Fast chlorophyll fluorescence rise kinetics as a high-throughput diagnostic tool for evaluating the capacity of 2-amino-3-methylhexanoic acid at inducing plant resistance against high temperature

Plant resistant induction is considered as a promising strategy for protecting crops against extreme high temperature (HT). However, a high-throughput method to accurately estimate the capacity of plant resistance inducers (PRIs) for HT resistance has not been developed. Here, we present a simple approach using fast chlorophyll fluorescence kinetics in Arabidopsis leaf discs to assess PRI efficacy in inducing HT resistance. Both 2-amino-3-methylhexanoic acid (AMHA) and salicylic acid (SA) significantly alleviated the temperature-dependent increase in the K-peak of the OJIP curve and variations in amplitude of heat-responsive JIP-test parameters within the elevated-temperature range of 25–42℃. The PI_ABS (performance index on absorption basis) and W_K (relative variable fluorescence at the K-step to the amplitude F_J - F_O) as two classical heat-responsive characteristic parameters were used to produce a novel hypersensitive parameter HT sensitivity indicator, PI_ABS/W_K (named H_s). Based on the correlation of logH_s with elevated temperatures, a model for quantifying the capacity of HT-resistance induction (called C_i) by AMHA or SA was established. A three-grade classification according to the C_i value was proposed as low (0< C_i ≤ 1℃), moderate (1℃ < C_i ≤ 2℃), and high res_istance (C_i > 2℃). AMHA at 1 µM and SA at 100 µM had C_i values of 2.49℃ and 4.09℃ in Arabidopsis plants, respectively, associated with their high level of HT resistance induction. Additionally, the EC₅₀ derived from the relative stimulation ratio (K_c) was also introduced as a quantitative index for measuring the ability of AMHA and SA to induce HT resistance. The EC₅₀ value of AMHA is about 0.1 µM in Arabidopsis and 0.35 µM in tomato, being much lower than that of SA (approximately 63 µM in Arabidopsis). Thus, AMHA is a more potent plant inducer than SA. The model was validated through additional experimental evidence, demonstrating its reliability and applicability. This study provides an expeditious high-throughput method for screening promising PRI candidates.