Nanoscale imaging of xyloglucan in plant cell walls

Abstract

Cell walls shape and protect plant cells and proper wall assembly is crucial for normal growth and development. Textbook diagrams of primary (growing) cell walls show that they are made up of cellulose in a matrix of xyloglucan, pectins, and various proteins; secondary cell walls have distinctive matrix polymers, including lignin. Despite years of research by numerous groups, there are still many unanswered questions about how cell walls are organized and how they incorporate new materials for growth, and different experimental techniques sometimes gave contrasting results (Cosgrove, 2014). To better understand the organization of the components of cell walls, better imaging techniques are required. Fluorescence microscopy lacks the resolution to see individual cellulose microfibrils or single polymer chains. Atomic force microscopy (AFM) and Field Emission Scanning Electron Microscopy (FESEM) offer vast improvements in resolution. Daniel Cosgrove has worked on various aspects of cell growth since he was a graduate student and has studied cell walls per se since 1990. For example, in 1992 his group discovered expansins (McQueen-Mason et al., 1992), a group of proteins that catalyze cell wall extension (Cosgrove, 2016) and are now known also from the microbial world (Cosgrove, 2017). Expansins are not enzymes that cleave polysaccharides, but instead disrupt noncovalent bonding between polysaccharides and thereby initiate wall loosening. Daniel mentioned that although there is a crystal structure for an expansin, its detailed mechanism of action remains enigmatic, in part because of gaps in our understanding of primary wall structure. Since 2009, he has been the director of a DOE-funded Center for Lignocellulose Structure and Formation at Penn State, which is part of a 6-campus consortium that studies plant cell walls with advanced physical and imaging techniques. For example, AFM enabled the Cosgrove group to image cellulose microfibril organization and movements in onion cells (Zhang et al., 2016, 2017). In the highlighted paper (Zheng et al., 2018), they turned their attention to xyloglucan. Although there have been many models for xyloglucan's role(s) in cell wall structure, it had not been experimentally established, at the nanometer scale, where exactly xyloglucan was located in the wall. Furthermore, an Arabidopsis mutant line that lacked detectable xyloglucan (Cavalier et al., 2008) had only subtle phenotypic defects, raising some questions about its importance for cell wall structure. The first author, Yunzhen, works for the DOE Center; she carried out all the FESEM experiments. Xuan is a 5th year graduate student who developed the methods they used for analyzing cellulose organization. Yuning, a former postdoc, and Edward, who started in the Cosgrove lab as an undergraduate and then stayed on as a technician after graduation, generated the CBM-nanogold probes used to detect binding to xyloglucan and cellulose. They wanted to know if xyloglucan was really in an extended form and bound to cellulose microfibril surfaces, as shown in textbooks, or was it coiled, the conformation it assumes in solution? As shown in the schematic in Figure 1, they used onion epidermal cells to image newly deposited cell wall components. To prepare onion epidermal peels for FESEM, the tissue must be dehydrated and superficial pectin molecules, which might obscure the cellulose microfibrils, must be removed by treatment with pectate lyase. By comparing the images of the cellulose microfibrils obtained with AFM and FESEM, they noticed that there was some distortion of the microfibrils in the FESEM images, likely due to the required dehydration step and pectate lyase treatment, whereas AFM images are from cell walls that are in water, and so presumably close to the in vivo situation. Nevertheless, the microfibrils were easily visualized with FESEM. They next treated the samples with substrate-specific endoglucanases, and quantified their effects on fiber bundle widths and pore sizes. Statistically significant changes were noted, indicating that the endoglucanase treatments had some consequences, but they could not unambiguously identify xyloglucan location or form. They next tried directly probing for xyloglucan, using an anti-xyloglucan antibody (LM15) and a secondary antibody conjugated to nanogold, but there were technical challenges in detection with standard FESEM. However, by using FESEM with backscattered electron detection, which highlights the nanogold signal, they were able to conclude that xyloglucan was located only partly on the surface of cellulose microfibrils. Why is backscattering an improvement? Daniel offered an analogy – regular FESEM is like shooting a bullet (a high-energy electron) at a brick wall and the detector senses the brick fragments that scatter in many directions, whereas backscattered electron detection is like shooting a bullet at a wall and the detector senses the bullet that ricochets directly backwards. Gold nuclei do this much better than carbon and oxygen and so gold particles are bright in such images. Finally, they used nanogold conjugates of carbohydrate binding module (CBM) proteins, namely CBM76, a small protein that binds xyloglucan, and CBM3, a protein that binds the hydrophobic face of cellulose. Small proteins are better probes than bulky antibody probes; the distance from xyloglucan to CBM76 should be 3 nanometers or less. By using these CBM-nanogold probes, in combination with xyloglucanase treatments, they concluded that at least some xyloglucan indeed assumes an extended conformation that binds to the hydrophobic surfaces of cellulose microfibrils, and that this binding appreciably prevents CBM3 binding to cellulose. They will expand their FESEM analyses to walls other than epidermal walls, as the orientations of cellulose microfibrils in the walls of other types of cell are transverse, and there might be differences. They hope to use FESEM and the CBM-nanogold probes to compare the walls of wild type Arabidopsis and the xyloglucan-deficient mutant line (Cavalier et al., 2008). They also want to develop nanogold probes for other wall components, such as pectins and glycoproteins. Using such techniques, researchers will be able to test quantitative models based on experimentally-derived nanoscale organization of wall components (and as a bonus, improve those textbook diagrams).

Plant phenotyping relevance

AFM/FESEMとナノゴールドプローブを用いて植物細胞壁の微細構造を可視化・定量する手法が中心であり、単なる細胞壁生物学の測定ではない。

Code and data availability

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