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

doi:10.21203/rs.3.rs-4918956/v1

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

https://doi.org/10.21203/rs.3.rs-4918956/v1

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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

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.

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.

2.1. 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 OD₆₀₀ of 1.8–2.0, resuspended with 10 mM 2-morpholinoethanesulphonic acid (MES), 10 mM MgCl₂ (pH 5.8), and 150 mM acetosyringone to an OD₆₀₀ 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.

3.1. 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. S1A. The McZFP1 gene contained an ORF of 489 bases and encoded a protein with 162 amino acids (Fig. S1A). Phylogenetic tree analysis of McZFP1 and Arabidopsis ZFP family proteins showed that McZFP1 clustered with AtZFP1 (Fig. 1A). 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. 1B, C). A conserved ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif was also identified in the McZFP1 protein (Fig. 1B, 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. 1D). 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. 2A). Furthermore, the McZFP1 gene was highly expressed in stems, followed by rhizomes, flowers, leaves, and adventitious roots (Fig. 2A). 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. 2B). 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. 3A, 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. 3C). 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. 4A, 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. 4C). 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. 4D). 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. 4E). 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. 5A–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. 5D–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. 2B). 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. 6A, B). By contrast, high NaCl concentrations strongly inhibited seed germination of the McZFP1 OE lines compared with WT (Fig. 6A, 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. 6C, 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. 7A, 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. 7C–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.

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. 2A). The McZFP1 gene had different responses to abiotic stressor (NaCl and mannitol) and hormone (ABA, GA, and ethylene) treatments (Fig. 2A). 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. 4A, 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. 4C–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. 2B). 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. 5A–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. 5D–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. 5F), 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. 2B). 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. 2B 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.

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.

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.

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. Plant J 72(3):474-490
Benyó D, Bató E, Faragó D, Rigó G, Domonkos I, Labhane N, Zsigmond L, Prasad M, Nagy I, Szabados L (2023) The zinc finger protein 3 of Arabidopsis thaliana regulates vegetative growth and root hair development. Front Plant Sci 14:1221519
Bernhardt C, Lee MM, Gonzalez A, Zhang F, Lloyd A, Schiefelbein J (2003) The bHLH genes GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3) specify epidermal cell fate in the Arabidopsis root. Development 130(26):6431-6439
Chang J, Yu T, Yang Q, Li C, Xiong C, Gao S, Xie Q, Zheng F, Li H, Tian Z, Yang C, Ye Z (2018) Hair , encoding a single C2H2 zinc finger protein, regulates multicellular trichome formation in tomato. Plant J 96(1):90-102
Charuvi D, Nevo R, Kaplan-Ashiri I, Shimoni E, Reich Z (2016) Studying the supramolecular organization of photosynthetic membranes within freeze-fractured leaf tissues by cryo-scanning electron microscopy. J Vis Exp (112):54066
Chen K, Li GJ, Bressan RA, Song CP, Zhu JK, Zhao Y (2020) Abscisic acid dynamics, signaling, and functions in plants. J Integr Plant Biol 62(1):25-54
Ciftci-Yilmaz S, Mittler R (2008) The zinc finger network of plants. Cell Mol Life Sci 65(7-8):1150-1160
Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16(6):735-743
Croteau RB, Davis EM, Ringer KL, Wildung MR (2005) (-)-Menthol biosynthesis and molecular genetics. Naturwissenschaften 92(12):562-577
Cui S, Suzaki T, Tominaga-Wada R, Yoshida S (2018) Regulation and functional diversification of root hairs. Semin Cell Dev Biol 83:115-122
Esch JJ, Chen MA, Hillestad M, Marks MD (2004) Comparison of TRY and the closely related At1g01380 gene in controlling Arabidopsis trichome patterning. Plant J 40(6):860-869
Fambrini M, Pugliesi C (2019) The dynamic genetic-hormonal regulatory network controlling the trichome development in leaves. Plants (Basel) 8(8):253
Feng Y, Xu P, Li B, Li P, Wen X, An F, Gong Y, Xin Y, Zhu Z, Wang Y, Guo H (2017) Ethylene promotes root hair growth through coordinated EIN3/EIL1 and RHD6/RSL1 activity in Arabidopsis. Proc Natl Acad Sci U S A 114(52):13834-13839
Feng Z, Bartholomew ES, Liu Z, Cui Y, Dong Y, Li S, Wu H, Ren H, Liu X (2021) Glandular trichomes: new focus on horticultural crops. Hortic Res 8(1):158
Gan L, Xia K, Chen JG, Wang S (2011) Functional characterization of TRICHOMELESS2, a new single-repeat R3 MYB transcription factor in the regulation of trichome patterning in Arabidopsis. BMC Plant Biol 11:176
Gan Y, Kumimoto R, Liu C, Ratcliffe O, Yu H, Broun P (2006) GLABROUS INFLORESCENCE STEMS modulates the regulation by gibberellins of epidermal differentiation and shoot maturation in Arabidopsis. Plant Cell 18(6):1383-1395
Gan Y, Liu C, Yu H, Broun P (2007) Integration of cytokinin and gibberellin signalling by Arabidopsis transcription factors GIS, ZFP8 and GIS2 in the regulation of epidermal cell fate. Development 134(11):2073-2081
Han G, Li Y, Qiao Z, Wang C, Zhao Y, Guo J, Chen M, Wang B (2021) Advances in the regulation of epidermal cell development by C2H2 zinc finger proteins in plants. Front Plant Sci 12:754512
Han G, Li Y, Yang Z, Wang C, Zhang Y, Wang B (2022) Molecular mechanisms of plant trichome development. Front Plant Sci 13:910228
Han G, Lu C, Guo J, Qiao Z, Sui N, Qiu N, Wang B (2020) C2H2 zinc finger proteins: master regulators of abiotic stress responses in plants. Front Plant Sci 11:115
Han G, Wei X, Dong X, Wang C, Sui N, Guo J, Yuan F, Gong Z, Li X, Zhang Y, Meng Z, Chen Z, Zhao D, Wang B (2020) Arabidopsis ZINC FINGER PROTEIN1 acts downstream of GL2 to repress root hair initiation and elongation by directly suppressing bHLH genes. Plant Cell 32(1):206-225
Han G, Yuan F, Guo J, Zhang Y, Sui N, Wang B (2019) AtSIZ1 improves salt tolerance by maintaining ionic homeostasis and osmotic balance in Arabidopsis. Plant Sci 285:55-67
Han X, Kui M, He K, Yang M, Du J, Jiang Y, Hu Y (2023) Jasmonate-regulated root growth inhibition and root hair elongation. J Exp Bot 74(4):1176-1185
He XF, Geng CA, Huang XY, Ma YB, Zhang XM, Chen JJ (2019) Chemical constituents from Mentha haplocalyx Briq. (Mentha canadensis L.) and their α-glucosidase inhibitory activities. Nat Prod Bioprospect 9(3):223-229
Hu Y, Schmidhalter U (2023) Opportunity and challenges of phenotyping plant salt tolerance. Trends Plant Sci 28(5):552-566
Huang L, Jiang Q, Wu J, An L, Zhou Z, Wong C, Wu M, Yu H, Gan Y (2020) Zinc finger protein 5 (ZFP5) associates with ethylene signaling to regulate the phosphate and potassium deficiency-induced root hair development in Arabidopsis. Plant Mol Biol 102(1-2):143-158
Huang L, Xu N, Wu J, Yang S, An L, Zhou Z, Wong CE, Wu M, Yu H, Gan Y (2024) GLABROUS INFLORESCENCE STEMS3 binds to and activates RHD2 and RHD4 genes to promote root hair elongation in Arabidopsis. Plant J 117(1):92-106
Huang R, Jiang S, Dai M, Shi H, Zhu H, Guo Z (2023) Zinc finger transcription factor MtZPT2-2 negatively regulates salt tolerance in Medicago truncatula. Plant Physiol 194(1):564-577
Huang Y, Du L, Wang M, Ren M, Yu S, Yang Q (2022) Multifaceted roles of zinc finger proteins in regulating various agronomic traits in rice. Front Plant Sci 13:974396
Huchelmann A, Boutry M, Hachez C (2017) Plant glandular trichomes: natural cell factories of high biotechnological interest. Plant Physiol 175(1):6-22
Johnson CS, Kolevski B, Smyth DR (2002) TRANSPARENT TESTA GLABRA2, a trichome and seed coat development gene of Arabidopsis, encodes a WRKY transcription factor. Plant Cell 14(6):1359-1375
Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K, Shinozaki K (1999) Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nat Biotechnol 17(3):287-291
Khan RA, Mohammad, Hurrah IM, Muzafar S, Jan S, Abbas N (2021) Transcriptional regulation of trichome development in plants: an overview. P INDIAN NATL SCI AC 87(1):36-47
Kim K, Jang YJ, Lee SM, Oh BT, Chae JC, Lee KJ (2014) Alleviation of salt stress by Enterobacter sp. EJ01 in tomato and Arabidopsis is accompanied by up-regulation of conserved salinity responsive factors in plants. Mol Cells 37(2):109-117
Kirik V, Simon M, Huelskamp M, Schiefelbein J (2004) The ENHANCER OF TRY AND CPC1 gene acts redundantly with TRIPTYCHON and CAPRICE in trichome and root hair cell patterning in Arabidopsis. Dev Biol 268(2):506-513
Kirik V, Simon M, Wester K, Schiefelbein J, Hulskamp M (2004) ENHANCER of TRY and CPC 2 (ETC2) reveals redundancy in the region-specific control of trichome development of Arabidopsis. Plant Mol Biol 55(3):389-398
Ku YS, Sintaha M, Cheung MY, Lam HM (2018) Plant hormone signaling crosstalks between biotic and abiotic stress responses. Int J Mol Sci 19(10):3206
Kumar D, Punetha A, Chauhan A, Suryavanshi P, Padalia RC, Kholia S, Singh S (2023) Growth, oil and physiological parameters of three mint species grown under saline stress levels. Physiol Mol Biol Plants 29(7):1061-1072
Kurowska M, Daszkowska-Golec A (2023) Molecular mechanisms of SNAC1 (Stress-responsive NAC1) in conferring the abiotic stress tolerance. Plant Sci 337:111894
Lang V, Mantyla E, Welin B, Sundberg B, Palva ET (1994) Alterations in water status, endogenous abscisic acid content, and expression of rab18 gene during the development of freezing tolerance in Arabidopsis thaliana. Plant Physiol 104(4):1341-1349
Larkin JC, Oppenheimer DG, Lloyd AM, Paparozzi ET, Marks MD (1994) Roles of the GLABROUS1 and TRANSPARENT TESTA GLABRA genes in Arabidopsis trichome development. Plant Cell 6(8):1065-1076
Lee MM, Schiefelbein J (1999) WEREWOLF, a MYB-related protein in Arabidopsis, is a position-dependent regulator of epidermal cell patterning. Cell 99(5):473-483
Leng B, Wang X, Yuan F, Zhang H, Lu C, Chen M, Wang B (2021) Heterologous expression of the Limonium bicolor MYB transcription factor LbTRY in Arabidopsis thaliana increases salt sensitivity by modifying root hair development and osmotic homeostasis. Plant Sci 302:110704
Li C, Mo Y, Wang N, Xing L, Qu Y, Chen Y, Yuan Z, Ali A, Qi J, Fernández V, Wang Y, Kopittke PM (2023) The overlooked functions of trichomes: water absorption and metal detoxication. Plant Cell Environ 46(3):669-687
Li M, Zhu Y, Li S, Zhang W, Yin C, Lin Y (2022) Regulation of phytohormones on the growth and development of plant root hair. Front Plant Sci 13:865302
Liu H, Tang X, Zhang N, Li S, Si H (2023) Role of bZIP transcription factors in plant salt stress. Int J Mol Sci 24(9):7893
Liu J, Wang H, Liu M, Liu J, Liu S, Cheng Q, Shen H (2021) Hairiness gene regulated multicellular, non-glandular trichome formation in pepper species. Front Plant Sci 12:784755
Liu Y, Khan AR, Gan Y (2022) C2H2 zinc finger proteins response to abiotic stress in plants. Int J Mol Sci 23(5):2730
Liu Y, Liu D, Hu R, Hua C, Ali I, Zhang A, Liu B, Wu M, Huang L, Gan Y (2017) AtGIS, a C2H2 zinc-finger transcription factor from Arabidopsis regulates glandular trichome development through GA signaling in tobacco. Biochem Biophys Res Commun 483(1):209-215
Liu Y, Liu D, Khan A, Liu B, Wu M, Huang L, Wu J, Song G, Ni H, Ying H, Yu H, Gan Y (2018) NbGIS regulates glandular trichome initiation through GA signaling in tobacco. Plant Mol Biol 98(1-2):153-167
Liu Y, Ma X, Li Y, Yang X, Cheng W (2024) Zinc Finger Protein8 (GhZFP8) regulates the initiation of trichomes in Arabidopsis and the development of fiber in cotton. Plants (Basel) 13(4):492
Long Y, Schiefelbein J (2020) Novel TTG1 mutants modify root-hair pattern formation in Arabidopsis. Front Plant Sci 11:383
Lyu T, Cao J (2018) Cys₂/His₂ Zinc-finger proteins in transcriptional regulation of flower development. Int J Mol Sci 19(9):2589
Ma Q, Xia Z, Cai Z, Li L, Cheng Y, Liu J, Nian H (2018) GmWRKY16 enhances drought and salt tolerance through an ABA-mediated pathway in Arabidopsis thaliana. Front Plant Sci 9:1979
Mishra A, Gupta P, Lal RK, Dhawan SS (2021) Assessing and integrating the transcriptome analysis with plant development, trichomes, and secondary metabolites yield potential in Mentha arvensis L. Plant Physiol Biochem 162:517-530
Mishra A, Lal RK, Chanotiya CS, Dhawan SS (2017) Genetic elaborations of glandular and non-glandular trichomes in Mentha arvensis genotypes: assessing genotypic and phenotypic correlations along with gene expressions. Protoplasma 254(2):1045-1061
Mittler R, Kim Y, Song L, Coutu J, Coutu A, Ciftci-Yilmaz S, Lee H, Stevenson B, Zhu JK (2006) Gain- and loss-of-function mutations in Zat10 enhance the tolerance of plants to abiotic stress. FEBS Lett 580(28-29):6537-6542
Moore K, Roberts LJ, 2nd (1998) Measurement of lipid peroxidation. Free Radic Res 28(6):659-671
Morohashi K, Zhao M, Yang M, Read B, Lloyd A, Lamb R, Grotewold E (2007) Participation of the Arabidopsis bHLH factor GL3 in trichome initiation regulatory events. Plant Physiol 145(3):736-746
Msanne J, Lin J, Stone JM, Awada T (2011) Characterization of abiotic stress-responsive Arabidopsis thaliana RD29A and RD29B genes and evaluation of transgenes. Planta 234(1):97-107
Payne CT, Zhang F, Lloyd AM (2000) GL3 encodes a bHLH protein that regulates trichome development in arabidopsis through interaction with GL1 and TTG1. Genetics 156(3):1349-1362
Qi X, Chen Z, Yu X, Li L, Bai Y, Fang H, Liang C (2022) Characterisation of the Mentha canadensis R2R3-MYB transcription factor gene McMIXTA and its involvement in peltate glandular trichome development. BMC Plant Biol 22(1):219
Qi X, Fang H, Yu X, Xu D, Li L, Liang C, Lu H, Li W, Chen Y, Chen Z (2018) Transcriptome analysis of JA signal transduction, transcription factors, and monoterpene biosynthesis pathway in response to Methyl Jasmonate elicitation in Mentha canadensis L. Int J Mol Sci 19(8):2364
Rai GK, Mishra S, Chouhan R, Mushtaq M, Chowdhary AA, Rai PK, Kumar RR, Kumar P, Perez-Alfocea F, Colla G, Cardarelli M, Srivastava V, Gandhi SG (2023) Plant salinity stress, sensing, and its mitigation through WRKY. Front Plant Sci 14:1238507
Reddy VA, Wang Q, Dhar N, Kumar N, Venkatesh PN, Rajan C, Panicker D, Sridhar V, Mao HZ, Sarojam R (2017) Spearmint R2R3-MYB transcription factor MsMYB negatively regulates monoterpene production and suppresses the expression of geranyl diphosphate synthase large subunit (MsGPPS.LSU). Plant Biotechnol J 15(9):1105-1119
Rushton DL, Tripathi P, Rabara RC, Lin J, Ringler P, Boken AK, Langum TJ, Smidt L, Boomsma DD, Emme NJ, Chen X, Finer JJ, Shen QJ, Rushton PJ (2012) WRKY transcription factors: key components in abscisic acid signalling. Plant Biotechnol J 10(1):2-11
Sakamoto H, Maruyama K, Sakuma Y, Meshi T, Iwabuchi M, Shinozaki K, Yamaguchi-Shinozaki K (2004) Arabidopsis Cys2/His2-type zinc-finger proteins function as transcription repressors under drought, cold, and high-salinity stress conditions. Plant Physiol 136(1):2734-2746
Schellmann S, Schnittger A, Kirik V, Wada T, Okada K, Beermann A, Thumfahrt J, Jürgens G, Hülskamp M (2002) TRIPTYCHON and CAPRICE mediate lateral inhibition during trichome and root hair patterning in Arabidopsis. EMBO J21(19):5036-5046
Schmidt JE, Gaudin ACM (2017) Toward an integrated root ideotype for irrigated systems. Trends Plant Sci 22(5):433-443
Shi X, Gu Y, Dai T, Wu Y, Wu P, Xu Y, Chen F (2018) Regulation of trichome development in tobacco by JcZFP8, a C2H2 zinc finger protein gene from Jatropha curcas L. Gene 658:47-53
Shibata M, Sugimoto K (2019) A gene regulatory network for root hair development. J Plant Res 132(3):301-309
Sun L, Zhang A, Zhou Z, Zhao Y, Yan A, Bao S, Yu H, Gan Y (2015) GLABROUS INFLORESCENCE STEMS3 (GIS3) regulates trichome initiation and development in Arabidopsis. New Phytol 206(1):220-230
Tominaga-Wada R, Wada T (2014) Regulation of root hair cell differentiation by R3 MYB transcription factors in tomato and Arabidopsis. Front Plant Sci 5:91
Tominaga R, Iwata M, Sano R, Inoue K, Okada K, Wada T (2008) Arabidopsis CAPRICE-LIKE MYB 3 (CPL3) controls endoreduplication and flowering development in addition to trichome and root hair formation. Development 135(7):1335-1345
van Zelm E, Zhang Y, Testerink C (2020) Salt tolerance mechanisms of plants. Annu Rev Plant Biol 71:403-433
Verma S, Negi NP, Pareek S, Mudgal G, Kumar D (2022) Auxin response factors in plant adaptation to drought and salinity stress. Physiol Plant 174(3):e13714
Vissenberg K, Claeijs N, Balcerowicz D, Schoenaers S (2020) Hormonal regulation of root hair growth and responses to the environment in Arabidopsis. J Exp Bot 71(8):2412-2427
Wada T, Tachibana T, Shimura Y, Okada K (1997) Epidermal cell differentiation in Arabidopsis determined by a Myb homolog, CPC. 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

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

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Abstract

Figures

Key Message

1. Introduction

2. Materials and methods

2.1. Plant materials and growth conditions

2.2. Stress treatments

2.3. Cloning and bioinformatics analysis of the McZFP1 gene

2.4. RNA isolation and qRT-PCR analysis

2.5. Subcellular localization of McZFP1

2.6. Transcriptional activation of McZFP1 in yeast cells

2.7. Microscopy

2.8. Measurement of physiological indexes

3. Results

3.1. McZFP1 isolation from M. canadensis and sequence analysis

3.2. Analysis of McZFP1 gene expression

3.3. Subcellular localization and transcriptional activity of McZFP1

3.4. McZFP1 overexpression inhibits trichome development in transgenic Arabidopsis

3.5. McZFP1 overexpression inhibits root hair development in transgenic Arabidopsis

3.6. McZFP1 overexpression reduces salt tolerance in transgenic Arabidopsis

3.7. McZFP1 overexpression in Arabidopsis reduces osmolyte accumulation and antioxidant enzyme activities

3.8. McZFP1 overexpression downregulates the expression of stress-responsive genes in transgenic Arabidopsis

4. Discussion

5. Conclusion

Declarations

References

Supplementary Files

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