MYB24 Negatively Regulates the Biosynthesis of Lignin and Capsaicin by Affecting the Expression of Key Genes in the Phenylpropanoid Metabolism Pathway in Capsicum chinense

The wide application of pepper is mostly related to the content of capsaicin, and phenylpropanoid metabolism and its branch pathways may play an important role in the biosynthesis of capsaicin. The expression level of MYB24, a transcription factor screened from the transcriptome data of the pepper fruit development stage, was closely related to the spicy taste. In this experiment, CcMYB24 was cloned from Hainan Huangdenglong pepper, a hot aromatic pepper variety popular in the world for processing, and its function was confirmed by tissue expression characteristics, heterologous transformation in Arabidopsis thaliana, and VIGS technology. The results showed that the relative expression level of CcMYB24 was stable in the early stage of pepper fruit development, and increased significantly from 30 to 50 days after flowering. Heterologous expression led to a significant increase in the expression of CcMYB24 and decrease in lignin content in transgenic Arabidopsis thaliana plants. CcMYB24 silencing led to a significant increase in the expression of phenylpropanoid metabolism pathway genes PAL, 4CL, and pAMT; lignin branch CCR1 and CAD; and capsaicin pathway CS, AT3, and COMT genes in the placenta of pepper, with capsaicin content increased by more than 31.72% and lignin content increased by 20.78%. However, the expression of PAL, pAMT, AT3, COMT, etc., in the corresponding pericarps did not change significantly. Although CS, CCR1, and CAD increased significantly, the relative expression amount was smaller than that in placental tissue, and the lignin content did not change significantly. As indicated above, CcMYB24 may negatively regulate the formation of capsaicin and lignin by regulating the expression of genes from phenylpropanoid metabolism and its branch pathways.


Introduction
Pepper is an annual or perennial plant of the genus Capsicum of Solanaceae. Because of its unique spicy flavor, the pepper is used in food [1], medical treatment [2], industry [3], environmental protection [4,5], military security [6], and other fields. Capsaicin, the main spice ingredient, is mainly synthesized in the placenta tissue of pepper fruit and accumulated in vacuoles of placenta epidermal cells. Its synthesis mainly involves, in phenylpropanoid metabolism, the capsaicin branch pathway (CBP) and branched chain fatty acid metabolism pathway [7,8].
As a ubiquitous and essential secondary metabolic pathway in plants, the phenylpropanoid metabolism pathway leads to the synthesis of downstream branch secondary metabolites such as capsaicin, lignin, flavonoids, and anthocyanins. Phenylalanine was

Cloning and Sequence Analysis of CcMYB24 Transcription Factors
The sequenced results showed that the full length ORF of the CcMYB24 r gene was 864 bp, encoding 287 aa. By analyzing the physicochemical properties of CcMYB24 protein, we found that it has the following essential characteristics: the molecular formula was C 2666 H 4470 N 864 O 1100 S 126 ; the molecular weight was 70,267.51 Da; the theoretical isoelectric point pI was 5.18; it does not contain a signal peptide and was a non-secretory protein; there was no transmembrane structure; and it was theoretically localized in the nucleus, indicating that this transcription factor may regulate gene transcription. In addition, the conserved structural domain was viewed using NCBI CDD and was found to be a typical R2R3-MYB structural domain. The evolutionary analysis revealed that the homologs of CcMYB24 were mainly CaMYB-like and CaMYB24 in Capsicum annuum and CbMYB24 in Capsicum baccatum ( Figure 1A). Conserved motifs and gene structure maps of the MEME predicted analysis proteins are shown in Figure 1B,C. capsaicin and lignin biosynthesis by affecting phenylpropanoid metabolism and the pression of branch genes.

Cloning and Sequence Analysis of CcMYB24 Transcription Factors
The sequenced results showed that the full length ORF of the CcMYB24 r gene w 864 bp, encoding 287 aa. By analyzing the physicochemical properties of CcMYB24 p tein, we found that it has the following essential characteristics: the molecular form was C2666H4470N864O1100S126; the molecular weight was 70,267.51 Da; the theoretical isoel tric point pI was 5.18; it does not contain a signal peptide and was a non-secretory prote there was no transmembrane structure; and it was theoretically localized in the nucle indicating that this transcription factor may regulate gene transcription. In addition, conserved structural domain was viewed using NCBI CDD and was found to be a typi R2R3-MYB structural domain. The evolutionary analysis revealed that the homologs CcMYB24 were mainly CaMYB-like and CaMYB24 in Capsicum annuum and CbMYB24 Capsicum baccatum ( Figure 1A). Conserved motifs and gene structure maps of the MEM predicted analysis proteins are shown in Figure 1B,C.

Differential Expression of CcMYB24 in Different Growth and Development Stages of Pepp
The qRT-PCR analysis of Huangdenglong pepper fruit showed that the expressi level of CcMYB24 was generally stable 10 to 30 days after flowering (Figure 2), but creased rapidly after 30 days. Compared with 30 days, the relative expression increas by about four times at 50 days after flowering. The relative expression level of CcMYB in placenta tissue was significantly higher than that in the pericarp. Because capsaicin mostly synthesized in placenta tissue, it is believed that this difference in expression related to the formation of spicy taste.

Differential Expression of CcMYB24 in Different Growth and Development Stages of Pepper
The qRT-PCR analysis of Huangdenglong pepper fruit showed that the expression level of CcMYB24 was generally stable 10 to 30 days after flowering ( Figure 2), but increased rapidly after 30 days. Compared with 30 days, the relative expression increased by about four times at 50 days after flowering. The relative expression level of CcMYB24 in placenta tissue was significantly higher than that in the pericarp. Because capsaicin is mostly synthesized in placenta tissue, it is believed that this difference in expression is related to the formation of spicy taste.
les 2023, 28, x FOR PEER REVIEW Figure 2. The relative expression levels of CcMYB24 in pepper at different Each value represents the mean ± standard of three replicates. a, b, c, d, differences in values at p < 0.05.

Heterologous Expression of the CcMYB24 Gene in A. thaliana
The seeds of infected A. thaliana were harvested and then tran to screen the T2-generation positive seedlings ( Figure 3C). Then, th transgenic plants were extracted, and the cDNA was synthesized PCR. Using pB-CcMYB24-R/F as primers, the actual length of the about 2044 bp (since the M13 site was located on both sides of th target segment, the actual band length was 1180 bp + 864 bp) i plants, while the target band was not amplified from wild-type A the same time, the PCR reaction was performed using A. thaliana c the expected bands could be observed by gel electrophoresis (Figu that the CcMYB24 gene has been transferred into the A. thaliana ge has occurred, allowing the next step of the experiment. The CcMYB24 in wild-type and transgenic A. thaliana showed that CcM pressed in transgenic plants ( Figure 3D). Lignin content determin generation transgenic A. thaliana and wild-type A. thaliana plants content was reduced by 29.47% in CcMYB24 transgenic A. thalian type A. thaliana ( Figure 3E).
It is speculated that the CcMYB24 inhibits phenylpropanoid l olism after being transferred into A. thaliana, so CcMYB24 may als in the lignin synthesis stage of pepper.

Heterologous Expression of the CcMYB24 Gene in A. thaliana
The seeds of infected A. thaliana were harvested and then transformed into the plates to screen the T 2 -generation positive seedlings ( Figure 3C). Then, the RNA of T 2 -generation transgenic plants were extracted, and the cDNA was synthesized by reverse transcription PCR. Using pB-CcMYB24-R/F as primers, the actual length of the amplified fragment was about 2044 bp (since the M13 site was located on both sides of the insertion point of the target segment, the actual band length was 1180 bp + 864 bp) in transgenic A. thaliana plants, while the target band was not amplified from wild-type A. thaliana ( Figure 3A). At the same time, the PCR reaction was performed using A. thaliana cDNA as a template, and the expected bands could be observed by gel electrophoresis ( Figure 3B), which indicates that the CcMYB24 gene has been transferred into the A. thaliana genome and transcription has occurred, allowing the next step of the experiment. The relative expression of CcMYB24 in wild-type and transgenic A. thaliana showed that CcMYB24 had been overexpressed in transgenic plants ( Figure 3D). Lignin content determination of the stems of T 2 -generation transgenic A. thaliana and wild-type A. thaliana plants showed that the lignin content was reduced by 29.47% in CcMYB24 transgenic A. thaliana compared with wild-type A. thaliana ( Figure 3E).
It is speculated that the CcMYB24 inhibits phenylpropanoid lignin branching metabolism after being transferred into A. thaliana, so CcMYB24 may also play an inhibitory role in the lignin synthesis stage of pepper.
The analysis of capsaicin content in pepper with different treatments showed that the content increased by 7.22% in the mature fruit placenta tissue of plants infected with blank vectors, while it decreased by 8% in the pericarp. The capsaicin content of plants infected with blank vectors was not significantly different from that of untreated plants ( Figure 5A). The changes of capsaicin in the pericarp and placenta of pepper after gene silencing treatment were different. The capsaicin content in the pericarp of pTRV2-CcMYB24 silenced plants increased by 21.33% compared with the untreated group, and increased by 31.72% in the placenta, respectively. There was no significant difference in lignin content in the placenta tissue and pericarp of fruit between the non-infected and blank vector-infected pepper, with an increase of 0.97% in the placenta and a decrease of 0.55% in the pericarp ( Figure 5B). However, in the plants silenced by pTRV2-CcMYB24, the lignin content in the pericarp and placenta tissue increased by 11.16% and 20.78%, respectively, indicating that CcMYB24 had a significant effect on the lignin content in the placenta tissue. The analysis of capsaicin content in pepper with different treatments showed that the content increased by 7.22% in the mature fruit placenta tissue of plants infected with blank vectors, while it decreased by 8% in the pericarp. The capsaicin content of plants infected with blank vectors was not significantly different from that of untreated plants ( Figure  5A). The changes of capsaicin in the pericarp and placenta of pepper after gene silencing treatment were different. The capsaicin content in the pericarp of pTRV2-CcMYB24 silenced plants increased by 21.33% compared with the untreated group, and increased by 31.72% in the placenta, respectively. There was no significant difference in lignin content in the placenta tissue and pericarp of fruit between the non-infected and blank vectorinfected pepper, with an increase of 0.97% in the placenta and a decrease of 0.55% in the pericarp ( Figure 5B). However, in the plants silenced by pTRV2-CcMYB24, the lignin content in the pericarp and placenta tissue increased by 11.16% and 20.78%, respectively, indicating that CcMYB24 had a significant effect on the lignin content in the placenta tissue.
Compared with the plants without silencing, the expression of PAL, 4CL, and pAMTrelated genes in the phenylpropanoid metabolism pathway in plants infected with pTRV2-CcMYB24 decreased in the pericarp and increased in the placenta. In the fruits and placentas of plants infected with pTRV2-CcMYB24, the expression of BCAT and KAS genes related to the branched-chain fatty acid pathway decreased; the expression of CCR1 and CAD genes related to the lignin pathway and CS, AT3, and COMT genes related to the capsaicin pathway increased; the capsaicin content increased; and the lignin content increased. It was speculated that CcMYB24 has an inhibitory effect on both capsaicin and lignin synthesis.

Discussion
Capsaicin biosynthesis is a branch of phenylpropanoid metabolism in pepper. Its synthesis is regulated by a series of structural genes and transcription factors, and MYB transcription factors are an important regulatory gene in plants [32]. In peppers, only the MYB [33,34], WRKY [35], and ERF [36] transcription factor families were found to be associated with the regulation of spiciness. MYBs are widely involved in the regulation of phenylpropanoid metabolism [22,37,[38][39][40] and gene expression in various branch pathways, such as the lignin [39], flavonoids [40], anthocyanidins [41,42], and proanthocyanidins [43] pathways. They are also involved in morphological formation regulation, stress, and other physiological activities.
Among plant MYBs, the most common are R2R3-MYB transcription factors, which Compared with the plants without silencing, the expression of PAL, 4CL, and pAMTrelated genes in the phenylpropanoid metabolism pathway in plants infected with pTRV2-CcMYB24 decreased in the pericarp and increased in the placenta. In the fruits and placentas of plants infected with pTRV2-CcMYB24, the expression of BCAT and KAS genes related to the branched-chain fatty acid pathway decreased; the expression of CCR1 and CAD genes related to the lignin pathway and CS, AT3, and COMT genes related to the capsaicin pathway increased; the capsaicin content increased; and the lignin content increased. It was speculated that CcMYB24 has an inhibitory effect on both capsaicin and lignin synthesis.

Discussion
Capsaicin biosynthesis is a branch of phenylpropanoid metabolism in pepper. Its synthesis is regulated by a series of structural genes and transcription factors, and MYB transcription factors are an important regulatory gene in plants [32]. In peppers, only the MYB [33,34], WRKY [35], and ERF [36] transcription factor families were found to be associated with the regulation of spiciness. MYBs are widely involved in the regulation of phenylpropanoid metabolism [22,[37][38][39][40] and gene expression in various branch pathways, such as the lignin [39], flavonoids [40], anthocyanidins [41,42], and proanthocyanidins [43] pathways. They are also involved in morphological formation regulation, stress, and other physiological activities.
Among plant MYBs, the most common are R2R3-MYB transcription factors, which regulate various biological processes such as tissue development, the abiotic stress response, and metabolism. Many genes and enzymes involved in capsaicin biosynthesis have been identified, cloned, and studied [44]. However, there are only a few studies on the MYB transcription factor of Capsicum chinense, and this study cloned the CcMYB24 transcription factor from the Hainan Huangdenglong pepper to view its conserved domain, which is the typical R2R3-MYB. It was subcellular in the nucleus through bioinformatics analysis, suggesting that it has a regulatory role. However, the research group speculated that the CcMYB24 gene was negatively correlated with the content of capsaicin, and CcMYB24 may affect the accumulation of capsaicin by regulating the synthesis of flavonoids, anthocyanins, and lignin. Hence, the heterologous expression of the CcMYB24 gene confirmed its function in A. thaliana and VIGS in pepper. Heterologous expression is often performed on the model plant A. thaliana to speculate on the function of plant genes [45,46].
In this experiment, the expression of genes related to phenylpropanoid metabolism in pTRV2-free plants did not change much compared with plants without gene silencing, consistent with previous studies [19]. Studies have shown that genes such as pAMT, BCAT, Ca4H, KAS, PAL, 4CL, CS, and AT3 were positively correlated with capsaicin synthesis [19,[47][48][49]. Compared with the plants without silencing, the expression of PAL, 4CL, and pAMT-related genes in the phenylpropanoid metabolism pathway in plants infected with pTRV2-CcMYB24 decreased in the pericarp, and it increased in the placenta, which may be related to the synthesis of capsaicin being mainly in the placenta. In the fruit and placenta of plants infected with pTRV2-CcMYB24, the expression of related genes BCAT and KAS in the branched-chain fatty acid pathway decreased; the expression of related genes CCR1 and CAD in the lignin pathway and CS, AT3, and COMT in the capsaicin pathway increased; the capsaicin content increased, and the lignin content increased. It is speculated that CcMYB24 has an inhibitory effect on both capsaicin synthesis and lignin synthesis. The effect of CcMYB24 may be to activate the synthesis of related substances in other metabolic branches of the phenylpropanoid pathway, and the specific function needs to be further tested for identification.

Experimental Materials
Hainan Huangdenglong pepper (Capsicum chinense Jacq.) was from the pepper research group of the College of Horticulture, Hainan University. The full seeds were selected and soaked in a 55 • C thermostat water bath for 20 min, soaked in 0.1% potassium permanganate for 15 min, rinsed under running water for 2 min, and soaked in room temperature water for 12 h. Then, the seeds were placed in a Petri dish covered with paper towels, poured with water, exposed to 28 • C light for 16 h and 22 • C dark for 8 h to promote germination, and then seeded in a hole dish; after being exposed to white light, they grew to about six true leaves and were transplanted in pots.

Wild-type Arabidopsis thaliana (Columbia) was granted by the College of Tropical
Crops, Hainan University. The seeds were placed in the refrigerated layer at low temperature and vernalized for 3 days, The seeds were evenly sprinkled on the nutrient soil and moisturized with plastic wrap. After the cotyledon was unfolded, the seeds were transplanted, and the seeds were exposed to light at 23 • C for 16 h and dark at 18 • C for 8 h, and the side branches grew more unopened buds in about four weeks for transformation.

Main Reagents
The

Extraction of Total RNA and Synthesis of the First Strand of cDNA
The total RNA from pepper young leaves was extracted using a FastPure ® Plant Total RNA Isolation Kit (RC401), and the concentration and purity of the extracted RNA were determined by Nanodrop ONE and agarose gel electrophoresis, and stored at −20 • C for later use. The first strand of cDNA was synthesized by reference to the HiScript ® III 1st Strand cDNA Synthesis Kit (R312).

Cloning of the CcMYB24 Transcription Factor
According to the protein annotation information in the genome of Capsicum chinense on NCBI, CcMYB24 (PHU01939.1), primer premier 5.0 was used to design the cloning primers CcMYB24-F/R (Appendix A), using cDNA as the template for PCR amplification, 2 × Phanta Max Master Mix 25 µL, CcMYB24-F 2 µL, CcMYB24-R 2 µL, cDNA 5 µL, and ddH 2 O 16 µL. There was predenaturation at 95 • C for 3 min, denaturation at 95 • C for 15 s, annealing at 52 • C for 15 s, extension at 72 • C for 30 s, 35 extension cycles, 72 • C, and final extension for 5 min, 4 • C storage. The amplification products were detected by 1% agarose gel electrophoresis. The fragments of interest were purified and recovered by Cowin Biotech Century's DNA Gel Extraction Kit, connected to the pCE2-TA vector by 5min TM TA/Blunt-Zero Cloning Mix, and then transformed into DH5α Competent Cell. The bacterial solution PCR was verified correctly and sent to Guangzhou Tianyihuiyuan Co., Ltd. for sequencing.

Heterologous Expression of CcMYB24 in A. thaliana
Based on cloning CcMYB24 in Hainan Huangdenglong pepper, ScaI and XBaI digestion sites on the pBI121 vector were selected and primers pB-CcMYB24-F/R with homologous arms were designed (Appendix A). The plasmid extracted from the sequenced CcMYB24 gene solution was used as a template to obtain fragments with homologous arms and digestion sites. The pBI121 no-load plasmid was linearized by endoenzyme digestion of ScaI and XBaI, followed by gel recovery. The insert with the homologous arm and digestion site was attached to the linearization vector. The homologous recombinant plasmid was converted into DH5α, single colonies were singled for PCR and sequencing verification, and the plasmid pBI121-CcMYB24 plant heterologous expression vector was extracted from the sequenced homologous recombinant plasmid to GV3101 Chemically Competent Cell.
The CcMYB24 gene was transformed with reference to A. thaliana inflorescence infection [50][51][52]. The total DNA of transgenic A. thaliana was extracted by the CTAB method. Positive plants identified correctly by PCR were continued in culture to collect T 2 seeds. T 2 -generation seeds were screened for resistance using 50 µg/mL Kan. The total RNA of T 2 -generation A. thaliana leaves was extracted, and the expression of the CcMYB24 gene in transgenic A. thaliana plants was detected by qRT-PCR; A. thaliana plants with high expression were selected. Wild-type A. thaliana plants up to eight weeks old and T 2 -generation transgenic A. thaliana plants with positive screening verification and high expression were dried at 80 • C, ground, and subjected to a 40-mesh sieve, and 5 mg was weighed for lignin assay.

VIGS of CcMYB24 in Hainan Huangdenglong Pepper
CcMYB24-specific fragments of about 400 bp were selected, the digestion sites BamHI and EcoRI were added at the 5 end of the upstream and downstream primers, and a homologous arm of the pTRV2 vector of about 20 bp was added, respectively, and the primer pT-CcMYB24-F/R with a homologous arm (Appendix A) was designed. The homologous recombination method of the pTRV2-CcMYB24 plant heterologous expression vector was obtained.
When the fruits reached the green ripening stage, the plants with the same growth were divided into three experimental groups; each experimental group had three peppers: one group was pTRV2 unloaded, one group was pTRV2-CcMYB24, and another group was a blank control. Except for the blank control group, all other groups needed to be mixed with pTRV1 without load and injected. An amount of 100 µL of pTRV2 no-loaded Agrobacterium, pTRV2-CcMYB24 Agrobacterium, and pTRV1 Agrobacterium was added to 10 mL of YEB liquid medium (25 µg/mL Kan, Rif and Gen) at 28 • C, 200 rpm, and cultured for 16-20 h to make the OD 600 value between 1.0 and 1.2. Then, to 1 mL of the cultured bacteria, 25 mL of IM induction medium was added at a ratio of 1:25 (NaH 2 PO 4 , glucose and MES) at 28 • C, 200 rpm, and cultured for 16 h, so that the OD 600 value was about 0.9. Then, the cultured bacteria were centrifuged at 4 • C, 3500 rpm for 10 min to collect the bacteria, 25 mL of MES was added to resuspend the bacteria, and they were centrifuged again at 4 • C for 10 min. Next, the bacteria were resuspended with 1/2 volume of MES to an OD 600 of about 1.0, and 50 µL of 200 mM AS was added per 25 mL pTRV1 (the final concentration of AS was 400 µM, and the bacteria OD 600 was about 2.0). pTRV1 was mixed with pTRV2 no-load and pTRV2-CcMYB24 at 1:1, respectively, so that the final concentration of AS was 200 µM, with an OD 600 of about 1.0, and was placed in the dark at about 23 • C for 3 h. In the placenta of the young fruit, about five days after injection of pepper flowers, the plants were dark treated at 16 • C for 24 h, then placed in light at 24 • C/16 h and dark at 22 • C/8 h to continue incubation. After the pepper was ripe, the content of capsaicin and lignin in the pericarp and placenta of the pepper, as well as the expression of genes related to phenylpropanoid metabolism, were measured. The genes and primers determined are shown in Appendix A. Actin was the internal reference, and the qRT-PCR reaction system and procedure were consistent with 4.6.

Statistical Analysis
All experiments were repeated three times. Data are presented as the mean ± standard error. Statistical comparisons of the data obtained were performed by SPSS. The data were statistically analyzed by using Student's t-test, with p < 0.05 being considered significant.

Conclusions
In this study, we analyzed the expression characteristics of the CcMYB24 gene, which was hypothesized to be negatively correlated with capsaicin content by the group in the previous stage. We confirmed its function by heterologous expression of the CcMYB24 gene in A. thaliana and VIGS in pepper. The results indicate that CcMYB24 may play an inhibitory role in the lignin synthesis pathway in A. thaliana. It is determined that CcMYB24 can inhibit the synthesis of capsaicin and lignin, but whether it also affects the expression of other branch-related genes of phenylpropanoid metabolism and the synthesis of metabolites remains to be further studied.
Author Contributions: S.Y. wrote the manuscript; S.Y., W.Z. and S.C. conceived and designed the experiments; S.Y. and W.Z. completed the experiments and data analysis; L.Z., D.W., P.S., C.H. and G.F. were helpful with some technology of the experiments; Q.D. and Z.W. led and guided the study; S.C. led and guided the study and revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are available from the authors. Table A1. The lowercase portion of the primer sequence above is the homology arm.