Cloning and Characterization of IbHQT1: A BAHD Acyltransferase Gene That Positively Regulates Chlorogenic Acid Biosynthesis in Sweet Potato

TL;DR

This paper identifies and characterizes a gene in sweet potato that increases the production of chlorogenic acid, an important plant compound.

Contribution

The study experimentally confirms the role of IbHQT1 in chlorogenic acid biosynthesis in sweet potato.

Findings

IbHQT1 encodes a protein with conserved motifs of the BAHD acyltransferase family.

Overexpression of IbHQT1 increases chlorogenic acid levels in sweet potato leaves and roots.

RNAi suppression of IbHQT1 reduces chlorogenic acid content in sweet potato.

Abstract

Background: Hydroxycinnamoyl-CoA quinate hydroxycinnamoyl-transferase (HQT) is an essential enzyme for chlorogenic acid (CGA) biosynthesis in plants. Multiple HQT-encoding genes potentially involved in CGA synthesis in sweet potato (Ipomoea batatas) have been predicted. However, the functions of these genes have not been verified. Methods: In this study, the gene IbHQT1 was isolated from the sweet potato cultivar ‘Nanshu-88’ and functionally characterized using transgenic technology. Results: IbHQT1 encodes a protein comprising 431 amino acids, with conserved HXXXD and DFGWG motifs characteristic of BAHD acyltransferase family members. A phylogenetic analysis indicated that IbHQT1 has a close evolutionary relationship with StHQT in Solanum tuberosum. According to qPCR data, IbHQT1 is highly expressed in young leaves, and its expression is affected by exogenous MeJA (100 µM), ABA (100 µM), GA3 (50 µM), and SA (100 µM). Analyses of cis-acting regulatory elements indicated that the IbHQT1 promoter contains multiple elements responsive to MeJA, ABA, SA, GA3, and light. In plants overexpressing IbHQT1, CGA contents in mature leaves and storage roots increased 1.30- to 1.44-fold and 1.28- to 1.43-fold, respectively. Conversely, in IbHQT1-RNAi lines, CGA contents in mature leaves and storage roots decreased by 16–38% and 18–40%, respectively. Conclusions: These findings indicate that IbHQT1 positively regulates CGA biosynthesis in sweet potato plants.

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1. Introduction

Secondary metabolism is crucial for plant growth, development, and environmental adaptation [1,2,3], with its products also having beneficial effects on humans (e.g., nutrition and health) [4]. Chlorogenic acid (CGA), a hydroxycinnamic acid derived from caffeic and quinic acids, is a common phenylpropanoid secondary metabolite in higher plants [5]. Previous studies revealed the biological effects of CGA, including antibacterial, antiviral, thrombolytic, antihypertensive, antineoplastic, and antioxidative effects [6,7]. Sweet potato leaves are rich in CGA, making them an important source of it [8]. Current research on CGA in sweet potato is mainly focused on its biological activity rather than its biosynthetic pathway and molecular regulatory mechanisms, which remain to be thoroughly characterized. Thus, elucidating the CGA biosynthetic pathway in sweet potato and identifying genes involved in CGA accumulation are crucial for breeding new varieties with high CGA contents.

CGA biosynthesis is regulated by various enzymes in the phenylalanine pathway, including phenylalanine ammonia lyase (PAL), cinnamic 4-hydroxylase, 4-hydroxycinnamoyl-CoA ligase (C4H), 4-coumarate-CoA ligase, p-coumarate 3′-hydroxylase, hydroxycinnamoyl-CoA shikimic acid/quinic acid hydroxycinnamoyl-transferase (HCT), and hydroxycinnamoyl-CoA quinate hydroxycinnamoyl-transferase (HQT). To date, three potential plant CGA biosynthetic pathways have been proposed in plants, among which the HQT-mediated pathway has received considerable attention (Figure 1) [9]. HQT genes belonging to the large plant BAHD acyltransferase superfamily play important roles in phenylpropanoid metabolism [10,11]. According to several studies, CGA biosynthesis in plants is affected by HQT expression levels. For example, HQT overexpression substantially increases the CGA content in tomato plants by 85%, whereas silencing HQT results in a 98% decrease in the CGA content [12]. Similarly, increasing HQT expression in Lonicera macranthoides reportedly increases leaf CGA levels by 60% [13]. In Cynara cardunculus, the CcHQT expression level is closely correlated with CGA synthesis and accumulation; suppressed HQT expression in C. cardunculus decreases leaf CGA contents by 82% [14,15]. In an earlier study on Taraxacum antungense (dandelion), TaHQT overexpression increased leaf CGA contents by 82.49% [16]. Collectively, these study findings reflect the importance of HQT genes for CGA biosynthesis in diverse plant species.

Notably, CGA is a natural phenolic acid metabolite that can enhance plant stress resistance [17,18]. For example, exogenous CGA can inhibit Aspergillus niger conidial germination and growth, thereby decreasing the incidence of tomato blight [17]. Comino et al. [19] determined that HQT expression influences plant responses to light stress. Specifically, an exposure to UV-light can upregulate HQT expression, leading to enhanced resistance to UV irradiation [19,20]. Additionally, HQT overexpression in tomato leads to a significant increase in the CGA content, thereby enhancing plant tolerance to oxidative stress and resistance to bacterial pathogens [20]. Considered together, these findings indicate that HQT expression is important for protecting plants from abiotic stress. Sweet potato, which is a high-quality food crop, has high leaf CGA contents; however, there are few reports describing sweet potato HQT-type genes that are positively correlated with CGA synthesis and accumulation.

In this study, we isolated a BAHD-type gene from Nanshu-88, which we designated IbHQT1, and clarified its function in CGA biosynthesis of sweet potato. On the basis of our findings, IbHQT1 overexpression in sweet potato can substantially increase the CGA concentration in mature leaves (ML) and storage roots (SR). Specifically, CGA levels in ML and SR were approximately 1.30- to 1.44-fold and 1.28- to 1.43-fold higher, respectively, in IbHQT1-overexpressing plants than in wild-type plants. Conversely, in plants in which IbHQT1 was silenced via RNA interference (RNAi), CGA concentrations in ML and SR decreased by 16–38% and 18–40%, respectively. The study results indicate that IbHQT1 in sweet potato encodes an enzyme that positively regulates CGA synthesis. The data provided herein lay a foundation for improving the cultivation of high-quality sweet potato varieties with high CGA contents.

2. Materials and Methods

2.1. Co-Expression Analysis

In the current study on sweet potato, IbHQT expression levels in various tissues were analyzed using data available online (https://doi.org/10.1038/s41598-022-06794-4, accessed on 16 August 2025) [21]. Differentially expressed genes from samples of young leaves (YL), mature leaves (ML), mature stems (MS), and storage roots (SR) were identified by the DESeq2 software (version 1.12.4) based on the criteria of log2 |Fold Change| > 2 and FDR < 0.001. Subsequently, the FPKM values of 11 IbHQTs genes were filtered and employed to construct the heatmap by TBtools-II [22].

2.2. Plant Cultivation, Treatments, and Sampling

Sweet potato variety Nanshu-88 bred by the Nanchong Academy of Agricultural Sciences was selected for this research. The seedlings derived from SR were cut off and transplanted into plastic containers, which were then incubated in a growth chamber at 25 ± 1 °C with a 16 h light/8 h dark cycle, and light intensity of ~200 μmol m^−2^ s^−1^ at China West Normal University, Nanchong City, Sichuan Province, China. Following a 2-week acclimatization period, five independent plants were set up as a single treatment and all leaves of each sample were completely sprayed with 50 mL of plant hormones on both sides: 100 µM abscisic acid (ABA), 100 µM methyl jasmonate (MeJA), 100 µM salicylic acid (SA), and 50 µM gibberellin (GA_3_). All the treatments were applied at the same time of day to minimize circadian effects, and the samples were collected at 0 (control time point), 3, 6, 12, 24, and 48 h post-treatment for subsequent analyses.

2.3. RNA Extraction, Expression Profiling and Quantitative PCR Assays

To analyze the expression level of IbHQT1, total RNA was extracted using a Plant Total RNA Isolation Kit (Tiangen, Beijing, China). Extracted RNA was treated with DNase I to eliminate residual DNA. Highly pure RNA (2 µg) served as the template for cDNA synthesis using a Reverse Transcriptase XL Kit (AMV) (Takara, Dalian, China). Quantitative PCR (qPCR) analyses were conducted in a reaction volume comprising 10 µL SYBR Green, 1.0 U ExTaq™ DNA polymerase (Takara, Dalian, China), forward and reverse primers (1.0 µM each), 1 µL cDNA, and RNase-free water for a final volume of 20 µL. A CFX96 cycler (Bio-Rad, Hercules, CA, USA) and the following program were used for the qPCR analysis: 94 °C for 5 min; 40 cycles of 94 °C for 15 s, 54 °C for 10 s, and 72 °C for 20 s. Ibtublin was selected as an internal reference to normalize expression data. Relative gene expression levels were calculated using the 2^−ΔΔCt^ method [23]. Three independent replicates were analyzed for each time point per treatment group.

2.4. Phylogenetic Analysis and Subcellular Localization Prediction of IbHQT1

The protein sequences of 8 HQTs and 7 HCTs from different species downloaded from NCBI database (www.ncbi.nlm.nih.gov/, accessed on 16 August 2025), as well as 11 IbHQTs from sweet potato, were aligned using ClustalW and DNAMAN. A phylogenetic analysis was performed according to the neighbor-joining method (1000 bootstrap replicates) using MEGA11. The subcellular localization of IbHQT1 was predicted by the WoLF PSORT website (https://wolfpsort.hgc.jp/, accessed on 18 August 2025).

2.5. IbHQT1 Promoter Cloning

To isolate the IbHQT1 promoter, sweet potato genomic DNA was extracted from fresh YL using the CTAB method [24]. Primers were designed according to the sweet potato genome (http://public-genomes-ngs.molgen.mpg.de/cgi-bin/hgGateway?db=ipoBat4, accessed on 10 August 2025). To prevent non-specific PCR amplification, forward primers were designed for the promoter region, whereas reverse primers were designed for the coding sequence (CDS). The IbHQT1 promoter was amplified by nested PCR using Pro Taq DNA polymerase (Aikerui, Changsha, China). PCR amplicons were analyzed by agarose gel electrophoresis, cloned into the pMD19-T vector (Takara, Dalian, China), and sequenced. The transcription start sites was predicted using the Softberry online resource (http://www.softberry.com/berry.phtml?topic=index&group=programs &subgroup=promoter, accessed on 10 August 2025). Cis-acting regulatory elements in the IbHQT1 promoter region were predicted using the PlantCARE online resource (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 10 August 2025). Primer details are provided in Table S1.

2.6. Gene Cloning and Vector Construction

The full-length IbHQT1 CDS was amplified by PCR using primer pair OEIbHQT1-F and OEIbHQT1-R (Table S1), which included NcoI and BstEII restriction sites, respectively. The amplified product was ligated into the pCAMBIA1301 vector to replace the GUS gene, thereby creating the plant overexpression vector pCAMBIA1301-IbHQT1. Additionally, a 250 bp fragment of IbHQT1 was amplified by PCR and inserted into the intermediate vector pHANNIBAL; the expression cassette was transferred into the pBin19 plasmid to construct the IbHQT1-RNAi vector. Recombinant vectors (pCAMBIA1301-IbHQT1 and pBin19-IbHQT1) along with the corresponding control vectors (pCAMBIA1301 and pBin19, respectively) were inserted into Agrobacterium tumefaciens strain EHA105 cells for the subsequent transformation of sweet potato.

2.7. Generation of Transgenic Sweet Potato

Transgenic plants in which IbHQT1 was overexpressed or silenced were generated as described by Mei et al. [25]. Briefly, 0.5 mL A. tumefaciens EHA105 suspension was added to 50 mL TY liquid medium, after which the shake culture was agitated until the optical density at 600 nm reached 0.8. The tender stem tips of sweet potato, cut from the SR, were immersed in the bacterial suspension for 8–10 h, subsequently rinsed in sterile water, and maintained under dim illumination for 24 h. The tender stem tips were then immersed in sterile water supplemented with 200 mg·L^−1^ cephalosporin for 1 h to prevent bacterial contamination. They were subsequently rinsed two or three times in sterilized water before being transplanted in a field and cultivated until SR formed.

2.8. PCR Analysis of Transgenic Sweet Potato

To confirm transgenic sweet potato plants were generated, genomic DNA was extracted from the leaves of seedlings derived from SR according to the CTAB method [24]. The extracted DNA served as a template for the detection of the hygromycin phosphotransferase gene Hygr, the neomycin phosphotransferase gene NPTII, and IbHQT1 (target gene). Subsequently, the IbHQT1 expression level was determined via qPCR as described by Yu et al. [26]. Primers used for molecular detection are listed in Table S1.

2.9. Liquid Chromatography-Based Analysis of CGA Contents

ML and SR from transgenic plants were desiccated to a consistent weight at 50 °C and then pulverized into a fine powder. After 0.4 g fine powder was transferred to a 10 mL centrifuge tube, 4 mL methanol was added. The mixture was ultrasonicated (40 W) in a 50 °C water bath for 30 min and then cooled to room temperature. The sample was centrifuged at 12,000 rpm for 10 min and then the supernatant was passed through a 0.22 μm filter and analyzed using an ultra-performance liquid chromatography (UPLC) system comprising a Waters BEH C18 column (2.1 mm × 50 mm, 1.7 μm), with a mobile phase composed of acetonitrile (A) and 0.1% H_3_PO_4_ (B). The elution gradient was set as follows: 0 to 4 min, 1% A increased to 6% A; 4 to 10 min, 6% A increased to 9% A; 10 to 16 min, 9% A increased to 18% A; 16 to 22 min, 18% A increased to 20% A; 22 to 23 min, 20% A increased to 90% A. The column temperature was maintained at 25 °C and the injection volume was 1 μL, with a flow rate of 0.25 mL/min. The detection wavelength was 325 nm. CGA was obtained from Sigma-Aldrich, St. Louis, MO, USA.

2.10. Statistical Analysis

All data are presented as means ± standard deviation (SD) of at least three independent experiments, with three replicates per experiment. The SPSS16.0 software was used for statistical analysis of differences between different samples, with significant differences of p < 0.05.

3. Results

3.1. IbHQT Expression Patterns in Sweet Potato

11 HQT-type genes in sweet potato are closely linked with CGA biosynthesis. An analysis of the co-expression of these 11 genes detected distinct expression patterns in various tissues. Specifically, IbHQT5, IbHQT6, IbHQT8, IbHQT9, and IbHQT11 were expressed at relatively low levels in YL, ML, MS, and SR, whereas IbHQT7 and IbHQT10 were highly expressed in MS and SR. IbHQT4 was expressed at high level in YL, but at relatively low levels in ML. By contrast, IbHQT1, IbHQT2, and IbHQT3 were expressed at high levels in both YL and ML. Notably; the IbHQT1 expression level was higher in YL than in the other tissues (Figure 2A). To more thoroughly characterize IbHQTs expression dynamics, we collected YL, ML, MS, and SR of sweet potatoes (Figure 2B), and used qPCR technology to analyze the expression patterns of these genes. On the basis of qPCR data, IbHQT1 was expressed in all examined sweet potato tissues, but it was more highly expressed in both YL and ML than in the other tissues (approximately 2.7-times higher than the corresponding expression levels in MS and SR), which expression pattern is completely consistent with the predicted results in the heatmap (Figure 2C). The expression patterns of IbHQT2 and IbHQT6 in YL, ML, MS, and SR shared similarity with IbHQT1, specifically showing higher expression level in YL and ML (Figure 2D,H). The expression levels of IbHQT3 and IbHQT9 were significantly higher in ML compared to other tissues (Figure 2E,K). The expression level of IbHQT4 was relatively high in MS and YL tissues (Figure 2F). IbHQT5’s expression does not exhibit significant tissue specificity (Figure 2G). The expression levels of IbHQT7 and IbHQT10 were significantly higher in SR and MS than in ML and YL (Figure 2I,L). IbHQT11 had a significantly higher expression level in MS than in SR, ML, and YL (Figure 2M). Among these genes, only the expression pattern of IbHQT1 matches the trend observed in the CGA content of these tissues (Figure 2N). Hence, we speculated that IbHQT1 may play an important role in CGA biosynthesis.

3.2. Isolation and Analysis of IbHQT1

The IbHQT1 CDS was amplified by PCR using primers IbHQT1-F and IbHQT1-R (Figure 3A) and then sequenced, which revealed that the CDS (1296 bp) encoded a protein consisting of 431 amino acids. According to a phylogenetic analysis, IbHQT1 has a close evolutionary relationship with StHQT in S. tuberosum (Figure 3B). Subcellular localization prediction results indicate that IbHQT1 was localized in the cytoplasm. Multiple alignments indicated that there were significant differences and low homology between IbHQT1 and other IbHQTs sequences in sweet potato. Motif scanning revealed that IbHQT1 has typical HQT protein structural characteristics, including conserved HXXXD and DFGWG motifs (Figure 3C), implying that it belongs to the BAHD acyltransferase family.

3.3. IbHQT1 Expression Patterns After Hormone Treatments

Exogenous application of specific concentrations of plant hormones can temporarily affect the expression level of IbHQT1. In this study, an exogenous MeJA treatment significantly increased the IbHQT1 expression level in leaves, with peak expression at 6 h post-treatment (3.67-times higher than the control expression level at 0 h), which was followed by a decrease in expression to pre-treatment levels at the 48 h time point (Figure 4A). Similarly, exogenous ABA significantly increased IbHQT1 expression in leaves between 3 and 12 h post-treatment (3.45 to 2.14-times higher than the control expression level at 0 h), with no notable changes between 12 and 24 h (Figure 4B). Exogenously applied GA_3_ clearly increased the IbHQT1 expression level, which peaked at 6 h post-treatment (2.32-times higher than the control level) and then decreased (Figure 4C). IbHQT1 expression in sweet potato leaves was sensitive to exogenous SA. More specifically, the IbHQT1 expression level initially increased, peaking at 3 h post-treatment (4.24-fold higher than the control level), after which it decreased before increasing again at 48 h (Figure 4D).

3.4. Analysis of the IbHQT1 Promoter Sequence

A 1916 bp DNA fragment was cloned from the genomic DNA of Nanshu-88 via nested PCR (Figure S1) and then sequenced (Figure 5). The transcription start site in the IbHQT1 promoter predicted using TSSP website (http://linux1.softberry.com/berry.phtml?topic=tssp&group=programs&subgroup=promoter, accessed on 10 August 2025) was 175 bp upstream of the start codon (ATG). PlantCARE website (http://www.dna.affrc.go.jp/PLACE/, accessed on 10 August 2025), which was used to identify possible cis-acting regulatory elements in the IbHQT1 promoter, detected many putative light-responsive cis-acting regulatory elements, suggesting that IbHQT1 expression may be regulated by light. These elements included an ATE-motif (AATTATTTTTTATT, position −116 to −129), a chs-CMA1a motif (TTACTTAA, position −1061 to −1068), a GT1-motif (GGTTAAT/GGTTAA, position −1797 to −1803), and two G-boxes (CACGTC/G, position −1851 to −1856 and −1899 to −1904). Notably, additional cis-acting regulatory elements were detected in the IbHQT1 promoter, including the following: anaerobic response element (ARE, AAACCA) at position −1526 to −1531, implying that IbHQT1 expression may be important for the response to anaerobic conditions; three ABA response elements (ABRE, ACGTG/CACGTG) at positions −1246 to −1250, −1252 to −1256, and −1900 to −1904, which was consistent with the observed responsiveness to ABA; two MeJA response-related elements at positions −1244 to −1247 (TGACG) and −1456 to −1460 (CGTCA); a GA_3_ response-related TATC-box at position −1272 to −1278; and three SA-responsive TCA-elements at positions −1643 to −1652 (TTTTTCTAAC), −134 to −142 (TTTGTACCAG), and −66 to −75 (TTTTTCTTCC). Considered together, these findings indicate that IbHQT1 expression may be induced by the exogenous application of various phytohormones, including MeJA, ABA, SA, and GA_3_. Additionally, the presence of transcription factor recognition elements in the IbHQT1 promoter, such as the MYB recognition element TAACCA and the MYC recognition element CATTTG, suggests that IbHQT1 expression may be influenced by these transcription factors.

3.5. Generation and Identification of IbHQT1-Overexpressing Transgenic Sweet Potato

To investigate the effect of IbHQT1 expression on CGA biosynthesis in sweet potato, we constructed IbHQT1 overexpression and RNAi recombinant vectors for the transformation of Nanshu-88 (Figure 6A–H). We detected the hygromycin resistance marker gene (Hygr) in IbHQT1-overexpressing lines as well as in empty vector control plants (Figure 6G,H). In addition, an IbHQT1 CDS with a partial NOS terminator (679 bp) and a 410 bp fragment of Hygr were detected exclusively in IbHQT1-overexpressing transgenic sweet potato plants. Statistical analysis revealed that 6 out of 36 IbHQT1-overexpressing samples tested positive. By contrast, an IbHQT1 CDS with a partial OCS terminator (489 bp) and a 618 bp fragment of NPTII were detected specifically in IbHQT1-RNAi transgenic plants (Figure S2). Of the 44 strains derived from RNAi-mediated IbHQT1 silencing, 7 strains tested positive. Consequently, IbHQT1 was successfully integrated into the Nanshu-88 genome.

3.6. IbHQT1 Expression Levels in IbHQT1-Overexpressing and IbHQT1-RNAi Transgenic Plants

To analyze IbHQT1 expression in transgenic plants, a qPCR analysis was performed for wild-type, empty vector (pCAMBIA1301) control, IbHQT1-overexpressing, and IbHQT1-RNAi plants. IbHQT1 expression levels in ML and SR did not differ significantly between wild-type plants and transgenic plants harboring the empty vector. However, IbHQT1 expression levels in ML were significantly higher in IbHQT1-overexpressing sweet potato plants than in wild-type and empty vector control plants. Specifically, IbHQT1 expression levels in ML were 5.23- to 14.23-times higher in OEHQT1-2, OEHQT1-5, OEHQT1-6, and OEHQT1-7 plants than in wild-type and empty vector control plants (Figure 7A). IbHQT1 expression levels in SR were 0.97- to 4.23-times higher in IbHQT1-overexpressing plants than in wild-type and empty vector control plants (Figure 7B). Furthermore, IbHQT1 expression levels in both ML (Figure 7C) and SR (Figure 7D) decreased significantly in IbHQT1-RNAi plants, ranging from 39–64% and 35–75%, respectively. These findings reflect the successful expression of IbHQT1 in transformed sweet potato plants.

3.7. IbHQT1 Overexpression Increased the CGA Content in Transgenic Sweet Potato Plants

To further explore the effect of IbHQT1 expression on CGA biosynthesis, we used a UPLC system to accurately measure the CGA content in wild-type, empty vector-transformed, and four independently transformed IbHQT1-overexpressing transgenic plants (Figure S3). ML CGA contents did not differ significantly between wild-type and empty vector-transformed sweet potato plants (6.81 and 7.42 mg/g dry weight, respectively). However, ML CGA contents in transgenic sweet potato plants overexpressing IbHQT1 (9.38, 9.23, 8.87, and 9.81 mg/g dry weight) were significantly higher than the corresponding contents in wild-type and empty vector-transformed plants (Figure 8A). A comparison of SR between IbHQT1-overexpressing plants and control plants revealed that SR CGA contents were significantly higher in four independently transformed IbHQT1-overexpressing transgenic plants (2.60, 2.76, 2.58, and 2.47 mg/g dry weight) than in wild-type and empty vector-transformed plants (1.93 and 1.86 mg/g dry weight, respectively) (Figure 8B). An examination of IbHQT1-RNAi lines, including RNAiHQT1-3, RNAiHQT1-7, RNAiHQT1-15, and RNAiHQT1-21, indicated that the CGA content decreased by 16–38% in ML and by 18–40% in SR (relative to the corresponding levels in wild-type plants). These findings suggest that overexpressing IbHQT1 enhances CGA synthesis and accumulation in sweet potato ML and SR.

4. Discussion

CGA, an important phenolic secondary metabolite in plants, plays an important role in plant growth, development, and biotic and abiotic stress resilience [27,28]. Furthermore, CGA has various biological activities and pharmacological properties, making it a promising compound for different applications (e.g., anti-inflammatory and antibacterial treatments as well as glycemic control) [29]. However, inherently low CGA concentrations in both medicinal plants and food crops are not conducive to the practical use of CGA [30]. Thus, genes encoding proteins that regulate CGA synthesis must be identified to elucidate the CGA biosynthetic pathway. Although sweet potato leaves contain substantial amounts of CGA [8], genes regulating CGA production and accumulation in sweet potato have not been thoroughly characterized. In plants, HQT is an important enzyme for CGA biosynthesis, catalyzing the conversion of caffeoyl-CoA and quinic acid to CGA [31]. In the current study, we analyzed the co-expression patterns of these 11 genes and verified the expression of a key gene, IbHQT1, by qPCR (Figure 2A,B). An analysis of expression patterns showed that IbHQT1 is more highly expressed in YL and ML than in MS and SR (Figure 2C). IbHQT1 expression patterns in YL, ML, MS, and SR were consistent with the results of the co-expression analysis. In addition, IbHQT1 was cloned from sweet potato variety Nanshu-88 (Figure 3A). A cluster analysis revealed the close relationship between IbHQT1 and StHQT1 in potato (Figure 3B). Protein sequence comparisons indicated that IbHQT1 includes two conserved motifs (HXXXD and DFGWG) common among BAHD acyltransferase family members (Figure 3C). Previous research showed that genes in the CGA biosynthetic pathway are responsive to exogenous phytohormones [26]. In the present study, IbHQT1 expression was induced by the exogenous application of MeJA, ABA, SA, and GA_3_. After 3 h of treatment with ABA and SA, IbHQT1 expression rapidly reached its maximum level, which was consistent with the expression patterns of genes directly induced by ABA and SA [32,33]. In contrast, IbHQT1 expression peaked 6 h after MeJA and GA_3_ treatment, a notably delayed response compared to ABA and SA, suggesting that the regulation of IbHQT1 by MeJA and GA_3_ was likely more intricate than that by ABA and SA. Additionally, an examination of the IbHQT1 promoter revealed multiple cis-acting regulatory elements and motifs associated with responses to MeJA, ABA, SA, and GA_3_. The elements in IbHQT1 promoter provides bioinformatics support for the possible regulatory role of hormones of MeJA, ABA, SA, and GA_3_. However, whether they indeed mediate functional responses to the aforementioned hormones in vivo still needs further experimental research to confirm.

We also genetically modified sweet potato plants to overexpress or silence IbHQT1 as described by Mei et al. [25] to elucidate the role of this gene in CGA synthesis. A genomic PCR analysis confirmed IbHQT1 was integrated into the sweet potato genome of transformed plants, whereas a qPCR analysis showed that IbHQT1 expression levels increased (6- to 12-fold) in independently transformed transgenic plants. CGA concentrations in the leaves and SR of sweet potato plants overexpressing IbHQT1 increased by 30–44% and 28–43%, respectively (relative to the corresponding levels in the wild-type control). In sweet potato plants in which IbHQT1 expression was suppressed, both IbHQT1 expression levels and CGA contents in ML and SR decreased significantly (Figure 8). These findings suggest that IbHQT1 expression is critical for CGA biosynthesis in sweet potato plants at the whole-tissue level. However, it is important to consider that CGA biosynthesis and accumulation are likely regulated in a cell type-specific manner. For instance, in plants, secondary metabolites synthesis often occurs in specific cell types, such as epidermal cells, glandular trichomes, or vascular parenchyma [34]. Our current approach, using whole-tissue homogenates for qPCR and metabolite analysis, averages signals across diverse cell types. This could mask potential cell-specific patterns of IbHQT1 expression or subcellular compartmentalization of CGA. Future studies utilizing techniques such as single-cell RNA sequencing will be crucial in pinpointing the precise cellular sites of IbHQT1 activity and CGA synthesis.

According to recent studies, CGA production in plants may be enhanced by overexpressing genes involved in the associated biosynthetic pathway [35,36]. For example, the overexpression of IbPAL1, which encodes an enzyme that catalyzes an early reaction in the CGA biosynthetic pathway, can promote CGA biosynthesis. However, this increase adversely affects sweet potato tuber enlargement, with significant decreases in the number of SR in IbPAL1-overexpressing sweet potato plants [37]. The HQT gene is considered a key gene in the synthesis pathway of phenylpropanoid compounds, and it can affect the metabolism of various phenylpropanoid derivatives. For example, overexpression of the NtHQT gene in tobacco not only enhances the synthesis of caffeoyl quinic acid but also promotes the accumulation of flavonols [38]. In this study, further research is needed to determine whether overexpression and RANi of IbHQT1 will affect the synthesis of these compounds.

Earlier studies determined that transcription factors can influence CGA synthesis. For example, overexpressing AtMYB11 and AtMYB12 in tobacco can significantly promote CGA accumulation. In transgenic tobacco plants overexpressing AtMYB11, the expression levels of both HQT and HCT reportedly increase, possibly reflecting the regulatory role of AtMYB11 [39]. Overexpressing LmMYB111 in tobacco and L. macranthoides results in the enhanced production of CGA and luteoloside [40]. A GARP-type transcription factor in sweet potato, IbGLK, can bind to the promoters of genes involved in CGA synthesis (e.g., IbHCT, IbHQT, IbC4H, and IbUGCT), thereby activating their transcription. In sweet potato leaves transiently transformed for the overexpression of IbGLK1, significant changes were observed in both CGA biosynthetic pathway gene expression levels and CGA contents [8]. This suggests that CGA biosynthesis in sweet potato plants may be modulated via the overexpression of certain transcription factor genes. In this study, we cloned and analyzed the IbHQT1 promoter sequence, which revealed the presence of MYB and MYC transcription factor recognition elements. These elements may represent target sequences for the regulated synthesis of CGA in sweet potato. To summarize, we isolated and functionally characterized IbHQT1 in terms of its contribution to CGA synthesis, while also identifying regulatory elements in the IbHQT1 promoter. The study findings provide a solid base for future research on the regulation of CGA synthesis and accumulation in sweet potato plants.

5. Conclusions

In the current study, IbHQT1 was cloned from sweet potato, and its function was characterized. A qPCR analysis revealed significant tissue-specific expression profiles, with higher expression levels in both ML and YL than in MS and SR. Additionally, IbHQT1 expression was induced by the exogenous plant hormones of MeJA (100 µM), ABA (100 µM), GA_3_ (50 µM), and SA (100 µM). IbHQT1 overexpression markedly enhanced CGA biosynthesis in sweet potato leaves and SR. These findings reflect the importance of IbHQT1 for CGA biosynthesis in sweet potato.