De novo Assembly and Functional Annotation of Henbit (Lamium amplexicaule) Transcriptome

Young Ji Choi¹Hyojung Son²Jaewon Lim²Siyoon Jeong¹Seongmin Oh¹Bomi Nam¹Kang-Yeol Yu¹Kyung Min Choi¹Myunghee Jung^2*Ha Yeun Song^1*

• ¹Advanced Research Center for Island Wildlife Biomaterials, Honam National Institute of Biological Resources, Mokpo-si, Republic of Korea
• ²Research and Development Center, Insilicogen Inc., Yongin-si 16954, Gyeonggi-do, Republic of Korea, yongin-si, Republic of Korea

Henbit, scientifically known as Lamium amplexicaule, is a winter annual weed from the Lamiaceae family, native to Europe, Asia, and North Africa. This plant holds considerable value in traditional medicine. The Lamiaceae family is frequently cited in ethnobotanical research as one of the most utilized plant families for medicinal purposes, with its potential medicinal properties and traditional uses being extensively studied (Alipieva et al., 2006, Bubueanu et al., 2019, Kachmar et al., 2021). For instance, a survey in Taza, Morocco identified the Lamiaceae family as the most frequently used for traditional medicine (Kachmar et al., 2021). Various Lamium species, particularly L. album and L. maculatum, have a long-standing history in folk and traditional medicine across cultures. L. album has traditionally been used as a blood tonic, anti-spasmodic, and antiinflammatory agent (Alipieva et al., 2006). In contrast, L. maculatum has been employed in Chinese folk medicine to treat trauma, fracture, and hypertension (Alipieva et al., 2006).Research has explored the haemostatic properties of butanolic extracts from these species, showing potential in blood clotting applications (Bubueanu et al., 2019). The medicinal uses of Lamium species are diverse, with L. album and L. purpureum being used in both human and veterinary traditional medicine, utilizing aerial parts and roots (Bubueanu et al., 2019). This plant contains several bioactive compounds, including flavonol glycosides (Nugroho et al., 2009), and iridoid glucosides such as lamalbid, sesamoside, and lamioside (Adema 1968, Kobayashi et al., 1986, Alipieva et al., 2003, Alipieva et al., 2007) and phytol, β-sitosterol, isorhamnetin, hydroxynervonic acid, and phenolic components have been isolated from L. amplexicaule (Ghoneim et al., 2018, Siham andRachid 2022).These compounds contribute to the plant's biological activities. Notably, L. amplexicaule has demonstrated promising antimicrobial properties, especially against methicillinresistant Staphylococcus aureus (MRSA) (Ghoneim et al., 2018). Compounds such as phytol, isorhamnetin, and 3,4-dihydroxy-methyl benzoate extracted from the plant showed significant anti-MRSA effects (Ghoneim et al., 2018). Additionally, the mechanism of action against the dehydro-squalene synthase enzyme was established, suggesting potential for developing new anti-MRSA candidates.In terms of plant agronomy, although L. amplexicaule is often regarded as a weed, it has attracted scientific interest due to its invasive nature, unique reproductive strategies, and role as an alternative host for agricultural pests. The species exhibits remarkable pheno-plasticity, particularly in its flower organs, with both cleistogamous (closed) and chasmogamous (open) flowers (Johnson et al., 2008). Researchers found, L. amplexicaule plant inhibited root and shoot growth of various species, including Lepidium sativum and Lolium multiflorum, with methyl caffeate identified as a phytotoxic substance with allelopathic activity (Jones et al., 2012, Sakamoto et al., 2019). Additionally, the L. amplexicaule has been identified as a host for the soybean cyst nematode (Heterodera glycines Ichinohe, SCN), a significant pest in soybean production (Ramarao et al., 2000, Johnson, et al., 2008). Furthermore, its attractive flowers, which draw pollinators and birds, combined with its ability to thrive in diverse climates, have made it a popular choice for landscaping, vegetation restoration, and ornamental gardening purposes (Binder et al., 2024, Stojanova et al., 2024, Zhou et al., 2024). Furthermore, plant-derived extracts containing high levels of phytotoxic compounds, such as methyl caffeate, were observed to suppress the growth of roots and shoots in various plant species, contributing to the allelopathic effect (Sakamoto et al., 2019).The stated objectives underscore the benefits and importance of cultivating/killing this plant for agricultural purposes and manufacturing nutraceutical products for industrial use. However, research into the genetic components, including genomic and transcriptomic aspects, remains scarce within this plant family. The scarcity of sequencing libraries in the NCBI public genetic database results in a dearth of published information for comprehending gene composition and identifying secondary metabolism-related genes in these plants. In the current genomics era, elucidating genetic elements for plants lacking a reference genome through de novo transcriptome assembly could offer a cost-efficient method to acquire preliminary data for any plant species. This research seeks to bridge the knowledge gap in genetic elements of the plant family by employing de novo transcriptome assembly techniques. Through the generation of a transcriptome, the scientists aim to reveal crucial information about secondary metabolism transcripts, potentially shedding light on the plant's applications in agriculture and industry. This strategy not only provides an economical solution for examining plants without a reference genome but also lays the groundwork for future studies on gene composition and secondary metabolism-related genes within the Lamiaceae family. In March 2023, Lamium amplexicaule was collected from Mokpo, Korea (34°76'N, 126°36'E).The plants were acclimated for two weeks in 12 cm diameter pots filled with culture soil, maintained at 25±2 °C under a 16-hour light/8-hour dark photoperiod. After acclimation, heat stress treatment was applied by placing two L. amplexicaule plants in a 35°C incubator (Multi-room Incubator, VISION) with a 16-hour light/8-hour dark photoperiod for three days, while three plants remained at 25°C as controls. A separate set of two plants was used for the salt stress treatment, 200 mL of seawater (salinity of 34 ‰) was applied daily for 14 days, and their physiological responses were monitored throughout the experiment (Figure 1). Post-treatment, leaves were collected for sampling. Control leaf samples were labeled C1, C2, and C3; those subjected to heat stress were labeled H1 and H2; and those subjected to salt stress were labeled S1 and S2 (Fig. 1). Fresh samples were immediately frozen in liquid nitrogen and stored at -80°C for subsequent experimental analyses. Total RNA was extracted from different tissue parts of L. amplexicaule using the Trizol method (Lian et al., 2024) and sequenced with the Illumina Next-Seq. The entire process was outsourced to Macrogen, South Korea The raw data obtained underwent filtration to exclude reads containing more than 5% Nbase content, reads with low-quality base counts exceeding 50%, and reads containing adapter contamination and repetitive sequences resulting from PCR amplification.Subsequently, the processed short read sequences were subjected to contig assembly with well optimized transcriptome assembler Trinity and translated with TransDecoder (Haas et al., 2013). Finally, the translated proteins sequences were subjected to homology search, with existing annotation databases (GO, KEGG, Uniprot) were employed to annotate the transcriptome function with Trinotate (Bryant et al., 2017). Further, Differential expression analysis was performed using the read count data of unigene expression from each sample, obtained through expression quantification. Transcript-level quantification was performed using Salmon, and differential expression analysis was conducted using edgeR (Robinson et al., 2010), which employs empirical Bayes methods to estimate gene-wise dispersion and improve statistical reliability, particularly under low-replicate conditions (Chen et al., 2014). Differentially expressed transcripts were filtered using a threshold of adjusted p-value (FDR) ≤ 0.05 and |log₂ fold change| ≥ 2. This study aimed to elucidate the key enzyme genes associated with plant secondary metabolites, adhering to the gene-to-metabolite principle (Osbourn, 2010). RNA was extracted from seven L. amplexicaule leaf samples, and the subsequent cDNA library was sequenced using the Illumina NextSeq high-throughput platform. After filtering, 2 GB of clean reads were obtained. In the absence of L. amplexicaule genomic data, Trinity software was utilized for short read assembly and clustering, eliminating redundancy and sequences with ≥95% similarity (Supplementary Figure 1). This process yielded 175,070 transcripts (Table 1A), with exhibiting an N50 length of 2,017 bp, lengths spanning 199 to 11,998 bp (Figure 2A), and an average length of 816 bp (Table 1A). To ensure the assembled transcriptome completeness the BUSCO (Benchmarking Universal Single-Copy Orthologs) was employed to assess gene completeness (Table 1D andFigure 2B), while coding region sequences (CDSs) were predicted for all unique transcripts, resulting in 81,194 complete CDSs (Table 1C). To optimize the identification of unique functional genes within the transcriptome, were annotated using multiple databases, including GO, KEGG, and Uniprot (Table 1B). Of the 175,070 unigenes, 115,450 (65.9%) were annotated in at least one database, with 116,454 (66.5%) annotated in the GO database and 96,557 (55.2%) in the KEGG database. Further, 102,502 (58.5%) transcripts were expressed across the three experimental groups, including control, heat stress, and salt stress. The expression and differential expression for both stresses were illustrated in Figure 2C-F. Based on the Trinotate annotation and KEGG pathway mapping, 6,595(5.85%) transcripts were assigned to 28 secondary metabolite pathways (only pathways with more than 10 annotated transcripts were considered), as shown in Supplementary Figure 2. In addition, functional categorization was performed using Mercator4, and the mapping results were visualized with MapMan (Bolger et al., 2021). This analysis focused on secondary metabolism, particularly the triterpenoid biosynthesis pathway, and the corresponding figures are provided as Supplementary Figure 3. Furthermore, to investigate the transcriptional behavior of core gene families involved in secondary metabolism, we focused on cytochrome P450 monooxygenases (PF00067.25) and UDP-glycosyltransferases (UGTs; PF00201.21), which play essential roles in triterpenoid and glycoside biosynthesis. A total of 159 CYP450 and 68 UGT genes were expressed under heat stress, and 166 CYP450 and 71 UGT genes under salt stress. Notably, DEG analysis revealed a stronger transcriptional response under salt stress (Table 2). These families are likely contributing to the biosynthesis of oxygenated and glycosylated triterpenoids, potentially linked to Lamium's antimicrobial and allelopathic properties. Many of these genes mapped to key KEGG pathways, including terpenoid backbone biosynthesis (map00900) and secondary metabolite biosynthesis (map00999), similar to functional modules reported in Aralia elata (Cheng et al., 2020). Functional validation may uncover novel genes involved in phytochemical production and stress resilience in L. amplexicaule.To further explore secondary metabolites (map00999), we generated a heatmap (Supplementary Figure 4) comprising 181 transcripts, 39 of which showed differential expression under salt or heat stress. Most transcripts were related to the terpenoid pathway, particularly triterpenoid biosynthesis (Kim et al., 2015). As explained in the introduction section, L. amplexicaule is known for its glycoside content with therapeutic potential. Many of the identified genes overlap with those found in the ginsenoside biosynthesis pathway, a well-characterized triterpenoid group with demonstrated clinical relevance (Mathiyalagan et al., 2024). Prior studies on Panax ginseng have highlighted the importance of functional group glycosylation (Kim et al., 2015) and enzymes such as dammarenediol synthase (Han et al., 2006) and β-amyrin synthase (Hou et al., 2021), which respond to environmental stresses and drive secondary metabolite biosynthesis. The availability of the complete genome from ginseng facilitates a more comprehensive elucidation of the ginsenoside biosynthesis process. It is well-established that plant scientists predominantly prefer transcriptome datasets for initial research, as advancements in sequencing and sequence assembly methods have been significantly updated to obtain complete transcript lengths and provide detailed insights into the transcripts present in plants. This dataset will facilitate plant scientists' understanding of the array of genes present in L. amplexicaule. The complete expression and differential expression data, along with annotations, were provided in Table 1. The significance of the data presented in this transcriptome analysis of Lamium amplexicaule encompasses several aspects: Firstly, it represents the initial comprehensive transcriptome analysis of L. amplexicaule, thereby providing valuable genetic information for this medicinally and agriculturally significant plant species. Secondly, it addresses the knowledge gap in genetic elements of the Lamiaceae family, facilitating comparative genomics and evolutionary studies. Additionally, it establishes a foundation for future research on gene functions, particularly those involved in secondary metabolism and antimicrobial properties. Furthermore, it enables targeted genetic improvement and utilization of L. amplexicaule for agricultural and industrial purposes. Moreover, it contributes to the understanding of L. amplexicaule's genetic architecture, which can inform strategies for weed management or cultivation for medicinal purposes. This data is of considerable value to researchers in plant genetics, pharmacology, agriculture, and related fields, as it provides a comprehensive genetic resource for further investigations into this species and its potential applications. This study has several limitations. First, only two biological replicates were used for each stress condition, limiting statistical power. Second, no qRT-PCR validation was performed to confirm gene expression patterns. Third, functional interpretation was focused mainly on triterpenoid and glycoside pathways. Additionally, while insights from Panax ginseng were referenced, they may not fully reflect the biology of L. amplexicaule. Lastly, the salt stress treatment (14 days of seawater) may not represent natural field conditions.