Abstract
Background
The homeodomain-leucine zipper I (HD-Zip I) transcription factor is a plant-specific protein that plays an essential role in the abiotic stress response of plants. Research on the HD-Zip I family in Salvia miltiorrhiza is still lacking.
Methods and Results
In this study, a total of 25 SmHD-Zip I proteins were identified. Their characterizations, phylogenetic relationships, conserved motifs, gene structures, and cis-elements were analyzed comprehensively using bioinformatics methods. Expression profiling revealed that SmHD-Zip I genes exhibited distinctive tissue-specific patterns and divergent responses to ABA, PEG, and NaCl stresses. SmHD-Zip12 responded the most strongly to ABA, PEG, and NaCl, so it was used for transgenic experiments. The overexpression of SmHD-Zip12 significantly increased the content of cryptotanshinone, dihydrotanshinone I, tanshinone I, and tanshinone IIA by 2.89-fold, 1.85-fold, 2.14-fold, and 8.91-fold compared to the wild type, respectively. Moreover, in the tanshinone biosynthetic pathways, the overexpression of SmHD-Zip12 up-regulated the expression levels of SmAACT, SmDXS, SmIDS, SmGGPPS, SmCPS1, SmCPS2, SmCYP76AH1, SmCYP76AH3, and SmCYP76AK1 compared with the wild type.
Conclusions
This study provides information the possible functions of the HD-Zip I family and lays a theoretical foundation for clarifying the functional mechanism of the SmHD-Zip12 gene in regulating the synthesis of tanshinone in S. miltiorrhiza.
Keywords: HD-Zip I family, Salvia miltiorrhiza, Abiotic stresses, Expression analysis, SmHD-Zip12, Tanshinone
Introduction
Transcription factors act as key components of transcriptional regulatory mechanisms, controlling gene expression in almost all processes of plant life (Franco-Zorrilla & Solano, 2017; Romani & Moreno, 2021; Manna et al., 2021). The homeodomain-leucine zipper I (HD-Zip I) is a plant-specific transcription factor that plays an important role in a plant’s response to diversity of stresses (Henriksson et al., 2005; Li et al., 2019c). HD-Zip I contains a highly-conserved homology domain (HD) and a leucine zipper (LZ) domain (Ariel et al., 2007; Bürglin & Affolter, 2016); the former can specifically bind to DNA and the latter serves as a dimerization motif (Ruberti et al., 1991; Elhiti & Stasolla, 2009). The HD-Zip I gene family has been extensively studied in plants, such as Arabidopsis thaliana (Henriksson et al., 2005), Nicotiana tabacum (Li et al., 2019b), Cucumis sativus (Liu et al., 2013), Sesamum indicum (Mehmood et al., 2022), Solanum lycopersicum (Zhang et al., 2014), and Chrysanthemum morifolium (Song et al., 2016).
HD-Zip I proteins are involved in plant growth and development and are associated with abiotic stress responses, such as drought stress, exposure to abscisic acid (ABA), and salinity stress (Johannesson et al., 2003; Hu et al., 2012; Shen et al., 2019; Gong et al., 2019; Hussain et al., 2021; Sharif et al., 2021). For example, ATHB7 and ATHB12 were strongly induced by water-deficit and ABA in A. thaliana (Ariel et al., 2007). In Oryza sativa, both the expression of OsSLI1 and Oshox22 were induced very quickly and dramatically by ABA, PEG, and salt treatment (Zhang et al., 2012; Huang et al., 2014). Studies have also shown that the overexpression of TaHDZipI-5 in bread wheat, encoding a stress-responsive HD-Zip I, increased the drought and frost tolerance of transgenic wheat lines (Yang et al., 2018). The overexpression of CaHDZ12 in tobacco enhanced the plant’s tolerance to osmotic stresses and sensitivity to ABA, and knocking out CaHDZ12 in chickpea increased the plant’s sensitivity to salt and drought stresses (Sen et al., 2017). In sunflowers, the HD-Zip I gene, HaHB11, responded to drought and salinity tolerance by closing stomata more rapidly and the plant lost less water because of this quick response (Cabello et al., 2017). The overexpression of the HD-Zip I family member, MdHB-7, ameliorated the restricted photosynthetic capacity and root damage caused by salt stress in apple (Zhao et al., 2021). Moreover, it has been reported that HD-Zip I genes mediate abiotic stress responses through ABA-mediated signal transduction pathways (Valdés et al., 2012; Zhang et al., 2012; Hussain et al., 2021). ZmHDZ10 overexpression in Arabidopsis positively regulated drought and salt tolerance, likely due to the expression of stress/ABA-responsive genes, including P5CS1, RD22, RD29B, and ABI1 (Zhao et al., 2014). In contrast, the overexpression of ZmHDZ1 reduced tolerance to salt stress and increased sensitivity to exogenous ABA, suggesting that ZmHDZ1 functions as a negative regulator in response to salt stress (Wang et al., 2017b). Although some HD-Zip I members have been functionally studied in plants, the response of the HD-Zip I gene family to abiotic stress in S. miltiorrhiza has not yet been studied.
S. miltiorrhiza is a well-known traditional Chinese medicine and is considered a model organism for medicinal plant research (Lu et al., 2020; Wang et al., 2021). Tanshinones are one of the pharmacological compounds in S. miltiorrhiza and have been shown to have a variety of antioxidant, antitumor, and antivirus properties (Su et al., 2015). Tanshinones are type of diterpene derived from isopentenyl diphosphate (IPP) and their isomer dimethylallyl diphosphates (DMAPP) synthesize through two different pathways in separate compartments: the mevalonate pathway (MVA pathway) in the cytosol and the methylerythritol phosphate pathway (MEP pathway) in the plastids (Jiang, Gao & Huang, 2019). An increasing number of medicines and health products use S. miltiorrhiza as a main ingredient, giving rise to increased market demand. However, the growth of S. miltiorrhiza has been seriously limited by drought, salinity, and other environmental stresses, decreasing the natural production of tanshinones to levels that cannot meet the clinical demand (Liu et al., 2011b; Wu et al., 2014; Wei et al., 2017; Jiang, Gao & Huang, 2019; Chen et al., 2021; Yu et al., 2021; Wang & Li, 2022). Therefore, research on the biosynthesis and regulation of tanshinones in S. miltiorrhiza is becoming increasingly relevant.
In this study, we identified 25 SmHD-Zip I genes. Various characteristics of these genes were analyzed, including the physicochemical parameters, phylogenetic relationships, conserved motifs, gene structure, and cis-element. Additionally, the expression patterns of SmHD-Zip I genes were analyzed in different tissues and ABA, PEG, and NaCl stress. Using these results, we investigated the functions of SmHD-Zip 12 in the hairy root of S. miltiorrhiza through transgenic experiments. This study provides useful information on HD-Zip I genes to serve as basis for further research into the functions of these genes.
Materials & Methods
Genome-wide identification of HD-Zip I family
The amino acid sequences of HD-Zip I proteins in A. thaliana were used as a query probe to search the S. miltiorrhiza genome by Blastp. Then, the Hidden Markov Model (HMM) profile of the homeodomain (PF00046) and the leucine zipper domain (PF02183) were downloaded from the PFAM database and used for the local HMM search using HMMER3.3 (Arce et al., 2011). The HD and LZ domain of S. miltiorrhiza HD-Zip I was confirmed using the Conserved Domain Database (CDD; https://www.ncbi.nlm.nih.gov/cdd/?term=).
Sequence analysis of SmHD-Zip I
The ExPASy server (Gasteiger et al., 2005) was used to predicate the physicochemical properties of SmHD-Zip I family members, including protein length, protein molecular weight (MW), theoretical isoelectric point (pI), instability index, aliphatic index, and grand average of hydropathicity (GRAVY). Then, subcellular localization was predicted according to the Plant-Ploc server (Chou & Shen, 2008).
Phylogenetic tree, motif distribution, gene structure analysis, and promoter cis-element analysis
The amino acid sequences of HD-Zip I proteins in A. thaliana were downloaded from the Arabidopsis Information Resource (TAIR) database (https://www.arabidopsis.org/). The amino acid sequences of HD-Zip I proteins in N. tabacum, O. sativa, Zea mays, and S. indicum were downloaded from the Plant Transcription Factor Database (PlantTFDB; http://planttfdb.gao-lab.org/). The phylogenetic tree of HD-Zip I proteins was constructed in MEGA-X using the neighbor-joining(NJ) method with 1,000 bootstrap replications (Kumar et al., 2018). The online software Multiple EM for Motif Elicitation (MEME; https://meme-suite.org/meme/) with a total of 20 motifs was used to investigate the conserved motifs of the SmHD-Zip I (Li et al., 2019b). The gene structure of all genes was displayed using the Gene Structure Display Server (GSDS; Hu et al., 2015). About 2,000 bp of the genomic DNA sequence of SmHD-Zip I genes, upstream of the starting codon (ATG), were selected for the analysis of cis-elements in the promoter using the Plant Care website (Lescot, 2002).
Plant materials and stress treatments
The S. miltiorrhiza cultivar ‘Huadan 2’ was used in this study. To analyze tissue-specific gene expressions, four tissues, including the root, stem, leaf, and flower, were collected from two-year-old S. miltiorrhiza plants at Shandong University of Traditional Chinese Medicine Medicinal Botanical Garden. The hairy roots of S. miltiorrhiza were used for abiotic stress tests. The hairy roots were cultured in 50 mL of liquid 6,7-V medium on an orbital shaker (Shandong, China) running at 120 rpm and then kept at 25 °C in darkness. The 18-day-old hairy roots were then treated with 100 µmol/L ABA, 20% PEG-6000, and 200 mmol/L NaCl, respectively. Samples were harvested at 0, 3, 6, and 9 h after treatments (Zhang et al., 2012; Bai et al., 2020). The samples were immediately frozen in liquid nitrogen and stored at −80 °C for RNA extraction. All treatments were replicated with at least three independent biological experiments.
RNA extraction and expression of SmHD-Zip I genes
Total RNA was isolated from all samples using the FastPure Plant Total RNA Isolation Kit (Vazyme, China); 1 µg of total RNA was used for reverse-transcription into the first strand of cDNA using the PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa, China). The TB Green Premix Ex Taq (TaKaRa, China) was used for real-time PCR according to the manufacturer’s instructions, and qRT-PCR was performed using a CFX96 Real-Time System (Bio-Rad, USA). The β-actin gene was chosen as the reference gene. Primers were designed using Primer3 and the NCBI database was used to identify their specificity. The primers used in this study are listed in Table S1. Relative expression was calculated using by the 2−ΔΔCt method (Schmittgen & Livak, 2008).
Subcellular localization of SmHD-Zip12
To determine the subcellular localization of SmHD-Zip12, the open reading frame (ORF) of SmHD-Zip12 was cloned into the pMDC202-GFP vector to form the pMDC202-SmHD-Zip12-GFP recombinant vector. The empty GFP vector and the fusion vector were transferred to Agrobacterium tumefaciens GV3101 and injected into tobacco leaf cells using a needleless syringe, respectively. The fluorescent signal was observed under the laser confocal microscope after two days of culture in the injected tobacco leaves under low light.
Plant expression vector construction
To produce the overexpression constructs of SmHD-Zip12, the ORF sequence was amplified and cloned into the pMDC202 vector using homologous recombination. The recombinant plasmids were transformed into Agrobacterium tumefaciens Ar.Qual, which was then introduced into S. miltiorrhiza leaf explants to obtain transgenic or wild type hairy roots. Four independent lines were used in these experiments for gene expression and metabolite analyses.
Determination of tanshinones contents
The hairy roots of the same transformation period were cultured in the 6,7-V liquid medium, taken out after 30 days of growth, and flash-frozen in liquid nitrogen at −80 °C for a high-performance liquid chromatography (HPLC) analysis. The extraction of tanshinones was conducted following the methods described by Li et al. (2019a) with modification. The hairy roots were freeze-dried for 48 h, then 0.25 g powdered hairy root was placed in a 50 mL centrifuge tube, and 25 mL methanol was added. The sample was then subjected to ultrasound for 50 min, at 100 W, 100 Hz, 50 °C and then centrifuged at 4,000 rpm for 10 min. The supernatant was removed and filtered through a 0.22 µm membrane, and the tanshinone content was measured using HPLC.
Data statistics and analysis
The statistical analysis was performed using IBM’s SPSS Statistics 22 software and the Student’s t-test. All experiments were repeated in triplicate. Significant differences were considered at p < 0.01 and p < 0.05.
Results
Identification of SmHD-Zip I
A total of 25 HD-Zip I proteins, containing the complete HD and LZ domains, were obtained from the S. miltiorrhiza genome and named SmHD-Zip1 to SmHD-Zip25. All these nucleotide sequences were uploaded to NCBI with GenBank accession numbers. The amino acid sequence length of SmHD-Zip I was found to vary from 121 aa (SmHD-Zip 1) to 471 aa (SmHD-Zip 10), with an average length of 239 aa. The predicted MW of the SmHD-Zip I proteins ranged from 13.89 to 53.52 kDa. The predicted pI of the SmHD-Zip I proteins ranged from 4.48 (SmHD-Zip 8) to 9.85 (SmHD-Zip 17). An analysis of the protein instability index showed that SmHD-Zip I proteins were unstable. The aliphatic index of the protein sequences ranged from 55.24 (SmHD-Zip 6) to 79.75 (SmHD-Zip 1). The predicted GRAVY of SmHD-Zip I proteins revealed hydrophilicity. The subcellular localization prediction indicated that five SmHD-Zip I proteins were localized to the chloroplast, whereas the remaining SmHD-Zip I proteins were located in the nucleus (Table S2).
Phylogenetic analysis of SmHD-Zip I
A phylogenetic tree was constructed of the sequence information of S. miltiorrhiza, A. thaliana, N. tabacum, O. sativa, Z. mays, and S. indicum HD-Zip I proteins (Table S3), to reveal the evolutionary relationships between the proteins. All HD-Zip I proteins were divided into 11 clades: α, β1, β2, γ, δ, ɛ, ζ, η, θ, φ1, and φ2 (Henriksson et al., 2005; Zhao et al., 2011; Li et al., 2019b; Fig. 1). SmHD-Zip I proteins were found in clades α, β1, β2, γ, ɛ, and η. Clades ɛ, η, and θ each contained five proteins. Clade β1 only contained the SmHD-Zip8 protein. These results provide some evidence for the functional prediction of HD-Zip I proteins in S. miltiorrhiza.
Figure 1. Phylogenetic tree of HD-Zip I proteins among S. miltiorrhiza, A. thaliana, N. tabacum, O. sativa, Z. mays, and S. indicum.
The phylogenetic tree was generated using the neighbor-joining (NJ) method in MEGA X with 1,000 bootstrap replicates. Proteins from S. miltiorrhiza, A. thaliana, N. tabacum, O. sativa, Z. mays, and S. indicum are denoted by red circles, green squares, blue diamonds, black diamonds, yellow triangles, and purple triangles, respectively.
Gene structure and conserved motifs analysis
To get insight into the structural diversity of SmHD-Zip I genes, the exon-intron structures were analyzed using GSDS online server. The results showed that the number of introns per SmHD-Zip I gene varied from one to five, except for SmHD-Zip14, which had no intron (Fig. 2B). SmHD-Zip10 contained the largest number of introns (5). Members of clades α and η had one to two introns, members of clade ɛ contained two to four introns, and member of clade γ contained one intron.
Figure 2. Phylogenetic relationships (A), gene structure (B), and conserved motifs (C) in SmHD-Zip I from S. miltiorrhiza.
(A) Phylogenetic tree of SmHD-Zip I. (B) Intron and exon structures of SmHD-Zip I. Yellow boxes indicate exons, blue boxes indicate untranslated 5′-and 3′-regions, and black lines indicate introns. (C) Conserved motifs. The motifs are displayed in different colored boxes. The sequence information for each motif is provided in Table S4.
The conserved motifs of the SmHD-Zip I proteins were analyzed and identified using the MEME online service. The predictive analysis showed that all 25 SmHD-Zip I proteins except SmHD-Zip1, SmHD-Zip12, SmHD-Zip16, SmHD-Zip23, and SmHD-Zip25, have motifs 1, 2, and 3. The results suggested that motif 1 and motif 3 encoded the HD domain, and motif 2 encoded the LZ domain (Fig. 2C, Table S4). In general, HD-Zip I protein members of the same clade possessed similar motifs. Members of clade α displayed motifs 8, 14, and 16, with SmHD-Zip5 additionally containing motif 19. Members of clade β2, SmHD-Zip11 and SmHD-Zip13, contained motif 15 and motif 17, with SmHD-Zip13 additionally containing motif 8. Motif 6 was displayed in clade θ and motif 7 was displayed in clade ɛ.
Cis-element analysis in the promoter region of SmHD-Zip I genes
To elucidate the possible regulatory mechanism of SmHD-Zip I genes, we used the Plant Care website to analyzed the promoter sequence of 2,000 bp of the genomic DNA sequence of SmHD-Zip I genes upstream of the starting codon (ATG; Table 1). We identified a total of 20 cis-elements. Among these cis-elements, six were responsive to hormones, four were responsive to stress, three were related to growth, and seven were associated with light responses. Among the hormone-related elements identified, ABRE (ABA-responsiveness) had the largest number, followed by CGTCA-motif (MeJA-responsiveness) and TGACG-motif (MeJA-responsiveness). TCA-element (SA-responsiveness), TGA-element (IAA-responsiveness), and P-box (IAA-responsiveness) were also found in the analyzed upstream region of the SmHD-Zip I gene. Many elements were identified that are related to stress response, including MBS, which is an MYB-binding site related to drought induction; 12 genes contained 1-2 MBS. ARE, LTR, and TC-rich repeats were also observed in the promoter region of the SmHD-Zip I gene. A few SmHD-Zip I genes contained growth-related elements, such as CAT-box (involved in meristem expression), circadian (involved in circadian rhythm control), and HD-Zip I (involved in differentiation of the palisade mesophyll cells). Light-responsive elements were also identified in the promoter region of all 25 SmHD-Zip I genes, except for the SmHD-Zip17 and SmHD-Zip19 gene, which contained the Box4 element. The distribution of these cis-elements in the promoter region of the SmHD-Zip I genes suggested that gene expression could be regulated at different growth stages and in response to environmental stimuli.
Table 1. Analysis of cis-elements in SmHD-Zip I genes.
| Functional class | Cis-element | Correlation | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | 25 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Hormone | ABRE | ABA-responsiveness | – | 1 | 3 | 1 | 3 | 1 | 1 | 2 | 0 | 6 | 3 | 6 | 0 | 3 | 2 | 11 | 0 | 1 | 3 | 2 | 0 | 7 | 4 | 4 | 3 |
| CGTCA-motif | MeJA-responsiveness | 3 | 2 | 5 | 0 | 1 | 3 | 1 | 1 | 2 | 5 | 1 | 2 | 1 | 2 | 4 | 0 | 0 | 3 | 1 | 0 | 0 | 0 | 2 | 3 | ||
| TGACG-motif | MeJA-responsiveness | 3 | 2 | 5 | 0 | 1 | 3 | 1 | 1 | 2 | 0 | 1 | 2 | 1 | 2 | 4 | 0 | 0 | 3 | 1 | 0 | 0 | 0 | 2 | 3 | ||
| TCA-element | SA-responsiveness | 1 | 1 | 2 | 2 | 0 | 1 | 3 | 0 | 1 | 0 | 1 | 1 | 1 | 2 | 0 | 0 | 0 | 3 | 0 | 0 | 0 | 0 | 0 | 0 | ||
| TGA-element | IAA-responsiveness | 0 | 0 | 2 | 1 | 0 | 0 | 0 | 2 | 0 | 2 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1 | ||
| P-box | GA-responsiveness | 0 | 0 | 2 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 1 | 0 | 0 | 1 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | ||
| Stress | ARE | anaerobic induction | – | 3 | 4 | 1 | 0 | 3 | 0 | 3 | 0 | 1 | 0 | 1 | 1 | 1 | 0 | 2 | 0 | 1 | 2 | 2 | 0 | 0 | 1 | 1 | 0 |
| LTR | low-temperature responsiveness | 2 | 3 | 0 | 1 | 3 | 1 | 0 | 0 | 0 | 0 | 1 | 1 | 4 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 0 | ||
| MBS | MYB binding site involved in drought-inducibility | 0 | 0 | 0 | 3 | 1 | 1 | 1 | 0 | 0 | 1 | 1 | 2 | 1 | 2 | 2 | 0 | 1 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | ||
| TC-rich repeats | defense and stress responsiveness | 0 | 0 | 0 | 0 | 2 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 1 | 1 | 0 | 0 | 1 | 0 | 1 | 1 | 0 | 0 | 1 | 0 | ||
| Development | CAT-box | meristem expression | – | 1 | 0 | 0 | 0 | 0 | 1 | 1 | 0 | 0 | 1 | 0 | 4 | 1 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 |
| circadian | circadian control | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 1 | 0 | 0 | 1 | 1 | 1 | 2 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 1 | 0 | ||
| HD-Zip 1 | differentiation of palisade mesophyll cells | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 2 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | ||
| Light | Box 4 | light responsiveness | – | 11 | 7 | 1 | 4 | 9 | 5 | 4 | 1 | 4 | 5 | 1 | 8 | 2 | 3 | 6 | 0 | 6 | 0 | 8 | 2 | 9 | 5 | 12 | 4 |
| G-Box | light responsiveness | 0 | 2 | 0 | 0 | 1 | 0 | 1 | 0 | 6 | 2 | 2 | 0 | 1 | 0 | 5 | 0 | 0 | 1 | 1 | 0 | 2 | 2 | 1 | 1 | ||
| G-box | light responsiveness | 0 | 0 | 3 | 2 | 1 | 1 | 0 | 0 | 0 | 1 | 3 | 0 | 2 | 1 | 5 | 0 | 2 | 1 | 2 | 0 | 6 | 2 | 2 | 4 | ||
| GT1-motif | light responsiveness | 0 | 0 | 0 | 1 | 4 | 0 | 2 | 3 | 1 | 1 | 2 | 0 | 0 | 0 | 1 | 0 | 3 | 0 | 0 | 1 | 2 | 0 | 0 | 1 | ||
| I-box | light responsiveness | 0 | 0 | 1 | 0 | 0 | 1 | 1 | 0 | 0 | 0 | 2 | 0 | 0 | 1 | 0 | 0 | 0 | 1 | 0 | 1 | 1 | 1 | 2 | 0 | ||
| MRE | light responsiveness | 1 | 0 | 0 | 0 | 3 | 0 | 1 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | ||
| TCT-motif | light responsiveness | 0 | 2 | 2 | 1 | 1 | 1 | 0 | 1 | 1 | 0 | 0 | 0 | 0 | 5 | 2 | 0 | 1 | 0 | 1 | 1 | 1 | 0 | 3 | 2 |
Expression profiles of SmHD-Zip I genes in different tissues
To study the potential biological functions of the SmHD-Zip I genes, we analyzed the expression profiles of SmHD-Zip I genes in different tissues, including the root, stem, leaf, and flower by qRT-PCR. As shown in Fig. 3, SmHD-Zip1, SmHD-Zip4, SmHD-Zip15, SmHD-Zip18, SmHD-Zip21, and SmHD-Zip23 genes had the highest expression levels in the root; SmHD-Zip7, SmHD-Zip11, SmHD-Zip13, and SmHD-Zip24 genes had the highest expression levels in the stem; SmHD-Zip9, SmHD-Zip10, SmHD-Zip14, SmHD-Zip17, and SmHD-Zip19 genes had the highest expression level in leaf; and SmHD-Zip2, SmHD-Zip3, SmHD-Zip5, SmHD-Zip6, SmHD-Zip8, SmHD-Zip12, SmHD-Zip16, SmHD-Zip20, SmHD-Zip22, and SmHD-Zip25 genes had the highest expression levels in the flower. The results indicated that HD-Zip I genes might play an important function in plant growth and development.
Figure 3. Expression analysis of the HD-Zip I genes in S. miltiorrhiza.
The 2−ΔΔCT method was used to calculate relative expression. Heatmap of the expression of SmHD-Zip I in different tissues, including the root, stem, leaf, and flower. –1.50 to 1.50 were artificially set with the color scale limits according to the normalized value. The gradient from blue to red represents low to high expression.
Expression analysis of SmHD-Zip I genes in response to ABA, PEG, and NaCl
To investigate whether SmHD-Zip I genes respond to abiotic stress, the expression patterns of SmHD-Zip I genes were analyzed in the hairy root of S. miltiorrhiza under different stress conditions including ABA, PEG, and NaCl.
As shown in Fig. 4A, the expression levels of three SmHD-Zip I genes (SmHD-Zip12, SmHD-Zip20, and SmHD-Zip25) significantly increased, by more than 3-fold, at 3 h, 6 h, and 9 h post-ABA treatment compared to the control. SmHD-Zip20 and SmHD-Zip25 expression reached a maximum level at 3 h that was more than 7-fold higher than the control. The SmHD-Zip12 gene responded to ABA the most strongly, with expression levels 80.00-fold higher than the control at 3 h post-ABA treatment. Compared with expression levels 3 h post-ABA treatment, the expression levels of SmHD-Zip12, SmHD-Zip20, and SmHD-Zip25 declined continuously at 6 h and 9 h post-ABA treatment, but remained higher than the control. At 9 h, the expression levels of the SmHD-Zip12, SmHD-Zip20, and SmHD-Zip25 were 40.01-fold, 4.74-fold, and 6.58-fold higher than the control, respectively. The expression levels of the genes of the other 22 SmHD-Zip I genes only slightly increased under ABA treatment, with expression levels less than 3-fold the control at all time points (Fig. S1A).
Figure 4. The expression patterns of SmHD-Zip I at 0, 3, 6, and 9 h after ABA (A), PEG (B), and NaCl (C) treatment.
Hairy roots were cultured in a 6,7-V liquid medium for 18 days before being treated. The 2−ΔΔCT method was used to calculate relative expression. SmActin was used as the internal standard. Asterisk (** and *) indicate significant differences compared to the control (0 h) at p < 0.01 and p < 0.05, respectively.
As shown in Fig. 4B, the expression levels of SmHD-Zip12 and SmHD-Zip16 significantly increased, by 3.66-fold and 16.18-fold, respectively, at 3 h post-PEG treatment compared to the control. At 6 h post-PEG treatment, SmHD-Zip12, SmHD-Zip16, and SmHD-Zip17 expression levels increased more than 3-fold compared to the control. At 9 h post-PEG treatment, the expression of the SmHD-Zip12 gene continued to increase to levels 25.43-fold higher than the control. The expression level of SmHD-Zip25 increased 3.16-fold compared to the control at 9 h. The expression levels of the genes of the other 21 SmHD-Zip I genes only slightly increased under PEG treatment, with expression levels less than 3-fold the control at all time points (Fig. S1B).
As shown in Fig. 4C, the expression levels of three genes (SmHD-Zip12, SmHD-Zip20, and SmHD-Zip25) significantly increased, by more than 3-fold, at 3 h post-NaCl treatment compared to the control. The expression levels of SmHD-Zip20 and SmHD-Zip25 increased 7.72-fold and 9.27-fold, respectively. SmHD-Zip20 reached its highest expression level at 3 h. At 3 h post-NaCl treatment, the expression level of SmHD-Zip 12 was 24.72-fold higher than the control. At 6 h post-NaCl treatment, the expression level of the SmHD-Zip12 gene reached a level 51.00-fold higher than the control. SmHD-Zip17 and SmHD-Zip25 expression peaked at 6 h post-NaCl treatment with expression levels more than 7-fold and 15-fold higher than the control, respectively. At 9 h post-NaCl treatment, the expression levels of SmHD-Zip12, SmHD-Zip20, and SmHD-Zip25 were lower compared to 6 h, but remained higher than the control. The expression level of the genes of the other 21 SmHD-Zip I genes only slightly increased under NaCl treatment, with expression levels less than 3-fold the control at all time points (Fig. S1C).
These results indicate that the SmHD-Zip 12 gene quickly and strongly responds to ABA, PEG, and NaCl.
Subcellular localization of SmHD-Zip12
Generally, the physiological function of a protein is linked to its intracellular location. The protein subcellular localization prediction result showed that the SmHD-Zip12 protein was localized in the nucleus (Table S2). To verify this prediction for the SmHD-Zip12 protein, the fusion protein vector pMDC202-SmHD-Zip12-GFP was constructed. As shown in Fig. 5, tobacco leaf epidermal cells with pMDC202-SmHD-Zip12-GFP fusion protein showed fluorescence signals only in the nucleus under microscope, whereas the control GFP protein was diffused throughout the cell, suggesting that SmHD-Zip12 is a nuclear-localized protein.
Figure 5. Subcellular localization analysis of SmHD-Zip12 in tobacco leaf cells.
The pMDC202-SmHD-Zip12-GFP (upper lane) and pMDC202-GFP (bottom lane) plasmids were transiently expressed in tobacco leaf cells. Fluorescence was observed using a confocal laser scanning microscope. The pictures show fluorescence, nucleus fluorescence, bright, and merge of four fields from left to right.
SmHD-Zip12 was involved in the tanshinone biosynthesis
To further study the functions of SmHD-Zip12, the SmHD-Zip12 over-expressed (OE) vector was constructed and transgenic hairy root lines were obtained. The desired transgenic lines were identified by PCR using rolB and SmHD-Zip12-specific primers, with a positive rate of 70.59% (Figs. S2A and S2B). Four SmHD-Zip12 over-expression lines(OE1, OE2, OE3, and OE4) were selected for further experiments based on SmHD-Zip12 expression level using qRT-PCR (Fig. S2C).
Cryptotanshinone (CT), dihydrotanshinone I (DT-I), tanshinone I (T-I), and tanshinone IIA (T-IIA) content in SmHD-Zip12 overexpression hairy roots were measured by HPLC (Fig. 6). The results showed that the overexpression of SmHD-Zip12 significantly increased CT, DT-I, T-I, and T-IIA contents, by 2.89-fold, 1.85-fold, 2.14-fold, and 8.91-fold, respectively compared with the wild type. In particular, the content of T-IIA in overexpression line 2 was 15.31-fold (365.48 µg/g DW) higher than the wild type (23.86 µg/g DW).
Figure 6. Cryptotanshinone, dihydrotanshinone I, tanshinone I, and tanshinone IIA content in hairy roots of S. miltiorrhiza, measured by HPLC.
Tanshinone concentration in the wild type (WT), empty vector (EV), and the four independent SmHD-Zip12-overexpressed (OE) transgenic hairy root lines. Asterisks (** and *) indicate significant differences compared to the control (WT) at p < 0.01 and p < 0.05, respectively.
The key enzyme genes in the biosynthesis pathway of tanshinones in overexpression SmHD-Zip12 hairy roots were analyzed by qRT-PCR (Fig. 7). The results showed that the relative expression levels of SmAACT, SmDXS, SmIDS, SmGGPPS, SmCPS1, SmCPS2, SmCYP76AH1, SmCYP76AH3, and SmCYP76AK1 in the overexpression lines were significantly up-regulated compared with the wild type. In particular, the expression levels of the SmGGPPS gene and SmCYP76AH3 gene in overexpression line 2 were 31.63-fold and 13.16-fold higher, respectively, compared with the wild type.
Figure 7. Relative expression analysis of nine tanshinone biosynthesis genes (SmAACT, SmDXS, SmIDS, SmGGPPS, SmCPS1, SmCPS2, SmCYP76AH1, SmCYP76AH3, and SmCYP76AK1) by qRT-PCR.
Asterisks (** and *) indicate significant differences compared with the wild type (WT) at p < 0.01 and p < 0.05, respectively. The solid and dashed lines represent one-step and multi-step reactions, respectively.
Discussion
HD-Zip I transcription factors are plant-specific proteins that play important roles in the growth of plants. There are 17 HD-Zip I genes in A. thaliana (Henriksson et al., 2005), 15 HD-Zip I genes in Eucalyptus grandis (Zhang et al., 2020), 20 HD-Zip I genes in Triticum aestivum (Yue et al., 2018), 16 HD-Zip I genes in Hordeum vulgare (Li et al., 2019d), and 30 HD-Zip I genes in Glycine max (Chen et al., 2014) , but little is known about HD-Zip I genes in S. miltiorrhiza. In this study, we identified 25 HD-Zip I genes in S. miltiorrhiza and then systematically analyzed their characteristics. Gene duplication and gene loss have resulted in different numbers of HD-Zip I genes during evolution (Que et al., 2018). The results of this study provide useful information for the functional characterization of SmHD-Zip I gene family.
Our conserved domain analysis showed that SmHD-Zip I proteins contained the conserved HD domain and the LZ domain, which is characteristic of HD-Zip I transcription factors (Liu et al., 2013; Zhang et al., 2014; Mehmood et al., 2022). The conserved motifs can reflect their protein-specific functions (Li et al., 2014; Liu et al., 2022). A motifs analysis showed that motifs 1 and 3 encoded the HD domain, which was involved in DNA binding (Zhang et al., 2014). Motif 2 encoded the LZ domain, which was involved in protein-protein interactions (Ruberti et al., 1991; Ariel et al., 2007). Similar results were found in Capsicum annuum (Zhang et al., 2021) and Medicago truncatula (Li et al., 2022). The evolutionary characteristics of the exon-intron structure have a role in the evolution of functions in paralogs (Boudet et al., 2001). The CDS was discontinuous due to the presence of introns. Furthermore, the loss or increase of introns may lead to changes in the length of gene introns (Zhang et al., 2020).
Cis-acting elements located upstream of transcription initiation sites control the regulation of gene expression (Hernandez-Garcia & Finer, 2014). ABRE (ABA-responsive element), as a decisive cis-element in most gene promoters, is involved in ABA regulation. ABRE belongs to the G-box family and contains an ACGT core, a sequence that is recognized by plant bZIP proteins (Gómez-Porras et al., 2007; Fujita et al., 2011). There are four ABREs in HA-HB4, one of which is responsible for the regulation of ABA, NaCl, and drought-related stress (Manavella et al., 2008). ABRE and MBS cis-acting elements that respond to hormones and environmental stresses were identified in the MdHB-1 gene promoter region (Wang et al., 2017a). In this study, ABREs were found in 20 SmHD-Zip I genes, which were also identified in M. truncatula (Li et al., 2022). TaHDZipI-3, TaHDZipI-4, and TaHDZipI-5 genes were characterized to bind to stress-related cis-elements and were differentially expressed under ABA treatment, cold treatment and water deficit (Harris et al., 2016; Mehmood et al., 2022). MBS cis-element was found in half of the SmHD-Zip I genes, and one to four LTR cis-elements were found in ten of the SmHD-Zip I genes, suggesting that SmHD-Zip I genes may be involved in the plant’s response to drought stress and low-temperature stress. In addition, growth-related elements and light-responsive elements were also found in SmHD-Zip I gene promoters. These results contribute to the understanding of the diverse transcriptional regulatory mechanisms of SmHD-Zip I genes in S. miltiorrhiza.
The HD-Zip I proteins play a role in the plant’s response to ABA and abiotic stresses (Gong et al., 2019). In M. truncatula, MtHB12 was highly inducible by salt, osmotic, and ABA stresses (Li et al., 2022). Overexpression of CaHDZ12 tobacco lines improved the plant’s tolerance to water deficit and salt stress and increased its sensitivity to ABA (Sen et al., 2017). In A. thaliana, ATHB12 was induced by ABA, water deficit, and NaCl (Shin et al., 2004; Valdés et al., 2012). A T-DNA insertion mutant of ATHB12 reduced sensitivity to ABA during plant germination (Son et al., 2010). OsHOX24 and OsHOX22 in rice, which are homologous genes of ATHB12, were highly expressed when subjected to stresses such as salinity and osmotic stress (Bhattacharjee, Khurana & Jain, 2016). OsHOX24 participates in the regulation of abiotic stress responses by regulating the expression of stress-responsive genes in rice (Bhattacharjee, Sharma & Jain, 2017). In this study, expression levels of HD-Zip I genes in S. miltiorrhiza were also induced under different stresses. Notably, the expression of SmHD-Zip12 increased under ABA, PEG, and NaCl stresses significantly more than the other genes. The expression level of SmHD-Zip12 was 80.00-fold higher at 3 h post-ABA exposure, 25.43-fold higher at 9 h post-PEG exposure, and 51.00-fold higher at 6 h post-NaCl exposure than under control conditions, respectively. Based on these results, we speculated that the SmHD-Zip12 gene participates in regulating abiotic stress.
Most transcription factors play the role of regulators in vivo and are often located in the nucleus (Wang et al., 2017b). GFP fusion protein in the leaves of tobacco showed that SmHD-Zip12 is localized exclusively in the nucleus, indicating that SmHD-Zip12 functions as a transcription factor. The results helped to further understand the mechanism of action of SmHD-Zip12. Studies have revealed the role of HD-Zip I genes in responses to abiotic stress (Manavella et al., 2006; Bhattacharjee, Sharma & Jain, 2017; Sen et al., 2017). In addition, abiotic stress could promote the accumulation of tanshinones (Zhao, Zhou & Wu, 2010; Liu et al., 2011a; Yang et al., 2012; Wei et al., 2016). Therefore, we speculated that HD-Zip I is closely related to tanshinone biosynthesis. In this study, the overexpression of SmHD-Zip12 significantly increased the contents of CT, DT-I, T-I, and T-IIA. Most of the key enzyme genes in the tanshinone synthesis pathway have now been cloned and identified. GGPPS is considered an important regulatory target in the tanshinone biosynthetic pathway, and overexpression of SmGGPPS could increase the content of tanshinones (Kai et al., 2011; Shi et al., 2016). CYP76AH3 plays a key role in the production of intermediates, and silencing SmCYP76AH3 reduced the content of tanshinones (Guo et al., 2016). In this study, most of the genes, such as SmAACT, SmDXS, SmIDS, SmGGPPS, SmCPS1, SmCPS2, SmCYP76AH1, SmCYP76AH3, and SmCYP76AK1, were up-regulated in SmHD-Zip12 overexpression lines, specifically the SmGGPPS and SmCYP76AH3 gene. Our results demonstrated that SmHD-Zip12 could regulate tanshinone biosynthesis through the activation of the key enzyme genes of the tanshinone synthesis pathway.
Conclusions
In the present study, a total of 25 HD-Zip I transcription factors were identified in S. miltiorrhiza. A phylogenetic analysis was performed and the gene structures, motifs, cis-elements, tissue-specific expression levels, and stress-induced expression levels were determined. Notably, SmHD-Zip12 was highly up-regulated after ABA, PEG, and NaCl stress. The over-expression of SmHD-Zip12 promoted tanshinone accumulation and the expression of the key enzyme genes in the tanshinone synthetic pathway. These results indicate that SmHD-Zip12 is a positive regulator of tanshinone biosynthesis in S. miltiorrhiza. This study provides abundant information for the future investigation of more potential HD-Zip I genes in S. miltiorrhiza.
Supplemental Information
Hairy roots were cultured in a 6,7-V liquid medium for 18 days before being treated. The 2−ΔΔCT method was used to be an evaluation of the relative expression. SmActin was used as the internal standard. ** and * indicate significant differences compared to the control (0 h) at p < 0.01 and p < 0.05, respectively.
Hairy roots were cultured in a 6,7-V liquid medium for 18 days before being treated. The 2−ΔΔCT method was used to be an evaluation of the relative expression. SmActin was used as the internal standard. ** and * indicate significant differences compared to the control (0 h) at p < 0.01 and textitp < 0.05, respectively.
M, DL-2000; 1, WT; 2, EV; 3-10, Overexpression of SmHD-Zip 12. (B)PCR analyses of transgenic hairy root lines by SmHD-Zip12-specific primers. M, DL-2000; N1: H2O; N2: WT-PC: Recombinant plasmid; 1–17: Overexpression of SmHD-Zip 12. (C)Relative expression analysis of SmHD-Zip 12 genes by qRT-PCR in in transgenic lines and WT. ** and * indicate significant differences compared with the wild type (WT) at p < 0.01 and p < 0.05, respectively.
Acknowledgments
We would like to thank the Youth Innovation Team of Higher Education Department of Shandong Province and the Sustainable Use Youth Innovation Team of Shandong University of Traditional Chinese Medicine for their support for this article.
Funding Statement
This work was supported by the National Natural Science Foundation of China (82003892); National Modern Agricultural Industry Technology System (CARS-21); the Natural Science Foundation of Shandong Province (ZR2021QH202); and the Traditional Chinese Medicine Science and Technology Project of Shandong Province (2021Q074). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Contributor Information
Xue Chen, Email: xchenaylli@sina.com.
Qian Liu, Email: cleanlq@163.com.
Additional Information and Declarations
Competing Interests
The authors declare there are no competing interests.
Author Contributions
Yanhong Bai conceived and designed the experiments, performed the experiments, analyzed the data, authored or reviewed drafts of the article, and approved the final draft.
Ying Zhou performed the experiments, prepared figures and/or tables, and approved the final draft.
Qiaoqi Lei performed the experiments, prepared figures and/or tables, and approved the final draft.
Yu Wang performed the experiments, prepared figures and/or tables, and approved the final draft.
Gaobin Pu analyzed the data, prepared figures and/or tables, and approved the final draft.
Zhenhua Liu analyzed the data, prepared figures and/or tables, and approved the final draft.
Xue Chen conceived and designed the experiments, analyzed the data, authored or reviewed drafts of the article, and approved the final draft.
Qian Liu conceived and designed the experiments, analyzed the data, authored or reviewed drafts of the article, and approved the final draft.
DNA Deposition
The following information was supplied regarding the deposition of DNA sequences:
Sequence data is available at NCBI accession numbers: OP607147-OP607171
Data Availability
The following information was supplied regarding data availability:
The raw data are available in the Supplemental File.
References
- Arce et al. (2011).Arce AL, Raineri J, Capella M, Cabello JV, Chan RL. Uncharacterized conserved motifs outside the HD-Zip domain in HD-Zip subfamily I transcription factors; a potential source of functional diversity. BMC Plant Biology. 2011;11:42. doi: 10.1186/1471-2229-11-42. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ariel et al. (2007).Ariel FD, Manavella PA, Dezar CA, Chan RL. The true story of the HD-Zip family. Trends in Plant Science. 2007;12:419–426. doi: 10.1016/j.tplants.2007.08.003. [DOI] [PubMed] [Google Scholar]
- Bai et al. (2020).Bai Z, Wu J, Huang W, Jiao J, Zhang C, Hou Z, Yan K, Zhang X, Han R, Liang Z, Zhang X. The ethylene response factor SmERF8 regulates the expression of SmKSL1 and is involved in tanshinone biosynthesis in Saliva miltiorrhiza hairy roots. Journal of Plant Physiology. 2020;244:153006. doi: 10.1016/j.jplph.2019.153006. [DOI] [PubMed] [Google Scholar]
- Bhattacharjee, Khurana & Jain (2016).Bhattacharjee A, Khurana JP, Jain M. Characterization of Rice Homeobox Genes, OsHOX22 and OsHOX24, and over-expression of OsHOX24 in transgenic Arabidopsis suggest their role in abiotic stress response. Frontiers in Plant Science. 2016;7:627. doi: 10.3389/fpls.2016.00627. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bhattacharjee, Sharma & Jain (2017).Bhattacharjee A, Sharma R, Jain M. Over-expression of OsHOX24 confers enhanced susceptibility to abiotic stresses in transgenic rice via modulating stress-responsive gene expression. Frontiers in Plant Science. 2017;8:628. doi: 10.3389/fpls.2017.00628. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Boudet et al. (2001).Boudet N, Aubourg S, Toffano-Nioche C, Kreis M, Lecharny A. Evolution of intron/exon structure of DEAD helicase family genes in Arabidopsis, Caenorhabditis, and Drosophila. Genome Research. 2001;11:2101–2114. doi: 10.1101/gr.200801. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bürglin & Affolter (2016).Bürglin TR, Affolter M. Homeodomain proteins: an update. Chromosoma. 2016;125:497–521. doi: 10.1007/s00412-015-0543-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cabello et al. (2017).Cabello JV, Giacomelli JI, Gómez MC, Chan RL. The sunflower transcription factor HaHB11 confers tolerance to water deficit and salinity to transgenic Arabidopsis and alfalfa plants. Journal of Biotechnology. 2017;257:35–46. doi: 10.1016/j.jbiotec.2016.11.017. [DOI] [PubMed] [Google Scholar]
- Chen et al. (2021).Chen J, Li N, Wang X, Meng X, Cui X, Chen Z, Ren H, Ma J, Liu H. Late embryogenesis abundant (LEA) gene family in Salvia miltiorrhiza: identification, expression analysis, and response to drought stress. Plant Signaling & Behavior. 2021;16:1891769. doi: 10.1080/15592324.2021.1891769. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chen et al. (2014).Chen X, Chen Z, Zhao H, Zhao Y, Cheng B, Xiang Y. Genome-wide analysis of soybean HD-Zip gene family and expression profiling under salinity and drought treatments. PLOS ONE. 2014;9:e87156. doi: 10.1371/journal.pone.0087156. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chou & Shen (2008).Chou K-C, Shen H-B. Cell-PLoc: a package of Web servers for predicting subcellular localization of proteins in various organisms. Nature Protocols. 2008;3:153–162. doi: 10.1038/nprot.2007.494. [DOI] [PubMed] [Google Scholar]
- Elhiti & Stasolla (2009).Elhiti M, Stasolla C. Structure and function of homodomain-leucine zipper (HD-Zip) proteins. Plant Signaling & Behavior. 2009;4:86–88. doi: 10.4161/psb.4.2.7692. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Franco-Zorrilla & Solano (2017).Franco-Zorrilla JM, Solano R. Identification of plant transcription factor target sequences. Biochimica et Biophysica Acta (BBA)—Gene Regulatory Mechanisms. 2017;1860:21–30. doi: 10.1016/j.bbagrm.2016.05.001. [DOI] [PubMed] [Google Scholar]
- Fujita et al. (2011).Fujita Y, Fujita M, Shinozaki K, Yamaguchi-Shinozaki K. ABA-mediated transcriptional regulation in response to osmotic stress in plants. Journal of Plant Research. 2011;124:509–525. doi: 10.1007/s10265-011-0412-3. [DOI] [PubMed] [Google Scholar]
- Gasteiger et al. (2005).Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch A. Protein identification and analysis tools on the ExPASy server. In: Walker JM, editor. The proteomics protocols handbook. Humana Press; Totowa: 2005. pp. 571–607. [DOI] [Google Scholar]
- Gómez-Porras et al. (2007).Gómez-Porras JL, Riaño Pachón DM, Dreyer I, Mayer JE, Mueller-Roeber B. Genome-wide analysis of ABA-responsive elements ABRE and CE3 reveals divergent patterns in Arabidopsis and rice. BMC Genomics. 2007;8:260. doi: 10.1186/1471-2164-8-260. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gong et al. (2019).Gong S, Ding Y, Hu S, Ding L, Chen Z, Zhu C. The role of HD—Zip class I transcription factors in plant response to abiotic stresses. Physiologia Plantarum. 2019;167:516–525. doi: 10.1111/ppl.12965. [DOI] [PubMed] [Google Scholar]
- Guo et al. (2016).Guo J, Ma X, Cai Y, Ma Y, Zhan Z, Zhou YJ, Liu W, Guan M, Yang J, Cui G, Kang L, Yang L, Shen Y, Tang J, Lin H, Ma X, Jin B, Liu Z, Peters RJ, Zhao ZK, Huang L. Cytochrome P450 promiscuity leads to a bifurcating biosynthetic pathway for tanshinones. The New Phytologist. 2016;210:525–534. doi: 10.1111/nph.13790. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Harris et al. (2016).Harris JC, Sornaraj P, Taylor M, Bazanova N, Baumann U, Lovell B, Langridge P, Lopato S, Hrmova M. Molecular interactions of the γ-clade homeodomain-leucine zipper class I transcription factors during the wheat response to water deficit. Plant Molecular Biology. 2016;90:435–452. doi: 10.1007/s11103-015-0427-6. [DOI] [PubMed] [Google Scholar]
- Henriksson et al. (2005).Henriksson E, Olsson ASB, Johannesson H, Johansson H, Hanson J, Engström P, Söderman E. Homeodomain leucine zipper class I genes in arabidopsis. Expression patterns and phylogenetic relationships. Plant Physiology. 2005;139:509–518. doi: 10.1104/pp.105.063461. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hernandez-Garcia & Finer (2014).Hernandez-Garcia CM, Finer JJ. Identification and validation of promoters and cis-acting regulatory elements. Plant Science. 2014;217–218:109–119. doi: 10.1016/j.plantsci.2013.12.007. [DOI] [PubMed] [Google Scholar]
- Hu et al. (2015).Hu B, Jin J, Guo A-Y, Zhang H, Luo J, Gao G. GSDS 2.0: an upgraded gene feature visualization server. Bioinformatics. 2015;31:1296–1297. doi: 10.1093/bioinformatics/btu817. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hu et al. (2012).Hu R, Chi X, Chai G, Kong Y, He G, Wang X, Shi D, Zhang D, Zhou G. Genome-wide identification, evolutionary expansion, and expression profile of homeodomain-leucine zipper gene family in poplar (Populus trichocarpa) PLOS ONE. 2012;7:e31149. doi: 10.1371/journal.pone.0031149. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Huang et al. (2014).Huang X, Duan M, Liao J, Yuan X, Chen H, Feng J, Huang J, Zhang H-S. OsSLI1, a homeodomain containing transcription activator, involves abscisic acid related stress response in rice (Oryza sativa L.) The Scientific World Journal. 2014;2014:809353. doi: 10.1155/2014/809353. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hussain et al. (2021).Hussain Q, Asim M, Zhang R, Khan R, Farooq S, Wu J. Transcription factors interact with ABA through gene expression and signaling pathways to mitigate drought and salinity stress. Biomolecules. 2021;11:1159. doi: 10.3390/biom11081159. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jiang, Gao & Huang (2019).Jiang Z, Gao W, Huang L. Tanshinones, critical pharmacological components in salvia miltiorrhiza. Frontiers in Pharmacology. 2019;10:202. doi: 10.3389/fphar.2019.00202. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Johannesson et al. (2003).Johannesson H, Wang Y, Hanson J, Engström P. The Arabidopsis thaliana homeobox gene ATHB5 is a potential regulator of abscisic acid responsiveness in developing seedlings. Plant Molecular Biology. 2003;51:719–729. doi: 10.1023/a:1022567625228. [DOI] [PubMed] [Google Scholar]
- Kai et al. (2011).Kai G, Xu H, Zhou C, Liao P, Xiao J, Luo X, You L, Zhang L. Metabolic engineering tanshinone biosynthetic pathway in Salvia miltiorrhiza hairy root cultures. Metabolic Engineering. 2011;13:319–327. doi: 10.1016/j.ymben.2011.02.003. [DOI] [PubMed] [Google Scholar]
- Kumar et al. (2018).Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution. 2018;35:1547–1549. doi: 10.1093/molbev/msy096. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lescot (2002).Lescot M. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Research. 2002;30:325–327. doi: 10.1093/nar/30.1.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Li et al. (2019a).Li W, Bai Z, Pei T, Yang D, Mao R, Zhang B, Liu C, Liang Z. SmGRAS1 and SmGRAS2 regulate the biosynthesis of tanshinones and phenolic acids in salvia miltiorrhiza. Frontiers in Plant Science. 2019a;10:1367. doi: 10.3389/fpls.2019.01367. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Li et al. (2019c).Li W, Dong J, Cao M, Gao X, Wang D, Liu B, Chen Q. Genome-wide identification and characterization of HD-ZIP genes in potato. Gene. 2019c;697:103–117. doi: 10.1016/j.gene.2019.02.024. [DOI] [PubMed] [Google Scholar]
- Li et al. (2022).Li X, Hou Y, Zhang F, Li M, Yi F, Kang J, Yang Q, Long R. Identification and characterization of stress responsive homeodomain leucine zipper transcription factors in Medicago truncatula. Molecular Biology Reports. 2022;49:3569–3581. doi: 10.1007/s11033-022-07197-4. [DOI] [PubMed] [Google Scholar]
- Li et al. (2019b).Li Y, Bai B, Wen F, Zhao M, Xia Q, Yang D-H, Wang G. Genome-wide identification and expression analysis of HD-ZIP I gene subfamily in Nicotiana tabacum. Genes. 2019b;10:575. doi: 10.3390/genes10080575. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Li et al. (2019d).Li Y, Xiong H, Cuo D, Wu X, Duan R. Genome-wide characterization and expression profiling of the relation of the HD-Zip gene family to abiotic stress in barley (Hordeum vulgare L.) Plant Physiology and Biochemistry. 2019d;141:250–258. doi: 10.1016/j.plaphy.2019.05.026. [DOI] [PubMed] [Google Scholar]
- Li et al. (2014).Li Z, Jiang H, Zhou L, Deng L, Lin Y, Peng X, Yan H, Cheng B. Molecular evolution of the HD-ZIP I gene family in legume genomes. Gene. 2014;533:218–228. doi: 10.1016/j.gene.2013.09.084. [DOI] [PubMed] [Google Scholar]
- Liu et al. (2011a).Liu D, Guo L, Huang L, Jin H, Wu L, Zeng Y, Zhang J, Yang Y. [Effects of soil water content on seedlings growth and active ingredients of Salvia miltiorrhiza] China Journal of Chinese Materia Medica. 2011a;36:321–325. [PubMed] [Google Scholar]
- Liu et al. (2011b).Liu H, Wang X, Wang D, Zou Z, Liang Z. Effect of drought stress on growth and accumulation of active constituents in Salvia miltiorrhiza Bunge. Industrial Crops and Products. 2011b;33:84–88. doi: 10.1016/j.indcrop.2010.09.006. [DOI] [Google Scholar]
- Liu et al. (2022).Liu K, Han X, Liang Z, Yan J, Cong P, Zhang C. Genome-wide identification, classification, and expression analysis of the HD-Zip transcription factor family in apple (Malus domestica Borkh.) International Journal of Molecular Sciences. 2022;23:2632. doi: 10.3390/ijms23052632. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Liu et al. (2013).Liu W, Fu R, Li Q, Li J, Wang L, Ren Z. Genome-wide identification and expression profile of homeodomain-leucine zipper class I gene family in Cucumis sativus. Gene. 2013;531:279–287. doi: 10.1016/j.gene.2013.08.089. [DOI] [PubMed] [Google Scholar]
- Lu et al. (2020).Lu X-Y, Liang X-Y, Li X, Shen P-X, Cao X-Y, Chen C, Song S-H, Wang D-H, Wang Z-Z, Zhang Y. Genome-wide characterisation and expression profiling of the LBD family in Salvia miltiorrhiza reveals the function of LBD50 in jasmonate signaling and phenolic biosynthesis. Industrial Crops and Products. 2020;144:112006. doi: 10.1016/j.indcrop.2019.112006. [DOI] [Google Scholar]
- Manavella et al. (2006).Manavella PA, Arce AL, Dezar CA, Bitton F, Renou J-P, Crespi M, Chan RL. Cross-talk between ethylene and drought signalling pathways is mediated by the sunflower Hahb-4 transcription factor. The Plant Journal. 2006;48:125–137. doi: 10.1111/j.1365-313X.2006.02865.x. [DOI] [PubMed] [Google Scholar]
- Manavella et al. (2008).Manavella PA, Dezar CA, Ariel FD, Chan RL. Two ABREs, two redundant root-specific and one W-box cis-acting elements are functional in the sunflower HAHB4 promoter. Plant Physiology and Biochemistry. 2008;46:860–867. doi: 10.1016/j.plaphy.2008.05.003. [DOI] [PubMed] [Google Scholar]
- Manna et al. (2021).Manna M, Thakur T, Chirom O, Mandlik R, Deshmukh R, Salvi P. Transcription factors as key molecular target to strengthen the drought stress tolerance in plants. Physiologia Plantarum. 2021;172:847–868. doi: 10.1111/ppl.13268. [DOI] [PubMed] [Google Scholar]
- Mehmood et al. (2022).Mehmood M, Khan MJ, Khan MJ, Akhtar N, Mughal F, Shah STA, Hyder MZ, Farrakh S, Sadiq I. Systematic analysis of HD-ZIP transcription factors in sesame genome and gene expression profiling of SiHD-ZIP class I entailing drought stress responses at early seedling stage. Molecular Biology Reports. 2022;49:2059–2071. doi: 10.1007/s11033-021-07024-2. [DOI] [PubMed] [Google Scholar]
- Que et al. (2018).Que F, Wang G-L, Li T, Wang Y-H, Xu Z-S, Xiong A-S. Genome-wide identification, expansion, and evolution analysis of homeobox genes and their expression profiles during root development in carrot. Functional & Integrative Genomics. 2018;18:685–700. doi: 10.1007/s10142-018-0624-x. [DOI] [PubMed] [Google Scholar]
- Romani & Moreno (2021).Romani F, Moreno JE. Molecular mechanisms involved in functional macroevolution of plant transcription factors. New Phytologist. 2021;230:1345–1353. doi: 10.1111/nph.17161. [DOI] [PubMed] [Google Scholar]
- Ruberti et al. (1991).Ruberti I, Sessa G, Lucchetti S, Morelli G. A novel class of plant proteins containing a homeodomain with a closely linked leucine zipper motif. The EMBO Journal. 1991;10:1787–1791. doi: 10.1002/j.1460-2075.1991.tb07703.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schmittgen & Livak (2008).Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative CT method. Nature Protocols. 2008;3:1101–1108. doi: 10.1038/nprot.2008.73. [DOI] [PubMed] [Google Scholar]
- Sen et al. (2017).Sen S, Chakraborty J, Ghosh P, Basu D, Das S. Chickpea WRKY70 regulates the expression of a homeodomain-leucine zipper (HD-Zip) I transcription factor CaHDZ12, which confers abiotic stress tolerance in transgenic tobacco and chickpea. Plant and Cell Physiology. 2017;58:1934–1952. doi: 10.1093/pcp/pcx126. [DOI] [PubMed] [Google Scholar]
- Sharif et al. (2021).Sharif R, Raza A, Chen P, Li Y, El-Ballat EM, Rauf A, Hano C, El-Esawi MA. HD-ZIP gene family: potential roles in improving plant growth and regulating stress-responsive mechanisms in plants. Genes. 2021;12:1256. doi: 10.3390/genes12081256. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shen et al. (2019).Shen W, Li H, Teng R, Wang Y, Wang W, Zhuang J. Genomic and transcriptomic analyses of HD-Zip family transcription factors and their responses to abiotic stress in tea plant (Camellia sinensis) Genomics. 2019;111:1142–1151. doi: 10.1016/j.ygeno.2018.07.009. [DOI] [PubMed] [Google Scholar]
- Shi et al. (2016).Shi M, Luo X, Ju G, Li L, Huang S, Zhang T, Wang H, Kai G. Enhanced diterpene tanshinone accumulation and bioactivity of transgenic salvia miltiorrhiza hairy roots by pathway engineering. Journal of Agricultural and Food Chemistry. 2016;64:2523–2530. doi: 10.1021/acs.jafc.5b04697. [DOI] [PubMed] [Google Scholar]
- Shin et al. (2004).Shin D, Koo YD, Lee J, Lee H, Baek D, Lee S, Cheon C-I, Kwak S-S, Lee SY, Yun D-J. Athb-12, a homeobox-leucine zipper domain protein from Arabidopsis thaliana, increases salt tolerance in yeast by regulating sodium exclusion. Biochemical and Biophysical Research Communications. 2004;323:534–540. doi: 10.1016/j.bbrc.2004.08.127. [DOI] [PubMed] [Google Scholar]
- Son et al. (2010).Son O, Hur Y-S, Kim Y-K, Lee H-J, Kim S, Kim M-R, Nam KH, Lee M-S, Kim B-Y, Park J, Park J, Lee S-C, Hanada A, Yamaguchi S, Lee I-J, Kim S-K, Yun D-J, Söderman E, Cheon C-I. ATHB12, an ABA-inducible homeodomain-leucine zipper (HD-Zip) protein of arabidopsis, negatively regulates the growth of the inflorescence stem by decreasing the expression of a gibberellin 20-oxidase gene. Plant and Cell Physiology. 2010;51:1537–1547. doi: 10.1093/pcp/pcq108. [DOI] [PubMed] [Google Scholar]
- Song et al. (2016).Song A, Li P, Xin J, Chen S, Zhao K, Wu D, Fan Q, Gao T, Chen F, Guan Z. Transcriptome-wide survey and expression profile analysis of putative chrysanthemum HD-zip I and II genes. Genes. 2016;7:19. doi: 10.3390/genes7050019. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Su et al. (2015).Su C-Y, Ming Q-L, Rahman K, Han T, Qin L-P. Salvia miltiorrhiza: traditional medicinal uses, chemistry, and pharmacology. Chinese Journal of Natural Medicines. 2015;13:163–182. doi: 10.1016/S1875-5364(15)30002-9. [DOI] [PubMed] [Google Scholar]
- Valdés et al. (2012).Valdés AE, Övernäs E, Johansson H, Rada-Iglesias A, Engström P. The homeodomain-leucine zipper (HD-Zip) class I transcription factors ATHB7 and ATHB12 modulate abscisic acid signalling by regulating protein phosphatase 2C and abscisic acid receptor gene activities. Plant Molecular Biology. 2012;80:405–418. doi: 10.1007/s11103-012-9956-4. [DOI] [PubMed] [Google Scholar]
- Wang et al. (2017a).Wang H, Jiang Y, Qi Y, Dai J, Liu Y, Zhu X, Liu C, Lü Y, Ren X. Identification and functional characterization of the MdHB-1 gene promoter sequence from Malus × domestica. Journal of Integrative Agriculture. 2017a;16:1730–1741. doi: 10.1016/S2095-3119(16)61548-4. [DOI] [Google Scholar]
- Wang et al. (2021).Wang K, Cheng Y, Yi L, He H, Zhan S, Yang P. Genome-wide identification of the tubby-like protein (TLPs) family in medicinal model plant Salvia miltiorrhiza. PeerJ. 2021;9:e11403. doi: 10.7717/peerj.11403. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wang et al. (2017b).Wang Q, Zha K, Chai W, Wang Y, Liu B, Jiang H, Cheng B, Zhao Y. Functional analysis of the HD-Zip I gene ZmHDZ1 in ABA-mediated salt tolerance in rice. Journal of Plant Biology. 2017b;60:207–214. doi: 10.1007/s12374-016-0413-9. [DOI] [Google Scholar]
- Wang & Li (2022).Wang S, Li Q. Genome-wide identification of the salvia miltiorrhiza SmCIPK gene family and revealing the salt resistance characteristic of SmCIPK13. International Journal of Molecular Sciences. 2022;23:6861. doi: 10.3390/ijms23126861. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wei et al. (2016).Wei T, Deng K, Liu D, Gao Y, Liu Y, Yang M, Zhang L, Zheng X, Wang C, Song W, Chen C, Zhang Y. Ectopic expression of DREB transcription factor, AtDREB1A, confers tolerance to drought in transgenic Salvia miltiorrhiza. Plant and Cell Physiology. 2016;57:1593–1609. doi: 10.1093/pcp/pcw084. [DOI] [PubMed] [Google Scholar]
- Wei et al. (2017).Wei T, Deng K, Zhang Q, Gao Y, Liu Y, Yang M, Zhang L, Zheng X, Wang C, Liu Z, Chen C, Zhang Y. Modulating AtDREB1C expression improves drought tolerance in salvia miltiorrhiza. Frontiers in Plant Science. 2017;8:52. doi: 10.3389/fpls.2017.00052. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wu et al. (2014).Wu Y, Liu C, Kuang J, Ge Q, Zhang Y, Wang Z. Overexpression of SmLEA enhances salt and drought tolerance in Escherichia coli and Salvia miltiorrhiza. Protoplasma. 2014;251:1191–1199. doi: 10.1007/s00709-014-0626-z. [DOI] [PubMed] [Google Scholar]
- Yang et al. (2012).Yang D, Ma P, Liang X, Wei Z, Liang Z, Liu Y, Liu F. PEG and ABA trigger methyl jasmonate accumulation to induce the MEP pathway and increase tanshinone production in Salvia miltiorrhiza hairy roots. Physiologia Plantarum. 2012;146:173–183. doi: 10.1111/j.1399-3054.2012.01603.x. [DOI] [PubMed] [Google Scholar]
- Yang et al. (2018).Yang Y, Luang S, Harris J, Riboni M, Li Y, Bazanova N, Hrmova M, Haefele S, Kovalchuk N, Lopato S. Overexpression of the class I homeodomain transcription factor TaHDZipI-5 increases drought and frost tolerance in transgenic wheat. Plant Biotechnology Journal. 2018;16:1227–1240. doi: 10.1111/pbi.12865. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yu et al. (2021).Yu W, Yu Y, Wang C, Zhang Z, Xue Z. Mechanism by which salt stress induces physiological responses and regulates tanshinone synthesis. Plant Physiology and Biochemistry. 2021;164:10–20. doi: 10.1016/j.plaphy.2021.04.011. [DOI] [PubMed] [Google Scholar]
- Yue et al. (2018).Yue H, Shu D, Wang M, Xing G, Zhan H, Du X, Song W, Nie X. Genome-wide identification and expression analysis of the HD-Zip gene family in wheat (Triticum aestivum L.) Genes. 2018;9:70. doi: 10.3390/genes9020070. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhang et al. (2020).Zhang J, Wu J, Guo M, Aslam M, Wang Q, Ma H, Li S, Zhang X, Cao S. Genome-wide characterization and expression profiling of Eucalyptus grandis HD-Zip gene family in response to salt and temperature stress. BMC Plant Biology. 2020;20:451. doi: 10.1186/s12870-020-02677-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhang et al. (2012).Zhang S, Haider I, Kohlen W, Jiang L, Bouwmeester H, Meijer AH, Schluepmann H, Liu C-M, Ouwerkerk PBF. Function of the HD-Zip I gene Oshox22 in ABA-mediated drought and salt tolerances in rice. Plant Molecular Biology. 2012;80:571–585. doi: 10.1007/s11103-012-9967-1. [DOI] [PubMed] [Google Scholar]
- Zhang et al. (2014).Zhang Z, Chen X, Guan X, Liu Y, Chen H, Wang T, Mouekouba LDO, Li J, Wang A. A genome-wide survey of homeodomain-leucine zipper genes and analysis of cold-responsive HD-Zip I members’ expression in tomato. Bioscience, Biotechnology, and Biochemistry. 2014;78:1337–1349. doi: 10.1080/09168451.2014.923292. [DOI] [PubMed] [Google Scholar]
- Zhang et al. (2021).Zhang Z, Zhu R, Ji X, Li HJ, Lv H, Zhang HY. Genome-wide characterization and expression analysis of the HD-ZIP gene family in response to salt stress in pepper. International Journal of Genomics. 2021;2021:1–14. doi: 10.1155/2021/8105124. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhao, Zhou & Wu (2010).Zhao J-L, Zhou L-G, Wu J-Y. Effects of biotic and abiotic elicitors on cell growth and tanshinone accumulation in Salvia miltiorrhiza cell cultures. Applied Microbiology and Biotechnology. 2010;87:137–144. doi: 10.1007/s00253-010-2443-4. [DOI] [PubMed] [Google Scholar]
- Zhao et al. (2021).Zhao S, Gao H, Jia X, Li X, Mao K, Ma F. The γ-clade HD-Zip I transcription factor MdHB-7 regulates salt tolerance in transgenic apple (Malus domestica) Plant and Soil. 2021;463:509–522. doi: 10.1007/s11104-021-04918-9. [DOI] [Google Scholar]
- Zhao et al. (2014).Zhao Y, Ma Q, Jin X, Peng X, Liu J, Deng L, Yan H, Sheng L, Jiang H, Cheng B. A novel maize homeodomain-leucine zipper (HD-Zip) I gene, Zmhdz10, positively regulates drought and salt tolerance in both rice and Arabidopsis. Plant & Cell Physiology. 2014;55:1142–1156. doi: 10.1093/pcp/pcu054. [DOI] [PubMed] [Google Scholar]
- Zhao et al. (2011).Zhao Y, Zhou Y, Jiang H, Li X, Gan D, Peng X, Zhu S, Cheng B. Systematic analysis of sequences and expression patterns of drought-responsive members of the HD-Zip gene family in maize. PLOS ONE. 2011;6:e28488. doi: 10.1371/journal.pone.0028488. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Hairy roots were cultured in a 6,7-V liquid medium for 18 days before being treated. The 2−ΔΔCT method was used to be an evaluation of the relative expression. SmActin was used as the internal standard. ** and * indicate significant differences compared to the control (0 h) at p < 0.01 and p < 0.05, respectively.
Hairy roots were cultured in a 6,7-V liquid medium for 18 days before being treated. The 2−ΔΔCT method was used to be an evaluation of the relative expression. SmActin was used as the internal standard. ** and * indicate significant differences compared to the control (0 h) at p < 0.01 and textitp < 0.05, respectively.
M, DL-2000; 1, WT; 2, EV; 3-10, Overexpression of SmHD-Zip 12. (B)PCR analyses of transgenic hairy root lines by SmHD-Zip12-specific primers. M, DL-2000; N1: H2O; N2: WT-PC: Recombinant plasmid; 1–17: Overexpression of SmHD-Zip 12. (C)Relative expression analysis of SmHD-Zip 12 genes by qRT-PCR in in transgenic lines and WT. ** and * indicate significant differences compared with the wild type (WT) at p < 0.01 and p < 0.05, respectively.
Data Availability Statement
The following information was supplied regarding data availability:
The raw data are available in the Supplemental File.