Characterization of MdMYB68, a suberin master regulator in russeted apples.

Introduction: Apple russeting is mainly due to the accumulation of suberin in the cell wall in response to defects and damages in the cuticle layer. Over the last decades, massive efforts have been done to better understand the complex interplay between pathways involved in the suberization process in model plants. However, the regulation mechanisms which orchestrate this complex process are still under investigation. Our previous studies highlighted a number of transcription factor candidates from the Myeloblastosis (MYB) transcription factor family which might regulate suberization in russeted or suberized apple fruit skin. Among these, we identified MdMYB68, which was co-expressed with number of well-known key suberin biosynthesis genes. Method: To validate the MdMYB68 function, we conducted an heterologous transient expression in Nicotiana benthamiana combined with whole gene expression profiling analysis (RNA-Seq), quantification of lipids and cell wall monosaccharides, and microscopy. Results: MdMYB68 overexpression is able to trigger the expression of the whole suberin biosynthesis pathway. The lipid content analysis confirmed that MdMYB68 regulates the deposition of suberin in cell walls. Furthermore, we also investigated the alteration of the non-lipid cell wall components and showed that MdMYB68 triggers a massive modification of hemicelluloses and pectins. These results were finally supported by the microscopy. Discussion: Once again, we demonstrated that the heterologous transient expression in N. benthamiana coupled with RNA-seq is a powerful and efficient tool to investigate the function of suberin related transcription factors. Here, we suggest MdMYB68 as a new regulator of the aliphatic and aromatic suberin deposition in apple fruit, and further describe, for the first time, rearrangements occurring in the carbohydrate cell wall matrix, preparing this suberin deposition.

MdMYB52 regulates lignin biosynthesis upon the suberization process in apple.

Our previous studies, comparing russeted vs. waxy apple skin, highlighted a MYeloBlastosys (Myb) transcription factor (MdMYB52), which displayed a correlation with genes associated to the suberization process. The present article aims to assess its role and function in the suberization process. Phylogenetic analyses and research against Arabidopsis thaliana MYBs database were first performed and the tissue specific expression of MdMYB52 was investigated using RT-qPCR. The function of MdMYB52 was further investigated using Agrobacterium-mediated transient overexpression in Nicotiana benthamiana leaves. An RNA-Seq analysis was performed to highlight differentially regulated genes in response MdMYB52. Transcriptomic data were supported by analytical chemistry and microscopy. A massive decreased expression of photosynthetic and primary metabolism pathways was observed with a concomitant increased expression of genes associated with phenylpropanoid and lignin biosynthesis, cell wall modification and senescence. Interestingly key genes involved in the synthesis of suberin phenolic components were observed. The analytical chemistry displayed a strong increase in the lignin content in the cell walls during MdMYB52 expression. More specifically, an enrichment in G-Unit lignin residues was observed, supporting transcriptomic data as well as previous work describing the suberin phenolic domain as a G-unit enriched lignin-like polymer. The time-course qPCR analysis revealed that the observed stress response, might be explain by this lignin biosynthesis and by a possible programmed senescence triggered by MdMYB52. The present work supports a crucial regulatory role for MdMYB52 in the biosynthesis of the suberin phenolic domain and possibly in the fate of suberized cells in russeted apple skins.

Handling wine pomace: The importance of drying to preserve phenolic profile and antioxidant capacity for product valorization.

Four different wine grape pomaces (GP) (Vitis vinifera) varieties, Auxerrois, Pinot Blanc, Gamay and Pinot Noir, and obtained from white, rosé or red wine vinification, were considered for possible valorization in food supplement industry. Stabilization of GP by drying is paramount prior to further processing in the valorization chain, as GP might suffer spoilage over time. The objectives of this work were therefore to: evaluate the effect of microbiological spoilage and drying on the polyphenol profile and antioxidant capacity of GP; define a drying procedure by comparing kinetics of freeze-drying (FD) and vacuum oven (VO) (at 60 °C and 40 °C). Microbiological spoilage led to significant losses (P < 0.01) of antioxidant capacity (40% to 87%) and total phenolic content (70% to 90%), while drying had no significant effect. FD and VO at 60 °C drying kinetics exhibited similar drying curves, and a dry weight (DW) plateau was reached by 48 hr. In contrast VO at 40 °C required 170 hr to reach similar DW values, pointing out the importance of temperature when opting for VO technology. Antioxidant capacity of GP extracts did not differ between drying methods. Interestingly, GPs from white and rosé wines (AUX, PB, and GAM) had up to 3.5 times higher content (P < 0.001) of total polyphenols compared to PN, obtained from red wine. These results reinforce the importance of drying of GP as a pretreatment, which otherwise could result in significant product degradation. Additionally, we propose white and rosé GP as more interesting sources for valorization, with higher phenolic content, compared to red wine GP.

Novel Insights from Comparative In Silico Analysis of Green Microalgal Cellulases.

The assumption that cellulose degradation and assimilation can only be carried out by heterotrophic organisms was shattered in 2012 when it was discovered that the unicellular green alga, Chlamydomonas reinhardtii (Cr), can utilize cellulose for growth under CO2-limiting conditions. Publications of genomes/transcriptomes of the colonial microalgae, Gonium pectorale (Gp) and Volvox carteri (Vc), between 2010⁻2016 prompted us to look for cellulase genes in these algae and to compare them to cellulases from bacteria, fungi, lower/higher plants, and invertebrate metazoans. Interestingly, algal catalytic domains (CDs), belonging to the family GH9, clustered separately and showed the highest (33⁻42%) and lowest (17⁻36%) sequence identity with respect to cellulases from invertebrate metazoans and bacteria, respectively, whereas the identity with cellulases from plants was only 27⁻33%. Based on comparative multiple alignments and homology models, the domain arrangement and active-site architecture of algal cellulases are described in detail. It was found that all algal cellulases are modular, consisting of putative novel cysteine-rich carbohydrate-binding modules (CBMs) and proline/serine-(PS) rich linkers. Two genes were found to encode a protein with a putative Ig-like domain and a cellulase with an unknown domain, respectively. A feature observed in one cellulase homolog from Gp and shared by a spinach cellulase is the existence of two CDs separated by linkers and with a C-terminal CBM. Dockerin and Fn-3-like domains, typically found in bacterial cellulases, are absent in algal enzymes. The targeted gene expression analysis shows that two Gp cellulases consisting, respectively, of a single and two CDs were upregulated upon filter paper addition to the medium.

Endothelial responses of the alveolar barrier in vitro in a dose-controlled exposure to diesel exhaust particulate matter.

BACKGROUND: During the last 250 years, the level of exposure to combustion-derived particles raised dramatically in western countries, leading to increased particle loads in the ambient air. Among the environmental particles, diesel exhaust particulate matter (DEPM) plays a special role because of its omnipresence and reported effects on human health. During recent years, a possible link between air pollution and the progression of atherosclerosis is recognized. A central effect of DEPM is their impact on the endothelium, especially of the alveolar barrier. In the present study, a complex 3D tetraculture model of the alveolar barrier was used in a dose-controlled exposure scenario with realistic doses of DEPM to study the response of endothelial cells. RESULTS: Tetracultures were exposed to different doses of DEPM (SRM2975) at the air-liquid-interface. DEPM exposure did not lead to the mRNA expression of relevant markers for endothelial inflammation such as ICAM-1 or E-selectin. In addition, we observed neither a significant change in the expression levels of the genes relevant for antioxidant defense, such as HMOX1 or SOD1, nor the release of pro-inflammatory second messengers, such as IL-6 or IL-8. However, DEPM exposure led to strong nuclear translocation of the transcription factor Nrf2 and significantly altered expression of CYP1A1 mRNA in the endothelial cells of the tetraculture. CONCLUSION: In the present study, we demonstrated the use of a complex 3D tetraculture system together with a state-of-the-art aerosol exposure equipment to study the effects of in vivo relevant doses of DEPM on endothelial cells in vitro. To the best of our knowledge, this study is the first that focuses on indirect effects of DEPM on endothelial cells of the alveolar barrier in vitro. Exposure to DEPM led to significant activation and nuclear translocation of the transcription factor Nrf2 in endothelial cells. The considerably low doses of DEPM had a low but measurable effect, which is in line with recent data from in vivo studies.

MdMyb93 is a regulator of suberin deposition in russeted apple fruit skins.

A comparison of the transcriptomes of russeted vs nonrusseted apple skins previously highlighted a tight relationship between a gene encoding an MYB-type transcription factor, MdMYB93, and some key suberin biosynthetic genes. The present work assesses the role of this transcription factor in the suberization process. A phylogenetic analysis of MdMYB93 and Arabidopsis thaliana MYBs was performed and the function of MdMYB93 was further investigated using Agrobacterium-mediated transient overexpression in Nicotiana benthamiana leaves. An RNA-Seq analysis was performed to highlight the MdMYB93-regulated genes. Ultraperformance liquid chromatography-triple time-of-flight (UPLC-TripleTOF) and GC-MS were used to investigate alterations in phenylpropanoid, soluble-free lipid and lipid polyester contents. A massive accumulation of suberin and its biosynthetic precursors in MdMYB93 agroinfiltrated leaves was accompanied by a remobilization of phenylpropanoids and an increased amount of lignin precursors. Gene expression profiling displayed a concomitant alteration of lipid and phenylpropanoid metabolism, cell wall development, and extracellular transport, with a large number of induced transcripts predicted to be involved in suberin deposition. The present work supports a major role of MdMYB93 in the regulation of suberin deposition in russeted apple skins, from the synthesis of monomeric precursors, their transport, polymerization, and final deposition as suberin in primary cell wall.

Multifunctional oxidosqualene cyclases and cytochrome P450 involved in the biosynthesis of apple fruit triterpenic acids.

Apple (Malus × domestica) accumulates bioactive ursane-, oleanane-, and lupane-type triterpenes in its fruit cuticle, but their biosynthetic pathway is still poorly understood. We used a homology-based approach to identify and functionally characterize two new oxidosqualene cyclases (MdOSC4 and MdOSC5) and one cytochrome P450 (CYP716A175). The gene expression patterns of these enzymes and of previously described oxidosqualene cyclases were further studied in 20 apple cultivars with contrasting triterpene profiles. MdOSC4 encodes a multifunctional oxidosqualene cyclase producing an oleanane-type triterpene, putatively identified as germanicol, as well as ß-amyrin and lupeol, in the proportion 82 : 14 : 4. MdOSC5 cyclizes 2,3-oxidosqualene into lupeol and ß-amyrin at a ratio of 95 : 5. CYP716A175 catalyses the C-28 oxidation of α-amyrin, ß-amyrin, lupeol and germanicol, producing ursolic acid, oleanolic acid, betulinic acid, and putatively morolic acid. The gene expression of MdOSC1 was linked to the concentrations of ursolic and oleanolic acid, whereas the expression of MdOSC5 was correlated with the concentrations of betulinic acid and its caffeate derivatives. Two new multifuntional triterpene synthases as well as a multifunctional triterpene C-28 oxidase were identified in Malus × domestica. This study also suggests that MdOSC1 and MdOSC5 are key genes in apple fruit triterpene biosynthesis.

Inflammation related responses of intestinal cells to plum and cabbage digesta with differential carotenoid and polyphenol profiles following simulated gastrointestinal digestion.

SCOPE: Plums/cabbages represent fruits/vegetables rich in carotenoids and polyphenols, and have been associated with anti-inflammatory properties. METHODS AND RESULTS: We tested four plum (Italian Plum, Plum 620, Ersinger, and Cherry Plum) and cabbage varieties (Duchy, Kalorama, Kale, Scots Kale) with contrasting carotenoid/polyphenol content for their capability to alter inflammation/oxidative stress following simulated gastrointestinal digestion. Digesta were exposed to Caco-2(TC-7) and to a triple-culture(Caco-2/HT-29-MTX (90:10 v/v) including THP-1 like macrophages), stimulated to induce inflammation (10 µg/mL LPS, 100 ng/mL TNF-α, 25 ng/mL IL-1-ß for 24 h, the last 18 h with digesta). Endpoints investigated included IL-6, IL-8, PGE-2, NO (all ELISA), NF-κB, MAPK, IL-6, IL-8, iNOS, Nrf2, COX-2 (real-time-PCR) and Nrf2 (immunostaining). IL-6 secretion was reduced in THP-1 cells by Scots Kale and Kalorama (up to 22%, p<0.05), and IL-8 secretion in the coculture (up to 35% in plums, p<0.05). This was accompanied by decreased NF-kB expressions in THP-1 cells (up to 30%, p<0.05). Nrf2 translocation to the nucleus was partly reduced by plums and cabbages (up to 40% (p<0.05). CONCLUSIONS: Some varieties, especially in the triple-culture, reduced inflammation, though this was unrelated to concentrations of carotenoids/polyphenols. The potential of phytochemical-rich fruits and vegetables to ameliorate gastrointestinal inflammation should be further investigated.

Iron uptake and homeostasis related genes in potato cultivated in vitro under iron deficiency and overload.

Potato is one of the most important staple food in the world because it is a good source of vitamin C, vitamin B6 but also an interesting source of minerals including mainly potassium, but also magnesium, phosphorus, manganese, zinc and iron to a lesser extent. The lack of iron constitutes the main form of micronutrient deficiency in the world, namely iron deficiency anemia, which strongly affects pregnant women and children from developing countries. Iron biofortification of major staple food such as potato is thus a crucial issue for populations from these countries. To better understand mechanisms leading to iron accumulation in potato, we followed in an in vitro culture experiment, by qPCR, in the cultivar Désirée, the influence of media iron content on the expression of genes related to iron uptake, transport and homeostasis. As expected, plantlets grown in a low iron medium (1 mg L(-1) FeNaEDTA) displayed a decreased iron content, a strong induction of iron deficiency-related genes and a decreased expression of ferritins. Inversely, plantlets grown in a high iron medium (120 mg L(-1) FeNaEDTA) strongly accumulated iron in roots; however, no significant change in the expression of our set of genes was observed compared to control (40 mg L(-1) FeNaEDTA).

Gene expression changes related to the production of phenolic compounds in potato tubers grown under drought stress.

Polyphenols represent a large family of plant secondary metabolites implicated in the prevention of various diseases such as cancers and cardiovascular diseases. The potato is a significant source of polyphenols in the human diet. In this study, we examined the expression of thirteen genes involved in the biosynthesis of polyphenols in potato tubers using real-time RT-PCR. A selection of five field grown native Andean cultivars, presenting contrasting polyphenol profiles, was used. Moreover, we investigated the expression of the genes after a drought exposure. We concluded that the diverse polyphenolic profiles are correlated to variations in gene expression profiles. The drought-induced variations of the gene expression was highly cultivar-specific. In the three anthocyanin-containing cultivars, gene expression was coordinated and reflected at the metabolite level supporting a hypothesis that regulation of gene expression plays an essential role in the potato polyphenol production. We proposed that the altered sucrose flux induced by the drought stress is partly responsible for the changes in gene expression. This study provides information on key polyphenol biosynthetic and regulatory genes, which could be useful in the development of potato varieties with enhanced health and nutritional benefits.

EgMYB2, a new transcriptional activator from Eucalyptus xylem, regulates secondary cell wall formation and lignin biosynthesis.

Summary EgMYB2, a member of a new subgroup of the R2R3 MYB family of transcription factors, was cloned from a library consisting of RNA from differentiating Eucalyptus xylem. EgMYB2 maps to a unique locus on the Eucalyptus grandis linkage map and co-localizes with a quantitative trait locus (QTL) for lignin content. Recombinant EgMYB2 protein was able to bind specifically the cis-regulatory regions of the promoters of two lignin biosynthetic genes, cinnamoyl-coenzyme A reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD), which contain MYB consensus binding sites. EgMYB2 was also able to regulate their transcription in both transient and stable expression assays. Transgenic tobacco plants over-expressing EgMYB2 displayed phenotypic changes relative to wild-type plants, among which were a dramatic increase in secondary cell wall thickness, and an alteration of the lignin profiles. Transcript abundance of genes encoding enzymes specific to lignin biosynthesis was increased to varying extents according to the position of individual genes in the pathway, whereas core phenylpropanoid genes were not significantly affected. Together these results suggest a role for EgMYB2 in the co-ordinated control of genes belonging to the monolignol-specific pathway, and therefore in the biosynthesis of lignin and the regulation of secondary cell wall formation.