**4. Discussion**

In this study, we found that the expression of *BAG9* was highly induced under heat stress in tomato. *Bag9* mutants reduced thermotolerance while overexpressing *BAG9* increased thermotolerance as reflected by antioxidant assays. We also found that BAG9 interacted with Hsp20 proteins in vitro and in vivo. Overexpressing *BAG9* enhanced the accumulation of Hsp proteins induced by heat, while the mutants had the opposite tendency. Thus, BAG9 played a crucial role in response to heat stress by regulating cellular redox homeostasis and the stability of heat shock proteins.

Similar to our study, the transcript levels of *OsBAGs* and *BAG* family members in grapes were significantly increased under heat exposure [48,49]. Considering that *BAG9* contained the HSE in the promoter region, it was selected to conduct further research for its potential significance in thermotolerance. BAG9 contained a conserved BAG domain and a CaM binding motif. The BAG domain combined with Hsc70 for decomposing incorrectly folded or translocated chloroplast proteins in *Arabidopsis* [50]. The phylogenetic analysis revealed that BAG9 was most close to OsBAG5, OsBAG6, AtBAG5, and AtBAG6. According to previous research and evolutionary relationships, we speculated that BAG9 may function in temperature protection, especially heat stress by binding with Hsps and maintaining cellular stability or involving in the Ca2+ sensing [28,48].

Various kinds of BAG proteins functioning in plant thermotolerance have been identified [28,29]. Heat shock-induced gene 1 (HSG1), a grape Bcl-2-associated athanogene, enhanced heat tolerance and activated *CONSTANS* (CO) expression in transgenic *Arabidopsis* plants [51]. In *Arabidopsis*, heat shock transcription factor (HsfA2) directly bound to HSE motif of *AtBAG6*, which dramatically increased its relative expression under heat stress [52]. AtBAG2 enhanced survival under heat by clearing ROS in plants [28]. AtBAG7 played a key role in mediating the heat-induced UPR pathway [29]. Studies in the BAG family showed that BAG9 stimulated burning symptoms under heat and reduced the thermotolerance of tomato, which did not occur in our experiment [53]. By overexpressing *BAG9* in *Arabidopsis*, the sensitivity to water scarcity, salinity, as well as ABA during the germination of seeds and the growth of seedlings were increased [54]. *BAG5b* (Solyc10g084170, namely *BAG9*) in leaves was activated by various adversity stimuli (extreme temperatures, salinity, and UV light) as well as treatment with phytohormones. Specifically, it improved the resistance to dark-induced leaf senescence by eliminating ROS and downgrading genes associated with leaf senescence [34].

In this study, *P*n, a typical indicator of photosystem I (PSI), was decreased in *bag9* mutants but was highly increased in *BAG9* overexpressing plants compared with WT plants. Similarly, *BAG9* overexpressing plants showed higher *Fv/Fm* values, and the mutants showed compromised *Fv/Fm* values than WT plants. Our results indicated that BAG9 promoted the stability of photosynthesis under heat exposure. Photosynthesis is a thermosensitive physiological process since the photochemical reactions and the carbon metabolism are susceptible to damage under heat exposure [55]. The disruption of the thylakoid membranes inhibits the rate of photosynthesis and PSII activity is also greatly reduced or even stopped under heat stress [56]. Chaperones protect and enhance photosynthesis under stressful environments [56]. The thermal resistance of photosystem II is upregulated by constitutive overexpression of a small Hsp, which suggests that sHsps prevent the damaging of photosynthetic apparatus from high temperature [57]. Hsp90 in the chloroplast was also an irreplaceable chaperone for protein translocating from the membrane into the organelles and served a significant role in heat resistance in photosynthetic organisms [58,59]. Similar to previous research, BAG9 served as a chaperone protein that may protect photosynthesis as shown in this study.

In previous studies, ROS is used as an indicator of plant resistance [60]. Overexpressing *AtBAG4* into the rice and exposing it to osmotic stress revealed that ROS accumulation was significantly reduced in its overexpressing plants [61]. The mutants *Atbag2* and *Atbag6* also showed higher ROS levels and less survival after heat treatment than WT [28]. Similar to the previous study, our results showed that *BAG9* overexpressing plants accumulated less ROS (H2O2, O2•−) and less protein carbonylation (which is a hallmark of protein oxidation), indicating a better resistance to high temperature. MDA is one of the products of ROS-induced membrane damage, whose amount represents the degree of cell membrane lipid peroxidation [60]. The continuous accumulation of MDA is positively correlated to high temperatures [62]. This study discovered that *BAG9* overexpressing plants showed less accumulation of MDA than WT, which indicated that BAG9 may protect biomembrane from being damaged under heat stress.

To mitigate elevated ROS-induced damage, plants have established a well-organized antioxidant-defense mechanism [62]. Antioxidants in plants have been classified into two main types: enzymatic and nonenzymatic antioxidants. The significant antioxidant enzymes in plant cells contain SOD, CAT, POD, and so on [63]. GSH and AsA are vital nonenzymatic antioxidants in plants. Meanwhile, APX, DHAR, and GR serve as significant enzymes in the AsA-GSH cycle [64]. Antioxidants are involved in multiple plant abiotic stresses, including heat stress [63]. Treating seedlings of *Broussonetia papyrifera* at high temperature, the activities of SOD, POD, and CAT were significantly increased [65]. The antioxidant enzyme activities in *Cruciferae* were closely related to high temperature, since its SOD, CAT, and GR activities under high-temperature (32 ◦C) stress were all higher than those of the control plants (20 ◦C) [66]. In *Brassica napus*, the developed activities of MDAR, DHAR, and GR under sub-high-temperature treatment (30 ◦C) elevated the levels of AsA and GSH, resulting in enhanced thermotolerance [67]. Similarly, our results illustrated that *BAG9* overexpressing plants upregulated the activities of antioxidant enzymes (SOD, CAT, POD, APX, DHAR, GR) and ratios of AsA/DHA and GSH/GSSG. All results indicated that the higher thermotolerance in *BAG9* overexpressing plants was probably achieved by enhanced activities of various antioxidants.

Hsps exist widely in plants to prevent stress from inducing damage to cells [68]. Previous studies showed that Hsp70 functioned in a chaperone cycle by Hsp70 chaperone systems [20]. BAG family is a kind of NEF that establishes direct interactions with the

ATPase domain of Hsp70 [25]. In tomato, results showed that BAG1 and BAG2 interacted with Hsp70 protein [69].

However, there have been no other Hsp–BAG interactions reported. In this study, we discovered that BAG9 interacted with Hsp20s (Hsp17.7A, Hsp17.7B, Hsp17.6B, Hsp17.6C) in the cytoplasm. Hsp20 is the predominant and most abundant class of proteins in many species induced by heat stress [70]. High temperature significantly induced the upregulation of *TaHsp17.4*, *TaHsp17.7A*, *TaHsp19.1*, and *TaHsp23.7* in wheat [71]. *OsHsp20* overexpressing plants had longer root length and higher germination rates than the control under heat and showed better resistance to high temperature [70]. Nonetheless, how BAG9 works under heat stress by interacting with Hsp20s requires further study.

Our results also witnessed the increase in the accumulation of Hsps (Hsp20, Hsp70, Hsp90, Hsp101) in *BAG9* overexpressing plants, indicating that BAG9 stimulated Hsps for enhancing thermotolerance. Hsp90 bound with Hsp70, establishing multiple complexes of chaperones and functioning well in sense signaling [72]. Hsp101, the most functional member in Hsp100, not only increased heat tolerance but also helped in recovery from heat shock [15]. However, the co-operations of BAG9, Hsp90, and Hsp101 need to be studied further.

#### **5. Conclusions**

In conclusion, we identified that BAG9 was involved in tomato thermotolerance. *BAG9* was highly induced under high temperature. *bag9* mutants were sensitive, while *BAG9* overexpressing plants were resistant under heat stress compared with WT. By analyzing the antioxidant and photosynthetic systems, we found that overexpressing *BAG9* may help in the removal of ROS and protect photosynthesis under heat stress. BAG9 interacted with Hsp20 proteins and protected Hsps accumulation under heat stress. In a word, BAG9 was probably significant for thermotolerance by regulating cellular redox homeostasis and the stability of heat shock proteins. Our findings further illustrated the functions of *BAGs* in adversity modulation, especially temperature stress.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antiox11081467/s1, Figure S1: Identification of *BAG9*-OE plants and *bag9* mutants; Figure S2: Analysis of *cis*-acting elements in the promoter region of *BAGs*; Table S1: Gene information of BAG family in tomato; Table S2: Primer sequences designed for RT-qPCR assays; Table S3: Primer sequences designed for pGBKT7 vectors construction; Table S4: Primer sequences designed for pGADT7 vectors construction; Table S5: Primer sequences designed for p2YC and p2YN vectors construction.

**Author Contributions:** J.Z. designed the research; H.H., C.L., C.Y., S.S. and Z.Q. performed the experiments; H.H. and J.Z. analyzed the data; J.Z., H.H., M.K.K. and C.L. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the National Key Research and Development Program of China (2019YFD1000300), the National Natural Science Foundation of China (31922078 and 31872089), the Public Projects of Zhejiang Province (LGN20C150011), the Fundamental Research Funds for the Central Universities (226-2022-00122), and the Starry Night Science Fund of Zhejiang University Shanghai Institute for Advanced Study (SN-ZJU-SIAS-0011).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article and Supplementary Materials.

**Conflicts of Interest:** The authors report no conflict of interest.
