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Article

Identification of the KNOX Gene Family in Salvia miltiorrhiza Revealing Its Response Characteristics to Salt Stress

by
Siqi Deng
1,†,
Wenjing Ma
1,†,
Chunxu Cui
1,†,
Shiqian Wang
1 and
Mei Jiang
1,2,*
1
Key Laboratory for Natural Active Pharmaceutical Constituents Research in Universities of Shandong Province, School of Pharmaceutical Sciences, Qilu University of Technology (Shandong Academy of Sciences), Ji’nan 250014, China
2
Shandong Engineering Research Center for Innovation and Application of General Technology for Separation of Natural Products, Shandong Analysis and Test Center, Qilu University of Technology (Shandong Academy of Sciences), Ji’nan 250014, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Plants 2025, 14(3), 348; https://doi.org/10.3390/plants14030348
Submission received: 17 December 2024 / Revised: 13 January 2025 / Accepted: 20 January 2025 / Published: 24 January 2025
(This article belongs to the Special Issue Bioinformatics and Functional Genomics in Modern Plant Science)
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Abstract

Salvia miltiorrhiza is a herbaceous plant that possesses significant medicinal value. Land salinization affects the growth of S. miltiorrhiza, resulting in a decline in its quality and yield. Knotted1-like homeobox (KNOX) genes are transcription factors that play important roles in plant growth and abiotic stress. The characteristics and functions of KNOX genes in S. miltiorrhiza remain unclear. Here, we identified ten KNOX genes in S. miltiorrhiza, all of which possess the characteristic four domains: KNOX1, KNOX2, ELK, and HD. These SmKNOXs were divided into two groups together with homologous genes. Cis-acting element analysis indicated all SmKNOXs contained elements associated with phytohormone, light, and stress response. The SmKNOXs show tissue-specific expression among roots, stems, leaves, and flowers. We assessed the response of the SmKNOXs to salt stress using quantitative RT-PCR analysis. Notably, SmKNOX4 expression significantly decreased within 24 h of salt exposure, while SmKNOX1, SmKNOX2, SmKNOX3, SmKNOX8, and SmKNOX9 showed significant increases. The expression of SmKNOX1, SmKNOX2, and SmKNOX3 was significantly positively correlated with that of their target genes, GA20ox1 and S11 MYB. These findings suggest that SmKNOXs and their target genes respond to salt stress, providing a foundation for studies of SmKNOXs and the potential genetic improvement of S. miltiorrhiza.

1. Introduction

Knotted1-like homeobox (KNOX) genes are a class of transcription factors that encode the homeodomain proteins, which belong to the three-amino-acid-loop-extension (TALE) superfamily. The KNOX gene family is a conserved gene family that contains four domains: KNOX1, KNOX2, homeodomain (HD), and ELK domain. The first KNOX gene, Knotted1 in maize, was the earliest identified in plants [1]. It was subsequently identified in many other plants, such as Arabidopsis [2], wheat [3], and Moso bamboo [4]. The KNOX gene plays an important role in plant growth and development. For example, studies on the loss-of-function mutant of the KNOX gene (OSH1) in rice revealed that KNOX genes play a vital role in the maintenance of the shoot apical meristem [5]. The Arabidopsis KNOX gene (STM) is involved in specifying floral meristem identity [6]. Studies on rice HOS59 mutants and overexpression lines have shown that the HOS59 gene has a negative regulatory effect on rice grain size and plant height [7].
Additionally, KNOX genes are implicated in plant responses to abiotic stresses. In Gossypium hirsutum, multiple KNOX genes were found to be involved in various stress responses. For example, silencing GhKNOX2 can enhance the salt tolerance of seedlings, while silencing GhKNOX10 and GhKNOX14 can reduce the tolerance of seedlings to salt stress [8]. Silencing GhKNOX4-A and GhKNOX22-D genes affects the growth and development of G. hirsutum seedlings by inducing oxidative stress in response to salt and drought stress [9]. In Populus alba × P. glandulosa, PagKNAT2/6b mediates changes in plant structure under drought conditions by directly targeting PagGA20ox1 [10]. Advances in bioinformatics and molecular biology techniques have enabled extensive studies demonstrating gene expression differences in KNOX genes in drought, salt, and other adversity stresses by transcriptome or qRT-PCR experiments [11,12,13]. For example, 19 KNOX genes were identified in D. huoshanense, and the expression of most DhKNOX genes showed a significant upward or downward trend under PEG-mediated drought conditions [11]. Although the role of KNOX in plant development and stress response has been partially elucidated, the characteristics and functions of the KNOX gene family in S. miltiorrhiza are unclear.
S. miltiorrhiza is a perennial upright herbaceous plant in the Lamiaceae family, valued for its thick, fleshy roots with significant medicinal value. Since ancient times, S. miltiorrhiza has been used as a traditional Chinese medicine and is known for its ability to promote blood circulation and remove blood stasis [14]. Pharmacological studies have shown that the extract of S. miltiorrhiza root has an ameliorative effect on microcirculatory disorders and target organ damage caused by ischemia–reperfusion [15]. During the cultivation process, S. miltiorrhiza faces a variety of abiotic stresses, such as salt and drought, which can negatively impact its quality and yield. Several versions of the S. miltiorrhiza genome have been published, including line 99-3 [16], line DSS3 [17], and line shh [18]. These genome versions facilitate the identification and functional study of genes in S. miltiorrhiza.
In this study, we conducted a genome-wide identification of all KNOX genes in S. miltiorrhiza. We analyzed the gene structure, conserved motifs, and phylogenetic relationships of these KNOX genes, while also exploring their differentiation and function. Furthermore, we examined the expression profiles of KNOX genes in different tissues and under salt stresses. Our findings contribute to a deeper understanding of KNOX genes and the utilization of these genes to optimize the growth and development of S. miltiorrhiza, enabling it to better cope with environmental stress.

2. Results

2.1. Identification of SmKNOX Gene Family of S. miltiorrhiza

A combination of conserved structural domains and homologous sequence comparison analysis was applied for gene family identification. Redundant sequences were removed, and a total of ten KNOX genes were identified in S. miltiorrhiza (Tables S1 and S2). The analysis of basic physical and chemical characteristics indicated that the S. miltiorrhiza KNOX encoded amino acids with lengths ranging from 260 aa (SmKNOX5) to 383 aa (SmKNOX4), while they had molecular weights ranging from 30,056.95 (SmKNOX5) to 43,083.98 (SmKNOX4) (Table 1). The theoretical isoelectric points ranged from 4.91 (SmKNOX6) to 6.50 (SmKNOX5), with all proteins being acidic. The instability index was more than 40, except SmKNOX10, indicating poor protein stability. The average hydrophilicity was less than 0, suggesting the proteins were hydrophobic. We performed subcellular localization prediction based on amino acid sequences using WoLF PSORT, and the results showed that all ten SmKNOX proteins were located in the nucleus. Then, we randomly selected one of them for experimental validation. The results showed that the signal of the empty vector pCAMBIA1300-35S-eGFP was mainly distributed in the cell membrane and nucleus (Figure 1). The fluorescent protein signal of the fusion vector pCAMBIA1300-35S-SmKNOX4-eGFP coincides with that of the nuclear localization. This indicates that the SmKNOX4 protein is located in the nucleus, consistent with the predictions.

2.2. SmKNOX Protein Sequence Alignment Analysis

A comprehensive multiple sequence alignment revealed that all ten SmKNOX proteins encompass the complete four domains characteristic of the KNOX family (Figure 2), namely, KNOX1, KNOX2, ELK, and HD. The KNOX1 and KNOX2 domains are located at the N-terminus of the protein, while ELK and HOX are located at the C-terminus of the protein. The HD domain, which is involved in DNA binding and homodimer formation [19], showed the highest level of conservation among the ten SmKNOX proteins, which is consistent with previous reports. In addition, the conserved domains of KNOX1, KNOX2, and ELK also exhibited high conservation. The ELK domain contains three conserved core sequences of “Glu(E)-Leu(L)-Lys(K)”, which may act as a nuclear localization peptide or participate in transcriptional repression [19,20]. In S. miltiorrhiza, except for SmKNOX6 and SmKNOX7, which are L-L-K, all others have the conserved core sequence of E-L-K.

2.3. Gene Structure and Conserved Domains Analysis

Further analysis of the gene structure and the conserved domains is shown in Figure 3. First, phylogenetic analysis based on the sequences of SmKNOX proteins was carried out, and the SmKNOX proteins were divided into four subfamilies (I, II, III, and IV) (Figure 3A). Then, the online software MEME (https://meme-suite.org/meme/tools/meme, accessed on 27 July 2024) was used to predict the motifs of SmKNOX proteins by finding ten motifs, and the results showed that each SmKNOX protein contained six or seven motifs (Figure 3B). The taxa in phylogenetic analysis had similar results in motif analysis. Some motifs were common to all members, such as motif2, motif3, and motif4, and some motifs were specific to subfamily members; for example, only II subfamily members included motif7 and motif9. Motif5, motif8, and motif10 were distributed only in subfamily IV, and motif6 was only not distributed in subfamily IV. In addition, only SmKNOX6 did not include motif1 (Figure 3B). The above results indicate that the functions of SmKNOXs between different subfamilies are similar but different. The detailed sequences of the different motifs are shown in Figure 3D.
By comparing the CDs of SmKNOX genes with the genome sequence of the corresponding gene, the UTR, CDs, and intron distribution of SmKNOX genes were analyzed in depth (Figure 3C). Members belonging to the same subfamily showed roughly similar exon/intron distribution patterns in terms of the exon length and the number of introns. SmKNOX1, classified as subfamily I, and SmKNOX4, classified as subfamily IV, had no UTR. In general, SmKNOX genes with close evolutionary relationships within the same subfamily showed consistency in gene structure and domain distribution, supporting the existence of a close evolutionary relationship between them.

2.4. Phylogenetic Analysis of SmKNOXs

To understand the evolutionary relationships of KNOX among S. miltiorrhiza and three other plant species, the sequences of the KNOX gene were analyzed among S. miltiorrhiza (10 SmKNOXs), Arabidopsis thaliana (8 AtKNATs), Oryza sativa (13 OsKNOXs), and G. hirsutum (6 GhKNOXs). Based on the sequence of multiple alignments, an unrooted tree was established with the maximum likelihood method in MEGA 11 (Figure 4). All KNOX proteins were divided into two subfamilies (Groups I and II), and then, group I was further divided into IA, IB, and IC. To predict the potential function of SmKNOXs, species with homologous genes to S. miltiorrhiza could be studied. In Group IA, six OsKNOXs, AtKNAT1, and GhKNOX14-A were clustered with two SmKNOX genes, which were SmKNOX2 and SmKNOX3. In Class IB, GhSTM3-A and AtSTM were clustered with SmKNOX1. In Class IC, two GhKNOXs, two OSKNOXs, and two AtKNATs were clustered with two SmKNOXs, which were SmKNOX6 and SmKNOX7. In Class II, five OSKNOXs, four AtKNATs, and two GhKNOXs were clustered with SmKNOX4, SmKNOX8, and SmKNOX9. GhSTM3-A had higher homology to SmKNOX1 (Figure 4). GhSTM3 affects the floral transition of cotton [8]. Meanwhile, SmKNOX1 had the highest relative expression level in flowers, suggesting SmKNOX1 may regulate flowering time and flower development.

2.5. Cis-Acting Element Analysis

Cis-acting elements play a crucial role in the transcriptional regulation of gene expression, acting as key factors in enabling genes to adapt to environmental changes and regulate growth and development. In this study, we analyzed the potential cis-acting elements in the 2000 bp sequence upstream of the SmKNOX genes, identifying a total of 264 elements (Figure 5). These cis-acting elements were categorized into four functional groups: phytohormone response, stress response, growth and development, and light response. Notably, no growth and development-related elements were predicted for SmKNOX2, SmKNOX6, and SmKNOX10, while all other genes contained elements from all four categories. The distribution of these elements varied, with 136 related to light response, 80 to hormones, 34 to stress, and 14 to growth and development. Among these, light-response- and phytohormone-related elements constituted the majority, underscoring the significant roles that light and plant hormones play in the regulation of SmKNOX genes.

2.6. The Expression Profiling of SmKNOXs Genes Among Tissues

To investigate the diverse functions of SmKNOX genes during the development of various tissues, we analyzed the gene expression profiles among root, stem, leaf, and flower by qPCR. Previous studies have indicated that KNOX genes belonging to group I are crucial in the formation of the shoot apical meristem and the development of inflorescence structures [6,21]. Consistently, in S. miltiorrhiza, genes within group I (SmKNOX1, SmKNOX2, SmKNOX3, SmKNOX5, SmKNOX6, SmKNOX7, and SmKNOX10) were almost not expressed in the leaf. Conversely, genes categorized under subgroup II (SmKNOX4, SmKNOX8, SmKNOX9) were expressed in all four tissues (Figure 6A–J). The cluster heat map revealed distinct expression patterns of SmKNOX genes in different tissues (Figure 6K). According to the expression profiles, SmKNOX2, SmKNOX3, SmKNOX6, and SmKNOX7 were grouped into a category where these genes were predominantly expressed in the stem. SmKNOX1 and SmKNOX10 were grouped into a category where these genes were predominantly expressed in the flower. SmKNOX5 and SmKNOX8 were clustered together with the highest expression in the root, whereas SmKNOX4 and SmKNOX9 were clustered together, showing elevated expression in the leaf. This study provides valuable insights for understanding the functional roles of these genes in tissue development.

2.7. Expression Analysis of SmKNOXs Under Salt Stress

To elucidate the significant role that KNOX genes in S. miltiorrhiza may play in responding to salt stress, we exposed the seedlings to 100 mM NaCl and monitored the changes in gene expression at intervals of 0 h, 3 h, 6 h, 24 h, and 48 h (Figure 7). Upon salt treatment, the expression patterns of SmKNOX1, SmKNOX2, and SmKNOX3 exhibited a similar up–down–up trend, peaking at 24 h with a significant increase compared to the 0 h baseline. The expression level of the SmKNOX8 gene consistently and significantly rose following salt exposure, reaching its maximum at 48 h. In contrast, the SmKNOX9 gene showed a substantial increase at 3 h post-treatment, after which, it gradually declined. On the other hand, SmKNOX4 expression significantly decreased after salt stress but recovered by the 48 h. The cluster heat map revealed that SmKNOX4, SmKNOX7, and SmKNOX8 formed a group with higher expression levels observed at 48 h. Another group, comprising SmKNOX1, SmKNOX2, SmKNOX3, and SmKNOX5, showed higher expression at 24 h. Lastly, the group including SmKNOX6, SmKNOX9, and SmKNOX10 had higher expression levels at 3 h. Overall, under salt stress, the expression levels of SmKNOX genes varied, with a significant decrease in SmKNOX4 and a significant increase in SmKNOX1, SmKNOX2, SmKNOX3, SmKNOX8, and SmKNOX9.

2.8. Expression Analysis of Target Genes of SmKNOXs Under Salt Stress

KNOX is a transcription factor that regulates plant growth and responses to abiotic stress by controlling the expression of downstream target genes [22]. According to previous studies, KNOX target genes include REVOLUTA, ABI3, GA20ox1, and S11 MYB [23,24,25,26]. We analyzed the 2000 bp upstream sequences of these target gene coding sequences (CDs) extracted from the S. miltiorrhiza genome. The results showed that all sequences contained the TGAC core binding motif of the KNOX protein (Table S3). The qPCR analysis revealed that, except S11 MYB-2, the expression levels of the other target genes were significantly upregulated under salt stress (Figure 8). Among these, ABI3, GA20ox1, S11 MYB-1, S11 MYB-2, and S11 MYB-3 reached their highest expression levels at 24 h and then decreased, while REVOLUTA-1 and REVOLUTA-2 showed peak expression levels at 48 h. Correlation analysis of SmKNOX genes and their target genes demonstrated that the expression of SmKNOX1 was significantly positively correlated with the expression of S11 MYB-2 (r ≥ 0.9, p ≤ 0.05). Similarly, the expression levels of SmKNOX2 and SmKNOX3 were significantly positively correlated with the expression of GA20ox1 and two S11 MYB genes (r ≥ 0.9, p ≤ 0.05). SmKNOX5 exhibited a significant positive correlation with the expression of ABI3, GA20ox1, and two S11 MYB genes (r ≥ 0.9, p ≤ 0.05). Additionally, SmKNOX1, SmKNOX2, and SmKNOX3 were significantly upregulated under salt stress. These results suggest that KNOX likely responds to salt stress and positively regulates the expression of downstream target genes, including GA20ox1 and S11 MYB.

2.9. Gene Cloning of SmKNOXs

To obtain the CDs of SmKNOX genes in S. miltiorrhiza, gene cloning was performed. By aligning these sequences with the reference genome, we found that the SmKNOX genes displayed single-nucleotide polymorphisms (SNPs) and insertions and deletions (indels), indicating individual variability among S. miltiorrhiza plants (Figures S1–S10). Based on alignment results, we categorized the 10 SmKNOX genes into three groups. The first group, including SmKNOX1, SmKNOX2, SmKNOX4, SmKNOX8, and SmKNOX10, had synonymous SNPs that did not alter the amino acid sequence. The second group, comprising SmKNOX5 and SmKNOX7, had non-synonymous SNPs that changed the amino acid sequence. The third group, which included SmKNOX3, SmKNOX6, and SmKNOX9, exhibited indels (Figure 9). Specifically, SmKNOX3 had a three-base deletion between 144 bp and 146 bp; SmKNOX6 had two indels with three bases inserted between 134 bp and 135 bp and twelve bases inserted between 366 bp and 377 bp; SmKNOX9 had a deletion of 114 bases between 157 bp and 270 bp. Despite these indels, they only affected the length of the amino acid sequence without impacting the subsequent encoded amino acids. This variability could be due to individual differences in S. miltiorrhiza plants or potential errors in genome annotation. The cloning results of SmKNOX genes in S. miltiorrhiza provide a reference sequence for further studies on these genes.

3. Discussion

The homeobox gene KNOX transcription factor family is a subset of the TALE supergene family, playing a crucial role in various biological processes such as plant growth and development [22,27]. To date, members of the KNOX gene family have been identified in multiple plants, including Arabidopsis, rice, and cotton, with their functions well characterized [2,6,7,8]. However, no KNOX genes have been reported in the medicinal plant S. miltiorrhiza. Additionally, previous research has mainly focused on the impact of KNOX genes on plant development, with limited studies examining their role in responding to abiotic stress. To better understand the potential functions of KNOX genes in S. miltiorrhiza, this study employed bioinformatics and molecular biology techniques to investigate SmKNOX genes. We examined the gene structure, phylogeny, expression patterns, and response to salt stress. These findings offer insights into the functions of KNOX genes in S. miltiorrhiza.
KNOX genes are typically classified into two subfamilies, class I and class II, based on their structural characteristics, expression patterns, and phylogenetic relationships [28,29]. In this study, we constructed a phylogenetic tree containing 10 SmKNOX genes from S. miltiorrhiza and 37 KNOX genes from various species. The results indicated that SmKNOX1, SmKNOX2, SmKNOX3, SmKNOX5, SmKNOX6, SmKNOX7, and SmKNOX10 in S. miltiorrhiza belong to class I. Different KNOX genes have distinct biological functions. Class I KNOX proteins are essential for the formation of shoot apical meristems, internode elongation, and inflorescence structure in angiosperms [6,21,30]. In S. miltiorrhiza, class I KNOX genes were highly expressed in flowers or stems but rarely in leaves, similar to the expression pattern observed in Arabidopsis and maize [27], where class I genes are mainly expressed in the shoot apex, stem, and inflorescence shoot apex. This suggests that class I KNOX proteins in S. miltiorrhiza may play a role in shoot apex and inflorescence development. In the phylogenetic tree, SmKNOX6, 7, and 10 of S. miltiorrhiza clustered with AtKNAT2, AtKNAT6, and GhKNOX2-A. Previous studies have shown that AtKNAT2 and AtKNAT6 negatively regulate the development of inflorescence structure [31,32]. However, in S. miltiorrhiza, SmKNOX6, and SmKNOX7 were expressed at higher levels in stems, and only SmKNOX10 was highly expressed in flowers. SmKNOX4, SmKNOX8, and SmKNOX9 in S. miltiorrhiza belong to class II. Class II KNOX genes play a significant role in vascular tissue development and lateral organ differentiation [7,33]. Based on the expression pattern, class II SmKNOX genes of S. miltiorrhiza show high expression levels in roots, stems, leaves, and flowers, which aligns with observations in rice, maize, and poplar [27]. Generally, class II KNOX genes are more broadly expressed in various tissues than class I KNOX genes. Phylogenetic analysis revealed that SmKNOX4 of S. miltiorrhiza clustered with AtKNAT3 and AtKNAT4. Previous studies have demonstrated that AtKNAT3 synergistically influences the biosynthesis of secondary cell walls, thereby altering the mechanical support strength of Arabidopsis stems [33,34]. This study provides a direction for further research on the function of KNOX genes in S. miltiorrhiza through phylogenetic relationship and expression pattern analysis, with future molecular biology experiments needed to verify their functions.
Abiotic stress, such as salt stress, can significantly reduce productivity and lower plant yields [35]. KNOX genes play a crucial role in responding to abiotic stress [36,37]. In this study, we assessed the response of the SmKNOX gene to salt stress using quantitative RT-PCR analysis. Notably, the expression of the SmKNOX4 gene decreased significantly within 24 h of salt stress, which may help to enhance the resistance of S. miltiorrhiza to salt stress. Conversely, other genes showed increased expression within 24 h, with SmKNOX1, SmKNOX2, SmKNOX8, and SmKNOX9 exhibiting significant upregulation, suggesting their broad mobilization in response to salt stress in S. miltiorrhiza. In G. hirsutum, silencing the GhKNOX14 gene decreases seed tolerance to salt [8]. Phylogenetic tree analysis revealed that SmKNOX2 is closely related to GhKNOX14. The expression of SmKNOX1 and 2 did not change notably at 3 h and 6 h of salt stress but significantly increased at 12 h before decreasing after 48 h. In G. hirsutum, silencing the GhKNOX4-A gene significantly impacts seedling growth under salt treatment [9]. Phylogenetic tree analysis revealed that SmKNOX8 and SmKNOX 9 are closely related to GhKNOX4-A. This indicates that these genes may have important roles in the salt stress response of S. miltiorrhiza. However, further systematic studies are needed to determine if their roles are identical to those of GhKNOX14 in G. hirsutum. In conclusion, the SmKNOX gene has a potential regulatory function in responding to salt stress, providing a foundation for future functional research on the SmKNOX gene and promoting the genetic improvement of S. miltiorrhiza under salt stress.

4. Materials and Methods

4.1. Plant Materials

S. miltiorrhiza plants were cultivated in the laboratory of Qilu University of Technology, Changqing District, Jinan City, Shandong Province, China (geospatial coordinates: E 116.8178, N 36.5645), and identified by Liu Wei, a researcher at Qilu University of Technology. Fifteen uniformly growing seedlings were selected for the salt stress experiment.

4.2. Gene Family Identification and Characterization

To identify the KNOX gene family members in S. miltiorrhiza, the S. miltiorrhiza genome sequence was obtained from the National Genomics Data Center with the accession number of GWHDOEA00000000 and described in a previously published paper [16]. We identified the KNOX gene family members based on the conserved structural domains and sequence homology. First, we downloaded the Hidden Markov Model (HMM) corresponding to four conserved structural domains from the Pfam database [38], which were the ELK domain (PF03789), KNOX1 domain (PF03790), KNOX2 domain (PF03791), and Homeobox KN domain (PF05920). The protein sequences of all genes within the S. miltiorrhiza genome were searched against these four HMMs separately by hmmsearch software (v. 3.3.2) under default parameters. Genes with four conserved structural domains at the same time were used as candidate KNOX gene family members. Then, we downloaded the protein sequences of A. thaliana’s KNOX genes from the UniprotKB database [39]. The protein sequences of all genes within the S. miltiorrhiza’s genome were against A. thaliana’s KNOX sequences by BLASTP software (v. 2.14.0) [40] at the parameter e-value ≤ 1 × 105. Finally, genes that met both the domains and the sequence homology were identified as members of the S. miltiorrhiz KNOX gene family.
The basic physicochemical properties of the protein sequences, including theoretical pI, instability index (II), and grand average of hydropathicity (GRAVY), were analyzed by ProtParam on the ExPASy website (https://www.expasy.org/, accessed on 27 July 2024). The structural features of the genes were analyzed and visualized by TBtools software (v. 2.136) [41] based on the annotation files of S. miltiorrhiza’s genome. Conserved structural domains of protein sequences were analyzed through the Multiple Expectation Maximization for motif Elicitation (MEME) website (https://meme-suite.org/meme/tools/meme, accessed on 27 July 2024) [42] and visualized by TBtools software (v. 2.136). To analyze the cis-acting elements, we obtained sequences located 2 kb upstream of the start codon (ATG) of the SmKNOX gene, which was subsequently analyzed using the PlantCARE tool (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 28 July 2024). Finally, we visualized the results using TBtools (v. 2.136).

4.3. Multiple Sequence Alignment and Phylogenetic Relationship Analysis

The sequences of the SmKNOX proteins of S. miltiorrhiza were subjected to sequence alignment by the ClustalW tool of MEGA software (v. 11) [43] under default parameters. The alignment results were visualized by GeneDoc software (v. 2.7). To analyze the phylogenetic relationships among SmKNOX proteins, the sequence alignment results were further used for phylogenetic tree construction using MEGA software (v. 11) [43]. The phylogenetic tree construction was performed using the maximum likelihood (ML) method with bootstrap replications set to 1000.
To analyze the phylogenetic relationships of KNOX genes in S. miltiorrhiza, A. thaliana, O. sativa, and G. hirsutum, we downloaded the protein sequences of KNOX genes belonging to A. thaliana, O. sativa, and G. hirsutum from Phytozome v13 website (https://phytozome-next.jgi.doe.gov/, accessed on 27 July 2024) [44], respectively. The protein sequences were subjected to alignment by the ClustalW tool of MEGA software (v. 11) under default parameters, and then, the sequence alignment results were further used for phylogenetic tree construction using MEGA software (v. 11). The phylogenetic tree construction was performed using the ML method with bootstrap replications set to 1000. Finally, the phylogenetic tree was visualized through the ChiPlo website (https://www.chiplot.online/, accessed on 28 July 2024) [45].

4.4. Tissue Differential Expression Analysis of SmKNOX Genes

S. miltiorrhiza plants were planted in the laboratory (School of Pharmaceutical Sciences, Qilu University of Technology) and grown under natural conditions for two years. During the flowering period, the root, stem, leaf, and flower of S. miltiorrhiza were collected from three separate plants. These samples were immediately frozen in liquid nitrogen and stored at −80 °C. The tissue expression profile of the SmKNOX gene was analyzed by qRT-PCR. The SmActin gene was selected as an internal reference gene according to a previous paper [46]. The primers are shown in Table S4. The relative expression of SmKNOXs among different tissues was analyzed using the 2−ΔΔCt method.

4.5. Stress Response Analysis of SmKNOX Genes

Fifteen annual seedlings with consistent growth were used for salt stress treatment. The S. miltiorrhiza seedlings were treated with salt stress using 100 mM NaCl. They were planted in soil and cultured in a climate chamber at 25 °C with a day-to-night ratio of 16:8. Leaf samples were collected from the S. miltiorrhiza plants at 0 h, 3 h, 6 h, 24 h, and 48 h during the salt stress treatment. The samples were stored immediately in a −80 °C refrigerator until use. Three biological replicates were used per treatment.
RNA was extracted using the M5 Plant RNeasy Complex Mini Kit (Mei5 Biotechnology Co., Ltd., Beijing, China). The integrity and concentration of RNA was checked using agarose electrophoresis gel and a NanoDrop 2000 spectrophotometer (Thermo Scientifc, Waltham, MA, USA), respectively. RNA was reverse transcribed into cDNA using the Hifair® II 1st Strand cDNA Synthesis SuperMix for qPCR (Yeasen Biotech, Shanghai, China) with Oligo (dT) as a primer. The qRT-PCR primers were designed using the IDT website and are shown in Table S4. The qRT-PCR experiments were performed using the Hieff qPCR SYBR Green Master Mix Kit (Yeasen Biotechnology, China) and detected by ABI QuantStudio 5 (Thermo Fisher, Waltham, MA, USA). The SmActin gene was selected as the internal reference gene as documented previously [46], and the relative expression of SmKNOXs among different treatments was analyzed by the 2−ΔΔCt method.

4.6. Gene Cloning Analysis of SmKNOX Genes

Gene cloning analysis was performed using pCAMBIA1300-35S-eGFP vectors, which were linearized by BamHI enzymes and SalI enzymes. The gene cloning primers consisted of two parts, the vector terminal homologous sequence and the gene sequence, and are shown in Table S5. The SmKNOX sequence was amplified from the cDNA by high-fidelity enzymes, and the amplified length was detected by agarose electrophoresis gel. Bands meeting the expected length were recovered using the DNA Gel Extraction Kit with Magnetic Beads (Beyotime, Shanghai, China) to obtain the purified sequence. The purified sequence was ligated to the linearized vector using a ClonExpress® II One Step Cloning Kit (Vazyme, Nanjing, China). The colonies were selected for PCR amplification and Sanger sequencing verification.

4.7. Subcellular Localization Analysis

The fusion vector pCAMBIA1300-35S-SmKNOX-EGFP was constructed. An empty pCAMBIA1300-35S-EGFP vector was used as a control. The vector was transformed into Agrobacterium GV3101 using the freeze–thaw method. After two weeks of tobacco seed germination, seedlings of uniform size were selected for transplanting and grown for four weeks in an incubation chamber at 25 °C, 16 h light/8 h dark. Agrobacterium tumefaciens was injected into the leaf epidermis of four-week-old tobacco for transient expression. The expression of the fusion protein was observed and photographed using laser confocal microscopy.

5. Conclusions

Here, the KNOX gene family was identified in S. miltiorrhiza, and a comprehensive analysis was conducted on gene structure, conserved domains, cis-acting elements, gene cloning, and expression patterns across different tissues and under salt stress conditions. A phylogenetic tree, incorporating 10 SmKNOX genes from S. miltiorrhiza and 27 KNOX genes from various species, alongside expression pattern analysis, revealed that group I KNOX proteins in S. miltiorrhiza may be involved in shoot apex and inflorescence development. Under salt stress, the expression levels of SmKNOX1, SmKNOX2, SmKNOX3, SmKNOX8, and SmKNOX9 increased significantly, while SmKNOX4 exhibited a marked decrease within 24 h of exposure. Furthermore, the expression of SmKNOX1, SmKNOX2, and SmKNOX3 was significantly positively correlated with that of their target genes, GA20ox1 and S11 MYB. In summary, SmKNOX1, SmKNOX2, and SmKNOX3 respond to salt stress and may further positively regulate target gene expression.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/plants14030348/s1: Table S1: The results of homologous sequence alignment; Table S2. The results of HMM search; Table S3: The prediction of SmKNOX protein core binding motifs upstream of target genes; Table S4: The primer sequences of qPCR used in this project; Table S5: The primer sequences of gene cloning used in this project; Figure S1: The gene cloning results of SmKNOX1; Figure S2: The gene cloning results of SmKNOX2; Figure S3: The gene cloning results of SmKNOX3; Figure S4: The gene cloning results of SmKNOX4; Figure S5: The gene cloning results of SmKNOX5; Figure S6: The gene cloning results of SmKNOX6; Figure S7: The gene cloning results of SmKNOX7; Figure S8: The gene cloning results of SmKNOX8; Figure S9: The gene cloning results of SmKNOX9; Figure S10: The gene cloning results of SmKNOX10.

Author Contributions

Formal analysis, S.D.; writing—original draft preparation, S.D. and W.M.; validation and visualization, W.M. and C.C.; software and visualization, S.W.; project administration and writing—review and editing, M.J. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Natural Science Foundation of Shandong Province [ZR2022QH084], the National Natural Science Foundation of China [82304656], the Talent Research Project from Qilu University of Technology (2023RCKY097), and the Innovation and Entrepreneurship Training Program for College Students in Shandong Province (S202410431004).

Data Availability Statement

Data is contained within the article or Supplementary Material.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Subcellular localization results. (A,B) represent empty vectors and SmKNOX4 proteins, respectively. From left to right, the images represent GFP fluorescence, RFP fluorescence, bright field, and merged images.
Figure 1. Subcellular localization results. (A,B) represent empty vectors and SmKNOX4 proteins, respectively. From left to right, the images represent GFP fluorescence, RFP fluorescence, bright field, and merged images.
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Figure 2. Multi-sequence alignment of conserved domains within the SmKNOX proteins. The KNOX1, KNOX2, ELK, and HD motifs are denoted by red, blue, green, and purple rectangles, respectively. The asterisk above the sequence represents the middle of the two numbers.
Figure 2. Multi-sequence alignment of conserved domains within the SmKNOX proteins. The KNOX1, KNOX2, ELK, and HD motifs are denoted by red, blue, green, and purple rectangles, respectively. The asterisk above the sequence represents the middle of the two numbers.
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Figure 3. Gene structure, conserved domain, and motif analysis of SmKNOXs proteins from S. miltiorrhiza. (A) The phylogenetic tree of all SmKNOXs proteins was constructed using the ML method. (B) Motifs in the SmKNOX proteins were identified by the MEME program. Motifs numbered 1–10 were colored differently. (C) The UTR, CDs, and intron organization of SmKNOXs. The green boxes represent UTRs, the yellow boxes represent CDs, and thin black lines represent introns. (D) The abscissa of the sequence logos refers to the amino acid with the highest frequency, and the ordinate represents the relative frequency of the corresponding amino acid.
Figure 3. Gene structure, conserved domain, and motif analysis of SmKNOXs proteins from S. miltiorrhiza. (A) The phylogenetic tree of all SmKNOXs proteins was constructed using the ML method. (B) Motifs in the SmKNOX proteins were identified by the MEME program. Motifs numbered 1–10 were colored differently. (C) The UTR, CDs, and intron organization of SmKNOXs. The green boxes represent UTRs, the yellow boxes represent CDs, and thin black lines represent introns. (D) The abscissa of the sequence logos refers to the amino acid with the highest frequency, and the ordinate represents the relative frequency of the corresponding amino acid.
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Figure 4. Unrooted phylogenetic tree of KNOX proteins from S. miltionhiza, A. thaliana, O. sativa, and G. hirsutum. The maximum likelihood (ML) phylogenetic tree was constructed using MEGA7.0 with 1000 bootstrap replicates. These KNOX proteins are divided into two groups (I–II) with three subgroups (IA, IB, and IC), distinguished by different colors. Genes from different species are represented by circles of different colors.
Figure 4. Unrooted phylogenetic tree of KNOX proteins from S. miltionhiza, A. thaliana, O. sativa, and G. hirsutum. The maximum likelihood (ML) phylogenetic tree was constructed using MEGA7.0 with 1000 bootstrap replicates. These KNOX proteins are divided into two groups (I–II) with three subgroups (IA, IB, and IC), distinguished by different colors. Genes from different species are represented by circles of different colors.
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Figure 5. Analysis of cis-acting element in SmKNOX genes. (A) Inspection of cis-acting elements in SmKNOX genes. The black line indicates the promoter length of the SmKNOX genes. (B) The total number of cis-acting elements in each group. (C) The type of cis-acting elements belonging to each group.
Figure 5. Analysis of cis-acting element in SmKNOX genes. (A) Inspection of cis-acting elements in SmKNOX genes. The black line indicates the promoter length of the SmKNOX genes. (B) The total number of cis-acting elements in each group. (C) The type of cis-acting elements belonging to each group.
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Figure 6. Expression profiles of SmKNOX genes in different tissues of S. miltiorrhiza. (A–J) Relative expression levels of SmKNOX genes in flowers, leaves, roots, and stems. The X-axis represents different tissues, and the Y-axis represents the relative expression levels normalized to the expression values of root. (K) A heatmap generated based on the relative expression levels of SmKNOX genes in different tissues. The color corresponds to the Z-score transformed from the expression levels of SmKNOX genes.
Figure 6. Expression profiles of SmKNOX genes in different tissues of S. miltiorrhiza. (A–J) Relative expression levels of SmKNOX genes in flowers, leaves, roots, and stems. The X-axis represents different tissues, and the Y-axis represents the relative expression levels normalized to the expression values of root. (K) A heatmap generated based on the relative expression levels of SmKNOX genes in different tissues. The color corresponds to the Z-score transformed from the expression levels of SmKNOX genes.
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Figure 7. Expression profiles of SmKNOX genes in S. miltiorrhiza under different salt stress periods. (A–J) Relative expression levels of SmKNOX genes after 0 h, 3 h, 6 h, 24 h, and 48 h of salt stress. The X-axis represents different periods, and the Y-axis represents the relative expression levels normalized to the expression values of 0 h. Error bars represent the standard error. Asterisks represent significant changes in expression compared with 0 h; specifically, *, **, and *** represent adjusted p < 0.05, p < 0.01, and p < 0.001, respectively. (K) Heatmap generated based on the relative expression levels of SmKNOX genes in different periods. The color corresponds to the Z-score transformed from the expression levels of SmKNOX genes.
Figure 7. Expression profiles of SmKNOX genes in S. miltiorrhiza under different salt stress periods. (A–J) Relative expression levels of SmKNOX genes after 0 h, 3 h, 6 h, 24 h, and 48 h of salt stress. The X-axis represents different periods, and the Y-axis represents the relative expression levels normalized to the expression values of 0 h. Error bars represent the standard error. Asterisks represent significant changes in expression compared with 0 h; specifically, *, **, and *** represent adjusted p < 0.05, p < 0.01, and p < 0.001, respectively. (K) Heatmap generated based on the relative expression levels of SmKNOX genes in different periods. The color corresponds to the Z-score transformed from the expression levels of SmKNOX genes.
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Figure 8. Expression profiles of target genes of SmKNOXs under different salt stress periods. (A–G) Relative expression levels of target genes after 0 h, 3 h, 6 h, 24 h, and 48 h of salt stress. The X-axis represents different periods, and the Y-axis represents the relative expression levels normalized to the expression values of 0 h. Error bars represent the standard error. Asterisks represent significant changes in expression compared with 0 h; specifically, * and *** represent adjusted p < 0.05, and p < 0.001, respectively. (H) Correlation of expression between SmKNOX and target genes. Numbers represent correlation values where the expression of two genes is significantly correlated (r ≥ 0.9, p ≤ 0.05).
Figure 8. Expression profiles of target genes of SmKNOXs under different salt stress periods. (A–G) Relative expression levels of target genes after 0 h, 3 h, 6 h, 24 h, and 48 h of salt stress. The X-axis represents different periods, and the Y-axis represents the relative expression levels normalized to the expression values of 0 h. Error bars represent the standard error. Asterisks represent significant changes in expression compared with 0 h; specifically, * and *** represent adjusted p < 0.05, and p < 0.001, respectively. (H) Correlation of expression between SmKNOX and target genes. Numbers represent correlation values where the expression of two genes is significantly correlated (r ≥ 0.9, p ≤ 0.05).
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Figure 9. The gene cloning results for SmKNOX3, SmKNOX6, and SmKNOX9. (A) The indel fragment of the SmKNOX3 gene. The first (B) and second (C) indel fragments of the SmKNOX6 gene. (D) The indel fragment of the SmKNOX9 gene. “R” represents the CDs extracted from the reference genome, and “C” represents the CDs obtained from gene cloning.
Figure 9. The gene cloning results for SmKNOX3, SmKNOX6, and SmKNOX9. (A) The indel fragment of the SmKNOX3 gene. The first (B) and second (C) indel fragments of the SmKNOX6 gene. (D) The indel fragment of the SmKNOX9 gene. “R” represents the CDs extracted from the reference genome, and “C” represents the CDs obtained from gene cloning.
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Table 1. Basic characteristics of the KNOX gene family in S. miltiorrhiza.
Table 1. Basic characteristics of the KNOX gene family in S. miltiorrhiza.
Gene NameNumber of Amino AcidsMolecular WeightpIInstability IndexGrand Average of Hydropathicity
SmKNOX134038,093.085.7357.33−0.56
SmKNOX235540,606.385.8645.66−0.849
SmKNOX335040,538.126.2255.54−1.065
SmKNOX438343,083.986.1559.2−0.72
SmKNOX526030,056.956.5052.1−0.717
SmKNOX627932,165.154.9142.07−0.71
SmKNOX728833,516.715.5045.65−0.863
SmKNOX836541,819.666.2561.99−0.924
SmKNOX937442,534.076.0353.65−0.941
SmKNOX1031836,265.814.9939.75−0.671
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Deng, S.; Ma, W.; Cui, C.; Wang, S.; Jiang, M. Identification of the KNOX Gene Family in Salvia miltiorrhiza Revealing Its Response Characteristics to Salt Stress. Plants 2025, 14, 348. https://doi.org/10.3390/plants14030348

AMA Style

Deng S, Ma W, Cui C, Wang S, Jiang M. Identification of the KNOX Gene Family in Salvia miltiorrhiza Revealing Its Response Characteristics to Salt Stress. Plants. 2025; 14(3):348. https://doi.org/10.3390/plants14030348

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Deng, Siqi, Wenjing Ma, Chunxu Cui, Shiqian Wang, and Mei Jiang. 2025. "Identification of the KNOX Gene Family in Salvia miltiorrhiza Revealing Its Response Characteristics to Salt Stress" Plants 14, no. 3: 348. https://doi.org/10.3390/plants14030348

APA Style

Deng, S., Ma, W., Cui, C., Wang, S., & Jiang, M. (2025). Identification of the KNOX Gene Family in Salvia miltiorrhiza Revealing Its Response Characteristics to Salt Stress. Plants, 14(3), 348. https://doi.org/10.3390/plants14030348

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