scholarly journals Three-Dimensional Imaging of Plant Cell Wall Deconstruction Using Fluorescence Confocal Microscopy

2020 ◽  
Vol 1 (2) ◽  
pp. 75-85
Author(s):  
Aya Zoghlami ◽  
Yassin Refahi ◽  
Christine Terryn ◽  
Gabriel Paës

Lignocellulosic biomass (LB) is recalcitrant to enzymatic hydrolysis due to its compact and complex cell wall structure. To identify the parameters behind LB recalcitrance, experimental data over hydrolysis time must be collected. Here, we describe a novel method to collect time-lapse images during cell wall deconstruction by enzymatic hydrolysis. The protocol includes instructions for sample preparation, layout of a custom designed incubation chamber and instructions for confocal time lapse acquisition. The protocol sets out a detailed plan where cross-sections of untreated and pretreated poplar samples are mounted in a sealed frame containing a buffer and an enzymatic cocktail. The sealed frame is then placed into an incubator to maintain the sample at a constant temperature of 50 °C, which is optimal for enzymatic reaction while avoiding enzymatic cocktail evaporation. Using lignin natural autofluorescence, confocal z-stacks of untreated and pretreated samples were acquired at regular time intervals during enzymatic hydrolysis for 24 h. Acquisition parameters were optimized to compromise between image resolution and reduced photo-bleaching. The acquired image might then be processed by further development of algorithms to extract precise quantitative information on cell wall deconstruction. This protocol is an important first step towards elucidating the underlying parameters of LB recalcitrance by allowing the acquisition of high-quality images of LB hydrolysis for extracting quantitative data on LB deconstruction.

2019 ◽  
Author(s):  
Sylvia L. Rivera ◽  
Akbar Espaillat ◽  
Arjun K. Aditham ◽  
Peyton Shieh ◽  
Chris Muriel-Mundo ◽  
...  

Transpeptidation reinforces the structure of cell wall peptidoglycan, an extracellular heteropolymer that protects bacteria from osmotic lysis. The clinical success of transpeptidase-inhibiting β-lactam antibiotics illustrates the essentiality of these cross-linkages for cell wall integrity, but the presence of multiple, seemingly redundant transpeptidases in many bacterial species makes it challenging to determine cross-link function precisely. Here we present a technique to covalently link peptide strands by chemical rather than enzymatic reaction. We employ bio-compatible click chemistry to induce triazole formation between azido- and alkynyl-D-alanine residues that are metabolically installed in the cell walls of Gram-positive and Gram-negative bacteria. Synthetic triazole cross-links can be visualized by substituting azido-D-alanine with azidocoumarin-D-alanine, an amino acid derivative that undergoes fluorescent enhancement upon reaction with terminal alkynes. Cell wall stapling protects the model bacterium Escherichia coli from β-lactam treatment. Chemical control of cell wall structure in live bacteria can provide functional insights that are orthogonal to those obtained by genetics.<br>


F1000Research ◽  
2016 ◽  
Vol 5 ◽  
pp. 119 ◽  
Author(s):  
Daniel J. Cosgrove

The growing cell wall in plants has conflicting requirements to be strong enough to withstand the high tensile forces generated by cell turgor pressure while selectively yielding to those forces to induce wall stress relaxation, leading to water uptake and polymer movements underlying cell wall expansion. In this article, I review emerging concepts of plant primary cell wall structure, the nature of wall extensibility and the action of expansins, family-9 and -12 endoglucanases, family-16 xyloglucan endotransglycosylase/hydrolase (XTH), and pectin methylesterases, and offer a critical assessment of their wall-loosening activity


2015 ◽  
Vol 66 (14) ◽  
pp. 4279-4294 ◽  
Author(s):  
Sivakumar Pattathil ◽  
Michael G. Hahn ◽  
Bruce E. Dale ◽  
Shishir P. S. Chundawat

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yanli Liu ◽  
Linlong Ma ◽  
Dan Cao ◽  
Ziming Gong ◽  
Jing Fan ◽  
...  

Abstract Background C. sinensis is an important economic crop with fluoride over-accumulation in its leaves, which poses a serious threat to human health due to its leaf consumption as tea. Recently, our study has indicated that cell wall proteins (CWPs) probably play a vital role in fluoride accumulation/detoxification in C. sinensis. However, there has been a lack in CWP identification and characterization up to now. This study is aimed to characterize cell wall proteome of C. sinensis leaves and to develop more CWPs related to stress response. A strategy of combined cell wall proteomics and N-glycoproteomics was employed to investigate CWPs. CWPs were extracted by sequential salt buffers, while N-glycoproteins were enriched by hydrophilic interaction chromatography method using C. sinensis leaves as a material. Afterwards all the proteins were subjected to UPLC-MS/MS analysis. Results A total of 501 CWPs and 195 CWPs were identified respectively by cell wall proteomics and N-glycoproteomics profiling with 118 CWPs in common. Notably, N-glycoproteomics is a feasible method for CWP identification, and it can enhance CWP coverage. Among identified CWPs, proteins acting on cell wall polysaccharides constitute the largest functional class, most of which might be involved in cell wall structure remodeling. The second largest functional class mainly encompass various proteases related to CWP turnover and maturation. Oxidoreductases represent the third largest functional class, most of which (especially Class III peroxidases) participate in defense response. As expected, identified CWPs are mainly related to plant cell wall formation and defense response. Conclusion This was the first large-scale investigation of CWPs in C. sinensis through cell wall proteomics and N-glycoproteomics. Our results not only provide a database for further research on CWPs, but also an insight into cell wall formation and defense response in C. sinensis.


Author(s):  
Liangjie Niu ◽  
Wei Wang

ABSTRACTAs the vital component of plant cell wall, proteins play important roles in stress response through modifying wall structure and involving in wall integrity signaling. However, the potential of cell wall proteins (CWPs) in improvement of crop stress tolerance has probably been underestimated. Recently, we have critically reviewed the predictors, databases and cross-referencing of subcellular locations of possible CWPs in plants (Briefings in Bioinformatics 2018;19:1130-1140). In this study, taking maize (Zea mays) as an example, we retrieved 1873 entries of probable maize CWPs recorded in UniProtKB. As a result, 863 maize CWPs are curated and classified as 59 kinds of protein families. By referring to GO annotation and gene differential expression in Expression Atlas, we highlight the potential of CWPs as defensive forwards in abiotic and biotic stress responses. In particular, several CWPs are found to play key roles in adaptation to many stresses. String analysis also reveals possibly strong interactions among many CWPs, especially those stress-responsive enzymes. The results allow us to narrow down the list of CWPs to a few specific proteins that could be candidates to enhance maize resistance.


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