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 log H 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 i stance ( 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 EC 50 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 EC 50 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. Flow chart illustrating the experimental procedure for quantifying the ability of PRIs at inducing HT resistance in plants using the ChlF rise kinetics OJIP • AMHA elicited plant defense mechanisms against HT by mitigating the damage to the OEC center of PSII. • A novel JIP-test parameter H s is hypersensitive to HT stress. • C i was presented as an indicator for quantifying the level of AMHA-induced resistance against HT. • EC 50 is an index to measure the ability of AMHA-inducing HT resistance. • This approach is reliable for predicting the activity of PRIs at inducing HT resistance.