*2.4. Texture Analysis*

The force required to shear *A. fistulosum* sheaths was evaluated using the 10-blade Allo–Kramer shearing compression cell, attached to the TMS-Pro Texture Press (Food Technology Corp., Sterling, VA, USA) in the Shand lab at the University of Saskatchewan. Calcium-treated plants received 100 mL soil drench of a 0.05 M CaCl2 solution every second day for four weeks. Each measurement was performed using three 4 cm sections of *A. fistulosum* sheaths, from the youngest leaf with the most developed sheath. The ligule and the epidermal cell layer were left intact. The full-scale load was 1000 N, the crosshead speed was 500 mm min−1. The shear force (in Newtons) required to shear 1 g of fresh sample was calculated using Equation (1) [46]. The shear force was measured in N g−<sup>1</sup> [46]. The experiment was repeated three independent times, with four replicates per trial (*n* = 12).

$$\text{Force Required to Shear Allium Shaths} = \frac{\text{peak shear force} \text{ (N)}}{\text{weight of shathts (g)}} \tag{1}$$

Equation (1). Force required to shear *Allium fistulosum* sheaths in N g<sup>−</sup>1.

#### *2.5. Water Loss in Pure Pectin Standards*

TIC Pretested Pectin HM (69–75% methylation) slow set (standardized with dextrose) was used as a model for pectin within *Allium* and GENU BETA pectin (55% methylation) (standardized with EU non-GM beet sucrose) was a model for *Arabidopsis* pectin. Treatments consisted of pectin solutions at concentrations of 4% and 8%, with and without 0.05 M of CaCl2 or 0.05 M of H3BO3. Overall, 12 different pectin solutions were examined with 4 replications per pectin solution treatment, and the experiment was repeated 3 times.

Gravimetric water loss on an analytical balance was recorded at hourly intervals over 0–6 h in the various pectin solutions. This timeline was selected following preliminary experiments. Approximately 1 g of each of the pectin solutions was evenly pipetted into Petri dishes from the center point. Throughout the course of the dehydration experiment, the lids were kept off the Petri dishes to allow for evaporation. The temperature remained constant at 23 ◦C, with a relative humidity of ~22%. The percentage water loss was measured as per Equation (2).

$$Percentage\ Water\ Loss = (1 - \left(\frac{T0\ weight}{T(x)weight}\right) \times 100) \tag{2}$$

Equation (2). Percentage water loss in pure pectin standards. Variable "*T*" is equal to the mass of the plant tissue at a specified time point.

#### *2.6. Allium Dehydration Stress Tolerance*

Dehydration tolerance was measured by first analyzing the percentage water loss over 16–18 h using 4 cm sections of *A. fistulosum* and *A. cepa* sheaths obtained from the youngest leaf with the most developed sheath. Both ends of the sheath were sealed with Vaseline® and weighed to obtain a T0 weight. The sheaths were then dehydrated under dark conditions at 23 ◦C and ~33% RH. Weights were recorded at 16 h and 18 h. Following dehydration, the sheaths were wrapped in moist Kim Wipes and placed in 50 mL Falcon tubes with the lid for 24 h. The percentage water loss was measured and calculated (Equation (2)).

Following dehydration, viability was assessed based on the presence/absence of protoplasmic streaming and staining using fluorescein diacetate (FDA). Epidermal cell layers were peeled from the sheath prior to viability analysis. Protoplasmic streaming was observed using a digital LEICA DM4 B microscope (Wetzlar, Germany) at 40× with a LEICA DFC7000 T camera (Wetzlar, Germany). Protoplasmic streaming was quantified as a percentage of viability across the epidermal cell layer (Equation (3)). The same microscope was used to visualize epidermal cell layers at 40× following FDA staining.

$$\text{Percent Cell Viabilities} = \frac{\text{number of cells with prototypical streamwise}}{\text{total number of cells}} \tag{3}$$

Equation (3). Percent cell viability based on protoplasmic streaming. Total cell count was measured by counting the number of cells within the frame at the 40× objective.

#### *2.7. Arabidopsis Dehydration Stress Tolerance*

Tolerance to dehydration stress was assessed by measuring percentage water loss every hour over 10 h in the total above ground biomass of the various two-week-old genotypes. An initial weight was recorded at 0 h and then every 2 h until 10 h. The percentage water loss was measured and calculated (Equation (2)). The samples were kept under dark conditions at 23 ◦C and ~33% RH. Prior to dehydration, the severed end of the shoot was sealed using Vaseline®. Following dehydration, plants were rehydrated in 2 mL sepia toned bottles with 100 μL of dH2O.

Viability following dehydration was assessed using electrical conductivity (Twin Compact Meter, Horiba, Japan) by first adding 1000 μL of dH2O to the bottles ~24 h following rehydration and then placing the bottles on the shaker for ~19 h at 23 ◦C. A second total electrical conductivity measurement was then taken after placement in a 100 ◦C dry bath for 10 min and then vortexed. Percentage electrolyte leakage (μS/cm) was measured and calculated (Equation (4)) [47].

$$Percent\ Efficiency\text{ }L\text{ }kage = \frac{\left(\left(\frac{Pre-Boil\\_Conductivity\ Value}{Post-Boil\\_Conductivity\ Value}\right) \times 100\right)}{T0\text{ }weight} \tag{4}$$

Equation (4). Percent electrolyte leakage in *Arabidopsis thaliana* above ground biomass following dehydration, where *T*0 was the time at 0 h.

The experiment was conducted under dark conditions to minimize the amount of water lost through open stomata. Stomatal closure was confirmed using the Suzuki Universal Micro-Printing (SUMP) method [48]. SUMP discs and SUMP liquid were used to take imprints of stomata, while a digital LEICA DM4 B microscope (Wetzlar, Germany) at 40× with a LEICA DFC7000 T camera (Wetzlar, Germany) attached assessed the imprints.

#### *2.8. Calcium Localization*

Calcium was spatially localized within single epidermal cell layers obtained from *A. fistulosum* using X-ray microscopy. Plants were grown and treated with calcium chloride as outlined above. The peeled epidermal layers were laid flat on Kapton tape and were used for data collection at the Advanced Photon Source (APS) beamline (20-ID: Sector 20—Insertion Device Beamline) (Lemont, IL, USA). Characteristics of the X-ray beam: incident energy—10 keV; sample scanning area—160 μm × 160 μm; scanning step size—1 μm; and dwell time—10 milliseconds per pixel. A total of 10 vertical scan maps were collected at a depth of every 2 μm into the sample [49].

Two-dimensional images were processed using PyMCA to fit the normalized average spectra of all points on the map and to generate the calcium distribution map (Version 5.3.1) [50]. OriginPro (Northhampton, MA, USA) was then used to plot the calcium map and to increase image quality by eliminating the saturated pixels. False color maps were produced using rainbow color scheme in which the blue-to-red color gradient shows low-to-high relative calcium concentrations.

The completed images were then analyzed using the "histogram" feature on ImageJ (Version 1.53a) to gain a greater understanding into differences in RGB pixels between control and calcium-treated epidermal cell layers.
