Biology subjects

Lyczakowski, J. J.

Publications and source records attributed to Lyczakowski, J. J..

7 recordsLinked to original sources

bioRxiv2024

Glucomannan engineering highlights the role of galactosyl modification in fine-tuning cellulose-glucomannan interaction in Arabidopsis cell walls

Widely found in most plant lineages, {beta}-mannans are structurally diverse polysaccharides that can bind to cellulose fibrils to form the complex polysaccharide architecture of the cell wall. Glucomannan backbones acquire variable patterns of galactosyl substitutions, depending on plant developmental stage and species. How the changes in polysaccharide structure influence its cell wall solubility or promote appropriate interaction with cellulose fibrils is poorly understood. Here, we show that fine-tuning of galactosyl modification on glucomannans is achieved by the differing acceptor recognition of mannan -galactosyltransferases (MAGTs). Biochemical analysis and 13C solid-state nuclear magnetic resonance spectroscopy of Arabidopsis with cell wall glucomannan engineered by MAGTs revealed that the degree of galactosylation strongly affects the interaction with cellulose. The findings indicate that plants tailor galactosyl modification on glucomannans for constructing an appropriate cell wall architecture, paving the way to convert properties of lignocellulosic biomass for better use.

bioengineering↗

bioRxiv2024

XAPT and XLPT enzymes modify the glucuronic acid side chains of tissue-specific xylans in Arabidopsis and Eucalyptus

* Polysaccharide structural complexity not only influences cell wall strength and extensibility, but also hinders pathogenic and biotechnological attempts to saccharify the wall. In certain species and tissues, glucuronic acid side chains on xylan exhibit arabinopyranose or galactose decorations whose genetic and evolutionary basis is completely unknown, impeding efforts to understand their function and engineer wall digestibility. * Genetics and polysaccharide profiling were used to identify the responsible loci in Arabidopsis and Eucalyptus from proposed candidates, while phylogenies uncovered a shared evolutionary origin. GH30-family endo-glucuronoxylanase activities were analysed by electrophoresis and their differing specificities were rationalised by phylogeny and structural analysis. * The newly identified xylan arabinopyranosyltransferases comprise an overlooked subfamily in the GT47-A family of Golgi glycosyltransferases, previously assumed to comprise mainly xyloglucan galactosyltransferases, highlighting an unanticipated adaptation of both donor and acceptor specificities. Further neofunctionalisation has produced a Myrtaceae-specific xylan galactosyltransferase. Simultaneously, GH30 endo-glucuronoxylanases have convergently adapted to overcome these decorations, suggesting a role for these structures in defence. The differential expression of glucuronoxylan-modifying genes across Eucalyptus tissues, however, hints at further functions. * Our results demonstrate the rapid adaptability of biosynthetic and degradative carbohydrate-active enzyme activities, providing insight into a plant-pathogen arms race and facilitating plant cell wall biotechnological utilisation.

plant biology↗

bioRxiv2022

Eudicot primary cell wall glucomannan is related in synthesis, structure and function to xyloglucan

The functional differences between plant cell wall hemicelluloses such as glucomannan, xylan and xyloglucan (XyG) remain unclear. These polysaccharides influence assembly and properties of the wall, perhaps by interacting with cellulose to affect the deposition and bundling of the fibrils. As the most abundant hemicellulose, XyG is considered important in eudicot primary cell walls (PCWs), but plants devoid of XyG show relatively mild phenotypes. We report here that a patterned {beta}-galactoglucomannan ({beta}-GGM) is widespread in PCW of eudicots and shows remarkable similarities to XyG. The sugar linkages forming the backbone and side chains of {beta}-GGM are analogous to those that make up XyG, and moreover, these linkages are formed by glycosyltransferases from the same CAZy families. Solid-state NMR indicated that {beta}-GGM shows low mobility in the cell wall, consistent with interaction with cellulose. Although Arabidopsis {beta}-GGM synthesis mutants show no obvious growth defects, genetic crosses between {beta}-GGM and XyG mutants produce exacerbated phenotypes compared to XyG mutants. These findings demonstrate a related role of these two similar but distinct classes of hemicelluloses in PCWs. This work will provide new avenues to study the roles of both {beta}-GGM and XyG in PCWs. One sentence summaryPatterned {beta}-GGM resembles xyloglucan in structure, biosynthesis and function.

plant biology↗

bioRxiv2021

Discovery of putative Golgi S-Adenosyl methionine transporters reveals the importance of plant cell wall polysaccharide methylation

Polysaccharide methylation, especially that of pectin, is a common and important feature of land plant cell walls. Polysaccharide methylation takes place in the Golgi apparatus and therefore relies on the import of S-adenosyl methionine (SAM) from the cytosol into the Golgi. However, to date, no Golgi SAM transporter has been identified in plants. In this work, we studied major facilitator superfamily members in Arabidopsis that we identified as putative Golgi SAM transporters (GoSAMTs). Knock-out of the two most highly expressed GoSAMTs led to a strong reduction in Golgi-synthesised polysaccharide methylation. Furthermore, solid-state NMR experiments revealed that reduced methylation changed cell wall polysaccharide conformations, interactions and mobilities. Notably, the NMR revealed the existence of pectin egg-box structures in intact cell walls, and showed that their formation is enhanced by reduced methyl-esterification. These changes in wall architecture were linked to substantial growth and developmental phenotypes. In particular, anisotropic growth was strongly impaired in the double mutant. The identification of putative transporters that import SAM into the Golgi lumen in plants provides new insights into the paramount importance of polysaccharide methylation for plant cell wall structure and function.

plant biology↗

bioRxiv2021

Two softwood GUX clades are responsible for distinct glucuronic acid patterns on xylan.

Wood of coniferous (softwood) trees, is a globally significant carbon sink and an important source of biomass for industrial applications. Despite its importance, very little is known about the genetic basis of softwood biosynthesis. Glucomannan and xylan are the main hemicelluloses in softwood secondary cell walls. Xylan interacts with the cellulose fibrils in a two-fold screw configuration. Moreover, we have shown that xylan GUX (GlucUronic acid substitution of Xylan)-dependent branching with glucuronic acid is critical for biomass recalcitrance. Here, we investigated the decoration patterns of xylan by softwood GUX enzymes. Using in vitro and in planta assays we demonstrate that two distinct clades of conifer GUX enzymes are active glucuronyltransferases. Interestingly, these enzymes have different specific activities, with one adding evenly spaced GlcA branches and the other one being also capable of glucuronidating two consecutive xyloses. Since xylan patterning might modulate xylan-cellulose and xylan-lignin interactions, our result further the understanding of softwood biosynthesis and can contribute to strategies aimed at modifying softwood cell wall properties.

plant biology↗

bioRxiv2020

Transformation of European ash (Fraxinus excelsior L.) callus as a starting point for understanding the molecular basis of ash dieback.

Plant genetic engineering requires transfer of genetic material into plant cells, a process that is frequently challenging for tree species. Here we report first successful genetic transformation of European ash (Fraxinus excelsior). The protocol relies on the use of Agrobacterium tumefaciens to transform callus tissue derived from embryos of F. excelsior. In our experiments, we use the {beta}-glucuronidase (GUS) reporter system to demonstrate transformation of ash callus tissue. Moreover, we describe an antibiotic-resistance based selection process enabling formation of stable transgenic callus lines. Since ash dieback threatens the long-term stability of many native F. excelsior populations, we hope that the transformation techniques described in this manuscript will facilitate rapid progress in uncovering the molecular basis of the disease and mechanisms used by trees to resist it.

plant biology↗

bioRxiv2019

Structural imaging of native cryo-preserved secondary cell walls reveals presence of macrofibrils composed of cellulose, lignin and xylan.

The woody secondary cell walls of plants are the largest repository of renewable carbon biopolymers on the planet. These walls are made principally from cellulose and hemicelluloses and are impregnated with lignin. Despite their importance as the main load bearing structure for plant growth, as well as their industrial importance as both a material and energy source, the precise arrangement of these constituents within the cell wall is not yet fully understood. We have adapted low temperature scanning electron microscopy (cryo-SEM) for imaging the nanoscale architecture of angiosperm and gymnosperm cell walls in their native hydrated state. Our work confirms that cell wall macrofibrils, cylindrical structures with a diameter exceeding 10 nm, are a common feature of the native hardwood and softwood samples. We have observed these same structures in Arabidopsis thaliana secondary cell walls, enabling macrofibrils to be compared between mutant lines that are perturbed in cellulose, hemicellulose and lignin formation. Our analysis indicates that the macrofibrils in Arabidopsis cell walls are composed, at least partially, of cellulose, xylan and lignin. This study is a useful additional approach for investigating the native nanoscale architecture and composition of hardwood and softwood secondary cell walls and demonstrates the applicability of Arabidopsis genetic resources to relate fibril structure with wall composition and biosynthesis.

plant biology↗