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Article

Transcriptomic Analysis Reveals the Biosynthesis Mechanism of Coixol Under Salicylic Acid Treatment

by
Yao Wang
1,†,
Hanli Ye
1,†,
Xuqin Luo
1,
Ziwei Li
1,
Chuanqi Zheng
2 and
Dali Sun
1,*
1
School of Public Health, Guizhou Medical University, Guiyang 561113, China
2
Qianxinan Research Institute of Agricultural and Forestry Sciences, Xingyi 562400, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2025, 11(3), 234; https://doi.org/10.3390/horticulturae11030234
Submission received: 16 January 2025 / Revised: 13 February 2025 / Accepted: 20 February 2025 / Published: 21 February 2025
(This article belongs to the Section Medicinals, Herbs, and Specialty Crops)
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Versions Notes

Abstract

:
Background: Coix (Coix lacryma-jobi L.) is cultivated as medicine and food homologous resources containing various active components. As one of its main ingredients, coixol possesses the biological activities of fever reduction, pain relief, tumor suppression, blood glucose, and pressure decrease. However, the biosynthesis mechanism of coixol in coix is still unclear. Methods: In this study, different dosages of salicylic acid (SA) were applied to coix plants, and the contents of coixol from different parts were detected by an ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). The biosynthesis pathway of coixol was determined by high-throughput transcriptome sequencing analysis and the genes were then verified by qRT-PCR. Results: SA treatment significantly increased plant height, root length, and fresh weight and increased the coixol contents in the root, stem, leaf, and seed. In total, eight enzyme-encoding genes were screened out as the key genes in the biosynthesis of coixol. The bioaccumulation of coixol was mainly through benzoxazinoid biosynthetic metabolic pathway (ko00402). Conclusions: These findings not only pointed the way for increasing the content of coixol in cultivation but also provided a reference for further elucidation of the gene functions involved in the bioaccumulation of coixol.

1. Introduction

Coix (Coix lacryma-jobi L.) belongs to the Poaceae family and is acclaimed as the “king of poaceae plant” due to its high nutritional and medicinal value. It originates from Asia and is widely planted in tropical monsoon climate regions such as China, India, and Myanmar with a cultivation history spanning over 6000 years [1,2]. China is one of the most important countries for coix origin and cultivation, with abundant resources and widespread cultivation across most provinces, particularly in Guangxi, Guizhou, and Yunnan [3,4,5,6,7,8]. As an important resource of medicine and food homology, coix seed is rich in protein, polysaccharides, terpenes, flavonoids, sterols, alkaloids, lactams, and other active ingredients with the functions of decreasing blood glucose levels, enhancing immunity, diuresis, dampness removal, protecting the spleen, and detoxifying [9,10,11,12,13]. The ethanolic extracts from coix seeds were discovered to inhibit the activity of uterine sarcoma cells and enhance their sensitivity to doxorubicin [14]. Additionally, coix polysaccharides were found to reduce blood glucose in a type II diabetes mouse model by modulating gut microbiota and activating the IGF1/PI3K/AKT pathway [15]. Coix seed oil was also shown to decrease lipid accumulation in rat liver through the inhibition of AMPK phosphorylation and down-regulation of the expression of SePP1/apoER2 [16]. Therefore, coix is a valuable resource in the fields of food and medicine.
Secondary metabolites play an important role in plants, helping them to adapt to different environmental conditions and provide defense against injury from pests, diseases, and other factors. On the other hand, these secondary metabolites are beneficial to human health and could be developed into functional food or be used as medicine. The secondary metabolites in coix mainly include sterols, phenolics, flavonoids, alkaloids, triterpenoids, tocopherols, lactams, lignans, benzoxazinoids (BXs), etc. [17,18]. Among them, BXs are widely detected in the Poaceae family, consisting of indole derivatives and their derivatives with a 2-hydroxy-2H-1,4-benzoxazin-3(4H) one skeleton. As one of the BX compounds, coixol is an important metabolite and a unique compound in coix plants with various physiological activities. Modern research has found that coixol has the function of cancer inhibition and an analgesic effect on the central nervous system. It can be used as an auxiliary treatment for cancer, hypertension, hyperlipidemia, fatty liver disease, rheumatoid arthritis, and other diseases [19,20]. Moreover, it could be used as a functional food due to the activities of strengthening the spleen, removing dampness, clearing heat, draining pus, and anti-aging [21,22,23,24]. At present, the product of “Kanglaite” injection with coix seed oil as the main component has been widely used in the clinical treatment of non-small-cell lung cancer, liver cancer, and other cancers [25].
The endogenous metabolites in this plant could be largely produced in response to the biotic or abiotic stresses [26,27]. Salicylic acid (SA) is an important plant hormone for regulating growth, development, maturation, and defense responses in plants. Treatment with SA led to a notable up-regulation of OsbZIP genes in rice, which were believed to enhance rice blast resistance through gene regulation, signal transduction, and hormone response [28]. Additionally, SA alleviated selenium (Se) toxicity in rice by maintaining selenium homeostasis, reducing oxidative stress, regulating methylglyoxal (MG) detoxification, and up-regulating gene expression related to reactive oxygen species (ROS) [29]. In safflower plants, SA treatment reduced the content of anthocyanins, flavonoids, and phenolic compounds to repair drought stress by modulating hormonal balance, activating defense responses, improving photosynthesis, and enhancing antioxidant capacity [30]. Studies have shown that the application of exogenous SA can significantly reduce physiological damage in plants and improve their stress resistance by increasing the contents of secondary metabolites [31,32]. Therefore, SA can be used as an external signal substance to enhance the defense response of crops and improve the active substances.
The technology of transcriptomics could be applied to analyze the gene expression in cells or tissues and provide information on the related functions of genes, which is widely used in plant systematic research [33]. Further, RNA-seq can comprehensively and dynamically detect the expression of targeted genes to verify the results of transcriptomics [34]. For example, transcriptome analysis revealed that methyl jasmonate (MeJA) improved the ripening quality of mango at a low temperature by regulating 10 MiERFs with 26 differentially expressed genes (DEGs) involved in ethylene biosynthesis, starch degradation, cell wall degradation, and sugar transport [35]. Genes involved in the flavonoid biosynthesis pathway in different tissues of sea buckthorn (Hippophae rhamnoides L.) revealed the expression patterns of these genes in different tissues by using transcriptome analysis [36].
In order to make clear whether SA could enhance the content of coixol and that it is the mechanism of biosynthesis, different dosages of SA were applied to coix plants during the growth period. Transcriptomics was applied to analyze the DEGs in different parts as well as its possible synthetic pathways. The qRT-PCR method was then used to confirm the expression levels of these genes. The results of this study could provide a new reference for the increase of coixol yield and promote the exploitation and utilization of coix resources.

2. Materials and Methods

2.1. SA Treatments

Coix seeds used in this study were obtained from Xingyi, Guizhou Province. Plump seeds were selected and soaked at 60 °C for 30 min to promote germination and prevent black smut disease. The seeds were then sown in large-sized horticultural plastic pots with a diameter of 25 cm and depth of 29 cm on 18 May 2023. During booting and flowering periods, SA solutions (1200 mL) of 1.0 mmol/L (low dose) and 1.5 mmol/L (high dose) were sprayed on the plants every 7 d. An equal amount of distilled water was applied to the control group until the maturity stage. Three replicates were conducted for each treatment group. Twenty-four hours after the last watering, samples of roots, stems, leaves, and seeds were collected, washed, and immediately stored in a −80 °C refrigerator for analysis.

2.2. Determination of Coixol Content

After the measurement of plant height, root length, and fresh weight, samples of roots, stems, and leaves were cut into pieces with scissors, and seeds were ground into power. They were then dried in a desiccator at 40 °C. After that, 0.1 g of the dried root, stem, leaf, and seed powder samples were weighed accurately in a centrifuge tube added with 5 mL of 80% methanol, ultrasonicated for 70 min, and centrifuged at 4000 r/min. The supernatant was collected and concentrated using a rotary evaporator, and then filtered with a 0.45 μm microporous filter into an injection vial for analysis.
Concentrations of coixol were determined with UPLC–MS/MS (QSight 210, PerkinElmer, Waltham, MA, USA) and separated by an Agilent ZORBAX SB-C18 column (250 mm × 4.6 mm, 5 μm, Agilent Technologies, Santa Clara, CA, USA). The chromatographic conditions were set as follows: mobile phase was acetonitrile and 0.1% formic acid solution (25/75, v/v), flow rate was 1.0 mL/min, column temperature was maintained at 25 °C, injection volume was 20 μL. The mass spectrometry conditions were as follows: ion source ESI+, electrospray voltage (eV) was 5500 V, atomizing gas pressure (GS1) was 50 psi, curtain gas pressure (CUR) was 25 psi, auxiliary heating gas pressure (GS2) was 60 psi, ion source temperature was 550 °C, inlet voltage (EP) was 10 V, and collision chamber outlet voltage (CXP) was 15 V.

2.3. RNA-Seq Library Preparation and Sequencing

To further understand the transcriptional regulatory mechanism of SA on the biosynthesis of coixol, roots (Root-C), stems (Stem-C), leaves (Leaf-C), and seeds (Seed-C) in the control group; roots (Root-L), stems (Stem-L), leaves (Leaf-L), and seeds (Seed-L) in the low-dose group; and roots (Root-H), stems (Stem-H), leaves (Leaf-H), and seeds (Seed-H) in the high-dose group were selected for transcriptome sequencing. Coix samples (100 mg) from the above groups were weighted and ground with liquid nitrogen. Total RNA was extracted using both ethanol precipitation and pBIOZOL plant total RNA extraction reagent methods [37]. Subsequently, the total RNA was identified and quantified using a Qubit 4 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and a Qsep400 high-throughput bio-fragment analyzer (BiOptic, Inc., Taiwan, China). After RNA samples passed the quality check, a sequencing library was constructed using the KAPA™ Single-Strand RNA Library Preparation Kit (Illumina, San Diego, CA, USA). The sequencing was performed on the Illumina HiseqX platform, and raw data (raw reads) were obtained.

2.4. De Novo Assembly, Functional Annotation, and Enrichment Analysis of DEGs

Raw data were filtered using the software fastp (Version 0.23.2, Shenzhen, Guangdong, China) to obtain clean reads and then assembled into transcripts using Trinity software (Version v2.13.2, Cambridge, MA, USA) [38]. After that, Corset was employed to cluster and remove redundancy from the assembled transcripts. The non-redundant transcript sequences were aligned against the Kyoto Encyclopedia of Genes and Genomes (KEGG), Non-Redundant Protein Sequence Database (NR), Swiss-Prot, Gene Ontology (GO), Clusters of Orthologous Groups/Eukaryotic Orthologous Groups (COG/KOG), and Translation of EMBL (Trembl) databases using DIAMOND software (Version v2.0.9, Tübingen, Germany), and the amino acid sequences were aligned with the Pfam database using HMMER software, resulting in annotation information from the seven major databases for the transcripts. Fragments Per Kilobase of transcripts per Million fragments mapped (FPKM) were employed as an indicator to measure gene expression levels [39]. Once the expression levels of each Unigene in the samples were determined, genes were functionally annotated to facilitate subsequent functional research. By comparing the gene expression levels between treatment groups, DEGs were identified under the conditions of fold change (FC) ≥ 1 and false discovery rate (FDR) < 0.05. GO enrichment analysis was carried out based on a comprehensive database describing gene functions and KEGG enrichment analysis was performed based on the major public databases related to the pathway.

2.5. qRT-PCR Analysis

To verify the reliability of gene expression based on the results of transcriptome sequencing, four notably up-regulated DEGs and four significantly down-regulated DEGs were selected for qRT-PCR analysis. Total RNA was extracted from coix using plant RNA extraction kit (Omega Bio-Tek. Inc., Norcross, GA, USA). cDNA was synthesized using Takara RR047A mRNA reverse-transcription kit (TaKaRa, Kusatsu, Shiga, Japan). Real-time fluorescent quantitative PCR (qRT-PCR) was conducted using Takara Premix Ex Taq TM II RR820A kit (TaKaRa, Kusatsu, Shiga, Japan) with GAPDH serving as the reference gene and detected by a BioRad CFX 96 quantitative PCR instrument (Bio-Rad Laboratories, Hercules, LA, USA). The experimental results were analyzed using the 2−ΔΔCt method [40]. The primers used in this study are listed in Table S1.

2.6. Statistical Analysis

Data obtained in this study are presented as the mean ± standard deviation (SD). Data analysis was performed using SPSS 27 statistical software by using one-way analysis of variance (ANOVA), followed by Tukey’s test. Differences were considered significant at p ≤ 0.05. All figures were created using Origin 2021 and GraphPad Prism 8 software programs.

3. Results

3.1. Effect of SA on Growth Indicators of Coix

The plant height of coix significantly increased after high doses of SA treatment (p < 0.05) compared to the control group. The height was 126.53 ± 4.16 cm in the control group and raised to 158.03 ± 14.32 cm in the high-dose group (Figure 1A). The root length of coix notably increased after low (58.40 ± 5.23 cm) and high (70.57 ± 1.50 cm) doses of SA treatment compared to the control group (39.20 ± 5.36 cm) (p < 0.05) (Figure 1B). Similarly, the fresh weight of coix treated with high doses of SA solution significantly increased compared to the control group (p < 0.05): 48.47 ± 1.60 g in the control group and 68.23 ± 8.30 g in the high-dose group (Figure 1C). These results indicated that SA treatment could increase the plant height, root length, and fresh weight of coix plants in a dose-dependent manner.

3.2. The Effect of SA on the Content of Coixol in Different Parts of Coix

In the roots, the content of coixol in the control group was 487.00 ± 8.52 mg/kg and 609.67 ± 24.31 mg/kg after high doses of SA treatment. Therefore, coixol levels notably increased after high doses of SA solution treatment compared to the control group (p < 0.05) (Figure 1D). The content of coixol significantly increased in stems after low and high doses of SA treatment compared to the control group (p < 0.01, p < 0.05). The concentration of coixol was 1.17 ± 0.12 mg/kg in the control group and raised to 5.91 ± 1.93 mg/kg and 4.12 ± 0.13 mg/kg after low and high doses of SA treatment, respectively (Figure 1E). Similarly, the content of coixol in leaves increased significantly after low and high-dose SA treatment (p < 0.05), with 15.07 ± 2.54 mg/kg in the control group enhanced to 23.73 ± 4.06 mg/kg and 23.54 ± 1.86 mg/kg in the low and high-dose groups (Figure 1F). In coix seeds, the content of coixol significantly increased with the increase of SA concentration in a dose-dependent manner. The content of coixol was 1.71 ± 0.50 mg/kg in the control group and increased to 3.60 ± 0.33 mg/kg in the low-dose group (p < 0.05) and 7.22 ± 0.58 mg/kg in the high-dose group (p < 0.01) (Figure 1G). Therefore, the application of the SA solution notably enhanced the contents of coixol in roots, stems, leaves, and seeds.

3.3. Transcriptome Analysis

After raw data filtering, sequencing error rate checking, and GC content distribution checking, clean reads were obtained for subsequent analysis. The 36 samples yielded 239.62 G of Clean Base with the effective data volume of each sample ranging from 5.79 to 7.58 G. The Q20 base percentage ranged from 97.92 to 98.53%, and the Q30 base percentage ranged from 94.26 to 95.73% with an average GC content of 54.24%. All clean reads were then assembled de novo using the Trinity program, producing 740,306 transcripts and 378,420 Unigenes with average lengths of 984 and 1406 bp for transcripts and Unigenes, respectively (Table 1).

3.4. Gene Function Annotation

The assembled Unigenes were annotated against seven major functional databases, including KEGG, Nr, Swiss-Prot, GO, KOG, TrEMBL, and Pfam with 378,420 Unigenes annotated in total. The proportion of Unigenes annotated by KEGG, Nr, Swiss-Prot, TrEMBL, KOG, GO, and Pfam were 49.14%, 68.43%, 44.43%, 66.85%, 48.47%, 58.48%, and 55.17%, respectively (Figure 2A). The gene homology species distribution showed that the highest proportion of Unigenes annotated to Quercus suber was 25.02%, followed by Miscanthus lutarioriparius (11.86%), Sorghum bicolor (11.46%), and Zea mays (6.67%) (Figure 2B). After GO annotation of the genes, the functions were divided into three categories: biological process (BP), cellular component (CC), and molecular function (MF). The highest number of Unigenes annotated to BP was in the cellular process (145,025) followed by the metabolic process (125,135). Unigenes annotated to CC included the cellular anatomical entity (179,697) and protein-containing complex (39,526). The second highest number of Unigenes annotated to MF included binding (131,281) and catalytic activity (113,051) (Figure 2C). The KOG classification showed that the largest number of Unigenes annotated to general function prediction was only 33,782, followed by post-translational modification, protein turnover, chaperones (22,583), and signal transduction mechanisms (17,215) (Figure 2D).

3.5. Differential Gene Expression Screening

DEGs were screened with the cutoff values of FC ≥ 1 and FDR < 0.05. Volcano plots showed that 58,561 and 63,241 DEGs were screened, out of which 29,009 and 25,175 were up-regulated and 29,552 and 38,066 were down-regulated in Root-L and Root-H, respectively, compared to Root-C. In coix stems, 13,870 and 28,948 DEGs were screened, out of which 6367 and 17,244 were up-regulated and 7503 and 11,704 were down-regulated in Stem-L and Stem-H, respectively, compared to Stem-C. In coix leaves, 26,445 and 25,846 DEGs were screened, out of which 11,350 and 11,477 were up-regulated and 15,095 and 14,369 were down-regulated in Leaf-L and Leaf-H, respectively, compared to Leaf-C. In coix seeds, 3602 and 2777 DEGs were screened, out of which 1641 and 993 were up-regulated and 1961 and 1784 were down-regulated in Seed-L and Seed-H, respectively, compared to Seed-C (Figure S1).

3.6. Differential Gene Expression GO Analysis

GO enrichment analysis was performed based on the functions of BP, CC, and MF. In coix roots, low and high doses of SA treatment enriched similar functions of these processes. The cellular process, metabolic process, and response to stimulus were the top 3 most enriched for the BP category, CC was concentrated in the cellular anatomical entity and protein-containing complex categories, and the MF category was mainly concentrated in binding and catalytic activity. The differential genes in coix stems, leaves, and seeds under SA treatment were primarily enriched in the same parts of BP, CC, and MF as those in the roots (Figure 3). These results revealed that the effects of SA treatment on different parts of the coix plant had certain commonalities, which could affect the growth and development of coix plants and their response to environmental stress.

3.7. Differential Gene Expression KEGG Analysis

KEGG enrichment analysis was conducted on differential genes from coix roots, stems, leaves, and seeds after being treated with low and high doses of the SA solution. In total, 50 KEGG pathways with the lowest Q-values from the enrichment analysis results were selected. As shown in Figure S2, the metabolic pathways in different parts of coix plants were primarily categorized into five major groups: cellular processes, environmental information processes, genetic information processing, metabolism, and organismal systems. In coix roots, the highest number of annotated genes was found in the metabolism, with the main DEGs annotated in metabolic pathways and secondary metabolite biosynthesis accounting for 48.03% and 26.89% in the Root-L vs. Root-C group, and 46.35% and 25.55% in the Root-H vs. Root-C group, respectively. The enrichment of plant–pathogen interactions in organismal systems was detected only in the Root-H vs. Root-C group with a proportion of 5.16%. Except for Stem-L vs. Stem-C, DEGs were mainly annotated in environmental information processes, genetic information processing, metabolism, and organism systems in stems, and leaves were consistent with roots, with proportions of 47.57% and 28.62% (Stem-L vs. Stem-C), 46.91% and 27.54% (Stem-H vs. Stem-C), 46.95% and 28.35% (Leaf-L vs. Leaf-C), 47.09% and 27.82% (Leaf-H vs. Leaf-C), 51.59% and 31.77% (Seed-L vs. Seed-C), and 51.33% and 29.35% (Seed-H vs. Seed-C), respectively.
As shown in Figure 4, the top 20 significantly enriched pathways are listed. In four parts of the coix plant, most DEGs were enriched in metabolic pathways, followed by the biosynthesis of secondary metabolites, while these two pathways had the smallest rich factor, indicating the lowest degree of enrichment. Pathways of zeatin biosynthesis and benzoxazinoid biosynthesis had the largest rich factor values in the Root-L and Root-H groups. In the stems, the pathway of benzoxazinoid biosynthesis had the largest rich factor value during low and high-dose SA treatment while exopolysaccharide biosynthesis owned the largest rich factor in leaves. Pathways of stilbenoid, diarylheptanoid, and gingerol biosynthesis had the largest rich factor values in the Seed-L group and photosynthesis-antenna proteins had the largest rich factor value in the Seed-H group. In summary, the effects of SA treatment on coix were primarily reflected in the regulation of gene expression, metabolic pathways, biosynthesis of secondary metabolites, environmental responses, plant–pathogen interactions, and photosynthesis, which could affect the growth, development, stress adaptability, and pathogen defense capabilities of the coix plant.

3.8. DEGs Related to the Biological Accumulation of Coixol Under SA Treatment

Based on the KEGG metabolic pathway enrichment analysis results, it was discovered that the accumulation of coixol primarily depended on benzoxazinoid biosynthesis (ko00402). Genes in coix roots, stems, leaves, and seeds exhibited differential expression in this pathway. Under SA treatment, eight enzyme-encoding genes played significant roles in the biosynthesis of coixol (Figure 5). Further analysis showed that six related enzymes, including indole-3-glycerol-phosphate lyase (BX1), indole-2-monooxygenase (BX2), indolin-2-one monooxygenase (BX3), 2,4-dihydroxy-1,4-benzoxazin-3-one-glucoside dioxygenase (BX6), 2,4,7-trihydroxy-1,4-benzoxazin-3-one-glucoside 7-O-methyltransferase (BX7), and UDP-glucosyltransferase (BX8/9), were involved in the biosynthesis of coixol in the roots. In total, 30 and 41 DEGs were involved in the Root-L and Root-H groups. Among them, 13 DEGs were commonly up-regulated and 11 DEGs were commonly down-regulated (Table 2). In the Stem-L group, 26 DEGs were detected, involving eight enzymes, which were BX1, BX2, BX3, 3-hydroxyindolin-2-one monooxygenase (BX4), 2-hydroxy-1,4-benzoxazin-3-one monooxygenase (BX5), BX6, BX7, and BX8/9, while the Stem-H group involved six enzymes, including BX1, BX2, BX3, BX6, BX7, and BX8/9, with 26 DEGs. In these two groups, four and ten DEGs were commonly up-regulated and down-regulated (Table S2). In the leaves, eight related enzymes named BX1, BX2, BX3, BX4, BX5, BX6, BX7, and BX8/9 were involved with 38 and 18 DEGs detected in the low and high-dose groups, respectively. Between them, three and eleven DEGs were commonly up-regulated and down-regulated (Table S3). The enzyme genes related to the biosynthesis of coixol in seed were BX2, BX3, BX7, and BX8/9, with four DEGs in the Seed-L group, and BX3, BX6, and BX8/9, with five DEGs in the Seed-H group. Among them, one DEG was commonly up-regulated and one DEG was commonly down-regulated (Table S4).

3.9. qRT-PCR

To validate the credibility of the RNA-seq data, the expression levels of eight coixol biosynthesis genes were analyzed by qRT-PCR in leaves. Cluster-204385.0, cluster-53781.1, cluster-234475.0, and cluster-190323.0 notably increased, while cluster-219989.1, cluster-219989.2, cluster-215188.0, and cluster-206382.0 significantly decreased (Figure 6). SA treatment significantly affected the expression of these genes, and the trends in gene expression detected by qRT-PCR were essentially consistent with the results of the transcriptome, indicating that the data were reliable.

4. Discussion

SA, one of the widely used endogenous signaling molecules in plants, has been identified as a plant hormone that plays important roles in disease resistance, cold tolerance, drought resistance, salt tolerance, fruit ripening, preservation after harvest, and seed germination [41,42,43,44]. There have been limited studies about the roles of SA in the biosynthesis of coixol, and its molecular mechanism is still unclear. Therefore, RNA-seq technology was applied to comparatively analyze the transcriptome in different parts of the coix plant after low and high doses of SA treatment.
Our study showed that SA treatment during the maturation period notably increased the plant height, root length, and fresh weight, revealing that SA treatment could promote the growth and development of the coix plant. The contents of coixol differed in four parts of the plant, with the highest concentrations detected in the roots, followed by the leaves, seeds, and stems. This might be due to their different roles in the physiological processes of the coix plant. For instance, the roots might be responsible for the absorption of water and nutrients while the leaves are involved in photosynthesis. Moreover, the roots are the main place for coixol accumulation, and the effect on the roots by SA was relatively larger than in other parts [45]. The coixol content in the stems and seeds was relatively low, while SA treatment resulted in a significant increase in coixol accumulation probably because SA treatment increased the metabolic activities in coix plants and further enhanced the coixol contents.
Plants have evolved a range of specialized or secondary metabolites to mitigate both biotic and abiotic stresses. Among them, benzoxazinoids (BXs) are an important group of defensive chemicals that are widely presented in the Poaceae family, such as corn, wheat, and rye [46,47,48]. BX and its metabolites DIMBOA, DIBOA, BOA, and MBOA (coixol) were demonstrated to achieve activities against diseases, pests, and allelopathy. They are considered crucial defensive metabolites in plants and are stored in the form of glycosides in the vacuoles of undamaged plant cells. Once the plant is attacked, they are broken down into the corresponding aglycones and sugars to exert their defensive role [46]. The biosynthetic pathway of BXs and related genes has been well studied in Poaceae species such as corn and wheat [49]. However, research on this pathway and its related genes in coix remains scarce. Coixol, a unique active ingredient found in the coix plant, has demonstrated a variety of physiological effects and has been used as an adjuvant therapeutic agent for various diseases such as cancer, diabetes, and rheumatoid arthritis [24,50]. In this study, the results of high-throughput sequencing showed that the biosynthesis of coixol mainly depended on the benzoxazinoid biosynthesis pathway, which was involved in eight related enzymes named BX1, BX2, BX3, BX4, BX5, BX6, BX7, and BX8/9. BX2 to BX5 are cytochrome P450-dependent monooxygenases, which is largely consistent with the benzoxazinoid biosynthesis pathway in corn [45,51]. The differential expression levels of genes related to coixol biosynthesis were observed in roots, stems, leaves, and seeds under SA treatment. Among them, the number of DEGs in roots was much higher than in other parts. With the increase of SA concentration, the number of DEGs increased along with the content of coixol. Leaves and stems treated with low concentrations of SA involved eight DEGs with down-regulated DEGs more than up-regulated ones. Stems treated with high concentrations of SA treatment only involved six related enzymes, while seeds involved few DEGs compared to other parts. Therefore, SA treatment could increase the expression levels of coixol biosynthesis-related genes in coix, thereby promoting the biosynthesis of coixol.
According to KEGG enrichment analysis, it was observed that under SA treatment, the DEGs in each group were not only enriched in the benzoxazinoid biosynthesis metabolic pathway stimulating the biological accumulation of coixol but were also significantly enriched in other metabolic pathways, leading to a series of metabolic activities. In coix root, DEGs in the Root-L group were significantly enriched in metabolic pathways (ko01100), the biosynthesis of secondary metabolites (ko01110), and isoquinoline alkaloids biosynthesis (ko00950), while DEGs in the Root-H group were significantly enriched in metabolic pathways (ko01100), tyrosine metabolism (ko00350), and isoquinoline alkaloid biosynthesis (ko00950). In the stems, DEGs in the Stem-L group were significantly enriched in plant–pathogen interactions (ko04626), plant hormone signal transduction (ko04075), and MAPK signaling pathway-piant (ko04016), while DEGs in the Stem-H group were significantly enriched in plant hormone signal transduction (ko04075), plant–pathogen interactions (ko04626), and the biosynthesis of secondary metabolites (ko01110). In leaves, DEGs in the Leaf-L group were enriched in plant hormone signal transduction (ko04075), alpha-linolenic acid metabolism (ko00592), and plant–pathogen interactions (ko04626), while DESs in the Leaf-H group were mainly enriched in the alpha-linolenic acid metabolism (ko00592), the biosynthesis of secondary metabolites (ko01110), and plant hormone signal transduction (ko04075). In seeds, DEGs in the Seed-L group were significantly enriched in the biosynthesis of secondary metabolites (ko01110); phenylalanine, tyrosine, and tryptophan biosynthesis (ko00400); and phenylpropanoid biosynthesis (ko00940), while DEGs in the Seed-H group were significantly enriched in photosynthesis (ko00195), photosynthesis-antenna proteins (ko00196), and metabolic pathways (ko01100). In summary, the activation of defense-related metabolic pathways, the regulation of plant hormone signal transduction, the enhancement of secondary metabolite biosynthesis, the improvement of the response to environmental stress, and the impact on photosynthesis were notably affected in coix plants by SA treatment. Pathways that could improve the disease resistance, stress tolerance, and growth regulation capabilities of coix plants contribute to further revelations of the molecular metabolic mechanisms of coix induced by SA.

5. Conclusions

In our study, low and high doses of SA treatment notably increased the plant height, root length, and fresh weight of coix plants in a dose-dependent manner. Moreover, this treatment promoted the accumulation of coixol in the roots, stems, leaves, and seeds. Transcriptome sequencing unearthed eight related enzymes in the benzoxazinoid biosynthetic metabolic pathway that indicated that this pathway might be the main route for the biosynthesis of coixol. The results of this study provided important data for the enhancement of coixol content in coix through genetic engineering and breeding, thereby increasing the agricultural economic value of coix.

Supplementary Materials

The following supporting information can be downloaded at: www.mdpi.com/article/10.3390/horticulturae11030234/s1, Figure S1: Volcano plot of DEGs in the differential parts of coix plant; Figure S2: Bar chart of KEGG enrichment of DEGs in different parts of coix plant; Table S1: qRT-PCR Primer sequence; Table S2: Enzymes and genes involved in the biosynthesis of coixol in stem; Table S3: Enzymes and genes involved in the biosynthesis of coixol in leaves; Table S4: Enzymes and genes involved in the biosynthesis of coixol in seeds.

Author Contributions

Conceptualization, C.Z. and D.S.; methodology, Y.W. and D.S.; validation, Y.W., H.Y., X.L., and Z.L.; formal analysis, Y.W.; investigation, C.Z.; writing—original draft preparation, Y.W.; writing—review and editing, D.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Guizhou Provincial Science and Technology Projects ([2022]151), the Central Government Support Fund for the Reform and Development of Local Universities ([2023]067), the Guizhou Provincial Foundation for Excellent Scholars Program (GCC [2023]076), and the earmarked fund for Guizhou modern agriculture research system (GZSTCYJSTX2025-1).

Data Availability Statement

All datasets obtained for this study are included in the manuscript/Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

References

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Horticulturae 11 00234 g001
Figure 1. Effects of SA on the growth indicators and coixol content in coix plants. (A) Plant height; (B) root length; (C) fresh weight; (D) coixol contents in roots, (E) stems, (F) leaves, and (G) seeds. Different lowercase letters represent significant differences between treatments (p < 0.05).
Figure 1. Effects of SA on the growth indicators and coixol content in coix plants. (A) Plant height; (B) root length; (C) fresh weight; (D) coixol contents in roots, (E) stems, (F) leaves, and (G) seeds. Different lowercase letters represent significant differences between treatments (p < 0.05).
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Figure 2. Coix gene functional annotation chart. (A) Unigene annotation statistics chart. (B) Unigene NR annotation pie chart root length. (C) Unigene GO classification bar chart. (D) Unigene KOG classification bar chart.
Figure 2. Coix gene functional annotation chart. (A) Unigene annotation statistics chart. (B) Unigene NR annotation pie chart root length. (C) Unigene GO classification bar chart. (D) Unigene KOG classification bar chart.
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Figure 3. Bar chart of GO enrichment of DEGs in different parts of coix plants.
Figure 3. Bar chart of GO enrichment of DEGs in different parts of coix plants.
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Figure 4. Scatter plot of KEGG enrichment of DEGs in different parts of coix plants.
Figure 4. Scatter plot of KEGG enrichment of DEGs in different parts of coix plants.
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Figure 5. DEGs associated with coixol biosynthesis under SA treatment. The red font represents the genes involved in qRT-PCR verification.
Figure 5. DEGs associated with coixol biosynthesis under SA treatment. The red font represents the genes involved in qRT-PCR verification.
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Figure 6. Validation of DEGs by qRT-PCR. The line chart displays the FPKM values of mRNA, and the histogram presents the relative values determined by qRT-PCR.
Figure 6. Validation of DEGs by qRT-PCR. The line chart displays the FPKM values of mRNA, and the histogram presents the relative values determined by qRT-PCR.
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Table 1. Assembly results statistics.
Table 1. Assembly results statistics.
TypeNumber (n)Mean Length (bp)N50 (bp)N90 (bp)Total Bases (n)
Transcript740,3069841778372728,126,344
Unigene378,42014062078619532,088,427
Table 2. Enzymes and genes involved in the biosynthesis of coixol in roots.
Table 2. Enzymes and genes involved in the biosynthesis of coixol in roots.
GroupsMetabolic PathwaykoIDEnzymeNumber of DEGs
Root-L vs. Root-CBenzoxazinoid biosynthesisko00402Indole-3-glycerol-phosphate lyase5
Indole-2-monooxygenase5
Indolin-2-one monooxygenase4
2,4-dihydroxy-1,4-benzoxazin-3-one-glucoside dioxygenase7
2,4,7-trihydroxy-1,4-benzoxazin-3-one-glucoside 7-O-methyltransferase1
UDP-glucosyltransferase8
Root-H vs. Root-CBenzoxazinoid biosynthesisko00402Indole-3-glycerol-phosphate lyase6
Indole-2-monooxygenase8
Indolin-2-one monooxygenase8
2,4-dihydroxy-1,4-benzoxazin-3-one-glucoside dioxygenase6
2,4,7-trihydroxy-1,4-benzoxazin-3-one-glucoside 7-O-methyltransferase2
UDP-glucosyltransferase11
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Wang, Y.; Ye, H.; Luo, X.; Li, Z.; Zheng, C.; Sun, D. Transcriptomic Analysis Reveals the Biosynthesis Mechanism of Coixol Under Salicylic Acid Treatment. Horticulturae 2025, 11, 234. https://doi.org/10.3390/horticulturae11030234

AMA Style

Wang Y, Ye H, Luo X, Li Z, Zheng C, Sun D. Transcriptomic Analysis Reveals the Biosynthesis Mechanism of Coixol Under Salicylic Acid Treatment. Horticulturae. 2025; 11(3):234. https://doi.org/10.3390/horticulturae11030234

Chicago/Turabian Style

Wang, Yao, Hanli Ye, Xuqin Luo, Ziwei Li, Chuanqi Zheng, and Dali Sun. 2025. "Transcriptomic Analysis Reveals the Biosynthesis Mechanism of Coixol Under Salicylic Acid Treatment" Horticulturae 11, no. 3: 234. https://doi.org/10.3390/horticulturae11030234

APA Style

Wang, Y., Ye, H., Luo, X., Li, Z., Zheng, C., & Sun, D. (2025). Transcriptomic Analysis Reveals the Biosynthesis Mechanism of Coixol Under Salicylic Acid Treatment. Horticulturae, 11(3), 234. https://doi.org/10.3390/horticulturae11030234

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