Metallochaperone protein OsHIPP53 reduces cadmium accumulation in rice (Oryza sativa L.) roots

Metallochaperone protein OsHIPP53 reduces cadmium accumulation in rice (Oryza sativa L.) roots | 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 Metallochaperone protein OsHIPP53 reduces cadmium accumulation in rice (Oryza sativa L.) roots Lin Wang, Changzhao Chen, Jing Huang, Renfang Shen, xiaofang zhu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7580354/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cadmium (Cd) pollution represents a widespread environmental issue in agricultural regions in China, adversely affecting crop productivity and threatening food safety. Heavy metal-associated isoprenylated plant proteins (HIPPs), a major class of metallochaperone proteins, are essential for plant adaptation to diverse biotic and abiotic stress conditions. This study characterizes a previously uninvestigated HIPP gene, OsHIPP53 , demonstrating its involvement in modulating Cd accumulation and tolerance in rice. Subcellular localization analysis revealed that OsHIPP53 is primarily localized at the plasma membrane and Cd exposure significantly induced its transcriptional level in root tissues. Heterologous expression of OsHIPP53 in Δycf1 yeast mutants conferred improved Cd resistance and reduced cellular Cd levels relative to yeast cells carrying the empty vector. Consistent with yeast findings, in rice, oshipp53 mutant lines ( oshipp53-1 and oshipp53-2 ) exhibited heightened Cd sensitivity, elevated root Cd concentrations, and restricted Cd translocation to the shoots. Conversely, overexpression lines ( OsHIPP53-OX-1 and OsHIPP53-OX-2 ) displayed greater Cd tolerance and Cd accumulation in the shoots. Taken together, these results suggested that OsHIPP53 functions in regulating Cd accumulation and tolerance in rice by facilitating the cellular efflux process of Cd. Accumulation cadmium (Cd) HIPPs OsHIPP53 tolerance translocation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Key message In the present study, we found that OsHIPP53 may be involved in the cellular efflux process of Cd and possesses the capability to modulate Cd tolerance in rice. Introduction Cadmium (Cd), the most common heavy metal contaminant in agricultural soils, is a toxic heavy metal extensively distributed within environmental systems, presents considerable carcinogenic risks to human populations (Han et al. 2025 ). Cd is non-degradable, exhibits a high degree of persistence within soil environments, and possesses significant mobility, which together contribute to its high toxicity (Alengebawy et al. 2021 ; Qin et al. 2021 ). Cd is toxic to plants in a variety of ways, including but not limited to cell structure, physiological metabolism, growth, development and reproduction and other life activities will be affected (Liu et al. 2024 ; Mushtaq et al. 2025 ). Cd impedes the uptake of essential macronutrients such as calcium, phosphorus, and potassium, as well as micronutrients including iron, manganese, and zinc, thereby causing poor plant development and even wilting or necrosis (Chen et al. 2017 ; Qin et al. 2020 ). Exposure to Cd environments damages the structure of chloroplasts in plants, leading to the inhibition of photosynthesis (Grajek et al. 2020 ; Kazmi et al. 2025 ). Cd stress also promotes the production of reactive oxygen species (ROS), which damage cell membranes and organelles, further disrupting cellular activities (Romero-Puertas et al. 2002 ; Luo et al. 2024 ). To counteract the detrimental effects of heavy metals like Cd, plants employ multiple physiological and molecular adaptation strategies (El Rasafi et al. 2022 ). A critical detoxification mechanism is reduction of cytoplasmic Cd concentration to maintain normal cellular function, including limiting Cd uptake, enhancing Cd efflux, immobilizing Cd in the cell wall, and compartmentalizing them into vacuoles (Ueno et al. 2011 ; Liu et al. 2014 ; Jutsz and Gnida 2015 ; Fu et al. 2019 ; Meng et al. 2022 ). Secondly, plants synthesize phytochelatins (PCs) to chelate free metal ions, thereby sequestering them and preventing their interaction with intracellular functional proteins, which helps mitigate Cd-induced cytotoxicity (Choppala et al. 2014 l et al. 2017 ). Thirdly, plants enhance oxidative stress responses by producing reactive oxygen species (ROS), including hydrogen peroxide (H₂O₂) and malondialdehyde (MDA), thereby promoting the synthesis of antioxidant compounds (Yu et al. 2013 ; Rizwan et al. 2016 ). In the process of plant responses to heavy metal stress, metallochaperone proteins frequently participate in these defense mechanisms and play critical roles in Cd detoxification and stress adaptation (Tehseen et al. 2010 ; Robinson and Winge 2010 ). HIPPs are a large family of metallochaperone proteins, defined by a conserved heavy metal-associated (HMA) domain that confers metal-binding capacity, making them crucial for metal ion homeostasis (Barr et al. 2023 ). Members of the HIPP family contribute significantly to plant adaptation under heavy metal exposure and diverse environmental stresses, and their physiological functions have been widely explored in model plants including Arabidopsis and rice (de Abreu-Neto et al. 2013 ). For example, AtCdI19 can directly bind Cd and enhance the growth of Arabidopsis under Cd stress (Suzuki et al. 2002 ). Additionally, the athipp20/21/22 triple mutant shows Cd sensitivity, including chlorosis and reduced biomass (Tehseen et al. 2010 ). In response to Cd stress, rice showed increased expression of several HIPP genes, including OsHIPP28 , OsHIPP41 , and OsHIPP21 (de Abreu-Neto et al. 2013 ). Other members such as OsHIPP9 , OsHIPP16 , OsHIPP17 , OsHIPP29 , and OsHIPP56 have been shown to be able to modulate Cd accumulation and contributed to Cd detoxification in rice (Zhang et al. 2020 ; Zhao et al. 2022 ; Cao et al. 2022 ; Xiong et al. 2023 ; Shi et al. 2023 ). In this study, we uncovered OsHIPP53 (LOC_Os03g22490), a previously unstudied member of the HIPP family in rice, and found that it is responsive to Cd stress. Phenotypic analyses revealed that oshipp53 mutant lines exhibited impaired growth under Cd stress, whereas OsHIPP53 overexpression lines exhibited improved growth under Cd stress. Based on these findings, we hypothesize that OsHIPP53 contributes to adaptation to Cd stress in rice. Therefore, this study was designed to elucidate the influence of OsHIPP53 on Cd tolerance and accumulation in rice. Materials and methods Gene Sequence and Phylogenetic Analysis of OsHIPP53 Sequence data, including both the full-length cDNA and protein sequences of OsHIPP53 and its related homologs, were collected from the Rice Genome Annotation Project ( https://rice.uga.edu ). Protein domain prediction for the OsHIPP53 was conducted using the NCBI Conserved Domain Database ( https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi ). All rice genes containing the conserved HMA domain were identified using TBtools software. MEGA 11 software was employed to perform multiple sequence alignment and construct a phylogenetic tree (bootstrapping test set to 1000 replicates). RNA Extraction and Gene Expression Analysis RNA extraction was performed with RNAiso Plus (Takara, Japan), followed by cDNA synthesis with a reverse transcription kit (Toyobo, Japan). The quantitative reverse transcription PCR (qRT-PCR) analysis was performed on a qRT-PCR system (CFX Opus 384, BIO-RAD) using SYBR Green (Toyobo, Japan) and the primers used are shown in Table 1 , and the relative gene expression levels were analyzed using the 2 −ΔΔCT method. To investigate the tissue-specific expression pattern of OsHIPP53 , samples were collected from various tissues at the seedling, tillering, booting, and flowering stages of wild-type Nipponbare (Nip) plants for RNA extraction. To investigate the time-response of OsHIPP53 to Cd stress, 2-week-old Nip seedlings were treated with 1 µM Cd for 0, 1, 3, 6, 12, and 24 hours. For dose-response analysis, 1-week-old seedlings were treated with 0, 1, 5, 10, and 25 µM Cd for 7 days. Table 1 The primers used in this work. Application Primer name Primer sequence 5’→3’ Gene expression analysis OsACTIN-qPCR-F CAACACCCCTGCTATGTACG OsACTIN-qPCR-R CATCACCAGAGTCCAACACAA OsHIPP53-qPCR-F CGAACCCGCCAAAGAAGCC OsHIPP53-qPCR-R TGCTCTTCTCGTCCTTTGCTC GUS analysis OsHIPP53-GUS-F ATCACTAGTAAAAGGTACCCAATATGTGCGTGAAGCCCAA OsHIPP53-GUS-R AGATCTACCATGGGGATCCGGATGGAGCGAGGCGGATTG Subcellular localization OsHIPP53-NGFP-F GAGCTGTACAAGGGTACCATGGCGTCTGCGGATGCG OsHIPP53-NGFP-R GTCGACTCTAGAGGATCCTCACATGACGCTGCAGGCGT Yeast heterologous expression OsHIPP53-pDR196-F CCGGGCTGCAGGAATTCATGGCGTCTGCGGATGCG OsHIPP53-pDR196-R CCCTCGAGGTCGACTCACATGACGCTGCAGGCGT OsNramp5-pDR196-F CCGGGCTGCAGGAATTCATGGAGATTGAGAGAGAGAGCAGT OsNramp5-pDR196-R CCCTCGAGGTCGACCTACCTTGGGAGCGGGATGTC Mutant identification OsHIPP53-mut-F TTTTATGTTTGGTTTCAGGC OsHIPP53-mut-R TCTTCTCGTCCTTTGCTC Over-expression OsHIPP53-OX-F CTAGAAAGCTTCTGCAGATGGCGTCTGCGGATGCG OsHIPP53-OX-R ACTAGTATTTAAATGTCGACCATGACGCTGCAGGCGTTG Yeast two-hybrid OsHIPP53-BD-F TCAGAGGAGGACCTGCATAATGGCGTCTGCGGATGCG OsHIPP53-BD-R ATGCGGCCGCTGCAGGTCGATCACATGACGCTGCAGGCGT OsNramp1-AD-F ATGGAGGCCAGTGAATTCATGGGGGTGACGAAGGCGGAG OsNramp1-AD-R AGCTCGAGCTCGATGGATCCCTACACGGGTGGCTCTTTGCTG OsNramp2-AD-F ATGGAGGCCAGTGAATTCATGGCGTCGCGCGACCTCGCCGAG OsNramp2-AD-R AGCTCGAGCTCGATGGATCCTCATGTGCTCTTTGTCATTGCTGAG OsNramp3-AD-F ATGGAGGCCAGTGAATTCATGAGCGGCCCAATGCAACGCT OsNramp3-AD-R AGCTCGAGCTCGATGGATCCCTAATCGAGATCAGAAGCAGTTCG OsNramp5-AD-F ATGGAGGCCAGTGAATTCATGGAGATTGAGAGAGAGAG OsNramp5-AD-R AGCTCGAGCTCGATGGATCCCTACCTTGGGAGCGGGATGT OsHMA1-AD-F ATGGAGGCCAGTGAATTCATGCAGCTCCTCACCGCCGCCTC OsHMA1-AD-R AGCTCGAGCTCGATGGATCCCTACAACGGAACAGCATCAACTAC OsHMA2-AD-F ATGGAGGCCAGTGAATTCATGGCGGCGGAGGGAGGGAG OsHMA2-AD-R AGCTCGAGCTCGATGGATCCTTATAGGCTGTTCTTACCAGCAATC OsABCG36-AD-F ATGGAGGCCAGTGAATTCATGGACGCGGCGGGGGAGAT OsABCG36-AD-R AGCTCGAGCTCGATGGATCCTCATCTCTTCTGGAAGTTGAACT OsCCX2-AD-F ATGGAGGCCAGTGAATTCATGGCGCTCCTGCGCAGGCG OsCCX2-AD-R AGCTCGAGCTCGATGGATCCCTATGCTGGCCACCAGGAGT OsIRT1-AD-F ATGGAGGCCAGTGAATTCATGGCGACGCCGCGGACACTG OsIRT1-AD-R AGCTCGAGCTCGATGGATCCTCACGCCCACTTGGCCATGACGG OsZIP1-AD-F ATGGAGGCCAGTGAATTCATGGCCAGGACGATGACGATGAG OsZIP1-AD-R AGCTCGAGCTCGATGGATCCTCAGTCCCAGATCATGACGACAGC OsZIP5-AD-F ATGGAGGCCAGTGAATTCATGGCGACGGCGGCGATGACC OsZIP5-AD-R AGCTCGAGCTCGATGGATCCTCACGCCCAGATGGCGATCATG OsZIP7-AD-F ATGGAGGCCAGTGAATTCATGGAGCGGTTCGTGCAGT OsZIP7-AD-R AGCTCGAGCTCGATGGATCCTCAGGCCCAGATTGCAAGG OsZIP9-AD-F ATGGAGGCCAGTGAATTCATGGCTTTCGATCTCAAGCTAACC OsZIP9-AD-R AGCTCGAGCTCGATGGATCCCAAAGCTATCCCAACCAGCAGC OsCAL1-AD-F ATGGAGGCCAGTGAATTCATGGCTCCGTCTCGTCGCATG OsCAL1-AD-R AGCTCGAGCTCGATGGATCCCTAGCAGACCTTCTTGCAGAAGC OsABCC2-AD-F ATGGAGGCCAGTGAATTCATGGGGCCATTCTTGATCACCTAC OsABCC2-AD-R AGCTCGAGCTCGATGGATCCTTATAGATTGTTTACCTCCCATATTTC OsABCC3-AD-F ATGGAGGCCAGTGAATTCATGCCCTTGGCCGCCGCACCGATG OsABCC3-AD-R AGCTCGAGCTCGATGGATCCCTACGTGTGCGTCGATCTCATCGTG OsABCC9-AD-F ATGGAGGCCAGTGAATTCATGCTCGGCCTGGCGCACCTCTC OsABCC9-AD-R AGCTCGAGCTCGATGGATCCTTACAGGTTGGATGAGCGATTGG OsDEF8-AD-F ATGGAGGCCAGTGAATTCATGGAGGCTTCACGCAAGGTGTTC OsDEF8-AD-R AGCTCGAGCTCGATGGATCCTCAGGGGCAGGGCTTGGTGCAC OsCd1-AD-F ATGGAGGCCAGTGAATTCATGGAGGTGTTCTACTACCTCGTG OsCd1-AD-R AGCTCGAGCTCGATGGATCCTTAAGGATTCAGTGGCTCATCTTC Luciferase complementation imaging OsHIPP53-nLUC-F CGAGCTCGGTACCCGGGATCCATGGCGTCTGCGGATGCG OsHIPP53-nLUC-R CGCGTACGAGATCTGGTCGACCATGACGCTGCAGGCGTTG OsNramp1-cLUC-F TACGCGTCCCGGGGCGGTACCATGGGGGTGACGAAGGCGGAG OsNramp1-cLUC-R TGTAGTCCATTTGTTGGATCCCTACACGGGTGGCTCTTTGCTG OsNramp2-cLUC-F TACGCGTCCCGGGGCGGTACCATGGCGTCGCGCGACCTCGCCGAG OsNramp2-cLUC-R TGTAGTCCATTTGTTGGATCCTCATGTGCTCTTTGTCATTGCTGAG OsNramp3-cLUC-F TACGCGTCCCGGGGCGGTACCATGAGCGGCCCAATGCAACGCT OsNramp3-cLUC-R TGTAGTCCATTTGTTGGATCCCTAATCGAGATCAGAAGCAGTTCG OsNramp5-cLUC-F TACGCGTCCCGGGGCGGTACCATGGAGATTGAGAGAGAGAGCAGT OsNramp5-cLUC-R TGTAGTCCATTTGTTGGATCCCTACCTTGGGAGCGGGATGTC OsHMA1-cLUC-F TACGCGTCCCGGGGCGGTACCATGCAGCTCCTCACCGCCGCCTC OsHMA1-cLUC-R TGTAGTCCATTTGTTGGATCCCTACAACGGAACAGCATCAACTAC OsHMA2-cLUC-F TACGCGTCCCGGGGCGGTACCATGGCGGCGGAGGGAGGGAG OsHMA2-cLUC-R TGTAGTCCATTTGTTGGATCCTTATAGGCTGTTCTTACCAGCAATC OsHMA3-cLUC-F TACGCGTCCCGGGGCGGTACCATGGCCGGAAAGGATGAGGCG OsHMA3-cLUC-R TGTAGTCCATTTGTTGGATCCTCATCCTTTCACTTCACCGGAG OsABCG36-cLUC-F TACGCGTCCCGGGGCGGTACCATGGACGCGGCGGGGGAGAT OsABCG36-cLUC-R TGTAGTCCATTTGTTGGATCCTCATCTCTTCTGGAAGTTGAACT OsCCX2-cLUC-F TACGCGTCCCGGGGCGGTACCATGGCGCTCCTGCGCAGGCG OsCCX2-cLUC-R TGTAGTCCATTTGTTGGATCCCTATGCTGGCCACCAGGAGTG OsIRT1-cLUC-F TACGCGTCCCGGGGCGGTACCATGGCGACGCCGCGGACACTG OsIRT1-cLUC-R TGTAGTCCATTTGTTGGATCCTCACGCCCACTTGGCCATGACGG OsZIP1-cLUC-F TACGCGTCCCGGGGCGGTACCATGGCCAGGACGATGACGATGAG OsZIP1-cLUC-R TGTAGTCCATTTGTTGGATCCTCAGTCCCAGATCATGACGACAGC OsZIP5-cLUC-F TACGCGTCCCGGGGCGGTACCATGGCGACGGCGGCGATGACC OsZIP5-cLUC-R TGTAGTCCATTTGTTGGATCCTCACGCCCAGATGGCGATCATG OsZIP7-cLUC-F TACGCGTCCCGGGGCGGTACCATGGAGCGGTTCGTGCAGT OsZIP7-cLUC-R TGTAGTCCATTTGTTGGATCCTCAGGCCCAGATTGCAAGG OsZIP9-cLUC-F TACGCGTCCCGGGGCGGTACCATGGCTTTCGATCTCAAGCTAACC OsZIP9-cLUC-R TGTAGTCCATTTGTTGGATCCCAAAGCTATCCCAACCAGCAGC OsCAL1-cLUC-F TACGCGTCCCGGGGCGGTACCATGGCTCCGTCTCGTCGCATG OsCAL1-cLUC-R TGTAGTCCATTTGTTGGATCCCTAGCAGACCTTCTTGCAGAAGC OsABCC3-cLUC-F TACGCGTCCCGGGGCGGTACCATGCCCTTGGCCGCCGCACCGATG OsABCC3-cLUC-R TGTAGTCCATTTGTTGGATCCCTACGTGTGCGTCGATCTCATCGTG OsABCC9-cLUC-F TACGCGTCCCGGGGCGGTACCATGCTCGGCCTGGCGCACCTCTC OsABCC9-cLUC-R TGTAGTCCATTTGTTGGATCCTTACAGGTTGGATGAGCGATTGG OsDEF8-cLUC-F TACGCGTCCCGGGGCGGTACCATGGAGGCTTCACGCAAGGTGTTC OsDEF8-cLUC-R TGTAGTCCATTTGTTGGATCCTCAGGGGCAGGGCTTGGTGCAC OsCd1-cLUC-F TACGCGTCCCGGGGCGGTACCATGGAGGTGTTCTACTACCTCGTG OsCd1-cLUC-R TGTAGTCCATTTGTTGGATCCTTAAGGATTCAGTGGCTCATCTTC GUS Staining The upstream promoter region of OsHIPP53 (about 2.5 kb) was cloned, and used for the construction of ProOsHIPP53 :GUS transgenic seedlings. The primers used are shown in Table 1 . ProOsHIPP53 :GUS transgenic seedlings were treated with or without 1 µM Cd for 1 day. Seedlings were stained using a GUS histochemical detection kit (Coolaber X-gluc, China) and incubated overnight at 37°C. The root cross-section slices were obtained using a vibratome (VT 1200S, Leica). Stained seedlings and tissues samples were observed and imaged using the flatbed scanner (Perfection V750 PRO, EPSON) and the microscope (AZ100, Nikon). Subcellular Localization To generate the recombinant plasmid for subcellular localization, the coding sequence of OsHIPP53 was cloned from Nip cDNA and ligated into the N-GFP vector using a seamless cloning strategy (TOROIVD One Step Fusion Cloning Mix, Japan). The primers used are shown in Table 1 . The recombinant vector was introduced into rice protoplasts to examine the subcellular localization of OsHIPP53 . The fluorescence signals were observed using the confocal microscope (LSM 710, Zeiss) with the excitation wavelength of 488 nm and the emission range of 495–556 nm. Heterologous Expression and Functional Characterization of OsHIPP53 in Yeast The coding sequence of OsHIPP53 and OsNramp5 were cloned and ligated to the pDR196 vector. The primers used are shown in Table 1 . The empty pDR196 vector, pDR196- OsHIPP53 , and pDR196- OsNramp5 were separately transformed into the Cd-sensitive yeast mutant Δycf1 . The transgenic yeast cells were incubated overnight in SD-U liquid medium at 30°C. Cell suspensions were serially diluted (OD 600 = 1, 10⁻¹, 10⁻², 10⁻³) with sterile water and spotted onto SD-U agar plates supplemented with varying concentrations of Cd. Then the plates were incubated at 30°C for 2 ~ 3 days. To further evaluate the growth dynamics, transgenic yeast strains were cultured in SD-U liquid medium containing 0 or 10 µM Cd at 30°C with 200 rpm shaking. Optical density at 600 nm (OD₆₀₀) was recorded at designated time points to construct growth curves. To further measure Cd accumulation, the yeast solution was incubated to saturation, then washed with sterile water, dried to constant weight and digested. Finally, the Cd content of yeast cells was determined by ICP-MS. Determination of Cd Content The sample to be determined were washed with sterile water and dried in the oven at 80°C for 2 ~ 3 days. After achieving constant dry weight, samples were digested with 2 mL HNO₃ at 120°C in a digestion oven until complete dissolution was achieved. Then the digested samples were diluted to a final volume of 10 mL, passed through filter paper, and analyzed for cd content by ICP-MS. Plant Materials and Growth Conditions The rice materials used in this study included the Nip, oshipp53 mutant lines ( oshipp53-1 and oshipp53-2 ), and OsHIPP53 overexpression lines ( OsHIPP53-OX-1 and OsHIPP53-OX-2 ) with Nip background. The oshipp53 mutant lines were generated by CRISPR-Cas9 technology, and the sgRNA sequence was 5’-CAGCAGCCTGAGGCTGCGCC-3’. To generate OsHIPP53 overexpression lines, the coding sequence of OsHIPP53 was cloned and ligated into the P-super-1300 vector. The knockout and gene overexpression vectors were constructed and introduced into Agrobacterium tumefaciens , then the knockout and overexpression lines were obtained by infecting the callus of Nip background with A. tumefaciens suspension. Genomic DNA was extracted from knockout lines and sequenced to confirm their genotypes. To identify the OsHIPP53 overexpression lines, the total RNA from overexpression transgenic plants was extracted and reversed to cDNA, and analyzing OsHIPP53 transcript levels through qRT-PCR. All primers used are shown in Table 1 . The seeds were germinated in sterile water at 37°C in a temperature-controlled incubator. After germination, seedlings were transferred to a 0.5 mM CaCl₂ solution and grown in darkness, then transferring the seedlings to 1/2 Kimura B nutrient solution (pH 5.6) after rooting for continued cultivation. When seedlings grew to about 1-week-old, uniform seedlings were selected for a 7-day treatment under the following conditions: control (-Cd, 1/2 Kimura B solution) and Cd treatment (+ Cd, 1/2 Kimura B solution containing 1 µM Cd). The nutrient solutions were replaced every two days. Growth conditions were a 14 h light/30°C and 10 h dark/23°C cycle, 400 µmol m⁻² s⁻¹ light intensity, and 60% relative humidity. Determination of Plant Growth Parameters Growth parameters were assessed by measuring root length and plant height with a ruler (cm), while root and shoot biomass were determined using an electronic balance (mg). Determination of Cd Content in Xylem Sap After 7 days of Cd treatment, the shoots were cut at approximately 2 cm above the root. Xylem sap was collected within 1 h after cutting, and the Cd concentration was determined by ICP-MS. Yeast Two Hybrid Assay The yeast two-hybrid assays were performed using Saccharomyces cerevisiae strain AH109 as the host system The coding sequence of OsHIPP53 was ligated into the pGBKT7 vector, while candidate interacting genes were ligated into the pGADT7 vector. The recombinant plasmids were co-transformed into AH109 yeast cells in different combinations. Then the cell suspensions were adjusted to OD 600 = 0.2 and spotted onto SD-Trp/-Leu (SD-T-L) and SD-Trp/-Leu/-Ade/-His (SD-T-L-A-H) solid medium, finally incubated at 30°C for 2 ~ 3 days. Luciferase Complementation Imaging Assay For luciferase complementation assays, the CDS of OsHIPP53 was ligated into the pCambia1300-nLUC vector, and the CDSs of candidate interacting proteins were ligated into the pCambia1300-cLUC vector. The recombinant constructs were first introduced into A. tumefaciens strain GV3101, then different pairwise combinations were co-infiltrated into Nicotiana benthamiana leaves. The LUC signals were observed using a plant live imaging system after 48 h of cultivation. Statistical Analysis Statistical analyses were carried out using student’s t-test. Significant differences ( p < 0.05) were indicated by asterisks. Results Gene Structure and Phylogenetic Analysis of OsHIPP53 Structural characterization of OsHIPP53 identified an 1,875 bp genomic sequence containing a 1,137 bp coding segment with three exons and two introns, producing a 378-amino acid protein (Supplementary Fig. 1A). Protein domain analysis indicated that OsHIPP53 contains a conserved HMA domain, a characteristic feature of the HIPP protein family. The evolutionary relationship of OsHIPP53 with its rice homologs was analyzed using their corresponding amino acid sequences. The results showed that OsHIPP53 is closely related to OsATX1 and OsCCH , suggesting that it may share similar biological functions with these proteins (Supplementary Fig. 1B). Expression Pattern of OsHIPP53 To investigate the expression profile of OsHIPP53 , we utilized quantitative reverse transcription polymerase chain reaction (qRT-PCR) to assess OsHIPP53 transcript abundance in different rice organs throughout plant development (Fig. 1 A). The qRT-PCR analysis indicated that OsHIPP53 exhibited low expression levels across all regions of the seedling. During the tillering phase, the expression of OsHIPP53 was observed to be most pronounced in the root system. As the plant progressed to the booting and heading stages, a notable upsurge in OsHIPP53 expression was detected in the basal regions and the aerial stems. Furthermore, GUS staining was conducted on the ProOsHIPP53 :GUS transgenic lines to investigate the tissue-specific expression of OsHIPP53 , as indicated by the staining outcomes. The GUS staining outcomes revealed that OsHIPP53 exhibited a constitutive expression pattern throughout various rice tissues, encompassing roots, basal regions, leaves, leaf sheaths, and panicles (Fig. 1 B-F). The expression of OsHIPP53 was induced by Cd To investigate whether the expression of OsHIPP53 responds to Cd, time-course and dose-response experiments were conducted using Nip seedlings, and the transcript level of OsHIPP53 was determined by qRT-PCR. In the roots, the expression of OsHIPP53 was significantly increased by Cd from 1 h onwards and reached a peak at 6 h (Fig. 2 A), while in the dose-response experiment, its expression reached a peak at 5 µM Cd treatment (Fig. 2 B). These results demonstrated that the expression of OsHIPP53 was enhanced in roots under Cd stress. However, in the shoots, the expression of OsHIPP53 did not increase under Cd treatment, instead it showed a decreasing trend (Fig. 2 C, D). Additionally, histochemical staining of ProOsHIPP53 :GUS transgenic seedlings exhibited a marked increase in GUS intensity in the roots after Cd exposure, further supporting that Cd treatment enhances the expression of OsHIPP53 in root tissues (Fig. 2 E, F). Subcellular localization of OsHIPP53 The recombinant vector 35S-OsHIPP53-N-GFP was employed to conduct subcellular localization analysis. The N-GFP control protein exhibited localization in both cytoplasmic and nuclear compartments, while the OsHIPP53-N-GFP fusion protein was specifically targeted at the plasma membrane. These results indicate that OsHIPP53 protein was a plasma membrane-localized protein (Fig. 3 ). The expression of OsHIPP53 confers enhanced resistance to Cd in yeast cells by diminishing the accumulation of Cd To evaluate the potential involvement of OsHIPP53 in Cd transport, the Δycf1 mutant yeast strain, deficient in the YCF1 gene and consequently compromised in its ability to sequester Cd into the vacuole, was utilized. The Δycf1 mutant was transformed with either the empty vector pDR196, recombinant vector pDR196- OsHIPP53 , or pDR196- OsNramp5 , respectively. Upon adjusting the optical density of the yeast solution, serial dilutions were subsequently applied onto SD-U solid medium supplemented with 0, 10, 20 µM of Cd (Fig. 4 A). The growth status of Δycf1 yeast cells transformed with various vectors was observed to be largely consistent on SD-U plates devoid of Cd. Conversely, on Cd-enriched SD-U medium, a discernible growth inhibition was noted in yeast cells expressing OsNramp5 compared to those with empty vector controls. This was in stark contrast to the significant growth enhancement observed in yeast cells expressing OsHIPP53 . To further verify the above findings, the growth of each transformed yeast strain was determined in SD-U liquid medium with or without 10 µM Cd. OD₆₀₀ values were measured at regular intervals to generate growth curves (Fig. 4 B, C). In the absence of Cd, all yeast strains exhibited similar growth curves. However, under Cd exposure, yeast cells expressing OsHIPP53 displayed significantly faster growth than those carrying the empty vector. Furthermore, all yeast strains were introduced into SD-U liquid medium supplemented with 10 µM Cd, following which the yeast were harvested subsequent to incubation until saturation, and the cellular Cd content was quantified (Fig. 4 D). The results showed that expression of OsHIPP53 led to a significant reduction in Cd accumulation in yeast cells, suggesting that it contributes to Cd tolerance by decreasing intracellular Cd levels. OsHIPP53 positively regulated Cd tolerance in rice To investigate the role of OsHIPP53 in Cd tolerance of rice plants, we generated both knockout and overexpression lines of OsHIPP53 as experimental materials. The knockout lines oshipp53-1 and oshipp53-2 were developed using CRISPR-Cas9 technology (Fig. 5 A). Additionally, OsHIPP53 was overexpressed in the Nip background to generate the transgenic lines OsHIPP53-OX-1 and OsHIPP53-OX-2 . One-week-old seedlings of Nip, oshipp53 mutant lines, and OsHIPP53 overexpression lines with uniform growth were moved to nutrient solution with or without 1 µM Cd for 7 d (Fig. 5 B). Under normal conditions, the Nip, oshipp53 mutant lines, and OsHIPP53 overexpression lines exhibited no significant differences in phenotype. However, under Cd stress, all genotypes exhibited growth inhibition in terms of shoot height, root length (Fig. 5 C), and biomass (Fig. 5 D), but the inhibition was greater in oshipp53 mutant lines than Nip, whereas the OsHIPP53 overexpression lines exhibited less inhibition. These findings suggested that OsHIPP53 positively contributed to Cd tolerance in rice. OsHIPP53 regulates Cd translocation from roots to shoots in rice To investigate the impact of OsHIPP53 on the Cd accumulation in rice, we determined the Cd content of rice seedlings treated in nutrient solution with 1 µM Cd for 7 d. The results showed that, Cd levels in the roots were elevated in oshipp53 mutant lines but decreased in OsHIPP53 overexpression lines relative to the Nip (Fig. 6 A). In contrast, Cd accumulation in the shoots was reduced in oshipp53 mutant lines and elevated in OsHIPP53 overexpression lines, displaying an inverse trend to that observed in the roots (Fig. 6 B). Further analysis showed that Cd concentration in the xylem sap was lower in oshipp53 mutant lines and higher in OsHIPP53 overexpression lines compared to Nip (Fig. 6 C). Taken together, these findings suggest that OsHIPP53 expression facilitates the translocation of Cd from roots to shoots in rice. OsHIPP53 operates independently of the known Cd responsible transporters To further investigate the mechanism by which OsHIPP53 influenced Cd tolerance and accumulation in rice, yeast two-hybrid assays were conducted to identify potential proteins that interact with OsHIPP53. Since HIPP was involved in the process of Cd efflux, therefore, we chose several metal transporter proteins that have transporter activity for Cd (OsNramp5, OsHMA3, OsCCX2, etc.) to verify whether OsHIPP53 could interact with these transporter proteins to function in Cd tolerance and accumulation. Nevertheless, according to the yeast two-hybrid assay, it appears that OsHIPP53 cannot interact with these Cd transporter proteins (Fig. 7 A). Consequently, we also employed Luciferase complementation imaging (LCI) to ascertain whether OsHIPP53 engages in interactions with these proteins. The co-expression of OsHIPP53-nLUC with the cLUC recombinant vectors of the candidate genes did not yield detectable luminescent signals, suggesting that OsHIPP53 does not form physical interactions with these transporters (Fig. 7 B). Discussion In recent years, active production activities such as mining, smelting, and chemical industries have significantly accelerated Cd pollution in agricultural soils (Moghimi Dehkordi et al. 2024 ). On one hand, increased Cd pollution exerts phytotoxic effects on rice, interfering with nutrient uptake and inhibiting normal growth and development of rice plants, ultimately leading to reduced yield and grain quality (Xia et al. 2024 ). On the other hand, the presence of Cd in paddy soils promotes the over-enrichment of Cd in rice grains and its entry into the human body through the food chain, posing a threat to human health (Clemens et al. 2013 ; Yang et al. 2018 ; Ali and Khan 2019 ). Among the various food sources for human beings, rice is considered to be the main route of Cd intake, about 3% of Cd in soil can be absorbed by rice and later ingested by human bodies (Song et al. 2017 ; Li et al. 2021 ). From this, it has become an urgent challenge to conduct safe agricultural production on Cd-contaminated land, especially to improve Cd tolerance and reduce Cd accumulation in rice. To date, 44 members of the HIPP family have been identified in rice, but limited numbers have been functionally characterized, particularly in relation to Cd homeostasis (khan et al. 2019 ). Knockout of OsHIPP29 and OsHIPP56 increases rice sensitivity to Cd and leads to elevated Cd accumulation (Zhang et al. 2020 ; Zhao et al. 2022 ). The mutants of OsHIPP42 and OsHIPP16 are also sensitive to Cd, while their Cd content is significantly reduced (Khan et al. 2020 ; Cao et al. 2022 ). Similarly, the oshipp17 mutant exhibits increased Cd accumulation under Cd stress but weaker growth inhibition (Shi et al. 2023 ). This may be attributable to the roles of OsHIPP42 , OsHIPP16 , and OsHIPP17 in regulating Cd distribution within rice. In addition, a previous study reported that OsHIPP9 primarily affects Cd uptake by roots during reproductive growth and influences Cd content in various rice tissues by mediating Cd retention in the nodes (Xiong et al. 2023 ). This study reported a novel HIPP family member, OsHIPP53 , which is ubiquitously expressed across various tissues and developmental stages of rice, suggesting a potential role throughout the whole process of rice growth. Under Cd stress, the expression of OsHIPP53 was significantly up-regulated in roots but decreased in shoots, indicating that OsHIPP53 might have different expression modes in the roots and shoots, and might mainly preform a function of regulating Cd tolerance in the roots. Based on these results, it was suggested that OsHIPP53 is a Cd-responsive gene, primarily involved in the detoxification process in roots of rice. As the metallochaperone, HIPP proteins lack transmembrane domains and are not classified as metal transporters (Xia et al. 2025 ). Although HIPP proteins cannot directly export metal ions out of the cell across membranes, they exhibit metal transport activity that enables them to chelate free metal ions and facilitate their intracellular trafficking (Zhao et al. 2022 ). In the Δycf1 yeast strain, the expression of OsHIPP53 significantly improved yeast growth under Cd stress, indicating that OsHIPP53 could alleviate Cd toxicity in yeast. In addition, Cd content in OsHIPP53 -expressing yeast cells was significantly reduced compared to the empty vector, suggesting a role for OsHIPP53 in promoting Cd efflux. Similar detoxification effects have been observed with other rice HIPP proteins, such as OsHIPP16 and OsHIPP42 , which also enhanced Cd tolerance in yeast (Khan et al. 2020 ; Cao et al. 2022 ). However, unlike OsHIPP16 and OsHIPP42 , the expression of OsHIPP53 led to a measurable reduction of Cd accumulation in yeast cells. This difference may be attributed to diverse Cd transport mechanisms among different HIPP family members. Moreover, subcellular localization analysis revealed that OsHIPP53 was localized at the plasma membrane, suggesting that it may transfer free Cd ions from the cytoplasm to the membrane, which is conducive to the efflux of Cd ions. To further investigate the function of OsHIPP53 in Cd detoxification and accumulation in rice, we generated both OsHIPP53 knockout and overexpression lines. Under Cd stress, oshipp53 mutant lines displayed weaker growth compared to the wild-type Nip, characterized by inhibited elongation and decreased biomass of both shoots and roots. In contrast, the growth inhibition was partially alleviated in the OsHIPP53 overexpressing lines. These results were in agreement with the findings from the yeast assay. Measurements of Cd accumulation following Cd treatment showed that, in the roots, oshipp53 mutant lines retained higher Cd levels than the wild type, while OsHIPP53 overexpression lines displayed significantly lower Cd content. However, in shoots, the Cd content of the oshipp53 mutant lines was lower than that of Nip, while that of the OsHIPP53 overexpressing lines was higher. Additionally, the Cd content in the xylem sap of the oshipp53 mutant lines was also low, while that of the OsHIPP53 overexpressing lines was high, indicating that the expression of OsHIPP53 promotes Cd transport to the shoots. Based on these results, it can be inferred that Cd detoxification mediated by OsHIPP53 in rice may be achieved by promoting Cd transfer to the aboveground parts for storage, thereby reducing Cd accumulation in roots. As the metallochaperone, HIPP family proteins tend to interact with metal transporters and participate in metal transport together. In rice, OsATX1 can capture Cu ions and deliver them to Cu transporters such as HMA4 , HMA5 , HMA6 , and HMA9 , and AtATX1 is also capable of interacting with the Cu ion transporter RAN1 to cooperatively regulate intracellular Cu homeostasis (Li et al. 2017 ; Zhang et al. 2018 ). Thus, in this study, we also tried to investigate whether OsHIPP53 could interact with other proteins. Based on the hypothesis that may be involved in Cd efflux process, we selected previously reported Cd transporter proteins to perform interaction assays. However, both yeast two hybrid and luciferase complementation assays showed no interaction between OsHIPP53 and Cd transporter proteins such as OsNramp5 , OsHMA3 , OsZIP5 , OsCCX2 , or OsABCG36 , etc. Meanwhile, it also has been shown that HIPP proteins could interact with transcription factors. For instance, in rice, OsHIPP17 has been reported to be able to interact with the zinc finger protein OsLOL3 , potentially contributing to regulation of Cd tolerance in rice jointly (Shi et al. 2023 ). Therefore, further studies are needed to uncover whether OsHIPP53 can interacts with other proteins to coordinate the response to Cd stress. Conclusion In conclusion, this study investigated the role of OsHIPP53 in regulating Cd tolerance and accumulation in rice. OsHIPP53 is localized to the plasma membrane and verified to have the ability to promote Cd ions transport. In rice, oshipp53 mutant lines were more sensitivity to Cd stress, as evidenced by growth inhibition, whereas OsHIPP53 overexpression lines enhanced Cd tolerance. Cd content analysis in rice revealed that OsHIPP53 promotes Cd translocation to the shoots, so that decreasing Cd accumulation in the roots. These findings suggest that OsHIPP53 is involved in regulating the tolerance, accumulation, and distribution of Cd in rice. Declarations Funding This study was supported by the National Key Research and Development Program of China (Grant Nos.2024YFD1900101; No. 2024YFD1501001), the Field Frontier Program of the Institute of Soil Science (ISSASIP2215), and the National Natural Science Foundation of China (Grant Nos. 42020104004 and 32472059). Competing Interests The authors have no relevant financial or non-financial interests to disclose. 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Environmental and Experimental Botany 193:104680. https://doi.org/10.1016/j.envexpbot.2021.104680 Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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12:30:30","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":82674,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7580354/v1/cac71c3e33c6550f4ac3d687.png"},{"id":93630778,"identity":"7ad0e372-e8b3-4c1c-97dd-6aef0fb63ebf","added_by":"auto","created_at":"2025-10-15 22:04:26","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":27675,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7580354/v1/bc0533a72d8963ee96242c71.png"},{"id":93630773,"identity":"7a16812f-8c4e-4b00-adc5-17f3ba1048b5","added_by":"auto","created_at":"2025-10-15 22:04:26","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":167640,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7580354/v1/48d72f100f1e559a483ef2d4.png"},{"id":93630782,"identity":"d1879d99-97df-429b-b2d2-027681887a17","added_by":"auto","created_at":"2025-10-15 22:04:26","extension":"xml","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":138658,"visible":true,"origin":"","legend":"","description":"","filename":"PCRED25010370structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7580354/v1/4cb666110060c0d0910310e5.xml"},{"id":93630781,"identity":"19e045fb-4585-4d6a-acba-570451f0e2b6","added_by":"auto","created_at":"2025-10-15 22:04:26","extension":"html","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":150896,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7580354/v1/a38267bb22c320c2be14fb03.html"},{"id":93631052,"identity":"62894f3a-b165-4ce8-b591-618aaae9fd5b","added_by":"auto","created_at":"2025-10-15 22:12:25","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":218840,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression pattern of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eOsHIPP53\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in diverse tissues of rice plants at different growth stages.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Relative expression levels of \u003cem\u003eOsHIPP53\u003c/em\u003e in different tissues at different growth stages. The expression level was determined by qRT-PCR. Data are means ± SD (n = 4). \u003cstrong\u003e(B-F)\u003c/strong\u003e GUS staining in the root (B), the basal region (C), the leaf (D), the leaf sheath (E) and the spikelet (F). Scale bars = 0.5 cm.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7580354/v1/807bfb0c99ae759bbbb3acf4.png"},{"id":93630758,"identity":"377c0129-e596-47ac-91b7-c0efaab6277e","added_by":"auto","created_at":"2025-10-15 22:04:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":143873,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression level of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eOsHIPP53\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e under Cd treatment. (A-D)\u003c/strong\u003e Time and dose-responses of\u003cem\u003eOsHIPP53\u003c/em\u003e to Cd in roots and shoots, data are means ± SD (n = 4). Asterisks indicate significant differences (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). \u003cstrong\u003e(E)\u003c/strong\u003e GUS staining of seedlings under normal and Cd treatment. Scale bars = 1 cm. \u003cstrong\u003e(F)\u003c/strong\u003e GUS staining of transverse sections of the root under normal and Cd treatment. Scale bars = 100 μm.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7580354/v1/d3d681e43ee1f125823ead97.png"},{"id":93681933,"identity":"f4fce67e-d2d5-4868-89a5-1a1dcfda38d0","added_by":"auto","created_at":"2025-10-16 12:29:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":131130,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe subcellular localization of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eOsHIPP53\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e by transient expression in rice protoplast. \u003c/strong\u003eScale bars = 20 μm.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7580354/v1/2a92264d88d7be3e52979ebf.png"},{"id":93630762,"identity":"e00584c4-51d6-49cb-859d-b740c89daba0","added_by":"auto","created_at":"2025-10-15 22:04:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":652702,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCd transport assay of\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e OsHIPP53\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e by heterologous expression in yeast cells.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Cd tolerance assay. The yeast liquid cultures of \u003cem\u003eΔycf1\u003c/em\u003e mutant cells transformed with pDR196 empty vectors, pDR196-\u003cem\u003eOsHIPP53\u003c/em\u003e and pDR196-\u003cem\u003eOsNramp5\u003c/em\u003e were grown and adjusted to the OD\u003csub\u003e600\u003c/sub\u003e = 1, then 6 μL aliquots of serial dilutions (1, 10\u003csup\u003e-1\u003c/sup\u003e, 10\u003csup\u003e-2\u003c/sup\u003e, 10\u003csup\u003e-3\u003c/sup\u003e) were plated on SD-U medium supplemented with 0, 10, 20 μM Cd at 30 °C for 2-3 days. \u003cstrong\u003e(B)\u003c/strong\u003e Growth curve of yeast cells. \u003cem\u003eΔycf1\u003c/em\u003e transformed with the empty vector pDR196, pDR196-\u003cem\u003eOsHIPP53\u003c/em\u003e and pDR196-\u003cem\u003eOsNramp5\u003c/em\u003e were cultured in SD-U liquid medium with or without 10 μM Cd. \u003cstrong\u003e(C)\u003c/strong\u003e Determination of Cd concentration in yeast cells. Data are means ± SD (n = 4). Asterisks indicate significant differences (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7580354/v1/9865005793a97e70abeca217.png"},{"id":93630765,"identity":"abd45697-0318-4888-bcdd-1c40fd7c9227","added_by":"auto","created_at":"2025-10-15 22:04:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":228946,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGrowth responses of the Nip, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eoshipp53\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant lines, and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eOsHIPP53\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e overexpression lines. (A)\u003c/strong\u003eSchematic diagram of \u003cem\u003eoshipp53\u003c/em\u003e mutants. \u003cem\u003eoshipp53-1\u003c/em\u003e had a 1bp deletion, and \u003cem\u003eoshipp53-2\u003c/em\u003e had a 1bp alteration and a 1bp deletion. \u003cstrong\u003e(B)\u003c/strong\u003ePhenotypes of the Nip, \u003cem\u003eoshipp53\u003c/em\u003e mutant lines, and \u003cem\u003eOsHIPP53\u003c/em\u003e overexpression lines under normal and 1 μM Cd treatment for 7 days. Scale bars = 5 cm. \u003cstrong\u003e(C-D)\u003c/strong\u003ePhenotypic statistics of the plant height, the root length (C), the shoot biomass, and the root biomass (D) of the Nip, \u003cem\u003eoshipp53\u003c/em\u003e mutant lines, and \u003cem\u003eOsHIPP53\u003c/em\u003e overexpression lines under normal and 1 μM Cd treatment for 7 days. Data are means ± SD (n = 4). Asterisks indicate significant differences (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7580354/v1/5dcc874e42ffe072384dcc99.png"},{"id":93631055,"identity":"b16ae773-a8c5-4075-bc1f-988fa177d4ac","added_by":"auto","created_at":"2025-10-15 22:12:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":113935,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCd content in various parts of rice.\u003c/strong\u003e Cd accumulation statistics in the roots \u003cstrong\u003e(A)\u003c/strong\u003e, the shoots \u003cstrong\u003e(B)\u003c/strong\u003e, and the xylem sap \u003cstrong\u003e(C)\u003c/strong\u003e of the Nip, \u003cem\u003eoshipp53\u003c/em\u003e mutant lines, and \u003cem\u003eOsHIPP53\u003c/em\u003e overexpression lines under 1 μM Cd treatment for 7 days. Data are means ± SD (n = 4). Asterisks indicate significant differences (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7580354/v1/a6b9542be2e1edd9c03061eb.png"},{"id":93631058,"identity":"c9727f1b-cc0b-40b3-aaad-ae0fefde8c06","added_by":"auto","created_at":"2025-10-15 22:12:26","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":588201,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eValidation of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eOsHIPP53\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e interacting proteins.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e The yeast two-hybrid experiment of \u003cem\u003eOsHIPP53\u003c/em\u003eand its potential interacting proteins. SD-T-L:SD-Trp/-Leu medium; SD-T-L-A-H:SD-Trp/-Leu/-Ade/-His medium. \u003cstrong\u003e(B)\u003c/strong\u003e The fluorescence enzyme complementary experiment of \u003cem\u003eOsHIPP53\u003c/em\u003e and its potential interacting proteins.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7580354/v1/33c3dc1f0101aa03776dc7a7.png"},{"id":94988332,"identity":"160b7926-df70-4d75-984b-4fa829b77daa","added_by":"auto","created_at":"2025-11-03 07:08:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3465854,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7580354/v1/a71151a4-e5fc-4532-b774-e1b3773c1699.pdf"},{"id":93630766,"identity":"7639e23c-0bfe-4dc9-9e08-93edfd14144f","added_by":"auto","created_at":"2025-10-15 22:04:26","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":144812,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7580354/v1/3f73e47efef8ac886377d3d8.docx"}],"financialInterests":"","formattedTitle":"Metallochaperone protein OsHIPP53 reduces cadmium accumulation in rice (Oryza sativa L.) roots","fulltext":[{"header":"Key message ","content":"\u003cp\u003eIn the present study, we found that \u003cem\u003eOsHIPP53\u003c/em\u003e may be involved in the cellular efflux process of Cd and possesses the capability to modulate Cd tolerance in rice.\u003c/p\u003e\n"},{"header":"Introduction","content":"\u003cp\u003eCadmium (Cd), the most common heavy metal contaminant in agricultural soils, is a toxic heavy metal extensively distributed within environmental systems, presents considerable carcinogenic risks to human populations (Han et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Cd is non-degradable, exhibits a high degree of persistence within soil environments, and possesses significant mobility, which together contribute to its high toxicity (Alengebawy et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Qin et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Cd is toxic to plants in a variety of ways, including but not limited to cell structure, physiological metabolism, growth, development and reproduction and other life activities will be affected (Liu et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Mushtaq et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Cd impedes the uptake of essential macronutrients such as calcium, phosphorus, and potassium, as well as micronutrients including iron, manganese, and zinc, thereby causing poor plant development and even wilting or necrosis (Chen et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Qin et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Exposure to Cd environments damages the structure of chloroplasts in plants, leading to the inhibition of photosynthesis (Grajek et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kazmi et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Cd stress also promotes the production of reactive oxygen species (ROS), which damage cell membranes and organelles, further disrupting cellular activities (Romero-Puertas et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Luo et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo counteract the detrimental effects of heavy metals like Cd, plants employ multiple physiological and molecular adaptation strategies (El Rasafi et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). A critical detoxification mechanism is reduction of cytoplasmic Cd concentration to maintain normal cellular function, including limiting Cd uptake, enhancing Cd efflux, immobilizing Cd in the cell wall, and compartmentalizing them into vacuoles (Ueno et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Jutsz and Gnida \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Fu et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Meng et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Secondly, plants synthesize phytochelatins (PCs) to chelate free metal ions, thereby sequestering them and preventing their interaction with intracellular functional proteins, which helps mitigate Cd-induced cytotoxicity (Choppala et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003el et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Thirdly, plants enhance oxidative stress responses by producing reactive oxygen species (ROS), including hydrogen peroxide (H₂O₂) and malondialdehyde (MDA), thereby promoting the synthesis of antioxidant compounds (Yu et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Rizwan et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In the process of plant responses to heavy metal stress, metallochaperone proteins frequently participate in these defense mechanisms and play critical roles in Cd detoxification and stress adaptation (Tehseen et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Robinson and Winge \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHIPPs are a large family of metallochaperone proteins, defined by a conserved heavy metal-associated (HMA) domain that confers metal-binding capacity, making them crucial for metal ion homeostasis (Barr et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Members of the HIPP family contribute significantly to plant adaptation under heavy metal exposure and diverse environmental stresses, and their physiological functions have been widely explored in model plants including \u003cem\u003eArabidopsis\u003c/em\u003e and rice (de Abreu-Neto et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). For example, \u003cem\u003eAtCdI19\u003c/em\u003e can directly bind Cd and enhance the growth of \u003cem\u003eArabidopsis\u003c/em\u003e under Cd stress (Suzuki et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Additionally, the \u003cem\u003eathipp20/21/22\u003c/em\u003e triple mutant shows Cd sensitivity, including chlorosis and reduced biomass (Tehseen et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In response to Cd stress, rice showed increased expression of several \u003cem\u003eHIPP\u003c/em\u003e genes, including \u003cem\u003eOsHIPP28\u003c/em\u003e, \u003cem\u003eOsHIPP41\u003c/em\u003e, and \u003cem\u003eOsHIPP21\u003c/em\u003e (de Abreu-Neto et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Other members such as \u003cem\u003eOsHIPP9\u003c/em\u003e, \u003cem\u003eOsHIPP16\u003c/em\u003e, \u003cem\u003eOsHIPP17\u003c/em\u003e, \u003cem\u003eOsHIPP29\u003c/em\u003e, and \u003cem\u003eOsHIPP56\u003c/em\u003e have been shown to be able to modulate Cd accumulation and contributed to Cd detoxification in rice (Zhang et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Cao et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Xiong et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Shi et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn this study, we uncovered \u003cem\u003eOsHIPP53\u003c/em\u003e (LOC_Os03g22490), a previously unstudied member of the HIPP family in rice, and found that it is responsive to Cd stress. Phenotypic analyses revealed that \u003cem\u003eoshipp53\u003c/em\u003e mutant lines exhibited impaired growth under Cd stress, whereas \u003cem\u003eOsHIPP53\u003c/em\u003e overexpression lines exhibited improved growth under Cd stress. Based on these findings, we hypothesize that \u003cem\u003eOsHIPP53\u003c/em\u003e contributes to adaptation to Cd stress in rice. Therefore, this study was designed to elucidate the influence of \u003cem\u003eOsHIPP53\u003c/em\u003e on Cd tolerance and accumulation in rice.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cb\u003eGene Sequence and Phylogenetic Analysis of\u003c/b\u003e \u003cb\u003eOsHIPP53\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSequence data, including both the full-length cDNA and protein sequences of \u003cem\u003eOsHIPP53\u003c/em\u003e and its related homologs, were collected from the Rice Genome Annotation Project (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://rice.uga.edu\u003c/span\u003e\u003cspan address=\"https://rice.uga.edu\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Protein domain prediction for the \u003cem\u003eOsHIPP53\u003c/em\u003e was conducted using the NCBI Conserved Domain Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). All rice genes containing the conserved HMA domain were identified using TBtools software. MEGA 11 software was employed to perform multiple sequence alignment and construct a phylogenetic tree (bootstrapping test set to 1000 replicates).\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eRNA Extraction and Gene Expression Analysis\u003c/h2\u003e\u003cp\u003eRNA extraction was performed with RNAiso Plus (Takara, Japan), followed by cDNA synthesis with a reverse transcription kit (Toyobo, Japan). The quantitative reverse transcription PCR (qRT-PCR) analysis was performed on a qRT-PCR system (CFX Opus 384, BIO-RAD) using SYBR Green (Toyobo, Japan) and the primers used are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, and the relative gene expression levels were analyzed using the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method. To investigate the tissue-specific expression pattern of \u003cem\u003eOsHIPP53\u003c/em\u003e, samples were collected from various tissues at the seedling, tillering, booting, and flowering stages of wild-type Nipponbare (Nip) plants for RNA extraction. To investigate the time-response of \u003cem\u003eOsHIPP53\u003c/em\u003e to Cd stress, 2-week-old Nip seedlings were treated with 1 \u0026micro;M Cd for 0, 1, 3, 6, 12, and 24 hours. For dose-response analysis, 1-week-old seedlings were treated with 0, 1, 5, 10, and 25 \u0026micro;M Cd for 7 days.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eThe primers used in this work.\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eApplication\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePrimer name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePrimer sequence 5\u0026rsquo;\u0026rarr;3\u0026rsquo;\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003eGene expression analysis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsACTIN-qPCR-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCAACACCCCTGCTATGTACG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsACTIN-qPCR-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCATCACCAGAGTCCAACACAA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsHIPP53-qPCR-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCGAACCCGCCAAAGAAGCC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsHIPP53-qPCR-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTGCTCTTCTCGTCCTTTGCTC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eGUS analysis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsHIPP53-GUS-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eATCACTAGTAAAAGGTACCCAATATGTGCGTGAAGCCCAA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsHIPP53-GUS-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAGATCTACCATGGGGATCCGGATGGAGCGAGGCGGATTG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSubcellular localization\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsHIPP53-NGFP-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGAGCTGTACAAGGGTACCATGGCGTCTGCGGATGCG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsHIPP53-NGFP-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGTCGACTCTAGAGGATCCTCACATGACGCTGCAGGCGT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003eYeast heterologous expression\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsHIPP53-pDR196-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCCGGGCTGCAGGAATTCATGGCGTCTGCGGATGCG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsHIPP53-pDR196-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCCCTCGAGGTCGACTCACATGACGCTGCAGGCGT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsNramp5-pDR196-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCCGGGCTGCAGGAATTCATGGAGATTGAGAGAGAGAGCAGT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsNramp5-pDR196-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCCCTCGAGGTCGACCTACCTTGGGAGCGGGATGTC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMutant identification\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsHIPP53-mut-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTTTTATGTTTGGTTTCAGGC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsHIPP53-mut-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTCTTCTCGTCCTTTGCTC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eOver-expression\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsHIPP53-OX-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCTAGAAAGCTTCTGCAGATGGCGTCTGCGGATGCG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsHIPP53-OX-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eACTAGTATTTAAATGTCGACCATGACGCTGCAGGCGTTG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"39\" rowspan=\"40\"\u003e\u003cp\u003eYeast\u003c/p\u003e\u003cp\u003etwo-hybrid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsHIPP53-BD-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTCAGAGGAGGACCTGCATAATGGCGTCTGCGGATGCG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsHIPP53-BD-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eATGCGGCCGCTGCAGGTCGATCACATGACGCTGCAGGCGT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsNramp1-AD-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eATGGAGGCCAGTGAATTCATGGGGGTGACGAAGGCGGAG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsNramp1-AD-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAGCTCGAGCTCGATGGATCCCTACACGGGTGGCTCTTTGCTG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsNramp2-AD-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eATGGAGGCCAGTGAATTCATGGCGTCGCGCGACCTCGCCGAG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsNramp2-AD-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAGCTCGAGCTCGATGGATCCTCATGTGCTCTTTGTCATTGCTGAG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsNramp3-AD-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eATGGAGGCCAGTGAATTCATGAGCGGCCCAATGCAACGCT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsNramp3-AD-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAGCTCGAGCTCGATGGATCCCTAATCGAGATCAGAAGCAGTTCG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsNramp5-AD-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eATGGAGGCCAGTGAATTCATGGAGATTGAGAGAGAGAG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsNramp5-AD-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAGCTCGAGCTCGATGGATCCCTACCTTGGGAGCGGGATGT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsHMA1-AD-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eATGGAGGCCAGTGAATTCATGCAGCTCCTCACCGCCGCCTC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsHMA1-AD-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAGCTCGAGCTCGATGGATCCCTACAACGGAACAGCATCAACTAC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsHMA2-AD-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eATGGAGGCCAGTGAATTCATGGCGGCGGAGGGAGGGAG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsHMA2-AD-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAGCTCGAGCTCGATGGATCCTTATAGGCTGTTCTTACCAGCAATC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsABCG36-AD-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eATGGAGGCCAGTGAATTCATGGACGCGGCGGGGGAGAT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsABCG36-AD-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAGCTCGAGCTCGATGGATCCTCATCTCTTCTGGAAGTTGAACT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsCCX2-AD-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eATGGAGGCCAGTGAATTCATGGCGCTCCTGCGCAGGCG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsCCX2-AD-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAGCTCGAGCTCGATGGATCCCTATGCTGGCCACCAGGAGT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsIRT1-AD-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eATGGAGGCCAGTGAATTCATGGCGACGCCGCGGACACTG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsIRT1-AD-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAGCTCGAGCTCGATGGATCCTCACGCCCACTTGGCCATGACGG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsZIP1-AD-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eATGGAGGCCAGTGAATTCATGGCCAGGACGATGACGATGAG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsZIP1-AD-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAGCTCGAGCTCGATGGATCCTCAGTCCCAGATCATGACGACAGC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsZIP5-AD-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eATGGAGGCCAGTGAATTCATGGCGACGGCGGCGATGACC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsZIP5-AD-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAGCTCGAGCTCGATGGATCCTCACGCCCAGATGGCGATCATG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsZIP7-AD-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eATGGAGGCCAGTGAATTCATGGAGCGGTTCGTGCAGT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsZIP7-AD-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAGCTCGAGCTCGATGGATCCTCAGGCCCAGATTGCAAGG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsZIP9-AD-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eATGGAGGCCAGTGAATTCATGGCTTTCGATCTCAAGCTAACC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsZIP9-AD-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAGCTCGAGCTCGATGGATCCCAAAGCTATCCCAACCAGCAGC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsCAL1-AD-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eATGGAGGCCAGTGAATTCATGGCTCCGTCTCGTCGCATG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsCAL1-AD-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAGCTCGAGCTCGATGGATCCCTAGCAGACCTTCTTGCAGAAGC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsABCC2-AD-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eATGGAGGCCAGTGAATTCATGGGGCCATTCTTGATCACCTAC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsABCC2-AD-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAGCTCGAGCTCGATGGATCCTTATAGATTGTTTACCTCCCATATTTC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsABCC3-AD-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eATGGAGGCCAGTGAATTCATGCCCTTGGCCGCCGCACCGATG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsABCC3-AD-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAGCTCGAGCTCGATGGATCCCTACGTGTGCGTCGATCTCATCGTG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsABCC9-AD-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eATGGAGGCCAGTGAATTCATGCTCGGCCTGGCGCACCTCTC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsABCC9-AD-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAGCTCGAGCTCGATGGATCCTTACAGGTTGGATGAGCGATTGG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsDEF8-AD-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eATGGAGGCCAGTGAATTCATGGAGGCTTCACGCAAGGTGTTC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsDEF8-AD-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAGCTCGAGCTCGATGGATCCTCAGGGGCAGGGCTTGGTGCAC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsCd1-AD-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eATGGAGGCCAGTGAATTCATGGAGGTGTTCTACTACCTCGTG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsCd1-AD-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAGCTCGAGCTCGATGGATCCTTAAGGATTCAGTGGCTCATCTTC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"39\" rowspan=\"40\"\u003e\u003cp\u003eLuciferase complementation imaging\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsHIPP53-nLUC-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCGAGCTCGGTACCCGGGATCCATGGCGTCTGCGGATGCG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsHIPP53-nLUC-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCGCGTACGAGATCTGGTCGACCATGACGCTGCAGGCGTTG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsNramp1-cLUC-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTACGCGTCCCGGGGCGGTACCATGGGGGTGACGAAGGCGGAG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsNramp1-cLUC-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTGTAGTCCATTTGTTGGATCCCTACACGGGTGGCTCTTTGCTG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsNramp2-cLUC-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTACGCGTCCCGGGGCGGTACCATGGCGTCGCGCGACCTCGCCGAG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsNramp2-cLUC-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTGTAGTCCATTTGTTGGATCCTCATGTGCTCTTTGTCATTGCTGAG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsNramp3-cLUC-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTACGCGTCCCGGGGCGGTACCATGAGCGGCCCAATGCAACGCT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsNramp3-cLUC-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTGTAGTCCATTTGTTGGATCCCTAATCGAGATCAGAAGCAGTTCG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsNramp5-cLUC-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTACGCGTCCCGGGGCGGTACCATGGAGATTGAGAGAGAGAGCAGT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsNramp5-cLUC-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTGTAGTCCATTTGTTGGATCCCTACCTTGGGAGCGGGATGTC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsHMA1-cLUC-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTACGCGTCCCGGGGCGGTACCATGCAGCTCCTCACCGCCGCCTC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsHMA1-cLUC-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTGTAGTCCATTTGTTGGATCCCTACAACGGAACAGCATCAACTAC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsHMA2-cLUC-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTACGCGTCCCGGGGCGGTACCATGGCGGCGGAGGGAGGGAG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsHMA2-cLUC-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTGTAGTCCATTTGTTGGATCCTTATAGGCTGTTCTTACCAGCAATC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsHMA3-cLUC-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTACGCGTCCCGGGGCGGTACCATGGCCGGAAAGGATGAGGCG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsHMA3-cLUC-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTGTAGTCCATTTGTTGGATCCTCATCCTTTCACTTCACCGGAG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsABCG36-cLUC-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTACGCGTCCCGGGGCGGTACCATGGACGCGGCGGGGGAGAT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsABCG36-cLUC-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTGTAGTCCATTTGTTGGATCCTCATCTCTTCTGGAAGTTGAACT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsCCX2-cLUC-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTACGCGTCCCGGGGCGGTACCATGGCGCTCCTGCGCAGGCG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsCCX2-cLUC-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTGTAGTCCATTTGTTGGATCCCTATGCTGGCCACCAGGAGTG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsIRT1-cLUC-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTACGCGTCCCGGGGCGGTACCATGGCGACGCCGCGGACACTG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsIRT1-cLUC-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTGTAGTCCATTTGTTGGATCCTCACGCCCACTTGGCCATGACGG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsZIP1-cLUC-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTACGCGTCCCGGGGCGGTACCATGGCCAGGACGATGACGATGAG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsZIP1-cLUC-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTGTAGTCCATTTGTTGGATCCTCAGTCCCAGATCATGACGACAGC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsZIP5-cLUC-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTACGCGTCCCGGGGCGGTACCATGGCGACGGCGGCGATGACC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsZIP5-cLUC-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTGTAGTCCATTTGTTGGATCCTCACGCCCAGATGGCGATCATG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsZIP7-cLUC-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTACGCGTCCCGGGGCGGTACCATGGAGCGGTTCGTGCAGT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsZIP7-cLUC-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTGTAGTCCATTTGTTGGATCCTCAGGCCCAGATTGCAAGG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsZIP9-cLUC-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTACGCGTCCCGGGGCGGTACCATGGCTTTCGATCTCAAGCTAACC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsZIP9-cLUC-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTGTAGTCCATTTGTTGGATCCCAAAGCTATCCCAACCAGCAGC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsCAL1-cLUC-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTACGCGTCCCGGGGCGGTACCATGGCTCCGTCTCGTCGCATG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsCAL1-cLUC-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTGTAGTCCATTTGTTGGATCCCTAGCAGACCTTCTTGCAGAAGC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsABCC3-cLUC-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTACGCGTCCCGGGGCGGTACCATGCCCTTGGCCGCCGCACCGATG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsABCC3-cLUC-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTGTAGTCCATTTGTTGGATCCCTACGTGTGCGTCGATCTCATCGTG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsABCC9-cLUC-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTACGCGTCCCGGGGCGGTACCATGCTCGGCCTGGCGCACCTCTC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsABCC9-cLUC-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTGTAGTCCATTTGTTGGATCCTTACAGGTTGGATGAGCGATTGG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsDEF8-cLUC-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTACGCGTCCCGGGGCGGTACCATGGAGGCTTCACGCAAGGTGTTC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsDEF8-cLUC-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTGTAGTCCATTTGTTGGATCCTCAGGGGCAGGGCTTGGTGCAC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsCd1-cLUC-F\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTACGCGTCCCGGGGCGGTACCATGGAGGTGTTCTACTACCTCGTG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOsCd1-cLUC-R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTGTAGTCCATTTGTTGGATCCTTAAGGATTCAGTGGCTCATCTTC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eGUS Staining\u003c/h3\u003e\n\u003cp\u003eThe upstream promoter region of \u003cem\u003eOsHIPP53\u003c/em\u003e (about 2.5 kb) was cloned, and used for the construction of \u003cem\u003eProOsHIPP53\u003c/em\u003e:GUS transgenic seedlings. The primers used are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. \u003cem\u003eProOsHIPP53\u003c/em\u003e:GUS transgenic seedlings were treated with or without 1 \u0026micro;M Cd for 1 day. Seedlings were stained using a GUS histochemical detection kit (Coolaber X-gluc, China) and incubated overnight at 37\u0026deg;C. The root cross-section slices were obtained using a vibratome (VT 1200S, Leica). Stained seedlings and tissues samples were observed and imaged using the flatbed scanner (Perfection V750 PRO, EPSON) and the microscope (AZ100, Nikon).\u003c/p\u003e\n\u003ch3\u003eSubcellular Localization\u003c/h3\u003e\n\u003cp\u003eTo generate the recombinant plasmid for subcellular localization, the coding sequence of \u003cem\u003eOsHIPP53\u003c/em\u003e was cloned from Nip cDNA and ligated into the N-GFP vector using a seamless cloning strategy (TOROIVD One Step Fusion Cloning Mix, Japan). The primers used are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The recombinant vector was introduced into rice protoplasts to examine the subcellular localization of \u003cem\u003eOsHIPP53\u003c/em\u003e. The fluorescence signals were observed using the confocal microscope (LSM 710, Zeiss) with the excitation wavelength of 488 nm and the emission range of 495\u0026ndash;556 nm.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHeterologous Expression and Functional Characterization of\u003c/b\u003e \u003cb\u003eOsHIPP53\u003c/b\u003e \u003cb\u003ein Yeast\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe coding sequence of \u003cem\u003eOsHIPP53\u003c/em\u003e and \u003cem\u003eOsNramp5\u003c/em\u003e were cloned and ligated to the pDR196 vector. The primers used are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The empty pDR196 vector, pDR196-\u003cem\u003eOsHIPP53\u003c/em\u003e, and pDR196-\u003cem\u003eOsNramp5\u003c/em\u003e were separately transformed into the Cd-sensitive yeast mutant \u003cem\u003eΔycf1\u003c/em\u003e. The transgenic yeast cells were incubated overnight in SD-U liquid medium at 30\u0026deg;C. Cell suspensions were serially diluted (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1, 10⁻\u0026sup1;, 10⁻\u0026sup2;, 10⁻\u0026sup3;) with sterile water and spotted onto SD-U agar plates supplemented with varying concentrations of Cd. Then the plates were incubated at 30\u0026deg;C for 2\u0026thinsp;~\u0026thinsp;3 days. To further evaluate the growth dynamics, transgenic yeast strains were cultured in SD-U liquid medium containing 0 or 10 \u0026micro;M Cd at 30\u0026deg;C with 200 rpm shaking. Optical density at 600 nm (OD₆₀₀) was recorded at designated time points to construct growth curves. To further measure Cd accumulation, the yeast solution was incubated to saturation, then washed with sterile water, dried to constant weight and digested. Finally, the Cd content of yeast cells was determined by ICP-MS.\u003c/p\u003e\n\u003ch3\u003eDetermination of Cd Content\u003c/h3\u003e\n\u003cp\u003eThe sample to be determined were washed with sterile water and dried in the oven at 80\u0026deg;C for 2\u0026thinsp;~\u0026thinsp;3 days. After achieving constant dry weight, samples were digested with 2 mL HNO₃ at 120\u0026deg;C in a digestion oven until complete dissolution was achieved. Then the digested samples were diluted to a final volume of 10 mL, passed through filter paper, and analyzed for cd content by ICP-MS.\u003c/p\u003e\n\u003ch3\u003ePlant Materials and Growth Conditions\u003c/h3\u003e\n\u003cp\u003eThe rice materials used in this study included the Nip, \u003cem\u003eoshipp53\u003c/em\u003e mutant lines (\u003cem\u003eoshipp53-1\u003c/em\u003e and \u003cem\u003eoshipp53-2\u003c/em\u003e), and \u003cem\u003eOsHIPP53\u003c/em\u003e overexpression lines (\u003cem\u003eOsHIPP53-OX-1\u003c/em\u003e and \u003cem\u003eOsHIPP53-OX-2\u003c/em\u003e) with Nip background. The \u003cem\u003eoshipp53\u003c/em\u003e mutant lines were generated by CRISPR-Cas9 technology, and the sgRNA sequence was 5\u0026rsquo;-CAGCAGCCTGAGGCTGCGCC-3\u0026rsquo;. To generate \u003cem\u003eOsHIPP53\u003c/em\u003e overexpression lines, the coding sequence of \u003cem\u003eOsHIPP53\u003c/em\u003e was cloned and ligated into the P-super-1300 vector. The knockout and gene overexpression vectors were constructed and introduced into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e, then the knockout and overexpression lines were obtained by infecting the callus of Nip background with \u003cem\u003eA. tumefaciens\u003c/em\u003e suspension. Genomic DNA was extracted from knockout lines and sequenced to confirm their genotypes. To identify the \u003cem\u003eOsHIPP53\u003c/em\u003e overexpression lines, the total RNA from overexpression transgenic plants was extracted and reversed to cDNA, and analyzing \u003cem\u003eOsHIPP53\u003c/em\u003e transcript levels through qRT-PCR. All primers used are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eThe seeds were germinated in sterile water at 37\u0026deg;C in a temperature-controlled incubator. After germination, seedlings were transferred to a 0.5 mM CaCl₂ solution and grown in darkness, then transferring the seedlings to 1/2 Kimura B nutrient solution (pH 5.6) after rooting for continued cultivation. When seedlings grew to about 1-week-old, uniform seedlings were selected for a 7-day treatment under the following conditions: control (-Cd, 1/2 Kimura B solution) and Cd treatment (+\u0026thinsp;Cd, 1/2 Kimura B solution containing 1 \u0026micro;M Cd). The nutrient solutions were replaced every two days. Growth conditions were a 14 h light/30\u0026deg;C and 10 h dark/23\u0026deg;C cycle, 400 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1; light intensity, and 60% relative humidity.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eDetermination of Plant Growth Parameters\u003c/h2\u003e\u003cp\u003eGrowth parameters were assessed by measuring root length and plant height with a ruler (cm), while root and shoot biomass were determined using an electronic balance (mg).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDetermination of Cd Content in Xylem Sap\u003c/h3\u003e\n\u003cp\u003eAfter 7 days of Cd treatment, the shoots were cut at approximately 2 cm above the root. Xylem sap was collected within 1 h after cutting, and the Cd concentration was determined by ICP-MS.\u003c/p\u003e\n\u003ch3\u003eYeast Two Hybrid Assay\u003c/h3\u003e\n\u003cp\u003eThe yeast two-hybrid assays were performed using \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e strain AH109 as the host system The coding sequence of \u003cem\u003eOsHIPP53\u003c/em\u003e was ligated into the pGBKT7 vector, while candidate interacting genes were ligated into the pGADT7 vector. The recombinant plasmids were co-transformed into AH109 yeast cells in different combinations. Then the cell suspensions were adjusted to OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.2 and spotted onto SD-Trp/-Leu (SD-T-L) and SD-Trp/-Leu/-Ade/-His (SD-T-L-A-H) solid medium, finally incubated at 30\u0026deg;C for 2\u0026thinsp;~\u0026thinsp;3 days.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eLuciferase Complementation Imaging Assay\u003c/h2\u003e\u003cp\u003eFor luciferase complementation assays, the CDS of \u003cem\u003eOsHIPP53\u003c/em\u003e was ligated into the pCambia1300-nLUC vector, and the CDSs of candidate interacting proteins were ligated into the pCambia1300-cLUC vector. The recombinant constructs were first introduced into \u003cem\u003eA. tumefaciens\u003c/em\u003e strain GV3101, then different pairwise combinations were co-infiltrated into \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves. The LUC signals were observed using a plant live imaging system after 48 h of cultivation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were carried out using student\u0026rsquo;s t-test. Significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were indicated by asterisks.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eGene Structure and Phylogenetic Analysis of\u003c/b\u003e \u003cb\u003eOsHIPP53\u003c/b\u003e\u003c/p\u003e\u003cp\u003eStructural characterization of \u003cem\u003eOsHIPP53\u003c/em\u003e identified an 1,875 bp genomic sequence containing a 1,137 bp coding segment with three exons and two introns, producing a 378-amino acid protein (Supplementary Fig.\u0026nbsp;1A). Protein domain analysis indicated that \u003cem\u003eOsHIPP53\u003c/em\u003e contains a conserved HMA domain, a characteristic feature of the HIPP protein family. The evolutionary relationship of \u003cem\u003eOsHIPP53\u003c/em\u003e with its rice homologs was analyzed using their corresponding amino acid sequences. The results showed that \u003cem\u003eOsHIPP53\u003c/em\u003e is closely related to \u003cem\u003eOsATX1\u003c/em\u003e and \u003cem\u003eOsCCH\u003c/em\u003e, suggesting that it may share similar biological functions with these proteins (Supplementary Fig.\u0026nbsp;1B).\u003c/p\u003e\u003cp\u003e\u003cb\u003eExpression Pattern of\u003c/b\u003e \u003cb\u003eOsHIPP53\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the expression profile of \u003cem\u003eOsHIPP53\u003c/em\u003e, we utilized quantitative reverse transcription polymerase chain reaction (qRT-PCR) to assess \u003cem\u003eOsHIPP53\u003c/em\u003e transcript abundance in different rice organs throughout plant development (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The qRT-PCR analysis indicated that \u003cem\u003eOsHIPP53\u003c/em\u003e exhibited low expression levels across all regions of the seedling. During the tillering phase, the expression of \u003cem\u003eOsHIPP53\u003c/em\u003e was observed to be most pronounced in the root system. As the plant progressed to the booting and heading stages, a notable upsurge in \u003cem\u003eOsHIPP53\u003c/em\u003e expression was detected in the basal regions and the aerial stems.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFurthermore, GUS staining was conducted on the \u003cem\u003eProOsHIPP53\u003c/em\u003e:GUS transgenic lines to investigate the tissue-specific expression of \u003cem\u003eOsHIPP53\u003c/em\u003e, as indicated by the staining outcomes. The GUS staining outcomes revealed that \u003cem\u003eOsHIPP53\u003c/em\u003e exhibited a constitutive expression pattern throughout various rice tissues, encompassing roots, basal regions, leaves, leaf sheaths, and panicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-F).\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe expression of\u003c/b\u003e \u003cb\u003eOsHIPP53\u003c/b\u003e \u003cb\u003ewas induced by Cd\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate whether the expression of \u003cem\u003eOsHIPP53\u003c/em\u003e responds to Cd, time-course and dose-response experiments were conducted using Nip seedlings, and the transcript level of \u003cem\u003eOsHIPP53\u003c/em\u003e was determined by qRT-PCR. In the roots, the expression of \u003cem\u003eOsHIPP53\u003c/em\u003e was significantly increased by Cd from 1 h onwards and reached a peak at 6 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), while in the dose-response experiment, its expression reached a peak at 5 \u0026micro;M Cd treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). These results demonstrated that the expression of \u003cem\u003eOsHIPP53\u003c/em\u003e was enhanced in roots under Cd stress. However, in the shoots, the expression of \u003cem\u003eOsHIPP53\u003c/em\u003e did not increase under Cd treatment, instead it showed a decreasing trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAdditionally, histochemical staining of \u003cem\u003eProOsHIPP53\u003c/em\u003e:GUS transgenic seedlings exhibited a marked increase in GUS intensity in the roots after Cd exposure, further supporting that Cd treatment enhances the expression of \u003cem\u003eOsHIPP53\u003c/em\u003e in root tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F).\u003c/p\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eSubcellular localization of OsHIPP53\u003c/h2\u003e\u003cp\u003eThe recombinant vector 35S-OsHIPP53-N-GFP was employed to conduct subcellular localization analysis. The N-GFP control protein exhibited localization in both cytoplasmic and nuclear compartments, while the OsHIPP53-N-GFP fusion protein was specifically targeted at the plasma membrane. These results indicate that \u003cem\u003eOsHIPP53\u003c/em\u003e protein was a plasma membrane-localized protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe expression of\u003c/b\u003e \u003cb\u003eOsHIPP53\u003c/b\u003e \u003cb\u003econfers enhanced resistance to Cd in yeast cells by diminishing the accumulation of Cd\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo evaluate the potential involvement of \u003cem\u003eOsHIPP53\u003c/em\u003e in Cd transport, the \u003cem\u003eΔycf1\u003c/em\u003e mutant yeast strain, deficient in the YCF1 gene and consequently compromised in its ability to sequester Cd into the vacuole, was utilized. The \u003cem\u003eΔycf1\u003c/em\u003e mutant was transformed with either the empty vector pDR196, recombinant vector pDR196-\u003cem\u003eOsHIPP53\u003c/em\u003e, or pDR196-\u003cem\u003eOsNramp5\u003c/em\u003e, respectively.\u003c/p\u003e\u003cp\u003eUpon adjusting the optical density of the yeast solution, serial dilutions were subsequently applied onto SD-U solid medium supplemented with 0, 10, 20 \u0026micro;M of Cd (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The growth status of \u003cem\u003eΔycf1\u003c/em\u003e yeast cells transformed with various vectors was observed to be largely consistent on SD-U plates devoid of Cd. Conversely, on Cd-enriched SD-U medium, a discernible growth inhibition was noted in yeast cells expressing OsNramp5 compared to those with empty vector controls. This was in stark contrast to the significant growth enhancement observed in yeast cells expressing \u003cem\u003eOsHIPP53\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further verify the above findings, the growth of each transformed yeast strain was determined in SD-U liquid medium with or without 10 \u0026micro;M Cd. OD₆₀₀ values were measured at regular intervals to generate growth curves (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C). In the absence of Cd, all yeast strains exhibited similar growth curves. However, under Cd exposure, yeast cells expressing \u003cem\u003eOsHIPP53\u003c/em\u003e displayed significantly faster growth than those carrying the empty vector.\u003c/p\u003e\u003cp\u003eFurthermore, all yeast strains were introduced into SD-U liquid medium supplemented with 10 \u0026micro;M Cd, following which the yeast were harvested subsequent to incubation until saturation, and the cellular Cd content was quantified (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The results showed that expression of \u003cem\u003eOsHIPP53\u003c/em\u003e led to a significant reduction in Cd accumulation in yeast cells, suggesting that it contributes to Cd tolerance by decreasing intracellular Cd levels.\u003c/p\u003e\u003cp\u003e\u003cb\u003eOsHIPP53\u003c/b\u003e \u003cb\u003epositively regulated Cd tolerance in rice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the role of \u003cem\u003eOsHIPP53\u003c/em\u003e in Cd tolerance of rice plants, we generated both knockout and overexpression lines of \u003cem\u003eOsHIPP53\u003c/em\u003e as experimental materials. The knockout lines \u003cem\u003eoshipp53-1\u003c/em\u003e and \u003cem\u003eoshipp53-2\u003c/em\u003e were developed using CRISPR-Cas9 technology (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Additionally, \u003cem\u003eOsHIPP53\u003c/em\u003e was overexpressed in the Nip background to generate the transgenic lines \u003cem\u003eOsHIPP53-OX-1\u003c/em\u003e and \u003cem\u003eOsHIPP53-OX-2\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOne-week-old seedlings of Nip, \u003cem\u003eoshipp53\u003c/em\u003e mutant lines, and \u003cem\u003eOsHIPP53\u003c/em\u003e overexpression lines with uniform growth were moved to nutrient solution with or without 1 \u0026micro;M Cd for 7 d (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Under normal conditions, the Nip, \u003cem\u003eoshipp53\u003c/em\u003e mutant lines, and \u003cem\u003eOsHIPP53\u003c/em\u003e overexpression lines exhibited no significant differences in phenotype. However, under Cd stress, all genotypes exhibited growth inhibition in terms of shoot height, root length (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), and biomass (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), but the inhibition was greater in \u003cem\u003eoshipp53\u003c/em\u003e mutant lines than Nip, whereas the \u003cem\u003eOsHIPP53\u003c/em\u003e overexpression lines exhibited less inhibition. These findings suggested that \u003cem\u003eOsHIPP53\u003c/em\u003e positively contributed to Cd tolerance in rice.\u003c/p\u003e\u003cp\u003e\u003cb\u003eOsHIPP53\u003c/b\u003e \u003cb\u003eregulates Cd translocation from roots to shoots in rice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the impact of \u003cem\u003eOsHIPP53\u003c/em\u003e on the Cd accumulation in rice, we determined the Cd content of rice seedlings treated in nutrient solution with 1 \u0026micro;M Cd for 7 d. The results showed that, Cd levels in the roots were elevated in \u003cem\u003eoshipp53\u003c/em\u003e mutant lines but decreased in \u003cem\u003eOsHIPP53\u003c/em\u003e overexpression lines relative to the Nip (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). In contrast, Cd accumulation in the shoots was reduced in \u003cem\u003eoshipp53\u003c/em\u003e mutant lines and elevated in \u003cem\u003eOsHIPP53\u003c/em\u003e overexpression lines, displaying an inverse trend to that observed in the roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Further analysis showed that Cd concentration in the xylem sap was lower in \u003cem\u003eoshipp53\u003c/em\u003e mutant lines and higher in \u003cem\u003eOsHIPP53\u003c/em\u003e overexpression lines compared to Nip (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Taken together, these findings suggest that \u003cem\u003eOsHIPP53\u003c/em\u003e expression facilitates the translocation of Cd from roots to shoots in rice.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eOsHIPP53 operates independently of the known Cd responsible transporters\u003c/h2\u003e\u003cp\u003eTo further investigate the mechanism by which \u003cem\u003eOsHIPP53\u003c/em\u003e influenced Cd tolerance and accumulation in rice, yeast two-hybrid assays were conducted to identify potential proteins that interact with OsHIPP53. Since HIPP was involved in the process of Cd efflux, therefore, we chose several metal transporter proteins that have transporter activity for Cd (OsNramp5, OsHMA3, OsCCX2, etc.) to verify whether OsHIPP53 could interact with these transporter proteins to function in Cd tolerance and accumulation. Nevertheless, according to the yeast two-hybrid assay, it appears that OsHIPP53 cannot interact with these Cd transporter proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Consequently, we also employed Luciferase complementation imaging (LCI) to ascertain whether OsHIPP53 engages in interactions with these proteins. The co-expression of OsHIPP53-nLUC with the cLUC recombinant vectors of the candidate genes did not yield detectable luminescent signals, suggesting that OsHIPP53 does not form physical interactions with these transporters (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn recent years, active production activities such as mining, smelting, and chemical industries have significantly accelerated Cd pollution in agricultural soils (Moghimi Dehkordi et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). On one hand, increased Cd pollution exerts phytotoxic effects on rice, interfering with nutrient uptake and inhibiting normal growth and development of rice plants, ultimately leading to reduced yield and grain quality (Xia et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). On the other hand, the presence of Cd in paddy soils promotes the over-enrichment of Cd in rice grains and its entry into the human body through the food chain, posing a threat to human health (Clemens et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ali and Khan \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Among the various food sources for human beings, rice is considered to be the main route of Cd intake, about 3% of Cd in soil can be absorbed by rice and later ingested by human bodies (Song et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). From this, it has become an urgent challenge to conduct safe agricultural production on Cd-contaminated land, especially to improve Cd tolerance and reduce Cd accumulation in rice.\u003c/p\u003e\u003cp\u003eTo date, 44 members of the HIPP family have been identified in rice, but limited numbers have been functionally characterized, particularly in relation to Cd homeostasis (khan et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Knockout of \u003cem\u003eOsHIPP29\u003c/em\u003e and \u003cem\u003eOsHIPP56\u003c/em\u003e increases rice sensitivity to Cd and leads to elevated Cd accumulation (Zhang et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The mutants of \u003cem\u003eOsHIPP42\u003c/em\u003e and \u003cem\u003eOsHIPP16\u003c/em\u003e are also sensitive to Cd, while their Cd content is significantly reduced (Khan et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Cao et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Similarly, the \u003cem\u003eoshipp17\u003c/em\u003e mutant exhibits increased Cd accumulation under Cd stress but weaker growth inhibition (Shi et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This may be attributable to the roles of \u003cem\u003eOsHIPP42\u003c/em\u003e, \u003cem\u003eOsHIPP16\u003c/em\u003e, and \u003cem\u003eOsHIPP17\u003c/em\u003e in regulating Cd distribution within rice. In addition, a previous study reported that \u003cem\u003eOsHIPP9\u003c/em\u003e primarily affects Cd uptake by roots during reproductive growth and influences Cd content in various rice tissues by mediating Cd retention in the nodes (Xiong et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This study reported a novel HIPP family member, \u003cem\u003eOsHIPP53\u003c/em\u003e, which is ubiquitously expressed across various tissues and developmental stages of rice, suggesting a potential role throughout the whole process of rice growth. Under Cd stress, the expression of \u003cem\u003eOsHIPP53\u003c/em\u003e was significantly up-regulated in roots but decreased in shoots, indicating that \u003cem\u003eOsHIPP53\u003c/em\u003e might have different expression modes in the roots and shoots, and might mainly preform a function of regulating Cd tolerance in the roots. Based on these results, it was suggested that \u003cem\u003eOsHIPP53\u003c/em\u003e is a Cd-responsive gene, primarily involved in the detoxification process in roots of rice.\u003c/p\u003e\u003cp\u003eAs the metallochaperone, HIPP proteins lack transmembrane domains and are not classified as metal transporters (Xia et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Although HIPP proteins cannot directly export metal ions out of the cell across membranes, they exhibit metal transport activity that enables them to chelate free metal ions and facilitate their intracellular trafficking (Zhao et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In the \u003cem\u003eΔycf1\u003c/em\u003e yeast strain, the expression of \u003cem\u003eOsHIPP53\u003c/em\u003e significantly improved yeast growth under Cd stress, indicating that \u003cem\u003eOsHIPP53\u003c/em\u003e could alleviate Cd toxicity in yeast. In addition, Cd content in \u003cem\u003eOsHIPP53\u003c/em\u003e-expressing yeast cells was significantly reduced compared to the empty vector, suggesting a role for \u003cem\u003eOsHIPP53\u003c/em\u003e in promoting Cd efflux. Similar detoxification effects have been observed with other rice HIPP proteins, such as \u003cem\u003eOsHIPP16\u003c/em\u003e and \u003cem\u003eOsHIPP42\u003c/em\u003e, which also enhanced Cd tolerance in yeast (Khan et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Cao et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, unlike \u003cem\u003eOsHIPP16\u003c/em\u003e and \u003cem\u003eOsHIPP42\u003c/em\u003e, the expression of \u003cem\u003eOsHIPP53\u003c/em\u003e led to a measurable reduction of Cd accumulation in yeast cells. This difference may be attributed to diverse Cd transport mechanisms among different HIPP family members. Moreover, subcellular localization analysis revealed that \u003cem\u003eOsHIPP53\u003c/em\u003e was localized at the plasma membrane, suggesting that it may transfer free Cd ions from the cytoplasm to the membrane, which is conducive to the efflux of Cd ions.\u003c/p\u003e\u003cp\u003eTo further investigate the function of \u003cem\u003eOsHIPP53\u003c/em\u003e in Cd detoxification and accumulation in rice, we generated both \u003cem\u003eOsHIPP53\u003c/em\u003e knockout and overexpression lines. Under Cd stress, \u003cem\u003eoshipp53\u003c/em\u003e mutant lines displayed weaker growth compared to the wild-type Nip, characterized by inhibited elongation and decreased biomass of both shoots and roots. In contrast, the growth inhibition was partially alleviated in the \u003cem\u003eOsHIPP53\u003c/em\u003e overexpressing lines. These results were in agreement with the findings from the yeast assay. Measurements of Cd accumulation following Cd treatment showed that, in the roots, \u003cem\u003eoshipp53\u003c/em\u003e mutant lines retained higher Cd levels than the wild type, while \u003cem\u003eOsHIPP53\u003c/em\u003e overexpression lines displayed significantly lower Cd content. However, in shoots, the Cd content of the \u003cem\u003eoshipp53\u003c/em\u003e mutant lines was lower than that of Nip, while that of the \u003cem\u003eOsHIPP53\u003c/em\u003e overexpressing lines was higher. Additionally, the Cd content in the xylem sap of the \u003cem\u003eoshipp53\u003c/em\u003e mutant lines was also low, while that of the \u003cem\u003eOsHIPP53\u003c/em\u003e overexpressing lines was high, indicating that the expression of \u003cem\u003eOsHIPP53\u003c/em\u003e promotes Cd transport to the shoots. Based on these results, it can be inferred that Cd detoxification mediated by \u003cem\u003eOsHIPP53\u003c/em\u003e in rice may be achieved by promoting Cd transfer to the aboveground parts for storage, thereby reducing Cd accumulation in roots.\u003c/p\u003e\u003cp\u003eAs the metallochaperone, HIPP family proteins tend to interact with metal transporters and participate in metal transport together. In rice, \u003cem\u003eOsATX1\u003c/em\u003e can capture Cu ions and deliver them to Cu transporters such as \u003cem\u003eHMA4\u003c/em\u003e, \u003cem\u003eHMA5\u003c/em\u003e, \u003cem\u003eHMA6\u003c/em\u003e, and \u003cem\u003eHMA9\u003c/em\u003e, and \u003cem\u003eAtATX1\u003c/em\u003e is also capable of interacting with the Cu ion transporter \u003cem\u003eRAN1\u003c/em\u003e to cooperatively regulate intracellular Cu homeostasis (Li et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Thus, in this study, we also tried to investigate whether \u003cem\u003eOsHIPP53\u003c/em\u003e could interact with other proteins. Based on the hypothesis that may be involved in Cd efflux process, we selected previously reported Cd transporter proteins to perform interaction assays. However, both yeast two hybrid and luciferase complementation assays showed no interaction between \u003cem\u003eOsHIPP53\u003c/em\u003e and Cd transporter proteins such as \u003cem\u003eOsNramp5\u003c/em\u003e, \u003cem\u003eOsHMA3\u003c/em\u003e, \u003cem\u003eOsZIP5\u003c/em\u003e, \u003cem\u003eOsCCX2\u003c/em\u003e, or \u003cem\u003eOsABCG36\u003c/em\u003e, etc. Meanwhile, it also has been shown that HIPP proteins could interact with transcription factors. For instance, in rice, \u003cem\u003eOsHIPP17\u003c/em\u003e has been reported to be able to interact with the zinc finger protein \u003cem\u003eOsLOL3\u003c/em\u003e, potentially contributing to regulation of Cd tolerance in rice jointly (Shi et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, further studies are needed to uncover whether \u003cem\u003eOsHIPP53\u003c/em\u003e can interacts with other proteins to coordinate the response to Cd stress.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, this study investigated the role of \u003cem\u003eOsHIPP53\u003c/em\u003e in regulating Cd tolerance and accumulation in rice. \u003cem\u003eOsHIPP53\u003c/em\u003e is localized to the plasma membrane and verified to have the ability to promote Cd ions transport. In rice, \u003cem\u003eoshipp53\u003c/em\u003e mutant lines were more sensitivity to Cd stress, as evidenced by growth inhibition, whereas \u003cem\u003eOsHIPP53\u003c/em\u003e overexpression lines enhanced Cd tolerance. Cd content analysis in rice revealed that \u003cem\u003eOsHIPP53\u003c/em\u003e promotes Cd translocation to the shoots, so that decreasing Cd accumulation in the roots. These findings suggest that \u003cem\u003eOsHIPP53\u003c/em\u003e is involved in regulating the tolerance, accumulation, and distribution of Cd in rice.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Key Research and Development Program of China (Grant Nos.2024YFD1900101; No. 2024YFD1501001), the Field Frontier Program of the Institute of Soil Science (ISSASIP2215), and the National Natural Science Foundation of China (Grant Nos. 42020104004 and 32472059).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLW\u003c/strong\u003e: conceptualization, methodology, investigation, validation, formal analysis, visualization, data curation, writing \u0026ndash; original draft, writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCZC\u003c/strong\u003e: methodology, investigation, validation, visualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJH\u003c/strong\u003e: methodology, investigation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRFS\u003c/strong\u003e: supervision, resources, project administration, funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eXFZ\u003c/strong\u003e: conceptualization, writing \u0026ndash; review \u0026amp; editing, supervision, resources, project administration, funding acquisition.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated or analyzed during the current study are available in the article and supplementary information files.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Prof. Guijie Lei for kindly providing the pDR196 vector.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlengebawy A, Abdelkhalek ST, Qureshi SR, Wang M-Q (2021) Heavy Metals and Pesticides Toxicity in Agricultural Soil and Plants: Ecological Risks and Human Health Implications. 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Environmental and Experimental Botany 193:104680. https://doi.org/10.1016/j.envexpbot.2021.104680\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Accumulation, cadmium (Cd), HIPPs, OsHIPP53, tolerance, translocation","lastPublishedDoi":"10.21203/rs.3.rs-7580354/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7580354/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCadmium (Cd) pollution represents a widespread environmental issue in agricultural regions in China, adversely affecting crop productivity and threatening food safety. Heavy metal-associated isoprenylated plant proteins (HIPPs), a major class of metallochaperone proteins, are essential for plant adaptation to diverse biotic and abiotic stress conditions. This study characterizes a previously uninvestigated \u003cem\u003eHIPP\u003c/em\u003e gene, \u003cem\u003eOsHIPP53\u003c/em\u003e, demonstrating its involvement in modulating Cd accumulation and tolerance in rice. Subcellular localization analysis revealed that \u003cem\u003eOsHIPP53\u003c/em\u003e is primarily localized at the plasma membrane and Cd exposure significantly induced its transcriptional level in root tissues. Heterologous expression of \u003cem\u003eOsHIPP53\u003c/em\u003e in \u003cem\u003eΔycf1\u003c/em\u003e yeast mutants conferred improved Cd resistance and reduced cellular Cd levels relative to yeast cells carrying the empty vector. Consistent with yeast findings, in rice, \u003cem\u003eoshipp53\u003c/em\u003e mutant lines (\u003cem\u003eoshipp53-1\u003c/em\u003e and \u003cem\u003eoshipp53-2\u003c/em\u003e) exhibited heightened Cd sensitivity, elevated root Cd concentrations, and restricted Cd translocation to the shoots. Conversely, overexpression lines (\u003cem\u003eOsHIPP53-OX-1\u003c/em\u003e and \u003cem\u003eOsHIPP53-OX-2\u003c/em\u003e) displayed greater Cd tolerance and Cd accumulation in the shoots. Taken together, these results suggested that \u003cem\u003eOsHIPP53\u003c/em\u003e functions in regulating Cd accumulation and tolerance in rice by facilitating the cellular efflux process of Cd.\u003c/p\u003e","manuscriptTitle":"Metallochaperone protein OsHIPP53 reduces cadmium accumulation in rice (Oryza sativa L.) roots","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-15 22:04:21","doi":"10.21203/rs.3.rs-7580354/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f3d1bcbe-01b5-4d73-bd0b-cf025299a13b","owner":[],"postedDate":"October 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-01T10:56:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-15 22:04:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7580354","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7580354","identity":"rs-7580354","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}