**1. Introduction**

Global warming exacerbates the occurrence of extreme weather, among which high temperature is a major environmental threat to crop yields [1]. Under heat stress, the ultrastructure and function of chloroplasts and mitochondria suffer damage, resulting in a burst of reactive oxygen species (ROS), such as singlet oxygen, superoxide anion, hydrogen peroxide, and hydroxyl [2]. The accumulation of ROS leads to the damage of nucleotides, membrane lipid peroxidation, and protein denaturation [3,4]. Furthermore, protein denaturation induced by high temperature results in oxidation, misfolding, and aggregation of proteins. The gathering of these proteins leads to cell death in the absence of chaperones, proteasomes, and autophagy systems [5].

Molecular chaperones help in maintaining protein homeostasis under heat by restoring the native conformation of proteins and preventing the aggregation of non-native proteins for later folding or assembling [6]. Five groups of molecular chaperones heat shock proteins

**Citation:** Huang, H.; Liu, C.; Yang, C.; Kanwar, M.K.; Shao, S.; Qi, Z.; Zhou, J. BAG9 Confers Thermotolerance by Regulating Cellular Redox Homeostasis and the Stability of Heat Shock Proteins in *Solanum lycopersicum*. *Antioxidants* **2022**, *11*, 1467. https://doi.org/10.3390/ antiox11081467

Academic Editor: Nafees A. Khan

Received: 17 June 2022 Accepted: 25 July 2022 Published: 27 July 2022

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(Hsps) have been identified, including small heat shock proteins (sHsps)/Hsp20, Hsp60, Hsp70, Hsp90, and Hsp100 [7,8]. They not only protect proteins, but also increase the stability of lipid membranes, membrane proteins such as the photosystems, and soluble proteins [9]. Small Hsps are distinguished from other Hsps since they work in an ATPindependent manner to form a complex with non-native proteins preventing the harmful aggregation of proteins under stress [10]. Overexpressing *OsHsp18.2* in *Arabidopsis* highly enhanced the activity of seeds and the percentage of germination under heat stress [11]. Hsp60 especially improved the thermotolerance of plastid proteins such as Rubisco and retarded cell death [12,13]. Hsp90 interacted with the FK506 binding proteins (FKBPs) regulating thermotolerance [14]. In Hsp100 class, Hsp101 exhibited significant heat resistance and functioned well in recovery from heat shock [15,16]. Co-operation between Hsp100 and heat stress-associated 32-KD protein (HSA32) promoted the effects of heat acclimation in rice seedlings [17].

Among Hsps, Hsp70 regulating mechanism has been widely researched [18]. The work of hsp70 is assisted by a large chaperone system [19–21]. Under cell stress, ATP hydrolysis is indispensable for the binding of Hsp70 to polypeptide chains in non-native protein structures [22]. J-proteins are significant components in the Hsp70 chaperone system, which involve in heat stress response by regulating ATP activity, thus enhancing the binding affinity of Hsp70 with unfolded peptides or other substrates [23]. Nucleotide exchange factors (NEFs) are also necessary co-chaperones in the Hsp70 system [24]. Bcl-2-associated athanogene (BAG) has been identified as a NEF chaperone family, which contains a BAG domain interacting with Hsp70 on its ATPase domain, influencing nucleotide exchange by assisting ATP to bind with Hsp70 and releasing ADP, enhancing protein quality control. The BAG family may establish an association between the Hsp chaperone system and its substrates [25].

As chaperones, the BAG family in plants plays various roles in response to multiple stresses such as heat, freezing, salinity, drought, and ultraviolet (UV) [26,27]. For temperature resistance, *Atbag2* or *Atbag6* mutants survived worse under heat [28]. Upon sensing heat, the processed AtBAG7 entered the nucleus from the endoplasmic reticulum (ER) to interact with WRKY29, initiating unfolded protein response (UPR) pathway to enhance thermotolerance [29,30]. For pathogen resistance, BAG6 activated autophagy by being cleaved by aspartyl protease (APCB1) upon recognizing an intrusive pathogen in *Arabidopsis thaliana* [31]. Similarly in rice, enhanced blight and blast resistance 1 (EBR1) targeted OsBAG4, ubiquitinating and degrading it for immunity regulation and extensive defense against disease [32]. For inhibiting senescence, the signal complex calmodulinlike motif (CaM)/AtBAG5/heat shock cognate 70 (Hsc70) upregulated a high level of Ca2+ in mitochondria to inhibit senescence [33]. Likewise in tomato, BAG2 and BAG5b improved the resistance to dark-induced leaf senescence [34]. Various abiotic stresses induced AtBAG4 and regulated ion channels and stomatal motion by interacting with and adjusting KAT1 [35,36].

Tomato is one of the main economic crops in protected cultivation. Heat stress deranges metabolic imbalance in tomato, highly decreasing the quality and production [37]. However, the mechanism of BAGs affecting the thermotolerance of tomato is unclear. To further explore the role of the BAG chaperone family under heat stress and its relationship with Hsps, we generated *BAG9* overexpressing lines and *bag9* mutants and treated them with high temperature. We observed the phenotypes and measured a range of resistance indicators. Results showed that *bag9* was more sensitive to heat stress compared to the wild type (WT), while *BAG9* overexpressing plants showed the opposite tendency. It indicated a positive regulatory effect of BAG9 in temperature tolerance.

#### **2. Materials and Methods**

#### *2.1. Phylogenetic Analysis and Structural Domain Prediction of BAG Family*

The amino-acid sequences of BAG family proteins in *Solanum lycopersicum*, *Arabidopsis thaliana*, *Oryza sativa*, and *Nicotiana tabacum* were obtained from the Ensembl Plants database (http://plants.ensembl.org (accessed on 4 May 2020)). The set of protein sequences was imported into the Molecular Evolutionary Genetics Analysis tool (MEGA 11) and multiplexed using the ClustalW method and exported in MEGA format. The phylogenetic tree was constructed using the maximum likelihood tree (ML) method and the bootstrap analysis was applied with 1000 replicates/iterations. Finally, the constructed phylogenetic tree was polished with Evolview (http://evolgenius.info (accessed on 5 May 2020)). Structural domains of the BAG family in tomato were analyzed using the native InterProScan program (http://www.ebi.ac.uk/interpro/ (accessed on 5 May 2020)). The structural domain sequences were obtained from the Pfam database and the structural schematics were manufactured using Domain Graph (DOG) software (http://dog.biocuckoo.org/ (accessed on 5 May 2020)).
