Overexpression of SlATG8f gene enhanced autophagy and pollen protection in tomato under heat stress | Scientific Reports

Scientific Reports volume 14, Article number: 26892 (2024) Cite this article

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Autophagy is a mechanism for the degradation of cellular components in eukaryotes and plays a critical role in plant responses to abiotic stress. As a core member of the autophagy process, ATG8’s role in how plants respond to heat stress remains unclear. To investigate the response of the tomato autophagy core member ATG8f to heat stress, we studied the key gene ATG8f and generated tomato lines overexpressing SlATG8f using the recombinant expression vector pBWA(V)HS. We observed that under heat stress, SlATG8f overexpression (OE) plants exhibited decreased heat tolerance compared to wild-type (WT) plants. Specifically, OE plants showed increased relative electrolyte leakage, reduced soluble solid content, elevated chlorophyll content, and higher autophagosome numbers, with less damage to chloroplasts and mitochondria. Additionally, expression of some ATG8 family genes and heat shock protein-related genes was upregulated. Moreover, SlATG8f overexpressing plants had higher pollen vitality and more intact pollen morphology. These results suggest that while SlATG8f overexpression renders plants more sensitive to heat, it helps mitigate high-temperature damage to tomato pollen by maintaining chloroplast integrity and interacting with heat shock proteins to respond to heat stress.

Global climate change and the increasing frequency of severe heatwaves are major environmental threats to plant growth and development1. Plants, which remain in a fixed location throughout their growth, are particularly vulnerable to heat stress(HS), leading to significant biochemical and physiological disruptions at both cellular and systemic levels2. To counteract these effects, plants have developed various strategies, such as altering leaf orientation, increasing antioxidant activity, and producing heat shock proteins3. Autophagy, a critical cellular process, plays a key role in how plants manage heat stress. For instance, NBR1-mediated selective autophagy targets and removes ubiquitinated protein aggregates, enhancing basic heat tolerance and facilitating the degradation of heat shock protein chaperones after heat exposure4;5. Additionally, ATG8 Interacting Protein 3 (ATI3) is crucial in the plant response to heat stress6. Given the importance of ATG8 in selective autophagy, understanding its role and the broader mechanisms involved is essential for developing strategies to improve plant resilience to heat stress6,7,8.

The ATG8 protein is essential for autophagosome formation. It binds to membrane lipids, specifically phosphatidylethanolamine (PE), with the assistance of ATG4 protease, forming the ATG8-PE complex9. This modification helps expand phagophores, form autophagosomes, and facilitates their fusion with vacuoles or lysosomes10;11. Other autophagy proteins, such as ATG1, ATG3, and ATG16, also play a role in modifying ATG8 during its lipidation with PE12. While yeast has a single ATG8 gene, higher eukaryotes, including mammals and plants, possess a large gene family13;14. Several homologous ATG8 genes have been identified: 9 in Arabidopsis thaliana, 5 in Zea mays, 7 in Oryza sativa and Solanum tuberosum each, and 11 in Glycine max15. At present, extensive research has been conducted on ATG8 family genes in relation to heat stress responses. Zhai et al.16 demonstrated that both silencing and overexpressing the CaATG8c gene increased plant susceptibility to heat. CaATG8e negatively regulates heat tolerance in both pepper and Arabidopsis17. Interestingly, overexpression of MdATG8i enhances heat tolerance in apple18, while heterologous expression of MaATG8f improves drought resistance in Arabidopsis19. In addition, ATG8 is known to have effects beyond autophagy. Recent research has demonstrated that under high-temperature stress, ATG8 localizes to the Golgi apparatus and integrates with the Golgi membrane, thereby facilitating the recovery of Golgi apparatus function and normal cellular operations20.

Typically, heat stress primarily affects crop yield or quality indirectly by influencing the normal development of reproductive organs, such as flowers, which are highly sensitive to elevated temperatures. Singh’s21 research found that excessive autophagy might accelerate the death of tobacco anther cells, leading to abortion of microspore development. Interestingly, Dundar et al.22 also discovered that autophagy plays a crucial role in cell death during the degeneration of anther wall cells, with autophagy-deficient mutants showing near-complete male sterility under moderate heat stress. These findings indicate a close relationship between autophagy, heat stress, and pollen development.

In our previous research, we identified seven ATG8 homologous genes in tomato23. Overexpression of SlATG8f induced the formation of autophagosomes within tomato fruits, increased autophagic flux, and promoted fruit maturation and quality24. To further investigate the role of the key autophagy gene SlATG8f in response to abiotic stress, we used the dwarf tomato ‘Mic-Tom’ wild type (WT) and SlATG8f overexpression (OE) lines. We analyzed and compared the changes in leaf and pollen development under HS in WT and OE plants to explore some functions of SlATG8f in HS response.

Photographic records were taken of plants subjected to heat stress and control plants were kept at normal temperature. The results are shown in Fig. 1. Compared to control plants at normal temperature, after heat treatment (42℃ for 7 h), wild-type plants exhibited wilting only in localized areas of the leaves, while SlATG8f OE plants showed varying degrees of wilting across their entire leaves, with severe wilting accompanied by leaf curling (Fig. 1A). Therefore, based on seedling phenotype changes, wild-type plants displayed stronger heat tolerance compared to SlATG8f OE plants. Additionally, regardless of whether it was under normal temperature conditions or after HS, the leaf soluble protein content of WT plants were slightly higher than those of SlATG8f OE plants. Under both normal conditions and thermal stress, the relative electrical conductivity of leaves from OE plants is higher than that of WT plants, whereas in CK7, it is significantly lower than that of WT plants. Chlorophyll b content decreased in both types of plants when not subjected to heat stress, with SlATG8f OE plants exhibiting lower levels than WT plants. However, after heat stress treatment, the chlorophyll b content of SlATG8f OE plants was significantly higher than that of WT plants. Following HS, leaf conductivity, soluble protein, and chlorophyll b content all significantly increased in different plant types (Fig. 2B).

Phenotypic and physiological changes of tomato seedlings and leaves. (A) Seedling phenotype, Scale (5 cm); (B) Physiological indicators (conductivity, soluble protein, chlorophyll content); The Error bars represent SD for three biological replicates, The letters on the bar chart indicate their level of significance, P < 0.05. Note: CK: 0 hours at normal temperature; CK7: 7 hours at normal temperature; H7: 7 hours at 42℃ heat stress.

To investigate the effect of heat stress on the pollen viability of different tomato plants, this study observed pollen viability using FDA staining (Fig. 2). The FDA staining results indicated that in the control group, the pollen viability of WT plants was higher than that of SlATG8f OE plants. At 3 days of heat treatment, the pollen viability of WT plants was slightly higher than that of SlATG8f OE plants, but after 7 days of heat treatment, the pollen viability of SlATG8f OE plants was higher than that of WT plants.

FDA staining to assess the pollen vitality of WT, ATG8f-OE plant. Vital pollen is stained greed; Scale bars, 100 μm. Note: 25℃ (0d) : Treatment at 25℃ for 0 days; 25℃ (3d): Treatment at 25℃ for 3 days; 35℃ (3d) : Treatment at 35℃ for 3 days; 25℃ (7d): Treatment at 25℃ for 7 days; 35℃ (7d) : Treatment at 35℃ for 7 days.

To investigate the effects of HS on the pollen development of different tomato plant lines, this study examined pollen morphology using scanning electron microscopy (Fig. 3). Scanning electron microscopy observations revealed that, in the control group, WT pollen had a larger volume compared to that of SlATG8f OE plants. After heat treatment, both WT and SlATG8f OE plant pollens exhibited deformities, which became more severe with prolonged treatment. At the same treatment time, the deformity degree was higher in WT pollen compared to SlATG8f OE pollen.

Scanning electron microscopy images of mature pollen grains from WT, ATG8f-OE tomato plants. Note: 25℃ (0d) : Treatment at 25℃ for 0 days; 25℃ (3d): Treatment at 25℃ for 3 days; 35℃ (3d) : Treatment at 35℃ for 3 days; 25℃ (7d): Treatment at 25℃ for 7 days; 35℃ (7d) : Treatment at 35℃ for 7 days.

To determine whether autophagy responds to heat stress, we analyzed the expression levels of the autophagy core gene ATG8 family members. Real-time fluorescence quantitative (qRT-PCR) was used to determine the expression levels of ATG8 family members in plant leaves after normal temperature control and heat treatment (42℃, 7 h) (Fig. 4). The results showed that the expression multiples of all ATG8 family members in the leaves of SlATG8f OE plants were significantly higher after heat treatment than those in WT plant leaves; the expression levels of SlATG8b, SlATG8c, SlATG8e, SlATG8f and SlATG8g in WT and SlATG8f OE plants were significantly up-regulated after heat treatment; SlATG8b was significantly down-regulated after heat treatment in WT plants, while it was significantly up-regulated in SlATG8f OE plants after heat treatment; the expression level of SlATG8d almost did not change after heat treatment in WT plants, while it was significantly up-regulated in SlATG8f OE plants after heat treatment.

Analysis of differential expression of autophagy-related genes in tomato plant leaves. The Error bars represent SD for three biological replicates, The letters on the bar chart indicate their level of significance, P < 0.05. Note: CK: 0 hours at normal temperature; CK7: 7 hours at normal temperature; H7: 7 hours at 42℃ heat stress.

To determine whether heat stress enhances autophagy activity in tomato flowers, this study analyzed the expression levels of the autophagy core gene family, ATG8 family members, in tomato flowers. Real-time fluorescence was quantitative (qRT-PCR) used to determine the expression levels of flower ATG8 family members in control and heat treated plants (Fig. 5). The results showed that, at all time points, the expression levels of ATG8 family members in pollen of SlATG8f OE plants were higher than those in WT; among them, the expression levels of SlATG8a, SlATG8c, and SlATG8d in pollen of WT and SlATG8f OE plants were significantly up-regulated after heat treatment; the expression level of SlATG8b first increased and then decreased after heat treatment in WT plants, while it was significantly up-regulated after heat treatment in SlATG8f OE plants; the expression levels of SlATG8e, SlATG8f, and SlATG8g in WT and SlATG8f OE plants were up-regulated after heat treatment and then decreased.

Analysis of differential expression of autophagy-related genes in tomato plant flowers. The Error bars represent SD for three biological replicates, The letters on the bar chart indicate their level of significance, P < 0.05. Note: CK0: Treatment at 25℃ for 0 days; CK3 : Treatment at 25℃ for 3 days; H3 : Treatment at 35℃ for 3 days; CK7 : Treatment at 25℃ for 7 days; H7 : Treatment at 35℃ for 7 days.

To investigate the changes in the expression levels of heat shock protein-responsive genes in WT and SlATG8f OE plants after heat treatment, this study measured the expression levels of heat shock protein-responsive genes in plant leaves after control and heat treatment (42℃, 7 h) (Fig. 6). The results showed that the expression levels of SlHSFA2, SlHSP20, SlHSP21, SlHSP70, and SlHSP90 were significantly up-regulated after heat treatment in both WT and SlATG8f OE plants, with most of the heat shock protein-responsive genes showing expression multiples increasing from several times to hundreds or thousands of times. Among them, the expression level of SlHSP21 in both WT and SlATG8f OE plants remained almost the same after control and heat treatment, without significant changes. Regardless of control or heat treatment, the expression levels of SlHSFA2, SlHSP20, SlHSP21, SlHSP70, and SlHSP90 in SlATG8f OE plant leaves were higher than those in WT plants. Unlike the above heat shock protein-responsive genes, SlHSFA3 showed higher expression levels in WT plants than in SlATG8f OE plants, both in control and heat treatment conditions; SlMYB21 exhibited downregulation in expression after heat treatment in WT plants, while it was up-regulated after heat treatment in SlATG8f OE plants.

The differential expression analysis of heat shock protein response genes in tomato plant leaves. The Error bars represent SD for three biological replicates, The letters on the bar chart indicate their level of significance, P < 0.05. Note: CK: 0 hours at normal temperature; CK7: 7 hours at normal temperature; H7: 7 hours at 42℃ heat stress.

To investigate the changes in the expression levels of heat shock protein response genes in flowers of WT and SlATG8f OE plants after heat treatment, this study determined the expression levels of heat shock protein response-related genes in flowers of control and heat-treated plants (Fig. 7). The results showed that in WT plant flowers, the expression levels of SlHSFA1a, SlHSFA2, SlHSFA3, SlHSP70, SlHSP90, SlMYB21, SlMYB26, and SlATG8f OE plant flowers increased first and then decreased with treatment time; the expression levels of SlHSFA2, SlHSFA3, and SlHSP90 in SlATG8f OE plant flowers increased first and then decreased with treatment time. The expression levels of SlHSP20 and SlHSP21 showed a trend of first decreasing, then increasing, and finally decreasing in both WT and SlATG8f OE plant flowers. In SlATG8f OE plant flowers, the expression levels of SlHSP70 and SlMYB26 first decreased and then increased; SlHSFA2 expression decreased in WT plant flowers, while SlMYB21 expression increased in SlATG8f OE plant flowers. The expression level of SlHSFA2 first increased, then decreased, and finally increased in SlATG8f OE plant flowers. Interestingly, the expression levels of SlHSP90 in WT and SlATG8f OE plant flowers were highest at 7 days in the control group and significantly higher than those in plant flowers after heat treatment.

The differential expression analysis of heat shock protein response genes in tomato plant flowers. The Error bars represent SD for three biological replicates, The letters on the bar chart indicate their level of significance, P < 0.05. Note: CK0: Treatment at 25℃ for 0 days; CK3 : Treatment at 25℃ for 3 days; H3 : Treatment at 35℃ for 3 days; CK7 : Treatment at 25℃ for 7 days; H7 : Treatment at 35℃ for 7 days.

To determine whether HS enhances autophagy activity in tomato leaves, we observed the number of autophagosomes in plant leaves through transmission electron microscopy and detected the key protein ATG8 involved in plant autophagosome formation using Western blotting (Fig. 8A). The Western blotting results showed that protein bands in the control group were relatively weak, while those in plant leaves after heat treatment were significantly stronger compared to the control group). Among them, the protein bands in WT plant leaves were the brightest after heat treatment, followed by those in SlATG8f OE plant leaves after heat treatment (Fig. 8B). These results indicate that HS significantly enhances plant autophagy activity.

To further understand the changes in the number of autophagosomes, we observed the number of autophagosomes in tomato plant leaves using transmission electron microscopy(Fig. 8C). The results showed that the number of autophagosomes in SlATG8f OE plant leaves was higher than that in the wild type under both control and heat treatment conditions. The number of autophagosomes in WT plant leaves and SlATG8f OE plant leaves was about twice that of the control group after heat treatment, and the degree of damage to chloroplasts and mitochondria in WT plant leaves was higher than that in SlATG8f OE plant leaves after heat treatment.

The change in autophagic activity of tomato plant leaves. (A) Analysis of ATG8 Protein by Western blotting (The image has been cropped and the original image is in the Supplementary Fig. 1). (B) Comparison of protein expression grayscale values. The Error bars represent SD for three biological replicates, The letters on the bar chart indicate their level of significance, P < 0.05. (C) Changes in autophagic activity of leaf; red arrows (autophagosomes), green arrows (chloroplasts), blue arrows (mitochondria). Note: CK: 0 hours at normal temperature; CK7: 7 hours at normal temperature; H7: 7 hours at 42℃ heat stress.

High temperatures negatively impact plant growth and development by inducing protein misfolding, denaturation, oxidation, and aggregation25. Autophagy is a ubiquitous process in eukaryotic cells responsible for degrading damaged proteins and organelles; thus, the degradation of damaged proteins via autophagy represents an alternative mechanism for plants to acquire heat tolerance26. In our study, overexpression of ATG8f in tomatoes decreased their heat tolerance (Fig. 1A). This finding is consistent with the results of Zhai et al.16. and Liang et al.17, suggesting that SlATG8f overexpression may disrupt ROS signaling, thereby leading to impaired responses to environmental stress. Interestingly, overexpression of MdATG8i in apples enhances heat tolerance18. Thus, further research is needed to elucidate the functions and mechanisms of ATG8 family genes in response to thermal stress. Concurrently, we observed that under normal conditions, OE plants display lower, soluble protein, and chlorophyll content compared to their counterparts (Fig. 1B). Nonetheless, following heat stress, OE plants exhibited a higher chlorophyll content than WT plants (Fig. 1B). Additionally, electron microscopic analysis revealed that OE plants suffered lesser chloroplast damage upon exposure to high temperature stress than WT plants (Fig. 1B). Based on these findings, we infer that overexpression of SlATG8f diminishes the thermal tolerance of plants, yet concurrently safeguards chloroplasts, facilitating plant adaptation to heat stress.

Programmed cell death (PCD) is a gene-regulated process that facilitates the elimination of specific cells, tissues, or entire organs. Research indicates that ATGs are involved in regulating PCD during petal senescence. Silencing ATG6 or PI3K in petunias accelerates petal senescence, reducing flower number and biomass27. Overexpression of ATG6/Beclin1 genes in tobacco and Arabidopsis leads to infertility and abortive microsporogenesis due to excessive autophagy28. This also explains why, in our study, WT pollen exhibits higher vitality and larger volume compared to OE pollen, which shows partial deformities (Figs. 2 and 3). During pollen development, heat stress disrupts the development and function of pollen and ovules. Heat stress causes premature degradation and abnormal vacuolation of endosperm cells in barley29 and Arabidopsis30, leading to the complete cessation of pollen development. However, autophagy can partially alleviate high-temperature damage to Arabidopsis pollen development22. In OE plants, enhanced autophagic activity results in greater heat tolerance of pollen. It can be inferred that the overexpression of SlATG8f affects the regulation of pollen PCD, but SlATG8f mitigates high-temperature damage to tomato pollen development by modulating autophagic activity.

To further investigate the mechanism by which ATG8f responds to heat stress, we performed a transcriptomic analysis of other members of the ATG8 gene family in tomatoes. Surprisingly, the expression levels of other ATG8 family genes varied differently under heat stress and also showed differential expression across various tissues (Figs. 4 and 5). This suggests that the ATG8 gene family exhibits contradictory behavior, with both mutually enhancing and inhibiting interactions among its members. We hypothesize that this complexity may be due to the large-scale expansion of the ATG8 gene family in plants, which occurred through multiple whole-genome duplications31. This expansion has led to the diversity of selective autophagy observed in plants14, thus contributing to these seemingly contradictory effects.

SlATG8f responds to heat stress by modulating the expression of other heat-related genes (HSPs/HSFs). Specifically, exposure toheat stress results in a dramatic induction of SlHSP20, SlHSP21, SlHSP90, and SlHSF2 in leaf tissues, with expression levels increasing by several hundred to thousands of times. Notably, the upregulation of some of these genes is more pronounced in OE lines compared to WT plants (Fig. 6). This observation aligns with previous research findings32. It is noteworthy that the expression and expression trend of SlHSP21 after heat stress are nearly identical in both WT and OE plants, which may be attributed to the unique role of HSP21. Prior studies have shown that HSP21 is the only plastid small HSP in Arabidopsis, essential for heat stress memory and localized in the chloroplast33, where it accumulates rapidly following heat stress34. Under heat stress conditions, HSP21 is critical for chloroplast function and early seedling development35. Reports also indicate that HSP21 can protect photosystem II (PSII) in tomatoes (Solanum lycopersicum) when exposed to excessive temperatures36. In our study, although OE plants exhibit greater heat stress damage to leaf tissues, autophagy provides some protection against chloroplast damage under heat stress, thus stabilizing the expression of SlHSP21 in OE plants. Therefore, we infer that SlATG8f may protect chloroplasts and stabilize SlHSP21 expression by promoting autophagy.

Unlike leaf organs, the expression levels of heat-related genes (HSPs/HSFs) in floral organs are not as pronounced and are relatively complex. However, overall, the expression of most heat-related genes (HSPs/HSFs) is significantly higher in OE flowers compared to WT plants. Notably, SlHSP20, SlHSP21, and SlHSP90 show higher expression levels in the control CK3/CK7 than under heat stress. These genes largely depend on the activation of HSFs under heat stress37. In our study, the expression of HSFA2 decreased in both WT and OE plants following heat stress (Fig. 6). Therefore, we speculate that in floral organs, autophagy has a minimal influence on enhancing heat tolerance by influencing the expression of heat-related genes (HSPs/HSFs).

The tomato (Solanum lycopersicum L.) variety ‘Micro-Tom’ used in the experiment originated from our research team (Wen Xu). It were stored in the Horticultural Laboratory of the College of Agriculture, Guizhou University24. According to conventional soilless substrate cultivation methods, wild-type and SlATG8f overexpressing plants were cultivated. When the seedlings had been transplanted for 20 days, nine plants of each genotype were transferred to a growth chamber maintained at a constant temperature of 42℃ for 7 h, while the control group was kept at 25℃38. Leaf samples were collected after treatment to measure chlorophyll content, cell membrane relative permeability, antioxidant enzyme activity, autophagy activity, soluble protein content, as well as the expression levels of ATG8 family members and heat shock protein-related genes.

When the first inflorescence appeared, nine selected plants were transferred to a 35℃ constant temperature light incubator for 7 days, with the control group maintained at 25℃22. Pollen samples were collected at 0 d, 3 d, and 7 d of treatment to investigate whether overexpression of SlATG8f affects pollen morphology, pollen viability, pollen germination, and the expression levels of ATG8 family members and heat shock protein-related genes under high-temperature stress.

The pro-ATG8f overexpression vector was constructed by connecting the coding region of ATG8f with pBWA(V)HS vector. The healthy cotyledons were inoculated with the Agrobacterium strain GV3101, transformed with pro35S-ATG8f. The expression level of SlATG8f in tomato SlATG8f-OE line was detected by qRT-PCR. Finally, the T2 generation is selected as the experimental material24.

The reference methods for conductivity measurement index Kang39, soluble protein index determination Cao40, and chlorophyll b index determination Cheng41.

After sampling, RNA was extracted using the Trizol reagent kit and reverse transcribed. The obtained cDNA was then analyzed using qRT-PCR. Specific primers were designed using Premier 5.0 and synthesized by a biological company. The gene names and primers were as follows (Supplementary Table 1). The expression levels of genes were determined by real-time fluorescence quantitative method using the TB Green Takara Premix Ex Taq™ II fluorescence quantitative reagent kit. Each sample was set with 3 replicates. According to previous studies, Actin (Solyc03g078400) was used as the reference gene42. The qRT-PCR reaction system and procedure were performed according to the recommendations of 2X Taq PCR Master Mix II(Takara). The relative gene expression level was calculated using the 2−ΔΔCT method43.

The number of leaf autophagosomes was analyzed by transmission electron microscopy.

Pollen morphology was directly observed by scanning electron microscopy, with the following specific experimental steps: ① Collect flowers and scatter pollen on a metal carrier coated with double-sided tape using tweezers. ② Spread the pollen evenly. ③ Spray gold powder on the pollen for 4 ~ 5 min using an Eiko IB5 ion sputter (Japan). ④ Observe the pollen morphology using a Hitachi Model TM-1000 scanning electron microscope (Japan).

Pollen viability was detected using the FDA staining method, with the following specific steps: ① Prepare a 2 mg/ml FDA stock solution, and dilute it to a 0.01% working solution with 0.5 mL of s ucrose when in use. ② Place a drop of FDA working solution on a microscope slide with open flowers, shake the stamens with tweezers to scatter the pollen in the staining solution, and incubate at room temperature in the dark for 1 h. ③ Observe and photograph under a fluorescence microscope.

The content of ATG8 complex protein in leaves was established by Western blot. Appropriate amount of wild type and transgenic tomato fresh tissue were ground into fine powder in liquid nitrogen. Then 5 times the volume of protein extraction reagent, incubated at 4℃ overnight. After centrifugation, the protein concentration in the sample was identified with the BCA protein detection kit. For SDS-PAGE electrophoresis, the protein sample is combined with the sample buffer at a ratio of 5:95, and then heated in boiling water at 95 to 100℃ for 5 min. Constant pressure electrophoresis was performed at the concentration gel 80 V and separation gel 120 V. 300 mA constant flow membrane, the transfer time is adapted according to the molecular weight of the target protein. The improved film was in addition to the blotting membrane sealer and closed at room temperature for 1 h. Add the primary antibody (Rabbit polyclonal to Actin (1:5000), Rabbit polyclonal to APG8A/ATG8A (1:1000); abcam) diluted with the primary antibody diluent (ASPEN) 4℃ overnight. Wash with TBST three times, 5 min each time. The second antibody (HRP-Goat anti Rabbit, 1:10,000, ASPEN) diluted with the secondary antibody diluent was added, incubated at room temperature for 30 min, and washed four times with TBST on a shaker at room temperature for 5 min each time. A freshly prepared ECL (ASPEN) mixture (A: B = 1:1) was in addition to the protein side of the membrane and exposed in a X-ray magazine (AX-II, Guangdong Yuehua Medical Instrument Factory Co.,Ltd.). The film is scanned and archived, and the AlphaEaseFC software processing system analyzes the optical density value of the target tape.

The experiment was conducted using a completely randomized design, with random sampling within each group and three replicates per group. Data were processed using Microsoft Excel 2019, statistical significance of the results was assessed with SPSS Statistics 23.0, and Duncan’s multiple range test was applied. Different lowercase letters in the numbers indicate significant differences at the (P < 0.05) level. Figures were plotted using Origin 2022, and data are presented as mean ± standard deviation.

The data presented in this study are available on request from the corresponding author.

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This work was supported by the National Natural Science Foundation of China (32260754) and Platform construction project of Engineering Research Center for Protected Vegetable Crops in Higher Learning Institutions of Guizhou Province (Qian Jiao Ji [2022] No. 040).

College of Agriculture, Guizhou University, Guiyang, 550025, China

Liu Song, Cen Wen, Zhuo He, Xingxue Zha, Qunmei Cheng & Wen Xu

Engineering Research Center for Protected Vegetable Crops in Higher Learning Institutions of Guizhou Province, Guiyang, 550025, China

Wen Xu

Institute of Edible Fungi Industry Technology Research, Guizhou University, Guiyang, 550025, China

Xingxue Zha

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Liu Song: Conceptualization, Writing original draft, Formal analysis. Cen Wen and Zhuo He: Software, Validation. Xingxue Zha: Software, Methodology, QunMei Cheng: Data curation. Wen Xu: Writing review & editing, Conceptualization, Funding acquisition, Project administration, Resources. All authors have read and agreed to the published version of the manuscript.

Correspondence to Wen Xu.

The authors declare no competing interests.

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Song, L., Wen, C., He, Z. et al. Overexpression of SlATG8f gene enhanced autophagy and pollen protection in tomato under heat stress. Sci Rep 14, 26892 (2024). https://doi.org/10.1038/s41598-024-77491-7

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Received: 17 June 2024

Accepted: 22 October 2024

Published: 06 November 2024

DOI: https://doi.org/10.1038/s41598-024-77491-7

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