*2.6. Experimental Treatments and Sample Collection*

The roots, stems, and leaves of R9 and B6 pepper seedlings (at the 4-to-6-leaf stage) grown under normal conditions were sampled in order to analyze expression of *CaHSP18.1a* in different tissues. For the thermotolerance treatment, R9 and B6 pepper seedlings (again, at the 4-to-6-leaf stage) were grown at 42 ◦C for 24 h, and root, stem, and leaf samples were collected from stress-treated seedlings at 0, 0.5, 1, 3, and 6 h post-treatment. For the drought stress treatment, the roots of R9 seedlings were soaked in 300 mM mannitol, and root, stem, and leaf samples were collected from stress-treated seedlings at 0, 3, 6, 12, and 24 h after treatment.

To analyze the function of *CaHSP18.1a* in response to pepper abiotic stress, silenced pepper seedlings and TRV2:00 pepper seedlings were grown at 42 ◦C for 24 h. For the drought and salt stresses, seedlings were treated with 300 mM mannitol and 300 mM NaCl for 24 h. Samples were collected and malondialdehyde (MDA) content, total chlorophyll content, and relative electrolyte leakage (REL) were determined. To identify the tolerance of *CaHSP18.1a*-overexpression in *Arabidopsis thaliana* in response to heat, salt, and drought stress, T3-generation *Arabidopsis thaliana* and wild-type lines were treated as described.

For heat stress, 2-week-old OE3 seedlings were treated at 42 ◦C for 24 h. For drought stress, water was withheld from 3-week-old transgenic *Arabidopsis* seedlings for 10 d. Samples were collected to measure the total chlorophyll contents, MDA content, REL, the activity levels of CAT, SOD, and ascorbic acid peroxidase (APX). For salt stress, the seeds of WT and transgenic lines were sown on MS medium with 0, 100, and 150 mM NaCl, and the roots lengths were measured after 10 days of treatment. The germination rate was determined after 6 d. The 3-week-old WT and transgenic plants were irrigated with 200 mM NaCl solution for 7 days, once every 2 days.
