Pectate Lyase (Aspergillus sp.)

Adventitious root (AR) formation is one of the most important developmental processes in vegetative propagation. Although genotypic differences in rose rooting ability are well known, the causal factors are not well understood. The rooting of two contrasting genotypes, ‘Herzogin Friederike’ and ‘Mariatheresia’, was compared following a multiscale approach. Using magnetic resonance imaging, we non-invasively monitored the inner structure of stem cuttings during initiation and progression of AR formation for the first time. Spatially resolved Fourier-transform infrared spectroscopy characterized the chemical composition of the tissues involved in AR formation. The results were validated through light microscopy and complemented by immunolabelling. The outcome demonstrated similarity of both genotypes in root primordia formation, which did not result in root protrusion through the shoot cortex in the difficult-to-root genotype ‘Mariatheresia’. The biochemical composition of the contrasting genotypes highlighted main differences in cell wall-associated components. Further spectroscopic analysis of 15 contrasting rose genotypes confirmed the biochemical differences between easy- and difficult-to-root groups. Collectively, our data indicate that it is not the lack of root primordia limiting AR formation in these rose genotypes, but the firmness of the outer stem tissue and/or cell wall modifications that pose a mechanical barrier and prevent root extension and protrusion.

Lignocellulose is a renewable but complex material exhibiting high recalcitrance to enzymatic hydrolysis, which is attributed, in part, to the presence of covalent linkages between lignin and polysaccharides in the plant cell wall. Glucuronoyl esterases from carbohydrate esterase family 15 (CE15) have been proposed as an aid in reducing this recalcitrance by cleaving ester bonds found between lignin and glucuronoxylan. In the Bacteroidota phylum, some species organize genes related to carbohydrate metabolism in polysaccharide utilization loci (PULs) which encode all necessary proteins to bind, deconstruct, and respond to a target glycan. Bioinformatic analyses identified CE15 members in some PULs that appear to not target the expected glucuronoxylan. Here, five CE15 members from such PULs were investigated with the aim of gaining insights on their biological roles. The selected targets were characterized using glucuronoyl esterase model substrates and with a new synthetic molecule mimicking a putative ester linkage between pectin and lignin. The CE15 enzyme from Phocaeicola vulgatus was structurally determined by X-ray crystallography both with and without carbohydrate ligands with galacturonate binding in a distinct conformation than that of glucuronate. We further explored whether these CE15 enzymes could act akin to pectin methylesterases on pectin-rich biomass but did not find evidence to support the proposed activity. Based on the evidence gathered, the CE15 enzymes in the PULs expected to degrade pectin could be involved in cleavage of uronic acid esters in rhamnogalacturonans.

Plant cell wall-derived oligosaccharides, i.e., damage-associated molecular patterns (DAMPs), could be generated after pathogen attack or during normal plant development, perceived by cell wall receptors, and can alter immunity and cell wall composition. Therefore, we hypothesised that xylo-oligosaccharides (XOS) could act as an elicitor and trigger immune responses. To test this, we treated Arabidopsis with xylobiose (XB) and investigated different parameters. XB-treatment significantly triggered the generation of reactive oxygen species (ROS), activated MAPK protein phosphorylation, and induced callose deposition. The combination of XB (DAMP) and flg22 a microbe-associated molecular pattern (MAMP) further enhanced ROS response and gene expression of PTI marker genes. RNA sequencing analysis revealed that more genes were differentially regulated after 30 min compared to 24 h XB-treated leaves, which correlated with ROS response. Increased xylosidase activity and soluble xylose level after 30 min and 3 h of XB-treatment were observed which might have weakened the DAMP response. However, an increase in total cell wall sugar and a decrease in uronic acid level was observed at both 30 min and 24 h. Additionally, arabinose, rhamnose, and xylose levels were increased in 30 min, and glucose was increased in 24 h compared to mock-treated leaves. The level of jasmonic acid, abscisic acid, auxin, and cytokinin were also affected after XB treatment. Overall, our data revealed that the shortest XOS can act as a DAMP, which triggers the PTI response and alters cell wall composition and hormone level.

The root nodule symbiosis with its global impact on nitrogen fertilization of soils is characterized by an intracellular colonization of legume roots by rhizobia. Although the symbionts are initially taken up by morphologically adapted root hairs, rhizobia persistently progress within a membrane-confined infection thread through several root cortical and later nodular cell layers. Throughout this transcellular passaging, rhizobia have to repeatedly pass host plasma membranes and cell walls. Here, we investigated this essential process and describe the concerted action of one of the symbiosis-specific pectin methyl esterases (SyPME1) and the nodulation pectate lyase (NPL) at the infection thread and transcellular passage sites. Their coordinated function mediates spatially confined pectin alterations in the cell-cell interface that result in the establishment of an apoplastic compartment where bacteria are temporarily released into and taken up from the subjacent cell. This process allows successful intracellular progression of infection threads through the entire root cortical tissue.

Background: The unicellular charophycean green alga Penium margaritaceum has emerged as an appealing experimental organism in plant cell wall and cell biology research. Innovative practical approaches in the manipulation and maintenance of this unicellular model alga are needed in order to probe the complexities of its subcellular and molecular machinery. Protoplast isolation and manipulation expedites a new range of experimental possibilities for Penium-based studies. These include enhanced means of isolation of subcellular components and macromolecules, application of intracellular probes for high resolution microscopy of live cells, transformation studies and analysis of the fundamental mechanisms of plant cell expansion and wall polymer deposition. Results: We present a methodology for enzyme-based digestion of the Penium cell wall and the isolation of protoplasts. The subcellular events associated with this technology are presented using multiple microscopy-based techniques. We also provide protocols for applying an array of intracellular dyes that can be used as markers for specific organelles and membrane microdomains in live cells. Finally, we present a protocol for the purification of a nuclei-rich fraction from protoplasts, which can be used for the isolation of nuclear DNA. Conclusion: Protoplast isolation, culturing and manipulation provide valuable means for molecular and cellular studies of Penium. The protocol described here offers a rapid and effective mechanism for fast and effective production of protoplasts. Subsequently, the protoplasts may be used for microscopy-based studies of specific subcellular components and the isolation of organelles and nuclear DNA. These methods offer a new practical methodology for future studies of this model organism in cell and molecular biology.

Using immunolocalization methods combined with monoclonal antibodies, the distribution of pectin and hemicellulose epitopes was examined in the secondary phloem of two diffuse porous (birch, aspen)- and two ring porous (oak, ash) hardwoods with a focus on sieve tube elements (SEs), companion cells (CCs), axial/ray parenchyma cells, and sclerenchyma cells (sclereids and phloem fibers). In all tree species, rhamnogalacturonan-I (RG-I), homogalacturonan (HG), and xyloglucan epitopes were common in cell walls of SEs, CCs, and axial/ray parenchyma cells. However, the amount of these epitopes varied greatly between cell types and between hardwood species. Apart from aspen, heteroxylan or/and heteromannan epitopes were detected in SEs, but were not detected in CCs and parenchyma cells. With sclerenchyma cells, RG-I, HG, and xyloglucan epitopes were common in compound middle lamellae (CML) of sclereids and phloem fibers. Except for oak, heteromannan epitopes were also detected in CML of sclereids. Distributional patterns of epitopes in CML of birch and ash sclereids varied greatly depending on anatomical structure of CML. Secondary cell walls of sclereids and phloem fibers revealed abundant heteroxylan epitopes, but showed no heteromannan epitopes. Some phloem fibers also showed sparse xyloglucan epitopes in secondary cell walls. Together, results suggest that there are great variations in distributional patterns of pectin and hemicellulose epitopes in hardwood phloem between cell types and between tree species.

Objective: Cupressaceae pollen has commonly been reported to be an important aeroallergen and causal factor of spring, autumn and winter pollinosis in many countries. The aim of this study was to compare of the structure and allergenic protein content of Cupressus arizonica Greene. , Cupressus sempervirens L. and Juniperus oxycedrus L. pollen in detail and contribute to Cupressaceae pollen allergen diagnosis and therapy studies in Turkey. Methods: The pollen structure were examined by LM and SEM. Pollen protein content was investigated by Bradford protein assay, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot analysis and two-dimensional polyacrylamide gel electrophoresis (2DE PAGE), respectively. Pectate lyase (PL) enzyme activities were compared. Immunoblotting was carried out by using extracts of the three taxa pollen collected from Turkey. Results: All three taxa was found very similar in terms of pollen morphology however, intine thickness was prominently different. Cupressus arizonica pollen extracts showed the lowest PL activity. Five sera specific IgE of all allergic subjects showed reaction with only C. arizonica pollen extracts. Conclusions: As a conclusion, the pollen structure, protein function or protein structure and isoforms of allergens could affects allergenic properties of the pollen. This study also may help to improve the Cupressaceae pollen allergen diagnosis and therapy.