Natural variation of AcEGY3 mediates chloroplastic ROS homeostasis to confer kiwifruit thermotolerance

Ling, Chengcheng; Yang, Jun; Xu, Jiale; Tang, Wei; Liu, Yunyan; Wang, Yingzhen; Li, Pengwei; He, Yan; Ouyang, Zhiyin; Chen, Shuqi; Xing, Fei; Wang, Xinxin; Liu, Pu; Liu, Yajing; Wang, Runze; Liu, Xiaofen; Yin, Xueren; Huo, Heqiang; Li, Dawei; Smalle, Jan; Liu, Yongsheng; Wang, Songhu

doi:10.1038/s41467-025-61593-5

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Published: 04 July 2025

Natural variation of AcEGY3 mediates chloroplastic ROS homeostasis to confer kiwifruit thermotolerance

Chengcheng Ling^1,2^na1,
Jun Yang^1,2^na1,
Jiale Xu¹,
Wei Tang¹,
Yunyan Liu¹,
Yingzhen Wang¹,
Pengwei Li ORCID: orcid.org/0000-0002-1146-0702¹,
Yan He¹,
Zhiyin Ouyang¹,
Shuqi Chen¹,
Fei Xing¹,
Xinxin Wang¹,
Pu Liu¹,
Yajing Liu¹,
Runze Wang¹,
Xiaofen Liu¹,
Xueren Yin¹,
Heqiang Huo³,
Dawei Li⁴,
Jan Smalle ORCID: orcid.org/0000-0002-6067-4619⁵,
Yongsheng Liu¹ &
…
Songhu Wang ORCID: orcid.org/0000-0002-7822-769X¹

Nature Communications volume 16, Article number: 6184 (2025) Cite this article

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Abstract

The genus of Actinidia consists of the popular kiwifruits consuming worldwide. Most kiwifruit species are naturally distributed in warm and moist environments. So, they are vulnerable to high levels of heat stress. Till now, genetic basis of kiwifruit thermotolerance has remained largely unexplored. Here, we uncover a natural variation responsible for thermotolerance in kiwifruit species. We reveal that thermotolerant kiwifruit species have increased expression of AcEGY3, a gene encoding a chloroplast-localized protein, which can promote expression of nuclear thermotolerance genes via H₂O₂-dependent retrograde signaling. We show that natural variation in the promoter of AcEGY3 constitute a binding site for the heat stress-inducible transcription factor AcGATA1. The increased expression of AcEGY3 is regulated by AcGATA1 and its possible interaction with another transcription factor AcHSFA2-2. This natural variation is absent from the thermosensitive kiwifruit species. Collectively, our results reveal genetic basis of kiwifruit thermotolerance and set the foundation for breeding thermotolerant kiwifruits cultivars.

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Introduction

The perennial woody kiwifruit species belong to the genus Actinidia. Their fruits are popular worldwide because of their appealing taste and the high contents of vitamin C, flavonoids, various minerals, and other bioactive compounds^1,2. The Actinidia genus includes 54 species, and four species (A. chinesis, A. deliciosa, A. eriantha, and A. arguta) have been bred into commercial kiwifruit cultivars². Most kiwifruit species are naturally distributed in warm, moist environments³ and are vulnerable to various abiotic stresses such as drought^4,5, high salinity⁶, waterlogging⁷, and extreme temperature^8,9,10. High temperature (heat stress) causes leaf curling, inhibits photosynthesis, reduces fruit quality and yields, and can lead to plant death^11,12,13. Due to global warming, heat stress is becoming a bigger issue for kiwifruit production, putting increased emphasis on breeding programs to improve the heat stress tolerance of this important crop. However, the genetic basis of kiwifruit thermotolerance has remained largely unexplored.

To survive heat stress, plants perceive the temperature increase^14,15,16,17 and transduce this signal into the nucleus to transcriptionally activate heat stress-responsive genes¹⁸. The transcriptional network of a heat stress response involves several key transcription factors, including HEAT SHOCK TRANSCRIPTION FACTOR A1s (HSFA1s)^19,20, HSFA2^21,22,23,24, HSFA3^25,26, and DREB2A^25,27. Genes encoding heat shock proteins (HSPs) functioning as molecular chaperones and reactive oxygen species (ROS)-scavenging enzymes are major targets of the above-mentioned transcription factors^18,28. In kiwifruit, a recent study showed that overexpression of AcHSFA2-1 enhanced heat stress tolerance by activating multiple genes, including AcHSP20s⁹.

Although ROS accumulation negatively impacts plant growth, low concentrations of ROS, especially hydrogen peroxide (H₂O₂), can act as secondary messengers and play a positive role in the acclimation to heat stress²⁹. Similar to cytosol and nucleus, mitochondria and chloroplasts also produce H₂O₂ during heat stress³⁰, which subsequently diffuses into the cytosol and nucleus^29,30,31. Heat-induced H₂O₂ plays a major role in the activation of HSFs and the resulting induction of HSP genes during the early phase of heat stress³². The increased cytosolic H₂O₂ causes the redox-dependent activation and translocation of HSFs and MBF1C from the cytosol to the nucleus^33,34,35, both of which cooperatively regulate the expression of many HSPs and other genes required for thermotolerance¹⁸. Cytosolic H₂O₂ also activates the kinase OXIDATIVE SIGNAL-INDUCIBLE1 (OXI1), which is required for activation of mitogen-activated protein kinases and downstream responses³⁶.

Investigation of natural variation (NV) has contributed substantially to understanding the genetic basis of agronomic traits and to the development of molecular markers for crop breeding³⁷. NV of thermotolerance has been found in many plant species, including Arabidopsis^38,39, wheat^40,41,42, rice^43,44,45, maize⁴⁶, tomato²³, and grapevine⁴⁷. Kiwifruit species are woody perennials characterized by long juvenile periods, and it may take up to 5 years to bloom in the field⁴⁸. The long crossing cycle caused by these long juvenile periods is an unavoidable obstacle for map-based cloning efforts aimed at the genetic improvement of kiwifruit cultivars⁴⁸. Because of this technical limitation, very few genes have been identified via genetic approaches^49,50,51.

In this study, through map-based cloning, we identify an NV in the promoter of the EGY3 gene that is responsible for the differential thermotolerance levels observed between the two species of kiwifruit. We find that heat stress-induced EGY3 expression reaches a higher level in the thermotolerant species, and that this elevated gene expression is due to the presence of a promoter binding site for the transcriptional activator GATA1. However, this NV is absent in the EGY3 promoter of the thermosensitive species. We show that the elevated expression of EGY3 under heat stress is caused by the coordinate binding of GATA1 and HSFA2-2 to the EGY3 promoter.

Results

Map-based cloning of the thermotolerance-promoting gene AcEGY3

A wild species of A. eriantha (named “MH-Z11”) is hypersensitive to heat stress (Fig. 1a), when compared to variety HY0809 derived from A. chinensis cv. “Hongyang.” In response to an 8-h treatment at 40 °C, MH-Z11 plants showed visible signs of heat stress sensitivity as evidenced by the curling and wilting of leaves, while most HY0809 leaves remained intact (Fig. 1a). After a recovery period of 2 days at 24 °C, most leaves of MH-Z11 were dead while 80% of HY0809 leaves survived (Fig. 1a, b). The net photosynthetic rate (Pn) of MH-Z11 was dramatically reduced after a 1 h heat treatment and hardly detected after an 8 h treatment, while that of HY0809 remained at about 60% of the 0 h control (Fig. 1c).

**Fig. 1: Map-based cloning of *AcEGY3* mediating thermotolerance of kiwifruit.**

To perform map-based cloning of gene variants that potentially mediate this thermotolerance variation, we constructed a backcross population between MH-Z11 and HY0809 (Supplementary Fig. 1a). Kiwifruit is a dioecious species. A female individual of MH-Z11 was crossed with a male HY0809 plant to generate F1 plants. F1 plants showed higher Pn values under heat stress than MH-Z11 and HY0809 (Supplementary Fig. 1b), suggesting that HY0809 has one or more dominant gene variants mediating thermotolerance. Next, a male plant of the F1 generation was backcrossed with MH-Z11 (female) to produce the backcross1 (BC1) population containing about 130 individuals (Supplementary Fig. 1a). In this population, we collected genomic DNA samples of individual plants whose Pn values were either less than 3 (<3) or more than 9 (>9) to build two mixed pools (Supplementary Fig. 1c), which were used for BSA sequencing. QTL mapping (Fig. 1d) was performed by using the GradePool-seq method⁵², based on the chromosome-level reference genome of HY (v3.0)⁵³. The highest ratio value was associated with a region of 380 kb on chromosome group 2 (Fig. 1d), containing 17 annotated genes (Fig. 1d). Sanger sequencing of the coding regions of these 17 genes from the two Kiwifruit species, did not reveal any mutations causing premature stop codons, frame shifts, insertions or deletions. We did find a few SNPs causing amino acid variations between MH-Z11 and HY0809. However, none of these variations were predicted to alter conserved domains of the respective proteins, suggesting that they do not cause functional differences.

Transcriptomic analyses (Supplementary Data 1) revealed that only one of the 17 genes (Actinidia02441) was induced by the heat stress treatment (highlighted in the red in Fig. 1e) and that there is a substantial difference in induction level between the MH-Z11 and HY0809 backgrounds (Fig. 1f). This gene-of-interest encodes a protein with high similarity to EGY3 from Arabidopsis^54,55, which mediates chloroplastic ROS homeostasis and retrograde signaling in response to salt stress. AcEGY3 (HY0809) and AeEGY3 (MH-Z11) share 98% identity at the protein sequence level. Similar to Arabidopsis EGY3 (AtEGY3), AcEGY3 and AeEGY3 have the conserved GNLR motif and are also missing the HEXXH and NPDG motifs that are important for proteolytic activity (Supplementary Fig. 1d). Also similar to Arabidopsis EGY3, AeEGY3 is localized in the chloroplast (Supplementary Fig. 2a). As the N-terminal signaling peptides of AcEGY3 and AeEGY3 are identical, we concluded that AcEGY3 is also most likely chloroplast localized. Considering that Arabidopsis EGY3 mediates ROS homeostasis in response to stress and that ROS play an indispensable role in thermotolerance³², we hypothesized that the different thermotolerance levels between MH-Z11 and HY0809 are caused by the different expression levels of AcEGY3 and AeEGY3 under heat stress conditions. In Arabidopsis, EGY3 promotes H₂O₂ accumulation in response to salt stress by increasing the stability of Cu/Zn-SOD2 (CSD2), which turns O₂^.− into H₂O₂ that induces the expression of genes needed for salt tolerance⁵⁴. In support of this hypothesis, we found that AeEGY3 shows a protein interaction with AeCSD2, as confirmed by co-immunoprecipitation (Co-IP) and luciferase complementation imaging (LCI) (Supplementary Fig. 2b, c). Besides, MH-Z11 accumulated less H₂O₂ but more O₂^.− under heat stress when compared to HY0809, suggesting that the dismutation of O₂^.− to H₂O₂ is attenuated in MH-Z11 plants (Supplementary Fig. 3a). This was also observed previously with the salt stress hypersensitive Arabidopsis egy3 loss of function mutant that accumulated less H₂O₂ but more O₂^.− than wild-type plants⁵⁴. The higher H₂O₂ level in HY0809 plants indeed corresponded with elevated SOD activity, indicating that EGY3 serves similar functions in the salt and heat stress tolerance of respectively Arabidopsis and Kiwifruit (Supplementary Fig. 3b). In addition to SOD, heat-stressed HY0809 plants also had higher ascorbate peroxidase (APX) activity when compared to MH-Z11 (Supplementary Fig. 3c). In contrast, the activity levels of peroxidase (POD) and catalase (CAT) were heat-induced to a higher level in MH-Z11 (Supplementary Fig. 3d, e).

Considering that H₂O₂ is a known inducer of HSP gene expression³², the lower H₂O₂ level in heat-stressed MH-Z11 suggests that it could be one of the reasons causing its heat stress sensitivity. Indeed, most differentially expressed HSPs showed higher levels in HY0809 than in MH-Z11 after a 1 h heat stress treatment (green frame in Supplementary Fig. 4a–d). Exogenous application of low concentrations of H₂O₂ could partially rescue the heat sensitivity and expression of HSPs in MH-Z11 (Supplementary Fig. 4e–h).

To further analyze the involvement of EGY3 in thermotolerance, we generated several independent AeEGY3-FLAG overexpression (OE) lines in the MH-Z11 background (Fig. 2a, b). These AeEGY3-OE lines were significantly more thermotolerant than MH-Z11, as indicated by the increased leaf survival rates and the higher Pn values under heat stress (Fig. 2c, d), thus confirming that the EGY3 expression level is positively correlated with heat stress tolerance. Indeed, a 1 h heat treatment increased the expression of HSP20 and HSP70-2 to a substantially higher level in all three OE lines when compared to MH-Z11 (Fig. 2e). AeEGY3-FLAG overexpression also resulted in increased H₂O₂ accumulation and SOD activities (Supplementary Fig. 5a, b). In addition to HSP genes, AeEGY3-FLAG overexpression also caused the upregulation of other H₂O₂-mediated retrograde-signal-induced genes, including ZT10, ZT12 (Supplementary Fig. 5c, d), and APX2 (Fig. 2f). We also used a H₂O₂-specific fluorescent probe, BES-H₂O₂-Ac, to visualize its accumulation in subcellular compartments. Under 1 h heat treatment, chloroplasts of mesophyll cells in MH-Z11 produced less H₂O₂ than those in HY0809, while AeEGY3-FLAG overexpression increased chloroplastic H₂O₂ accumulation in MH-Z11 (Supplementary Fig. 5e).

**Fig. 2: *EGY3* is required for kiwifruit thermotolerance.**

To obtain conclusive proof that the EGY3 expression level is a key determinant of kiwifruit thermotolerance, we used RNA interference (RNAi) to silence AcEGY3 in HY0809 and obtained three RNAi lines in which AcEGY3 expression was significantly decreased, even under heat stress conditions (Fig. 2g, j). As expected, all three lines were more sensitive to heat stress compared to HY0809, as indicated by the wilting leaves, reduced survival rates of leaves, and lower Pn values (Fig. 2h, i). Moreover, HSP and APX2 gene expression was downregulated in these RNAi lines, thus confirming the important role of EGY3 in promoting the retrograde signaling-based induction of stress tolerance genes (Fig. 2j). Collectively, these results reveal a positive role for EGY3 in kiwifruit thermotolerance and that the differential expression of AcEGY3 and AeEGY3 is responsible for the thermotolerant difference between HY0809 and MH-Z11.

AcEGY3 promoter contains a GATA binding site, which is absent in AeEGY3

To determine the underlying cause for the differential heat stress induction of AcEGY3 and AeEGY3, we fused the promoters (pro) of both genes to the coding region of luciferase (LUC) and introduced the resulting proEGY3^HY0809(proEGY3^HY)::LUC and proEGY3^MHZ11(proEGY3^MH)::LUC constructs into HY0809. The transgenic plants for both promoter fusion constructs had very low luminescence intensities under control conditions (Fig. 3a, b). In response to a 37 °C treatment, proEGY3^HY::LUC plants displayed remarkably stronger luminescence signals and LUC activities compared to the proEGY3^MH::LUC plants (Fig. 3a, b), which is consistent with the different expression levels of the corresponding endogenous EGY3 genes in HY0809 and MH-Z11 (Fig. 1f). These results indicated that the expression level difference between AcEGY3 and AeEGY3 is caused by one or more differences at the promoter level.

**Fig. 3: AcGATA1 promotes *AcEGY3* expression and HY0809 thermotolerance.**

Sequence analysis of proEGY3^HY and proEGY3^MH revealed that the two promoters share high DNA sequence similarity, except for a 492 bp fragment (NV) in proEGY3^HY which is absent in proEGY3^MH (red frame in Supplementary Fig. 6a and Fig. 3c). We randomly selected two groups of 30 plants each from the BC1 populations, in which this NV was either present (+) or absent (−), and checked their Pn values and EGY3 expression levels under heat stress. Our results show that +NV plants showed significantly higher Pn values and EGY3 expression than −NV plants (Supplementary Fig. 6g), indicating that this NV correlates with the segregating EGY3 expression and thermotolerance levels in the BC1 populations, which is consistent with the map-based cloning results (Fig. 1d).

The NV contains a cis element for the transcription factor GATA (Fig. 3c). Weighted correlation network analysis (WGCNA) (Supplementary Fig. 7a) was performed by using physiological results including H₂O₂ contents, SOD, CAT, and APX activities (Supplementary Fig. 3) as phenotypic input data. The results indicated that AcEGY3 is present in the blue module related to H₂O₂, SOD, and APX activities (Supplementary Fig. 7b) and shows a co-expression pattern with several transcription factor genes including Actinidia15578, annotated as AcGATA1 (Supplementary Fig. 7c). qRT-PCR analysis revealed that AcGATA1 expression is significantly induced by heat stress (Fig. 3d). Dual-luciferase reporter assays (DLRAs) showed that AcGATA1 activates proEGY3^HY but not proEGY3^MH (Fig. 3e), suggesting that AcGATA1 indeed binds the GATA cis element in proEGY3^HY. Electrophoretic Mobility Shift Assays (EMSAs) confirmed that AcGATA1 directly binds a probe containing the cis element “TGATAG” from HY0809 (HY-GATA-Box, HY-G-Box) but not the mutant probe (HY-G-Box-mut), of which the element was replaced with “CCCCCC” (Fig. 3f). Mutation of the G-box in proEGY3^HY (proEGY3^HYmut) inhibited its interaction with AcGATA1 (Supplementary Fig. 6c), while addition of this G-box to proEGY3^MH promoted its interaction with AeGATA1 (Supplementary Fig. 6d, e). Moreover, HY plants containing the proEGY3^HYmut::LUC transgene showed significantly lower luminescence intensities and LUC activities under heat stress compared to HY0809 plants containing proEGY3^HY::LUC (Supplementary Fig. 6b). These results show that the presence of the GATA binding site (G-box) is responsible for the elevated heat-induced AcEGY3 expression in HY0809 plants. Consistently, HY0809 transgenic lines overexpressing GATA1 (AcGATA1OE-1, -2, and -3) had significantly increased AcEGY3 mRNA levels (Fig. 3g, i), and enhanced thermotolerance compared to HY0809 control plants (Fig. 3g), as indicated by the increased leaf survival rates after a 12 h-long treatment at 42 °C (Fig. 3h).

AcGATA1 possibly interacts with AcHSFA2-2 to enhance AcEGY3 expression

According to our WGCNA results (Supplementary Fig. 7c), AcEGY3 was also co-expressed with an HSF-encoding gene (DTZ79_20g03210 in the A. eriantha genome⁵⁶ and Ach20g247541 in the A. chinensis genome²), with high sequence similarity to AcHSFA2-1⁹, and thereby we named it AcHSFA2-2. DLRAs showed that AcHSFA2-2 activates both proEGY3^HY and proEGY3^MHZ11 (Fig. 4a–d). The cis element “TTCTAGAC” (HSF binding site, H-Box) is present in both promoters of AeEGY3 and AcEGY3 (Fig. 3c), and EMSAs confirmed that AcHSFA2-2 and AeHSFA2-2 directly bind to DNA probes containing the respective H-Box sequences from proEGY3^HY and proEGY3^MHZ11 (Fig. 4e). Interestingly, DLRAs showed that co-expression of AcGATA1 and AcHSFA2-2 significantly enhanced the transcriptional activity of proEGY3^HY (Fig. 4f, g), but not that of proEGY3^MH (Supplementary Fig. 6d). However, the modified promoter proEGY3^MHZ11-G-box, which contains an artificial G-box, also displayed enhanced transcriptional activity in response to co-expression of AeGATA1 and AeHSFA2-2 (Supplementary Fig. 6e). Next, we observed a protein interaction between AcGATA1 and AcHSFA2-2 by LCI (Fig. 4h) and Co-IP assays (Fig. 4i). These results indicated that the higher expression of AcEGY3 under heat stress is caused by the coordinate binding of AcHSFA2-2 and AcGATA1 to the AcEGY3 promoter in HY0809 plants, which is absent in MH-Z11 plants as the corresponding AeEGY3 promoter doesn’t have a GATA1 binding site. However, due to the difficulty of generating high-order mutants in kiwifruit, we don’t have genetic evidence to confirm the interactions of AcGATA1 or AcHSFA2-2 with AcEGY3.

**Fig. 4: AcGATA1 possibly interacts with AcHSFA2-2 to cooperatively promote *AcEGY3* expression.**

AeHSFA2-2-FLAG overexpression in MH-Z11 substantially increased AeEGY3 expression and leaf survival rates after an 8 h treatment at 40 °C (Fig. 5a–c), indicating that AeHSFA2-2 acts as a limiting factor in this thermotolerance mechanism. To test if heat stress-induced changes in ROS homeostasis impact AeHSFA2-2 function, we analyzed the effects of H₂O₂ treatment and found that this causes the oligomerization (dimerization and trimerization) of AeHSFA2-2-FLAG in vivo, which was diminished by the antioxidant DTT (Fig. 5d). Moreover, methyl Viologen (MV), a herbicide inducing ROS in chloroplasts, enhanced but DMTU, a H₂O₂ scavenger, impaired the protein interaction between nLUC-AeHSFA2-2 and cLUC-AeHSFA2-2 (Fig. 5e). Both DMTU and DTT treatment impaired AeHSFA2-2-induced transcription activation of proEGY3^MH::LUC (Fig. 5f). These results indicate that stress-induced H₂O₂ enhances AeHSFA2-2 oligomerization and transcriptional activity, leading to increased expression of its target genes. In addition to EGY3, AeHSFA2-2 targets the promoter of AeHSP70-2 (proAeHSP70-2) (Fig. 5g), and EMSA confirmed that AeHSFA2-2 binds a probe containing the H-box from proAeHSP70-2 (Fig. 5h). AeHSP70-2 expression was also significantly increased in AeHSFA2-2-FLAG overexpressing plants (Fig. 5i). These results suggest that both AeEGY3 and AeHSP70-2 are transcriptionally regulated by AeHSFA2-2. However, we don’t have genetic evidence to confirm the interaction of AeHSFA2 with either AeEGY3 or AeHSP70-2.

**Fig. 5: Overexpression of *AeHSFA2-2* enhances *AeEGY3* expression, and H₂O₂-induced polymerization of AeHSFA2-2 promotes its transcription activity.**

The GATA-box natural variation is present in the EGY3 promoters of other thermotolerant kiwifruit species

To determine whether the AcEGY3 promoter GATA variant is also present in other kiwifruit species, we designed a pair of primers encompassing the GATA binding site of this promoter, and PCR assays on the genomic DNAs of a range of species revealed that, in addition to HY, this natural variant potentially also exists in JinTao (JT, A. chinensis) and MiLiang (ML, A. deliciosa), but is absent in RuanZao (RZ, A. arguta), and White (HT, A. eriantha) (Fig. 6a). Similarly to MH-Z11, plants of RZ, and HT were more sensitive to heat stress and had lower stress-induced EGY3 expression, when compared to HY, JT, and ML (Fig. 6b, c). Next, we cloned the EGY3 promoters from DongHong (DH, A. chinensis), JT, ML, BiYu (BY, A. chinensis), ShanLi (SL, A. rufa), and sequencing revealed that the GATA binding site “TGATAG” is indeed highly conserved in these promoters (Supplementary Fig. 6f). In addition, we identified varieties of A. eriantha containing the GATA promoter variation, including MH-JX02, MH-LP4, and MH-HT16 (Fig. 6d). We randomly picked MH-JX02 for further analyses, and this revealed that MH-JX02 plants are more tolerant to heat stress (Fig. 6e, f), that their heat-induced AeEGY3 expression is higher compared to MH-Z11 (Fig. 6f), and that AeHSFA2-2 and AeGATA1 can cooperatively activate the corresponding promoter resulting in high transcription activity (Fig. 6g).

**Fig. 6: Thermotolerance level of kiwifruit species correlates with the presence of a GATA-box in their respective *EGY3* promoters.**

Discussion

Kiwifruit plants favor warm and moist environments but are sensitive to extreme temperatures, with heat stress inhibiting their growth, fruit quality, and yield, and potentially also causing plant death^11,12,13. Prior to this study, we only knew that the AcHSFA2-1 gene is involved in promoting its thermotolerance⁹. Here, we have identified a NV in kiwifruit thermotolerance by comparing the thermosensitive variety MH-Z11 with the thermotolerant HY0809 (Fig. 1). Through BSA sequencing and QTL mapping of a backcross population between HY0809 and MH-Z11, combined with transcriptomic analyses, we revealed a NV in the promoter of EGY3 that was previously shown to promote salt and oxidative stress tolerance in Arabidopsis⁵⁴. The corresponding EGY3 genes from MH-Z11 and HY0809, respectively, AeEGY3 and AcEGY3, are both induced by heat stress. However, the strength of this stress induction is substantially higher for AcEGY3 (Fig. 1f). Overexpression and RNAi-based gene suppression experiments revealed that the level of EGY3 expression is positively correlated with thermotolerance (Fig. 2), implying that this differential gene expression between AeEGY3 and AcEGY3 is responsible for the difference in heat stress tolerance between MH-Z11 and HY0809.

Chloroplasts are widely considered as one of the main sites for heat stress damage in plants³⁰. Accordingly, they serve as a key heat stress sensing location to trigger a response that includes retrograde signaling aimed at nuclear gene expression changes that promote thermotolerance⁵⁷. Chloroplast is one of the major organelles producing H₂O₂, and this chloroplastic H₂O₂ acts as a retrograde signal, which is induced under heat stress conditions⁵⁸. This increased H₂O₂ production is a consequence of the increased formation of the superoxide radical O₂^.−, as a result of thermal damage to Photosystem I⁵⁹. CSD is required for the subsequent dismutation of O₂^.−. However, heat stress also potentially causes accelerated CSD degradation, and in Arabidopsis, this was shown to be counteracted by the EGY3 protein that binds and stabilizes CSD2^54,55. Accordingly, the egy3 mutant accumulates more O₂^.− but less H₂O₂ than wild-type plants under stress conditions^54,55, which is similar to what was observed for the thermosensitive MH-Z11 plants (Supplementary Fig. 3). Our preliminary data showed that AeEGY3 could also interact with AeCSD2 in kiwifruit (Supplementary Fig. 2b, c). Moreover, exogenous application of low concentrations of H₂O₂ rescued the sensitivity of the egy3 mutant to salt stress⁵⁴ and that of MH-Z11 to heat stress (Supplementary Fig. 4). Therefore, the low levels of H₂O₂ in heat-stressed MH-Z11 are most likely caused by the low heat stress-inducibility of the AeEGY3 gene. This notion is supported by our results that overexpression of AeEGY3 in MH-Z11 significantly increased H₂O₂ accumulation and thermotolerance (Supplementary Fig. 5a and Fig. 2). In addition to the increased expression of HSP-encoding genes (Fig. 2e), AeEGY3 overexpression enhanced chloroplastic H₂O₂ accumulation (Supplementary Fig. 5e) and caused elevated transcript levels of other retrograde-signal-responsive genes, such as APX2, ZAT10, and ZAT12 (Fig. 2f, Supplementary Fig. 5c, d). The same gene set was also induced by EGY3 overexpression in Arabidopsis⁵⁴. These results indicate that, in addition to its role in the salt stress response, EGY3 plays a crucial role in thermotolerance, suggesting that this protein is a shared player in plant adaptation to multiple abiotic stresses.

Sequence comparison revealed a 492 bp region in the AcEGY3 promoter which is absent in the promoter of AeEGY3 (Supplementary Fig. 6a). This NV constitutes a binding site for the heat stress-inducible transcription factor AcGATA1 (Fig. 3). AcGATA1 enhances the heat-stress induction of AcEGY3, through protein interaction and cooperation with the transcription factor HSFA2-2 that serves to promote a base-level of heat stress induction for both AcEGY3 and AeEGY3 (Fig. 4). HSFs usually form hetero- or homo-oligomers to fulfill their transcription activity^22,60,61, and our results indicated that H₂O₂ promotes the oligomerization of AeHSFA2-2 to enhance its transcription activity (Fig. 5e–g). Our results also revealed AeHSP70-2 as another target of AeHSFA2-2 (Fig. 5h–j). Therefore, heat stress-induced H₂O₂ could constitute a feedforward mechanism in that it promotes the expression of stress tolerance genes, but also further elevates EGY3 levels by activating the transcription factor that induces the EGY3 gene, thus generating even more EGY3-dependent H₂O_2, resulting in a further boosting of stress tolerance gene expression.

A hypothetical model that can explain thermotolerance difference between the MH-Z11 and HY0809 kiwifruit species is summarized in Fig. 6h. While heat stress induces both the expression of AcEGY3 and AeEGY3 in the respective HY0809 and MH-Z11 plants, this induction is stronger in HY0809 because the AcHSFA2-2-mediated induction is enhanced through a cooperative protein interaction with AcGATA1, which does not occur in MH-Z11. As a result, there is more EGY3 accumulation in HY0809 plants, leading to higher H₂O₂ production and elevated expression of stress tolerance genes. Moreover, this increased H₂O₂ accumulation promotes the oligomerization of HSFA2-2, which enhances its transcription activity, thus further increasing the expression of its target genes EGY3 and HSP70-2, which contribute to thermotolerance.

NVs are valuable resources for understanding and improving the genetic basis of agronomic traits. The thermotolerance-promoting NV is first identified between two species A. chinesis and A. eriantha, but it also exists between A. deliciosa and A. argute (Fig. 6a–c). Therefore, this variation could be a useful marker for the interspecific hybrid among these four species. In addition, the variation exists among different accessions of A. eriantha (Fig. 6d–f), suggesting it might also be useful for intraspecific hybridization in A. eriantha. Together, our results uncover possible interplays among transcription factors AcGATA1, AcHSFA2-2, and their target gene AcEGY3 in promoting kiwifruit thermotolerance. It provides a valuable avenue for molecular markers-assisted hybrid breeding programs aimed at improving the thermotolerance of economically important kiwifruit varieties.

Methods

Plant materials and heat stress treatment

An offspring line of A. chinensis cv. “Hong Yang” (HY0809), A. eriantha “MH-Z11” (a wild species), A. arguta cv. “Ruanzao” (RZ), A. deliciosa cv. “Miliang” (ML), A. chinensis cv. “Jintao” (JT), A. eriantha cv. “White” (HT) and A. eriantha “MH-JX02” (a wild species) were used in this study. Kiwifruit vines were planted in the orchards of the Anhui Agricultural University, Hefei, China. Healthy branches from kiwifruit vines were harvested and cultured in water for new sprouts to come out in the greenhouse. The 3–5 cm sprouts were surface-sterilized for tissue culture. The sterilized sprouts were subjected to an initial culture for bud induction on MS medium supplemented with 3% sucrose, 6-benzylaminopurine (BAP; 1.0 mg L⁻¹) and indole-3-butyric acid (IBA; 1.0 mg L⁻¹) solidified with 0.8% agar at pH 5.8. The new buds were subsequently further propagated on MS medium with zeatin 3.0 mg/L + BAP 1.0 mg/L + IBA 0.1 mg/L, and finally, rooting was induced on half-strength MS medium containing 1.0 mg L⁻¹ IBA. These tissue-cultured seedlings were then transferred into fertilized soils and grown in an environmentally controlled growth chamber (95% humidity, 16 h light:8 h dark, 24 °C) for about 2 months.

Sixty-day-old plants (about 40 cm tall) were used for heat stress treatments. Kiwifruit plants were transferred into a growth chamber (95% humidity) with the temperature set at 25 °C for 2 h, and then increased to 40 or 42 °C for 8 h. To document the impact of heat stress on intact plants, photographs were taken, and the survival rates of leaves were calculated after a 2-day recovery period at 24 °C.

Exogenous application of H₂O₂ before heat stress

Four hours before the treatments, a control group of MH-Z11 seedlings was sprayed with distilled water, and the other six groups were sprayed with freshly prepared 50, 100, 200, 500, 600, and 1200 µM H₂O₂, respectively. Leaves were sprayed on both sides until water dripping (around 150–200 mL solution applied for each pot). Heat treatments were performed in a climatic chamber according to the above-mentioned protocol. Photographs were taken, and the survival rates of leaves were calculated after a 2-day recovery (24 °C). For qRT-PCR analysis of gene expression, mature leaves were harvested 1 h after the heat stress treatment and immediately frozen in Liquid nitrogen, and then stored at −80 °C.

Vector construction and genetic transformation

The coding DNA sequences (CDS) of the respective EGY3, GATA, and HSFA2-2 genes were amplified using cDNA prepared from HY0809 and MH-Z11 leaf tissues, and fused with Flag-tag or eGFP-tag, then cloned into the pCAMBIA1302 vector to generate AeEGY3-FLAG-OE, AcGATA1-eGFP-OE, and AeHSFA2-2-FLAG-OE. The PCR-amplified cDNA fragments of AcEGY3 were inserted into the pHB vector⁶² to produce the RNAi construct 35S::AcEGY3-RNAi. The promoters (encompassing 2000 bp upstream of the start codon ATG) of AcEGY3 and AeEGY3 were fused with the luciferase tag and then inserted into the pCAMBIA1302 vector to produce the constructs proEGY3^HY::LUC and proEGY3^MHZ11::LUC.

A. tumefaciens strain EHA105, containing the recombinant plasmids, was used to infect explants of A. chinensis or A. eriantha according to methods^63,64. Petioles of A. chinensis were cut into 1 cm long pieces and used as infection materials, and transgenic calli were screened by incubation on growth medium containing 40 mg/L hygromycin. For A. eriantha transformation, 2 cm long leaf strips were inoculated with EHA105 containing the target vector and transferred to selection medium containing 40 mg/L hygromycin and Timentin. After 4 weeks, green callus nodules were formed along the edges of leaf strips. These calli developed into shoots after two subcultures. The presence of transgenes and their expression levels in regenerated shoots were determined by PCR, western blot, and qRT-PCR analyses. The primers used for vector construction and identification of transgenic lines are listed in Supplementary Data 2.

Measurements of net photosynthetic rate

The net photosynthetic rate of mature leaves was measured at the four time points of heat stress treatments (0, 1, 3, and 8 h), using a LI-6800 portable photosynthesis system (Li-Cor Inc., Lincoln, NE, USA). The Li-6800 chamber was set at a light intensity of 10000Lux and an 80% relative humidity. Per plant line, a minimum of 9 plants each from three independent experiments were measured.

For the backcross population, the net photosynthetic rates of individual plants were measured at 39–40 °C in the open field. The Li-6800 chamber was set to maintain light intensity at 160,000 Lux and relative humidity at 60%. All individual plants were tested in three independent experiments.

BSA sequencing and QTL analysis

The backcross populations (about 130 strains) were evaluated by measuring net photosynthetic rate (Pn) at 39–40 °C in the open field. The Li-6800 chamber was set to maintain the light intensity at 160,000 Lux and the chamber’s relative humidity at 60%. Each individual plant was measured in three independent experiments. The plants with Pn <3 or >9 were collected into two groups (two pools), respectively. For each pool, the genomic DNAs of individual plants were extracted from fresh leaf tissue of backcross populations using a Plant genome DNA extraction kit (BioFit), and equal amounts of DNA were mixed into a pool for sequencing. A sequencing library was constructed with an insert size of 400–500 bp for a single index according to the protocol of TruSeq DNA PCR-free prep kit (Illumina® platforms). Based on the reference genome sequence (A. chinensis Hong Yang v3 Genome), rapid mapping of QTLs was performed using GradedPool-Seq and the Radit (SNP/InDel) test for differences between the two mixed pools⁵².

RNA extraction and qRT-PCR analysis

Total RNAs were extracted at different time points from heat-treated mature leaves using the NucleoSpin RNA plant kit (TianGen, Beijing, China). qRT-PCR was used to validate the transcriptome data by analyzing the expression levels of HSPs, EGY3, APX2, and FSD2. Transgenic Kiwifruit leaves were also identified by qRT-PCR. Relative gene expression levels were analyzed using the 2^−ΔΔCt method⁶⁵. To standardize the data, kiwifruit Actin was used as the internal control. The primers used for qPCR are listed in Supplementary Data 2.

ROS assay

To determine H₂O₂ and O₂^– levels, 3,3′-diaminobenzidine (DAB) and Nitroblue tetrazolium (NBT) histochemical stainings were performed by using commercial DAB and NBT solutions (Coolaber, Beijing). Mature leaves were immersed in DAB or NBT staining solution and infiltrated under a vacuum for 20 min. Before photography, samples were incubated for 8 h at room temperature in the dark, and then 95%-100% (v/v) ethanol was used for decoloring.

H₂O₂ in chloroplasts was visualized by using a H₂O₂-specific fluorescent probe, BES-H₂O₂-Ac (Wako, #024-18751). Plants of HY0809, MH-Z11, and AeEGY3-OE lines were treated at 40 °C for 0 h (control) and 1 h. The mature leaves were immersed in 100 μM BES-H₂O₂-Ac for vacuum infiltration and incubated for 20 min.

Analysis of antioxidant enzyme activities

Protein extractions were prepared by first grinding a 0.1 g leaf sample with liquid nitrogen, followed by the addition of ice-cold 50 mM potassium phosphate buffer (pH 7.5) containing 0.5 mM EDTA and 1% polyvinylpolypyrrolidone for the detection of SOD, POD, and CAT assays. For APX detection, an additional 1 mM AsA was incorporated into the buffer. The extraction was centrifuged at 12,000 × g for 20 min at 4 °C, and the supernatant was harvested for enzyme assays. The activity of SOD was determined by the reduction of NBT and was measured at 560 nm (A₅₆₀)⁶⁶. APX activity was measured by recording A₂₉₀, which reflects the decrease in absorbance of ascorbate (Ɛ₂₉₀ = 2.8 L mmol⁻¹ cm⁻¹), according to the method⁶⁷. POD uses H₂O₂ to oxidize guaiacol to produce a brown product, which has a light absorption value at 470 nm. The POD activity was measured by monitoring A₄₇₀ at intervals of 30 s within a 3-min-long period⁶⁸. CAT activity was assayed using the measurement of H₂O₂ consumption at 240 nm for 1 min⁶⁹.

RNA sequencing and WGCNA analysis

Sixty-day-old plants of HY0809 and MH-Z11 were treated with 40 °C for 8 h. Mature leaves were harvested at 0 h (before the temperature increase), 1, 3, and 8 h. Each time point included three biological replicates. All harvested leaves were immediately frozen in liquid nitrogen and stored at −80 °C for RNA sequencing. Total RNAs of 32 samples were extracted and sequenced by Shanghai MajorBio Bio-pharm Technology Co. (China). The raw data of RNA sequencing was uploaded to the NCBI with the registration number PRJNA1017621.

The WGCNA package (v.1.72) with default parameters was utilized to generate co-expression network modules using the FPKM values of DEGs⁷⁰. We performed automatic network construction (blockwise Modules) using the default parameters, except for the soft threshold power set at 28, the TOM type was set as signed, the mergeCutHeight at 0.35, and the minModuleSize at 100, to construct co-expression modules. The eigengene value for each module was computed in order to investigate its correlation with phenotypic data (such as, H₂0₂, SOD, CAT, APX, PRO, MDA and Vc) during heat treatment. The transcriptional regulatory networks were constructed by merging the Pearson correlation coefficient between transcription factors and structural genes in the same module. The visualization of the networks was performed using Cytoscape software (v.3.7.2, USA)⁷¹.

Subcellular localization

The coding regions of AcEGY3 without a stop codon and eGFP without ATG were used to create AcEGY3-eGFP by using fusion PCR. The fused fragment was inserted into the pCAMBIA1302 vector to produce 35S::AcEGY3-eGFP. Agrobacteria (EHA105) containing 35S::AcEGY3-eGFP or 35S::eGFP were injected into leaves of 1-month-old tobacco, according to the method⁶². After incubation at 25 °C in darkness for 12 h, followed by 16 h light/8 h darkness for 2 days, fluorescence signals were determined by confocal laser scanning microscopy (Zeiss LSM 700, Germany).

Dual-luciferase assay

Promoter sequences were first analyzed with PLANTPAN 3.0 software⁷² to identify the cis elements. For the Dual-luciferase assay, the promoters of AcEGY3 and AeEGY3 were cloned into the pGreen0800-Luc vector to obtain the reporters: proEGY3^HY::LUC and proEGY3^MHZ11::LUC. The ORFs of AcGATA1 and AeHSFA2-2 were inserted into the pGreenII-62sk vector to obtain effectors: 35S::AcGATA1 and 35S::AeHSFA2-2, respectively. The multiple combinations of the agrobacteria carrying effector or reporter constructs were co-injected into tobacco leaves, and then incubated for 2–3 d at 23 °C. Photographs were taken using a live plant imaging system (Tanon 5200). The promoter activities expressed as a ratio (LUC/REN) were measured using a Dual-luciferase Kit (YEASEN, Shanghai, China), and determined using a microporous plate light detector (Berthold Centro LB960).

Electrophoretic mobility shift assay (EMSA)

The full-length CDSs of AeHSFA2-2, AcHSFA2-2, and AcGATA1 were cloned into the pGEX4T1 vector, and the AeHSFA2-2-GST, AeHSFA2-2-GST, and AcGATA1-GST recombinant proteins were expressed in E. coli BL21 and purified using a BeaverBeads GSH kit (Beaver, Suzhou). DNA probes of both proEGY3^HY and proEGY3^MHZ11 were labeled with biotin using an EMSA Probe Biotin Labelling Kit (Beyotime), and the same sequences without labels (50- and 100-fold of labeled probes) were used as competitors. The fusion proteins with the probes were incubated at 25 °C for 20 min in a binding buffer. The GST protein alone was used as a negative control. The proteins and biotin-labeled DNA probes were incubated at room temperature for 20 min and were separated by a pre-run polyacrylamide gel. The binding reactions were transferred to a Nylon membrane, and the transferred DNA was crosslinked to the membrane by a commercial UV-light cross-linker (SCIENTZ 03-II, NINGBO SCIENTZ Biotech. Co LTD, China). The biotin-labeled DNA was detected by chemiluminescence⁷³.

Luciferase complementation imaging (LCI) assay

The ORFs of HSFA2-2 and GATA were cloned into JW771 (NLUC) and JW772 (CLUC) vectors obtained from a previous study⁷⁴, yielding the GATA-CLUC and HSFA2-2-NLUC constructs for the LCI assay. The empty vector was used as a negative control. Agrobacterium GV3101 harboring the above constructs was infiltrated into 1-month-old tobacco leaves, followed by incubation for 2–3 d at 23 °C. The leaves were subsequently treated with 1 mM luciferin, and the resulting luciferase signals were taken by using a live plant imaging system (Tanon 5200).

Co-immunoprecipitation (Co-IP)

The Co-IPs were performed according to a previous study⁵⁴. Briefly, ORFs of AcGATA and AcHSFA2-2 were cloned into the pRI101 vectors, containing eGFP-tag or HA-tag, respectively. The recombinant vectors were introduced into Agrobacterium GV3101. Two different pairs of GV3101 mixtures (GATA-eGFP + HSFA2-2-HA and eGFP + HSFA2-2-HA) were used to co-infiltrate tobacco leaves for the transient expression analyses. Total proteins were extracted for Co-IP assays with anti-HA beads, and immunoblot analysis was performed using anti-HA and anti-GFP antibodies, respectively. For immunoblot analysis, total extracted proteins were separated by native-PAGE (for Fig. 5d only) or SDS-PAGE (for the rest blots) and transferred to nitrocellulose membranes (Hybond C-Extra; GE). The membranes were probed with the different antibodies. Anti-Actin antibodies were purchased from Agrisera (#AS132640). Anti-GFP antibodies were purchased from Chengdu Zen-Bioscience Co., Ltd (#300943). Anti-HA (#ABT2040) and Anti-FLAG (#ABT2010) antibodies were purchased from Abbkine (USA).

Statistical analysis

All experiments have been repeated independently at least three times, and all data analyses were based on three biological replicates. Statistical parameters, including the exact value of n and statistical significance, are highlighted in the figures and figure Legends. The significant differences were calculated by two-tailed t-test (Microsoft Excel 365 and GraphPad Prism 9) or one-way ANOVA with Duncan’s multiple-range test (SPSS Statistics, IBM).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

Data supporting the findings of this work are available within this paper and its Supplementary Information files. RNA sequencing data and raw data of BSA sequencing generated in this study have been deposited at the Sequence Read Archive database in NCBI under accession PRJNA1017621 and PRJNA1195612. The mRNA sequences of AcEGY3 and AeEGY3 were deposited at GeneBank in NCBI under accession PV815682 and PV815681. The materials included in the study are available from the corresponding authors upon request. Source data are provided with this paper.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant Nos. U23A20204 to YS.L. and 32472703 to S.W.) and Anhui Province Key Laboratory of Quality Biology of Horticultural Crops. We thank Dr. Jianhua Zhu (Anhui Agricultural University) for critical reading of the manuscript.

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These authors contributed equally: Chengcheng Ling, Jun Yang.

Authors and Affiliations

Anhui Province Key Laboratory of Horticultural Crop Quality Biology, School of Horticulture, Anhui Agricultural University, Hefei, China
Chengcheng Ling, Jun Yang, Jiale Xu, Wei Tang, Yunyan Liu, Yingzhen Wang, Pengwei Li, Yan He, Zhiyin Ouyang, Shuqi Chen, Fei Xing, Xinxin Wang, Pu Liu, Yajing Liu, Runze Wang, Xiaofen Liu, Xueren Yin, Yongsheng Liu & Songhu Wang
College of Food and Bioengineering, Bengbu University, Bengbu, China
Chengcheng Ling & Jun Yang
Department of Environmental Horticulture, Mid-Florida Research and Education Center, University of Florida, Apopka, FL, USA
Heqiang Huo
CAS Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, The Innovative Academy of Seed Design, Chinese Academy of Sciences, Wuhan, China
Dawei Li
University of Kentucky, Lexington, KY, USA
Jan Smalle

Authors

Chengcheng Ling
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Jun Yang
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Jiale Xu
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Wei Tang
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Yunyan Liu
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Yingzhen Wang
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Pengwei Li
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Yan He
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Zhiyin Ouyang
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Shuqi Chen
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Fei Xing
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Xinxin Wang
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Pu Liu
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Yajing Liu
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Runze Wang
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Xiaofen Liu
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Heqiang Huo
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Dawei Li
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Jan Smalle
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Yongsheng Liu
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Songhu Wang
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Contributions

S.W., C.L., H.H., X.Y. and YS.L. designed the experiments. W.T. and S.W. constructed the backcross population. C.L., J.Y., J.X., YY.L., Y.W., PW.L., Y.H., Z.O., S.C., F.X., X.W., P.L., YJ.L., R.W., X.L. and D.L. conducted the experiments. C.L., J.Y., and S.W. analyzed the data. S.W., C.L., J.Y. and J.S. wrote the manuscript.

Corresponding author

Correspondence to Songhu Wang.

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Nature Communications thanks Christine Foyer and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

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Ling, C., Yang, J., Xu, J. et al. Natural variation of AcEGY3 mediates chloroplastic ROS homeostasis to confer kiwifruit thermotolerance. Nat Commun 16, 6184 (2025). https://doi.org/10.1038/s41467-025-61593-5

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Received: 09 December 2024
Accepted: 26 June 2025
Published: 04 July 2025
DOI: https://doi.org/10.1038/s41467-025-61593-5