Genome-wide identification of the NRAMP gene family in kiwifruit and the response of AcNRAMP7 to cadmium stress

Genome-wide identification of the NRAMP gene family in kiwifruit and the response of AcNRAMP7 to cadmium stress | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genome-wide identification of the NRAMP gene family in kiwifruit and the response of AcNRAMP7 to cadmium stress Xuechun Li, Hanbing Cai, Ping Tian, Ke Zhao, Jiaqiong Wan, Tuo Yin, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6967468/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Dec, 2025 Read the published version in BMC Plant Biology → Version 1 posted 15 You are reading this latest preprint version Abstract The NRAMP gene family acts on a wide range of divalent metal ions in plants and plays a key role in Cd accumulation and resistance in plants. In this study, we identified 12 AcNRAMP genes from the kiwifruit genome that were phylogenetically categorized into four groups. Chromosome distribution analysis revealed that 11 of the 12 AcNRAMP genes were unevenly distributed on seven chromosomes. The AcNRAMP gene promoter cis-elements were mainly categorized into hormone-associated cis-acting elements and environmental stress-associated cis-acting elements. In addition, expression profiling revealed that three genes were significantly differentially expressed in the roots and that two genes were markedly differentially expressed in the leaves. Based on the AcNRAMP family analysis by RT-PCR, we hypothesized that there might be a mechanism by which members of the AcNRAMP gene family respond to Cd stress: after being subjected to high concentrations of Cd stress, the expression of AcNRAMP7 and AcNRAMP10 was downregulated in roots, whereas the expression of AcNRAMP6 was upregulated in leaves, the concentration of Cd translocated to the root cells was reduced, and the environmental Cd in the environment decreased the degree of damage to plant root cells, which in turn led to an increase in Cd tolerance in kiwifruit plants and resulted in slower root growth. The results of this study are crucial, as they lay the foundation for further exploration of the regulatory functions of AcNRAMPs in kiwifruit in response to Cd stress. kiwifruit NRAMP cadmium stress expression pattern analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction The Kiwi fruit is a perennial deciduous vine of the genus Actinidia in the Actinidiaceae family. It has high nutritional, medical, and health value and is known as the king of fruit, the crown of vitamin C, Green Bank (New Zealand), and Beauty Fruit (Japan). Kiwifruit has a large planting area in China and is widely loved by people for its unique flavor. Its roots, stems, and leaves can also be used as medicines; thus, it is a Chinese medicinal material worthy of vigorous development and utilization (Jia et al., 2012). It can also be used as an adjuvant drug to treat hypertension, coronary heart disease, cancer, and other diseases (Song et al., 1984). The phenolic compounds in kiwi fruits have good antioxidant effects, and the saponins have strong hypoglycemic effects. However, with rapid industrial development, the intensified use of pesticides, and the lack of environmental remediation resources, large amounts of heavy metals have begun to enter the environment, with cadmium pollution in arable soil being the most serious (Yin et al., 2020). The total rate of heavy metal pollution in agricultural soil in China has reached 19.4%. Among all kinds of pollution, cadmium pollution accounts for the highest percentage, with a rate of excess points of 7.0%, making it one of the most crucial soil pollutants in China (Xiao et al., 2015). Cadmium is easily absorbed by plants. When cadmium accumulates to a certain extent in plants, it affects their regular growth, inhibits their photosynthesis, interferes with their normal metabolic process, reduces their transpiration, and accelerates their growth during aging (Wang et al., 2020. Shakirova et al. reported that cadmium could inhibit mitosis in root cells, cause chromosome aberrations in plants, and affect crop growth (Shakirova et al., 2016). Shakirova et al. reported that cadmium can exacerbate the production of reactive oxygen species (ROS) in crops and damage the antioxidant system of plants (Shakirova et al., 2016). Cadmium is also very harmful to animals and humans. Cadmium enters the human body through the food chain, causing great harm to the human kidney, liver, brain, and bones and having strong carcinogenic effects (Gao et al., 2017). Plants have evolved many heavy metal stress tolerance mechanisms, including strict regulation of the uptake, efflux, storage, detoxification, and transport of heavy metals and intracellular chelation to minimize the toxicity of heavy metals (Geng et al., 2011). Multiple gene family members play vital roles in heavy metal transport regulation (Singh et al., 2011). Among many gene families, NRAMPs play a key role in Cd uptake and accumulation in plants (Gao et al., 2020). The NRAMP gene was first isolated from mice in 1993 (Bozzi et al., 2021) and was identified as a divalent metal transporter protein in bacteria, fungi, insects, plants, and mammals (Pinner et al., 1997; Gunshin et al., 1997). In addition, NRAMP proteins are involved in the transport of various divalent cations in plants, such as Fe 2+ , Mn 2+ , Cu 2+ , Pb 2+ , and Cd 2+ . To date, the NRAMP gene family has been studied in tobacco (Gunshin et al., 1997), P. trichocarpa (Zheng et al., 2023), poplar (Ma et al., 2023), potato (Yu et al., 2022), Arabidopsis (Tian et al., 2021), and tea trees (Maser et al., 2001). In Arabidopsis, AtNRAMP6 is an intracellular cadmium transporter protein, whereas AtNRAMP1 is located in the root plasma membrane and acts as a Mn transporter protein (Li et al., 2021). There are seven NRAMP members in rice ( Oryza sativa ), among which OsNRAMP5 is a plasma membrane-localized transporter protein. Knocking out OsNRAMP5 reduces Mn and Cd concentrations in roots, shoots, and grains (Cailliatte et al., 2009). However, the transport mechanism of the NRAMP protein family in kiwifruit is still unknown. This study aimed to identify 12 NRAMPs , analyze their physicochemical properties, phylogenetics, gene structures, domains, conserved motifs, cis-regulatory elements, chromosomal locations, and gene replication, and investigate the expression patterns of kiwifruit in response to cadmium stress. Our study results lay the foundation for further exploration of the regulatory function of AcNRAMPs in the kiwifruit cadmium stress response. 2. Materials and methods 2.1 Identification and analysis of the physicochemical properties of the NRAMP genes in the kiwifruit genome NRAMP sequences were downloaded from the TAIR website (https://www.arabidopsis.org/Six Arabidopsis). From the kiwifruit genome database (http://kiwifruitgenome.org/organism/5), the genome data of kiwifruit ( Actinidia chinensis Hong Yang v3), the genome data of Arabidopsis thaliana from the Ensembal Plants database (https://plants.ensembl.org/index.html), and the early tea tree genome data were obtained from the Tea Tree Genome Database (http://tpia.teaplant.org/download.html). To identify the NRAMP gene in the kiwifruit 'Hongyang', conserved domain (Cation_efflux, Pfam number: PF01566) sequence information was obtained from Li (Yang et al., 2014). Based on the downloaded data, the conserved domain NRAMP (Cation_efflux, Pfam No. PF01566) was searched, and the results were screened for protein sequence number (Li et al., 2021) with an E value <1×10-5. Madeira F . Next, via the BLASTP program of BLAST software, the protein sequence of the Arabidopsis NRAMP gene family was used as a reference to perform BLAST on the kiwifruit genomic protein data, and the protein sequences in the kiwifruit genomic protein data were compared with the protein sequences of the Arabidopsis NRAMP gene family. For high values (E value <1×10 -5), the above steps were repeated. Finally, the sequence numbers obtained in the above two steps were merged. The merged protein sequence numbers were extracted from the kiwifruit genomic protein data via the TBtools tool and saved as *.fasta files (Madeira et al., 2019). The conserved domain database (CDD) tool of the NCBI database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and the online software PFAM (http://pfam.xfam.org/search #tabview=Additional file 1) further confirmed the existence of the NRAMP domain (Chen et al., 2020). The identified NRAMP gene families were renamed according to their chromosomal locations, and the naming format was as follows: NRAMP +serial number. The physicochemical properties were analyzed via ProtParam (http://www.ExPASy. org/tools/protparam.html) online software to determine the molecular weight (MW), isoelectric point (PI), number of amino acids, and aliphatic index (Marchler-Bauer et al., 2017). Finally, the Plant-mPLoc (Plant-mPLoc server (sjtu.edu.cn)) online tool was used for subcellular localization. 2.2 Phylogenetic analysis of AcNRAMPs To understand the evolutionary relationships among the NRAMP gene families in kiwifruit, Camellia japonica , and A. thaliana . The identified NRAMP protein sequences of kiwifruit, C. japonica , and A. thaliana were merged, sequence alignment was performed via MEGA11.0 software, trimming was performed via TBtools software, and a system was subsequently constructed via MEGA11.0 software and the maximum likelihood (ML) method of the IQtree Developmental Tree (Xi et al., 2023). The online software Chiplot (http://www.evolgenius.info/evolview/#/treeview) was used to visualize the evolutionary tree. Finally, the 12 kiwifruit NRAMP proteins were grouped based on the grouping of the Arabidopsis NRAMP proteins (Tian et al., 2004). 2.3 Gene structure, domain, and conserved motif analysis TBtools software was used to analyze the gene structure of each gene based on the kiwifruit genome annotation file (GFF3) (Madeira et al., 2019). The structural domain was subsequently predicted via the CD search online tool of the NCBI database (http://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) with the default parameters (Anderson et al., 2011). The conserved motifs of AcNRAMP proteins were analyzed via MEME (http://meme-suite.org/) online software (Brown et al., 2013). Thus, the differences among AcNRAMP family members were analyzed. The above results were visualized via TBtools software (Madeira et al., 2019). 2.4 Chromosomal positioning, gene replication, and collinearity Chromosome position information was obtained from the genome annotation file (GFF3), and chromosome position mapping was performed via TBtools software (Madeira et al., 2019). Gene duplication events were subsequently analyzed via MCScanX, and the collinear relationships between the AcNRAMP gene family members of Arabidopsis and kiwifruit were analyzed via TBtools software. 2.5 Analysis of cis-acting elements Gene IDs were obtained based on identification. The upstream 2000 bp nucleotide sequence of each gene was extracted from the genome annotation files (GFF3 format) via TBtools software and the online analysis software PlantCARE (https://bioinformatiAc.psb.ugent.be/webtools/plantcare/html/) for promoter cis-acting element analysis (Lescot et al., 2002). 2.6 Three-dimensional AcNRAMP protein model prediction and multiple sequence alignment Based on the protein sequences of the AcNRAMP genes extracted from the kiwifruit genome-wide protein sequence files, the proteins of the 12 AcNRAMP genes were predicted via a protein model via the WISS-MODEL database (https://swissmodel.ExPASy.org/). Multiple sequence alignment was performed via MEGA11 software, and the sequences were visualized via GeneDoc software. 2.7 GO functional analysis of the AcNRAMP genes The protein sequence of the AcNRAMP gene family was extracted from the kiwifruit genome-wide protein sequence file, and the protein sequences of the AcNRAMP gene family were extracted from the kiwifruit genome database (http://kiwifruitgenome.org/organism/5). GO function searches were performed for proteins of the 12 AcNRAMP genes for gene function annotation. TBtools was subsequently used to perform GO functional enrichment analysis on the NRAMP genes of kiwifruit, and the online tool biorender (https://app.biorender.com/) was used for mapping and visualization. 2.8 Transcriptome data-based gene expression analysis of AcNRAMPs The transcriptome sequencing data used the kiwifruit Hongyang genome (V3) (http://kiwifruitgenome.org/organism/3) as the reference genome and hissat2 for sequence alignment. The expression status of each gene is understood in detail via comparison analysis with the reference genome. Differential expression analysis between RCd_vs_RCK and LCd_vs_LCK was performed via the DESeqR software package (1.18.0). The expression profiles of the AcNRAMP genes in the leaves and roots were obtained from transcriptome data. The expression profile of the AcNRAMP gene was drawn based on the FPKM value, and the genes with significant differential expression were screened. The screening thresholds were FDR1, or log 2 FC<-1. The expression of the AcNRAMP gene was normalized, and TBtools software was used to visualize the AcNRAMP gene expression data. 2.9 Expression analysis of AcNRAMP under cadmium stress The oligonucleotide primers used to target AcNRAMP were designed via Primer Premier 5 (Appendix 1). First-strand cDNA was synthesized via a PrimeScriptTM series RT kit (TaKaRa). The ABI PRISM 7500 Real-Time PCR System was used for quantitative real-time PCR (qRT-PCR) and detection by Weiwei Genomics, Inc. The qPCR program was as follows: 95 °C for 3 min; 40 cycles of 95 °C for 15 s, 57 °C for 20 s, and 72 °C for 20 s; and a melting process at 60-95 °C to generate melting curves. For normalization, the Actinidia07041 gene was used as an internal control (Appendix 1). The 2 -ΔΔCt method was used to calculate the relative expression of the AcNRAMP gene (Pfaffl et al., 2001). The data are presented as the means ± standard deviations. Statistical analysis was performed via one-way analysis of variance (ANOVA) via GraphPad Prism 10.0 software. 3. Results 3.1 Identification and analysis of the physicochemical properties of AcNRAMP proteins Based on the protein sequence of the Arabidopsis NRAMP gene family as a reference, BLAST and hmmSearch were performed on the genomic protein data of kiwifruit, the kiwifruit NRAMP gene family was predicted via CD search, and Pfam and incomplete or nonexistent domains were removed to form a total of. Twelve NRAMP genes were identified. These genes were named AcNRAMP1-AcNRAMP11 according to their chromosomal locations from top to bottom, where AcNRAMP12 was not located on the chromosome. Further analysis of the sequence revealed that the lengths of the NRAMP genes were quite different, ranging from 129 ( AcNRAMP10 ) to 1,291 ( AcNRAMP3 ) amino acids, and the molecular weights were in the range of 13,897.89 ( AcNRAMP10 ) to 141577.95 (AcNRAMP3 ) kDa. The pi range was 4.43 ( AcNRAMP10 ) to 9.01 ( AcNRAMP5 ), the instability coefficient was between 27.17 ( AcNRMP2 ) and 41.79 ( AcNRAMP10 ), and in particular, the only genes greater than 40 were AcNRAMP3 , AcNRAMP1 and AcNRAMP10 , indicating that in addition to the proteins of the above three genes, other genes also had different values. The other AcNRAMPs were all unstable proteins. In terms of amino acid composition, the average aliphatic index was 110.39, which was conducive to increasing the thermal stability of globular proteins. The subcellular localization predictions of the Plant-mPLoc tool revealed that, with the exception of the AcNRAMP1 gene, which is located in the chloroplast and nucleus, the AcNRAMP3 and AcNRAMP4 genes are located in the chloroplast. However, the other genes are located in the cell membrane (Additional file 1). 3.2 Phylogenetic analysis of the AcNRAMP gene family To elucidate the evolutionary relationship between the kiwifruit NRAMP gene family genes and other NRAMP gene family genes, we aligned 12 NRAMP protein sequences from kiwifruit, 11 NRAMP protein sequences from tea plants, and six from A. thaliana and successfully constructed a phylogenetic tree (Figure 1). According to the clustering results, the kiwifruit NRAMP gene family could be divided into four groups, namely, Group 1, Group 2, Group 3, and Group 4. The NRAMP gene family was the most distributed in the first group, with four gene families in kiwi fruit, three gene families from Jasmine chinensis, and two gene families from Arabidopsis. The number of AcNRAMP genes was highest in Group 1 and Group 2, with four AcNRAMPs each. Groups 3 and 4 each have two AcNRAMPs . Furthermore, the Arabidopsis NRAMP gene was not present in the second group. 3.3 Gene structure, domains, and conserved motif analysis of the AcNRAMP gene family To further study the sequence characteristics of the AcNRAMP protein, this study used the MEME online tool to analyze the motif composition of this protein (Figure 2-A). The results revealed that ten motifs were identified in AcNRAMP proteins, and the conservation of these ten motifs (motifs 1, 2, 3, 4, 5, 6, 7, 8, and 10) was high; however, the same motif was used at different positions in the protein sequence, which may be related to the structure and function of the proteins. However, Motif 9 was absent in the AcNRAMP gene, and the AcNRAMP10 gene had only one Motif 5. The differences in the distribution of conserved motifs among the AcNRAMP gene family may also be affected by gene structure and domains. The distribution of gene structure is the key to studying evolutionary characteristics within a gene family. We performed sequence alignment of the 12 AcNRAMP genes and analyzed the gene structure based on the genome annotation files (Figure 2-C). The results revealed that the gene structure of most of the AcNRAMP gene family was conserved. There were 0-11 introns in the 12 AcNRAMP gene family members. Except for AcNRAMP11 , AcNRAMP7 , and AcNRAMP8 , which have no exons, the other genes all contain 1--3 exons, indicating that although the introns and exons of this gene family have some differences, the gene structure is also the same. The relative conservation was distinct, especially for each gene. In addition, this study also analyzed the conserved domains. Among the 12 AcNRAMP proteins, only two genes, AcNRAMP4 and AcNRAMP3 , had the NRAMP gene family domain, and the remaining nine genes all had the SLC5-6-like_sbd superfamily domain. (Figure 2-B). 3.4 Chromosomal location and gene replication analysis of the AcNRAMP gene To understand the chromosomal distribution and genome-wide density of the kiwifruit AcNRAMP genes, TBtools software was used to perform positioning analysis on the 29 chromosomes of the kiwifruit genome (Figure 3C). Our results revealed that 11 of the 12 genes were distributed on chromosomes, whereas AcNRAMP12 was not located on chromosomes and was free elsewhere. Most chromosomes do not have the AcNRAMP gene. The other 11 Ac NRAMP genes are Ac NRAMP1 , Ac NRAMP2 , Ac NRAMP3 , Ac NRAMP4 , Ac NRAMP5 , AcNRAMP6 , Ac NRAMP7 , Ac NRAMP8 , Ac NRAMP9 , Ac NRAMP10 , and Ac NRAMP11 , which are distributed on Chr5, Chr9, Chr18, Chr21, Chr26, Chr26, Chr27, Chr27, Chr29, Chr29), and Chr29. Moreover, chromosome length is not directly related to the number of genes. For example, chromosome 29 is 12 Mb in length and has three AcNRAMP genes, whereas chromosome 5 is 20 Mb and has only one AcNRAMP gene. Gene duplication events are critical for the evolution of family members. Therefore, to explore the evolutionary pattern of the NRAMP gene family in the kiwifruit genome, gene duplication analysis was performed for the 12 AcNRAMP genes (Figure 3A). In our study, 13 pairs of fragmented and repetitive genes were identified, and they were distributed on seven different chromosomes. In particular, the number of genes on chromosome 29 was high, with three AcNRAMP genes and four gene duplication events on this chromosome. These fragment repeats may play a key role in the gene duplication events of the AcNRAMP family. This study also analyzed the NRAMP collinear relationships among the three species of kiwi, Arabidopsis , and grape (Figure 3C). The AcNRAMP genes were screened from the collinearity files for collinearity visualization. The results revealed nine pairs of homologous genes in kiwifruit, Arabidopsis , and grape. In the literature, the evolutionary relationships of Arabidopsis and dicots are closer than those of monocots. 3.5 Cis-acting element analysis of the AcNRAMP genes The upstream 2000-base sequences of the 12 AcNRAMP gene family members were analyzed for cis-acting elements via PlantCARE and TBtools software. The results revealed that the promoter region of each AcNRAMP gene presented multiple stress- or hormone-related cis-elements (Figure 4). The conserved DNA modules involved in the light response and the elements essential for anaerobic induction were distributed in almost every gene. Hormone-related cis-acting elements, such as jasmonate ( MeJA ), gibberellin (GA), salicylic acid , and gibberellic acid, are second only to response elements involved in anaerobic induction. However, among the 12 AcNRAMP genes, only five AcNRAMP genes did not have MeJA response elements; 58% of the genes in this gene family had MeJA response elements in their promoters; and there were few GA response elements in this gene family. It only appeared in five AcNRAMP genes, but among these five AcNRAMP genes, except for the AcNRAMP10 gene, which contained 2 GA response elements, each gene contained only 1 GA response element (Figure 4). However, compared with other genes, there were relatively more cis-acting elements involved in the gibberellic acid response, e.g., one element in both AcNRAMP8 and AcNRAMP11 and three elements in AcNRAMP3 , AcNRAMP6 , AcNRAMP5 , and AcNRAMP10 . All four genes were present, which might be due to the interaction between these six genes and the cis-acting elements involved in the gibberellic acid response. Next are environmental stress response elements, such as low temperature, drought, defense and stress, and anaerobic-induced response elements. These types of response elements are distributed in each gene; for example, the response elements in the AcNRAMP4 gene participate in defense and stress responses. There are four response elements: one response element involved in the low-temperature response; four response elements in the AcNRAMP10 gene participate in the drought response; and two in the AcNRAMP5 gene participate in the low-temperature response. The distributions of other response elements, such as cis-regulatory elements involved in endosperm expression, auxin response elements, protein binding sites, zein metabolism regulation response elements, and circadian rhythm response elements, were relatively low. In short, in addition to the conserved DNA modules involved in the light response, most promoter homeopathic elements are involved in abiotic stress and hormone responses and regulate gene expression and substance metabolism in plants to develop stress tolerance. This phenomenon improves the adaptability of kiwifruit to cadmium stress. 3.6 Three-dimensional AcNRAMP protein model prediction and multiple sequence alignment The WISS-MODEL tool was used to confirm the structures of the AcNRAMP proteins. All AhNRAMP proteins were well modeled via the 5m87.1 template (Figure 5A and Additional file 2). The sequence identities were 23.54%-35.27%, the GMQE values were in the range of 0.2--0.59, and the overall QMEANDisCo score was in the range of 0.42--0.63 (Additional file 2). These data indicated that the three-dimensional model prediction quality of AcNRAMP proteins was high. Multiple sequence alignment revealed that all the AcNRAMP proteins presented considerable homology (Figure 5B). In addition to AcNRAMP 2, other AcNRAMP s contain the NRAMP family domain DPGN (essential for metal binding). Except for AcNRAMP10 , all AcNRAMPs contain the typical conserved amino acid residue GQSSTITGTYAGQY(/F)V(/I)MQ(/G/E)GFL, where the last five residues (MQGFL), which is a unique structural feature among the NRAMP metal transporter proteins, are retained (Cellier et al., 2021). 3.7 GO functional analysis of the AcNRAMP genes To further describe the biological functions of the AcNRAMP gene family, this study used the kiwifruit database to annotate the proteins of 12 AcNRAMP genes for gene function, and the online tool GraphPad Prism 10.2.3 was used for drawing and visualization (Figure 5C). Among the 12 genes, 12 AcNRAMP genes were annotated and assigned to three categories: molecular function (MF), biological process (BP), and cellular component (CC). In particular, in terms of molecular function, transmembrane transporter activity, metal ion transmembrane transporter activity, inorganic cation transmembrane transporter activity, single-atom cation transmembrane transporter activity, substrate-specific transmembrane transporter activity, substrate-specific transporter activity, etc., the enrichment level of the AcNRAMP genes was high, indicating that the molecular function of the AcNRAMP gene family plays a crucial role in regulation. However, among the other CCs, the overall composition of the membrane, diaphragm, membrane, organelle, and intracellular organelle fractions was enriched, and the number of enriched AcNRAMP genes was higher. In terms of biological processes, the genes enriched in AcNRAMPs were associated with transition metal ion transport, ion transport, metal ion transport, cation transport, divalent metal ion transport, inorganic cation transmembrane transport, cation transmembrane transport, and the active regulation of biological processes. During growth and development, the transport of metal ions affects the morphogenesis of plants. The GO functions of the AcNRAMP genes indicate that the AcNRAMP gene family adapts plants to different environments by regulating plant metal tolerance. In addition, the number of enriched genes in each category was significantly greater than 11, indicating that the same gene may play different roles in different functional items. 3.8 Tissue-specific expression profiles of AcNRAMP genes When plants are under stress, plant cells respond to environmental fluctuations by continuously changing their gene expression, and these continuous changes are regulated by a multilevel network of gene families. Therefore, to understand the expression pattern of the AcNRAMP gene family under cadmium stress, this study analyzed the transcriptome data of kiwifruit plants after cadmium stress. The results revealed 12 AcNRAMPs under cadmium stress, including five downregulated genes and seven upregulated genes in leaves, as well as nine downregulated genes and three upregulated genes in roots. Compared with those in the control group, three genes were significantly differentially expressed in the roots. The two genes AcNRAMP7 , AcNRAMP10 , and AcNRAMP11 were significantly differentially expressed in the leaves. AcNRAMP6 and AcNRAMP11 (Figure 6) imply that these genes may be crucial in fighting cadmium toxicity. The expression level of the AcNRAMP5 gene was 0 in the control group and was upregulated to a certain extent after being subjected to cadmium stress. Those with relatively high expression levels in leaves, AcNRAMP6 , presented relatively low expression levels in roots. 3.9 Analysis of AcNRAMP gene expression under cadmium stress In this study, we conducted cadmium treatment on kiwifruit seedlings under consistent growth conditions. To elucidate the expression pattern of AcNRAMP under Cd stress, we analyzed the expression of five DEGs under Cd stress via RT-qPCR and only the transcripts of three kiwifruit NRAMP family members (Figure 7B). The other two differential genes might be due to the primers; after many attempts, no product was amplified. The qRT-PCR data revealed that AcNRAMP6 expression in the leaves of the RCK and RCd groups significantly increased. However, the RCK and RCd groups presented significant decreases in AcNRAMP7 and AcNRAMP10 in the roots, indicating that the levels of AcNRAMP7 and AcNRAMP10 in the RCK group and the RCd group significantly decreased under Cd stress. The stronger the response was, the higher the expression level was. Under cadmium treatment, AcNRAMP7 and AcNRAMP10 presented different responses in roots than did AcNRAMPs in leaves, suggesting that there is an intricate interaction between the level of Cd stress and the regulation of gene expression. This observation revealed the complex regulatory mechanism controlling AcNRAMP expression under Cd stress. These findings further emphasize the necessity of specific molecular responses of these transition metal transporters in kiwifruit under different stresses. 4. Discussions Plant metal tolerance proteins are membrane-divalent transporter proteins. As part of the response to specific metal toxicity, they play a crucial role in the special transport of different heavy metal ions. In addition to their key function in maintaining mineral plants, changes in their transcript levels can also be considered environmental biomarkers for examining heavy metal pollution levels. Dong et al. studied rice under cadmium stress and reported that the growth of rice plants was significantly inhibited after one week of cadmium treatment. In this study, the leaf edges of kiwifruit seedlings subjected to 10 μM CdCl 2 and 100 μM CdCl 2 presented different degrees of yellowing and browning, especially in response to 100 μM CdCl 2 . Some phenolic compounds in the treatment group caused the damaged tissues to turn brown. Plant tissue browning and discoloration are the most intuitive responses of plants to stress, and to a certain extent, they can reflect the cadmium tolerance of plants (Figure 7A). Zhang et al. studied the physiological and phenotypic status of Chuanqiong under different concentrations of cadmium stress. They reported that, under 20 mg/L cadmium treatment, the plants in the treatment group presented obvious toxicity symptoms, including yellowing, wilting, sensitization, and browning. As cadmium stress intensifies, so does the degree of browning. At this time, kiwifruit seedlings were severely injured by cadmium toxicity, indicating that it was difficult for kiwifruit seedlings to withstand cadmium toxicity under 100 μM CdCl 2 treatment (Figure 7A), which was consistent with previous study results in other plants. The plant genome contains many genes with different functions. The NRAMP gene family comprises complete membrane transporter proteins in plants, such as potato, tea, and Brassica napus (Tian et al., 2021; Liu et al., 2023; Migeon et al., 2010; Meng et al., 2010). Regulating the response to various metal ions by mediating gene expression. In addition, they participate in many biological mechanisms, including photosynthesis, protein function, metabolism, and response to environmental stress (Nevo et al., 2006). However, no information has focused on the genome-wide identification of the NRAMP family in kiwifruit or the effects of cadmium stress on the transcript levels of genes known to be expressed in response to metal stress. Therefore, this study conducted a bioinformatics analysis of the kiwifruit NRAMP gene family, including phylogenetic information, conserved motif information, gene structure information, expression data, and information on cis-acting elements. The number of AcNRAMPs differed from that in Arabidopsis and tea. This may be due to the distant relationship between the AcNRAMP of 'Hongyang' and them. Among them, AcNRAMP3 and AcNRAMP4 were the largest, with lengths of 1291 AA and 1230 AA, respectively, molecular weights of 141577.95 kDa and 135045.72 kDa, and instability coefficients of 40.88 and 38.65, respectively. AcNRAMP10 was the shortest, with a length of 129 AA, a molecular weight of 13897.89, and an instability coefficient of 41.79. In biological systems, variation in protein length within gene families such as NRAMPs is not uncommon. These variations can lead to functional diversity among gene family members, allowing them to adapt to different environmental conditions and biotic interactions (Yin et al., 2023). Certain aspects of gene profiles, such as the arrangement, location, and number of exons and introns, are considered decisive factors for understanding genome function and evolution (Bari et al., 2021) Bari MA. In this study, the gene structure of AcNRAMPs included 4--11 introns. Except for AcNRAMP7 , AcNRAMP8 , and AcNRAMP11 , which do not have exons, the other genes all contain 1--3 exons. In the clustering tree, the members of the same cluster presented similar numbers of exons and introns, and all presented larger introns and smaller exons. Previous studies have demonstrated several characteristics of plant genes through high expression levels. These features include abundant longer introns, a less compact gene structure, larger primary transcripts, etc., and the opposite trend was observed in animal genes. For example, genes in yeast, Thaler, and mice share a regular feature, that is, fewer introns, which may be due to the rapid activation of genes in response to a wide range of environmental stresses (Jeffares et al., 2008). In this study, ten motifs were also screened via the MEME tool. Conserved motif analysis revealed that similar genes were clustered in the same group and that there were multiple motifs in specific proteins. This finding illustrates that these genes might have special functions. Motif 5 was present in all the AcNRAMPs , and this motif was always present between motifs 3 and 2, whereas the other motifs presented different patterns. The existence of the complete motif represents the evolutionary function and structural conservation of AcNRAMPs . Studies in rice have shown that OsNRAMP5 is a plasma membrane-localized transporter protein that facilitates the uptake of Fe, Mn, Cd, and lead (Pb). The knockout of OsNRAMP5 significantly reduces the concentrations of Mn and Cd in roots, shoots, and grains but promotes the transport of Cd from roots to shoots (Bari et al., 2021). Among the 12 AcNRAMP protein sequences, AcNRAMP2 and AcNRAMP10 lacked the structural characteristics of NRAMP proteins. The remaining ten AcNRAMP proteins all contained all the structural characteristics of the NRAMP proteins. They contain conserved motifs. Numerous studies have shown that these two domains are critical for the metal transport activity of NRAMP transporters. Next, the AcNRAMP protein was better modeled via the same 3D model template, 5m87.1A. The 5m87.1A template is the crystal structure of the ScanDMT divalent metal transporter protein. This model is a close prokaryotic homolog of the NRAMP family, which functions to transport divalent transition metal ions. Multiple sequence alignment revealed that the AcNRAMP proteins presented considerable homology. These analogous structures indicated that AcNRAMPs could have physiological functions similar to those of ScaDMT. Analysis of the cis-elements revealed that a variety of elements located 2000 bp upstream of the promoter region are involved in various functions. This provides a better understanding of gene transcription and expression, as well as their responses to environmental stress. This study revealed that the AcNRAMP gene had more elements essential for anaerobic induction than did the other genes. Hormone-related cis-acting elements, such as jasmonate (MeJA), gibberellin (GA), salicylic acid, and gibberellic acid, are second only to response elements involved in anaerobic induction. The distribution of cis-acting elements involved in the gibberellic acid response is relatively greater; there are 1--3 elements each in AcNRAMP3 , AcNRAMP5 , AcNRAMP6 , AcNRAMP8 , AcNRAMP10 , and AcNRAMP11 . Formula effect element interaction causes. These elements have also been reported in the NRAMP gene of Ziping ( Spirodela polyrhiza ) (Chen et al., 2021). In addition, gibberellin is heavily involved in the rice defense response to abiotic environmental stress by increasing salicylic acid production (Bari et al., 2021). The published literature indicates that salicylic acid is vital in managing plant adaptations to abiotic stresses, especially heavy metal toxicity. This is achieved by maintaining membrane stability and integrity and scavenging ROS. Therefore, it is reasonable to predict that gibberellin enhances cadmium tolerance by increasing glucose metabolism, promoting mitotic activity, and increasing RNA content (Emamverdian et al., 2020). Emamverdian A. These plant hormones can regulate the transcription of NRAMP genes, potentially affecting their functions in metal ion transport and stress response in plants. Specifically, these genes may be involved in NRAMP gene expression regulation in response to various environmental stimuli, such as biotic and abiotic stresses. In our molecular biology study, the distribution of AcNRAMP gene family members on seven chromosomes was significantly unbalanced, whereas no such genes were detected on the remaining 22 chromosomes. In addition, we found that most of these genes were derived from fragment duplication events, which highlights the vital role of fragment duplication in promoting gene family expansion. This result is different from that of potato and poplar (Tian et al., 2021; Siqin et al., 2023; Migeon et al., 2010), and the other results were similar. Transcriptomic analysis revealed the expression patterns of the NRAMP genes in roots under cadmium stress. Many studies have analyzed NRAMP expression in different species under different stresses. Genes from the same subfamily have similar expression levels in the same tissue (Shirazi et al., 2023). For example, the AcNRAMP6 and AcNRAMP11 genes in kiwifruit leaves were located in the Geoup2 branch. Similarly, in rice, OsNRAMP6 and OsNRAMP5 are involved in Fe and Mn uptake. In addition, the functions of AtNRAMP3 and AtNRAMP4 have been confirmed in the transfer of Fe and Mn (Bari et al., 2021; Jeffares et al., 2008; Emamverdian et al., 2020; Shirazi et al., 2023; Rasheed et al., 2024) Rasheed A. These reports support the involvement of the NRAMP gene in metal transport in plant tissues. In our study, we focused on the different expression patterns of AcNRAMPs in the presence of cadmium contamination. The results revealed that the expression of AcNRAMP7 and AcNRAMP10 in roots was significantly downregulated under Cd stress; the expression of AcNRAMP6 was markedly upregulated in leaves, suggesting that these genes may be involved in Cd transport. As such, we speculated that after high-concentration Cd stress, the expression level of AcNRAMP6 was upregulated, and the expression levels of AcNRAMP7 and AcNRAMP10 were downregulated. The three genes whose expression significantly differed may have contributed to the reduced amount of Cd entry into root cells, resulting in the effect of Cd in the environment on plants. The destructive power of root cells decreased, which in turn increased the tolerance of kiwifruit plants to Cd. 5. Conclusions Notably, this study provides the first elaboration of Ac N RAMP gene family information in the kiwifruit genome. Gene family analysis revealed that there were more metal stress and hormone response elements and that these elements played a critical role in the metal stress response. The expression patterns revealed that two genes of the NRAMP gene family were differentially expressed in kiwifruit roots and that one gene was differentially expressed in the leaves. In addition, the regulatory mechanism of AcNRAMP family members in response to Cd stress is as follows: after high-concentration Cd stress, the expression levels of AcNRAMP7 and AcNRAMP10 are downregulated (Figure 8). As a result, the amount of Cd entry into root cells is reduced, the Cd concentration in roots is reduced, the damage caused by Cd in the environment to kiwifruit root cells is reduced, and the tolerance of kiwifruit plants is improved. This study lays the foundation for mechanistic studies of the uptake and transport of heavy metal elements in kiwifruit. Declarations Acknowledgments The authors thank Prof./Dr. Deqiang Zhang, Beijing Forestry University, for his critical reading of the manuscript. Data availability statement All the data generated or analyzed in this study are included in this article. Kiwifruit genome annotation files can be accessed at http://kiwifruitgenome.org/. RNA-Seq data under drought stress can be found. The login link is https://www.ncbi.nlm.nih.gov/sra/PRJNA1246740. The RNA-Seq data are publicly available at the National Center for Biotechnology Information. The other data presented in this study are available in the Supplementary Materials. Authorship contribution statement Xuechun Li: conceptualization, investigation, formal analysis, writing-original draft preparation, and software. Hanbing Cai: Methodology, formal analysis, and visualization. Tuo Yin: Software, Data curation. Ke Zhao: Validation. Ping Tian: Validation. Jiaqiong Wan: Writing-review. Hanyao Zhang: Review and editing, supervision, funding acquisition. Xiaozhen Liu: Methodology, Writing-review and editing, Funding acquisition. All the authors have read and approved the final manuscript. Ethics declarations Ethics approval and consent to participate Not applicable. 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. Funding This study is supported by the Yunnan Academician (Expert) Workstation Project (202305AF150020), the Agricultural Joint Key Projects in Yunnan Province (202301BD070001-003), the Rural Revitalization Science and Technology Project-Rural Revitalization Industry Key Technology Integration Demonstration Project (202304BP090005), the Yunnan First-class Construction Discipline of Forestry Science of Southwest Forestry University and the Start-up Fund Project of Doctoral Research at Southwest Forestry University. The funders had no role in the design of the study, the collection, analysis, or interpretation of the data, or the writing of the manuscript. References Jia Z, Zeng H. Medicinal value of kiwifruit[J]. Modern Distance Education of Chinese Traditional Medicine,2012, 10(04):118.https://doi.org/10.3969/j.issn.1672-2779.2012.04.081 Song PJ, Zhang L, Ding L. Cancer preventive effects of Chinese kiwifruit juice-(II) Blockade of nitrosamine synthesis in simulated gastric juice in vitro by Ames test[J]. Journal of Nutrition, 1984(03):241-246.https: //doi.org/10.13325/j.cnki.acta.untr.sin.1984.03.006 Yin M, Tang H, Yang D, et al. Remediation experiments of different varieties of red hemp in heavily and slightly cadmium-polluted cropland[J]. Journal of Agricultural Environmental Science,2020,39(10):2267-2276. https://doi.org/10.11654/jaes.2020-0257 Xiao Q, Wang J, Zheng X, et al. Differential protein expression in rice roots in response to cadmium stress[J]. Journal of Ecology, 2015, 35(24):8276-8283.https://doi.org/10. 5846/stxb201403230517 Wang B, Liu Y, Wang H, et al. Contamination and health risk assessment of lead, arsenic, cadmium, and aluminum from a total diet study of Jilin Province,/China.[J]. Food Science & Nutrition, 2020, 8(10):5631-5640.https: //doi.org/10.1002/fsn3.1851 Shakirova FM, Allagulova ChR, Maslennikova DR, et al. Salicylic acid-induced protection against cadmium toxicity in wheat plants. Environmental & Experimental Botany 2016:19-28.https://doi.org/10.1016/j.envexpbot. 2015.08.002 Gao Y, Cheng Q, Zhang M, et al. Research on remediation technology of cadmium-contaminated soil[J]. Biotechnology Bulletin, 2017,33(10):103-110. https://doi.org/10. 13560/j.cnki.biotech.bull.1985.2017-0336 Geng HX, Wang L. Cadmium: Toxic effects on placental and embryonic development[J]. Environmental Toxicology and Pharmacology, 2019, 67:102-107.https://doi.org/10. 13560/10. 1016/j.etap.2019.02.006 Singh RK, Anandhan S, Singh S, Patade VY, Ahmed Z, Pande V. Metallothionein-like gene from Cicer microphyllum is regulated by multiple abiotic stresses. Protoplasma. 2011 Oct;248(4):839-47.https://doi.org/10.1007/s00709-010-0249-y Gao Y, Yang F, Liu J, Xie W, Zhang L, Chen Z, Peng Z, Ou Y, Yao Y. (2020). Genome-wide identification of metal tolerance protein genes in Populus trichocarpa and their roles in response to various heavy metal stresses. International Journal of Molecular Sciences. 21:1680. https://doi.org/10.3390/ijms21051680 Bozzi AT, Gaudet R. Molecular mechanism of NRAMP-family transition metal transport. Journal of Molecular Biology. 2021, 433, 166991.https://doi.org/10.1016/j.jmb.2021.166991 Vidal SM, Malo D, Vogan K, Skamene E, Gros P. Natural resistance to infection with intracellular parasites: isolation of a candidate for Bcg. Cell. 1993 May 7;73(3):469-85. https://doi.org/10.1016/0092-8674(93)90135-d Pinner E, Gruenheid S, Raymond M, Gros P. Functional complementation of the yeast divalent cation transporter family SMF by NRAMP2 , a member of the mammalian natural resistance-associated macrophage protein family. J Biol Chem. 1997 Nov 14;272(46):28933-8.https://doi.org/10.1074/jbc.272.46.28933 Gunshin H, MacKenzie B, Berger UV, Gunshin Y, Romero MF, Boron WF, Nussberger S, Gollan JL, Hediger MA. Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature Cell Biology. 1997,388,482-488.https://doi.org/10.1038/41343 Zheng X, Jiang J, Wang C, Hua Y, Huang H, Xu Y, Wei P, Tao J, Cao P, Kang Z, Li X, Gao Q, Chen Q. NRAMP6c plays a key role in plant cadmium accumulation and resistance in tobacco ( Nicotiana tabacum L.). Ecotoxicol Environ Saf. 2024 Feb;271:115885. https://doi.org/10.1016/j. ecoenv.2023.115885 Ma X, Yang H, Bu Y, et al. Genome-wide identification of the NRAMP gene family in Populus trichocarpa and their function as heavy metal transporters. Ecotoxicology and Environmental Safety. 2023;261:115110.https://doi.org/10.1016/j.ecoenv.2023.115110 Yu W, Deng S, Chen X, Cheng Y, Li Z, Wu J, Zhu D, Zhou J, Cao Y, Fayyaz P, Shi W, Luo Z. PcNRAMP1 Enhances Cadmium Uptake and Accumulation in Populus × canescens . Int J Mol Sci. 2022 Jul 8;23(14):7593. https://doi.org/10.3390/ijms23147593 Tian W, He G, Qin L, Li D, Meng L, Huang Y, He T. Genome-wide analysis of the NRAMP gene family in potato ( Solanum tuberosum ): Identification, expression analysis and response to five heavy metals stress. Ecotoxicol Environ Saf. 2021 Jan 15;208:111661. https://doi.org/10.1016 /j.ecoenv.2020.111661 Mäser P, Thomine, S, Schroeder, J.I, Ward, J.M, Hirschi, K, Sze, H, Talke, I.N, Amtmann, A, Maathuis, F.J, Sanders, D, et al. Phylogenetic relationships within cation transporter families of Arabidopsis. Plant Physiology.2001,126,1646-1667.https://doi.org/10.1104/pp.126.4.1646 Li J, Duan Y, Han Z, et al. Genome-wide identification and expression analysis of the NRAMP family genes in tea plant ( Camellia sinensis ). Plants (Basel). 2021;10(6):1055. Published 2021 May 25.https://doi.org/10.3390/plants10061055 Cailliatte R, Lapeyre B, Briat J, Mari S, Curie C. The NRAMP6 metal transporter contributes to cadmium toxicity. Biochemistry Journal.2009,422,217-228.https://doi.org/10.1042/BJ20090655 Yang M, Zhang Y, Zhang L, Hu J, Zhang X, Lu K, Dong H, Wang D, Zhao F.J, Huang C.F, et al. OsNRAMP5 contributes to manganese translocation and distribution in rice shoots. Journal of Experimental Botany.2014,65,4849-4861.https://doi.org/10.1093/jxb/eru259 Li J, Duan Y, Han Z, Shang X, Zhang K, Zou Z, Ma Y, Li F, Fang W, Zhu X. Genome-Wide Identification and Expression Analysis of the NRAMP Family Genes in Tea Plant ( Camellia sinensis ). Plants (Basel). 2021 May 25;10(6):1055. https://doi.org/10.3390/plants10061055 Madeira F, Park YM, Lee J, Buso N, Gur T, Madhusoodanan N, Basutkar P, Tivey ARN, Potter SC, Finn RD, Lopez R. The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Research. 2019 Jul 2;47(W1):W636-W641.https://doi.org/10.1093/nar/gkz268 Chen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, Xia R. TBtools: an integrative toolkit developed for interactive analyses of big biological data. Molecular Plant. 2020 Aug 3;13(8):1194-1202. doi: 10.1016/j.molp.2020.06.009.https://doi.org/10.1016/j.molp.2020.06.009 Marchler-Bauer A, Bo Y, Han L, He J, Lanczycki CJ, Lu S, Chitsaz F, Derbyshire MK, Geer RC, Gonzales NR, Gwadz M, Hurwitz DI, Lu F, Marchler GH, Song JS, Thanki N, Wang Z, Yamashita RA, Zhang D, Zheng C, Geer LY, Bryant SH. CDD/SPARCLE: functional classification of proteins via subfamily domain architectures. Nucleic Acids Res. 2017 Jan 4;45(D1):D200-D203.https://doi.org/10.1093/nar/gkw1129 Xi D, Yin T, Han P, Yang X, Zhang M, Du C, Zhang H, Liu X. Genome-wide identification of sweet orange WRKY transcription factors and analysis of their expression in response to infection by Penicillium digitatum . Current Issues In Molecular Biology. 2023 Feb 3;45(2):1250-1271. doi: 10.3390/cimb45020082.https://doi.org/10.3390/cimb45020082 Tian C, Wan P, Sun S, Li J, Chen M. Genome-wide analysis of the GRAS gene family in rice and Arabidopsis. Plant Molecular Biology. 2004 Mar;54(4):519-32. doi: 10.1023/B: PLAN.0000038256.89809.57.https://doi.org/10.1023/B:PLAN.0000038256.89809.57 Marchler-Bauer A, Lu S, Anderson JB, Chitsaz F, Derbyshire MK, DeWeese-Scott C, Fong JH, Geer LY, Geer RC, Gonzales NR, Gwadz M, Hurwitz DI, Jackson JD, Ke Z, Lanczycki CJ, Lu F, Marchler GH, Mullokandov M, Omelchenko MV, Robertson CL, Song JS, Thanki N, Yamashita RA, Zhang D, Zhang N, Zheng C, Bryant SH. CDD: a Conserved Domain Database for the functional annotation of proteins. Nucleic Acids Res. 2011 Jan;39(Database issue): D225-9. .https://doi.org/10.1093/nar/gkq1189 Brown P, Baxter L, Hickman R, Beynon J, Moore JD, Ott S. MEME-LaB: motif analysis in clusters. Bioinformatics. 2013 Jul 1;29(13):1696-7. https://doi.org/10.1093/bioinformatics/btt248 Lescot M, Déhais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rouzé P, Rombauts S. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002 Jan 1;30(1):325-7.https://doi.org/10. 1093/nar/30.1.32 Wang L, Cui X, Cheng H, Chen F, Wang J, Zhao X, Lin C, Pu X. A review of soil cadmium contamination in China including a health risk assessment. Environmental Science and Pollution Research. 2015，22(21):16441-52.https://doi.org/10.1007/s11356-015-5273-1 Tian W, He G, Qin L, Li D, Meng L, Huang Y, He T. Genome-wide analysis of the NRAMP gene family in potato ( Solanum tuberosum ): Identification, expression analysis and response to five heavy metals stress. Ecotoxicol Environ Saf. 2021 Jan 15;208:111661.https://doi.org/10.1016/J.ecoenv.2020.111661 Liu, S. , Long, T. , Chen, Z. , Liu, J. , Cui, W. , & Leng, H. , et al. (2023). Genome-wide identification of nramp family genes in Populus trichocarpa and their roles in transport of heavy metals. Tree Genetics & Genomes(6), 19.https://doi.org/10.1007/s11295-023-01629-3 Migeon A, Blaudez D, Wilkins O, Montanini B, Campbell MM, Richaud P, Thomine S, Chalot M. Genome-wide analysis of plant metal transporters, with an emphasis on poplar. Cellular and Molecular Life Science. 2010, 67(22):3763-84.https://doi.org/10.1007/s00018-010-0445-0 Meng JG, Zhang XD, Tan SK, Zhao KX, Yang ZM. Genome-wide identification of Cd-responsive NRAMP transporter genes and analysis of the expression of NRAMP 1 mediated by miR167 in Brassica napus. Biometals. 2017 Dec;30(6):917-931.https://doi.org/10.1007/s00018-010-0445-0 Nevo Y, Nelson N. The NRAMP family of metal-ion transporters. Biochimica et Biophysica Acta. 2006, 1763(7):609-20.https://doi.org/10.1007/s00018-010-0445-0 Yin S, Zhao L, Liu J, Sun Y, Li B, Wang L, Ren Z, Chen C. Pangenome Analysis of WOX Gene Family and Function Exploration of CsWOX9 in Cucumber. International Journal of Molecular Sciences. 2023, 17;24(24):17568.https://doi.org/10.3390/ijms242417568 Bari MA, El-Shehawi AM, Elseehy MM, Naheen NN, Rahman MM, Kabir AH. Molecular characterization and bioinformatics analysis of transporter genes associated with Cd-induced phytotoxicity in rice ( Oryza sativa L.). Plant Physiol Biochem. 2021 Oct;167:438-448. https://doi.org/10.1016/j.plaphy.2021.08.024 Jeffares DC, Penkett CJ, Bähler J. Rapidly regulated genes are intron poor. Trends in Genetics. 2008, 24(8):375-8.https://doi.org/10.1016/j.tig.2008.05.006 Emamverdian A, Ding Y, Mokhberdoran F. The role of salicylic acid and gibberellin signaling in plant responses to abiotic stress with an emphasis on heavy metals. Plant Signaling and Behavior 2020, 2;15(7):1777372.https://doi.org/10.1080/15592324.2020.1777372 Shirazi Z, Khakdan F, Rafiei F, Balalami MY, Ranjbar M. Genome-wide identification and expression profile analysis of metal tolerance protein-encoding gene family in Eucalyptus grandis under metal stresses. BMC Plant Biol. 2023 May 6;23(1):240.https://doi. org/10.1186/s12870-023-04240-9 Rasheed A, Al-Huqail AA, Ali B, Alghanem SMS, Shah AA, Azeem F, Rizwan M, Al-Qthanin RN, Soudy FA. Molecular characterization of genes involved in tolerance of cadmium in Triticum aestivum (L.) under Cd stress. J Hazard Mater. 2024 Feb 15;464:132955. https://doi.org/10.1016/J.jhazmat.2023.132955 Chen Y, Zhao X, Li G, Kumar S, Sun Z, Li Y, Guo W, Yang J, Hou H. Genome-Wide Identification of the Nramp Gene Family in Spirodela polyrhiza and Expression Analysis under Cadmium Stress. International Journal of Molecular Sciences. 2021, 15;22(12):6414.https://doi. org/10.3390/ijms22126414 Cellier M, Prive G, Belouchi A, Kwan T, Rodrigues V, Chia W, Gros P. NRAMP defines a family of membrane proteins. Proceedings of the National Academy of Sciences of the United States of America, 1995, 92 , 10089–10093.https://doi.org/10.1073/pnas.92.22.10089 Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research. 2001, 29 , e45.https://doi.org/10.1093/nar/29.9.e45 Additional Declarations No competing interests reported. Supplementary Files Additionalfile1.xlsx Supplemental material Supplementary Tables: Additional file 1. Physicochemical properties of AcABF genes. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6967468","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":486651873,"identity":"d6cdeaa6-3205-4652-8a2e-d7f3ec3beb73","order_by":0,"name":"Xuechun Li","email":"","orcid":"","institution":"Southwest Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Xuechun","middleName":"","lastName":"Li","suffix":""},{"id":486651875,"identity":"016f880a-1ab9-4730-9bfc-40e4e8d7cbf4","order_by":1,"name":"Hanbing Cai","email":"","orcid":"","institution":"Ministry of Education, Southwest Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Hanbing","middleName":"","lastName":"Cai","suffix":""},{"id":486651878,"identity":"7289110e-c4da-42b0-b9e6-22b314a2f13b","order_by":2,"name":"Ping Tian","email":"","orcid":"","institution":"National Forest and Grassland Administration, Southwest Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Ping","middleName":"","lastName":"Tian","suffix":""},{"id":486651882,"identity":"f96385db-05ec-42aa-b8d8-7884c86ddc89","order_by":3,"name":"Ke Zhao","email":"","orcid":"","institution":"Ministry of Education, Southwest Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Ke","middleName":"","lastName":"Zhao","suffix":""},{"id":486651885,"identity":"4ff6e306-74f1-433d-a45e-b0fadd9b9ac6","order_by":4,"name":"Jiaqiong Wan","email":"","orcid":"","institution":"Yunnan Academy of Agricultural Science","correspondingAuthor":false,"prefix":"","firstName":"Jiaqiong","middleName":"","lastName":"Wan","suffix":""},{"id":486651886,"identity":"2e726c1c-887c-46a6-9a86-526391a7caea","order_by":5,"name":"Tuo Yin","email":"","orcid":"","institution":"Southwest Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Tuo","middleName":"","lastName":"Yin","suffix":""},{"id":486651889,"identity":"40e1fc68-a10a-4603-8278-1e64d54cb726","order_by":6,"name":"Hanyao Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBElEQVRIiWNgGAWjYDACCSBmbAAzGR8wMMiBGAZEa2EGKjUmTQubBFFa5Gc3P3v4dYddnsHxw8cqf7YZJDawN2+TYKi5g1ML45xj5sayZ5KLDc6kpd3mBWnhOVYmwXDsGU4tzBIJZtKSbcyJGw7kmN1m3PYnsUEix0yCseEwTi1sEunfgFrqEzecf2NW+HMb0Bb5N/i18ADNlPzYdjhxw40cMwZekBYJHvxaJCRyyqQZ244nzrzxLFma95+BcRtPWrFFwjHcWuRnpG+T/NlWndh3Pvngxx9nDGT72Q9vvPGhBrcWcBDwAAmFAzDfgYgEvBqAAf0DZF0DAVWjYBSMglEwcgEAzI5XE6AGtnQAAAAASUVORK5CYII=","orcid":"","institution":"Southwest Forestry University","correspondingAuthor":true,"prefix":"","firstName":"Hanyao","middleName":"","lastName":"Zhang","suffix":""},{"id":486651892,"identity":"d227a602-d9e9-46e4-888d-eb7549e61a41","order_by":7,"name":"Xiaozhen Liu","email":"","orcid":"","institution":"Southwest Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Xiaozhen","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2025-06-24 15:53:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6967468/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6967468/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-025-07865-0","type":"published","date":"2025-12-04T15:57:34+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87059073,"identity":"fc23734a-18d8-4f60-ab69-c7be1b54a992","added_by":"auto","created_at":"2025-07-18 16:27:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":306507,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree of the \u003cem\u003eNRAMP\u003c/em\u003e gene family genes from Arabidopsis, kiwifruit, and tea tree. The phylogenetic tree was constructed via the maximum likelihood method via bootstrap values of 1,000 repeats. Black represents 1,000 repeats with bootstrap values \u0026gt; 90; gray represents 1,000 repeats with 90 \u0026gt; bootstrap values \u0026gt; 70; white and gray represent bootstrap values \u0026lt; 70.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6967468/v1/da403adb5bd2f427c6413a98.png"},{"id":87059071,"identity":"3b1e3b30-c715-4107-8e9c-1b8131938807","added_by":"auto","created_at":"2025-07-18 16:27:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":130456,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic relationships, gene structure, and domain and conserved motif distributions of the \u003cem\u003eAcNRAMP\u003c/em\u003e gene family genes. (A) Distribution of conserved \u003cem\u003eAcNRAMP\u003c/em\u003e motifs. The motifs are represented by colored boxes, and the black line represents the relative length of the proteins. (B) The conserved domain of the \u003cem\u003eAcNRAMP\u003c/em\u003e gene. (C) The exon-intron structure of the \u003cem\u003eAcNRAMP\u003c/em\u003egene.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6967468/v1/de95edf1af798b7e9d866149.png"},{"id":87059077,"identity":"22c4bc04-7258-49e9-8707-fff45bb1e06d","added_by":"auto","created_at":"2025-07-18 16:27:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":253566,"visible":true,"origin":"","legend":"\u003cp\u003eA Collinearity analysis of the \u003cem\u003eAcNRAMP\u003c/em\u003egene in kiwifruit. The gray lines represent all duplicated genes, and the light blue lines represent the \u003cem\u003eNRAMP\u003c/em\u003e gene pairs with fragmentary duplications. The heatmap and line map show the gene density. The density of the line map increases from blue to white to red. The yellow rectangles are chromosomes, and the chromosome name is displayed between each chromosome and the gene density. Figure 3B, Collinear distributions of kiwifruit, \u003cem\u003eArabidopsis\u003c/em\u003e, and grape. The blue line links the \u003cem\u003eAcNRAMP\u003c/em\u003egene with the collinear relationship between \u003cem\u003eArabidopsis \u003c/em\u003eand grapes. The gray lines link the collinear relationships of other genes. Ac-, At-, and Vv- represent the chromosomes of kiwifruit, \u003cem\u003eArabidopsis\u003c/em\u003e, and grape, respectively, followed by the sequence number of the chromosome. Figure 3 Cschematically shows the distribution of the\u003cem\u003e AcNRAMP\u003c/em\u003e genes on the seven chromosomes of kiwifruit. The gene name is indicated in black letters on the right side. The chromosomal region where the \u003cem\u003eAcNRAMP\u003c/em\u003e gene is located is indicated by the indicator line on the right. The chromosome number can be observed in the left area of each chromosome (Chr), and the chromosome number is indicated with blue letters.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6967468/v1/1087e9d536abd7f62e5654ac.png"},{"id":87059079,"identity":"47afe88c-4643-4df0-a8ef-1e4017a756af","added_by":"auto","created_at":"2025-07-18 16:27:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":298727,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of cis-acting elements in the 2000 bp upstream sequences of the 12 \u003cem\u003eAcNRAMP\u003c/em\u003egene family genes. Different cis-acting elements are indicated in various colors.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6967468/v1/32c1280c231ca2ae64cff692.png"},{"id":87059084,"identity":"d8a10a2c-4381-4b81-ab17-69bc2727241f","added_by":"auto","created_at":"2025-07-18 16:27:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":802484,"visible":true,"origin":"","legend":"\u003cp\u003e5A shows a schematic diagram of the 3D structure of the kiwifruit \u003cem\u003eAcNRAMP\u003c/em\u003e protein predicted by SWISS-MODEL. Figure 5B, Sequence alignment of \u003cem\u003eAcNRAMP\u003c/em\u003e. Identical residues are highlighted in black, homologous residues are highlighted in gray, and the canonical conserved amino acid residues GQSSTITGTYAGQY(/F)V(/I)MQ(/G/E)GFL are highlighted in red. The \u003cem\u003eNRAMP\u003c/em\u003e family domain DPGN is highlighted in yellow. Figure 5C, Annotation results of GO with\u003cem\u003e AcNRAMPs\u003c/em\u003e. The cellular component represents the cellular component, the molecular function represents the molecular function, and the biological process represents the biological process, represented in rose, green, and orange, respectively. The X-axis represents the number of gene functions, and the y-axis represents the annotation function of the gene.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6967468/v1/e7ac0990b859c7852e35cbe7.png"},{"id":87059573,"identity":"f1f8abdf-e0e8-476c-871b-9b6ec8876963","added_by":"auto","created_at":"2025-07-18 16:35:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":95591,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap of the expression profiles of 12 \u003cem\u003eAcNRAMP\u003c/em\u003e genes under cadmium stress. Boxes with different colors represent different log\u003csub\u003e2\u003c/sub\u003e(FPKM) values, and the expression level gradually increases from light to light color and then to dark color. CK represents the control group, RCd represents the roots under cadmium stress, and LCd represents the leaves under cadmium stress.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6967468/v1/e0514c364ce999f2de1be16f.png"},{"id":87059571,"identity":"d1a141dd-7afe-47dc-b09c-3e252a9ea775","added_by":"auto","created_at":"2025-07-18 16:35:31","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":432495,"visible":true,"origin":"","legend":"\u003cp\u003eA Morphological changes in the leaves of kiwifruit plants under cadmium stress. After 10 μM and 100 μM CdCl2 treatment, the morphology of the leaves and roots changed at 30 days. After 10 μM CdCl2 treatment, there were significantly fewer roots than after 0 μM CdCl2 treatment, and the leaf edge turned significantly yellow; after 100 μM CdCl2 treatment, there were also remarkably fewer roots than after 10 μM CdCl2 treatment. The area of the yellowed leaf edge also became significantly larger. Figure 7B, Differential response of\u003cem\u003e AcNRAMP\u003c/em\u003e expression in kiwifruit seedlings under cadmium stress. Three\u003cem\u003eAcNRAMPs\u003c/em\u003e are shown in the figure. The donors were \u003cem\u003eAcNRAMP6\u003c/em\u003e, \u003cem\u003eAcNRAMP7, \u003c/em\u003eand \u003cem\u003eAcNRAMP10\u003c/em\u003e. The RCK and RCd values shown on the horizontal axis were divided into the control group and the cadmium-treated group; the values on the vertical axis are expressed as qRT-PCR results from triplicate samples. The average value. ** denotes a significant difference at p\u0026lt;0.01; ns denotes a nonsignificant difference.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6967468/v1/0a819cacbe21e2632d85c34e.png"},{"id":87059575,"identity":"6b559816-34a4-4f72-bac3-41794766e709","added_by":"auto","created_at":"2025-07-18 16:35:31","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":284363,"visible":true,"origin":"","legend":"\u003cp\u003eMechanistic diagram of the\u003cem\u003e AcNRAMP\u003c/em\u003eresponse of kiwifruit seedlings under cadmium stress.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6967468/v1/6a4b686dfc81df25ddc43a6d.png"},{"id":97724022,"identity":"7f302fec-c06f-49e3-aa11-77b96c8899df","added_by":"auto","created_at":"2025-12-08 16:11:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3517098,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6967468/v1/26ad96ed-bb59-4aec-a354-60abb12c13b8.pdf"},{"id":87059570,"identity":"c295194e-7cbc-4baa-b45a-2538754b5226","added_by":"auto","created_at":"2025-07-18 16:35:31","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11287,"visible":true,"origin":"","legend":"\u003cp\u003eSupplemental material\u003c/p\u003e\n\u003cp\u003eSupplementary Tables: Additional file 1. Physicochemical properties of \u003cem\u003eAcABF\u003c/em\u003e genes.\u003c/p\u003e","description":"","filename":"Additionalfile1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6967468/v1/8bbae81422805ba12556c824.xlsx"},{"id":87059074,"identity":"b6db74eb-2a80-4806-a651-a280b477ecdd","added_by":"auto","created_at":"2025-07-18 16:27:31","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10931,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6967468/v1/ce31796b4642cf9c2ba694f0.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-wide identification of the NRAMP gene family in kiwifruit and the response of AcNRAMP7 to cadmium stress","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e\u003cstrong\u003eThe\u003c/strong\u003e Kiwi fruit is a perennial deciduous vine of the genus Actinidia in the Actinidiaceae family. It has high nutritional, medical, and health value and is known as the king of fruit, the crown of vitamin C, Green Bank (New Zealand), and Beauty Fruit (Japan). Kiwifruit has a large planting area in China and is widely loved by people for its unique flavor. Its roots, stems, and leaves can also be used as medicines; thus, it is a Chinese medicinal material worthy of vigorous development and utilization (Jia et al., 2012). It can also be used as an adjuvant drug to treat hypertension, coronary heart disease, cancer, and other diseases (Song et al., 1984). The phenolic compounds in kiwi fruits have good antioxidant effects, and the saponins have strong hypoglycemic effects.\u003c/p\u003e\n\u003cp\u003eHowever, with rapid industrial development, the intensified use of pesticides, and the lack of environmental remediation resources, large amounts of heavy metals have begun to enter the environment, with cadmium pollution in arable soil being the most serious\u0026nbsp;(Yin et al., 2020). The total rate of heavy metal pollution in agricultural soil in China has reached 19.4%. Among all kinds of pollution, cadmium pollution accounts for the highest percentage, with a rate of excess points of 7.0%, making it one of the most crucial soil pollutants in China\u0026nbsp;(Xiao et al., 2015). Cadmium is easily absorbed by plants. When cadmium accumulates to a certain extent in plants, it affects their regular growth, inhibits their photosynthesis, interferes with their normal metabolic process, reduces their transpiration, and accelerates their growth during aging\u0026nbsp;(Wang et al., 2020. Shakirova et al. reported that cadmium could inhibit mitosis in root cells, cause chromosome aberrations in plants, and affect crop growth\u0026nbsp;(Shakirova et al., 2016). Shakirova et al. reported that cadmium can exacerbate the production of reactive oxygen species (ROS) in crops and damage the antioxidant system of plants\u0026nbsp;(Shakirova et al., 2016). Cadmium is also very harmful to animals and humans. Cadmium enters the human body through the food chain, causing great harm to the human kidney, liver, brain, and bones and having strong carcinogenic effects\u0026nbsp;(Gao et al., \u0026nbsp;2017).\u003c/p\u003e\n\u003ch6\u003ePlants have evolved many heavy metal stress tolerance mechanisms, including strict regulation of the uptake, efflux, storage, detoxification, and transport of heavy metals and intracellular chelation to minimize the toxicity of heavy metals\u0026nbsp;(Geng et al., 2011). Multiple gene family members play vital roles in heavy metal transport regulation (Singh et al., 2011). Among many gene families,\u003cem\u003e\u0026nbsp;NRAMPs\u003c/em\u003e play a key role in Cd uptake and accumulation in plants\u0026nbsp;(Gao et al., 2020). The \u003cem\u003eNRAMP\u003c/em\u003e gene was first isolated from mice in 1993\u0026nbsp;(Bozzi et al., 2021)\u0026nbsp;and was identified as a divalent metal transporter protein in bacteria, fungi, insects, plants, and mammals\u0026nbsp;(Pinner\u0026nbsp;et al., 1997;\u0026nbsp;Gunshin et al., 1997). In addition, \u003cem\u003eNRAMP\u003c/em\u003e proteins are involved in the transport of various divalent cations in plants, such as Fe\u003csup\u003e2+\u003c/sup\u003e, Mn\u003csup\u003e2+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e, Pb\u003csup\u003e2+\u003c/sup\u003e, and Cd\u003csup\u003e2+\u003c/sup\u003e.\u003c/h6\u003e\n\u003cp\u003eTo date, the \u003cem\u003eNRAMP\u003c/em\u003e gene family has been studied in tobacco (Gunshin et al., 1997), \u003cem\u003eP. trichocarpa\u003c/em\u003e (Zheng et al., 2023), poplar\u0026nbsp;(Ma et al., 2023), potato\u0026nbsp;(Yu et al., 2022), Arabidopsis\u0026nbsp;(Tian et al., 2021), and tea trees\u0026nbsp;(Maser et al., 2001). In Arabidopsis, \u003cem\u003eAtNRAMP6\u003c/em\u003e is an intracellular cadmium transporter protein, whereas \u003cem\u003eAtNRAMP1\u003c/em\u003e is located in the root plasma membrane and acts as a Mn transporter protein\u0026nbsp;(Li et al., 2021). There are seven \u003cem\u003eNRAMP\u003c/em\u003e members in rice (\u003cem\u003eOryza sativa\u003c/em\u003e), among which \u003cem\u003eOsNRAMP5\u003c/em\u003e is a plasma membrane-localized transporter protein. Knocking out \u003cem\u003eOsNRAMP5\u003c/em\u003e reduces Mn and Cd concentrations in roots, shoots, and grains\u0026nbsp;(Cailliatte et al., 2009). However, the transport mechanism of the \u003cem\u003eNRAMP\u003c/em\u003e protein family in kiwifruit is still unknown.\u003c/p\u003e\n\u003cp\u003eThis study aimed to identify 12\u003cem\u003e\u0026nbsp;NRAMPs\u003c/em\u003e, analyze their physicochemical properties, phylogenetics, gene structures, domains, conserved motifs, cis-regulatory elements, chromosomal locations, and gene replication, and investigate the expression patterns of kiwifruit in response to cadmium stress. Our study results lay the foundation for further exploration of the regulatory function of \u003cem\u003eAcNRAMPs\u003c/em\u003e in the kiwifruit cadmium stress response.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e2.1 Identification and analysis of the physicochemical properties of the \u003cem\u003eNRAMP\u003c/em\u003e genes in the kiwifruit genome\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNRAMP\u003c/em\u003e sequences were downloaded from the TAIR website (https://www.arabidopsis.org/Six Arabidopsis). From the kiwifruit genome database (http://kiwifruitgenome.org/organism/5), the genome data of kiwifruit (\u003cem\u003eActinidia chinensis\u003c/em\u003e Hong Yang v3), the genome data of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e from the Ensembal Plants database (https://plants.ensembl.org/index.html), and the early tea tree genome data were obtained from the Tea Tree Genome Database (http://tpia.teaplant.org/download.html). To identify the \u003cem\u003eNRAMP\u003c/em\u003e gene in the kiwifruit \u0026apos;Hongyang\u0026apos;, conserved domain (Cation_efflux, Pfam number: PF01566) sequence information was obtained from Li (Yang et al., 2014).\u003c/p\u003e\n\u003cp\u003eBased on the downloaded data, the conserved domain \u003cem\u003eNRAMP\u003c/em\u003e (Cation_efflux, Pfam No. PF01566) was searched, and the results were screened for protein sequence number (Li et al., 2021) with an E value \u0026lt;1\u0026times;10-5. Madeira F . Next, via the BLASTP program of BLAST software, the protein sequence of the Arabidopsis \u003cem\u003eNRAMP\u003c/em\u003e gene family was used as a reference to perform BLAST on the kiwifruit genomic protein data, and the protein sequences in the kiwifruit genomic protein data were compared with the protein sequences of the Arabidopsis \u003cem\u003eNRAMP\u003c/em\u003e gene family. For high values (E value \u0026lt;1\u0026times;10 -5), the above steps were repeated. Finally, the sequence numbers obtained in the above two steps were merged. The merged protein sequence numbers were extracted from the kiwifruit genomic protein data via the TBtools tool and saved as *.fasta files (Madeira\u0026nbsp;et al., 2019). The conserved domain database (CDD) tool of the NCBI database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and the online software PFAM (http://pfam.xfam.org/search #tabview=Additional file\u0026nbsp;1) further confirmed the existence of the \u003cem\u003eNRAMP\u003c/em\u003e domain\u0026nbsp;(Chen et al., 2020). The identified \u003cem\u003eNRAMP\u003c/em\u003e gene families were renamed according to their chromosomal locations, and the naming format was as follows: \u003cem\u003eNRAMP\u003c/em\u003e+serial number. The physicochemical properties were analyzed via ProtParam (http://www.ExPASy. org/tools/protparam.html) online software to determine the molecular weight (MW), isoelectric point (PI), number of amino acids, and aliphatic index (Marchler-Bauer et al., 2017). Finally, the Plant-mPLoc (Plant-mPLoc server (sjtu.edu.cn)) online tool was used for subcellular localization.\u003c/p\u003e\n\u003cp\u003e2.2\u0026nbsp;Phylogenetic analysis of \u003cem\u003eAcNRAMPs\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo understand the evolutionary relationships among the \u003cem\u003eNRAMP\u003c/em\u003e gene families in kiwifruit,\u003cem\u003e\u0026nbsp;Camellia japonica\u003c/em\u003e, and \u003cem\u003eA. thaliana\u003c/em\u003e. The identified \u003cem\u003eNRAMP\u003c/em\u003e protein sequences of kiwifruit, \u003cem\u003eC. japonica\u003c/em\u003e, and \u003cem\u003eA. thaliana\u003c/em\u003e were merged, sequence alignment was performed via MEGA11.0 software, trimming was performed via TBtools software, and a system was subsequently constructed via MEGA11.0 software and the maximum likelihood (ML) method of the IQtree Developmental Tree (Xi et al., 2023). The online software Chiplot (http://www.evolgenius.info/evolview/#/treeview) was used to visualize the evolutionary tree. Finally, the 12 kiwifruit \u003cem\u003eNRAMP\u003c/em\u003e proteins were grouped based on the grouping of the Arabidopsis \u003cem\u003eNRAMP\u003c/em\u003e proteins (Tian et al., 2004).\u003c/p\u003e\n\u003cp\u003e2.3 Gene structure, domain, and conserved motif analysis\u003c/p\u003e\n\u003cp\u003eTBtools software was used to analyze the gene structure of each gene based on the kiwifruit genome annotation file (GFF3) (Madeira\u0026nbsp;et al., 2019). The structural domain was subsequently\u0026nbsp;predicted via the CD search online tool of the NCBI\u0026nbsp;database (http://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) with the default parameters\u0026nbsp;(Anderson et al., \u0026nbsp;2011). The conserved motifs of \u003cem\u003eAcNRAMP\u003c/em\u003e proteins were analyzed via MEME (http://meme-suite.org/)\u0026nbsp;online software\u0026nbsp;(Brown et al., 2013). Thus, the differences among \u003cem\u003eAcNRAMP\u003c/em\u003e family members were analyzed. The above results were visualized via TBtools software\u0026nbsp;(Madeira\u0026nbsp;et al., 2019).\u003c/p\u003e\n\u003cp\u003e2.4 Chromosomal positioning, gene replication, and collinearity\u003c/p\u003e\n\u003cp\u003eChromosome position information was obtained from the genome annotation file (GFF3), and chromosome position mapping was performed via TBtools software (Madeira\u0026nbsp;et al., 2019). Gene duplication events were subsequently analyzed via MCScanX, and the collinear relationships between the \u003cem\u003eAcNRAMP\u003c/em\u003e gene family members of Arabidopsis and kiwifruit were analyzed via TBtools software.\u003c/p\u003e\n\u003cp\u003e2.5 Analysis of cis-acting elements\u003c/p\u003e\n\u003cp\u003eGene IDs were obtained based on identification. The upstream 2000 bp nucleotide sequence of each gene was extracted from the genome annotation files (GFF3 format) via TBtools software and the online analysis software PlantCARE (https://bioinformatiAc.psb.ugent.be/webtools/plantcare/html/) for promoter cis-acting element analysis (Lescot et al., 2002).\u003c/p\u003e\n\u003cp\u003e2.6 Three-dimensional \u003cem\u003eAcNRAMP\u003c/em\u003e protein model prediction and multiple sequence alignment\u003c/p\u003e\n\u003cp\u003eBased on the protein sequences of the \u003cem\u003eAcNRAMP\u003c/em\u003e genes extracted from the kiwifruit genome-wide protein sequence files, the proteins of the 12 \u003cem\u003eAcNRAMP\u003c/em\u003e genes were predicted via a protein model via the WISS-MODEL database (https://swissmodel.ExPASy.org/). Multiple sequence alignment was performed via MEGA11 software, and the sequences were visualized via GeneDoc software.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;2.7 GO functional analysis of the \u003cem\u003eAcNRAMP\u003c/em\u003e genes\u003c/p\u003e\n\u003cp\u003eThe protein sequence of the\u003cem\u003e\u0026nbsp;AcNRAMP\u003c/em\u003e gene family was extracted from the kiwifruit genome-wide protein sequence file, and the protein sequences of the \u003cem\u003eAcNRAMP\u0026nbsp;\u003c/em\u003egene family were extracted from the kiwifruit genome database (http://kiwifruitgenome.org/organism/5). GO function searches were performed for proteins of the 12 \u003cem\u003eAcNRAMP\u003c/em\u003e genes for gene function annotation. TBtools was subsequently used to perform GO functional enrichment analysis on the \u003cem\u003eNRAMP\u003c/em\u003e genes of kiwifruit, and the online tool biorender (https://app.biorender.com/) was used for mapping and visualization.\u003c/p\u003e\n\u003cp\u003e2.8 Transcriptome data-based gene expression analysis of\u003cem\u003e\u0026nbsp;AcNRAMPs\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe transcriptome sequencing data used the kiwifruit Hongyang genome (V3) (http://kiwifruitgenome.org/organism/3) as the reference genome and hissat2 for sequence alignment. The expression status of each gene is understood in detail via comparison analysis with the reference genome. Differential expression analysis between RCd_vs_RCK and LCd_vs_LCK was performed via the DESeqR software package (1.18.0). The expression profiles of the AcNRAMP genes in the leaves and roots were obtained from transcriptome data. The expression profile of the \u003cem\u003eAcNRAMP\u003c/em\u003e gene was drawn based on the FPKM value, and the genes with significant differential expression were screened. The screening thresholds were FDR\u0026lt;0.05, log 2 FC \u0026gt;1, or log 2 FC\u0026lt;-1. The expression of the \u003cem\u003eAcNRAMP\u003c/em\u003e gene was normalized, and TBtools software was used to visualize the\u003cem\u003e\u0026nbsp;AcNRAMP\u003c/em\u003e gene expression data.\u003c/p\u003e\n\u003cp\u003e2.9 Expression analysis of \u003cem\u003eAcNRAMP\u003c/em\u003e under cadmium stress\u003c/p\u003e\n\u003cp\u003eThe oligonucleotide primers used to target \u003cem\u003eAcNRAMP\u003c/em\u003e were designed via Primer Premier 5 (Appendix 1). First-strand cDNA was synthesized via a PrimeScriptTM series RT kit (TaKaRa). The ABI PRISM 7500 Real-Time PCR System was used for quantitative real-time PCR (qRT-PCR) and detection by Weiwei Genomics, Inc. The qPCR program was as follows: 95 \u0026deg;C for 3 min; 40 cycles of 95 \u0026deg;C for 15 s, 57 \u0026deg;C for 20 s, and 72 \u0026deg;C for 20 s; and a melting process at 60-95 \u0026deg;C to generate melting curves. For normalization, the \u003cem\u003eActinidia07041\u003c/em\u003e gene was used as an internal control (Appendix 1). The 2\u003csup\u003e\u0026nbsp;-\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method was used to calculate the relative expression of the \u003cem\u003eAcNRAMP\u003c/em\u003e gene (Pfaffl et al., 2001). The data are presented as the means \u0026plusmn; standard deviations. Statistical analysis was performed via one-way analysis of variance (ANOVA) via GraphPad Prism 10.0 software.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e3.1 Identification and analysis of the physicochemical properties of \u003cem\u003eAcNRAMP\u003c/em\u003e proteins\u003c/p\u003e\n\u003cp\u003eBased on the protein sequence of the Arabidopsis \u003cem\u003eNRAMP\u003c/em\u003e gene family as a reference, BLAST and hmmSearch were performed on the genomic protein data of kiwifruit, the kiwifruit \u003cem\u003eNRAMP\u003c/em\u003e gene family was predicted via CD search, and Pfam and incomplete or nonexistent domains were removed to form a total of. Twelve \u003cem\u003eNRAMP\u003c/em\u003e genes were identified. These genes were named \u003cem\u003eAcNRAMP1-AcNRAMP11\u003c/em\u003e according to their chromosomal locations from top to bottom, where \u003cem\u003eAcNRAMP12\u003c/em\u003e was not located on the chromosome. Further analysis of the sequence revealed that the lengths of the \u003cem\u003eNRAMP\u003c/em\u003e genes were quite different, ranging from 129 (\u003cem\u003eAcNRAMP10\u003c/em\u003e) to 1,291 (\u003cem\u003eAcNRAMP3\u003c/em\u003e) amino acids, and the molecular weights were in the range of 13,897.89 (\u003cem\u003eAcNRAMP10\u003c/em\u003e) to 141577.95 \u003cem\u003e(AcNRAMP3\u003c/em\u003e) kDa. The pi range was 4.43 (\u003cem\u003eAcNRAMP10\u003c/em\u003e) to 9.01 (\u003cem\u003eAcNRAMP5\u003c/em\u003e), the instability coefficient was between 27.17 (\u003cem\u003eAcNRMP2\u003c/em\u003e) and 41.79 (\u003cem\u003eAcNRAMP10\u003c/em\u003e), and in particular, the only genes greater than 40 were \u003cem\u003eAcNRAMP3\u003c/em\u003e, \u003cem\u003eAcNRAMP1\u003c/em\u003e and \u003cem\u003eAcNRAMP10\u003c/em\u003e, indicating that in addition to the proteins of the above three genes, other genes also had different values. The other\u003cem\u003e\u0026nbsp;AcNRAMPs\u003c/em\u003e were all unstable proteins.\u003c/p\u003e\n\u003cp\u003eIn terms of amino acid composition, the average aliphatic index was 110.39, which was conducive to increasing the thermal stability of globular proteins. The subcellular localization predictions of the Plant-mPLoc tool revealed that, with the exception of the\u003cem\u003e\u0026nbsp;AcNRAMP1\u003c/em\u003e gene, which is located in the chloroplast and nucleus, the \u003cem\u003eAcNRAMP3\u003c/em\u003e and\u003cem\u003e\u0026nbsp;AcNRAMP4\u003c/em\u003e genes are located in the chloroplast. However, the other genes are located in the cell membrane (Additional file\u0026nbsp;1).\u003c/p\u003e\n\u003cp\u003e3.2 Phylogenetic analysis of the \u003cem\u003eAcNRAMP\u003c/em\u003e gene family\u003c/p\u003e\n\u003cp\u003eTo elucidate the evolutionary relationship between the kiwifruit \u003cem\u003eNRAMP\u003c/em\u003e gene family genes and other \u003cem\u003eNRAMP\u003c/em\u003e gene family genes, we aligned 12 \u003cem\u003eNRAMP\u003c/em\u003e protein sequences from kiwifruit, 11 \u003cem\u003eNRAMP\u003c/em\u003e protein sequences from tea plants, and six from \u003cem\u003eA. thaliana\u003c/em\u003e and successfully constructed a phylogenetic tree (Figure 1). According to the clustering results, the kiwifruit \u003cem\u003eNRAMP\u003c/em\u003e gene family could be divided into four groups, namely, Group 1, Group 2, Group 3, and Group 4. The \u003cem\u003eNRAMP\u003c/em\u003e gene family was the most distributed in the first group, with four gene families in kiwi fruit, three gene families from Jasmine chinensis, and two gene families from Arabidopsis. The number of\u003cem\u003e\u0026nbsp;AcNRAMP\u003c/em\u003e genes was highest in Group 1 and Group 2, with four \u003cem\u003eAcNRAMPs\u003c/em\u003e each. Groups 3 and 4 each have two \u003cem\u003eAcNRAMPs\u003c/em\u003e. Furthermore, the Arabidopsis \u003cem\u003eNRAMP\u003c/em\u003e gene was not present in the second group.\u003c/p\u003e\n\u003cp\u003e3.3 Gene structure, domains, and conserved motif analysis of the \u003cem\u003eAcNRAMP\u003c/em\u003e gene family\u003c/p\u003e\n\u003cp\u003eTo further study the sequence characteristics of the \u003cem\u003eAcNRAMP\u003c/em\u003e protein, this study used the MEME online tool to analyze the motif composition of this protein (Figure 2-A). The results revealed that ten motifs were identified in \u003cem\u003eAcNRAMP\u003c/em\u003e proteins, and the conservation of these ten motifs (motifs 1, 2, 3, 4, 5, 6, 7, 8, and 10) was high; however, the same motif was used at different positions in the protein sequence, which may be related to the structure and function of the proteins. However, Motif 9 was absent in the \u003cem\u003eAcNRAMP\u003c/em\u003e gene, and the \u003cem\u003eAcNRAMP10\u003c/em\u003e gene had only one Motif 5. The differences in the distribution of conserved motifs among the \u003cem\u003eAcNRAMP\u003c/em\u003e gene family may also be affected by gene structure and domains. The distribution of gene structure is the key to studying evolutionary characteristics within a gene family. We performed sequence alignment of the 12 \u003cem\u003eAcNRAMP\u003c/em\u003e genes and analyzed the gene structure based on the genome annotation files (Figure 2-C). The results revealed that the gene structure of most of the \u003cem\u003eAcNRAMP\u003c/em\u003e gene family was conserved. There were 0-11 introns in the 12 \u003cem\u003eAcNRAMP\u003c/em\u003e gene family members. Except for \u003cem\u003eAcNRAMP11\u003c/em\u003e,\u003cem\u003e\u0026nbsp;AcNRAMP7\u003c/em\u003e, and \u003cem\u003eAcNRAMP8\u003c/em\u003e, which have no exons, the other genes all contain 1--3 exons, indicating that although the introns and exons of this gene family have some differences, the gene structure is also the same. The relative conservation was distinct, especially for each gene. In addition, this study also analyzed the conserved domains. Among the 12 \u003cem\u003eAcNRAMP\u003c/em\u003e proteins, only two genes, \u003cem\u003eAcNRAMP4\u003c/em\u003e and \u003cem\u003eAcNRAMP3\u003c/em\u003e, had the \u003cem\u003eNRAMP\u003c/em\u003e gene family domain, and the remaining nine genes all had the SLC5-6-like_sbd superfamily domain. (Figure 2-B).\u003c/p\u003e\n\u003cp\u003e3.4\u003cstrong\u003e\u0026nbsp;Chromosomal location and gene replication analysis of the\u003cem\u003e\u0026nbsp;AcNRAMP\u0026nbsp;\u003c/em\u003egene\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo understand the chromosomal distribution and genome-wide density of the kiwifruit \u003cem\u003eAcNRAMP\u003c/em\u003e genes, TBtools software was used to perform positioning analysis on the 29 chromosomes of the kiwifruit genome (Figure 3C). Our results revealed that 11 of the 12 genes were distributed on chromosomes, whereas \u003cem\u003eAcNRAMP12\u003c/em\u003e was not located on chromosomes and was free elsewhere. Most chromosomes do not have the \u003cem\u003eAcNRAMP\u003c/em\u003e gene. The other 11 Ac\u003cem\u003eNRAMP\u003c/em\u003e genes are \u003cem\u003eAc\u003c/em\u003e\u003cem\u003eNRAMP1\u003c/em\u003e, \u003cem\u003eAc\u003cem\u003eNRAMP2\u003c/em\u003e\u003c/em\u003e, \u003cem\u003eAc\u003c/em\u003e\u003cem\u003eNRAMP3\u003c/em\u003e, Ac\u003cem\u003eNRAMP4\u003c/em\u003e, \u003cem\u003eAc\u003c/em\u003e\u003cem\u003eNRAMP5\u003c/em\u003e, \u003cem\u003eAcNRAMP6\u003c/em\u003e, \u003cem\u003eAc\u003cem\u003eNRAMP7\u003c/em\u003e\u003c/em\u003e, \u003cem\u003eAc\u003c/em\u003e\u003cem\u003eNRAMP8\u003c/em\u003e, Ac\u003cem\u003eNRAMP9\u003c/em\u003e, \u003cem\u003eAc\u003c/em\u003e\u003cem\u003eNRAMP10\u003c/em\u003e, and \u003cem\u003eAc\u003c/em\u003e\u003cem\u003eNRAMP11\u003c/em\u003e, which are distributed on Chr5, Chr9, Chr18, Chr21, Chr26, Chr26, Chr27, Chr27, Chr29, Chr29), and Chr29. Moreover, chromosome length is not directly related to the number of genes. For example, chromosome 29 is 12 Mb in length and has three \u003cem\u003eAcNRAMP\u003c/em\u003e genes, whereas chromosome 5 is 20 Mb and has only one \u003cem\u003eAcNRAMP\u003c/em\u003e gene.\u003c/p\u003e\n\u003cp\u003eGene duplication events are critical for the evolution of family members. Therefore, to explore the evolutionary pattern of the \u003cem\u003eNRAMP\u003c/em\u003e gene family in the kiwifruit genome, gene duplication analysis was performed for the 12 \u003cem\u003eAcNRAMP\u003c/em\u003e genes (Figure 3A). In our study, 13 pairs of fragmented and repetitive genes were identified, and they were distributed on seven different chromosomes. In particular, the number of genes on chromosome 29 was high, with three \u003cem\u003eAcNRAMP\u003c/em\u003e genes and four gene duplication events on this chromosome. These fragment repeats may play a key role in the gene duplication events of the \u003cem\u003eAcNRAMP\u003c/em\u003e family.\u003c/p\u003e\n\u003cp\u003eThis study also analyzed the \u003cem\u003eNRAMP\u003c/em\u003e collinear relationships among the three species of kiwi, \u003cem\u003eArabidopsis\u003c/em\u003e, and grape (Figure 3C). The \u003cem\u003eAcNRAMP\u003c/em\u003e genes were screened from the collinearity files for collinearity visualization. The results revealed nine pairs of homologous genes in kiwifruit, \u003cem\u003eArabidopsis\u003c/em\u003e, and grape. In the literature, the evolutionary relationships of \u003cem\u003eArabidopsis\u003c/em\u003e and dicots are closer than those of monocots.\u003c/p\u003e\n\u003cp\u003e3.5 Cis-acting element analysis of the \u003cem\u003eAcNRAMP\u003c/em\u003e genes\u003c/p\u003e\n\u003cp\u003eThe upstream 2000-base sequences of the 12 \u003cem\u003eAcNRAMP\u003c/em\u003e gene family members were analyzed for cis-acting elements via PlantCARE and TBtools software. The results revealed that the promoter region of each \u003cem\u003eAcNRAMP\u003c/em\u003e gene presented multiple stress- or hormone-related cis-elements (Figure 4). The conserved DNA modules involved in the light response and the elements essential for anaerobic induction were distributed in almost every gene. Hormone-related cis-acting elements, such as jasmonate (\u003cem\u003eMeJA\u003c/em\u003e), gibberellin (GA), salicylic \u003cem\u003eacid\u003c/em\u003e, and gibberellic acid, are second only to response elements involved in anaerobic induction. However, among the 12 \u003cem\u003eAcNRAMP\u003c/em\u003e genes, only five \u003cem\u003eAcNRAMP\u003c/em\u003e genes did not have MeJA response elements; 58% of the genes in this gene family had MeJA response elements in their promoters; and there were few GA response elements in this gene family. It only appeared in five \u003cem\u003eAcNRAMP\u003c/em\u003e genes, but among these five \u003cem\u003eAcNRAMP\u003c/em\u003e genes, except for the \u003cem\u003eAcNRAMP10\u003c/em\u003e gene, which contained 2 GA response elements, each gene contained only 1 GA response element (Figure 4). However, compared with other genes, there were relatively more cis-acting elements involved in the gibberellic acid response, e.g., one element in both \u003cem\u003eAcNRAMP8\u003c/em\u003e and\u003cem\u003e\u0026nbsp;AcNRAMP11\u003c/em\u003e and three elements in \u003cem\u003eAcNRAMP3\u003c/em\u003e, \u003cem\u003eAcNRAMP6\u003c/em\u003e, \u003cem\u003eAcNRAMP5\u003c/em\u003e, and\u003cem\u003e\u0026nbsp;AcNRAMP10\u003c/em\u003e. All four genes were present, which might be due to the interaction between these six genes and the cis-acting elements involved in the gibberellic acid response. Next are environmental stress response elements, such as low temperature, drought, defense and stress, and anaerobic-induced response elements. These types of response elements are distributed in each gene; for example, the response elements in the \u003cem\u003eAcNRAMP4\u003c/em\u003e gene participate in defense and stress responses. There are four response elements: one response element involved in the low-temperature response; four response elements in the\u003cem\u003e\u0026nbsp;AcNRAMP10\u0026nbsp;\u003c/em\u003egene participate in the drought response; and two in the \u003cem\u003eAcNRAMP5\u003c/em\u003e gene participate in the low-temperature response. The distributions of other response elements, such as cis-regulatory elements involved in endosperm expression, auxin response elements, protein binding sites, zein metabolism regulation response elements, and circadian rhythm response elements, were relatively low. In short, in addition to the conserved DNA modules involved in the light response, most promoter homeopathic elements are involved in abiotic stress and hormone responses and regulate gene expression and substance metabolism in plants to develop stress tolerance. This phenomenon improves the adaptability of kiwifruit to cadmium stress.\u003c/p\u003e\n\u003cp\u003e3.6 Three-dimensional \u003cem\u003eAcNRAMP\u003c/em\u003e protein model prediction and multiple sequence alignment\u003c/p\u003e\n\u003cp\u003eThe WISS-MODEL tool was used to confirm the structures of the \u003cem\u003eAcNRAMP\u003c/em\u003e proteins. All \u003cem\u003eAhNRAMP\u003c/em\u003e proteins were well modeled via the 5m87.1 template (Figure 5A and\u0026nbsp;Additional file\u0026nbsp;2). The sequence identities were 23.54%-35.27%, the GMQE values were in the range of 0.2--0.59, and the overall QMEANDisCo score was in the range of 0.42--0.63 (Additional file\u0026nbsp;2). These data indicated that the\u0026nbsp;three-dimensional model prediction quality of \u003cem\u003eAcNRAMP\u003c/em\u003e proteins was high.\u003c/p\u003e\n\u003cp\u003eMultiple sequence alignment revealed that all the \u003cem\u003eAcNRAMP\u003c/em\u003e proteins presented considerable homology (Figure 5B). In addition to \u003cem\u003eAcNRAMP\u003c/em\u003e2, other \u003cem\u003eAcNRAMP\u003c/em\u003es contain the \u003cem\u003eNRAMP\u003c/em\u003e family domain DPGN (essential for metal binding). Except for \u003cem\u003eAcNRAMP10\u003c/em\u003e, all \u003cem\u003eAcNRAMPs\u003c/em\u003e contain the typical conserved amino acid residue GQSSTITGTYAGQY(/F)V(/I)MQ(/G/E)GFL, where the last five residues (MQGFL), which is a unique structural feature among the \u003cem\u003eNRAMP\u003c/em\u003e metal transporter proteins, are retained (Cellier et al., 2021).\u003c/p\u003e\n\u003cp\u003e3.7 GO functional analysis of the \u003cem\u003eAcNRAMP\u003c/em\u003e genes\u003c/p\u003e\n\u003cp\u003eTo further describe the biological functions of the \u003cem\u003eAcNRAMP\u003c/em\u003e gene family, this study used the kiwifruit database to annotate the proteins of 12 \u003cem\u003eAcNRAMP\u003c/em\u003e genes for gene function, and the online tool GraphPad Prism 10.2.3 was used for drawing and visualization (Figure 5C). Among the 12 genes, 12 \u003cem\u003eAcNRAMP\u003c/em\u003e genes were annotated and assigned to three categories: molecular function (MF), biological process (BP), and cellular component (CC). In particular, in terms of molecular function, transmembrane transporter activity, metal ion transmembrane transporter activity, inorganic cation transmembrane transporter activity, single-atom cation transmembrane transporter activity, substrate-specific transmembrane transporter activity, substrate-specific transporter activity, etc., the enrichment level of the \u003cem\u003eAcNRAMP\u003c/em\u003e genes was high, indicating that the molecular function of the \u003cem\u003eAcNRAMP\u003c/em\u003e gene family plays a crucial role in regulation. However, among the other CCs, the overall composition of the membrane, diaphragm, membrane, organelle, and intracellular organelle fractions was enriched, and the number of enriched \u003cem\u003eAcNRAMP\u003c/em\u003e genes was higher. In terms of biological processes, the genes enriched in \u003cem\u003eAcNRAMPs\u003c/em\u003e were associated with transition metal ion transport, ion transport, metal ion transport, cation transport, divalent metal ion transport, inorganic cation transmembrane transport, cation transmembrane transport, and the active regulation of biological processes. During growth and development, the transport of metal ions affects the morphogenesis of plants. The GO functions of the \u003cem\u003eAcNRAMP\u003c/em\u003e genes indicate that the \u003cem\u003eAcNRAMP\u003c/em\u003e gene family adapts plants to different environments by regulating plant metal tolerance. In addition, the number of enriched genes in each category was significantly greater than 11, indicating that the same gene may play different roles in different functional items.\u003c/p\u003e\n\u003cp\u003e3.8 Tissue-specific expression profiles of \u003cem\u003eAcNRAMP\u003c/em\u003e genes\u003c/p\u003e\n\u003cp\u003eWhen plants are under stress, plant cells respond to environmental fluctuations by continuously changing their gene expression, and these continuous changes are regulated by a multilevel network of gene families. Therefore, to understand the expression pattern of the \u003cem\u003eAcNRAMP\u003c/em\u003e gene family under cadmium stress, this study analyzed the transcriptome data of kiwifruit plants after cadmium stress. The results revealed 12 \u003cem\u003eAcNRAMPs\u003c/em\u003e under cadmium stress, including five downregulated genes and seven upregulated genes in leaves, as well as nine downregulated genes and three upregulated genes in roots. Compared with those in the control group, three genes were significantly differentially expressed in the roots. The two genes \u003cem\u003eAcNRAMP7\u003c/em\u003e, \u003cem\u003eAcNRAMP10\u003c/em\u003e, and \u003cem\u003eAcNRAMP11\u003c/em\u003e were significantly differentially expressed in the leaves. \u003cem\u003eAcNRAMP6\u003c/em\u003e and \u003cem\u003eAcNRAMP11\u003c/em\u003e (Figure 6) imply that these genes may be crucial in fighting cadmium toxicity. The expression level of the \u003cem\u003eAcNRAMP5\u003c/em\u003e gene was 0 in the control group and was upregulated to a certain extent after being subjected to cadmium stress. Those with relatively high expression levels in leaves, \u003cem\u003eAcNRAMP6\u003c/em\u003e, presented relatively low expression levels in roots.\u003c/p\u003e\n\u003cp\u003e3.9\u0026nbsp;\u003cstrong\u003eAnalysis of \u003cem\u003eAcNRAMP\u003c/em\u003e gene expression under cadmium stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, we conducted cadmium treatment on kiwifruit seedlings under consistent growth conditions. To elucidate the expression pattern of\u003cem\u003e\u0026nbsp;AcNRAMP\u003c/em\u003e under Cd stress, we analyzed the expression of five DEGs under Cd stress via RT-qPCR and only the transcripts of three kiwifruit \u003cem\u003eNRAMP\u003c/em\u003e family members (Figure 7B). The other two differential genes might be due to the primers; after many attempts, no product was amplified. The qRT-PCR data revealed that AcNRAMP6 expression in the leaves of the RCK and RCd groups significantly increased. However, the RCK and RCd groups presented significant decreases in \u003cem\u003eAcNRAMP7\u003c/em\u003e and \u003cem\u003eAcNRAMP10\u003c/em\u003e in the roots, indicating that the levels of \u003cem\u003eAcNRAMP7\u003c/em\u003e and \u003cem\u003eAcNRAMP10\u003c/em\u003e in the RCK group and the RCd group significantly decreased under Cd stress. The stronger the response was, the higher the expression level was. Under cadmium treatment, \u003cem\u003eAcNRAMP7\u003c/em\u003e and \u003cem\u003eAcNRAMP10\u003c/em\u003e presented different responses in roots than did \u003cem\u003eAcNRAMPs\u003c/em\u003e in leaves, suggesting that there is an intricate interaction between the level of Cd stress and the regulation of gene expression. This observation revealed the complex regulatory mechanism controlling \u003cem\u003eAcNRAMP\u003c/em\u003e expression under Cd stress. These findings further emphasize the necessity of specific molecular responses of these transition metal transporters in kiwifruit under different stresses.\u003c/p\u003e"},{"header":"4. Discussions","content":"\u003cp\u003ePlant metal tolerance proteins are membrane-divalent transporter proteins. As part of the response to specific metal toxicity, they play a crucial role in the special transport of different heavy metal ions. In addition to their key function in maintaining mineral plants, changes in their transcript levels can also be considered environmental biomarkers for examining heavy metal pollution levels. Dong et al. studied rice under cadmium stress and reported that the growth of rice plants was significantly inhibited after one week of cadmium treatment. In this study, the leaf edges of kiwifruit seedlings subjected to 10 \u0026mu;M CdCl\u003csub\u003e2\u003c/sub\u003e and 100 \u0026mu;M CdCl\u003csub\u003e2\u003c/sub\u003e presented different degrees of yellowing and browning, especially in response to 100 \u0026mu;M CdCl\u003csub\u003e2\u003c/sub\u003e. Some phenolic compounds in the treatment group caused the damaged tissues to turn brown. Plant tissue browning and discoloration are the most intuitive responses of plants to stress, and to a certain extent, they can reflect the cadmium tolerance of plants (Figure 7A). Zhang et al. studied the physiological and phenotypic status of Chuanqiong under different concentrations of cadmium stress. They reported that, under 20 mg/L cadmium treatment, the plants in the treatment group presented obvious toxicity symptoms, including yellowing, wilting, sensitization, and browning. As cadmium stress intensifies, so does the degree of browning. At this time, kiwifruit seedlings were severely injured by cadmium toxicity, indicating that it was difficult for kiwifruit seedlings to withstand cadmium toxicity under 100 \u0026mu;M CdCl\u003csub\u003e2\u003c/sub\u003e treatment (Figure 7A), which was consistent with previous study results in other plants.\u003c/p\u003e\n\u003cp\u003eThe plant genome contains many genes with different functions. The \u003cem\u003eNRAMP\u003c/em\u003e gene family comprises complete membrane transporter proteins in plants, such as potato, tea, and \u003cem\u003eBrassica napus\u003c/em\u003e (Tian et al., 2021; Liu et al., 2023; Migeon et al., 2010; Meng et al., 2010). Regulating the response to various metal ions by mediating gene expression. In addition, they participate in many biological mechanisms, including photosynthesis, protein function, metabolism, and response to environmental stress (Nevo et al., 2006). However, no information has focused on the genome-wide identification of the \u003cem\u003eNRAMP\u003c/em\u003e family in kiwifruit or the effects of cadmium stress on the transcript levels of genes known to be expressed in response to metal stress. Therefore, this study conducted a bioinformatics analysis of the kiwifruit \u003cem\u003eNRAMP\u003c/em\u003e gene family, including phylogenetic information, conserved motif information, gene structure information, expression data, and information on cis-acting elements. The number of \u003cem\u003eAcNRAMPs\u003c/em\u003e differed from that in \u003cem\u003eArabidopsis\u003c/em\u003e and tea. This may be due to the distant relationship between the \u003cem\u003eAcNRAMP\u0026nbsp;\u003c/em\u003eof \u0026apos;Hongyang\u0026apos; and them. Among them, \u003cem\u003eAcNRAMP3\u003c/em\u003e and\u003cem\u003e\u0026nbsp;AcNRAMP4\u003c/em\u003e were the largest, with lengths of 1291 AA and 1230 AA, respectively, molecular weights of 141577.95 kDa and 135045.72 kDa, and instability coefficients of 40.88 and 38.65, respectively. \u003cem\u003eAcNRAMP10\u003c/em\u003e was the shortest, with a length of 129 AA, a molecular weight of 13897.89, and an instability coefficient of 41.79. In biological systems, variation in protein length within gene families such as \u003cem\u003eNRAMPs\u003c/em\u003e is not uncommon. These variations can lead to functional diversity among gene family members, allowing them to adapt to different environmental conditions and biotic interactions (Yin et al., 2023).\u003c/p\u003e\n\u003cp\u003eCertain aspects of gene profiles, such as the arrangement, location, and number of exons and introns, are considered decisive factors for understanding genome function and evolution\u0026nbsp;(Bari et al., 2021)\u0026nbsp;Bari MA. In this study, the gene structure of \u003cem\u003eAcNRAMPs\u003c/em\u003e included 4--11 introns. Except for \u003cem\u003eAcNRAMP7\u003c/em\u003e, \u003cem\u003eAcNRAMP8\u003c/em\u003e, and \u003cem\u003eAcNRAMP11\u003c/em\u003e, which do not have exons, the other genes all contain 1--3 exons. In the clustering tree, the members of the same cluster presented similar numbers of exons and introns, and all presented larger introns and smaller exons. Previous studies have demonstrated\u0026nbsp;several\u0026nbsp;characteristics of plant genes through high expression levels. These features include abundant longer introns, a less compact gene structure, larger primary transcripts, etc., and the opposite trend was observed in animal genes. For example, genes in yeast, Thaler, and mice share a\u0026nbsp;regular\u0026nbsp;feature, that is, fewer introns, which may be due to the rapid activation of genes in response to a wide range of environmental stresses\u0026nbsp;(Jeffares et al., 2008). In this study,\u0026nbsp;ten\u0026nbsp;motifs were also screened via the MEME tool. Conserved motif analysis revealed that similar genes were clustered in the same group and that there were multiple motifs in specific proteins. This finding illustrates that these genes might have special functions. Motif 5 was present in all the \u003cem\u003eAcNRAMPs\u003c/em\u003e, and this motif was always present between motifs 3 and 2, whereas the other motifs presented different patterns. The existence of the complete motif represents the evolutionary function and structural conservation of \u003cem\u003eAcNRAMPs\u003c/em\u003e. Studies in rice have shown that \u003cem\u003eOsNRAMP5\u003c/em\u003e is a plasma membrane-localized transporter protein that facilitates the uptake of Fe, Mn, Cd, and lead (Pb). The knockout of \u003cem\u003eOsNRAMP5\u003c/em\u003e significantly reduces the concentrations of Mn and Cd in roots, shoots, and grains but promotes the transport of Cd from roots to shoots\u0026nbsp;(Bari et al., 2021).\u003c/p\u003e\n\u003cp\u003eAmong the 12 \u003cem\u003eAcNRAMP\u003c/em\u003e protein sequences, \u003cem\u003eAcNRAMP2\u003c/em\u003e and \u003cem\u003eAcNRAMP10\u003c/em\u003e lacked the structural characteristics of \u003cem\u003eNRAMP\u003c/em\u003e proteins. The remaining ten \u003cem\u003eAcNRAMP\u003c/em\u003e proteins all contained all the structural characteristics of the \u003cem\u003eNRAMP\u003c/em\u003e proteins. They contain conserved motifs. Numerous studies have shown that these two domains are critical for the metal transport activity of \u003cem\u003eNRAMP\u003c/em\u003e transporters. Next, the\u003cem\u003e\u0026nbsp;AcNRAMP\u003c/em\u003e protein was better modeled via the same 3D model template, 5m87.1A. The 5m87.1A template is the crystal structure of the ScanDMT divalent metal transporter protein. This model is a close prokaryotic homolog of the \u003cem\u003eNRAMP\u003c/em\u003e family, which functions to transport divalent transition metal ions. Multiple sequence alignment revealed that the \u003cem\u003eAcNRAMP\u003c/em\u003e proteins presented considerable homology. These analogous structures indicated that \u003cem\u003eAcNRAMPs\u003c/em\u003e could have physiological functions similar to those of ScaDMT.\u003c/p\u003e\n\u003cp\u003eAnalysis of the cis-elements revealed that a variety of elements located 2000 bp upstream of the promoter region are involved in various functions. This provides a better understanding of gene transcription and expression, as well as their responses to environmental stress. This study revealed that the \u003cem\u003eAcNRAMP\u003c/em\u003e gene had more elements essential for anaerobic induction than did the other genes. Hormone-related cis-acting elements, such as jasmonate (MeJA), gibberellin (GA), salicylic acid, and gibberellic acid, are second only to response elements involved in anaerobic induction. The distribution of cis-acting elements involved in the gibberellic acid response is relatively greater; there are 1--3 elements each in \u003cem\u003eAcNRAMP3\u003c/em\u003e, \u003cem\u003eAcNRAMP5\u003c/em\u003e, \u003cem\u003eAcNRAMP6\u003c/em\u003e, \u003cem\u003eAcNRAMP8\u003c/em\u003e, \u003cem\u003eAcNRAMP10\u003c/em\u003e, and \u003cem\u003eAcNRAMP11\u003c/em\u003e. Formula effect element interaction causes. These elements have also been reported in the \u003cem\u003eNRAMP\u003c/em\u003e gene of Ziping (\u003cem\u003eSpirodela polyrhiza\u003c/em\u003e) (Chen et al., 2021). In addition, gibberellin is heavily involved in the rice defense response to abiotic environmental stress by increasing salicylic acid production\u0026nbsp;(Bari et al., 2021). The published literature indicates that salicylic acid is vital in managing plant adaptations to abiotic stresses, especially heavy metal toxicity. This is achieved by maintaining membrane stability and integrity and scavenging ROS. Therefore, it is reasonable to predict that gibberellin enhances cadmium tolerance by increasing glucose metabolism, promoting mitotic activity, and increasing RNA content\u0026nbsp;(Emamverdian et al., 2020). Emamverdian A. These plant hormones can regulate the transcription of \u003cem\u003eNRAMP\u003c/em\u003e genes, potentially affecting their functions in metal ion transport and stress response in plants. Specifically, these genes may be involved in \u003cem\u003eNRAMP\u003c/em\u003e gene expression regulation in response to various environmental stimuli, such as biotic and abiotic stresses.\u003c/p\u003e\n\u003cp\u003eIn our molecular biology study, the distribution of \u003cem\u003eAcNRAMP\u003c/em\u003e gene family members on seven chromosomes was significantly unbalanced, whereas no such genes were detected on the remaining 22 chromosomes. In addition, we found that most of these genes were derived from fragment duplication events, which highlights the vital role of fragment duplication in promoting gene family expansion. This result is different from that of potato and poplar\u0026nbsp;(Tian et al., 2021; Siqin et al., 2023; Migeon et al., 2010), and the other results were similar.\u003c/p\u003e\n\u003cp\u003eTranscriptomic analysis revealed the expression patterns of the \u003cem\u003eNRAMP\u003c/em\u003e genes in roots under cadmium stress. Many studies have analyzed \u003cem\u003eNRAMP\u003c/em\u003e expression in different species under different stresses. Genes from the same subfamily have similar expression levels in the same tissue (Shirazi et al., 2023). For example, the \u003cem\u003eAcNRAMP6\u003c/em\u003e and \u003cem\u003eAcNRAMP11\u003c/em\u003e genes in kiwifruit leaves were located in the Geoup2 branch. Similarly, in rice, \u003cem\u003eOsNRAMP6\u003c/em\u003e and \u003cem\u003eOsNRAMP5\u003c/em\u003e are involved in Fe and Mn uptake. In addition, the functions of \u003cem\u003eAtNRAMP3\u0026nbsp;\u003c/em\u003eand \u003cem\u003eAtNRAMP4\u0026nbsp;\u003c/em\u003ehave been confirmed in the transfer of Fe and Mn\u0026nbsp;(Bari et al., 2021;\u0026nbsp;Jeffares et al., 2008;\u0026nbsp;Emamverdian et al., 2020;\u0026nbsp;Shirazi et al., 2023;\u0026nbsp;Rasheed et al., 2024)\u0026nbsp;Rasheed A. These reports support the involvement of the \u003cem\u003eNRAMP\u003c/em\u003e gene in metal transport in plant tissues. In our study, we focused on the different expression patterns of \u003cem\u003eAcNRAMPs\u003c/em\u003e in the presence of cadmium contamination. The results revealed that the expression\u0026nbsp;of\u003cem\u003e\u0026nbsp;AcNRAMP7\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;AcNRAMP10\u003c/em\u003e in roots was significantly downregulated under Cd stress; the expression of \u003cem\u003eAcNRAMP6\u003c/em\u003e was markedly upregulated in leaves, suggesting that these genes may be involved in Cd transport.\u0026nbsp;As such, we speculated that after high-concentration Cd stress, the expression level of \u003cem\u003eAcNRAMP6\u003c/em\u003e was upregulated, and the expression levels of \u003cem\u003eAcNRAMP7\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;AcNRAMP10\u003c/em\u003e were downregulated. The three genes whose expression significantly differed may have contributed to the reduced amount of Cd entry into root cells, resulting in the effect of Cd in the environment on plants. The destructive power of root cells decreased, which in turn increased the tolerance of kiwifruit plants to Cd.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eNotably, this study provides the first elaboration of\u0026nbsp;\u003cem\u003eAc\u003c/em\u003e\u003cem\u003eN\u003c/em\u003e\u003cem\u003eRAMP\u003c/em\u003e gene family information in the kiwifruit genome. Gene family analysis revealed that there were more metal stress and hormone response elements and that these elements played a critical role in the metal stress response. The expression patterns revealed that two genes of the \u003cem\u003eNRAMP\u0026nbsp;\u003c/em\u003egene family were differentially expressed in kiwifruit roots and that one gene was differentially expressed in the leaves. In addition, the regulatory mechanism of \u003cem\u003eAcNRAMP\u003c/em\u003e family members in response to Cd stress is as follows: after high-concentration Cd stress, the expression levels of \u003cem\u003eAcNRAMP7\u0026nbsp;\u003c/em\u003eand \u003cem\u003eAcNRAMP10\u003c/em\u003e are downregulated (Figure 8). As a result, the amount of Cd entry into root cells is reduced, the Cd concentration in roots is reduced, the damage caused by Cd in the environment to kiwifruit root cells is reduced, and the tolerance of kiwifruit plants is improved. This study lays the foundation for mechanistic studies of the uptake and transport of heavy metal elements in kiwifruit.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eThe authors thank Prof./Dr. Deqiang Zhang, Beijing Forestry University, for his critical reading of the manuscript.\u003c/p\u003e\n\u003ch2\u003eData availability statement\u003c/h2\u003e\n\u003cp\u003eAll the data generated or analyzed in this study are included in this article. Kiwifruit genome annotation files can be accessed at http://kiwifruitgenome.org/. RNA-Seq data under drought stress can be found. The login link is https://www.ncbi.nlm.nih.gov/sra/PRJNA1246740. The RNA-Seq data are publicly available at the National Center for Biotechnology Information. The other data presented in this study are available in the Supplementary Materials.\u003c/p\u003e\n\u003ch2\u003eAuthorship contribution statement\u003c/h2\u003e\n\u003cp\u003eXuechun Li: conceptualization, investigation, formal analysis, writing-original draft preparation, and software. Hanbing Cai: Methodology, formal analysis, and visualization. Tuo Yin: Software, Data curation. Ke Zhao: Validation. Ping Tian: Validation. Jiaqiong Wan: Writing-review. Hanyao Zhang: Review and editing, supervision, funding acquisition. Xiaozhen Liu: Methodology, Writing-review and editing, Funding acquisition. All the authors have read and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eEthics declarations\u003c/h2\u003e\n\u003ch3\u003eEthics approval and consent to participate\u003c/h3\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e\n\u003cp\u003eThe 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.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis study is supported by the Yunnan Academician (Expert) Workstation Project (202305AF150020), the Agricultural Joint Key Projects in Yunnan Province (202301BD070001-003), the Rural Revitalization Science and Technology Project-Rural Revitalization Industry Key Technology Integration Demonstration Project (202304BP090005), the Yunnan First-class Construction Discipline of Forestry Science of Southwest Forestry University and the Start-up Fund Project of Doctoral Research at Southwest Forestry University. The funders had no role in the design of the study, the collection, analysis, or interpretation of the data, or the writing of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJia Z, Zeng H. Medicinal value of kiwifruit[J]. Modern Distance Education of Chinese Traditional Medicine,2012, 10(04):118.https://doi.org/10.3969/j.issn.1672-2779.2012.04.081\u003c/li\u003e\n\u003cli\u003eSong PJ, Zhang L, Ding L. Cancer preventive effects of Chinese kiwifruit juice-(II) Blockade of nitrosamine synthesis in simulated gastric juice in vitro by Ames test[J]. Journal of Nutrition, 1984(03):241-246.https: //doi.org/10.13325/j.cnki.acta.untr.sin.1984.03.006\u003c/li\u003e\n\u003cli\u003eYin M, Tang H, Yang D, et al. Remediation experiments of different varieties of red hemp in heavily and slightly cadmium-polluted cropland[J]. Journal of Agricultural Environmental Science,2020,39(10):2267-2276. https://doi.org/10.11654/jaes.2020-0257\u003c/li\u003e\n\u003cli\u003eXiao Q, Wang J, Zheng X, et al. Differential protein expression in rice roots in response to cadmium stress[J]. Journal of Ecology, 2015, 35(24):8276-8283.https://doi.org/10. 5846/stxb201403230517\u003c/li\u003e\n\u003cli\u003eWang B, Liu Y, Wang H, et al. Contamination and health risk assessment of lead, arsenic, cadmium, and aluminum from a total diet study of Jilin Province,/China.[J]. Food Science \u0026amp; Nutrition, 2020, 8(10):5631-5640.https: //doi.org/10.1002/fsn3.1851\u003c/li\u003e\n\u003cli\u003eShakirova FM, Allagulova ChR, Maslennikova DR, et al. Salicylic acid-induced protection against cadmium toxicity in wheat plants. Environmental \u0026amp; Experimental Botany 2016:19-28.https://doi.org/10.1016/j.envexpbot. 2015.08.002\u003c/li\u003e\n\u003cli\u003eGao Y, Cheng Q, Zhang M, et al. Research on remediation technology of cadmium-contaminated soil[J]. Biotechnology Bulletin, 2017,33(10):103-110. https://doi.org/10. 13560/j.cnki.biotech.bull.1985.2017-0336\u003c/li\u003e\n\u003cli\u003eGeng HX, Wang L. Cadmium: Toxic effects on placental and embryonic development[J]. Environmental Toxicology and Pharmacology, 2019, 67:102-107.https://doi.org/10. 13560/10. 1016/j.etap.2019.02.006\u003c/li\u003e\n\u003cli\u003eSingh RK, Anandhan S, Singh S, Patade VY, Ahmed Z, Pande V. Metallothionein-like gene from Cicer microphyllum is regulated by multiple abiotic stresses. Protoplasma. 2011 Oct;248(4):839-47.https://doi.org/10.1007/s00709-010-0249-y\u003c/li\u003e\n\u003cli\u003eGao Y, Yang F, Liu J, Xie W, Zhang L, Chen Z, Peng Z, Ou Y, Yao Y. (2020). Genome-wide identification of metal tolerance protein genes in \u003cem\u003ePopulus trichocarpa\u003c/em\u003e and their roles in response to various heavy metal stresses. International Journal of Molecular Sciences. 21:1680. https://doi.org/10.3390/ijms21051680\u003c/li\u003e\n\u003cli\u003eBozzi AT, Gaudet R. Molecular mechanism of NRAMP-family transition metal transport. Journal of Molecular Biology. 2021, 433, 166991.https://doi.org/10.1016/j.jmb.2021.166991\u003c/li\u003e\n\u003cli\u003eVidal SM, Malo D, Vogan K, Skamene E, Gros P. Natural resistance to infection with intracellular parasites: isolation of a candidate for Bcg. Cell. 1993 May 7;73(3):469-85. https://doi.org/10.1016/0092-8674(93)90135-d\u003c/li\u003e\n\u003cli\u003ePinner E, Gruenheid S, Raymond M, Gros P. Functional complementation of the yeast divalent cation transporter family SMF by \u003cem\u003eNRAMP2\u003c/em\u003e, a member of the mammalian natural resistance-associated macrophage protein family. J Biol Chem. 1997 Nov 14;272(46):28933-8.https://doi.org/10.1074/jbc.272.46.28933\u003c/li\u003e\n\u003cli\u003eGunshin H, MacKenzie B, Berger UV, Gunshin Y, Romero MF, Boron WF, Nussberger S, Gollan JL, Hediger MA. Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature Cell Biology. 1997,388,482-488.https://doi.org/10.1038/41343\u003c/li\u003e\n\u003cli\u003eZheng X, Jiang J, Wang C, Hua Y, Huang H, Xu Y, Wei P, Tao J, Cao P, Kang Z, Li X, Gao Q, Chen Q. \u003cem\u003eNRAMP6c\u003c/em\u003e plays a key role in plant cadmium accumulation and resistance in tobacco (\u003cem\u003eNicotiana tabacum\u003c/em\u003e L.). Ecotoxicol Environ Saf. 2024 Feb;271:115885. https://doi.org/10.1016/j. ecoenv.2023.115885\u003c/li\u003e\n\u003cli\u003eMa X, Yang H, Bu Y, et al. Genome-wide identification of the NRAMP gene family in \u003cem\u003ePopulus trichocarpa\u003c/em\u003e and their function as heavy metal transporters. Ecotoxicology and Environmental Safety. 2023;261:115110.https://doi.org/10.1016/j.ecoenv.2023.115110\u003c/li\u003e\n\u003cli\u003eYu W, Deng S, Chen X, Cheng Y, Li Z, Wu J, Zhu D, Zhou J, Cao Y, Fayyaz P, Shi W, Luo Z. \u003cem\u003ePcNRAMP1\u003c/em\u003e Enhances Cadmium Uptake and Accumulation in \u003cem\u003ePopulus\u003c/em\u003e \u0026times; \u003cem\u003ecanescens\u003c/em\u003e. Int J Mol Sci. 2022 Jul 8;23(14):7593. https://doi.org/10.3390/ijms23147593\u003c/li\u003e\n\u003cli\u003eTian W, He G, Qin L, Li D, Meng L, Huang Y, He T. Genome-wide analysis of the NRAMP gene family in potato (\u003cem\u003eSolanum tuberosum\u003c/em\u003e): Identification, expression analysis and response to five heavy metals stress. Ecotoxicol Environ Saf. 2021 Jan 15;208:111661. https://doi.org/10.1016 /j.ecoenv.2020.111661\u003c/li\u003e\n\u003cli\u003eM\u0026auml;ser P, Thomine, S, Schroeder, J.I, Ward, J.M, Hirschi, K, Sze, H, Talke, I.N, Amtmann, A, Maathuis, F.J, Sanders, D, et al. Phylogenetic relationships within cation transporter families of Arabidopsis. Plant Physiology.2001,126,1646-1667.https://doi.org/10.1104/pp.126.4.1646\u003c/li\u003e\n\u003cli\u003eLi J, Duan Y, Han Z, et al. Genome-wide identification and expression analysis of the NRAMP family genes in tea plant (\u003cem\u003eCamellia sinensis\u003c/em\u003e). Plants (Basel). 2021;10(6):1055. Published 2021 May 25.https://doi.org/10.3390/plants10061055\u003c/li\u003e\n\u003cli\u003eCailliatte R, Lapeyre B, Briat J, Mari S, Curie C. The\u003cem\u003e NRAMP6\u003c/em\u003e metal transporter contributes to cadmium toxicity. Biochemistry Journal.2009,422,217-228.https://doi.org/10.1042/BJ20090655\u003c/li\u003e\n\u003cli\u003eYang M, Zhang Y, Zhang L, Hu J, Zhang X, Lu K, Dong H, Wang D, Zhao F.J, Huang C.F, et al. \u003cem\u003eOsNRAMP5\u003c/em\u003e contributes to manganese translocation and distribution in rice shoots. Journal of Experimental Botany.2014,65,4849-4861.https://doi.org/10.1093/jxb/eru259\u003c/li\u003e\n\u003cli\u003eLi J, Duan Y, Han Z, Shang X, Zhang K, Zou Z, Ma Y, Li F, Fang W, Zhu X. Genome-Wide Identification and Expression Analysis of the NRAMP Family Genes in Tea Plant (\u003cem\u003eCamellia sinensis\u003c/em\u003e). Plants (Basel). 2021 May 25;10(6):1055. https://doi.org/10.3390/plants10061055\u003c/li\u003e\n\u003cli\u003eMadeira F, Park YM, Lee J, Buso N, Gur T, Madhusoodanan N, Basutkar P, Tivey ARN, Potter SC, Finn RD, Lopez R. The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Research. 2019 Jul 2;47(W1):W636-W641.https://doi.org/10.1093/nar/gkz268\u003c/li\u003e\n\u003cli\u003eChen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, Xia R. TBtools: an integrative toolkit developed for interactive analyses of big biological data. Molecular Plant. 2020 Aug 3;13(8):1194-1202. doi: 10.1016/j.molp.2020.06.009.https://doi.org/10.1016/j.molp.2020.06.009\u003c/li\u003e\n\u003cli\u003eMarchler-Bauer A, Bo Y, Han L, He J, Lanczycki CJ, Lu S, Chitsaz F, Derbyshire MK, Geer RC, Gonzales NR, Gwadz M, Hurwitz DI, Lu F, Marchler GH, Song JS, Thanki N, Wang Z, Yamashita RA, Zhang D, Zheng C, Geer LY, Bryant SH. CDD/SPARCLE: functional classification of proteins via subfamily domain architectures. Nucleic Acids Res. 2017 Jan 4;45(D1):D200-D203.https://doi.org/10.1093/nar/gkw1129\u003c/li\u003e\n\u003cli\u003eXi D, Yin T, Han P, Yang X, Zhang M, Du C, Zhang H, Liu X. Genome-wide identification of sweet orange WRKY transcription factors and analysis of their expression in response to infection by \u003cem\u003ePenicillium digitatum\u003c/em\u003e. Current \u003cem\u003eIssues\u003c/em\u003e In Molecular Biology. 2023 Feb 3;45(2):1250-1271. doi: 10.3390/cimb45020082.https://doi.org/10.3390/cimb45020082\u003c/li\u003e\n\u003cli\u003eTian C, Wan P, Sun S, Li J, Chen M. Genome-wide analysis of the GRAS gene family in rice and Arabidopsis. Plant Molecular Biology. 2004 Mar;54(4):519-32. doi: 10.1023/B: PLAN.0000038256.89809.57.https://doi.org/10.1023/B:PLAN.0000038256.89809.57\u003c/li\u003e\n\u003cli\u003eMarchler-Bauer A, Lu S, Anderson JB, Chitsaz F, Derbyshire MK, DeWeese-Scott C, Fong JH, Geer LY, Geer RC, Gonzales NR, Gwadz M, Hurwitz DI, Jackson JD, Ke Z, Lanczycki CJ, Lu F, Marchler GH, Mullokandov M, Omelchenko MV, Robertson CL, Song JS, Thanki N, Yamashita RA, Zhang D, Zhang N, Zheng C, Bryant SH. CDD: a Conserved Domain Database for the functional annotation of proteins. Nucleic Acids Res. 2011 Jan;39(Database issue): D225-9. .https://doi.org/10.1093/nar/gkq1189\u003c/li\u003e\n\u003cli\u003eBrown P, Baxter L, Hickman R, Beynon J, Moore JD, Ott S. MEME-LaB: motif analysis in clusters. Bioinformatics. 2013 Jul 1;29(13):1696-7. https://doi.org/10.1093/bioinformatics/btt248\u003c/li\u003e\n\u003cli\u003eLescot M, D\u0026eacute;hais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rouz\u0026eacute; P, Rombauts S. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002 Jan 1;30(1):325-7.https://doi.org/10. 1093/nar/30.1.32\u003c/li\u003e\n\u003cli\u003eWang L, Cui X, Cheng H, Chen F, Wang J, Zhao X, Lin C, Pu X. A review of soil cadmium contamination in China including a health risk assessment. Environmental Science and Pollution Research. 2015，22(21):16441-52.https://doi.org/10.1007/s11356-015-5273-1\u003c/li\u003e\n\u003cli\u003eTian W, He G, Qin L, Li D, Meng L, Huang Y, He T. Genome-wide analysis of the NRAMP gene family in potato (\u003cem\u003eSolanum tuberosum\u003c/em\u003e): Identification, expression analysis and response to five heavy metals stress. Ecotoxicol Environ Saf. 2021 Jan 15;208:111661.https://doi.org/10.1016/J.ecoenv.2020.111661\u003c/li\u003e\n\u003cli\u003eLiu, S. , Long, T. , Chen, Z. , Liu, J. , Cui, W. , \u0026amp; Leng, H. , et al. (2023). Genome-wide identification of nramp family genes in\u003cem\u003e Populus trichocarpa \u003c/em\u003eand their roles in transport of heavy metals. Tree Genetics \u0026amp; Genomes(6), 19.https://doi.org/10.1007/s11295-023-01629-3\u003c/li\u003e\n\u003cli\u003eMigeon A, Blaudez D, Wilkins O, Montanini B, Campbell MM, Richaud P, Thomine S, Chalot M. Genome-wide analysis of plant metal transporters, with an emphasis on poplar. Cellular and Molecular Life Science. 2010, 67(22):3763-84.https://doi.org/10.1007/s00018-010-0445-0\u003c/li\u003e\n\u003cli\u003eMeng JG, Zhang XD, Tan SK, Zhao KX, Yang ZM. Genome-wide identification of Cd-responsive NRAMP transporter genes and analysis of the expression of \u003cem\u003eNRAMP\u003c/em\u003e\u003cem\u003e \u003c/em\u003e\u003cem\u003e1\u003c/em\u003e mediated by miR167 in Brassica napus. Biometals. 2017 Dec;30(6):917-931.https://doi.org/10.1007/s00018-010-0445-0\u003c/li\u003e\n\u003cli\u003eNevo Y, Nelson N. The NRAMP family of metal-ion transporters. Biochimica et Biophysica Acta. 2006, 1763(7):609-20.https://doi.org/10.1007/s00018-010-0445-0\u003c/li\u003e\n\u003cli\u003eYin S, Zhao L, Liu J, Sun Y, Li B, Wang L, Ren Z, Chen C. Pangenome Analysis of \u003cem\u003eWOX\u003c/em\u003e Gene Family and Function Exploration of \u003cem\u003eCsWOX9\u003c/em\u003e in Cucumber. International Journal of Molecular Sciences. 2023, 17;24(24):17568.https://doi.org/10.3390/ijms242417568\u003c/li\u003e\n\u003cli\u003eBari MA, El-Shehawi AM, Elseehy MM, Naheen NN, Rahman MM, Kabir AH. Molecular characterization and bioinformatics analysis of transporter genes associated with Cd-induced phytotoxicity in rice (\u003cem\u003eOryza sativa\u003c/em\u003e L.). Plant Physiol Biochem. 2021 Oct;167:438-448. https://doi.org/10.1016/j.plaphy.2021.08.024\u003c/li\u003e\n\u003cli\u003eJeffares DC, Penkett CJ, B\u0026auml;hler J. Rapidly regulated genes are intron poor. Trends in Genetics. 2008, 24(8):375-8.https://doi.org/10.1016/j.tig.2008.05.006\u003c/li\u003e\n\u003cli\u003eEmamverdian A, Ding Y, Mokhberdoran F. The role of salicylic acid and gibberellin signaling in plant responses to abiotic stress with an emphasis on heavy metals. Plant Signaling and Behavior 2020, 2;15(7):1777372.https://doi.org/10.1080/15592324.2020.1777372\u003c/li\u003e\n\u003cli\u003eShirazi Z, Khakdan F, Rafiei F, Balalami MY, Ranjbar M. Genome-wide identification and expression profile analysis of metal tolerance protein-encoding gene family in Eucalyptus grandis under metal stresses. BMC Plant Biol. 2023 May 6;23(1):240.https://doi. org/10.1186/s12870-023-04240-9\u003c/li\u003e\n\u003cli\u003eRasheed A, Al-Huqail AA, Ali B, Alghanem SMS, Shah AA, Azeem F, Rizwan M, Al-Qthanin RN, Soudy FA. Molecular characterization of genes involved in tolerance of cadmium in \u003cem\u003eTriticum aestivum\u003c/em\u003e (L.) under Cd stress. J Hazard Mater. 2024 Feb 15;464:132955. https://doi.org/10.1016/J.jhazmat.2023.132955\u003c/li\u003e\n\u003cli\u003eChen Y, Zhao X, Li G, Kumar S, Sun Z, Li Y, Guo W, Yang J, Hou H. Genome-Wide Identification of the \u003cem\u003eNramp\u003c/em\u003e Gene Family in \u003cem\u003eSpirodela polyrhiza\u003c/em\u003e and Expression Analysis under Cadmium Stress. International Journal of Molecular Sciences. 2021, 15;22(12):6414.https://doi. org/10.3390/ijms22126414\u003c/li\u003e\n\u003cli\u003eCellier M, Prive G, Belouchi A, Kwan T, Rodrigues V, Chia W, Gros P. NRAMP defines a family of membrane proteins. Proceedings of the National Academy of Sciences of the United States of America, 1995, \u003cem\u003e92\u003c/em\u003e, 10089\u0026ndash;10093.https://doi.org/10.1073/pnas.92.22.10089\u003c/li\u003e\n\u003cli\u003ePfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. \u003cem\u003eNucleic Acids Research.\u003c/em\u003e 2001, \u003cem\u003e29\u003c/em\u003e, e45.https://doi.org/10.1093/nar/29.9.e45\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"kiwifruit, NRAMP, cadmium stress, expression pattern analysis","lastPublishedDoi":"10.21203/rs.3.rs-6967468/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6967468/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe \u003cem\u003eNRAMP\u003c/em\u003e gene family acts on a wide range of divalent metal ions in plants and plays a key role in Cd accumulation and resistance in plants. In this study, we identified 12 \u003cem\u003eAcNRAMP\u003c/em\u003e genes from the kiwifruit genome that were phylogenetically categorized into four groups. Chromosome distribution analysis revealed that 11 of the 12 \u003cem\u003eAcNRAMP\u003c/em\u003egenes were unevenly distributed on seven chromosomes. The \u003cem\u003eAcNRAMP\u003c/em\u003e gene promoter cis-elements were mainly categorized into hormone-associated cis-acting elements and environmental stress-associated cis-acting elements. In addition, expression profiling revealed that three genes were significantly differentially expressed in the roots and that two genes were markedly differentially expressed in the leaves. Based on the \u003cem\u003eAcNRAMP\u003c/em\u003e family analysis by RT-PCR, we hypothesized that there might be a mechanism by which members of the \u003cem\u003eAcNRAMP\u003c/em\u003egene family respond to Cd stress: after being subjected to high concentrations of Cd stress, the expression of \u003cem\u003eAcNRAMP7\u003c/em\u003e and \u003cem\u003eAcNRAMP10\u003c/em\u003e was downregulated in roots, whereas the expression of \u003cem\u003eAcNRAMP6 \u003c/em\u003ewas upregulated in leaves, the concentration of Cd translocated to the root cells was reduced, and the environmental Cd in the environment decreased the degree of damage to plant root cells, which in turn led to an increase in Cd tolerance in kiwifruit plants and resulted in slower root growth. The results of this study are crucial, as they lay the foundation for further exploration of the regulatory functions of \u003cem\u003eAcNRAMPs\u003c/em\u003e in kiwifruit in response to Cd stress.\u003c/p\u003e","manuscriptTitle":"Genome-wide identification of the NRAMP gene family in kiwifruit and the response of AcNRAMP7 to cadmium stress","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-18 16:27:26","doi":"10.21203/rs.3.rs-6967468/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-01T12:15:09+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-28T00:24:45+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-23T07:57:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-22T07:23:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-22T07:20:22+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-13T09:18:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"265813820990426095228289278398197621570","date":"2025-08-13T01:01:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"316538476833558200192938963189473986340","date":"2025-08-13T00:21:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"142307188323183062713289391685630498651","date":"2025-08-12T23:16:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"110534312469357643350838689536164147311","date":"2025-08-07T06:52:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"143442894558421436418657576843837543906","date":"2025-07-25T21:24:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-16T13:49:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-04T14:27:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-04T14:25:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-06-24T15:47:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9c9d9140-4685-48d7-b114-466c1578c029","owner":[],"postedDate":"July 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-08T16:05:07+00:00","versionOfRecord":{"articleIdentity":"rs-6967468","link":"https://doi.org/10.1186/s12870-025-07865-0","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2025-12-04 15:57:34","publishedOnDateReadable":"December 4th, 2025"},"versionCreatedAt":"2025-07-18 16:27:26","video":"","vorDoi":"10.1186/s12870-025-07865-0","vorDoiUrl":"https://doi.org/10.1186/s12870-025-07865-0","workflowStages":[]},"version":"v1","identity":"rs-6967468","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6967468","identity":"rs-6967468","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}