In this study, the seeds of maize B73 Inbred Line were obtained from Anhui Agricultural University, China. The seeds germinated on the wet cotton cloth at 28°C for 3 days, and then the germinated seeds were transplanted to nutritious soil and grew at 28°C with long-day conditions of 15 h (550 μmol m^-2s^-1) and 9 h of dark and the relative humidity of 60% for the whole growth and developmental period. At the V4 developmental period (there is a visible ring in the leaf base of the fourth leaf), the maize seedlings were transferred to a dark incubator with 42°C treatment for 0, 4, and 8 h. Moreover, according to the previous methods (Hu et al., 2021), the seedlings were subjected to drought and salt treatments (200 mM NaCl) for 0, 4, and 8 h, respectively. The treated seedling roots, stems and leaves were collected after the three stress treatments, respectively. All these collected tissues were stored in liquid nitrogen to further extract RNA. In addition, Arabidopsis ecotype Columbia (Col) was used as original materials for genetic transformation to obtain transgenic plants and perform further functional analysis. All Arabidopsis plants were grown in the controlled chamber (22°C,16h light (120 μmol m^-2s^-1)/8 h darkness, and 60% relative humidity).

The full-length CDS sequence of ZmNAC074 was extracted from the NCBI database (http://www.ncbi.nlm.nih.gov). The orthologous protein sequences in rice and Arabidopsis were retrieved from Phytozome (https://phytozome-next.jgi.doe.gov/) and NCBI (http://www.ncbi.nlm.nih.gov) (Zhang et al., 2022). Furthermore, the molecular weight and theoretical isoelectric point (PI) of ZmNAC074 protein were calculated in Expasy (http://web.expasy.org/compute_pi/) (Khan et al., 2018). The multiple amino acid sequences were calibrated and compared using ClustalW program. The phylogenetic tree was constructed using MEGA 7.0 software with the neighbor-joining method and 1000 bootstrap replicates in this study. The conserved motifs were predicted using online MEME website (http://alternate.meme-suite.org/tools/meme). In addition, the structure of these genes was analyzed by GSDS website (http://gsds.cbi.pku.edu.cn/) referring to our previous methods (Hu et al., 2021). The amino acid sequences of ZmNAC074 and OsNTL3 proteins were further compared using DNAMAN software. The transmembrane domain of ZmNAC074 was predicted using TMHHM 2.0 (https://services.healthtech.dtu.dk/service.php?TMHMM-2.0). The three dimensional (3D) structural model of ZmNAC074 protein was predicted using the I-TASSER server (https://seq2fun.dcmb.med.umich.edu//I-TASSER/) (Yang and Zhang, 2015; Zhang et al., 2017; Zheng et al., 2021). The predicted model was edited and annotated using the PyMOL software. Moreover, the functional associations of several NAC MTFs and stress response-associated proteins in Arabidopsis and rice were predicted to construct their protein protein interaction networks by the STRING website (https://cn.string-db.org/), respectively.

The total RNA was isolated with Trizol reagent (TIANGEN, China) (Gai et al., 2020). The first strand of cDNA was synthesized from 2μg of total RNA using the FastKing RT Kit (TIANGEN, China). Quantitative real time-PCR (qRT-PCR) was performed using Light-CykerR96 real-time PCR system (BioRad, USA). The reaction conditions are as follows: preincubation at 95°C for 600 s, and then performing 40 cycles of 95°C for 10s, 55°C for 30s and 72°C for 30s. In maize and Arabidopsis, the relative gene expression data were calculated using the 2^−△△Ct method with the expression of ZmActin and AtActin as internal controls, respectively. All qRT-PCR analyses were performed by three biological replicates. The primer sequences used were listed in Supplementary Table S1 .

The full-length CDS sequence of ZmNAC074 was amplified by PCR and inserted into the PHB vector driven by the cauliflower mosaic virus (CaMV) 35S promoter to generate a recombinant vector, which was confirmed via double digestion validation with Hind III and Xba I enzymes and sequencing. The constructed vector was further transformed into Arabidopsis ecotype Col-0 using the floral dipping method mediated by Agrobacterium tumefaciens strain GV3101. The seeds of T₀ and T₁ generations were screened with 1/2 MS media containing 30 mg/L hygromycin and identified by RT-PCR. Three T₃ homozygotes of ZmNAC074 transgenic overexpression lines (line 1, line 2, and line 9) were ultimately selected for further functional analysis (Gai et al., 2020).

The three-week-old transgenic Arabidopsis and WT Arabidopsis seedlings were treated at 42°C for 24h and 48h, respectively (Huang et al., 2019). Then, these two kinds of Arabidopsis plants with different treatment time were restored to normal environmental conditions growing for one week. The determination of physiological characteristics and related gene expression was performed just at the end of heat stress treatments.

Hydrogen peroxide (H₂O₂) in leaves was determined with 3, 3’-diaminobenzidine (DAB) solution. According to the previously described methods (Daudi and O’brien, 2012), the leaves were immersed in 1.0 mg/mL DAB-HCl solution (add the mixed solution of 0.05% (v/v) Tween 20 and 8 times the volume of 200 mM Na₂HPO₄ to adjust the PH of DAB-HCl solution to 7.0) for 8 h at 25 °C and dark. Then place the stained leaves in a beaker containing 95% (v/v) alcohol and carefully place them in a boiling water bath (95°C) for 15 ± 5 minutes until the brown spots appear on the leaves and there are no green areas (Ali et al., 2020). Further, the histochemical staining of superoxide radical (O₂ ^-) was mentioned as previously described (Xia et al., 2009; Liu et al., 2012), but with slight modifications. The leaves were incubated in 0.1% nitro blue tetrazolium (NBT) with 50 mM potassium phosphate buffer (pH 7.8) at 25°C for 12 h. Then, the same decolorization method mentioned above was used for bleaching and photography.

The sample pretreatment methods for some physiological indexs (MDA, soluble protein and proline) in this experiment are as follows: weigh 0.1g of leaves, add 900 mL of physiological saline, mechanically homogenize in an ice-water bath, centrifuge at 10,000 rpm for 10 min and take the sample supernatant for subsequent experiments. The activities of superoxide dismutase (SOD) and catalase (CAT) were determined using xanthine-xanthine oxidase method and ammonium molybdate spectrophotometric method, respectively (Hadwan and Abed, 2016). SOD and CAT activities were calculated by the absorbance value obtained at the wavelength of 550 nm and 405 nm, respectively. Moreover, the total antioxidant capacity (T-AOC) was determined by colorimetry at 520nm as directed by the manufacturer. In addition, the H₂O₂ content was determined with molybdenic acid colorimetry. H₂O₂ bound with molybdenic acid to form a complex (Zhou et al., 2021), which was measured by using spectrophotometer at the wavelength of 405nm. Then, the content of H₂O₂ was calculated from the measured absorbance. Malondialdehyde (MDA) content was determined using the thiobarbituric acid (TBA) assay (Aguilar Diaz De Leon and Borges, 2020). For this, 0.1mL of the sample supernatant was mixed with 4.1mL of the mixed solution containing TBA configured according to the manufacturer’s instructions and heated at 95°C for 40min. The reaction mixture was taken to determine the absorbance at 532nm and calculate the content of MDA. The content of soluble protein was determined by Coomassie Brilliant Blue method (Goldring, 2019) through adding 50μL the sample supernatant to 3mL Coomassie Brilliant Blue dye reagent, which was mixed and incubated at 25°C for 10 minutes. The graph of absorbance readings and soluble protein content at 595nm was plotted. The content of soluble protein was calculated by using the absorbance value of the reaction mixture and the graph. Furthermore, the proline content in leaves was determined by acid ninhydrin method (Shabnam et al., 2016). 0.5mL of sample supernatant mixed with 2mL of 1.25% (v/v) ninhydrin in 2mL glacial acetic acid at 100°C for 30 min. After cooling, the reaction mixture was taken to determine the absorbance at 520 nm. Moreover, to identify the content of photosynthetic pigments, 1g leaf tissue (ground with liquid nitrogen) was treated with 80% (v/v) acetone. The absorbance was measured at 470nm, 645nm, and 633nm, respectively (Zengin, 2013). Using the absorbance value, the contents of chlorophyll and carotenoid were calculated, respectively. All analyses were performed for three biological replicates. All the above kits or reagents were purchased from Nanjing Jiancheng Bioengineering Institute. Spectrophotometer (Shanghai Youke instrument Co., Ltd., UV752N) was used for all measurements.

Statistical analysis was carried out using the SPSS 20 software. The data were analyzed by one-way ANOVA and the means were compared by Duncan’s multiple range test. Compared with the control group, P < 0.05 is considered to be statistically significant and P < 0.01 extremely significant.

The open reading frame (ORF) of ZmNAC074 is consisted of 1556 bp, which encodes 517 amino acids (AA) with a molecular weight of 57.5 kDa and a theoretical isoelectric point (PI) of 5.49. It is suggested that ZmNAC074 protein is a kind of acidic protein and under the condition of isoelectric point (PI=5.49), this protein shows its minimum solubility, viscosity, osmotic pressure and swelling property. The phylogenetic tree of NAC proteins in maize, rice, and Arabidopsis revealed that ZmNAC074 protein shares a close evolutionary relationship with rice OsNTL3 protein ( Figure 1A ). In addition, AtNAC089, AtNAC60 and AtNTL8 also belong to the same branch with ZmNAC074, indicating that the evolutionary relationships among them are relatively conserved. Information of corresponding genes encoding these NAC proteins in plants was listed in Supplementary Table S2 . According to sequence characteristics of these NAC proteins, 10 conserved motifs (motif 1-10) were predicted using the MEME program ( Figure 1B ). All NAC proteins contain motifs 1-4. Except for AtNAC089, AtNAC60, and AtNTL8, all other proteins contain motif 5. It is worth noting that ZmNAC074 and OsNTL3 contain the same motif distribution (motif 1-5). Annotations of the motifs are listed in Supplementary Table S3 . In addition, the corresponding genes encoding these NAC proteins (including ZmNAC074, OsNTL3, AtNAC089, AtNAC60 and AtNTL8) in the same branch share a common feature in gene structure, namely only containing three introns ( Figure 1C ). Moreover, the N-terminus and C-terminus of ZmNAC074 protein contain a NAC domain (10-136 AA) and a transmembrane domain (488-510 AA), respectively ( Figure 1D ). Moreover, the amino acid sequence alignment between ZmNAC074 and OsNTL3 showed that their NAC domains share high similarity, indicating that ZmNAC074 may have a similar function to OsNTL3 ( Figure 1E ). In addition, according to the TMHMM prediction, ZmNAC074 protein only contains one transmembrane domain, which is distributed at the C-terminus of ZmNAC074 protein ( Figure 1F ). The positions of the N- terminus (represented by the white letter N) and the C- terminus (represented by the white letter C) of ZmNAC074 protein are marked in the picture of the 3D model ( Figure 1G ). In addition, the middle region of the two labeled amino acids (ALA-488 and TYR-510) represents the transmembrane domain of this protein. Moreover, the α-helix and β-strand are depicted as red and yellow ribbon diagrams, respectively, and loops as green lines.

To examine whether ZmNAC074 responds to heat stress and other abiotic stresses, the expression pattern of ZmNAC074 was evaluated under heat (42°C), salt (200mM NaCl), and drought stress treatments for 0, 4, and 8 h in maize B73 inbred line, respectively. The results showed that ZmNAC074 was expressed in roots, stems, and leaves under normal conditions, but its expression level was the highest in leaves in comparison to other tissues ( Figure 2 ). The expression level of ZmNAC074 was significantly upregulated following heat, drought and salt stress treatments, especially under heat stress treatment, respectively ( Figures 2A–C ). After heat stress, the expression level of ZmNAC074 in three different tissues increased gradually with the prolongation of stress time ( Figure 2A ). After drought stress, the expression levels of ZmNAC074 in stem and root tissues gradually increased, peaked at 4h, and then decreased ( Figure 2B ). After salt stress, the expression levels of ZmNAC074 in stem and root tissues decreased at 4h and then increased at 8h ( Figure 2A ). Overall, these results demonstrate that ZmNAC074 can respond to heat, drought, and salt stress, but the response to heat stress is particularly strong. Therefore, the regulatory role of ZmNAC074 in heat stress tolerance was focused on in the subsequent study.

To investigate the potential functions of ZmNAC074 under heat stress, ZmNAC074 was successfully introduced into Arabidopsis driven by CaMV 35S promoter ( Figures 3A–B ) (Zuo et al., 2022). The schematic diagram of constructed vector containing ZmNAC074 under the control of CaMV 35S promoter was displayed ( Figure 3C ). The recombinant vector was further transformed into Arabidopsis and 16 ZmNAC074-overexpressed transgenic Arabidopsis lines were obtained in this study. The amplification map showed that the exogenous ZmNAC074 has been integrated into the genome of 15 transgenic Arabidopsis lines ( Figure 3D ). Quantitative RT-PCR analysis showed that there was no significant difference in the expression of ZmNAC074 among line 1, line 2, and line 9 when they were three weeks old ( Figure 3E ). Moreover, when growing under normal conditions, there was no visible phenotypic difference between the three lines and WT Arabidopsis ( Figure 3F ). Therefore, line 1, line 2 and line 9 can be selected as three biological repeats for further exploration.

To further verify whether ZmNAC074 is associated with heat resistance, three-week-old WT Arabidopsis and selected transgenic Arabidopsis lines were subjected to heat stress treatments for 24h and 48h, respectively. No visible difference was observed between WT Arabidopsis and transgenic Arabidopsis lines under normal conditions ( Figure 4A ). However, under heat stress conditions, after all treated plants were transferred to normal growth environmental conditions and recovered for one week, the WT Arabidopsis leaves showed serious wilting symptoms, whereas numerous transgenic Arabidopsis leaves remained green ( Figure 4A ). Notably, the phenotypic difference between WT Arabidopsis and transgenic Arabidopsis plants under heat stress treatment for 48h was more significant than that for 24h ( Figure 4A ). According to statistics, after heat treatment for 24h, the survival ratio of transgenic Arabidopsis lines were 1.16-1.32 times higher than that of WT Arabidopsis plants, respectively. In contrast, after heat treatment for 48h, the survival ratio of all transgenic Arabidopsis lines was more than 70%, whereas that of WT Arabidopsis was less than 50% ( Figure 4B ). The results indicated that transgenic Arabidopsis exhibited more obvious thermotolerance than WT Arabidopsis.

Furthermore, to investigate whether the heat tolerance phenotype of transgenic Arabidopsis lines was resulted from alteration of ROS homeostasis, the accumulation of ROS in transgenic Arabidopsis lines and WT Arabidopsis plants under normal and heat stress conditions was compared, respectively. Qualitative staining by DAB and NBT and quantitative measurement showed that the staining of H₂O₂ and superoxide anion (O₂ ^-) and the accumulation of H₂O₂ in leaf tissues of transgenic Arabidopsis lines were indistinguishable from those in WT Arabidopsis plants grown under normal conditions ( Figures 4C–E ). However, after heat stress treatment, the staining of H₂O₂ and superoxide anion (O₂ ^-) was more evident and the accumulation of H₂O₂ was revealed to significantly increase in leaf tissues of transgenic Arabidopsis lines and WT Arabidopsis plants than that of corresponding control plants growing under normal conditions ( Figures 4C–E ). Although the accumulation of ROS in all plants increased with the prolongation of heat stress treatment time, the staining of H₂O₂ and superoxide anion (O₂ ^-) in leaf tissues of transgenic Arabidopsis lines was less evident than that of WT Arabidopsis plants when treated with heat stress treatment for 24h and 48h, respectively. Accordingly, the transgenic Arabidopsis lines showed lower H₂O₂ accumulation levels than WT Arabidopsis plants. Thus, these results suggest that the cellular oxidative damage of transgenic Arabidopsis lines is lesser than that of WT Arabidopsis plants. Further, to detect the ability of plants to scavenge ROS, the activities of related SOD and CAT antioxidant enzymes and total antioxidant capacity (T-AOC) were evaluated in this study. Under normal conditions, there were no obvious differences in the SOD and CAT activities and T-AOC between the WT Arabidopsis and transgenic Arabidopsis lines. However, with the extension of heat stress time, the SOD and CAT activities and T-AOC of all plants were exhibited to increase, but the SOD and CAT activities and T-AOC of transgenic Arabidopsis lines were much higher than those of WT Arabidopsis plants ( Figures 4F–H ). Thus, these results suggest that heat stress resistance of ZmNAC074-overexpressed transgenic Arabidopsis lines may be resulted from their enhanced ROS-scavenging capability.

Moreover, MDA (Malondialdehyde) can severely damage plant cell membranes, and thereby the degree of MDA accumulation is usually regarded as an important indicator of membrane-lipid peroxidation (Davey et al., 2005). In this study, no differences were observed in accumulation of MDA contents between all plants under normal conditions. However, after heat stress treatment, MDA contents of transgenic Arabidopsis lines were significantly lower than in WT Arabidopsis plants ( Figure 4I ). Therefore, these results suggest that overexpression of ZmNAC074 in transgenic Arabidopsis may weaken the membrane lipid peroxidation and enhance the stability of cell membrane under heat stress, thereby contributing to slow down the oxidative damage caused by heat stress. In addition, compared with WT Arabidopsis plants, heat stress increased the accumulation of osmotic adjustment substances, including soluble proteins and proline in transgenic Arabidopsis lines ( Figures 4J, K ). Moreover, without heat stress treatment, the leaf chlorophyll and carotenoid contents of transgenic Arabidopsis lines were comparable to those of WT Arabidopsis plants ( Figures 4L, M ). Interestingly, after 24h of heat stress treatment, there was no significant difference in chlorophyll content between transgenic Arabidopsis lines and WT Arabidopsis plants, but the carotenoid content of transgenic Arabidopsis lines was 1.05-1.08 times higher than that of WT Arabidopsis plants. Moreover, after 48h of heat stress treatment, the leaf chlorophyll and carotenoid contents of transgenic Arabidopsis lines were 1.14-1.17 and 1.11-1.13 times higher than those of WT Arabidopsis plants, respectively. Therefore, these results demonstrate that transgenic Arabidopsis lines can enhance heat stress tolerance by retaining more chlorophyll and carotenoids to protect photosystem II under heat stress.

To elucidate the possible regulatory roles of ZmNAC074 in heat stress tolerance, the expression levels of ZmNAC074 and heat stress responsive genes have been characterized by qRT-PCR ( Figure 5A ). There was no significant difference in the expression level of ZmNAC074 among three transgenic Arabidopsis lines under normal conditions and after heat stress treatment for 24h. However, after 48h of heat stress treatment, the expression level of ZmNAC074 in line 9 was higher than those in line 1 and line 2. It was worth noting that with the prolongation of heat stress treatment time, the expression level of ZmNAC074 gradually increased. In addition, the expression levels of 11 heat stress responsive genes (AtHsp70-4, AtHsp101, AtHsp18.2, AtHsfA1a-e, AtHsfA2, AtHsfA3, AtHsfA7a, and AtbZIP60) were detected ( Figures 5B–L ). Under normal conditions, except that the expression levels of AtHsfA1b and AtHsfA2 genes in transgenic Arabidopsis lines were significantly higher than those in WT Arabidopsis plants, there was no significant difference in the expression levels of other genes between transgenic Arabidopsis lines and WT Arabidopsis plants under normal conditions. In contrast, under heat stress treatment, except that the expression level of AtHsfA3 gene in transgenic Arabidopsis lines was lower than that in WT Arabidopsis plants, the expression levels of other genes in transgenic Arabidopsis lines were higher than those in WT Arabidopsis plants. Thus, these results suggest that ZmNAC074 may enhance heat stress tolerance by regulating heat stress responsive genes.

To further detect the ability of the ZmNAC074-overexpressed transgenic Arabidopsis to scavenge ROS, the expression levels of some antioxidant-related genes (AtAPX1-8 and AtGPX1-8) between transgenic Arabidopsis lines and WT Arabidopsis plants under normal conditions and heat stress treatments were determined, respectively ( Figures 6A–P ). Under normal conditions, there were no significant differences in the expression levels of most antioxidant-related genes between transgenic Arabidopsis lines and WT Arabidopsis plants, except for AtAPX3, AtAPX7, AtAPX8, AtGPX1, and AtGPX2. However, under heat stress treatments, the expression levels of most antioxidant-related genes in transgenic Arabidopsis lines were significantly higher than those in WT Arabidopsis plants. Noticeably, the expression levels of AtGPX2 in three transgenic Arabidopsis lines was lower than that in WT Arabidopsis plants after heat stress treatment for 24h, and only transgenic line 9 exhibited higher AtGPX2 expression level than WT Arabidopsis plants after heat stress treatment for 48h. Interestingly, the expression levels of most antioxidant-related genes were the highest in transgenic line 9 and the lowest in transgenic line 2. Taken together, these results suggest that overexpression of ZmNAC074 in transgenic Arabidopsis can upregulate the expression levels of antioxidant-related genes under heat stress treatments.

To reveal the potential interaction effects of ZmNAC074 in response to heat stress, the possible interactions between some NAC MTFs and stress response-associated proteins in Arabidopsis and rice were integrated, especially OsNTL3 and AtNAC089, which are closely related to ZmNAC074 protein. The analysis of protein protein interaction network in Arabidopsis showed that bZIP family members, especially bZIP60, may be the central regulators of functional association network, which can interact with three NAC MTFs (including AtNAC089, AtNAC62, and AtNAC103) ( Figure 7A ). Further, the analysis of protein protein interaction network in rice displayed that BIP2 and BIP4 can interact with OsNTL3 ( Figure 7B ). Moreover, BIP2 and BIP4 are also closely related to bZIP family members, indicating the interaction between HSR-associated proteins and bZIP proteins. These networks are extremely complex, and there may be a variety of hierarchical regulation of NAC proteins in plants under heat or ER stress, such as the possibility of the interaction between OsNTL3 and BIP2, BIP2 and BZIP50 (a transcription factor involved in ER stress response), and BZIP50 and HSFA3 (a transcriptional regulator that specifically binds DNA of heat shock promoter elements). Moreover, the protein information involved in the figures was described as shown in Supplementary Table S4 . Overall, these results suggest that NAC MTFs, including ZmNAC074 protein, may interact with UPR- and HSR-associated proteins to enhance stress tolerance.

Studies have shown that under abiotic stresses, membrane-bound transcription factor (MTF) can be transferred into the nucleus and become an active TF (Seo and Kim, 2008), such as TaNTL1 and OsNTL3 (Liu et al., 2020; Sun et al., 2022). A previous study has shown that ZmNAC074 (also known as ZmNTL1) can be localized to plasma membrane and that the GFP-ZmNTL1-△TM (lacking the transmembrane motif) fusion construct product can be co-localized with nuclear marker (Wang et al., 2016). Moreover, ZmNAC074 protein shares high similarity of amino acid sequence with the reported orthologous OsNTL3 protein in rice, suggesting a similar functional role can exist in between ZmNAC074 and OsNTL3. Therefore, we speculate that ZmNAC074, like OsNTL3, can be localized to the plasma membrane under normal conditions and translocate to the nucleus in response to heat stress or other stresses (Liu et al., 2020). For another, ZmNAC074 may play a crucial role in response to heat, drought, and salt stress, especially heat stress. In addition, under these three kinds of various stresses, the expression of ZmNAC074 in leaves was changed highly and significantly compared with other tissues, indicating that ZmNAC074 may play a vital role through the tissue-specific expression in leaves.

Plants have evolved a variety of morphological, physiological and biochemical mechanisms to cope with heat stress, and the corresponding indicators have been used to evaluate heat resistance (Guihur et al., 2022). Phenotypically, the thermotolerance of transgenic Arabidopsis was significantly enhanced after heat stress. The enhancement of the adaptability of these transgenic Arabidopsis plants to heat stress was inseparable from the synchronous adaptation changes of external morphology and biochemical levels (Lephatsi et al., 2021). For example, levels of lipid oxidation markers such as MDA are typically reduced in plants designed as heat-resistant (Davey et al., 2005; Alché, 2019), while soluble proteins and proline, a penetrant or a free radical scavenger, increase (Alvarez et al., 2022; Ghosh et al., 2022). Therefore, the overexpression of exogenous ZmNAC074 may enhance tolerance to heat stress through decreasing lipid peroxidation, maintaining the stability of plant membrane structure and inducing proline and soluble protein biosynthesis to enhance osmotic potential in transgenic Arabidopsis.

Furthermore, the photosynthetic organelles of plants are highly sensitive to heat stress treatment and plants with higher chlorophyll content can be considered to exhibit greater heat tolerance (Hu et al., 2020; Muhammad et al., 2020). Carotenoids can be used as a stress-protective metabolite and mitigate harmful stress-induced ROS (Kang et al., 2018; Felemban et al., 2019; Guihur et al., 2022). After heat stress treatment, the content of carotenoids in transgenic Arabidopsis was higher than that in WT Arabidopsis, probably because the overexpression of ZmNAC074 in Arabidopsis may promote the synthesis of carotenoid production related enzymes under heat stress. However, there was no significant difference in chlorophyll content between WT Arabidopsis and transgenic Arabidopsis plants under heat stress after 24 h, but the chlorophyll content of transgenic Arabidopsis was significantly higher than that of WT Arabidopsis after 48 h, which may be due to the slight damage caused by heat stress in a short time was not enough for ZmNAC074 to activate numerous related chlorophyll degradation genes. In any case, further study is deserved to excavate the exact mechanisms underlying the positive role of ZmNAC074 in regulating heat stress response.

ROS is similar to a double-edged sword and ROS homeostasis is vital for plant growth, development and acclimatization against stresses (Sachdev et al., 2021). Excessive ROS generated by heat stress are toxic molecules capable of causing oxidative damage to proteins, nucleic acids, carbohydrates, cell membranes, lipids, and other sites of cells (Huchzermeyer et al., 2022). However, plants have evolved an antioxidant defense system including diverse antioxidants like SOD and CAT, which can protect them against oxidative damage (Baxter et al., 2014). Consistently, in this study, H₂O₂ levels and O₂ ^- contents were lower while SOD and CAT activities were higher in transgenic Arabidopsis than WT Arabidopsis under heat stress treatments, indicating that increased SOD and CAT activities resulted from ZmNAC074 overexpression in transgenic Arabidopsis is associated with heat stress tolerance through ROS-mediated stress responsive signaling pathway.

Further, APXs and GPXs can protect cells from stress-induced oxidative damage and play a key role in plant development and growth (Bela et al., 2015; Li et al., 2019; Singh et al., 2022). In this study, the significantly up-regulated expression of AtAPX1-8 and AtGPX1-8 in transgenic Arabidopsis suggested that ZmNAC074 may enhance the ability of antioxidants to scavenge excessive ROS by up-regulating these antioxidant enzyme genes, thus enhancing the ability to resist heat stress. Notably, several genes (AtAPX3, AtAPX7, AtAPX8, and AtGPX1) in transgenic Arabidopsis were expressed at higher levels than in WT Arabidopsis under normal conditions. We speculate that ZmNAC074 may trigger antioxidant defense system through signaling before heat stress. Under heat stress treatments, the antioxidant response was elevated, further increasing the activity of antioxidant enzymes. This protective mechanism may appear to cause more limited damage to cellular components in transgenic Arabidopsis lines in comparison to WT Arabidopsis plants. In conclusion, these results have evidenced that overexpression of ZmNAC074 can trigger the expression levels of numerous stress-associated genes in transgenic Arabidopsis. These genes can be considered as direct or indirect downstream genes of ZmNAC074, and further works are required to reveal the signal transduction pathways associated with ZmNAC074 during heat stress.

Previous studies have demonstrated that heat shock response (HSR) in cytoplasm and unfolded protein response (UPR) in ER act as two evolutionarily conserved systems that protect plants from heat stress (Li et al., 2020b). Heat stress induces HSR, in which heat shock transcription factors (HSFs) are a class of conservative transcription factors and can bind to heat shock cis-regulatory elements in promoter regions of HSP genes and promote the expression of HSP genes (Hayes et al., 2021; Li and Howell, 2021). HSPs, as molecular chaperones, promote the correct folding and aggregation of proteins (Hayes et al., 2021). In this study, the expression levels of most HSP and HSF genes in transgenic Arabidopsis were more highly expressed in the transgenic Arabidopsis lines, indicating that overexpression of ZmNAC074 confers enhanced heat stress tolerance of transgenic Arabidopsis by up-regulating the expression levels of these heat stress responsive genes. Therefore, ZmNAC074 may be a candidate gene for genetic engineering in generating crops to enhance heat tolerance.

Previous studies have revealed that AtbZIP60 can encode an UPR-associated transcription factor, and respond to NaCl and tunicamycin (TM) and thereby induce endoplasmic reticulum (ER) stress and enhance the abiotic stress tolerance in Arabidopsis (Li et al., 2017). Moreover, the close relationship between plant NAC proteins and ER stress has been revealed (Yu et al., 2022). For instance, three Arabidopsis NAC TFs (including AtNAC062, AtNAC089, and AtNAC103) can be activated to respond to ER stress and regulate different UPR-associated gene clusters (Sun et al., 2013; Yang et al., 2014a; Yang et al., 2014b). Notably, it has been reported that OsNTL3 (orthologous with ZmNAC074) directly binds to the promoter of OsbZIP74 (orthologous with AtbZIP60) and regulates its expression in response to heat stress (Liu et al., 2020). Thus, in this study, the expression level of AtbZIP60 in transgenic Arabidopsis was significantly higher than that in WT Arabidopsis under heat stress, indicating that ZmNAC074 may act on AtbZIP60 to alleviate UPR caused by heat stress to enhance heat stress tolerance. Moreover, the protein protein interaction networks further revealed that AtNAC089 (orthologous with ZmNAC074) interacted with AtbZIP60 in Arabidopsis and OsNTL3 may also affect OsbZIP50 (orthologous to AtbZIP60) by interacting with BiP2 or BiP4 in rice, implying that ZmNAC074 may function in heat stress-induced UPR-associated regulatory pathways. However, further investigations are needed to clarify the detailed mechanisms of the interactions between ZmNAC074 and these bZIP proteins in regulating stress responses in maize.

Taken together, we successfully constructed transgenic Arabidopsis plants overexpressing ZmNAC074 under the control of CaMV 35S promoter in this study. As expected, the transgenic Arabidopsis plants exhibited enhanced tolerance to heat stress, which might be associated with modulating HSR, UPR and ROS homeostasis as shown in the proposed model in this study ( Figure 8 ). Therefore, overexpression of ZmNAC074 may confer thermotolerance in transgenic Arabidopsis through the involvement in multiple diverse regulatory pathways. However, the molecular mechanisms underlying heat stress response still remain to be further elucidated.