*3.3. Heat Tolerance Index*

Pea cultivars significantly varied in HTI, calculated from the relative seed yield of cultivars under control and heat stress, with values ranging from 0.35 to 1.25 (Figure 4A). Generally, upright cultivars with dark green canopies had a greater HTI, with the smallest HTI in normal leafed vining cultivars with dark green canopies (Figure 4B). Cultivars with greater (>1) HTI included CDC Meadow, TMP 15 2013, CDC Golden, Naparynk and TMP 15181 (Figure 4A). Heat tolerance index was negatively correlated with canopy temperature (Figure 5C), positively correlated with lamina wax concentration and WBI (Figure 5E,F).

**Figure 4.** Heat tolerance index by cultivars (**A**), and canopy type (**B**) of 24 pea cultivars grown in four field environments (control and stress) in western Canada. N = 8 for each cultivar in panel A; and N = 12 for type 1, 20 for type 2, 12 for type 3, 16 for type 4, 24 for type 5, and 8 for canopy type 6 in panel B. Error bars are standard errors of the mean. In panel B, canopy types labeled with same letters are not significantly different at *p* < 0.05. Legend for canopy type: n, normal leaf; sl, semi-leafless; u, upright habit; v, vining habit; bg, bright-green color; dg, dark-green color.

**Figure 5.** Matrix plot showing correlation between canopy temperature (CT) and lamina wax (**A**), CT and WBI (**B**), CT and HTI (**C**), Lamina wax and WBI (**D**), Lamina wax and HTI (**E**), and WBI and HTI (**F**) of 24 pea cultivars grown in four field environments (control and stress) at Rosthern and Saskatoon, western Canada, in 2014−16. Each symbol is a cultivar averaged over 16 observations (four environments by four replications). \*, \*\*, and \*\*\* indicates significant at 0.05, 0.01, and 0.001 probability levels, respectively. HTI, heat tolerance index; WBI, water band index.

*3.4. Phenotypic Correlation among Pigments, Wax, Vegetation Indices, Canopy Temperature, and Heat Tolerance Index*

Leaf spectral reflectance is mainly affected by pigment and wax compositions and concentrations. Our result showed that most vegetation indices had significant correlation with pigment and wax concentrations, canopy temperature and HTI (Table 3 and Figure 5). The PRI and GNDVI had similar trends, correlating positively with chlorophyll, carotenoid, and anthocyanin concentrations, and negatively with chlorophyll a/b ratio. In contrast, NPCI had the opposite pattern to PRI and GNDVI. Correlations among lamina wax concentration, WBI, canopy temperature, and HTI were of specific interest and illustrated in Figure 5. Water band index correlated positively with lamina wax concentration, and negatively with canopy temperature. Finally, Heat tolerance index was negatively correlated with canopy temperature (Figure 5C), and positively correlated with lamina wax concentration and WBI (Figure 5E,F).

**Table 3.** Pearson correlation test showing associations among canopy temperature, total chlorophyll, chlorophyll a/b ratio, carotenoid, anthocyanin, wax, photochemical reflectance index (PRI), green normalized vegetation index (GNDVI), water band index (WBI), and normalized pigment chlorophyll ratio index (NPCI) of 24 pea cultivars grown under field conditions under control (upper right diagonal) and stress (lower left diagonal) environments averaged over two environmental levels and four replications. 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.


\*, \*\*, and \*\*\* indicates significant level at the 0.05, 0.01, and 0.001 probability levels, respectively.

#### **4. Discussion**

#### *4.1. Leaf Pigment Concentrations as Heat Resistance Traits*

We found that heat stress and the significant cultivar by environment interaction lowered chlorophyll a and chlorophyll b concentrations in leaf lamina and petiole, with the reduction more pronounced in heat sensitive cultivars. Photosyntheric pigments are prone to heat and other environmental stresses. Recently, Giordano et al. [38] reviewed the reduction of photosynthetic pigments in response to heat stress, and such reduction led to the reduction of photosynthetic activities related to photosystem II. Cultivars that were able to maintain chlorophyll concentration under heat stress had greater HTI values, and therefore greater heat resistance, implying that chlorophyll concentration was likely linked to plant heat response. As chlorophyll is an integral component in light absorption and transfer; chlorophyll loss or degradation leads to reduced photosynthesis and coupled oxidative damage which consequently reduces growth and yield [7,11]. Under heat and excess radiation stress, chlorophyll loss arises either due to limited biosynthesis caused by enzyme malfunctioning [39], or due to rapid degradation caused by heat and radiation damage. Chlorophyll loss also occurs naturally in senescing plants, and stress induces tissue senescence [40].

Interestingly, chlorophyll a/b ratio increased under heat stress in both leaf lamina and petiole in our research, likely due to rapid chlorophyll b degradation compared to that of chlorophyll a, suggesting a differential susceptibility in light-harvesting chlorophyll a/b-binding protein complex [41]. Although chlorophyll a/b ratio changes were associated with plant heat response, the literature is inconsistent in how chlorophyll a/b ratio changes with stress in crops. Feng et al. [42] found decreased chlorophyll a/b ratio was associated with heat tolerance in wheat, but Cui et al. [43] reported the opposite on a cool season perennial grass tall fescue (*Festuca arundinacea*). While the optimal range of chlorophyll a/b ratio needs further study, we noted that pea cultivars with either high (>4.0), or low (<2.5) chlorophyll a/b ratio had low heat tolerance indices (Figures 1C and 5A), suggesting damage at the antenna complex or reaction center, respectively, as reported by Feng et al. [42]. We found, generally, that upright cultivars with dark green canopies had low chlorophyll a/b ratio, and greater HTI than vining cultivars with bright green canopies (Figures 2C and 4B), inferring that upright canopies were less stressed.

Leaf lamina carotenoid concentration had a similar pattern as chlorophyll concentration and decreased due to the heat stress (Figures 1C and 3D). In published research, carotenoid biosynthesis and accumulation were influenced by multiple factors including light and temperature stresses [44]. Although heat stress resulted in a decreased concentration of carotenoid, there was a significant difference among the pea cultivars. Cultivars better able to maintain relatively stable carotenoid concentration under heat stress had greater HTI (Figures 1D and 4A), implying that greater or maintained carotenoid concentration reduced heat damage on pea seed yield. Carotenoids are antenna pigments and have direct influence on photosynthesis, their two major roles being light harvesting during photosynthesis, and minimizing photo-oxidative damage of chlorophyll molecules by dissipating excess energy in the form of heat [10,45] by the Mehler-ascorbate-peroxidase cycle at Photosystem I [46].

Anthocyanin concentration increased with heat-stress (Table 2; Figure 1E), a pattern opposite to chlorophyll and carotenoid concentrations. Anthocyanin production is enhanced in response to most environmental stresses including cold, heat, drought, and light [47]. However, stressful environments also trigger formation of reactive oxygen species and free radicals [48]. To protect plants from the damaging effects of reactive oxygen species, high levels of anti-oxidants are required, and anthocyanins fulfill such a protective role [47]. Anthocyanins protect chloroplasts by reducing incident light, and they have an anti-oxidant role through scavenging reactive oxygen species [49]. Unlike chlorophyll and carotenoid, anthocyanin concentration was greater in petiole than lamina, and anthocyanin concentration declined in leaf lamina during reproduction, indicating anthocyanin biosynthesis was growth-stage dependent and younger leaves produced more. Anthocyanins also function like sunscreen for leaves, where anthocyanins form a layer and damaging radiation does not penetrate internal sensitive tissue. In addition to heat and UV protection, increased anthocyanin concentration under heat stress is associated with enhanced water uptake and decreased transpiration [7].
