Determination of Free Proline in Plants.

Proline metabolism has been associated with the induction of reactive oxygen species (ROS), antioxidant enzymes, and the control of cellular redox status. Moreover, proline accumulation is a highly evolutionarily conserved response to diverse abiotic stresses in plants. Thus, proline quantification has been helpful in abiotic stress research as a stress marker. The need for a reliable, fast, and simple method to detect proline in plant tissues is a powerful resource to imply the physiological status of plants under abiotic stress. This chapter summarizes the main strategies for proline extraction and quantification, highlighting their limitations and advantages, and recommends and details a specific protocol for proline extraction and quantification. The chapter provides a friendly version of this protocol with notes useful for researchers to perform the protocol.

The K+ transporter NPF7.3/NRT1.5 and the proton pump AHA2 contribute to K+ transport in Arabidopsis thaliana under K+ and NO3- deficiency.

Nitrate (NO3 -) and potassium (K+) are distributed in plants via short and long-distance transport. These two pathways jointly regulate NO3 - and K+ levels in all higher plants. The Arabidopsis thaliana transporter NPF7.3/NRT1.5 is responsible for loading NO3 - and K+ from root pericycle cells into the xylem vessels, facilitating the long-distance transport of NO3 - and K+ to shoots. In this study, we demonstrate a protein-protein interaction of NPF7.3/NRT1.5 with the proton pump AHA2 in the plasma membrane by split ubiquitin and bimolecular complementation assays, and we show that a conserved glycine residue in a transmembrane domain of NPF7.3/NRT1.5 is crucial for the interaction. We demonstrate that AHA2 together with NRT1.5 affects the K+ level in shoots, modulates the root architecture, and alters extracellular pH and the plasma membrane potential. We hypothesize that NRT1.5 and AHA2 interaction plays a role in maintaining the pH gradient and membrane potential across the root pericycle cell plasma membrane during K+ and/or NO3 - transport.

Exploring Toxoplasma gondii´s Biology within the Intestinal Epithelium: intestinal-derived models to unravel sexual differentiation.

A variety of intestinal-derived culture systems have been developed to mimic in vivo cell behavior and organization, incorporating different tissue and microenvironmental elements. Great insight into the biology of the causative agent of toxoplasmosis, Toxoplasma gondii, has been attained by using diverse in vitro cellular models. Nonetheless, there are still processes key to its transmission and persistence which remain to be elucidated, such as the mechanisms underlying its systemic dissemination and sexual differentiation both of which occur at the intestinal level. Because this event occurs in a complex and specific cellular environment (the intestine upon ingestion of infective forms, and the feline intestine, respectively), traditional reductionist in vitro cellular models fail to recreate conditions resembling in vivo physiology. The development of new biomaterials and the advances in cell culture knowledge have opened the door to a next generation of more physiologically relevant cellular models. Among them, organoids have become a valuable tool for unmasking the underlying mechanism involved in T. gondii sexual differentiation. Murine-derived intestinal organoids mimicking the biochemistry of the feline intestine have allowed the generation of pre-sexual and sexual stages of T. gondii for the first time in vitro, opening a window of opportunity to tackling these stages by "felinizing" a wide variety of animal cell cultures. Here, we reviewed intestinal in vitro and ex vivo models and discussed their strengths and limitations in the context of a quest for faithful models to in vitro emulate the biology of the enteric stages of T. gondii.

Spectral phasor analysis reveals altered membrane order and function of root hair cells in Arabidopsis dry2/sqe1-5 drought hypersensitive mutant.

Biological membranes allow the regulation of numerous cellular processes, which are affected when unfavorable environmental factors are perceived. Lipids and proteins are the principal components of biological membranes. Each lipid has unique biophysical properties, and, therefore the lipid composition of the membrane is critical to maintaining the bilayer structure and functionality. Membrane composition and integrity are becoming the focus of studies aiming to understand how plants adapt to its environment. In this study, using a combination of di-4-ANEPPDHQ fluorescence and spectral phasor analysis, we report that the drought hypersensitive/squalene epoxidase (dry2/sqe1-5) mutant with reduced major sterols such as sitosterol and stigmasterol in roots presented higher membrane fluidity than the wild type. Moreover, analysis of endomembrane dynamics showed that vesicle formation was affected in dry2/sqe1-5. Further analysis of proteins associated with sterol rich micro domains showed that dry2/sqe1-5 presented micro domains function altered.