Redox response of iron-sulfur glutaredoxin GRXS17 activates its holdase activity to protect plants from heat stress

Living organisms use a large panel of mechanisms to protect themselves from environmental stress. Particularly, heat stress induces misfolding and aggregation of proteins which are guarded by chaperone systems. Here, we examine the function the glutaredoxin GRXS17, a member of thiol reductases families in the model plant Arabidopsis thaliana. GRXS17 is a nucleocytosolic monothiol glutaredoxin consisting of an N-terminal thioredoxin (TRX)-domain and three CGFS-active site motif-containing GRX-domains that coordinate three iron-sulfur (Fe-S) clusters in a glutathione (GSH)-dependent manner. As a Fe-S cluster-charged holoenzyme, GRXS17 is likely involved in the maturation of cytosolic and nuclear Fe-S proteins. In addition to its role in cluster biogenesis, we showed that GRXS17 presents both foldase and redox-dependent holdase activities. Oxidative stress in combination with heat stress induces loss of its Fe-S clusters followed by subsequent formation of disulfide bonds between conserved active site cysteines in the corresponding TRX domains. This oxidation leads to a shift of GRXS17 to a high-MW complex and thus, activates its holdase activity. Moreover, we demonstrate that GRXS17 is specifically involved in plant tolerance to moderate high temperature and protects root meristematic cells from heat-induced cell death. Finally, we showed that upon heat stress, GRXS17 changes its client proteins, possibly to protect them from heat injuries. Therefore, we propose that the iron-sulfur cluster enzyme glutaredoxin GRXS17 is an essential guard to protect proteins against moderate heat stress, likely through a redox-dependent chaperone activity. All in all, we reveal the mechanism of an Fe-S cluster-dependent activity shift, turning the holoenzyme GRXS17 into a holdase that prevents damage caused by heat stress.


GRXS17 releases its Fe-S cluster and oligomerizes under combined oxidative and heat stress
When purified under anaerobic conditions, GRXS17 adopts a dimeric form coordinating up to three Fe-S clusters together with six molecules of GSH (Knuesting et al., 2015). As Fe-S clusters are highly sensitive to oxidation, we sought to examine the structural modifications of GRXS17 upon transfer to aerobic conditions. As expected, anaerobically reconstituted GRXS17 Fe-S cluster shows 110 a circular dichroism (CD) spectrum typical for the presence of [2Fe-2S] clusters ( Figure 1A). When exposed to air, the spectral footprint of the Fe-S cluster was progressively destabilized by oxygen and finally lost, confirming the sensitivity of the Fe-S cluster for oxidation ( Figure 1A).
In parallel to the loss of the Fe-S cluster, from size-exclusion chromatography (SEC) assay, we observed a progressive shift of GRXS17 to a high-MW form of >250 kDa after 3 h of exposure to 115 air. After 12 h, a high-MW complex of a higher range was observed ( Figure 1B). Similar data were observed by native gel electrophoresis and non-reducing SDS-PAGE ( Figure 1E-F). As expected, holo-GRXS17 migrates, on non-reducing native gel, as a single band likely corresponding to a dimeric form. However, after storage under aerobic conditions for more than 12 h, oligomers were formed, underscoring the high sensitivity of holo-GRXS17 to oxygen ( Figure 1E-F). Interestingly, 120 the size of the HMW-complexes was somehow lower under non-reducing SDS-PAGE conditions than on a native gel, suggesting that both weak-bond and disulfide-bond interactions are formed under oligomerization. All in all, we showed that the presence of the Fe-S cluster prevents the oligomerization of GRXS17.
To further examine the stability of the Fe-S cluster under oxidative conditions, we exposed the 125 holoGRXS17 to physiological concentrations (0.2-1 mM) of H 2 O 2. As previously shown (Knuesting et al., 2015), the UV-VIS spectrum of the reconstituted GRXS17 exhibited absorbance peaks at 320 and 410 nm, characteristic for [2Fe-2S] clusters. Upon H 2 O 2 treatment, the Fe-S cluster is lost in a dose-dependent manner (~50% loss in 12 and 5 min at 0.2 mM and 1 mM, respectively), confirming the sensitivity of the Fe-S cluster to oxidation ( Figure 1C-D). As the high temperature 130 was previously shown to destabilize Fe-S clusters in other proteins (Yan et al., 2013), we also monitor its stability at high temperatures. Both 35°C and 43°C temperatures had little effect on the stability of the cluster after short-term treatments, indicating that the Fe-S cluster is not heat sensitive ( Figure 1C-D and Supplemental Figure 1). However, when combining H 2 O 2 treatment with physiological high-temperatures, an accelerated loss of the Fe-S cluster (~50% loss in 3 min at 135 0.2 mM H 2 O 2 ) was observed, showing that higher temperatures potentiate the oxidative effect on Fe-S cluster stability. As expected, extensive oligomerization of GRXS17 was observed by native gel and non-reducing SDS-PAGE gels after H 2 O 2 treatment, while high temperature exposure had less impact on oligomerization of GRXS17 ( Figure 2E-F).

GRXS17 oligomerizes through intermolecular disulfides and noncovalent interactions
We further explored GRXS17 oligomerization after loss of Fe-S clusters using SEC ( Figure 2). As expected, HMW complexes (~900-1000 kDa) were observed in the native apoGRXS17 ( Figure 2A, blue line). When separating these fractions on a non-reducing SDS-PAGE gel, a laddering profile 145 was observed, suggesting that the HMW complexes are partially dissociated by SDS and that the remaining HMW bands contain disulfide-linked GRXS17 oligomers ( Figure 2B). These data indicate that native GRXS17 oligomerizes through non-covalent interactions and intermolecular disulfides. Consistently, incubation of the native GRXS17 with DTT prior to SEC led to a decrease of the ~900-1000 kDa peak and a concomitant increase of the 110 kDa peak, likely corresponding 150 to homodimeric GRXS17 ( Figure 2A). Furthermore, SDS-PAGE analyses indicate that the peaks corresponding to oligomeric and dimeric GRXS17 consist of non-covalently interacting monomers ( Figure 2B).
In order to analyze the role of the active-site cysteines during the oligomerization, mutated forms of GRXS17 were analyzed by SEC. Interestingly, the TRX-domain C33S mutant behaved similarly to 155 reduced wild-type GRXS17 ( Figure 2C), namely exhibiting comparable peaks at ~900-1000 kDa and 110 kDa. Moreover, the distribution between these peaks was not influenced by DTT treatment prior to SEC, indicating that Cys33 plays a major role in the oligomerization, very likely by forming intermolecular disulfide bonds ( Figure 2C). Strikingly, mutation of all active site cysteines completely abolishes oligomerization, as GRXS17-C33/179/309/416S exclusively elutes as a dimer 160 ( Figure 2D).
Finally, to verify the impact of Cys mutations on the GRXS17 structure, we analyzed secondary structure changes of GRXS17 and GRXS17-C33/179/309/416S with circular dichroism spectroscopy (Supplemental Figure 2). The general structures (α-helices, β -strands and turn) remains similar between both proteins, indicating that the C33/179/309/416S mutations does not 165 induce major secondary structure changes of GRXS17. The effect of Cys oxidation on the conformation of GRXS17 was also determined. Treatment with 400 µM H 2 O 2 resulted in a minor change in secondary structure composition of wild-type GRXS17, but not for the GRXS17-C33/179/309/416S variant (Supplemental Figure 2A). This minor change in secondary structure composition of GRXS17 upon oxidation can correspond to either a local or a global conformational 170 change, which is likely to involve the partial unfolding of α -helices and the formation of β -strands.
As this conformational change was not observed for the quadruple Cys variant GRXS17-C33/179/309/416S, we can conclude that this structural change depends on the oxidation of the catalytic cysteines and the formation of disulfide bonds in wild-type GRXS17.
To summarize, we showed that recombinant GRXS17 is able to oligomerize by forming 175 intermolecular disulfides in which the active site C33 of the TRX-domain plays a major role. In addition to being involved in Fe-S cluster coordination, C179, C309 and C416 seems to be important for maintaining non-covalent interactions between protomers. Finally, the active site residues C33, C179, C309 and C416 are not involved in the dimerization process.

180
A holo-apo redox switch of GRXS17 activates holdase activity upon oxidation Molecular chaperone activities are often associated with oligomerization and are related to thermotolerance (Lee et al., 2009). Therefore, the potential role of GRXS17 as a molecular chaperone was analyzed using different chaperone substrates. In order to monitor holdase activity, we used mitochondrial citrate synthase (CS) as a client protein (Figure 3). We analyzed the effect of 185 GRXS17 on the high-temperature-induced aggregation of CS, a classical assay to monitor the activity of molecular chaperones in vitro (Voth et al., 2014). Incubation of CS with reduced or oxidized apo-GRXS17 prevented thermal aggregation at 43°C ( Figure 3A). Increasing the apo-GRXS17/CS ratio progressively decreased CS aggregation which was fully prevented at a molar ratio of 0.5:1 apo-GRXS17:CS ( Figure 3B). This effect was stronger than described for holdases 190 from various organisms (e.g., HSP33, Get3, CnoX, TDX) (Jakob et al., 1999;Lee et al., 2009;Voth et al., 2014;Goemans et al., 2018). In contrast, in the absence of GRXS17 or in the presence of an excess of ovalbumin, CS significantly aggregates after 15 min ( Figure 3A). Interestingly, comparable holo-GRXS17/CS ratios did not prevent CS aggregation, indicating that the holdase activity of GRXS17 is specifically associated with the apo-GRXS17 form ( Figure 3A). 195 Subsequently, we verified that the Fe-S cluster in the holo-GRXS17 is stable at 43°C (Supplemental We also monitored the foldase activity of GRXS17 by using glucose-6-phosphate dehydrogenase (G6PDH) as a substrate. To distinguish between foldase and disulfide reductase activities, we used urea-denatured Cys-free G6PDH. Increasing the apo-GRXS17:G6PDH ratio progressively restored 200 the G6PDH activity ( Figure 3C-D). This reactivation was significantly faster in the presence of GRXS17 with nearly 36% recovery after 3 h at a 10:1 ratio of GRXS17:G6PDH. Reduced or oxidized holo-GRXS17 similarly restored G6PDH activity, indicating that the foldase activity of GRXS17 is independent of the presence or absence of the Fe-S cluster ( Figure 3C-D). We also could show that these activities are ATP-independent (Supplemental Figure 3). Collectively, our 205 data show that GRXS17 can adopt both a holdase and a foldase activity in vitro, and that the holdase activity is specific for the apo-form.

Oligomerisation determines the holdase activity of GRXS17
The capacity of apo-GRXS17 to oligomerize depends on both disulfide bonds involving active site 210 Cys residues and non-covalent electrostatic interactions ( Figure 2). In order to uncover forms that are associated with chaperone activity, we tested the chaperone activity of the Cys/Ser-variants of GRXS17 using cytosolic malate dehydrogenase (cytMDH1) as substrate ( Figure 4A). We found that GRXS17(C33S) has holdase activity for those forms with a high oligomerization state ( Figure 2C and Figure 4A). In the oligomerization-abolished GRXS17-C33/179/309/416S variant ( Figure 2D) 215 no holdase activity was observed ( Figure 4A). This suggested that the holdase activity depends on the active-site cysteines. To further demonstrate that oligomerization is required for holdase activity, we incubated reduced and oxidized apo-GRXS17 with a low concentration of the chaotropic agent guanidinium hydrochloride (GdnHCl), which abolishes weak-bonds like hydrophobic and electrostatic interactions ( Figure 4B). We found that the holdase activity of reduced GRXS17 is 220 inhibited with GdnHCl, while the holdase activity of oxidized GRXS17 is preserved. Finally, we tested the role of the cysteines in GRXS17 foldase activity ( Figure 4C). We found that GRXS17 Cys mutations does not negatively affect GRXS17 foldase activity, suggesting that disulfide-linked oligomerization is not required for its foldase activity.

GRXS17 is involved in tolerance to moderate heat stress
To reveal the physiological significance of the redox-dependent chaperone activity of GRXS17, we explored the possible role of GRXS17 in plant thermotolerance. Therefore, we subjected grxs17 mutant plants to different heat-stress regimes (Yeh et al., 2012). The impact of heat stress on shoot meristem viability was first quantified by monitoring the appearance of new leaves. The viability of 230 the grxs17 mutant was similar to wild-type plants for both Short-term Acquired Thermotolerance (SAT) and Long-term Acquired Thermotolerance (LAT) regimes ( Figure 5A-B). In contrast, a mutant lacking the heat-shock protein 101 (HSP101) chaperone was found to be hypersensitive to all heat-stress regimes (Yeh et al., 2012), indicating that GRXS17 does not overlap with HSP101 functions in SAT and LAT. Nevertheless, compared to wild-type plants, grxs17 mutants were 235 significantly more sensitive to a moderate heat stress regime (TMHT: Tolerance to Moderated High Temperature) ( Figure 5C), indicating that GRXS17 is specifically involved in this heat stress regime. The thermosensitive phenotype of grxs17 was fully complemented by overexpressing the full-length GRXS17:GFP fusion, indicating that the TMHT phenotype of grxs17 is due to the loss of the GRXS17 function and that the attached GFP does not perturb the function of the protein 240 ( Figure 5C and Supplemental Figures 4 and 5). Importantly, the overexpression of the GRXS17-C33S:GFP fusion was able to restore the wild-type phenotype, while the expression of the GRXS17-C33/179/309/416S:GFP variant failed to complement the mutant phenotype ( Figure  In order to study the impact of high temperature on root, wee performed a similar experiment by following the root development after the TMHT regime ( Figure 6). Plants were grown at 20°C for 6 days before the temperature was shifted to 30°C. For root growth, 30°C was used instead of 35°C because this latter temperature affected to much root growth of the wild-type plants (not shown).
We observed that growth of the primary root of wild-type seedlings tends to be accelerated after the 250 temperature shift, while root growth of the grxs17 mutant almost completely stopped, confirming the sensitivity of the mutant to moderate heat stress. We also observed a dramatic decrease of emerged lateral root number in the grxs17 ( Figure 6B). When the grxs17 mutant was complemented with the GRXS17-GFP fusion protein, the mutant behaves like wild-type. On the other hand, GRXS17-C33/179/309/416S:GFP fails to complement the grxs17 mutant ( Figure 6A-B). The 255 structure of the root apical meristem (RAM) was observed at 11 days of growth (after 5 days of heat stress). Consistently, the grxs17 mutant presented a significantly shorter RAM compared to wildtype and the GRXS17-GFP complemented lines, showing a high impact of heat stress on the meristematic activity of the mutant ( Figure 6C-E). Propidium iodide (PI) staining was used to monitor cell death in meristematic zones (Truernit and Haseloff, 2008). While we did not observe 260 any cell death in the RAM, massive cell death was observed in the lateral root primordia in grxs17 ( Figure 6G-I). This suggested that GRXS17 prevents heat stress injuries at an early stage of meristem establishment, which we confirmed in a germination experiment under heat stress using the grxs17 mutant. While the mutant seeds are able to germinate properly, they stop growing at early plantlet stage and harbor a very short root. Importantly, the structure of the RAM looks 265 disorganized and shows extensive cell death (Supplemental Figure 6). The GRXS17-C33/179/309/416S:GFP complemented lines failed to complement the shorter RAM of the grxs17 mutant and did not prevent cell death in the lateral root initials, indicating the key role of Cys in GRXS17 functions ( Figure 6F and J). In addition, we also observed perturbations in the root hair morphology in these latter plants, which were not observed in any other conditions, suggesting a 270 deleterious effect of GRXS17-C33/179/309/416S:GFP overexpression in root hairs ( Figure 6J, inset).
Altogether, these results highlight the role of GRXS17 in the tolerance of plants to moderate heat stress, acting to protect both shoot and root meristems. Importantly, the active site cysteines of GRXS17 seems to play an essential role in root growth and thermotolerance. 275 GRXS17 oligomerizes in planta and associates with a different set of proteins upon shifting to a higher temperature In addition to in vitro assays, we also explored GRXS17 oligomerization in vivo. Total soluble protein extracts prepared from Arabidopsis seedlings grown at standard temperature (20°C) and 280 exposed to heat stress (35°C for 2 h) were fractionated by SEC (Figure 7). Western-blot analyses of eluted fractions revealed a peak containing GRXS17 eluting at ~100-150 kDa (lanes 23-25), corresponding to a molecular weight of a GRXS17 dimeric form (~110 kDa) (Knuesting et al., 2015). Moreover, at 20°C, a GRXS17 signal was also detected in fractions (lanes 18-21) corresponding to an HMW (~380 kDa) form ( Figure 7). Interestingly, plants subjected to heat stress accumulated higher levels of HMW forms eluting in the range from SEC column (lanes 16-18), corresponding to 520 kDa ( Figure 7). These data suggest that similarly to in vitro analyses ( Figure 1 and 2), GRXS17 forms, HMW-complexes in vivo, although the size of these complexes are not fully similar. Also, we cannot rule out that GRXS17 oligomeres associate with other proteins.
Finally, we sought to identify GRXS17 partners by using immunoprecipitation coupled to mass-290 spectrometry determination (IP-MS). To this end, grxs17 mutant plants were transformed with a Pr35S::GRXS17:FLAG-HA construct. The expression of the construct was verified by immunoblotting and its functionality was assessed by complementation of the phenotype under TMHT (Supplemental Figure 7). Total protein extracts from GRXS17:FLAG-HA expressing plants grown at 20°C or shifted for 2 h to 35°C were collected for IP-MS analyses. Non-specific GRXS17 295 contaminants were identified within the same experiments using grxs17 mutant plants expressing a GRXS17 protein devoid of the FLAG-tag. We employed a label-free quantitative proteomic approach to identify proteins significantly enriched after IP by comparison with the control samples.
Five biological replicates were analyzed by mass spectrometry after FLAG immunoprecipitation from total protein extracts of plants expressing GRXS17:FLAG-HA or non-tagged GRXS17 300 (control) grown at 20°C or shifted for 2 h to 35°C. GRXS17 was the only protein significantly enriched in the tagged samples after IP (P<0.05, Bonferroni corrected Student T-test). In addition, we identified 23 proteins from plants grown at 20°C and 12 proteins from plants shifted to 35°C that were present and quantified in at least 3 of the 5 replicates from the tagged strain and not found in any of the 5 replicates from control non-tagged plants (Table 1). Using this all-or-nothing 305 approach, several likely interactants such as protein disulfide isomerase (PDIL) and the antioxidant enzyme monodehydroascorbate reductase (MDAR) were identified at 20°C. A different set of interaction partners was identified after a 2 h temperature shift from 20°C to 35°C, and only 4 proteins were identified in both conditions (Table 1). Among them, the nucleo-cytoplasmic BolA2 was always present (Supplemental Table 1). This interaction is consistent with previous studies that 310 reported the interaction of GRXS17 with BolA2 (Couturier et al., 2014). Finally, to identify GRXS17 partners in HMW complexes, total protein extracts from plants grown at 20°C or shifted for 2 h to 35°C were first separated by SEC, and HMW fractions containing GRXS17:FLAG-HA were collected for IP-MS analysis. However, a simple subtractive analysis between the samples failed to identify a similar set of proteins as in the previous approach, suggesting that those proteins 315 are not involved in HMW complexes (Supplemental Table 1). However, BolA2 was identified after IP-MS in HMW fractions, indicating that BolA2 is a major GRXS17 oligomer interactant associated with the molecular chaperone activity of GRXS17.

Apo-GRXS17 switches to holdase activity upon stress
During periods of heat stress, living organisms need to rapidly adapt to cope with the damaging effects of increasing temperatures. The sessile nature of plants makes them particularly vulnerable to heat stress. As in other organisms also in plants, a set of heat-shock proteins acts as molecular 325 chaperones to prevent damage caused by protein inactivation and aggregation. Most functions of heat-shock proteins are heat-inducible, but their activation mechanism is not well understood (Hartl et al., 2011). Here, we demonstrated that the Fe-S cluster-harboring GRXS17 becomes a holdase after exposure to heat in oxidizing conditions. This switch happens within minutes by a redox and temperature destabilization of the Fe-S cluster, and subsequent disulfide bond formation ( Figure 8). 330 The redox-switch of Fe-S cluster-containing GRX has previously been shown to occur under oxidizing conditions in other GRX. In these cases, the active site Cys are involved in the Fe-S cluster which inhibits the thiol-reducing activity, the loss of the Fe-S cluster restoring the activity (Lillig et al., 2005;Berndt et al., 2007;Rouhier et al., 2007;Bandyopadhyay et al., 2008;Riondet et al., 2012). In contrast, the apo-GRXS17 of this study is inactive as a thiol reductase, as also reported 335 for another class-II GRX (Bandyopadhyay et al., 2008;Knuesting et al., 2015;Moseler et al., 2015).
This lack of thiol reductase activity was proposed to be linked to the specificity of the active site and the low reactivity for GSH (Moseler et al., 2015;Begas et al., 2017).
Interestingly, the active-site cysteines of GRXS17 are crucial for the holdase redox switch as they coordinate both the Fe-S cluster, and respond to an oxidizing environment, such as aerobic 340 conditions, H 2 O 2 exposure, or heat stress. As such, GRXS17 may act as a sensor for cellular redox changes. In fact, high temperatures by itself do not destabilize the Fe-S cluster, but the simultaneous Proteins that combine chaperone function with a redox activity previously have been described. For example, plant thiol reductase proteins TRXh3, TDX and NTRC exhibit chaperone activities triggered by oxidative stress and heat shock exposure, leading to a shift of the protein structure from 355 a low-MW form to high-MW complexes (Lee et al., 2009;Chae et al., 2013;Park et al., 2009).
Also, overoxidation of nucleophilic cysteine of 2-Cys PRX triggers oligomerization and induces chaperone activity (Liebthal et al., 2018). GRXS17 is different as it has no oxidoreductase activity, but it has a much more efficient holdase activity than the thiol oxidoreductases ( Figure 3 and Lee et al., 2009;Chae et al., 2013;Park et al., 2009). Remarkably, the key to induce holdase chaperone 360 activity is the release of the redox-dependent Fe-S cluster. To our knowledge, this is the first example of an FeS protein that turns into a chaperone upon switching between holo-and apo-form.
In E. coli, a similar example is hsp33 which coordinates zinc via conserved cysteines (Jakob et al., 1999;Jakob et al., 2000;Winter et al., 2008). Upon oxidation with H 2 O 2 , disulfide bonds are formed between the coordinating cysteines and zinc is released, activating HSP33 holdase activity. 365 Here, zinc coordination, oxidation and activation state of hsp33 are directly linked to the redox state of the environment (Jakob et al., 1999;Jakob et al., 2000;Winter et al., 2008). As another example in yeast, the cytosolic ATPase Get3 turns into an effective ATP-independent chaperone when oxidized (Voth et al., 2014). Furthermore, the holdase activity of E. coli chaperedoxin CnoX is activated by chlorination upon bleach treatment to protect substrates from irreversible oxidation 370 (Goemans et al., 2018). Whether GRXS17 acts by protecting its interactors from overoxidation remains to be determined.

GRXS17 exhibits a constitutive foldase activity
GRXS17 is likely acting as a housekeeping chaperone involved in folding cytosolic (or nuclear) 375 nascent peptides or in refolding misfolded peptides. We found that the foldase activity of GRXS17 is independent of ATP (Supplemental Figure 3). This clearly differs from most prokaryotic (e.g. GroEL-GroES) and eukaryotic foldases (e.g. hsp70, hsp90) which need ATP for their foldase activity (Mayer, 2010;Hartl et al., 2011). Nevertheless, a number of chaperones have been identified that promote folding in the absence of high-energy cofactors (Stull et al., 2016;Horowitz 380 et al., 2018). As an example, the small ATP-independent chaperone Spy from E. coli uses longrange electrostatic interactions and short-range hydrophobic interactions to bind to its unfolded client proteins and drive their folding (Koldewey et al., 2016).
Protein disulfide isomerase (PDI), or cyclophilin, which catalyze disulfide formation and isomerization of disulfides, exhibit folding activities through their active site Cys (Park et al., 2013;385 Ali Khan and Mutus, 2014). GRXS17 functions differently, as the foldase activity of GRXS17 is independent of its active-site cysteines ( Figure 4). Nevertheless, two additional cysteines are present in the first and third GRXS17 domains, and their involvement in GRXS17-foldase activity cannot be ruled out. Future experiments will have to be performed to decipher the mechanistic of the foldase activity of GRXS17. 390

GRXS17 associates with different proteins under high temperature
In eukaryotic organisms grown under non-stress conditions, the cytosolic compartment is a highly reducing environment (Schwarzländer et al., 2008). However, in heat-stressed plants, intracellular oxidation triggered by ROS accumulation was largely documented (Choudhury et al., 2017;395 Dickinson et al., 2018). This is consistent with measurements of the glutathione redox potential (E GSH ) monitored in plant expressing the GRX1-roGFP2 construct in the cytosol and nucleus and subjected to high temperature (Supplemental Figure 8). Therefore, in regard to changes in redox status depending on the temperature, it is likely that the major GRXS17 form is the Fe-S clustercontaining dimeric form under standard growth temperatures, but a significant proportion of 400 GRXS17 switches to HMW complexes in plants subjected to heat stress (Figure 7). Although the difference in the size of the HMW complexes observed in recombinant GRXS17 and in plant extract will have to be further explored, the switch to HMW complexes appears to be necessary for plant survival under heat stress. As the grxs17 mutant is highly sensitive to moderate high temperature regimes, the mutant can only be complemented by the GRXS17-Cys variants that are 405 still able to oligomerize (GRXS17 and GRXS17-C33S) and not by GRXS17-C33/179/309/416S GRXS17 was previously proposed to improve thermo-, drought-and oxidative-tolerance by enhancing ROS scavenging capacities and expression of Heat-Shock Proteins. This might occur by modulation of gene expression, enzyme activity or protection of antioxidant enzymes (Wu et al., 415 2012(Wu et al., 415 , 2017Hu et al., 2017). Our data suggest an additional function of GRXS17 through its chaperone activity. The GRXS17 partner protein identification resulted in a low overlap between proteins immunoprecipitated at 20°C and those identified at a higher temperature, suggesting that the set of interacting proteins is completely changed under heat stress. Although it is tempting to suggest that the proteins we identified in immunoprecipitation experiments are protected by 420 GRXS17, further experiments will need to validate these partners and the biological significance of these interactions. Importantly, our observations showing that GRXS17 maintains plant viability under moderate heat stress conditions and prevents cell death in founder cells of root primordia under high temperature might indicate a role in protecting key actors of meristem function. Possible candidates that we found in our IP are MPK4 and MKK2 which play roles in temperature signalling 425 pathways (Zhao et al., 2017).
Finally, we propose that the unique properties of GRXS17 among the redox-regulated chaperones (e.g., 2-Cys PRX, TRXh3 or TDX) comes from its inducible holdase function (i.e. Fe-S cluster release under stress), which is not seen for other heat stress-related chaperones (Stull et al., 2016).
Homologs of GRXS17 are present in all plants and in most other organisms (Couturier et al., 2009), 430 and their functions in iron metabolism are well documented (Encinar del Dedo et al., 2015;Berndt and Lillig, 2017). It is likely that the switch to holdase chaperone activity, which we described here, could be conserved among different kingdoms of life. Thus, unlike other Fe-S cluster-containing proteins in which cluster release generally impairs protein function (Lill, 2009;Klinge et al., 2007; Berndt and Lillig, 2017), GRXS17 uses this oxidation-induced switch to activate holdase 435 functionality, and in this way, protects plants from heat-induced damage.
Sterilized seeds were plated onto ½ MS agar medium (2.2 g/L Murashige and Skoog, 0.5 g/L MES, 1% plant agar (w/v), adjusted to pH 5.8) and stratified 48 h at 4°C in the dark. Seedling were grown 445 under 120 µmol.m -2 .s -1 photosynthetic flux at 22°C and 16 h light/8 h dark cycles. Those growth conditions were systematically used, unless otherwise indicated.

Transformation of plants
Plasmids were transferred into Arabidopsis via Agrobacterium tumefaciens strain GV3101 using the described methods (Clough and Bent, 1998). Transformants were selected on ½ MS medium plates containing 50 μ g ml -1 hygromycin and checked by PCR using appropriate primers (Supplemental Table 2). 470

Production and purification of recombinant proteins
All proteins were produced by induction in Escherichia coli BL21(DE3). Cultures of 2 L of a ampicillin-resistant (100 mg.mL -1 ) colony were grown at 37 °C and induced by 100 µM isopropylβ -D-galactopyranoside (IPTG) in the exponential phase (DO=0.5). Bacteria were harvested by 475 centrifugation 3 h after induction and stored at -80°C before protein extraction. Pellets were resuspended in buffer (50mM Tris-HCl pH 7.0, 100 mM NaCl, and 1 tablet anti-protease cocktail (Roche) for 25 mL), extracted using a cell disrupter (Constant Systems Limited, Northants, UK) and purified with a commercial kit according to manufacturer's instructions (His-bind buffer kit, Millipore). 480

In vitro characterization of holo-GRXS17
Chemical reconstitution of holo-GRXS17 was performed under strictly anaerobic conditions in an anaerobic vinyl tent (COY Laboratory Products, Grass Lake, U.S.A.) according to Freibert et al. (2018). In brief, 100 μ M GRXS17 were reduced in reconstitution buffer (50 mM Tris-HCl, pH 8, 485 150 mM NaCl, 10% glycerol (v/v)) in the presence of 2 mM GSH and 2 mM DTT for 3 h at room temperature. For reconstitution, the GRXS17 concentration was diluted to 20 μ M and ferric ammonium citrate was added to a final concentration of 400 μ M. After 5 min of incubation, lithium sulfide (400 μ M) were added followed by a 3-h incubation. Excess iron ammonium citrate and lithium sulfide was removed by desalting using a NAP™ 25 column (GE Healthcare). 490 Subsequently, the ISC were characterized using a CD-spectrophotometer (J-815, Jasco). The reconstituted GRXS17 was transferred to a CD cuvette, which was sealed airtight. Oxidative treatment of the holo-GRXS17 was performed with air oxygen (21%) for the indicated time. The analytical SEC was carried out under strict anaerobic conditions. The gel filtration was performed with the ÄKTA Prime Plus System (GE Healthcare) coupled to a Superdex200 10/300 GL column 495 (GE Healthcare). In each case, 50 μ g of GRXS17 were applied into the equilibrated column (50 mM Tris-HCl pH 8, 150 mM NaCl, 10% glycerol (v/v)) and separated at a flow rate of 0.5 mL/min.
The column was calibrated with the Molecular Weight standard 29-669 kDa (Sigma-Aldrich).

Confocal Laser-Scanning Microscopy and cell death observation
Confocal microscopic observations were carried out using the Axio observer Z1 microscope with 510 the LSM 700 scanning module and the ZEN 2010 software (Zeiss). GFP and propidium iodide (PI) were excited using the 488nm argon-ion laser and collected at 500-550 nm (GFP) or 600-656 nm (PI). Excitation of roGFP2 was performed at 488 and 405 nm and a bandpass (BP 490-555 nm) emission filter was used to collect roGFP2 signal. For background subtraction, signal was recovered using a BP 420-480 nm emission filter during excitation at 405 nm. Pictures analyses and 515 quantifications were performed as previously described (Schwarzländer et al., 2008), using the public domain image analysis program ImageJ 1.52i (https://imagej.nih.gov/ij/). For measurement of cell death in roots, seedlings were stained with 10 µg/mL PI (Sigma-Aldrich) for 5 min before imaging.

Size-exclusion chromatography
Gel filtration experiments were performed using an ÄKTA Fast Protein Liquid Chromatography (FPLC) system (Amersham Biosciences) at 4°C. The absorbance was monitored at 280 nm. The column was calibrated using the molecular weigth standard (29-669 kDa). For recombinant GRXS17 (wild-type and mutated forms), a Superose 6 column was used, equilibrated with a 50 mM 525 Tris-HCl (pH 7.5) buffer containing 150 mM NaCl and 5 mM MgCl 2 at a flow rate of 0.5 ml min -1 .
All recombinant protein samples were dialyzed before application. For reducing conditions, samples were incubated with 100 mM of DTT and dialyzed against the above-described buffer supplemented with 5 mM of DTT to maintain the proteins in their reduced state.
For separation of total proteins extracts, a HiPrep column Sephacryl S-300 HR (GE Healthcare Life 530 Sciences) was used, equilibrated with a 50 mM Tris-HCl (pH 7.5) buffer containing 150 mM NaCl, 5 mM MgCl 2 , 10% (v/v) glycerol and 0.1% NP40 at a flow rate of 0.5 ml min -1 . Samples were extracted in the same buffer containing 10 µM of MG132 and anti-protease cocktail (Roche) at a concentration of 1 tablet for 10 mL. Extraction is realized with a ratio of 1 g of plant powder in two volumes of buffer. 535

Protein separation by gel electrophoresis and immunoblotting
Precast SDS-PAGE or native gels were used according to the manufacturer's instructions (Biorad).
For immunoblot analysis, gels were transferred to nitrocellulose membranes. Rabbit polyclonal antibodies against GRXS17 (1:25,000) were used for protein Western blotting. Goat anti-rabbit 540 antibodies conjugated to horseradish peroxidase (1:10,000) were used as secondary antibodies and revealed with enhanced chemiluminescence reagents (Immobilon Western Chemiluminescent HRP Substrate, Millipore).

Thermotolerance assays
For the thermotolerance assays, we applied three different protocols: LAT (Long-Term Acquired Thermotolerance), SAT (Short-Term Acquired Thermotolerance) and TMHT (Thermotolerance to Moderately High Temperatures) as defined by (Yeh et al., 2012). The seeds germinated and grown in horizontal plates containing medium MS (4.4 g/L Murashige and Skoog, 1% (w/v) sucrose, pH 5.8). The percentage of viability was calculated by counting the plants that recovered leaf growth 550 after heat stress. For examining the impact of heat stress on roots, seeds were grown on vertical plates and root growth was daily measured using ImageJ.

Quantitative Mass Spectrometry Analyses 565
Chemicals and enzymes: Proteomics grade Trypsin and ProteaseMax surfactant were purchased from Promega (Charbonnieres, France). Reversed-phase C18 spin columns, precolumns, and analytical columns were all obtained from Thermo Scientific (Les Ulis, France). Solvents and ionpairing agents were certified LC-MS grade and all other chemicals were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France) with the highest purity available. 570 Sample preparation: Immunoprecipitated samples were incubated in the presence of 0.02% (v/v) ProteaseMax with 5 mM DTT for 20 min at 40°C. Free cysteines were further carbamidomethylated by adding 11 mM iodoacetamide for 30 min at room temperature in darkness. Proteins were digested overnight at 35°C with modified porcine trypsin in a 1:50 (w/w) enzyme:substrate ratio.
The digestion was stopped by the addition of 0.1% trifluoroacetic acid and peptide mixtures were 575 centrifuged for 10 min at 21,500xg at 4°C. Tryptic peptides present in supernatants were then subjected to desalting using reversed-phase C18 spin columns as recommended by the supplier.
After elution, desalted peptides were concentrated with a SpeedVac (Eppendorf) and prepared in 3% acetonitrile, 0.1% formic acid (solvent A) were added to obtain a final protein concentration of 0.27 µg/µL based on the initial concentration. were performed with precursor mass tolerances of 20 and 4.5 ppm, respectively. The minimum peptide length was set to seven amino acids and specificity for protein digestion was restricted to 615 trypsin/P cleavage with a maximum of two missed cleavages per peptides. Methionine oxidation and N-terminal acetylation were specified as variable modifications, carbamidomethylation of cysteines was specified as a fixed modification. The peptide and protein false discovery rate (FDR) was set to 1%. To increase protein identification, a match between runs was systematically performed (Match time window 0.7 min; alignment time window: 20 min) and for label-free 620 quantification of immuno-precipitated proteins, the LFQ algorithm and normalization were chosen and unique and razor peptides were used.
MaxQuant output was further processed and analyzed using Perseus software (version 1.6.07).
Protein quantification was performed using the ProteinGroups.txt file after removing reverse and contaminant proteins and the Log2 transformation of quantitative data. Proteins showing at least 625 two unique and razor peptides and having quantitative data in at least three biological replicates were considered for statistical analysis of each temperature condition using a Benjamini-Hochbergcorrected Student T-test. Proteins showing a FDR < 0.05 were considered significantly enriched.
For both temperature conditions, proteins having quantitative data in biological samples expressing the tagged GRXS17 but not in the five corresponding control samples were treated separately. In 630 this case, only proteins showing quantitative data in three out of the five biological replicates with at least two unique and razor peptides from samples expressing tagged GRXS17 were considered as significant.

Circular Dichroism 635
In preparation for CD analysis, samples of GRXS17 were buffer-exchanged into 20 mM sodium phosphate, 100 mM NaF, pH 7.3, using Bio-Spin ® 6 columns, adjusted to 4 μ M, and treated with 400 μ M of either TCEP or H 2 O 2 for 1 h at 25°C. Samples were centrifuged and vacuum-degassed for 10 min prior to analysis by CD. CD spectra were collected at 20°C using a J-715 spectropolarimeter (JASCO) with a 0.1 mm path-length cuvette, from a range of 250-185 nm in 1.0 640 nm sampling and spectrum averaged over 8 repeats. The spectra of the buffer alone with TCEP/H 2 O 2 was subtracted prior to the conversion of data to units of mean residue ellipticity.
Secondary structure content was estimated using the online server, BeStSel (Micsonai et al., 2015).
For monitoring change in secondary structure over time, CD spectra of 250-190 nm were collected at 100 nm min -1 . The secondary structure change of GRXS17 following the addition of a 100-fold 645 excess of H 2 O 2 was measured as the change in [θ] at 222 nm over a course of 65 min, spectra collected every 41 s. In Prism (GraphPad) a one-phase decay model was fitted to these data, and a rate of conformational change was determined.      Heat Stress (HS)), ISC are released and GRXS17 switches to an apo-dimeric form with a foldase 765 activity. Dimers can spontaneously associate to oligomeric forms and further oxidation induces oligomeric structure through formation of intermolecular disulfide bonds at active-site Cys residues.
In this form, GRXS17 acquires holdase activities and provides thermotolerance. The oligomeric form can be reduced to recover the apo-dimeric GRXS17 and the Fe-S cluster is reassembled.    Holo-GRXS17 or apo-GRXS17 in their reduced or oxidized forms were incubated with CS at a 0.5:1 ratio at 43°C. Apo-GRXS17 but not holo-GRXS17 protects CS from temperature-induced precipitation. (B) Different ratios of apo-GRXS17/MDH were incubated at 55°C. Similar results were found by incubating apo-GRXS17/CS at 43°C. (C-D) GRXS17 foldase activity was measured by reactivation of the glucose 6-phosphate dehydrogenase (G6PDH). (C) Holo-GRXS17 or apo-GRXS17 in their reduced or oxidized forms were incubated with G6PDH at a 5:1 ratio at 30°C. Apo-GRXS17 and holo-GRXS17 support reactivation of G6PDH. (D) The ratio of apo-GRXS17/G6PDH was varied. As controls, substrates were incubated alone (Control)  GRXS17 and Cys-mutated GRXS17 proteins holdase activity was measured towards cytMDH1 heat-induced precipitation by light scattering at 0.5:1 ratios at 55°C. CytMDH1 was incubated alone (Control), with ovalbumin or with GRXS17 proteins. (B) GRXS17 proteins reduced by 5 mM DTT (green) or oxidized with 1 mM H 2 O 2 (brown) in absence or presence (respectively yellow and light blue) of 20 mM guanidinium hydrochloride (GdnHCl) as chaotropic reagent. (C) GRXS17 foldase activity was determined by measuring the glucose-6 phosphate dehydrogenase (G6PDH) activity after denaturation with urea at a 5:1 ratio. CytMDH1 was incubated alone (Control), with ovalbumin or with GRXS17 proteins. Data are representative of three independent experiments. Positive control Ovalbumin S17 Red S17 Red + GuaHcl S17 Ox S17 Ox + GuaHcl   Size-exclusion chromatography analysis (Sephacryl S300 HR) of crude protein extracts from Arabidopsis seedlings cultivated 14 days at 20°C and further treated for 2 h at 35°C. The GRXS17 protein was detected after western bloting and immunodetection using an anti-GRXS17 serum. Numbered lines correspond to the protein fractions. Protein size of the respective fractions was obtained after calibration using marker proteins. Under reducing conditions, the holo-GRXS17 forms a dimer which coordinates an iron-sulfur cluster (ISC). In this form, GRXS17 can transfer ISC to cytosolic and nuclear target proteins (e.g., BolA). Under oxidative conditions (H 2 O 2 , O 2 , Heat Stress (HS)), ISC are released and GRXS17 switches to an apo-dimeric form with a foldase activity. Dimers can spontaneously associate to oligomeric forms and further oxidation induces oligomeric structure through formation of intermolecular disulfide bonds at active-site Cys residues. In this form, GRXS17 acquires holdase activities and provides thermotolerance. The oligomeric form can be reduced to recover the apo-dimeric GRXS17 and the Fe-S cluster and the FeS cluster is reassembled. Estimation of secondary structure content from the obtained CD spectra was performed in BeStSel (Micsonai et al., 2015), and the difference in structural content between the reduced and oxidized forms of GRXS17 determined. Panel B plots the subtraction of the TCEP-treated GRXS17 secondary structure composition from that of the H 2 O 2 -treated sample, thereby indicating the relative change in secondary structure content upon oxidation. Samples of GRXS17 were treated with TCEP or H 2 O 2 at 400 µM (molar ratio of 100:1 compound to protein). GRXS17 foldase activity was measured by reactivation of denaturated glucose-6 phosphate dehydrogenase (G6PDH) at a 5:1 ratio at 30°C. GRXS17 (yellow), but not ovalbumin (red) reactivates G6PDH (dark blue). Addition of ATP (1 mM) to GRXS17 does not significantly modify the foldase activity (green). Data are representative of three independent experiments.  Coomassie Col-0 grxs17 +GRXS17-1 +GRXS17-2 +GRXS17-3 +GRXS17-4 grxs17 +GRXS17m1-1 +GRXS17m1-2 +GRXS17m1-3 +GRXS17m1-4 Col-0 grxs17 +GRXS17-1 +GRXS17-2 +GRXS17-3 +GRXS17-4 +GRXS17-C33S-1 +GRXS17-C33S-2 +GRXS17-C33S-3 +GRXS17-C33S-4