Identification of Dioscorea opposite Thunb. CDPK gene family reveals that DoCDPK20 is related to heat resistance

Identification of CDPK genes, phylogenetic analysis, and sequence analyses of Dioscorea opposite Thunb_•

To obtain the sequences of the Dioscorea opposite Thunb. CDPK gene family, we searched from pre-transcriptomic sequencing of yam tubers (Gao et al., 2022). The Pfam database was used to acquire the Hidden Markov Model (HMM) file (PF00069) for the CDPK domain (Table 1) (Punta et al., 2004). Then, the yam transcriptome was compared to BLASTp using HMMER 3.0 (E-value set to 0.01) of the model as a sample. For the validation of the candidate gene CDPK, we used the online program PFAM, which contained Conserved Domains Database Search and SMART tool (Table 1). Prediction of open reading frames (ORFs) was performed via the website (https://www.ncbi.nlm.nih.gov/gorf/gorf.html) and follow-up tests.

We used the online ExPasy program (http://www.expasy.org/tools/) to predict the physical properties of CDPK members, such as length, molecular weight (MW), and isoelectric point (pI) (Wang et al., 2019). We used an online tool, the MEME website, to determine the motif, to annotate it with InterPro Scan, set the parameters to 0 or 1 occurrence per sequence (up to 10 subjects), and set the length of each motif to 5–50 aa (Table 1) (Bailey et al., 2015).

The amino acid sequence of DoCDPK of the ORF was compared with the rice CDPK and we used the Clustal Omega tool to perform multiple sequence alignment (Table 1). According to the above results, we used the maximum likelihood of MEGAX to construct a phylogenetic tree with 1,000 bootstrap replications of maximum likelihood (Tamura et al., 2011) and used ITOL to draw (Table 1).

Plant materials and growth conditions

The yam (Dioscorea opposite Thunb.) cultivar Dahechangyv was used as plant material. First, yam sterile seedlings were obtained from young stem tips of plants that were grown in the field for 2 months. They were rinsed under running water for 30 min, disinfected with 75% alcohol for 30 s, soaked with 0.1% Hgcl₂ for 8 min, and washed with sterile water three to four times. Next, these explants were inoculated in MS medium. Then, these sterile seedlings were placed in a lighted incubator (25 °C, 2000 Lux, 16 h of light, 8 h of dark incubation). Using previous methods as references (Weckwerth, Ehlert & Romeis, 2014; Tian et al., 2016; Chang et al., 2011), yam sterile seedlings were selected for uniform growth and placed in either a −4 °C low temperature incubator or a 42 °C incubator. We collected yam leaves at 0, 1, 2, 4, 8, and 12 h; snap-froze them in liquid nitrogen; and stored them at −80 °C for gene expression analysis.

Analysis of CDPK gene expression

The RNA of all samples were extracted using Trizol (TIANgel, Beijing, China) according to the manufacturer’s descriptions. The quality of the RNA samples were tested using NanoDrop2000c (Thermo Fisher Scientific, Waltham, MA, USA) and gel electrophoresis. BiomarkerScript III RT Master Mix (Biomarker, Beijing, China) was used to perform reverse transcription. We used the TB Green Premix Ex Taq II kit (TaKaRa, Dalian, China) to implement qRT-PCR on the FTC-3000P (Funglyn Biotech, Richmond, ON, Canada). The 18S-rRNA gene is an actin gene. Primer Premier 5.0 software was used to design the primers based on transcriptomics sequencing data. The primers and reaction procedure are shown in Table S1. The experiment was set up with three replications. All qRT-PCR data were calculated using 2^−ΔΔCt.

Cloning DoCDPK20 and constructing pPZP221-DoCDPK20 plant expression vector

PCR amplification was performed using ORF special primers of DoCDPK20 (Table S1). The ORF of DoCDPK20 was connected into the pGM-T vector (PGM-T Vector Linking Kit; TaKaRa, Dalian, China) and sequenced for confirmation. The ORF of DoCDPK20 was amplified using the specific primers DoCDPK20-Z with restriction enzyme cleavage sites (Kpn I and BamH I), and the amplification product was connected into the pPZP221-35S-NOS vector. Table S1 contains the primer sequences. The pPZP221-DoCDPK20 plant expression vector was obtained, and Agrobacterium tumefaciens EHA101 (Weidi Biotechnology, Shanghai, China) was transformed using the freeze-thaw method.

Preparation of transgenic tobacco plants that overexpressing DoCDPK20

The Agrobacterium-mediated transformation method was used to transform wild-type tobacco (Nicotiana tabacum L., labeled WT) with Agrobacterium containing the plant expression vector pPZP221-DoCDPK20. Positive seedlings were screened for resistance using MS medium (containing 6-BA 1.0 mg/L, gentamicin 50 mg/L, and Cefotaxim 500 mg/L). Transgenic plants were grown on 1/2 MS medium (containing gentamicin 50 mg/L and Cefotaxim 500 mg/L). RNA and DNA assays were performed using the Hi-DNAsecure Plant Kit (TIANGEN, Beijing, China). Transgenic tobacco plants with high expression (labeled T2, T3, and T7) were selected for the next experiments.

Phenotypic observation and photosynthesis-related and physiological index measurements of transgenic tobacco

Transgenic and WT tobacco plants were washed from the culture medium and transferred to the substrate for 2 weeks. For follow-up experiments, we selected transgenic and WT tobacco plants with uniform growth. Under normal growth conditions, transgenic and WT tobacco plants were used as the control group. Under high temperature stress for 8 h, these plants were used as the experimental group. The morphological changes of the transgenic and WT tobacco plants were photographed and recorded at each stage. We selected plants with inverted trifoliate leaves (mature leaves) and used a photosynthesis meter (CIRAS-3, Hansatech, Norfolk, UK) to measure the gas exchange parameters in fully unfolded leaves, including photosynthetic rate (Pn), intercellular CO₂ concentration (Ci), stomatal conductance (Gs), and transpiration rate (Tr). Pn/Tr is the water use efficiency (WUE) (Nieva et al., 1999). Fast chlorophyll fluorescence induction curves were measured using a continuous excitation fluorometer (M-PEA, Hansatech, Norfolk, UK) and set up with five replicates. The first true leaves were collected for histochemical staining observation. Under high temperature treatment, hydrogen peroxide (H₂O₂) and superoxide anion (O²⁻) levels were tested using 3,3-diaminobenzidine (DAB) and nitroblue tetrazolium chloride (NBT) colouring, respectively (Zhao, Liu & Xu, 2009; Hans, Zhang & Wei, 1997). The fully expanded inverted third and fourth blades were sampled, snap-frozen in liquid nitrogen, and stored at −80 °C for subsequent experiments. Peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) activity, and malondialdehyde (MDA), proline (Pro) and soluble protein content, were measured, respectively, and the experiment was set up with three biological replicates (Li, 2000).

Statistical analysis

The gene expression, photosynthetic parameter, and physiological indicator data were tested at the p < 0.05 level. Data statistics were conducted using IBM SPSS Statistics 25 (IBM, Armonk, NY, USA) and Excel 2010 (Microsoft Corporation, Redmond, WA, USA).

Identification of DoCDPKs in Dioscorea opposite Thunb

Twenty-nine DoCDPKs were obtained from Dioscorea opposite Thunb. and labeled DoCDPK1-DoCDPK29 based on blastp comparison of CDPK protein sequences from other plants. Among the 29 DoCDPKs, the cDNA length of ORFs were predicted to range from 930 bp (DoCDPK29) to 1,827 bp (DoCDPK5). To further investigate the characteristics of DoCDPKs with complete sequences, the pI and MW of 29 DoCDPK proteins were calculated. The pI ranged from 5.5 (DoCDPK1) to 8.6 (DoCDPK22) and MW ranged from 35.11 kDa (DoCDPK29) to 67.67 kDa (DoCDPK5) (Table 2).

Phylogenetic analysis of DoCDPKs

To understand the phylogenetic relationships of CDPK proteins, the sequences of CDPK proteins in rice (Oryza sativa) and yam (Dioscorea opposite Thunb.) were used to construct phylogenetic trees. All CDPKs were divided into four groups. Group I included 11 OsCDPKs and six DoCDPKs. Group II contained eight OsCDPKs and five DoCDPKs. Group III consisted of eight OsCDPKs and 11 DoCDPKs. Group IV was comprised of two OsCDPKs and seven DoCDPKs (Fig. 1). To further analyze the structure of the DoCDPK proteins, we identified their conserved motifs based on the MEME databases. Ten conserved motifs were identified (named Motif 1–10), length between 21 and 50 amino acids (Fig. 2). The Group I proteins contained motifs 7, 5, 4, 1, 2, 3, 6, 9, and 8; Group II proteins contained motifs 7, 5, 4, 1, 2, and 3; Group III proteins contained motifs 2 and 3; and Group IV proteins contained motifs 4, 1, 2, 3, and 6 (Fig. 2A). Notably, each DoCDPK contained five to 10 conserved motifs, and each gene sequence contained motifs 2 and 3.

The DoCDPK gene expression pattern of temperature stresses

To investigate the possible role of DoCDPK under low (−4 °C) and high (42 °C) temperature stress, we performed qRT-PCR assay in yam leaves. We randomly selected two genes from each group and monitored these gene expression trends (Fig. 3). DoCDPK1 and DoCDPK6 were selected from Group I, DoCDPK7 and DoCDPK10 from Group II; DoCDPK16 and DoCDPK17 from Group III, and DoCDPK20 and DoCDPK22 from Group IV. Under low temperature stress, DoCDPK1, 6, and 7 were down-regulated compared with the control (0 h), and DoCDPK16 was opposite (Fig. 3A). The remaining four DoCDPK genes were responsive to low temperature stimulation and down-regulated after 1 h of treatment (Fig. 3A). Among them, DoCDPK10 and DoCDPK20 increased 4.5 and 6.9-fold at 12 and 8 h of low temperature treatment, respectively, compared to the control (0 h) (Fig. 3A). Genes in Group I were significantly repressed under low-temperature stress. DoCDPK7 and DoCDPK10 in Group II were significantly repressed and induced under low temperature stress, respectively. The selected genes in Group III and Group IV were both significantly induced to be highly expressed (Fig. 3A). Under high temperature stress, DoCDPK6, 7, 10, 16, and 22 were induced to express higher values at 1, 4, 4, 8 and 4 h, respectively (Fig. 3B). Similarly, under low temperature stress, DoCDPK1 expression was inhibited (Fig. 3B). DoCDPK17 and DoCDPK20 gene expression levels were similarly down-regulated at 1 h, the former reaching a high value at 4 h and 3.9 times higher than the control (0 h), and the latter reaching a high value at 8 h and 8.4 times higher than the control (0 h) (Fig. 3B). DoCDPK1 and DoCDPK6 of Group I were significantly repressed and induced under high temperature stress, respectively. The genes in Group II, Group III, and Group IV were significantly induced (Fig. 3B). However, the changes of gene expression were more pronounced in Group IV when compared to the other groups (Fig. 3). In summary, DoCDPK20 showed the maximum change in the level of expression at 8 h after high temperature stress (Fig. 3). Thus, we speculated that DoCDPK20 may be a significant gene in high temperature stress response and conducted the following functional analysis.

Phenotypic changes in DoCDPK20 transgenic tobacco under high temperature stress

To determine whether the gene enhances the heat tolerance of plants, we used by the pPZP221 plant expression vector to expand the ORF cDNA of DoCDPK20 from yam tuber and overexpress in Nicotiana tabacum L. We obtained eight transgenic tobacco strains and selected three transgenic strains (T2, T3, and T7) to characterize the heat-induced phenotypes. Overexpression of DoCDPK20 notably improved the high temperature tolerance in tobacco (Fig. 4). Under normal growth conditions, there was no significant difference in morphology between WT and overexpression of DoCDPK20 transgenic tobacco. In plants overexpressing DoCDPK20, the newly developed leaves of the transgenic tobacco grew normally, while the WT foliage crumpled and rolled up after 8 h of high temperature treatment. These results indicate that the transgenic tobacco exhibited healthier morphological characteristics than WT tobacco plants under high temperature treatment.

Effect of heat treatment on photosynthesis-related indicators in DoCDPK20-overexpressing plants

To investigate the causes of phenotypic changes in transgenic tobacco and WT plants, we performed photosynthesis analysis under thermal stress. After 8 h of high temperature treatment, Pn, Gs, Ci, and Tr were significantly higher in overexpressed DoCDPK20 plants than WT plants. After heat treatment, WUE was significantly higher in WT than transgenic tobacco (Fig. 5A). After 8 h of high temperature treatment, Pn, Gs, Ci, and Tr in transgenic tobacco increased compared with WT tobacco. The Tr of transgenic tobacco was 1.4 times higher than WT, and the WUE of transgenic tobacco was 0.8 times lower than WT tobacco. Under high temperature treatment, the rapid chlorophyll fluorescence induction kinetic curves of DoCDPK20 overexpression and WT tobacco leaves showed a typical 0-J-I-P curve. Fluorescence rose from the lowest level (Fo or O) to the highest level (Fm or P) in three different stages, and the two points were named J and I, respectively.

After high-temperature stress, the I and P points of the leaves showed a significant decrease compared with the control, and the difference was more pronounced in WT than transgenic tobacco plants (Fig. 5B). As the heat stress prolonged, the maximum photochemical efficiency (Fv/Fm) significantly decreased, but there was no significant difference between transgenic tobacco and WT at different treatment stages. PIabs is a comprehensive parameter that reflects plant photosynthetic performance. Under high temperature treatment, PIabs was significantly lower than the control and significantly higher in transgenic tobacco than WT tobacco, with transgenic tobacco PIabs elevated 1.2-fold compared to WT tobacco (Fig. 5C). These results suggest that overexpression of DoCDPK20 improved the heat tolerance of transgenic tobacco.

Changes in physiological indicators of heat tolerance in DoCDPK20-overexpressed plants

In order to further analyze the causes of phenotypic changes in transgenic tobacco and WT plants, DAB and NBT staining were performed at 0 and 8 h under heat treatment, respectively. For DAB staining, the foliage of both transgenic tobacco and WT plants started to colour after 8 h of heat treatment, with WT darkening more compared to the transgenic leaves. These results suggest that WT tobacco accumulated more H₂O₂ than transgenic tobacco leaves (Fig. 6A). For NBT staining, the leaves of transgenic tobacco and WT plants started to colour after 8 h of heat treatment, with the WT becoming significantly darker than the transgenic tobacco leaves. These results indicate that WT tobacco accumulated more O²⁻ (Fig. 6A). In summary, DoCDPK20 overexpression delayed the accumulation of H₂O₂ and O²⁻ in the leaves. This may be because CDPK enhances the resistance of plants and protects them from damage caused by accumulation of peroxides and active oxygen.

To better understand the differences between the overexpression of DoCDPK20 and WT plants, the activities of SOD, POD, and CAT, and the contents of soluble sugar, Pro, and MDA were tested under high-temperature stress. SOD, POD, and CAT are known as protective enzyme systems that can scavenge ROS and other superoxide radicals from plants. Our results showed that the activities of SOD, POD, and CAT showed the same trend. Under normal growth conditions, there was no difference in the enzyme activities of transgenic and WT tobacco plants. After 8 h of heat treatment, the enzyme activity of the transgenic tobacco increased significantly, and was significantly higher than the control (Fig. 6B). The activities of SOD, POD, and CAT in transgenic tobacco were 1.1, 1.6, and 1.2 times higher than WT, respectively, at 8 h of high temperature treatment. Compared with the control, the content of soluble sugar and Pro in transgenic tobacco and WT plants increased under heat treatment, and transgenic tobacco plants had significantly higher levels than WT (Fig. 6B). The soluble sugar and Pro content of the transgenic tobacco was higher than that of the WT tobacco at 8 h of high temperature treatment. The MDA content significantly increased in transgenic and WT tobacco plants under heat treatment, and WT tobacco plants had significantly higher levels compared to transgenic tobacco. The MDA content of transgenic tobacco was 0.6 times lower than that of WT at 8 h of high temperature treatment. These results demonstrated that the increase of SOD and POD activity protected plant cell membrane from high temperature damage. The increase of soluble sugar and Pro content increased the concentration of plant cytosol. Compared to transgenic tobacco, WT accumulated more H₂O₂ and MDA, which can cause more damage to plants. In summary, the high temperature resistance of transgenic tobacco plants improved.

Changes in DoCDPK20 gene expression in transgenic tobacco for heat tolerance

To further investigate the mechanism of heat resistance of DoCDPK20 overexpression, we measured DoCDPK20 gene expression in transgenic tobacco plants. Our results illustrate that DoCDPK20 gene expression was significantly elevated under heat treatment conditions. The overexpression of DoCDPK20 plants was significantly higher than WT tobacco at different stages (Fig. 7). The expression of DoCDPK20 gene was 7.2-fold higher in WT and 12.0-fold higher in transgenic tobacco compared to the control (0 h). At 8 h of high temperature treatment, the expression of DoCDPK20 gene in transgenic tobacco was 1.7-fold higher than in WT tobacco (Fig. 7). These results suggest that DoCDPK20 plays an important role in high temperature conditions.