Introduction

Global warming has led to an increasingly widespread spatial distribution of droughts worldwide, with frequent occurrences in local areas1,2. Drought is one of the major natural disasters that plants face during growth and development. As the drought stress gradient increases, the germination rate, germination index, and vitality index of seeds all show a declining trend, and severe water stress can severely inhibit seed germination3. For plants in the growth stage, drought can disrupt the water balance within the plant, alter physiological indicators and morphological characteristics, affect normal growth, and cause wilting or even death4,5.

To adapt to drought conditions, plants have evolved various survival mechanisms, one of which is to enhance tolerance to water deficiency by regulating the expression of drought-resistant genes6. Drought-resistant genes are mainly divided into structural genes and regulatory genes. Transcription factors are regulatory genes that can control the expression levels of various functional genes related to drought resistance within the plant7. Several transcription factors have been proven to be involved in regulating plant responses to drought stress, such as NAC, bZIP, MYB, WRKY, and AP2/ERF8,9,10,11. MYB is an important class of transcription factors, playing crucial roles in plant growth and development, secondary metabolism, cell differentiation, cell cycle, and stress responses12,13. MYB transcription factor family members are ubiquitous in plants, composed of three parts: DNA binding domain, transcription activation domain, and negative regulatory domain14. In the DNA binding domain, a highly conserved amino acid sequence forms the unique structural domain of MYB genes. The MYB domain typically contains 1–4 incomplete repeat sequences (R) composed of 51–53 amino acid residues and spacer sequences, with a tryptophan residue or other hydrophobic group regularly appearing approximately every 18 amino acid residues15. Based on the location and number of repeat sequences, the MYB transcription factor family can be divided into four subfamilies: 1R-MYB, R2R3-MYB, 3R-MYB (R1R2R3), and 4R-MYB16,17. Studies have shown that when plants are subjected to drought stress, MYB transcription factors regulate the expression of certain genes through signaling networks, allowing the plant to produce a series of physiological and biochemical responses that serve a drought-resistant function18,19,20. For example, FtMYB9 and FtMYB13 can activate different stress response signals in tartary buckwheat (Fagopyrum tataricum (L.) Gaertn), increasing the response to ABA and thus improving plant drought resistance21,22. Under drought stress, rice overexpressing OsMYB1 or OsMYB6 genes showed higher CAT and SOD activities, lower REL and MDAcontent, and significantly improved drought resistance compared to wild-type rice23,24.

Honeysuckle, the dried flower buds or partially opened flowers of Lonicera japonica Thunb. from the family Caprifoliaceae, is a widely used traditional Chinese medicine with various pharmacological effects, such as clearing heat and detoxifying, antioxidation, and anti-inflammatory properties25. Honeysuckle contains a variety of active ingredients, with over 140 compounds isolated and identified, which can be used to treat various diseases such as SARS coronavirus, influenza A virus, and H1 N126,27,28. Flavonoids are one of the main effective components of honeysuckle and an important indicator of the quality of honeysuckle medicinal materials29. Flavonoid compounds play an important role in plant responses to abiotic stress. The biosynthesis of flavonoid compounds has been widely studied, originating from the general phenylpropanoid pathway and then being catalyzed by a series of enzymes30. Studies have shown that MYB transcription factors can regulate the accumulation of flavonoid compounds in plants and exhibit different specificities for target genes in different plants31,32. In Arabidopsis, transcription factors AtMYB11, AtMYB12, and AtMYB111can activate genes encoding chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), and flavonol synthase (FLS), together determining flavonol content33,34,35,36. An R2R3-MYB transcription factor AgMYB2 in purple celery can interact with bHLH proteins, promoting the synthesis of anthocyanins in Arabidopsis, producing deep purple leaves and flowers, and helping to elucidate the molecular mechanism of anthocyanin biosynthesis in purple celery37. Overexpression of CcMYB12from Cirsium setidens in Arabidopsis activates the expression of endogenous flavonol biosynthetic genes, leading to increased flavonol accumulation38. However, MYB transcription factors can also have inhibitory effects on flavonoid biosynthesis, such as MYB165 and MYB194in poplar39.

In this study, we identified three MYB transcription factors associated with drought stress response and flavonoid biosynthesis in the honeysuckle genome. Utilizing transcriptome sequencing, phylogenetic analysis, and correlation studies, we ascertained their subcellular localization and ectopically expressed them in Arabidopsis to assess their impact on drought resistance and flavonoid content. Our findings revealed that these MYB transcription factors can bolster plant drought tolerance and elevate flavonoid accumulation. This research offers both theoretical and technical foundations for further investigating the roles of honeysuckle MYB transcription factors, optimizing cultivation practices, ensuring the consistent quality of honeysuckle medicinal materials, and enhancing germplasm.

Results

Identification and drought stress response analysis of honeysuckle MYB transcription factors

The HMM model PF00249 of MYB was downloaded from the Pfam database. After a series of screenings, 356 MYB transcription factors were obtained from the honeysuckle genome and named, including 203 1R-MYB (LjMYB1R1-LjMYB1R203), 146 R2R3-MYB (LjMYB1-LjMYB146), 3 3R-MYB (LjMYB3R1-LjMYB3R3), and 4 4R-MYB (LjMYB4R1-LjMYB4R4). All identified MYBs have conserved MYB domains. A neighbor-joining phylogenetic tree was constructed for Arabidopsis and honeysuckle MYB proteins.

As shown in Fig. 1 A and R-MYB proteins from the two species were mainly divided into six groups - CCA1-like, CPC-like, TRF-like, TBP-like, I-box-like, and GARP. The GARP subgroup is the largest and can be further divided into RR, MYB-CC, GLK, LUX, HHO, KANADI, and GARP-related. CPC-like subgroup transcription factors are involved in regulating plant cell morphogenesis and secondary metabolism. Flower and fruit formation is regulated by the I-box-like subgroup, and TBP subgroup proteins can bind to plant telomeric sequences. GARP subgroup members are related to various physiological processes and stress responses in plants. As shown in Fig. 1B, honeysuckle and Arabidopsis R2R3-MYB, 3R-MYB, and 4R-MYB are divided into 22 subgroups.

Fig. 1
figure 1

Phylogenetic tree of honeysuckle and Arabidopsis MYB proteins. (A) 1R-MYB protein phylogenetic tree; (B) Phylogenetic tree of R2R3-MYB, 3R-MYB, and 4R-MYB proteins.: Arabidopsis MYB transcription factors, Honeysuckle MYB transcription factors. After determining the MYB transcription factors in honeysuckle, the next step is to identify which of these transcription factors respond to drought stress. Through the analysis of transcriptome data, the expression levels of 104 MYB genes changed significantly during drought stress treatment. The expression trend of these genes was clustered, as shown in Fig. 2. As the drought time prolonged, the expression levels of 21 genes remained elevated, 27 genes decreased, 34 genes first increased and then decreased, and 22 genes first decreased and then increased.

Full size image

After determining the MYB transcription factors in honeysuckle, the next step is to identify which of these transcription factors respond to drought stress. Through the analysis of transcriptome data, the expression levels of 104 MYB genes changed significantly during drought stress treatment. The expression trend of these genes was clustered, as shown in Fig. 2. As the drought time prolonged, the expression levels of 21 genes remained elevated, 27 genes decreased, 34 genes first increased and then decreased, and 22 genes first decreased and then increased.

Fig. 2
figure 2

MYB genes responsive to drought stress in honeysuckle genome and their expression trends. (A) Gene expression levels at 0 days of drought stress treatment. (B) Gene expression levels at 3 days of drought stress treatment. (C) Gene expression levels at 6 days of drought stress treatment. (D) Gene expression levels at 9 days of drought stress treatment. The data were processed with column-standardization and normalized scaling.

Full size image

Determination of the content of active ingredients in honeysuckle under drought stress

To understand the impact of drought stress on the active ingredients in honeysuckle and establish a connection between MYB transcription factors and active ingredients, this study measured the content of 11 active ingredients in honeysuckle. These 11 active ingredients belong to three major categories: flavonoids, phenolic acids, and iridoid glycosides. The analysis of the linear equation and methodological investigation results showed that the method adopted in this study had good repeatability, with R2 of the linear equation ≥ 0.999, sample recovery rate meeting the requirements, strong stability of the test solution within 24 h, and high precision of the instrument. Figure 3 shows the integration results of high-performance liquid chromatography, with good separation for all components. As shown in Fig. 3, under drought stress, the content of 11 components in honeysuckle plants mostly showed an increasing trend, with only the content of loganin acid decreasing. Among flavonoid components, the content of luteoloside changed the most, increasing by 1.37 times, and chlorogenic acid also increased by 1.35 times. In phenolic acid components, the content of cryptochlorogenic acid changed the most, increasing by 1.32 times, and the content of neochlorogenic acid increased by 1.18 times. Meanwhile, chlorogenic acid and neochlorogenic acid are also important indicators for evaluating the quality of honeysuckle. Overall, the changes in flavonoid components were more significant than those in phenolic acid components. Therefore, this study focused on flavonoid components for subsequent experiments.

Fig. 3
figure 3

HPLC chromatogram of honeysuckle mixed reference solution. (A) 240 nm; (B) 327 nm; (C) 3500 nm. (1) neochlorogenic acid; (2) loganin acid; (3) chlorogenic acid; (4) cryptochlorogenic acid; (5) loganin; (6) forsythoside A; (7) chlorogenic acid; (8) luteoloside; (9) isochlorogenic acid B; (10) isochlorogenic acid A; (11) isochlorogenic acid C.

Full size image

MYB transcription factors involved in the regulation of flavonoid biosynthesis

The biosynthesis of flavonoids is closely related to MYB transcription factors. 104 drought-responsive MYB transcription factors were identified from 356 MYB transcription factors and were used to construct an NJ phylogenetic tree with known transcription factors involved in flavonoid biosynthesis to screen for MYB transcription factors related to flavonoid components in honeysuckle. Known MYB transcription factors are mainly divided into three categories: those related to anthocyanins, proanthocyanidins, and flavonols (Fig. 4A). Among the 104 candidate genes, 18 genes related to flavonoid biosynthesis were identified, with the highest number (10) related to anthocyanins, and 4 each related to flavonols and proanthocyanidins. Thus, we further screened 18 MYB genes related to both drought stress response and flavonoid biosynthesis from the 104 genes. To explore the mechanism of these genes, this study identified 11 key genes in the flavonoid biosynthesis process from the honeysuckle genome. Using two-month-old honeysuckle as the material, the expression levels of these genes in roots, stems, and leaves were measured (Fig. 4B). LjMYB144, F3’H, LjMYB1R190, and LjMYB145 had the highest expression levels in roots, suggesting that they might play a major role in roots. ANS, F3H, ANR, FLS, and LAR might play an important role in stems, while the remaining genes had significantly higher expression levels in leaves than in the other two tissues. As the degree of drought deepened, the changing trends of different genes in different tissues varied (Fig. 4C, D and E). CHI and LjMYB8 were continuously upregulated in different tissues, suggesting that they might have a positive regulatory effect on drought stress. The changing trends of LjMYB1R129, LjMYB63, LjMYB145, LjMYB1R22, FLS, and LjMYB131 were first elevated and then decreased, suggesting that they might play a role in the early or middle stages of drought stress. Overall, most genes played a protective role in the early stages of stress by upregulating or downregulating their expression levels. The expression levels of different genes varied in the same tissue, and the expression levels of the same gene varied in different tissues. Plants regulate gene expression levels in different time periods and tissues to cope with stress. Combining all results, we performed a correlation analysis of the 18 target genes and key genes in the flavonoid biosynthesis process (Fig. 4F). The results indicated that PAL2. exhibited a negative correlation with LjMYB1R19, while F3H was negatively correlated with LjMYB145. In contrast, CHI demonstrated a positive correlation with LjMYB8. Additionally, CHS showed a negative correlation with both LjMYB1R43 and LjMYB63, but a positive correlation with LjMYB98 and LjMYB3. Furthermore, FNS was positively correlated with LjMYB63 and negatively correlated with LjMYB8, LjMYB145, and LjMYB3. Based on the positively correlated MYB transcription factors, we selected LjMYB3, LjMYB8, and LjMYB63 for further experiments due to their highest correlation coefficients.

Fig. 4
figure 4

Identification of MYB transcription factors involved in regulating flavonoid biosynthesis. (A) Phylogenetic tree of all candidate MYB transcription factors and other flavonoid biosynthesis-related factors; (B) Tissue expression profiles of the candidate genes; (C) Expression trends of the candidate genes in roots; (D) Expression trends of the candidate genes in stems; (E) Expression trends of the candidate genes in leaves; (F) Co-expression analysis of L. japonica MYB genes and key genes in flavonoid biosynthesis under 0.3 mol‧L−1 mannitol solution treatment. Correlation analysis based on gene expression changes in L. japonica leaves after drought stress treatment (Pearson’s correlation test, *p < 0.05, **p < 0.01).

Full size image

Subcellular localization of MYB transcription factors

Many transcription factors exert their regulatory functions mainly in the cell nucleus. The nuclear localization signals of LjMYB3, LjMYB8, and LjMYB63 predict that they may be located in the cell nucleus. To determine the subcellular localization of the candidate genes, their ORFs (without stop codons) were fused with the CaMV 35S-containing pBWA(V)HS-GLosgfp vector (Fig. 5A). Using Agrobacterium-mediated transformation, the recombinant proteins were transiently expressed in tobacco cells. Confocal microscopy showed that GFP protein was located throughout the cell, while LjMYB3-GFP was located in both cytoplasm and nucleus, and bothLjMYB8-GFP and LjMYB63-GFP were located in the nucleus (Fig. 5B).

Fig. 5
figure 5

Subcellular localization images. (A) Structure of 35 S: LjMYB-GFP; (B) Subcellular localization images of the target genes. Green fluorescent protein: excitation light 488 nm, emission light 510 nm. Chlorophyll: excitation light 640 nm, emission light 675 nm.

Full size image

Identification of positive Arabidopsis plants

The target genes were cloned and fused with the PBI 121 overexpression vector. The recombinant vector was transferred to Arabidopsis plants using the Arabidopsis flower dipping method. Harvested seeds were sterilized and screened on 1/2 MS medium containing 50 mg‧L−1 kanamycin. In the selective medium, transgenic seedlings grew normally, while the remaining seedlings had yellow leaves and gradually died (Fig. 6A). After PCR verification, the target gene was not cloned from wild-type Arabidopsis. We obtained 11 LjMYB3 (LjMYB3-Line-1 to LjMYB3-Line-11), 15 LjMYB8 (LjMYB8-Line-1 to LjMYB8-Line-16), and 11 LjMYB63 (LjMYB63-Line-1 to LjMYB63-Line-11) transgenic plants (Fig. 6B, C and D). The relative gene expression levels of the obtained transgenic plants were measured by qRT-PCR, using the least-expressed line as a control group and the 2−ΔΔCt method for calculation. Two lines with the highest relative expression levels for each gene were selected for phenotypic observation. Finally, we chose LjMYB3-Line-3, LjMYB3-Line-5, LjMYB8-Line-1, LjMYB8-Line-15, LjMYB63-Line-8, and LjMYB63-Line-10.

Fig. 6
figure 6

Identification of transgenic plants. (A) Positive Arabidopsis plants; (B) Expression levels of the LjMYB3 gene in transgenic plants; (C) Expression levels of the LjMYB8 gene in transgenic plants; (D) Expression levels of the LjMYB63 gene in transgenic plants. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Full size image

Performance of transgenic plants under drought stress

After obtaining T3 seeds of transgenic Arabidopsis, we observed the germination rate of seeds on 1/2 MS medium containing different concentrations of mannitol and recorded the data daily. As shown in Fig. 7 A, in the absence of mannitol, both wild-type and transgenic Arabidopsis grew normally, and there was no significant change in germination rate. When the mannitol concentration was increased to 0.3 mol‧L−1, the germination rate and growth status of both wild-type and transgenic Arabidopsis were severely affected, but the latter was less affected than the former. By analyzing the daily germination rate, the germination rate of wild-type and transgenic seeds was above 98% in the absence of mannitol, and the peak germination period was mainly concentrated between the first and second days (Fig. 7B). As the mannitol concentration increased, the germination rate of seeds decreased. On 0.1 mol‧L−1 mannitol medium, the germination rate of seeds was slightly affected, with the wild-type seed germination rate dropping to 90% and the transgenic seed germination rate remaining above 95% (Fig. 7 C). When the mannitol concentration increased to 0.2 mol‧L−1, the germination rate of wild-type seeds was inhibited, approximately 70%, while in transgenic seeds, except for LjMYB3-Line-5 with a germination rate of 85%, the others were all above 85% (Fig. 7D). In addition, the number of seeds germinating between the first and second days decreased, and the number of seeds germinating between the second and third days increased. On 0.3 mol‧L−1 mannitol medium, the germination rate of Arabidopsis seeds was greatly inhibited, with the wild-type seed germination rate dropping to 40%, LjMYB3 germination rate ranging from 47 to 54%, LjMYB8 lines germination rate ranging from 56 to 58%, and LjMYB63 lines having the highest germination rate ranging from 70 to 74% (Fig. 7E). There was no obvious peak period for seed germination. The data comparison showed that under drought stress, the germination rate of transgenic Arabidopsis was higher than that of wild-type Arabidopsis, with LjMYB63 series having the highest germination rate.

Drought stress not only inhibits seed germination but also affects root growth. To explore the effect of drought stress on seed root length, we transferred wild-type and transgenic Arabidopsis seeds with a root length of 0.5 cm to 1/2 MS medium containing different concentrations of mannitol, placed vertically, and recorded root length after one week of natural growth. As shown in Fig. 8A, with the intensification of drought, the root length of Arabidopsis seeds was inhibited. When the mannitol concentration increased to 0.3 mol‧L−1, all seeds except LjMYB63-Lines almost stopped root growth, and the number of lateral roots increased, with leaves starting to turn red. After two weeks of drought treatment on Arabidopsis plants (Fig. 8B and C), it was evident that transgenic lines had higher drought resistance than wild-type plants. After analysis, the selected LjMYB3, LjMYB8, and LjMYB63 all enhanced plant tolerance to drought stress.

Fig. 7
figure 7

Germination rate of Arabidopsis seeds under drought stress. (A) Germination of Arabidopsis seeds under 0 mol‧L−1 and 0.3 mol‧L−1 mannitol stress. Photos were taken on the fifth day after treatment; (B) Daily germination rate of Arabidopsis seeds without mannitol stress; (C) Daily germination rate of Arabidopsis seeds under 0.1 mol‧L−1 mannitol stress; (D) Daily germination rate of Arabidopsis seeds under 0.2 mol‧L−1 mannitol stress; (E) Daily germination rate of Arabidopsis seeds under 0.3 mol‧L−1 mannitol stress.

Full size image
Fig. 8
figure 8

Phenotypic changes of Arabidopsis under drought stress. (A) Root length of Arabidopsis seeds under 0 mol‧L−1 and 0.3 mol‧L−1 mannitol stress. Photos were taken on the seventh day after treatment; (B) Phenotype of untreated Arabidopsis; (C) Phenotype of Arabidopsis after 24 h of 0.3 mol‧L−1 mannitol stress.

Full size image

Determination of total flavonoid content

Using rutin as a control, the total flavonoid content in wild-type and transgenic Arabidopsis was measured (linear equation: y = 0.5991x − 0.0056, R2 = 0.9993). The results are shown in Fig. 9A, the total flavonoid content in transgenic Arabidopsis was higher than that in wild-type Arabidopsis, with the highest total flavonoid content in LjMYB63-Lines, which was 2.2 times that of wild-type. Combining the biosynthetic pathway of flavonoid compounds, the transcription levels of 7 flavonoid biosynthesis genes in wild-type and transgenic Arabidopsis were analyzed by qRT-PCR (Fig. 9B). The results showed that transcription factor LjMYB3 inhibited the expression of DFR and ANS genes, while upregulated the expression of other genes, especially CHS and CHI genes. Transcription factor LjMYB8 can upregulate the expression of all genes, with the expression level of CHI gene being upregulated the most, reaching more than 28 times. Except for the FLS gene, transcription factor LjMYB63 can promote the expression of other genes, with the regulation of CHS and CHI being more than 25 times. Based on the previous research results, we speculate that LjMYB3 transcription factor can promote the expression of CHS and FLS genes, thereby increasing the content of flavanol compounds. LjMYB8 and LjMYB63 can promote the expression of CHS and CHI genes, increase the content of downstream related products, and thus enhance the content of proanthocyanidins and anthocyanin compounds.

Fig. 9
figure 9

Measurement of total flavonoid content and expression levels of related genes. (A) Measurement of total flavonoid content; (B) Transcription levels of flavonoid biosynthesis genes in wild-type and transgenic Arabidopsis. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Full size image

Discussion

Water scarcity is a serious problem faced by the world today, and most plants cannot grow and develop normally in arid environments40. Therefore, breeding drought-resistant plants is of great significance for protecting the ecology and plant diversity. When plants are subjected to drought stress, they resist and adapt to drought in different ways. In recent years, transcription factors regulating the expression of genes related to plant resistance and stress have received increasing attention. MYB is one of the very common transcription factor families in plants, and a large number of studies have shown that R2R3-MYB has a huge contribution to plant growth and response to adversity stress. Honeysuckle plays a very important role in traditional Chinese medicine. With the completion of the whole genome sequencing of honeysuckle, it provides a shortcut for studying the function of various gene families in honeysuckle and breeding new varieties. So far, 256 MYB genes have been identified from Arabidopsis, 195 from grape, 569 from tobacco, and 490 from wheat. It is well known that gene duplication and loss events during evolution have led to differences in the number of the same genes between different species41. In this study, 356 MYB transcription factors were screened from the honeysuckle genome, including 203 1R-MYB and 146 R2R3-MYB. The MYB gene family has functional diversity, and there are multiple MYB genes in the honeysuckle genome.Through sequence analysis, we found that these genes all have the conserved DNA structural domains unique to MYB. This indicates that the MYB transcription factors in honeysuckle are highly homologous with those in other species.

MYB transcription factors can participate in the drought stress response process through various pathways. The TaMYB33 gene in wheat can increase the salt and drought tolerance of Arabidopsis, and its expression is induced by PEG, NaCl, and ABA42. PtrMYB94cooperates with ABA signaling to improve the drought resistance of transgenic poplar43. Through transcriptome analysis, we found that 104 of the 356 MYB transcription factors in honeysuckle respond to drought stress. This result indicates that the response of honeysuckle to drought stress is the result of the joint action of many MYB transcription factors, and MYB transcription factors play an important role in this process.

Many secondary metabolites play specific physiological functions in plant defense responses and basic adaptations to various adverse environments, as well as their potential toxicity to plant cells themselves, which means that their biosynthesis, transport, and storage processes are usually subject to strict regulation44. Therefore, MYB is indispensable in the regulatory network controlling development, secondary metabolism, and defense responses to biotic and abiotic stresses. Under drought stress, plants usually increase the content of secondary metabolites by regulating the synthesis of organic substances, changing the enzyme activities involved in metabolic pathways, and enhancing sensitization. Overexpression of the soybean GmMYB12gene can increase the production of downstream flavonoids and the expression of flavonoid biosynthesis pathway-related genes, thereby improving the resistance to drought stress during seed germination, root development, and growth18. By measuring the active ingredients of honeysuckle after drought treatment, we can easily find that the content of secondary metabolites such as flavonoids and iridoid glycosides has increased significantly. From this, we can infer that honeysuckle resists drought by increasing the content of flavonoids and iridoid glycosides and other secondary metabolites.

Flavonoids are important indicators for measuring the quality of honeysuckle. Currently, a large number of studies have shown that MYB transcription factors can participate in the regulation of flavonoid biosynthesis. MdMYB1, MdMYBA, MdMYB10, and MYB110a are key factors regulating anthocyanin synthesis in various tissues of apple (Malus domestica), where MdMYB1 and MdMYBA are mainly involved in the synthesis of anthocyanins in fruit skin, while anthocyanin synthesis in fruit and leaves is regulated by MdMYB10, and the expression of MYB110ais related to the synthesis of anthocyanins in the outer layer of fruit tissue45,46. Since the genetic transformation method of honeysuckle is not mature, we overexpressed the three target genes in Arabidopsis. The results show that the total flavonoid content in transgenic Arabidopsis is significantly higher than that in the wild-type, indicating that LjMYB3, LjMYB8, and LjMYB63 can increase the flavonoid content in Arabidopsis, with LjMYB63 showing the best effect. The mechanism of MYB transcription factors in increasing plant flavonoid content is complex, and one of them is to regulate the expression of key genes. Overexpression of AN1 in tomato will lead to the up-regulation of the expression of structural genes CHS, CHI, and DFR, resulting in the accumulation of a large amount of anthocyanins in tomato fruit47. GmMYB176 in soybean regulates isoflavone biosynthesis by activating the expression of chalcone synthase genes48. These results further confirm that the LjMYB3 transcription factor can promote the expression of CHS and FLS genes, while LjMYB8 and LjMYB63 can promote the expression of CHS and CHI genes, thereby increasing the flavonoid content in Arabidopsis.

In this study, we explored the response mechanism of MYB transcription factors in honeysuckle (Lonicera japonica) under drought stress and functionally validated it by using Arabidopsis (Arabidopsis thaliana) as a model plant. However, the limitations associated with the experimental setup were not sufficiently discussed in the study, especially the potential impact of the imperfections in the genetic transformation system of honeysuckle on the results of the study. The genetic transformation technology of honeysuckle is still in its infancy, and a stable and efficient transformation system has not yet been developed. Although studies have attempted to establish a genetic transformation system for honeysuckle through Agrobacterium-mediated methods, these methods still have many challenges, such as low transformation efficiency, as well as a long transformation cycle. These limitations make functional validation in honeysuckle itself a major challenge, which in turn limits direct confirmation of the role of MYB transcription factors in vivo.

As a model species for plant research, Arabidopsis thaliana provides abundant resources for the study of plant gene function and genetic regulation due to its small genome, easy operation, short life cycle and clear genetic background. However, there are great differences in genome structure, growth environment and physiological characteristics between Arabidopsis and many crops, which limits the direct application of its research results in crops. In addition, Arabidopsis thaliana mainly grows in temperate regions and has limited adaptability to the environment, which makes the environmental adaptability mechanism studied in Arabidopsis thaliana may not be applicable to other plants, especially those growing in extreme environments. Despite these limitations, through gene homology analysis, transcriptome and metabolomics analysis and functional verification experiments, the research results of Arabidopsis thaliana can be extended to honeysuckle or other closely related species. For example, by comparing the gene sequences of Arabidopsis thaliana and Lonicera japonica, it was found that the MYB gene in Lonicera japonica had high homology with the AtMYB15 gene in Arabidopsis thaliana, indicating that the MYB gene function studied in Arabidopsis thaliana may also have similar functions in Lonicera japonica. In addition, transcriptome and metabolomics techniques were used to study the gene expression and metabolite changes of Lonicerae Japonicae Flos at different developmental stages, and compared with the transcriptome and metabolomics data of Arabidopsis thaliana. It can be found that Lonicerae Japonicae Flos has similar gene expression patterns and metabolic pathways to Arabidopsis thaliana. Gene function verification experiments in honeysuckle, such as gene knockout, overexpression, etc., can further verify the applicability of gene functions and regulatory mechanisms found in Arabidopsis thaliana.

In summary, LjMYB63 found in the honeysuckle genome can effectively improve plant drought resistance and flavonoid content. This study systematically analyzed the MYB transcription factors in honeysuckle to improve stress tolerance and active ingredients, providing a theoretical basis for breeding new honeysuckle varieties and explaining the quality of honeysuckle from a genetic perspective. However, we still do not know the specific functions of MYB in other secondary metabolites and other gene families in honeysuckle. In addition, the genetic transformation system of honeysuckle is not yet mature. Therefore, these issues require further exploration in the future.

Materials and methods

Materials

The variety of honeysuckle used in this study is “Hua Jin 2”, which is planted in the Medicinal Plant Garden of Shandong University of Traditional Chinese Medicine and has obtained the permission for use. Uniformly grown honeysuckle plants were transferred to a greenhouse and watered once every three days to ensure sufficient water. Two weeks later, a large amount of 0.5 mol‧L−1 mannitol solution was used for irrigation, and their leaves were collected on days 0, 3, 6, and 9 after irrigation for transcriptome sequencing. There were seven plant materials in each group, with a total of five groups.Arabidopsis thaliana used in this study was the Columbia wild type strain, which was kindly provided by the research group. All plant materials used were identified by Professor Yongqing Zhang.

Methods

Identification and phylogenetic analysis of MYB transcription factors

The MYB HMM model PF00249 was downloaded from the Pfam database (http://pfam.xfam.org/), and the hmmsearch software was used to screen MYB candidate genes in the honeysuckle genome, with an E-value cut-off of 1e-5. Candidate genes were submitted to the National Microbiology Data Center (https://nmdc.cn/analyze) for CD-hit to remove redundant sequences (identity: 0.9, threads: 10). The NCBI (https://www.ncbi.nlm.nih.gov) and SMART (http://smart.embl-heidelberg.de/) databases were used to verify whether candidate genes have the conserved MYB domain. Arabidopsis MYB protein sequences were obtained from the TAIR database (https://www.arabidopsis.org/). The NJ phylogenetic tree of honeysuckle MYB and Arabidopsis MYB was constructed using MEGA-X software. The honeysuckle MYB transcription factors were grouped according to the Arabidopsis MYB classification method. The registration numbers of all the genes involved are listed in Table S1.

Determination of the content of active ingredients in honeysuckle under drought stress

Chlorogenic acid, Loganin, Quercetin-3-O-galactoside, Luteolin-7-O-glucoside, Lonicerin, Neochlorogenic acid, Loganin acid, 4-Dicaffeoylquinic Acid, Isochlorogenic acid A, Isochlorogenic acid B, and Isochlorogenic acid C were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Formic acid and acetonitrile were of HPLC grade and obtained from Fisher Company (Waltham, MA, USA).

The chromatographic column was ZORBAXSB-C18 (250 mm × 4.6 mm, 5 μm), with the column temperature maintained at 30 °C. The flow rate was set at 1.0 mL/min, and the injection volume was 20 µL. The mobile phase consisted of 0.1% formic acid (A) and acetonitrile (B), with the following gradient: 0–10 min, 8–10% B; 10–20 min, 10–15% B; 20–30 min, 15% B; 30–50 min, 15–25% B; 50–70 min, 25–100% B. Detection wavelengths were 240 nm, 327 nm, and 350 nm. Plant materials were honeysuckle leaves on days 0 and 9 after mannitol solution treatment, and the processing method referred to previous research.

Identification of MYB transcription factors involved in flavonoid biosynthesis

Plant materials were two-month-old honeysuckle seedlings, irrigated with 0.3 mol‧L−1 mannitol solution, and samples of roots, stems, and leaves were collected separately at 0, 1, 3, 8, 12, and 24 h. Eleven key genes involved in flavonoid biosynthesis were identified from the honeysuckle genome, and their expression levels and correlations with key flavonoid biosynthesis genes were determined using real-time fluorescence quantitative PCR. Total RNA was extracted from plant materials using an RNA isolation reagent kit (Takara, Dalian, China), and RNA concentration was measured using SimpliNano 29,061,711. The 2−ΔΔCt method was used to calculate relative gene expression levels. The results were analyzed using TBtools (https://www.tbtools.com/home), SPSS 22.0 (https://spss.mairuan.com/), and Chiplot (https://www.chiplot.online/). All of the primer sequences have been listed in Table S2.

Subcellular localization analysis

The target genes LjMYB3, LjMYB8, and LjMYB63 were amplified and connected to the pBWA(V)HS-GLosgfp vector, respectively, to construct recombinant vectors. The recombinant vectors were transformed into Agrobacterium tumefaciens strain GV3101. Agrobacterium was injected into tobacco leaves, and the plants were cultured for three days. The results were observed using a laser confocal microscope. At least six independent tobacco leaves were injected in each experiment, and three repeated experiments were performed to verify the localization. Representative photos were selected.

Arabidopsis genetic transformation

The overexpression vector PBI121 was linearized by BamHI and SacI double digestion, and the vector fragment was recovered by the gel recovery kit and connected with the target gene amplified fragment by T4 ligase. The recombinant vector pBI121-LjMYB was transformed into E.coli, and the positive colonies were screened by PCR colony verification and sequenced. The plasmid was extracted and transformed into Agrobacterium GV3101. The Arabidopsis thaliana plants were transformed by Agrobacterium-mediated flower-flocculation method, and the T0 generation seeds of overexpressing Arabidopsis thaliana were harvested. The T0 generation seeds of Arabidopsis thaliana were disinfected with 75% ethanol and evenly spread on a 1/2 MS solid screening medium containing 50 mg·L−1Kan. Purified at 4 °C for 3 d, cultured at 24 °C, 16 h light/8 h dark. The positive plants were transplanted into the nutrient soil, and the leaves were verified by real-time fluorescence quantitative PCR. The two lines with the highest relative gene expression were selected for subsequent research. T1 generation seeds were harvested after ripening, and T3 homozygous lines were obtained after 3 generations of screening.

Drought tolerance analysis of transgenic Arabidopsis

To verify the function of the target gene during seed germination, T3 seeds of wild-type and transgenic Arabidopsis were disinfected five times with 75% alcohol, each time for 1 min. The disinfected seeds were placed on sterile filter paper to dry and then transferred to 1/2 MS medium containing 0, 0.1, 0.2, and 0.3 mol‧L−1 mannitol using a sterile bamboo stick. After storage at 4 °C for three days, the seeds were placed in a growth environment at 24 °C with 16 h of light and 8 h of darkness, and germination rates were recorded daily, with photos taken on day 5. Simultaneously, disinfected seeds were transferred to 1/2 MS medium, stored at 4 °C for three days, and then vertically placed in the medium. Once the roots reached 0.5 cm in length, the seedlings were transferred to medium containing different concentrations of mannitol and grown vertically for one week. Root length was recorded and analyzed using ImageJ (https://imagej.en.softonic.com/). All operations were performed under sterile conditions, with four replicates per group, totaling five groups.

To verify the drought resistance effect of the target gene during plant growth and development, two-week-old wild-type and T3 transgenic Arabidopsis plants with uniform growth were selected and irrigated with a 0.3 mol‧L−1 mannitol solution. Phenotypic changes were observed 24 h later, and photographs were taken to record the observations. Four plants were chosen for each line, with three biological replicates.

Flavonoid-related gene expression and flavonoid content determination

Three-week-old wild-type and T3 transgenic Arabidopsis plants with uniform growth were selected and placed in liquid nitrogen for subsequent gene expression analysis. Seven key enzyme genes involved in flavonoid biosynthesis were selected from the Arabidopsis genome, and their relative expression levels were detected by qRT-PCR. All of the primer sequences have been listed in Table S2. The flavonoid content in wild-type and transgenic Arabidopsis was detected using a plant flavonoid content detection kit.