Imaging plant cell walls using fluorescent stains: The beauty is in the details.

Plants continuously face various environmental stressors throughout their lifetime. To be able to grow and adapt in different environments, they developed specialized tissues that allowed them to maintain a protected yet interconnected body. These tissues undergo specific primary and secondary cell wall modifications that are essential to ensure normal plant growth, adaptation and successful land colonization. The composition of cell walls can vary among different plant species, organs and tissues. The ability to remodel their cell walls is fundamental for plants to be able to cope with multiple biotic and abiotic stressors. A better understanding of the changes taking place in plant cell walls may help identify and develop new strategies as well as tools to enhance plants' survival under environmental stresses or prevent pathogen attack. Since the invention of microscopy, numerous imaging techniques have been developed to determine the composition and dynamics of plant cell walls during normal growth and in response to environmental stimuli. In this review, we discuss the main advances in imaging plant cell walls, with a particular focus on fluorescent stains for different cell wall components and their compatibility with tissue clearing techniques. Lay Description: Plants are continuously subjected to various environmental stresses during their lifespan. They evolved specialized tissues that thrive in different environments, enabling them to maintain a protected yet interconnected body. Such tissues undergo distinct primary and secondary cell wall alterations essential to normal plant growth, their adaptability and successful land colonization. Cell wall composition may differ among various plant species, organs and even tissues. To deal with various biotic and abiotic stresses, plants must have the capacity to remodel their cell walls. Gaining insight into changes that take place in plant cell walls will help identify and create novel tools and strategies to improve plants' ability to withstand environmental challenges. Multiple imaging techniques have been developed since the introduction of microscopy to analyse the composition and dynamics of plant cell walls during growth and in response to environmental changes. Advancements in plant tissue cleaning procedures and their compatibility with cell wall stains have significantly enhanced our ability to perform high-resolution cell wall imaging. At the same time, several factors influence the effectiveness of cleaning and staining plant specimens, as well as the time necessary for the process, including the specimen's size, thickness, tissue complexity and the presence of autofluorescence. In this review, we will discuss the major advances in imaging plant cell walls, with a particular emphasis on fluorescent stains for diverse cell wall components and their compatibility with tissue clearing techniques. We hope that this review will assist readers in selecting the most appropriate stain or combination of stains to highlight specific cell wall components of interest.

Structural and biochemical characterization of Arabidopsis alcohol dehydrogenases reveals distinct functional properties but similar redox sensitivity.

Alcohol dehydrogenases (ADHs) are a group of zinc-binding enzymes belonging to the medium-length dehydrogenase/reductase (MDR) protein superfamily. In plants, these enzymes fulfill important functions involving the reduction of toxic aldehydes to the corresponding alcohols (as well as catalyzing the reverse reaction, i.e., alcohol oxidation; ADH1) and the reduction of nitrosoglutathione (GSNO; ADH2/GSNOR). We investigated and compared the structural and biochemical properties of ADH1 and GSNOR from Arabidopsis thaliana. We expressed and purified ADH1 and GSNOR and determined two new structures, NADH-ADH1 and apo-GSNOR, thus completing the structural landscape of Arabidopsis ADHs in both apo- and holo-forms. A structural comparison of these Arabidopsis ADHs revealed a high sequence conservation (59% identity) and a similar fold. In contrast, a striking dissimilarity was observed in the catalytic cavity supporting substrate specificity and accommodation. Consistently, ADH1 and GSNOR showed strict specificity for their substrates (ethanol and GSNO, respectively), although both enzymes had the ability to oxidize long-chain alcohols, with ADH1 performing better than GSNOR. Both enzymes contain a high number of cysteines (12 and 15 out of 379 residues for ADH1 and GSNOR, respectively) and showed a significant and similar responsivity to thiol-oxidizing agents, indicating that redox modifications may constitute a mechanism for controlling enzyme activity under both optimal growth and stress conditions.

Acquisition of hypoxia inducibility by oxygen sensing N-terminal cysteine oxidase in spermatophytes.

N-terminal cysteine oxidases (NCOs) use molecular oxygen to oxidise the amino-terminal cysteine of specific proteins, thereby initiating the proteolytic N-degron pathway. To expand the characterisation of the plant family of NCOs (plant cysteine oxidases [PCOs]), we performed a phylogenetic analysis across different taxa in terms of sequence similarity and transcriptional regulation. Based on this survey, we propose a distinction of PCOs into two main groups. A-type PCOs are conserved across all plant species and are generally unaffected at the messenger RNA level by oxygen availability. Instead, B-type PCOs appeared in spermatophytes to acquire transcriptional regulation in response to hypoxia. The inactivation of two A-type PCOs in Arabidopsis thaliana, PCO4 and PCO5, is sufficient to activate the anaerobic response in young seedlings, whereas the additional removal of B-type PCOs leads to a stronger induction of anaerobic genes and impairs plant growth and development. Our results show that both PCO types are required to regulate the anaerobic response in angiosperms. Therefore, while it is possible to distinguish two clades within the PCO family, we conclude that they all contribute to restrain the anaerobic transcriptional programme in normoxic conditions and together generate a molecular switch to toggle the hypoxic response.

A synthetic switch based on orange carotenoid protein to control blue-green light responses in chloroplasts.

Synthetic biology approaches to engineer light-responsive systems are widely used, but their applications in plants are still limited due to the interference with endogenous photoreceptors and the intrinsic requirement of light for photosynthesis. Cyanobacteria possess a family of soluble carotenoid-associated proteins named orange carotenoid proteins (OCPs) that, when activated by blue-green light, undergo a reversible conformational change that enables the photoprotection mechanism that occurs on the phycobilisome. Exploiting this system, we developed a chloroplast-localized synthetic photoswitch based on a protein complementation assay where two nanoluciferase fragments were fused to separate polypeptides corresponding to the OCP2 domains. Since Arabidopsis (Arabidopsis thaliana) does not possess the prosthetic group needed for the assembly of the OCP2 complex, we first implemented the carotenoid biosynthetic pathway with a bacterial ß-carotene ketolase enzyme (crtW) to generate keto-carotenoid-producing plants. The photoswitch was tested and characterized in Arabidopsis protoplasts and stably transformed plants with experiments aimed to uncover its regulation by a range of light intensities, wavelengths, and its conversion dynamics. Finally, we applied the OCP-based photoswitch to control transcriptional responses in chloroplasts in response to green light illumination by fusing the two OCP fragments with the plastidial SIGMA FACTOR 2 and bacteriophage T4 anti-sigma factor AsiA. This pioneering study establishes the basis for future implementation of plastid optogenetics to regulate organelle responses upon exposure to specific light spectra.

A Synthetic Oxygen Sensor for Plants Based on Animal Hypoxia Signaling.

Due to the involvement of oxygen in many essential metabolic reactions, all living organisms have developed molecular systems that allow adaptive physiological and metabolic transitions depending on oxygen availability. In mammals, the expression of hypoxia-response genes is controlled by the heterodimeric Hypoxia-Inducible Factor. The activity of this transcriptional regulator is linked mainly to the oxygen-dependent hydroxylation of conserved proline residues in its α-subunit, carried out by prolyl-hydroxylases, and subsequent ubiquitination via the E3 ligase von Hippel-Lindau tumor suppressor, which targets Hypoxia-Inducible Factor-α to the proteasome. By exploiting bioengineered versions of this mammalian oxygen sensor, we designed and optimized a synthetic device that drives gene expression in an oxygen-dependent fashion in plants. Transient assays in Arabidopsis (Arabidopsis thaliana) mesophyll protoplasts indicated that a combination of the yeast Gal4/upstream activating sequence system and the mammalian oxygen sensor machinery can be used effectively to engineer a modular, oxygen-inducible transcriptional regulator. This synthetic device also was shown to be selectively controlled by oxygen in whole plants when its components were expressed stably in Arabidopsis seedlings. We envision the exploitation of our genetically encoded controllers to generate plants able to switch gene expression selectively depending on oxygen availability, thereby providing a proof of concept for the potential of synthetic biology to assist agricultural practices in environments with variable oxygen provision.

Combined off-pump minimally invasive coronary artery bypass grafting and mitral valve repair with NeoChord via a left anterolateral small thoracotomy.

Off-pump direct coronary artery bypass grafting through a left anterior small thoracotomy is widely utilized for minimally invasive myocardial revascularization. More recently, a novel technique for transapical off-pump mitral valve repair with the NeoChord device is demonstrating its efficacy. This report describes a case of an 84-year-old male patient with coronary artery disease involving the left anterior descending coronary artery and acute severe mitral regurgitation secondary to posterior leaflet flail who underwent both off-pump coronary artery bypass and mitral valve repair with the transapical implantation of artificial chordae using the NeoChord DS 1000 system through the same anterolateral small thoracotomy.