<scp>HTD</scp> : a targetome database for plant physiology and regulation in <scp>HPPD</scp> family

4-Hydroxyphenylpyruvate dioxygenase (HPPD) represents a pivotal enzyme in the metabolic processes of plants, exerting a decisive influence on the catabolism of the amino acid tyrosine. It is located within the plastids of plant cells, where it functions as a catalyst for the conversion of 4-hydroxyphenylpyruvate to homogentisate (HGA; Raspail et al., 2011). This reaction represents the initial committed step in the biosynthetic pathway that culminates in the formation of plastoquinone and tocopherols (Ren et al., 2011). These compounds are indispensable for the optimal functioning of photosynthesis and the protection of plants from oxidative stress. Given its pivotal function in these processes, the regulation of HPPD is rigorously controlled at the transcriptional, posttranscriptional, and posttranslational levels. The HPPD protein has become the subject of extensive research, with the aim of understanding its structure and function, its regulation in different plant species, and its evolutionary history. Furthermore, studies on HPPD contribute to the broader understanding of plant physiology, particularly with regard to the mechanisms of stress tolerance (Ji et al., 2016; Frelin et al., 2017), the regulation of secondary metabolism (Dreesen et al., 2018; Y. Yang et al., 2024), and the interplay between metabolic pathways that are essential for plant growth and survival (Diaz-Tielas et al., 2019). Overall, HPPD represents a vital node in plant metabolic networks, with implications ranging from basic botanical research to practical applications in crop management and improvement. In recent years, research on the role of HPPD in plant metabolism and physiology has seen a notable expansion (Concepcion et al., 2021; Kim et al., 2021; Park et al., 2022; Wang et al., 2025). For example, the CRISPR-Cas9-mediated gene editing of the OsHPPD 3′ UTR resulted in the creation of new rice lines exhibiting resistance to HPPD-inhibiting herbicides (Wu et al., 2023). A fluorescent biosensor targeting HPPD was developed for noninvasive and real-time imaging of plant abiotic stress responses, thereby facilitating the detection of HPPD expression in plants under various stresses, including drought, salinity, and extreme temperatures (Fu et al., 2022). An efficient germline-specific evolution system was employed to generate herbicide-resistant HPPD variants, which could be utilized in crop breeding (Wang et al., 2024). Despite recent advances, challenges remain in understanding the molecular mechanisms underlying HPPD in plant stress tolerance, as well as in developing artificial evolution and functional interpretation of HPPD. A specialized database on the HPPD family could integrate information from multiple dimensions, thereby offering novel insights and perspectives. Here, we present the HPPD Targetome Database (HTD), a comprehensive database that covers the molecular, genomic, and biological aspects of the HPPD family. The database contains more than 18 164 enzyme entries across 11 037 species, including plants, animals, fungi, and bacteria. HTD provides comprehensive data on proteomics and genomics, including gene ontology annotations and gene expression profiles. Furthermore, the database contains information on metabolic pathway analysis and protein interaction networks, thereby providing an integrated view of the biological functions and interactions. Additionally, the HTD platform has been expanded to include a collection of chemical inhibitors for HPPD, along with their associated physicochemical properties and thermodynamic parameters. The database is freely available online at https://chemyang.ccnu.edu.cn/ccb/database/HTD/. The transcript and protein sequences were obtained from the National Center for Biotechnology Information (NCBI) GenBank and UniProt databases, respectively. The sequence data are extracted by querying the aforementioned databases with the relevant keywords. In this instance, the protein or gene name was employed as the search term (4-hydroxyphenylpyruvate dioxygenase, hppd, hpd). A deep protein language mode ESM-1b was employed to predict the potential missense variants (Rives et al., 2021). The protein structures are retrieved from the RCSB PDB, our research group, or modeled by AlphaFold2 (Jumper et al., 2021). Gene functions are annotated using the Gene Ontology database. The gene expression profiles of the microarray data were obtained from NCBI GEO database using the advanced R package geoquery. The KEGG REST API was used to retrieve specific types of pathway data. The protein interaction network was obtained from the STRING database. The chemical inhibitors were sourced from a combination of literature in Web of Science, our research group, and the BindingMOAD database. The literature was retrieved by combining keywords, including ((‘4-Hydroxyphenylpyruvate dioxygenase’ OR HPPD) and inhibit*). The chemical structures and thermodynamic parameters were collated from the literature. The backend of the HTD platform was developed using LNMP environment (Linux, Nginx, MySQL, and PHP). We used PHP for server-side scripting and built the interactive interface using Bootstrap and jQuery. The MySQL server was used as data management, and NGL Viewer as the molecular viewer (Rose et al., 2018). Gene and protein sequence alignments were performed using NCBI Blast (v.2.2.28+) (Camacho et al., 2009). HTD is freely available online to users. We recommend using a modern web browser that supports the HTML5 standard, such as Firefox, Chrome, Safari, Edge. There is no login requirement to access data in HTD. HTD is dedicated to the development of a comprehensive and user-friendly multi-omics data platform centered on the HPPD family (Fig. 1). To this end, we have developed dedicated modules dedicated to HPPD-related proteins, encompassing domains, such as the transcriptome, proteome, gene function and expression, metabolic pathways, and interaction networks. At present, the HTD platform contains 18 164 protein entries from 11 037 species. Genetic variations have been identified from transcriptome data or predicted using a deep protein language model (Rives et al., 2021). Furthermore, 223 crystal structures have been curated, complemented by additional structures generated through AlphaFold2 modeling. The database also includes over 2000 chemical interactions with experimentally determined thermodynamic parameters. A total of 17 277 genes have been assigned gene ontology annotations, while 2953 gene expression profiles have been collated from the NCBI GEO database. Moreover, 8476 KEGG pathways have been identified, underscoring the role of HPPD in tyrosine catabolism and its interconnections with broader metabolic networks. In order to construct protein interaction networks, 8009 interaction networks were retrieved from the STRING database. The HTD platform incorporates a suite of diverse tools designed to facilitate comprehensive analysis of protein sequences and structures. A web-based user interface has been developed for the purpose of facilitating the retrieval and presentation of the data (Fig. 2). The HTD platform offers users the opportunity to examine the evolutionary patterns of the HPPD protein across species, tracing its conservation, divergence, and potential functional adaptations (Fig. 2a–c). For example, a multiple sequence alignment demonstrated that Medicago sativa HPPD (MsHPPD) exhibits a high degree of similarity with HPPD sequences from other species, including Medicago truncatula, Lactuca sativa, and Arabidopsis thaliana (Jiang et al., 2017). Furthermore, phylogenetic analysis showed that MsHPPD clusters with HPPD sequences from dicotyledonous plants, indicating its evolutionary proximity to other dicot HPPDs. The diversity of isozymes across species provides a valuable opportunity to investigate evolutionary divergence in stress resistance. The comprehensive coverage of HPPD sequences across 11 037 species offered by HTD also presents the potential to identify novel isozymes and functional variants, thereby paving the way for new research directions in enzyme evolution and ecological adaptation. The application of gene ontology annotations to HPPD-related proteins, in conjunction with gene expression profiles, enables comprehensive investigations into the mechanisms governing HPPD regulation at the transcriptional and posttranscriptional levels. Users may examine the factors that regulate HPPD expression across species and contexts, including developmental stages or environmental stress (Fig. 2d). An innovative bioorthogonal fluorescence labeling and imaging technique was developed specifically for the specific purpose of visualizing the distribution and cellular uptake of HPPD in plants without interfering with normal plant growth (Zeng et al., 2023). The technique has been employed to monitor the real-time expression and localization of HPPD in Arabidopsis thaliana under a range of abiotic stress conditions, including high temperatures, drought, salinity, and heavy metal exposure. The findings indicated a significant correlation between HPPD expression levels and stress exposure, thereby substantiating the enzyme's involvement in plant stress resistance. To elucidate the molecular mechanisms underlying the stress response, we sought to investigate the interaction network of HPPD during this process and identified dozens of key interacting proteins. This discovery provides a foundation for elucidating the mechanisms underlying the stress response associated with HPPD. HTD facilitates multi-level investigations into the impact of alterations in HPPD expression, genetic variations, or chemical interactions affect the overall metabolic equilibrium, rendering it a valuable instrument for systems biology research (Fig. 2e–f). For example, the rational redesign of the Arabidopsis thaliana HPPD enzyme results in the production of hydroxyphenylacetate (HPA) rather than the native product, HGA by altering its catalytic pathway (Lin et al., 2021). A number of HPPD mutants, including the S267W variant, were created with the objective of selectively producing HPA. Moreover, the crystal structure of the S267W mutant furnished crucial structural insights, corroborating the alternative product pathway. These findings establish a robust platform for enzyme engineering, thereby enabling the design of biocatalysts with novel functions. By integrating transcriptomic, proteomic, and metabolic pathway data, HTD facilitates the exploration of the role of HPPD within broader metabolic networks. The incorporation of KEGG pathways, particularly those pertaining to tyrosine metabolism, allows users to visualize the role of HPPD within pivotal metabolic pathways, including its connections to the biosynthesis of vital metabolites, such as plastoquinone and tocopherols. HPPD has gained significant attention as the target of a class of herbicides known as HPPD inhibitors (Santucci et al., 2017; Lin et al., 2019; T-L. Yang et al., 2024), which cause a characteristic bleaching of plant tissues, ultimately leading to plant death. The selectivity of HPPD inhibitors is of paramount importance for ensuring the safety of these inhibitors in crops. Targetome structure-based design represents a promising approach for the development of highly selective inhibitors, with a foundation in the structures from diverse species (Lin et al., 2023). HTD's collection of three-dimensional structures provides indispensable data for investigating the structure–function relationship of HPPD enzymes (Fig. 2g,h). These data allow users to investigate the impact of structural variations on inhibitor binding, which is crucial for the discovery of herbicide-resistant crops. The mesotrione-resistant variants of the HPPD enzyme were developed through directed evolution (Qian & Shi, 2024). Four critical mutations (P329S, T331A, K335E, and G412S) in the cotton HPPD gene were identified as conferring resistance to the herbicide mesotrione while preserving the enzyme's native activity. The combination of three or four of these mutations exhibited significantly higher resistance than the single mutations, demonstrating a synergistic effect. These findings establish a rationale for employing gene-editing techniques to incorporate these advantageous mutations into crops, thus enhancing herbicide resistance and optimizing weed management strategies. The HTD platform represents a transformative resource for the study of the evolution, function, and regulation of the HPPD family in diverse range of species. HTD offers unparalleled opportunities for understanding HPPD's role in evolutionary biology, metabolic engineering, and enzyme regulation by integrating a wide range of omics data. Although the HTD provides a comprehensive platform, there are some potential limitations that could be addressed to enhance its utility and robustness in the future. The current version of the HTD database is deficient in robust analytical workflows. The integration of commonly used bioinformatics tools would facilitate the discovery of evolutionarily conserved domains and pathways specific to certain species. The implementation of regular updates that integrate new data would ensure the currency and relevance of the database to ongoing research. We hope that HTD will serve as the central repository for functional research in HPPD family, thereby facilitating advancements in plant stress response, crop breeding, and herbicide discovery. This work was supported by the National Key Research and Development Program of China (no.: 2023YFD1700500), the National Natural Science Foundation of China (nos.: 22377031 and 22007035), self-determined research funds of CCNU from the colleges' basic research and operation of MOE (no.: CCNU24JCPT023), the Hubei Provincial Department of Education Science and Technology Plan Project (no.: 2024CSA063), the Postdoctoral Fellowship Program (Grade B) of China Postdoctoral Science Foundation (no.: GZB20230250). None declared. G-FY and H-YL contributed to the study conception and design. L-CM, H-YL, X-HY, L-JC, J-HM, and Y-TX contributed to the data collection. FW constructed the online website. L-CM wrote the draft manuscript, and G-FY and H-YL revised the manuscript. All authors read and approved the final manuscript. L-CM and FW contributed equally to this work. HTD database is available online at https://chemyang.ccnu.edu.cn/ccb/database/HTD/. The New Phytologist Foundation remains neutral with regard to jurisdictional claims in maps and in any institutional affiliations.