TaZFP 23, a new Cys2/His2-type zinc-finger protein, is a regulator of wheat (Triticum aestivum L.) growth and abiotic stresses

Plant material and growth conditions

The experimental materials used in this study included wheat (Triticum aestivum L. Fielder), Nicotiana benthamiana, and Arabidopsis thaliana (ecotype Columbia 0, Col-0). Wheat and Arabidopsis were grown in a growth chamber at 22 °C with a photoperiod of 16 h light/8 h dark and a humidity of 80%. Nicotiana benthamiana was grown in a growth chamber at 25 °C with a photoperiod of 16 h light/8 h dark and a humidity of 80%.

After surface sterilization and cold stratification at 4 °C for 2–3 days, seeds of wild-type Arabidopsis (WT) and Arabidopsis overexpressing TaZFP23 were sown on 1/2 MS agar plates. Approximately 7–10 days later, seedlings were transferred to potting soil and placed in a growth chamber at 22 °C. After 14 days, uniformly growing seedlings from each line were subjected to stress treatments. The stress treatments included watering with 350 mM NaCl solution every 4 days, drought stress (cessation of watering), and normal growth conditions. After 10 days of stress treatment, basal leaves from each line were harvested, rapidly frozen in liquid nitrogen, and stored at −80 °C for subsequent physiological, biochemical, and molecular analyses. Survival rates were recorded after 14 days of stress treatment. The experiment was conducted with three replicates, each consisting of 20 seedlings.

RNA extraction and cDNA synthesis

Total RNA was extracted from wheat using the RNA isolater Total RNA Extraction Reagent from Vazyme Biotech. The RNA concentration was measured prior to storage at −80 °C using a Nanodrop 2000 instrument. For reverse transcription, the extracted RNA was treated with the HiScript II Reverse Transcriptase Kit from Vazyme Biotech, and cDNA synthesis was carried out following the kit’s protocol.

Obtaining and cloning of TaZFP23 gene sequence in wheat

The coding region, promoter, and amino acid sequence of the wheat TaZFP23 gene were downloaded from the Ensembl Plants database (http://plants.ensembl.org/). Specific cloning primers for TaZFP23 were designed using Primer 5 software: pCAMBIA2300-TaZFP38F:ggtacccggggatcctctagaATGGCCGTGGAGGCGGTTCTTGAA; pCAMBIA2300-TaZFP38R:agctcctcctcctcctctagaCGCCGCGAGCATGAGCCTCG.

Bioinformatics analysis of TaZFP23 protein in wheat

The amino acid sequence of TaZFP23 protein was submitted to ProtParam (http://web.expasy.org/protparam/) to predict its physicochemical properties. The conserved protein domains of TaZFP23 were analyzed using the SMART database (http://smart.embl-heidelberg.de/). The hydrophobicity of the TaZFP23 protein was predicted using ProtScale (https://www.expasy.org/resources/protscale). The transmembrane structure domains of TaZFP23 protein were predicted using the TMHMM-2.0 tool (https://services.healthtech.dtu.dk/services/TMHMM-2.0/). Subcellular localization of TaZFP23 protein was predicted using the Plant-mPLoc tool (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/). Signal peptide prediction for TaZFP23 was performed using SignalP 5.0 (https://services.healthtech.dtu.dk/service.php?SignalP-5.0). The 1,500 bp upstream sequence of the wheat TaZFP23 gene was submitted to PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) to predict cis-acting regulatory elements.

Subsequently, homologous protein sequences of TaZFP23 from various species were retrieved through alignment in Ensembl Plants. These sequences, along with the amino acid sequence of TaZFP23, were aligned using MUSCLE for sequence alignment. An evolutionary tree was constructed using MEGA7, employing the neighbor-joining method to analyze the evolutionary relationships among the TaZFP23 homologs from different species.

Gene construct, transformation and expression analysis and transcription self activation

After purifying the PCR-amplified target fragment of TaZFP23, it was ligated into the XbaI-digested pCAMBIA2300-GFP vector for recombination. A one-step cloning kit from Novogene Corporation (Nanjing, China) was employed to construct the fusion expression vector. Subsequently, the plasmid was transformed into Agrobacterium tumefaciens.

Tobacco plants were cultivated in a growth chamber. After approximately two days, they were ready to be used for transient expression experiments. The bacterial culture was adjusted to an OD600 of approximately 0.3 by adding injection buffer. Using a syringe, the bacterial culture was injected into tobacco leaves and excess culture was carefully removed. The injected tobacco leaves were then incubated in darkness for two days. Subsequently, the injected tobacco leaf epidermis was examined to determine the subcellular localization of the targeted protein.

Yeast single colonies were inoculated into five mL of SD/-Trp liquid medium and incubated overnight at 30 °C with shaking at 180 rpm. 200 µL of the culture was transferred to a 1.5 mL microcentrifuge tube and centrifuged at room temperature at 12,000 rpm for 15 s. The supernatant was discarded, and 500 µL of sterile water was added to the pellet, followed by vortexing to resuspend the cells. This washing step was repeated twice. The OD600 of the yeast culture was adjusted to 1, and 3 µL of the culture was spotted onto SD/-Trp-Ade-His agar plates. The plates were then incubated at 30 °C for 4 days.

The germination rate of seeds under NaCl mannitol and ABA treatments

Seeds of two homozygous lines, one overexpressing TaZFP23 and the other a wild-type Arabidopsis, were harvested concurrently and subjected to surface sterilization and cold stratification at 4 °C for 2–3 days. The seeds were then sown on 1/2 MS solid medium containing varying concentrations of NaCl (0 mM, 80 mM, 120 mM), mannitol (0 mM, 200 mM, 250 mM), and ABA (0 µM, 0.5 µM, 1 µM). The plates were placed in a plant growth chamber at 22 °C under a 16-hour light/8-hour dark cycle for normal growth. Daily observations of the germination rate should be conducted using a microscope (SMZ-171; Motic). Each treatment group consists of three replicates, with 36 seeds per replicate. The eyepiece and objective lensof microscope should be adjusted as needed based on the specific circumstances.

Measurements of survival rates and contents of proline and malondialdehyde

Before measuring the survival rates and contents of proline contents and malondialdehyde (MDA), Arabidopsis (two-week-old) seedlings were treated until clear differences in wilting were observed under drought conditions. About 0.1 g of plant leaves were taken, and MDA and proline contents were determined using an assay kit (Sangon Biotech, Shanghai, China) based on the manufacturer’s protocols. All measurements were carried out with three biological replicates, and statistical analysis was carried out using one-way ANOVA.

Enzyme activities assay

For enzyme activities assay, the enzyme extraction was performed from leaves of plants treated under drought conditions. Superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) were measured according to previously described methods using an assay kit (Sangon Biotech, Shanghai, China). All extractions were obtained with three biological replicates.

Data analysis

In this study, physiological parameters and seed germination rates were quantified and analyzed statistically. The physiological indicators, including (specific indicators such as survival rate (%), Pro, MDA, POD, CAT, SOD), were measured and expressed as means ± standard deviation (SD). Seed germination rates were calculated as the percentage of seeds that successfully germinated over the total number of seeds planted. All measurements were carried out with three biological replicates. Statistical analyses were conducted using one-way analysis of variance (ANOVA) to compare differences among treatment groups, with t-test applied for multiple comparisons. bars = 5 cm, Error bars indicate SD. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001.

Cloning of TaZFP23 into pCAMBIA2300 and transformation of its GFP-fusion

The Q-type C2H2 zinc finger protein gene sequences from rice and Arabidopsis were subjected to BLAST analysis on PlantGDB, leading to the identification of the EST sequence of C2H2 zinc finger protein gene TaZFP23 in wheat. The TaZFP23 EST sequence was then aligned using Ensemble Plants to identify the specific sequence information (100% nucleotide similarity) of TaZFP23 (TraesCS5B02G076400). The expression vector for the TaZFP23-GFP fusion protein was constructed (Fig. 1A). The full-length coding sequence (CDS) of TaZFP23 is 720 bp and it was cloned with high-fidelity enzymes using wheat cDNA as a template (Fig. 1B). The TaZFP23 CDS was subsequently inserted into the pCAMBIA2300 vector through one-step cloning. The clone was transformed into DH5α and positive clones were identified by colony PCR (Fig. 1C). All positive clones displayed the expected gene size. Following Sanger sequencing, the alignment of the target sequence with the sequencing results led to the generation of the expression vector pCAMBIA2300-GFP::TaZFP23 for the TaZFP23-GFP fusion protein.

Analysis of the physicochemical properties of the TaZFP23 protein

The amino acid sequence of the TaZFP23 protein was submitted to the ProtParam website (https://web.expasy.org/protparam/) for the prediction of its physicochemical properties (Table 1). It has a relative molecular weight of 24.43 kDa, a total number of 3,394 atoms, an isoelectric point of 7.07, a molecular formula of C₁₀₄₅H₁₆₈₆N₃₁₄O₃₃₇S₁₂, and an instability index of 57.38. These parameters suggested that the protein encoded by TaZFP23 is an unstable one. The protein’s lipid solubility coefficient is 68.12 with a total average hydropathy index of −0.283, indicating that the TaZFP23 protein is likely a hydrophilic protein.

Analysis and prediction of TaZFP23 protein domain, hydrophobicity, transmembrane region, signal peptide, and subcellular localization

The structural domain analysis of the TaZFP23 protein was conducted using SMART (Simple Modular Architecture Research Tool, http://smart.embl-heidelberg.de). This analysis showed that the TaZFP23 protein contains two C2H2 zinc finger domains at positions 85–107 and 147–169, indicating that TaZFP23 belongs to the C2H2-type transcription factor (Fig. 2A).

Hydrophobicity plays a crucial role in studying the spatial and transmembrane structure of proteins. The hydrophobicity analysis of the TaZFP23 protein is shown in Fig. 2B, with most sequence positions scoring below 0. There is a significant peak at amino acid positions 40–50, suggesting that the protein is likely hydrophilic. Transmembrane domains of proteins are typically hydrophobic regions that play a significant role in maintaining their tertiary structure. TMHMM analysis of the TaZFP23 protein revealed that it does not have any transmembrane regions (Fig. 2C), indicating that it is a non-transmembrane protein.

Promoter analysis of TaZFP23 gene

Transcription factors can regulate transcription by binding to cis-elements in the promoter sequences. The cis-elements in the upstream 1,500 bp promoter sequence of TaZFP23 predicted using PlantCARE included ABRE, MBS, CGTCA-motif, LTR, TGA-element, G-box, ARE, and others (Table 2). These cis-elements are closely related to hormone and abiotic stress responses, suggesting that TaZFP23 may play a role in abiotic stress responses and plant hormone signal transduction.

Evolutionary analysis of TaZFP23 protein

Based on data in Ensemble Plant, homologous proteins of TaZFP23 were identified in rice, maize, barley, Arabidopsis, potato, tomato, and soybean. The results of the phylogenetic analysis reveal that the evolutionary relationship of TaZFP23 protein is closer to those in rice, barley, and maize, all of which belong to the Poaceae family (Fig. 3). The sequence similarity alignment showed that TaZFP23 and its homologous proteins all contain two C2H2 zinc finger domains and an EAR motif (Fig. 4). Furthermore, the C2H2 zinc finger domains of TaZFP23 protein harbor a highly conserved QALGGH sequence, suggesting its classification into the Q-type C2H2 zinc finger protein subfamily.

Subcellular localization and transcriptional activity analysis of TaZFP23

Tobacco leaves injected with the expression vector carrying the fusion of TaZFP23 and GFP protein (pCAMBIA2300-GFP::TaZFP23) were used as the experimental group, while tobacco leaves injected with the control vector containing GFP tag (pCAMBIA2300) were used as the control group to detect the subcellular localization of the TaZFP23 protein. As shown in Fig. 4A, green fluorescence was observed throughout the cells in the control group, while green fluorescence was observed only in the nucleus of cells expressing TaZFP23, indicating that TaZFP23 protein is localized in the nucleus.

As a member of the Q-type C2H2 transcription factor family, TaZFP23 was examined for its potential transcriptional activation activity by cloning it into the pGBKT7 vector. Both the empty pGBKT7 vector and the recombinant plasmid pGBKT7-TaZFP23 were transformed into the yeast strain AH109 using the PEG/LiAc transformation method. Their activation activity was assessed on SD-III (SD/-Trp-His-Ade) deficient medium. As illustrated in Fig. 4B, yeast cells transformed with the empty pGBKT7 vector and pGBKT7-TaZFP23 were able to grow on SD-I (SD-Trp/Ade) medium. However, colonies were absent on the SD-III deficient medium, suggesting that the TaZFP23 protein may not possess self-activation activity.

Germination rates of TaZFP23 overexpressing Arabidopsis seeds under some abiotic stress treatments

Physiological indicators such as seed germination rate and seedling emergence rate are routinely used to assess stress tolerance ability of plants. The germination rates of wild-type Arabidopsis (WT) and Arabidopsis overexpressing TaZFP23 (OE-5 and OE-31) were assessed under different concentrations of NaCl (80 mmol/L, 150 mmol/L), mannitol (200 mmol/L, 250 mmol/L), and ABA (0.5 µmol/L, 1 µmol/L) treatments. Results from the assessments showed that under normal conditions, there was little difference in the germination rates among WT, OE-5, and OE-31 lines. However, under NaCl treatment, significant differences in germination rates were observed between WT and OE-5, OE-31 lines, with the most significant differences seen on the 2nd day with 80 mmol/L NaCl treatment and on the 3rd and 4th days with 120 mmol/L NaCl treatment (Figs. 5A and 5B). These results suggested that overexpression of TaZFP23 reduced the salt tolerance of Arabidopsis.

Under mannitol treatment, the germination rates of WT, OE-5, and OE-31 lines were all inhibited. The most significant differences were observed on the 2nd day with 200 mmol/L and 250 mmol/L mannitol treatments (Figs. 5C and 5D), indicating a negative regulatory role of TaZFP23 on seed germination under drought conditions in Arabidopsis.

Following ABA treatment, the germination rate of WT was significantly higher than that of the TaZFP23 overexpressing lines. The most significant differences were observed on the 2nd day with 0.5 µmol/L ABA treatment and on the 3rd day with 1 µmol/L ABA treatment (Figs. 5E and 5F). These results showed that overexpression of TaZFP23 increased the sensitivity of seed germination to ABA in Arabidopsis.

Stress resistance analysis of TaZFP23 overexpressing Arabidopsis under NaCl and drought stress treatments

Comparisons in the growth status and survival rates between the wild-type (WT) and Arabidopsis lines overexpressing TaZFP23 under salt stress (300 mM NaCl) treatment were conducted. Varying degrees of chlorosis were observed in the leaves of Arabidopsis plants after 14 days of treatments with 300 mM NaCl (Figs. 6A and 6B). Significant differences in survival rates between WT (82.1%) and OE-5 (43.8%) or OE-31 (36.5%) were detected, indicating that overexpression of TaZFP23 reduced the salt tolerance of Arabidopsis. Moreover, most WT plants retained green leaves after 14 days of drought stress treatment, whereas the majority of OE-5 and OE-31 plants exhibited leaf wilting and eventual death (Figs. 6C and 6D). The survival rate of WT was 70.8%, while the survival rates of OE-5 and OE-31 plants were 34.4% and 29%, respectively. The significant differences in survival rates suggested that overexpression of TaZFP23 reduced the drought tolerance of Arabidopsis. It is well established that stress conditions can trigger lipid peroxidation in plants, leading to the accumulation of malondialdehyde (MDA) (Fang et al., 2022). Under non-stress conditions, no statistically significant differences in MDA content were detected among the wild-type (WT), OE-5, and OE-31 lines. However, when subjected to drought and salt stress, MDA levels in the leaves of the WT, OE-5, and OE-31 lines exhibited significant increases compared to the wild type (Fig. 6F). These results indicate that the overexpression of TaZFP23 is correlated with elevated lipid peroxidation levels and more severe cellular damage in Arabidopsis under conditions of drought and salt stress.

Moreover, the activity of peroxidase (POD) and catalase (CAT) enzymes was analyzed. Theseare important protective enzymes in plant defense systems, reflecting metabolic changes in plants over time, and closely related to photosynthesis and respiration.

The activities of peroxidase (POD) and catalase (CAT) were investigated due to their established roles in photosynthesis and respiration, and because CAT activity serves as an indicator of metabolic levels and resistance to various stressors. The analysis revealed that enzyme activities were elevated in all three lines studied. However, the activities of both enzymes were significantly lower in the lines overexpressing TaZFP23 compared to the wild type under drought and salt stress (Fig. 6H). Additionally, superoxide dismutase (SOD) activity increased in all three lines under these stress conditions. Notably, SOD activity in the leaves of the two lines overexpressing TaZFP23 was significantly lower than that observed in the wild type (Fig. 6I). Collectively, these findings suggest that the overexpression of TaZFP23 diminishes the tolerance of Arabidopsis to drought and NaCl stress.