Trihelix is a class of transcription factors unique to plants that play a major role in abiotic and biotic stress responses, seed isolate development, floral organ morphogenesis, and plant photomorphogenesis. Nevertheless, the Trihelix transcription factor family in Gardenia jasminoides (G. jasminoides) has not been systematically characterized. In this study, 11 GjTrihelix genes were identified from the G. jasminoides genome, unevenly distributed across five chromosomes, and can be classified into four subfamilies: GT-1, GT-2, SIP and SH4. Gene structure and functional motif analyses revealed high conservation within the same subfamily. Cis-acting element analysis showed that these genes are closely related to hormone responses, stress responses, and growth and development processes. Intraspecific synteny analysis showed a segmental duplication between GjTrihelix-3 and GjTrihelix-10. Interspecific collinearity analysis revealed that G. jasminoides shared 21 collinear gene pairs with soybean, compared with five pairs with Arabidopsis and 13 with Populus, indicating greater syntenic block conservation between G. jasminoides and soybean. Transcriptome data analysis demonstrated distinct spatiotemporal expression specificity of this gene family. Several genes were constitutively expressed in fruits; GjTrihelix-1 and GjTrihelix-3 were predominantly expressed in green fruits, while GjTrihelix-11 was highly expressed in red fruits. Under melatonin treatment, five GjTrihelix genes showed significant up-regulation and obvious transcriptional suppression of another five genes. Following infection by Botryosphaeria dothidea, GjTrihelix-5 and GjTrihelix-7 were progressively induced and peaked at 72 h. qRT-PCR results indicated that most GjTrihelix genes were highly expressed in leaves, while GjTrihelix-11 was highly expressed in flowers. Most GjTrihelix genes were significantly down-regulated under NaCl, ABA, GA3 and IAA stresses. This study provides new insights into the potential association of the Trihelix transcription factor family in G. jasminoides growth, development, and stress adaptation, offering theoretical references for stress-resistant G. jasminoides breeding.

KNOX (KNOTTED1-like HOMEOBOX) belongs to a class of important homeobox genes, which encode the homeodomain proteins binding to the specific element of target genes, and widely participate in plant growth and development, secondary metabolite synthesis, biotic and abiotic stresses responses. However, genome-wide identification of the KNOX gene family has not been reported in E. ulmoides. In this study, 8 KNOX proteins were identified based on genomic database of E. ulmoides. Phylogenetic analysis showed that the EuKNOX proteins were distributed in five subgroups based on homology with KNOX proteins in Arabidopsis, these genes were unevenly distributed across six chromosomes. Gene structure analysis revealed that EuKNOX genes contained between three and four introns. The number of introns within members of the same evolutionary branch was generally consistent. Prediction of cis-regulatory elements indicated that the EuKNOX genes were involved in hormone response, light response and stress response. RNA-seq data revealed tissue-specific expression patterns, among which five EuKNOXs were highly expressed in the xylem. The expression levels of most EuKNOXs were higher in female flowers than in male flowers. Functional analysis suggested that EuKNOXs contribute to bark, fruit and leaf development, Eu-rubber biosynthesis and leaf-color formation. qRT-PCR analysis demonstrated that EuKNOXs may act as positive regulators of NaCl and GA3 treatments, however, EuKNOXs may act as negative regulators of MeJA treatments, EuKNOXs showed a trend of first increasing, then decreasing and then increasing under drought and ABA treatment, especially, EuKNOX5 was induced 343-fold at 24 h under NaCl stress, suggesting that KNOX genes in E. ulmoides regulate plant growth and respond to different stresses by following different hormone signaling pathways, which laid a valuable foundation for further understanding the function of KNOX genes in multiple stress responses and phytohormone crosstalk in E. ulmoides.

Artemisia argyi Levl. et Vant., a traditional Chinese medicinal herb, is highly regarded for its therapeutic properties, including antimicrobial, anti-inflammatory, analgesic, and anticancer effects. However, the quality of A. argyi varies considerably due to factors such as climate, cultivation practices, and harvest timing. This study investigated the volatile compound profiles of A. argyi from six different regions using an electronic nose (E-nose) and headspace gas chromatography-ion mobility spectrometry (HS-GC-IMS). A total of 106 volatile compounds were identified by HS-GC-IMS, and terpenoids accounted for 39.62 % of the total, underscoring their importance in the chemical composition of A. argyi. E-nose sensor responses indicated that W1W and W2W exhibited the highest sensitivity, with the strongest signals observed in samples from Qichun (QC). By integrating variable importance in projection (VIP ≥ 1) and OPLS-DA results (P < 0.05), 28 key volatile compounds and 2 E-nose sensors (W1W and W2W) were identified as significant contributors to regional variations. Correlation analysis between HS-GC-IMS and E-nose data revealed 15 significantly differentiated compounds, six of which were common to both datasets, indicating their potential as reliable markers for geographic origin identification. These findings provide a methodological reference for the differentiation and quality control of A. argyi from diverse geographical regions.

Rubia cordifolia L. is routinely employed as a traditional Chinese medicinal herb in clinical practice. As research on R. cordifolia advances, the identification of stable internal reference genes has become essential for gene expression studies. A comprehensive literature search reveals that the screening of internal reference genes for R. cordifolia has been reported neither domestically nor internationally. This study employed quantitative real-time PCR (RT-qPCR) to assess the expression stability of several candidate internal reference genes in R. cordifolia . The identification of internal reference genes that are stably expressed across different tissues and under various stress conditions in R. cordifolia has laid an important foundation for gene expression studies in this plant. This study profiled the expression stability of 12 candidate internal reference genes in R. cordifolia across various tissues and under multiple abiotic stresses (NaCl, dehydration, SA, MeJA, ABA), leveraging a suite of algorithms (GeNorm, NormFinder, BestKeeper, ∆Ct, and RefFinder) for comprehensive evaluation.Analysis of the 12 candidate internal reference genes was performed using RefFinder, a computational tool that calculates a comprehensive stability ranking based on weighted geometric means. The collective analysis of candidate internal reference genes using geNorm, Normfinder, BestKeeper, and ΔCt methods identified hnRNP, UBQ, HIS3.3, PP2A , and PTBP2 as the five most stable genes. In contrast, ACT7 and EF-1α were found to be the least stable. Consistent with the findings from the four individual algorithms, RefFinder also identified hnRNP, HIS3.3, PTBP2, PP2A , and RPL5 as the top five most stable genes. Moreover,to further validate the reliability of the selected internal reference genes, we examined the expression patterns of the mevalonate kinase ( MVK) gene across a range of tissues and under a variety of treatment conditions, Using an inappropriate internal reference genes as a control can lead to significant bias in the results. This study systematically evaluated the expression stability of internal reference genes in R. cordifolia , as a fundamental step toward reliable functional genomics research in this species. The findings pave the way for accurately elucidating the roles of its genes.

IntroductionIsodon rubescens f. lushanensis, a typical form of I. rubescens, is characterized by its natural deficiency in oridonin and the presence of a unique ent-kaurane diterpenoid, lushanrubescensin.MethodsTo elucidate the molecular basis underlying the distinct chemical profiles between I. rubescens f. lushanensis and I. rubescens (Hemsl.) Hara, we conducted a comprehensive analysis utilizing high-quality genomic and transcriptomic data of I. rubescens f. lushanensis.ResultsThrough a genome-wide survey, our study identified 83 terpene synthase (TPS) genes, including 13 TPS-C and 9 TPS-e/f subfamily genes, which are implicated in diterpenoid biosynthesis. By integrating conserved motif analysis, differential expression profiling (FPKM), and RT-qPCR validation, we successfully screened key candidate genes. Subsequent heterologous expression in Saccharomyces cerevisiae enabled the functional characterization of five diterpene synthase genes, specifically CPS and KSL enzymes, involved in the central modules of diterpenoid synthesis.DiscussionThese findings not only expand the repertoire of known biosynthetic genes in I. rubescens but also offer valuable insights into the divergent biosynthetic pathways of oridonin and lushanrubescensin, paving the way for future metabolic engineering and synthetic biology studies.

Isodonis Excisoidis Herba (IEH) is a newly discovered herbal medicine used to treat esophageal cancer, chronic pharyngitis, and hepatitis, and ent-kaurane diterpenoids are its main active components. However, systematic studies on the chemical profile of ent-kaurane diterpenoids are lacking. In this study, UHPLC-LTQ-Orbitrap-MS was performed to investigate the fragmentation behaviors of three different types of ent-kaurane diterpenoids from IEH. Bridgehead-unsubstituted 7,20-epoxy-ent-kaurane diterpenoids yielded ions with typical losses of R7H, R1H, R14H, CH2O, CO, and R6H. The [M + NH4 - NH3 - R20]+ precursor ions at 331.1895 and the characteristic ions at m/z 313.1792, 295.1686, 285.1842, 277.1581, 267.1737, and 249.1632 were the most possible fragmentation pathways for bridgehead-substituted 7,20-epoxy-ent-kaurane diterpenoids. Fragmentation with the successive loss of multiple 18, 42, or 60 Da occurring in the OH groups and OAc groups is characteristic of 7,20-non-epoxy-kaurane diterpenoids. Using accurate mass measurements for each precursor ion and the subsequent fragmented ions, a total of 94 ent-kaurane diterpenoids were identified or tentatively characterized in IEH, including 48 potentially new ent-kaurane diterpenoids.

Gypsophila Radix (Chinese: ShanYin ChaiHu) is a traditional Chinese medicine derived from the dried roots of three Caryophyllaceae species: Silene jenisseensis, Arenaria juncea, and Gypsophila licentiana. Clinically, it is used as an adjuvant therapy for hepatic fibrosis in chronic hepatitis B. However, no reports have yet described the chloroplast genomes of these species. This study aimed to sequence and characterize the chloroplast genomes of these three species and to explore their phylogenetic relationships within Caryophyllaceae. The complete chloroplast genomes of S. jenisseensis, A. juncea, and G. licentiana were sequenced and annotated. Comparative genomic analyses were performed among these species and with representative members of the same family to identify genomic variation, simple sequence repeats (SSRs), and repetitive sequences. The chloroplast genome of S. jenisseensis, G. licentiana and A. juncea were 161,759 bp, 152,676 bp, and 134,812 bp in length, respectively, showing considerable size variation. A. juncea contained 46 SSRs and 16 repetitive sequences; G. licentiana contained 83 SSRs and 47 repetitive sequences; while, S. jenisseensis had the highest numbers, with 125 SSRs and 50 repetitive sequences, Phylogenetic analysis revealed that these three newly sequenced chloroplast genomes formed well supported subgroups within their respective genera: Silene, Gypsophila, and Arenaria, confirming the monophyly of these genera. The comparative analysis of repetitive sequence and SSRs provide valuable insights for developing chloroplast genome-based markers. This study enriches the chloroplast genomic resources of Caryophyllaceae and lays a foundation for species identification, evolution studies and the genetic resource utilization of Caryophyllaceae.

BACKGROUND: Isodon rubescensis used in traditional Chinese medicine and has drawn attention as a rich source of oridonin, a compound reported to possess medicinal properties. Differential transcriptome analysis is known to be an effective method for gene selection of diterpene oridonin biosynthetic pathways, however, the MeJA-induced genetic responses of I. rubescens have not yet been analyzed. RESULTS: In this study, comparative transcriptome analysis of JY (with oridonin) and LS (no oridonin) lines of I. rubescens with or without a methyl jasmonate (MeJA) treatment revealed 43,115 assembled unigenes, 11,253 of which were differentially expressed. We also identified a total of 5,070 statistically significant differentially expressed unigenes which respond to MeJA including 397 transcription factor (TF) unigenes, such as bHLH, ERF, NAC, and MYB. Furthermore, assignments with KEGG pathways identified 18 of 53 unigenes referring to the diterpenoid biosynthesis, 10 cytochrome P450 unigenes, and three different expression TF unigenes were speculated that these genes could regulate the synthesis of oridonin based on MeJA induced. Finally, 18 unigenes were validated by qRT-PCR, and their expression levels were matched with the sequence data. In addition, Single Nucleotide Polymorphisms (SNPs) were analyzed and 14,609 simple sequence repeats (SSRs) were detected in I. rubescens, providing further support for future genetic variation and marker-assisted selection. CONCLUSIONS: This is the first time to study the response of related genes to MeJA treatment in I. rubescens, and some new genes were putatively involved in oridonin biosynthesis in I. rubescens. Our results provides a valuable gene resource to functionally characterize of response to MeJA and oridonin biosynthesis in I. rubescens.

Sinomenium acutum, a traditional medicinal plant, has been utilized for millennia to alleviate various forms of rheumatic pain symptoms. The structurally diverse benzylisoquinoline alkaloids (BIAs) found in S. acutum are the primary contributors to its therapeutic efficacy, with sinomenine being the principal bioactive constituent. In this study, we employed an integrated transcriptomic and metabolomic approach to investigate BIA biosynthesis in S. acutum. Transcriptome sequencing, functional annotation, and differential gene expression analysis were combined with metabolite profiling to predict biosynthetic pathways of structurally diverse BIAs and screen candidate genes. Metabolomic analysis revealed significant stem-enriched accumulation of BIAs compared to leaves. Furthermore, we proposed a biosynthetic pathway of sinomenine and hypothesized that 34 key candidate genes, including cytochrome P450 (CYP450s), reductases, 2-oxoglutarate-dependent dioxygenases (2-ODDs), and O-methyltransferases (O-MTs), might be involved in its biosynthetic process. This study provides a foundation for understanding the biosynthesis of structurally diverse BIA compounds in S. acutum and offers critical insights for future characterization of functional genetic elements.

Rubia cordifolia is a well-known plant used in oriental medicine plant, and is also serves as the primary traditional source of plant red dyestuffs. With the current depletion of natural resources of R. cordifolia, it is critical to conduct cultivation studies on the R. cordifolia. Here, we report on the dynamic growth characteristics and secondary metabolite accumulation of cultivated R. cordifolia, as well as the discovery of important genes involved in anthraquinone biosynthesis. The results showed that R. cordifolia grows better in sunny environments than in shaded environments, and its triennials better than its biennials, base on the biomass and the concentration of the primary components purpurin and mollugin. The dynamic accumulation of purpurin and mollugin content suggested that 30 June to 15 October is a fair window for harvesting R. cordifolia, and the possibility of a specific transition connection during the purpurin and mollugin biosynthesis process. Furthermore, we sequenced R. cordifolia using SMRT technology for the first time and obtained 45,925 full-length transcripts, 564 alternative splicing events, 3182 transcription factors, 6454 SSRs, and 6361 lncRNAs. We hypothesized an anthraquinone biosynthetic pathway and found 280 full-length transcripts that may be involved in anthraquinone biosynthesis in R. cordifolia. In addition, RT-qPCR was used to detect the relative expression levels of 12 candidate ungenes in the above- and underground parts of R. cordifolia. Above all, our findings have crucial implications for the field management of cultivation and harvesting of cultivated R. cordifolia, and also provide useful genetic information for clarifying the potential genes involved in anthraquinone biosynthesis.

Flavonoids with great medicinal value play an important role in plant individual growth and stress resistance. Flavonol synthase (FLS) is one of the key enzymes to synthesize flavonoids. However, there is no information available about FLS family in Eucommia ulmoides, an ancient and precious plant with great economic value. In this study, twelve EuFLS genes were identified and classified into two distinct subgroups based on their phylogenetic trees, these genes were unevenly distributed across eight chromosomes. Gene structure analysis revealed that EuFLS genes contained between two and four introns. The number of introns within members of the same evolutionary branch was generally consistent. The EuFLS promoters region contained a substantial number of hormone-responsive, stress-responsive, and light-responsive. RNA-seq data revealed tissue-specific expression patterns, EuFLS2 and EuFLS9 displayed the highest expression levels in leaves, whereas EuFLS4 the peak expression level in the xylem. The majority of EuFLS genes showed higher expression levels in red leaves and male flowers; furthermore, these genes contributed to leaf development and rubber biosynthesis. qRT-PCR analysis showed that most EuFLS genes downregulation under ABA and SA treatments. EuFLSs displayed divergent expression trends under MeJA treatment. While drought stress significantly induced the expression of most EuFLSs, especially, EuFLS9 was induced 50-fold at 3 h, suggesting that FLS genes in E. ulmoides regulate plant growth and respond to different stresses by following different hormone signaling pathways, which laid a valuable foundation for further understanding the function of FLS genes in multiple stress responses and phytohormone crosstalk in E. ulmoides.

Chalcone synthetase(CHS) is a key enzyme in the flavonoid biosynthesis pathway. To investigate the functions of CHS gene in Eucommia ulmoides, this study conducted a comprehensive identification of EuCHS gene family members based on the E. ulmoides genome data by using bioinformatics methods. Systematic analyses were performed on their physicochemical properties, evolutionary relationships, conserved motifs, gene structures, chromosomal localization, promoter cis-acting elements, and expression patterns, aiming to provide a theoretical foundation for further research on the functions of EuCHS genes. In this study, a total of four EuCHS gene family members were identified, named EuCHS1 to EuCHS4. The encoded amino acids ranges from 385 to 397 aa with relative molecular weights between 42 315.75 and 43 336.97, theoretical isoelectric points ranging from 5.72 to 8.06, instability index ranging from 39.35 to 53.82, and aliphatic indices from 90.91 to 94.25. All EuCHS proteins were hydrophilic, and subcellular localization analysis showed that they were located in the cytoplasm. Evolutionary analysis indicated that the EuCHS family members were divided into two subfamilies, Group Ⅰ and Group Ⅲ, with each containing two EuCHS members. Gene structure analysis showed that all EuCHS contained two exons. Cis-acting element analysis indicated that EuCHS family members might be involved in various biological processes, including growth and development, hormone regulation, and abiotic stress responses. Transcriptome and quantitative reverse transcription-polymerase chain reaction(qRT-PCR) data analysis showed that EuCHS exhibited tissue-specific expression patterns, with most genes showing the highest expression levels in leaves, particularly during the early stages of leaf development. These genes participated in the formation of E. ulmoides gum and the leaf color of ` Ziye' E. ulmoides, as well as in floral organ development. Under drought stress, EuCHS1, EuCHS2, and EuCHS4 were upregulated, while EuCHS3 was downregulated. Gibberellic acid(GA_3) treatment suppressed EuCHS3 expression but induced EuCHS4 upregulation. Under abscisic acid(ABA) treatment, EuCHS1 and EuCHS3 were downregulated, while EuCHS2 and EuCHS4 exhibited an initial increase followed by a decrease in expression. This study identified four EuCHS gene family members with significant conservation and classified them into two subfamilies, Group I and Group Ⅲ. EuCHS genes displayed distinct expression differences across various tissues and developmental stages and were involved in abiotic stress responses in E. ulmoides. This study provides a theoretical basis for further exploring the functions of the CHS genes in E. ulmoides.

This study explored the growth-promoting effect and mechanism of the endophytic bacterium Kocuria rosea on Rehmannia glutinosa, aiming to provide a scientific basis for the development of green bacterial fertilizer. R. glutinosa 'Jinjiu' was treated with K. rosea, and the shoot parameters including leaf length, leaf width, plant width, and stem diameter were measured every 15 days. After 120 days, the shoots and roots were harvested. The root indicators(root number, root length, root diameter, root fresh weight, root dry weight, root volume, and root vitality) and secondary metabolites(catalpol, rehmannioside A, rehmannioside D, verbascoside, and leonuride) were determined. The R. glutinosa growth-promoting mechanism of K. rosea was discussed from the effect of K. rosea on the nutrient element content in R. glutinosa and rhizosphere soil and the genome information of this plant. After application of K. rosea, the maximum increases in leaf length, leaf width, plant width, and stem diameter were 35.67%(60 d), 25.39%(45 d), 40.17%(60 d), and 113.85%(45 d), respectively. The root number, root length, root diameter, root volume, root fresh weight, root dry weight, and root viability increased by 41.71%, 45.10%, 48.61%, 94.34%, 101.55%, 147.61%, and 42.08%, respectively. In addition, the content of rehmannioside A and verbascoside in the root of R. glutinosa increased by 76.67% and 69.54%, respectively. K. rosea promoted the transformation of nitrogen(N), phosphorus(P), and potassium(K) in the rhizosphere soil into the available state. Compared with that in the control, the content of available N(54.60 mg·kg~(-1)), available P(1.83 μmol·g~(-1)), and available K(83.75 mg·kg~(-1)) in the treatment with K. rosea increased by 138.78%, 44.89%, and 14.34%, respectively. The content of N, P, and K in the treatment group increased by 293.22%, 202.63%, and 23.80% in the roots and by 23.60%, 107.23%, and 134.53% in the leaves of R. glutinosa, respectively. K. rosea carried the genes related to colonization(rbsB, efp, bcsA, and gmhC), N, P, and K metabolism(narG, narH, narI, nasA, nasB, GDH2, pyk, aceB, ackA, CS, ppa, ppk, ppk2, pstS, pstA, pstB, and pstC), and indole-3-acetic acid and zeatin synthesis(iaaH and miaA). Further studies showed that K. rosea could colonize the roots of R. glutinosa and secrete indole-3-acetic acid(3.85 μg·mL~(-1)) and zeatin(0.10 μg·mL~(-1)). In summary, K. rosea promotes the growth of R.ehmannia glutinosa by enhancing the nutrient uptake, which provides a theoretical basis for the development of plant growth-promoting microbial products.

BACKGROUND:Rabdosiae rubescentis herba (Isodon rubescens) is widely used as a folk medicine to treat esophageal cancer and sore throat in China. Its germplasm resources are abundant in China, with I. rubescens (Hemsl.) Hara and I. rubescens f. lushanensis as 2 typical forms. I. rubescens (Hemsl.) Hara is featured by biosynthesis of the diterpenoid oridonin with strong anticancer activity, while I. rubescens f. lushanensis produces another diterpenoid with anticancer activity, lushanrubescensin. However, the biosynthetic pathways of both still need to be fully understood. In particular, little is known about the genetic background of I. rubescens f. lushanensis. FINDINGS:We used Pacific Biosciences (PacBio) single-molecule real-time and Nanopore Ultra-long sequencing platforms, respectively, and obtained 139.07 Gb of high-quality data, with a sequencing depth of about 328×. We also obtained a high-quality reference genome for I. rubescens f. lushanensis, with a genome size of 349 Mb and a contig N50 of 28.8 Mb. The heterozygosity of the genome is 1.7% and the repeatability is 83.43%. In total, 34,865 protein-coding genes were predicted. Moreover, we found that most of the variant or unique genes in the diterpenoid synthesis pathways of I. rubescens f. lushanensis and I. rubescens (Hemsl.) Hara were enriched in diterpene synthases. CONCLUSIONS:We provide the first genome sequence and gene annotation for the I. rubescens f. lushanensis, which provides molecular evidence for understanding the chemotypic differences of I. rubescens.

Objective:Lilium brownii var. viridulum (LB) and L. lancifolium (LL) are the main sources of medicinal lily (Lilii Bulbus, Baihe in Chinese) in China. However, the functional components of these two species responsible for the treatment efficacy are yet not clear. In order to explore the therapeutic material basis of Lilii Bulbus, we selected L. davidii var. willmottiae (LD) only used for food as the control group to analyze the differences between LD and the other two (LB and LL). Methods:Metabolome and transcriptome were carried out to investigate the differences of active components in LD vs LB and LD vs LL. Data of metabolome and transcriptome was analysed using various analysis methods, such as principal component analysis (PCA), hierarchical cluster analysis (HCA), and so on. Differentially expressed genes (DEGs) were enriched through KEGG and GO enrichment analysis. Results:The PCA and HCA of the metabolome indicated the metabolites were clearly separated and varied greatly in LL and LB contrasted with LD. There were 318 significantly differential metabolites (SDMs) in LD vs LB group and 298 SDMs in LD vs LL group. Compared with LD group, the significant up-regulation of steroidal saponins and steroidal alkaloids were detected both in LB and LL groups, especially in LB group. The HCA of transcriptome indicated that there was significant difference in LB vs LD group, while the difference between LL and LD varied slightly. Additionally, 47 540 DEGs in LD vs LB group and 18 958 DEGs in LD vs LL group were identified. Notably, CYP450s involving in the biosynthesis of steroidal saponins and steroidal alkaloids were detected, and comparing with LD, CYP724, CYP710A, and CYP734A1 in LB and CYP90B in LL were all up-regulated. Conclusion:This study suggested that steroidal saponins and steroidal alkaloids maybe the representative functional components of Lilii Bulbus, which can provide new insights for Lilii Bulbus used in the research and development of classic famous formula.

Benzylisoquinoline alkaloids (BIAs) represent a significant class of secondary metabolites with crucial roles in plant physiology and substantial potential for clinical applications. CYP82 genes are involved in the formation and modification of various BIA skeletons, contributing to the structural diversity of compounds. In this study, Corydalis yanhusuo , a traditional Chinese medicine rich in BIAs, was investigated to identify the catalytic function of CYP82s during BIA formation. Specifically, 20 CyCYP82-encoding genes were cloned, and their functions were identified in vitro. Ten of these CyCYP82s were observed to catalyze hydroxylation, leading to the formation of protopine and benzophenanthridine scaffolds. Furthermore, the correlation between BIA accumulation and the expression of CyCYP82s in different tissues of C. yanhusuo was assessed their. The identification and characterization of CyCYP82s provide novel genetic elements that can advance the synthetic biology of BIA compounds such as protopine and benzophenanthridine, and offer insights into the biosynthesis of BIAs with diverse structures in C. yanhusuo .

SINA (Seven in absentia) E3 ubiquitin ligases are a family of RING (really interesting new gene) E3 ubiquitin ligases, and they play a crucial role in regulating plant growth and development, hormone response, and abiotic and biotic stress. However, there is little research on the SINA gene family in U. rhynchophylla. In this study, a total of 10 UrSINA genes were identified from the U. rhynchophylla genome. The results of multiple sequence alignments and chromosomal locations show that 10 UrSINA genes were unevenly located on 22 chromosomes, and each UrSINA protein contained a SINA domain at the N-terminal and RING domains at the C-terminal. Synteny analysis showed that there are no tandem duplication gene pairs and there are four segmental gene pairs in U. rhynchophylla, contributing to the expansion of the gene family. Furthermore, almost all UrSINA genes contained the same gene structure, with three exons and two introns, and there were many cis-acting elements relating to plant hormones, light responses, and biotic and abiotic stress. The results of qRT-PCR show that most UrSINA genes were expressed in stems, with the least expression in roots; meanwhile, most UrSINA genes and key enzyme genes were responsive to ABA and MeJA hormones with overlapping but different expression patterns. Co-expression analysis showed that UrSINA1 might participate in the TIA pathway under ABA treatment, and UrSINA5 and UrSINA6 might participate in the TIA pathway under MeJA treatment. The mining of UrSINA genes in the U. rhynchophylla provided novel information for understanding the SINA gene and its function in plant secondary metabolites, growth, and development.

Artemisia argyi is a traditional herbal medicine plant, and its folium artemisia argyi is widely in demand due to moxibustion applications globally. The Auxin/indole-3-acetic acid (Aux/IAA, or IAA) gene family has critical roles in the primary auxin-response process, with extensive involvement in plant development and stresses, controlling various essential traits of plants. However, the systematic investigation of the Aux/IAA gene family in A. argyi remains limited. In this study, a total of 61 Aux/IAA genes were comprehensively identified and characterized. Gene structural analysis indicated that 46 Aux/IAA proteins contain the four typical domains, and 15 Aux/IAA proteins belong to non-canonical IAA proteins. Collinear prediction and phylogenetic relationship analyses suggested that Aux/IAA proteins were grouped into 13 distinct categories, and most Aux/IAA genes might experience gene loss during the tandem duplication process. Promoter cis-element investigation indicated that Aux/IAA promoters contain a variety of plant hormone response and stress response cis-elements. Protein interaction prediction analysis demonstrated that AaIAA26/29/7/34 proteins are possibly core members of the Aux/IAA family interaction. Expression analysis in roots and leaves via RNA-seq data indicated that the expression of some AaIAAs exhibited tissue-specific expression patterns, and some AaIAAs were involved in the regulation of salt and saline-alkali stresses. In addition, RT-qPCR results indicated that AaIAA genes have differential responses to auxin, with complex response patterns in response to other hormones, indicating that Aux/IAA may play a role in connecting auxin and other hormone signaling pathways. Overall, these findings shed more light on AaIAA genes and offer critical foundational knowledge toward the elucidation of their function during plant growth, stress response, and hormone networking of Aux/IAA family genes in A. argyi.

Trihelix transcription factors play important roles in plant light responses,growth and development,and stress responses.However,Trihelix has not yet been reported in Eucommia ulmoides.In this study,bioinformatics methods were used to comprehensively identify and analyze the expression patterns of the Trihelix gene family in E.ulmoides,aiming to provide a basis for further functional studies of EuGTs genes.A total of 9 Trihelix gene family members were identified in E.ulmoides,encoding proteins with 339 to 883 amino acids,with isoelectric points ranging from 5.13 to 9.39 and relative molecular weights between 36 992.06 and 97 871.61.Subcellular localization results showed that only EuGT-2 was localized in chloroplasts,while the others were located in the nucleus.The Trihelix gene family was categorized into six subfamilies:GT-1,GT-2,SH4,SIP1,GTγ,and GTδ.EuGTs were distributed among three subfamilies:SH4,GT-1,and GT-2,containing 1,6,and 2 Trihelix proteins,respectively,with 2 to 17 exons.The promoters of EuGTs contained various cis-acting elements related to hormones,stress,photoperiod,and growth and development.Collinearity analysis revealed 5 collinear gene pairs between E.ulmoides and Arabidopsis thaliana,and 14 collinear gene pairs between E.ulmoides and Populus.Expression pattern analysis showed that EuGTs exhibited tissue-specific expression:EuGT-1,EuGT-2 had the highest expression levels in leaves,EuGT-4,EuGT-6,EuGT-9 had the highest transcriptional levels in marginal peel,and EuGT-5、EuGT-8 were predominantly expressed in the xylem.As leaves developed,EuGTs showed a trend of asynchronous changes.No significant differences in EuGTs expression were observed between male and female flowers,with high expression levels mainly during the induction stage of flowering.The qRT-PCR analysis indicated that most EuGTs genes were most highly expressed in the leaves of E.ulmoides,while EuGT-5 was highly expressed in the stems.Under 200 mmol·L-1 NaCl treatment,most EuGTs genes exhibited an initial increase followed by a decrease in expression,significantly responding to salt stress.This study provides important genetic resources for further exploration of EuGTs gene functions and germplasm innovation in E.ulmoides.

BACKGROUND:Plant-specific TIFY proteins play crucial roles in regulating plant growth, development, and various stress responses. However, there is no information available about this family in Artemisia argyi, a well-known traditional medicinal plant with great economic value. RESULTS:A total of 34 AaTIFY genes were identified, including 4 TIFY, 22 JAZ, 5 PPD, and 3 ZML genes. Structural, motif scanning, and phylogenetic relationships analysis of these genes revealed that members within the same group or subgroup exhibit similar exon-intron structures and conserved motif compositions. The TIFY genes were unevenly distributed across the 15 chromosomes. Tandem duplication events and segmental duplication events have been identified in the TIFY family in A. argyi. These events have played a crucial role in the gene multiplication and compression of different subfamilies within the TIFY family. Promoter analysis revealed that most AaTIFY genes contain multiple cis-elements associated with stress response, phytohormone signal transduction, and plant growth and development. Expression analysis of roots and leaves using RNA-seq data revealed that certain AaTIFY genes showed tissue-specific expression patterns, and some AaTIFY genes, such as AaTIFY19/29, were found to be involved in regulating salt and saline-alkali stresses. In addition, RT-qPCR analysis showed that TIFY genes, especially AaTIFY19/23/27/29, respond to a variety of hormonal treatments, such as MeJA, ABA, SA, and IAA. This suggested that TIFY genes in A. argyi regulate plant growth and respond to different stresses by following different hormone signaling pathways. CONCLUSION:Taken together, our study conducted a comprehensive identification and analysis of the TIFY gene family in A. argyi. These findings suggested that TIFY might play an important role in plant development and stress responses, which laid a valuable foundation for further understanding the function of TIFY genes in multiple stress responses and phytohormone crosstalk in A. argyi.