Physiology, anatomy, and cell membrane thermostability selection of leafy radish (Raphanus sativus var. oleiformis Pers.) with different tolerance under heat stress

• Line 9911-9 was able to tolerant high temperature through diminishing both stomatal and non-stomatal limitations.
• The stomatal limitation was reduced by increasing occupancy of total stomata apertures on leaf mainly through enlarging stomatal aperture as well as developing more xylem vessels.
• The non-stomatal limitation was decreased by scavenging ROS through inducing the activities of SOD and CAT within the first 24 h.
• Cell membrane thermostability of cotyledon was a reliable and selective index for heat-tolerant breeding in leafy radish.

Leafy radish (Raphanus sativus L. var. oleiformis Pers.), a temperate crop, is usually heat-sensitive. Line 9911-9 is a heat-tolerant line bred for summer vegetables in Taiwan. Understanding the heat tolerance mechanism of Line 9911-9 may facilitate future breeding programs. We conducted a comparative study on growing plants of heat-tolerant Line 9911-9, moderately tolerant Line 9611, and heat-sensitive ‘Taichung No. 1’ at 40/35 °C and 25/20 °C to elucidate the heat tolerance mechanism of leafy radish. After 28-day of acclimation, the maintenance of net photosynthetic rate (Pn) and chlorophyll fluorescence (Fv/Fm) accompanying a double increase of stomatal conductance (gs) and transpiration rate (E) in Line 9911-9 revealed that the heat-tolerant line had adjusted physiologically to be more efficient in heat dispersing. Morphologically, Line 9911-9 developed more xylem vessels, an enlarged stomatal aperture by 5-fold, and an increased stomatal opening to 93%, which contributed 6-fold increase in the occupancy of total stomata aperture on leaf without altering stomata density under high temperature stress. Furthermore, the occupancy was correlated with gs and E with a coefficient of 0.87 and 0.98, respectively, confirming that stomatal factors play a crucial role in heat tolerance in leafy radish. Regarding the heat shock response, Line 9911-9 could have sustained over 9 days at 40/35 °C, whereas Line 9611 was maintained over 3 days, with ‘Taichung No. 1’ for only 1 day. Line 9911-9 was able to maintain its cellular membrane stability by inducing the activities of superoxide dismutase (SOD) and catalase (CAT) to scavenge reactive oxygen species (ROS) within the first 24 h and detoxify ROS unbalance under high temperature stress. These results indicated that Line 9911-9 was able to lower the non-stomatal limitation effect on Pn. A high correlation (R2 = 0.89) between cotyledon relative injury at 50 °C for 30 min in 21 leafy radish accessions and their yield reduction ratio under high temperature stress suggests that the CMT of cotyledon may be used as a reliable index for selecting heat-tolerant leafy radish.