The plastid metalloprotease FtsH6 and small heat shock protein HSP21 jointly regulate thermomemory in Arabidopsis

Acquired tolerance to heat stress is an increased resistance to elevated temperature following a prior exposure to heat. The maintenance of acquired thermotolerance in the absence of intervening stress is called ‘thermomemory' but the mechanistic basis for this memory is not well defined. Here we show that Arabidopsis HSP21, a plastidial small heat shock protein that rapidly accumulates after heat stress and remains abundant during the thermomemory phase, is a crucial component of thermomemory. Sustained memory requires that HSP21 levels remain high. Through pharmacological interrogation and transcriptome profiling, we show that the plastid-localized metalloprotease FtsH6 regulates HSP21 abundance. Lack of a functional FtsH6 protein promotes HSP21 accumulation during the later stages of thermomemory and increases thermomemory capacity. Our results thus reveal the presence of a plastidial FtsH6–HSP21 control module for thermomemory in plants.

and Col-0 seedlings were subjected to 44°C for 45 min. The seedlings were then transferred to normal growth condition and photographed seven days later. (b) Seedling survival was scored seven days after the recovery period; data are represented as percentage of the initial viable seedlings. Means ± SD are given (n = 7 plates with ~ 50 seedlings each).

Supplementary Figure 4. Protein level and sequence comparison of HSP21 in Arabidopsis accessions N13, Col-0 and Tsu-0. (a)
Immunoblot analyses of HSP21 during the memory phase (one and three days after priming) shown on the right of each section. Note the much higher abundance of HSP21 protein level in N13 compared to Col-0 and Tsu-0 at day 4 into the memory phase. kDa, kilo Dalton. Bands shown for Col-0 seedlings have been rearranged for presentation purpose. The original blot image is shown in Supplementary Fig. 10. (b) Comparison of the HSP21 protein sequences (deduced from cDNA sequences) in accessions N13, Col-0 and Tsu-0. The threonine 77 in Col-0 is changed to alanine in N13 and Tsu-0 due to an ACC to GCC transition. (c) HSP21 expression in N13, Col-0 and Tsu-0 seedlings during the memory phase (24 h and 48 h) compared to unprimed controls. FC, fold change. Error bars indicate means ± SD of three independent biological replicates each containing a pool of ~100 seedlings.

Met A S S S S A L S F P L S N I P T C S K K S Q Q F Q K P A S L S K S S H T H K P S L K T Q T L H H K L T K R N L L S L T T A L G F T S A L G T V L A H P A K A E P E A P I E A T S N R Met S Y S R F L Q H L K E N E V K K V D L I E N G T V A I V E I S N P V Stop b) Alignment of FtsH6 amino acid sequences from Arabidopsis accessions.
The following Arabidopsis accessions were included in our preliminary screen for differences in thermomemory: Col-0, N13, Cvi-0, C24, Ct, Can-0, Lip-0, Mdn-1, Mh-0, Mt-0, Aitba-1, Abd-0, Bl-1, Bur-0, Dog-4, Tsu-0, St-0, Kas-1, Yo-0, Xan-1, Kondara, Ler-0, Ko-2, Ri-0, Fei-0, Leo-1, Yeg-1, Ste-0, ICE1, Kin-0, Le-0, Oy-0, N14, Sah-0, Berk, Hiro, Ita, HEK2, Stepn, Shign, and Borsk2.
Amino acids divergent from Col-0 are highlighted in red. Conservative substitutions are indicated as ´:´ in the Col-0 sequence, while non-conservative exchanges are indicated by a free space. Asterisks (*) denote amino acids identical to Col-0.

Supplementary Figure 7. The effect of cycloheximide on the accumulation of FtsH6 protein in
Arabidopsis accession Col-0. (a) Immunoblot analyses of FtsH6 protein in Col-0 at days 2, 3 and 4 of the memory phase upon cycloheximide (CHX) and mock (0.1% DMSO) treatment. CHX and mock treatment was applied to seedlings at 6 h into the memory phase, as shown schematically. The seedlings were harvested for immunoblotting at days 2, 3 or 4. kDa, kilo Dalton. (b) Signals of the immunoblot analysis were quantified using ImageJ and normalized to the amount of RbcL in the same samples. Mean ± SD are given (n = 3; independent biological replicates each representing a pool of ~120 seedlings grown on 6 plates; gel blots used for the quantification are shown in Supplementary Fig. 10). Asterisks indicate significant difference in the level of FtsH6 between CHX-and mock-treated samples at each indicated time point (p < 0.05; Student´s t-test).

Supplementary Figure 9.
Supplementary Figure 9. Immunodetection of HSP101 protein in Col-0 and ftsh6. Immunoblot analysis of HSP101 protein was performed in Col-0 and ftsh6 seedlings at days 2, 3 and 4 of the memory phase, using anti-HSP101 antibody (Abcam). Note the decline of HSP101 protein level at day 4 of the memory phase with no difference between Col-0 and ftsh6. RbcL, ribulose-1,5-bis-phosphate carboxylase/ oxygenase large subunit (loading control). kDa, kilo Dalton.