*3.1. Treatment Effects on Pigment, Wax and Vegetation Indices*

The main treatment effects of ESL, canopy type and cultivar significantly affected all pigment and wax traits. The one exception was petiole anthocyanin which was not significantly affected by cultivar. Vegetation indices including PRI, GNDVI, WBI, and NPCI were significantly affected by all main effect treatment factors. The interaction of ESL by type impacted all traits except petiole anthocyanin and the majority of the vegetation indices. Only PRI, WBI and NPCI were affected by the interaction of ESL by type. The ESL by cultivar interaction affected lamina chlorophyll a, lamina and petiole chlorophyll b, lamina carotenoid, lamina and petiole wax concentration, GNDVI, WBI and NPCI, but not other traits (Table 2).

For ESL, compared to the control, stress decreased mean lamina chlorophyll a and chlorophyll b concentrations by 22.8, and 34.9%, respectively. In contrast, stress increased the corresponding chlorophyll a/b ratio, anthocyanin, and bulk wax concentrations by 23.9, 24.5, and 28.4%, respectively (Table 2). The increased chlorophyll a/b ratio under stress resulted from a greater reduction in chlorophyll b concentration than in chlorophyll a (Table 2; Figure 1A,B).



\*, \*\*, and \*\*\* indicates significant at 0.05, 0.01, and 0.001 probability levels, respectively. NS, not significantly different at 0.05 probability level. For each variable within each raw, means labeled by same letter are not significantly different at 0.05 probability level. For each stress level, N = 192 from 24 cultivars, two environments and four replications. GNDVI, green normalized difference vegetation index; NDVI, normalized difference vegetation index; NPCI, normalized pigment chlorophyll ratio index.; PRI, photochemical reflectance index; WBI, water band index.

As reproduction proceeded from early flowering to pod filling, the leaf lamina chlorophyll a, chlorophyll b, carotenoid, and wax concentrations increased by 20, 18, 5, and 39%, respectively, and the corresponding anthocyanin concentration decreased by 20%. Similarly, petiole chlorophyll a, chlorophyll b, carotenoid, and wax concentrations increased by 28, 10, 6, and 53%, respectively (Figure 2). Generally, leaf lamina had >32% greater chlorophyll and carotenoid concentrations than those found in the petiole. On the other hand, higher anthocyanin and wax concentrations were found in the petioles compared with the leaf lamina (Table 2). Under both control and stress conditions, cultivars with dark green canopies, including Superscout, Rally, MPG87, Mini, and CDC Vienna, were associated with greater lamina chlorophyll and carotenoid concentrations. In contrast, the bright green cultivars including TMP15116, Naparnyk, CDC Sage, and Torsdag had less (<32 μg cm<sup>−</sup>2) lamina chlorophyll and carotenoid concentrations (Figure 1A,B,D).

**Figure 1.** Mean lamina pigment and wax concentrations of 24 pea cultivars grown in control (blue) and stress (red) conditions, with (**A**) chlorophyll a, (**B**) chlorophyll b, (**C**) chlorophyll a/b ratio, (**D**) carotenoid, (**E**) anthocyanin and (**F**) wax concentrations (μg cm−2). Each bar represents the mean values, and error bars are the standard error of the mean. For each bar, N = 16 (two environments × four replications × two growth stage samples) for each of control or stress condition. The LSD values for each of the stress levels is shown in the figure. The control conditions are 2014 late seeding date at Rosthern and 2016 normal seeding date at Saskatoon; and the stress conditions are 2015 late seeding date at Rosthern and Saskatoon, Canada.

**Figure 2.** Chlorophyll a, b, a/b, carotenoid, anthocyanin and wax concentrations (μg cm−<sup>2</sup> of tissue) in leaf lamina and petiole at early flowering and flower termination stages of pea grown in six field environments in Saskatchewan, Canada (2014 to 2016). Each bar is the mean value averaged over 24 cultivars, six environments and four replications per environment, i.e., N = 576 for each variable.

Generally, for chlorophyll and carotenoid concentrations under both control and heat stress, the normal leafed vining cultivars with dark green canopies had the greatest chlorophyll a, chlorophyll b, and carotenoid concentrations; but the least chlorophyll and carotenoid concentrations were found in normal leafed vining cultivars with bright green color (Figure 3A,B,D). Under control and heat stress, bright green cultivars had a higher chlorophyll a/b ratio than dark green cultivars regardless of the growth habit and leaf type (Figure 3C). Under control conditions, normal leafed vining cultivars with bright green canopies had lower anthocyanin concentration than all other types; but under heat stress this type had a relatively greater anthocyanin concentration than other types (Figure 3E). For leaf wax, under control conditions, semi-leafless cultivars had the same wax concentration regardless of their canopy habit and color. The lowest wax concentrations were found in normal leafed vining cultivars with bright green canopies (Figure 3F). Under heat stress, upright semi-leafless cultivars with dark green canopies had the greatest wax concentrations, and normal leafed vining cultivars had the lowest wax concentrations (Figure 3F).

**Figure 3.** Box plot showing the interaction effects of heat stress and canopy type on: chlorophyll a (**A**), chlorophyll b (**B**), chlorophyll a/b ratio (**C**), carotenoid (**D**), anthocyanin (**E**), wax (**F**) concentrations (μg cm−2); and vegetation indices: PRI (**G**), NPCI (**H**), and WBI (**I**) measured on pea leaf stipules under control and stress environments at Rosthern and Saskatoon, Canada, 2014–16. The size of box represents 50% of the middle data, the line in the middle of the box is the median, and the whiskers represent the range of the data. Boxes labeled with same letters within trait are not significantly different at *p* < 0.05. N = 24 for type 1, 40 for type 2, 24 for type 3, 32 for type 4, 48 for type 5, and 16 for type 6. The control conditions are 2014 late seeding date at Rosthern and 2016 normal seeding date at Saskatoon; and the stress conditions are 2015 late seeding date at Rosthern and Saskatoon, Canada.; Canopy type legend: n, sl, u, v, bg, and dg represents normal leaf, semi-leafless; upright habit, vining habit, bright-green color, and dark-green color, respectively; and vegetation indices: PRI, photochemical reflectance index; NPCI, normalized pigment chlorophyll ratio index; WBI, water band index.

#### *3.2. Response of Vegetation Indices*

The control environment had greater NDVI and GNDVI values than the stress and intermediate environments. Although the values of most vegetation indices were in the 'normal' range for healthy vegetation, heat stress and control treatments differed (Table 2). For PRI, under both control and heat stress, dark green cultivars had greater PRI than bright green cultivars regardless of leaf type and canopy habit (Figure 3G). Under both control and stress conditions, normal leafed vining cultivars with bright green canopies had greater NCPI, suggesting more stress than all other canopy combinations (Figure 3H). For WBI, for the control, semi-leafless upright cultivars with dark green canopies had a greater WBI than vining cultivars regardless of leaf type and canopy color. Furthermore, under heat stress, semi-leafless upright cultivars with dark green canopies had the greatest WBI, inferring a high leaf water content compared to all other canopy types. For WBI and heat stress, the cultivar ranking matched with the cultivar ranking for wax concentration. Water band index is associated with leaf water content, so the greater WBI value in upright and semi-leafless cultivars implied that these cultivars maintained greater leaf water content under heat stress.
