The auxin-regulated CrRLK1L kinase ERULUS controls cell wall composition during root hair tip growth

Root hairs facilitate a plant’s ability to acquire soil anchorage and nutrients. Root hair growth is regulated by the plant hormone auxin and dependent on localized synthesis, secretion and modification of the root hair tip cell wall. However, the exact well wall regulators in root hairs controlled by auxin have yet to be determined. In this study, we describe the characterization of ERULUS (ERU), an auxin-induced Arabidopsis receptor-like kinase whose expression is directly regulated by ARF7 and ARF19 transcription factors. ERU belongs to the Catharanthus roseus RECEPTOR-LIKE KINASE 1-LIKE (CrRLK1L) subfamily of putative cell wall sensor proteins. Imaging of a fluorescent fusion protein revealed that ERU is localized to the apical root hair plasma membrane. ERU regulates cell wall composition in root hairs and modulates pectin dynamics through negative control of pectin methylesterase (PME) activity. Mutant eru (-/-) root hairs accumulate de-esterified homogalacturonan and exhibit aberrant pectin Ca²⁺ binding site oscillations and increased PME activity. Up to 80% of the eru root hair phenotype is rescued by pharmacological supplementation with a PME inhibiting catechin extract. ERU transcription is altered in specific cell wall-related root hair mutants, suggesting it is a target for feedback regulation. Loss of ERU alters the phosphorylation status of FERONIA and H⁺-ATPases 1/2, regulators of apoplastic pH. Furthermore, H⁺-ATPases 1/2 and ERU are differentially phosphorylated in response to auxin. We conclude that ERULUS is a key auxin-controlled regulator of cell wall composition and pectin dynamics during root hair tip growth

In vivo measurement of the Young’s modulus of the cell wall of single root hairs

Root hairs are cells from the root epidermis that grow as long tubular bulges perpendicular to the root. They can grow in a variety of mechanical or chemical environments. Their mechanical properties are mainly due to their stiff cell wall which also constitutes a physical barrier between the cell and its environment. Thus, it is essential to be able to quantify the cell wall mechanical properties and their adaptation to environmental cues. Here, we present a technique we developed to measure the Young’s (elastic) modulus of the root hair cell wall. In essence, using custom-made glass microplates as cantilevers of calibrated stiffness, we are able to measure the force necessary to bend a single living root hair. From these experiments one can determine the stiffness and Young’s modulus of the root hair cell wall

The Kinase ERULUS Controls Pollen Tube Targeting and Growth in Arabidopsis thaliana

In this paper, we describe the role of the receptor-like kinase ERULUS (ERU) in PT growth of Arabidopsis thaliana. In silico analysis and transcriptional reporter lines revealed that ERU is only expressed in pollen and root hairs (RHs), making it a tip growth-specific kinase. Deviations from Mendelian inheritance were observed in the offspring of self-pollinated heterozygous eru plants. We found that in vivo eru PT targeting was disturbed, providing a possible explanation for the observed decrease in eru fertilization competitiveness. Extracellular calcium perception and intracellular calcium dynamics lie at the basis of in vivo pollen tube (PT) tip growth and guidance. In vitro, ERU loss-of-function lines displayed no obvious PT phenotype, unless grown on low extracellular calcium ([Ca2+]ext) medium. When grown at 12 the normal [Ca2+]ext, eru PTs grew 37% slower relative to WT PTs. Visualization of cytoplasmic [Ca2+]cyt oscillations using the Yellow Cameleon 3.6 (YC3.6) calcium sensor showed that, unlike in WT PTs, eru apical [Ca2+]cyt oscillations occur at a lower frequency when grown at lower [Ca2+]ext, consistent with the observed reduced growth velocity. Our results show that the tip growth-specific kinase ERULUS is involved in regulating Ca2+-dependent PT growth, and most importantly, fertilization efficiency through successful PT targeting to the ovules

Automated quantification of fluorescence signals in bivalve hemocytes (Mytilus edulis) as a proxy for micro-and-nanoplastic toxicity assessment

Plastic particles are prevalent in the environment, and the risks they pose especially to aquatic organisms are of growing concern. A range of responses attributed to micro-and-nanoplastic particle (MPs/NPs) exposure has been documented across various levels of biological organization, extending from cellular to population-level endpoints. However, systematic analyses of effect endpoints at the cellular level, which are considered a sensitive response variable to plastic particle exposure, are still lacking. It has been established that plastic particles can be internally distributed in organisms through various ways, such as translocation and penetration of biological barriers. In particular, particles with dimensions in the order of nanometers (i.e. nanoplastics, NPs, <1000 nm) are able to enter cells via endocytotic pathways (i.e. phagocytosis). These cellular processes have been highlighted as integral factors that influence key physiological functions including cellular immunity. Hemocytes are responsible for cell-mediated immunity in bivalve mollusks such as the mussel Mytilus edulis, which is highly susceptible to NP exposure due to its sedentary and filter-feeding lifestyle. The hemocytes circulate within the hemolymph and can cross all epithelial boundaries, acting as phagocytes against foreign particles, including plastic particles. Assessing the different physicochemical properties of MPs/NPs, such as polymer type, size, shape, and surface charge, is crucial as these parameters may directly affect the cellular uptake mechanisms as well as cytotoxicity. Hence, this study aims to validate and apply a microscopy-based method for determining whether MPs/NPs exposure induces apoptosis/necrosis (cellular death) in the hemocyte of mussels M. edulis as a proxy for micro-and-nanoplastic risk assessment. An in-vitro approach will be applied to characterize and interpret the response profiles under environmentally relevant exposure conditions and in assessing differential effects linked to characteristics of the plastic particles (type, size, shape). The apoptosis and necrosis signal quantification will be carried out through dual staining approach using an apoptosis detection kit (Biovision) and by the complementary application of fluorescence microscopy and ImageJ, an open-source image processing program. The results will provide insights into the underlying mechanisms associated with different physicochemical characteristics of MPs/NPs at a cellular level which is crucial for fate and toxicity assessment in aquatic organisms

Perturbation of Auxin Homeostasis and Signaling by PINOID Overexpression Induces Stress Responses in Arabidopsis

Under normal and stress conditions plant growth require a complex interplay between phytohormones and reactive oxygen species (ROS). However, details of the nature of this crosstalk remain elusive. Here, we demonstrate that PINOID (PID), a serine threonine kinase of the AGC kinase family, perturbs auxin homeostasis, which in turn modulates rosette growth and induces stress responses in Arabidopsis plants. Arabidopsis mutants and transgenic plants with altered PID expression were used to study the effect on auxin levels and stress-related responses. In the leaves of plants with ectopic PID expression an accumulation of auxin, oxidative burst and disruption of hormonal balance was apparent. Furthermore, PID overexpression led to the accumulation of antioxidant metabolites, while pid knockout mutants showed only moderate changes in stress-related metabolites. These physiological changes in the plants overexpressing PID modulated their response toward external drought and osmotic stress treatments when compared to the wild type. Based on the morphological, transcriptome, and metabolite results, we propose that perturbations in the auxin hormone levels caused by PID overexpression, along with other hormones and ROS downstream, cause antioxidant accumulation and modify growth and stress responses in Arabidopsis. Our data provide further proof for a strong correlation between auxin and stress biology