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Germination of pollen grains is a crucial step in plant reproduction. However, the molecular mechanisms involved remain unclear. We investigated the role of PECTIN METHYLESTERASE48 (PME48), an enzyme implicated in the remodeling of pectins in Arabidopsis (Arabidopsis thaliana) pollen. A combination of functional genomics, gene expression, in vivo and in vitro pollen germination, immunolabeling, and biochemical analyses was used on wild-type andAtpme48mutant plants. We showed thatAtPME48is specifically expressed in the male gametophyte and is the second most expressedPMEin dry and imbibed pollen grains. Pollen grains from homozygous mutant lines displayed a significant delay in imbibition and germination in vitro and in vivo. Moreover, numerous pollen grains showed two tips emerging instead of one in the wild type. Immunolabeling and Fourier transform infrared analyses showed that the degree of methylesterification of the homogalacturonan was higher inpme48−/− pollen grains. In contrast, the PME activity was lower inpme48−/−, partly due to a reduction of PME48 activity revealed by zymogram. Interestingly, the wild-type phenotype was restored inpme48−/− with the optimum germination medium supplemented with 2.5 mM calcium chloride, suggesting that in the wild-type pollen, the weakly methylesterified homogalacturonan is a source of Ca²⁺ necessary for pollen germination. Although pollen-specific PMEs are traditionally associated with pollen tube elongation, this study provides strong evidence that PME48 impacts the mechanical properties of the intine wall during maturation of the pollen grain, which, in turn, influences pollen grain germination.

Current agricultural practices rely heavily on synthetic fertilizers, which not only consume a lot of energy but also disrupt the ecological balance. The overuse of synthetic fertilizers has led to soil degradation. In a more sustainable approach, alternative methods based on biological interactions, such as plant growth-promoting bacteria (PGPRs), are being explored. PGPRs, which include both symbiotic and free-living bacteria, form mutualistic relationships with plants by enhancing nutrient availability, producing growth regulators, and regulating stress responses. This study investigated the potential of Sphingomonas sediminicola Dae20, an α-Proteobacteria species commonly found in the rhizosphere, as a beneficial PGPR. We observed that S. sediminicola Dae20 stimulated the root system and growth of three different plant species in the Brassicaceae family, including Arabidopsis thaliana, mustard, and rapeseed. The bacterium produced auxin, nitric oxide, siderophores and showed ACC deaminase activity. In addition to activating an auxin response in the plant, S. sediminicola Dae20 exhibited the ability to modulate other plant hormones, such as abscisic acid, jasmonic acid and salicylic acid, which are critical for plant development and defense responses. This study highlights the multifunctional properties of S. sediminicola Dae20 as a promising PGPR and underscores the importance of identifying effective and versatile beneficial bacteria to improve plant nutrition and promote sustainable agricultural practices.

Despite an ever-increasing interest for the use of pectin-derived oligogalacturonides (OGs) as biological control agents in agriculture, very little information exists-mainly for technical reasons-on the nature and activity of the OGs that accumulate during pathogen infection. Here we developed a sensitive OG profiling method, which revealed unsuspected features of the OGs generated during infection of Arabidopsis thaliana with the fungus Botrytis cinerea. Indeed, in contrast to previous reports, most OGs were acetyl-and methylesterified, and 80% of them were produced by fungal pectin lyases, not by polygalacturonases. Polygalacturonase products did not accumulate as larger size OGs but were converted into oxidized GalA dimers. Finally, the comparison of the OGs and transcriptomes of leaves infected with B. cinerea mutants with reduced pectinolytic activity but with decreased or increased virulence, respectively, identified candidate OG elicitors. In conclusion, OG analysis provides insights into the enzymatic arms race between plant and pathogen and facilitates the identification of defense elicitors. oligogalacturonides | plant-pathogen interaction | Arabidopsis thaliana | Botrytis cinerea | pectin lyase T he cell wall forms the first line of defense in the interaction of plants with their microbial environment. Cell walls consist of complex polysaccharide networks, which combine multiple functional properties such as strength (to resist turgor pressure), ex-tensibility (to allow growth), as well as protection against microbial attack. Such protection involves so called \"basal immunity,\" which is triggered upon recognition of pathogen-associated molecular patterns but also of plant-derived molecules associated with infection, referred to as damage-associated molecular patterns (DAMPs). The latter include cell wall-derived oligosaccharides with elicitor activity (also called oligosaccharins) (1) that are produced during infection with microbes and, in particular, necrotrophic bacteria and fungi, which feed on cell walls and have large arsenals of cell wall-degrading enzymes (2). A major source of cell wall-derived DAMPs are pectins. The pectic polymer, homogalacturonan (HG) for example, represents in Arabidopsis 20% of the wall of growing cells. HG is a linear polymer of α-1-4-linked galacturonic acid (GalA) residues secreted in a methylesterified (on C6) and often acetylesterified (on C2 and/or C3) form (3), which can be enzymatically deacetylesterified by pectin acetylesterases and demethylesterified by pectin methyl-esterases (PMEs) in muro (4). Demethylesterification exposes the polymer to degradation by polygalacturonases (PGs) and pectate lyases (PLs, that cleave unmethylesterified HG by β-elimination, thus generating an unsaturated bond at the nonreducing end), which can be of plant or fungal origin. HG demethylesterification plays a key role in the control of cell wall rheology underlying plant growth (5). In addition, HG turnover generates oligogalacturonides (OGs)

Background Kv10.1 potassium channel has been shown to be involved in breast cancer luminal cell proliferation and survival as well as migration in basal cell model in normoxia. Moreover, it is clearly established that solid tumors present a hypoxic center. Currently, few information is available about the involvement of the Kv10.1 channel in hypoxic context in breast cancer progression. Methods We explored the role of Kv10.1 in the hypoxia-induced aggressiveness promotion of different breast cancer cell models: luminal A (MCF-7 cells), HER2-overexpressing (SKBr3 cells) and basal subtype (MDA-MB-231 cells) by using complementary approaches of cell biology assay, western blot and qPCR. Results Our results show that Kv10.1 expression and function are related to hypoxia and HIF-1α. Under hypoxia, we demonstrated that Kv10.1 is a major actor of the migration and it regulates the hypoxia-induced EMT in the three cells models by promoting the mesenchymal phenotype. Finally, after RNAseq comparison of cells expressing or not the channel in hypoxia condition, we highlighted the involvement of the β1-integrin / FAK, AKT and ERK pathways in the MDA-MB-231 breast cancer cell aggressiveness. Conclusion We demonstrated, for the first time, the impact of Kv10.1 channel in hypoxia-induced aggressivity of breast cancer cells demonstrating its potential use as a complementary target to fight against metastasis development.

Lignans, phenolic plant secondary metabolites, are derived from the phenylpropanoid biosynthetic pathway. Although, being investigated for their health benefits in terms of antioxidant, antitumor, anti-inflammatory and antiviral properties, the role of these molecules in plants remains incompletely elucidated; a potential role in stress response mechanisms has been, however, proposed. In this study, a non-targeted metabolomic analysis of the roots, stems, and leaves of wild-type and PLR1-RNAi transgenic flax, devoid of (+) secoisolariciresinol diglucoside ((+) SDG)—the main flaxseed lignan, was performed using 1H-NMR and LC-MS, in order to obtain further insight into the involvement of lignan in the response of plant to osmotic stress. Results showed that wild-type and lignan-deficient flax plants have different metabolic responses after being exposed to osmotic stress conditions, but they both showed the capacity to induce an adaptive response to osmotic stress. These findings suggest the indirect involvement of lignans in osmotic stress response.

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.

Pancreatic diseases, such as pancreatitis or pancreatic ductal adenocarcinoma, are characterized by the presence of activated pancreatic stellate cells (PSCs). These cells represent key actors in the tumor stroma, as they actively participate in disease development and progression: reprograming these PSCs into a quiescent phenotype has even been proposed as a promising strategy for restoring the hallmarks of a healthy pancreas. Since TRPM7 channels have been shown to regulate hepatic stellate cells proliferation and survival, we aimed to study the role of these magnesium channels in PSC activation and proliferation. PS-1 cells (isolated from a healthy pancreas) were used as a model of healthy PSCs: quiescence or activation were induced using all-trans retinoic acid or conditioned media of pancreatic cancer cells, respectively. The role of TRPM7 was studied by RNA silencing or by pharmacological inhibition. TRPM7 expression was found to be correlated with the activation status of PS-1 cells. TRPM7 expression was able to regulate proliferation through modulation of cell cycle regulators and most importantly p53, via the PI3K/Akt pathway, in a magnesium-dependent manner. Finally, the analysis of TCGA database showed the overexpression of TRPM7 in cancer-associated fibroblasts. Taken together, we provide strong evidences that TRPM7 can be considered as a marker of activated PSCs.

The external seed coat cell layer of certain species is specialized in the production and extrusion of a polysaccharide matrix called mucilage. Variations in the content of the released mucilage have been mainly associated with genetically regulated physiological modifications. Understanding the mucilage extrusion process in crop species is of importance to gain deeper insight into the complex cell wall biosynthesis and dynamics. In this study, we took advantage of the varying polysaccharide composition and the size of the flax mucilage secretory cells (MSCs) to study mucilage composition and extrusion in this species of agricultural interest. We demonstrate herein that flax MSCs are structured in four superimposed layers and that rhamnogalacturonans I (RG I) are firstly synthesized, in the upper face, preceding arabinoxylan and glucan synthesis in MSC lower layers. Our results also reveal that the flax mucilage release originates from inside MSC, between the upper and deeper layers, the latter collaborating to trigger polysaccharide expansion, radial cell wall breaking and mucilage extrusion in a peeling fashion. Here, we provide evidence that the layer organization and polysaccharide composition of the MSCs regulate the mucilage release efficiency like a peeling mechanism. Finally, we propose that flax MSCs may represent an excellent model for further investigations of mucilage biosynthesis and its release.

Pancreatic ductal adenocarcinoma (PDAC) is the main and the deadliest form of pancreatic cancer. This is a major problem of public health since it will become the second leading cause of death by cancer in the next few years, mainly due to the lack of efficient therapies. Transient Receptor Potential Cation Channel Subfamily M Member 7 (TRPM7) protein, a cation channel fused with a serine/threonine kinase domain is overexpressed in PDAC and associated with a low survival. In this work, we aim to study the role of kinase domain on pancreatic cell fates by using a model of kinase domain deletion by CRISPR-Cas9. PANC-1 and MIA PaCa-2 PDAC cell lines were used and kinase domain was deleted by CRISPR-Cas9 strategy. Kinase domain deletion (ΔK) was validated by RT-qPCR and western blots. The effect of kinase domain deletion on channel function was studied by patch-clamp and Mn 2+ -quenching. The cell phenotype was studied by MTT and cell migration/invasion assays. Finally, the role of kinase domain was studied in vivo in xenografted mice. Here we show that TRPM7 kinase domain is required to maintain a mesenchymal phenotype in PDAC cells. We also demonstrated that TRPM7 and PAK1 interact in the same protein complexes. Moreover, TRPM7 kinase domain is required for carcinogenesis and cancer cell dissemination in vivo. Intriguingly, the role of TRPM7 kinase is cell specific and may depend on the KRAS oncogene mutation status. In conclusion, TRPM7 kinase domain is required to maintain a mesenchymal and aggressive phenotype in PDAC cells, and it could be a promising target against PDAC.

Fusarium oxysporum is the one of the most common and impactful pathogens of flax. Cultivars of flax that show resistance to this pathogen have previously been identified. To better understand the mechanisms that are responsible for this resistance, we conducted time-lapse analysis of one susceptible and one resistant cultivar over a two-week period following infection. We also monitored changes in some metabolites. The susceptible cultivar showed a strong onset of symptoms from 6 to 8 days after inoculation, which at this time point, was associated with changes in metabolites in both cultivars. The resistant cultivar maintained its height and normal photosynthetic capacity but showed a reduced growth of its secondary stems. This resistance was correlated with the containment of the pathogen at the root level, and an increase in some metabolites related to the phenylpropanoid pathway.