Drought-induced 19 gene FvDi19-3 from woodland strawberry enhances drought and salt tolerance in transgenic Arabidopsis

Drought-induced 19 gene FvDi19-3 from woodland strawberry enhances drought and salt tolerance in transgenic Arabidopsis | 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 Drought-induced 19 gene FvDi19-3 from woodland strawberry enhances drought and salt tolerance in transgenic Arabidopsis Jingjing Kong, Keli Qiu, Junyong Zhou, Debao Li, Lijuan Lu, Mao Liu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5719169/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Apr, 2025 Read the published version in Plant Cell Reports → Version 1 posted 5 You are reading this latest preprint version Abstract Di19 (drought-induced 19) proteins play a crucial role in regulating plant development and various stress responses. However, a systematic identification and functional analysis of the Di19 gene family members in woodland strawberry has yet to be conducted. In this study, we identified four Di19 genes in woodland strawberry, and analyzed the phylogenetic tree, conserved protein domains, and gene structure. Cis -elements suggested that FvDi19s may be involved in plant development and stress responses. Gene expression analysis revealed diverse expression patterns of FvDi19s under different stress conditions, and overexpression of FvDi19s enhanced drought and salt tolerance in yeast. Transgenic and stress tolerance assays indicated FvDi19-3 overexpression in Arabidopsis enhanced plant drought and salt tolerance by promoting stomatal closure, improving the plant's ability to scavenge reactive oxygen species and the expression of drought or salt-responsive genes. Furthermore, the LUC/REN ratio indicated that FvWRKY42 and FvMYB114 can activate the expression of FvDi19-3 , and expression of these three genes is dependent on the ABA signaling pathway. In conclusion, our study characterized the Di19 gene family in woodland strawberry and investigated the biological functions of FvDi19-3 in drought and salt tolerance, providing a basis for further functional studies of FvDi19s in responses to abiotic stress. FvDi19-3 woodland strawberry drought and salt stresses stomatal aperture ROS-scavenging system 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 Key message FvDi19-3 enhances drought and salt tolerance in Arabidopsis by promoting stomatal closure, improving the ability to scavenge reactive oxygen species, and increasing the expression of drought- or salt-responsive genes. 1. Introduction Drought and salinity are key abiotic stress factors that affect the growth and development of plants in nature environment (Shinozaki and Yamaguchi-Shinozaki 2006; Zhang, et al. 2021). Over the course of evolution, plants have developed multiple signal transduction systems respond various environmental stimuli, thereby enhancing their adaptability to environmental changes and minimizing stress-related damage, which ensures normal growth and development (Joshi, et al. 2016). When specific sensing elements located on the exterior of the plant cell membrane capture the stress signal, they rapidly transmit these stimuli to second messenger within the cell, such as Ca 2+ , ROS, and NO, which in turn activate regulatory genes, including CDPKs, MAPKs, and ribosomal protein kinases, etc (Munns 2005). Subsequently, these activated regulatory genes trigger a variety of transcription factors (TFs) through phosphorylation or dephosphorylation. Changes in TF activity can influence the expression of downstream functional genes, resulting in a series of physiological and biochemical reactions, such as maintaining internal homeostasis and degrading toxic substances, thereby ensuring the sustained growth and development of plants under abiotic drought and salt stresses (Joshi, et al. 2016; Li, et al. 2021; Zhang, et al. 2021). Numerous transcription factors play critical roles in the mechanisms of salt and drought tolerance in plants. For instance, the MdHB7-like, a HD-Zip transcription factor, has been demonstrated to positively regulate salt tolerance in apple by promoting autophagic activity and Na + efflux (Yang, et al. 2023). The rose transcription factor RcbHLH59 regulated the accumulation of callose by adjusting the expression of RcPR4 and RcPR5 , thereby facilitating Na⁺/K⁺ balance to achieve salt tolerance (Su, et al. 2023). In addition, transcription factors BcWRKY33A and BcHSFA4A interact to positively regulate the transcription and expression of salt stress-related genes BcZAT12 and BcHSP17.6A , resulting in enhanced salt tolerance in non-heading Chinese cabbage (Wang, et al. 2022). In cucumber, the NAC-type transcription factor CsATAF1 directly regulates the expression of CsDREB2C , CsCu-ZnSOD and CsABI5 , thereby improving drought tolerance by enhancing the scavenging capacity of ROS in transgenic plants (Wang, et al. 2018). CaWRKY1 and CaWRKY41 directly bind to the CaCIPK3 promoter, positively regulating drought resistance through the modulation of the MeJA signaling pathway and antioxidant defense system via the CBL-CIPK network (Ma, et al. 2021). In grapevine, VlbZIP30 enhances drought resistance by directly activating VvNAC17 , which regulate three peroxidase genes: VvPRX4 , VvPRX72 , and VvPRX N1 (Tu, et al. 2020). The Di19 (Drought-induced protein 19) transcription family represents a novel type of Cys2/His2 zinc-finger protein, comprising zf-di19 and di19_C domains. Several studies have reported that this family of transcription factors is associated with drought and salt tolerance across various species (Milla, et al. 2006). In Arabidopsis , AtDi19-1 and AtDi19-3 rely on the ABA signaling pathway to regulate drought tolerance. AtDi19-1 acts as a positive regulator, whereas AtDi19-3 may function as a negative regulator (Gosti 1995; Liu, et al. 2013; Milla, et al. 2006; Qin, et al. 2014). In rice, overexpression of OsDi19-4 increases drought tolerance by enhancing ROS scavenging activity (Wang, et al. 2014; Wang, et al. 2016). OsDi19-5 interacts with the caleosin OsClo5 to form a heterodimer that represses the transcription of two target genes OsUSP and OsMST , thereby negatively regulating salt tolerance (Jing, et al. 2021). In soybean, GmDi19-5 interacts with the E3 ubiquitin ligase GmPUB21, and both function as negative regulators under drought and salinity stresses (Yang, et al. 2023). Calcium-dependent protein kinase (CDPK)-mediated serine phosphorylation of GhDi19-1/-2 enhances sensitivity to high salinity and augments the response to ABA in cotton (Qin, et al. 2016). TaDi19A exhibits sensitivity to drought and salt through ABA signaling, while homologous genes PtDi19-2 and PtDi19-7 increase drought tolerance via an ABA-dependent pathway (Li, et al. 2010; Wu, et al. 2022). In mango, MiDil9-4B promotes earlier flowering and enhances drought and salt resistance in transgenic Arabidopsis (Zhu, et al. 2023). However, members of Di19 gene family have not yet been reported in strawberry. Strawberry is an economically significant berry plant cultivated globally. Drought and salt stress severely impact its growth and yield (Grant, et al. 2010; Liao, et al. 2018). Fragaria vesca , a diploid woodland strawberry (2n=2x=14), is widely recognized as a model plant for strawberry research (Castillejo, et al. 2020). In this study, we conducted a comprehensive survey of the Di19 gene family members in woodland strawberry, focusing on their sequence features, phylogenetic relationships, chromosomal distribution, and cis -acting elements. Additionally, we investigated the expression patterns of the FvDi19 genes under various stress conditions and assessed the stress-related phenotypes following the heterologous transformation of yeast. Furthermore, we performed analyses the subcellular localization and functional verification of FvDi19-3 . These results not only provide a foundation for a deeper understanding of the FvDi19 gene functions but also provide a new perspective for its future application in strawberry stress-resistance breeding. 2. Materials and methods 2.1 Identification of Di19 family members The genome sequences and annotations for strawberry and grape were obtained from the Genome Database in Rosaceae (GDR) (https://www.rosaceae.org/). Genome sequence information for rice, millet, soybean, maize, and poplar was retrieved from the Plant Genomics Resource (Phytozome v13) (https://phytozome-next.jgi.doe.gov/). Protein sequence information for all AtDi19s was obtained from the TAIR database (https://www.arabidopsis.org/). Subsequently, the protein sequences of Di19 in strawberry, rice, millet, soybean, maize, grape, and poplar were acquired using AtDi19s protein sequences as queries via BLASTP program (E-value≤1x10 -5 ). Hidden Markov Model (HMM) profiles for the zf-Di19 domain (PF05605), Di19_C (PF14571), and ZnF_C2H2 motifs were utilized to search strawberry proteome sequences via the HMMER 3.0 software with an E-value cut-off of 1x10 -5 . The specific sequences containing both zf-Di19 and Di19_C domains were identified as putative Di19s. The resulting Di19 protein sequences were validated using the SMART (http://smart.embl-heidelberg.de/) and InterProScan databases (https://www.ebi.ac.uk/interpro/). The molecular weight (MW), isoelectric point (pI), aliphatic index, and grand average of hydropathicity (GRAVY) of Di19 proteins were predicted using ExPASy (https://web.expasy.org/compute_pi/). The WoLF PSORT (https://wolfpsort.hgc.jp/) was used to predict the Di19s subcellular localization. 2.2 Gene structure, conserved motif and phylogenetic analysis The Gene Structure Display Server program (GSDS v.2.0) (https://gsds.gao-lab.org/) was used to analyze the structure of strawberry Di19 genes, including the 5' UTR, intron, exon and 3' UTR (Hu, et al. 2015). The conserved motifs were predicted using the Multiple Expectation Maximization for Motif Elicitation (MEME) tool (Bailey, et al. 2015). Default parameters were employed, with the maximum number of motifs set to 10. Multiple sequence alignment of protein sequences from strawberry, Arabidopsis , grape, rice, soybean, and maize was conducted using MUSCLE 3.8.1551 software, followed by trimming of the alignment to remove gaps using TrimAL 1.4.1. Subsequently, a phylogenetic tree was constructed using the Neighbor-Joining algorithm in MEGA 10.1 software, with the bootstrap values set to 1000 replicates to ensure statistical reliability (Kumar, et al. 2018). The gene structure, conserved motif map, and phylogenetic tree were generated through TBtools analysis and visualized with Adobe Illustrator 2020 software (Chen, et al. 2023). 2.3 Chromosome distribution, gene duplication and collinearity analysis The chromosomal location information for FvDi19 genes was obtained from the Strawberry Genome Database, and a distribution map was generated using TBtools (Chen, et al. 2023). The MCSCanX software was employed to analyze the tandem and segmental duplication of the FvDi19 genes using the default parameters (Wang, et al. 2012). Comparative syntenic analysis of Di19 genes between strawberry and Arabidopsis was conducted using MCSCanX (Wang, et al. 2012). 2.4 Identification of cis-regulatory elements of FvDi19s To predict the cis -acting regulatory elements in the promoter of FvDi19 s (p FvDi19s ), 2000 bp upstream of the Di19 genes promoter regions were extracted and submitted to PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (Lescot, et al. 2002). A visual display of the functional elements was provided by TBtools. 2.5 Plant Materials and Stress Treatments The woodland strawberry ecotype ‘Ruegen’ was generously provided by Jiangsu Academy of Agricultural Sciences. Following normal germination, the strawberry seedlings were planted in pots containing a mixture of soil and vermiculite (1:4) and grown in a phytotron under strictly controlled conditions (16/8-h light/dark cycle, 22±2°C, 12000 lux light intensity and 65% relative humidity) (Li, et al. 2023). To simulate abiotic stress, seedlings with similar growth status were treated with 200 mM NaCl, 20% PEG6000 to induce salt and drought stress, respectively. Additionally, solutions of 40 µmol/L abscisic acid (ABA), jasmonic acid (MeJA), and salicylic acid (SA) were sprayed to induce hormonal stress. Specimens (leaves) were collected at specified time points (0 h, 1 h, 2 h, 4 h, 8 h, 12 h, or 24h) following the application of stress and hormones. The ecotype Col-0 of Arabidopsis was used in this study. The di19 mutant (SALK 088814) was obtained from the Nottingham Arabidopsis Stock Centre. Arabidopsis seeds were sterilized using 12% sodium hypochlorite and seeded onto 1/2 Murashige and Skoog (1/2 MS) medium. The plates were stored at 4°C for 2 days and then placed vertically under standard conditions. For assays of drought and salt stresses, 4-day-old seedlings were transplanted onto 1/2 MS medium containing 250 mM mannitol and 130 mM NaCl, respectively. After 7 days of vertical growth, the seedlings were photographed and measured. Then, the samples were promptly frozen in liquid nitrogen and stored at -80℃ (Dong, et al. 2020). Each assay included three biological replicates, with each replicate containing at least four plants. 2.6 Vector construction and plant transformation The vectors were constructed using a PCR-based infusion cloning system ( pEASY ® -Uni Seamless Cloning and Assembly Kit, Transgene , Beijing). The fusion vectors, pYES2-NTB-FvDi19-1 , pYES2-NTB-FvDi19-2 , pYES2-NTB-FvDi19-3 and pYES2-NTB-FvDi19-like , were obtained by inserting the coding sequences of the four FvDi19s into the pYES2-NTB yeast vector following the provided instructions. These vectors were then separately transferred into the INVSC1 yeast strain. The coding sequence of FvDi19-3 without/with the stop codon was inserted into the pMDC43/pGBKT7 vector with/without the GFP reporter gene to generate the 35S:GFP-FvDi19-3/pGBKT7-FvDi19-3 construct, respectively. The di19 mutants were identified using the triple primer method, with specific primers detailed in Supplementary Table S4. Agrobacterium tumefaciens GV3101 was used to introduce 35S:GFP-FvDi19-3 plasmids into Col-0 or di19 mutants, and the transformants were selected on 1/2 MS medium containing hygromycin (30 µg/ml). Homozygous T3 offspring were used in the following experiments. 2.7 Functional analysis of FvDi19s in yeast cell under drought and salt stress The five transformed yeast cells were plated onto SD-Ura medium. Positive colonies were then diluted with SD-Ura solution and cultivated at 29°C until the OD 600 value reached 1.2-1.4. Following this, the yeast cells were centrifuged and cleaned with SD-Ura liquid medium devoid of a carbon source and cultured with shaking for three hours. After centrifugation, the cells were cultured in SG-Ura containing 2% galactose for 8-12 hours, with the OD 600 adjusted to 1.0. Subsequently, the yeast cell suspensions were diluted (10 0 , 10 -1 , 10 -2 , 10 -3 ) and plated onto SG-Ura agar plates containing various concentrations of mannitol (0.75 M, 1 M and 1.5 M) or NaCl solution (0.25 M, 0.5 M, 0.75 M and 1.0 M) (Chen, et al. 2023). 2.8 Subcellular Localization Analysis The 35S:GFP-FvDi19-3 plasmid and the nuclear marker NLS-RFP were co-transformed into Arabidopsis protoplasts using a polyethylene glycol-mediated transient expression system with the empty vector (pMDC43-GFP) as a control. The transformed protoplasts were incubated in the dark for 12-18 hours. GFP signals were observed using a laser scanning confocal microscope (LSM 880, Zeiss, Germany). 2.9 Physiological observations and measurements Stomatal morphology was examined using a microscope (M165FC, Lecia), and the images were analyzed using the ImageJ software (National Institutes of Health). The relative water content (RWC) was calculated using the formula: (FW-DW)/(TW-DW) × 100% (FW: fresh weight; DW: dry weight; TW: thick water weight) (Zhang, et al. 2022). The H 2 O 2 and O 2 - were detected by histochemical staining using 3,3'-diaminobenzidine (DAB) and nitro-blue tetrazolium (NBT), respectively (Liang, et al. 2022). The content of malondialdehyde (MDA), proline, and superoxide dismutase (SOD) were measured using detection kits from Nanjing Jiancheng Bioengineering Institute, following the provided protocols. 2.10 RNA extraction and quantitative real-time PCR (RT-qPCR) analysis Total RNA was extracted from strawberry leaves using MolPure® Plant Plus RNA Kit (Yeasen Biotechnology, Shanghai, Co., Ltd.) following the manufacturer’s protocols. The quality and purity of RNA were assessed by 1% agarose gel and a Nanodrop One microvolume UV-Vis spectrophotometer (Thermo Scientific), with A260/280 ratios ranging from 1.8 to 2.1 prior to reverse transcription. The Hifair® Ⅱ 1st Strand cDNA Synthesis Kit (Yeasen Biotechnology, Shanghai, Co., Ltd.) was used to reverse transcribe total RNA into cDNA, and all cDNAs were stored at -20℃. Specific primers were designed and validated using Primer Premier 5 software and NCBI-Primer Blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/), with FvActin employed as a reference gene for RT-qPCR analysis (Table S4). The qRT-PCR was carried out using the Hieff® qPCR SYBR Green Master Mix (High Rox Plus) (Yeasen Biotechnology, Shanghai, Co., Ltd.). The PCR reaction program was executed in the ABI7500 thermal cycler (Applied Biosystems) and the amplification process was performed according to the instructions. Three biological and three technical replicates were performed. Data were calculated using the 2 - △△ CT method. 2.11 Dual-luciferase assay The coding sequences of FvMYB63 , FvMYB82 , FvMYB114 , and FvWRKY42 were cloned into the pCambia1300 vector under the CaMV35S promoter to generate effector constructs, and the 2000 bp promoter regions of FvDi19-3 were cloned into pGreen II 0800 vectors to generate reporter. The resulting LUC-FvDi19-3 fusion protein construct, along with recombinant constructs 35S-FvMYB6 , 35S-FvMYB82 , 35S-FvMYB114 , and 35S-FvWRKY42 , were individually co-transformed into Agrobacterium GV3101 (pSoup-19). The bacterial cultures were combined at a 1:1 volume ratio with an osmotic buffer before being injected into the leaves of tobacco. Three days after infiltration, the effector and reporter assay system (Promega, Beijing, China) was utilized for experimentation according to previous report (Yu, et al. 2024). Each experiment included three biological replicates. 3. Results 3.1 Comprehensive analysis of the Di19 gene family A total of four FvDi19 genes were identified in strawberry, all of which contain the zf-Di19, Di19-C and ZnF-C2H2 conserved domain (Fig.1A-B). Except for FvDi19-1 , which contains four exons, all three other members contain five exons (Fig.1C). Motif analysis revealed that FvDi19-1 and FvDi19-3 contained seven motifs and FvDi19-2 and FvDi19-like contained eight motifs (Fig.1D and Table S1). This suggested a difference in gene structure among the four members of the FvDi19 gene family. The basic characterizations of the 4 FvDi19s , such as their position, protein length, cDNA length, MW, pI, aliphatic index, GRAVY and predicted subcellular location were listed in the Table 1. Table 1. Characterization of FvDi19s identified in woodland strawberry Gene name Gene ID Chr Position Protein sequences length (aa) cDNA length (bp) MW (Da) pI Grand average of hydropathicity (GRAVY) Aliphatic in dex Predicted subcellular location FvDi19-1 FvH4_6g07090.t1 Fvb6 4275176-4278714 195 588 21.81059 5.10 -0.513 75.49 Nuclear FvDi19-2 FvH4_4g19190.t1 Fvb4 22899486-22902190 213 642 23.89478 5.05 -0.547 74.13 Nuclear FvDi19-3 FvH4_5g06340.t1 Fvb5 3722050-3726963 203 612 22.37110 5.60 -0.328 72.12 Nuclear FvDi19-like FvH4_3g08710.t1 Fvb3 5089946-5093625 231 696 26.06925 5.93 -0.426 79.83 Nuclear cytoplasm Moreover, seven Di19 genes were identified in Arabidopsis , alongside seven in rice, seven in maize, six in millet, fifteen in soybean, six in grapes, and eight in poplar. In total, these 60 Di19 proteins, along with the four FvDi19s , were compiled to construct a comprehensive phylogenetic tree, resulting in their classification into five groups (Fig S1 and Table S2). The Di19 proteins in group Ⅱ contained the fewest numbers, with a mere four proteins, while the group I had the highest representation, encompassing 18 Di19 proteins. Notably, group I was exclusively composed of Di19 proteins from dicotyledonous plants, whereas the other four subgroups encompassed both dicotyledonous and monocotyledonous plants. Previous studies have shown that silencing GmDi19-5 and AtDi19-3 enhances salinity tolerance, suggesting that genes within this group may share similar functions (Qin, et al. 2014; Yang, et al. 2023). Additionally, the four FvDi19s were located on four different chromosomes, suggesting that these genes may have different biological functions. Collinearity analysis revealed that strawberry FvDi19-1 and FvDi19-2 exhibited collinearity within the species, with FvDi19-1 being linked to AtDi19-1 , AtDi19-3 , AtDi19-4 , and AtDi19-6 , while FvDi19-2 was connected to AtDi19-3 , AtDi19-4 , and AtDi19-7 across species (Fig. S2) In plant, gene transcription is regulated by cis- elements that serve as binding sites for TFs. To analyze the ci s-elements, 2000bp upstream promoter regions of FvDi19 s were scanned. Five types of ci s-elements were predicted, including MYB-related, development, hormone, light, and stress response elements (Fig.1E-F), suggesting that FvDi19s might have related functions. 3.2 FvDi19s were induced by drought, salt and various hormones The induced expression patterns of drought, salt, MEJA, ABA, and SA of FvDi19s were further explored because the p FvDi19s contain abundant cis -acting elements in response to stress and hormones, and MBS was associated with drought (Fig.1E-F) (Huang, et al. 2024). After NaCl and PEG treatments, the expression of FvDi19-1 , FvDi19-3 , and FvDi19-like genes tended to increase, with FvDi19-3 showing the highest up-regulation. However, the expression level of FvDi19-2 was repressed by NaCl and PEG treatment (Fig.2A-B), which might perform different function under NaCl and PEG stress compared to other FvDi19 s. Upon to ABA and MeJA stress, all the FvDi19 s were upregulated, with FvDi19–3 was slightly up-regulated (Fig.2C-D). When treated by SA, only FvDi19–2 was down regulated, while other three genes expression level was increased, but the increase was not significant except for FvDi19-like (Fig.2E). To further verify the function of FvDi19 genes in stress response, we examined the function of FvDi19s in yeast under drought and salt conditions. The findings indicated that the control ( pYES2-NTB ) and four FvDi19 -transformed yeast strains ( pYES2-NTB-FvDi19-1 , pYES2-NTB-FvDi19-2 , pYES2-NTB-FvDi19-3 , pYES2-NTB-FvDi19-like ) exhibited normal growth patterns on the SG-Ura medium, indicating that overexpression of FvDi19s in INVSc1 strains did not impair their growth under standard conditions (Fig.3A). Under drought condition, we observed the bacterial solution of the control exhibited growth under conditions of SG-Ura supplemented with 0.75 M, 1 M, and 1.5 M mannitol, but its growth activity decreased with the increase of mannitol concentration and failed to thrive at 10 -3 -fold dilution on the SG-Ura plate containing 1.5 M mannitol (Fig.3B-D). However, the growth pattern of any concentration bacterial solution from the experimental group ( pYES2-NTB-FvDi19s ) still exhibited growth on the SG-Ura plate with various mannitol and pYES2-NTB-FvDi19-3 showed the strongest ability to survive (Fig.3B-D). Under salt stress, compared with the control, overexpression of four FvDi19 s induced a tolerance to salt stress on the SG-Ura plate containing 0.25 M, 0.5 M, 0.75M and 1 M NaCl (Fig.3E-H). Interestingly, pYES2-NTB-FvDi19-3 showed the strongest survival ability under drought and salt condition. These findings demonstrated that FvDi19-3 could enhance the drought and salt tolerance in yeast by a wide margin. 3.3 FvDi19-3 encoded a transcription factor and located in nucleus The FvDi19-3 was demonstrated responsiveness to various stress treatments at the mRNA level in the aerial part and in yeast, these reinforced the need for us to investigate its potential role in the stress response. The function of genes in transcriptional regulation was contingent upon their position within the cellule. To ascertain the subcellular localization of FvDi19-3, the control 35S::GFP (empty vector) and recombinant plasmid FvDi19-3::GFP were transiently expressed in Arabidopsis mesophyll protoplasts (Fig.4A). As shown in Fig.4B, the control (35S::GFP) showed green fluorescence in the nucleus and cell membrane, while the FvDi19-3::GFP was mainly located in the nucleus of cells, further confirming the FvDi19-3 was a transcription factor. Furthermore, FvDi19-3 exhibited transactivation activity in yeast cells (Fig.4C). Based on these findings, we proposed that FvDi19-3 encodes a bona fide transcription factor. 3.4 Overexpression of FvDi19-3 enhanced drought and salt tolerance in transgenic Arabidopsis To ascertain the biological function of FvDi19-3 in drought and salt tolerance, overexpressing FvDi19-3 transgenic Arabidopsis plants were generated, and three high-expression homozygous T3 plants were then selected for the experiments (Fig.5A). As shown in Figure 5B-D, the root length and fresh weight of Col-0 and three FvDi19-3 - OE seedlings did not exhibit significant differences when grown on MS medium without mannitol or NaCl, however, three overexpression plants showed longer root length and heavier fresh weight compared with those Col-0 Arabidopsis under drought and salt condition (Fig.5B-D). Furthermore, seedlings of ten-day-old Col-0 and three overexpression lines were transferred into a soil mixture and grown under standard conditions for three weeks. As shown in Fig.6A, there were no discernible differences in growth between the Col-0 and transgenic Arabidopsis under normal conditions, however, following a two-week drought or salt treatment, the majority of Col-0 plant leaves exhibited wilting or turned white, whereas the leaves of the transgenic Arabidopsis displayed minimal wilting or whitening. Moreover, the survival rate and relative water content (RWC) of the three overexpression lines were significantly higher than Col-0 (Fig.6B-C). Additionally, three FvDi19-3-OE plants exhibited significantly narrower stomatal apertures compared to Col-0 under stress conditions, this observation was consistent with the phenomenon that drought and salt stressed leaves from the FvDi19-3-OE plants maintained a higher relative water content than Col-0 (Fig. 6C). These results suggested that FvDi19-3 may regulate water loss by modulating stomatal aperture, thereby enhancing drought and salt tolerance in Arabidopsis . 3.5 Overexpression FvDi19-3 increased the capacity of the ROS-scavenging system It has been demonstrated that drought or salt stress leads to the accumulation of reactive oxygen species (ROS), including hydrogen peroxide (H 2 O 2 ) and oxygen (O 2 - ) (Wang, et al. 2018). As shown in Fig.7A, under normal conditions, the leaves of Col-0 and the three transgenic lines exhibited minimal or no staining with DAB and NBT. In contrast, after exposure to drought and salt stresses, the leaves were displayed significant brown staining with DAB and blue staining with NBT, with the Col-0 leaves showing stronger staining than the three overexpression plants, which indicated that the H 2 O 2 and O 2 - contents in transgene Arabidopsis were lower than Col-0 under drought and salt stresses. Consistent with this observation, we also found that FvDi19-3 overexpression plants exhibited reduced malondialdehyde (MDA) content, along with elevated proline accumulation and increased activities of antioxidant enzyme such as superoxide dismutase (SOD) under drought and salt stresses (Fig. 7B-D). However, there were no discernible differences in the physiological and biochemical parameters between Col-0 and FvDi19-3 overexpression plants under control conditions (Fig. 7B-D). To gain further insight into the molecular mechanisms underlying FvDi19-3 -enhanced drought and salt stress tolerance, we examined the expression patterns of drought-related ( DREB2A , RD29B , P5CS1 ) and salt-related ( SOS2 , EDR15 , NHX1 ) marker genes by qRT-PCR (Fig.7E-J). The expression of these genes did not change significantly in Col-0 and transgenic plants under normal conditions, however, following drought and salt treatments, we observed a notable increase in the expression of genes associated with drought or salt response, respectively. In conclusion, these results suggested that the overexpression of FvDi19-3 enhances the drought and salt resistance by increasing ROS-scavenging capacity and the expression levels of stress resistance genes in Arabidopsis . 3.6 Overexpression of FvDi19-3 partially rescue di19 tolerance to drought and salt stresses in Arabidopsis It has been previously demonstrated that the Arabidopsis di19 , a loss-of-function mutant of the Di19 gene, exhibits sensitivity to drought and salt stresses (Liu, et al. 2013). Considering the facts that FvDi19-3 is the Arabidopsis di19 homologs in strawberry and over-expression FvDi19-3 can enhanced the drought and salt tolerance in Arabidopsis , we then proceeded to ascertain whether FvDi19-3 could rescue the drought and salt sensitivity phenotype of di19 in Arabidopsis . di19 mutants were identified as previous report (Fig. S3 and Table. S4), and three complementary lines were isolated for drought and salt stresses (Fig. 8A). As shown in Fig. 8B-D, root length and fresh weight of Col-0 , di19 and three complementary plants did not exhibit significant differences when grown on MS medium without mannitol or NaCl. In response to drought and salt stresses, the root length and fresh weight of di19 were significantly reduced compared to Col-0 , consistent with previous reports (Liu, et al. 2013). Nevertheless, over-expression of FvDi19-3 was found to nearly rescue the sensitivity of di19 to drought and salt stresses, with root length and fresh weight comparable to those of Col-0 (Fig. 8B-D). Besides, following drought and salt treatments in soil mixture, the di19 mutant was significantly more sensitive to drought and salt stresses than the Col-0 . Notably, the complementary plants demonstrated a significant rescue effect on the di19 mutants' sensitivity to drought and salt stress, with both survival rates and RWC being higher than the di19 mutants (Fig.9A-C). Furthermore, no significant difference was observed in the stomatal apertures of di19 mutants among control, drought and salt stress conditions, however, stomatal apertures were reduced in Col-0 and the three complementary plants under both drought and salt stresses (Fig. 9D-E). This further supports the result that FvDi19-3 enhances the drought and salt tolerance of di19 mutants through stomatal reactivation. The accumulation of H 2 O 2 , O 2 - and MDA in the leaves of di19 mutants was higher than that in Col-0 and the three transgenic plants (Fig. 10A-B). In contrast, proline content and SOD enzyme activity were obviously increased in Col-0 and the three transgenic plants compared to di19 under drought and salt stresses (Fig. 10C-D). The expression level of drought-related genes ( DREB2A , RD29B , P5CS1 ) and salt-related genes ( SOS2 , EDR15 , NHX1 ) in the di19 mutants were significantly lower than those in Col-0 , while the expression levels in the complemented lines were comparable to those in Col-0 (Fig. 10E-J). These results indicate that FvDi19-3 can complement drought and salt tolerance of di19 mutants, sharing the similar function to Di19 in Arabidopsis . 3.7 FvDi19-3 confers ABA sensitivity to Arabidopsis plants To ascertain whether the enhanced drought and salt resistance observed in the FvDi19-3 overexpressing Arabidopsis is associated with ABA, seeds from transgenic and Col-0 plants were germinated on 1/2 MS medium with or without ABA. No significant differences in germination rates were observed among the Col-0 , di19 mutant, overexpressed and complementary seeds when grown without ABA (Fig.11A). However, when germinated on 1/2 MS medium containing 0.5 µM or 1 µM ABA, all types of Arabidopsis exhibited a notable decline the germination rates (Fig. 11A-B). Besides, the germination rates of the FvDi19-3-OE lines were significantly lower than those of the Col-0 , while the di19 plants demonstrated a higher germination rate compared to the Col-0 , this suggests that FvDi19-3 can mitigate the insensitivity phenotype observed in the di19 mutant (Fig. 11A-B). It is well documented that ABA exerts an inhibitory effect on plant root growth, therefore, the differences in ABA-induced inhibition of root growth were also observed. As shown in Fig.11C, the exogenous application of ABA resulted in a more pronounced inhibition of root growth in the FvDi19-3-OE seedlings when compared to the Col-0 and di19 seedlings, and overexpression of FvDi19-3 in di19 mutants significantly reduced the root length of the mutants (Fig.11C-D). This finding suggests that FvDi19-3 can increase ABA sensitivity in Arabidopsis . Additionally, the expression of ABA signaling genes, such as AtABI3 and AtABI5 , were substantially higher in the overexpression lines compared to those in wild-type and di19 plants under salt and drought conditions (Fig. S4). These results indicate that FvDi19-3 may play a role in the response to ABA-mediated drought and salt stresses. 3.8 FvMYB114 and FvWRKY42 can activate the promoter activity of FvDi19-3 Promoter element analysis revealed that the FvDi19-3 promoter region contains binding sites (MYB and W-box elements) for MYB and WRKY transcription factors (Fig.1F), it warrants further investigation to determine whether specific MYB and WRKY transcription factors associated with drought and salt stresses in strawberry can regulate the expression of the FvDi19-3 . Previous studies have demonstrated that strawberry FvMYB63, FvMYB82 and FvMYB114 can positively regulate the salt tolerance of transgenic Arabidopsis (Li, et al. 2023; Li, et al. 2022; Wang, et al. 2024). Additionally, FvWRKY42 has been shown to enhance both salt and drought tolerance in transgenic Arabidopsis (Wei, et al. 2018). As shown in Fig.12, we employed a dual luciferase assay to investigate whether these transcription factors (FvMYB63, FvMYB82, FvMYB114 and FvWRKY42) can regulate the expression of the FvDi19-3 . The results indicated that FvMYB114 and FvWRKY42 significantly activated the expression of FvDi19-3 , whereas FvMYB63 and FvMYB82 did not exhibit a significant activation effect on FvDi19-3 , similar to the empty control. This suggests that FvMYB114 and FvWRKY42 may function as upstream regulatory genes of FvDi19-3 , playing a positive regulatory role in response to drought and salt stresses. 4. Discussion Di19 proteins are members of the C2H2 transcription factor superfamily, and have been shown to play roles in plant growth, development, and stress response (Jiang, et al. 2022; Zhao, et al. 2022; Zhu, et al. 2023). Although the Di19 gene family has been identified in several plant species, including Arabidopsis (Liu, et al. 2013), rice (Wang, et al. 2014), maize (Zhang, et al. 2019), soybeans (Feng, et al. 2015) and others, there is a paucity of research on the Di19 genes in woodland strawberry. To gain a deeper insight into the potential biological functions of Di19 genes in strawberry, an investigation was conducted using bioinformatics tools, in conjunction with analyses of phylogeny, gene structure, motif composition, chromosomal distribution, duplication events, synteny, cis- elements and expression profiles. In this study, we have identified four highly conserved Di19 genes in woodland strawberry. Prior research has shown that many species have a limited number of Di19 family members, such as Arabidopsis (7) (Milla, et al. 2006), poplar (8) (Wu, et al. 2022), rice (7) (Wang, et al. 2014), maize (7) (Zhao, et al. 2022), millet (6) (Xiao, et al. 2023), soybean (15) (Jiang, et al. 2022) , and grapes (6) (Table. S2). The relatively small number of FvDi19 s in woodland strawberry may be related to the genome size of the species (Edger, et al. 2019). As shown in Table 1, the Di19 proteins was encoded by small multigene, which were found to be low molecular weight, acidic, and hydrophilic proteins. The analysis of the gene structure and protein domains indicated that the FvDi19s exhibited comparable gene structures, protein lengths, and motif compositions, which suggested that the FvDi19 members are subject to evolutionary conservation (Fig.1). Cis -elements in the promoter region are instrumental in regulating gene expression (Wei, et al. 2023). In the event of environmental stress, such as drought or salinity, the plant will respond by triggering a series of chemical reactions. These reactions activate transcription factors (TFs), which subsequently bind to specific elements within the DNA sequence to regulate the gene expression (Zhao, et al. 2022). In the promoter region of FvDi19 genes, five types of elements, associated with development, hormone, light, stress and MYB-related functions were identified (Fig.1E-F). Notably, the promoter region of FvDi19 genes displayed a high density of MYB and MYC cis -acting elements. MYB transcription factors are widely distributed across plant species and bind to MYB elements to modulate the plant’s response to external stimulus (Pireyre and Burow 2015). The promoter region of PtDi19-2/7 in poplar contains both MYB and MYC cis- elements, and its expression can be induced by PEG, NaCl, ABA and cold stresses (Wu, et al. 2022). These findings suggest that FvDi19 genes may play a significant role in stress response. Previous studies have also demonstrated that Di19 genes exhibit varying expression levels under multiple stresses in different plants (Feng, et al. 2015; Jiang, et al. 2022; Wang, et al. 2014; Wu, et al. 2022). Analysis via qRT-PCR has shown that the transcripts of FvDi19 genes are elevated in response to drought and salt except FvDi19-2 (Fig.2). This differential expression suggests that FvDi19-2 may have distinct roles in strawberry growth and development compared to other FvDi19s . It is noteworthy that FvDi19s expression were also induced by MeJA and ABA, in accordance with the preceding reports on rice, wheat and soybean (Du, et al. 2023; Feng, et al. 2015; Wang, et al. 2016). However, ABA treatment did not significantly influence the expression levels of AtDi19 , indicating that the regulation of dehydration and salt stress by Di19 is independent of ABA (Milla, et al. 2006). These expression patterns of Di19 genes may reflect functional differences in various plant species. Besides, FvDi19s have the potential to enhance drought and salt tolerance in yeast (Fig.3), further demonstrating the role of the FvDi19 gene in response to abiotic stress. Interestingly, FvDi19-3 exhibited the most pronounced ability to augment tolerance to drought and salt stresses compared to other FvDi19 genes in yeast, which may be associated with its elevated expression level under an inducible expression mode (Fig.2). However, the biological functions of FvDi19-3 in relation to drought and salt tolerance require further validation. An increasing body of evidence indicates that the heterologous expression of genes from non-model plants in model plants affects plant performance under stress conditions (Abid, et al. 2022). Previous studies have shown that the overexpression of Di19 in plants can either enhance or decrease their resilience to environmental stressors (Xiao, et al. 2023; Yang, et al. 2023; Zhang, et al. 2019). For instance, SiDi19-3 has been found to improve the salt tolerance of Foxtail Millet and Arabidopsis (Xiao, et al. 2023), whereas the ectopic expression of GmDi19 – 15 in Arabidopsis leads in reduced drought tolerance (Jiang, et al. 2022). In general, orthologous genes or closely related genes tend to have many similar functions. As can be illustrated in the phylogenetic tree (Fig. S1), both GmDi19-5 and AtDi19-3 from group Ⅰ are associated with reduce salt tolerance, while ZmDi19-1 and OsDi19-4 from group V have been shown to enhance stress tolerance in plants (Feng, et al. 2015; Qin, et al. 2014; Wang, et al. 2014; Zhang, et al. 2019). In our study, overexpression of FvDi19-3 in both Col-0 and di19 Arabidopsis resulted in increased drought and salt tolerance (Fig. 5-10), which is consistent with the function of the GmDi19-15 gene within the same group (Jiang, et al. 2022). Besides, FvDi19-3 could promote stomatal closure in transgenic Arabidopsis , and stomatal aperture in the di19 mutant remains insensitive under drought and salt stresses (Fig.6C-E, Fig.9C-E). Closed stomata reduce transpiration and water loss from plant leaves, thereby enhancing plant drought and salt tolerance (Hou, et al. 2024). As is well known, drought and salt stresses disrupt ROS homeostasis, leading to the overproduction of ROS in plants, which has detrimental effects on for DNA, proteins, and lipids (Mishra, et al. 2023; Xu, et al. 2023). Generally, excessive ROS can disrupt antioxidant systems and the integrity of cell membranes, resulting in the accumulation of MDA (Gill and Tuteja 2010). The elevated activity of proline and SOD enzymes effectively degrades ROS in living cells, thereby safeguarding cellular membranes and reducing lipid peroxidation (Wang, et al. 2020). In comparison to Col-0 , exposure to drought and salt stresses resulted in significantly reduced levels of H 2 O 2 , O 2 - , and MDA in the overexpression line. Conversely, the proline content and SOD enzyme activity were found to be elevated, indicating that oxidative damage is mitigated in the overexpression line (Figure 7A-D). Besides, these ROS substances showed opposite expression level in di19 mutant, which could be partially alleviated by complementing lines (Fig. 10A-D). These results indicate that FvDi19-3 transgenic plants may possess enhanced ROS scavenging activity compared to Col-0 and di19 plants, resulting in reduced membrane lipid peroxidation and an improved antioxidant defense system under drought and salt stresses. In plants, the processes of drought and salt stress are complex, involving the coordinated action of numerous genes. Previous research has identified a variety of genes responsive to drought stress, such as AtDREB2A , AtRD29B , and AtP5CS1 , as well as those responsive to salt stress, including AtEDR15 , AtNHX1 , and AtSOS2 (Fu, et al. 2023; Pan, et al. 2024; Yang, et al. 2019). These genes operate through distinct pathways, ultimately enhancing the plant's resilience to drought and saline conditions. For instance, DREB transcription factors promote the expression of specific stress-responsive genes, thereby increasing drought tolerance through an ABA-mediated pathway. The RD29B gene has been recognized as a target of DREB transcription factors, while AtP5CS1 is known to facilitate proline synthesis (Verma, et al. 2019; Virlouvet, et al. 2014). Additionally, SOS2 is involved in the export of sodium ions from cells to maintain ionic balance, and NHX1 assists in the uptake of sodium ions into vacuoles (Yang, et al. 2019). Our RT-qPCR analysis demonstrated that a significant number of genes related to drought and salt stress exhibited up-regulation in transgenic lines subjected to these stresses, showing notable differences compared to control conditions. In summary, FvDi19-3 , along with several drought and salt stress-responsive genes, worked synergistically to enhance the resilience of Arabidopsis against drought and salt stresses. It has been demonstrated that ABA plays a pivotal role in enabling plants to withstand a multitude of abiotic stresses (Raghavendra, et al. 2010; Yu, et al. 2020). OsDi19-4 functions as a downstream regulator of OsCDPK14 , positively influencing ABA responses by modulating the expression of ABA-responsive genes in rice (Wang, et al. 2016). Overexpression of PtDi19-2 and PtDi19-7 conferred drought tolerance in Arabidopsis by promoting ABA-induced stomatal closure (Wu, et al. 2022). In our study, two ABA-responsive elements (ABREs) were identified in the promoter regions of FvDi19-3 (Fig.1F). FvDi19-3 was significantly induced by exogenous ABA (Fig. 2C) and overexpression of FvDi19-3 resulted in transgenic Arabidopsis hypersensitive to ABA. Previous study also shown that dehydration-responsive element-binding proteins (DREBs or DRE core) play significant role in regulating water deficit stress responses in ABA-dependent pathways (Zhao, et al. 2020). The DREB family member AtDREB2A was induced in transgenic Arabidopsis under drought and salt stresses (Fig.11A-D, 10E), further indicating FvDi19-3 is involved in drought and salt stress responses through ABA-dependent pathways. The transcription factors FvWRKY42 and FvMYB114 have been shown to enhance the salt and drought tolerance of transgenic Arabidopsis (Li, et al. 2023; Wei, et al. 2018). These factors can bind to the promoter of FvDi19-3 , thereby activating the expression of the FvDi19-3 gene. Furthermore, the expression of FvWRKY42 and FvMYB114 is dependent on the ABA signaling pathway, providing additional evidence that FvDi19-3 contributes to drought and salt tolerance in Arabidopsis via this pathway. However, further research is necessary to elucidate the interconnections among these three genes and to clarify the specific regulatory mechanisms of FvDi19-3 in drought and salt resistance. 5. Conclusion The present study identified 4 Di19 genes in woodland strawberry and conducted a comprehensive analysis of their phylogeny, gene structure, motif composition, collinearity relationships and cis -element. qRT-PCR analysis of FvDi19 genes indicate that FvDi19 s may play a significant role in responses to abiotic stresses and hormones. Overexpression of FvDi19s enhanced the resistance of yeast to drought and salt stresses, among which FvDi19-3 showed the strongest tolerance. LUC/REN assay indicated FvWRKY42 and FvMYB114 can bind to the promoter of FvDi19-3 and activate the expression of FvDi19-3 gene, and the expression of these three genes depends on the ABA signaling pathway. Furthermore, transgenic and stress tolerance assays indicated FvDi19-3 overexpression in Arabidopsis enhanced plant drought and salt tolerance by promoting stomatal closure, improving the plant's ability to scavenge reactive oxygen species and the expression of drought or salt-responsive genes (Fig.13). Nevertheless, the precise function of the FvDi19-3 gene must be corroborated in future studies employing contemporary genome editing and functional genomics techniques, particularly in the context of strawberry development and stress responses. In conclusion, the results of this study contribute to a deeper understanding of the specific functions of the FvDi19-3 genes and provide a valuable new resource for the genetic improvement of strawberry stress resistance. Declarations Acknowledgments This work was supported by Anhui Provincial Key Research and Development Plan (202104f06020004), Open Funding of National Engineering Laboratory of Crop Stress Resistance Breeding (NELCOF20210103). CRediT authorship contribution statement Jingjing Kong: Writing - original draft. Keli Qiu: Methodology, Data curation. Debao Li: Methodology, Data curation. Junyong Zhou: Formal analysis, Data curation. Lijuan Lu: Software, Methodology. Mao Liu: Formal analysis, Data curation. 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Trends Plant Sci 25(11):1117-1130. https://doi.org/10.1016/j.tplants.2020.06.008. Zhang H, Zhu J, Gong Z, Zhu J-K (2021) Abiotic stress responses in plants. Nature Reviews Genetics 23(2):104-119. https://doi.org/10.1038/s41576-021-00413-0. Zhang T, Li Y, Kang Y, Wang P, Li W, Yu W, Wang J, Wang J, Song X, Jiang X, Zhou Y (2022) The Dendrobium catenatum DcCIPK24 increases drought and salt tolerance of transgenic Arabidopsis . Industrial Crops and Products 187:115375. https://doi.org/10.1016/j.indcrop.2022.115375. Zhang X, Cai H, Lu M, Wei Q, Xu L, Bo C, Ma Q, Zhao Y, Cheng B (2019) A maize stress-responsive Di19 transcription factor, ZmDi19-1 , confers enhanced tolerance to salt in transgenic Arabidopsis . Plant Cell Reports 38(12):1563-1578. https://doi.org/10.1007/s00299-019-02467-1. Zhao L, Li Y, Li Y, Chen W, Yao J, Fang S, Lv Y, Zhang Y, Zhu S (2022) Systematical Characterization of the Cotton Di19 Gene Family and the Role of GhDi19-3 and GhDi19-4 as Two Negative Regulators in Response to Salt Stress. Antioxidants 11(11):2225. https://doi.org/10.3390/antiox11112225. Zhao Q, Fan Z, Qiu L, Che Q, Wang T, Li Y, Wang Y (2020) MdbHLH130, an Apple bHLH Transcription Factor, Confers Water Stress Resistance by Regulating Stomatal Closure and ROS Homeostasis in Transgenic Tobacco. Frontiers in Plant Science 11:543696. https://doi.org/10.3389/fpls.2020.543696. Zhao Y, Xu L, Huang Y, Wu H, Zhang X, Hu X, Ma Q (2022) Identification and Characterization of the Core Region of ZmDi19-5 Promoter Activity and Its Upstream Regulatory Proteins. International Journal of Molecular Sciences 23(13):7390. https://doi.org/10.3390/ijms23137390. Zhu J, Du D, Li Y, Zhang Y, Hu WL, Chen L, He X, Xia L, Mo X, Xie F, Luo C (2023) Isolation of three MiDi19-4 genes from mango, the ectopic expression of which confers early flowering and enhances stress tolerance in transgenic Arabidopsis . Planta 258(1):14. https://doi.org/10.1007/s00425-023-04172-6. Supplementary Files Fig.S1.tif Fig. S1: Unrooted phylogenetic tree for Di19 proteins family from Arabidopsis, rice, millet, soybean, maize, grape, and poplar, and woodland strawberry. A neighbor-joining (NJ) phylogenetic tree was constructed by MEGA Ⅹ with 1000 bootstrap replicates. Fig.S2.tif Fig. S2: Chromosomal distribution and gene duplications of Di19 gene family in F. vesca . (A). Chromosomal locations of FvDi19s . The scale on the left is in mega-bases (B). Collinearity analysis of the FvDi19 gene members in the woodland strawberry genome. The red line represents the segmentally duplicated pairs. (C). The synteny analysis of the Di19 genes in F. vesca and Arabidopsis . Gray lines in the background indicated collinear blocks within F. vesca and Arabidopsis genomes. The collinearity of Di19 gene pairs were linked with green lines. Fig.S3.tif Fig. S3: Identification of the Arabidopsis di19 mutants. Fig.S4.tif Fig. S4: The expression levels of AtABI3 and AtABI5 genes in Col-0, di19, FvDi19-3-OE and FvDi19-3-com under normal, drought and salt stresses. Data were means ± SD (n=3). Significant differences in mean values were indicated by an asterisk: **P < 0.01 (Student’s t -test). SupplementTables.xlsx Cite Share Download PDF Status: Published Journal Publication published 07 Apr, 2025 Read the published version in Plant Cell Reports → Version 1 posted Editorial decision: Minor revisions 13 Feb, 2025 Reviewers agreed at journal 13 Jan, 2025 Reviewers invited by journal 09 Jan, 2025 First submitted to journal 30 Dec, 2024 Editor assigned by journal 27 Dec, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-5719169","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":399995279,"identity":"019f312e-1517-48e9-967e-2aa8e058eb46","order_by":0,"name":"Jingjing Kong","email":"","orcid":"","institution":"Anhui Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jingjing","middleName":"","lastName":"Kong","suffix":""},{"id":399995280,"identity":"c792e362-9f12-4937-baab-8a6a93317a6e","order_by":1,"name":"Keli Qiu","email":"","orcid":"","institution":"Anhui Agriculture University: Anhui Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Keli","middleName":"","lastName":"Qiu","suffix":""},{"id":399995281,"identity":"a3339303-696e-4386-a720-e442af8156f9","order_by":2,"name":"Junyong Zhou","email":"","orcid":"","institution":"Anhui Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Junyong","middleName":"","lastName":"Zhou","suffix":""},{"id":399995282,"identity":"99b878e6-047f-4d95-bb00-1fb19e179fa3","order_by":3,"name":"Debao Li","email":"","orcid":"","institution":"Anhui Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Debao","middleName":"","lastName":"Li","suffix":""},{"id":399995283,"identity":"7d8fcd91-cdc3-49e8-8105-c79748395e05","order_by":4,"name":"Lijuan Lu","email":"","orcid":"","institution":"Anhui Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Lijuan","middleName":"","lastName":"Lu","suffix":""},{"id":399995284,"identity":"45605130-47d4-498a-9e45-ee54443cf21f","order_by":5,"name":"Mao Liu","email":"","orcid":"","institution":"Anhui Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mao","middleName":"","lastName":"Liu","suffix":""},{"id":399995285,"identity":"cae53d01-f065-43c0-8574-9fd26e5f9c94","order_by":6,"name":"Shufang Zhu","email":"","orcid":"","institution":"Anhui Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Shufang","middleName":"","lastName":"Zhu","suffix":""},{"id":399995286,"identity":"1f658f78-b427-403d-a9da-1caa93a72203","order_by":7,"name":"Zhiyuan Ning","email":"","orcid":"","institution":"Anhui Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Zhiyuan","middleName":"","lastName":"Ning","suffix":""},{"id":399995287,"identity":"4dd76a97-72e5-476a-b85d-bc9302bc190c","order_by":8,"name":"Qibao Sun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAx0lEQVRIiWNgGAWjYDACCRDxQ8KOn5n54APitTD22CRLtrMlG5BgC1sa44bzPGYCROngn91jJvmD5zCz8WEGMwaGGptowpbcOWMmzWNxmM/sMEPaA4ZjabkNBPXcyDGTZgDaAtRy3ICx4TBhLfJALZI/2A4zbm5mbJMgSosBUIsED8j7zMxsxGkxvJFWbM0LDGSJw2zMBgnE+EXuRvLGmz9AUdl//uODDzU2RHifgcNEAs5OIKwcBNgffyBO4SgYBaNgFIxYAAAO6zyqADVZFAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0009-0002-3919-8408","institution":"Anhui Academy of Agricultural Sciences","correspondingAuthor":true,"prefix":"","firstName":"Qibao","middleName":"","lastName":"Sun","suffix":""}],"badges":[],"createdAt":"2024-12-27 05:38:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5719169/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5719169/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00299-025-03481-2","type":"published","date":"2025-04-07T16:05:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":73448640,"identity":"505fe7d5-78b9-4788-8065-a37fa6cd86c4","added_by":"auto","created_at":"2025-01-10 05:30:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2251101,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene structure, conserved domain, motif and phylogenetic analysis of\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eFvDi19s in F. vesca\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A). The phylogenetic tree of all FvDi19s proteins was constructed using neighbor-joining method with 1000 bootstrap.\u003c/p\u003e\n\u003cp\u003e(B). The gene structure analysis of \u003cem\u003eFvDi19s\u003c/em\u003e. Green block diagrams represent the 5′ and 3′ non-coding regions, yellow block diagrams and black lines represent exons and introns, respectively. The length indicates the size of exon and intron.\u003c/p\u003e\n\u003cp\u003e(C). Domain analysis of all the\u003cem\u003e \u003c/em\u003eFvDi19\u003cem\u003e \u003c/em\u003eproteins. Green block diagrams represent zf-Di19, red block diagrams represent Di19-C and blue block diagrams represent ZnF-C2H2.\u003c/p\u003e\n\u003cp\u003e(D). Conserved motifs of the \u003cem\u003eFvDi19\u003c/em\u003e identified by MEME. The gray lines represent the non-conserved sequences, and each motif is indicated by a colored box numbered at top right.\u003c/p\u003e\n\u003cp\u003e(E). Visual map of the promoter analysis of the \u003cem\u003eFvDi19\u003c/em\u003e genes based on \u003cem\u003ecis\u003c/em\u003e-elements. The different color boxes represent the various cis-regulatory elements. Some cis-regulatory element may overlap with others.\u003c/p\u003e\n\u003cp\u003e(F).\u003cstrong\u003e \u003c/strong\u003eStatistical summary of \u003cem\u003ecis\u003c/em\u003e-elements in the promoter regions of \u003cem\u003eFvDi19s.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5719169/v1/608682a24c74f15f87cab993.png"},{"id":73446795,"identity":"b3723e86-afdc-4645-bd3d-265a68505e20","added_by":"auto","created_at":"2025-01-10 05:06:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1938617,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression patterns of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFvDi19\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes\u003c/strong\u003e \u003cstrong\u003ein response to abiotic and hormone stress by qRT-PCR.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGene expression patterns under 25% PEG-6000 (A), 300 mM NaCl (B), 40 µmol/L ABA(C), 40 µmol/L MeJA (D), and 40 µmol/L SA (E) treatment, respectively. Data is shown as mean ±SD. Student’s t test: *\u003cem\u003ep\u003c/em\u003e-value \u003cem\u003e\u0026lt; \u003c/em\u003e0.05; **\u003cem\u003ep\u003c/em\u003e-value \u003cem\u003e\u0026lt; \u003c/em\u003e0.01; ***\u003cem\u003ep\u003c/em\u003e-value \u003cem\u003e\u0026lt; \u003c/em\u003e0.001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5719169/v1/68f000293096a8d63a9b85da.png"},{"id":73446797,"identity":"af446478-5b9e-4d42-aa13-8ac67221cd41","added_by":"auto","created_at":"2025-01-10 05:06:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":13196594,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe function analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFvDi19 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egenes under drought and salt stress in yeast strain INVSC1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A). The growth of control and \u003cem\u003eFvDi19 \u003c/em\u003eyeast under unstress condition.\u003c/p\u003e\n\u003cp\u003e(B-D). The function analysis of control and \u003cem\u003eFvDi19 \u003c/em\u003egenes under different drought stress in yeast strain INVSC1.\u003c/p\u003e\n\u003cp\u003e(E-H). The function analysis of control and \u003cem\u003eFvDi19 \u003c/em\u003egenes under different salt stress in yeast strain INVSC1.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5719169/v1/31a7e3fbe33ca192454cf30e.png"},{"id":73446801,"identity":"a754ad64-a270-4afd-8e56-0dfc16c5f7bd","added_by":"auto","created_at":"2025-01-10 05:06:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6893412,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSubcellular localization and transcription activator analysis of FvDi19-3 genes in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eF. vesca\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A). Schematic diagram of FvDi19-3 construct in subcellular localization assay, the yellow box represents the gene constructed onto the vector. 35 S: cauliflower mosaic virus 35 S promoter; NOS: nopaline synthase gene (NOS) terminator; RB: right border; LB: left border; GFP: Green fluorescent protein.\u003c/p\u003e\n\u003cp\u003e(B). Subcellular localization of the fusion protein FvDi19-3-GFP in \u003cem\u003eArabidopsis\u003c/em\u003e mesophyll protoplasts. Cells were observed by confocal microscopy. Cells were observed by confocal microscopy. Scale bars = 20 μm.\u003c/p\u003e\n\u003cp\u003e(C). Transcription activator analysis of \u003cem\u003eFvDi19-3\u003c/em\u003e. Transformed yeast cells were grown on SD/-Trp medium with or without X-α-Gal and AbA.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5719169/v1/58b7e5808091beecb2e41896.png"},{"id":73446800,"identity":"3cea7cdc-0378-49d7-8ae9-e911342c8b1a","added_by":"auto","created_at":"2025-01-10 05:06:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":20863180,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFvDi19-3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e confers drought and salt tolerance in transgenic\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A). Relative expression of \u003cem\u003eFvDi19-3\u003c/em\u003e genes in\u003cem\u003e Col-0\u003c/em\u003e and the indicated over-expressing lines. \u003cem\u003eActin\u003c/em\u003e was used as an internal control.\u003c/p\u003e\n\u003cp\u003e(B). Four-day-old and three transgenic \u003cem\u003eArabidopsis\u003c/em\u003e were grown on MS medium were transferred to MS medium without or with 250 mM mannitol and 130 mM NaCl, respectively. Photographs were taken 9 d after transfer. \u003cem\u003eBars\u003c/em\u003e= 1 cm.\u003c/p\u003e\n\u003cp\u003e(C-D). Root length (C) and Fresh weight (D) were measured after 8 d. Data are means ± SD (n\u0026gt;20). \u003cem\u003eBars\u003c/em\u003e = 1 cm.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5719169/v1/8b8cf97d6aa5032c6aac3c2e.png"},{"id":73448638,"identity":"a6c09fa1-69f6-4690-ade6-ca8e0e9cdbe5","added_by":"auto","created_at":"2025-01-10 05:30:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":15080947,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe phenotype of transgenic\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e Arabidopsis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e under drought and salt stresses in soil.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Phenotypes of \u003cem\u003eCol-0\u003c/em\u003e and three transgenic\u003cem\u003e Arabidopsis \u003c/em\u003eplants under normal, drought and salt conditions. \u003cem\u003eBars\u003c/em\u003e = 5 cm\u003c/p\u003e\n\u003cp\u003e(B-C). The survival rate (B) and relative water content (C) were measured. \u003cem\u003e*P \u0026lt; 0.05 \u003c/em\u003eand \u003cem\u003e**P \u0026lt; 0.01\u003c/em\u003e compared with \u003cem\u003eCol-0\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e(D-E) Representative images and stomatal aperture of \u003cem\u003eCol-0\u003c/em\u003e and three transgenic\u003cem\u003e Arabidopsis \u003c/em\u003eplants under normal, drought and salt conditions.\u003cem\u003eBars\u003c/em\u003e = 20 µm\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5719169/v1/49c7ad4ad43f1703a2d5a056.png"},{"id":73446818,"identity":"3ff48de1-9318-452d-9532-4a0cc19c2b92","added_by":"auto","created_at":"2025-01-10 05:06:38","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":8728928,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFvDi19-3 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ein \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e leads to reduced ROS-scavenging ability under drought and salt stress.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A). DAB staining for H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and NBT staining for superoxide O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e in \u003cem\u003eCol-0 \u003c/em\u003eand three overexpression\u003cem\u003e Arabidopsis \u003c/em\u003eplants under normal, drought and salt stresses.\u003c/p\u003e\n\u003cp\u003e(B-D). Quantitative measurement of MDA (B), Proline (C) and SOD (D) content. Data were means ± SD (n=3). Significant differences in mean values were indicated by an asterisk: \u003cem\u003e**P \u0026lt; 0.01\u003c/em\u003e (Student’s \u003cem\u003et\u003c/em\u003e-test).\u003c/p\u003e\n\u003cp\u003e(E-J). The expression levels of drought and salt stress-related genes in \u003cem\u003eCol-0 \u003c/em\u003eand\u003cem\u003e \u003c/em\u003ethree overexpression \u003cem\u003eArabidopsis\u003c/em\u003e plants under normal, drought and salt stresses. Data were means ± SD (n=3). Significant differences in mean values were indicated by an asterisk: \u003cem\u003e**P \u0026lt; 0.01\u003c/em\u003e (Student’s \u003cem\u003et\u003c/em\u003e-test).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-5719169/v1/91efddc4710b7faa7bd9bc89.png"},{"id":73448670,"identity":"7a7ae1d0-c3e1-4b5d-88f6-16d560421a87","added_by":"auto","created_at":"2025-01-10 05:31:30","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":21266398,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFvDi19-3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e can partially rescue\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e di19\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e tolerance to drought and salt stress in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A). Relative expression of \u003cem\u003eFvDi19-3\u003c/em\u003e gene in \u003cem\u003edi19\u003c/em\u003e and the indicated complemented lines.\u003cem\u003e Actin\u003c/em\u003e was used as an internal control.\u003c/p\u003e\n\u003cp\u003e(B). Four-day-old \u003cem\u003eCol-0\u003c/em\u003e,\u003cem\u003e di19 \u003c/em\u003eand three complemented \u003cem\u003eArabidopsis\u003c/em\u003ewere grown on MS medium were transferred to 1/2 MS medium with or without 250 mM mannitol and 130 mM NaCl, respectively. Photographs were taken 8 d after transfer. \u003cem\u003eBars\u003c/em\u003e= 1 cm.\u003c/p\u003e\n\u003cp\u003e(C-D). Root length (C) and Fresh weight (D) were measured after 8 d. Data are means ± SD (n\u0026gt;20). \u003cem\u003eBars\u003c/em\u003e = 1 cm.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-5719169/v1/5a9e70750745a69cf0974038.png"},{"id":73446853,"identity":"5e0865bf-cdc2-465b-9e2b-9166bc99a6c3","added_by":"auto","created_at":"2025-01-10 05:06:39","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":37935847,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe phenotype of complemented\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e Arabidopsis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e under drought and salt stresses in soil.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Phenotypes of \u003cem\u003eCol-0\u003c/em\u003e,\u003cem\u003e di19\u003c/em\u003e and three complemented\u003cem\u003e Arabidopsis \u003c/em\u003eplants under normal, drought and salt conditions. \u003cem\u003eBars\u003c/em\u003e = 5 cm\u003c/p\u003e\n\u003cp\u003e(B-C). The survival rate (B) and relative water content (C) were measured. \u003cem\u003e*P \u0026lt; 0.05 \u003c/em\u003eand \u003cem\u003e**P \u0026lt; 0.01\u003c/em\u003e compared with \u003cem\u003eCol-0\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e(D-E) Representative images and stomatal aperture of \u003cem\u003eCol-0\u003c/em\u003e,\u003cem\u003edi19 \u003c/em\u003eand three complemented\u003cem\u003e Arabidopsis \u003c/em\u003eplants under normal, drought and salt conditions.\u003cem\u003e Bars\u003c/em\u003e = 20 µm\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-5719169/v1/e1de27e7749b767b9cc1c5b2.png"},{"id":73448672,"identity":"0a2487ad-5c70-4f94-a630-dbb3c7b7ab58","added_by":"auto","created_at":"2025-01-10 05:31:30","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":13079858,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe physiological parameters of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCol-0\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e,\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e di19 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand three complemented\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e Arabidopsis \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eplants under different conditions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A). DAB staining for H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and NBT staining for superoxide O\u003csub\u003e2\u003c/sub\u003e- in \u003cem\u003eCol-0\u003c/em\u003e,\u003cem\u003e di19 \u003c/em\u003eand three complemented\u003cem\u003e Arabidopsis \u003c/em\u003eplants under normal, drought and salt stresses.\u003c/p\u003e\n\u003cp\u003e(B-D). Quantitative measurement of MDA (B), Proline (C) and SOD (D) content. Data\u003c/p\u003e\n\u003cp\u003ewere means ± SD (n=3). Significant differences in mean values were indicated by an asterisk: \u003cem\u003e**P \u0026lt; 0.01\u003c/em\u003e (Student’s \u003cem\u003et\u003c/em\u003e-test).\u003c/p\u003e\n\u003cp\u003e(E-J). The expression levels of drought and salt stress-related genes in \u003cem\u003eCol-0, di19 \u003c/em\u003eand\u003cem\u003e \u003c/em\u003ethree complemented \u003cem\u003eArabidopsis\u003c/em\u003e plants under normal, drought and salt stresses. Data were means ± SD (n=3). Significant differences in mean values were indicated by an asterisk: \u003cem\u003e**P \u0026lt; 0.01\u003c/em\u003e (Student’s \u003cem\u003et\u003c/em\u003e-test).\u003c/p\u003e","description":"","filename":"Figure10.png","url":"https://assets-eu.researchsquare.com/files/rs-5719169/v1/cb959d66580f0941caaf2466.png"},{"id":73448642,"identity":"2a618653-4ec0-45c1-8325-5dd2f614482d","added_by":"auto","created_at":"2025-01-10 05:30:37","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":31131766,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eFvDi19-3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003econfers ABA sensitivity to \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e plants.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A). Seed germination between \u003cem\u003eCol-0, di19\u003c/em\u003e, overexpressed and complemented lines under 0.5 μM and 1μM ABA treatment.\u003c/p\u003e\n\u003cp\u003e(B) Root elongation between \u003cem\u003eCol-0, di19\u003c/em\u003e, overexpressed and complemented lines under 35 μM treatment.\u003c/p\u003e\n\u003cp\u003e(C). The statistics of germination rates under control, 0.5 μM and 1μM ABA treatment.\u003c/p\u003e\n\u003cp\u003e(D) The statistics of primary root length under control and 35 μM ABA treatment. The data were means ± SD from three independent replications.\u003cem\u003e *p \u0026lt; 0.05\u003c/em\u003e, \u003cem\u003e**p \u0026lt; 0.01 \u003c/em\u003e(Student’s \u003cem\u003et\u003c/em\u003e-test).\u003c/p\u003e","description":"","filename":"Figure11.png","url":"https://assets-eu.researchsquare.com/files/rs-5719169/v1/cbe3c83ccea9469e11d3595d.png"},{"id":73449224,"identity":"73df77ea-82ca-49a4-aca1-4ab7492027fa","added_by":"auto","created_at":"2025-01-10 05:38:37","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":1185368,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTransactivation assays of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFvDi19-3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in tobacco.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A). Schematic diagrams of the effector and reporter constructs used in the dual-luciferase reporter assay.\u003c/p\u003e\n\u003cp\u003e(B) The effector and reporter assay in tobacco leaves demonstrated the transactivation effect of FvICE1, FvMYB63\u003cem\u003e,\u003c/em\u003e FvMYB82, FvMYB114 and FvWRKY42\u003cem\u003e \u003c/em\u003eon the promoter of \u003cem\u003eFvDi19-3\u003c/em\u003e. Values are means ± SD from three independent experiments; statistical significance was determined by Student’s \u003cem\u003et\u003c/em\u003e-test (*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure12.png","url":"https://assets-eu.researchsquare.com/files/rs-5719169/v1/372805a978d2f345a682d74a.png"},{"id":73448674,"identity":"93f4e588-4152-48c6-a766-bf25eaa9ea66","added_by":"auto","created_at":"2025-01-10 05:31:32","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":271759,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProposed working model of the mechanism of drought and salt tolerance regulated by\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e FvDi19-3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in woodland strawberry.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure13.png","url":"https://assets-eu.researchsquare.com/files/rs-5719169/v1/dd6b3c3da3583e36b5640137.png"},{"id":80558928,"identity":"efde2a04-3d2f-4b9a-b10b-52a0ed81879a","added_by":"auto","created_at":"2025-04-14 16:17:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":158858379,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5719169/v1/a8b1a5c0-413f-4a21-ae2f-dc307a74102c.pdf"},{"id":73446812,"identity":"c696ae44-79d9-4381-abcf-c5fc5943cf66","added_by":"auto","created_at":"2025-01-10 05:06:37","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1687512,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S1:\u003c/strong\u003e Unrooted phylogenetic tree for Di19 proteins family from Arabidopsis, rice, millet, soybean, maize, grape, and poplar, and woodland strawberry. A neighbor-joining (NJ) phylogenetic tree was constructed by MEGA Ⅹ with 1000 bootstrap replicates.\u003c/p\u003e","description":"","filename":"Fig.S1.tif","url":"https://assets-eu.researchsquare.com/files/rs-5719169/v1/0eb7c6dbfaa79e2c420aba64.tif"},{"id":73446846,"identity":"e629fc57-8168-493f-9e46-08bb9697bf0b","added_by":"auto","created_at":"2025-01-10 05:06:39","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":8077756,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S2: Chromosomal distribution and gene duplications of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eDi19\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egene family in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eF. vesca\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A). Chromosomal locations of \u003cem\u003eFvDi19s\u003c/em\u003e. The scale on the left is in mega-bases\u003c/p\u003e\n\u003cp\u003e(B). Collinearity analysis of the \u003cem\u003eFvDi19\u003c/em\u003e gene members in the woodland strawberry genome. The red line represents the segmentally duplicated pairs.\u003c/p\u003e\n\u003cp\u003e(C). The synteny analysis of the \u003cem\u003eDi19\u003c/em\u003e genes in \u003cem\u003eF. vesca\u003c/em\u003e and \u003cem\u003eArabidopsis\u003c/em\u003e. Gray lines in the background indicated collinear blocks within \u003cem\u003eF. vesca\u003c/em\u003eand \u003cem\u003eArabidopsis\u003c/em\u003e genomes. The collinearity of \u003cem\u003eDi19\u003c/em\u003e gene pairs were linked with green lines.\u003c/p\u003e","description":"","filename":"Fig.S2.tif","url":"https://assets-eu.researchsquare.com/files/rs-5719169/v1/d1f85c77a67d0c6c92320fde.tif"},{"id":73446803,"identity":"e7242946-55df-481c-b247-58b39bb3a739","added_by":"auto","created_at":"2025-01-10 05:06:37","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1780776,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S3: \u003c/strong\u003eIdentification of the \u003cem\u003eArabidopsis\u003c/em\u003e \u003cem\u003edi19\u003c/em\u003e mutants.\u003c/p\u003e","description":"","filename":"Fig.S3.tif","url":"https://assets-eu.researchsquare.com/files/rs-5719169/v1/1f4a9c29a84f22120938210a.tif"},{"id":73446824,"identity":"91c52c90-e8e8-46fa-8f14-bcd3dc530211","added_by":"auto","created_at":"2025-01-10 05:06:38","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":4948476,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S4:\u003c/strong\u003e The expression levels of \u003cem\u003eAtABI3\u003c/em\u003e and \u003cem\u003eAtABI5 \u003c/em\u003egenes in \u003cem\u003eCol-0, di19, FvDi19-3-OE \u003c/em\u003eand\u003cem\u003e FvDi19-3-com \u003c/em\u003eunder normal, drought and salt stresses. Data were means ± SD (n=3). Significant differences in mean values were indicated by an asterisk: \u003cem\u003e**P \u0026lt; 0.01\u003c/em\u003e (Student’s \u003cem\u003et\u003c/em\u003e-test).\u003c/p\u003e","description":"","filename":"Fig.S4.tif","url":"https://assets-eu.researchsquare.com/files/rs-5719169/v1/e8fb01e8345644a54b68ef81.tif"},{"id":73448665,"identity":"e3f80492-8e08-49a9-9314-0e01920eb539","added_by":"auto","created_at":"2025-01-10 05:31:01","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":25061,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementTables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5719169/v1/233306381053b1a0769032d8.xlsx"}],"financialInterests":"","formattedTitle":"Drought-induced 19 gene FvDi19-3 from woodland strawberry enhances drought and salt tolerance in transgenic Arabidopsis","fulltext":[{"header":"Key message","content":"\u003cp\u003e\u003cem\u003eFvDi19-3\u003c/em\u003e enhances drought and salt tolerance in \u003cem\u003eArabidopsis\u003c/em\u003e by promoting stomatal closure, improving the ability to scavenge reactive oxygen species, and increasing the expression of drought- or salt-responsive genes.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eDrought and salinity are key abiotic stress factors that affect the growth and development of plants in nature environment\u0026nbsp;(Shinozaki and Yamaguchi-Shinozaki 2006; Zhang, et al. 2021). Over the course of evolution, plants have developed multiple signal transduction systems respond various environmental stimuli, thereby enhancing their adaptability to environmental changes and minimizing stress-related damage, which ensures normal growth and development (Joshi, et al. 2016). When specific sensing elements located on the exterior of the plant cell membrane capture the stress signal, they rapidly transmit these stimuli to second messenger within the cell, such as Ca\u003csup\u003e2+\u003c/sup\u003e, ROS, and NO, which in turn activate regulatory genes, including CDPKs, MAPKs, and ribosomal protein kinases, etc (Munns 2005). Subsequently, these activated regulatory genes trigger a variety of transcription factors (TFs) through phosphorylation or dephosphorylation. Changes in TF activity can influence the expression of downstream functional genes, resulting in a series of physiological and biochemical reactions, such as maintaining internal homeostasis and degrading toxic substances, thereby ensuring the sustained growth and development of plants under abiotic drought and salt stresses (Joshi, et al. 2016; Li, et al. 2021; Zhang, et al. 2021).\u003c/p\u003e\n\u003cp\u003eNumerous transcription factors play critical roles in the mechanisms of salt and drought tolerance in plants. For instance, the MdHB7-like, a HD-Zip transcription factor, has been demonstrated to positively regulate salt tolerance in apple by promoting autophagic activity and Na\u003csup\u003e+\u003c/sup\u003e efflux (Yang, et al. 2023).\u0026nbsp;The rose transcription factor RcbHLH59 regulated the accumulation of callose by adjusting the expression of \u003cem\u003eRcPR4\u003c/em\u003e and \u003cem\u003eRcPR5\u003c/em\u003e, thereby facilitating Na⁺/K⁺ balance to achieve salt tolerance\u0026nbsp;(Su, et al. 2023).\u0026nbsp;In addition,\u0026nbsp;transcription factors BcWRKY33A and BcHSFA4A interact to positively regulate the transcription and expression of salt stress-related genes\u003cem\u003e\u0026nbsp;BcZAT12\u003c/em\u003e and \u003cem\u003eBcHSP17.6A\u003c/em\u003e, resulting in enhanced salt tolerance in non-heading Chinese cabbage\u0026nbsp;(Wang, et al. 2022). In cucumber, the NAC-type transcription factor CsATAF1 directly regulates the expression of \u003cem\u003eCsDREB2C\u003c/em\u003e, \u003cem\u003eCsCu-ZnSOD\u003c/em\u003e and\u003cem\u003e\u0026nbsp;CsABI5\u003c/em\u003e, thereby improving drought tolerance by enhancing the scavenging capacity of ROS in transgenic plants\u0026nbsp;(Wang, et al. 2018). \u003cem\u003eCaWRKY1\u003c/em\u003e and \u003cem\u003eCaWRKY41\u003c/em\u003e directly bind to the \u003cem\u003eCaCIPK3\u003c/em\u003e promoter, positively regulating drought resistance through the modulation of the MeJA signaling pathway and antioxidant defense system via the CBL-CIPK network\u0026nbsp;(Ma, et al. 2021). In grapevine, \u003cem\u003eVlbZIP30\u003c/em\u003e enhances drought resistance by directly activating \u003cem\u003eVvNAC17\u003c/em\u003e, which regulate three peroxidase genes: \u003cem\u003eVvPRX4\u003c/em\u003e, \u003cem\u003eVvPRX72\u003c/em\u003e, and \u003cem\u003eVvPRX N1\u003c/em\u003e (Tu, et al. 2020).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Di19 (Drought-induced protein 19) transcription family represents a novel type of Cys2/His2 zinc-finger protein, comprising zf-di19 and di19_C domains. Several studies have reported that this family of transcription factors is associated with drought and salt tolerance across various species (Milla, et al. 2006). In \u003cem\u003eArabidopsis\u003c/em\u003e, \u003cem\u003eAtDi19-1\u003c/em\u003e and \u003cem\u003eAtDi19-3\u003c/em\u003e rely on the ABA signaling pathway to regulate drought tolerance. \u003cem\u003eAtDi19-1\u003c/em\u003e acts as a positive regulator, whereas \u003cem\u003eAtDi19-3\u003c/em\u003e may function as a negative regulator (Gosti 1995; Liu, et al. 2013; Milla, et al. 2006; Qin, et al. 2014). In rice, overexpression of \u003cem\u003eOsDi19-4\u003c/em\u003e increases drought tolerance by enhancing ROS scavenging activity (Wang, et al. 2014; Wang, et al. 2016). OsDi19-5 interacts with the caleosin OsClo5 to form a heterodimer that represses the transcription of two target genes \u003cem\u003eOsUSP\u003c/em\u003e and \u003cem\u003eOsMST\u003c/em\u003e, thereby negatively regulating salt tolerance (Jing, et al. 2021). In soybean, GmDi19-5 interacts with\u0026nbsp;the E3 ubiquitin ligase GmPUB21, and both function as negative regulators under drought and salinity stresses\u0026nbsp;(Yang, et al. 2023). Calcium-dependent protein kinase (CDPK)-mediated serine phosphorylation of GhDi19-1/-2 enhances sensitivity to high salinity and augments the response to ABA in cotton\u0026nbsp;(Qin, et al. 2016). \u003cem\u003eTaDi19A\u0026nbsp;\u003c/em\u003eexhibits sensitivity to drought and salt through ABA signaling, while homologous genes \u003cem\u003ePtDi19-2\u003c/em\u003e and \u003cem\u003ePtDi19-7\u003c/em\u003e increase drought tolerance via an ABA-dependent pathway\u0026nbsp;(Li, et al. 2010; Wu, et al. 2022). In mango, \u003cem\u003eMiDil9-4B\u0026nbsp;\u003c/em\u003epromotes earlier flowering and enhances drought and salt resistance in transgenic \u003cem\u003eArabidopsis\u0026nbsp;\u003c/em\u003e(Zhu, et al. 2023). However, members of Di19 gene family have not yet been reported in strawberry.\u003c/p\u003e\n\u003cp\u003eStrawberry is an economically significant berry plant cultivated globally. Drought and salt stress severely impact its growth and yield (Grant, et al. 2010; Liao, et al. 2018). \u003cem\u003eFragaria vesca\u003c/em\u003e, a diploid woodland strawberry (2n=2x=14), is widely recognized as a model plant for strawberry research (Castillejo, et al. 2020). In this study, we conducted a comprehensive survey of the Di19 gene family members in woodland strawberry, focusing on their sequence features, phylogenetic relationships, chromosomal distribution, and \u003cem\u003ecis\u003c/em\u003e-acting elements. Additionally, we investigated the expression patterns of the \u003cem\u003eFvDi19\u003c/em\u003e genes under various stress conditions and assessed the stress-related phenotypes following the heterologous transformation of yeast. Furthermore, we performed analyses the subcellular localization and functional verification of \u003cem\u003eFvDi19-3\u003c/em\u003e. These results not only provide a foundation for a deeper understanding of the \u003cem\u003eFvDi19\u003c/em\u003e gene functions but also provide a new perspective for its future application in strawberry stress-resistance breeding.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.1 Identification of Di19 family members\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genome sequences and annotations for strawberry and grape were obtained from the Genome Database in Rosaceae (GDR) (https://www.rosaceae.org/). Genome sequence information for rice, millet, soybean, maize, and poplar was retrieved from the Plant Genomics Resource (Phytozome v13) (https://phytozome-next.jgi.doe.gov/). Protein sequence information for all AtDi19s was obtained from the TAIR database (https://www.arabidopsis.org/). Subsequently, the protein sequences of Di19 in strawberry, rice, millet, soybean, maize, grape, and poplar were acquired using AtDi19s protein sequences as queries via BLASTP program (E-value\u0026le;1x10\u003csup\u003e-5\u003c/sup\u003e). Hidden Markov Model (HMM) profiles for the zf-Di19 domain (PF05605), Di19_C (PF14571), and ZnF_C2H2 motifs were utilized to search strawberry proteome sequences via the HMMER 3.0 software with an E-value cut-off of 1x10\u003csup\u003e-5\u003c/sup\u003e. The specific sequences containing both zf-Di19 and Di19_C domains were identified as putative Di19s. The resulting Di19 protein sequences were validated using the SMART (http://smart.embl-heidelberg.de/) and InterProScan databases (https://www.ebi.ac.uk/interpro/). The molecular weight (MW), isoelectric point (pI), aliphatic index, and grand average of hydropathicity (GRAVY) of Di19 proteins were predicted using ExPASy (https://web.expasy.org/compute_pi/). The WoLF PSORT (https://wolfpsort.hgc.jp/) was used to predict the Di19s subcellular localization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.2 Gene structure, conserved motif and phylogenetic analysis \u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Gene Structure Display Server program (GSDS v.2.0) (https://gsds.gao-lab.org/) was used to analyze the structure of strawberry Di19 genes, including the 5\u0026apos; UTR, intron, exon and 3\u0026apos; UTR (Hu, et al. 2015). The conserved motifs were predicted using the Multiple Expectation Maximization for Motif Elicitation (MEME) tool (Bailey, et al. 2015). Default parameters were employed, with the maximum number of motifs set to 10. Multiple sequence alignment of protein sequences from strawberry, \u003cem\u003eArabidopsis\u003c/em\u003e, grape, rice, soybean, and maize was conducted using MUSCLE 3.8.1551 software, followed by trimming of the alignment to remove gaps using TrimAL 1.4.1. Subsequently, a phylogenetic tree was constructed using the Neighbor-Joining algorithm in MEGA 10.1 software, with the bootstrap values set to 1000 replicates to ensure statistical reliability (Kumar, et al. 2018). The gene structure, conserved motif map, and phylogenetic tree were generated through TBtools analysis and visualized with Adobe Illustrator 2020 software (Chen, et al. 2023).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.3\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eChromosome distribution, gene duplication and collinearity analysis \u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe chromosomal location information for \u003cem\u003eFvDi19\u003c/em\u003e genes was obtained from the Strawberry Genome Database, and a distribution map was generated using TBtools (Chen, et al. 2023). The MCSCanX software was employed to analyze the tandem and segmental duplication of the \u003cem\u003eFvDi19\u003c/em\u003e genes using the default parameters (Wang, et al. 2012). Comparative syntenic analysis of \u003cem\u003eDi19 \u003c/em\u003egenes between strawberry and \u003cem\u003eArabidopsis\u003c/em\u003e was conducted using MCSCanX (Wang, et al. 2012).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.4 Identification of cis-regulatory elements\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e \u003cem\u003eof FvDi19s\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo predict the\u003cem\u003e cis\u003c/em\u003e-acting regulatory elements in the promoter of \u003cem\u003eFvDi19\u003c/em\u003es (p\u003cem\u003eFvDi19s\u003c/em\u003e), 2000 bp upstream of the \u003cem\u003eDi19\u003c/em\u003e genes promoter regions were extracted and submitted to PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (Lescot, et al. 2002). A visual display of the functional elements was provided by TBtools. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.5\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e \u003cem\u003ePlant Materials and Stress Treatments\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe woodland strawberry ecotype \u0026lsquo;Ruegen\u0026rsquo; was generously provided by Jiangsu Academy of Agricultural Sciences. Following normal germination, the strawberry seedlings were planted in pots containing a mixture of soil and vermiculite (1:4) and grown in a phytotron under strictly controlled conditions (16/8-h light/dark cycle, 22\u0026plusmn;2\u0026deg;C, 12000 lux light intensity and 65% relative humidity) (Li, et al. 2023). To simulate abiotic stress, seedlings with similar growth status were treated with 200 mM NaCl, 20% PEG6000 to induce salt and drought stress, respectively. Additionally, solutions of 40 \u0026micro;mol/L abscisic acid (ABA), jasmonic acid (MeJA), and salicylic acid (SA) were sprayed to induce hormonal stress. Specimens (leaves) were collected at specified time points (0 h, 1 h, 2 h, 4 h, 8 h, 12 h, or 24h) following the application of stress and hormones. \u003c/p\u003e\n\u003cp\u003eThe ecotype \u003cem\u003eCol-0 \u003c/em\u003eof \u003cem\u003eArabidopsis\u003c/em\u003e\u003cem\u003e \u003c/em\u003ewas used in this study. The \u003cem\u003edi19\u003c/em\u003e mutant (SALK 088814) was obtained from the Nottingham \u003cem\u003eArabidopsis\u003c/em\u003e Stock Centre. \u003cem\u003eArabidopsis\u003c/em\u003e seeds were sterilized using 12% sodium hypochlorite and seeded onto 1/2 Murashige and Skoog (1/2 MS) medium. The plates were stored at 4\u0026deg;C for 2 days and then placed vertically under standard conditions. For assays of drought and salt stresses, 4-day-old seedlings were transplanted onto 1/2 MS medium containing 250 mM mannitol and 130 mM NaCl, respectively. After 7 days of vertical growth, the seedlings were photographed and measured. Then, the samples were promptly frozen in liquid nitrogen and stored at -80℃ (Dong, et al. 2020). Each assay included three biological replicates, with each replicate containing at least four plants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.6 Vector construction and plant transformation\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe vectors were constructed using a PCR-based infusion cloning system (\u003cem\u003epEASY\u003c/em\u003e\u003csup\u003e\u0026reg;\u003c/sup\u003e-Uni Seamless Cloning and Assembly Kit, \u003cem\u003eTransgene\u003c/em\u003e, Beijing). The fusion vectors, \u003cem\u003epYES2-NTB-FvDi19-1\u003c/em\u003e, \u003cem\u003epYES2-NTB-FvDi19-2\u003c/em\u003e, \u003cem\u003epYES2-NTB-FvDi19-3\u003c/em\u003e and \u003cem\u003epYES2-NTB-FvDi19-like\u003c/em\u003e, were obtained by inserting the coding sequences of the four \u003cem\u003eFvDi19s\u003c/em\u003e into the \u003cem\u003epYES2-NTB\u003c/em\u003e yeast vector following the provided instructions. These vectors were then separately transferred into the INVSC1 yeast strain. The coding sequence of \u003cem\u003eFvDi19-3\u003c/em\u003e without/with the stop codon was inserted into the pMDC43/pGBKT7 vector with/without the GFP reporter gene to generate the 35S:GFP-FvDi19-3/pGBKT7-FvDi19-3 construct, respectively. The \u003cem\u003edi19\u003c/em\u003e mutants were identified using the triple primer method, with specific primers detailed in Supplementary Table S4. \u003cem\u003eAgrobacterium\u003c/em\u003e tumefaciens GV3101 was used to introduce \u003cem\u003e35S:GFP-FvDi19-3 \u003c/em\u003eplasmids into \u003cem\u003eCol-0\u003c/em\u003e or \u003cem\u003edi19\u003c/em\u003e mutants, and the transformants were selected on 1/2 MS medium containing hygromycin (30 \u0026micro;g/ml). Homozygous T3 offspring were used in the following experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.7 Functional analysis of FvDi19s in yeast cell under drought and salt stress\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe five transformed yeast cells were plated onto SD-Ura medium. Positive colonies were then diluted with SD-Ura solution and cultivated at 29\u0026deg;C until the OD\u003csub\u003e600\u003c/sub\u003e value reached 1.2-1.4. Following this, the yeast cells were centrifuged and cleaned with SD-Ura liquid medium devoid of a carbon source and cultured with shaking for three hours. After centrifugation, the cells were cultured in SG-Ura containing 2% galactose for 8-12 hours, with the OD\u003csub\u003e600\u003c/sub\u003e adjusted to 1.0. Subsequently, the yeast cell suspensions were diluted (10\u003csup\u003e0\u003c/sup\u003e, 10\u003csup\u003e-1\u003c/sup\u003e, 10\u003csup\u003e-2\u003c/sup\u003e, 10\u003csup\u003e-3\u003c/sup\u003e) and plated onto SG-Ura agar plates containing various concentrations of mannitol (0.75 M, 1 M and 1.5 M) or NaCl solution (0.25 M, 0.5 M, 0.75 M and 1.0 M) (Chen, et al. 2023). \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.8 Subcellular Localization Analysis \u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 35S:GFP-FvDi19-3\u003cem\u003e \u003c/em\u003eplasmid and the nuclear marker NLS-RFP were co-transformed into \u003cem\u003eArabidopsis\u003c/em\u003e protoplasts using a polyethylene glycol-mediated transient expression system with the empty vector (pMDC43-GFP) as a control. The transformed protoplasts were incubated in the dark for 12-18 hours. GFP signals were observed using a laser scanning confocal microscope (LSM 880, Zeiss, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.9 Physiological observations and measurements \u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStomatal morphology was examined using a microscope (M165FC, Lecia), and the images were analyzed using the ImageJ software (National Institutes of Health). The relative water content (RWC) was calculated using the formula: (FW-DW)/(TW-DW) \u0026times; 100% (FW: fresh weight; DW: dry weight; TW: thick water weight) (Zhang, et al. 2022). The H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e were detected by histochemical staining using 3,3\u0026apos;-diaminobenzidine (DAB) and nitro-blue tetrazolium (NBT), respectively (Liang, et al. 2022). The content of malondialdehyde (MDA), proline, and superoxide dismutase (SOD) were measured using detection kits from Nanjing Jiancheng Bioengineering Institute, following the provided protocols.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.10 RNA extraction and quantitative real-time PCR (RT-qPCR) analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from strawberry leaves using MolPure\u0026reg; Plant Plus RNA Kit (Yeasen Biotechnology, Shanghai, Co., Ltd.) following the manufacturer\u0026rsquo;s protocols. The quality and purity of RNA were assessed by 1% agarose gel and a Nanodrop One microvolume UV-Vis spectrophotometer (Thermo Scientific), with A260/280 ratios ranging from 1.8 to 2.1 prior to reverse transcription. The Hifair\u0026reg; Ⅱ 1st Strand cDNA Synthesis Kit (Yeasen Biotechnology, Shanghai, Co., Ltd.) was used to reverse transcribe total RNA into cDNA, and all cDNAs were stored at -20℃.\u003c/p\u003e\n\u003cp\u003eSpecific primers were designed and validated using Primer Premier 5 software and NCBI-Primer Blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/), with \u003cem\u003eFvActin\u003c/em\u003e employed as a reference gene for RT-qPCR analysis (Table S4). The qRT-PCR was carried out using the Hieff\u0026reg; qPCR SYBR Green Master Mix (High Rox Plus) (Yeasen Biotechnology, Shanghai, Co., Ltd.). The PCR reaction program was executed in the ABI7500 thermal cycler (Applied Biosystems) and the amplification process was performed according to the instructions. Three biological and three technical replicates were performed. Data were calculated using the 2\u003csup\u003e-\u003c/sup\u003e\u003csup\u003e△△\u003c/sup\u003e\u003csup\u003eCT\u003c/sup\u003e method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e2.11 Dual-luciferase assay\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe coding sequences of \u003cem\u003eFvMYB63\u003c/em\u003e, \u003cem\u003eFvMYB82\u003c/em\u003e, \u003cem\u003eFvMYB114\u003c/em\u003e, and\u003cem\u003e FvWRKY42\u003c/em\u003e were cloned into the pCambia1300 vector under the CaMV35S promoter to generate effector constructs, and the 2000 bp promoter regions of \u003cem\u003eFvDi19-3\u003c/em\u003e were cloned into pGreen II 0800 vectors to generate reporter. The resulting \u003cem\u003eLUC-FvDi19-3\u003c/em\u003e fusion protein construct, along with recombinant constructs \u003cem\u003e35S-FvMYB6\u003c/em\u003e, \u003cem\u003e35S-FvMYB82\u003c/em\u003e, \u003cem\u003e35S-FvMYB114\u003c/em\u003e, and \u003cem\u003e35S-FvWRKY42\u003c/em\u003e, were individually co-transformed into Agrobacterium GV3101 (pSoup-19). The bacterial cultures were combined at a 1:1 volume ratio with an osmotic buffer before being injected into the leaves of tobacco. Three days after infiltration, the effector and reporter assay system (Promega, Beijing, China) was utilized for experimentation according to previous report (Yu, et al. 2024). Each experiment included three biological replicates.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3.1 Comprehensive analysis of the Di19 gene family\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of four \u003cem\u003eFvDi19\u003c/em\u003e genes were identified in strawberry, all of which contain the zf-Di19, Di19-C and ZnF-C2H2 conserved domain (Fig.1A-B). Except for \u003cem\u003eFvDi19-1\u003c/em\u003e, which contains four exons, all three other members contain five exons (Fig.1C). Motif analysis revealed that \u003cem\u003eFvDi19-1\u003c/em\u003e and \u003cem\u003eFvDi19-3\u003c/em\u003e contained seven motifs and \u003cem\u003eFvDi19-2\u003c/em\u003e and \u003cem\u003eFvDi19-like\u003c/em\u003e contained eight motifs (Fig.1D and Table S1). This suggested a difference in gene structure among the four members of the \u003cem\u003eFvDi19\u003c/em\u003e gene family. The basic characterizations of the 4 \u003cem\u003eFvDi19s\u003c/em\u003e, such as their position, protein length, cDNA length, MW, pI, aliphatic index, GRAVY and predicted subcellular location were listed in the Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1. Characterization of \u003cem\u003eFvDi19s\u003c/em\u003e identified in\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ewoodland strawberry\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"871\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGene name\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGene ID\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 56px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eChr\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePosition\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eProtein\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003esequences length (aa)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ecDNA length\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e(bp)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMW (Da)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u003cstrong\u003epI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 103px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGrand average of hydropathicity (GRAVY)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAliphatic in\u003c/strong\u003e\u003cstrong\u003edex\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePredicted\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003esubcellular\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003elocation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eFvDi19-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 136px;\"\u003e\n \u003cp\u003eFvH4_6g07090.t1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003eFvb6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e4275176-4278714\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003e195\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e588\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e21.81059\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e5.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e-0.513\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e75.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003eNuclear\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eFvDi19-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 136px;\"\u003e\n \u003cp\u003eFvH4_4g19190.t1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003eFvb4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e22899486-22902190\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003e213\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e642\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e23.89478\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e5.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e-0.547\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e74.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003eNuclear\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eFvDi19-3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 136px;\"\u003e\n \u003cp\u003eFvH4_5g06340.t1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003eFvb5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e3722050-3726963\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003e203\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e612\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e22.37110\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e5.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e-0.328\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e72.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003eNuclear\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 62px;\"\u003e\n \u003cp\u003eFvDi19-like\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" style=\"width: 136px;\"\u003e\n \u003cp\u003eFvH4_3g08710.t1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56px;\"\u003e\n \u003cp\u003eFvb3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003e5089946-5093625\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003e231\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 66px;\"\u003e\n \u003cp\u003e696 \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 67px;\"\u003e\n \u003cp\u003e26.06925\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e5.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e-0.426\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003e79.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003eNuclear\u0026nbsp;\u003c/p\u003e\n \u003cp\u003ecytoplasm\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eMoreover, seven \u003cem\u003eDi19\u0026nbsp;\u003c/em\u003egenes were identified in \u003cem\u003eArabidopsis\u003c/em\u003e, alongside seven in rice, seven in maize, six in millet, fifteen in soybean, six in grapes, and eight in poplar. In total, these 60 Di19 proteins, along with the four \u003cem\u003eFvDi19s\u003c/em\u003e, were compiled to construct a comprehensive phylogenetic tree, resulting in their classification into five groups (Fig S1 and Table S2). The Di19 proteins in group Ⅱ contained the fewest numbers, with a mere four proteins, while the group I had the highest representation, encompassing 18 Di19 proteins. Notably, group I was exclusively composed of Di19 proteins from dicotyledonous plants, whereas the other four subgroups encompassed both dicotyledonous and monocotyledonous plants. Previous studies have shown that silencing \u003cem\u003eGmDi19-5\u003c/em\u003e and \u003cem\u003eAtDi19-3\u003c/em\u003e enhances salinity tolerance, suggesting that genes within this group may share similar functions (Qin, et al. 2014; Yang, et al. 2023). Additionally, the four \u003cem\u003eFvDi19s\u003c/em\u003e were located on four different chromosomes, suggesting that these genes may have different biological functions. Collinearity analysis revealed that strawberry \u003cem\u003eFvDi19-1\u003c/em\u003e and \u003cem\u003eFvDi19-2\u003c/em\u003e exhibited collinearity within the species, with\u003cem\u003e\u0026nbsp;FvDi19-1\u003c/em\u003e being linked to \u003cem\u003eAtDi19-1\u003c/em\u003e, \u003cem\u003eAtDi19-3\u003c/em\u003e, \u003cem\u003eAtDi19-4\u003c/em\u003e, and \u003cem\u003eAtDi19-6\u003c/em\u003e, while \u003cem\u003eFvDi19-2\u003c/em\u003e was connected to \u003cem\u003eAtDi19-3\u003c/em\u003e, \u003cem\u003eAtDi19-4\u003c/em\u003e, and \u003cem\u003eAtDi19-7\u003c/em\u003e across species (Fig. S2)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn plant, gene transcription is regulated by \u003cem\u003ecis-\u003c/em\u003eelements that serve as binding sites for TFs. To analyze the \u003cem\u003eci\u003c/em\u003es-elements, 2000bp upstream promoter regions of\u003cem\u003e\u0026nbsp;FvDi19\u003c/em\u003es were scanned. Five types of \u003cem\u003eci\u003c/em\u003es-elements were predicted, including MYB-related, development, hormone, light, and stress response elements (Fig.1E-F), suggesting that \u003cem\u003eFvDi19s\u003c/em\u003e might have related functions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 \u003cem\u003eFvDi19s were induced by drought, salt and various hormones\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe induced expression patterns of drought, salt, MEJA, ABA, and SA of \u003cem\u003eFvDi19s\u003c/em\u003e were further explored because the p\u003cem\u003eFvDi19s\u003c/em\u003e contain abundant \u003cem\u003ecis\u003c/em\u003e-acting elements in response to stress and hormones, and MBS was associated with drought (Fig.1E-F) (Huang, et al. 2024). After NaCl and PEG treatments, the expression of\u003cem\u003e\u0026nbsp;FvDi19-1\u003c/em\u003e, \u003cem\u003eFvDi19-3\u003c/em\u003e, and \u003cem\u003eFvDi19-like\u003c/em\u003e genes tended to increase, with\u003cem\u003e\u0026nbsp;FvDi19-3\u0026nbsp;\u003c/em\u003eshowing the highest up-regulation. However, the expression level of \u003cem\u003eFvDi19-2\u0026nbsp;\u003c/em\u003ewas repressed by NaCl and PEG treatment (Fig.2A-B), which might perform different function under NaCl and PEG stress compared to other \u003cem\u003eFvDi19\u003c/em\u003es. Upon to ABA and MeJA stress, all the \u003cem\u003eFvDi19\u003c/em\u003es were upregulated, with \u003cem\u003eFvDi19\u0026ndash;3\u0026nbsp;\u003c/em\u003ewas slightly up-regulated (Fig.2C-D). When treated by SA, only \u003cem\u003eFvDi19\u0026ndash;2\u0026nbsp;\u003c/em\u003ewas down regulated, while other three genes expression level was increased, but the increase was not significant except for \u003cem\u003eFvDi19-like\u0026nbsp;\u003c/em\u003e(Fig.2E).\u003c/p\u003e\n\u003cp\u003eTo further verify the function of \u003cem\u003eFvDi19\u003c/em\u003e genes in stress response, we examined the function of \u003cem\u003eFvDi19s\u003c/em\u003e in yeast under drought and salt conditions. The findings indicated that the control (\u003cem\u003epYES2-NTB\u003c/em\u003e) and four \u003cem\u003eFvDi19\u003c/em\u003e-transformed yeast strains (\u003cem\u003epYES2-NTB-FvDi19-1\u003c/em\u003e,\u003cem\u003e\u0026nbsp;pYES2-NTB-FvDi19-2\u003c/em\u003e,\u003cem\u003e\u0026nbsp;pYES2-NTB-FvDi19-3\u003c/em\u003e,\u003cem\u003e\u0026nbsp;pYES2-NTB-FvDi19-like\u003c/em\u003e) exhibited normal growth patterns on the SG-Ura medium, indicating that overexpression of \u003cem\u003eFvDi19s\u003c/em\u003e in INVSc1 strains did not impair their growth under standard conditions (Fig.3A). Under drought condition, we observed the bacterial solution of the control exhibited growth under conditions of SG-Ura supplemented with 0.75 M, 1 M, and 1.5 M mannitol, but its growth activity decreased with the increase of mannitol concentration and failed to thrive at 10\u003csup\u003e-3\u003c/sup\u003e-fold dilution on the SG-Ura plate containing 1.5 M mannitol (Fig.3B-D). However, the growth pattern of any concentration bacterial solution from the experimental group (\u003cem\u003epYES2-NTB-FvDi19s\u003c/em\u003e) still exhibited growth on the SG-Ura plate with various mannitol and \u003cem\u003epYES2-NTB-FvDi19-3\u003c/em\u003e showed the strongest ability to survive (Fig.3B-D). Under salt stress, compared with the control, overexpression of four \u003cem\u003eFvDi19\u003c/em\u003es induced a tolerance to salt stress on the SG-Ura plate containing 0.25 M, 0.5 M, 0.75M and 1 M NaCl (Fig.3E-H). Interestingly, \u003cem\u003epYES2-NTB-FvDi19-3\u003c/em\u003e showed the strongest survival ability under drought and salt condition. These findings demonstrated that \u003cem\u003eFvDi19-3\u0026nbsp;\u003c/em\u003ecould enhance the drought and salt tolerance in yeast by a wide margin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3\u003cem\u003e\u0026nbsp;FvDi19-3 encoded a transcription factor and located in nucleus\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eFvDi19-3\u003c/em\u003e was demonstrated responsiveness to various stress treatments at the mRNA level in the aerial part and in yeast, these reinforced the need for us to investigate its potential role in the stress response. The function of genes in transcriptional regulation was contingent upon their position within the cellule. To ascertain the subcellular localization of FvDi19-3, the control 35S::GFP (empty vector) and\u0026nbsp;recombinant plasmid FvDi19-3::GFP were transiently expressed in \u003cem\u003eArabidopsis\u003c/em\u003e mesophyll protoplasts (Fig.4A). As shown in Fig.4B, the control (35S::GFP) showed green fluorescence in the nucleus and cell membrane, while the FvDi19-3::GFP was mainly located in the nucleus of cells, further confirming the FvDi19-3 was a transcription factor. Furthermore, \u003cem\u003eFvDi19-3\u003c/em\u003e exhibited transactivation activity in yeast cells (Fig.4C). Based on these findings, we proposed that \u003cem\u003eFvDi19-3\u003c/em\u003e encodes a bona fide transcription factor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 \u003cem\u003eOverexpression of FvDi19-3 enhanced drought and salt tolerance in transgenic Arabidopsis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo ascertain the biological function of \u003cem\u003eFvDi19-3\u003c/em\u003e in drought and salt tolerance, overexpressing \u003cem\u003eFvDi19-3\u0026nbsp;\u003c/em\u003etransgenic \u003cem\u003eArabidopsis\u003c/em\u003e plants were generated, and three high-expression homozygous T3 plants were then selected for the experiments (Fig.5A). As shown in Figure 5B-D, the root length and fresh weight of\u003cem\u003e\u0026nbsp;Col-0\u003c/em\u003e and three \u003cem\u003eFvDi19-3\u003c/em\u003e-\u003cem\u003eOE\u003c/em\u003e seedlings did not exhibit significant differences when grown on MS medium without mannitol or NaCl, however, three overexpression plants showed longer root length and heavier fresh weight compared with those \u003cem\u003eCol-0\u003c/em\u003e \u003cem\u003eArabidopsis\u003c/em\u003e under drought and salt condition (Fig.5B-D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurthermore, seedlings of ten-day-old \u003cem\u003eCol-0\u003c/em\u003e and three overexpression lines were transferred into a soil mixture and grown under standard conditions for three weeks. As shown in Fig.6A, there were no discernible differences in growth between the \u003cem\u003eCol-0\u003c/em\u003e and transgenic \u003cem\u003eArabidopsis\u003c/em\u003e under normal conditions, however, following a two-week drought or salt treatment, the majority of \u003cem\u003eCol-0\u003c/em\u003e plant leaves exhibited wilting or turned white, whereas the leaves of the transgenic \u003cem\u003eArabidopsis\u003c/em\u003e displayed minimal wilting or whitening. Moreover, the survival rate and relative water content (RWC) of the three overexpression lines were significantly higher than \u003cem\u003eCol-0\u003c/em\u003e (Fig.6B-C). Additionally, three \u003cem\u003eFvDi19-3-OE\u003c/em\u003e plants exhibited significantly narrower stomatal apertures compared to \u003cem\u003eCol-0\u003c/em\u003e under stress conditions,\u0026nbsp;this observation was consistent with the phenomenon that drought and salt stressed leaves from the \u003cem\u003eFvDi19-3-OE\u003c/em\u003e plants maintained a higher relative water content than \u003cem\u003eCol-0\u003c/em\u003e (Fig. 6C). These results suggested that \u003cem\u003eFvDi19-3\u003c/em\u003e may regulate water loss by modulating stomatal aperture, thereby enhancing drought and salt tolerance in \u003cem\u003eArabidopsis\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Overexpression\u003cem\u003e\u0026nbsp;FvDi19-3\u003c/em\u003e increased the capacity of the ROS-scavenging system\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt has been demonstrated that drought or salt stress leads to the accumulation of reactive oxygen species (ROS), including hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) and oxygen (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e) (Wang, et al. 2018). As shown in Fig.7A, under normal conditions, the leaves of \u003cem\u003eCol-0\u003c/em\u003e and the three transgenic lines exhibited minimal or no staining with DAB and NBT. In contrast,\u0026nbsp;after exposure to drought and salt stresses, the leaves were\u0026nbsp;displayed significant brown staining with DAB and blue staining with NBT, with the \u003cem\u003eCol-0\u003c/em\u003e leaves showing stronger staining than the three overexpression plants, which indicated that the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e contents in transgene \u003cem\u003eArabidopsis\u003c/em\u003e were lower than \u003cem\u003eCol-0\u0026nbsp;\u003c/em\u003eunder drought and salt stresses. Consistent with this observation, we also found that \u003cem\u003eFvDi19-3\u003c/em\u003e overexpression plants exhibited reduced malondialdehyde (MDA) content, along with elevated proline accumulation and increased activities of antioxidant enzyme such as superoxide dismutase (SOD) under drought and salt stresses (Fig. 7B-D). However, there were no discernible differences in the physiological and biochemical parameters between \u003cem\u003eCol-0\u003c/em\u003e and \u003cem\u003eFvDi19-3\u003c/em\u003e overexpression plants under control conditions (Fig. 7B-D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo gain further insight into the molecular mechanisms underlying \u003cem\u003eFvDi19-3\u003c/em\u003e-enhanced drought and salt stress tolerance, we examined the expression patterns of drought-related (\u003cem\u003eDREB2A\u003c/em\u003e, \u003cem\u003eRD29B\u003c/em\u003e, \u003cem\u003eP5CS1\u003c/em\u003e) and salt-related (\u003cem\u003eSOS2\u003c/em\u003e, \u003cem\u003eEDR15\u003c/em\u003e, \u003cem\u003eNHX1\u003c/em\u003e) marker genes by qRT-PCR (Fig.7E-J). The expression of these genes did not change significantly in \u003cem\u003eCol-0\u003c/em\u003e and transgenic plants under normal conditions, however, following drought and salt treatments, we observed a notable increase in the expression of genes associated with drought or salt response, respectively. In conclusion, these results suggested that the overexpression of \u003cem\u003eFvDi19-3\u003c/em\u003e enhances the drought and salt resistance by increasing ROS-scavenging capacity and the expression levels of stress resistance genes in \u003cem\u003eArabidopsis\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 \u003cem\u003eOverexpression of FvDi19-3 partially rescue di19 tolerance to drought and salt stresses in Arabidopsis\u003c/em\u003e\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt has been previously demonstrated that the \u003cem\u003eArabidopsis di19\u003c/em\u003e, a loss-of-function mutant of the \u003cem\u003eDi19\u003c/em\u003e gene, exhibits sensitivity to drought and salt stresses (Liu, et al. 2013). Considering the facts that \u003cem\u003eFvDi19-3\u003c/em\u003e is the \u003cem\u003eArabidopsis di19\u0026nbsp;\u003c/em\u003ehomologs in strawberry and over-expression \u003cem\u003eFvDi19-3\u003c/em\u003e can enhanced the drought and salt tolerance in \u003cem\u003eArabidopsis\u003c/em\u003e, we then proceeded to ascertain whether \u003cem\u003eFvDi19-3\u003c/em\u003e could rescue the drought and salt sensitivity phenotype of \u003cem\u003edi19\u003c/em\u003e in \u003cem\u003eArabidopsis\u003c/em\u003e. \u003cem\u003edi19\u0026nbsp;\u003c/em\u003emutants were identified as previous report (Fig. S3 and Table. S4), and three complementary lines were isolated for drought and salt stresses (Fig. 8A). As shown in Fig. 8B-D, root length and fresh weight of \u003cem\u003eCol-0\u003c/em\u003e, \u003cem\u003edi19\u003c/em\u003e and three complementary plants did not exhibit significant differences when grown on MS medium without mannitol or NaCl. In response to drought and salt stresses, the root length and fresh weight of\u003cem\u003e\u0026nbsp;di19\u003c/em\u003e were significantly reduced compared to \u003cem\u003eCol-0\u003c/em\u003e, consistent with previous reports (Liu, et al. 2013).\u0026nbsp;Nevertheless, over-expression of \u003cem\u003eFvDi19-3\u003c/em\u003e was found to nearly rescue the sensitivity of\u003cem\u003e\u0026nbsp;di19\u003c/em\u003e to drought and salt stresses, with root length and fresh weight comparable to those of \u003cem\u003eCol-0\u003c/em\u003e (Fig. 8B-D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBesides, following drought and salt treatments in soil mixture, the\u003cem\u003e\u0026nbsp;di19\u003c/em\u003e mutant was significantly more sensitive to drought and salt stresses than the \u003cem\u003eCol-0\u003c/em\u003e. Notably, the complementary plants demonstrated a significant rescue effect on the \u003cem\u003edi19\u0026nbsp;\u003c/em\u003emutants\u0026apos; sensitivity to drought and salt stress, with both survival rates and RWC being higher than the\u003cem\u003e\u0026nbsp;di19\u003c/em\u003e mutants (Fig.9A-C). Furthermore, no significant difference was observed in the stomatal apertures of \u003cem\u003edi19\u0026nbsp;\u003c/em\u003emutants among control, drought and salt stress conditions, however, stomatal apertures were reduced in \u003cem\u003eCol-0\u0026nbsp;\u003c/em\u003eand the three complementary plants under both drought and salt stresses (Fig. 9D-E). This further supports the result that \u003cem\u003eFvDi19-3\u0026nbsp;\u003c/em\u003eenhances the drought and salt tolerance of \u003cem\u003edi19\u003c/em\u003e mutants through stomatal reactivation. The accumulation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e and MDA in the leaves of \u003cem\u003edi19\u0026nbsp;\u003c/em\u003emutants\u003cem\u003e\u0026nbsp;\u003c/em\u003ewas higher than that in \u003cem\u003eCol-0\u0026nbsp;\u003c/em\u003eand the three transgenic plants (Fig. 10A-B). In contrast, proline content and SOD enzyme activity were obviously increased in \u003cem\u003eCol-0\u0026nbsp;\u003c/em\u003eand the three transgenic plants compared to \u003cem\u003edi19\u0026nbsp;\u003c/em\u003eunder drought and salt stresses (Fig. 10C-D). The expression level of drought-related genes (\u003cem\u003eDREB2A\u003c/em\u003e, \u003cem\u003eRD29B\u003c/em\u003e, \u003cem\u003eP5CS1\u003c/em\u003e) and salt-related genes (\u003cem\u003eSOS2\u003c/em\u003e, \u003cem\u003eEDR15\u003c/em\u003e, \u003cem\u003eNHX1\u003c/em\u003e) in the \u003cem\u003edi19\u003c/em\u003e mutants were significantly lower than those in \u003cem\u003eCol-0\u003c/em\u003e, while the expression levels in the complemented lines were comparable to those in \u003cem\u003eCol-0\u003c/em\u003e (Fig. 10E-J). These results indicate that \u003cem\u003eFvDi19-3\u003c/em\u003e can complement drought and salt tolerance of \u003cem\u003edi19\u003c/em\u003e mutants, sharing the similar function to\u003cem\u003e\u0026nbsp;Di19\u0026nbsp;\u003c/em\u003ein \u003cem\u003eArabidopsis\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.7 \u003cem\u003eFvDi19-3\u003c/em\u003e confers ABA sensitivity to \u003cem\u003eArabidopsis\u003c/em\u003e plants\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo ascertain whether the enhanced drought and salt resistance observed in the \u003cem\u003eFvDi19-3\u0026nbsp;\u003c/em\u003eoverexpressing \u003cem\u003eArabidopsis\u003c/em\u003e is associated with ABA, seeds from transgenic and \u003cem\u003eCol-0\u003c/em\u003e plants were germinated on 1/2 MS medium with or without ABA. No significant differences in germination rates were observed among the \u003cem\u003eCol-0\u003c/em\u003e, \u003cem\u003edi19\u003c/em\u003e mutant, overexpressed and complementary seeds when grown without ABA (Fig.11A). However, when germinated on 1/2 MS medium containing 0.5 \u0026micro;M or 1 \u0026micro;M ABA, all types of\u003cem\u003e\u0026nbsp;Arabidopsis\u0026nbsp;\u003c/em\u003eexhibited a notable decline the germination rates (Fig. 11A-B). Besides, the germination rates of the \u003cem\u003eFvDi19-3-OE\u003c/em\u003e lines were significantly lower than those of the \u003cem\u003eCol-0\u003c/em\u003e, while the \u003cem\u003edi19\u003c/em\u003e plants demonstrated a higher germination rate compared to the\u003cem\u003e\u0026nbsp;Col-0\u003c/em\u003e, this suggests that \u003cem\u003eFvDi19-3\u003c/em\u003e can mitigate the insensitivity phenotype observed in the \u003cem\u003edi19\u003c/em\u003e mutant (Fig. 11A-B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt is well documented that ABA exerts an inhibitory effect on plant root growth, therefore, the differences in ABA-induced inhibition of root growth were also observed.\u0026nbsp;As shown in Fig.11C, the exogenous application of ABA resulted in a more pronounced inhibition of root growth in the \u003cem\u003eFvDi19-3-OE\u003c/em\u003e seedlings when compared to the \u003cem\u003eCol-0\u003c/em\u003e and \u003cem\u003edi19\u003c/em\u003e seedlings, and overexpression of \u003cem\u003eFvDi19-3\u003c/em\u003e in \u003cem\u003edi19\u003c/em\u003e mutants significantly reduced the root length of the mutants (Fig.11C-D). This finding suggests that \u003cem\u003eFvDi19-3\u0026nbsp;\u003c/em\u003ecan increase ABA sensitivity in \u003cem\u003eArabidopsis\u003c/em\u003e. Additionally, the expression of ABA signaling genes, such as \u003cem\u003eAtABI3\u003c/em\u003e and \u003cem\u003eAtABI5\u003c/em\u003e, were substantially higher in the overexpression lines compared to those in wild-type and \u003cem\u003edi19\u0026nbsp;\u003c/em\u003eplants under salt and drought conditions (Fig. S4).\u0026nbsp;These results indicate that \u003cem\u003eFvDi19-3\u003c/em\u003e may play a role in the response to ABA-mediated drought and salt stresses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.8 \u003cem\u003eFvMYB114\u003c/em\u003e and \u003cem\u003eFvWRKY42\u0026nbsp;\u003c/em\u003ecan activate the promoter activity of \u003cem\u003eFvDi19-3\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePromoter element analysis revealed that the \u003cem\u003eFvDi19-3\u003c/em\u003e promoter region contains binding sites (MYB and W-box elements) for MYB and WRKY transcription factors (Fig.1F), it warrants further investigation to determine whether specific MYB and WRKY transcription factors associated with drought and salt stresses in strawberry can regulate the expression of the \u003cem\u003eFvDi19-3\u003c/em\u003e. Previous studies have demonstrated that strawberry \u003cem\u003eFvMYB63, FvMYB82\u003c/em\u003e and \u003cem\u003eFvMYB114\u003c/em\u003e can positively regulate the salt tolerance of transgenic \u003cem\u003eArabidopsis\u003c/em\u003e (Li, et al. 2023; Li, et al. 2022; Wang, et al. 2024). Additionally, \u003cem\u003eFvWRKY42\u003c/em\u003e has been shown to enhance both salt and drought tolerance in transgenic \u003cem\u003eArabidopsis\u003c/em\u003e (Wei, et al. 2018). As shown in Fig.12, we employed a dual luciferase assay to investigate whether these transcription factors (FvMYB63, FvMYB82, FvMYB114 and FvWRKY42) can regulate the expression of the \u003cem\u003eFvDi19-3\u003c/em\u003e. The results indicated that \u003cem\u003eFvMYB114\u003c/em\u003e and \u003cem\u003eFvWRKY42\u003c/em\u003e significantly activated the expression of\u003cem\u003e\u0026nbsp;FvDi19-3\u003c/em\u003e, whereas \u003cem\u003eFvMYB63\u0026nbsp;\u003c/em\u003eand \u003cem\u003eFvMYB82\u003c/em\u003e did not exhibit a significant activation effect on \u003cem\u003eFvDi19-3\u003c/em\u003e, similar to the empty control. This suggests that\u003cem\u003e\u0026nbsp;FvMYB114\u003c/em\u003e and \u003cem\u003eFvWRKY42\u003c/em\u003e may function as upstream regulatory genes of \u003cem\u003eFvDi19-3\u003c/em\u003e, playing a positive regulatory role in response to drought and salt stresses.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eDi19 proteins are members of the C2H2 transcription factor superfamily, and have been shown to play roles in plant growth, development, and stress response (Jiang, et al. 2022; Zhao, et al. 2022; Zhu, et al. 2023). Although the \u003cem\u003eDi19\u003c/em\u003e gene family has been identified in several plant species, including \u003cem\u003eArabidopsis\u0026nbsp;\u003c/em\u003e(Liu, et al. 2013), rice (Wang, et al. 2014), maize (Zhang, et al. 2019), soybeans (Feng, et al. 2015) and others, there is a paucity of research on the \u003cem\u003eDi19\u003c/em\u003e genes in woodland strawberry. To gain a deeper insight into the potential biological functions of \u003cem\u003eDi19\u003c/em\u003e genes in strawberry, an investigation was conducted using bioinformatics tools, in conjunction with analyses of phylogeny, gene structure, motif composition, chromosomal distribution, duplication events, synteny,\u003cem\u003e\u0026nbsp;cis-\u003c/em\u003eelements and expression profiles.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study, we have identified four highly conserved \u003cem\u003eDi19\u003c/em\u003e genes in woodland strawberry. Prior research has shown that many species have a limited number of Di19 family members, such as \u003cem\u003eArabidopsis\u003c/em\u003e (7) (Milla, et al. 2006), poplar (8) (Wu, et al. 2022), rice (7) (Wang, et al. 2014), maize (7) (Zhao, et al. 2022), millet (6) (Xiao, et al. 2023), soybean (15) (Jiang, et al. 2022)\u0026nbsp;, and \u003cem\u003egrapes\u0026nbsp;\u003c/em\u003e(6) (Table. S2). The relatively small number of \u003cem\u003eFvDi19\u003c/em\u003es in woodland strawberry may be related to the genome size of the species\u0026nbsp;(Edger, et al. 2019). As shown in Table 1, the Di19 proteins was encoded by small multigene, which were found to be low molecular weight, acidic, and hydrophilic proteins. The analysis of the gene structure and protein domains indicated that the \u003cem\u003eFvDi19s\u003c/em\u003e exhibited comparable gene structures, protein lengths, and motif compositions, which suggested that the \u003cem\u003eFvDi19\u003c/em\u003e members are subject to evolutionary conservation (Fig.1).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCis\u003c/em\u003e-elements in the promoter region are instrumental in regulating gene expression (Wei, et al. 2023). In the event of environmental stress, such as drought or salinity, the plant will respond by triggering a series of chemical reactions. These reactions activate transcription factors (TFs), which subsequently bind to specific elements within the DNA sequence to regulate the gene expression (Zhao, et al. 2022). In the promoter region of \u003cem\u003eFvDi19\u003c/em\u003e genes, five types of elements, associated with development, hormone, light, stress and MYB-related functions were identified (Fig.1E-F). Notably, the promoter region of \u003cem\u003eFvDi19\u003c/em\u003e genes displayed a high density of MYB and MYC \u003cem\u003ecis\u003c/em\u003e-acting elements. MYB transcription factors are widely distributed across plant species and bind to MYB elements to modulate the plant\u0026rsquo;s response to external stimulus (Pireyre and Burow 2015). The promoter region of \u003cem\u003ePtDi19-2/7\u003c/em\u003e in poplar contains both MYB and MYC \u003cem\u003ecis-\u003c/em\u003eelements, and its expression can be induced by PEG, NaCl, ABA and cold stresses (Wu, et al. 2022). These findings suggest that \u003cem\u003eFvDi19\u003c/em\u003e genes may play a significant role in stress response. Previous studies have also demonstrated that \u003cem\u003eDi19\u0026nbsp;\u003c/em\u003egenes\u003cem\u003e\u0026nbsp;\u003c/em\u003eexhibit varying expression levels under multiple stresses in different plants (Feng, et al. 2015; Jiang, et al. 2022; Wang, et al. 2014; Wu, et al. 2022). Analysis via qRT-PCR has shown that the transcripts of \u003cem\u003eFvDi19\u003c/em\u003e genes are elevated in response to drought and salt except \u003cem\u003eFvDi19-2\u003c/em\u003e (Fig.2). This differential expression suggests that \u003cem\u003eFvDi19-2\u003c/em\u003e may have distinct roles in strawberry growth and development compared to other \u003cem\u003eFvDi19s\u003c/em\u003e. It is noteworthy that \u003cem\u003eFvDi19s\u0026nbsp;\u003c/em\u003eexpression were also induced by\u003cem\u003e\u0026nbsp;\u003c/em\u003eMeJA and ABA, in accordance with the preceding reports on rice, wheat and soybean (Du, et al. 2023; Feng, et al. 2015; Wang, et al. 2016). However, ABA treatment did not significantly influence the expression levels of \u003cem\u003eAtDi19\u003c/em\u003e, indicating that the regulation of dehydration and salt stress by\u003cem\u003e\u0026nbsp;Di19\u0026nbsp;\u003c/em\u003eis independent of ABA\u003cem\u003e\u0026nbsp;\u003c/em\u003e(Milla, et al. 2006). These expression patterns of \u003cem\u003eDi19\u0026nbsp;\u003c/em\u003egenes may reflect functional differences in various plant species. Besides, \u003cem\u003eFvDi19s\u0026nbsp;\u003c/em\u003ehave the potential to enhance drought and salt tolerance in yeast (Fig.3), further demonstrating the role of the \u003cem\u003eFvDi19\u003c/em\u003e gene in response to abiotic stress. Interestingly, \u003cem\u003eFvDi19-3\u003c/em\u003e exhibited the most pronounced ability to augment tolerance to drought and salt stresses compared to other \u003cem\u003eFvDi19\u003c/em\u003e genes in yeast, which may be associated with its elevated expression level under an inducible expression mode (Fig.2). However, the biological functions of \u003cem\u003eFvDi19-3\u0026nbsp;\u003c/em\u003ein relation to drought and salt tolerance require further validation.\u003c/p\u003e\n\u003cp\u003eAn increasing body of evidence indicates that the heterologous expression of genes from non-model plants in model plants affects plant performance under stress conditions (Abid, et al. 2022). Previous studies have shown that the overexpression of \u003cem\u003eDi19\u003c/em\u003e in plants can either enhance or decrease their resilience to environmental stressors (Xiao, et al. 2023; Yang, et al. 2023; Zhang, et al. 2019). For instance, \u003cem\u003eSiDi19-3\u003c/em\u003e has been found to improve the salt tolerance of \u003cem\u003eFoxtail Millet\u003c/em\u003e and \u003cem\u003eArabidopsis\u0026nbsp;\u003c/em\u003e(Xiao, et al. 2023), whereas the ectopic expression of \u003cem\u003eGmDi19\u003c/em\u003e\u0026ndash;\u003cem\u003e15\u0026nbsp;\u003c/em\u003ein \u003cem\u003eArabidopsis\u0026nbsp;\u003c/em\u003eleads in reduced drought tolerance (Jiang, et al. 2022). In general, orthologous genes or closely related genes tend to have many similar functions. As can be illustrated in the phylogenetic tree (Fig. S1), both \u003cem\u003eGmDi19-5\u003c/em\u003e and\u003cem\u003e\u0026nbsp;AtDi19-3\u003c/em\u003e from group Ⅰ are associated with reduce salt tolerance,\u003cem\u003e\u0026nbsp;\u003c/em\u003ewhile\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eZmDi19-1\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;OsDi19-4\u003c/em\u003e from group V\u003cem\u003e\u0026nbsp;\u003c/em\u003ehave been shown to enhance stress tolerance in plants (Feng, et al. 2015; Qin, et al. 2014; Wang, et al. 2014; Zhang, et al. 2019). In our study, overexpression of \u003cem\u003eFvDi19-3\u003c/em\u003e in both \u003cem\u003eCol-0\u003c/em\u003e and \u003cem\u003edi19\u003c/em\u003e \u003cem\u003eArabidopsis\u003c/em\u003e resulted in increased drought and salt tolerance (Fig. 5-10), which is consistent with the function of the \u003cem\u003eGmDi19-15\u003c/em\u003e gene within the same group\u0026nbsp;(Jiang, et al. 2022). Besides, \u003cem\u003eFvDi19-3\u003c/em\u003e could promote stomatal closure in transgenic\u003cem\u003e\u0026nbsp;Arabidopsis\u003c/em\u003e, and stomatal aperture in the \u003cem\u003edi19\u0026nbsp;\u003c/em\u003emutant remains insensitive under drought and salt stresses (Fig.6C-E, Fig.9C-E). Closed stomata reduce transpiration and water loss from plant leaves, thereby enhancing plant drought and salt tolerance\u0026nbsp;(Hou, et al. 2024).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs is well known, drought and salt stresses disrupt ROS homeostasis, leading to the overproduction of ROS in plants, which has detrimental effects on for DNA, proteins, and lipids (Mishra, et al. 2023; Xu, et al. 2023). Generally, excessive ROS can disrupt antioxidant systems and the integrity of cell membranes, resulting in the accumulation of MDA (Gill and Tuteja 2010). The elevated activity of proline and SOD enzymes effectively degrades ROS in living cells, thereby safeguarding cellular membranes and reducing lipid peroxidation (Wang, et al. 2020). In comparison to\u003cem\u003e\u0026nbsp;Col-0\u003c/em\u003e, exposure to drought and salt stresses resulted in significantly reduced levels of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e, and MDA in the overexpression line. Conversely, the proline content and SOD enzyme activity were found to be elevated, indicating that oxidative damage is mitigated in the overexpression line (Figure 7A-D). Besides, these ROS substances showed opposite expression level in \u003cem\u003edi19\u003c/em\u003e mutant, which could be partially alleviated by complementing lines (Fig. 10A-D). These results indicate that \u003cem\u003eFvDi19-3\u003c/em\u003e transgenic plants may possess enhanced ROS scavenging activity compared to \u003cem\u003eCol-0\u003c/em\u003e and \u003cem\u003edi19\u003c/em\u003e plants, resulting in reduced membrane lipid peroxidation and an improved antioxidant defense system under drought and salt stresses.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn plants, the processes of drought and salt stress are complex, involving the coordinated action of numerous genes. Previous research has identified a variety of genes responsive to drought stress, such as \u003cem\u003eAtDREB2A\u003c/em\u003e, \u003cem\u003eAtRD29B\u003c/em\u003e, and \u003cem\u003eAtP5CS1\u003c/em\u003e, as well as those responsive to salt stress, including \u003cem\u003eAtEDR15\u003c/em\u003e, \u003cem\u003eAtNHX1\u003c/em\u003e, and\u003cem\u003e\u0026nbsp;AtSOS2\u003c/em\u003e (Fu, et al. 2023; Pan, et al. 2024; Yang, et al. 2019). These genes operate through distinct pathways, ultimately enhancing the plant\u0026apos;s resilience to drought and saline conditions. For instance, DREB transcription factors promote the expression of specific stress-responsive genes, thereby increasing drought tolerance through an ABA-mediated pathway. The \u003cem\u003eRD29B\u003c/em\u003e gene has been recognized as a target of DREB transcription factors, while \u003cem\u003eAtP5CS1\u003c/em\u003e is known to facilitate proline synthesis (Verma, et al. 2019; Virlouvet, et al. 2014). Additionally, \u003cem\u003eSOS2\u0026nbsp;\u003c/em\u003eis involved in the export of sodium ions from cells to maintain ionic balance, and \u003cem\u003eNHX1\u003c/em\u003e assists in the uptake of sodium ions into vacuoles (Yang, et al. 2019). Our RT-qPCR analysis demonstrated that a significant number of genes related to drought and salt stress exhibited up-regulation in transgenic lines subjected to these stresses, showing notable differences compared to control conditions. In summary, \u003cem\u003eFvDi19-3\u003c/em\u003e, along with several drought and salt stress-responsive genes, worked synergistically to enhance the resilience of\u003cem\u003e\u0026nbsp;Arabidopsis\u003c/em\u003e against drought and salt stresses.\u003c/p\u003e\n\u003cp\u003eIt has been demonstrated that ABA plays a pivotal role in enabling plants to withstand a multitude of abiotic stresses (Raghavendra, et al. 2010; Yu, et al. 2020). \u003cem\u003eOsDi19-4\u003c/em\u003e functions as a downstream regulator of \u003cem\u003eOsCDPK14\u003c/em\u003e, positively influencing ABA responses by modulating the expression of ABA-responsive genes in rice (Wang, et al. 2016). Overexpression of \u003cem\u003ePtDi19-2\u003c/em\u003e and\u003cem\u003e\u0026nbsp;PtDi19-7\u003c/em\u003e conferred drought tolerance in \u003cem\u003eArabidopsis\u003c/em\u003e by promoting ABA-induced stomatal closure (Wu, et al. 2022). In our study, two ABA-responsive elements (ABREs) were identified in the promoter regions of \u003cem\u003eFvDi19-3\u0026nbsp;\u003c/em\u003e(Fig.1F). \u003cem\u003eFvDi19-3\u003c/em\u003e was significantly induced by exogenous ABA (Fig. 2C) and overexpression of \u003cem\u003eFvDi19-3\u003c/em\u003e resulted in transgenic \u003cem\u003eArabidopsis\u003c/em\u003e hypersensitive to ABA. Previous study also shown that dehydration-responsive element-binding proteins (DREBs or DRE core) play significant role in regulating water deficit stress responses in ABA-dependent pathways (Zhao, et al. 2020).\u0026nbsp;The \u003cem\u003eDREB\u003c/em\u003e family member \u003cem\u003eAtDREB2A\u003c/em\u003e was induced in transgenic \u003cem\u003eArabidopsis\u0026nbsp;\u003c/em\u003eunder drought and salt stresses (Fig.11A-D, 10E), further indicating \u003cem\u003eFvDi19-3\u003c/em\u003e is involved in drought and salt stress responses through ABA-dependent pathways. The transcription factors FvWRKY42 and FvMYB114 have been shown to enhance the salt and drought tolerance of transgenic\u003cem\u003e\u0026nbsp;Arabidopsis\u0026nbsp;\u003c/em\u003e(Li, et al. 2023; Wei, et al. 2018). These factors can bind to the promoter of \u003cem\u003eFvDi19-3\u003c/em\u003e, thereby activating the expression of the \u003cem\u003eFvDi19-3\u003c/em\u003e gene. Furthermore, the expression of \u003cem\u003eFvWRKY42\u003c/em\u003e and \u003cem\u003eFvMYB114\u003c/em\u003e is dependent on the ABA signaling pathway, providing additional evidence that \u003cem\u003eFvDi19-3\u0026nbsp;\u003c/em\u003econtributes to drought and salt tolerance in \u003cem\u003eArabidopsis\u003c/em\u003e via this pathway. However, further research is necessary to elucidate the interconnections among these three genes and to clarify the specific regulatory mechanisms of \u003cem\u003eFvDi19-3\u003c/em\u003e in drought and salt resistance.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe present study identified 4 \u003cem\u003eDi19\u003c/em\u003e genes in woodland strawberry and conducted a comprehensive analysis of their phylogeny, gene structure, motif composition, collinearity relationships and \u003cem\u003ecis\u003c/em\u003e-element. qRT-PCR analysis of \u003cem\u003eFvDi19\u003c/em\u003e genes indicate that \u003cem\u003eFvDi19\u003c/em\u003es may play a significant role in responses to abiotic stresses and hormones. Overexpression of \u003cem\u003eFvDi19s\u0026nbsp;\u003c/em\u003eenhanced the resistance of yeast to drought and salt stresses, among which \u003cem\u003eFvDi19-3\u003c/em\u003e showed the strongest tolerance. LUC/REN assay indicated \u003cem\u003eFvWRKY42\u003c/em\u003e and \u003cem\u003eFvMYB114\u003c/em\u003e can bind to the promoter of \u003cem\u003eFvDi19-3\u003c/em\u003e and activate the expression of \u003cem\u003eFvDi19-3\u003c/em\u003e gene, and the expression of these three genes depends on the ABA signaling pathway. Furthermore, transgenic and stress tolerance assays indicated\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eFvDi19-3\u003c/em\u003e overexpression in \u003cem\u003eArabidopsis\u0026nbsp;\u003c/em\u003eenhanced plant drought and salt tolerance by promoting stomatal closure, improving the plant\u0026apos;s ability to scavenge reactive oxygen species and the expression of drought or salt-responsive genes (Fig.13). Nevertheless, the precise function of the \u003cem\u003eFvDi19-3\u003c/em\u003e gene must be corroborated in future studies employing contemporary genome editing and functional genomics techniques, particularly in the context of strawberry development and stress responses. In conclusion, the results of this study contribute to a deeper understanding of the specific functions of the \u003cem\u003eFvDi19-3\u003c/em\u003e genes and provide a valuable new resource for the genetic improvement of strawberry stress resistance.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Anhui Provincial Key Research and Development Plan (202104f06020004), Open Funding of National Engineering Laboratory of Crop Stress Resistance Breeding (NELCOF20210103).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJingjing Kong:\u0026nbsp;\u003c/strong\u003eWriting - original draft. \u003cstrong\u003eKeli Qiu:\u003c/strong\u003e Methodology, Data curation. \u003cstrong\u003eDebao Li:\u003c/strong\u003e Methodology, Data curation.\u003cstrong\u003e\u0026nbsp;Junyong Zhou:\u003c/strong\u003e Formal analysis, Data curation. \u003cstrong\u003eLijuan Lu:\u003c/strong\u003e Software, Methodology. \u003cstrong\u003eMao Liu:\u003c/strong\u003e Formal analysis, Data curation. \u003cstrong\u003eShufang Zhu:\u003c/strong\u003e Writing - review \u0026amp; editing. \u003cstrong\u003eZhiyuan Ning:\u003c/strong\u003e Formal analysis. \u003cstrong\u003eQibao Sun:\u0026nbsp;\u003c/strong\u003eWriting - review \u0026amp; editing, Funding acquisition.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbid M, Gu S, Zhang Y, Sun S, Li Z, Bai D, Sun L, Qi X, Zhong Y, Fang J (2022) Comparative transcriptome and metabolome analysis reveal key regulatory defense networks and genes involved in enhanced salt tolerance of \u003cem\u003eActinidia\u003c/em\u003e (kiwifruit). 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Planta 258(1):14. https://doi.org/10.1007/s00425-023-04172-6.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"FvDi19-3, woodland strawberry, drought and salt stresses, stomatal aperture, ROS-scavenging system","lastPublishedDoi":"10.21203/rs.3.rs-5719169/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5719169/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDi19 (drought-induced 19) proteins play a crucial role in regulating plant development and various stress responses. However, a systematic identification and functional analysis of the \u003cem\u003eDi19\u003c/em\u003egene family members in woodland strawberry has yet to be conducted. In this study, we identified four \u003cem\u003eDi19\u003c/em\u003e genes in woodland strawberry, and analyzed the phylogenetic tree, conserved protein domains, and gene structure. \u003cem\u003eCis\u003c/em\u003e-elements suggested that \u003cem\u003eFvDi19s\u003c/em\u003e may be involved in plant development and stress responses. Gene expression analysis revealed diverse expression patterns of\u003cem\u003eFvDi19s\u003c/em\u003e under different stress conditions, and overexpression of \u003cem\u003eFvDi19s\u003c/em\u003eenhanced drought and salt tolerance in yeast. Transgenic and stress tolerance assays indicated\u003cem\u003e FvDi19-3\u003c/em\u003e overexpression in \u003cem\u003eArabidopsis \u003c/em\u003eenhanced plant drought and salt tolerance by promoting stomatal closure, improving the plant's ability to scavenge reactive oxygen species and the expression of drought or salt-responsive genes. Furthermore, the LUC/REN ratio indicated that \u003cem\u003eFvWRKY42\u003c/em\u003eand \u003cem\u003eFvMYB114\u003c/em\u003e can activate the expression of \u003cem\u003eFvDi19-3\u003c/em\u003e, and expression of these three genes is dependent on the ABA signaling pathway. In conclusion, our study characterized the\u003cem\u003e Di19 \u003c/em\u003egene family in woodland strawberry and investigated the biological functions of\u003cem\u003e FvDi19-3\u003c/em\u003e in drought and salt tolerance, providing a basis for further functional studies of \u003cem\u003eFvDi19s\u003c/em\u003e in responses to abiotic stress.\u003c/p\u003e","manuscriptTitle":"Drought-induced 19 gene FvDi19-3 from woodland strawberry enhances drought and salt tolerance in transgenic Arabidopsis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-10 05:06:31","doi":"10.21203/rs.3.rs-5719169/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revisions","date":"2025-02-13T07:26:10+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-01-14T00:41:53+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-01-09T14:00:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell Reports","date":"2024-12-31T04:02:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-12-27T06:48:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"79dd627b-d376-4957-9b3a-a5658f5c8479","owner":[],"postedDate":"January 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-04-14T16:10:55+00:00","versionOfRecord":{"articleIdentity":"rs-5719169","link":"https://doi.org/10.1007/s00299-025-03481-2","journal":{"identity":"plant-cell-reports","isVorOnly":false,"title":"Plant Cell Reports"},"publishedOn":"2025-04-07 16:05:00","publishedOnDateReadable":"April 7th, 2025"},"versionCreatedAt":"2025-01-10 05:06:31","video":"","vorDoi":"10.1007/s00299-025-03481-2","vorDoiUrl":"https://doi.org/10.1007/s00299-025-03481-2","workflowStages":[]},"version":"v1","identity":"rs-5719169","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5719169","identity":"rs-5719169","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}