A C2H2-type zinc finger protein from Mentha canadensis, McZFP1, negatively regulates epidermal cell patterning and salt tolerance

A C2H2-type zinc finger protein from Mentha canadensis, McZFP1, negatively regulates epidermal cell patterning and salt tolerance | 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 A C2H2-type zinc finger protein from Mentha canadensis, McZFP1, negatively regulates epidermal cell patterning and salt tolerance Yang Bai, Xiaowei Zheng, Yichuan Xu, Li Li, Xiwu Qi, Xu Yu, Chun Qin, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4918956/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract C2H2-type zinc finger protein (C2H2-ZFP) transcription factors play evident roles in regulating plant growth and development and abiotic stress responses. However, the role of C2H2-ZFP from Mentha canadensis remains uncertain. Here, we identified the multifunctional C2H2-ZFP gene McZFP1 from M. canadensis based on phylogenetic analysis. The McZFP1 gene was highly expressed in stems, responding to abiotic stress and phytohormone treatments. McZFP1 localized in the nucleus and showed no transcriptional self-activation activity. McZFP1 overexpression in Arabidopsis thaliana significantly reduced the number of trichomes and root hairs, root hair length, and salt stress tolerance. Further study revealed that McZFP1 overexpression increased the expression of negative regulator genes and decreased that of positive regulator genes to inhibit plant trichome and root hair development. Malondialdehyde accumulation was promoted, but the proline content and catalase, superoxide dismutase, and peroxidase activities were reduced and the expression of stress-response genes was inhibited in McZFP1 overexpression lines under salt treatment, thereby compromising plant salt tolerance. Overall, these results indicate that McZFP1 is a novel C2H2-ZFP transcription factor that plays negative roles in trichome and root hair development and salt stress tolerance. Mentha canadensis McZFP1 Trichome Root hair Salt stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Key Message McZFP1 negatively regulate plant trichome and root hair development by altering the gene expression of key regulator, and plant salt tolerance by reducing ROS scavenging and osmotic substance biosynthesis abilities. 1. Introduction Mint ( Mentha canadensis L.) is a widely cultivated medicinal herb and spice crop with a high essential oil content. Mint oil, which is composed of mostly monoterpenes and a small amount of sesquiterpenes, has high economic value (He et al. 2019 ). Mint oil is produced and stored in glandular trichomes. In plants, trichomes are unicellular or multicellular appendages originating from epidermal cells that are usually divided into two types: non-glandular and glandular trichomes. They play important roles in secondary metabolite synthesis and biotic and abiotic stress defense (Huchelmann et al. 2017 ; Li et al. 2023 ). The development mechanism of unicellular trichomes is understood more deeply and thoroughly than that of multicellular trichomes (Han et al. 2022 ). Genetic factors and environmental factors play essential roles in plant trichome development (Khan et al. 2021 ). Arabidopsis has been systematically studied as a model for unicellular trichome development. In Arabidopsis, the gene regulatory network is formed by positive regulators R2R3-MYB transcription factors (TFs) (GLABRA1, GL1/MYB23), WD40 repeat proteins (TRANSPARENT TESTA GLABRA1, TTG1), basic helix–loop–helix (bHLH) TFs (GL3/ENHANCER of GLABRA3, EGL3), homeodomain-leucine zipper (HD-Zip) TFs (GL2), WRKY TFs (TTG2), C2H2 zinc finger proteins (ZFPs), and negative regulators R3 MYBs, controlling cell fate determination and trichome initiation (Han et al. 2022 ; Wang et al. 2019 ). These TFs also play key roles in the development of multicellular trichomes in plants, such as tomato ( Solanum lycopersicum ), cucumber ( Cucumis sativus ), sweet wormwood ( Artemisia annua ), and tobacco ( Nicotiana tabacum ) (Feng et al. 2021 ). Phytohormones, such as gibberellins (GAs), jasmonic acid (JA), cytokinin (CK), brassinosteroids (BRs), auxin, ethylene, abscisic acid (ABA), and salicylic acid (SA), are also involved in plant trichome development, especially GA (Fambrini and Pugliesi 2019 ; Han et al. 2022 ; Yu et al. 2022 ). Different from plant trichomes derived from epidermal cells of aerial tissues, root hairs are unicellular extensions from root epidermal cells that function in anchorage, nutrient and water acquisition, and soil microbe interactions (Schmidt and Gaudin 2017 ). In Arabidopsis, the development regulation networks of both types of epidermal cell outgrowths share some of the same TFs, which play two completely opposite roles. Positive regulators in trichome formation, GL3/EGL3/TTG1/GL2, negatively regulate root hair formation; however, trichome formation-related negative relators, R3 MYBs, positively regulate root hair formation (Li et al. 2022 ; Vissenberg et al. 2020 ). R2R3-MYB TFs (e.g., WEREWOLF [WER]) and bHLH TFs (e.g., ROOT HAIRDEFECTIVE 6 [RHD6] and its orthologues) are also involved in root hair development (Vissenberg et al. 2020 ). Phytohormones ethylene, auxin, JA, strigolactone, and CK are positive regulators of root hair development, whereas BR and ABA are negative regulators (Cui et al. 2018 ; Li et al. 2022 ). Soil salinization is an environmental stressor that restricts seed germination, crop growth, and productivity (Hu and Schmidhalter 2023 ). To adapt to salt stress, plants have evolved strategies to alter and regulate their cellular physiology and biochemical processes, phenotypic structures, and signaling pathways (van Zelm et al. 2020 ; Zhao et al. 2020 ). Upon salt stress, plants use a salt excretion mechanism to eliminate excess salt, produce antioxidants and osmotic substances, and activate a series of salt stress-related genes to mitigate the impact of salinity (Zhou et al. 2024 ). Transcriptional gene regulation plays a crucial role in plant responses to salt stress. TFs, such as AREB/ABF (ABA-responsive element (ABRE)-binding protein/ABRE-binding factor), NAC (NAM, ATAF1/2, and CUC2), DREB (dehydration responsive element-binding protein), MYB (Myeloblastosis), ARF (auxin response factor), C2H2-ZFP, and WRKY, regulate target gene expression during plant salt stress responses by binding to DNA binding domains on target gene promoters (Kurowska and Daszkowska-Golec 2023 ; Liu et al. 2022 , 2023 ; Rai et al. 2023 ; Verma et al. 2022 ; Wang et al. 2021 ; Warsi et al. 2021 ). Phytohormones, especially ABA, are responsible for abiotic stress responses and tolerance due to various TFs (Chen et al. 2020 ; Ku et al. 2018 ). ZFPs constitute one of the largest TF families in eukaryotes and are classified into C2H2, C2HC, C2HC5, C3HC4, CCCH, C4, C4HC3, C6, and C8 groups based on the number and location of cysteine (C) and/orhistidine (H) residues (Ciftci-Yilmaz and Mittler 2008 ; Huang et al. 2022 ). Among them, the C2H2-ZFP sub-family accounts for a large proportion of the ZFP family and has been extensively studied. Most plant C2H2-ZFPs have a conserved zinc finger domain (CX2-4CX3FX5LX2HX3-5H) that contains a plant-specific conserved QALGGH sequence (Huang et al. 2022 ). Many studies have demonstrated that C2H2-ZFPs act in combination with other TFs or plant signaling hormones to regulate plant growth and development processes, such as flower development, seed development and germination, trichome formation, and root hair development, and to respond to stressors, such as drought, high salt, cold, high light, osmotic, and oxidative stress (Han et al. 2020 , 2021 ; Huang et al. 2022 ; Liu et al. 2022 ; Lyu and Cao 2018 ). In Arabidopsis, C2H2-ZFPs, including AtGIS, AtGIS2, AtGIS3, AtZFP1, AtZFP5, AtZFP6, and AtZFP8, have been reported to positively regulate trichome development via GA or cytokinin signaling pathways (Gan et al. 2006 , 2007 ; Sun et al. 2015 ; Zhang et al. 2018 , 2020 ; Zhou et al. 2011 , 2013 ). In Arabidopsis, C2H2-ZFPs, including AtZFP3 and AtZP1, negatively regulate root hair development, while AtZFP5 and AtGIS3 play positive roles via ethylene and GA or cytokinin signaling pathways (An et al. 2012 ; Benyó et al. 2023 ; Han et al. 2020 ; Huang et al. 2020 , 2024 ). AtZFP3, AtSIZ1, AtZAT10, and AtAZF2 positively regulate salt tolerance in Arabidopsis by scavenging reactive oxygen species (ROS), increasing the levels of osmotic adjustment substances, or regulating the ABA signaling pathway (Han et al. 2019 ; Huang et al. 2023 ; Sakamoto et al. 2004 ; Zhang et al. 2016 ). Glandular trichome development and density have been significantly correlated with mint oil content, and environmental factors, such as salt stress, are limiting factors for essential oil production (Kumar et al. 2023 ; Mishra et al. 2017 , 2021 ). However, only a few MYB/WRKY-type TFs, including McMIXTA, MsMYB, MsGSW2, and MhGSW2, have been reported to regulate glandular trichome development (Qi et al. 2022 ; Reddy et al. 2017 ; Xie et al. 2021 ). To date, no C2H2-ZFPs from M. canadensis have been reported to regulate trichome or root hair formation or salt tolerance. In the current study, the C2H2-ZFP gene McZFP1 from M. canadensis was cloned and investigated. Expression analysis showed that McZFP1 expression was higher in shoots than in other tissues and was inhibited by salt (NaCl), GA, and ethylene. Arabidopsis plants heterologously expressing the McZFP1 gene inhibited trichome and root hair development and reduced salt tolerance. The results suggest that the McZFP1 gene negatively regulates trichome development, root hair development, and salt tolerance in transgenic Arabidopsis. 2. Materials and methods 2.1. Plant materials and growth conditions Mentha canadensis and tobacco plants were cultured in a mixture of nutrient soil and vermiculite (2:1, v:v). The growth conditions were maintained at 23–25°C with a 16-h light/8-h dark photoperiod. Arabidopsis seeds were sterilized with 70% alcohol for 4 min, washed four times with sterile water, and sown on 1/2 Murashige and Skoog (MS) medium (1% sucrose and 1% agar). After 2 days of stratification at 4°C, the plants were transferred to a growth chamber (200 µmol m –2 s –1 light intensity, 16-h light/8-h dark photoperiod, 22°C). 2.2. Stress treatments For stress treatments, 3-week-old water-cultured M. canadensis seedlings were separately transferred to MS medium containing 300 mM mannitol, 150 mM NaCl, 100 µM GA, 200 µM 1-aminocyclopropane-1-carboxylic acid (ACC, a precursor substance for ethylene synthesis), 200 µM ABA, and 300 mM mannitol and treated for 0, 2, 4, 8, 12, and 24 h. The leaves and adventitious roots of treated plants were harvested and frozen in liquid nitrogen for subsequent total RNA isolation. For the root length assay, sterilized seeds of the wild-type (WT) and overexpression (OE) lines were germinated and cultivated in 1/2 MS medium for 4 days, and uniform seedlings were transferred to 1/2 MS medium containing 0 or 150 mM NaCl. After 2 weeks of cultivation, the roots were photographed, and the root length was measured using ImageJ software. For the Arabidopsis seed germination assay, seeds were sterilized and sown on 1/2 MS plates containing 0, 150, or 200 mM NaCl and cultivated for 15 days. Seed germination was defined as the rupture of the testa concomitant with radicle protrusion. 2.3. Cloning and bioinformatics analysis of the McZFP1 gene The transcript sequences of the McZFP1 gene were amplified from the cDNA of M. canadensis using a pair of specific primers (Table S1 ). A phylogenetic tree was constructed using the neighbor-joining method in MEGA7 with 1000 bootstrap replicates, and the protein sequences of Arabidopsis ZFPs were obtained from PlantTFDB ( http://planttfdb.gao-lab.org/index.php ). Multiple alignments of McZFP1 and its homologs in Arabidopsis was performed using DNAMAN. The MEME combinatorial tool was used for the motif search. 2.4. RNA isolation and qRT-PCR analysis Various plant tissues were prepared for the isolation of total RNA using the Eastep® Super Total RNA Extraction Kit (Promega, Beijing, China), according to the manufacturer’s instructions. Total RNA was used to produce cDNA via the HiScript III 1st Strand cDNA Synthesis Kit (+ gDNA wiper) (Vazyme, Nanjing, China). qRT-PCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme) on a CFX96 Real-Time PCR Detection System (Bio-Rad, Boston, MA, USA) in accordance with the manufacturer’s instructions. The McACT gene and AtACT2 gene were used as the reference genes for normalizing the gene expression in M. canadensis and Arabidopsis , respectively. The primers used for qRT-PCR are listed in Supplemental Table S1 . 2.5. Subcellular localization of McZFP1 The McZFP1 coding sequence (CDS) was introduced into the pGate8-GFP vector to produce 35S::McZFP1–GFP , which was then transformed into Agrobacterium strain GV3101. To evaluate McZFP1 localization within cells, the Agrobacterium GV3101 colony carrying the 35S::McZFP1–GFP construct was cultured to an OD 600 of 1.8–2.0, resuspended with 10 mM 2-morpholinoethanesulphonic acid (MES), 10 mM MgCl 2 (pH 5.8), and 150 mM acetosyringone to an OD 600 of 0.8–1.0, and then infiltrated into Nicotiana benthamiana leaves. After 2–3 days of cultivation, N. benthamiana leaves were collected to observe the fluorescence imaging. The 35S::McZFP1–GFP construct was introduced into WT plants using the floral dip method to determine the subcellular localization of McZFP1 in Arabidopsis seedlings (Clough and Bent 1998 ). DAPI (4’,6-diamidino-2-phenylindole, 1 µg/mL) was used to incubate tobacco leaves or transgenic Arabidopsis seedlings to locate the nucleus. Primers used for the construction of 35S::McZFP1–GFP are listed in Supplemental Table S1 . 2.6. Transcriptional activation of McZFP1 in yeast cells The open reading frame (ORF) of McZFP1 was ligated into the pGBKT7 (BD) vector at the EcoR1 site to generate pGBKT7–McZFP1 ( BD–McZFP1 ). The BD vector and pGBKT7–AtSIZ1 ( BD–AtSIZ1 ) (Leng et al. 2021 ) were used as empty and positive controls, respectively. These three vectors were separately transformed into yeast strain Y2HGold using the Yeast Transformation Kit (Coolaber Science & Technology, Beijing, China). The transformed yeast cells were cultured on SD/-Trp and SD/-Trp/-His/-Ade medium at 30°C for 2–3 days; their transcriptional activities were evaluated according to the yeast growth status. The primers used for BD–McZFP1 construction are listed in Supplemental Table S1 . 2.7. Microscopy Photographs of yeasts on medium, seedlings, and germinated seeds were acquired using a digital camera. The GUS-stained tissues and root hairs were photographed using a stereoscope (DVM6a, Leica, Wetzlar, Germany). A confocal laser scanning microscope (LSM900, Zeiss, Oberkochen, Germany) was used for green fluorescent protein (GFP) and DAPI imaging in plant tissues. The excitation and emission wavelengths during observation were 405 and 538–632 nm, respectively, for DAPI and 488 and 493–536 nm, respectively, for GFP. Trichomes were photographed using cryo-scanning electron microscopy (cryo-SEM, SU8010, Hitachi, Tokyo, Japan) according to Charuvi et al. ( 2016 ) with modifications. Briefly, leaf samples were mounted in a cylindrical plug of a copper sample holder and rapidly frozen in liquid nitrogen at − 210°C for 2 min, transferred to the cold stage of a preparation chamber, and etched. The cold stage temperature was raised from − 140°C to − 70°, held for 10 min, and then returned to − 140°C. After etching, platinum sputtering was conducted at 10 mA for 60 s. Images were acquired using cryo-SEM on a cryo-preparation system (PP3010T, Quorum, Tokyo, Japan) at an accelerating voltage of 3 kV. For root hairs, the primary roots of 7-day-old Arabidopsis seedlings cultivated in 1/2 MS medium were observed, and approximately 7 mm of the root tip was photographed to determine the number and length of root hairs. 2.8. Measurement of physiological indexes Four-day-old Arabidopsis WT and OE plants after normal and salt treatments for 2 weeks were used to determine related physiological indexes. The proline and malondialdehyde (MDA) contents were determined using the Proline Assay Kit and Plant Malondialdehyde (MDA) Assay Kit, respectively. The Catalase (CAT) Assay Kit, Total Superoxide Dismutase (T-SOD) Assay Kit, and Peroxidase Assay Kit were used to determine the CAT, SOD, and POD activities, respectively. The kits used above were purchased from Nanjing Jiancheng (Nanjing, China), and each physiological index was determined according to the manufacturer’s instructions. 3. Results 3.1. McZFP1 isolation from M. canadensis and sequence analysis In this study, the M. canadensis McZFP1 gene was identified using a BLASTP search on the reported NCBI transcriptome data (SRP132644) with the AtZFP1 amino acid sequence as a query (Qi et al. 2018 ). The DNA sequence of McZFP1 is depicted in Fig. S1 A. The McZFP1 gene contained an ORF of 489 bases and encoded a protein with 162 amino acids (Fig. S1 A). Phylogenetic tree analysis of McZFP1 and Arabidopsis ZFP family proteins showed that McZFP1 clustered with AtZFP1 (Fig. 1 A). Amino acid sequence analysis and sequence alignment of McZFP1 with the 11 homologous C2H2-ZFPs from Arabidopsis revealed that McZFP1 contained a conserved zinc finger domain (CX2-4CX3FX5LX2HX3-5H) with a plant-specific conserved QALGGH sequence (Fig. 1 B, C). A conserved ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif was also identified in the McZFP1 protein (Fig. 1 B, C). Phylogenetic tree analysis of McZFP1 and its homologs in other plants showed that McZFP1 had a close relationship with ShZFP1-like from Salvia hispanica (Fig. 1 D). These results suggest that McZFP1 is a C2H2-type ZFP protein. 3.2. Analysis of McZFP1 gene expression Tissue expression patterns were examined to characterize the potential function of the McZFP1 gene in M. canadensis . McZFP1 was expressed in the examined tissues, including leaves, stems, flowers, rhizomes, and adventitious roots, according to qRT-PCR analysis (Fig. 2 A). Furthermore, the McZFP1 gene was highly expressed in stems, followed by rhizomes, flowers, leaves, and adventitious roots (Fig. 2 A). To further analyze the potential roles of the McZFP1 gene in the plant response to abiotic stress and hormones, we measured its expression levels in leaves and adventitious roots with NaCl, mannitol, ABA, GA, and ACC treatments. When treated with 150 mM NaCl, McZFP1 expression was inhibited at 2, 4, 12, and 24 h post-treatment (hpt) but did not change significantly at 8 hpt in leaves and was inhibited at 2, 4, and 12 hpt and enhanced at 24 hpt in adventitious roots. Under 300 mM mannitol treatment, McZFP1 expression was markedly induced in leaves at 2 hpt and increased in adventitious roots by about 5–7-fold within 8–24 h, reaching a peak at 12 hpt. Under ABA treatment, McZFP1 expression increased at 2–8 hpt in leaves but decreased at 2–12 hpt and increased at 24 hpt in adventitious roots. When treated with GA, McZFP1 expression decreased in leaves at 2–12 hpt and increased in adventitious roots at 2 and 4 hpt. Under ACC treatment, McZFP1 expression significantly decreased in leaves and adventitious roots at 2–24 hpt (Fig. 2 B). Among the five treatments, McZFP1 expression was mainly inhibited by NaCl and ACC treatment but enhanced by mannitol treatment. It was induced in leaves but repressed in adventitious roots under ABA treatment and induced in adventitious roots but repressed in leaves under GA treatment. These results suggest that the McZFP1 gene plays different regulatory roles in stress and hormone responses. 3.3. Subcellular localization and transcriptional activity of McZFP1 For subcellular localization of McZFP1, the 35S::McZFP1–GFP plasmid was constructed and transformed into Agrobacterium strain GV3101 and then transiently expressed in N. benthamiana leaves using the Agrobacterium -mediated transient expression system. The leaves were stained with the nucleus marker DAPI for 2 h before fluorescence microscopy detection. Furthermore, the 35S::McZFP1–GFP construct was transformed into Arabidopsis WT plants, and the subcellular location of McZFP1 was observed in transgenic seedlings after DAPI staining. The GFP and DAPI fluorescence patterns overlapped under a confocal microscope, indicating that McZFP1 localized in the nucleus (Fig. 3 A, B). These results suggest that McZFP1 is expressed within the nucleus. We further used a yeast system to identify the transcriptional activation activity of McZFP1. The pGBKT7–McZFP1 vector was constructed and transformed into yeast strain Y2H. Yeast cells containing pGBKT7–McZFP1 (BD–McZFP1), pGBKT7 (BD vector, empty control), and pGBKT7–AtSIZ1 (BD–AtSIZ1, positive control) all grew normally on SD/-Trp medium. When cultured on SD/-Trp/-His/-Ade medium, BD–McZFP1 failed to grow normally (Fig. 3 C). These results suggest that McZFP1 has no transcriptional self-activation activity in yeast cells. 3.4. McZFP1 overexpression inhibits trichome development in transgenic Arabidopsis To determine whether McZFP1 regulates trichome development, the 35S::McZFP1–GFP construct was introduced into Arabidopsis WT plants to generate McZFP1 transgenic OE plants. Five homozygous McZFP1 OE lines were obtained, and two showing higher McZFP1 expression (OE2 and OE3) were used in function analysis (Fig. S2 ). The WT and McZFP1 OE lines were grown under normal conditions. The two McZFP1 OE lines had fewer trichomes in rosette leaves than WT (Fig. 4 A, B). Previous studies have demonstrated that some C2H2-ZFP genes and MBW complex-related genes play positive roles in trichome initiation in Arabidopsis, while some CPC-type R3 MYB genes play negative roles (Han et al. 2022 ). We examined the expression levels of related genes in rosette leaves of Arabidopsis WT and McZFP1 OE transgenic plants. C2H2-ZFP genes, including AtZFP1 , AtZFP8 , AtGIS , AtGIS2 , and AtGIS3 , were significantly inhibited in McZFP1 OE plants compared with WT plants, while AtZFP5 and AtZFP6 were slightly affected (Fig. 4 C). MBW complex-related genes, including AtGL3 , AtEGL3 , AtTTG2 , and AtGL1 , showed lower expression in McZFP1 OE plants than in WT plants, while AtTTG1 showed higher expression, and AtGL2 had no obvious changes (Fig. 4 D). The expression levels of CPC-type R3 MYB genes, namely AtTRY , AtTCL1 , and AtETC2 , were increased, whereas those of AtETC1 , AtETC3 , and AtCPC were decreased, and AtTCL2 was almost unchanged (Figs. 4 E). These results indicate that increased McZFP1 expression decreases the trichome number in Arabidopsis by inhibiting trichome-initiation gene expression and elevating trichome-inhibition gene expression. 3.5. McZFP1 overexpression inhibits root hair development in transgenic Arabidopsis C2H2-ZFPs function not only in trichome development but also in root hair formation. To determine whether McZFP1 plays a role in root hair development, we observed the root hair phenotypes of WT and McZFP1 OE plants. McZFP1 overexpression in Arabidopsis led to a reduction in root hair number and length compared with WT plants (Fig. 5 A–C). To further elucidate the role of McZFP1 in root hair formation, the expression levels of genes involved in root hair initiation and development (e.g., negative root hair-development regulator genes AtTTG1 , AtWER , AtGL3 , AtGL2 , AtZP1 , and AtZFP3 and positive regulator genes AtZFP5 , AtGIS3 , AtCPC , AtTRY , AtETC1 , AtETC2 , AtETC3 , AtRHD6 , AtRSL1 , AtRHD2 , AtRHD4 , AtRSL2 , AtRSL4 , AtLRL1 , AtLRL2 , AtLRL3 , AtEIL1 , and AtEIN3 ) were measured in the roots of McZFP1 OE and WT seedlings (Fig. 5 D–F). Among these negative regulator genes, AtTTG1 , AtWER , AtZP1 , and AtZFP3 were significantly upregulated, while AtGL3 and AtGL2 were not changed in the McZFP1 OE lines compared with WT. Among these positive regulator genes, AtZFP5 , AtGIS3 , AtCPC , AtTRY , AtETC1 , AtETC2 , AtETC3 , AtRHD6 , AtRHD2 , AtRHD4 , AtRSL4 , AtLRL3 , AtEIL1 , and AtEIN3 were downregulated, while AtRSL1 , AtRSL2 , and AtLRL1 were slightly upregulated. AtLRL2 remained consistent in the McZFP1 OE lines and WT. These results suggest that McZFP1 overexpression promotes the expression of most examined negative regulator genes and inhibits the expression of most tested positive regulator genes in root hair development, reducing the number and length of root hairs in the McZFP1 OE lines. 3.6. McZFP1 overexpression reduces salt tolerance in transgenic Arabidopsis McZFP1 responded to NaCl and mannitol treatment (Fig. 2 B). To investigate the role of McZFP1 in plant tolerance to salt or drought stress, we treated plants at the germination and seedling stages with salt and mannitol, respectively. We first checked the germination rate of WT and McZFP1 OE seeds upon salt treatment. No obvious differences were observed between WT and McZFP1 OE lines grown on 1/2 MS (Fig. 6 A, B). By contrast, high NaCl concentrations strongly inhibited seed germination of the McZFP1 OE lines compared with WT (Fig. 6 A, B). Under 150 mM NaCl treatment, the germination rate of McZFP1 OE lines was lower than that of WT at the early germination stage. When treated with 200 mM NaCl, the germination rate of McZFP1 OE lines was lower than that of WT throughout the germination stage. We further measured the root lengths of WT and McZFP1 OE seedlings under normal and salt stress conditions. No significant differences were observed between WT and McZFP1 OE seedlings under normal conditions. However, when treated with 150 mM NaCl, McZFP1 OE seedlings showed shorter roots than WT seedlings (Fig. 6 C, D). We also performed mannitol treatment on WT and McZFP1 OE seeds, and no obvious differences were observed under treatment with 300 mM mannitol (Fig. S3). ABA plays a crucial role in the salt stress response. ABA treatment led to seed germination phenotypes similar to the NaCl treatment (Fig. S4). These results suggest that McZFP1 overexpression reduces the salt tolerance of transgenic Arabidopsis. 3.7. McZFP1 overexpression in Arabidopsis reduces osmolyte accumulation and antioxidant enzyme activities To determine whether McZFP1-mediated salt intolerance was associated with the alteration in osmolyte accumulation and ROS homeostasis, the proline and MDA contents and SOD, CAT, and POD enzyme activities were detected in WT and McZFP1 OE plants under normal and salt stress conditions. Under control conditions, the proline and MDA contents and SOD, CAT, and POD enzyme activities were comparable in WT and McZFP1 OE plants. When treated with 150 mM NaCl, the proline content in McZFP1 OE plants decreased compared with WT, while the MDA content was enhanced (Fig. 7 A, B). Salt stress significantly increased SOD, CAT, and POD enzyme activities, and the increases were more pronounced in WT plants than in McZFP1 OE plants (Fig. 7 C–E). These results indicate that McZFP1 overexpression reduces the osmoregulation and antioxidant capacity of transgenic Arabidopsis plants under drought stress. 3.8. McZFP1 overexpression downregulates the expression of stress-responsive genes in transgenic Arabidopsis To investigate the mechanisms by which McZFP1 regulates salt stress-responsive genes in plants, we further analyzed the expression levels of some well-studied abiotic stress-responsive genes, including AtDREB1A , AtCOR15A , AtCOR15B , AtRD29A , AtRD29B , and AtRAB18 , in transgenic Arabidopsis. The expression levels of these genes did not differ between McZFP1 OE and WT plants under normal growth conditions. However, these stress-related genes showed low expression in McZFP1 OE plants compared with WT plants under 150 mM NaCl treatment. These data suggest that McZFP1 overexpression inhibits the expression of stress-responsive genes in transgenic Arabidopsis, which may explain the decreased tolerance of McZFP1 OE transgenic plants. 4. Discussion Mentha canadensis is widely used in industrial production due to its essential oils. Mint oil is biosynthesized and stored in glandular trichomes (Croteau et al. 2005 ). Root hairs facilitate nutrient acquisition and environmental interactions, which contribute to plant root anchorage, stress resistance, growth, and development (Han et al. 2023 ; Shibata and Sugimoto 2019 ). Salt stress restricts plant growth and development and is a non-negligible factor affecting mint oil production (Kumar et al. 2023 ). Therefore, evaluating trichome and root hair development and the salt stress response in M. canadensis may promote mint growth and development for higher essential oil production. C2H2-ZFPs have been reported to play different roles in regulating plant trichome and root hair development and salt stress adaptation (Han et al. 2021 ; Huang et al. 2022 ; Liu et al. 2022 ). However, few studies on trichome and root hair development, salt stress response, and C2H2-ZFP functions in M. canadensis have been reported. In this study, a novel C2H2-ZFP TF was identified from M. canadensis and named McZFP1 based on phylogenetic analysis (Fig. 1 ). Further analysis revealed that McZFP1 negatively regulated trichome formation, root hair development, and salt tolerance in transgenic Arabidopsis. The C2H2 ZFP family generally contains a conserved zinc finger domain, and most plant C2H2-ZFPs have a specific conserved QALGGH sequence in the zinc finger domain (Huang et al. 2022 ). Domain and multiple sequence alignment analyses showed that McZFP1 contained a typical zinc finger domain with a plant-specific QALGGH motif (Fig. 1 ). The EAR domain has also been found in many C2H2-ZFPs, which function as repressors (Xie et al. 2019 ). We found an EAR motif at the end of the C terminus in McZFP1 (Fig. 1 ). Thus, McZFP1 from M. canadensis is a new, typical member of the plant C2H2-ZFPs, suggesting that it may have a conserved function. The spatiotemporal expression patterns of genes are generally presumed to reflect their potential roles in plant growth and development or environmental stimulus responses. In our study, the expression level of McZFP1 was higher in stems than in leaves, flowers, adventitious roots, and rhizomes of M. canadensis , implying that McZFP1 plays different roles in their development or functions (Fig. 2 A). The McZFP1 gene had different responses to abiotic stressor (NaCl and mannitol) and hormone (ABA, GA, and ethylene) treatments (Fig. 2 A). NaCl treatment mainly inhibited McZFP1 expression, while mannitol treatment mainly induced it, suggesting that McZFP1 negatively regulates plant salt tolerance and positively regulates drought tolerance. The responses of McZFP1 upon ABA, GA, and ethylene treatment suggest that it may play roles in the complex vital movements mediated by these hormones. Subcellular localization and transcriptional activity assays showed that McZFP1 localized in the nucleus and possessed no transcriptional self-activating activity (Fig. 3 ). The gene expression patterns and protein characteristics of McZFP1 indicate that it responds to environmental and hormone stimuli and has roles in plant development and abiotic stress responses. C2H2-ZFPs from several plant species, including Arabidopsis, tomato, tobacco, cotton, cucumber, pepper, and Jatropha curcas , have been reported to play positive roles in trichome initiation (Chang et al. 2018 ; Liu et al. 2018 , 2021 , 2024 ; Shi et al. 2018 ; Zhou et al. 2011 ). In Arabidopsis, the MBW complex (AtGL1–AtGL3/AtEGL3–AtTTG1) induces the expression of AtGL2 and AtTTG2 to initiate trichome formation, while R3 MYBs move from a trichome precursor cell to its neighboring cell to compete with AtGL1 and interact with AtGL3 or AtEGL3, disrupting the functionality of the activator MBW complex and thus inhibiting trichome initiation (Wang et al. 2019 ). Therefore, AtGL1, AtGL3, AtEGL3, AtTTG1, AtGL2, and AtTTG2 are positive regulators in trichome development, while AtTRY, AtTCL1, AtETC1, AtETC2, AtETC3, and AtCPC are negative regulators (Gan et al. 2011 ; Han et al. 2021 ; Johnson et al. 2002 ; Larkin et al. 1994 ; Morohashi et al. 2007 ; Payne et al. 2000 ; Schellmann et al. 2002 ; Walker et al. 1999 ; Wester et al. 2009 ). Several Arabidopsis C2H2-ZFPs, such as AtGIS, AtGIS2, AtGIS3, AtZFP1, AtZFP5, AtZFP6, and AtZFP8, act upstream of the MBW complex and positively regulate trichome initiation (Gan et al. 2006 , 2007 ; Sun et al. 2015 ; Zhang et al. 2018 , 2020 ; Zhou et al. 2011 , 2013 ). To regulate the trichome development network, AtZFP6 acts upstream of AtZFP5 and AtGIS, and AtZFP5/AtGIS3 acts upstream of AtGIS, AtGIS2, AtZFP8, AtGL1, and AtGL3 (Sun et al. 2015 ; Zhou et al. 2011 , 2012 , 2013 ). In this study, we investigated the role of McZFP1 in trichome formation in Arabidopsis because no transgenic mint line has been obtained. McZFP1 overexpression produced fewer trichomes in Arabidopsis than in WT, differing from results reported for C2H2-ZFPs (Fig. 4 A, B). To understand the reason for this phenotype, we examined the gene expression levels of C2H2-ZFP genes, MBW complex-related positive regulator genes, and negative regulator R3 MYB genes. Five of seven positive C2H2-ZFP regulator genes ( AtZFP1 , AtZFP8 , AtGIS , AtGIS2 , and AtGIS3 ) and five of seven MBW complex-related activator genes ( AtGL3 , AtEGL3 , AtGL1 , AtMYB5 , and AtTTG2 ) were significantly downregulated in McZFP1 OE lines, while three of seven R3 MYB repressor genes ( AtTRY , AtTCL1 , and AtETC2 ) were upregulated (Fig. 4 C–E). Thus, elevated McZFP1 expression may reduce the trichome number in Arabidopsis by elevating trichome-inhibition gene expression but inhibiting trichome-initiation gene expression. GA can induce the expression of some C2H2-ZFPs and plays a dominant role in C2H2-ZFP-mediated regulation of trichome development (Gan et al. 2006 , 2007 ; Liu et al. 2017 , 2018 ). In this study, McZFP1 expression was inhibited by GA treatment (Fig. 2 B). We speculate that McZFP1 may play a negative role in GA-mediated regulation of trichome development, but this relationship requires further investigation. Root hairs are tubular polarized outgrowths of a trichoblast, which are developed from specialized root epidermal cells and regulated by a well-defined cellular differentiation program (Han et al. 2023 ; Shibata and Sugimoto 2019 ). C2H2-ZFPs play contrasting roles in root hair development. In Arabidopsis, both AtZFP5 and AtGIS3 positively regulate root hair development, while AtZFP3 and AtZP1 play negative roles (Benyó et al. 2023 ; Han et al. 2020 ; Huang et al. 2020 , 2024 ). In the present study, the McZFP1 OE lines showed fewer and shorter root hairs compared with WT, suggesting that McZFP1 plays a negative role in root hair development (Fig. 5 A–C). The MBW complex and R3 MYBs are also found to play opposite roles in the regulation of root hair formation in Arabidopsis. In root hair development, the MBW complex (AtWER–AtGL3–AtTTG1) induces AtGL2 expression in non-hair cells to suppress root hair initiation, while R3 MYBs compete with AtWER for binding to AtGL3, thereby suppressing MBW complex activity and AtGL2 expression and initiating hair cell specification (Tominaga-Wada and Wada 2014 ). AtWER , AtGL3 , AtTTG1 , and AtGL2 play crucial negative roles in root hair development, and their mutation generally leads to ectopic root hair formation (Bernhardt et al. 2003 ; Lee and Schiefelbein 1999 ; Long and Schiefelbein 2020 ; Wang et al. 2010 ). Five R3 MYBs ( AtCPC , AtETC1 , AtETC2 , AtETC3 , and AtTRY ) contribute to root hair development, and their overexpression promotes root hair formation (Esch et al. 2004 ; Kirik et al. 2004 ; Kirik et al. 2004 ; Schellmann et al. 2002 ; Tominaga et al. 2008 ; Wada et al. 1997 ). After cell fate specification, several bHLH TFs act downstream of AtGL2 or CPC-type R3 MYBs to form a regulatory network and regulate root hair initiation ( AtRHD6 and AtRSL1 ) and elongation ( AtRSL2 , AtRSL4 , AtLRL1 , AtLRL2 , and AtLRL3 ) in Arabidopsis (Masucci and Schiefelbein, 1994; Menand et al., 2007; Karas et al., 2009; Yi et al., 2010; Bruex et al., 2012; Pires et al., 2013; Lin et al., 2015). AtZFP5 positively controls root hair development by directly promoting AtCPC expression (An et al. 2012 ; Huang et al. 2020 ). AtGIS3 binds to and activates AtRHD2 and AtRHD4 genes to promote root hair elongation in Arabidopsis (Huang et al. 2024 ). AtZP1 acts downstream of AtGL2 to inhibit root hair development as a repressor of AtRHD6 , AtRSL2 , and AtRSL4 (Han et al. 2020 ). AtZFP3 functions as a repressor in root hair development by inhibiting the activity of key regulatory genes, such as AtRSL4 (Benyó et al. 2023 ). In the McZFP1 OE lines, the higher gene expression levels of negative regulators, including AtTTG1 , AtWER , AtZP1 , and AtZFP3 , and the lower gene expression levels of positive regulators, including AtZFP5 , AtGIS3 , AtCPC , AtETC1 , AtETC2 , AtETC3 , AtTRY , AtRHD6 , AtRHD2 , AtRHD4 , AtRSL4 , and AtLRL3 , were detected compared with WT (Fig. 5 D–F). These data suggest that McZFP1 overexpression inhibits root hair development in Arabidopsis by enhancing negative root hair-development gene expression but decreasing positive root hair-development gene expression. AtZFP5 and AtGIS3 mediate ethylene signals to regulate root hair development (An et al. 2012 ; Huang et al. 2020 , 2024 ). Ethylene-activated TF ETHYLENE-INSENSITIVE 3 (EIN3)/EIN3-LIKE 1 (EIL1) physically interacts with RHD6/ RHD6-LIKE 1 (RSL1) to enhance root hair initiation by regulating a subset of core root hair genes in Arabidopsis (Feng et al. 2017 ). We found that both the AtEIN3 and AtEIL1 genes showed lower expression levels in the McZFP1 OE lines than in WT (Fig. 5 F), indicating that McZFP1 is involved in the ethylene signaling pathway to regulate root hair development. Further research is needed to explain this inference. Salt stress significantly inhibits plant seed germination, growth, and development (Zhou et al. 2024 ). C2H2-ZFPs play extensive roles in the plant response to salt stress (Liu et al. 2022 ). In Arabidopsis, constitutive AtZFP3 expression enhances proline accumulation and stress-related gene expression to improve plant salt tolerance (Zhang et al. 2016 ). In rice, OsZFP179 contributes to the ROS scavenging system and osmotic substance biosynthesis, thus enabling salt stress resistance (Zhang et al. 2018 ). C2H2-ZFPs can cope with salt stress through ABA-dependent and -independent signaling pathways (Liu et al. 2022 ). Sweet potato IbZFP1 has been demonstrated to promote salt tolerance by regulating the ABA signaling pathway and osmotic substance accumulation (Wang et al. 2016 ). Arabidopsis AtZAT10 does not respond to ABA treatment and enhances plant salt tolerance by maintaining ionic balance (Mittler et al. 2006 ). However, a few C2H2-ZFPs play negative roles in regulating plant salt stress tolerance. A C2H2-type ZFP ( MtZPT2-2 ) in Medicago truncatula has been reported to negatively regulate plant salt tolerance by regulating antioxidant defense and Na + homeostasis (Huang et al. 2023 ). In this study, a negative C2H2-type ZFP regulator, McZFP1, was demonstrated in plant salt tolerance. McZFP1 gene expression in M. canadensis was compromised under 150 mM NaCl treatment (Fig. 2 B). To further investigate its role in the plant salt stress response, we employed germination rate and root length assays. McZFP1 overexpression in Arabidopsis significantly reduced the seed germination rate and seedling root length compared with WT under NaCl treatment (Fig. 6 ), suggesting that McZFP1 plays a negative role in plant salt stress responses. Proline, as an osmolyte and a potent antioxidant and programmed cell death inhibitor, is one of the most important indicators of plant stress tolerance (Yoshiba et al. 1997 ). Salt stress produces excessive ROS accumulation and breaks ROS homeostasis, thus compromising lipid membrane functions and ultimately causing oxidative damage to plant cells (Zhou et al. 2024 ). The MDA content is used to indicate cell membrane lipid peroxidation and changes in plants under stress (Moore and Roberts 1998 ). An antioxidative defense system containing SOD, CAT, and POD has evolved in plants to scavenge ROS or inhibit their harmful effects on biomolecules (Wang et al. 2024 ). We further found that the proline and MDA contents and SOD, CAT, and POD activities in WT and McZFP1 OE plants were comparable under normal conditions. However, under 150 mM NaCl treatment, the proline content in McZFP1 OE plants was lower than that in WT plants, while the MDA content in McZFP1 OE plants was higher. The antioxidant enzyme activity in McZFP1 OE plants was also lower (Fig. 7 ). These results suggest that McZFP1 overexpression reduces the ROS scavenging ability by reducing the antioxidant enzyme activity, thereby decreasing plant salt tolerance. In Arabidopsis, a series of salt stress-responsive genes, including AtDREB1A , AtCOR15A , AtCOR15B , AtRD29A , AtRD29B , and AtRAB18 , are induced by abiotic stress, making them marker genes in the abiotic stress response (Kasuga et al. 1999 ; Kim et al. 2014 ; Ma et al. 2018 ; Msanne et al. 2011 ; Yang et al. 2019 ). We examined the expression levels of these genes in Arabidopsis WT and McZFP1 OE plants under normal and NaCl treatment. Their expression in McZFP1 OE plants was lower than in WT plants under salt stress (Fig. 8 ), partly explaining the decreased tolerance of McZFP1 OE transgenic plants to salt stress. These aforementioned stress-responsive genes are induced by ABA and generally considered ABA-responsive marker genes (Lang et al. 1994 ; Msanne et al. 2011 ; Rushton et al. 2012 ; Wilhelm and Thomashow 1993 ). In our study, McZFP1 responded to ABA treatment, and McZFP1 OE seeds had a reduced germination rate under ABA treatment compared with that of WT (Figs. 2 B and S4 ). Overall, these results suggest that McZFP1 negatively regulates plant salt tolerance by compromising ROS scavenging and osmotic substance biosynthesis abilities and inhibiting stress-related gene expression. Moreover, the ABA signaling pathway may play a role in the McZFP1-regulated salt stress response, which requires further investigation. 5. Conclusion This study demonstrated that McZFP1, a newly characterized C2H2-type ZFP TF from M. canadensis , responded to salt and drought stress and phytohormones, such as GA, ethylene, and ABA. In contrast to the reported C2H2-type ZFPs, McZFP1 negatively regulated trichome and root hair development and salt tolerance in transgenic Arabidopsis plants. Further investigations revealed that McZFP1 overexpression mediated a complex gene expression regulatory network involved in epidermal cell patterning to negatively regulate trichome and root hair formation. In addition, McZFP1 overexpression reduced ROS scavenging and osmotic substance biosynthesis abilities and stress-related gene expression, leading to compromised plant salt tolerance. Phytohormone signals may be involved in McZFP1-mediated trichome and root hair development and salt tolerance. Declarations Acknowledgments We thank LetPub (www.letpub.com.cn) for its linguistic assistance during the preparation of this manuscript. Authorship contributions Yang Bai: Conceptualization, Writing - Original Draft, Writing - review & editing. Xiaowei Zheng: Writing - Original Draft, Investigation. Yichuan Xu: Investigation. Li Li: Writing - Original Draft, Investigation. Xiwu Qi: Formal analysis. Xu Yu: Resources. Chun Qin: Investigation. Dongmei Liu: Writing - review & editing. Zequn Chen: Resources. Chengyuan Liang: Project administration. Funding This research was supported by the National Science Foundation of China [grant numbers 32100313], the Natural Science Foundation of the Jiangsu Province [grant numbers BK20210164], and Jiangsu Key Laboratory for the Research and Utilization of Plant Resources [grant numbers JSPKLB202029] to YB; the National Science Foundation of China [grant numbers 32370397] to CYL; and the National Science Foundation of China [grant numbers 32200302] to XY. Data availability Data will be made available on request. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References An L, Zhou Z, Sun L, Yan A, Xi W, Yu N, Cai W, Chen X, Yu H, Schiefelbein J, Gan Y (2012) A zinc finger protein gene ZFP5 integrates phytohormone signaling to control root hair development in Arabidopsis. 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Science 277(5329):1113-1116 Walker AR, Davison PA, Bolognesi-Winfield AC, James CM, Srinivasan N, Blundell TL, Esch JJ, Marks MD, Gray JC (1999) The TRANSPARENT TESTA GLABRA1 locus, which regulates trichome differentiation and anthocyanin biosynthesis in Arabidopsis, encodes a WD40 repeat protein. Plant Cell 11(7):1337-1350 Wang F, Tong W, Zhu H, Kong W, Peng R, Liu Q, Yao Q (2016) A novel Cys2/His2 zinc finger protein gene from sweetpotato, IbZFP1 , is involved in salt and drought tolerance in transgenic Arabidopsis. Planta 243(3):783-797 Wang P, Liu WC, Han C, Wang S, Bai MY, Song CP (2024) Reactive oxygen species: multidimensional regulators of plant adaptation to abiotic stress and development. J Integr Plant Biol 66(3):330-367 Wang S, Barron C, Schiefelbein J, Chen JG (2010) Distinct relationships between GLABRA2 and single-repeat R3 MYB transcription factors in the regulation of trichome and root hair patterning in Arabidopsis. New Phytol 185(2):387-400 Wang X, Niu Y, Zheng Y (2021) Multiple functions of MYB transcription factors in abiotic stress responses. Int J Mol Sci 22(11):6125 Wang Z, Yang Z, Li F (2019) Updates on molecular mechanisms in the development of branched trichome in Arabidopsis and nonbranched in cotton. Plant Biotechnol J 17(9):1706-1722 Warsi MK, Howladar SM, Alsharif MA (2021) Regulon: an overview of plant abiotic stress transcriptional regulatory system and role in transgenic plants. Braz J Biol 83:e245379 Wester K, Digiuni S, Geier F, Timmer J, Fleck C, Hülskamp M (2009) Functional diversity of R3 single-repeat genes in trichome development. Development 136(9):1487-1496 Wilhelm KS, Thomashow MF (1993) Arabidopsis thaliana cor15b , an apparent homologue of cor15a , is strongly responsive to cold and ABA, but not drought. Plant Mol Biol 23(5):1073-1077 Xie L, Yan T, Li L, Chen M, Ma Y, Hao X, Fu X, Shen Q, Huang Y, Qin W, Liu H, Chen T, Hassani D, Kayani SL, Rose JKC, Tang K (2021) The WRKY transcription factor AaGSW2 promotes glandular trichome initiation in Artemisia annua . J Exp Bot 72(5):1691-1701 Xie M, Sun J, Gong D, Kong Y (2019) The roles of Arabidopsis C1-2i subclass of C2H2-type zinc-finger transcription factors. Genes (Basel) 10(9):653 Yang R, Hong Y, Ren Z, Tang K, Zhang H, Zhu JK, Zhao C (2019) A role for PICKLE in the regulation of cold and salt stress tolerance in Arabidopsis . Front Plant Sci 10:900 Yoshiba Y, Kiyosue T, Nakashima K, Yamaguchi-Shinozaki K, Shinozaki K (1997) Regulation of levels of proline as an osmolyte in plants under water stress. Plant Cell Physiol 38:1095-1102 Yu D, Li X, Li Y, Ali F, Li F, Wang Z (2022) Dynamic roles and intricate mechanisms of ethylene in epidermal hair development in Arabidopsis and cotton. New Phytol 234(2):375-391 Zhang A, Liu D, Hua C, Yan A, Liu B, Wu M, Liu Y, Huang L, Ali I, Gan Y (2016) The Arabidopsis gene zinc finger protein 3 ( ZFP3 ) Is involved in salt stress and osmotic stress response. PloS One 11(12):e0168367 Zhang A, Liu Y, Yu C, Huang L, Wu M, Wu J, Gan Y (2020) Zinc Finger Protein 1 (ZFP1) Is Involved in trichome initiation in Arabidopsis thaliana . Agriculture 10(12):645 Zhang N, Yang L, Luo S, Wang X, Wang W, Cheng Y, Tian H, Zheng K, Cai L, Wang S (2018) Genetic evidence suggests that GIS functions downstream of TCL1 to regulate trichome formation in Arabidopsis . BMC Plant Biol 18(1):63 Zhang Z, Liu H, Sun C, Ma Q, Bu H, Chong K, Xu Y (2018) A C2H2 zinc-finger protein OsZFP213 interacts with OsMAPK3 to enhance salt tolerance in rice. J Plant Physiol 229:100-110 Zhao C, Zhang H, Song C, Zhu JK, Shabala S (2020) Mechanisms of plant responses and adaptation to soil salinity. Innovation (Camb) 1(1):100017 Zhou H, Shi H, Yang Y, Feng X, Chen X, Xiao F, Lin H, Guo Y (2024) Insights into plant salt stress signaling and tolerance. J Genet Genomics 51(1):16-34 Zhou Z, An L, Sun L, Gan Y (2012) ZFP5 encodes a functionally equivalent GIS protein to control trichome initiation. Plant Signal Behav 7(1):28-30 Zhou Z, An L, Sun L, Zhu S, Xi W, Broun P, Yu H, Gan Y (2011) Zinc finger protein5 is required for the control of trichome initiation by acting upstream of zinc finger protein8 in Arabidopsis. Plant Physiol 157(2):673-682 Zhou Z, Sun L, Zhao Y, An L, Yan A, Meng X, Gan Y (2013) Zinc Finger Protein 6 ( ZFP6 ) regulates trichome initiation by integrating gibberellin and cytokinin signaling in Arabidopsis thaliana . New Phytol 198(3):699-708 Supplementary Files SupplementaryMaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4918956","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":343441866,"identity":"77f289d6-e9e3-4ae0-b703-41aed6df78e6","order_by":0,"name":"Yang Bai","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYBACAwbGBjCDn735wIEPP0jRItlzLPHgzB6itMAYN3KMD3OwEaHFnP1wm8TPHbUMkjNyPhxm4GGQ5xc7gF+LZU9im2TvmeMM/DxvNxwusGAwnDk7gYDDDiS2SfC2HWOQbM/dcHgGD0OCwW1CWs4/bJP8C9RicCDnwWEeNmK03Ehsk+Ztq2EwOJHDQJwWyxkPm61l2w7wAAPZABjIEoT9Ys6f/vDm27Y6OWBUPv7w4YeNPL80AS1AwCLBAHQSlCNBUDkIMH9gYKgjSuUoGAWjYBSMUAAA431KkHWa5L4AAAAASUVORK5CYII=","orcid":"","institution":"Institute of Botany Jiangsu Province and Chinese Academy of Sciences","correspondingAuthor":true,"prefix":"","firstName":"Yang","middleName":"","lastName":"Bai","suffix":""},{"id":343441867,"identity":"46d1d74f-096a-4f42-9683-43012ecf0a65","order_by":1,"name":"Xiaowei Zheng","email":"","orcid":"","institution":"Institute of Botany Jiangsu Province and Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xiaowei","middleName":"","lastName":"Zheng","suffix":""},{"id":343441868,"identity":"4a63bba0-e227-4bb1-aeb1-6f7afd2b39cd","order_by":2,"name":"Yichuan Xu","email":"","orcid":"","institution":"Institute of Botany Jiangsu Province and Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yichuan","middleName":"","lastName":"Xu","suffix":""},{"id":343441869,"identity":"66c918c9-1003-47bd-bc67-c774e15321dc","order_by":3,"name":"Li Li","email":"","orcid":"","institution":"Institute of Botany Jiangsu Province and Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Li","suffix":""},{"id":343441870,"identity":"f78b8940-44aa-402b-a03b-acd7e14626dd","order_by":4,"name":"Xiwu Qi","email":"","orcid":"","institution":"Institute of Botany Jiangsu Province and Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xiwu","middleName":"","lastName":"Qi","suffix":""},{"id":343441871,"identity":"1de73d75-79f1-494b-b96b-d2c4655b565c","order_by":5,"name":"Xu Yu","email":"","orcid":"","institution":"Institute of Botany Jiangsu Province and Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xu","middleName":"","lastName":"Yu","suffix":""},{"id":343441872,"identity":"14e05e3b-a8aa-4609-946c-2b672063190b","order_by":6,"name":"Chun Qin","email":"","orcid":"","institution":"Institute of Botany Jiangsu Province and Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Chun","middleName":"","lastName":"Qin","suffix":""},{"id":343441873,"identity":"4f547b42-d0b7-4ba8-aa1f-b2ed0a0ec564","order_by":7,"name":"Dongmei Liu","email":"","orcid":"","institution":"Institute of Botany Jiangsu Province and Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Dongmei","middleName":"","lastName":"Liu","suffix":""},{"id":343441874,"identity":"b52e0c63-31c2-4d47-b3ba-6ad33a03e6d8","order_by":8,"name":"Zequn Chen","email":"","orcid":"","institution":"Institute of Botany Jiangsu Province and Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Zequn","middleName":"","lastName":"Chen","suffix":""},{"id":343441875,"identity":"569fee64-7867-4a28-9093-9240373e047a","order_by":9,"name":"Chengyuan Liang","email":"","orcid":"https://orcid.org/0000-0002-4902-4914","institution":"Institute of Botany Jiangsu Province and Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Chengyuan","middleName":"","lastName":"Liang","suffix":""}],"badges":[],"createdAt":"2024-08-15 11:19:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4918956/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4918956/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":64900811,"identity":"c798c5e6-5f1f-4c7f-81a5-51a5afc6a180","added_by":"auto","created_at":"2024-09-20 07:58:44","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":8093516,"visible":true,"origin":"","legend":"\u003cp\u003eBioinformatic analysis of McZFP1. (A) Phylogenetic analysis of McZFP1 and Arabidopsis ZFPs. McZFP1 is marked with an orange solid circle. (B) Phylogenetic relationship and motif patterns of McZFP1 and 11 Arabidopsis homologous C2H2-ZFP proteins. (C) Sequence alignment of McZFP1 with 11 homologous C2H2-ZFP proteins from Arabidopsis using DNAMAN. The conserved C2H2 zinc finger domain (CX2-4CX3FX5LX2HX3-5H)is marked with a green line. The conserved signature motif (QALGGH) is marked with a red rectangular box. The EAR motif signature is marked with a yellow rectangular box. (D) Phylogenetic relationships of McZFP1 and its homologs from different plants using MEGA and ClustalX software. The scale bar indicates the diversity distance.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4918956/v1/0826b4d5f13aacfa7c24f577.jpg"},{"id":64901444,"identity":"8a1e2398-c373-4799-b2f3-37285a866978","added_by":"auto","created_at":"2024-09-20 08:06:44","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2612328,"visible":true,"origin":"","legend":"\u003cp\u003eExpression analysis of \u003cem\u003eMcZFP1\u003c/em\u003ein various tissues and under different treatments. (A) Expression profiles of \u003cem\u003eMcZFP1\u003c/em\u003ein \u003cem\u003eMentha canadensis\u003c/em\u003e. AR, adventitious roots; S, stems; R, rhizomes; L, leaves; F, flowers. (B) Time-course expression levels of \u003cem\u003eMcZFP1\u003c/em\u003e in leaves and adventitious roots under treatment with 150 mM NaCl, 300 mM mannitol, 200 μM ABA, 100 μM GA, and 200 μM ACC.\u003cem\u003e \u003c/em\u003eThe values were standardized using the \u003cem\u003eM. canadensis McACT \u003c/em\u003egene. Data are the means of three independent experiments ± standard deviations (SD). The different letters indicate significant differences (one-way ANOVA, Tukey–Kramer test, P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4918956/v1/74160c99b87c9a7538fd279a.jpg"},{"id":64900808,"identity":"aff739aa-321e-4168-8dad-9d8707091f1e","added_by":"auto","created_at":"2024-09-20 07:58:44","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4384924,"visible":true,"origin":"","legend":"\u003cp\u003eSubcellular localization and self-activation verification of McZFP1. (A) Expression of the McZFP1-GFP fusion protein in tobacco epidermal cells stained with DAPI. Bar = 100 μm. (B) McZFP1-GFP fusion protein expressed in Arabidopsis roots and stained with DAPI. Bar = 20 μm. (C) Self-activation verification of McZFP1. BD-McZFP1 was used as the experimental group; BD-AtSIZ1 was set as the positive control; BD vector was used as the empty control.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4918956/v1/a5cd6bfc1343a56ecf879b09.jpg"},{"id":64900807,"identity":"e38dddf2-06f8-45ec-9a0d-d02ba1ceeb8f","added_by":"auto","created_at":"2024-09-20 07:58:44","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4592910,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eMcZFP1\u003c/em\u003e overexpression modulates trichome development in \u003cem\u003eA. thaliana\u003c/em\u003e under normal conditions. (A) Leaf trichome phenotypes of WT and \u003cem\u003eMcZFP1\u003c/em\u003e transgenic OE lines by Cryo-SEM. (B) Trichome densities on rosette leaves of the WT and \u003cem\u003eMcZFP1 \u003c/em\u003eOE lines. The values are the mean ± SD. (C–E) Relative expression levels of the trichome formation-regulating TF genes. RNA was isolated from rosetteleaves of 4-week-old WT and \u003cem\u003eMcZFP1\u003c/em\u003e OE lines and used for RT-qPCR analysis. The \u003cem\u003eAtACT2\u003c/em\u003e gene was used as a housekeeping gene. Transcript abundance was measured using RT-qPCR, and the values are relative to WT. Data represent the means ± SD of three biological replicates. Different letters indicate significant differences according to one-way analysis of variance (ANOVA) with Tukey’s post-test method (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4918956/v1/914c6aa1eaf4f63609445f26.jpg"},{"id":64900805,"identity":"def3265a-274b-487c-98df-08b88dac3f5c","added_by":"auto","created_at":"2024-09-20 07:58:44","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5240142,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eMcZFP1\u003c/em\u003e regulates root hair development in transgenic Arabidopsis. (A) Root hair phenotypes of WT and \u003cem\u003eMcZFP1\u003c/em\u003e OE lines grown on 1/2 MS medium. Bar = 500 μm. (B) Average root hair number of WT and \u003cem\u003eMcZFP1\u003c/em\u003e OE lines. Error bars indicate SD (n = 20). (C) Average root hair length of WT and \u003cem\u003eMcZFP1\u003c/em\u003eOE lines. Error bars indicate SD (n = 20), and more than 1000 root hairs from 20 plants for each line were measured. (D, E) Expression profiles of root hair development-related genes. RNA was isolated from roots of 7-day-old WT and \u003cem\u003eMcZFP1\u003c/em\u003e OE seedlings for qRT-PCR analysis. The values were standardized using the \u003cem\u003eAtACT2\u003c/em\u003e gene. Expression values are relative to WT, and data represent the means ± SD of three biological replicates. Different letters indicate significant differences at P \u0026lt; 0.05, as determined by one-way analysis of variance (ANOVA) with Tukey’s post-test.\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4918956/v1/b73eb6d6188df9bf2d73a8ae.jpg"},{"id":64901442,"identity":"a18ab395-c9be-4bde-8b36-56c56d4c9594","added_by":"auto","created_at":"2024-09-20 08:06:44","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3688854,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eMcZFP1\u003c/em\u003e overexpression reduces salt tolerance in transgenic Arabidopsis. (A) Phenotypes of WT and \u003cem\u003eMcZFP1\u003c/em\u003e OE seeds germinated in 1/2 MS with 0, 150, and 200 mM NaCl for 7 days. (B) The germination rate of WT and \u003cem\u003eMcZFP1\u003c/em\u003e transgenic seeds germinated in 1/2 MS with 0, 150, and 200 mM NaCl. (C) Root phenotype of WT and \u003cem\u003eMcZFP1\u003c/em\u003e OE seedlings treated with 0 and 150 mM NaCl for 14 days. (D) Root length of WT and \u003cem\u003eMcZFP1\u003c/em\u003e OE seedlings treated with 0 and 150 mM NaCl for 14 days. Data are the means and SE of more than 30 replicates. Different letters indicate significant differences at P \u0026lt; 0.05, as determined by one-way analysis of variance (ANOVA) with Tukey’s post-test.\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4918956/v1/484bd72a46fcb47a7594198b.jpg"},{"id":64900804,"identity":"900b5d1c-6bb5-4a20-b8e8-efad0159c0d7","added_by":"auto","created_at":"2024-09-20 07:58:44","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2236653,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eMcZFP1\u003c/em\u003egene overexpression reduced osmolyte accumulation and antioxidant enzyme activity in transgenic Arabidopsis plants. (A, B) Proline and MDA contents of WT and \u003cem\u003eMcZFP1\u003c/em\u003e OE lines under normal and salt stress conditions. (C–E) SOD, CAT, and POD activities of WT and \u003cem\u003eMcZFP1\u003c/em\u003e OE lines under normal and 150 mM NaCl stress conditions. Data are the means and SE of three replicates, and significant differences at P \u0026lt; 0.05 are indicated by different letters according to one-way analysis of variance (ANOVA) with Tukey’s post-test.\u003c/p\u003e","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4918956/v1/58e4132ee91c103d809ddd96.jpg"},{"id":64901920,"identity":"2f199981-ab27-4015-a061-8ad86b600b9f","added_by":"auto","created_at":"2024-09-20 08:14:44","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2423755,"visible":true,"origin":"","legend":"\u003cp\u003eExpression levels of stress-responsive genes in WT and transgenic Arabidopsis plants. (A–D) Transcript levels of the \u003cem\u003eAtDREB1A\u003c/em\u003e, \u003cem\u003eAtCOR15A\u003c/em\u003e, \u003cem\u003eAtCOR15B\u003c/em\u003e, \u003cem\u003eAtRD29A\u003c/em\u003e, \u003cem\u003eAtRD29B\u003c/em\u003e, and \u003cem\u003eAtRAB18\u003c/em\u003e genes under control and salt stress conditions. The values were standardized using the \u003cem\u003eAtACT2\u003c/em\u003e gene. Expression values are relative to WT, and data represent the means ± SD of three biological replicates. Different letters indicate significant differences at P \u0026lt; 0.05, as determined by one-way analysis of variance (ANOVA) with Tukey’s post-test.\u003c/p\u003e","description":"","filename":"Fig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4918956/v1/f7c870da6814c1c5039af890.jpg"},{"id":65096483,"identity":"d1584abe-4504-418b-9207-85d47a961311","added_by":"auto","created_at":"2024-09-23 14:43:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":17043030,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4918956/v1/2203919b-7feb-4146-9fa0-c2bfe45553bc.pdf"},{"id":64900813,"identity":"24bfbdeb-1f10-4698-839d-625a259eba05","added_by":"auto","created_at":"2024-09-20 07:58:44","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2091844,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4918956/v1/644e2dc0d547bae135992f38.docx"}],"financialInterests":"","formattedTitle":"A C2H2-type zinc finger protein from Mentha canadensis, McZFP1, negatively regulates epidermal cell patterning and salt tolerance","fulltext":[{"header":"Key Message","content":"\u003cp\u003eMcZFP1 negatively regulate plant trichome and root hair development by altering the gene expression of key regulator, and plant salt tolerance by reducing ROS scavenging and osmotic substance biosynthesis abilities.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eMint (\u003cem\u003eMentha canadensis\u003c/em\u003e L.) is a widely cultivated medicinal herb and spice crop with a high essential oil content. Mint oil, which is composed of mostly monoterpenes and a small amount of sesquiterpenes, has high economic value (He et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Mint oil is produced and stored in glandular trichomes. In plants, trichomes are unicellular or multicellular appendages originating from epidermal cells that are usually divided into two types: non-glandular and glandular trichomes. They play important roles in secondary metabolite synthesis and biotic and abiotic stress defense (Huchelmann et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The development mechanism of unicellular trichomes is understood more deeply and thoroughly than that of multicellular trichomes (Han et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Genetic factors and environmental factors play essential roles in plant trichome development (Khan et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Arabidopsis has been systematically studied as a model for unicellular trichome development. In Arabidopsis, the gene regulatory network is formed by positive regulators R2R3-MYB transcription factors (TFs) (GLABRA1, GL1/MYB23), WD40 repeat proteins (TRANSPARENT TESTA GLABRA1, TTG1), basic helix\u0026ndash;loop\u0026ndash;helix (bHLH) TFs (GL3/ENHANCER of GLABRA3, EGL3), homeodomain-leucine zipper (HD-Zip) TFs (GL2), WRKY TFs (TTG2), C2H2 zinc finger proteins (ZFPs), and negative regulators R3 MYBs, controlling cell fate determination and trichome initiation (Han et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These TFs also play key roles in the development of multicellular trichomes in plants, such as tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e), cucumber (\u003cem\u003eCucumis sativus\u003c/em\u003e), sweet wormwood (\u003cem\u003eArtemisia annua\u003c/em\u003e), and tobacco (\u003cem\u003eNicotiana tabacum\u003c/em\u003e) (Feng et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Phytohormones, such as gibberellins (GAs), jasmonic acid (JA), cytokinin (CK), brassinosteroids (BRs), auxin, ethylene, abscisic acid (ABA), and salicylic acid (SA), are also involved in plant trichome development, especially GA (Fambrini and Pugliesi \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Han et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Yu et al. \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDifferent from plant trichomes derived from epidermal cells of aerial tissues, root hairs are unicellular extensions from root epidermal cells that function in anchorage, nutrient and water acquisition, and soil microbe interactions (Schmidt and Gaudin \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In Arabidopsis, the development regulation networks of both types of epidermal cell outgrowths share some of the same TFs, which play two completely opposite roles. Positive regulators in trichome formation, GL3/EGL3/TTG1/GL2, negatively regulate root hair formation; however, trichome formation-related negative relators, R3 MYBs, positively regulate root hair formation (Li et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Vissenberg et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). R2R3-MYB TFs (e.g., WEREWOLF [WER]) and bHLH TFs (e.g., ROOT HAIRDEFECTIVE 6 [RHD6] and its orthologues) are also involved in root hair development (Vissenberg et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Phytohormones ethylene, auxin, JA, strigolactone, and CK are positive regulators of root hair development, whereas BR and ABA are negative regulators (Cui et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSoil salinization is an environmental stressor that restricts seed germination, crop growth, and productivity (Hu and Schmidhalter \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). To adapt to salt stress, plants have evolved strategies to alter and regulate their cellular physiology and biochemical processes, phenotypic structures, and signaling pathways (van Zelm et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Upon salt stress, plants use a salt excretion mechanism to eliminate excess salt, produce antioxidants and osmotic substances, and activate a series of salt stress-related genes to mitigate the impact of salinity (Zhou et al. \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Transcriptional gene regulation plays a crucial role in plant responses to salt stress. TFs, such as AREB/ABF (ABA-responsive element (ABRE)-binding protein/ABRE-binding factor), NAC (NAM, ATAF1/2, and CUC2), DREB (dehydration responsive element-binding protein), MYB (Myeloblastosis), ARF (auxin response factor), C2H2-ZFP, and WRKY, regulate target gene expression during plant salt stress responses by binding to DNA binding domains on target gene promoters (Kurowska and Daszkowska-Golec \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Rai et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Verma et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Warsi et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Phytohormones, especially ABA, are responsible for abiotic stress responses and tolerance due to various TFs (Chen et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ku et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eZFPs constitute one of the largest TF families in eukaryotes and are classified into C2H2, C2HC, C2HC5, C3HC4, CCCH, C4, C4HC3, C6, and C8 groups based on the number and location of cysteine (C) and/orhistidine (H) residues (Ciftci-Yilmaz and Mittler \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Among them, the C2H2-ZFP sub-family accounts for a large proportion of the ZFP family and has been extensively studied. Most plant C2H2-ZFPs have a conserved zinc finger domain (CX2-4CX3FX5LX2HX3-5H) that contains a plant-specific conserved QALGGH sequence (Huang et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Many studies have demonstrated that C2H2-ZFPs act in combination with other TFs or plant signaling hormones to regulate plant growth and development processes, such as flower development, seed development and germination, trichome formation, and root hair development, and to respond to stressors, such as drought, high salt, cold, high light, osmotic, and oxidative stress (Han et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Lyu and Cao \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In Arabidopsis, C2H2-ZFPs, including AtGIS, AtGIS2, AtGIS3, AtZFP1, AtZFP5, AtZFP6, and AtZFP8, have been reported to positively regulate trichome development via GA or cytokinin signaling pathways (Gan et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2006\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In Arabidopsis, C2H2-ZFPs, including AtZFP3 and AtZP1, negatively regulate root hair development, while AtZFP5 and AtGIS3 play positive roles via ethylene and GA or cytokinin signaling pathways (An et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Beny\u0026oacute; et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Han et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). AtZFP3, AtSIZ1, AtZAT10, and AtAZF2 positively regulate salt tolerance in Arabidopsis by scavenging reactive oxygen species (ROS), increasing the levels of osmotic adjustment substances, or regulating the ABA signaling pathway (Han et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Sakamoto et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGlandular trichome development and density have been significantly correlated with mint oil content, and environmental factors, such as salt stress, are limiting factors for essential oil production (Kumar et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Mishra et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, only a few MYB/WRKY-type TFs, including McMIXTA, MsMYB, MsGSW2, and MhGSW2, have been reported to regulate glandular trichome development (Qi et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Reddy et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Xie et al. \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). To date, no C2H2-ZFPs from \u003cem\u003eM. canadensis\u003c/em\u003e have been reported to regulate trichome or root hair formation or salt tolerance. In the current study, the C2H2-ZFP gene \u003cem\u003eMcZFP1\u003c/em\u003e from \u003cem\u003eM. canadensis\u003c/em\u003e was cloned and investigated. Expression analysis showed that \u003cem\u003eMcZFP1\u003c/em\u003e expression was higher in shoots than in other tissues and was inhibited by salt (NaCl), GA, and ethylene. Arabidopsis plants heterologously expressing the \u003cem\u003eMcZFP1\u003c/em\u003e gene inhibited trichome and root hair development and reduced salt tolerance. The results suggest that the \u003cem\u003eMcZFP1\u003c/em\u003e gene negatively regulates trichome development, root hair development, and salt tolerance in transgenic Arabidopsis.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Plant materials and growth conditions\u003c/h2\u003e \u003cp\u003e \u003cem\u003eMentha canadensis\u003c/em\u003e and tobacco plants were cultured in a mixture of nutrient soil and vermiculite (2:1, v:v). The growth conditions were maintained at 23\u0026ndash;25\u0026deg;C with a 16-h light/8-h dark photoperiod. Arabidopsis seeds were sterilized with 70% alcohol for 4 min, washed four times with sterile water, and sown on 1/2 Murashige and Skoog (MS) medium (1% sucrose and 1% agar). After 2 days of stratification at 4\u0026deg;C, the plants were transferred to a growth chamber (200 \u0026micro;mol m\u003csup\u003e\u0026ndash;2\u003c/sup\u003e s\u003csup\u003e\u0026ndash;1\u003c/sup\u003e light intensity, 16-h light/8-h dark photoperiod, 22\u0026deg;C).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Stress treatments\u003c/h2\u003e \u003cp\u003eFor stress treatments, 3-week-old water-cultured \u003cem\u003eM. canadensis\u003c/em\u003e seedlings were separately transferred to MS medium containing 300 mM mannitol, 150 mM NaCl, 100 \u0026micro;M GA, 200 \u0026micro;M 1-aminocyclopropane-1-carboxylic acid (ACC, a precursor substance for ethylene synthesis), 200 \u0026micro;M ABA, and 300 mM mannitol and treated for 0, 2, 4, 8, 12, and 24 h. The leaves and adventitious roots of treated plants were harvested and frozen in liquid nitrogen for subsequent total RNA isolation. For the root length assay, sterilized seeds of the wild-type (WT) and overexpression (OE) lines were germinated and cultivated in 1/2 MS medium for 4 days, and uniform seedlings were transferred to 1/2 MS medium containing 0 or 150 mM NaCl. After 2 weeks of cultivation, the roots were photographed, and the root length was measured using ImageJ software. For the Arabidopsis seed germination assay, seeds were sterilized and sown on 1/2 MS plates containing 0, 150, or 200 mM NaCl and cultivated for 15 days. Seed germination was defined as the rupture of the testa concomitant with radicle protrusion.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Cloning and bioinformatics analysis of the \u003cem\u003eMcZFP1\u003c/em\u003e gene\u003c/h2\u003e \u003cp\u003eThe transcript sequences of the \u003cem\u003eMcZFP1\u003c/em\u003e gene were amplified from the cDNA of \u003cem\u003eM. canadensis\u003c/em\u003e using a pair of specific primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). A phylogenetic tree was constructed using the neighbor-joining method in MEGA7 with 1000 bootstrap replicates, and the protein sequences of Arabidopsis ZFPs were obtained from PlantTFDB (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://planttfdb.gao-lab.org/index.php\u003c/span\u003e\u003cspan address=\"http://planttfdb.gao-lab.org/index.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Multiple alignments of McZFP1 and its homologs in Arabidopsis was performed using DNAMAN. The MEME combinatorial tool was used for the motif search.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. RNA isolation and qRT-PCR analysis\u003c/h2\u003e \u003cp\u003e Various plant tissues were prepared for the isolation of total RNA using the Eastep\u0026reg; Super Total RNA Extraction Kit (Promega, Beijing, China), according to the manufacturer\u0026rsquo;s instructions. Total RNA was used to produce cDNA via the HiScript III 1st Strand cDNA Synthesis Kit (+\u0026thinsp;gDNA wiper) (Vazyme, Nanjing, China). qRT-PCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme) on a CFX96 Real-Time PCR Detection System (Bio-Rad, Boston, MA, USA) in accordance with the manufacturer\u0026rsquo;s instructions. The \u003cem\u003eMcACT\u003c/em\u003e gene and \u003cem\u003eAtACT2\u003c/em\u003e gene were used as the reference genes for normalizing the gene expression in \u003cem\u003eM. canadensis\u003c/em\u003e and \u003cem\u003eArabidopsis\u003c/em\u003e, respectively. The primers used for qRT-PCR are listed in Supplemental Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Subcellular localization of McZFP1\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eMcZFP1\u003c/em\u003e coding sequence (CDS) was introduced into the pGate8-GFP vector to produce \u003cem\u003e35S::McZFP1\u0026ndash;GFP\u003c/em\u003e, which was then transformed into \u003cem\u003eAgrobacterium\u003c/em\u003e strain GV3101. To evaluate McZFP1 localization within cells, the \u003cem\u003eAgrobacterium\u003c/em\u003e GV3101 colony carrying the \u003cem\u003e35S::McZFP1\u0026ndash;GFP\u003c/em\u003e construct was cultured to an OD\u003csub\u003e600\u003c/sub\u003e of 1.8\u0026ndash;2.0, resuspended with 10 mM 2-morpholinoethanesulphonic acid (MES), 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e (pH 5.8), and 150 mM acetosyringone to an OD\u003csub\u003e600\u003c/sub\u003e of 0.8\u0026ndash;1.0, and then infiltrated into \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves. After 2\u0026ndash;3 days of cultivation, \u003cem\u003eN. benthamiana\u003c/em\u003e leaves were collected to observe the fluorescence imaging. The \u003cem\u003e35S::McZFP1\u0026ndash;GFP\u003c/em\u003e construct was introduced into WT plants using the floral dip method to determine the subcellular localization of McZFP1 in Arabidopsis seedlings (Clough and Bent \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). DAPI (4\u0026rsquo;,6-diamidino-2-phenylindole, 1 \u0026micro;g/mL) was used to incubate tobacco leaves or transgenic Arabidopsis seedlings to locate the nucleus. Primers used for the construction of \u003cem\u003e35S::McZFP1\u0026ndash;GFP\u003c/em\u003e are listed in Supplemental Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Transcriptional activation of McZFP1 in yeast cells\u003c/h2\u003e \u003cp\u003eThe open reading frame (ORF) of \u003cem\u003eMcZFP1\u003c/em\u003e was ligated into the \u003cem\u003epGBKT7\u003c/em\u003e (BD) vector at the EcoR1 site to generate \u003cem\u003epGBKT7\u0026ndash;McZFP1\u003c/em\u003e (\u003cem\u003eBD\u0026ndash;McZFP1\u003c/em\u003e). The BD vector and \u003cem\u003epGBKT7\u0026ndash;AtSIZ1\u003c/em\u003e (\u003cem\u003eBD\u0026ndash;AtSIZ1\u003c/em\u003e) (Leng et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) were used as empty and positive controls, respectively. These three vectors were separately transformed into yeast strain Y2HGold using the Yeast Transformation Kit (Coolaber Science \u0026amp; Technology, Beijing, China). The transformed yeast cells were cultured on SD/-Trp and SD/-Trp/-His/-Ade medium at 30\u0026deg;C for 2\u0026ndash;3 days; their transcriptional activities were evaluated according to the yeast growth status. The primers used for \u003cem\u003eBD\u0026ndash;McZFP1\u003c/em\u003e construction are listed in Supplemental Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Microscopy\u003c/h2\u003e \u003cp\u003ePhotographs of yeasts on medium, seedlings, and germinated seeds were acquired using a digital camera. The GUS-stained tissues and root hairs were photographed using a stereoscope (DVM6a, Leica, Wetzlar, Germany). A confocal laser scanning microscope (LSM900, Zeiss, Oberkochen, Germany) was used for green fluorescent protein (GFP) and DAPI imaging in plant tissues. The excitation and emission wavelengths during observation were 405 and 538\u0026ndash;632 nm, respectively, for DAPI and 488 and 493\u0026ndash;536 nm, respectively, for GFP. Trichomes were photographed using cryo-scanning electron microscopy (cryo-SEM, SU8010, Hitachi, Tokyo, Japan) according to Charuvi et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) with modifications. Briefly, leaf samples were mounted in a cylindrical plug of a copper sample holder and rapidly frozen in liquid nitrogen at \u0026minus;\u0026thinsp;210\u0026deg;C for 2 min, transferred to the cold stage of a preparation chamber, and etched. The cold stage temperature was raised from \u0026minus;\u0026thinsp;140\u0026deg;C to \u0026minus;\u0026thinsp;70\u0026deg;, held for 10 min, and then returned to \u0026minus;\u0026thinsp;140\u0026deg;C. After etching, platinum sputtering was conducted at 10 mA for 60 s. Images were acquired using cryo-SEM on a cryo-preparation system (PP3010T, Quorum, Tokyo, Japan) at an accelerating voltage of 3 kV. For root hairs, the primary roots of 7-day-old Arabidopsis seedlings cultivated in 1/2 MS medium were observed, and approximately 7 mm of the root tip was photographed to determine the number and length of root hairs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Measurement of physiological indexes\u003c/h2\u003e \u003cp\u003eFour-day-old Arabidopsis WT and OE plants after normal and salt treatments for 2 weeks were used to determine related physiological indexes. The proline and malondialdehyde (MDA) contents were determined using the Proline Assay Kit and Plant Malondialdehyde (MDA) Assay Kit, respectively. The Catalase (CAT) Assay Kit, Total Superoxide Dismutase (T-SOD) Assay Kit, and Peroxidase Assay Kit were used to determine the CAT, SOD, and POD activities, respectively. The kits used above were purchased from Nanjing Jiancheng (Nanjing, China), and each physiological index was determined according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1. \u003cem\u003eMcZFP1\u003c/em\u003e isolation from \u003cem\u003eM. canadensis\u003c/em\u003e and sequence analysis\u003c/h2\u003e \u003cp\u003eIn this study, the \u003cem\u003eM. canadensis McZFP1\u003c/em\u003e gene was identified using a BLASTP search on the reported NCBI transcriptome data (SRP132644) with the AtZFP1 amino acid sequence as a query (Qi et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The DNA sequence of \u003cem\u003eMcZFP1\u003c/em\u003e is depicted in Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA. The \u003cem\u003eMcZFP1\u003c/em\u003e gene contained an ORF of 489 bases and encoded a protein with 162 amino acids (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Phylogenetic tree analysis of McZFP1 and Arabidopsis ZFP family proteins showed that McZFP1 clustered with AtZFP1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Amino acid sequence analysis and sequence alignment of McZFP1 with the 11 homologous C2H2-ZFPs from Arabidopsis revealed that McZFP1 contained a conserved zinc finger domain (CX2-4CX3FX5LX2HX3-5H) with a plant-specific conserved QALGGH sequence (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C). A conserved ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif was also identified in the McZFP1 protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C). Phylogenetic tree analysis of McZFP1 and its homologs in other plants showed that McZFP1 had a close relationship with ShZFP1-like from \u003cem\u003eSalvia hispanica\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). These results suggest that McZFP1 is a C2H2-type ZFP protein.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Analysis of \u003cem\u003eMcZFP1\u003c/em\u003e gene expression\u003c/h2\u003e \u003cp\u003eTissue expression patterns were examined to characterize the potential function of the \u003cem\u003eMcZFP1\u003c/em\u003e gene in \u003cem\u003eM. canadensis\u003c/em\u003e. \u003cem\u003eMcZFP1\u003c/em\u003e was expressed in the examined tissues, including leaves, stems, flowers, rhizomes, and adventitious roots, according to qRT-PCR analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Furthermore, the \u003cem\u003eMcZFP1\u003c/em\u003e gene was highly expressed in stems, followed by rhizomes, flowers, leaves, and adventitious roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). To further analyze the potential roles of the \u003cem\u003eMcZFP1\u003c/em\u003e gene in the plant response to abiotic stress and hormones, we measured its expression levels in leaves and adventitious roots with NaCl, mannitol, ABA, GA, and ACC treatments. When treated with 150 mM NaCl, \u003cem\u003eMcZFP1\u003c/em\u003e expression was inhibited at 2, 4, 12, and 24 h post-treatment (hpt) but did not change significantly at 8 hpt in leaves and was inhibited at 2, 4, and 12 hpt and enhanced at 24 hpt in adventitious roots. Under 300 mM mannitol treatment, \u003cem\u003eMcZFP1\u003c/em\u003e expression was markedly induced in leaves at 2 hpt and increased in adventitious roots by about 5\u0026ndash;7-fold within 8\u0026ndash;24 h, reaching a peak at 12 hpt. Under ABA treatment, \u003cem\u003eMcZFP1\u003c/em\u003e expression increased at 2\u0026ndash;8 hpt in leaves but decreased at 2\u0026ndash;12 hpt and increased at 24 hpt in adventitious roots. When treated with GA, \u003cem\u003eMcZFP1\u003c/em\u003e expression decreased in leaves at 2\u0026ndash;12 hpt and increased in adventitious roots at 2 and 4 hpt. Under ACC treatment, \u003cem\u003eMcZFP1\u003c/em\u003e expression significantly decreased in leaves and adventitious roots at 2\u0026ndash;24 hpt (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Among the five treatments, \u003cem\u003eMcZFP1\u003c/em\u003e expression was mainly inhibited by NaCl and ACC treatment but enhanced by mannitol treatment. It was induced in leaves but repressed in adventitious roots under ABA treatment and induced in adventitious roots but repressed in leaves under GA treatment. These results suggest that the \u003cem\u003eMcZFP1\u003c/em\u003e gene plays different regulatory roles in stress and hormone responses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Subcellular localization and transcriptional activity of McZFP1\u003c/h2\u003e \u003cp\u003eFor subcellular localization of McZFP1, the \u003cem\u003e35S::McZFP1\u0026ndash;GFP\u003c/em\u003e plasmid was constructed and transformed into \u003cem\u003eAgrobacterium\u003c/em\u003e strain GV3101 and then transiently expressed in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves using the \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transient expression system. The leaves were stained with the nucleus marker DAPI for 2 h before fluorescence microscopy detection. Furthermore, the \u003cem\u003e35S::McZFP1\u0026ndash;GFP\u003c/em\u003e construct was transformed into Arabidopsis WT plants, and the subcellular location of McZFP1 was observed in transgenic seedlings after DAPI staining. The GFP and DAPI fluorescence patterns overlapped under a confocal microscope, indicating that McZFP1 localized in the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). These results suggest that McZFP1 is expressed within the nucleus.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further used a yeast system to identify the transcriptional activation activity of McZFP1. The \u003cem\u003epGBKT7\u0026ndash;McZFP1\u003c/em\u003e vector was constructed and transformed into yeast strain Y2H. Yeast cells containing \u003cem\u003epGBKT7\u0026ndash;McZFP1\u003c/em\u003e (BD\u0026ndash;McZFP1), \u003cem\u003epGBKT7\u003c/em\u003e (BD vector, empty control), and \u003cem\u003epGBKT7\u0026ndash;AtSIZ1\u003c/em\u003e (BD\u0026ndash;AtSIZ1, positive control) all grew normally on SD/-Trp medium. When cultured on SD/-Trp/-His/-Ade medium, BD\u0026ndash;McZFP1 failed to grow normally (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). These results suggest that McZFP1 has no transcriptional self-activation activity in yeast cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4. \u003cem\u003eMcZFP1\u003c/em\u003e overexpression inhibits trichome development in transgenic Arabidopsis\u003c/h2\u003e \u003cp\u003eTo determine whether \u003cem\u003eMcZFP1\u003c/em\u003e regulates trichome development, the \u003cem\u003e35S::McZFP1\u0026ndash;GFP\u003c/em\u003e construct was introduced into Arabidopsis WT plants to generate \u003cem\u003eMcZFP1\u003c/em\u003e transgenic OE plants. Five homozygous \u003cem\u003eMcZFP1\u003c/em\u003e OE lines were obtained, and two showing higher \u003cem\u003eMcZFP1\u003c/em\u003e expression (OE2 and OE3) were used in function analysis (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The WT and \u003cem\u003eMcZFP1\u003c/em\u003e OE lines were grown under normal conditions. The two \u003cem\u003eMcZFP1\u003c/em\u003e OE lines had fewer trichomes in rosette leaves than WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePrevious studies have demonstrated that some C2H2-ZFP genes and MBW complex-related genes play positive roles in trichome initiation in Arabidopsis, while some CPC-type R3 MYB genes play negative roles (Han et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). We examined the expression levels of related genes in rosette leaves of Arabidopsis WT and \u003cem\u003eMcZFP1\u003c/em\u003e OE transgenic plants. C2H2-ZFP genes, including \u003cem\u003eAtZFP1\u003c/em\u003e, \u003cem\u003eAtZFP8\u003c/em\u003e, \u003cem\u003eAtGIS\u003c/em\u003e, \u003cem\u003eAtGIS2\u003c/em\u003e, and \u003cem\u003eAtGIS3\u003c/em\u003e, were significantly inhibited in \u003cem\u003eMcZFP1\u003c/em\u003e OE plants compared with WT plants, while \u003cem\u003eAtZFP5\u003c/em\u003e and \u003cem\u003eAtZFP6\u003c/em\u003e were slightly affected (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). MBW complex-related genes, including \u003cem\u003eAtGL3\u003c/em\u003e, \u003cem\u003eAtEGL3\u003c/em\u003e, \u003cem\u003eAtTTG2\u003c/em\u003e, and \u003cem\u003eAtGL1\u003c/em\u003e, showed lower expression in \u003cem\u003eMcZFP1\u003c/em\u003e OE plants than in WT plants, while \u003cem\u003eAtTTG1\u003c/em\u003e showed higher expression, and \u003cem\u003eAtGL2\u003c/em\u003e had no obvious changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The expression levels of CPC-type R3 MYB genes, namely \u003cem\u003eAtTRY\u003c/em\u003e, \u003cem\u003eAtTCL1\u003c/em\u003e, and \u003cem\u003eAtETC2\u003c/em\u003e, were increased, whereas those of \u003cem\u003eAtETC1\u003c/em\u003e, \u003cem\u003eAtETC3\u003c/em\u003e, and \u003cem\u003eAtCPC\u003c/em\u003e were decreased, and \u003cem\u003eAtTCL2\u003c/em\u003e was almost unchanged (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). These results indicate that increased \u003cem\u003eMcZFP1\u003c/em\u003e expression decreases the trichome number in Arabidopsis by inhibiting trichome-initiation gene expression and elevating trichome-inhibition gene expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.5. \u003cem\u003eMcZFP1\u003c/em\u003e overexpression inhibits root hair development in transgenic Arabidopsis\u003c/h2\u003e \u003cp\u003eC2H2-ZFPs function not only in trichome development but also in root hair formation. To determine whether \u003cem\u003eMcZFP1\u003c/em\u003e plays a role in root hair development, we observed the root hair phenotypes of WT and \u003cem\u003eMcZFP1\u003c/em\u003e OE plants. \u003cem\u003eMcZFP1\u003c/em\u003e overexpression in Arabidopsis led to a reduction in root hair number and length compared with WT plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further elucidate the role of \u003cem\u003eMcZFP1\u003c/em\u003e in root hair formation, the expression levels of genes involved in root hair initiation and development (e.g., negative root hair-development regulator genes \u003cem\u003eAtTTG1\u003c/em\u003e, \u003cem\u003eAtWER\u003c/em\u003e, \u003cem\u003eAtGL3\u003c/em\u003e, \u003cem\u003eAtGL2\u003c/em\u003e, \u003cem\u003eAtZP1\u003c/em\u003e, and \u003cem\u003eAtZFP3\u003c/em\u003e and positive regulator genes \u003cem\u003eAtZFP5\u003c/em\u003e, \u003cem\u003eAtGIS3\u003c/em\u003e, \u003cem\u003eAtCPC\u003c/em\u003e, \u003cem\u003eAtTRY\u003c/em\u003e, \u003cem\u003eAtETC1\u003c/em\u003e, \u003cem\u003eAtETC2\u003c/em\u003e, \u003cem\u003eAtETC3\u003c/em\u003e, \u003cem\u003eAtRHD6\u003c/em\u003e, \u003cem\u003eAtRSL1\u003c/em\u003e, \u003cem\u003eAtRHD2\u003c/em\u003e, \u003cem\u003eAtRHD4\u003c/em\u003e, \u003cem\u003eAtRSL2\u003c/em\u003e, \u003cem\u003eAtRSL4\u003c/em\u003e, \u003cem\u003eAtLRL1\u003c/em\u003e, \u003cem\u003eAtLRL2\u003c/em\u003e, \u003cem\u003eAtLRL3\u003c/em\u003e, \u003cem\u003eAtEIL1\u003c/em\u003e, and \u003cem\u003eAtEIN3\u003c/em\u003e) were measured in the roots of \u003cem\u003eMcZFP1\u003c/em\u003e OE and WT seedlings (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eD\u0026ndash;F). Among these negative regulator genes, \u003cem\u003eAtTTG1\u003c/em\u003e, \u003cem\u003eAtWER\u003c/em\u003e, \u003cem\u003eAtZP1\u003c/em\u003e, and \u003cem\u003eAtZFP3\u003c/em\u003e were significantly upregulated, while \u003cem\u003eAtGL3\u003c/em\u003e and \u003cem\u003eAtGL2\u003c/em\u003e were not changed in the \u003cem\u003eMcZFP1\u003c/em\u003e OE lines compared with WT. Among these positive regulator genes, \u003cem\u003eAtZFP5\u003c/em\u003e, \u003cem\u003eAtGIS3\u003c/em\u003e, \u003cem\u003eAtCPC\u003c/em\u003e, \u003cem\u003eAtTRY\u003c/em\u003e, \u003cem\u003eAtETC1\u003c/em\u003e, \u003cem\u003eAtETC2\u003c/em\u003e, \u003cem\u003eAtETC3\u003c/em\u003e, \u003cem\u003eAtRHD6\u003c/em\u003e, \u003cem\u003eAtRHD2\u003c/em\u003e, \u003cem\u003eAtRHD4\u003c/em\u003e, \u003cem\u003eAtRSL4\u003c/em\u003e, \u003cem\u003eAtLRL3\u003c/em\u003e, \u003cem\u003eAtEIL1\u003c/em\u003e, and \u003cem\u003eAtEIN3\u003c/em\u003e were downregulated, while \u003cem\u003eAtRSL1\u003c/em\u003e, \u003cem\u003eAtRSL2\u003c/em\u003e, and \u003cem\u003eAtLRL1\u003c/em\u003e were slightly upregulated. \u003cem\u003eAtLRL2\u003c/em\u003e remained consistent in the \u003cem\u003eMcZFP1\u003c/em\u003e OE lines and WT. These results suggest that \u003cem\u003eMcZFP1\u003c/em\u003e overexpression promotes the expression of most examined negative regulator genes and inhibits the expression of most tested positive regulator genes in root hair development, reducing the number and length of root hairs in the \u003cem\u003eMcZFP1\u003c/em\u003e OE lines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.6. \u003cem\u003eMcZFP1\u003c/em\u003e overexpression reduces salt tolerance in transgenic Arabidopsis\u003c/h2\u003e \u003cp\u003e \u003cem\u003eMcZFP1\u003c/em\u003e responded to NaCl and mannitol treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). To investigate the role of \u003cem\u003eMcZFP1\u003c/em\u003e in plant tolerance to salt or drought stress, we treated plants at the germination and seedling stages with salt and mannitol, respectively. We first checked the germination rate of WT and \u003cem\u003eMcZFP1\u003c/em\u003e OE seeds upon salt treatment. No obvious differences were observed between WT and \u003cem\u003eMcZFP1\u003c/em\u003e OE lines grown on 1/2 MS (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). By contrast, high NaCl concentrations strongly inhibited seed germination of the \u003cem\u003eMcZFP1\u003c/em\u003e OE lines compared with WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). Under 150 mM NaCl treatment, the germination rate of \u003cem\u003eMcZFP1\u003c/em\u003e OE lines was lower than that of WT at the early germination stage. When treated with 200 mM NaCl, the germination rate of \u003cem\u003eMcZFP1\u003c/em\u003e OE lines was lower than that of WT throughout the germination stage. We further measured the root lengths of WT and \u003cem\u003eMcZFP1\u003c/em\u003e OE seedlings under normal and salt stress conditions. No significant differences were observed between WT and \u003cem\u003eMcZFP1\u003c/em\u003e OE seedlings under normal conditions. However, when treated with 150 mM NaCl, \u003cem\u003eMcZFP1\u003c/em\u003e OE seedlings showed shorter roots than WT seedlings (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D). We also performed mannitol treatment on WT and \u003cem\u003eMcZFP1\u003c/em\u003e OE seeds, and no obvious differences were observed under treatment with 300 mM mannitol (Fig. S3). ABA plays a crucial role in the salt stress response. ABA treatment led to seed germination phenotypes similar to the NaCl treatment (Fig. S4). These results suggest that \u003cem\u003eMcZFP1\u003c/em\u003e overexpression reduces the salt tolerance of transgenic Arabidopsis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.7. \u003cem\u003eMcZFP1\u003c/em\u003e overexpression in Arabidopsis reduces osmolyte accumulation and antioxidant enzyme activities\u003c/h2\u003e \u003cp\u003eTo determine whether McZFP1-mediated salt intolerance was associated with the alteration in osmolyte accumulation and ROS homeostasis, the proline and MDA contents and SOD, CAT, and POD enzyme activities were detected in WT and \u003cem\u003eMcZFP1\u003c/em\u003e OE plants under normal and salt stress conditions. Under control conditions, the proline and MDA contents and SOD, CAT, and POD enzyme activities were comparable in WT and \u003cem\u003eMcZFP1\u003c/em\u003e OE plants. When treated with 150 mM NaCl, the proline content in \u003cem\u003eMcZFP1\u003c/em\u003e OE plants decreased compared with WT, while the MDA content was enhanced (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B). Salt stress significantly increased SOD, CAT, and POD enzyme activities, and the increases were more pronounced in WT plants than in \u003cem\u003eMcZFP1\u003c/em\u003e OE plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003eC\u0026ndash;E). These results indicate that \u003cem\u003eMcZFP1\u003c/em\u003e overexpression reduces the osmoregulation and antioxidant capacity of transgenic Arabidopsis plants under drought stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.8. \u003cem\u003eMcZFP1\u003c/em\u003e overexpression downregulates the expression of stress-responsive genes in transgenic Arabidopsis\u003c/h2\u003e \u003cp\u003eTo investigate the mechanisms by which \u003cem\u003eMcZFP1\u003c/em\u003e regulates salt stress-responsive genes in plants, we further analyzed the expression levels of some well-studied abiotic stress-responsive genes, including \u003cem\u003eAtDREB1A\u003c/em\u003e, \u003cem\u003eAtCOR15A\u003c/em\u003e, \u003cem\u003eAtCOR15B\u003c/em\u003e, \u003cem\u003eAtRD29A\u003c/em\u003e, \u003cem\u003eAtRD29B\u003c/em\u003e, and \u003cem\u003eAtRAB18\u003c/em\u003e, in transgenic Arabidopsis. The expression levels of these genes did not differ between \u003cem\u003eMcZFP1\u003c/em\u003e OE and WT plants under normal growth conditions. However, these stress-related genes showed low expression in \u003cem\u003eMcZFP1\u003c/em\u003e OE plants compared with WT plants under 150 mM NaCl treatment. These data suggest that \u003cem\u003eMcZFP1\u003c/em\u003e overexpression inhibits the expression of stress-responsive genes in transgenic Arabidopsis, which may explain the decreased tolerance of \u003cem\u003eMcZFP1\u003c/em\u003e OE transgenic plants.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e \u003cem\u003eMentha canadensis\u003c/em\u003e is widely used in industrial production due to its essential oils. Mint oil is biosynthesized and stored in glandular trichomes (Croteau et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Root hairs facilitate nutrient acquisition and environmental interactions, which contribute to plant root anchorage, stress resistance, growth, and development (Han et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Shibata and Sugimoto \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Salt stress restricts plant growth and development and is a non-negligible factor affecting mint oil production (Kumar et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, evaluating trichome and root hair development and the salt stress response in \u003cem\u003eM. canadensis\u003c/em\u003e may promote mint growth and development for higher essential oil production. C2H2-ZFPs have been reported to play different roles in regulating plant trichome and root hair development and salt stress adaptation (Han et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, few studies on trichome and root hair development, salt stress response, and C2H2-ZFP functions in \u003cem\u003eM. canadensis\u003c/em\u003e have been reported. In this study, a novel C2H2-ZFP TF was identified from \u003cem\u003eM. canadensis\u003c/em\u003e and named \u003cem\u003eMcZFP1\u003c/em\u003e based on phylogenetic analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Further analysis revealed that \u003cem\u003eMcZFP1\u003c/em\u003e negatively regulated trichome formation, root hair development, and salt tolerance in transgenic Arabidopsis.\u003c/p\u003e \u003cp\u003eThe C2H2 ZFP family generally contains a conserved zinc finger domain, and most plant C2H2-ZFPs have a specific conserved QALGGH sequence in the zinc finger domain (Huang et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Domain and multiple sequence alignment analyses showed that McZFP1 contained a typical zinc finger domain with a plant-specific QALGGH motif (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The EAR domain has also been found in many C2H2-ZFPs, which function as repressors (Xie et al. \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). We found an EAR motif at the end of the C terminus in McZFP1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Thus, McZFP1 from \u003cem\u003eM. canadensis\u003c/em\u003e is a new, typical member of the plant C2H2-ZFPs, suggesting that it may have a conserved function. The spatiotemporal expression patterns of genes are generally presumed to reflect their potential roles in plant growth and development or environmental stimulus responses. In our study, the expression level of \u003cem\u003eMcZFP1\u003c/em\u003e was higher in stems than in leaves, flowers, adventitious roots, and rhizomes of \u003cem\u003eM. canadensis\u003c/em\u003e, implying that \u003cem\u003eMcZFP1\u003c/em\u003e plays different roles in their development or functions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The \u003cem\u003eMcZFP1\u003c/em\u003e gene had different responses to abiotic stressor (NaCl and mannitol) and hormone (ABA, GA, and ethylene) treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). NaCl treatment mainly inhibited \u003cem\u003eMcZFP1\u003c/em\u003e expression, while mannitol treatment mainly induced it, suggesting that \u003cem\u003eMcZFP1\u003c/em\u003e negatively regulates plant salt tolerance and positively regulates drought tolerance. The responses of \u003cem\u003eMcZFP1\u003c/em\u003e upon ABA, GA, and ethylene treatment suggest that it may play roles in the complex vital movements mediated by these hormones. Subcellular localization and transcriptional activity assays showed that McZFP1 localized in the nucleus and possessed no transcriptional self-activating activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The gene expression patterns and protein characteristics of \u003cem\u003eMcZFP1\u003c/em\u003e indicate that it responds to environmental and hormone stimuli and has roles in plant development and abiotic stress responses.\u003c/p\u003e \u003cp\u003eC2H2-ZFPs from several plant species, including Arabidopsis, tomato, tobacco, cotton, cucumber, pepper, and \u003cem\u003eJatropha curcas\u003c/em\u003e, have been reported to play positive roles in trichome initiation (Chang et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Shi et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In Arabidopsis, the MBW complex (AtGL1\u0026ndash;AtGL3/AtEGL3\u0026ndash;AtTTG1) induces the expression of AtGL2 and AtTTG2 to initiate trichome formation, while R3 MYBs move from a trichome precursor cell to its neighboring cell to compete with AtGL1 and interact with AtGL3 or AtEGL3, disrupting the functionality of the activator MBW complex and thus inhibiting trichome initiation (Wang et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Therefore, AtGL1, AtGL3, AtEGL3, AtTTG1, AtGL2, and AtTTG2 are positive regulators in trichome development, while AtTRY, AtTCL1, AtETC1, AtETC2, AtETC3, and AtCPC are negative regulators (Gan et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Han et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Johnson et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Larkin et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Morohashi et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Payne et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Schellmann et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Walker et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Wester et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Several Arabidopsis C2H2-ZFPs, such as AtGIS, AtGIS2, AtGIS3, AtZFP1, AtZFP5, AtZFP6, and AtZFP8, act upstream of the MBW complex and positively regulate trichome initiation (Gan et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2006\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). To regulate the trichome development network, AtZFP6 acts upstream of AtZFP5 and AtGIS, and AtZFP5/AtGIS3 acts upstream of AtGIS, AtGIS2, AtZFP8, AtGL1, and AtGL3 (Sun et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In this study, we investigated the role of \u003cem\u003eMcZFP1\u003c/em\u003e in trichome formation in Arabidopsis because no transgenic mint line has been obtained. \u003cem\u003eMcZFP1\u003c/em\u003e overexpression produced fewer trichomes in Arabidopsis than in WT, differing from results reported for C2H2-ZFPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). To understand the reason for this phenotype, we examined the gene expression levels of C2H2-ZFP genes, MBW complex-related positive regulator genes, and negative regulator R3 MYB genes. Five of seven positive C2H2-ZFP regulator genes (\u003cem\u003eAtZFP1\u003c/em\u003e, \u003cem\u003eAtZFP8\u003c/em\u003e, \u003cem\u003eAtGIS\u003c/em\u003e, \u003cem\u003eAtGIS2\u003c/em\u003e, and \u003cem\u003eAtGIS3\u003c/em\u003e) and five of seven MBW complex-related activator genes (\u003cem\u003eAtGL3\u003c/em\u003e, \u003cem\u003eAtEGL3\u003c/em\u003e, \u003cem\u003eAtGL1\u003c/em\u003e, \u003cem\u003eAtMYB5\u003c/em\u003e, and \u003cem\u003eAtTTG2\u003c/em\u003e) were significantly downregulated in \u003cem\u003eMcZFP1\u003c/em\u003e OE lines, while three of seven R3 MYB repressor genes (\u003cem\u003eAtTRY\u003c/em\u003e, \u003cem\u003eAtTCL1\u003c/em\u003e, and \u003cem\u003eAtETC2\u003c/em\u003e) were upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u0026ndash;E). Thus, elevated \u003cem\u003eMcZFP1\u003c/em\u003e expression may reduce the trichome number in Arabidopsis by elevating trichome-inhibition gene expression but inhibiting trichome-initiation gene expression. GA can induce the expression of some C2H2-ZFPs and plays a dominant role in C2H2-ZFP-mediated regulation of trichome development (Gan et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2006\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In this study, \u003cem\u003eMcZFP1\u003c/em\u003e expression was inhibited by GA treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). We speculate that \u003cem\u003eMcZFP1\u003c/em\u003e may play a negative role in GA-mediated regulation of trichome development, but this relationship requires further investigation.\u003c/p\u003e \u003cp\u003eRoot hairs are tubular polarized outgrowths of a trichoblast, which are developed from specialized root epidermal cells and regulated by a well-defined cellular differentiation program (Han et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Shibata and Sugimoto \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). C2H2-ZFPs play contrasting roles in root hair development. In Arabidopsis, both AtZFP5 and AtGIS3 positively regulate root hair development, while AtZFP3 and AtZP1 play negative roles (Beny\u0026oacute; et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Han et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In the present study, the \u003cem\u003eMcZFP1\u003c/em\u003e OE lines showed fewer and shorter root hairs compared with WT, suggesting that \u003cem\u003eMcZFP1\u003c/em\u003e plays a negative role in root hair development (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eA\u0026ndash;C). The MBW complex and R3 MYBs are also found to play opposite roles in the regulation of root hair formation in Arabidopsis. In root hair development, the MBW complex (AtWER\u0026ndash;AtGL3\u0026ndash;AtTTG1) induces AtGL2 expression in non-hair cells to suppress root hair initiation, while R3 MYBs compete with AtWER for binding to AtGL3, thereby suppressing MBW complex activity and \u003cem\u003eAtGL2\u003c/em\u003e expression and initiating hair cell specification (Tominaga-Wada and Wada \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). \u003cem\u003eAtWER\u003c/em\u003e, \u003cem\u003eAtGL3\u003c/em\u003e, \u003cem\u003eAtTTG1\u003c/em\u003e, and \u003cem\u003eAtGL2\u003c/em\u003e play crucial negative roles in root hair development, and their mutation generally leads to ectopic root hair formation (Bernhardt et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Lee and Schiefelbein \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Long and Schiefelbein \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Five R3 MYBs (\u003cem\u003eAtCPC\u003c/em\u003e, \u003cem\u003eAtETC1\u003c/em\u003e, \u003cem\u003eAtETC2\u003c/em\u003e, \u003cem\u003eAtETC3\u003c/em\u003e, and \u003cem\u003eAtTRY\u003c/em\u003e) contribute to root hair development, and their overexpression promotes root hair formation (Esch et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Kirik et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Kirik et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Schellmann et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Tominaga et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Wada et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). After cell fate specification, several bHLH TFs act downstream of \u003cem\u003eAtGL2\u003c/em\u003e or CPC-type R3 MYBs to form a regulatory network and regulate root hair initiation (\u003cem\u003eAtRHD6\u003c/em\u003e and \u003cem\u003eAtRSL1\u003c/em\u003e) and elongation (\u003cem\u003eAtRSL2\u003c/em\u003e, \u003cem\u003eAtRSL4\u003c/em\u003e, \u003cem\u003eAtLRL1\u003c/em\u003e, \u003cem\u003eAtLRL2\u003c/em\u003e, and \u003cem\u003eAtLRL3\u003c/em\u003e) in Arabidopsis (Masucci and Schiefelbein, 1994; Menand et al., 2007; Karas et al., 2009; Yi et al., 2010; Bruex et al., 2012; Pires et al., 2013; Lin et al., 2015). \u003cem\u003eAtZFP5\u003c/em\u003e positively controls root hair development by directly promoting \u003cem\u003eAtCPC\u003c/em\u003e expression (An et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). AtGIS3 binds to and activates \u003cem\u003eAtRHD2\u003c/em\u003e and \u003cem\u003eAtRHD4\u003c/em\u003e genes to promote root hair elongation in Arabidopsis (Huang et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). \u003cem\u003eAtZP1\u003c/em\u003e acts downstream of \u003cem\u003eAtGL2\u003c/em\u003e to inhibit root hair development as a repressor of \u003cem\u003eAtRHD6\u003c/em\u003e, \u003cem\u003eAtRSL2\u003c/em\u003e, and \u003cem\u003eAtRSL4\u003c/em\u003e (Han et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). \u003cem\u003eAtZFP3\u003c/em\u003e functions as a repressor in root hair development by inhibiting the activity of key regulatory genes, such as AtRSL4 (Beny\u0026oacute; et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In the \u003cem\u003eMcZFP1\u003c/em\u003e OE lines, the higher gene expression levels of negative regulators, including \u003cem\u003eAtTTG1\u003c/em\u003e, \u003cem\u003eAtWER\u003c/em\u003e, \u003cem\u003eAtZP1\u003c/em\u003e, and \u003cem\u003eAtZFP3\u003c/em\u003e, and the lower gene expression levels of positive regulators, including \u003cem\u003eAtZFP5\u003c/em\u003e, \u003cem\u003eAtGIS3\u003c/em\u003e, \u003cem\u003eAtCPC\u003c/em\u003e, \u003cem\u003eAtETC1\u003c/em\u003e, \u003cem\u003eAtETC2\u003c/em\u003e, \u003cem\u003eAtETC3\u003c/em\u003e, \u003cem\u003eAtTRY\u003c/em\u003e, \u003cem\u003eAtRHD6\u003c/em\u003e, \u003cem\u003eAtRHD2\u003c/em\u003e, \u003cem\u003eAtRHD4\u003c/em\u003e, \u003cem\u003eAtRSL4\u003c/em\u003e, and \u003cem\u003eAtLRL3\u003c/em\u003e, were detected compared with WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eD\u0026ndash;F). These data suggest that \u003cem\u003eMcZFP1\u003c/em\u003e overexpression inhibits root hair development in Arabidopsis by enhancing negative root hair-development gene expression but decreasing positive root hair-development gene expression. AtZFP5 and AtGIS3 mediate ethylene signals to regulate root hair development (An et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Ethylene-activated TF ETHYLENE-INSENSITIVE 3 (EIN3)/EIN3-LIKE 1 (EIL1) physically interacts with RHD6/ RHD6-LIKE 1 (RSL1) to enhance root hair initiation by regulating a subset of core root hair genes in Arabidopsis (Feng et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). We found that both the \u003cem\u003eAtEIN3\u003c/em\u003e and \u003cem\u003eAtEIL1\u003c/em\u003e genes showed lower expression levels in the \u003cem\u003eMcZFP1\u003c/em\u003e OE lines than in WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eF), indicating that McZFP1 is involved in the ethylene signaling pathway to regulate root hair development. Further research is needed to explain this inference.\u003c/p\u003e \u003cp\u003eSalt stress significantly inhibits plant seed germination, growth, and development (Zhou et al. \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). C2H2-ZFPs play extensive roles in the plant response to salt stress (Liu et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In Arabidopsis, constitutive \u003cem\u003eAtZFP3\u003c/em\u003e expression enhances proline accumulation and stress-related gene expression to improve plant salt tolerance (Zhang et al. \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In rice, OsZFP179 contributes to the ROS scavenging system and osmotic substance biosynthesis, thus enabling salt stress resistance (Zhang et al. \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). C2H2-ZFPs can cope with salt stress through ABA-dependent and -independent signaling pathways (Liu et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Sweet potato \u003cem\u003eIbZFP1\u003c/em\u003e has been demonstrated to promote salt tolerance by regulating the ABA signaling pathway and osmotic substance accumulation (Wang et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Arabidopsis \u003cem\u003eAtZAT10\u003c/em\u003e does not respond to ABA treatment and enhances plant salt tolerance by maintaining ionic balance (Mittler et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). However, a few C2H2-ZFPs play negative roles in regulating plant salt stress tolerance. A C2H2-type ZFP (\u003cem\u003eMtZPT2-2\u003c/em\u003e) in \u003cem\u003eMedicago truncatula\u003c/em\u003e has been reported to negatively regulate plant salt tolerance by regulating antioxidant defense and Na\u003csup\u003e+\u003c/sup\u003e homeostasis (Huang et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In this study, a negative C2H2-type ZFP regulator, McZFP1, was demonstrated in plant salt tolerance. \u003cem\u003eMcZFP1\u003c/em\u003e gene expression in \u003cem\u003eM. canadensis\u003c/em\u003e was compromised under 150 mM NaCl treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). To further investigate its role in the plant salt stress response, we employed germination rate and root length assays. \u003cem\u003eMcZFP1\u003c/em\u003e overexpression in Arabidopsis significantly reduced the seed germination rate and seedling root length compared with WT under NaCl treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003e), suggesting that \u003cem\u003eMcZFP1\u003c/em\u003e plays a negative role in plant salt stress responses. Proline, as an osmolyte and a potent antioxidant and programmed cell death inhibitor, is one of the most important indicators of plant stress tolerance (Yoshiba et al. \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Salt stress produces excessive ROS accumulation and breaks ROS homeostasis, thus compromising lipid membrane functions and ultimately causing oxidative damage to plant cells (Zhou et al. \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The MDA content is used to indicate cell membrane lipid peroxidation and changes in plants under stress (Moore and Roberts \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). An antioxidative defense system containing SOD, CAT, and POD has evolved in plants to scavenge ROS or inhibit their harmful effects on biomolecules (Wang et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). We further found that the proline and MDA contents and SOD, CAT, and POD activities in WT and \u003cem\u003eMcZFP1\u003c/em\u003e OE plants were comparable under normal conditions. However, under 150 mM NaCl treatment, the proline content in \u003cem\u003eMcZFP1\u003c/em\u003e OE plants was lower than that in WT plants, while the MDA content in \u003cem\u003eMcZFP1\u003c/em\u003e OE plants was higher. The antioxidant enzyme activity in \u003cem\u003eMcZFP1\u003c/em\u003e OE plants was also lower (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These results suggest that \u003cem\u003eMcZFP1\u003c/em\u003e overexpression reduces the ROS scavenging ability by reducing the antioxidant enzyme activity, thereby decreasing plant salt tolerance. In Arabidopsis, a series of salt stress-responsive genes, including \u003cem\u003eAtDREB1A\u003c/em\u003e, \u003cem\u003eAtCOR15A\u003c/em\u003e, \u003cem\u003eAtCOR15B\u003c/em\u003e, \u003cem\u003eAtRD29A\u003c/em\u003e, \u003cem\u003eAtRD29B\u003c/em\u003e, and \u003cem\u003eAtRAB18\u003c/em\u003e, are induced by abiotic stress, making them marker genes in the abiotic stress response (Kasuga et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Kim et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Ma et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Msanne et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). We examined the expression levels of these genes in Arabidopsis WT and \u003cem\u003eMcZFP1\u003c/em\u003e OE plants under normal and NaCl treatment. Their expression in \u003cem\u003eMcZFP1\u003c/em\u003e OE plants was lower than in WT plants under salt stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e8\u003c/span\u003e), partly explaining the decreased tolerance of \u003cem\u003eMcZFP1\u003c/em\u003e OE transgenic plants to salt stress. These aforementioned stress-responsive genes are induced by ABA and generally considered ABA-responsive marker genes (Lang et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Msanne et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Rushton et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Wilhelm and Thomashow \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). In our study, \u003cem\u003eMcZFP1\u003c/em\u003e responded to ABA treatment, and \u003cem\u003eMcZFP1\u003c/em\u003e OE seeds had a reduced germination rate under ABA treatment compared with that of WT (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). Overall, these results suggest that \u003cem\u003eMcZFP1\u003c/em\u003e negatively regulates plant salt tolerance by compromising ROS scavenging and osmotic substance biosynthesis abilities and inhibiting stress-related gene expression. Moreover, the ABA signaling pathway may play a role in the McZFP1-regulated salt stress response, which requires further investigation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study demonstrated that McZFP1, a newly characterized C2H2-type ZFP TF from \u003cem\u003eM. canadensis\u003c/em\u003e, responded to salt and drought stress and phytohormones, such as GA, ethylene, and ABA. In contrast to the reported C2H2-type ZFPs, \u003cem\u003eMcZFP1\u003c/em\u003e negatively regulated trichome and root hair development and salt tolerance in transgenic Arabidopsis plants. Further investigations revealed that \u003cem\u003eMcZFP1\u003c/em\u003e overexpression mediated a complex gene expression regulatory network involved in epidermal cell patterning to negatively regulate trichome and root hair formation. In addition, \u003cem\u003eMcZFP1\u003c/em\u003e overexpression reduced ROS scavenging and osmotic substance biosynthesis abilities and stress-related gene expression, leading to compromised plant salt tolerance. Phytohormone signals may be involved in McZFP1-mediated trichome and root hair development and salt tolerance.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e We thank LetPub (www.letpub.com.cn) for its linguistic assistance during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship contributions\u0026nbsp;\u003c/strong\u003eYang Bai: Conceptualization, Writing - Original Draft, Writing - review \u0026amp; editing. Xiaowei Zheng: Writing - Original Draft, Investigation. Yichuan Xu: Investigation. Li Li: Writing - Original Draft, Investigation. Xiwu Qi: Formal analysis. Xu Yu:\u0026nbsp;Resources. Chun Qin: Investigation. Dongmei Liu: Writing - review \u0026amp; editing. Zequn Chen: Resources. Chengyuan Liang: Project administration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis research was supported by the National Science Foundation of China [grant numbers 32100313], the Natural Science Foundation of the Jiangsu Province [grant numbers BK20210164], and Jiangsu Key Laboratory for the Research and Utilization of Plant Resources [grant numbers JSPKLB202029] to YB; the National Science Foundation of China [grant numbers 32370397] to CYL; and the National Science Foundation of China [grant numbers 32200302] to XY.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003eData will be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAn L, Zhou Z, Sun L, Yan A, Xi W, Yu N, Cai W, Chen X, Yu H, Schiefelbein J, Gan Y (2012) A zinc finger protein gene \u003cem\u003eZFP5\u003c/em\u003e integrates phytohormone signaling to control root hair development in Arabidopsis. 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New Phytol 198(3):699-708\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Mentha canadensis, McZFP1, Trichome, Root hair, Salt stress","lastPublishedDoi":"10.21203/rs.3.rs-4918956/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4918956/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eC2H2-type zinc finger protein (C2H2-ZFP) transcription factors play evident roles in regulating plant growth and development and abiotic stress responses. However, the role of C2H2-ZFP from \u003cem\u003eMentha canadensis\u003c/em\u003e remains uncertain. Here, we identified the multifunctional C2H2-ZFP gene \u003cem\u003eMcZFP1\u003c/em\u003e from \u003cem\u003eM. canadensis\u003c/em\u003e based on phylogenetic analysis. The \u003cem\u003eMcZFP1\u003c/em\u003e gene was highly expressed in stems, responding to abiotic stress and phytohormone treatments. McZFP1 localized in the nucleus and showed no transcriptional self-activation activity. \u003cem\u003eMcZFP1\u003c/em\u003e overexpression in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e significantly reduced the number of trichomes and root hairs, root hair length, and salt stress tolerance. Further study revealed that \u003cem\u003eMcZFP1\u003c/em\u003e overexpression increased the expression of negative regulator genes and decreased that of positive regulator genes to inhibit plant trichome and root hair development. Malondialdehyde accumulation was promoted, but the proline content and catalase, superoxide dismutase, and peroxidase activities were reduced and the expression of stress-response genes was inhibited in \u003cem\u003eMcZFP1\u003c/em\u003e overexpression lines under salt treatment, thereby compromising plant salt tolerance. Overall, these results indicate that McZFP1 is a novel C2H2-ZFP transcription factor that plays negative roles in trichome and root hair development and salt stress tolerance.\u003c/p\u003e","manuscriptTitle":"A C2H2-type zinc finger protein from Mentha canadensis, McZFP1, negatively regulates epidermal cell patterning and salt tolerance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-20 07:58:39","doi":"10.21203/rs.3.rs-4918956/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"eef1aa8d-422f-4db5-b1d4-c00156b80937","owner":[],"postedDate":"September 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-23T14:35:40+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-20 07:58:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4918956","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4918956","identity":"rs-4918956","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}