While human extracellular vesicles (EVs) have attracted a big deal of interest and have been extensively characterized over the last years, plant-derived EVs and nanovesicles have earned less attention and have remained poorly investigated. Although a series of investigations already revealed promising beneficial health effects and drug delivery properties, adequate (pre)clinical studies are rare. This fact might be caused by a lack of sources with appropriate qualities. Our study introduces plant cell suspension culture as a new and well controllable source for plant EVs. Plant cells, cultured in vitro, release EVs into the growth medium which could be harvested for pharmaceutical applications. In this investigation we characterized EVs and nanovesicles from distinct sources. Our findings regarding secondary metabolites indicate that these might not be packaged into EVs in an active manner but enriched in the membrane when lipophilic enough, since apparently lipophilic compounds were associated with nanovesicles while more hydrophilic structures were not consistently found. In addition, protein identification revealed a possible explanation for the mechanism of EV cell wall passage in plants, since cell wall hydrolases like 1,3-β-glucosidases, pectinesterases, polygalacturonases, β-galactosidases and β-xylosidase/α-L-arabinofuranosidase 2-like are present in plant EVs and nanovesicles which might facilitate cell wall transition. Further on, the identified proteins indicate that plant cells secrete EVs using similar mechanisms as animal cells to release exosomes and microvesicles.

Summary: 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.

Frost stress severely affects agriculture and agroforestry worldwide. Although many studies about frost hardening and resistance have been published, most of them focused on the aboveground organs and only a minority specifically targets the roots. However, roots and aboveground tissues have different physiologies and stress response mechanisms. Climate models predict an increase in the magnitude and frequency of late-frost events, which, together with an observed loss of soil insulation, will greatly decrease plant primary production due to damage at the root level. Molecular and metabolic responses inducing root cold hardiness are complex. They involve a variety of processes related to modifications in cell wall composition, maintenance of the cellular homeostasis and the synthesis of primary and secondary metabolites. After a summary of the current climatic models, this review details the specificity of freezing stress at the root level and explores the strategies roots developed to cope with freezing stress. We then describe the level to which roots can be frost hardy, depending on their age, size category and species. After that, we compare the environmental signals inducing cold acclimation and frost hardening in the roots and aboveground organs. Subsequently, we discuss how roots sense cold at a cellular level and briefly describe the following signal transduction pathway, which leads to molecular and metabolic responses associated with frost hardening. Finally, the current options available to increase root frost tolerance are explored and promising lines of future research are discussed.

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. 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. 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.

Russeting is characterized by a particular rough and brown phenotype, which is mainly due to the accumulation of suberin in the inner part of the epidermal cell walls. In our previous bulk transcriptomic analysis, comparing fully russeted, and waxy apple varieties, showed, in apple fruit skin, a massive decreased expression of cutin, wax and some pentacyclic triterpene biosynthesis genes in the russeted varieties, with an expected concomitant enhanced expression of the suberin biosynthetic genes. In the present work, we performed a deep investigation of the aliphatic composition of the cutin, suberin, waxes, and triterpenes in the waxy and russeted patches of the semi-russeted apple variety “Cox Orange Pippin.” A targeted gene expression profiling was performed to validate candidate genes which were identified in our previous work and might be involved in the respective metabolic pathways. Our results showed that a decrease of cuticular waxes, ursolic acid and oleanolic acid, accompanied by an accumulation of alkyl-hydroxycinamates and betulinic acid, occurs in the russeted patches. The suberin monomer composition is characterized by specific occurrence of 20, 22, and 24 carbon aliphatic chains, whereas cutin is mainly represented by common C16 and C18 aliphatic chains. This work depicts, for the first time in apple, the complex composition of suberin, cutin, waxes and triterpenes, and confirms the strong interplay between these epidermal polymers in apple fruit skin.

Abiotic constraints are a source of concern in agriculture, because they can have a strong impact on plant growth and development, thereby affecting crop yield. The response of plants to abiotic constraints varies depending on the type of stress, on the species and on the organs. Although many studies have addressed different aspects of the plant response to abiotic stresses, only a handful has focused on the role of the cell wall. A targeted approach has been used here to study the expression of cell wall-related genes in different organs of alfalfa plants subjected for four days to three different abiotic stress treatments, namely salt, cold and heat stress. Genes involved in different steps of cell wall formation (cellulose biosynthesis, monolignol biosynthesis and polymerization) have been analyzed in different organs of Medicago sativa L. Prior to this analysis, an in silico classification of dirigent/dirigent-like proteins and class III peroxidases has been performed in Medicago truncatula and M. sativa. The final goal of this study is to infer and compare the expression patterns of cell wall-related genes in response to different abiotic stressors in the organs of an important legume crop.