Figure S1. Induction of autophagy by thermopriming is disrupted in the atg5 mutant. (a). Accumulation of autophagosomes and autophagic bodies during the thermorecovery phase in Col‐0 and atg5 mutant. Col‐0 and atg5 (SAIL_129B07) mutants carrying the GFP‐ATG8a construct were exposed to the priming heat stimulus. After priming, seedlings were returned to normal growth condition at 22°C and subsequently visualized by fluorescence confocal microscopy for GFP signal at days 3 and 4 after priming (during the thermorecovery phase). Plants were treated with ConA (1 μM) for 4 h prior to confocal imaging. Microscopic images show representative areas of the leaf epidermis of Col‐0/ GFP‐ATG8a (left panel) and atg5/GFP‐ATG8a (right panel) plants at day 3 of the recovery phase; scale bars, 5 μm. The priming experiment for imaging was carried out two times showing similar autophagic body accumulation in the primed Col‐0 seedling leaves and no autophagic body in the atg5 seedling leaves. Similar observations were made at day 4. (b). Immunoblot analysis showing autophagy flux in unprimed and primed atg5/GFP‐ATG8a seedlings at different time points of the recovery phase. The GFP‐ATG8a fusion and free GFP form are indicated. 35S:GFP seedlings were used as a positive control to detect free GFP. Immunodetection was performed using anti‐GFP antibody (upper panels). RbcL, Ribulose‐1,5‐bis‐phosphate carboxylase/oxygenase large subunit, Ponceau‐stained (loading control; lower panels). Figure S2. Phenotype of 5‐day‐old atg mutants and wild‐type (Col‐0) seedlings under normal growth condition (22°C). Figure S3. Acquired thermotolerance of Col‐0 and atg mutant seedlings. Growth condition and HS regimes are schematically shown; 5‐day‐old seedlings were subjected to HS priming stimulus (thermopriming). Following a 90‐min recovery (growth at 22°C) seedlings were subjected to severe HS (44°C for 100 min). After the HS, seedlings were transferred to normal growth condition (22°C) and photographed after 8 days. Figure S4. Immunoblot analysis of HSP17.6, HSP101 and HSP90 proteins in atg5 and atg5/sid2 mutants during the thermorecovery phase. The seedlings were harvested for immunoblotting at days 2, 3 and 4 into the thermorecovery phase. Figure S5. Immunoblot analysis of HSP70, psbD and Rubisco proteins in atg5–1 mutant and Col‐0 seedlings during the thermorecovery phase. The seedlings were harvested for immunoblotting at days 2, 3 and 4 after thermopriming, as shown schematically. Immunodetection was performed using anti‐HSP70, anti‐psbD, anti‐Rubisco (large subunit) antibodies (top panels). Coomassie brilliant blue‐stained was shown as loading control (bottom panel). Figure S6. Co‐localization of the thermomemory‐related HSPs with ATG8A labelled autophagosomes. Transient co‐expression of GFP‐ATG8A with HSP90.2‐RFP, HSP101‐RFP, HSP17.6‐RFP, and RFP empty vector (RFP:EV) as control in N. benthamiana leaves. Confocal micrographs show single optical sections of GFP signal in green, RFP signal in red and the overlay indicating co‐localization in yellow. The intensity plots represent relative GFP and RFP fluorescence signals along the dotted line connecting points a‐b, c‐d, e‐f, and gh. Note, the overlapping of ATG8A fluorescence intensity peak with the fluorescence intensity peak of HSPs‐RFP. Images are representative of two biological replicates each consists of at least 10 images. Figure S7. Uncropped gel blot presented in Figure 3d. M, molecular weight marker. Numbers indicate protein molecular weights in kilo Dalton. Figure S8. Uncropped gel blots presented in Figure 4c and 4f. . Labelling above each image indicates the corresponding Figure and experimental condition. M, molecular weight marker. Numbers left of images indicate protein molecular weights in kilo Dalton. Table S1. Expression of ATG genes during the memory phase compared to unprimed controls. Data indicate fold change compared to controls. The qRT‐PCR experiments were performed with three independent biological replications. Table S2. qRT‐PCR primer sequences. Table S3. Primer sequences for cloning