Transcriptome analysis reveals the potential mechanism of altering viability, yield, and isoquinoline alkaloids in Coptis chinensis through Cunninghamia lanceolata understory cultivation

The dried rhizomes of Coptis chinensis hold significance in Chinese medicine. Monocropping C. chinensis under the shade of a manmade scaffold, the primary planting mode, poses a threat to the ecological balance. In recent years, the practice of C. chinensis–Cunninghamia lanceolata understory cultivation has gained prevalence in southwest China. However, there is no evidence to suggest that understory cultivation enhances the viability, yield, or isoquinoline alkaloid content of C. chinensis . This study examined the physiological properties, yield indicators, and isoquinoline alkaloid content to investigate variations in C. chinensis in response to understory cultivation. Transcriptome analysis was conducted to elucidate potential mechanisms driving these alterations. The results indicate that understory cultivation significantly enhances the viability, yield, and levels of epiberberine, palmatine, and berberine in C. chinensis while reducing coptisine content. Transcriptomic analyses identified 2062 upregulated and 1853 downregulated genes in the understory cultivation system. Pathways such as “phenylpropanoid biosynthesis,” “zeatin biosynthesis,” “photosynthesis,” “tyrosine metabolism,” “isoquinoline alkaloid biosynthesis,” and “starch and sucrose metabolism” exhibited significant enrichment of differentially expressed genes (DEGs). DEGs involved in these pathways were thoroughly analyzed. INV , BGL-2 , BGL-4 , SPS-2 , AMY-3 , Psb B , Psb R , Psb S , Psa D , Psa E , Psa H , Psa O , Pet C , Pet H , deta , and b exhibited significant positive correlations with plant fresh weight, aboveground fresh weight, and underground fresh weight. 6-OMT-2 and COMT1-3 displayed significant positive correlations with coptisine content, but negative correlations with epiberberine, palmatine, and berberine content. ZOG-1 , ZOG-3 , TAT , PPO , POD-13 and UGT 73C5-1 showed noteworthy positive correlations with berberine content. Conversely, MIFH , POD-4 , POD-5 , and POD-8 displayed significant positive correlations with epiberberine, palmatine, and berberine content. POD-5 , and POD-7 were significantly negatively correlated with coptisine content. Furthermore, gene expression levels determined by qRT–PCR aligned with the transcriptomic sequencing results, confirming the


Introduction
Coptis chinensis Franch., a member of the Ranunculaceae family, stands as a pivotal medicinal plant [1].The dried rhizomes of C. chinensis, known as "Weilian" in Chinese, found widespread use for purging fire, detoxication, and their antioxidant properties [2].The primary active components of C. chinensis include isoquinoline alkaloids, such as epiberberine, palmatine, berberine, and coptisine [3].With an annual output of 4000 tons, C. chinensis is extensively cultivated in southwest China [4], and its utilization has expanded into functional foods, beverages, and various products [5], leading to a significant rise in demand.However, the prevailing practice of monocropping C. chinensis under the shade of manmade scaffolds has been detrimental to the environment [6].Continuous monocropping cultivation has resulted in a large-scale production reduction and diminished the quality of C. chinensis [4].
Understory cultivation emerges as a crucial agroforestry practice fostering increased biodiversity, the ecological balance, and enhanced crop quality [7,8].The complementarity in traits, timing, and spatial utilization between crops in the understory cultivation system plays a pivotal role in elevating crop quality [9,10].In the case of C. chinensis, the tall trees Cunninghamia lanceolata (Lamb.)Hooks, provide natural shade for C. chinensis, obviating the need for artificial scaffolds.Therefore, the C. chinensis-C.lanceolata understory cultivation (UC) pattern has been adopted in Lichuan, Hubei province.A prior study demonstrated that understory cultivation systems could enhance the yield and quality of Fritillaria hupehensis [11].However, it remains uncertain whether this approach can similarly improve the yield of isoquinoline alkaloids, lacking the scientific evidence.
In recent years, integrative analyses combining physiological-chemical properties and transcriptomic sequencing have been employed to identify genes associated with yield and accumulation of secondary metabolite, predicting their functions [12].Liu et al. analyzed the C. chinensis genome [13], and Chen et al. identified the genes linked to isoquinoline alkaloid biosynthesis [1].However, the crucial genes governing the yield and isoquinoline alkaloid biosynthesis in response to understory cultivation remain poorly understood.
This study evaluated the physiological properties, yield indicators, and isoquinoline alkaloid content to discern alterations in C. chinensis under understory cultivation with C. lanceolata.Subsequent transcriptomic sequencing reveled gene expression variations crucial for enhancing viability, yield, and isoquinoline alkaloid production.Pearson's correlation analysis identified candidate genes associated with the response to understory cultivation, predicting the relationships between gene expression and yield indicators/isoquinoline alkaloid content.These findings of novel insights into potential mechanisms that enhance the viability, yield, and isoquinoline alkaloid content of C. chinensis under understory cultivation systems.

Plant materials and experimental design
The experiment took place in Jianzhuxi, Lichuan, Hubei Province, China (108° 33′ 21″ E, 30° 23′ 38′′ N, altitude 1530 m).Two cropping systems, C. chinensis monocropping (MC) and UC, were investigated.In the UC system, C. chinensis seedlings were planted within a 20-yearold C. lanceolata plantation with a plant row spacing of 2 × 3 m.In the MC system, C. chinensis seedlings were planted in the shade of a man-made scaffold using wooden stakes.C. chinensis seedlings, graded before testing, comprised 2-year-old healthy seedlings of similar sizes (± 20% errors).On May 18, 2021, C. chinensis seedlings were sown with a row spacing of 10 × 10 cm in both the MC and UC systems.The experiment, conducted in three replicates for MC and UC systems, had a plot size of 6.67 m 2 (1.50 × 4.45 m), and all plots were maintained following a conventional management model.
On February 13, 2022, young leaves were collected from three independent C. chinensis plants and combined as one biological duplicate for the UC and MC systems, respectively.Each system underwent three biological replicates.Leaves, frozen in liquid nitrogen and stored at − 80 °C, were used for RNA extraction.In addition, on June 13, 2022, 10 individual plants were randomly chosen from the UC and MC systems to measure plant height and fresh weight (yield indicators).Subsequently, fresh roots and plants were harvested.Fresh roots, washed and dried at 60 °C, were used to measure isoquinoline alkaloid contents, while fresh leaves utilized for measuring physiological properties.

Physiological property, plant height, and yield indicator measurements
Traditionally, C. chinensis rhizomes are harvested 5 years after planting for use in Chinese medicine [6].This study selected plant fresh weight, aboveground fresh weight, and underground fresh weight as yield indicators. Plant height was measured using a 30 cm ruler, and plant, aboveground, and underground fresh weights were measured with an electronic (maximum range: 200 g).Values were averaged across 10 plants.Chlorophyll a (Chl a), chlorophyll b (Chl b), Chl a + Chl b (Chl), carotenoid (Car), peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), soluble protein, and soluble sugar content were measured following the procedures outlined by Li et al. [13].Starch and sucrose content, sucrose phosphate synthase (SPS), and sucrose synthase (SS, synthetic direction) activities were evaluated as described by Shi et al. [14].

Isoquinoline alkaloid content measurements
The isoquinoline alkaloids, including epiberberine, palmatine, and berberine, were prepared following previously established procedures [2].The isoquinoline alkaloid contents were determined using a high-performance liquid chromatography (HPLC) system (Agilent 1260, Agilent Technologies, Germany) equipped with an Agilent C-18 chromatographic column (5 μm, 4.6 × 250 mm).The mobile phase and HPLC conditions were set according to the protocol outlined by Liu et al. [2].

Transcriptome analysis
Transcriptomic sequencing was performed by Shanghai Majorbio Bio-Pharm Biotechnology Co. Ltd. (Shanghai, China).Total RNA was extracted from C. chinensis leaves following the instructions for TRIzol ® Reagent (Invitrogen, USA).RNA purification and concentration detection were performed as previously described [11].The construction of cDNA libraries utilized high-quality RNA, adhering to the manufacturer's instructions on an Illumina ® Stranded mRNA Prep (Illumina, San Diego, CA, USA).Sequencing was performed using the Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA).HISAT2 software was employed to align the clean reads with the reference C. chinensis genome [15], and aligned reads were assembled using StringTie software [16].
Differential expression analysis was conducted using DESeq2 [17], with gene expression levels calculated as fragments per kilobase million.Genes meeting the criteria |log 2 (fold change)| ≥ 1 and false discovery rate ≤ 0.05 were considered significantly differentially expressed genes (DEGs).The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed using KOBAS software [18].Transcription factor gene families (TFs) were evaluated using the PlantTFDB Database website (http:// plant tfdb.cbi.pku.edu.cn/).
All raw sequencing data were submitted to the Genome Sequence Archive database (https:// ngdc.cncb.ac.cn/ gsub/) under the BioProject accession number CRA012562.

Quantitative reverse transcription-polymerase chain reaction analysis
Eight genes involved in isoquinoline alkaloid biosynthesis and the starch and sucrose metabolism pathways were randomly selected for real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR).The internal reference gene used was 18S rRNA [19].Primers were designed at https:// bioin fo.ut.ee/ prime r3-0.4.0/, and the sequences are listed in Additional file 2: Table S1.qRT-PCR was performed as described in a previous study [19].The 2 −△△CT method was used to calculate the relative expression levels with three biological and three technical replicates analyzed.

Statistical analyses
Statistical analyses were conducted using SPSS software version 19.0.Significant differences were assessed using a one-way analysis of variance and Duncan's multiple range test (p < 0.05).Column graphs and Pearson's correlation heat maps of gene expression levels, physiological properties, plant height, yield indicators, and isoquinoline alkaloid content were plotted using Origin Pro 2021 software.The TBTools software (version 1.068) was employed to illustrate the DEG heatmaps.

Physiological properties, plant height, and yield indicators of C. chinensis in response to understory cultivation
In this study, we measured physiological properties and yield indicators to explore how C. chinensis responds to understory cultivation (Figs. 1, 2, Table 1).The UC group exhibited significantly higher Chl, Chl a, Chl b, and Car contents compared to the MC group (p < 0.05).In addition, POD, SOD, and SS enzyme activities, soluble protein and sugar content, starch and sucrose content, and MDA content exhibited significant increases in the UC group.Specifically, POD activity, SOD activity, SS activity, MDA content, and soluble sugar content rose by 62.08%, 102.68%, 43.86%, 154.86%, and 66.82% in UC, respectively, compared to MC.In addition, plant height, plant fresh weight, aboveground fresh weight, and underground fresh weight in the UC treatment saw significant increases of 101.29%, 44.68%, 40.26%, and 52.55%, respectively.These results indicate that understory cultivation increases the viability and yield of C. chinensis.

Transcriptome sequencing, functional annotation, and differentially expressed gene analysis in C. chinensis
Illumina sequencing was employed to assess gene expression levels in the UC and MC leaf samples.An overview of the sequencing data is presented in Additional file 2: Table S2.The UC and MC samples yielded a total of 43,287,359 and 43,533,923 clean reads, with 86.58% and 86.94% uniquely mapped to the C. chinensis genomes, respectively.Moreover, the Q30 average values for the UC and MC samples were 94.56% and 94.65%, respectively, indicating satisfactory quality of transcriptome sequencing.
Functional annotations based on the GO databases were utilized to obtain comprehensive information on the assembled unigenes (Additional file 1: Fig. S1).A total of 3915 DEGs were classified into "biological process, " "cellular component, " and "molecular function." In the biological process category, DEGs (1856) were predominantly enriched in the "catalytic activity" GO term.For the cellular component category, the majority of unigenes (1438) were annotated in the "cell part" GO term.In the molecular function category, the "cellular process" (1206) and "metabolic process" (1123) were the most common.

Differentially expressed genes in photosynthesis
After understory cultivation, a total of 22 DEGs related to the photosynthetic pathway (map00195) were identified (Fig. 6, Additional file 2: Table S5).Compared with MC,

Differentially expressed genes in the tyrosine metabolism and isoquinoline alkaloid biosynthesis
Genes involved in tyrosine metabolism and isoquinoline alkaloid biosynthesis were found to be altered by understory cultivation.Eight DEGs were identified in tyrosine metabolism and isoquinoline alkaloid biosynthesis pathways (Fig. 7A, Additional file 2: Table S6).
Compared with MC, one gene each encoding tyrosine aminotransferase (TAT), tyrosine/dopa decarboxylase 2 (TYDC2), and polyphenol oxidase (PPO) was significantly upregulated in UC in both the tyrosine metabolism and isoquinoline alkaloid biosynthesis pathways.
In addition, the content of the four isoquinoline alkaloids was measured using HPLC (Fig. 7B-D).The epiberberine, palmatine, and berberine contents significantly increased by 41.27%, 27.80%, and 7.99%, respectively, in UC, whereas the coptisine level significantly decreased by 8.86% compared to MC.

Differentially expressed genes in the starch and sucrose metabolism
In the "UC_vs_MC" comparison, 16 DEGs were identified in the starch and sucrose metabolism pathways (Fig. 8, Additional file 2: Table S7).The results showed that one, one, four, two, three, and one genes encoding beta-fructofuranosidase (INV), alpha-glucosidase (AGL), beta-glucosidase (BGL), SPS, alpha-amylase (AMY), and Fig. 6 DEGs in the photosynthesis pathway.Red frames denote upregulated genes and green frames denote downregulated genes in "UC_vs_MC".The relative expression levels of DEGs were calculated by log 2 ratio isoamylase (ISA) were significantly upregulated in UC compared to MC.

Validation of transcriptomic data using qRT-PCR
To validate the transcriptome results, eight genes were randomly selected for qRT-PCR analysis (Additional file 2: Table S10).As expected, the gene expression levels calculated using the 2 −△△CT were consistent with the RNA sequencing (RNA-seq) results (Fig. 10).In addition, linear regression analysis validated the reliability and accuracy of the RNA-seq results (Additional file 1: Fig. S4 and Additional file 2: Table S11).

Discussion
In the present study, chlorophyll and carotenoid content, plant height, and fresh weight were significantly higher in UC than in MC.Plant height and fresh weight are important growth and yield parameters [20], and chlorophyll and carotenoids play essential roles in light energy absorption and photosynthesis [21].The substantial increase in these indicators under UC indicates a significant enhancement in the photosynthetic efficiency and overall yield of C. chinensis.Understory cultivation with C. lanceolata improves C. chinensis viability.In general, MDA is used to measure membrane peroxidation damage, and its value increases when crops are under stress [22].Higher MDA levels in the MC indicated that C. chinensis in the understory cultivation system may have been subjected to stress.These stressors may cause cells in C. chinensis to produce superoxide-free radicals and induce antioxidant enzymes to reduce oxidative damage by preventing  [12,23].Under UC, there is a significant increase in SOD enzyme activity, catalyzing the disproportionation of superoxide-free radical to generate more H 2 O 2 and O 2 [22].Subsequently, POD enzyme activity increases to clear excess H 2 O 2 [24].Alterations in antioxidant enzyme activities improved C. chinensis viability in response to intercropping.In addition, soluble sugars, soluble proteins, and starch have multiple cellular functions, including stress responses [25,26].This study observed a significant increase in soluble sugar, soluble protein, sucrose, and starch contents in the UC system.These findings suggest that these components may play crucial roles in enhancing the viability of C. chinensis under understory cultivation system.
Transcriptomic analyses revealed significant enrichment of DEGs in key pathways such as "phenylpropanoid biosynthesis, " "zeatin biosynthesis, " "photosynthesis, " and "starch and sucrose metabolism, " indicating their crucial roles in regulating the viability and yield of C. chinensis.Phenylpropanoids, specialized secondary metabolites, are known for their critical involvement in biotic and abiotic stress responses [27].Wu et al. [28] emphasized the significance of phenylpropanoid biosynthesis in tea defense metabolism and its positive impact on enhancing plant viability in understory cultivation systems.Notably, genes participating in phenylpropanoid biosynthesis, including one 4CL, two COMT1, and thirteen POD genes, exhibited upregulation in the UC group.The observed increase in POD activity further suggests the crucial involvement of these genes in enhancing the viability of C. chinensis [29][30][31].Photosynthesis, a fundamental process influencing crop biomass and yield [32], was also impacted in response to understory cultivation.Twenty genes, including Psb B, Psb O, Psb R, Psb S, Psa D, Psa E, Psa F, Psa G, Psa H, Psa K, Psa N, Psa O, Pet C, Pet E, Pet F, gamma, delta, and b, identified in the photosynthesis pathway, exhibited upregulation.These genes may play pivotal roles in the regulation of photosynthesis and yield in C. chinensis [31,32].Furthermore, sucrose and starch metabolism, crucial for providing nutrients and energy for crop yield and stress responses [33], demonstrated significant alterations in the UC.SS and SPS play crucial roles in the transformation of fructose and glucose into sucrose [34].SS activity exhibited a substantial increase, and SPS activity showed a slight elevation, indicating their essential roles in regulating sucrose and starch metabolism.The expression of genes associated with sucrose and starch metabolism, including INV, AGL, BGL, SPS, AMY, and ISA, was significantly altered in UC, indicating their potential roles in enhancing yield and viability.Cytokinins, hormones crucial for plant growth and development primarily produced mainly through zeatin biosynthesis [35], also exhibited significant alterations in the UC.In particular, CYP 735A can catalyze trans-zeatin biosynthesis, whereas CKX catalyzes zeatin glycosylation to regulate active cytokinins [36].Genes such as CYP 735A, CKX5, CKX3, ZOG, and UGT 73C5 significantly differentially expressed, indicating their potential the growth and development of C. chinensis.In addition, the DEGs annotated various TFs, including ERF/DREB, WRKY, HD-ZIP, MYB, MIKC C4-GATArelated, GARP_G2-like, SBP, DOF, and AP2, may play pivotal roles in enhancing the viability and yield of the understory cultivation system.
Isoquinoline alkaloids, such as coptisine, epiberberine, palmatine, and berberine, constitute the primary phytochemicals in C. chinensis [1].This study revealed that epiberberine, palmatine, and berberine concentrations notably elevated in the UC compared to the MC, while coptisine exhibited a contrasting trend.The cultivation method positively influenced the levels of epiberberine, palmatine, and berberine, while diminishing coptisine content in C. chinensis.In isoquinoline alkaloid biosynthesis, key enzymes such as TAT, TYDC, and PPO play crucial roles.TAT facilitates the conversion of tyrosine to generate 4-hydroxyphenylpyruvate [37], while PPO oxidizes l-tyrosine to generate L-DOPA [38].TYDC is pivotal in catalyzing the conversion of tyrosine and L-DOPA to form dopamine [1].This study noted significant upregulation of TAT , TYDC2, and PPO genes in UC, correlating with the increased levels of in epiberberine, palmatine, and berberine.These findings suggest that these genes contribute positively to the accumulation of these phytochemicals.O-Methyltransferases (OMTs) have been proposed as essential enzymes in isoquinoline alkaloid biosynthesis [39,40].Interestingly, our study noted a significant downregulation of 6-OMT and SMT in UC, consistent with the observed decrease in coptisine.This implies a potential positive regulatory role for these genes in coptisine accumulation.In addition, since isoquinoline alkaloids derive from tyrosine the enrichment of genes related to tyrosine metabolism in our transcriptomic results suggests the involvement of tyrosine metabolism in isoquinoline alkaloid biosynthesis in C. chinensis.Notably, the upregulation of HPPD and MIFH in UC indicates their potential significance in isoquinoline alkaloid biosynthesis under understory cultivation conditions.
Pearson's correlation analyses, a widely employed method for predicting relationships between gene expression and traits [11,40], have been instrumental in elucidating various biological phenomena.In a study by Wang et al. [40], Pearson's r values were calculated between candidate gene expression levels and metabolite intensity, identifying four genes potentially responsible for raffinose biosynthesis.In our current study, a multitude of genes, including INV, BGL-2, BGL-4, SPS-2, AMY-3, Psb B, Psb R, Psb S, Psa D, Psa E, Psa H, Psa O, Pet C, Pet H, deta, and b, exhibited significant positive correlations with plant fresh weight, aboveground fresh weight, and underground fresh weight.This further substantiates the pivotal roles these DEGs may play in yield accumulation.Interestingly, 6-OMT-2 and COMT1-3 demonstrated significant positive correlations with coptisine content but negative correlations with epiberberine, palmatine, and berberine content.This suggests a potential positive role for these genes in coptisine accumulation but a negative impact on the accumulation of the latter three isoquinoline alkaloids.Similarly, ZOG-1, ZOG-3, TAT , PPO, POD-13, and UGT 73C5-1 exhibited significant positive correlations with berberine content, while MIFH, POD-4, POD-5, and POD-8 showed significant positive correlations with epiberberine, palmatine, and berberine content.Conversely, POD-5 and POD-7 displayed significant negative correlations with coptisine content.These intricate relationships highlight the complex roles these DEGs may play in isoquinoline alkaloid accumulation.Notably, SOD activity, POD activity, and MDA content were significantly and positively correlated with epiberberine, palmatine, and berberine content.Likewise, Chl a, Chl b, Chl, Cars, soluble protein, soluble sugar content, starch content, sucrose content, and SS activity exhibited significant positive correlations with individual plant, aboveground, and underground fresh weights.These findings underscore the multifaceted interplay between gene expression levels and physiological properties in the growth and isoquinoline alkaloid accumulation of C. chinensis.However, to unravel the intricacies of these relationships, future studies should focus on the functional characterization of these genes through molecular cloning, protein expression, and biochemical assays.Such endeavors will contribute to a deeper understanding of how C. chinensis responds to understory cultivations.

Fig. 1
Fig. 1 Phenotypic changes of C. chinensis in MC and UC systems

Fig. 2
Fig. 2 Physiological properties of C. chinensis in UC and MC systems.All the data in the figure are presented as the mean ± standard error of the mean (SEM).Different letters in each system represent significant differences at p < 0.05.Chl a chlorophyll a, Chl b chlorophyll b, Chl chlorophyll a + chlorophyll b, Car carotenoid 20 of those genes encoding photosystem II CP47 reaction center protein (Psb B), oxygen-evolving enhancer protein 1 (Psb O), photosystem II 10 kDa polypeptide (Psb R), and photosystem II 22 kDa protein (Psb S) in the photosystem II reaction center, photosystem I reaction center subunit proteins (Psa D, Psa E, Psa F, Psa G, Psa H, Psa K, Psa N, and Psa O), cytochrome b6-f complex iron-sulfur subunit (Pet C), plastocyanin A (Pet E), and ferredoxin-3

Fig. 7
Fig. 7 Isoquinoline alkaloid contents and DEGs in tyrosine metabolism and the isoquinoline alkaloid biosynthesis pathway.A DEGs in tyrosine metabolism and the isoquinoline alkaloid biosynthesis pathway.Red frames indicate upregulated genes and green frames indicate downregulated genes in "UC_vs_MC".The relative expression levels of DEGs were calculated using the log 2 ratio.B-E Isoquinoline alkaloid contents in the UC and MC systems

Fig. 8 Fig. 9
Fig. 8 DEGs in the starch and sucrose metabolism pathways.Red frames indicate upregulated genes and blue frames indicate up-and down-regulated genes in "UC_vs_MC".The relative expression levels of DEGs were calculated using the log 2 ratio

Fig. 10
Fig. 10 qRT-PCR validation of eight DEGs involved in the isoquinoline alkaloid biosynthesis and starch and sucrose metabolism pathways.Data in the figure are presented as mean ± SEM. **p < 0.01

Table 1
Plant height and yield indicators of C. chinensis in UC and MC systemsThe values in the table are expressed as mean ± SEM.Different letters within each system represent indicate differences at p < 0.