Functional Analysis of Cellulose and Xyloglucan in the Walls of Stomatal Guard Cells of Arabidopsis  

Abstract

Stomatal guard cells are pairs of specialized epidermal cells that control water and CO 2 exchange between the plant and the environment. To fulfill the functions of stomatal opening and closure that are driven by changes in turgor pressure, guard cell walls must be both strong and flexible, but how the structure and dynamics of guard cell walls enable stomatal function remains poorly understood. To address this question, we applied cell biological and genetic analyses to investigate guard cell walls and their relationship to stomatal function in Arabidopsis (Arabidopsis thaliana). Using live-cell spinning disk confocal microscopy, we measured the motility of cellulose synthase (CESA)-containing complexes labeled by green fluorescent protein (GFP)-CESA3 and observed a reduced proportion of GFP-CESA3 particles colocalizing with microtubules upon stomatal closure. Imaging cellulose organization in guard cells revealed a relatively uniform distribution of cellulose in the open state and a more fibrillar pattern in the closed state, indicating that cellulose microfibrils undergo dynamic reorganization during stomatal movements. In cesa3 je5 mutants defective in cellulose synthesis and xxt1 xxt2 mutants lacking the hemicellulose xyloglucan, stomatal apertures, changes in guard cell length, and cellulose reorganization were aberrant during fusicoccin-induced stomatal opening or abscisic acid-induced stomatal closure, indicating that sufficient cellulose and xyloglucan are required for normal guard cell dynamics. Together, these results provide new insights into how guard cell walls allow stomata to function as responsive mediators of gas exchange at the plant surface.

Integrating cell biology, image analysis, and computational mechanical modeling to analyze the contributions of cellulose and xyloglucan to stomatal function

“…9 Using spinning disk confocal microscopy of fluorescent protein (FP)-labeled CESAs in young tissues, we found that after stomatal closure, CSCs moved faster and there was less colocalization between CSCs and MTs. 1 We also detected drastically lower CSC density in guard cells in older tissues, suggesting that wall mechanical requirements for mature guard cells are largely fulfilled by existing cellulose that was previously deposited. 1 Cellulose in guard cells is arranged radially, 11,12 and xyloglucan is a hemicellulose that modulates wall mechanics 13 and is present in guard cell walls.…”

“…1 We also detected drastically lower CSC density in guard cells in older tissues, suggesting that wall mechanical requirements for mature guard cells are largely fulfilled by existing cellulose that was previously deposited. 1 Cellulose in guard cells is arranged radially, 11,12 and xyloglucan is a hemicellulose that modulates wall mechanics 13 and is present in guard cell walls. 4 To test whether cellulose and xyloglucan regulate the anisotropic deformation of guard cells during stomatal movements, 14,15 we analyzed mutants deficient in cellulose or lacking detectable xyloglucan, finding that cellulose restricts, whereas xyloglucan facilitates longitudinal expansion and contraction in guard cells, thus regulating stomatal aperture.…”

“…4 To test whether cellulose and xyloglucan regulate the anisotropic deformation of guard cells during stomatal movements, 14,15 we analyzed mutants deficient in cellulose or lacking detectable xyloglucan, finding that cellulose restricts, whereas xyloglucan facilitates longitudinal expansion and contraction in guard cells, thus regulating stomatal aperture. 1 Cellulose and xyloglucan are also required for the reorganization of cellulose in guard cells between a diffuse pattern in the open state and extensive bundles in the close state. 1 The use of these mutants has allowed us to dissect the contributions of individual wall components to stomatal dynamics.…”

“…1 Cellulose and xyloglucan are also required for the reorganization of cellulose in guard cells between a diffuse pattern in the open state and extensive bundles in the close state. 1 The use of these mutants has allowed us to dissect the contributions of individual wall components to stomatal dynamics.…”

“…16 To gauge colocalization for MTs and CSCs, FP-CESA particles were detected using Imaris software (Bitplane, Zurich, Switzerland) and the particles that overlapped with MT fluorescence were initially manually selected 1 (Fig. 1a).…”