Quantitative confocal imaging method for analyzing cellulose dynamics during cell wall regeneration in Arabidopsis mesophyll protoplasts

Abstract

The network structure of cellulose fibrils provides mechanical properties to the primary cell wall, thereby determining the shapes and growth patterns of plant cells. Despite intensive studies, the construction process of the network structure in muro remains largely unknown, mainly due to the lack of a robust, straightforward technique to evaluate network configuration. Here, we developed a quantitative confocal imaging method for general use in the study of cell wall dynamics in protoplasts derived from Arabidopsis leaf mesophyll cells. Confocal imaging of regenerating cell walls in protoplasts stained with Calcofluor allowed us to visualize the cellulose network, comprising strings of bundled cellulosic fibrils. Using image analysis techniques, we measured several metrics including total length, which is a measure of the spread of the cellulose network. The total length increased during cell wall regeneration. In a proof-of-concept experiment using microtubule-modifying agents, oryzalin, an inhibitor of microtubule polymerization, inhibited the increase in total length and caused abnormal orientation of the network, as shown by the decrease in the average angle of the cellulose with respect to the cell long axis. Taxol, a microtubule stabilizer, stimulated the bundling of cellulose fibrils, as shown by the increase in skewness in the fluorescence intensity distribution of Calcofluor, and inhibited the increase in total length. These results demonstrate the validity of this method for quantitative imaging of the cellulose network, providing an opportunity to gain insight into the dynamic aspects of cell wall regeneration. K E Y W O R D SArabidopsis thaliana, cell wall, cellulose microfibrils, imaging, protoplast, quantitative image analysis, regeneration

Rab10 regulates tubular endosome formation through KIF13A/B motors

“…2H). Maximum intensity z-projected images were binarized and then skeletonized by LpxLineExtract, which is invoked by Lpx_Filter2d plug-ins (filter=lineFilters, linemode=lineExtract) in the LPixel ImageJ plugins package (https://lpixel.net/services/research/ lpixel-imagej-plugins/) (Kuki et al, 2017). The >5 µm skeletonized lines in the images were extracted by using the analyze particles function in Fiji.…”

A Genomic Perspective on the Evolutionary Diversity of the Plant Cell Wall

“…For example, staining with Pontamine Fast Scarlet 4B, a dye that fluoresces in the presence of cellulose, facilitates the imaging of cellulose dynamics, and has revealed that cellulose bundles rotate in a transverse to longitudinal direction during cell expansion [ 28 , 29 ]. Additionally, we recently developed an imaging technique that can be used to quantitatively evaluate the network of cellulose microfibrils [ 30 , 31 ]. By combining this quantitative imaging technique with a high-yield cell-wall regeneration procedure, we successfully quantified the total length, mean intensity, skewness of intensity distribution, and coefficient of variation of regenerating cellulose microfibrils in protoplasts derived from Arabidopsis leaf mesophyll cells [ 31 ].…”

“…Additionally, we recently developed an imaging technique that can be used to quantitatively evaluate the network of cellulose microfibrils [ 30 , 31 ]. By combining this quantitative imaging technique with a high-yield cell-wall regeneration procedure, we successfully quantified the total length, mean intensity, skewness of intensity distribution, and coefficient of variation of regenerating cellulose microfibrils in protoplasts derived from Arabidopsis leaf mesophyll cells [ 31 ]. Furthermore, by adopting a quantitative imaging approach using a xyloglucan-deficient xxt1/xxt2 mutant of Arabidopsis thaliana , we showed that the absence of xyloglucans has almost no influence on either the structure of the cellulose microfibril network or protoplast stability in regenerating protoplasts, thereby indicating that xyloglucan does not directly contribute to the initial assembly of the cellulose network or the mechanical strength of the cell wall of protoplasts [ 32 ].…”

Dynamics of structural polysaccharides deposition on the plasma-membrane surface of plant protoplasts during cell wall regeneration

“…Cultured protoplast is one of the suitable materials to allow investigation of the dynamics of primary cell wall formation, e.g., arrangements of cellulose microfibrils and the sequence of polysaccharide synthesis, as well as compositional changes in cell walls during cell division [11][12][13][14][15][16][17]. Protoplasts, which lack cell walls, are appropriate for investigation of primary wall formation from initiation to the final stages.…”

“…Previous reports have investigated cell wall formation by protoplasts of herbaceous plants [11,[14][15][16][17]24], but few comparable studies have been undertaken on protoplasts of woody plants [12], which are important for biomass utilization. Therefore, the current study aimed to clarify the occurrence of cellulose microfibrils (CMFs) in protoplasts of white birch callus under the stress culture condition by in situ observation.…”