Sweet cherry genome‑wide Identification of the NHX gene family and functional dissection of PavNHX37 a key salt tolerance gene

Sweet cherry genome‑wide Identification of the NHX gene family and functional dissection of PavNHX37 a key salt tolerance gene | 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 Sweet cherry genome‑wide Identification of the NHX gene family and functional dissection of PavNHX37 a key salt tolerance gene Zhigang Guo, Yifan Zhou, Songling Han, Yali Zou, Xiaojuan An This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9596831/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 10 You are reading this latest preprint version Abstract Background Soil salinization severely limits sweet cherry ( Prunus avium L.) growth and productivity, yet the NHX (Na⁺/H⁺ antiporter) gene family, which is central to salt tolerance, remains uncharacterized in this species. Results Here, we identified 37 PavNHX genes unevenly distributed across eight chromosomes. All PavNHX proteins were predicted to localize to the plasma membrane except PavNHX21 (vacuolar), and their secondary structures were dominated by α-helices and random coils. Phylogenetic analysis divided the family into three groups, with Group I containing both sweet cherry and Arabidopsis homologs, indicating evolutionary conservation. Expression profiling revealed diverse tissue-specific patterns, and qRT-PCR under salt stress identified PavNHX37 as the most significantly upregulated gene. Subcellular localization confirmed PavNHX37 at the plasma membrane. Stable overexpression of PavNHX37 in Nicotiana benthamiana dramatically improved salt tolerance: transgenic lines exhibited superior growth, higher proline accumulation, elevated SOD、POD、CAT activities, and reduced MDA content compared to wild-type plants under 150 mM NaCl. Conclusion This study provides the first genome-wide characterization of the NHX gene family in sweet cherry and demonstrates that PavNHX37 functions as a key positive regulator of salt tolerance. These findings establish a theoretical foundation and genetic resources for improving salt tolerance in sweet cherry. Sweet cherry NHX gene family Salt stress Bioinformatics analysis Enzyme activity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 1. Introduction Sweet cherry ( Prunus avium L.) is a commercially important fruit tree of the Rosaceae family. It is widely cultivated due to the rich nutrition and economic value of its fruits. However, sweet cherry exhibits moderate sensitivity to salinity, and soil salinization has become a major factor limiting the expansion of cultivation and the stability of yield, particularly in arid and semiarid regions [ 1 ]. Soil salinization is a key abiotic stress factor restricting global agricultural productivity, affecting an estimated 1 billion hectares of cultivated land worldwide [ 2 ]. High concentrations of salt in the soil disrupt cellular ion homeostasis, induce osmotic stress, and trigger excessive accumulation of reactive oxygen species (ROS), ultimately inhibiting plant growth, reducing yield, and even leading to plant death [ 3 – 4 ]. The Na⁺/H⁺ exchanger gene family plays a critical role in maintaining cellular ion homeostasis under salt stress by mediating the transmembrane transport of Na⁺ and H⁺ [ 5 ]. NHX proteins are divided into two subgroups: those localized to the plasma mem-brane (PM) and those localized to the endosomal or vacuolar membrane. These two sub-groups reduce Na⁺ toxicity in the cytoplasm and maintain K⁺/Na⁺ balance by either extruding Na⁺ out of the cell or sequestering Na⁺ into the vacuole, respectively [ 6 ]. Overexpression of AtNHX1 enhances salt tolerance in Arabidopsis thaliana [ 7 – 8 ], while OsNHX1 and OsNHX2 contribute to salt adaptation in rice by regulating Na⁺ sequestration into the vacuole [ 9 ]. In woody plants, MdNHX from apple ( Malus domestica L.) and PpNHX2 from peach ( Prunus persica L.) have been shown to improve salt tolerance when heterologously expressed in tobacco [ 10 – 11 ]. High salt concentrations disrupt cellular ion homeostasis, induce osmotic stress, and trigger excessive accumulation of reactive oxygen species (ROS) such as superoxide anion (O₂⁻), hydrogen peroxide (H₂O₂), and hydroxyl radical (-OH) [ 12 ]. These ROS cause oxidative damage to lipids, proteins, and nucleic acids, leading to membrane lipid peroxidation, metabolic disorders, and ultimately growth inhibition or even plant death [ 13 ]. In response, plants activate their antioxidant defense system to alleviate ROS induced damage, in which superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) play key roles: SOD catalyzes the dismutation of O₂⁻ into H₂O₂ and O₂, while POD and CAT further scavenge H₂O₂ to prevent oxidative injury [ 14 ]. Malondialdehyde (MDA), a product of lipid peroxidation, is widely used as a key indicator of membrane damage under stress [ 15 ]. Moreover, under salt stress, osmotic regulatory substances such as proline rapidly accumulate in plant cells, lowering the cellular osmotic potential to facilitate water uptake and protecting enzymes and membrane structures [ 16 ]. With the publication of the sweet cherry genome sequence [ 17 ], genome wide identification and functional analysis of gene families have become feasible. To date, several stress responsive gene families, such as WRKY and AP2/ERF, have been identified in sweet cherry; however, the NHX family, which plays a central role in ion homeostasis regulation, has not yet been reported [ 18 – 19 ]. Given the conserved function of NHX genes in ion transport and the unique salt response mechanism of sweet cherry, a comprehensive analysis of the NHX family is necessary to fill this research gap. Through genome wide homology screening and domain validation, a total of 37 NHX family members were identified in sweet cherry. Their molecular characteristics, chromosomal distribution, phylogenetic relationships, gene structures, cis acting elements, and expression patterns in different tissues and under salt stress were systematically analyzed. Furthermore, the subcellular localization of the salt responsive gene PavNHX37 was verified, and its function in regulating the antioxidant system was analyzed in stably overexpressing transgenic tobacco plants. This study reveals the molecular mechanism of salt tolerance in sweet cherry and provides genetic resources for the genetic improvement of salt tolerant varieties. 2. Materials and Methods 2.1. Plant Materials Plant materials used in this study were in vitro plantlets of sweet cherry cultivar ‘Tieton’ ( Prunus avium L. ), preserved in the Laboratory of Fruit Tree Physiology and Molecular Biology, Tianshui Normal University. The cultivar was originally obtained and formally identified from the university’s Fruit Tree Germplasm Resource Bank. No wild plant materials were sampled. All experimental procedures involving plants were performed in accordance with institutional guidelines and relevant national regulations of China. Sweet cherry plantlets were subcultured on MS medium containing 1.0 mg/L 6-BA, 0.1 mg/L IBA, 30 g/L sucrose and 7 g/L agar (pH 5.8). Using the paper bridge method, plantlets were exposed to NaCl solutions at concentrations of 0, 50, 100 and 150 mmol/L for 24 h. Leaves were collected with three biological replicates, snap-frozen in liquid nitrogen, and stored at − 80°C for subsequent RNA extraction and reverse transcription. Nicotiana benthamiana seeds were conserved in the same laboratory. Plants were grown in a controlled environment with a 3:1 (v/v) mixture of nutrient soil and vermiculite, maintained at 25 ± 2°C under a 16 h light/8 h dark photoperiod. Four-week-old seedlings with consistent and vigorous growth were selected for subcellular localization analysis. For genetic transformation, tobacco seeds were surface-sterilized: rinsed with sterile water for 1 min, immersed in 75% ethanol for 40 s, treated with 5% sodium hypochlorite for 10 s, and washed 4–5 times with sterile water. Sterilized seeds were sown on MS medium supplemented with 30 g/L sucrose and 7 g/L agar (pH 5.8). Seedlings with two cotyledons were used for subsequent transformation experiments. 2.2. Sweet Cherry NHX Gene Family Identification and Physicochemical Property Analysis The accession numbers of NHX genes were obtained from the Arabidopsis genome database ( https://www.arabidopsis.org ), and the coding sequence of each gene was retrieved from NCBI. The sweet cherry genome annotation file was downloaded from the NCBI genome database ( https://www.ncbi.nlm.nih.gov/datasets/genome/ ). The CDS sequences of the sweet cherry genome were converted into protein sequences and simplified using TBtools-Ⅱ v2.382. Homology alignment was performed between the protein sequences of Arabidopsis and sweet cherry, followed by a secondary alignment of the preliminarily screened protein sequences using the BLAST module of NCBI. The gene IDs of the retained sequences were recorded, and their amino acid sequences were extracted. Functional domain screening was conducted using NCBI-CDD ( https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi ). Candidate genes lacking the specific domains of NHX genes were excluded. Through this screening, a total of 37 NHX gene family members in sweet cherry were identified. Based on the selected NHX amino acid sequences, their basic information, including amino acid number, molecular weight, isoelectric point, and hydrophilicity, was obtained using ExPASy ( https://web.expasy.org/protparam/ ). 2.3. Sweet Cherry NHX Gene Family Chromosomal Localization Subcellular Localization and Protein Secondary Structure The chromosomal positions of sweet cherry NHX genes were predicted and visualized using TBtools-Ⅱ v2.382. Subcellular localization of the NHX gene family was predicted using WoLF PSORT ( https://www.genscript.com/wolf-psort.html ). Protein secondary structure analysis of the NHX gene family was performed using NPSA ( https://npsa.lyon.inserm.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html ). 2.4. Sweet Cherry NHX Gene Family Phylogenetic Tree Construction and Synteny Analysis A phylogenetic tree was constructed using the One Step Build a ML Tree function in TBtools-Ⅱ with the neighbor-joining (NJ) method. The tree was then beautified using the ChiPlot tool ( https://www.chiplot.online/#BioPlot ). Genome and annotation files for synteny analysis were downloaded from the NCBI genome database ( https://www.ncbi.nlm.nih.gov/datasets/genome/ ) for grape ( Vitis vinifera L.), Arabidopsis thaliana, apple ( Malus domestica L.), woodland strawberry ( Fragaria vesca ), and peach ( Prunus persica L.). Synteny analysis of sweet cherry NHX genes was performed and visualized using the synteny tool in TBtools-Ⅱ. 2.5. Sweet Cherry NHX Gene Family Gene Structure Motif and Cis-Acting Element Analysis Gene structure prediction was performed using TBtools‑Ⅱ software. Conserved motif analysis was carried out using the MEME website ( http://meme-suite.org/ ) with the number of motifs set to 10. Conserved protein domains were analyzed using the NCBI‑CDD website ( https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi ). The promoter sequences (2000bp upstream of the start codon) of NHX gene family members were extracted. Cis‑acting element screening was completed using the PlantCARE website ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ), and visualization was performed using TBtools‑Ⅱ software. 2.6. Sweet Cherry NHX Gene Family Codon Usage Bias Analysis and Protein-Protein Interaction Network Analysis Codon usage bias analysis was performed using R software (version 4.3.3) with the following bioinformatics and statistical packages: seqinr (v4.2‑25) for biological sequence manipulation, Biostrings (v2.68‑1) for codon parsing of CDS sequences, and ggplot2 (v3.4‑4) for visualization. The protein sequences of the sweet cherry NHX family were used to construct a protein‑protein interaction network via the STRING database ( https://string-db.org/ ). 2.7. Sweet Cherry NHX Gene Family Tissue-Specific Expression Profile Analysis Using published sweet cherry tissue expression data [ 17 ] the corresponding gene accession numbers of NHX protein members were retrieved. The obtained data were organized using Excel 2007 and subsequently visualized using the ChiPlot tool. 2.8. Sweet Cherry NHX Gene Family qRT-PCR Analysis Primers were synthesized by Shanghai Bioengineering Technology Co., Ltd. (Table 1 ). The PCR reaction system (total 20µL) consisted of 7µL ddH₂O, 1µL cDNA, 2µL forward and reverse primers, and 10µL 2× SYBR TaKaRa Premix Ex Taq II. The PCR amplification conditions were 35 cycles as follows: 95°C for 30s, 95°C for 10s, 58°C for 30s, and 72°C for 20s. Three biological replicates and three technical replicates were performed to ensure the reliability of the results. Relative expression levels were calculated using the 2 ⁻ΔΔCt method. Table 1 Primers used in the experiment. Use Names of primers Primer of sequence (5′-3′) Gene-amplification pCAMBIA2300- PavNHX37 -F GAGAACACGGGGGACGAGCTCATGGACATTGGTTTCCTGGAGA pCAMBIA2300- PavNHX37 -R CACCATGGTGTCGACTCTAGACTCTACTATCTGCTGCTCCCCAA Plant identification 35s-F GACGCACAATCCCACTATCC qRT-PCR PavNHX03 -F GCCGTCCAGACTGACTTGAA PavNHX03 -R GTGTCACCCCACGCAATTTC PavNHX05 -F GATCCCTACGCTGCTCCTTC PavNHX05- R GGAGACATTGGACTCGGTGG PavNHX08 -F GTGTCCTTGTTGGTTTCGGC PavNHX08 -R GAGAAGTTTCAGGTCCGCCA PavNHX09 -F TCCTGGCGGAGAATTTGCAT PavNHX09- R TCGAACGTCATGCACCTCAA PavNHX17 -F GGTGATGGCCTTGAGGGTAC PavNHX17 -R TGTGAATGACGCTGTGCTCT PavNHX19 -F TCTCGGATCTGCGTTGCTTT PavNHX19 -R GTGGTCGCAAAAACTGGCAT PavNHX22 -F AGCTCAACCAAAAACGACGC PavNHX22 -R CCGCCATAGTCTTCCTCGTC PavNHX25 -F GGTTGCCACCATGATCCAGA PavNHX25 -R GATGTTGGTGGCTGTTGCTG PavNHX29 -F GCCCTGGTCATAGGCATCTC PavNHX29 -R TTCCATCGTGACCGTTCGTT PavNHX30 -F CCTTGCTCAGTCGGTGTTCT PavNHX30 -R CCTCCCAAAAACAGCACAGC PavNHX36 -F TGGAAAGCCAGAACCTAGCG PavNHX36 -R AAGCGCAACAAAAGCCTCAG PavNHX37 -F ACTCAGGCAGCATCATGTCC PavNHX37 -R CGGCCATATCCCACTTTGGA pavEF1-a1-F TGAGAGGCTGACTGTGCTGTTC pavEF1-a1-R GGAGTAGTGGCATCCATCTTGTT 2.9. PavNHX37 Overexpression Vector Construction and Nicotiana benthamiana Subcellular Localization The overexpression vector pCAMBIA2300 ‑PavNHX37 ‑GFP was constructed. Primers were designed with restriction sites ( Sac I and Xba I) (Table 1 ). The construct was then transformed into Agrobacterium tumefaciens strain GV3101. After overnight shaking, the bacterial suspension was stored at -80°C. The Agrobacterium cells harboring the fusion vector were collected by centrifugation after overnight shaking, washed twice with sterile water, and centrifuged again, and the supernatant was discarded. The bacterial pellet was resuspended in resuspension buffer (10mM 2-ethanesulfonic acid, MES pH 5.8, 10mM MgCl₂, 200µM Acetosyringone, AS) to an OD₆₀₀ of 0.6–0.8, followed by shaking at 180rpm at 28°C in darkness for 2–3h. The resuspended bacterial suspension was injected into tobacco leaves that had been water‑deprived for two days using a needleless syringe. After dark culture for 24h, the plants were transferred to normal light conditions for an additional 24h. Subcellular localization of the pCAMBIA2300‑ PavNHX37 ‑GFP fusion protein was observed under a laser confocal scanning microscope (LSM710, Carl Zeiss, Germany). 2.10. Nicotiana benthamiana Stable Genetic Transformation and Identification Healthy and sterile Nicotiana benthamiana test-tube plantlets were selected, and their leaves were cut into 5mm × 5mm pieces. These leaf pieces were placed on pre-culture medium and cultured in darkness for 2–3 days. The pre-cultured leaf pieces were immersed in an Agrobacterium solution containing the overexpression vector pCAMBIA230- PavNHX37 -GFP. After removing excess bacterial solution, the leaf pieces were transferred to co-culture medium and cultured in darkness for another 3 days. Following co-cultivation, the leaf pieces were transferred to selection medium and cultured under light conditions, with subculture to fresh medium every 1–2 weeks. Once new shoots regenerated, they were transferred to rooting medium. After roots had developed, leaf samples were collected to test the positivity of transgenic plants. After obtaining the overexpressing tobacco plants, leaves were randomly selected from different plants and individually labeled. Genomic DNA was extracted from the leaves using the TIANGEN Plant Genomic DNA Extraction Kit (Beijing, China). The extracted DNA was used as a template for PCR amplification with gene-specific primers. The amplification products were detected by agarose gel electrophoresis to complete the molecular positive identification of the overexpressing tobacco plants. 2.11. Overexpressing PavNHX37 Tobacco Enzyme Activity Assay Tobacco leaf tissue samples from wild-type (WT) and overexpressing (OE) lines treated for 72 h under different conditions were collected. The samples were flash-frozen in liquid nitrogen and ground into powder. Then 0.1g of powder was weighed and pre-cooled extraction buffer (50mmol/L Tris(hydroxymethyl)aminomethane, Tris-HCl pH 8.0, 1mmol/L Ethylenediaminetetraacetic acid, EDTA, 1mmol/L Phenylmethylsulfonyl fluoride, PMSF, 1% Polyvinylpyrrolidone, PVP) was added. After homogenization on ice, the mixture was centrifuged at 12 000r/min for 20min at 4°C. The supernatant was collected as the crude enzyme extract. The activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) as well as the contents of malondialdehyde (MDA) and proline (Pro) were determined using commercial assay kits purchased from Suzhou Grace Biotechnology Co., Ltd. 2.12. Data Processing and Statistical Analysis All experimental data were statistically analyzed using SPSS 23.0 software. One-way analysis of variance (ANOVA) was performed followed by an ANOVA test ( P < 0.05). Graphs were generated using Origin Pro 2024 software. All measured data were derived from three independent biological replicates. 3. Result 3.1. Sweet Cherry pavNHX Gene Family Identification and Secondary Structure Analysis A total of 37 NHX family genes were identified in sweet cherry through homology analysis and were named PavNHX01 - PavNHX37 . These genes were distributed on eight chromosomes of sweet cherry. Among them, ten genes were located on chromosomes 2 and 6, followed by five genes on chromosomes 1 and 4, three genes on chromosome 5, two genes on chromosome 8, and only one gene on chromosomes 3 and 7 (Fig. 1 ). The number of amino acids encoded by NHX members ranged from 150 to 1223, with substantial variation even among genes within the same subfamily. The molecular weights of the proteins ranged from 15,971.08 to 132,067.04 Da, the theoretical isoelectric points (pI) ranged from 4.6 to 9.14, and the instability indices ranged from 24.71 to 49.37. The hydrophilicity values ranged from 0.060 to 1.127, with higher values indicating stronger hydrophilicity. Subcellular localization prediction showed that all genes were localized to the plasma membrane except PavNHX21 , which was localized to the vacuole. The atomic composition of PavNHX36 could not be calculated due to ambiguous bases in the analyzed sequence (Table 2 ). The secondary structure of sweet cherry NHX family proteins was dominated by α‑helices (42.00%–68.68%) and random coils (19.95%–39.27%), followed by β‑turns (1.18%–4.55%) and extended strands (8.67%–16.64%) ( Table 3 ). Table 2 physical and chemical properties of PavNHX gene family. gen ID gen name Location of Chromosome Amino acid Molecular weight/ KD PI Instability index: Aliphatic index: hydropathicity Formula Subcelluar localization FUN_001618-T1 PavNHX01 chr1 551 60730.92 8.19 45.79 107.35 0.499 C 2790 H 4348 N 696 O 768 S 25 Plasma membrane FUN_003199-T1 PavNHX02 chr1 816 88278.88 8.9 37.5 109.31 0.41 C 4008 H 6449 N 1041 O 1115 S 38 Plasma membrane FUN_004934-T1 PavNHX03 chr1 1148 127491.96 5.94 39.62 102.97 0.096 C 5785 H 9083 N 1499 O 1669 S 36 Plasma membrane FUN_005739-T2 PavNHX04 chr1 255 27578.24 4.6 29.27 102.43 0.089 C 1228 H 1957 N 317 O 391 S 5 Plasma membrane FUN_005739-T1 PavNHX05 chr1 591 63803.82 5.78 29.29 122.52 0.581 C 2907 H 4675 N 731 O 827 S 22 Plasma membrane FUN_008475-T1 PavNHX06 chr2 765 84271.72 7.26 38.9 112.09 0.399 C 3863 H 6115 N 973 O 1070 S 30 Plasma membrane FUN_008684-T1 PavNHX07 chr2 765 84271.72 7.26 38.9 112.09 0.399 C 3863 H 6115 N 973 O 1070 S 30 Plasma membrane FUN_008760-T1 PavNHX08 chr2 841 92082.37 6.21 38.34 102.69 0.179 C 4099 H 6556 N 1092 O 1221 S 44 Plasma membrane FUN_011354-T1 PavNHX09 chr2 912 100683.18 5.99 34.46 98.12 0.084 C 4496 H 7133 N 1195 O 1326 S 48 Plasma membrane FUN_012393-T1 PavNHX10 chr2 813 90061.57 9.13 40.34 109.91 0.264 C 4100 H 6548 N 1072 O 1134 S 33 Plasma membrane FUN_012394-T1 PavNHX11 chr2 1223 132067.04 5.13 41.37 104.88 0.06 C 5854 H 9452 N 1596 O 1801 S 33 Plasma membrane FUN_012399-T1 PavNHX12 chr2 685 75828.07 5.95 37.33 111.53 0.416 C 3426 H 5461 N 881 O 962 S 45 Plasma membrane FUN_012409-T1 PavNHX13 chr2 292 32247.32 8.78 32.53 124.28 0.623 C 1502 H 2375 N 367 O 393 S 12 Plasma membrane FUN_012716-T2 PavNHX14 chr2 150 15971.08 7.67 24.71 126.67 1.127 C 743 H 1164 N 176 O 193 S 10 Plasma membrane FUN_012716-T1 PavNHX15 chr2 816 90019.67 6.71 44.19 117.34 0.39 C 4064 H 6546 N 1076 O 1139 S 41 Plasma membrane FUN_017854-T1 PavNHX16 chr3 572 61910.63 5.64 31.76 117.55 0.622 C 2832 H 4498 N 698 O 797 S 26 Plasma membrane FUN_031503-T1 PavNHX17 chr4 540 59315.46 5.53 40.03 99.69 0.427 C 2725 H 4153 N 675 O 761 S 24 Plasma membrane FUN_031504-T1 PavNHX18 chr4 824 90950.62 7.32 45.92 109.3 0.331 C 4111 H 6568 N 1072 O 1154 S 46 Plasma membrane FUN_032382-T1 PavNHX19 chr4 540 59315.46 5.53 40.03 99.69 0.427 C 2725 H 4153 N 675 O 761 S 24 Plasma membrane FUN_035179-T1 PavNHX20 chr4 551 61702.79 7.21 37.22 112.96 0.58 C 2853 H 4446 N 702 O 758 S 31 Plasma membrane FUN_035478-T1 PavNHX21 chr4 525 58253.25 7.18 32.95 115.89 0.601 C 2702 H 4195 N 665 O 726 S 20 Vacuole FUN_024568-T1 PavNHX22 chr5 856 92668.69 5.5 35.91 108.9 0.355 C 4161 H 6647 N 1105 O 1200 S 41 Plasma membrane FUN_025222-T1 PavNHX23 chr5 790 87368.57 6.23 37.18 109.59 0.331 C 3979 H 6257 N 1025 O 1118 S 32 Plasma membrane FUN_025223-T1 PavNHX24 chr5 800 86656.83 8.79 40.99 114.7 0.415 C 3915 H 6337 N 1045 O 1093 S 35 Plasma membrane FUN_018802-T1 PavNHX25 chr6 808 87403.31 8.75 36.16 108.13 0.351 C 3932 H 6343 N 1057 O 1110 S 38 Plasma membrane FUN_018803-T1 PavNHX26 chr6 758 83582.65 6.19 40.16 107.57 0.365 C 3795 H 5999 N 963 O 1072 S 41 Plasma membrane FUN_018804-T1 PavNHX27 chr6 754 83357.53 8.06 37.76 105.46 0.343 C 3790 H 5972 N 984 O 1045 S 41 Plasma membrane FUN_018805-T1 PavNHX28 chr6 759 83622.04 7.15 49.37 111.73 0.389 C 3804 H 6048 N 966 O 1066 S 39 Plasma membrane FUN_018806-T1 PavNHX29 chr6 696 77297.88 7.87 48.48 105.42 0.312 C 3465 H 5553 N 907 O 980 S 53 Plasma membrane FUN_018816-T1 PavNHX30 chr6 757 83456.67 6.37 45.41 114.32 0.39 C 3794 H 6037 N 967 O 1069 S 36 Plasma membrane FUN_021849-T1 PavNHX31 chr6 858 94339.05 6.13 35.5 110.83 0.371 C 4320 H 6791 N 1105 O 1194 S 32 Plasma membrane FUN_021851-T1 PavNHX32 chr6 848 91916.5 9.14 39.94 107.84 0.294 C 4159 H 6695 N 1117 O 1163 S 30 Plasma membrane FUN_022777-T1 PavNHX33 chr6 782 84719.33 8.73 43.09 114.46 0.38 C 3822 H 6192 N 1030 O 1072 S 31 Plasma membrane FUN_022826-T1 PavNHX34 chr6 543 59396.33 6.51 36.53 107.96 0.57 C 2743 H 4269 N 665 O 760 S 21 Plasma membrane FUN_039269-T1 PavNHX35 chr7 539 59876.29 8.83 34.46 113.91 0.545 C 2770 H 4330 N 690 O 744 S 21 Plasma membrane FUN_029070-T1 PavNHX36 chr8 616 66511.05 5.72 35.9 116.93 0.574 - Plasma membrane FUN_031269-T1 PavNHX37 chr8 785 85778.87 7.58 35.07 118.78 0.523 C 3952 H 6290 N 994 O 1080 S 24 Plasma membrane Table 2 Secondary structure analysis of PavNHX protein. Gen name Alpha helix Beta turn Extended strand Random coil PavNHX01 48.82 3.09 13.07 35.03 PavNHX02 45.1 3.68 14.58 36.64 PavNHX03 55.66 3.22 10.19 30.92 PavNHX04 57.25 1.18 11.37 30.2 PavNHX05 59.73 2.03 10.15 28.09 PavNHX06 46.54 4.18 15.42 33.86 PavNHX07 44 4.04 14.98 36.98 PavNHX08 42 4.17 15.57 38.27 PavNHX09 68.68 2.7 8.67 19.95 PavNHX10 44.16 3.94 15.13 36.78 PavNHX11 46.04 3.9 16.1 33.96 PavNHX12 45.84 4.38 16.64 33.14 PavNHX13 43.75 3.43 15.44 37.38 PavNHX14 62.67 3.33 12.67 21.33 PavNHX15 60.31 1.92 11.19 26.57 PavNHX16 44.3 3.88 14.81 37.01 PavNHX17 51.11 3.52 14.07 31.3 PavNHX18 51.11 3.52 14.07 31.3 PavNHX19 44.75 4.12 15.62 35.5 PavNHX20 46.86 3.81 14.76 34.56 PavNHX21 44.28 3.39 14.6 37.73 PavNHX22 45.44 3.42 14.68 36.46 PavNHX23 45.62 3.5 15.5 35.38 PavNHX24 44.31 3.71 15.22 36.76 PavNHX25 44.69 3.98 16.45 34.88 PavNHX26 44.33 4.09 16.09 35.49 PavNHX27 44.01 4.35 16.07 35.57 PavNHX28 44.12 4.49 16.38 35.01 PavNHX29 45.69 4.02 16.24 34.05 PavNHX30 42.54 4.55 14.57 38.34 PavNHX31 45.91 3.71 15.6 34.78 PavNHX32 43.04 3.77 13.92 39.27 PavNHX33 49.36 3.5 14 33.15 PavNHX34 49.35 3.53 13.54 33.58 PavNHX35 59.58 2.44 12.18 25.81 PavNHX36 55.54 3.44 11.97 29.04 PavNHX37 47.91 3.27 13.43 35.39 3.2. Sweet Cherry pavNHX Gene Family Evolutionary Analysis To further understand the evolutionary characteristics of the NHX gene family, a phylogenetic tree was constructed using NHX family members from sweet cherry and Arabidopsis thaliana (Fig. 2 ). The family members were divided into three groups. Group I contained eight sweet cherry genes and eight Arabidopsis genes. Group II and Group III consisted entirely of sweet cherry NHX family members, with six and twenty-three members respectively. Among the three groups, only Group I contained Arabidopsis NHX family members, indicating that NHX gene family members are highly conserved within the groups. 3.3. Sweet Cherry pavNHX Gene Family Protein Structure and Motif Analysis The conserved structural domains of the sweet cherry NHX gene family were analyzed online using the MEME software, and the family structures were compared and localized. Among the members, PavNHX03 , PavNHX04 , PavNHX18 , PavNHX17 , and PavNHX33 contained only one motif. PavNHX22 , PavNHX20 , PavNHX08, PavNHX07 , PavNHX21 , PavNHX32 , PavNHX31 , PavNHX02 , PavNHX24 , PavNHX23 , PavNHX30 , PavNHX10 , PavNHX12 , PavNHX13 , PavNHX16 , PavNHX06 , PavNHX26 , PavNHX28 , and PavNHX27 all shared the same motifs, indicating a high degree of conservation and suggesting that their functions are largely similar. Furthermore, PavNHX37 , PavNHX01 , and PavNHX35 contained only three motifs, while PavNHX34 contained only two motifs (Fig. 3 a). Protein structure analysis of the sweet cherry NHX gene family revealed that these genes contain 0 to 22 introns. PavNHX25 contained no intron, while the longest intron was found in PavNHX12 . Additionally, PavNHX05 , PavNHX35 , PavNHX15 , PavNHX14 , PavNHX17 , PavNHX33 , PavNHX34 , PavNHX01 , PavNHX09 , PavNHX26 , PavNHX32 , PavNHX24 , and PavNHX23 possessed exons, whereas the remaining genes lacked exons. Notable differences in exon length and position were observed, which may contribute to functional divergence among these genes (Fig. 3 b). 3.4. Sweet Cherry pavNHX Gene Family Promoter Cis-Acting Element Prediction Among the 37 predicted cis-acting promoter elements of the sweet cherry NHX gene family, 32 response elements were identified. All NHX gene family members contained the following elements: a cis-acting regulatory element involved in MeJA responsiveness, a cis-acting element involved in salicylic acid responsiveness, a cis-acting element involved in abscisic acid responsiveness, a MYB binding site involved in drought inducibility, a cis-acting element involved in auxin responsiveness, a cis-acting regulatory element root specific, an auxin-responsive element, a MYB-like binding site in flavonoid biosynthetic genes regulation, and a cis-acting regulatory element related to meristem specific activation. Different members contained different response elements. In addition to the elements listed above, PavNHX01 , PavNHX11 , and PavNHX27 contained a cis-acting regulatory element involved in zein metabolism regulation. PavNHX04 and PavNHX05 lacked the cis-acting element involved in low-temperature responsiveness. PavNHX23 and PavNHX12 contained the fewest response elements, only 12 elements, whereas PavNHX01 contained the most with 23 elements (Fig. 4 ). 3.5. Sweet Cherry pavNHX Gene Family Tissue-Specific Expression Analysis To elucidate the functions of PavNHX genes in sweet cherry growth and development, the expression patterns of 37 PavNHX genes across 15 tissues and fruit developmental stages were analyzed. After Log₂(FPKM + 1) normalization, a heatmap was generated (Fig. 5 ). The results showed that the PavNHX gene family exhibited significant tissue-specific and stage-specific expression characteristics. All members could be clearly divided into two major expression clusters. The first cluster was a high-expression cluster, containing genes such as PavNHX24 , PavNHX23 , PavNHX34 , and PavNHX18 . These genes maintained consistently high expression levels in vegetative organs including annual shoots, young leaves, and mature leaves. Among them, PavNHX24 and PavNHX34 showed the highest expression levels and stable expression in most tissues. The second cluster was a low-expression cluster, containing genes such as PavNHX22 , PavNHX30 , PavNHX29 , and PavNHX06 . These genes exhibited extremely low expression in most vegetative organs but displayed distinct targeted expression characteristics: PavNHX29 was significantly upregulated at stem stages 2–3; PavNHX30 and PavNHX22 showed elevated expression in middle-to-late fruit development stages; PavNHX06 and PavNHX08 accumulated a small amount of expression in floral organs. In addition, some genes such as PavNHX04 and PavNHX16 maintained low expression levels across all 15 tissues and stages without obvious tissue preference. 3.6. Sweet Cherry pavNHX Gene Family Synteny Analysis Intraspecific synteny analysis of the sweet cherry NHX gene family revealed three pairs of duplicated genes located on chromosomes 1, 7, 6, and 2. However, no linked genes were found on chromosomes 3, 4, 5, and 8 (Fig. 6 ). To further elucidate the phylogenetic mechanism of the sweet cherry NHX gene family as well as the relationship between paralogy and orthology, synteny analysis was performed between sweet cherry and five representative species. The results showed that sweet cherry shared 16 syntenic gene pairs with grape berry, 17 with Arabidopsis thaliana, 12 with apple, 22 with woodland strawberry, and 30 with peach (Fig. 7 ). Some NHX genes were found to be associated with more than two syntenic gene pairs, while others had no syntenic counterpart. These results indicate that syntenic relationships exist both within the sweet cherry NHX gene family and between sweet cherry and other species, and that some family genes are closely linked but not entirely orthologous. 3.7. Sweet Cherry NHX Gene Family Codon Usage Bias Analysis The relative synonymous codon usage (RSCU) of the 37 sweet cherry NHX genes was analyzed (Fig. 8 ). RSCU > 1 indicates preferred codons. RSCU < 1 indicates non-preferred codons. RSCU = 1 indicates no usage bias. For leucine (Leu) and serine (Ser) which have two codon families each they were analyzed separately as Leu1/Leu2 and Ser1/Ser2. All 22 amino acids showed varying degrees of synonymous codon usage bias. The total RSCU values varied among amino acids. Leu1 had the highest total RSCU value significantly higher than Leu2. The total RSCU values of alanine (Ala) arginine (Arg) glycine (Gly) proline (Pro) threonine (Thr) and valine (Val) were all close to 4. The total RSCU values of asparagine (Asn) aspartic acid (Asp) cysteine (Cys) glutamine (Gln) glutamic acid (Glu) histidine (His) isoleucine (Ile) lysine (Lys) methionine (Met) phenylalanine (Phe) threonine (Thr) tryptophan (Trp) and tyrosine (Tyr) were all close to 2. In addition, the rare codons showed specific RSCU values. For example, the RSCU value of Trp’s UGG was approximately 2. Overall, the preferred codons of the 37 sweet cherry NHX genes all ended with U or C while non-preferred codons tended to end with A or G. This indicates that the synonymous codon usage of these genes exhibits a significant bias toward U or C endings. 3.8. Sweet Cherry NHX Gene Family Protein‑Protein Interaction Network Analysis The interaction network analysis tool was used to predict the interaction relationships among sweet cherry PavNHX proteins (Fig. 9 ). The PavNHX gene family members formed a complete interaction network. Thirty members showed interaction connections while seven members were isolated nodes indicating extensive interactions among family members. PavNHX35, PavNHX22 and PavNHX06 were the core nodes in the network. These nodes had the largest size and the darkest color with interaction strength close to the highest threshold value of 0.900. They also exhibited dense connections with many surrounding genes making them key hubs in the regulatory network. PavNHX19, PavNHX36 and PavNHX14 showed intermediate node sizes and interaction strengths. In contrast PavNHX12, PavNHX07 and PavNHX27 had the smallest node sizes and the lightest colors with relatively low interaction strength. These genes had only single connections with a few genes and were located at the periphery of the interaction network. 3.9. Sweet Cherry NHX Gene Family Member Expression Analysis To further analyze the expression patterns of gene members twelve genes were selected based on the gene family phylogenetic tree for qRT-PCR analysis. The qRT-PCR results showed that the expression levels of six sweet cherry NHX gene family members decreased with increasing salt concentration. Compared with the control PavNHX03 , PavNHX08 and PavNHX17 showed significantly reduced expression under high salt stress among which pavNHX17 decreased by 10-fold. The relative expression levels of PavNHX19 and PavNHX36 did not change significantly under the salt stress. In addition, the relative expression trends of PavNHX09 and PavNHX25 first decreased and then increased. Furthermore, we found that the relative expression of PavNHX37 increased significantly reaching 8.9-fold that of the control (Fig. 10 ). 3.10. PavNHX37 Sequence Analysis and Subcellular Localization The PavNHX37 gene is located on sweet cherry chromosome 8 at positions 207796 to 211408. Its full length is 1991 bp. The CDS coding region is 1656 bp in length encoding 551 amino acids (Fig. 11 a). A conserved Na⁺/H⁺ exchanger domain is present at amino acid positions 34 to 455. The fusion expression vector pCAMBIA2300- PavNHX37 -GFP was used (Fig. 11 b). The constructed fusion expression vector and the pCAMBIA2300-GFP negative control were injected into tobacco leaves via Agrobacterium. The results showed that the negative control was expressed in both the nucleus and the plasma membrane whereas PavNHX37 was localized to the plasma membrane. This result is consistent with the subcellular localization prediction (Fig. 11 c). 3.11. PavNHX37 Overexpressing Tobacco Functional Verification Positive transgenic lines were screened by genomic PCR using gene-specific primers. As shown in Fig. 12 a a specific band of approximately 1500 bp was successfully amplified in most candidate overexpression lines. This band was consistent with the positive control (P0) and the expected size of the target gene fragment. In contrast wild-type (WT) plants showed no such specific band. Four lines OE-8 OE-13 OE-14 and OE-17 failed to amplify the target gene fragment and were therefore excluded from subsequent experiments. The expression levels of the target gene in wild-type and overexpressing lines under different NaCl treatments were detected by qRT-PCR (Fig. 12 c). Under normal control conditions (0 mM NaCl) the basal expression level of the target gene was very low in wild-type plants. All tested overexpression lines showed significantly elevated gene expression levels approximately 40-fold higher than that of the wild-type ( P < 0.05). This confirmed that the exogenous gene had been successfully overexpressed. Wild-type plants and the selected overexpression lines were treated with 0 50 100 and 150 mM NaCl solutions. Under non-stress conditions (0 mM NaCl) no significant differences in growth status or morphological characteristics were observed between wild-type and overexpression lines (Fig. 12 b). This indicated that overexpression of the target gene did not affect normal plant growth and development. With increasing NaCl concentration wild-type plants showed obvious growth inhibition leaf yellowing and wilting. In contrast the overexpression lines exhibited significantly enhanced salt tolerance. Under 50 mM NaCl treatment the overexpression lines showed greater growth vigor larger leaves and higher biomass compared to wild-type plants. Under moderate (100 mM NaCl) and severe (150 mM NaCl) salt stress the growth advantage of the OE lines became more pronounced. Notably even under 150 mM NaCl treatment the OE lines maintained good leaf condition and vigorous growth whereas wild-type plants displayed severe growth dwarfism and leaf senescence. 3.12. Overexpressing PavNHX37 Tobacco Enzyme Activity Assay The enzyme activity assay results after NaCl treatment of overexpressing tobacco showed that proline accumulation in overexpressing plants was significantly higher than that in wild-type plants while MDA content was significantly lower. This indicated enhanced osmotic regulation capacity and reduced oxidative damage. Meanwhile the activities of SOD POD and CAT antioxidant enzymes were significantly increased in overexpressing plants. Under 150 mM NaCl treatment the activities of these three enzymes reached 3.0-fold 3.2-fold and 1.2-fold those of wild-type plants respectively. Overexpression of the target gene significantly enhanced salt tolerance in tobacco plants by strengthening osmotic protection reducing oxidative damage and increasing reactive oxygen species scavenging capacity (Fig. 13 ). 4. Discussion In this study 37 NHX gene family members were identified in sweet cherry for the first time through genome‑wide homology alignment combined with domain validation. This number is significantly higher than that in cotton which has 24 members [ 11 ]. The 37 PavNHX genes were unevenly distributed on eight chromosomes. Chromosomes 2 and 6 each contained 10 members suggesting that these chromosomes may be enriched regions for the NHX gene family [ 21 ]. The amino acid lengths (150–1223 amino acids) and molecular weights (15.97–132.07 kD) of PavNHX proteins varied considerably. Their secondary structures were dominated by α‑helices (42.00%–68.68%) and random coils (19.95%–39.27%). This is highly consistent with the structural features of NHX proteins as transmembrane transporters: α‑helices typically form transmembrane domains to mediate ion transmembrane transport while random coils may participate in protein‑protein or protein‑molecule interactions [ 22 ]. Furthermore, subcellular localization prediction showed that all PavNHX members were localized to the plasma membrane except PavNHX21 which was localized to the vacuole. This result is consistent with the classification of the oat NHX family into plasma membrane (PM) vacuolar (Vac) and endosomal (Endo) subfamilies [ 23 ]. Phylogenetic analysis divided the PavNHX family into three groups. Group I contained eight sweet cherry members and eight Arabidopsis members. This suggests that genes in this group are highly conserved during evolution and may perform basic functions in plant ion transport [ 5 ]. Group II (6) and Group III (22) contained only sweet cherry members. They are speculated to be sweet-cherry-specific expanded subfamilies which is similar to the conserved subfamily results of the mung bean NHX family [ 24 ]. Synteny analysis showed that sweet cherry shared 30 syntenic gene pairs with peach which belongs to the same Prunus genus. This number is significantly higher than the numbers with grape (16) and Arabidopsis (17). This pattern is consistent with the phylogenetic relationships among these species [ 25 ]. The tissue expression profiles of PavNHX genes showed significant specificity. High-expression cluster genes such as PavNHX24 and PavNHX34 were consistently highly expressed in vegetative organs including annual shoots and young leaves. Low-expression cluster genes such as PavNHX29 and PavNHX30 were upregulated at specific stages of stem development or during late fruit maturation. In tobacco NtNHX1-3 showed high expression in leaves but low or no expression in other tissues [ 26 ]. As photosynthetic organs and primary sites of salt stress response leaves require sustained ion transport capacity to maintain physiological functions [ 27 ]. Promoter cis-element analysis revealed that all PavNHX genes contained MeJA responsiveness elements salicylic acid responsiveness elements ABA responsiveness elements and drought-inducible MYB binding sites. Salt stress often occurs together with drought stress. ABA as a core stress-responsive hormone can maintain ion balance by regulating NHX gene expression [ 28 ]. This is consistent with the salt-stress response patterns of PavNHX genes observed in this study. qRT-PCR analysis of 12 representative PavNHX genes under salt stress revealed distinct expression patterns. Six genes including PavNHX03 and PavNHX17 showed decreased expression with increasing salt concentration possibly due to feedback inhibition. PavNHX37 expression under 150 mM salt stress was 8.9-fold that of the control making it the most significantly salt-stress-responsive gene. This finding is similar to results in quinoa [ 29 ]. Following salt stress treatment wild-type tobacco exhibited varying degrees of wilting and chlorosis while overexpressing tobacco lines did not. Enzyme activity assays showed significantly increased activities of SOD POD and CAT decreased MDA content and increased proline accumulation. Proline accumulation is one of the important physiological responses of plants to stress [ 30 ]. These results suggest that PavNHX37 may enhance salt tolerance through a dual mechanism: on one hand maintaining ion homeostasis via Na⁺/H⁺ exchange and on the other hand regulating the antioxidant system to scavenge ROS and accumulating osmolytes to alleviate stress damage [ 31 ]. Furthermore, although PavNHX37 is not a core node in the protein-protein interaction network it interacts with multiple family members. This implies that PavNHX37 may participate in a complex ion transport regulatory network and optimize salt tolerance through synergistic actions with other NHX proteins [ 32 ]. 5. Conclusions In this study, 37 NHX gene family members were systematically identified for the first time in sweet cherry. Their molecular characteristics, phylogenetic relationships, and tissue-specific expression patterns were elucidated, and PavNHX37 was screened as a key gene regulating salt tolerance. Overexpression of PavNHX37 in tobacco significantly enhanced salt tolerance by maintaining ion homeostasis and increasing antioxidant enzyme activities. This study provides a theoretical basis and genetic resources for improving salt tolerance in sweet cherry. Declarations Aknowledgements We sincerely thank all colleagues in the laboratory for their valuable discussions and technical support. Meanwhile, we extend our heartfelt gratitude to the editor and reviewers for their rigorous evaluation of this manuscript and their constructive comments for its improvement. Authors’ contributions Z.G, Y.Z., and S.H. conceived and designed the research. X.A., Z.G., Y.Z., S.H performed the experiments. X.A. and Z.G. performed the data analysis and wrote the manuscript. S.H. and Z.G. supervised the research. X.A. revised the manuscript. All authors read and approved the final manuscript. Funding This research was funded by Central guidance for local science and technology development fund projects, grant number 25ZYJE001. Data availability The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author. Ethics approval and consent to participate The authors declare that no studies involving human participants or animals were conducted for this article. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9596831","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":640197815,"identity":"4725edb7-6822-4ced-b508-b46d07240f41","order_by":0,"name":"Zhigang Guo","email":"","orcid":"","institution":"Tianshui Normal University","correspondingAuthor":false,"prefix":"","firstName":"Zhigang","middleName":"","lastName":"Guo","suffix":""},{"id":640197816,"identity":"065ff6e0-1a11-419b-a633-a81efefdfdc7","order_by":1,"name":"Yifan Zhou","email":"","orcid":"","institution":"Tianshui Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yifan","middleName":"","lastName":"Zhou","suffix":""},{"id":640197817,"identity":"7b83c982-6f9f-4bb2-bfcb-14e00e1e441a","order_by":2,"name":"Songling Han","email":"","orcid":"","institution":"Tianshui Normal University","correspondingAuthor":false,"prefix":"","firstName":"Songling","middleName":"","lastName":"Han","suffix":""},{"id":640197818,"identity":"88058d1c-0cab-49de-8338-327575230558","order_by":3,"name":"Yali Zou","email":"","orcid":"","institution":"Tianshui Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yali","middleName":"","lastName":"Zou","suffix":""},{"id":640197819,"identity":"f2652049-8f63-4088-9c0e-b20bf991921a","order_by":4,"name":"Xiaojuan An","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIie2RsQrCMBCGUwKdTrtGEPURrgSKQ8AHcYkImXRydSg4uBRcBV+mcNDVNYNDHkE3R5vJsTcK5pv/j7v/TohE4kdB+1ZQSEmBr7waM52cc4dsJbvmzuAdFooVL24NHgAINIFAcTTrQUU9OqthSVDRqA2ic/t60PHbVscpFY0tZjUNK3O/qTXk/WInQMVSsJ9S9vUBJVcpvbPxyKCoP7LldJn5XRVfuSouROF5NIz6QuTfD1pGPCIDM5hIJBL/ygcVQTuzZ4AjmQAAAABJRU5ErkJggg==","orcid":"","institution":"Tianshui Normal University","correspondingAuthor":true,"prefix":"","firstName":"Xiaojuan","middleName":"","lastName":"An","suffix":""}],"badges":[],"createdAt":"2026-05-03 01:38:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9596831/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9596831/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109462364,"identity":"62b982c8-1407-449a-a2d1-dfe23cd87b08","added_by":"auto","created_at":"2026-05-18 11:10:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":958155,"visible":true,"origin":"","legend":"\u003cp\u003eChromosomal localization of 37 \u003cem\u003ePavNHX\u003c/em\u003e genes on eight sweet cherry chromosomes\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9596831/v1/60f3eec2de846a00a9fd077d.png"},{"id":109462383,"identity":"9b898afb-9e5a-4355-b12f-0401024c4926","added_by":"auto","created_at":"2026-05-18 11:10:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":816582,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree analysis of the NHX gene family in sweet cherry.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9596831/v1/4ce558450e6a234a352f2de0.png"},{"id":109462376,"identity":"310c702a-4b94-4337-8c5f-9bf08eb30ba2","added_by":"auto","created_at":"2026-05-18 11:10:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1056430,"visible":true,"origin":"","legend":"\u003cp\u003eConserved motifs and gene structures of \u003cem\u003ePavNHX\u003c/em\u003e family genes in sweet cherry. (a) Conserved protein motifs; ten distinct motifs are colored as indicated, scale=amino acids; (b) Gene structure; CDS (blue boxes), UTR (red boxes), introns (gray lines), scale=base pairs (bp).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9596831/v1/ec9088c88fd11f1b3713f693.png"},{"id":109759750,"identity":"54c23d1a-7f11-48ac-ab6f-79fd6c406eaa","added_by":"auto","created_at":"2026-05-22 07:27:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1227121,"visible":true,"origin":"","legend":"\u003cp\u003eCis-acting regulatory elements in \u003cem\u003ePavNHX\u003c/em\u003e promoters. The 2000 bp upstream regions of \u003cem\u003ePavNHX\u003c/em\u003e genes contain diverse hormone-, light-, and stress-responsive elements. Scale bar: position relative to the transcription start site (bp).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9596831/v1/80ca06989666793189844fe4.png"},{"id":109462386,"identity":"d4517c86-65c4-4970-b0eb-4db1a8b9fc16","added_by":"auto","created_at":"2026-05-18 11:10:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":975469,"visible":true,"origin":"","legend":"\u003cp\u003eExpression heatmap of 37 \u003cem\u003ePavNHX\u003c/em\u003e genes across 15 sweet cherry tissues and fruit developmental stages.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-9596831/v1/b15c0d3a82f08f0dab43939a.png"},{"id":109462380,"identity":"3250bf1e-cb94-4a7a-8629-67d4cf3a59b1","added_by":"auto","created_at":"2026-05-18 11:10:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":854359,"visible":true,"origin":"","legend":"\u003cp\u003eIntraspecific synteny analysis of the sweet cherry NHX gene family. Circular plot of sweet cherry chromosomes showing \u003cem\u003ePavNHX\u003c/em\u003e gene positions, collinear blocks (gray lines), and segmental duplication pairs (colored lines).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-9596831/v1/cc2799d66a52f35119031071.png"},{"id":109462365,"identity":"12ba3cf5-78ce-4ae6-b66e-942a62c619d3","added_by":"auto","created_at":"2026-05-18 11:10:53","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3238387,"visible":true,"origin":"","legend":"\u003cp\u003eSynteny analysis of NHX genes between Prunus avium and five plant species. Chromosomes of P. avium (top) and other species (bottom) are shown as horizontal bars. Pink lines highlight syntenic NHX gene pairs; gray lines indicate other collinear genomic regions.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-9596831/v1/b93958b9394112dd6ef4b40d.png"},{"id":109800205,"identity":"d9f54ea3-5983-46ba-bcfb-30352e410076","added_by":"auto","created_at":"2026-05-22 15:36:51","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1528606,"visible":true,"origin":"","legend":"\u003cp\u003eCodon usage bias of \u003cem\u003ePavNHX\u003c/em\u003e genes.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-9596831/v1/f788cfe9b647cc14aed2fe6c.png"},{"id":109462381,"identity":"0e106308-3b43-499e-9600-c1fc5c252592","added_by":"auto","created_at":"2026-05-18 11:10:55","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":832780,"visible":true,"origin":"","legend":"\u003cp\u003eInteraction network of \u003cem\u003ePavNHX\u003c/em\u003e genes. Nodes represent individual \u003cem\u003ePavNHX\u003c/em\u003e genes; edges represent predicted functional interactions. Edge thickness indicates interaction confidence (low to highest, 0.150–0.900). Genes with thicker edges show stronger predicted associations.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-9596831/v1/ee64b19334c8d58af57cd44a.png"},{"id":109462445,"identity":"2d4abeb1-35cd-4836-b60f-f882157dc337","added_by":"auto","created_at":"2026-05-18 11:10:56","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":763685,"visible":true,"origin":"","legend":"\u003cp\u003eExpression profiles of selected \u003cem\u003ePavNHX\u003c/em\u003e genes under different NaCl concentrations. The relative expression levels of 12 \u003cem\u003ePavNHX\u003c/em\u003e genes were analyzed by qRT-PCR under 0, 50, 100, and 150 mM NaCl treatments. Each bar represents the mean ± standard error of three biological replicates. Different lowercase letters above the bars indicate significant differences (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05) according to one-way ANOVA followed by Tukey’s multiple range test.\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-9596831/v1/f1f01555f413709259dd1032.png"},{"id":109462444,"identity":"218f97ad-b33f-4988-8c73-25305c7905b3","added_by":"auto","created_at":"2026-05-18 11:10:56","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":1231921,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization and subcellular localization of \u003cem\u003ePavNHX37\u003c/em\u003e. (a) Gene structure of PavNHX37 on Chr8, showing gDNA, CDS, and conserved Na⁺/H⁺ exchanger domain; (b) Schematic of the 35S::\u003cem\u003ePavNHX37\u003c/em\u003e::GFP fusion and control vectors; (c) Subcellular localization in tobacco. Scale bar = 50μm.\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-9596831/v1/22cec6def5f962b5e6571b3a.png"},{"id":109462382,"identity":"b3c99654-7df5-4ede-b955-1357ccd4a65c","added_by":"auto","created_at":"2026-05-18 11:10:55","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":6050683,"visible":true,"origin":"","legend":"\u003cp\u003eSalt tolerance of\u003cem\u003e PavNHX37\u003c/em\u003e-overexpressing tobacco plants. (a) PCR identification of transgenic lines; (b) Phenotypes of WT and OE plants under 0–150 mM NaCl treatments (scale bar = 2cm); (c) Relative \u003cem\u003ePavNHX37\u003c/em\u003e expression in WT and OE lines under salt stress. Data are mean ± SE (n=3); different letters denote significant differences (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"floatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-9596831/v1/0d826d4a790137436e6d309c.png"},{"id":109462379,"identity":"d8962d79-5148-40b5-804b-fab0403cbec0","added_by":"auto","created_at":"2026-05-18 11:10:55","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":3132969,"visible":true,"origin":"","legend":"\u003cp\u003ePhysiological responses of \u003cem\u003ePavNHX37\u003c/em\u003e-overexpressing tobacco under salt stress. Pro, MDA, H₂O₂ contents, and SOD, POD, CAT activities in WT and OE plants under 0–150 mM NaCl treatments (mean ± SE, n=3). Asterisks denote significant differences (*\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, ****\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001).\u003c/p\u003e","description":"","filename":"floatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-9596831/v1/7ab97b910c7d59aadf1212e4.png"},{"id":109760296,"identity":"faf288d1-b719-49a4-b411-112de2c45b85","added_by":"auto","created_at":"2026-05-22 07:28:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":21789182,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9596831/v1/94a0f400-a828-4c8f-af1c-f0ea68d45794.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sweet cherry genome‑wide Identification of the NHX gene family and functional dissection of PavNHX37 a key salt tolerance gene","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSweet cherry (\u003cem\u003ePrunus avium\u003c/em\u003e L.) is a commercially important fruit tree of the Rosaceae family. It is widely cultivated due to the rich nutrition and economic value of its fruits. However, sweet cherry exhibits moderate sensitivity to salinity, and soil salinization has become a major factor limiting the expansion of cultivation and the stability of yield, particularly in arid and semiarid regions [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Soil salinization is a key abiotic stress factor restricting global agricultural productivity, affecting an estimated 1\u0026nbsp;billion hectares of cultivated land worldwide [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. High concentrations of salt in the soil disrupt cellular ion homeostasis, induce osmotic stress, and trigger excessive accumulation of reactive oxygen species (ROS), ultimately inhibiting plant growth, reducing yield, and even leading to plant death [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe Na⁺/H⁺ exchanger gene family plays a critical role in maintaining cellular ion homeostasis under salt stress by mediating the transmembrane transport of Na⁺ and H⁺ [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. NHX proteins are divided into two subgroups: those localized to the plasma mem-brane (PM) and those localized to the endosomal or vacuolar membrane. These two sub-groups reduce Na⁺ toxicity in the cytoplasm and maintain K⁺/Na⁺ balance by either extruding Na⁺ out of the cell or sequestering Na⁺ into the vacuole, respectively [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Overexpression of \u003cem\u003eAtNHX1\u003c/em\u003e enhances salt tolerance in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], while \u003cem\u003eOsNHX1\u003c/em\u003e and \u003cem\u003eOsNHX2\u003c/em\u003e contribute to salt adaptation in rice by regulating Na⁺ sequestration into the vacuole [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In woody plants, MdNHX from apple (\u003cem\u003eMalus domestica\u003c/em\u003e L.) and \u003cem\u003ePpNHX2\u003c/em\u003e from peach (\u003cem\u003ePrunus persica\u003c/em\u003e L.) have been shown to improve salt tolerance when heterologously expressed in tobacco [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. High salt concentrations disrupt cellular ion homeostasis, induce osmotic stress, and trigger excessive accumulation of reactive oxygen species (ROS) such as superoxide anion (O₂⁻), hydrogen peroxide (H₂O₂), and hydroxyl radical (-OH) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. These ROS cause oxidative damage to lipids, proteins, and nucleic acids, leading to membrane lipid peroxidation, metabolic disorders, and ultimately growth inhibition or even plant death [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In response, plants activate their antioxidant defense system to alleviate ROS induced damage, in which superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) play key roles: SOD catalyzes the dismutation of O₂⁻ into H₂O₂ and O₂, while POD and CAT further scavenge H₂O₂ to prevent oxidative injury [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Malondialdehyde (MDA), a product of lipid peroxidation, is widely used as a key indicator of membrane damage under stress [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Moreover, under salt stress, osmotic regulatory substances such as proline rapidly accumulate in plant cells, lowering the cellular osmotic potential to facilitate water uptake and protecting enzymes and membrane structures [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWith the publication of the sweet cherry genome sequence [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], genome wide identification and functional analysis of gene families have become feasible. To date, several stress responsive gene families, such as WRKY and AP2/ERF, have been identified in sweet cherry; however, the NHX family, which plays a central role in ion homeostasis regulation, has not yet been reported [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Given the conserved function of NHX genes in ion transport and the unique salt response mechanism of sweet cherry, a comprehensive analysis of the NHX family is necessary to fill this research gap. Through genome wide homology screening and domain validation, a total of 37 NHX family members were identified in sweet cherry. Their molecular characteristics, chromosomal distribution, phylogenetic relationships, gene structures, cis acting elements, and expression patterns in different tissues and under salt stress were systematically analyzed. Furthermore, the subcellular localization of the salt responsive gene \u003cem\u003ePavNHX37\u003c/em\u003e was verified, and its function in regulating the antioxidant system was analyzed in stably overexpressing transgenic tobacco plants. This study reveals the molecular mechanism of salt tolerance in sweet cherry and provides genetic resources for the genetic improvement of salt tolerant varieties.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Plant Materials\u003c/h2\u003e \u003cp\u003ePlant materials used in this study were in vitro plantlets of sweet cherry cultivar \u0026lsquo;Tieton\u0026rsquo; (\u003cem\u003ePrunus avium L.\u003c/em\u003e), preserved in the Laboratory of Fruit Tree Physiology and Molecular Biology, Tianshui Normal University. The cultivar was originally obtained and formally identified from the university\u0026rsquo;s Fruit Tree Germplasm Resource Bank. No wild plant materials were sampled. All experimental procedures involving plants were performed in accordance with institutional guidelines and relevant national regulations of China.\u003c/p\u003e \u003cp\u003eSweet cherry plantlets were subcultured on MS medium containing 1.0 mg/L 6-BA, 0.1 mg/L IBA, 30 g/L sucrose and 7 g/L agar (pH 5.8). Using the paper bridge method, plantlets were exposed to NaCl solutions at concentrations of 0, 50, 100 and 150 mmol/L for 24 h. Leaves were collected with three biological replicates, snap-frozen in liquid nitrogen, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for subsequent RNA extraction and reverse transcription.\u003c/p\u003e \u003cp\u003e \u003cem\u003eNicotiana benthamiana\u003c/em\u003e seeds were conserved in the same laboratory. Plants were grown in a controlled environment with a 3:1 (v/v) mixture of nutrient soil and vermiculite, maintained at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C under a 16 h light/8 h dark photoperiod. Four-week-old seedlings with consistent and vigorous growth were selected for subcellular localization analysis.\u003c/p\u003e \u003cp\u003eFor genetic transformation, tobacco seeds were surface-sterilized: rinsed with sterile water for 1 min, immersed in 75% ethanol for 40 s, treated with 5% sodium hypochlorite for 10 s, and washed 4\u0026ndash;5 times with sterile water. Sterilized seeds were sown on MS medium supplemented with 30 g/L sucrose and 7 g/L agar (pH 5.8). Seedlings with two cotyledons were used for subsequent transformation experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Sweet Cherry NHX Gene Family Identification and Physicochemical Property Analysis\u003c/h2\u003e \u003cp\u003eThe accession numbers of NHX genes were obtained from the Arabidopsis genome database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.arabidopsis.org\u003c/span\u003e\u003cspan address=\"https://www.arabidopsis.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and the coding sequence of each gene was retrieved from NCBI. The sweet cherry genome annotation file was downloaded from the NCBI genome database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/datasets/genome/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/datasets/genome/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The CDS sequences of the sweet cherry genome were converted into protein sequences and simplified using TBtools-Ⅱ v2.382. Homology alignment was performed between the protein sequences of Arabidopsis and sweet cherry, followed by a secondary alignment of the preliminarily screened protein sequences using the BLAST module of NCBI. The gene IDs of the retained sequences were recorded, and their amino acid sequences were extracted. Functional domain screening was conducted using NCBI-CDD (\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). Candidate genes lacking the specific domains of NHX genes were excluded. Through this screening, a total of 37 NHX gene family members in sweet cherry were identified. Based on the selected NHX amino acid sequences, their basic information, including amino acid number, molecular weight, isoelectric point, and hydrophilicity, was obtained using ExPASy (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/protparam/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Sweet Cherry NHX Gene Family Chromosomal Localization Subcellular Localization and Protein Secondary Structure\u003c/h2\u003e \u003cp\u003eThe chromosomal positions of sweet cherry NHX genes were predicted and visualized using TBtools-Ⅱ v2.382. Subcellular localization of the NHX gene family was predicted using WoLF PSORT (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genscript.com/wolf-psort.html\u003c/span\u003e\u003cspan address=\"https://www.genscript.com/wolf-psort.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Protein secondary structure analysis of the NHX gene family was performed using NPSA (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://npsa.lyon.inserm.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html\u003c/span\u003e\u003cspan address=\"https://npsa.lyon.inserm.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Sweet Cherry NHX Gene Family Phylogenetic Tree Construction and Synteny Analysis\u003c/h2\u003e \u003cp\u003eA phylogenetic tree was constructed using the One Step Build a ML Tree function in TBtools-Ⅱ with the neighbor-joining (NJ) method. The tree was then beautified using the ChiPlot tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.chiplot.online/#BioPlot\u003c/span\u003e\u003cspan address=\"https://www.chiplot.online/#BioPlot\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGenome and annotation files for synteny analysis were downloaded from the NCBI genome database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/datasets/genome/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/datasets/genome/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for grape (\u003cem\u003eVitis vinifera\u003c/em\u003e L.), Arabidopsis thaliana, apple (\u003cem\u003eMalus domestica\u003c/em\u003e L.), woodland strawberry (\u003cem\u003eFragaria vesca\u003c/em\u003e), and peach (\u003cem\u003ePrunus persica\u003c/em\u003e L.). Synteny analysis of sweet cherry NHX genes was performed and visualized using the synteny tool in TBtools-Ⅱ.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Sweet Cherry NHX Gene Family Gene Structure Motif and Cis-Acting Element Analysis\u003c/h2\u003e \u003cp\u003eGene structure prediction was performed using TBtools‑Ⅱ software. Conserved motif analysis was carried out using the MEME website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://meme-suite.org/\u003c/span\u003e\u003cspan address=\"http://meme-suite.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with the number of motifs set to 10. Conserved protein domains were analyzed using the NCBI‑CDD website (\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). The promoter sequences (2000bp upstream of the start codon) of NHX gene family members were extracted. Cis‑acting element screening was completed using the PlantCARE website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and visualization was performed using TBtools‑Ⅱ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Sweet Cherry NHX Gene Family Codon Usage Bias Analysis and Protein-Protein Interaction Network Analysis\u003c/h2\u003e \u003cp\u003eCodon usage bias analysis was performed using R software (version 4.3.3) with the following bioinformatics and statistical packages: seqinr (v4.2‑25) for biological sequence manipulation, Biostrings (v2.68‑1) for codon parsing of CDS sequences, and ggplot2 (v3.4‑4) for visualization.\u003c/p\u003e \u003cp\u003eThe protein sequences of the sweet cherry NHX family were used to construct a protein‑protein interaction network via the STRING database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org/\u003c/span\u003e\u003cspan address=\"https://string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Sweet Cherry NHX Gene Family Tissue-Specific Expression Profile Analysis\u003c/h2\u003e \u003cp\u003eUsing published sweet cherry tissue expression data [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] the corresponding gene accession numbers of NHX protein members were retrieved. The obtained data were organized using Excel 2007 and subsequently visualized using the ChiPlot tool.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Sweet Cherry NHX Gene Family qRT-PCR Analysis\u003c/h2\u003e \u003cp\u003ePrimers were synthesized by Shanghai Bioengineering Technology Co., Ltd. (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The PCR reaction system (total 20\u0026micro;L) consisted of 7\u0026micro;L ddH₂O, 1\u0026micro;L cDNA, 2\u0026micro;L forward and reverse primers, and 10\u0026micro;L 2\u0026times; SYBR TaKaRa Premix Ex Taq II. The PCR amplification conditions were 35 cycles as follows: 95\u0026deg;C for 30s, 95\u0026deg;C for 10s, 58\u0026deg;C for 30s, and 72\u0026deg;C for 20s. Three biological replicates and three technical replicates were performed to ensure the reliability of the results. Relative expression levels were calculated using the 2\u003csup\u003e⁻ΔΔCt\u003c/sup\u003e method.\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\u003ePrimers used in the experiment.\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\u003eUse\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNames of primers\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePrimer of sequence (5\u0026prime;-3\u0026prime;)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGene-amplification\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epCAMBIA2300-\u003cem\u003ePavNHX37\u003c/em\u003e-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGAGAACACGGGGGACGAGCTCATGGACATTGGTTTCCTGGAGA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epCAMBIA2300-\u003cem\u003ePavNHX37\u003c/em\u003e-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCACCATGGTGTCGACTCTAGACTCTACTATCTGCTGCTCCCCAA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlant identification\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e35s-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGACGCACAATCCCACTATCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"25\" rowspan=\"26\"\u003e \u003cp\u003eqRT-PCR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX03\u003c/em\u003e-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGCCGTCCAGACTGACTTGAA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX03\u003c/em\u003e-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGTGTCACCCCACGCAATTTC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX05\u003c/em\u003e-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGATCCCTACGCTGCTCCTTC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX05-\u003c/em\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGGAGACATTGGACTCGGTGG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX08\u003c/em\u003e-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGTGTCCTTGTTGGTTTCGGC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX08\u003c/em\u003e-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGAGAAGTTTCAGGTCCGCCA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX09\u003c/em\u003e-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTCCTGGCGGAGAATTTGCAT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX09-\u003c/em\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTCGAACGTCATGCACCTCAA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX17\u003c/em\u003e-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGGTGATGGCCTTGAGGGTAC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX17\u003c/em\u003e-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTGTGAATGACGCTGTGCTCT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX19\u003c/em\u003e-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTCTCGGATCTGCGTTGCTTT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX19\u003c/em\u003e-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGTGGTCGCAAAAACTGGCAT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX22\u003c/em\u003e-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAGCTCAACCAAAAACGACGC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX22\u003c/em\u003e-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCCGCCATAGTCTTCCTCGTC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX25\u003c/em\u003e-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGGTTGCCACCATGATCCAGA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX25\u003c/em\u003e-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGATGTTGGTGGCTGTTGCTG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX29\u003c/em\u003e-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGCCCTGGTCATAGGCATCTC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX29\u003c/em\u003e-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTTCCATCGTGACCGTTCGTT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX30\u003c/em\u003e-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCCTTGCTCAGTCGGTGTTCT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX30\u003c/em\u003e-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCCTCCCAAAAACAGCACAGC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX36\u003c/em\u003e-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTGGAAAGCCAGAACCTAGCG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX36\u003c/em\u003e-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAAGCGCAACAAAAGCCTCAG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX37\u003c/em\u003e-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eACTCAGGCAGCATCATGTCC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX37\u003c/em\u003e-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCGGCCATATCCCACTTTGGA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epavEF1-a1-F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTGAGAGGCTGACTGTGCTGTTC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epavEF1-a1-R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGGAGTAGTGGCATCCATCTTGTT\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 \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. \u003cem\u003ePavNHX37\u003c/em\u003e Overexpression Vector Construction and Nicotiana benthamiana Subcellular Localization\u003c/h2\u003e \u003cp\u003eThe overexpression vector pCAMBIA2300\u003cem\u003e‑PavNHX37\u003c/em\u003e‑GFP was constructed. Primers were designed with restriction sites (\u003cem\u003eSac\u003c/em\u003eI and \u003cem\u003eXba\u003c/em\u003eI) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The construct was then transformed into Agrobacterium tumefaciens strain GV3101. After overnight shaking, the bacterial suspension was stored at -80\u0026deg;C.\u003c/p\u003e \u003cp\u003eThe Agrobacterium cells harboring the fusion vector were collected by centrifugation after overnight shaking, washed twice with sterile water, and centrifuged again, and the supernatant was discarded. The bacterial pellet was resuspended in resuspension buffer (10mM 2-ethanesulfonic acid, MES pH 5.8, 10mM MgCl₂, 200\u0026micro;M Acetosyringone, AS) to an OD₆₀₀ of 0.6\u0026ndash;0.8, followed by shaking at 180rpm at 28\u0026deg;C in darkness for 2\u0026ndash;3h. The resuspended bacterial suspension was injected into tobacco leaves that had been water‑deprived for two days using a needleless syringe. After dark culture for 24h, the plants were transferred to normal light conditions for an additional 24h. Subcellular localization of the pCAMBIA2300‑\u003cem\u003ePavNHX37\u003c/em\u003e‑GFP fusion protein was observed under a laser confocal scanning microscope (LSM710, Carl Zeiss, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Nicotiana benthamiana Stable Genetic Transformation and Identification\u003c/h2\u003e \u003cp\u003eHealthy and sterile Nicotiana benthamiana test-tube plantlets were selected, and their leaves were cut into 5mm \u0026times; 5mm pieces. These leaf pieces were placed on pre-culture medium and cultured in darkness for 2\u0026ndash;3 days. The pre-cultured leaf pieces were immersed in an Agrobacterium solution containing the overexpression vector pCAMBIA230-\u003cem\u003ePavNHX37\u003c/em\u003e-GFP. After removing excess bacterial solution, the leaf pieces were transferred to co-culture medium and cultured in darkness for another 3 days. Following co-cultivation, the leaf pieces were transferred to selection medium and cultured under light conditions, with subculture to fresh medium every 1\u0026ndash;2 weeks. Once new shoots regenerated, they were transferred to rooting medium. After roots had developed, leaf samples were collected to test the positivity of transgenic plants.\u003c/p\u003e \u003cp\u003eAfter obtaining the overexpressing tobacco plants, leaves were randomly selected from different plants and individually labeled. Genomic DNA was extracted from the leaves using the TIANGEN Plant Genomic DNA Extraction Kit (Beijing, China). The extracted DNA was used as a template for PCR amplification with gene-specific primers. The amplification products were detected by agarose gel electrophoresis to complete the molecular positive identification of the overexpressing tobacco plants.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11. Overexpressing \u003cem\u003ePavNHX37\u003c/em\u003e Tobacco Enzyme Activity Assay\u003c/h2\u003e \u003cp\u003eTobacco leaf tissue samples from wild-type (WT) and overexpressing (OE) lines treated for 72 h under different conditions were collected. The samples were flash-frozen in liquid nitrogen and ground into powder. Then 0.1g of powder was weighed and pre-cooled extraction buffer (50mmol/L Tris(hydroxymethyl)aminomethane, Tris-HCl pH 8.0, 1mmol/L Ethylenediaminetetraacetic acid, EDTA, 1mmol/L Phenylmethylsulfonyl fluoride, PMSF, 1% Polyvinylpyrrolidone, PVP) was added. After homogenization on ice, the mixture was centrifuged at 12 000r/min for 20min at 4\u0026deg;C. The supernatant was collected as the crude enzyme extract. The activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) as well as the contents of malondialdehyde (MDA) and proline (Pro) were determined using commercial assay kits purchased from Suzhou Grace Biotechnology Co., Ltd.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12. Data Processing and Statistical Analysis\u003c/h2\u003e \u003cp\u003eAll experimental data were statistically analyzed using SPSS 23.0 software. One-way analysis of variance (ANOVA) was performed followed by an ANOVA test (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Graphs were generated using Origin Pro 2024 software. All measured data were derived from three independent biological replicates.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Result","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Sweet Cherry \u003cem\u003epavNHX\u003c/em\u003e Gene Family Identification and Secondary Structure Analysis\u003c/h2\u003e \u003cp\u003eA total of 37 NHX family genes were identified in sweet cherry through homology analysis and were named \u003cem\u003ePavNHX01\u003c/em\u003e-\u003cem\u003ePavNHX37\u003c/em\u003e. These genes were distributed on eight chromosomes of sweet cherry. Among them, ten genes were located on chromosomes 2 and 6, followed by five genes on chromosomes 1 and 4, three genes on chromosome 5, two genes on chromosome 8, and only one gene on chromosomes 3 and 7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The number of amino acids encoded by NHX members ranged from 150 to 1223, with substantial variation even among genes within the same subfamily. The molecular weights of the proteins ranged from 15,971.08 to 132,067.04 Da, the theoretical isoelectric points (pI) ranged from 4.6 to 9.14, and the instability indices ranged from 24.71 to 49.37. The hydrophilicity values ranged from 0.060 to 1.127, with higher values indicating stronger hydrophilicity. Subcellular localization prediction showed that all genes were localized to the plasma membrane except \u003cem\u003ePavNHX21\u003c/em\u003e, which was localized to the vacuole. The atomic composition of \u003cem\u003ePavNHX36\u003c/em\u003e could not be calculated due to ambiguous bases in the analyzed sequence (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe secondary structure of sweet cherry NHX family proteins was dominated by α‑helices (42.00%\u0026ndash;68.68%) and random coils (19.95%\u0026ndash;39.27%), followed by β‑turns (1.18%\u0026ndash;4.55%) and extended strands (8.67%\u0026ndash;16.64%) (\u003cb\u003eTable\u0026nbsp;3\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ephysical and chemical properties of \u003cem\u003ePavNHX\u003c/em\u003e gene family.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\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 \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003egen ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003egen name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLocation of Chromosome\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAmino acid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMolecular weight/ KD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eInstability index:\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eAliphatic index:\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003ehydropathicity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eFormula\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eSubcelluar localization\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_001618-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX01\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e551\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e60730.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e8.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e45.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e107.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.499\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e2790\u003c/sub\u003eH\u003csub\u003e4348\u003c/sub\u003eN\u003csub\u003e696\u003c/sub\u003eO\u003csub\u003e768\u003c/sub\u003eS\u003csub\u003e25\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_003199-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX02\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e816\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e88278.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e8.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e37.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e109.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e4008\u003c/sub\u003eH\u003csub\u003e6449\u003c/sub\u003eN\u003csub\u003e1041\u003c/sub\u003eO\u003csub\u003e1115\u003c/sub\u003eS\u003csub\u003e38\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_004934-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX03\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1148\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e127491.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e39.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e102.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.096\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e5785\u003c/sub\u003eH\u003csub\u003e9083\u003c/sub\u003eN\u003csub\u003e1499\u003c/sub\u003eO\u003csub\u003e1669\u003c/sub\u003eS\u003csub\u003e36\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_005739-T2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX04\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e255\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e27578.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e29.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e102.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.089\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e1228\u003c/sub\u003eH\u003csub\u003e1957\u003c/sub\u003eN\u003csub\u003e317\u003c/sub\u003eO\u003csub\u003e391\u003c/sub\u003eS\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_005739-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX05\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e591\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e63803.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e29.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e122.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.581\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e2907\u003c/sub\u003eH\u003csub\u003e4675\u003c/sub\u003eN\u003csub\u003e731\u003c/sub\u003eO\u003csub\u003e827\u003c/sub\u003eS\u003csub\u003e22\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_008475-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX06\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e765\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e84271.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e38.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e112.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.399\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e3863\u003c/sub\u003eH\u003csub\u003e6115\u003c/sub\u003eN\u003csub\u003e973\u003c/sub\u003eO\u003csub\u003e1070\u003c/sub\u003eS\u003csub\u003e30\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_008684-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX07\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e765\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e84271.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e38.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e112.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.399\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e3863\u003c/sub\u003eH\u003csub\u003e6115\u003c/sub\u003eN\u003csub\u003e973\u003c/sub\u003eO\u003csub\u003e1070\u003c/sub\u003eS\u003csub\u003e30\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_008760-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX08\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e841\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e92082.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e38.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e102.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.179\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e4099\u003c/sub\u003eH\u003csub\u003e6556\u003c/sub\u003eN\u003csub\u003e1092\u003c/sub\u003eO\u003csub\u003e1221\u003c/sub\u003eS\u003csub\u003e44\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_011354-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX09\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e912\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e100683.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e34.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e98.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.084\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e4496\u003c/sub\u003eH\u003csub\u003e7133\u003c/sub\u003eN\u003csub\u003e1195\u003c/sub\u003eO\u003csub\u003e1326\u003c/sub\u003eS\u003csub\u003e48\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_012393-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX10\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e813\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e90061.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e9.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e40.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e109.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.264\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e4100\u003c/sub\u003eH\u003csub\u003e6548\u003c/sub\u003eN\u003csub\u003e1072\u003c/sub\u003eO\u003csub\u003e1134\u003c/sub\u003eS\u003csub\u003e33\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_012394-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX11\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1223\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e132067.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e41.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e104.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e5854\u003c/sub\u003eH\u003csub\u003e9452\u003c/sub\u003eN\u003csub\u003e1596\u003c/sub\u003eO\u003csub\u003e1801\u003c/sub\u003eS\u003csub\u003e33\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_012399-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX12\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e685\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e75828.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e37.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e111.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.416\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e3426\u003c/sub\u003eH\u003csub\u003e5461\u003c/sub\u003eN\u003csub\u003e881\u003c/sub\u003eO\u003csub\u003e962\u003c/sub\u003eS\u003csub\u003e45\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_012409-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX13\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e292\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e32247.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e8.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e32.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e124.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.623\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e1502\u003c/sub\u003eH\u003csub\u003e2375\u003c/sub\u003eN\u003csub\u003e367\u003c/sub\u003eO\u003csub\u003e393\u003c/sub\u003eS\u003csub\u003e12\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_012716-T2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX14\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e15971.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e24.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e126.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.127\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e743\u003c/sub\u003eH\u003csub\u003e1164\u003c/sub\u003eN\u003csub\u003e176\u003c/sub\u003eO\u003csub\u003e193\u003c/sub\u003eS\u003csub\u003e10\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_012716-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX15\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e816\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e90019.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e44.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e117.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e4064\u003c/sub\u003eH\u003csub\u003e6546\u003c/sub\u003eN\u003csub\u003e1076\u003c/sub\u003eO\u003csub\u003e1139\u003c/sub\u003eS\u003csub\u003e41\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_017854-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX16\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e572\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e61910.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e31.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e117.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.622\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e2832\u003c/sub\u003eH\u003csub\u003e4498\u003c/sub\u003eN\u003csub\u003e698\u003c/sub\u003eO\u003csub\u003e797\u003c/sub\u003eS\u003csub\u003e26\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_031503-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX17\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e540\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e59315.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e40.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e99.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.427\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e2725\u003c/sub\u003eH\u003csub\u003e4153\u003c/sub\u003eN\u003csub\u003e675\u003c/sub\u003eO\u003csub\u003e761\u003c/sub\u003eS\u003csub\u003e24\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_031504-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX18\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e824\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e90950.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e45.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e109.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.331\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e4111\u003c/sub\u003eH\u003csub\u003e6568\u003c/sub\u003eN\u003csub\u003e1072\u003c/sub\u003eO\u003csub\u003e1154\u003c/sub\u003eS\u003csub\u003e46\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_032382-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX19\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e540\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e59315.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e40.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e99.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.427\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e2725\u003c/sub\u003eH\u003csub\u003e4153\u003c/sub\u003eN\u003csub\u003e675\u003c/sub\u003eO\u003csub\u003e761\u003c/sub\u003eS\u003csub\u003e24\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_035179-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX20\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e551\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e61702.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e37.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e112.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e2853\u003c/sub\u003eH\u003csub\u003e4446\u003c/sub\u003eN\u003csub\u003e702\u003c/sub\u003eO\u003csub\u003e758\u003c/sub\u003eS\u003csub\u003e31\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_035478-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX21\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e525\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e58253.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e32.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e115.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.601\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e2702\u003c/sub\u003eH\u003csub\u003e4195\u003c/sub\u003eN\u003csub\u003e665\u003c/sub\u003eO\u003csub\u003e726\u003c/sub\u003eS\u003csub\u003e20\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eVacuole\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_024568-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX22\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e856\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e92668.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e35.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e108.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.355\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e4161\u003c/sub\u003eH\u003csub\u003e6647\u003c/sub\u003eN\u003csub\u003e1105\u003c/sub\u003eO\u003csub\u003e1200\u003c/sub\u003eS\u003csub\u003e41\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_025222-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX23\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e790\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e87368.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e37.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e109.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.331\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e3979\u003c/sub\u003eH\u003csub\u003e6257\u003c/sub\u003eN\u003csub\u003e1025\u003c/sub\u003eO\u003csub\u003e1118\u003c/sub\u003eS\u003csub\u003e32\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_025223-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX24\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e86656.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e8.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e40.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e114.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.415\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e3915\u003c/sub\u003eH\u003csub\u003e6337\u003c/sub\u003eN\u003csub\u003e1045\u003c/sub\u003eO\u003csub\u003e1093\u003c/sub\u003eS\u003csub\u003e35\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_018802-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX25\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e808\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e87403.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e8.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e36.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e108.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.351\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e3932\u003c/sub\u003eH\u003csub\u003e6343\u003c/sub\u003eN\u003csub\u003e1057\u003c/sub\u003eO\u003csub\u003e1110\u003c/sub\u003eS\u003csub\u003e38\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_018803-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX26\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e758\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e83582.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e40.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e107.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.365\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e3795\u003c/sub\u003eH\u003csub\u003e5999\u003c/sub\u003eN\u003csub\u003e963\u003c/sub\u003eO\u003csub\u003e1072\u003c/sub\u003eS\u003csub\u003e41\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_018804-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX27\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e754\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e83357.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e8.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e37.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e105.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.343\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e3790\u003c/sub\u003eH\u003csub\u003e5972\u003c/sub\u003eN\u003csub\u003e984\u003c/sub\u003eO\u003csub\u003e1045\u003c/sub\u003eS\u003csub\u003e41\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_018805-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX28\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e759\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e83622.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e49.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e111.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.389\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e3804\u003c/sub\u003eH\u003csub\u003e6048\u003c/sub\u003eN\u003csub\u003e966\u003c/sub\u003eO\u003csub\u003e1066\u003c/sub\u003eS\u003csub\u003e39\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_018806-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX29\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e696\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e77297.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e48.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e105.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.312\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e3465\u003c/sub\u003eH\u003csub\u003e5553\u003c/sub\u003eN\u003csub\u003e907\u003c/sub\u003eO\u003csub\u003e980\u003c/sub\u003eS\u003csub\u003e53\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_018816-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX30\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e757\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e83456.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e45.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e114.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e3794\u003c/sub\u003eH\u003csub\u003e6037\u003c/sub\u003eN\u003csub\u003e967\u003c/sub\u003eO\u003csub\u003e1069\u003c/sub\u003eS\u003csub\u003e36\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_021849-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX31\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e858\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e94339.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e35.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e110.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.371\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e4320\u003c/sub\u003eH\u003csub\u003e6791\u003c/sub\u003eN\u003csub\u003e1105\u003c/sub\u003eO\u003csub\u003e1194\u003c/sub\u003eS\u003csub\u003e32\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_021851-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX32\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e848\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e91916.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e9.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e39.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e107.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.294\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e4159\u003c/sub\u003eH\u003csub\u003e6695\u003c/sub\u003eN\u003csub\u003e1117\u003c/sub\u003eO\u003csub\u003e1163\u003c/sub\u003eS\u003csub\u003e30\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_022777-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX33\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e782\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e84719.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e8.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e43.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e114.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e3822\u003c/sub\u003eH\u003csub\u003e6192\u003c/sub\u003eN\u003csub\u003e1030\u003c/sub\u003eO\u003csub\u003e1072\u003c/sub\u003eS\u003csub\u003e31\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_022826-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX34\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e543\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e59396.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e36.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e107.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e2743\u003c/sub\u003eH\u003csub\u003e4269\u003c/sub\u003eN\u003csub\u003e665\u003c/sub\u003eO\u003csub\u003e760\u003c/sub\u003eS\u003csub\u003e21\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_039269-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX35\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e539\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e59876.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e8.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e34.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e113.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.545\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e2770\u003c/sub\u003eH\u003csub\u003e4330\u003c/sub\u003eN\u003csub\u003e690\u003c/sub\u003eO\u003csub\u003e744\u003c/sub\u003eS\u003csub\u003e21\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_029070-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX36\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e616\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e66511.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e35.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e116.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.574\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUN_031269-T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePavNHX37\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003echr8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e785\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e85778.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e35.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e118.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.523\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eC\u003csub\u003e3952\u003c/sub\u003eH\u003csub\u003e6290\u003c/sub\u003eN\u003csub\u003e994\u003c/sub\u003eO\u003csub\u003e1080\u003c/sub\u003eS\u003csub\u003e24\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003ePlasma membrane\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSecondary structure analysis of \u003cem\u003ePavNHX\u003c/em\u003e protein.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGen name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAlpha helix\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBeta turn\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eExtended strand\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRandom coil\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e48.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e35.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e45.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e36.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e55.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e30.92\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e57.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e30.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e59.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e28.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e46.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e33.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e36.98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e38.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e68.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e19.95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e44.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e36.78\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e46.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e33.96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e45.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e33.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e43.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e37.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e62.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e21.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e26.57\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e44.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e37.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e51.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e31.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e51.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e31.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e44.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e35.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e46.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e34.56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e44.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e37.73\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e45.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e36.46\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e45.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e35.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e44.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e36.76\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e44.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e34.88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e44.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e35.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e44.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e35.57\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e44.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e35.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e45.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e34.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e38.34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e45.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e34.78\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e43.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e39.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e49.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e33.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e49.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e33.58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e59.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e25.81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e55.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e29.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePavNHX37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e47.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e35.39\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 \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Sweet Cherry \u003cem\u003epavNHX\u003c/em\u003e Gene Family Evolutionary Analysis\u003c/h2\u003e \u003cp\u003eTo further understand the evolutionary characteristics of the NHX gene family, a phylogenetic tree was constructed using NHX family members from sweet cherry and Arabidopsis thaliana (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The family members were divided into three groups. Group I contained eight sweet cherry genes and eight Arabidopsis genes. Group II and Group III consisted entirely of sweet cherry NHX family members, with six and twenty-three members respectively. Among the three groups, only Group I contained Arabidopsis NHX family members, indicating that NHX gene family members are highly conserved within the groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Sweet Cherry \u003cem\u003epavNHX\u003c/em\u003e Gene Family Protein Structure and Motif Analysis\u003c/h2\u003e \u003cp\u003eThe conserved structural domains of the sweet cherry NHX gene family were analyzed online using the MEME software, and the family structures were compared and localized. Among the members, \u003cem\u003ePavNHX03\u003c/em\u003e, \u003cem\u003ePavNHX04\u003c/em\u003e, \u003cem\u003ePavNHX18\u003c/em\u003e, \u003cem\u003ePavNHX17\u003c/em\u003e, and \u003cem\u003ePavNHX33\u003c/em\u003e contained only one motif. \u003cem\u003ePavNHX22\u003c/em\u003e, \u003cem\u003ePavNHX20\u003c/em\u003e, \u003cem\u003ePavNHX08, PavNHX07\u003c/em\u003e, \u003cem\u003ePavNHX21\u003c/em\u003e, \u003cem\u003ePavNHX32\u003c/em\u003e, \u003cem\u003ePavNHX31\u003c/em\u003e, \u003cem\u003ePavNHX02\u003c/em\u003e, \u003cem\u003ePavNHX24\u003c/em\u003e, \u003cem\u003ePavNHX23\u003c/em\u003e, \u003cem\u003ePavNHX30\u003c/em\u003e, \u003cem\u003ePavNHX10\u003c/em\u003e, \u003cem\u003ePavNHX12\u003c/em\u003e, \u003cem\u003ePavNHX13\u003c/em\u003e, \u003cem\u003ePavNHX16\u003c/em\u003e, \u003cem\u003ePavNHX06\u003c/em\u003e, \u003cem\u003ePavNHX26\u003c/em\u003e, \u003cem\u003ePavNHX28\u003c/em\u003e, and \u003cem\u003ePavNHX27\u003c/em\u003e all shared the same motifs, indicating a high degree of conservation and suggesting that their functions are largely similar. Furthermore, \u003cem\u003ePavNHX37\u003c/em\u003e, \u003cem\u003ePavNHX01\u003c/em\u003e, and \u003cem\u003ePavNHX35\u003c/em\u003e contained only three motifs, while \u003cem\u003ePavNHX34\u003c/em\u003e contained only two motifs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eProtein structure analysis of the sweet cherry NHX gene family revealed that these genes contain 0 to 22 introns. \u003cem\u003ePavNHX25\u003c/em\u003e contained no intron, while the longest intron was found in \u003cem\u003ePavNHX12\u003c/em\u003e. Additionally, \u003cem\u003ePavNHX05\u003c/em\u003e, \u003cem\u003ePavNHX35\u003c/em\u003e, \u003cem\u003ePavNHX15\u003c/em\u003e, \u003cem\u003ePavNHX14\u003c/em\u003e, \u003cem\u003ePavNHX17\u003c/em\u003e, \u003cem\u003ePavNHX33\u003c/em\u003e, \u003cem\u003ePavNHX34\u003c/em\u003e, \u003cem\u003ePavNHX01\u003c/em\u003e, \u003cem\u003ePavNHX09\u003c/em\u003e, \u003cem\u003ePavNHX26\u003c/em\u003e, \u003cem\u003ePavNHX32\u003c/em\u003e, \u003cem\u003ePavNHX24\u003c/em\u003e, and \u003cem\u003ePavNHX23\u003c/em\u003e possessed exons, whereas the remaining genes lacked exons. Notable differences in exon length and position were observed, which may contribute to functional divergence among these genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Sweet Cherry \u003cem\u003epavNHX\u003c/em\u003e Gene Family Promoter Cis-Acting Element Prediction\u003c/h2\u003e \u003cp\u003eAmong the 37 predicted cis-acting promoter elements of the sweet cherry NHX gene family, 32 response elements were identified. All NHX gene family members contained the following elements: a cis-acting regulatory element involved in MeJA responsiveness, a cis-acting element involved in salicylic acid responsiveness, a cis-acting element involved in abscisic acid responsiveness, a MYB binding site involved in drought inducibility, a cis-acting element involved in auxin responsiveness, a cis-acting regulatory element root specific, an auxin-responsive element, a MYB-like binding site in flavonoid biosynthetic genes regulation, and a cis-acting regulatory element related to meristem specific activation. Different members contained different response elements. In addition to the elements listed above, \u003cem\u003ePavNHX01\u003c/em\u003e, \u003cem\u003ePavNHX11\u003c/em\u003e, and \u003cem\u003ePavNHX27\u003c/em\u003e contained a cis-acting regulatory element involved in zein metabolism regulation. \u003cem\u003ePavNHX04\u003c/em\u003e and \u003cem\u003ePavNHX05\u003c/em\u003e lacked the cis-acting element involved in low-temperature responsiveness. \u003cem\u003ePavNHX23\u003c/em\u003e and \u003cem\u003ePavNHX12\u003c/em\u003e contained the fewest response elements, only 12 elements, whereas \u003cem\u003ePavNHX01\u003c/em\u003e contained the most with 23 elements (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Sweet Cherry \u003cem\u003epavNHX\u003c/em\u003e Gene Family Tissue-Specific Expression Analysis\u003c/h2\u003e \u003cp\u003eTo elucidate the functions of \u003cem\u003ePavNHX\u003c/em\u003e genes in sweet cherry growth and development, the expression patterns of 37 \u003cem\u003ePavNHX\u003c/em\u003e genes across 15 tissues and fruit developmental stages were analyzed. After Log₂(FPKM\u0026thinsp;+\u0026thinsp;1) normalization, a heatmap was generated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The results showed that the \u003cem\u003ePavNHX\u003c/em\u003e gene family exhibited significant tissue-specific and stage-specific expression characteristics. All members could be clearly divided into two major expression clusters. The first cluster was a high-expression cluster, containing genes such as \u003cem\u003ePavNHX24\u003c/em\u003e, \u003cem\u003ePavNHX23\u003c/em\u003e, \u003cem\u003ePavNHX34\u003c/em\u003e, and \u003cem\u003ePavNHX18\u003c/em\u003e. These genes maintained consistently high expression levels in vegetative organs including annual shoots, young leaves, and mature leaves. Among them, \u003cem\u003ePavNHX24\u003c/em\u003e and \u003cem\u003ePavNHX34\u003c/em\u003e showed the highest expression levels and stable expression in most tissues. The second cluster was a low-expression cluster, containing genes such as \u003cem\u003ePavNHX22\u003c/em\u003e, \u003cem\u003ePavNHX30\u003c/em\u003e, \u003cem\u003ePavNHX29\u003c/em\u003e, and \u003cem\u003ePavNHX06\u003c/em\u003e. These genes exhibited extremely low expression in most vegetative organs but displayed distinct targeted expression characteristics: \u003cem\u003ePavNHX29\u003c/em\u003e was significantly upregulated at stem stages 2\u0026ndash;3; \u003cem\u003ePavNHX30\u003c/em\u003e and \u003cem\u003ePavNHX22\u003c/em\u003e showed elevated expression in middle-to-late fruit development stages; PavNHX06 and \u003cem\u003ePavNHX08\u003c/em\u003e accumulated a small amount of expression in floral organs. In addition, some genes such as \u003cem\u003ePavNHX04\u003c/em\u003e and \u003cem\u003ePavNHX16\u003c/em\u003e maintained low expression levels across all 15 tissues and stages without obvious tissue preference.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Sweet Cherry \u003cem\u003epavNHX\u003c/em\u003e Gene Family Synteny Analysis\u003c/h2\u003e \u003cp\u003eIntraspecific synteny analysis of the sweet cherry NHX gene family revealed three pairs of duplicated genes located on chromosomes 1, 7, 6, and 2. However, no linked genes were found on chromosomes 3, 4, 5, and 8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). To further elucidate the phylogenetic mechanism of the sweet cherry NHX gene family as well as the relationship between paralogy and orthology, synteny analysis was performed between sweet cherry and five representative species. The results showed that sweet cherry shared 16 syntenic gene pairs with grape berry, 17 with Arabidopsis thaliana, 12 with apple, 22 with woodland strawberry, and 30 with peach (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Some NHX genes were found to be associated with more than two syntenic gene pairs, while others had no syntenic counterpart. These results indicate that syntenic relationships exist both within the sweet cherry NHX gene family and between sweet cherry and other species, and that some family genes are closely linked but not entirely orthologous.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Sweet Cherry NHX Gene Family Codon Usage Bias Analysis\u003c/h2\u003e \u003cp\u003eThe relative synonymous codon usage (RSCU) of the 37 sweet cherry NHX genes was analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). RSCU\u0026thinsp;\u0026gt;\u0026thinsp;1 indicates preferred codons. RSCU\u0026thinsp;\u0026lt;\u0026thinsp;1 indicates non-preferred codons. RSCU\u0026thinsp;=\u0026thinsp;1 indicates no usage bias. For leucine (Leu) and serine (Ser) which have two codon families each they were analyzed separately as Leu1/Leu2 and Ser1/Ser2. All 22 amino acids showed varying degrees of synonymous codon usage bias. The total RSCU values varied among amino acids. Leu1 had the highest total RSCU value significantly higher than Leu2. The total RSCU values of alanine (Ala) arginine (Arg) glycine (Gly) proline (Pro) threonine (Thr) and valine (Val) were all close to 4. The total RSCU values of asparagine (Asn) aspartic acid (Asp) cysteine (Cys) glutamine (Gln) glutamic acid (Glu) histidine (His) isoleucine (Ile) lysine (Lys) methionine (Met) phenylalanine (Phe) threonine (Thr) tryptophan (Trp) and tyrosine (Tyr) were all close to 2. In addition, the rare codons showed specific RSCU values. For example, the RSCU value of Trp\u0026rsquo;s UGG was approximately 2. Overall, the preferred codons of the 37 sweet cherry NHX genes all ended with U or C while non-preferred codons tended to end with A or G. This indicates that the synonymous codon usage of these genes exhibits a significant bias toward U or C endings.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.8. Sweet Cherry NHX Gene Family Protein‑Protein Interaction Network Analysis\u003c/h2\u003e \u003cp\u003eThe interaction network analysis tool was used to predict the interaction relationships among sweet cherry PavNHX proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). The PavNHX gene family members formed a complete interaction network. Thirty members showed interaction connections while seven members were isolated nodes indicating extensive interactions among family members. PavNHX35, PavNHX22 and PavNHX06 were the core nodes in the network. These nodes had the largest size and the darkest color with interaction strength close to the highest threshold value of 0.900. They also exhibited dense connections with many surrounding genes making them key hubs in the regulatory network. PavNHX19, PavNHX36 and PavNHX14 showed intermediate node sizes and interaction strengths. In contrast PavNHX12, PavNHX07 and PavNHX27 had the smallest node sizes and the lightest colors with relatively low interaction strength. These genes had only single connections with a few genes and were located at the periphery of the interaction network.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.9. Sweet Cherry NHX Gene Family Member Expression Analysis\u003c/h2\u003e \u003cp\u003eTo further analyze the expression patterns of gene members twelve genes were selected based on the gene family phylogenetic tree for qRT-PCR analysis. The qRT-PCR results showed that the expression levels of six sweet cherry NHX gene family members decreased with increasing salt concentration. Compared with the control \u003cem\u003ePavNHX03\u003c/em\u003e, \u003cem\u003ePavNHX08\u003c/em\u003e and \u003cem\u003ePavNHX17\u003c/em\u003e showed significantly reduced expression under high salt stress among which \u003cem\u003epavNHX17\u003c/em\u003e decreased by 10-fold. The relative expression levels of \u003cem\u003ePavNHX19\u003c/em\u003e and \u003cem\u003ePavNHX36\u003c/em\u003e did not change significantly under the salt stress. In addition, the relative expression trends of \u003cem\u003ePavNHX09\u003c/em\u003e and \u003cem\u003ePavNHX25\u003c/em\u003e first decreased and then increased. Furthermore, we found that the relative expression of \u003cem\u003ePavNHX37\u003c/em\u003e increased significantly reaching 8.9-fold that of the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.10. \u003cem\u003ePavNHX37\u003c/em\u003e Sequence Analysis and Subcellular Localization\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003ePavNHX37\u003c/em\u003e gene is located on sweet cherry chromosome 8 at positions 207796 to 211408. Its full length is 1991 bp. The CDS coding region is 1656 bp in length encoding 551 amino acids (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003ea). A conserved Na⁺/H⁺ exchanger domain is present at amino acid positions 34 to 455. The fusion expression vector pCAMBIA2300-\u003cem\u003ePavNHX37\u003c/em\u003e-GFP was used (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eb). The constructed fusion expression vector and the pCAMBIA2300-GFP negative control were injected into tobacco leaves via Agrobacterium. The results showed that the negative control was expressed in both the nucleus and the plasma membrane whereas \u003cem\u003ePavNHX37\u003c/em\u003e was localized to the plasma membrane. This result is consistent with the subcellular localization prediction (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.11. \u003cem\u003ePavNHX37\u003c/em\u003e Overexpressing Tobacco Functional Verification\u003c/h2\u003e \u003cp\u003ePositive transgenic lines were screened by genomic PCR using gene-specific primers. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003ea a specific band of approximately 1500 bp was successfully amplified in most candidate overexpression lines. This band was consistent with the positive control (P0) and the expected size of the target gene fragment. In contrast wild-type (WT) plants showed no such specific band. Four lines OE-8 OE-13 OE-14 and OE-17 failed to amplify the target gene fragment and were therefore excluded from subsequent experiments.\u003c/p\u003e \u003cp\u003eThe expression levels of the target gene in wild-type and overexpressing lines under different NaCl treatments were detected by qRT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003ec). Under normal control conditions (0 mM NaCl) the basal expression level of the target gene was very low in wild-type plants. All tested overexpression lines showed significantly elevated gene expression levels approximately 40-fold higher than that of the wild-type (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This confirmed that the exogenous gene had been successfully overexpressed.\u003c/p\u003e \u003cp\u003eWild-type plants and the selected overexpression lines were treated with 0 50 100 and 150 mM NaCl solutions. Under non-stress conditions (0 mM NaCl) no significant differences in growth status or morphological characteristics were observed between wild-type and overexpression lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003eb). This indicated that overexpression of the target gene did not affect normal plant growth and development. With increasing NaCl concentration wild-type plants showed obvious growth inhibition leaf yellowing and wilting. In contrast the overexpression lines exhibited significantly enhanced salt tolerance. Under 50 mM NaCl treatment the overexpression lines showed greater growth vigor larger leaves and higher biomass compared to wild-type plants. Under moderate (100 mM NaCl) and severe (150 mM NaCl) salt stress the growth advantage of the OE lines became more pronounced. Notably even under 150 mM NaCl treatment the OE lines maintained good leaf condition and vigorous growth whereas wild-type plants displayed severe growth dwarfism and leaf senescence.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.12. Overexpressing \u003cem\u003ePavNHX37\u003c/em\u003e Tobacco Enzyme Activity Assay\u003c/h2\u003e \u003cp\u003eThe enzyme activity assay results after NaCl treatment of overexpressing tobacco showed that proline accumulation in overexpressing plants was significantly higher than that in wild-type plants while MDA content was significantly lower. This indicated enhanced osmotic regulation capacity and reduced oxidative damage. Meanwhile the activities of SOD POD and CAT antioxidant enzymes were significantly increased in overexpressing plants. Under 150 mM NaCl treatment the activities of these three enzymes reached 3.0-fold 3.2-fold and 1.2-fold those of wild-type plants respectively. Overexpression of the target gene significantly enhanced salt tolerance in tobacco plants by strengthening osmotic protection reducing oxidative damage and increasing reactive oxygen species scavenging capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study 37 NHX gene family members were identified in sweet cherry for the first time through genome‑wide homology alignment combined with domain validation. This number is significantly higher than that in cotton which has 24 members [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The 37 \u003cem\u003ePavNHX\u003c/em\u003e genes were unevenly distributed on eight chromosomes. Chromosomes 2 and 6 each contained 10 members suggesting that these chromosomes may be enriched regions for the NHX gene family [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The amino acid lengths (150\u0026ndash;1223 amino acids) and molecular weights (15.97\u0026ndash;132.07 kD) of \u003cem\u003ePavNHX\u003c/em\u003e proteins varied considerably. Their secondary structures were dominated by α‑helices (42.00%\u0026ndash;68.68%) and random coils (19.95%\u0026ndash;39.27%). This is highly consistent with the structural features of NHX proteins as transmembrane transporters: α‑helices typically form transmembrane domains to mediate ion transmembrane transport while random coils may participate in protein‑protein or protein‑molecule interactions [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Furthermore, subcellular localization prediction showed that all \u003cem\u003ePavNHX\u003c/em\u003e members were localized to the plasma membrane except \u003cem\u003ePavNHX21\u003c/em\u003e which was localized to the vacuole. This result is consistent with the classification of the oat NHX family into plasma membrane (PM) vacuolar (Vac) and endosomal (Endo) subfamilies [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePhylogenetic analysis divided the \u003cem\u003ePavNHX\u003c/em\u003e family into three groups. Group I contained eight sweet cherry members and eight Arabidopsis members. This suggests that genes in this group are highly conserved during evolution and may perform basic functions in plant ion transport [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Group II (6) and Group III (22) contained only sweet cherry members. They are speculated to be sweet-cherry-specific expanded subfamilies which is similar to the conserved subfamily results of the mung bean NHX family [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Synteny analysis showed that sweet cherry shared 30 syntenic gene pairs with peach which belongs to the same Prunus genus. This number is significantly higher than the numbers with grape (16) and Arabidopsis (17). This pattern is consistent with the phylogenetic relationships among these species [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe tissue expression profiles of \u003cem\u003ePavNHX\u003c/em\u003e genes showed significant specificity. High-expression cluster genes such as \u003cem\u003ePavNHX24\u003c/em\u003e and \u003cem\u003ePavNHX34\u003c/em\u003e were consistently highly expressed in vegetative organs including annual shoots and young leaves. Low-expression cluster genes such as \u003cem\u003ePavNHX29\u003c/em\u003e and \u003cem\u003ePavNHX30\u003c/em\u003e were upregulated at specific stages of stem development or during late fruit maturation. In tobacco \u003cem\u003eNtNHX1-3\u003c/em\u003e showed high expression in leaves but low or no expression in other tissues [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. As photosynthetic organs and primary sites of salt stress response leaves require sustained ion transport capacity to maintain physiological functions [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Promoter cis-element analysis revealed that all \u003cem\u003ePavNHX\u003c/em\u003e genes contained MeJA responsiveness elements salicylic acid responsiveness elements ABA responsiveness elements and drought-inducible MYB binding sites. Salt stress often occurs together with drought stress. ABA as a core stress-responsive hormone can maintain ion balance by regulating NHX gene expression [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This is consistent with the salt-stress response patterns of \u003cem\u003ePavNHX\u003c/em\u003e genes observed in this study.\u003c/p\u003e \u003cp\u003eqRT-PCR analysis of 12 representative \u003cem\u003ePavNHX\u003c/em\u003e genes under salt stress revealed distinct expression patterns. Six genes including \u003cem\u003ePavNHX03\u003c/em\u003e and \u003cem\u003ePavNHX17\u003c/em\u003e showed decreased expression with increasing salt concentration possibly due to feedback inhibition. \u003cem\u003ePavNHX37\u003c/em\u003e expression under 150 mM salt stress was 8.9-fold that of the control making it the most significantly salt-stress-responsive gene. This finding is similar to results in quinoa [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Following salt stress treatment wild-type tobacco exhibited varying degrees of wilting and chlorosis while overexpressing tobacco lines did not. Enzyme activity assays showed significantly increased activities of SOD POD and CAT decreased MDA content and increased proline accumulation. Proline accumulation is one of the important physiological responses of plants to stress [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. These results suggest that \u003cem\u003ePavNHX37\u003c/em\u003e may enhance salt tolerance through a dual mechanism: on one hand maintaining ion homeostasis via Na⁺/H⁺ exchange and on the other hand regulating the antioxidant system to scavenge ROS and accumulating osmolytes to alleviate stress damage [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Furthermore, although \u003cem\u003ePavNHX37\u003c/em\u003e is not a core node in the protein-protein interaction network it interacts with multiple family members. This implies that \u003cem\u003ePavNHX37\u003c/em\u003e may participate in a complex ion transport regulatory network and optimize salt tolerance through synergistic actions with other NHX proteins [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn this study, 37 NHX gene family members were systematically identified for the first time in sweet cherry. Their molecular characteristics, phylogenetic relationships, and tissue-specific expression patterns were elucidated, and \u003cem\u003ePavNHX37\u003c/em\u003e was screened as a key gene regulating salt tolerance. Overexpression of \u003cem\u003ePavNHX37\u003c/em\u003e in tobacco significantly enhanced salt tolerance by maintaining ion homeostasis and increasing antioxidant enzyme activities. This study provides a theoretical basis and genetic resources for improving salt tolerance in sweet cherry.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe sincerely thank all colleagues in the laboratory for their valuable discussions and technical support. Meanwhile, we extend our heartfelt gratitude to the editor and reviewers for their rigorous evaluation of this manuscript and their constructive comments for its improvement.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZ.G, Y.Z., and S.H. conceived and designed the research. X.A., Z.G., Y.Z., S.H performed the experiments. X.A. and Z.G. performed the data analysis and wrote the manuscript. S.H. and Z.G. supervised the research. X.A. revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by Central guidance for local science and technology development fund projects, grant number 25ZYJE001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no studies involving human participants or animals were conducted for this article. All methods complied with applicable guidelines and regulations, and Tianshui Normal University approved every experimental protocol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAras S, Eşitken A, Karakurt Y. The Comprehensive Responses of Young Sweet Cherry Trees Under Moderate Saline Conditions Depending on the Different Rootstocks. 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Nucleic Acids Res. 2021;49:D605\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Sweet cherry, NHX gene family, Salt stress, Bioinformatics analysis, Enzyme activity","lastPublishedDoi":"10.21203/rs.3.rs-9596831/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9596831/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eSoil salinization severely limits sweet cherry (\u003cem\u003ePrunus avium\u003c/em\u003e L.) growth and productivity, yet the NHX (Na⁺/H⁺ antiporter) gene family, which is central to salt tolerance, remains uncharacterized in this species.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eHere, we identified 37 \u003cem\u003ePavNHX\u003c/em\u003e genes unevenly distributed across eight chromosomes. All PavNHX proteins were predicted to localize to the plasma membrane except \u003cem\u003ePavNHX21\u003c/em\u003e (vacuolar), and their secondary structures were dominated by α-helices and random coils. Phylogenetic analysis divided the family into three groups, with Group I containing both sweet cherry and \u003cem\u003eArabidopsis\u003c/em\u003e homologs, indicating evolutionary conservation. Expression profiling revealed diverse tissue-specific patterns, and qRT-PCR under salt stress identified \u003cem\u003ePavNHX37\u003c/em\u003e as the most significantly upregulated gene. Subcellular localization confirmed \u003cem\u003ePavNHX37\u003c/em\u003e at the plasma membrane. Stable overexpression of \u003cem\u003ePavNHX37\u003c/em\u003e in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e dramatically improved salt tolerance: transgenic lines exhibited superior growth, higher proline accumulation, elevated SOD、POD、CAT activities, and reduced MDA content compared to wild-type plants under 150 mM NaCl.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis study provides the first genome-wide characterization of the NHX gene family in sweet cherry and demonstrates that \u003cem\u003ePavNHX37\u003c/em\u003e functions as a key positive regulator of salt tolerance. These findings establish a theoretical foundation and genetic resources for improving salt tolerance in sweet cherry.\u003c/p\u003e","manuscriptTitle":"Sweet cherry genome‑wide Identification of the NHX gene family and functional dissection of PavNHX37 a key salt tolerance gene","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-18 11:10:14","doi":"10.21203/rs.3.rs-9596831/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-20T06:39:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-19T02:34:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"237118411690206008785292822970313598733","date":"2026-05-14T04:02:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"336897422566845183530355898009085644387","date":"2026-05-13T14:12:22+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-09T07:00:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"262870844504744509998316267204515202642","date":"2026-05-09T00:51:45+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-05-08T12:05:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-08T07:19:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-05-08T00:46:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2026-05-08T00:41:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bbfe5b2c-e2b0-4615-8b75-08ce38c2804b","owner":[],"postedDate":"May 18th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-20T06:39:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-19T02:34:49+00:00","index":18,"fulltext":""},{"type":"reviewerAgreed","content":"237118411690206008785292822970313598733","date":"2026-05-14T04:02:57+00:00","index":17,"fulltext":""},{"type":"reviewerAgreed","content":"336897422566845183530355898009085644387","date":"2026-05-13T14:12:22+00:00","index":16,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-09T07:00:40+00:00","index":15,"fulltext":""},{"type":"reviewerAgreed","content":"262870844504744509998316267204515202642","date":"2026-05-09T00:51:45+00:00","index":14,"fulltext":""},{"type":"reviewersInvited","content":"5","date":"2026-05-08T12:05:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-08T07:19:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-05-08T00:46:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2026-05-08T00:41:08+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-20T06:54:19+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-18 11:10:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9596831","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9596831","identity":"rs-9596831","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}