AvERF73 positively regulates waterlogging tolerance in kiwifruit by participating in hypoxia response and mevalonate pathway

Abstract

Waterlogging stress is one of the greatest environmental threats to kiwifruit growth and development. ERF-VII proteins have been demonstrated to play pivotal roles in regulating plant tolerance to waterlogging. Nevertheless, the genome-wide role of ERF-VII in kiwifruit waterlogging stress tolerance remains unclear. Here, we report the function and regulatory network of an ERF-VII transcription factor located to the nucleus, AvERF73, in kiwifruit waterlogging tolerance. Overexpression of AVERF73 in Arabidopsis thaliana and A. chinensis cv. Hongyang enhanced waterlogging tolerance in transgenic plants. Furthermore, we performed transcriptome analysis (RNA-seq) and DNA affinity purification sequencing (DAP-seq) to explore the regulatory mechanism of AvERF73. RNA-seq coupled with DAP-seq showed that AvERF73 might directly activate AcNAC022 involved in the "cellular response to hypoxia" process and AcHMGS1 involved in the mevalonate pathway to respond to waterlogging, which were also confirmed by a dual-luciferase reporter assay. Based on our results, we propose a putative working model for controlling waterlogging tolerance by AvERF73 in kiwifruit.

Headnote

Received 22 March 2023; Accepted 11 May 2023; Available online 2 February 2024

ABSTRACT

Waterlogging stress is one of the greatest environmental threats to kiwifruit growth and development. ERF-VII proteins have been demonstrated to play pivotal roles in regulating plant tolerance to waterlogging. Nevertheless, the genome-wide role of ERF-VII in kiwifruit waterlogging stress tolerance remains unclear. Here, we report the function and regulatory network of an ERF-VII transcription factor located to the nucleus, AvERF73, in kiwifruit waterlogging tolerance. Overexpression of AVERF73 in Arabidopsis thaliana and A. chinensis cv. Hongyang enhanced waterlogging tolerance in transgenic plants. Furthermore, we performed transcriptome analysis (RNA-seq) and DNA affinity purification sequencing (DAP-seq) to explore the regulatory mechanism of AvERF73. RNA-seq coupled with DAP-seq showed that AvERF73 might directly activate AcNAC022 involved in the "cellular response to hypoxia" process and AcHMGS1 involved in the mevalonate pathway to respond to waterlogging, which were also confirmed by a dual-luciferase reporter assay. Based on our results, we propose a putative working model for controlling waterlogging tolerance by AvERF73 in kiwifruit.

Keywords: Actinidia; ERF-VII, Waterlogging stress; RNA-seq; DAP-seq

1. Introduction

Waterlogging stress is a serious environmental threat limiting plant growth and development and reducing the yield and quality of many crops, such as bitter melon, peanut, soybean and cotton (Tamang et al., 2014; Peng et al., 2020; Zhang et al., 2021; Zeng et al., 2022). Kiwifruit (Actinidia spp.), native to China, is popular worldwide because of its exotic taste and high vitamin C content. However, waterlogging stress, caused by frequent heavy rainfall and poor drainage in the orchard, is one of the main factors limiting the growth and development of kiwifruit, especially in southern China (Li et al., 2021). Kiwifruit comprises 54 species and 21 varieties (Huang and Liu, 2014). A. chinensis and A. deliciosa are extensively cultivated worldwide, but they are generally considered to be sensitive to waterlogging (Zhang et al, 2015). Therefore, it is imperative to explore waterlogging mechanisms and improve the waterlogging tolerance of kiwifruit.

The morphological and physiological responses of plants to waterlogging have been thoroughly studied (Sairam et al., 2008; Fukao et al., 2019). When subjected to waterlogging stress, plant aerobic respiration is inhibited and anaerobic fermentation is triggered owing to oxygen deprivation (Ruperti et al., 2019), which adversely impacts multiple metabolic processes and ultimately causes plant death (Kaur et al., 2020). Once plant perceives a stress signal, its internal genes will immediately respond. Many studies have shown that waterlogging-tolerant and -sensitive plants exhibit different molecular responses to waterlogging stress (Butsayawarapat et al., 2019). Recent research has shown that the waterlogging-tolerant chrysanthemum cultivar has higher ethylene production and expression of genes regulated in hormone response, N-end rule pathway, and ROS signaling than the waterlogging-sensitive chrysanthemum cultivar (Zhao et al, 2018). Additionally, the waterlogging-tolerant cucumber Zaoer№ maintained a more efficient carbohydrate metabolism and had higher gene expression involved in the ethylene pathway and formation of adventitious roots than the waterlogging-sensitive Pepino' (Xu et al., 2017).

Transcription factors (TFs), as the key transcriptional regulators, play crucial roles in initiating downstream gene expression to positively respond to environmental stress, such as WRKY (Wang et al., 2021; Lu et al., 2023; Dong et al., 2024; Wang et al., 2024), MYB (Ge et al., 2023; Mao et al., 2023), bHLH (Cui et al., 2018) and bZIP (Tu et al., 2020). Ethylene-responsive factors (ERFs), a plant-specific transcription factor family, share a highly conserved AP2 domain with a length of approximately 60-70 amino acids (Licausi et al., 2013a) and specifically bind to the ethylene-responsive element GCC-box (Ohme-Takagi and Shinshi, 1995). ERFs play a crucial role in crops resistance to abiotic stress (An et al., 2020; Qu et al., 2020; Chen et al., 2022). The group VII ethylene-responsive factors (ERF-VIIs) play a vital role in the hypoxia response in various plants (Nakano et al., 2006; Licausi et al., 2013b). In the model plant Arabidopsis thaliana, all five ERF-VII members play essential roles in waterlogging tolerance (Licausi et al., 2010; Xie et al., 2019). When oxygen is sufficient, AtERF-VII proteins are constantly degraded through the N-terminal rule because of their conserved MC motif (MCGGAI/L), which is recognized by the NERP pathway enzymes. However, under hypoxic condition, these ERF-VII proteins accumulate and positively regulate the expression of downstream hypoxia response genes (Licausi et al., 2013b; Tang et al, 2021a; Xie et al., 2021). In recent years, members of the ERF-VII group have been identified in other plant species, including oilseed rape (Lv et al., 2016), wheat (Wei et al., 2019), barley (Yin et al, 2019), petunia (Yu et al, 2019) and maize (Luan et al., 2020), and their positive function in waterlogging stress has been confirmed. In Chrysanthemum morifolium, CmERFS transcriptionally activates CmRAP2.3 gene expression, which enhances waterlogging tolerance by decreasing ROS levels (Li et al., 2022a).

Several ERF-VII genes have been identified in kiwifruit. A. deliciosa RAP2.3, a gene homologous to AtRAP2.3, was sharply induced under waterlogging stress and enhanced waterlogging tolerance of transgenic tobacco (Pan et al., 2019). Additionally, two ERF-VII members, AcERF74 and AcERF75 were isolated from A. chinensis, which respond to waterlogging stress by activating alcoholic fermentation (Liu et al., 2022). However, the reported ERF-VIIs were isolated from A. deliciosa or A. chinensis which show poor tolerance to waterlogging stress. It remains uncertain whether these genes can improve the waterlogging tolerance of kiwifruit. Therefore, it is necessary to identify key ERF-VII members in waterlogging-tolerant kiwifruit. Our previous study screened a waterlogging-tolerant genotype, KR5, from A. valvata, which survived well under longterm waterlogging treatment (Li et al., 2020). In previous study, we performed transcriptome-wide identification and expression analysis of ERF family in A. valvata during waterlogging stress, and found AvERF73 was specifically induced in waterlogging tolerant kiwifruit genotypes, but relatively low in the sensitive genotypes (Bai et al., 2021; Li et al., 2022b). Here, we investigated the role of AVERF73 in regulating waterlogging tolerance using overexpression technology and explored the molecular mechanism of AvERF73 using high-throughput sequencing. Our study provides new insights into the function of AvERF73 in regulating the waterlogging-tolerance mechanism in kiwifruit.

2. Materials and methods

2.1. Gene isolating and sequence analysis

The coding sequences (CDS) of AVERF73 was amplified from KRS (A. valvata) root cDNA using specific primers (Table 51). Online tool Expasy (https://web.expasy.org) was utilized to assess the molecular weight, isoelectric point, instability index and grand average of hydropathicity of the AvERF73 protein. The predicted protein sequences were aligned using the Clustal W method with default settings. The Neighbor-joining method with 1 000 bootstrap repeats was used to construct phylogenetic trees via MEGA 7.0.

2.2. Subcellular localization

To investigate subcellular localization, AvERF73 CDS lacking the stop code was amplified using specific primers (Table S1). The PCR product was subsequently inserted to pBI121-GFP, which was double-digested using restriction enzymes Xba I and BamH I. The fusion plasmid 35S-AvERF73-GFP and empty plasmid 35S-GFP (negative control) were then introduced into GV3101 strain of Agrobacterium tumefaciens. Positive colonies verified by PCR were transiently expressed in tobacco leaves for 2-3 а, and the GFP fluorescence were observed using a confocal laser scanning microscope (FV1000; Olympus, Tokyo, Japan).

2.3. Arabidopsis transformation and waterlogging treatment

The CDS of AVERF73 was cloned by PCR using a specific primer pair (Table S1) modified with Xba I and BamH I restriction sites. The AvERF73 CDS was cloned into a CaMV35S-driven pBI121 vector. The fusion plasmid was subsequently transformed into GV3101. The Agrobacterium-mediated transformation of Arabidopsis was conducted using the floral-dip method (Davis et al., 2009). PCR and qRT-PCR were used to screen positive transgenic plants. Three positive T3 transgenic lines of Arabidopsis and WT plants were subjected to waterlogging. In brief, five-week-old potted plants of transgenic lines and WT plants were placed in blue plastic basket (45 cm x 35 cm x 16 cm) and waterlogged to 2 cm of soil surface. Plant samples were collected on 0 d and 9 а after waterlogging treatment. Each sample consisted of three duplicates, with three seedlings per replication.

2.4. Kiwifruit transformation and waterlogging treatment

Micropropagated plantlets of A. chinensis Hongyang were cultured on Murashige and Skoog (MS) culture medium with 30 g-L^-1 sucrose and 7.2 g^-L agar (pH 5.8). AVERF73 CDS was inserted into pBI121-GFP vector and transformed into EHA105 strain. Agrobacterium harboring the fusion plasmid was transformed into 'Hongyang' leaves via the leaf disc method (Wang et al, 2007). PCR and qRT-PCR were used to screen positive transgenic plants. Two positive lines with high AvERF73 expression level were selected for further analyses. Transgenic lines (4- to 5-leaf stage) and WT kiwifruit plants were transferred to MS medium containing 0.7 mg-L- IBA for root culture. After rooting, the tissue culture plantlets were planted in pots containing a 2:1:1 mixture of peat moss, vermiculite, and perlite. Two-month-old plants were used to conduct waterlogging treatment according to the method described above. Each sample consisted of three duplicates, with three seedlings per replication.

2.5. Determination of physiological indices

The fresh weight (FW) and dry weight (DW) of leaves were manually measured before and after treatment according to the previously described method (Li et al., 2020), then the relative water content (RWC) was calculated by the following formula [RWC = (FW-DW)/FW x 100%]. O> generation rate, as well as H,0, and MDA contents were examined using Superoxide Anion assay kit, Hydrogen Peroxide assay kit and Plant Malondialdehyde assay kits, respectively (Nanjing Jiancheng Bioengineering Institute, China). The chlorophyll fluorescence of Arabidopsis plants and kiwifruit leaves were photographed using an IMAGING-PAM chlorophyll fluorometer (LRLB0187; Walz, Effeltrich, Germany), at the same time, F,/F,, was determined using Imaging Win-GegE (v 2.41a) software.

2.6. RNA extraction, cDNA synthesis and qRT-PCR

RNAprep Pure Plant Plus Kit (TIANGEN Biotech, Beijing, China) was used to extract plant total RNA. Subsequently, total RNA was reverse transcribed into cDNA according to the instructions of the First Strand CDNA Synthesis Kit (TOYOBO, Osaka, Japan). NovoStart® SYBR qPCR SuperMix Plus kit (Novoprotein, Suzhou, China) was used for qRT-PCR. Each sample consisted of three duplicates, and all reactions were analyzed using the 2"""T method.

2.7. RNA-seg and data analysis

Root samples of OE53 and WT kiwifruit plants waterlogged for 1 day were collected for transcriptome sequencing by the Novaseg6000 sequencing instrument (Bluescape Scientific, Hebei, China). The Hisat2 tool was used to map sequencing reads to "Hongyang reference genome (https://kiwifruitgenome.org). DEGs were defined as genes with P < 0.05 and |log,(Fold Change)| > 1. Enrichment analysis of DEGs function was conducted using the GO database. The threshold for significant GO enrichment was FDR <0.05.

2.8. DAP-seq and data analysis

DAP-seg was conducted as previously stated (O'Malley et al., 2016; Bartlett et al., 2017). Briefly, genomic DNA (gDNA) of the roots from WT Hongyang and OE53 plants waterlogged for 1 day was equal mixed and fragmented to construct sequencing libraries. The AvERF73 coding sequence was inserted into pFN19K HaloTag T7 SP6 Flexi vector, and then the fusion plasmid was expressed utilizing the TNT SP6 Coupled Wheat Germ Extract System (Promega, Madison, WI, USA) and purified and captured utilizing Magne Halo tag beads (Promega, Madison, Wisconsin, USA). The AvERF73-bound beads with gDNA libraries were incubated for 2 h at 25 °C. Then, the eluted DNA fragments were sequenced in two technical replicates using Illumina NovaSeq instrument. The beads without the addition of AvERF73 protein were used as input libraries (negative control). BWA-MEM tool was used to match the DAP-seq reads to the "Hongyang reference genome version 3 (Vasimuddin et al, 2019). MACS (Zhang et al, 2008) was used to obtain the peaks. Furthermore, the peaks from the two replicates with P < 0.05 were merged using IDR (Li et al, 2011). The conserved motifs in the peaks area were analyzed using MEME-ChIP software (Machanick and Bailey, 2011). Peaks were annotated using HOMER software (Heinz et al., 2010).

2.9. Luciferase reporter assay

The AvERF73 CDS was cloned into pBI121 to generate effector constructs. The 146 bp promoter sequences containing the target gene promoter GCCGCC cis-elements were inserted into pGreenlI 0800-LUC vectors to generate reporter plasmids. The effector and reporter plasmids were subsequently introduced into GV3101 (pSoup) strain. A mixed bacterial solution of TF and promoter cultures (3:1) was used to infiltrate tobacco leaves for induction analysis. After 48-72 h, LUC/REN ratio was determined using a Dual-Luciferase Reporter Gene kit (YENSEN, China). An empty effector plus a promoter reporter was utilized as the control.

2.10. Statistical analysis

Each experiment was conducted in triplicate. The statistical analysis was performed utilizing OriginPro 2022 v 9.9.0.220 (OriginLab Corporation, Northampton, MA, USA) and LSD test (Р < 0.05) was used determine the significant differences. The primers were designed using Primer version 5 software (Table S1).

3. Results

3.1. Cloning of AVERF73 and sequence analysis

We previously identified a waterlogging-induced ERF-VII gene in A. valvata KR5, named AvERF73. This gene has a full-length open reading frame (ORF) of 834 bp and was predicted to encode 277 amino acids with a predicted molecular weight (MW) of 31.23 kD, isoelectric point (IP) of 6.78, instability index of 49.90 and grand average of hydropathicity of -0.916 (Table S expression pattern of AVERF73 in different tissues showed that it was most abundant in the root (Fig. 1, a). In addition, the neighbor-joining phylogenetic tree showed that the protein sequences of AVERF73 were most closely related to those of AcERF74 (Fig. 1, b). Sequence analysis revealed AVERF73 protein contains a unique AP2 conserved domain, and its AP2 domain exists two conserved sites, Alal4 and Asp19 (Fig. 1, c), which further demonstrated that AvERF73 belongs to the ERF transcription family. In addition, AVERF73 was fused with the green fluorescent protein (GFP) to investigate its subcellular localization in vivo, and GFP fluorescence was detected in the nucleus (Fig.

3.2. Heterologous expression of AVERF73 improves waterlogging tolerance of Arabidopsis

To investigate the function of AvERF73 gene, we overexpressed it in A. thaliana and obtained three transgenic lines OE2-3, OE4-3 and OE7-2 (Fig. S1). Five-week-old wild-type (WT) and transgenic plants were subjected to waterlogging. We observed, compared with transgenic lines, WT plants suffered more seriously damaged after waterlogging for 9 days (Fig. 2, a). Under waterlogging stress, fresh weight and leaf relative water content (RWC) of WT plants was significantly decreased (Fig. 2, c, d), and O; generation rate and the H₂O₂ and MDA contents were significantly increased (Fig. 2, f, g, h). Imaging investigation showed that chlorophyll fluorescence was lower in the WT plant leaves (Fig. 2, b-e). Collectively, these results confirm that overexpression of AvERF73 enhances waterlogging tolerance in A. thaliana.

3.3. Overexpression of AVERF73 enhances the waterlogging tolerance of kiwifruit

To further confirm the role of AVERF73 in kiwifruit, two overexpressing kiwifruit lines, OE53 and OE65, were generated by transforming explants produced from leaf strips of A. chinensis 'Hongyang' (Fig. S2). When subjected to waterlogging for 10 d, the transgenic lines and WT plants showed distinct differences in phenotype and physiology. The leaves of WT plants wilted and chlorophyll fluorescence decreased under waterlogging stress; however, the transgenic lines performed well (Fig. 3, a-c, d). We also determined RWC of the leaves and found that transgenic lines had higher RWC (Fig. 3, b). In addition, the roots of the WT plants showed obvious symptoms after 10 d of waterlogging stress, but roots of the transgenic line performed well (Fig. 3, е). The roots of the WT plants had higher H,0, and MDA contents than those of the transgenic lines (Fig. 3, f and g). Our findings suggest that AvERF73 plays a beneficial role in response to waterlogging stress.

3.4. Transcriptomic analysis to identify genes regulated by AvERF73

To better clarify the molecular mechanisms regulated by AvERF73 under waterlogging stress, after waterlogging treatment for 1 d, we collected the roots of OE53 with better waterlogging tolerance and WT kiwifruit plants, and performed transcriptome sequencing (RNA-seq). Each sample consisted of three biological replicates. Both qRT-PCR assay and correlation analysis demonstrated that transcriptome data were accurate and reliable (Fig. S3). Compared to the WT plants, 6 865 differentially expressed genes (DEGs), including 2 769 up- and 4 096 downregulated genes, were identified in OE53 (Fig. 4, a; Table 53). GO enrichment analysis revealed that 6 865 differentially expressed genes (DEGs) were significantly enriched in 145 biology processes (Table S4).

As AvERF73is a positive regulator of waterlogging tolerance in kiwifruit, we focused on the up-regulated genes in OE53. We found that the "cellular response to hypoxia" was significantly enrichedin up-regulated genes (Fig. 4, b). Compared to WT plants, 50 genes involved in the "cellular response to hypoxia" were significantly induced in OE53 plants under waterlogging stress (Table S5). These hypoxia response genes (HRGs) contained many transcription factors, such as LOB, WRKY, zinc finger protein and NAC (Fig. 4, c). Because hypoxia is the main limiting factor for plant growth under waterlogging stress, we speculated that AvERF73 might regulate the expression of HRGs to resist waterlogging stress.

In addition, the up-regulated genes were significantly enriched in terpenoid compounds and sterol biosynthesis processes (Fig. 4, b). For the production of terpenoid compounds and sterols in higher plants, there are two biosynthetic pathways: the mevalonate (MVA) pathway in the cytoplasm and the methylerythritol phosphate/deoxyxylulose phosphate (MEP/DOXP) pathway in the organelle (Bohlmann et al., 1998; Tholl, 2006). We found that genes encoding key enzymes of the MVA pathway were more highly expressed in OE53 plants subjected to waterlogging stress for 1 d than in WT plants (Fig. 4, d). Terpenoids and sterols play positive roles in the adaptation to adverse environments for plants (Vriet et al., 2013; Tahri et al., 2022). We speculate that AVERF73 accumulates the terpenoids and sterols to cope with waterlogging stress by activating the MVA pathway.

3.5. Genome-wide binding targets of AVERF73

To identify AvERF73-binding target genes, we constructed gDNA libraries taking OE53 and WT kiwifruit roots as the materials and performed DAP-seq. Two replicates, DAP_1 and DAP_2, were observed, with 3 173 and 3 238 peaks, respectively, corresponding to 3 744 unique genes (Fig. 54). Merging two replicates, we obtained 3 962 peaks (Table S6), of which 220 were located in the promoter region, 98 in 5' untranslated regions (UTRs), 204 in introns, 2 605 in exons, 22 in 3' UTR, 91 in downstream, and 722 were in intergenic (Fig. 5, a). We further analyzed the binding motifs of AVERF73 protein using the MEME-CHIP tool and found one DNA motif, GCC-box ('GCCGCC'), with strong significance (Fig. 5, b). As a transcription factor, AvERF73 regulates the expression of downstream target genes by binding to a motif located in the promoter region. As described above, we identified 6 865 DEGs regulated by AvERF73 overexpression and obtained 220 AvERF73-binding peaks (corresponding to 218 unique genes, Table S7) located in the promoter regions using DAP-seq. Furthermore, 42 potential direct target genes (DTGs), comprising 16 up- and 26 down-regulated genes, were identified by comparing DEGs in OE53 to AvERF73 potential targets (Fig. 5, с; Table S8). As AVERF73 is a positive regulator of kiwifruit waterlogging tolerance, we focused on 16 up-regulated DTGs (Fig. 5, d).

3.6. AvERF73 is directly involved in the hypoxia response and MVA pathway

RNA-seq analysis revealed that AvERF73 may enhance the waterlogging tolerance of kiwifruit by upregulating the expression level of genes involved in "cellular response to hypoxia" and MVA pathway (Fig. 4). To further unveil putative DTGs of AvERF73, we performed a Venn diagram analysis. Strikingly, we found that among the 50 up-regulated genes in the "cellular response to hypoxia" process, only one candidate gene, Actinidia06591 (a NAC gene), was identified as a potential direct target of AVERF73 (Fig. 6, a). One prior study identified 142 NAC genes (ACNAC001-142) in A. chinensis 'Hongyang' (Jia et al., 2021). Based on the evolution analysis, we named Actinidia06591 as AcNACO22 (Fig. 55). Similarly, among the 17 up-regulated genes involved in the MVA pathway, only one gene, Actinidia27412 (a HMGS gene), was identified as a potential direct target of AVERF73, named АсНМС51 (Fig. 6, с). Our DAP-seq results revealed a clear AvERF73-binding peak in the promoters of AcNACO22 and AcHMGS1 (Fig. 56). Additionally, compared to WT plants, expression levels of ACNACO22 and AcHMGS1 were higher in OE53 and OE65 (Fig. 6, b-d). We further conducted a dual-luciferase reporter assay, and found that AcNACO22 and AcHMGS1 showed a positive relationship with AvERF73 protein (Fig. 6, e). We speculated that the expression of AcNACO22 and AcHMGS1 is regulated by AVERF73.

4. Discussion

Waterlogging stress is a major factor affecting the growth and development of kiwifruit. Unfortunately, there are few reports on the molecular mechanisms underlying waterlogging tolerance in kiwifruit. ERF TFs, the plant specific TF family, have been proven to be essential for plants to cope with adverse environmental conditions, including drought (Pan et al., 2012), cold (Sun et al., 2022), salt (Schmidt et al., 2013) and high temperature (Magar et al, 2022). The ERF family can be divided into 12 groups (Nakano et al., 2006). Many studies have shown that the seventh group of members of the ERF family, ERF-VIIs, plays a crucial role in the ability of plants to resist waterlogging stress (van Veen et al., 2014; Alpuerto et al., 2016). In our previous study, 26 ERFVII TFs were identified in the waterlogging-tolerant kiwifruit KR5 (A. valvata) (Bai et al, 2021). qRT-PCR results showed that AvERF73 had higher expression in KR5 plants under waterlogging stress (Bai et al., 2021). Evolution analysis revealed that AvERF73 was most closely related to AcERF74 (Fig. 1, b and с). Moreover, recent research has reported that AcERF74 plays a pivotal role in the response of kiwifruit to waterlogging stress (Liu et al., 2022). Based on this, we speculated that AvERF73 might be a positive regulator of the waterlogging tolerance in kiwifruit.

The overexpression of ERF genes in plants induces tolerance to abiotic stress (Rehman and Mahmood, 2015; Xie et al., 2019). To test whether AVERF73 was involved in the waterlogging tolerance, we overexpressed AvERF73 in A. thaliana and kiwifruit 'Hongyang'. Compared to WT plants, AvERF73-overexpressing lines (both A. thaliana and kiwifruit) showed better waterlogging tolerance (Figs. 2 and 3). After waterlogging, the transgenic plants showed significantly higher RWC of leaves than the WT plants. The chlorophyll fluorescence parameter, Fy/Fm, is a trustworthy indication of the photosynthetic ability of stressed plants (Lin et al., 2022). We found that the Fy/Fm ratio of the transgenic plants remained steady under waterlogging stress; but the F/Fm of the WT plants decreased sharply after waterlogging. In addition, waterlogging stress accumulates excessive ROS in plants, damaging plant metabolism (Lin et al., 2021). Os , H,0, and MDA are key oxidate stress indicators that reflect the degree of injury caused by stresses (Phukan et al., 2016). Under waterlogging stress, we observed that transgenic plants suffered slighter oxidate stress. These results verified the positive role of AVERF73 in the waterlogging response.

TFs, key regulator of signal transduction, play a critical role in regulating defense gene expression by directly binding to the promoter region of targets (Xu et al., 2011). Although research has focused on the function of ERF-VIIs, how waterlogging stress signaling interacts with ERF-VIIs to resist stress remains largely unclear. Here, to investigate the regulatory mechanism by which AvERF73 is involved in the waterlogging response, we first conducted RNA-seg to identify DEGs between AvERF73-overexpressing line OE53 and WT kiwifruit plants treated for 1 d, and 6 865 DEGs were identified in OE53 (Fig. 4, a). We then performed DAP-seq to identify AvERF73-binding genes. Core binding sequences and key targets of many TFs have been identified by DAP-seq, including LBD (Han et al., 2021), NAC (D'Inca et al., 2021), MYB (Tang et al, 2021b) and WRKY (Wang et al., 2022). Importantly, the publication of A. chinensis genome provides a foundation for DAP sequencing (Wu et al., 2019). Here, we identified the binding element of AvERF73 with the core sequence of 'GCCGCC,' which is similar to the reported ERF binding motif GCC box (Fig. 5, b) (Hao et al., 1998). Combining DAP-seq and RNA-seq, 42 overlapping genes (16 up- and 26 down-regulated genes) were designated as AVERF73 putative DTGs involved in the response to waterlogging stress in kiwifruit (Fig. 5, с; Table 58).

O2 deficiency is the main factor affecting plant growth under waterlogging stress (Bailey-Serres et al., 2012). HRGs can be induced under low-oxygen conditions, resulting in metabolic adaptation at both the cellular and organismal level. ERF-VIIs can activate HRGs to resist waterlogging stress (Bailey-Serres et al., 2012). In our study, the genes up-regulated in OE53 were significantly enriched in "cellular response to hypoxia" process (Fig. 4, b). In a comparison of up-regulated putative DTGs and upregulated genes participated in "cellular response to hypoxia", we only found one candidate target gene, AcNAC022 with a distinct AvERF73-binding peak situated its promoter 375 bp upstream of start codon (Fig. 6, a; Fig. S6, a). NAC TFs have been demonstrated to be crucial for other plants to cope with waterlogging stress. For example, A. thaliana ANAC102 is an important regulator of seed germination under flooding condition (Christianson et al., 2009). AcNACO022 showed a much higher expression level in AVERF73overexpressing lines than in WT plants (Fig. 6, b). Moreover, we demonstrated that AvERF73 can directly activate AcNAC022 gene expression (Fig. 6, е).

Terpenoids can eliminate excess ROS to cope with abiotic stresses (Grote et al, 2006; Lee et al., 2015). Plant sterols are important components of the membrane lipid bilayer and play crucial roles in abiotic stress responses (Rogowska and Szakiel, 2020; Du et al., 2022). Plant terpenoids and sterols are synthesized via the MVA pathway in the cytoplasm and the MEP/DOXP pathway in organelle (Bohlmann et al., 1998; Tholl, 2006). In the present study, overexpression of AVERF73 in kiwifruits significantly up-regulated the expression of key genes in the MVA pathway (Fig. 4, b-d). AcHMGS1 (Actinidia08251), an important gene in the MVA pathway, was designated as a putative direct target gene by comparing the RNA-seq and DAP-seq results (Fig. 6, с and д). HMGS, catalyzing the condensation of acetoacetyl-CoA to produce HMG-CoA, has been proven to be important for plant resistance to abiotic stress (Liu et al., 2019). Dual-luciferase reporter assay further demonstrated that AvERF73 directly activate the expression of АСНМС51 (Fig. 6, e).

5. Conclusions

Our data demonstrated that AVERF73 was an important regulator of waterlogging tolerance in kiwifruit and we proposed a putative working model (Fig. 7). On the one hand, overexpression of AvERF73 can activate the expression of AcNAC022 to cope with hypoxic conditions caused by waterlogging stress. On the other hand, overexpression of AVERF73 might regulate the MVA pathway to synthesize terpenoid compounds and sterols by directly activating the expression of AcHMGS1. However, the mechanism by which the interaction of AvERF73 with AcNAC022 and AcHMGS1 regulates waterlogging tolerance in kiwifruit requires further exploration. Our results provide candidate genes and new insight into the regulatory network of waterlogging tolerance in kiwifruit.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This study was funded by the National Key Research and Development Program (Grant No. 2022YFD1600700), Major Science and Technology Projects of Henan Province (Grant No. 221100110400), the China Agriculture Research System of MOF and MARA (Grant No. CARS-26), Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Science (Grant No. CAAS-ASTIP-2023-ZFRI-03), Yunnan Science and Technology Program (Grant No. 202205AF150043), and Sichuan Science and Technology Program (Grant No. 2021YFN0060).

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at https://doi.org/10.1016/j.hpj.2023. 05.021.

Footnote

1 These authors contributed equally to this work.

* Corresponding authors. Tel.: [phone].

E-mail addresses: [email protected]; [email protected]

Peer review under responsibility of Chinese Society of Horticultural Science (CSHS) and Institute of Vegetables and Flowers (IVF), Chinese Academy of Agricultural Sciences (CAAS)

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