Theanine, a unique nonproteinogenic natural amino acid predominantly accumulated in tea plants (Camellia sinensis), is a key determinant of tea flavor and quality, with broad applications. Theanine synthase (CsTSI) is a key enzyme in theanine biosynthesis. However, the transcriptional regulation of CsTSI, particularly by bHLH transcription factors, remains largely unexplored. Through coexpression network screening and tissue-specific qPCR, we identified CsbHLH18 as a putative regulator of CsTSI. Yeast one-hybrid (Y1H) and dual-luciferase reporter assays confirmed the direct interaction between CsbHLH18 and the CsTSI promoter. An electrophoretic mobility shift assay (EMSA) further identified the specific binding motif of CsbHLH18 as "CAAATG". Transient overexpression and virus-induced gene silencing (VIGS) in tea plants demonstrated that CsbHLH18 positively regulates theanine biosynthesis by activating CsTSI transcription. These findings elucidate a previously uncharacterized molecular mechanism underlying theanine biosynthesis and provide theoretical insights into molecular breeding and cultivation of high-quality tea varieties.

McORA activates McHDR transcription to enhance aphid-induced (E)-β-farnesene biosynthesis, conferring increased aphid resistance in German chamomile and providing a target for genetic improvement. German chamomile (Matricaria chamomilla L.) is an annual medicinal plant whose essential oil is rich in sesquiterpenoids, including (E)-β-farnesene (EβF), chamazulene, nerolidol and α-bisabolol, which collectively exhibit antibacterial, anti-inflammatory, hypoglycemic, hypolipidemic, spasmolytic, and sedative activities. However, German chamomile is highly susceptible to aphid infestation, resulting in stunted growth, leaf chlorosis and curling, and premature senescence. Here, we demonstrate that EβF production can be induced by aphid-feeding, and that it exerts a repellent effect on aphids, as shown by Y-tube olfactometer choice assays. To elucidate the regulatory mechanism underlying EβF biosynthesis, we cloned the promoter of McHDR, a key gene in the EβF pathway, using Fusion Primer and Nested Integrated PCR (FPNI-PCR). Correlation analysis and yeast one-hybrid (Y1H) library screening identified McORA as a transcription factor that regulates McHDR, and full-length McORA cDNA was obtained using 5’/3’-RACE. McORA specifically bound the CAACA motif within the McHDR promoter and activated its transcription, as confirmed by Y1H point-to-point assays, dual-luciferase reporter assays and electrophoretic mobility shift assays (EMSA). Finally, transgenic hairy roots and virus-induced gene silencing (VIGS) in German chamomile leaves verified that McORA positively regulates McHDR expression and EβF accumulation, and aphid infestation experiments under natural conditions showed that McORA silencing significantly reduces aphid resistance. Taken together, these findings provide experimental evidence that EβF functions as an effective aphid-repellent and, for the first time, elucidate McORA-mediated transcriptional regulation of McHDR and its influence on EβF biosynthesis in German chamomile, thereby highlighting a potential genetic engineering strategy to enhance aphid resistance.

Clinopodium chinense (Benth.) Kuntze is the botanical source of the traditional Chinese medicine “Duanxueliu”, whose pharmacological activities are largely attributed to the oleanane-type triterpenoid saponin Clinopodiside A. Beta-amyrin synthase (bAS) catalyzes the first committed step in oleanane-type triterpenoids biosynthesis. However, the genetic basis underlying Clinopodiside A biosynthesis remains largely unknown, particularly in non-model medicinal plants. Here, we quantified Clinopodiside A contents and analyzed transcriptomes across ten tissues and developmental stages of C. chinense. Weighted gene co‑expression network analysis (WGCNA) identified the MEgreen module, which was strongly correlated with Clinopodiside A accumulation (r = 0.89, p < 0.001), and contained a candidate bAS gene, designated CcbAS. The full-length coding sequence of CcbAS is 2289 bp and encodes a protein of 763 amino acids. High-Pressure Liquid Chromatography (HPLC) and quantitative real-time PCR (qPCR) analyses revealed highly consistent tissue-specific and developmental patterns between CcbAS expression and Clinopodiside A accumulation. Subcellular localization in Nicotiana benthamiana showed that CcbAS is localized in the cytoplasm and associated with the plasma membrane. To overcome the lack of stable transformation systems, we established two rapid, complementary Agrobacterium-based platforms (transient overexpression and VIGS) in C. chinense. Functional validation demonstrated that CcbAS overexpression increased Clinopodiside A accumulation by 2.18‑fold, while VIGS‑mediated silencing reduced it by 53.06%. These findings identify CcbAS as a key gene involved in Clinopodiside A biosynthesis and establish an efficient platform for rapid functional characterization of biosynthetic genes in C. chinense, providing a foundation for pathway elucidation and metabolic engineering.

German chamomile (Matricaria chamomilla L.) is a traditional medicinal aromatic plant, and the sesquiterpenoids in its flowers have important medicinal value. The (E)-β-farnesene (EβF) is one of the active sesquiterpenoid components and is also a major component of aphid alarm pheromones. In this study, two EβF synthase (βFS) genes (McβFS1 and McβFS2), were cloned from German chamomile. Subcellular localization analysis showed that both McβFS1 and McβFS2 were localized in the cytoplasm and nucleus. Tissue-specific expression analysis revealed that McβFS1 and McβFS2 were expressed in all flower stages, with the highest levels observed during the tubular flower extension stage. Prokaryotic expression and enzyme activity results showed that McβFS1 and McβFS2 possess catalytic activity. Overexpression of McβFS1 and McβFS2 in the hairy roots of German chamomile led to the accumulation of EβF, demonstrating enzyme activity in vivo. The promoters of McβFS1 and McβFS2 were cloned and analyzed. After treating German chamomile with methyl jasmonate (MeJA) and methyl salicylate (MeSA), the transcription levels of McβFS1 and McβFS2 were found to be regulated by both hormones. In addition, feeding experiments showed that aphid infestation upregulated the expression levels of McβFS1 and McβFS2. Our study provides valuable insights into the biosynthesis of EβF, laying a foundation for further research into its metabolic pathways.

The essential oil of German chamomile (Matricaria chamomilla L.) is rich in sesquiterpenoids, which contribute to its extensive applications in the pharmaceutical, tea, and cosmetics industries. Sesquiterpenoids are synthesized through the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways. In this study, we identified and cloned genes encoding for three key enzymes involved in these pathways: 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), 1-deoxy-D-xylulose 5-phosphate synthase (DXS), and 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) from German chamomile. Expression analysis revealed that McDXS2a, McDXS2b, McDXR, and McHMGR were highly expressed during flower development. Sampling was performed on the whole flower before the initial flowering stage, and subsequently, disk florets and ray florets were sampled separately for further analysis. Subcellular localization analysis indicated that McDXS1, McDXS2a, McDXS2b, and McDXR localized to the chloroplasts, while McHMGR localized to the cytoplasm. Overexpressing these genes in German chamomile hairy roots significantly increased sesquiterpenoid accumulation, confirming their functional roles. Transcription factors associated with these genes were identified and cloned, providing a data reference for further investigation into their regulatory mechanisms. Additionally, precursor feeding experiments demonstrated that the MEP pathway play an important role in the biosynthesis of sesquiterpenoids in German chamomile. These findings provide a basis for future research on the biosynthetic regulation of sesquiterpenoids.

German chamomile (Matricaria chamomilla L.) is a traditional aromatic medicinal plant, its flower contains volatile aromatic oil (essential oil). The main sesquiterpene components of the essential oil are (E)-β-farnesene, chamazulene, and α-bisabolol, these components have significant medicinal value and are used in food, cosmetics, and pharmaceuticals. However, the German chamomile genome has not yet been cataloged in any database; consequently, research on the intricate regulatory network and interaction mechanisms among proteins in German chamomile remains limited. Furthermore, no study has thus far developed a yeast cDNA library for German chamomile. Therefore, we constructed a homogenized yeast cDNA library using different tissues of German chamomile, this yeast cDNA library had a titer of 1.444 × 108 colony-forming units/mL, an average insert size of > 1,000 bp, and a positive rate of 100

Background German chamomile ( Matricaria chamomilla L.) is an important medicinal plant, and the essential oils in the flowers have various biological activities. Genetic transformation systems are important for plant quality improvement and molecular research. To the best of our knowledge, a genetic transformation system has not yet been reported for German chamomile. Results In this study, we developed Agrobacterium -mediated transformation protocols for German chamomile callus tissues. This involved optimizing key parameters, such as hygromycin and cefotaxime concentrations, bacterial density, and infection and co-culture durations. We also performed gas chromatography–mass spectrometry analysis to identify volatile compounds in non-transgenic and transgenic callus and hairy root tissues. Furthermore, to compare and verify the callus transformation system of German chamomile, we transferred McFPS to the hairy roots of German chamomile. The results showed that the optimal conditions for Agrobacterium -mediated callus tissue transformation were as follows: explant, petiole; cefotaxime concentration, 300 mg/L; hygromycin concentration, 10 mg/L; and bacterial solution concentration, OD 600 = 0.6; callus transformation efficiency was the highest when the co-culture time was 3 days. Conclusions Establishment of a high-efficiency callus transformation system will lay the foundation for gene function identification in German chamomile.

The essential oil of German chamomile (Matricaria recutita L.) is widely used in food, cosmetics, and the pharmaceutical industry. α-Bisabolol is the main active substance in German chamomile. Farnesyl diphosphate synthase (FPS) and α-bisabolol synthase (BBS) are key enzymes related to the α-bisabolol biosynthesis pathway. However, little is known about the α-bisabolol biosynthesis pathway in German chamomile, especially the transcription factors (TFs) related to the regulation of α-bisabolol synthesis. In this study, we identified MrFPS and MrBBS and investigated their functions by prokaryotic expression and expression in hairy root cells of German chamomile. The results suggest that MrFPS is the key enzyme in the production of sesquiterpenoids, and MrBBS catalyzes the reaction that produces α-bisabolol. Subcellular localization analysis showed that both MrFPS and MrBBS proteins were located in the cytosol. The expression levels of both MrFPS and MrBBS were highest in the extension period of ray florets. Furthermore, we cloned and analyzed the promoters of MrFPS and MrBBS. A large number of cis-acting elements related to light responsiveness, hormone response elements, and cis-regulatory elements that serve as putative binding sites for specific TFs in response to various biotic and abiotic stresses were identified. We identified and studied TFs related to MrFPS and MrBBS, including WRKY, AP2, and MYB. Our findings reveal the biosynthesis and regulation of α-bisabolol in German chamomile and provide novel insights for the production of α-bisabolol using synthetic biology methods.

Serine carboxypeptidase-like acyltransferases (SCPL-ATs) play a vital role in the diversification of plant metabolites. Galloylated flavan-3-ols highly accumulate in tea (Camellia sinensis), grape (Vitis vinifera), and persimmon (Diospyros kaki). To date, the biosynthetic mechanism of these compounds remains unknown. Herein, we report that two SCPL-AT paralogs are involved in galloylation of flavan-3-ols: CsSCPL4, which contains the conserved catalytic triad S-D-H, and CsSCPL5, which has the alternative triad T-D-Y. Integrated data from transgenic plants, recombinant enzymes, and gene mutations showed that CsSCPL4 is a catalytic acyltransferase, while CsSCPL5 is a non-catalytic companion paralog (NCCP). Co-expression of CsSCPL4 and CsSCPL5 is likely responsible for the galloylation. Furthermore, pull-down and co-immunoprecipitation assays showed that CsSCPL4 and CsSCPL5 interact, increasing protein stability and promoting post-translational processing. Moreover, phylogenetic analyses revealed that their homologs co-exist in galloylated flavan-3-ol- or hydrolyzable tannin-rich plant species. Enzymatic assays further revealed the necessity of co-expression of those homologs for acyltransferase activity. Evolution analysis revealed that the mutations of the CsSCPL5 catalytic residues may have taken place about 10 million years ago. These findings show that the co-expression of SCPL-ATs and their NCCPs contributes to the acylation of flavan-3-ols in the plant kingdom.

Pigeonpea (Cajanus cajan) is an important legume food crop and plays a crucial role in a secure food supply in many developing countries. Several previous studies have suggested that pigeonpea has great potential for phosphorus (P) deficiency tolerance, but little is known about the underlying mechanism. In this study, the physiological and molecular responses of pigeonpea roots to phosphate (Pi) starvation were investigated through integrating phenotypic, genomic, transcriptomic, metabolomic, and lipidomic analyses. The results showed that low-Pi treatment increased total root length, root surface area, and root acid phosphatase activity, and promoted the secretion of organic acids (e.g. citric acids, piscidic acids, and protocatechuic acids) and the degradation of phospholipids and other P-containing metabolites in the roots of pigeonpea. Consistent with the morphological, physiological, and biochemical changes, a large number of genes involved in these Pi-starvation responses were significantly upregulated in Pi-deficient pigeonpea roots. Among these Pi-starvation response genes upregulated by low-Pi treatment, four gene families were expanded through recent tandem duplication in the pigeonpea genome, namely phosphate transporter 1 (PHT1), phosphoethanolamine/phosphocholine phosphatase (PECP), fasciclin-like arabinogalactan protein (FLA), and glutamate decarboxylase (GAD). These gene families may be associated with Pi uptake from the soil, phospholipid recycling, root morphological remodeling, and regulation of organic acid exudation. Taken together, our results suggest that pigeonpea employs complex Pi-starvation responses to strengthen P acquisition and utilization during low-Pi stress. This study provides new insights into the genome evolution and P deficiency adaptation mechanism of pigeonpea.

Lilium lancifolium is an important economic crop in Huoshan county of Anhui province, China. Continuous cropping obstacles serious affect the yield and quality of L. lancifolium . At present, the effect of the continuous cropping of L. lancifolium on soil fungal community structure is not clear. In this study, Illumina MiSeq was used to study the fungi of the rhizosphere soil associated with L. lancifolium subjected to three treatments: no continuous cropping, continuous cropping for 3 years, and continuous cropping for 5 years. The results showed that continuous cropping of L. lancifolium could increase the fungal richness and diversity in the rhizosphere to different degrees. Ascomycota was the dominant phylum, and its abundance increased after continuous cropping. In addition, the abundance of beneficial fungi, such as Chaetomium , decreased, and the abundance of harmful fungi, such as Fusarium and Colletotrichum, greatly increased with the duration of continuous cropping. Overall, continuous cropping changed the composition of soil fungal communities, reduced the abundance of beneficial fungi, and increased the abundance of harmful fungi. Thus, continuous cropping increased the potential for soil-borne diseases and endangered the bulb growth of L. lancifolium .

近年来,卷丹百合的连作问题愈发突出,严重限制了百合产业的可持续发展.连作所产生的障碍主要表现在:土壤病虫害严重,植株的生长受限,生理功能逐渐衰退、品质变差.基于此,针对卷丹百合重茬病的主要成因、危害及其作用机理进行了研究,重点探讨了改进卷丹百合的种植模式、施肥方法和土壤病虫害的防治等这类治理措施产生的积极作用.将近年来在连作障碍防治方面具有广泛应用价值的几种方法作一综述.

茶是世界流行的三大无酒精饮料之一,由茶树叶片加工而成,因其中含有三大茶树特征代谢产物(儿茶素、茶氨酸和咖啡碱)而具有独特风味.转录因子不仅在植物生长发育中起重要调控作用,也影响植物的次级代谢产物合成,而茶树中关于转录因子调控儿茶素,茶氨酸和咖啡碱的报道较少.本研究结合茶树全器官转录组数据与茶树基因组数据重构转录本,获得大量新基因和新转录本,进而结合茶树不同器官中儿茶素、茶氨酸和咖啡碱的含量差异,分别建立茶树中儿茶素、茶氨酸和咖啡碱与差异表达基因之间的相关性,筛选鉴定与茶树中儿茶素、茶氨酸和咖啡碱相关的基因及转录因子,并分别对与茶树中儿茶素、茶氨酸和咖啡碱合成相关的3条转录因子WAKY、bZIP、BES基因进行克隆,结果表明,其ORF框大小分别为1 011 bp,1 692 bp和867 bp,与转录组分析结果一致.

German chamomile (Matricaria chamomilla L.) is one of the most ancient medicinal species in the world and terpenoids from their flowers have important medicinal value. We cloned three sesquiterpene synthase genes, McGDS1, McGDS2 and McGDS3, and performed sequence alignment and phylogenetic analysis. The encoded proteins possess three conserved structural features: an RRxxxxxxxxW motif, an RxR motif, and a DDxxD motif. McGDS1, McGDS2 and McGDS3 were confirmed to be (E)-farnesene synthase, germacrene D synthase, and germacrene A synthase, respectively. Subcellular localization revealed diffuse GFP reporter-gene signals in the cytoplasm and nucleus. qPCR indicated that McGDS1, McGDS2 and McGDS3, were more highly expressed in young flowers than in old flowers and the expression was highly correlated with amounts of the end-product essential oils ((E)-β-farnesene, germacrene D and β-elemene), with coefficients of 0.76, 0.83 and 0.68, respectively. We also established a transformation system for chamomile hairy roots. The overexpression of McGDS1, McGDS2 and McGDS3 resulted in γ-muurolene accumulation in hairy roots. The activity of three aphid alarm pheromones here forms the molecular basis for the study of the biosynthesis and regulation of volatile terpenes. Transformation of chamomile hairy roots provides a simple system in which to study terpene biosynthesis in chamomile.

Background Matricaria recutita (German chamomile) and Chamaemelum nobile (Roman chamomile) belong to the botanical family Asteraceae. These two herbs are not only morphologically distinguishable, but their secondary metabolites – especially the essential oils present in flowers are also different, especially the terpenoids. The aim of this project was to preliminarily identify regulatory mechanisms in the terpenoid biosynthetic pathways that differ between German and Roman chamomile by performing comparative transcriptomic and metabolomic analyses. Results We determined the content of essential oils in disk florets and ray florets in these two chamomile species, and found that the terpenoid content in flowers of German chamomile is greater than that of Roman chamomile. In addition, a comparative RNA-seq analysis of German and Roman chamomile showed that 54% of genes shared >75% sequence identity between the two species. In particular, more highly expressed DEGs (differentially expressed genes) and TF (transcription factor) genes, different regulation of CYPs (cytochrome P450 enzymes), and rapid evolution of downstream genes in the terpenoid biosynthetic pathway of German chamomile could be the main reasons to explain the differences in the types and levels of terpenoid compounds in these two species. In addition, a phylogenetic tree constructed from single copy genes showed that German chamomile and Roman chamomile are closely related to Chrysanthemum nankingense. Conclusion This work provides the first insights into terpenoid biosynthesis in two species of chamomile. The candidate unigenes related to terpenoid biosynthesis will be important in molecular breeding approaches to modulate the essential oil composition of Matricaria recutita and Chamaemelum nobile.

Free amino acids, including theanine, glutamine and glutamate, contribute greatly to the pleasant taste and multiple health benefits of tea. Amino acids in tea plants are mainly synthesized in roots and transported to new shoots, which are significantly affected by nitrogen (N) level and forms. However, the regulatory amino acid metabolism genes have not been systemically identified in tea plants. Here, we investigated the dynamic changes of free amino acid contents in response to N deficiency and forms in tea plant roots, and systemically identified the genes associated amino acid contents in individual metabolism pathways. Our results showed that glutamate-derived amino acids are the most dynamic in response to various forms of N and N deficiency. We then performed transcriptomic analyses of roots treated with N deficiency and various forms of N, and differentially expressed amino acid metabolic genes in each pathway were identified. The analyses on expression patterns and transcriptional responses of metabolic genes to N treatments provided novel insights for the molecular basis of high accumulation of theanine in tea plant root. These analyses also identified potential regulatory genes in dynamic amino acid metabolism in tea plant root. Furthermore, our findings indicated that the dynamic expression levels of CsGDH, CsAlaDC, CsAspAT, CsSDH, CsPAL, CsSHMT were highly correlated with changes of amino acid contents in their corresponding pathways. Herein, this study provides comprehensive insights into transcriptional regulation of amino acid metabolism in response to nitrogen deficiency and nitrogen forms in tea plant root.

Growth factor receptor bound protein-7 (Grb7) is a multi-domain signaling adaptor protein that regulates various cellular functions acting as an adaptor protein in relaying signal transduction. Although several studies indicated that Grb7 amplifies EGFR-mediated signaling in cancers, the detailed regulatory mechanism of whether and how Grb7 is involved in EGFR-mediated lung cancer progression remains unclear. Here, we demonstrate that EGF-regulated Grb7 phosphorylation promotes lung cancer progression through phosphorylation of STAT3. Intrinsically, EGF/EGFR signal is required for the formation of Grb7/STAT3 complex as well as its nuclear accumulation. Once in the nucleus, STAT3 interacts with EPHA4 promoter, which in turn affects the gene expression level of EPHA4 through transcriptional regulation. Functionally, EphA4 together with EGFR promotes cancer migration, proliferation, and anchorage-independent growth. Our study reveals a novel mechanism in which Grb7 contribute to lung cancer malignancies through its interaction with STAT3 that leads to sequential regulation of EPHA4 gene expression in an EGF/EGFR signal-dependent manner.

Focal adhesion kinase (FAK) mediates vital cellular pathways during development. Despite its necessity, how FAK regulates and integrates with other signals during early embryogenesis remains poorly understood. We found that the loss of Fak1a impaired epiboly, convergent extension and hypoblast cell migration in zebrafish embryos. We also observed a clear disturbance in cortical actin at the blastoderm margin and distribution of yolk syncytial nuclei. In addition, we investigated a possible link between Fak1a and a well-known gastrulation regulator, Wnt5b, and revealed that the overexpression of fak1a or wnt5b could cross-rescue convergence defects induced by a wnt5b or fak1a antisense morpholino (MO), respectively. Wnt5b and Fak1a were shown to converge in regulating Rac1 and Cdc42, which could synergistically rescue wnt5b and fak1a morphant phenotypes. Furthermore, we generated several alleles of fak1a mutants using CRISPR/Cas9, but those mutants only revealed mild gastrulation defects. However, injection of a subthreshold level of the wnt5b MO induced severe gastrulation defects in fak1a mutants, which suggested that the upregulated expression of wnt5b might complement the loss of Fak1a. Collectively, we demonstrated that a functional interaction between Wnt and FAK signalling mediates gastrulation cell movements via the possible regulation of Rac1 and Cdc42 and subsequent actin dynamics.

In mammalian cells, extracellular vesicles (EVs) derived from the endosomal system carry many different kinds of bioactive molecule to deliver to recipient cells in a paracrine or endocrine manner. EVs can mediate local and systemic intercellular communications, including reeducating stromal cells, remodeling the architecture of the tumor microenvironment, modulating cancer metabolism and metastases, or even conferring drug resistance. Because the molecular and functional characteristics of prostate cancer (PCa) evolve over time, the bioactive molecule profiles/signatures of tumor-derived EVs (TDEs) reflect the real-time status of cancer cells. TDEs appear to be valuable diagnostic and prognostic biomarkers as well as potential therapeutic vehicles, suggesting their essential role in precision medicine of disease management. We summarized critical aspects of TDEs in PCa and discussed their potential clinical applications.

In mammalian cells, extracellular vesicles (EVs) derived from the endosomal system carry many different kinds of bioactive molecule to deliver to recipient cells in a paracrine or endocrine manner. EVs can mediate local and systemic intercellular communications, including reeducating stromal cells, remodeling the architecture of the tumor microenvironment, modulating cancer metabolism and metastases, or even conferring drug resistance. Because the molecular and functional characteristics of prostate cancer (PCa) evolve over time, the bioactive molecule profiles/signatures of tumor-derived EVs (TDEs) reflect the real-time status of cancer cells. TDEs appear to be valuable diagnostic and prognostic biomarkers as well as potential therapeutic vehicles, suggesting their essential role in precision medicine of disease management. We summarized critical aspects of TDEs in PCa and discussed their potential clinical applications.