Introduction

Global warming has led to frequent high-temperature events, and heat stress has become an important issue affecting food security1,2. Heat is one of the main abiotic stressors that restrict plant growth and yield formation3,4,5. Research has shown that rising temperatures have significant adverse effects on plant growth, including accelerating plant transpiration, leading to water loss, affecting plant growth and metabolism, reducing plant photosynthetic capacity, and affecting nutrient absorption and growth5,6. Plants have developed complex transcriptional regulatory networks to resist high temperatures, and transcription factors play an important role in this network by activating or repressing target genes.

Transcription factor (TF) genes account for a large proportion of the plant genome. For example, there are over 2100 transcription factor genes in Arabidopsis thaliana and over 2300 transcription factor genes in rice, which can be divided into the MYB, NAC, AP2/ERF2, and WRKY families based on their protein structure7. As one of the largest transcription factor superfamilies, WRKY transcription factors are widely distributed in plants, and all possess the WRKYGQK sequence as well as a C2H2- or C2HC-type zinc-finger motif8. Numerous studies have shown that WRKY proteins regulate plant growth and development, aging, secondary metabolism, and environmental responses via specific interactions with the cis-acting element W-box (TTGACC/T) of target genes9,10. In terms of high-temperature stress, AtWRKY39 confers heat tolerance when overexpressed in Arabidopsis11, and the TaWRKY33 protein can positively regulate high-temperature tolerance in Arabidopsis12. AtWRKY25/26/33 have also been shown to positively regulate heat tolerance, and many WRKY genes have been found to respond to high temperatures13.

Pinellia ternata is a herb belonging to the Araceae family that is widely distributed in China and Southeast Asia. Its tubers contain alkaloids, organic acids, and polysaccharides14,15, and are used for medicinal purposes. Modern pharmacological studies have shown that P. ternata has many medicinal properties, including analgesic, anxiolytic, antitussive, and anticancer effects16. The suitable temperature for the growth of P. ternata is 15–25 °C. When exposed to high temperatures during growth, P. ternata is susceptible to withering, a phenomenon known as “sprout tumble” (ST)17,18. ST formation in P. ternata shortens its growth period, which is a key limiting factor in tuber production19. Therefore, it is important to analyze the ST mechanism and delay the process of ST for improving the yield of P. ternata . Thus far, only the functions of PtSAD and PtsHSP in P. ternata in response to high temperatures have been reported20,21, and the transcriptional regulatory network of P. ternata related to ST is still largely unknown.

In this study, we isolated WRKY genes from P. ternata based on the full-length transcriptome and identified a WRKY gene, PtWRKY2, that was significantly induced by high temperatures. Moreover, the function of PtWRKY2 in high-temperature tolerance was investigated. These data provide new insights into ST mechanisms at the transcriptional level, which could contribute to the genetic improvement of P. ternata.

Results

Identification and phylogenetic tree analysis of PtWRKY genes

Based on the reported transcriptome data for P. ternata19, five PtWRKY genes with serial numbers i1_LQ_Pts1_c14283/f5p11/2027, i1_LQ_Pts1_c61466/f1p0/1914, i1_LQ_Pts1_c39338/f1p8/1965, i2_LQ_Pts1_c2682/f1p6/2136, and i1_HQ_Pts1_c10843/f5p4/1677 were identified and annotated as PtWRKY1-5. The predicted coding proteins ranged from 350 amino acids (aa) (PtWRKY2) to 515 aa (PtWRKY1), with molecular masses from 37.79 to 55.07 kD. The isoelectric points varied from 6.1 (PtWRKY5) to 10.15 (PtWRKY2) (Table S2). The five PtWRKYs contained five conserved domains, in which the typical WRKYGQK motif and C2HC-type zinc-finger motif appeared, while the C2H2-type zinc-finger motif appeared in PtWRKY3-5 (Fig. S1).

Phylogenetic analysis indicated that the 25 WRKYs from P. ternata, Oryza sativa, and Arabidopsis could be divided into six subclasses. PtWRKY1, AtWRKY25, AtWRKY26, and AtWRKY33 clustered into Group I, whereas PtWRKY2, together with the closely related AtWRKY30, AtWRKY53, and OsWRKY72, were categorized as Group III. PtWRKY3 and PtWRKY4, together with the closest AtWRKY6, belonged to Group IIb and PtWRKY5 clustered with Group IId (Fig. 1).

Figure 1
figure 1

Phylogenetic analysis of the WRKY proteins of P. ternata, Oryza sativa, and Arabidopsis was performed using MEGA7.0, with a bootstrap setting of 1000.

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Expression pattern analysis of PtWRKY genes

The expression profiles of the five PtWRKY genes were investigated using quantitative real-time PCR. The five PtWRKY genes existed in all tissues of P. ternata, with PtWRKY1, PtWRKY3, PtWRKY4, and PtWRKY5 highly expressed in the roots, and PtWRKY2 highly expressed in the leaves (Fig. 2A). In terms of their heat responses, we observed that only the PtWRKY2 transcription level significantly increased within 24 h of treatment, with an expression peak at 12 h (nearly 200-fold induction). However, the expression levels of the other four PtWRKY genes significantly decreased under stress treatment (Fig. 2B). These results implied that the induction of PtWRKY2 might participate in the growth regulation of P. ternata at high temperatures.

Figure 2
figure 2

Expression analysis of PtWRKY genes in P. ternata using RT-qPCR. (A) Expression profiles of PtWRKY genes in the roots, petioles, leaves, and tubers of two-month-old P. ternata plants. (B) Expression of PtWRKY genes in response to 42 °C heat stress. Values are presented as means ± SD (n = 3). Different letters indicate significant differences at P < 0.05.

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Transcription factor characteristics of PtWRKY2

To identify the biological functions of PtWRKY2 in response to high temperatures, PtWRKY2 was selected for further study. First, the PtWRKY2-GFP and GFP vectors were extracted for subcellular localization analysis. The cells were then transformed into tobacco epidermal cells for observation under a fluorescence microscope. As shown in Fig. 3A, the PtWRKY2-GFP fusion proteins were found only in the cell nucleus, whereas the control GFP signals were widely distributed throughout the cell, indicating that PtWRKY2 encodes a nuclear protein.

Figure 3
figure 3

PtWRKY2 characterization. (A) Subcellular localization of PtWRKY2 in tobacco leaf epidermal cells. Scale bar = 20 μm. (B) Transactivation of PtWRKY2. The construct of pGBKT7-PtWRKY2 was transformed into yeast strain Y2HGold and examined on SD/ − Trp and SD/ − Trp/ − His/ − Ade/X-α-gal plates. (C) Dual-LUC assay of PtWRKY2 with three repeats of the W-box (TTGACY) and mW-box (TTTAAY) elements. W-box-0800, mW-box-0800, and pGreenII 0800-LUC empty vectors were transiently expressed in tobacco leaves along with PtWRKY2-62-SK. The LUC signal was captured at 72 h post-transfection.

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Second, to confirm the transcriptional activity of PtWRKY2, the plasmids pGBKT7-PtWRKY2, pGBKT7-53 (positive control), and pGBKT7 (negative control) were used for a transcriptional activation assay in Y2HGold yeast cells. The results revealed that all yeast cells transformed with the three vectors grew normally on SD/-Trp plates. Only the positive control grew on the SD/-Trp/-His/-Ade plate, and turned blue with the addition of X-α-gal, whereas the transformants containing pGBKT7-PtWRKY2 and pGBKT7 both failed to grow (Fig. 3B), which suggests that the PtWRKY2 protein did not have transcriptional activation activity.

WRKY transcription factors regulate gene expression by specifically binding to W-box elements within the target gene promoter22. To further explore whether PtWRKY2 could bind to W-box, the plasmid PtWRKY2-62-SK was grouped with W-box-0800, mW-box-0800, and pGreenII-0800, respectively. The three combined plasmids were injected into tobacco epidermal cells for the dual-luciferase reporter assay. As shown in Fig. 3C, LUC luminescence signals were produced only when the PtWRKY2-62-SK and W-box-0800 vectors were co-transformed. These results suggest that PtWRKY2 is a typical WRKY protein that specifically binds to W-box elements.

Overexpression of PtWRKY2 enhanced the heat tolerance of transgenic Arabidopsis

To confirm the function of PtWRKY2, we obtained PtWRKY2-overexpressing lines in Arabidopsis (OE1-2, OE6-3, and OE7-8) (Fig. 4A), which were used for further analyses in combination with WT Arabidopsis. In the seed germination assay, the germination rates of the WT and transgenic lines reached 100% under normal conditions. When exposed to heat stress for two days (2DHS), the germination rate exhibited a downward trend, with a > 90% rate in the transgenic lines and a 73.6% rate in the WT lines. After 3DHS treatment, the germination rate of OE6-3 reached 48.1%, followed by 36.2% in OE7-8, and 31.5% in OE1-2, while only 12.8% of the WT seeds germinated (Fig. 4B–D). The effects of PtWRKY2 on root length were also evaluated using the above Arabidopsis lines. As shown in Fig. 4E,F, the root length of the transgenic lines was greater than that of the WT plants under normal conditions, and root growth was inhibited under high temperature conditions. Notably, root length inhibition was less in the transgenic lines than in the WT plants.

Figure 4
figure 4

Germination and root elongation analyses of wild-type (WT) and transgenic Arabidopsis under 42 °C heat stress. (A) PtWRKY2 expression levels in WT and three T3 generation transgenic Arabidopsis plants based on RT-PCR. (B) Germination of WT and three PtWRKY2 transgenic lines on 1/2MS medium exposed to 42 °C heat stress for 0, 2, or 3 days. (C) Distribution diagram of WT and three PtWRKY2 transgenic lines on a plate. (D) Seed germination rates of WT and transgenic plants within 14 days. Root length (E) and statistical analysis (F) of WT and three transgenic Arabidopsis grown on 1/2MS medium exposed to 42 °C heat stress for 1 day and captured after 10 days. HS represents heat stress. OE1-2, OE6-3, and OE7-8 represent three independent pure transgenic lines. Different letters indicate significant differences at P < 0.05.

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The heat tolerance of the transgenic and WT Arabidopsis plants was further evaluated by exposure to heat stress. After 1DHS, the survival rate of the WT line plants was only 31.8%, whereas the survival rates of the OE1-2, OE6-3, and OE7-8 lines were 76.9%, 80.3%, and 81.9%, respectively (Fig. 5). These results indicate that the overexpression of PtWRKY2 in Arabidopsis could significantly enhance heat tolerance.

Figure 5
figure 5

Heat tolerance of WT and transgenic Arabidopsis plants. Growth observation (A) and survival rate statistics (B) of WT and transgenic Arabidopsis under 42 °C heat stress for 1 day. Different letters indicate significant differences at P < 0.05. 1DHS represents 1 d of heat stress.

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Overexpression of PtWRKY2 improved the ROS scavenging capacity of Arabidopsis under high temperature conditions

It is well known that plant senescence induced by abiotic stress is usually associated with the accumulation of reactive oxygen species (ROS), particularly H2O2 and O2-. The levels of H2O2 and O2- in the leaves were detected by DAB and NBT staining. The PtWRKY2-overexpressing lines exhibited lighter histochemical staining than the WT leaves under heat stress conditions, whereas no significant difference was observed under control conditions (Fig. 6A,B). The activities of PtCAT, PtSOD, and PtPOD were determined in transgenic and WT Arabidopsis. The results revealed that the PtCAT, PtSOD, and PtPOD activities were similar in the WT and transgenic Arabidopsis under normal conditions. When exposed to high temperatures, the activities increased in both WT and transgenic lines; however, the increment in transgenic plants was higher than that in WT plants (Fig. 6). These results suggest that PtWRKY2 induces the activities of PtCAT, PtSOD, and PtPOD, thereby reducing ROS accumulation in transgenic Arabidopsis.

Figure 6
figure 6

Detection of reactive oxygen species (ROS) accumulation and antioxidant enzyme activity in WT and transgenic Arabidopsis plants under heat stress. Leaves of 3-week-old Arabidopsis plants grown under normal or heat stress conditions were sampled for (A) DAB and (B) nitro blue tetrazolium (NBT) staining and (C–E) catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) activities. Different letters indicate significant differences at P < 0.05.

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Overexpression of PtWRKY2 altered the transcriptional expression levels of heat shock associated genes

Because the overexpression of PtWRKY2 enhanced the high-temperature tolerance of Arabidopsis, the expression profiles of heat shock-associated genes were analyzed in the WT and transgenic lines under high-temperature conditions. The expression levels of five heat shock-related transcription factor-encoding genes, HSFA1A, HSFA7A, bZIP28, DREB2A, and DREB2B, were significantly higher in the PtWRKY2 overexpressed lines compared to that in the WT plants. Moreover, three genes related to antioxidant protection (POD34, CAT1, and SOD1) were upregulated in the transgenic lines. In addition, the overexpression of PtWRKY2 increased the transcript levels of the heat shock protein-coding genes HSP70 and HSP17.4 (Fig. 7). These results suggest that PtWRKY2 enhances the expression of heat shock-associated genes, thereby conferring heat tolerance to plants.

Figure 7
figure 7

Expression of heat shock-associated genes in WT and transgenic Arabidopsis plants under heat stress. * indicates significant differences compared to WT (P < 0.05).

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Discussion

WRKY transcription factors are widely distributed in plants and have been identified in many species, including Arabidopsis23, rice24, and wheat25, etc. WRKY proteins play an important regulatory role in many life processes and stress responses, and act as an important part of the plant stress signal transduction pathway26,27,28. In the present study, we identified five PtWRKY genes and analyzed the function of PtWRKY2 in response to heat stress, which could be helpful in understanding the transcriptional regulation of ST in P. ternata under high-temperature stress.

WRKY expression is usually tissue specific and is often affected by stress, thereby playing a role in regulating various biological processes in plants. In peppers, CaWRKY40 is induced by heat stress and functions as a positive regulator of heat stress tolerance29. In potatoes, the leaf-specific genes StWRKY016, StWRKY045, and StWRKY055 act as regulators of heat stress30. In accordance with these reports, PtWRKY2 was highly expressed in the leaves of P. ternata and significantly upregulated at high temperatures. Furthermore, PtWRKY2 is closely related to AtWRKY30, which enhances thermotolerance in wheat31, which suggests that PtWRKY2 most likely plays an important role for P. ternata in terms of the response to heat stress. In addition to heat stress-related WRKY genes in Arabidopsis13, WRKYs have been shown to function in high temperature tolerance in other species. For example, overexpression of OsWRKY11 increases the heat resistance of rice, whereas TaWRKY1 and TaWRKY33 confer heat tolerance in Arabidopsis 32,33. Here, the overexpression of PtWRKY2 in Arabidopsis increased seed germination, root elongation, and seedling survival at high temperatures, suggesting that PtWRKY2 positively regulates thermotolerance. Basing on the phylogenetic analysis, PtWRKY2 is also grouped together with AtWRKY53 and OsWRKY72, which is related with the drought stress signal34,35. Thereby, it is speculated that PtWRKY2 probably also functions in the drought stress of P. ternata.

Heat stress often leads to the excessive accumulation of ROS in plants, causing oxidative damage and leading to plant senescence36. In P. ternata, ROS accumulation and antioxidant enzyme activity fluctuate under heat stress37,38, suggesting that dynamic ROS accumulation is closely related to senescence. Previous studies have revealed that some WRKYs participate in the ROS clearance pathway; for example, transgenic rice plants overexpressing OsWRKY2 displayed increased ROS accumulation, whereas AtWRKY57 could confer ROS clearance39,40. Here, we found that overexpression of PtWRKY2 contributes to ROS elimination in Arabidopsis, which suggests that PtWRKY2 enhances thermotolerance, possibly by regulating ROS clearance.

When plants encounter heat stress, a transcriptional network is activated to regulate the expression of thermoresponsive genes41. It has reported that HSF1s usually play a major role in these signaling networks42. In addition, DREB2A and bZIP28 are important heat shock response-related genes; mutant plants are usually hypersensitive to heat stress43,44. The upregulation of these genes (HSFA1A, DREB2A, and bZIP28) in PtWRKY2 transgenic plants suggested that PtWRKY2 may act as an important regulator in heat shock signal networks. Heat shock proteins (HSPs) are induced by high temperatures, and act as molecular chaperones that enhance thermotolerance45. A previous study revealed that WRKY family members could induce the expression of certain HSPs46, and we found HSP70 and HSP17.4 were both induced in the PtWRKY2 overexpressing lines, further suggesting that PtWRKY2 enhances thermotolerance, most likely by increasing the transcription of certain HSPs. Coinciding with the high activities of CAT, SOD, and POD, the expression of CAT1, SOD1 and POD34 was upregulated in the transgenic lines under heat stress, which is in line with the findings of Arabidopsis MEKK147. Collectively, the transcriptional regulatory function of PtWRKY2 in response to heat stress was explored; however, the location of PtWRKY2 in the signaling networks remains unclear and requires further research.

Materials and methods

Plant materials and growth conditions

P. ternata tubers (1 cm in diameter) were selected from the Experimental Farm of Huaibei Normal University (N 33°16′, E 116°23′, altitude: 340 m) and planted in potting soil. The potted plants were kept in a phytotron with a 16 h photoperiod and 35 µmol m−2 s−1 light intensity at 25 °C. When P. ternata reached the three-leaf stage, with a height of approximately 15 cm, its leaves, petioles, tubers, and roots were collected. To induce high-temperature stress, the three-leaf-stage seedlings were exposed to temperatures of 42 °C, while the photoperiod and light density remained unchanged. Whole plants were collected after 0, 4, 12, and 24 h of high-temperature stress. Each sample consisted of three plants, and three biological replicates were used for each treatment.

Col-0 background Arabidopsis was used as the wild-type (WT) line, and all Arabidopsis seeds were vernalized for 3 days and sown in a sterilized mixture with three parts nutrient soil and one part vermiculite. The seedlings were exposed to a 16 h light (50 µmol m−2 s−1) and 8 h dark cycle at 23 °C.

Identification and bioinformatics analysis of WRKY family proteins in P. ternata

The WRKY protein in P. ternata was searched in our previous transcriptome data19 based on the sequences of WRKY conserved domains in Arabidopsis. Candidate WRKY genes of P. ternata were obtained by BLASTP analysis using a hidden Markov model of WRKY. The molecular weights (MWs) and isoelectric points (pIs) of the WRKY proteins were predicted using the ProtParam tool (https://web.expasy.org/protparam/). The domains and conserved domains of the PtWRKY proteins were analyzed using Pfam (http://pfam.xfam.org/) and MEME (http://meme-suite.org/index.html). Finally, TBtools software was used to produce the visualization diagram. The WRKY proteins from Arabidopsis and Oryza were downloaded from the NCBI database48, and the phylogenetic tree of WRKY proteins in P. ternata, Arabidopsis, and Oryza was established using MEGA7.0 software and the Maximum Likelihood method, with the bootstrap setting as 1000.

Subcellular localization assay

The coding sequence of PtWRKY2 was amplified and then transformed into the vector pCAMBIA1302 for constructing a fusion plasmid, named 35S-PtWRKY2-GFP. The resulting 35S-PtWRKY2-GFP plasmid and empty pCAMBIA1302 control were transformed into tobacco epidermal cells refering to a previously published method22. GFP signals from the tobacco epidermal cells were captured under a fluorescence microscope (PA53 FS6, Motic, China).

Transcriptional activation activity assay

The full-length coding sequence of PtWRKY2 was cloned and fused to the GAL4 DNA-binding domain (DB) in the pGBKT7 vector to generate a recombinant vector, pGBKT7-PtWRKY2. The pGBKT7-53 plasmid and pGBKT7 empty vector were used as positive and negative controls, respectively. All plasmids were transformed into the Y2HGold yeast strain, which were subsequently cultured on SD/-Trp and SD/-Trp/-His/-Ade plates with or without X-α-gal. After 3–5 days’ incubation of the yeast cells at 30 °C, the transactivation activity of PtWRKY2 was evaluated using a previously reported method28.

Dual-luciferase activity assay

A dual-luciferase activity assay of PtWRKY2 and W-box was performed according to a previously reported method22. Briefly, the PtWRKY2 coding sequence was amplified and transformed into the pGreenII62-SK vector to produce a fusion vector, PtWRKY2-62-SK. The three repeats of the W-box (TTGACY) and mW-box (TTTAAY) were synthesized by oligonucleotide sequencing and cloned into the vector pGreenII-0800 to generate W-box-0800 and mW-box-0800, respectively. Using pGreenII-0800 as a control, the plasmid PtWRKY2-62-SK was co-transformed into N. benthamiana leaves with W-box-800, mW-box-800, and pGreenII-0800, respectively. After 72 h transfection, LUC signals were captured in the leaves using a multi-chemiluminescent imaging system (Tanon 5200, China). The activity of LUC/REN was determined using the Dual-LUC Assay Kit (Yeasen, China).

Plasmid construction and acquisition of transgenic plant material

The 1053 bp coding sequence of PtWRKY2 was amplified using its specific primers (Table S1), and introduced into the multiple cloning sites behind the CaMV 35S promoter in the pCAMBIA1301a vector; the result was an overexpression vector of the PtWRKY2 construct. The recombinant plasmid was transformed into Col-0 Arabidopsis via the Agrobacterium-mediated floral-dip method49. The T3 homozygous lines were obtained for subsequent experiments.

High temperature tolerance analysis of transgenic plants

Newly harvested seeds from the WT and T3 pure transgenic lines were selected for germination experiments. The seeds were sown on 1/2 MS solid media after surface sterilization, and treated with 4 °C for 3 days’ vernalization. Thereafter, the seeds were kept at 23 °C for 1 day and subsequently exposed to a temperature of 42 °C for 0, 2, and 3 days. Next, seeds were cultured at 23 °C and captured at 14 days for germination rate calculations. For the root length experiments, the seeds were germinated on 1/2 MS solid media at 23 °C. When the root length reached approximately 0.5 cm, half of the germinated seeds were exposed to temperatures of 42 °C for 1 day, while the others were kept at 23 °C; the root length was measured after 10 days of growth. For the seedling survival rate assay, three-week-old WT and transgenic Arabidopsis plants were exposed to temperatures of 42 °C for 24 h, and subsequently transferred to a 23 °C environment for the survival rate calculation. Each experiment was repeated thrice.

Determination of ROS and antioxidant enzyme activity

Three-week-old WT and transgenic Arabidopsis plants were exposed to temperatures of 42 °C for 12 h, and the leaves were collected for reactive oxygen species (ROS) and antioxidant enzyme activity detection. The accumulation of hydrogen peroxide (H2O2) and superoxide anion (O2−) in the leaves were analyzed via staining with 3,3′-DAB and nitro blue tetrazolium (NBT), respectively. The catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) activities were measured using a previously reported method50.

RNA extraction and quantitative real-time PCR

Total RNA was extracted from P. ternata or Arabidopsis samples using TRIzol reagent (Invitrogen), following a previously reported procedure18,50. The RNA was then used as a template for cDNA synthesis using the MonScript™ RT III × All-In-One transcription kit, which was obtained from Monad (Chuzhou, China). Quantitative real-time PCR was performed using SYBR Premix (Roche) on a LightCycler 96 system (Roche, Basel, Switzerland). Pt18S and AtTUB2 were selected as internal references for P. ternata and Arabidopsis, respectively. Each assay was run with three biological replicates, and the relative expression levels were calculated based on the 2−△△CT method. The primer sequences used in this study are listed in Table S1.

Statistical analysis

Data were analyzed using SPSS Statistics 22 software and the data presented in the figures represent the means ± SD values of three biological replicates. Statistical significance was assessed using Duncan’s multiple range test or the Student’s t-test, with significance set at P < 0.05.

Ethical statement

The authors declare that all the plant experiments/protocols were performed with relevant institutional, national, and international guidelines and legislation.

Conclusions

In summary, five PtWRKYs were identified from the transcriptome data while PtWRKY2 was induced and the other four PtWRKYs were suppressed under high temperatures. The overexpression of PtWRKY2 significantly improved the heat tolerance of Arabidopsis, inluding seed germination, root growth and survival rate. RT-qPCR revealed that PtWRKY2 could up-regulate heat shock-associated genes, and decreased the ROS accumulation under high temperature, thereby to enhance the heat tolerance in Arabidopsis. This study firstly identified the function of PtWRKY2 and laid a foundation for further exploring the transcriptional regulation of PtWRKYs in the heat response of P. ternata.