*4.2. Wax as Heat Resistance Trait*

While the roles of epicuticular wax as a drought tolerance trait have been extensively reported in cereals and brassica crops [19,22,23,50], a heat avoidance role for wax has rarely been addressed. We found significant variation, ranging from 23 to 53 μg cm−2, in lamina and petiole bulk wax concentrations under heat and control conditions. Wax composition and concentration shows variation within and across crop species [19,22,23]. Our results showed that compared to the control, heat-stress resulted in a 28% increase in total leaf wax concentration. Moreover, during reproduction, from early flowering to full seed stage, wax concentration increased by >45% in heat and control environments (Figure 2). Part of this wax increase can be due to a reduction in leaf expansion during the season as crops experience diminished water supply, and part of this increase is likely due to increased induction of leaf wax biosynthesis. Overall, our results indicate that genetic factors (cultivar), plant age and heat stress jointly contributed to effects on leaf wax biosynthesis. In addition to heat stress, various stresses such as drought, cold, salinity, and mechanical damage have each contributed to increased wax load in crops [19,23].

For heat avoidance, epicuticular wax has two major roles. First, guarding leaves and stems from radiation and heat loads by reflecting ultraviolet, visible and infrared wavelengths. In a pilot study in which extra wax was applied to pea leaf surfaces under field conditions, we recorded radiation reflectance in the visible and near-infrared region and found reflectance here was positively associated with wax concentration [51]. Second, by minimizing water loss through reduced stomatal and residual (i.e., non-stomatal) transpiration, several groups associated epicuticular wax with improved drought tolerance [22,52,53]. Drought and heat stress usually occur together, and drought stress aggravates plant heat stress. Heat stress can be moderated if the plant is able to maintain and conserve sufficient water in leaves and tissues for transpirational cooling while minimizing non-transpirational losses. Our results showed that greater wax concentration was generally associated with a cooler canopy temperature, and a higher heat tolerance index (Figure 5A,E).

We discovered that upright canopies have an advantage in stress, an important finding in pea where leaf type determines the fate of upright crops to stay upright or lodge and suffer high temperatures early in vegetative growth. Upright canopies have also been linked to lower canopy temperatures versus lodged canopies in wheat [54]. Our pea cultivars with upright growth habits and semi-leafless leaf type, both stress hardy traits, were also associated with higher wax concentration under heat stress (Figure 3F). Wax accumulation was positively associated with WBI in both control and heat stress conditions; WBI is a proxy for leaf water content, indicating that leaf surface wax minimized water loss (Table 3). Thus, leaf wax indirectly functioned as a heat tolerance trait because sufficient water supply was able to moderate heat stress by 2 ◦C [51]. Similarly, Camarillo-Castillo et al. [17] reported the importance of leaf epicuticular wax in enhancing light reflecting both in the visible and near infrared regions, which likely contribute to the dissipation of heat and excess energy [52]. Generally, glaucousness or waxy leaves were associated with high water potential that contributed to cooler canopy [19]. We concluded that greater lamina and petiole wax concentrations minimized heat stress by guarding pea from excess radiation and heat, and they also helped maintain leaf water content by lowering residual transpiration.
