*4.1. Mechanical Changes and Calcium Localization*

The structural role that pectin plays in the cell wall is critical to a plant's ability to withstand stress [4]. Within the cell wall, pectin is far from the only critical component. However, what sets pectin apart from other components of the cell wall is the unique ability for HG and RG-II to form complex structures through cross-linkages to other elements [5,7]. Both the rheological results and those obtained from the analysis of shear force are suggestive that structural changes occurred because of calcium and boron application. Furthermore, the addition of these elements likely resulted in the formation of "egg-box" structures and RG-II dimers, as they are known to influence cell wall integrity, including rigidity and tensile strength [6,7,61].

The notion that calcium–HG cross-links form within *A. fistulosum* epidermal cell walls because of calcium application is further supported by images obtained from the Advanced Photon Source (Lemont, IL, USA) using the 20-ID beamline, the X-ray microprobe technique and X-ray fluorescence. These images clearly show an accumulation of calcium around the cell walls of epidermal cells obtained from calcium treated *A. fistulosum* and are a key piece of the puzzle in the narrative of the formation of "egg-box" structures. Within plant cells, pectin is only found within the cell wall and is most highly concentrated in the middle lamella and the primary cell wall [4]. Thus, the localization of calcium to the cell wall/middle lamella region and the increased shear force suggest that the application of calcium to *A. fistulosum* results in the formation of calcium–HG cross-links. Our next step was to analyze how the likely formation of these structures, in addition to boron and PMEIs, would influence dehydration stress resistance.
