Development Of Live-cell Fluorescence Microscopy Methods For The Investigation Of Cell Wall Structure

The cell wall is a network of flexible polysaccharide polymers that acts as the main skeleton outside the cell membrane of plants,bacteria and fungi.It plays an important role in maintaining cell shape,resistance to physical and chemical stress and controlling cell growth.So far,methods for researching the main components and their relative abundance within cell walls have been established.High-resolution imaging techniques such as AFM,TEM and FESEM play leading roles in analyzing cell wall ultrastructure.However,these methods are generally not adequate to explore the original structure of cell walls in vivo.In this study,we utilized the advantages of optical microscopy,especially its ability for direct observation of living cells,to explore new methods for studying the original structure of cell walls noninvasively.The main components of plant cell walls are cellulose,hemicellulose,pectin,lignin and a diverse set of proteins.In order to visualize specific components of the wall,we use 14 kinds of small molecule size fluorescent dyes as labels.Excitation and emission spectra of 9kinds of cell wall polysaccharides mixed with these dyes were measured to predict the staining specificity.Importantly,we compared the dyes usability for d STORM on polysaccharides and onion epidermal cell walls.4 dyes showed a high potential for using as the specific labels for super resolution imaging of plant cell walls.Immunofluorescence labeling with antibodies directed against specific wall polymers were taken as the reference.Moreover,the resolution differences of methods such as confocal imaging,TIRF microscopy,d STORM imaging,d STORM performance after HAWK pretreatment,SRRF microscopy were compared.The results show that using the selected cell wall dyes combined with the current advanced super-resolution imaging technology can improve the resolution of plant cell wall imaging to a certain level.The resolution limit afforded by these dyes was found to be around 50 nm,offering a 4 times improvement over the conventional fluorescence microscopy.By comparing the excitation and emission spectra of some fluorescent dyes,we found that there is overlap between the excitation spectra of trypan blue,malachite green,and BHQ3 and the emission spectra of the plasma membrane dye FM4-64.It was found that a fluorescence quenching phenomenon in which FRET mechanism would be involved can occur between these dyes.The fluorescent intensity of FM4-64-labeled onion epidermal cell protoplasts,Gram-negative bacteria,and Gram-positive bacteria were decreased in correlation with increasing concentrations of trypan blue.This indicates that the fluorescence quenching effect commonly occur in biological media.Importantly,results showed that the structure of the cell wall has a large effect on the quenching efficiency.Three quenchers with different molecular size were used to compare the quenching efficiency of FM4-64 labeled Arabidopsis cell wall mutants and plants treated with cell wall synthesis inhibitors.These experiments were complemented with cellulose microfibrils density measurements by confocal microscopy,cell wall thickness measurement by TEM and cell wall nanoscale spatial structure determination by AFM.All results illustrated that the fluorescence quenching efficiency reflects differences in cell wall porosity.Using the quenching assay,we explored the relationship between cell wall porosity and cell length and root length of drought-treated Arabidopsis.The results indicate that increased porosity is not a precondition but a result of cell extension,thereby providing new insight on the mechanism of plant organ growth.The pectin contents in cell wall,degree of pectin methylesterification and the enzymes involved in the synthesis and structural regulation of pectic polysaccharides play important roles in the response of plants to cell wall remodeling under drought stress.By applying the quenching assay to fungi,we discovered a strong effect of cell wall structure modifying-chemicals on the efficacy of antifungal drugs.Higher cell wall porosity facilitated the action of antifungal drugs in Saccharomyces cerevisiae,presumably by enabling higher rates of drug uptake.