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HSP70-4 and farnesylated AtJ3 constitute a specific HSP70/HSP40-based chaperone machinery essential for prolonged heat stress tolerance in Arabidopsis

https://doi.org/10.1016/j.jplph.2021.153430Get rights and content

Abstract

AtJ3 (J3)—a member of the Arabidopsis cytosolic HSP40 family—harbors a C-terminal CaaX motif for farnesylation, which is exclusively catalyzed by protein farnesyltransferase (PFT). Previously, prolonged incubation at 37 °C for 4 d was found to be lethal to the heat-intolerant 5 (hit5) mutant lacking PFT and transgenic j3 plants expressing a CaaX-abolishing J3C417S construct, indicating that farnesylated J3 is essential for heat tolerance in plants. Given the role of HSP40s as cochaperones of HSP70s, the thermal sensitivity of five individual cytosolic HSP70 (HSP70-1 to HSP70-5) knockout mutants was tested in this study. Only hsp70-4 was sensitive to the prolonged heat treatment like hit5 and j3. The bimolecular fluorescence complementation (BiFC) assay revealed that HSP70-4 interacted with J3 and J3C417S in vivo at normal (23 °C) and high (37 °C) temperatures. At 23 °C, both HSP70-4–J3 and HSP70-4–J3C417S BiFC signals were uniformly distributed across the cell. However, following treatment at 37 °C, HSP70-4–J3, but not HSP70-4–J3C417S, BiFC signals were detected as discernable foci. These heat-induced HSP70-4–J3 BiFC foci were localized in heat stress granules (HSGs). In addition, hsp70-4 and J3C417S accumulated more insoluble proteins than the wild type. Thus, farnesylated J3 dictates the chaperone function of HSP70-4 in HSGs. Collectively, this study identified the first HSP70/HSP40-type chaperone machinery playing a crucial role in protecting plants against prolonged heat stress, and demonstrated the significance of protein farnesylation in its protective function.

Introduction

Plants are sessile organisms, and their growth and development are sensitive to diverse environmental stresses. Heat stress resulting from exposure to above-optimum temperatures can severely reduce crop productivity. Therefore, an understanding of plant responses to heat stress is imperative for devising strategies to improve crop yield and ensure food security (Bita and Gerats, 2013; Ortiz-Bobea et al., 2019).

Heat can disrupt hydrogen bonds and nonpolar hydrophobic interactions, which maintain both the secondary and tertiary protein structures; thus, protein denaturation is a direct and immediate detrimental effect of heat stress. Denatured proteins expose hydrophobic regions, promoting protein aggregation, altering proteostasis, and producing cytotoxicity. Members of the protein chaperone heat shock protein 70 kDa (HSP70) family act at the frontline of defense against protein aggregation (Rosenzweig et al., 2019). Central to the chaperone cycle of HSP70s is the nucleotide-controlled switch between the ATP- and ADP-bound states. In their ATP-bound state, the HSP70 and its substrate can bind, but with low affinity. Subsequent hydrolysis of ATP results in the ADP-bound state of HSP70, which can then stably bind to the exposed hydrophobic stretches of denatured proteins, stabilize the substrate in the intermediate conformation, and catalyze the folding reaction. Finally, after ADP is replaced by ATP, the substrate is released because of its low affinity for ATP-bound HSP70 (Alderson et al., 2016; Clerico et al., 2019). Although the molecular mechanism of HSP70 chaperone function is well characterized, the physiological roles of specific HSP70 family members remain unclear. For instance, genomic analysis had previously revealed five HSP70s (AtHSP70-1 to AtHSP70-5) to be localized in the cytoplasm of Arabidopsis (Lin et al., 2001). The reasons for—and importance of—the requirement for multiple HSP70s remain unclear, and a functionally specific HSP70 for plant survival under heat stress—assuming it exists—is yet to be identified.

Another aspect to be considered when deducing the physiological roles of HSP70s is that the proteins must invariably associate with heat shock protein 40 kDa (HSP40) family members to realize their function (Kampinga and Craig, 2010; Craig and Marszalek, 2017). HSP40s are a family of modular multidomain proteins characterized by the presence of a J-domain. Consequently, HSP40s are also designated as J-domain proteins (Miernyk, 2001; Rajan and D’Silva, 2009). As mentioned earlier, the conversion of ATP to ADP is essential for the stable binding of HSP70s to the substrates. However, the intrinsic ATPase activity of HSP70s is extremely low, and although the interaction of HSP70 with the substrate can stimulate ATPase activity to a small extent, it is inadequate for substrate trapping. ATPase activity can be fully stimulated only when the substrate and HSP40 are present simultaneously (Kityk et al., 2018). In addition, HSP40s can bind to substrates independent of HSP70 (Hageman et al., 2010). HSP40 and HSP70 bind to different regions of the same substrate; therefore, HSP40s are likely responsible for the initial contact with substrates and their delivery to HSP70s. Interestingly, HSP40s outnumber HSP70s in all organisms studied thus far. Arabidopsis contains 18 HSP70s and over 100 HSP40s (J-domain proteins) (Lin et al., 2001; Rajan and D’Silva, 2009). Thus, at least some HSP70s likely partner with multiple HSP40s (Pulido and Leister, 2017). These findings suggest that a unique HSP70–HSP40 pair facilitates a specific process at a distinct location within the cell to play a critical physiological role. Nevertheless, the key question of whether there is an HSP70–HSP40 pair functionally specific to heat tolerance in plants remains unresolved.

Recently, the Arabidopsis heat-intolerant 5 (hit5) mutant lacking protein farnesyltransferase (PFT) was shown to be sensitive to prolonged heat stress. Specifically, incubation at 37 °C for 4 d was lethal to hit5 but not to wild type (WT) seedlings (Wu et al., 2017). PFT catalyzes the covalent attachment of a 15-carbon unsaturated farnesyl lipid to proteins harboring a C-terminal CaaX motif (Running, 2014; Wang and Casey, 2016). Therefore, at least one PFT substrate is involved in heat tolerance in plants. Subsequently, Arabidopsis AtJ3 (At3g44110, hereafter termed J3)—an HSP40 with the CaaX motif—was shown to mediate the farnesylation-dependent heat-intolerant phenotype of hit5 (Wu et al., 2019). These findings provide vital clues into search for a potential HSP70–HSP40 pair crucial for heat tolerance in plant. Here, we describe the experimental evidence of such a HSP70–HSP40 pair.

Section snippets

Plant materials and growth conditions

The j3 (SALK_141625), hsp70-1 (SALK_135531), hsp70-2 (SALK_085076), hsp70-3 (SALK_018731), hsp70-4 (CS850152), and hsp70-5 (CS809145) T-DNA insertion mutants were obtained from the Arabidopsis Biological Resource Center (ABRC; Columbus, OH, USA). The hit5 mutant (mutation at At5G40280) has been described by Wu et al. (2017). Homozygous mutant lines were confirmed by PCR-based genotyping (Supplementary Fig. S1). The transgenic j3 line expressing the J3C417S construct was provided by Dr. Peter

HSP70-4 is required for plant tolerance to prolonged heat stress at 37 °C

Arabidopsis J3 is localized in the cytosol and nucleus (Yang et al., 2010; Shen et al., 2011). HSP70-1 (At5G02500), HSP70-2 (At5g02490), HSP70-3 (At3g09440), HSP70-4 (At3g12580), and HSP70-5 (At1g16030) encode cytosolic/nuclear HSP70s (Lin et al., 2001; Leng et al., 2017). Given that incubation at 37 °C for 4 d is lethal to the j3 null mutant but not to WT (Wu et al., 2019), a knockout mutation of HSP70 that specifically requires J3 as a partner for plant heat tolerance should give rise to a

Discussion

HSPs were originally discovered based on their role in heat stress response (hence the name “heat stress”), and it is therefore intuitive to associate their function with heat tolerance. Subsequently, numerous studies showed that the upregulation of particular HSP genes could enhance thermotolerance in plants (for review, see Haq et al., 2019). However, these studies only reveal the beneficial effects of HSPs on plants grown at high temperatures. To demonstrate the essential role of an HSP for

Conclusion

HSP70-4/AtJ3 is the first HSP70/HSP40-type chaperone machinery demonstrated to play a crucial role in protecting plants against prolonged heat stress. This study also reveals that protein farnesylation regulates HSP70-4/J3 function. These results will assist the research community to explore farnesylation-mediated and HSP70/HSP40-based stress responses in plants.

Funding

This work was supported by the Ministry of Science and Technology, Taiwan (MOST 108-2311-B-008-004-MY3 to S.-J. Wu).

CRediT authorship contribution statement

Tzu-Yun Wang: Investigation, Methodology, Software, Validation, Formal analysis, Writing - original draft, Project administration. Jia-Rong Wu: Methodology, Validation, Resources. Ngoc Kieu Thi Duong: Investigation, Validation. Chung-An Lu: Methodology, Supervision. Ching-Hui Yeh: Methodology, Supervision. Shaw-Jye Wu: Conceptualization, Investigation, Writing - original draft, Writing - review & editing, Project administration, Funding acquisition.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The authors are grateful to Dr. Peter Brodersen (University of Copenhagen, Denmark) for providing the J3C417S transgenic line.

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