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.
In their natural environment, plants are exposed to recurrent, sometimes irregular, environmental changes. Appropriate responses to environmental cues are required as plants cannot change their location on imposition of stress. Plants have an inherent ability to survive certain levels of stress (called basal tolerance), a characteristic that varies between species and genotypes. In addition, plants have the ability to acquire tolerance to otherwise lethal stresses and experimental evidence indicates the existence of a molecular ‘memory’ that enables them to withstand stress better if previously confronted with the same or a similar type of stress1,2. Pre-exposure to stress, called priming, induces the configuration of a new cellular state (through molecular and biochemical recasting) that is different from the (pre-stress) naive situation and allows the plant to respond in a superior manner to subsequent stimuli (called triggering). The intervening time, during which plants experience a non-stress situation, is called the ‘memory’ phase and can range from several hours to days, or even generations. However, the molecular machinery that underlies stress memory in plants is so far largely unknown.
An important environmental factor that often impairs plant growth, survival and productivity is heat. Heat stress affects the integrity of the proteome by causing misfolding and/or denaturation of proteins, thereby negatively affecting cell viability. Misfolded proteins are toxic to the cell and must be refolded, degraded or delivered to distinct quality control compartments that sequester potentially harmful misfolded proteins3.
Heat shock proteins (HSPs) are molecular chaperones that perform major roles in protecting the proteome against environmental stresses4. They are grouped into different families, based on their molecular masses: HSP70, HSP90, HSP100/ClpB (Hsp101) and small HSP (sHSP) families. SHSPs with a monomer molecular mass ranging from 12 to 42 kDa belong to an evolutionary conserved family harbouring a common α-crystallin domain located in the C-terminal part5. They occur in all eukaryotic organisms but are particularly abundant in plants, such as Arabidopsis thaliana and rice (Oryza sativa) which have 19 and 23 sHSPs, respectively, while there are 10 sHSPs in humans, 4 in Drosophila melanogaster, and 1 or 2 in bacteria6,7. The activity of sHSPs is independent of ATP and they are unable to refold non-native proteins, however, sHSPs provide immediate protection against unfavourable conditions by selectively binding to unfolded proteins and facilitating their subsequent refolding by other, ATP-dependent chaperones6,7,8,9,10. Hence, sHSPs play a critical role in the protein quality control system. Heat stress-induced expression of HSP genes is mainly controlled by heat shock transcription factors (HSFs) although other transcription factors also play a role11,12. In plants, sHSPs are targeted to different cellular compartments, including the cytosol, chloroplasts, mitochondria, and the endoplasmic reticulum13,14. Organelle-targeted sHSPs are unique to plants, the only known exception being mitochondrion-targeted sHSP22 in Drosophila15,16. Several sHSPs were shown to have important functions in the response of plants to heat stress and in acquired heat stress tolerance6,10,17.
The nuclear-encoded chloroplast protein HSP21 (monomer size 21 kDa, also known as HSP25.3-P (ref. 18)) is a plastid-localized sHSP in Arabidopsis19. It harbours a unique region towards its N terminus with several conserved methionine residues proposed to recognize hydrophobic stretches in partially unfolded proteins, and is mainly associated with thylakoid membranes20,21. Under conditions of oxidative stress the methionines are oxidized, and this process is reversed by a plastid-localized methionine sulfoxide reductase, which restores the chaperone activity of HSP21 (ref. 22). Under optimal growth conditions, HSP21 expression is largely restricted to pollen grains but its expression rapidly and strongly increases in most organs on high-temperature stress21,23. Overexpression of HSP21 in transgenic Arabidopsis plants enhances tolerance towards heat stress when imposed together with high light, a combination known to cause oxidative stress, while thermotolerance was not significantly altered in a low-light regime19; however, HSP21 is essential for chloroplast and early seedling development in the presence of heat stress21. In tomato (Solanum lycerpersicum), HSP21 was reported to protect photosystem II (PSII) on exposure of the plant to excessive temperature24.
Expression of HSP21 is controlled by Heat shock factor A2 (HsfA2), a master transcriptional regulator of thermomemory in Arabidopsis. and the hsfa2 knockout mutant is defective in thermomemory25. HSP21 transcript is absent 24 h after heat priming in the hsfa2 mutant while it is still highly abundant in wild-type plants. Absence of HSP21 transcript is also evident in the rof1 knockout mutant whose defective thermomemory phenotype resembles that of hsfa2 (ref. 26). ROF1 (AtFKBP62) encodes an FK506-binding protein-type peptidyl prolyl cis/trans isomerase. Similar to HsfA2, ROF1 is required for extending the duration of acquired thermotolerance (thermomemory), but not for its induction26.
Besides chaperones, proteases play a regulatory role in maintaining proteome homoeostasis in most subcellular compartments. Four major families of proteases; Clp, FtsH, DegP (in chloroplasts) and Lon protease (in chloroplasts and mitochondria)27, have been characterized in Arabidopsis and act as central elements of chloroplast and mitochondrial protein quality control systems28.
A relatively well-characterized family of proteases is the FtsH (filamentation temperature sensitive) family. FtsH proteases consist of an N-terminal transmembrane domain followed by a hydrophilic region containing an AAA (ATPase associated with various cellular activities) and a protease domain belonging to the M41 peptidase family, which carries a zinc-binding motif and therefore is categorized into the family of zinc metalloproteases29,30,31. Most bacteria have a single gene encoding FtsH, while 12 FtsH genes are present in the genome of Arabidopsis thaliana. In Arabidopsis, three proteins (FtsH3, FtsH4 and FtsH10) are targeted to mitochondria, eight (FtsH1, FtsH2, FtsH5 to FtsH9 and FtsH12) enter the chloroplast, while FtsH11 is dually targeted to both mitochondria and chloroplasts27,28,29,32,33. In addition, four proteolytically inactive FtsHs (designated as FtsHi) are targeted to chloroplasts34. FtsH6 has high-sequence similarity to FtsH2 and FtsH8 (refs 32, 35). Involvement of FtsH6 in degradation of the PSII light harvesting proteins Lhcb1 and Lhcb3 during dark-induced senescence or high-light treatment has been demonstrated in vitro36, however, additional studies have so far not revealed an in vivo function of FtsH6 in high light acclimation or any other biological process32,35. Notably, FtsH6 is expressed at very low levels in plants grown at normal growth temperature, but is highly induced after heat treatment in Arabidopsis (Arabidopsis eFP Browser; http://bar.utoronto.ca/efp_arabidopsis) and other species across the plant kingdom including the dicot crops rapeseed (Brassica napus37) and tomato (Solanum lycopersicum38), and the monocot crops wheat (Triticum aestivum39) and sorghum (Sorghum bicolor40). Thus, heat inducibility of FtsH6 expression appears to be evolutionary conserved in plants suggesting an important role of this metalloprotease in the response to heat stress.
Here we show that the sole plastidic sHSP, HSP21, plays a crucial role in extended thermomemory in Arabidopsis. We provide evidence that the ability to maintain high levels of HSP21 protein after priming determines the duration of memory in genetically modified plants as well as natural accessions of Arabidopsis with contrasting thermomemory capacity. Furthermore, we show that HSP21 abundance during the memory phase is negatively regulated by heat-induced plastid-localized metalloprotease FtsH6. Our results thus demonstrate the presence of a plastidial FtsH6–HSP21 control module for thermomemory in plants.
Identification of thermomemory-associated genes
To study thermomemory, 5-day-old seedlings of Arabidopsis Col-0 plants were subjected to an established thermomemory protocol (see Methods; Fig. 1a). Seedlings of both primed and unprimed plants became pale green and turned white after 4 days of heat stress/triggering stimulus. However, 7 days after the triggering, primed but not unprimed plants started to generate new leaves and grow further (Fig. 1a). To identify genes associated with thermomemory, we performed transcript profiling using Affymetrix ATH1 microarrays comparing Col-0 seedlings 4, 8, 24 and 48 h after the priming stimulus (that is, during the memory phase) with control plants (unprimed). Considering a two-fold cut-off on fold changes (FC), thermomemory-associated genes were selected as follows: genes whose expression was induced 4 h after priming and remained high at all examined time points until 48 h into the memory phase (60 genes), and genes whose expression was downregulated at 4 h after the priming stimulus and remained low until 48 h (19 genes) (Supplementary Data 1).
Upregulated genes were classified into several gene ontology (GO) functional categories, among them ‘response to stress’ (that is, high temperature, hydrogen peroxide, high light and oxidative stress) and ‘protein folding’ were the most enriched categories (Fig. 1b). Of the 60 upregulated genes, 17 encode for HSPs (Supplementary Data 1). Among the top thermomemory upregulated genes was HSP21 (AT4G27670), which encodes a small HSP (21 kDa monomer size) localized in plastids of both roots and leaves13. As shown in Fig. 1c,d, transcript levels of HSP21 as well as its protein abundance remain high until 48 h into the thermomemory phase suggesting a role for this HSP in the maintenance of extended thermomemory in Arabidopsis.
HSP21 confers priming-induced thermotolerance
To investigate whether HSP21 plays a role in heat stress priming and memory, we first generated transgenic Arabidopsis lines with enhanced or reduced HSP21 expression (Supplementary Fig. 1a–d) and subjected 5-day-old HSP21 transgenic and Col-0 seedlings to our standard priming and triggering stimulus protocol. As shown in Supplementary Fig. 2 there were no significant differences in the development of the HSP21 transgenic plants and Col-0 before priming as well as after 3 and 4 days of recovery, respectively. HSP21-amiRNA and 35S:HSP21 transgenic plants recovered and generated new leaves after a heat stress treatment given 3 days after priming, similar to Col-0 plants (Fig. 2a). Although there was no difference in the recovery rate (Fig. 2b) among HSP21 transgenics and Col-0 seedlings, the seedling fresh weight was significantly lower in HSP21-amiRNA than Col-0 seedlings, but higher in 35S:HSP21 (Fig. 2c). When the memory phase was extended to 4 days, the proportion of highly damaged seedlings (indicated as weak and dead) was significantly increased in Col-0. Notably, HSP21-amiRNA plants exhibited a more severe phenotype with significantly less fresh weight than Col-0, while 35S:HSP21 plants showed a higher survival rate and seedling fresh weight under this condition (Fig. 2a, top panel, b, and c). Our data thus clearly indicate that HSP21 is required for Arabidopsis plants to retain the memory of a prior exposure to heat stress.
To investigate the specificity of the role of HSP21 for thermomemory, we tested whether altering HSP21 expression has an effect on basal and acquired heat stress tolerance (see Methods section for details). As shown in Fig. 2a (lower middle and bottom panels), HSP21 overexpression and knockdown plants were similar in acquired and basal heat stress tolerance to Col-0 control plants suggesting that HSP21 is specifically required for maintenance of thermomemory.
Hypocotyl elongation assays have previously been employed for testing the effect of temperature on seedling growth41,42. Therefore, we next analysed hypocotyl growth in the HSP21 transgenic plants to test for changes in the thermomemory. To this end 4-day-old etiolated seedlings were subjected to the following temperature regime: 1.5 h, 37 °C (priming stimulus); 2 days recovery at 22 °C; and 45 min, 44 °C (triggering stimulus). As control, 6-day-old etiolated seedlings were subjected to 45 min, 44 °C without pre-adaptation. Hypocotyl elongation was quantified five days after the triggering stimulus. As shown in Fig. 2d, all genotypes failed to elongate their hypocotyl after treatment at 44 °C when no pre-treatment was given. However, wild-type and HSP21 overexpression seedlings retained the ability for hypocotyl elongation when 44 °C was given 2 days after a priming stimulus. In contrast, HSP21-amiRNA seedlings showed a severe block of hypocotyl elongation under this condition (Fig. 2d,e) further supporting the model that HSP21 is crucial for extended memory maintenance.
HSP21 is more abundant in strong memory accession N13
Accessions of Arabidopsis offer an excellent tool to explore natural diversification of a biological process of interest. Thus, to identify an accession with a better thermomemory than Col-0 we performed a screen (performed twice) with 40 additional Arabidopsis accessions (see Methods section) for differences in heat stress priming and memory. We identified N13 as a strong memory accession, which survives high-temperature stress (90 min, 44 °C), given 3 or 4 days after the priming treatment, significantly better than the Col-0 control. As displayed in Fig. 3a, both Col-0 and N13 survived heat stress applied 3 days after the priming treatment; the fresh weight of seedlings was significantly lower in Col-0 compared to N13 when analysed 14 days after the heat stress triggering stimulus. When 4 days was applied as the memory time, we observed a higher number of damaged seedlings for Col-0, whereas N13 seedlings recovered significantly better from severe repeated heat stress (Fig. 3a–c) demonstrating that N13 was better able to respond to the heat priming stimulus and to memorise the past heat stress experience. This conclusion was supported by analysing hypocotyl elongation in N13 and Col-0 under the condition of heat stress. Hypocotyl elongation after the triggering heat stress was more prominent in N13 than Col-0 (Fig. 3d,e). We did not observe any significant difference between N13 and Col-0 seedlings for basal heat stress tolerance, when 7-day-old seedlings were subjected to 45 min, 44 °C (Supplementary Fig. 3).
To investigate whether variation in HSP21 level contributes to the differential thermomemory performance of N13 and Col-0, we analysed its transcript and protein abundance in the two accessions at different time points along the memory phase. HSP21 protein was undetectable in both, Col-0 and N13 in unprimed conditions, but as seen in Fig. 4a, pronounced accumulation of HSP21 to the same level was evident in both accessions already 1.5 h after the first phase of the priming stimulus (1.5 h, 37 °C). However, during the thermomemory phase, HSP21 protein level progressively decreased to about 25% of its heat stress-induced level in Col-0 by day 4, but remained at above 80% of its induced level in N13 (Fig. 4a,b). At day 4, HSP21 protein was 3.4-fold more abundant in N13 than in Col-0 (Fig. 4b). Our data thus support a model where HSP21 plays an important role in the molecular machinery of thermomemory.
We then compared HSP21 expression in Col-0 and N13 during the memory phase. HSP21 was significantly higher expressed in Col-0 than N13 at 8 h and 24 h after the priming, but this difference vanished at the later time points (2, 3 and 4 days), suggesting that the diminished HSP21 protein in Col-0 is not correlated with HSP21 transcript level (Fig. 4c).
To investigate whether the more rapid decrease of HSP21 protein level in Col-0 compared to N13 during the thermomemory phase is due to an amino acid sequence polymorphism(s), which might alter protein structure and/or stability, we compared the amino acid sequences of HSP21 from the accessions. To strengthen our analysis, we included one more accession, Tsu-0, with a similar pattern of HSP21 accumulation and thermomemory behaviour as Col-0 (Supplementary Fig. 4). Sequence analysis of complementary DNA (cDNA) from the selected accessions identified one polymorphic site as amino acid codon 77, which in Col-0 encodes for threonine (ACC) but for alanine (GCC) in both, N13 and Tsu-0 (Supplementary Fig. 4a and b). Furthermore, HSP21 transcript levels during the recovery/memory phase were similar in the three examined accessions (Supplementary Fig. 4c). Therefore, we concluded that the amino acid polymorphism in the N13 protein sequence is unlikely to be responsible for higher accumulation/stability of HSP21 and consequently strong thermomemory.
Taken together, the results suggest that HSP21 protein abundance during the thermomemory is controlled by regulatory mechanisms at the translational and/or post-translation level.
Cycloheximide blocks decline of HSP21 in Col-0 during memory
To test whether high levels of HSP21 protein at later memory time points in accession N13 require de novo protein synthesis, we used cycloheximide (CHX) to inhibit protein translation. To this end, seedlings of both Col-0 and N13 were treated with CHX at 6 h after priming (when HSP21 level was high; Fig. 4a,b), and HSP21 protein level was analysed by immunoblotting at days 3 and 4 into the memory phase. As shown in Fig. 5a,b, CHX treatment did not inhibit the induction of HSP21 in N13 revealing that de novo translation is not the primary cause for accumulation of HSP21 in N13 at days 3 and 4 of the memory phase. Interestingly, CHX treatment of Col-0 seedlings resulted in significantly higher accumulation of HSP21, more evident at day 4 of the memory phase, indicating the presence of an unknown protein in Col-0, possibly a protease, whose de novo translation is required for HSP21 degradation at a later stage of the memory phase.
FtsH6 metalloprotease affects HSP21 level during memory
In an attempt to understand how HSP21 is degraded at the later stage of the memory phase, we first examined our thermomemory Affymetrix transcriptome data for expression of major nuclear-encoded (plastid) chloroplast proteases including stromal ATP-dependent Clp proteases, lumenal DegPs and the FtsH metalloproteases27,29. Interestingly, of 39 genes for plastid proteases the expression of only 1 gene encoding an FtsH metalloprotease (AT5G15250; FtsH6) was enhanced at 4 h into the memory phase and remained high until 24 h (Fig. 5c and Supplementary Data 2). FtsH6 (filamentation temperature-sensitive H6) is a nuclear-encoded chloroplast zinc- and ATP-dependent protease associated with the thylakoid membrane. Expression of FtsH6 is strongly induced by heat in Arabidopsis and other plant species including rapeseed37, wheat39, sorghum40 and tomato38.
To test a potential involvement of metalloproteases for HSP21 degradation, we treated Col-0 seedlings with the metalloprotease inhibitor 1,10-phenanthroline and detected HSP21 protein by immunoblotting at days 3 and 4 of the memory phase. As shown in Fig. 5d,e, treatment with 1,10-phenanthroline largely blocked the late thermomemory decrease of HSP21 protein level. The sustained induction of FtsH6 expression during the thermomemory phase as well as inhibition of HSP21 degradation on treatment with 1,10-phenanthroline suggest FtsH6 as a candidate protease involved in the degradation of HSP21 during the memory phase in Col-0.
To investigate whether variation in FtsH6 is responsible for differential abundance of HSP21 protein in N13 and Col-0, and thus their contrasting thermomemory behaviour, we first compared expression of FtsH6 between Col-0 and N13 during the memory phase (until 3 days) by quantative reverse trancription–PCR (Fig. 6a). In Col-0, the expression of FtsH6 was drastically induced on priming treatment and remained high until day 2 of the memory phase. We observed a similar induction pattern for FtsH6 in N13, albeit with a more rapid decline at the later time points. However, as we show below, N13 FtsH6 transcript is non-functional.
Next we detected FtsH6 protein by immunoblot analysis in N13 and Col-0 seedlings at different time points of the memory phase and compared it with control plants (unprimed). As shown in Fig. 6b, FtsH6 was undetectable in both Col-0 and N13 under unprimed condition. In Col-0, FtsH6 protein was abundant until day 3 of the memory phase in accordance with its higher transcript levels. Intriguingly, however, no FtsH6 protein was detected in N13 during the entire memory phase, even at an earlier time point (4 h) when its transcript was still abundant. Comparing the FtsH6 coding sequences of N13 and Col-0, we identified several nucleotide polymorphisms (Supplementary Fig. 5a), including a single-nucleotide deletion (nucleotide ‘G’ at position 379 of the Col-0 coding sequence is deleted in N13) resulting in a frame-shift mutation and a premature stop codon in the N13 sequence. This observation explains the absence of FtsH6 protein in N13. Only a few amino acid substitutions (0 to 3 residues) were found in FtsH6 proteins of the 30 other accessions we tested for heat stress priming and triggering compared with Col-0 (see above; Supplementary Fig. 5b); no genome sequences were available for the remaining 9 accessions.
This finding provides further evidence for FtsH6 as a potential protease involved in the regulation of HSP21 turnover during thermomemory. Lack of functional FtsH6 in N13 could be responsible for the higher accumulation of HSP21 and therefore enhanced thermomemory in this accession.
If FtsH6 is a key protease for degradation of HSP21 during the memory phase, the decrease in HSP21 level at later stage of thermomemory phase should be retarded in an ftsh6 knockout mutant. Therefore, we compared HSP21 protein level of wild type and the ftsh6 mutant (Salk_012429; Col-0 background) at different stages of thermomemory phase. As shown in Fig. 6c,d, protein level of HSP21 was identical in both genotypes after the first step of priming treatment (1.5 h, 37 °C) and until 8 h of the thermomemory phase. However, at later stages (24 h, 2, 3 and 4 days) the level of HSP21 was significantly higher in the ftsh6 mutant than in Col-0 (about 1.8-fold higher in ftsh6 than Col-0 at days 2, 3 and 4; Fig. 6d). Our data thus show that FtsH6 is involved in the degradation of HSP21 at the later stages of the memory phase. A slightly higher accumulation of HSP21 protein was observed after 1,10-phenanthroline treatment (Fig. 5d,e) compared to the ftsh6 mutant, suggesting that other metalloproteases are also involved in controlling the level of HSP21 later in the memory phase.
We next subjected 5-day-old ftsh6 mutant and Col-0 seedlings to our priming and triggering stimulus protocol. ftsh6 mutants revealed a slightly better thermomemory behaviour than Col-0, measured by seedling survival rate and fresh weight (Supplementary Fig. 6a–c). However, ftsh6 plants had similar basal and acquired heat stress tolerance as Col-0 control plants. We also determined the role of FtsH6 for thermomemory by analysing hypocotyl elongation after priming and triggering and observed that hypocotyl elongation was much less affected by the triggering heat stress in the ftsh6 mutant than the wild type (Col-0; Supplementary Fig. 6d and e), underscoring the contribution of FtsH6 to restricting the thermomemory by controlling HSP21 abundance.
To test whether the effect of CHX treatment on the induction of HSP21 protein level in the Col-0 accession (Fig. 5a,b) is due to the inhibition of de novo synthesis of FtsH6, Col-0 seedlings were treated with CHX at 6 h after priming and FtsH6 protein level was analysed by immunoblotting at days 2, 3 and 4 into the memory phase. As illustrated in Supplementary Figure 7a and b, treatment with CHX resulted in a significant reduction of the level of FtsH6 during thermomemory phase. This experiment shows that degradation of HSP21 during the memory phase in Col-0 correlates with enhanced FtsH6 protein synthesis.
Finally, we overexpressed FtsH6 from Col-0 in the N13 accession (35S:FtsH6Col-0/N13 plants; Supplementary Fig. 8a and b) and tested the effect of this on the thermomemory. Both, the seedling survival assay (Fig. 7a–c) and the hypocotyl elongation assay (Fig. 7d,e) revealed a decreased thermomemory on FtsH6 overexpression in the N13 accession. Furthermore, HSP21 protein level declined much faster in the 35S:FtsH6Col-0/N13 than in N13 empty-vector (EV) control plants during the thermomemory phase (Fig. 7f), supporting our conclusion that FtsH6 plays a key role in determining HSP21 abundance during memory. In accordance, we did not detect a difference between 35S:FtsH6Col-0/N13 overexpressors and control plants when testing basal and acquired heat stress tolerance (Supplementary Fig. 8c).
Although the importance of stress priming and memory in bacteria and plants is increasingly recognized1,2, the details of the molecular mechanisms underlying the stress memory phenomenon remain largely unexplored, in particular in plants. Experimental evidence indicates that plants have an ‘epigenetic’ memory, which may involve changes at the chromatin level including histone modifications and DNA methylation, which both occur in the response to stress43,44,45,46,47. Another mechanism for stress priming in plants could be RNA polymerase II stalling at gene promoters, which was suggested as a memory mark after drought stress in Arabidopsis48. The accumulation of inactive transcription factors (or cofactors) after a priming stimulus, and their activation on experiencing the triggering stimulus, may represent a further molecular mechanism of priming and memory49,50,51. Currently, however, details about how transcription factors affect thermomemory in plants are largely missing, although HsfA2 has been identified as a key element in this25. HsfA2 regulates the expression of a number of HSP genes52,53 and in a transcription regulatory cascade is located downstream of the NAC transcription factor JUNGBRUNNEN1 (JUB1), which like HSFA2 shows high expression during the thermomemory phase54,55. Hsa32 encoding a heat shock-associated protein of 32 kDa has also been shown to be a key component of thermomemory in Arabidopsis by interacting with HSP101 (refs 41, 56). In addition, microRNAs (miRNAs), which cause the degradation of mRNAs or translational inhibition, contribute to modulating the priming of stress responses in plants57. Finally, priming and stress memory might involve metabolic changes that are maintained throughout the memory phase, thus allowing a more rapid response of the plant to an upcoming new stress58,59,60.
Our results presented here provide evidence for a role of the plastid-localized sHSP HSP21–FtsH6 control module for thermomemory, whereby HSP21 protein abundance is negatively controlled by heat stress-induced FtsH6 (see model in Fig. 8). Previous studies have indicated a role of HSP21 in protecting photosystem II against oxidative and heat stress, a function that may be achieved by HSP21’s ability to maintain chloroplast function through an interaction with pTAC5, a nucleoid protein that interacts with the plastid-encoded RNA polymerase-dependent transcription complex, an important component of the chloroplast transcription machinery19,21. This capacity of HSP21 might be key to the role it has for extending the thermomemory. Notably, the abundance of cytosolic HSP101 is not affected during the thermomemory phase when FtsH6 is mutated (Supplementary Fig. 9), suggesting a specific role of FtsH6 for the plastidic component of plant thermomemory.
Importantly, we discovered genetic diversity of thermomemory-related HSP21 accumulation in two natural accessions of Arabidopsis contrasting in their thermomemory capacity. HSP21 protein abundance remained elevated for longer in accession N13, which we show has strong thermomemory, than in Col-0, which has a weak thermomemory. Using a combined pharmacological (protease inhibitor) and transcriptomic approach, we identified plastid-localized metalloprotease encoded by FtsH6 to be functionally involved in regulating the abundance of HSP21, in particular towards the end of the thermomemory phase. Notably, FtsH6 is mutated in N13, precluding the formation of FtsH6 protein, as confirmed by western blot analysis, while in Col-0, FtsH6 protein is readily detected after heat stress priming (Fig. 6b). Expressing FstH6 from Col-0 in N13 leads to a faster decline of HSP21 protein abundance during the thermomemory phase, concomitant with a reduced thermomemory compared with the N13 control line (Fig. 7), providing further evidence for the important role of FtsH6 in determining the thermomemory (Fig. 8).
A biological function for FtsH6 has not been identified previously and ftsh6 knockout mutants did not show a detectable phenotype under the experimental conditions applied earlier35,36, while FtsH1, FtsH2, FtsH5 and FtsH8 are involved in the degradation of photodamaged D1 protein of photosystem II, together with lumenal Deg proteases (reviewed in Wagner et al.29). In contrast to other FtsH genes, FtsH6 is rapidly and highly induced by heat stress in Arabidopsis (Fig. 5c) and other species including dicot and monocot crops37,38,39,40, indicating evolutionary conservation and ecological relevance of FtsH6 heat inducibility. In accordance with this, our data presented here demonstrate a role of FtsH6 in regulating the stability of HSP21 after a priming heat stress, although the molecular mechanism through which this occurs is not clear at present. Whether FtsH6 has a similar function in other plant species, including crops, needs to be determined in the future.
Although FtsH6 plays an important role in regulating HSP21 protein level it is likely not the only metalloprotease involved, as indicated by the fact that 1,10-phenanthroline treatment triggers a higher accumulation of HSP21 than knocking out FtsH6 (cf. Figs 5e and 6d). This finding is in accordance with the observation that ftsh6 has only a mild thermomemory effect in the survival assay, albeit a much stronger effect is observed when the mutant is tested in the hypocotyl elongation assay (Supplementary Fig. 6d and e). Finding further molecular processes controlling HSP21 protein level during the thermomemory phase will therefore remain an important task for the future.
Both HSP21 and FtsH6 are strongly induced by heat stress. HSP21 is a target of HsfA2 (ref. 52), a heat-inducible transcription factor that plays a crucial role for setting the thermomemory in Arabidopsis25. It is also upregulated by HsfA1a/b61, indicating that heat-induced expression of HSP21 is under the control of multiple heat-associated transcription factors. However, a direct upstream regulator of FtsH6 has not been reported so far, suggesting that a currently unknown upstream transcription factor(s) controls expression of FtsH6 during heat stress. We thus propose that coordinated expressional regulation of the HSP21 and FtsH6 genes by different transcription factors contributes to the fine-tuning of the thermomemory. Future research has to uncover the details of this transcriptional regulatory network that underlies heat stress priming and that helps the plant to configure its thermomemory.
In conclusion, our work demonstrates a crucial role of chloroplasts (or plastids in general) for the duration of the thermomemory in plants, which involves the plastid sHSP HSP21 in combination with FtsH6 metalloprotease as central components: continued high levels of HSP21 protein abundance after a priming heat stress promote the extension of the memory phase (Fig. 8). The level of HSP21 at the later phase of the thermomemory is negatively controlled by FtsH6, which is missing in the strong thermomemory accession N13, but produced on heat stress in the weak thermomemory accession Col-0. Besides HSP21, FtsH6 may interact with other proteins to determine the extent of the thermomemory, but such further targets remain unknown at present. The analysis of the proteome of FtsH6-modified lines (knockouts or overexpressors) at different time points throughout the memory phase may be a fruitful approach to identify additional targets of FtsH6. Furthermore, as natural variation exists for the HSP21–FtsH6 control module, addressing the selective forces that maintain this variation, as well as possible trade-offs that might be associated with an extended thermomemory, are further interesting aspects to study in the future. In addition, considering the involvement of plastids in establishing the thermomemory it is an interesting observation that chloroplast translation involving chloroplast ribosomal protein S1 is required for the heat stress-induced expression of HsfA2; however, whether ribosomal protein S1 is also required for thermomemory was not tested62.
Regulating the abundance of chaperones such as HSP21 after a priming heat stress could be a general mechanism for setting the temporal frame for maintaining thermomemory (or the memory to any other type of stress). Similarly, Hsa32 and large HSP HSP101 have recently been shown to form a regulatory interaction circuit in the cytosol that controls the abundance of HSP101 by Hsa32 and thereby thermomemory56. We propose that the control of HSP (or, more general, chaperone protein) abundance is a key mechanism for determining the duration of the memory phase. Future research will unravel the molecular details of the cellular networks that control the abundance and functionality of heat stress-related chaperones as key components of the plant’s thermomemory.
Plant material and growth conditions
The following Arabidopsis thaliana (L.) Heynh. 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. Seeds were obtained from the Arabidopsis thaliana Resource Centre for Genomics (INRA, France; http://dbsgap.versailles.inra.fr/publiclines), or from Ellen Zuther and Roosa Laitinen, Max Planck Institute of Molecular Plant Physiology, Potsdam, Germany. Surface-sterilized seeds were germinated in Petri dishes containing an identical volume of Murashige-Skoog agar medium supplemented with 1% sucrose (w/v)) and seedlings were grown under a diurnal cycle of 16 h light (120 μE m−2 s−1) at 22 °C and 8 h dark at 22 °C.
Generation of transgenic plants
Constructs were generated by PCR and restriction enzyme-mediated cloning. Sequences of oligonucleotides (Eurofins MWG Operon; Ebersberg, Germany) are given in Supplementary Table 1. PCR-generated amplicons were checked by DNA sequence analysis (MWG or Seqlab). To generate HSP21 knockdown plants we engineered amiRNAs by replacing the original miR319a/miR319a* sequence in plasmid pRS300 (ref. 63) with HSP21-specific amiRNA sequences. To this end, three different amiRNAs (amiRNA1: 5′-TTAGTATCTAACATTTGTCGC-3′; amiRNA2: 5′-TGTATCTAACATTTGTCGCAT-3′; amiRNA3: 5′-TATAATGTTGATCGAGTCCTA-3′) were designed using ‘WMD3: Web MicroRNA Designer 3’ to target different sites of the HSP21 transcript (Supplementary Fig. 1). The amiRNA constructs were subsequently cloned via added PmeI-PacI sites into pGreen0229-35S plant transformation vector64 behind the cauliflower mosaic virus (CaMV) 35S promoter. For 35S:HSP21, the HSP21 open reading frame was amplified by PCR from Arabidopsis Col-0 leaf cDNA, inserted into pCR2.1 vector using TA cloning kit (Invitrogen), and then cloned via added PmeI–PacI sites into pGreen0229-35S. The 35S:FtsH6 construct was generated in the same way. Constructs were transformed by floral dip into Arabidopsis using Agrobacterium tumefaciens.
Heat stress treatments
All heat stress treatments were performed in a water bath or incubator, in the dark. Thermomemory experiments were performed as described65. For the priming heat stress, seedlings were subjected to a heat regime of 1.5 h, 37 °C; 1.5 h recovery at 22 °C; and 45 min, 44 °C. After the priming heat stress treatment, seedlings were returned to normal growth condition for 3–4 days (recovery or memory phase) and then subjected to heat stress/triggering stimulus, that is, 1.5 h, 44 °C, a treatment normally lethal to plants that have not experienced a priming stimulus. For acquisition of thermotolerance, plants were initially heated to 37 °C for 90 min, returned to normal growth condition for 90 min, before finally heating to 44 °C for 100 min. Basal thermotolerance was assayed by heating plants to 44 °C without prior treatment at 37 °C. After heat treatments, plants were returned to normal growth conditions until analysis.
The quantitative hypocotyl elongation assay was adopted from ref. 42. In our assay, 4-day-old vertically grown, etiolated seedlings were primed at 37 °C for 90 min, then subjected to recovery for 2 days at 22 °C (memory phase), and finally subjected to the triggering heat stress (44 °C, 45 min). The priming heat treatment was omitted in control experiments. Hypocotyl length was measured 5 days after the heat stress.
Immunoblotting and signal quantification
Total proteins from Arabidopsis seedlings were prepared by phenol-based method66. Tissue powder was homogenized in 500 μl 0.7 M sucrose/0.5 M Tris/50 mM EDTA/0.1 M potassium chloride, pH 9.4, containing 2% (v/v) 2-mercaptoethanol and cOmplete Protease Inhibitor Cocktail (Roche; diluted to 1 × final concentration) added before use. The homogenate was mixed with 500 μl phenol and centrifuged at 20,000g (centrifuge 5427 R, Eppendorf) for 10 min at 4 °C. The upper, phenol phase was taken, and proteins were precipitated with the addition of 1 ml of 0.1 M ammonium acetate in methanol and overnight incubation at −20 °C. The protein pellet obtained following centrifugation was washed sequentially with methanolic ammonium. The protein pellets were dissolved in 1% SDS. The protein concentration was determined with the BCA Protein Assay kit (Thermo Fischer Scientific).
Proteins were resolved by SDS polyacrylamide gel electrophoresis. For immunoblot analysis, proteins were blotted to Protan nitrocellulose membrane (Sigma-Aldrich). Rabbit polyclonal antibodies were used in the following dilutions (v/v): anti-Hsp21 (Abcam; product number: ab80175), 1:3,000; anti-FtsH6 (Agrisera; AS05094A), 1:1,000; anti-HSP101 (Abcam; ab80121), 1:1,000. IRDye 800CW-conjugated goat anti-rabbit IgG(H+L) antibody was used as secondary antibody at 1:10,000 dilution (LI-COR Biosciences; 926-32211). Capturing bands of interest was done using the Odyssey Infrared Imaging System (LI-COR Biosciences), followed by quantifying band density with ImageJ software (www.imagej.net).
In all western blot experiments, loading controls originated from the same gel. For HSP21 and HSP101, the gels were cut into two parts. For HSP21, the lower part was used for immunodetection and the upper part was used for quantification of the protein (RbcL) loading, while for HSP101, the upper part was used for immunodetection and the lower part was used for quantification of RbcL loading. For FtsH6, the membrane was stripped after immunodetection and then stained with Ponceau S (Sigma) for loading control. For the comparisons between Arabidopsis Col-0 and N13 accessions, experiments were done in parallel in identical incubation solutions and with identical incubation times and conditions. Gel and immunoblot images have been cropped for presentation. Full-size images are presented in Supplementary Fig. 10.
Five-day-old primed seedlings were transferred to liquid culture medium containing 10 μM cycloheximide, 150 μM 1,10-phenanthroline or 0.1% (v/v) dimethylsulphoxide (mock) as control. The seedlings were harvested at the indicated time points and analysed by immunoblotting.
RNA isolation and microarray analysis
Global gene expression analysis by Affymetrix was performed in one experiment. The RNeasy Plant Mini kit (Qiagen, Hilden, Germany) was used to extract total RNA from primed and unprimed Col-0 seedlings at different time points (4, 8, 24 and 48 h) into the memory phase. Each sample was a pool of ∼160 seedlings. A 3-μg aliquot of quality-checked total RNA was then processed for use in Affymetrix ATH1 microarray hybridizations. Probe preparation and Affymetrix ATH1 microarray hybridizations were performed by ATLAS Biolabs (Berlin, Germany). Data analysis was performed as reported67. GO annotation was done using the Singular Enrichment Analysis tool from AgriGO (http://bioinfo.cau.edu.cn/agriGO)68 with the 60 upregulated genes (corrected P-value smaller than 0.0015).
Quantative reverse trancription–PCR
Synthesis of cDNA and quantative reverse trancription–PCR using SYBR Green were done as described67,69. Data were analysed using the comparative Ct method70 with ACTIN2 (AT3G18780) as the reference gene. Briefly, the Ct value of each gene was normalized to the Ct value of the reference gene, revealing delta Ct (dCt). The level of expression was expressed as the difference between an arbitrary value of 40 and dCt, so that a high 40-dCt value indicates high gene expression level. Primers were designed using QuantPrime (www.quantprime.de). PCR reactions were run on an ABI PRISM 7900HT sequence detection system (Applied Biosystems Applera, Darmstadt, Germany). Primers were obtained from Eurofins MWG Operon; sequences are given in Supplementary Table 1.
Microarray expression data are available from the NCBI Gene Expression Omnibus (GEO) repository (www.ncbi.nlm.nih.gov/geo/) under accession number GSE72949. The authors declare that all other data supporting the findings of this study are available within the article and its supplementary information files or are available from the corresponding author on request.
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We thank the Deutsche Forschungsgemeinschaft (DFG) for funding of the Collaborative Research Centre 973 ‘Priming and Memory of Organismic Responses to Stress’ (www.sfb973.de), Christiane Funks (Umea University, Sweden) for providing homozygous ftsh6 mutant seeds, Ellen Zuther and Roosa Laitinen for providing seeds of Arabidopsis accessions, Karin Koehl and colleagues for plant care, and Rouhollah Barahimipour for technical help with immunoblotting. We thank Isabel Bäurle and Anna Stief from our department for establishing the heat priming protocol employed in this study.
The authors declare no competing financial interests.
Supplementary Figures 1 - 10 and Supplementary Table 1 (PDF 1808 kb)
Expression of thermomemory-associated genes (60 up- and 19 down-regulated genes) during the memory phase compared to unprimed controls. (XLSX 20 kb)
Expression of nuclear-encoded, plastid localized proteases during the memory phase compared to unprimed controls. FC, fold change. (XLSX 13 kb)
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Sedaghatmehr, M., Mueller-Roeber, B. & Balazadeh, S. The plastid metalloprotease FtsH6 and small heat shock protein HSP21 jointly regulate thermomemory in Arabidopsis. Nat Commun 7, 12439 (2016). https://doi.org/10.1038/ncomms12439
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