Background

In recent years, due to the development of industry and the influence of human activities, the problem of soil pollution has become prominent. Heavy metal pollution is one of the main pollution sources, and Cd pollution accounts for the largest proportion [1, 2]. Cd stress can cause serious harm to plants, causing physiological dysfunction and nutritional disorders [3], leading to cell damage, destruction of photosynthetic systems, protein degradation, DNA damage mutations, and ultimately inhibiting plant growth [4,5,6]. The growth of crops under Cd stress will lead to a series of problems, such as serious yield reduction and quality reduction [7]. In addition, many studies have shown that crops and vegetables grown on heavy metal contaminated soil will accumulate a large number of heavy metals [8, 9], and the consumption of agricultural products with excessive Cd will induce many diseases including a variety of cancers, which is a serious threat to human health [10, 11].

Potato holds an important position in the world's food industry. It is the primary 'grain and vegetable' crop in China, and its healthy development is of great significance [12]. It is reported that the natural accumulation of Cd in potato tubers is much higher than that in fruits and grains [13]. Therefore, scholars have begun to pay attention to the impact of Cd pollution on potato, have committed to the analysis of potato Cd response mechanism, and have sought solutions [14, 15]. The cell wall of higher plants is the first line of defense against external stress. Previous studies have found that plants will promote the combination of ionic Cd and cell wall components, blocking Cd2+ in the cell wall and preventing it from entering the cytoplasm, thereby protecting intracellular metabolic activities [16, 17]. Lignin is an important part of the cell wall, and its accumulation can reduce external damage to plants [18, 19]. Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in its synthesis pathway, which is responsible for catalyzing the conversion of cinnamaldehydes (coniferyl aldehyde, cinnamaldehyde, and coumaraldehyde) into corresponding alcohols, thereby promoting lignin accumulation and participating in plant response to stress [20, 21]. Studies have shown that overexpression of the SaCAD gene can improve the tolerance of Arabidopsis to Cd [22]. Kim et al. found that a gene encoding CAD was highly expressed in sweet potato induced by low temperature and other environmental stresses [23]. Park et al. found that CAD activity in rice was significantly induced by UV treatment, suggesting that CAD plays an important role in rice resistance to UV radiation [24]. Hu et al. showed that CAD can enhance plant resistance to adversity by promoting the accumulation of lignin [25]. Our recent transcriptome analysis found that a CAD gene can participate in the response of potato to Cd stress through a targeted regulatory relationship with miRNA, so it is speculated that the CAD family may play an important role in the response of potato to Cd stress [26]. Therefore, we carried out member identification and bioinformatics analysis of the potato CAD family, and explored the expression patterns of StCAD family members after Cd stress, focusing on the changes of physiological indicators closely related to CAD genes, which laid a foundation for further clarifying the function of the StCAD family. At the same time, it can provide new ideas for later research on Cd tolerance breeding and heavy metal stress relief of potato.

Materials and methods

Plant materials and Cd stress treatment

In this experiment, potato 'Atlantic' tissue culture seedlings were used as experimental materials. Plantlets were grown in MS medium in a culture box, at a temperature of 21 °C, a photoperiod of 16 h light:8 h dark, and a light intensity of 3000 Lx for 20 days of treatment, the seedlings were transferred to a liquid medium supplemented with CdCl2 (5 mmol/L). Plant roots were collected before Cd treatment (T0) and at 12 (T1), 24 (T2), and 48 (T3) h after Cd treatment, washed three times with deionized water, quickly frozen in liquid nitrogen, and then stored in a freezer at -80℃. There were three biological replicates at each time point.

Identification of StCAD gene family members

The Hidden Markov Models (HMM) of cinnamyl alcohol dehydrogenase (CAD) protein domains ADH_N and ADH_zinc_N, PF08240 and PF00107, were downloaded from the Pfam database (http://www.sanger.ac.uk/Software/Pfam/). Using PF08240 and PF00107 as probes, the HMMER3.0 software package was used to search the protein sequences predicted by the potato reference genome (http://spuddb.uga.edu/) and the potato transcriptome constructed by our laboratory. The search results were submitted to the NCBI conserved domain database (http://www.ncbi.nlm.nih.gov/cdd/) for verification. The potato reference genome sequence and gff3 structure annotation file were downloaded from the Ensemble database (http://plants.ensembl.org/Solanum_tuberosum/Info/Index) to verify the CAD gene family members and subsequent analysis.

Bioinformatics analysis of StCAD gene

Mega 7 software [27] was used to construct the phylogenetic tree of StCAD family members by the ML method, bootstrap 1000 times, and the family members were divided into groups according to the branch of the phylogenetic tree. The amino acid sequences of each gene were submitted to the ExPASy website (https://web.expasy.org/protparam/) to output various physical and chemical properties, and the subcellular localization analysis was performed using the CELLO website (http://cello.life.nctu.edu.tw/). The motif analysis was performed by the meme tool (http://alternate.meme-suite.org/tools/meme) to identify conserved motifs. The gff3 file of the StCAD gene was input into TBtools software [28] to realize the visualization of chromosome position and exon–intron structure. The 2-kb promoter sequence upstream of the StCAD gene coding region was extracted and submitted to the PlantCARE database for cis-acting element prediction and visualization based on TBtools.

qRT-PCR analysis of StCAD gene in response to Cd stress

According to the sequencing results of previous studies (NCBI accession number: SRP314907) [26], the expression patterns of some StCAD genes under Cd stress were analyzed by qRT-PCR. Samples were taken at 0 h, 12 h, 24 h and 48 h under Cd stress. According to the manufacturer's instructions, the total RNA of the samples was extracted by RNAout kit (160,906–50, Tiandz, Beijing), RNA-gel in Additional file 1. Reverse transcription of mRNA was performed using the kit FastKing gDNA Dispelling RT SuperMix provided by Tiangen Biochemical Technology Co., Ltd. (Beijing, China), and actin was used as the mRNA reference gene. The obtained cDNA was detected on a fluorescence quantitative PCR instrument using the TB Green Premix Ex Taq II kit, and the relative expression level was calculated according to the 2−ΔΔCt method [29]. Each group had 3 biological replicates, and each reaction had 3 technical replicates. The primer sequence is shown in Additional file 2.

Measurement of physiological indices

The cinnamyl alcohol dehydrogenase (CAD) activity, peroxidase (POD) activity, and lignin content were determined using the kits BC4170, BC0090 and BC4200 provided by Beijing Solarbio Science Technology Co., Ltd (Beijing, China). The specific prescription determination method refers to the instructions in the kit.

Data analysis

SPSS 19.0 software was used for variance analysis. Least significant difference (LSD) and Student–Newman–Keuls (SNK) methods were used to investigate the difference at the P ≤ 0.05 level. Data analysis and mapping were performed using Origin 2018 software.

Results

Identification of StCAD gene family members

Through protein sequence analysis and verification, 50 members of the potato CAD family were finally obtained, named StCAD1–StCAD50, with isoelectric points ranging from 5.31 to 9.18. Most CAD proteins (76%) have isoelectric points less than 7, suggesting that they may be acidic proteins. The molecular weight is between 20,848.99 to 46,165.99 Dalton; the amino acid sequence size ranges from 190 to 433 aa. The total number of atoms is between 2923 and 6480. Most of the instability coefficient (92%) was below 40, suggesting that it was a stable protein. Subcellular localization prediction of all genes showed that all genes were located in the cytoplasm (Table 1).

Table 1 Physicochemical properties and subcellular localization of proteins encoded by StCADs
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Evolutionary analysis of the StCAD gene family

Phylogenetic analysis of the potato CAD family revealed that 50 genes belong to 6 subfamilies, of which the first subfamily includes 20 genes, the second subfamily includes 3 genes, the third subfamily includes 8 genes, the fourth subfamily includes 2 genes, the fifth subfamily includes 16 genes, and the sixth subfamily includes 1 gene. There are 9 differentially upregulated genes in the CAD family obtained by previous sequencing, which are in the first subfamily and the third subfamily. The 50 genes of potato CAD family are closely related to the 24 genes of Arabidopsis CAD family (Fig. 1 and Additional file 3).

Fig. 1
figure 1

Phylogenetic tree of potato StCADs gene family

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Structural characteristics analysis of StCAD gene family

The intron–exon and motif visualization analysis were performed using the software TBtools. Figure 2 shows that the number and length of exons in the StCAD family are different, and the number of exons varies from 3 to 10. Among them, StCAD40, StCAD43, StCAD45, StCAD49, and StCAD50 have the largest number of exons, 10, and StCAD2, StCAD4, StCAD5, StCAD7, StCAD9, and StCAD13 have the least number of exons, only 3.

Fig. 2
figure 2

Gene structure and motif analysis of StCADs in potato. The different line colors of gene IDs showed the classification of CADs based on phylogeny tree

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Conserved motif analysis of the StCAD protein showed that the members of the StCAD family were highly conserved at the 5' end. The conserved motifs contained in the family members were Motif 1, Motif 2, Motif 3 and Motif 5. In the same subgroup, the number and type of protein-conserved motifs were similar. Among them, subfamily I contained all conserved motifs except Motif 9 which contained more conserved motifs than other subfamilies. We speculate that the gene structure of subfamily I may be more complex than other subfamilies. In addition, some motifs have obvious subfamily specificity. Motif 1 and Motif 10 only appeared in Subfamily I and Subfamily VI, and Motif 7 only appeared in Subfamily I (Fig. 2 and Additional file 4).

Chromosome localization of the StCAD gene family

Chromosome localization analysis of potato CAD family genes showed that each gene was irregularly distributed on 12 chromosomes. Among them, chromosomes 3, 4, 9, 11, and 12 were more distributed. Five genes were distributed on chromosomes 3, 9, and 12, respectively. Six genes were distributed on chromosome 4, and 13 genes were distributed on chromosome 11. The tandem repeat gene clusters were formed on chromosomes 3, 4, 9, and 11 (Fig. 3).

Fig. 3
figure 3

Mapping of the StCADs gene family on potato chromosomes. The different line colors of gene IDs showed the classification of CADs based on phylogeny tree

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Cis-element analysis of StCAD gene promoter related to stress

In this study, a 2-kb sequence upstream of the translation initiation site of the CAD gene was extracted and submitted to the PlantCare online website for cis-element prediction. Figure 4 shows 18 major abiotic stress response elements. The results showed that the 50 CAD genes contained multiple phytohormone response elements, including auxin response element (TGA-element), auxin response element (AuxRR-core), salicylic acid response element (TCA-element), gibberellin response element (GARE-motif), abscisic acid responsive element (ABRE);: methyl jasmonate response element (TGACG-motif and CGTCA-motif); methyl jasmonate response element. These CAD genes were mainly distributed in subfamilies I, III and V. In addition, most CAD genes were also found to contain cis-elements in response to stress and stress signals, such as stress response element (STRE) in 28 genes and antioxidant response element (ARE) in 39 genes. In summary, the StCAD family has potential functions in regulating hormones and stress, especially closely related to abiotic stress.

Fig. 4
figure 4

Analysis of cis-acting elements of potato CAD family genes. STRE: stress response element; MRE: metal response element; ARE: antioxidant response element; GC-motif: hypoxia response element; LTR: low temperature response element; DRE: dehydration response element; WRE3: trauma-induced response element; WUN-motif: trauma-induced response element; MBSI: defense and pressure response components; TGA-element: auxin response element; AuxRR-core: auxin response element; TCA-element: salicylic acid response element; GARE-motif: gibberellin response element; ABRE: abscisic acid responsive element; TGACG-motif: methyl jasmonate response element; CGTCA-motif: methyl jasmonate response element; O2-site: zein metabolic regulatory elements; circadian: circadian response element

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Analysis of expression patterns of the StCAD gene family in response to Cd stress

Based on the previous research results, the potato CAD gene family may respond to abiotic stress. There are 9 differentially upregulated genes in the CAD family obtained by pre-sequencing, which are in the first subfamily and the third subfamily. Therefore, we analyzed the expression patterns of subfamily I and III. Cd stress was performed on potatoes, and samples were taken at different time points. The expression characteristics of StCAD in plants were verified by real-time PCR (Fig. 5 and Additional file 5). Except for StCAD9, StCAD10 and StCAD27, other genes were upregulated after Cd stress. StCAD3 changed the most at 12 h of Cd stress, which was 16.61 times higher than that before stress. StCAD6 changed the most at 24 h of Cd stress, which was 12.18 times higher. StCAD28 changed the most at 48 h of Cd stress, which was 15.41 times higher. The expression levels of StCAD7, StCAD8, StCAD14, StCAD15, StCAD16 and StCAD19 did not change significantly after stress. Based on this, we concluded that potato CAD gene family members have undergone functional differentiation during evolution.

Fig. 5
figure 5

Expression changes of CAD family genes in potato under cadmium stress

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Analysis of physiological characteristics related to the StCAD gene

The activities of POD, CAD and lignin in potato were significantly changed after Cd stress. The activities of POD, CAD and lignin in potato were significantly changed after Cd stress. The POD activity increased with the increase of Cd stress time, and the POD activity of the T3 treatment was significantly higher than that of other treatments. CAD activity increased significantly after Cd stress and CAD activity of the T0 treatment was significantly lower than that of other treatments. The lignin content showed a gradually increasing trend, and the lignin content of the T0 treatment was the lowest, while that of the T3 treatment was significantly higher than that of T0 and T1 (Fig. 6A). To explore the relationship between the StCAD gene and related physiological indicators, correlation analysis was conducted on the StCAD gene, POD and CAD activities and lignin content, and the results showed that except for StCAD9, StCAD10 and StCAD27, the other genes were positively correlated with physiological indicators (Fig. 6B).

Fig. 6
figure 6

Analysis of StCAD gene related physiological characteristics. A: StCAD gene related physiological indicators; B: correlation between gene expressions value of StCADs and physiological indexes

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Discussion

Lignin is widely regarded as an important secondary metabolite involved in plant stress resistance, which can enhance the mechanical strength and stress resistance of plants, is conducive to the transport of minerals and water in plants and the defense of an adverse external environment, and plays an important role in the process of plant growth [30, 31]. The biosynthesis of lignin is very complex, involving many enzymes and multi-step reactions [32, 33]. Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin synthesis, which can catalyze the last step of the lignin specific synthesis pathway and play an important regulatory function in lignin biosynthesis [34]. Many studies have shown that CAD family genes are closely related to lignin biosynthesis and lignin deposition [35,36,37].

In this study, members of the potato CAD family were identified, and gene structure, protein physicochemical form, phylogenetic evolution and expression pattern under Cd stress were analyzed. In recent years, scholars at home and abroad have gradually paid more attention to CAD gene families. CAD genes have been identified in a variety of plants such as Arabidopsis thaliana, and their number and species have been determined [38]. The Arabidopsis CAD family contains 12 genes, including 18 in grape, 12 in rice, and 17 in alfalfa. In addition, 12 CAD genes in the Arabidopsis CAD family were found, and 6 of them could catalyze 5 cinnamaldehydes to produce cinnamyl alcohol, which further promoted the synthesis of lignin [39]. We have identified 50 CAD family genes in potato, which is relatively large compared with other species, which may be related to the large potato genome and gene replication [40]. The analysis of physical and chemical properties showed that StCADs may be a class of acidic proteins, and most of the stability coefficient (92%) was below 40, suggesting that StCADs were a class of stable proteins. Subcellular localization prediction revealed that all the genes were located in the cytoplasm, indicating that they play a role in the cytoplasm. Evolutionary analysis and gene structure analysis showed that genes with close genetic relationship often had similar gene structures, which may be due to the fragment replication of CAD family members in the process of evolution, which played an important role in the amplification of family members. The study of the multiple replications of potato CAD family genes can help us understand the process of potato polyploidy better. The number of exons/introns was diverse between StCAD family members. It was stated that intron number can impact the speed of gene expression and genes with less number of introns can leave the nucleus faster to start the translation process [41, 42]. Based on this, StCAD family members have had different evolutionary processes and their genetic structure has been affected.

The cis-element analysis of potato CAD family genes showed that the promoter region was rich in cis-elements related to plant response to stress, including metal response elements, salicylic acid response elements and abscisic acid response elements, suggesting that it may play an important role in response to abiotic stress. There is evidence that the CAD activity in Miscanthus sinensis increases when it is subjected to cold stress [43]. Liu et al. found that drought could induce CmCAD2 and CmCAD3 expression and promote lignin biosynthesis in melon [44]. CAD activity and gene expression in barley leaves were induced to increase after cold and freezing treatment [45]. Previous studies have shown that CAD is involved in the regulation of low temperature stress by abscisic acid, which improves the cold resistance of sweet potato, and IbCAD1 is also involved in the mechanical damage response regulated by jasmonic acid and salicylic acid [23]. In our study, the expression pattern showed that most CAD genes were upregulated after Cd stress, indicating that StCAD family genes responded to Cd stress, which was consistent with previous studies. Many studies have shown that CAD family genes help plants resist stress by participating in lignin synthesis. Jourdes et al. found that the lignin content of plants was greatly reduced after double mutation of the AtCAD4 and AtCAD5 genes in Arabidopsis thaliana, and even lodging stems appeared [46]. Eudes et al. found that AtCAD1 played a compensatory role in lignin synthesis and participated in the regulation of lignification in AtCAD4 and AtCAD5 double mutants of Arabidopsis thaliana [47]. In the study of Populus tomentosa, it was found that the PtCAD9 gene may be involved in the defense mechanism of lignin [48]. In this study, after Cd stress, most of the genes in StCAD were upregulated, and CAD, POD activity and lignin content were significantly increased, moreover, most CAD genes were positively correlated with lignin content, which was similar to previous studies, indicating that CAD gene expression was closely related to lignin content. The StCAD family plays an important role in potato response to Cd stress, which is conducive to the improvement of potato Cd stress tolerance.

Conclusions

A total of 50 potato StCAD genes were identified, and subcellular localization prediction found that all genes were located in the cytoplasm, the 50 genes were divided into 6 subfamilies, and the homologous genes had similar structures. Chromosomal localization analysis found that all genes were irregularly distributed on 12 chromosomes, and cis-element analysis found that the StCAD family has potential functions in regulating hormones and stress, especially closely related to abiotic stress. Analysis of the expression patterns of CAD genes under Cd stress showed that most of the genes were upregulated after Cd stress, and the related activities of CAD, POD and lignin were also significantly increased, indicating that the StCAD family responded to Cd stress and played an important defense role in potato response to Cd stress, although the downstream regulation mechanism of transcription level still needs to be further studied. The genes that play an active role in the response of potato to cadmium stress were obtained in our study, which provides a new idea for the analysis of the mechanism of potato response to cadmium stress, and is very beneficial to the development of cadmium-tolerant potato breeding in the future.