaning stress in a cost-effective way. Their negative repercussions on public health and the environment have motivated heavy restrictions, and a consequent search for alternative feed ingredients/supplements. One of such alternatives is Laminaria digitata, a brown seaweed whose prebiotic components that can help weaned piglets deal with nutritional stress, by improving their gut health and immune status. However, their recalcitrant cell walls have antinutritional properties, for which alginate lyase supplementation is a possible solution. By evaluating ileal metabolism as influenced by dietary seaweed and enzyme supplementation, we aim at discovering how the weaned piglet adapts to them and what are their effects on this important segment of the digestive system.

Identification of new potential molecular actors related to fiber quality in flax through Omics.

One of the biggest challenges for a more widespread utilization of plant fibers is to better understand the different molecular factors underlying the variability in fineness and mechanical properties of both elementary and scutched fibers. Accordingly, we analyzed genome-wide transcription profiling from bast fiber bearing tissues of seven different flax varieties (4 spring, 2 winter fiber varieties and 1 winter linseed) and identified 1041 differentially expressed genes between varieties, of which 97 were related to cell wall metabolism. KEGG analysis highlighted a number of different enriched pathways. Subsequent statistical analysis using Partial Least-Squares Discriminant Analysis showed that 73% of the total variance was explained by the first 3 X-variates corresponding to 56 differentially expressed genes. Calculation of Pearson correlations identified 5 genes showing a strong correlation between expression and morphometric data. Two-dimensional gel proteomic analysis on the two varieties showing the most discriminant and significant differences in morphometrics revealed 1490 protein spots of which 108 showed significant differential abundance. Mass spectrometry analysis successfully identified 46 proteins representing 32 non-redundant proteins. Statistical clusterization based on the expression level of genes corresponding to the 32 proteins showed clear discrimination into three separate clusters, reflecting the variety type (spring-/winter-fiber/oil). Four of the 32 proteins were also highly correlated with morphometric features. Examination of predicted functions for the 9 (5 + 4) identified genes highlighted lipid metabolism and senescence process. Calculation of Pearson correlation coefficients between expression data and retted fiber mechanical measurements (strength and maximum force) identified 3 significantly correlated genes. The genes were predicted to be connected to cell wall dynamics, either directly (Expansin-like protein), or indirectly (NAD(P)-binding Rossmann-fold superfamily protein). Taken together, our results have allowed the identification of molecular actors potentially associated with the determination of both in-planta fiber morphometrics, as well as ex-planta fiber mechanical properties, both of which are key parameters for elementary fiber and scutched fiber quality in flax.

Isoprene-Emitting Tobacco Plants Are Less Affected by Moderate Water Deficit under Future Climate Change Scenario and Show Adjustments of Stress-Related Proteins in Actual Climate.

Isoprene-emitting plants are better protected against thermal and oxidative stresses, which is a desirable trait in a climate-changing (drier and warmer) world. Here we compared the ecophysiological performances of transgenic isoprene-emitting and wild-type non-emitting tobacco plants during water stress and after re-watering in actual environmental conditions (400 ppm of CO2 and 28 °C of average daily temperature) and in a future climate scenario (600 ppm of CO2 and 32 °C of average daily temperature). Furthermore, we intended to complement the present knowledge on the mechanisms involved in isoprene-induced resistance to water deficit stress by examining the proteome of transgenic isoprene-emitting and wild-type non-emitting tobacco plants during water stress and after re-watering in actual climate. Isoprene emitters maintained higher photosynthesis and electron transport rates under moderate stress in future climate conditions. However, physiological resistance to water stress in the isoprene-emitting plants was not as marked as expected in actual climate conditions, perhaps because the stress developed rapidly. In actual climate, isoprene emission capacity affected the tobacco proteomic profile, in particular by upregulating proteins associated with stress protection. Our results strengthen the hypothesis that isoprene biosynthesis is related to metabolic changes at the gene and protein levels involved in the activation of general stress defensive mechanisms of plants.

A molecular study of Italian ryegrass grown on Martian regolith simulant.

In the last decade, the exploration of deep space has become the objective of the national space programs of many countries. The International Space Exploration Coordination Group has set a roadmap whose long-range strategy envisions the expansion of human presence in the solar system to progress with exploration and knowledge and to accelerate innovation. Crewed missions to Mars could be envisaged by 2040. In this scenario, finding ways to use the local resources for the provision of food, construction materials, propellants, pharmaceuticals is needed. Plants are important resources for deep space manned missions because they produce phytochemicals of pharmaceutical relevance, are sources of food and provide oxygen which is crucial in bioregenerative life support systems. Growth analysis and plant biomass yield have been previously evaluated on Martian regolith simulants; however, molecular approaches employing gene expression analysis and proteomics are still missing. The present work aims at filling this gap by providing molecular data on a representative member of the Poaceae, Lolium multiflorum Lam., grown on potting soil and a Martian regolith simulant (MMS-1). The molecular data were complemented with optical microscopy of root/leaf tissues and physico-chemical analyses. The results show that the plants grew for 2 weeks on regolith simulants. The leaves were bent downwards and chlorotic, the roots developed a lacunar aerenchyma and small brownish deposits containing Fe were observed. Gene expression analysis and proteomics revealed changes in transcripts related to the phenylpropanoid pathway, stress response, primary metabolism and proteins involved in translation and DNA methylation. Additionally, the growth of plants slightly but significantly modified the pH of the regolith simulants. The results here presented constitute a useful resource to get a comprehensive understanding of the major factors impacting the growth of plants on MMS-1.

State-of-the-art methodological investigation of carotenoid activity and metabolism - from organic synthesis via metabolism to biological activity - exemplified by a novel retinoid signalling pathway.

Carotenoids are the most abundant lipophilic secondary plant metabolites and their dietary intake has been related to a large number of potential health benefits relevant for humans, including even reduced total mortality. An important feature is their potential to impact oxidative stress and inflammatory pathways, by interacting with transcription factors. For example, they may act as precursors of bioactive derivatives activating nuclear hormone receptor mediated signalling. These bioactive derivatives, originating e.g. from ß-carotene, i.e. retinoids / vitamin A, can activate the nuclear hormone receptors RARs (retinoic acid receptors). Due to new analytical insights, various novel metabolic pathways were recently outlined to be mediated via distinct nuclear hormone receptor activating pathways that were predicted and further confirmed. In this article, we describe old and novel metabolic pathways from various carotenoids towards novel ligands of alternative nuclear hormone receptors. However, to fully elucidate these pathways, a larger array of techniques and tools, starting from organic synthesis, lipidomics, reporter models, classical in vitro and in vivo models and further omics-approaches and their statistical evaluation are needed to comprehensively and conclusively study this topic. Thus, we further describe state-of-the-art techniques from A to Ω elucidating carotenoid biological mediated activities and describe in detail required materials and methods needed - in practical protocol form - for the various steps of carotenoid investigations.

Deep into the Apoplast: Grapevine and Plasmopara viticola Proteomes Reveal the Secret Beneath Host and Pathogen Communication at 6 h After Contact.

The apoplast is the first hub of plant-pathogen communication where pathogen effectors are recognized by plant defensive proteins and cell receptors, thus activating signal transduction pathways. As a result of this first contact, the host triggers a defense response that involves the modulation of extra- and intracellular proteins. In grapevine-pathogen interactions, little is known about the trafficking between extra- and intracellular spaces. Grapevine is an economically important crop that relies on heavy fungicide use to control several diseases, and a deeper knowledge on the activation of its immune response is crucial to define new control strategies. In this study, we focused on the first 6 h postinoculation with Plasmopara viticola to evaluate grapevine proteome modulation in the apoplast. The in planta P. viticola proteome was also assessed to enable a deeper understanding of plant-pathogen communication. Our results showed that several plant mechanisms are triggered in the tolerant grapevine cultivar Regent after inoculation, such as oomycete recognition, plant cell wall modifications, reactive oxygen species signaling, and secretion of proteins to disrupt oomycete structures. On the other hand, P. viticola proteins related to development and virulence were the most predominant. This pioneer study highlights the early dynamics of cellular communication in grapevine defense that leads to the successful establishment of an incompatible interaction.

An in-planta comparative study of Plasmopara viticola proteome reveals different infection strategies towards susceptible and Rpv3-mediated resistance hosts.

Plasmopara viticola, an obligate biotrophic oomycete, is the causal agent of one of the most harmful grapevine diseases, downy mildew. Within this pathosystem, much information is gathered on the host, as characterization of pathogenicity and infection strategy of a biotrophic pathogen is quite challenging. Molecular insights into P. viticola development and pathogenicity are just beginning to be uncovered, mainly by transcriptomic studies. Plasmopara viticola proteome and secretome were only predicted based on transcriptome data. In this study, we have identified the in-planta proteome of P. viticola during infection of a susceptible ('Trincadeira') and a Rpv3-mediated resistance ('Regent') grapevine cultivar. Four hundred and twenty P. viticola proteins were identified on a label-free mass spectrometry-based approach of the apoplastic fluid of grapevine leaves. Overall, our study suggests that, in the compatible interaction, P. viticola manipulates salicylic-acid pathway and isoprenoid biosynthesis to enhance plant colonization. Furthermore, during the incompatible interaction, development-associated proteins increased while oxidoreductases protect P. viticola from ROS-associated plant defence mechanism. Up to our knowledge this is the first in-planta proteome characterization of this biotrophic pathogen, thus this study will open new insights into our understanding of this pathogen colonization strategy of both susceptible and Rpv3-mediated resistance grapevine genotypes.

Proteomic responses of two spring wheat cultivars to the combined water deficit and aphid (Metopolophium dirhodum) treatments.

In the field, plants usually have to face the combined effects of abiotic and biotic stresses. In our study, two spring wheat cultivars-Septima and Quintus-were subjected to three water regimes [70%, 50%, and 40% soil water capacity (SWC)], aphid (Metopolophium dirhodum) infestation, or the combination of both stresses, i.e., water deficit (50%, 40% SWC) and aphids. The study has a 2 × 3 × 2 factorial design with three biological replicates. In the present study, the results of proteomic analysis using 2D-DIGE followed by MALDI-TOF/TOF protein identification are presented. Water deficit but also aphid infestation led to alterations in 113 protein spots including proteins assigned to a variety of biological processes ranging from signaling via energy metabolism, redox regulation, and stress and defense responses to secondary metabolism indicating a long-term adaptation to adverse conditions. The absence of specific proteins involved in plant response to herbivorous insects indicates a loss of resistance to aphids in modern wheat cultivars during the breeding process and is in accordance with the "plant vigor hypothesis." Septima revealed enhanced tolerance with respect to Quintus as indicated by higher values of morphophysiological characteristics (fresh aboveground biomass, leaf length, osmotic potential per full water saturation) and relative abundance of proteins involved in mitochondrial respiration and ATP biosynthesis.

Untargeted Metabolomics Sheds Light on the Secondary Metabolism of Fungi Triggered by Choline-Based Ionic Liquids.

Fungal secondary metabolites constitute a rich source of yet undiscovered bioactive compounds. Their production is often silent under standard laboratory conditions, but the production of some compounds can be triggered simply by altering the cultivation conditions. The usage of an organic salt - ionic liquid - as growth medium supplement can greatly impact the biosynthesis of secondary metabolites, leading to higher diversity of compounds accumulating extracellularly. This study examines if such supplements, specifically cholinium-based ionic liquids, can support the discovery of bioactive secondary metabolites across three model species: Neurospora crassa, Aspergillus nidulans, and Aspergillus fumigatus. Enriched organic extracts obtained from medium supernatant revealed high diversity in metabolites. The supplementation led apparently to increased levels of either 1-aminocyclopropane-1-carboxylate or α-aminoisobutyric acid. The extracts where bioactive against two major foodborne bacterial strains: Staphylococcus aureus and Escherichia coli. In particular, those retrieved from N. crassa cultures showed greater bactericidal potential compared to control extracts derived from non-supplemented cultures. An untargeted mass spectrometry analysis using the Global Natural Product Social Molecular Networking tool enabled to capture the chemical diversity driven by the ionic liquid stimuli. Diverse macrolides, among other compounds, were putatively associated with A. fumigatus; whereas an unexpected richness of cyclic (depsi)peptides with N. crassa. Further studies are required to understand if the identified peptides are the major players of the bioactivity of N. crassa extracts, and to decode their biosynthesis pathways as well.

Proteomics Evidence of a Systemic Response to Desiccation in the Resurrection Plant Haberlea rhodopensis.

Global warming and drought stress are expected to have a negative impact on agricultural productivity. Desiccation-tolerant species, which are able to tolerate the almost complete desiccation of their vegetative tissues, are appropriate models to study extreme drought tolerance and identify novel approaches to improve the resistance of crops to drought stress. In the present study, to better understand what makes resurrection plants extremely tolerant to drought, we performed transmission electron microscopy and integrative large-scale proteomics, including organellar and phosphorylation proteomics, and combined these investigations with previously published transcriptomic and metabolomics data from the resurrection plant Haberlea rhodopensis. The results revealed new evidence about organelle and cell preservation, posttranscriptional and posttranslational regulation, photosynthesis, primary metabolism, autophagy, and cell death in response to desiccation in H. rhodopensis. Different protective intrinsically disordered proteins, such as late embryogenesis abundant (LEA) proteins, thaumatin-like proteins (TLPs), and heat shock proteins (HSPs), were detected. We also found a constitutively abundant dehydrin in H. rhodopensis whose phosphorylation levels increased under stress in the chloroplast fraction. This integrative multi-omics analysis revealed a systemic response to desiccation in H. rhodopensis and certain targets for further genomic and evolutionary studies on DT mechanisms and genetic engineering towards the improvement of drought tolerance in crops.

Proteomic Studies of Roots in Hypoxia-Sensitive and -Tolerant Tomato Accessions Reveal Candidate Proteins Associated with Stress Priming.

Tomato (Solanum lycopersicum L.) is a vegetable frequently exposed to hypoxia stress induced either by being submerged, flooded or provided with limited oxygen in hydroponic cultivation systems. The purpose of the study was to establish the metabolic mechanisms responsible for overcoming hypoxia in two tomato accessions with different tolerance to this stress, selected based on morphological and physiological parameters. For this purpose, 3-week-old plants (plants at the juvenile stage) of waterlogging-tolerant (WL-T), i.e., POL 7/15, and waterlogging-sensitive (WL-S), i.e., PZ 215, accessions were exposed to hypoxia stress (waterlogging) for 7 days, then the plants were allowed to recover for 14 days, after which another 7 days of hypoxia treatment was applied. Root samples were collected at the end of each time-point and 2D-DIGE with MALDI TOF/TOF, and expression analyses of gene and protein-encoded alcohol dehydrogenase (ADH2) and immunolabelling of ADH were conducted. After collating the obtained results, the different responses to hypoxia stress in the selected tomato accessions were observed. Both the WL-S and WL-T tomato accessions revealed a high amount of ADH2, which indicates an intensive alcohol fermentation pathway during the first exposure to hypoxia. In comparison to the tolerant one, the expression of the adh2 gene was about two times higher for the sensitive tomato. Immunohistochemical analysis confirmed the presence of ADH in the parenchyma cells of the cortex and vascular tissue. During the second hypoxia stress, the sensitive accession showed a decreased accumulation of ADH protein and similar expression of the adh2 gene in comparison to the tolerant accession. Additionally, the proteome showed a greater protein abundance of glyceraldehyde-3-phosphate dehydrogenase in primed WL-S tomato. This could suggest that the sensitive tomato overcomes the oxygen limitation and adapts by reducing alcohol fermentation, which is toxic to plants because of the production of ethanol, and by enhancing glycolysis. Proteins detected in abundance in the sensitive accession are proposed as crucial factors for hypoxia stress priming and their function in hypoxia tolerance is discussed.

Identification of Novel Candidate Genes Involved in Apple Cuticle Integrity and Russeting-Associated Triterpene Synthesis Using Metabolomic, Proteomic, and Transcriptomic Data.

Apple russeting develops on the fruit surface when skin integrity has been lost. It induces a modification of fruit wax composition, including its triterpene profile. In the present work, we studied two closely related apple varieties, 'Reinette grise du Canada' and 'Reinette blanche du Canada', which display russeted and non-russeted skin phenotypes, respectively, during fruit development. To better understand the molecular events associated with russeting and the differential triterpene composition, metabolomics data were generated using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) and combined with proteomic and transcriptomic data. Our results indicated lower expression of genes linked to cuticle biosynthesis (cutin and wax) in russet apple throughout fruit development, along with an alteration of the specialized metabolism pathways, including triterpene and phenylpropanoid. We identified a lipid transfer protein (LTP3) as a novel player in cuticle formation, possibly involved in the transport of both cutin and wax components in apple skin. Metabolomic data highlighted for the first time a large diversity of triterpene-hydroxycinnamates in russeted tissues, accumulation of which was highly correlated with suberin-related genes, including some enzymes belonging to the BAHD (HXXXD-motif) acyltransferase family. Overall, this study increases our understanding about the crosstalk between triterpene and suberin pathways.

Leaf necrosis resulting from downregulation of poplar glycosyltransferase UGT72A2.

Reactive species (RS) causing oxidative stress are unavoidable by-products of various plant metabolic processes, such as photosynthesis, respiration or photorespiration. In leaves, flavonoids scavenge RS produced during photosynthesis and protect plant cells against deleterious oxidative damages. Their biosynthesis and accumulation are therefore under tight regulation at the cellular level. Glycosylation has emerged as an essential biochemical reaction in the homeostasis of various specialized metabolites such as flavonoids. This article provides a functional characterization of the Populus tremula x P. alba (poplar) UGT72A2 coding for a UDP-glycosyltransferase that is localized in the chloroplasts. Compared with the wild type, transgenic poplar lines with decreased expression of UGT72A2 are characterized by reduced growth and oxidative damages in leaves, as evidenced by necrosis, higher content of glutathione and lipid peroxidation products as well as diminished soluble peroxidase activity and NADPH to NADP+ ratio under standard growing conditions. They furthermore display lower pools of phenolics, anthocyanins and total flavonoids but higher proanthocyanidins content. Promoter analysis revealed the presence of cis-elements involved in photomorphogenesis, chloroplast biogenesis and flavonoid biosynthesis. The UGT72A2 is regulated by the poplar MYB119, a transcription factor known to regulate the flavonoid biosynthesis pathway. Phylogenetic analysis and molecular docking suggest that UGT72A2 could glycosylate flavonoids; however, the actual substrate(s) was not consistently evidenced with either in vitro assays nor analyses of glycosylated products in leaves of transgenic poplar overexpressing or downregulated for UGT72A2. This article provides elements highlighting the importance of flavonoid glycosylation regarding protection against oxidative stress in poplar leaves and raises new questions about the link between this biochemical reaction and regulation of the redox homeostasis system.

Plant Proteoforms Under Environmental Stress: Functional Proteins Arising From a Single Gene.

Proteins are directly involved in plant phenotypic response to ever changing environmental conditions. The ability to produce multiple mature functional proteins, i.e., proteoforms, from a single gene sequence represents an efficient tool ensuring the diversification of protein biological functions underlying the diversity of plant phenotypic responses to environmental stresses. Basically, two major kinds of proteoforms can be distinguished: protein isoforms, i.e., alterations at protein sequence level arising from posttranscriptional modifications of a single pre-mRNA by alternative splicing or editing, and protein posttranslational modifications (PTMs), i.e., enzymatically catalyzed or spontaneous modifications of certain amino acid residues resulting in altered biological functions (or loss of biological functions, such as in non-functional proteins that raised as a product of spontaneous protein modification by reactive molecular species, RMS). Modulation of protein final sequences resulting in different protein isoforms as well as modulation of chemical properties of key amino acid residues by different PTMs (such as phosphorylation, N- and O-glycosylation, methylation, acylation, S-glutathionylation, ubiquitinylation, sumoylation, and modifications by RMS), thus, represents an efficient means to ensure the flexible modulation of protein biological functions in response to ever changing environmental conditions. The aim of this review is to provide a basic overview of the structural and functional diversity of proteoforms derived from a single gene in the context of plant evolutional adaptations underlying plant responses to the variability of environmental stresses, i.e., adverse cues mobilizing plant adaptive mechanisms to diminish their harmful effects.

Gene expression and metabolite analysis in barley inoculated with net blotch fungus and plant growth-promoting rhizobacteria.

Net blotch, caused by the ascomycete Drechslera teres, can compromise barley production. Beneficial bacteria strains are of substantial interest as biological agents for plant protection in agriculture. Belonging to the genus Paraburkholderia, a bacterium, referred to as strain B25, has been identified as protective for barley against net blotch. The strain Paraburkholderia phytofirmans (strain PsJN), which has no effect on the pathogen's growth, has been used as control. In this study, the expression of target genes involved in cell wall-related processes, defense responses, carbohydrate and phenylpropanoid pathways was studied under various conditions (with or without pathogen and/or with or without bacterial strains) at different time-points (0-6-12-48 h). The results show that specific genes were subjected to a circadian regulation and that the expression of most of them increased in barley infected with D. teres and/or bacterized with the strain PsJN. On the contrary, a decreased gene expression was observed in the presence of strain B25. To complement and enrich the gene expression analysis, untargeted metabolomics was carried out on the same samples. The data obtained show an increase in the production of lipid compounds in barley in the presence of the pathogen. In addition, the presence of strain B25 leads to a decrease in the production of defense compounds in this crop. The results contribute to advance the knowledge on the mechanisms occurring at the onset of D. teres infection and in the presence of a biocontrol agent limiting the severity of net blotch in barley.

The Cell Wall Proteome of Craterostigma plantagineum Cell Cultures Habituated to Dichlobenil and Isoxaben.

The remarkable desiccation tolerance of the vegetative tissues in the resurrection species Craterostigma plantagineum (Hochst.) is favored by its unique cell wall folding mechanism that allows the ordered and reversible shrinking of the cells without damaging neither the cell wall nor the underlying plasma membrane. The ability to withstand extreme drought is also maintained in abscisic acid pre-treated calli, which can be cultured both on solid and in liquid culture media. Cell wall research has greatly advanced, thanks to the use of inhibitors affecting the biosynthesis of e.g., cellulose, since they allowed the identification of the compensatory mechanisms underlying habituation. Considering the innate cell wall plasticity of C. plantagineum, the goal of this investigation was to understand whether habituation to the cellulose biosynthesis inhibitors dichlobenil and isoxaben entailed or not identical mechanisms as known for non-resurrection species and to decipher the cell wall proteome of habituated cells. The results showed that exposure of C. plantagineum calli/cells triggered abnormal phenotypes, as reported in non-resurrection species. Additionally, the data demonstrated that it was possible to habituate Craterostigma cells to dichlobenil and isoxaben and that gene expression and protein abundance did not follow the same trend. Shotgun and gel-based proteomics revealed a common set of proteins induced upon habituation, but also identified candidates solely induced by habituation to one of the two inhibitors. Finally, it is hypothesized that alterations in auxin levels are responsible for the increased abundance of cell wall-related proteins upon habituation.