Functional characterization of FvCAMTA1in salt stress response of Fraxinus velutina

The calmodulin-binding transcription activator (CAMTA) family plays crucial roles in calcium-mediated abiotic stress responses in plants. This study isolated and functionally characterized FvCAMTA1, a CAMTA gene from the salt-tolerant woody species Fraxinus velutina. Promoter analysis identified salt-responsive cis-elements, with a 157-bp core region sufficient for basal promoter activity and upstream sequences enhancing transcriptional activation under salt stress. FvCAMTA1 was predominantly expressed in leaves and rapidly induced by NaCl treatment. The heterologous overexpression of FvCAMTA1 in Arabidopsis significantly enhanced salt tolerance, resulting in higher germination rates, improved root elongation, and increased fresh weight, whereas the camta5 mutant exhibited heightened sensitivity. Yeast two-hybrid screening identified 46 proteins interacting with FvCAMTA1, including FvWRKY7 and FvPP2C60, interactions subsequently confirmed by bimolecular fluorescence complementation and luciferase complementation assays. Our findings demonstrate that FvCAMTA1 acts as a positive regulator in the salt stress adaptation of woody plants through calcium signaling and transcriptional networks, providing a valuable candidate gene for molecular breeding of stress-resistant trees.

Introduction

Calmodulin (CaM), an essential Ca²⁺ signal sensor in plant cells, is a highly conserved calcium-binding protein that widely participates in plant growth, development, and environmental adaptation processes (Furio et al., 2020). Molecular interaction studies have identified over 90 calmodulin-binding transcription factors, including members of the CAMTA, MYB, bZIP, NAC, CBP60, WRKY, and MADS-box families (Park et al., 2005; Finkler et al., 2007; Popescu et al., 2007; Reddy et al., 2011; Singh et al., 2013; Zeng et al., 2015). Among these, the calmodulin-binding transcription activator (CAMTA) family has been confirmed as core regulatory components in plant Ca²⁺/CaM signaling pathways due to their unique calcium responsiveness (Yang et al., 2015; Kakar et al., 2018). Research on CAMTA transcription factors reveals their multifaceted roles in plant growth and abiotic stress responses through calcium signaling, including plant hormone response regulation, drought stress response, cold stress mediation, and immune response regulation. NtER1, a CAMTA family member, is induced by ethylene and participates in regulating plant senescence (Yang and Klee, 2000); Arabidopsis CAMTA1 mediates auxin signaling pathways to respond to heat stress (Galon et al., 2010a, 2010b).

The camta1 mutant exhibits reduced photosynthetic efficiency, water use efficiency, and relative water content under drought conditions, with significant alterations in gene expression related to osmoregulation, apoptosis, photosynthesis, and DNA methylation, suggesting AtCAMTA1’s involvement in drought recovery mechanisms (Pandey et al., 2013). The Atcamta1/camta3 double mutant shows significantly reduced freezing tolerance, indicating synergistic regulation of cold responses by AtCAMTA1 and AtCAMTA3 (Doherty et al., 2009) Calcium-bound AtCAMTA3/SR1 activates plant immune responses through protein binding (Feys et al., 2001; Zhang et al., 2012). AtCAMTA5 and AtCAMTA6 participate in responses to cold (Kidokoro et al., 2017), water deprivation (Méndez-Gómez et al., 2024), and salt (Shkolnik et al., 2019; Hau et al., 2024). Despite evidence showing salt stress-induced Ca²⁺ signaling in Arabidopsis (Bouche et al., 2005; Kim et al., 2009; Mahajan et al., 2009), molecular mechanisms decoding calcium signals and downstream pathways in woody plants remain largely unknown.

Fraxinus velutina, a deciduous tree native to southwestern North America, exhibits rapid growth and exceptional salinity tolerance. Owing to these traits, it has been widely introduced for cultivation in saline soils of China’s Yellow River Delta (Mao et al., 2017). Nevertheless, the molecular mechanisms underpinning the high salt tolerance in F. velutina remain largely elusive. Our previous transcriptomic comparison between salt-tolerant F. velutina and salt-sensitive F. chinensis identified 316 salt-induced genes, including FvCAMTA1. This gene exhibits significant induction kinetics under NaCl treatment and structural conservation with herbaceous homologs. Here, we present comprehensive analyses of FvCAMTA1 promoter architecture, expression dynamics, and functional validation through transgenic approaches. By integrating promoter truncation assays, and Arabidopsis transformation, this work provides mechanistic insights into CAMTA-regulated woody plant salt adaptation.

Materials and methods

Plant materials and treatments

Three-month old Fraxinus velutina tissue culture seedlings of variety ‘Lu Xiaowu 6’ and ‘Qing Bi’ from the Shandong Provincial Key Laboratory of Tree Breeding were used. Salt treatments involved hydroponic culture with 100-/300-mM NaCl solutions for 0, 1, 4, and 12 h. Then, fresh leaves were flash-frozen in liquid nitrogen for RNA extraction. Arabidopsis Col-0, camta5 mutants, and transgenic lines were maintained under controlled conditions (16 h light/8 h dark, 22°C).

Gene cloning and vector construction

Specific primers containing NdeI/KpnI sites amplified the coding sequence from leaf cDNA. The product was cloned into pMD18-T (TaKaRa) and verified by Sanger sequencing. For promoter analysis, three fragments (−1,084 to +1, −307 to +1, −157 to +1) were amplified using genome walking techniques and inserted into pPZP211-GUS vectors. Primers used for gene cloning are shown in Supplementary Table 1.

Bioinformatic analysis

NCBI CD-Search, InterPro, and DNAMAN 9.0 identified conserved domains. NetWheels predicted α-helix patterns in CaMBD regions. Phylogenetic trees were constructed using MEGA5.1 (Neighbor-Joining method). ProtScale and SOPMA analyzed hydrophobicity and secondary structure.

Tobacco transient expression and Arabidopsis transformation

Agrobacterium-mediated transient assays used 5-week-old N. benthamiana leaves. Infiltration buffer contained 10 mM MgCl₂, 10 mM MES (pH 5.6), and 100 μM acetosyringone. GUS staining (75 mM Na₃PO₄ pH 7.0, 0.05 mM K₃[Fe(CN)₆], 50 μg/mL X-Gluc) followed 48 h post-infiltration. Stable Arabidopsis transformation employed the floral dip method. T3 homozygous lines were selected on 50 mg/L kanamycin.

Salt stress treatment

Arabidopsis Col-0, camta5 mutants, and transgenic line were used for salt stress treatment. Germination assays scored seeds on 1/2MS medium with 0/200/300 mM NaCl after 7 days. Root elongation measurements were done at 21-day treatments. Hydroponic experiments quantified fresh weight after 30-day 200 mM NaCl exposure. Three biological replicates were conducted for all assays.

RNA extraction, qRT-PCR, yeast two-hybrid, BiFC, and LCA

Total RNA was extracted with the RNAprep Pure Plant Kit (#DP441, polysaccharides and polyphenolics-rich, Tiangen Biotech, Beijing, China). 2 µg of total RNA was used to synthesize the first-strand cDNA with the PrimeScript™ 1st Strand cDNA Synthesis Kit (Takara, Shiga, Japan). The cDNA reaction mixture was diluted five times, and 5 µl was used in the 20-µl PCR reaction. The PCR reactions included a pre-incubation step at 95°C for 2 min followed by 45 cycles of denaturation at 95°C for 15 s, annealing at 54°C for 30 s, and extension at 72°C for 30 s. All reactions were performed in the QuantStudio™ 5 Food Safety Real-Time PCR System using TB Green Fast qPCR Mix (Takara) and ROX reference dye. Each experiment had nine replicates (three technical replicates for each biological replicate). The relative expression level was calculated using the 2^^−ΔΔCt method.

Yeast two-hybrid screening and analysis were performed as methods mentioned by Chen and Wei (2022). A cDNA library was created by seeding of Fraxinus velutina with 150 mmol/L NaCl treated for 12 h. Bimolecular fluorescence complementation was performed as methods mentioned by Goto-Yamada et al. (2018). Luciferase complementation assay (LCA) was performed by methods mentioned by Zhou et al. (2018).

All primers used are listed in Supplementary Table 1.

Microscopy and statistical analysis

Confocal imaging (Leica TCS SP5) visualized signals in transformed protoplasts. ImageJ quantified GUS staining intensity. Data were analyzed using SPSS 18.0 with ANOVA and Duncan’s multiple range tests (p<0.05).

Results

Molecular characterization of FvCAMTA1

The 2,772-bp FvCAMTA1 ORF encodes 923 amino acids with a 104.33-kDa molecular weight and pI 7.37. Conserved domain analysis identified the N-terminal CG-1 DNA-binding domain (residues 30-130) with a nuclear localization signal; the TIG interaction domain (residues 140-250); ANK repeats (residues 260-380); and C-terminal IQ motifs (Ile763-Arg815): IQ1 (763IQHAFRKYETK773), IQ2 (782IQYRFRTWKMR792), and IQ3 (805IQAAVRGFQVR815) (Figures 1A, B). NetWheels modeling revealed an amphipathic α-helix structure in the CaMBD region (residues 826-843), with hydrophobic residues clustered at 0°-180° and hydrophilic residues (Arg838, Lys839, Arg841) at 180°-360°, consistent with classical CaMBD patterns (Figure 1C). Secondary structure prediction showed 45.50% α-helix, 15.17% β-sheet, 9.10% β-turn, and 30.23% random coils (Figure 1D).

Figure 1

Figure 1. The domains and secondary structures of FVCAMTA1. (A) Multiple comparisons of FVCAMTA1 with CaMBD sequences from other species; (B) The a-helix diagram of the CaMBD sequence of FVCAMTA1. (C) The predicted results of the domains of CAMTAS; (D) The predicted results of the secondary structure of the FVCAMTA1 protein.

Neighbor-joining tree (Saitou and Nei 1987) of CAMTAs from multiple species grouped FvCAMTA1 with Arabidopsis AtCAMTA5 (82% identity) and tomato SlCAMTA2 (Figure 2). These results indicating FvCAMTA1 is a member of CATMA family.

Figure 2

Figure 2. Construction of the phylogenetic tree of FVCAMTAI. The phylogenetic tree was constructed using the Neighbor-Joining method; the number of Bootstrap replications was 1000.

Promoter of FvCAMTA1 contains salt-response elements

Through genome walking techniques, we obtained a 1,231-bp promoter sequence upstream of the FvCAMTA1 ATG start codon (Figure 3A). Bioinformatic analysis using NNPP database identified three potential promoter regions located at positions 148–198 bp, 925–975 bp, and 1,076-1,126 bp relative to the transcription start site. PlantCARE analysis revealed conserved core promoter elements including TATA-box (essential for transcription initiation) and CAAT-box (enhancer element), which conform to eukaryotic promoter structural characteristics. Functional motif analysis identified comprehensive regulatory elements: Hormone response elements, light response elements, abiotic stress elements, and MYB/MYC binding sites were identified (Figure 3B), suggesting complex regulatory mechanisms under both developmental and stress conditions.

Figure 3

Figure 3. The FVCAMTA1 promoter analysis. (A) The promoter sequence cloned in this study. The dotted box indicates three potential promoter sites predicted by the NNPP online database. (B) Predicted motifs by PlantCARE.

To investigate salt-responsive promoter regions, we performed Agrobacterium-mediated transient transformation of salt-stressed tobacco leaves. Three promoter fragments (−1,084 to +1: 1,084 bp; −307 to +1: 307 bp; −157 to +1: 157 bp) were cloned into pPZP211-GUS vector (Figure 4). GUS staining showed that the 157-bp fragment produced consistent blue staining in salt-treated leaves, indicating that this minimal region contains essential cis-elements (Figure 4A). The 1,084-bp fragment showed significantly deeper staining intensity compared with the 157-bp fragment under identical conditions (Figure 4C). In the 307-bp fragment and controls, there was no detectable GUS activity in either empty vector or buffer-treated controls (Figures 4B, D–F). This suggests that whereas the 157-bp core region maintains basic promoter functionality, upstream sequences (particularly between −1,084 and −157 bp) contain enhancer elements critical for full transcriptional activation under salt stress.

Figure 4

Figure 4. GUS staining of promoters of different lengths in tobacco leaf after salt stress treatment. (A–C) demonstrate the expression of FVCAMTA1::GUS constructs driven by promoter regions of 157 bp, 307 bp, and 1084 bp in tobacco leaves. (D) Represents the negative control injected with a blank vector. (E) depicts samples subjected to salt stress without blank vector injection. (F) Blank vector-injected controls under salt stress.

Functional validation and of FvCAMTA1 in transgenic Arabidopsis

To clarify the contribution of FvCAMTA1 to salt tolerance, FvCAMTA1 overexpression lines were generated through Agrobacterium-mediated transformation. Given 82% amino acid identity between FvCAMTA1 and Arabidopsis AtCAMTA5, camta5 mutants and wild type were selected as controls (Figure 5). Under 200 mM NaCl stress, germination rates at day 5 showed camta5 mutants (42.3 ± 3.5%) significantly lower than the wild type (58.7 ± 4.1%, p<0.05), whereas the OE-6 line reached 72.6 ± 5.2% (Figure 5A). At 300 mM NaCl, camta5 mutants (23.4 ± 2.8%) remained significantly lower than the wild type (29.7 ± 5.1%, p<0.05), with OE-6 at 45.3 ± 4.7%. Root phenotyping showed OE-6 primary root length (4.2 cm) significantly longer than the wild type (2.9 cm, p<0.01) under 200 mM stress, versus camta5 mutants (1.1 cm) (Figure 5C). Long-term 200 mM NaCl treatment showed camta5 mutants’ fresh weight lower than the wild type, whereas OE-6 exceeded wild-type levels (Figure 5B), confirming FvCAMTA1’s positive regulatory role in salt stress responses.

Figure 5

Figure 5. The salt-tolerant phenotypes of camta5 mutant and FVCAMTA1 overexpression plants (A) After 5 days of germination on 1/2 MS medium with different concentrations of NaCl, the germination number on 1/2 MS medium without NaCl was used as the control to calculate *the relative germination rate of different materials; (B) After germination on 1/2 MS medium without NaCl for 7 days, they were transferred to the nutrient soil and treated with a 200 mM NaCl aqueous solution for 14-30 days. The fresh weight of the aboveground part of the WT plants on the 30th day was used as the control to calculate the relative fresh weight at different times and for different materials. (C) The root phenotypes of different materials after 14 days of cultivation on 1/2 MS medium with 200 mM NaCl. Representation: mean ± standard deviation, *represents p< 0.05, **represents p<0.01, ***represents p<0.001, Student's t-test, n = 20.

FvCAMTA1 majorly expressed in leaves and positively regulates resistance of salt stress

To clarify the response of FvCAMTA1 under salt stress, tissue expression specificity and salt stress treatment were performed. FvCAMTA1 is expressed at the lowest level in the stem, followed by the root, which is 2.1 times that of the stem. It is expressed at the highest level in the leaves, being 3.2 times that of the stem (Supplementary Figure 1). Under the treatment of 100-mmol/L NaCl solution, the expression level of FvCAMTA1 shows a trend of rapid increase followed by a decrease. At 1 h, the expression level of FvCAMTA1 is the highest, 2.236 times that of 0 h (Figure 6). Then, it rapidly decreases and reaches the same level as 0 h at 12 h. Under the treatment of 300 mmol/L NaCl solution, the expression trend of FvCAMTA1 is similar to that of 100 mmol/L NaCl treatment, but the expression level is significantly higher than that of 100 mmol/L NaCl treatment. At 1 h, its expression level is 3.8 times that of 0 h, and at 12 h, the expression level is still significantly higher than 0 h, being 4.1 times that of 0 h. In the salt-tolerant variety ‘Lu Xiaowu 6’, the expression level of FvCAMTA1 is higher than that in ‘Qing Bi’. After 1 h of treatment with 300 mmol/L NaCl solution, the expression level of FvCAMTA1 in ‘Lu Xiaowu 6’ is 6 times that of 0 h, and at 12 h, it is still 4.1 times that of 0 h (Figure 6).

Figure 6

Figure 6. Expression of FVCAMTA1 in WT and salt-tolerant cultivar ‘lila6' under 100 mmol/L and 300 mmol/L NaCl treatment. *represents a significant level of p<0.05, **represents a significant level of p<0.01.

Yeast two-hybrid screening identified multiple interacting proteins FvCAMTA1

To confirm how FvCAMTA1 induced salt resistance, Y2H were performed (Supplementary Figure 2). A total of 46 interacting proteins were screened from cDNA library of salt-stressed Fraxinus velutina (Supplementary Table 2). To confirm Y2H screening results, Y2H analysis, BiFC analysis, and LCA were performed between FvCAMTA1 and two genes: FvPP2C60 andFvWRKY7. Y2H results showed that FvCAMTA1 strongly interacted with FvWRKY7 and weakly interacted with FvPP2C60 (Figure 7A). LCA and BiFC analysis showed that FvCAMTA1 interacted with FvPP2C and FvWRKY7 (Figures 7B, C).

Figure 7

Figure 7. Y2H, LCA and BiFC analysis shown FVCAMTA1 interacts with FvWRKY7 and FvPP2C60. (A) Yeast two-hybrid (Y2H) assays confirmed interactions between FVCAMTA1 and FvWRKY7/FvPP2C60.TDO: SD/-Leu/-Trp/-His medium with 5 mM 3AT; QDO: SD/-Leu/-Trp/-His/-Ade medium; Numbers indicate dilution factors; "-": pGBKT7-FvCAMTA1+pGADT7 (negative control); "+": PGBKT7-53 +pGADT7-T (positive control); "1": pGBKT7-FvCAMTA1+pGADT7-FvWRKY7; "2": pGBKT7-FVCAMTA1+pGADT7-FvPP2C60. (B) Tobacco luciferase complementation assays (LCA) validated FVCAMTA1 interactions with FVWRKY7 and FvPP2C60. Upper-left quadrant: Agrobacterium mixture carrying pCAMBIA 1300-nLUC-FVCAMTA1 and pCAMBIA1300-cLUC-FvWRKY7/FvPP2C60; Upper-right and lower-left quadrants: Negative controls with one partner + empty vector (e.g., pCAMBIA 1300-nLUC-FvCAMTA1 + pCAMBIA1300- CLUC);Lower-right quadrant: Double empty vector control. (C) Bimolecular fluorescence complementation (BIFC) in tobacco epidermis demonstrated FVCAMTA1 interactions with FvWRKY7 and FvPP2C60.Row 1: PSPYNE-35S empty vector (negative control); Row 2: PSPYNE-35S-FVCAMTA1;Column 1: PSPYCE-35S empty vector (negative control);* Columns 2-4*: PSPYCE-35S-FvWRKY7, pSPYCE-35S-FvPP2C60;Scale bar = 50 μm.

Discussion

Structural and promoter architecture of CAMTA1

CAMTA transcription factors are evolutionarily conserved across eukaryotes, with documented roles in Populus trichocarpa (Li et al., 2015), Gossypium (Zhang et al., 2016), and Phaseolus vulgaris (Büyük et al., 2019). Our study successfully cloned the full-length FvCAMTA1 promoter containing typical eukaryotic promoter elements (TATA-box at −28 bp, CAAT-box at −98 bp) and multiple stress-responsive motifs. Functional validation confirmed that the 157-bp core promoter region maintains expression capability, whereas upstream elements enhance transcriptional activity. Galon et al. (2010a) demonstrated salt concentration-dependent GUS expression in AtCAMTA1 promoter studies. Prasad et al. (2016) revealed CAMTA3/SR1’s regulatory roles in salt stress through RNA-seq. Our transient expression assays confirmed FvCAMTA1’s salt-responsive activation, with expression levels positively correlated with stress severity and cultivar tolerance, aligning with Büyük et al.’s (2019) findings in common beans.

The 1,231-bp FvCAMTA1 promoter contains overlapping regulatory modules for hormonal crosstalk and abiotic stress. The 157-bp core region suffices for basal expression, but upstream elements (−1,084 to −157) amplify salt responsiveness. This modular organization resembles AtCAMTA1’s promoter, where auxin-responsive elements mediate thermotolerance (Galon et al., 2010a). The promoter sequence also contains plant hormone response element, light response element, and salt stress response element TC-rich repeats. Hormone response elements include salicylic acid (SA) response element TCA-element, abscisic acid (ABA) response element ABRE, jasmonic acid (JA) response element TGACG-motif and CGTCA-motif, ethylene response element ERE, and auxin response element TGA-element. SA, ABA, JA, and ethylene were reported to respond to salt stress (van Zelm et al., 2020; Zhang et al., 2022; Park et al., 2022; Liu et al., 2022; Gao et al., 2025). The abundance of ABRE and ARE motif implies integration with ABA-dependent and anaerobic pathways during salt adaptation.

FvCAMTA1 positively regulates salt resistance

Transgenic Arabidopsis assays confirm FvCAMTA1’s positive regulatory role in salt tolerance. Enhanced germination and root growth in overexpression lines mirror OsCAMTA4’s function in rice (Ding et al., 2020) whereas camta5 hypersensitivity highlights genetic specificity.

Expression levels of FvCAMTA1 were induced by salt stress (Figure 6). In the salt-tolerant variety ‘Lu Xiaowu 6’, the expression of FvCAMTA1 was significantly higher than that in wild-type ‘Qing Bi’ under salt stress. These results indicate that FvCAMTA1 positively regulated salt resistance.

The promoter of FvCAMTA1 in ‘Qing Bi’ and ‘Lu Xiaowu 6’ showed a motif difference (Supplementary Figure 3). MYB and TC-rich repeats were missing in ‘Qing Bi’, whereas the G-box was missing in ‘Lu Xiaowu 6’. These might result into a different response of FvCAMTA1 between two varieties.

FvCAMTA1 is expressed in leaves and might contribute to biotic stress

FvCAMTA1 expression in leaves suggests roles in photosynthetic tissue protection, contrasting with root-predominant AtCAMTA1. However, shared drought response mechanisms exist: FvCAMTA1 induction correlates with AtCAMTA1’s regulation of osmotic adjustment genes (Pandey et al., 2013). This functional overlap implies conserved stress response networks despite divergent expression gradients.

Y2H screening identified 46 interact proteins of FvCAMTA1. Among them, FvWRKY7 and FvPP2C60 were confirmed by Y2H, BiFC, and LCA. WRKY7 were members of the NRT1/PTR FAMILY 6.4, the WRKY7 transcription factor, ribosomal proteins, metallothionein, early light-induced protein (ELIP), and several proteins of unknown function. In Myrothamnus flabellifolia, the overexpression of MfWRKY7 exhibited a longer root length, better growth performance, higher contents of leaf water, and chlorophyll and osmolyte accumulation under salt stress compared with the wild type (Xu et al., 2025). Overexpression of Arabidopsis thaliana AtWRKY7 enhanced resistance to Pseudomonas syringae and black spot disease (Kim et al., 2006); OsWRKY7 was the potential candidate gene for resistance to panicle blast in rice (Sureshkumar et al., 2019). The interaction between FvCAMTA1 and FvWRKY7 suggests that they may cooperatively regulate plant salt responses. Furthermore, FvCAMTA1 was found to interact with FvPP2C60. In Arabidopsis thaliana, protein phosphatase 2C (PP2C) participates in the ABA signaling pathway and positively regulates salt stress (Yang et al., 2024).

In conclusion, we report the functional characterization of FvCAMTA1 in woody plants, demonstrating its dual role as a calcium sensor and transcriptional activator in salt stress networks. Comprehensive promoter analysis and transgenic validation collectively establish FvCAMTA1 as a positive regulator of salt tolerance in Arabidopsis thaliana. These findings provide molecular insights into CAMTA-mediated stress adaptation and offer biotechnological potential for forest tree improvement The function of FvCAMTA1 in salt stress response will be further verified in Fraxinus velutina in future studies.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Author contributions

LPY: Validation, Supervision, Resources, Funding acquisition, Conceptualization, Writing – review & editing, Project administration, Writing – original draft, Formal analysis, Visualization, Data curation, Methodology. DG: Writing – original draft, Writing – review & editing. YW: Data curation, Formal analysis, Methodology, Writing – review & editing. CS: Data curation, Writing – review & editing. TL: Methodology, Writing – review & editing. BW: Resources, Writing – review & editing. JY: Writing – review & editing, Validation. FR: Writing – review & editing, Data curation. LJY: Funding acquisition, Writing – review & editing, Supervision.

Funding

The author(s) declare financial support was received for the research and/or publication of this article. This study was supported by The Taishan Scholar Award Program funding as the first supporter funding and The “New Variety Selection for Salt-Tolerant Ecological Economic Tree Species” project of the Agricultural Superior Variety Project in Shandong Province (2023LZGC012).

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2025.1669043/full#supplementary-material

Supplementary Table 1 | Primers used in experiment.

Supplementary Table 2 | Genes screened by Y2H

References

Bouche, N., Yellin, A., Snedden, W. A., and Fromm, H. (2005). Plant-specific calmodulin-binding proteins. Annu. Rev. Plant Biol. 56, 435–466. doi: 10.1146/annurev.arplant.56.032604.144224

PubMed Abstract | Crossref Full Text | Google Scholar

Büyük, İ., İlhan, E., Şener, D., Özsoy, A. U., and Aras, S. (2019). Genome-wide identification of CAMTA gene family members in Phaseolus vulgaris L. and their expression profiling during salt stress. Mol. Biol. Rep. 46, 2721–2732. doi: 10.1007/s11033-019-04716-8

PubMed Abstract | Crossref Full Text | Google Scholar

Chen, Q. and Wei, T. (2022). Membrane and nuclear yeast two-hybrid systems. Methods Mol. Biol. (Clifton N.J.) 2400, 93–104. doi: 10.1007/978-1-0716-1835-6_10

PubMed Abstract | Crossref Full Text | Google Scholar

Ding, G., Lei, G. J., Yamaji, N., Yokosho, K., Mitani-Ueno, N., Huang, S., et al. (2020). Vascular Cambium-Localized AtSPDT Mediates Xylem-to-Phloem Transfer of Phosphorus for Its Preferential Distribution in Arabidopsis. Mol Plant. 13(1), 99–111. doi: 10.1016/j.molp.2019.10.002

PubMed Abstract | Crossref Full Text | Google Scholar

Doherty, C. J., Van Buskirk, H. A., Myers, S. J., and Thomashow, M. F. (2009). Roles for Arabidopsis CAMTA transcription factors in cold-regulated gene expression and freezing tolerance. Plant Cell 21, 972–984. doi: 10.1105/tpc.108.063958

PubMed Abstract | Crossref Full Text | Google Scholar

Feys, B. J., Moisan, L. J., Newman, M. A., and Parker, J. E. (2001). Direct interaction between the Arabidopsis disease resistance signaling proteins, EDS1 and PAD4. EMBO J. 20, 5400–5411. doi: 10.1093/emboj/20.19.5400

PubMed Abstract | Crossref Full Text | Google Scholar

Finkler, A., Ashery-Padan, R., and Fromm, H. (2007). CAMTAs: Calmodulin-binding transcription activators from plants to human. FEBS Lett. 581, 3893–3898. doi: 10.1016/j.febslet.2007.07.051

PubMed Abstract | Crossref Full Text | Google Scholar

Furio, R. N., Martínez-Zamora, G. M., Salazar, S. M., Coll, Y., Perato, S. M., Martos, G. G., et al. (2020). Role of calcium in the defense response induced by brassinosteroids in strawberry plants. Sci. Hortic. 261, 109010. doi: 10.1016/j.scienta.2019.109010

Crossref Full Text | Google Scholar

Galon, Y., Aloni, R., Nachmias, D., et al. (2010a). Calmodulin-binding transcription activator 1 mediates auxin signaling and responds to stresses in Arabidopsis. Planta 232, 165–178. doi: 10.1007/s00425-010-1153-6

PubMed Abstract | Crossref Full Text | Google Scholar

Galon, Y., Aloni, R., Nachmias, D., Snir, O., Feldmesser, E., Scrase-Field, S., et al (2010b). How calmodulin binding transcription activators (CAMTAs) mediate auxin responses. Plant Signaling Behav. 5, 1311–1314. doi: 10.4161/psb.5.10.13158

PubMed Abstract | Crossref Full Text | Google Scholar

Gao, X., Li, M., Gong, Q., Li, G., Yu, H., Dong, X., et al. (2025). Hydrogen-Sulfide-Mediated PpAOS3-JA Module Provides Insight into Salt Stress Resistance in Peach. Plants (Basel). 14(10), 1477. doi: 10.3390/plants14101477

PubMed Abstract | Crossref Full Text | Google Scholar

Goto-Yamada, S., Hikino, K., Nishimura, M., Nakagawa, T., and Mano, S. (2018). Bimolecular fluorescence complementation with improved gateway-compatible vectors to visualize protein-protein interactions in plant cells. Methods Mol. Biol. (Clifton N.J.) 1794, 245–258. doi: 10.1007/978-1-4939-7871-7_16

PubMed Abstract | Crossref Full Text | Google Scholar

Hau, B., Symonds, K., Teresinski, H., Janssen, A., Duff, L., Smith, M., et al. (2024). Arabidopsis calmodulin-like proteins CML13 and CML14 interact with calmodulin-binding transcriptional activators and function in salinity stress response. Plant Cell Physiol. 65, 282–300. doi: 10.1093/pcp/pcad152

PubMed Abstract | Crossref Full Text | Google Scholar

Kakar, K. U., Nawaz, Z., Cui, Z., Cao, P., Jin, J., Shu, Q., et al. (2018). Evolutionary and expression analysis of CAMTA gene family in Nicotiana tabacum yielded insights into their origin, expansion and stress responses. Sci Rep. 10 (1), 4635. doi: 10.1038/s41598-018-28148-9

PubMed Abstract | Crossref Full Text | Google Scholar

Kidokoro, S., Yoneda, K., Takasaki, H., Takahashi, F., Shinozaki, K., and Yamaguchi-Shinozaki, K. (2017). Different cold-signaling pathways function in the responses to rapid and gradual decreases in temperature. Plant Cell. 29, 760–774. doi: 10.1105/tpc.16.00669

PubMed Abstract | Crossref Full Text | Google Scholar

Kim, K. C., Fan, B., and Chen, Z. (2006). Pathogen-induced Arabidopsis WRKY7 is a transcriptional repressor and enhances plant susceptibility to Pseudomonas syringae. Plant Physiol. 142, 1180–1192. doi: 10.1104/pp.106.082487

PubMed Abstract | Crossref Full Text | Google Scholar

Kim, D., Kim, C. H., Moon, J. I., Chung, Y. G., Chang, M. Y., Han, B. S., et al. (2009). Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell 4, 472–476. doi: 10.1016/j.stem.2009.05.005

PubMed Abstract | Crossref Full Text | Google Scholar

Li, Q., Yeh, T. F., Yang, C., Song, J., Chen, Z. Z., Sederoff, R. R., et al. (2015). Populus trichocarpa. Methods Mol Biol. 1224:357–63. doi: 10.1007/978-1-4939-1658-0_28

PubMed Abstract | Crossref Full Text | Google Scholar

Liu, Z., Ma, C., Hou, L., Wu, X., Wang, D., Zhang, L., et al. (2022). Exogenous SA affects rice seed germination under salt stress by regulating na⁺/K⁺ Balance and endogenous GAs and ABA homeostasis. Int. J. Mol. Sci. 23, 3293. doi: 10.3390/ijms23063293

PubMed Abstract | Crossref Full Text | Google Scholar

Mahajan, A., Yuan, C., Lee, H., Chen, E. S., Wu, P. Y., Tsai, M. D., et al. (2009). Structure and function of the phosphothreonine-specific FHA domain. Sci. Signaling 2, ra12. doi: 10.1126/scisignal.151re12

PubMed Abstract | Crossref Full Text | Google Scholar

Mao, P., Tang, Q., Cao, B., Liu, J., Shao, H., Cai, Z., et al. (2017). Eco-physiological adaptability in mixtures of robinia pseudoacacia and Fraxinus velutina and coastal eco-engineering. Ecol. Eng. 106, 109–115. doi: 10.1016/j.ecoleng.2017.05.021

Crossref Full Text | Google Scholar

Méndez-Gómez, M., Sierra-Cacho, D., Jiménez-Morales, E., and Guzmán, P. (2024). Modulation of early gene expression responses to water deprivation stress by the E3 ubiquitin ligase ATL80: implications for retrograde signaling interplay. BMC Plant Biol. 24, 180. doi: 10.1186/s12870-024-04872-5

PubMed Abstract | Crossref Full Text | Google Scholar

Pandey, A., Gordon, D. M., Pain, J., Stemmler, T. L., Dancis, A., Pain, D., et al. (2013). Frataxin directly stimulates mitochondrial cysteine desulfurase by exposing substrate-binding sites, and a mutant Fe-S cluster scaffold protein with frataxin-bypassing ability acts similarly. J. Biol. Chem. 288, 36773–36786. doi: 10.1074/jbc.M113.525857

PubMed Abstract | Crossref Full Text | Google Scholar

Park, S. J., Park, S., Kim, Y., Hyeon, D. Y., Park, H., Jeong, J., et al. (2022). Ethylene responsive factor34 mediates stress-induced leaf senescence by regulating salt stress-responsive genes. Plant Cell Environ. 45(6), 1719–1733. doi: 10.1111/pce.1431

PubMed Abstract | Crossref Full Text | Google Scholar

Park, C. Y., Lee, J. H., Yoo, J. H., Moon, B. C., Choi, M. S., Kang, Y. H., et al. (2005). WRKY group IId transcription factors interact with calmodulin. FEBS Lett. 579, 1545–1550. doi: 10.1016/j.febslet.2005.01.057

PubMed Abstract | Crossref Full Text | Google Scholar

Popescu, S. C., Popescu, G. V., Bachan, S., Zhang, Z., Seay, M., Gerstein, M., et al. (2007). Differential binding of calmodulin-related proteins to their targets revealed through high-density Arabidopsis protein microarrays. Proc. Natl. Acad. Sci. United States America 104, 4730–4735. doi: 10.1073/pnas.0611615104

PubMed Abstract | Crossref Full Text | Google Scholar

Prasad, K. V. S. K., Abdel-Hameed, A. A. E., Xing, D., and Reddy, A. S. N. (2016). Global gene expression analysis using RNA-seq uncovered a new role for SR1/CAMTA3 transcription factor in salt stress. Sci. Rep. 6, 27021. doi: 10.1038/srep27021

PubMed Abstract | Crossref Full Text | Google Scholar

Reddy, A. S., Ali, G. S., Celesnik, H., and Day, I. S. (2011). Coping with stresses: Roles of calcium- and calcium/calmodulin-regulated gene expression. Plant Cell 23, 2010–2032. doi: 10.1105/tpc.111.084988

PubMed Abstract | Crossref Full Text | Google Scholar

Saitou, N. and Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 4(4), 406–25. doi: 10.1093/oxfordjournals.molbev.a040454

PubMed Abstract | Crossref Full Text | Google Scholar

Shkolnik, D., Finkler, A., Pasmanik-Chor, M., and Fromm, H. (2019). CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR 6: A key regulator of na⁺ Homeostasis during germination. Plant Physiol. 180, 1101–1118. doi: 10.1104/pp.19.00119

PubMed Abstract | Crossref Full Text | Google Scholar

Singh, V. P., Rubinstein, J., Arvanitis, D. A., Ren, X., Gao, X., Haghighi, K., et al. (2013). Abnormal calcium cycling and cardiac arrhythmias associated with the human Ser96Ala genetic variant of histidine-rich calcium-binding protein. J. Am. Heart Assoc. 2, e000460. doi: 10.1161/JAHA.113.000460

PubMed Abstract | Crossref Full Text | Google Scholar

Sureshkumar, V., Dutta, B., Kumar, V., Prakash, G., Mishra, D. C., Chaturvedi, K. K., et al. (2019). RiceMetaSysB: a database of blast and bacterial blight responsive genes in rice and its utilization in identifying key blast-resistant WRKY genes. Database: J. Biol. Database Curation 2019, baz015. doi: 10.1093/database/baz015

PubMed Abstract | Crossref Full Text | Google Scholar

van Zelm, E., Zhang, Y., and Testerink, C. (2020). Salt tolerance mechanisms of plants. Annu. Rev. Plant Biol. 71, 403–433. doi: 10.1146/annurev-arplant-050718-100005

PubMed Abstract | Crossref Full Text | Google Scholar

Xu, W., Xiang, X., Chen, D., Song, L., Tang, R., Zhou, X., et al. (2025). MfWRKY7.2 of woody resurrection plant Myrothamnus flabellifolia is involved in tolerance to drought and salt stress. Gene 961, 149548. doi: 10.1016/j.gene.2025.149548

PubMed Abstract | Crossref Full Text | Google Scholar

Yang, S. A. and Klee, C. B. (2000). Low affinity Ca²⁺-binding sites of calcineurin B mediate conformational changes in calcineurin A. Biochemistry 39, 16147–16154. doi: 10.1021/bi001321q

PubMed Abstract | Crossref Full Text | Google Scholar

Yang, G., Li, Z., Rong, M., Yu, R., Zhang, Q., Wang, G., et al. (2024). Comparative transcriptome analysis to identify the important mRNA and lncRNA associated with salinity tolerance in alfalfa. PeerJ 12, e18236. doi: 10.7717/peerj.18236

PubMed Abstract | Crossref Full Text | Google Scholar

Yang, Y., Sun, T., Xu, L., Pi, E., Wang, S., Wang, H., et al. (2015). Genome-wide identification of CAMTA gene family members in Medicago truncatula and their expression during root nodule symbiosis and hormone treatments. Front. Plant Sci. 6. doi: 10.3389/fpls.2015.00459

PubMed Abstract | Crossref Full Text | Google Scholar

Zeng, H. Q., Wang, G. P., Wang, H. Z., Lin, J. X., and Du, L. Q. (2015). Functions of calmodulin-binding transcription activators (CAMTAs) in plants. Plant Physiol. J. 51, 633–641.

Google Scholar

Zhang, G., Fang, X., Guo, X., Li, L., Luo, R., Xu, F., et al. (2012). The oyster genome reveals stress adaptation and complexity of shell formation. Nature 490, 49–54. doi: 10.1038/nature11413

PubMed Abstract | Crossref Full Text | Google Scholar

Zhang, T., Zhao, Y. L., Zhao, J. H., Wang, S., Jin, Y., Chen, Z. Q., et al. (2016). Cotton plants export microRNAs to inhibit virulence gene expression in a fungal pathogen. Nat Plants. 2(10), 16153. doi: 10.1038/nplants.2016.153

PubMed Abstract | Crossref Full Text | Google Scholar

Zhang, Q., Liu, Y., Jiang, Y., Li, A., Cheng, B., and Wu, J. (2022). OsASR6 enhances salt stress tolerance in rice. Int. J. Mol. Sci. 23, 9340. doi: 10.3390/ijms23169340

PubMed Abstract | Crossref Full Text | Google Scholar

Zhou, Z., Bi, G., and Zhou, J. M. (2018). Luciferase complementation assay for protein-protein interactions in plants. Curr. Protoc. Plant Biol. 3, 42–50. doi: 10.1002/cppb.20066

PubMed Abstract | Crossref Full Text | Google Scholar