Lycorine (Lycoris radiata)-a unique natural medicine on breast cancer.

Breast cancer (BC) is one of the most common types of cancer among women worldwide. Lycorine (Lycoris radiata), a small molecule derived from the traditional Chinese herb Amaryllidaceae plants, has appeared potential effect on inhibiting the growth of cancer cells and inducing apoptosis in various types of cancer with minor side effects. To discuss the therapeutic effects and molecular mechanisms of lycorine on BC established by lycorine-treated S180 tumour-bearing mice in vivo. Furthermore, both the mitotic and microtubule assembly dynamics genes were performed by qPCR assays, and the protein expression associated with mitotic arrest was investigated by western blot. Lycorine was demonstrated to reduce sarcoma growth of S180 tumour-bearing mice and inhibit the proliferation of MCF-7 cells in concentration-dependent manner. Moreover, lycorine induced M phase cell cycle arrest via interfering with the mitotic apparatus regulated the expression of 20 genes and 15 proteins in cell cycle progression. Furthermore, this study confirmed that the potential effect of lycorine on BC might be mediated by cell cycle arrest in M phase for the first time. These results would be the consequence of exploitation of lycorine as a potential drug for BC therapy, however further preclinical and clinical studies are still needed.

Ascorbate peroxidase catalyses synthesis of protocatechualdehyde from p-hydroxybenzaldehyde in Lycoris aurea.

Protocatechualdehyde is a plant natural phenolic aldehyde and an active ingredient with important bioactivities in traditional Chinese medicine. Protocatechualdehyde is also a key intermediate in the synthesis of Amaryllidaceae alkaloids for supplying the C6-C1 skeleton. However, the biosynthesis of protocatechualdehyde in plants remains obscure. In this study, we measured the protocatechualdehyde contents in the root, bulb, scape and flower of the Amaryllidaceae plant Lycoris aurea (L'Hér.) Herb., and performed the correlation analysis between the protocatechualdehyde contents and the transcriptional levels of the phenolic oxidization candidate protein encoding genes. We found that a novel ascorbate peroxidase encoded by the contig_24999 in the L. aurea transcriptome database had potential role in the biosynthesis of protocatechualdehyde. The LauAPX_24999 gene was then cloned from the cDNA of the scape of L. aurea. The transient expression of LauAPX_24999 protein in Arabidopsis protoplasts demonstrated that LauAPX_24999 protein was localized in the cytoplasm, thus belonging to Class II L-ascorbate peroxidase. Subsequently, LauAPX_24999 protein was heterogenously expressed in Escherichia coli, and identified that LauAPX_24999 biosynthesized protocatechualdehyde from p-hydroxybenzaldehyde using L-ascorbic acid as the electron donor. The protein structure modelling and molecular docking indicated that p-hydroxybenzaldehyde could access to the active pocket of LauAPX_24999 protein, and reside at the δ-edge of the heme group while L-ascorbic acid binds at the γ-heme edge. To our knowledge, LauAPX_24999 is the first enzyme discovered in plants able to biosynthesize protocatechualdehyde from p-hydroxybenzaldehyde, and offers a competent enzyme resource for the biosynthesis of Amaryllidaceae alkaloids via synthetic biology.

Distinct fungal communities affecting opposite galanthamine accumulation patterns in two Lycoris species.

Lycoris radiata is the main source of galanthamine, a clinical drug used in Alzheimer's disease; however, the galanthamine content in L. radiata is low. Lycoris aurea is another Lycoris species with high galanthamine content. Fungal endophytes can enhance plant secondary metabolite accumulation; thus, we compared the fungal communities in these two Lycoris species to identify certain fungal taxa in L. aurea capable of enhancing galanthamine accumulation. Several fungal endophytes, which were enriched in, exclusively isolated from L. aurea, or showed significant correlations with galanthamine, were demonstrated to enhance the accumulation of only galanthamine but no other Amaryllidaceae alkaloids (AAs) in L. radiata. These fungal endophytes mainly upregulated the downstream genes in the biosynthesis pathways of AAs in L. radiata, suggesting that they may allocate more precursors for galanthamine biosynthesis. This study demonstrated that fungal endophytes from L. aurea with higher galanthamine content can specifically enhance the accumulation of this medicinal alkaloid in other Lycoris species, thereby increasing the galanthamine source and reducing galanthamine separation and purification costs. This study broadens our understanding of the complex interactions between plant secondary metabolites and fungal endophytes.

Lycorine inhibits Ang II-induced heart remodeling and inflammation by suppressing the PI3K-AKT/NF-κB pathway.

BACKGROUND: Ang II induces hypertensive heart failure (HF) via hemodynamic and non-hemodynamic actions. Lycorine (LYC) is an alkaloid derived from Lycoris bulbs, and it possesses anti-cardiovascular disease-related activities. Herein, we explored the potential LYC-mediated regulation of Ang II-induced HF. METHODS: Over 4 weeks, we established a hypertensive HF mouse model by infusing Ang II into C57BL/6 mice using a micro-osmotic pump. For the final two weeks, mice were administered LYC via intraperitoneal injection. The LYC signaling network was then deduced using RNA sequencing. RESULTS: LYC administration strongly suppressed hypertrophy, myocardial fibrosis, and cardiac inflammation. As a result, it minimized heart dysfunction while causing no changes in blood pressure. The Nuclear Factor kappa B (NF-κB) network/phosphoinositol-3-kinase (PI3K)-protein kinase B (AKT) was found to be a major modulator of LYC-based cardioprotection using RNA sequencing study. We further confirmed that in cultured cardiomyocytes and mouse hearts, LYC reduced the inflammatory response and downregulated the Ang II-induced PI3K-AKT/NF-κB network. Moreover, PI3K-AKT or NF-κB axis depletion in cardiomyocytes completely abrogated the anti-inflammatory activities of LYC. CONCLUSION: Herein, we demonstrated that LYC safeguarded hearts in Ang II -stimulated mice by suppressing the PI3K-AKT/NF-κB-induced inflammatory responses. Given the evidence mentioned above, LYC is a robust therapeutic agent for hypertensive HF.

Identification of transcription factor genes responsive to MeJA and characterization of a LaMYC2 transcription factor positively regulates lycorine biosynthesis in Lycoris aurea.

Jasmonates (JAs) are among the main phytohormones, regulating plant growth and development, stress responses, and secondary metabolism. As the major regulator of the JA signaling pathway, MYC2 also plays an important role in plant secondary metabolite synthesis and accumulation. In this study, we performed a comparative transcriptome analysis of Lycoris aurea seedlings subjected to methyl jasmonate (MeJA) at different treatment times. A total of 31,193 differentially expressed genes (DEGs) were identified by RNA sequencing. Among them, 732 differentially expressed transcription factors (TFs) comprising 51 TF families were characterized. The most abundant TF family was WRKY proteins (80), followed by AP2/ERF-EFR (67), MYB (59), bHLH (52), and NAC protein (49) families. Subsequently, by calculating the Pearson's correlation coefficient (PCC) between the expression level of TF DEGs and the lycorine contents, 41 potential TF genes (|PCC| >0.8) involved in lycorine accumulation were identified, including 36 positive regulators and 5 negative regulators. Moreover, a MeJA-inducible MYC2 gene (namely LaMYC2) was cloned on the basis of transcriptome sequencing. Bioinformatic analyses revealed that LaMYC2 proteins contain the bHLH-MYC_N domain and bHLH-AtAIB_like motif. LaMYC2 protein is localized in the cell nucleus, and can partly rescue the MYC2 mutant in Arabidopsis thaliana. LaMYC2 protein could interact with most LaJAZs (especially LaJAZ3 and LaJAZ4) identified previously. Transient overexpression of LaMYC2 increased lycorine contents in L. aurea petals, which might be associated with the activation of the transcript levels of tyrosine decarboxylase (TYDC) and phenylalanine ammonia lyase (PAL) genes. By isolating the 887-bp-length promoter fragment upstream of the start codon (ATG) of LaTYDC, we found several different types of E-box motifs (CANNTG) in the promoter of LaTYDC. Further study demonstrated that LaMYC2 was indeed able to bind the E-box (CACATG) present in the LaTYDC promoter, verifying that the pathway genes involved in lycorine biosynthesis could be regulated by LaMYC2, and that LaMYC2 has positive roles in the regulation of lycorine biosynthesis. These findings demonstrate that LaMYC2 is a positive regulator of lycorine biosynthesis and may facilitate further functional research of the LaMYC2 gene, especially its potential regulatory roles in Amaryllidaceae alkaloid accumulation in L. aurea.

Reciprocal natural hybridization between Lycoris aurea and Lycoris radiata (Amaryllidaceae) identified by morphological, karyotypic and chloroplast genomic data.

BACKGROUND: Hybridization is considered as an important model of speciation, but the evolutionary process of natural hybridization is still poorly characterized in Lycoris. To reveal the phylogenetic relationship of two new putative natural hybrids in Lycoris, morphological, karyotypic and chloroplast genomic data of four Lycoris species were analyzed in this study. RESULTS: Two putative natural hybrids (2n = 18 = 4 m + 5t + 6st + 3 T) possessed obvious heterozygosity features of L. radiata (2n = 22 = 10t + 12st) and L. aurea (2n = 14 = 8 m + 6 T) in morphology (e.g. leaf shape and flower color), karyotype (e.g. chromosome numbers, CPD/DAPI bands, 45S rDNA-FISH signals etc.) and chloroplast genomes. Among four Lycoris species, the composition and structure features of chloroplast genomes between L. radiata and the putative natural hybrid 1 (L. hunanensis), while L. aurea and the hybrid 2, were completely the same or highly similar, respectively. However, the features of the cp genomes between L. radiata and the hybrid 2, while L. aurea and the hybrid 1, including IR-LSC/SSC boundaries, SSRs, SNPs, and SNVs etc., were significantly different, respectively. Combining the karyotypes and cp genomes analysis, we affirmed that the natural hybrid 1 originated from the natural hybridization of L. radiata (♀) × L. aurea (♂), while the natural hybrid 2 from the hybridization of L. radiata (♂) × L. aurea (♀). CONCLUSION: The strong evidences for natural hybridization between L. radiata (2n = 22) and L. aurea (2n = 14) were found based on morphological, karyotypic and chloroplast genomic data. Their reciprocal hybridization gave rise to two new taxa (2n = 18) of Lycoris. This study revealed the origin of two new species of Lycoris and strongly supported the role of natural hybridization that facilitated lineage diversification in this genus.

"Structure-function" analysis using starches isolated from Lycoris chinensis bulbs of different developmental stages.

In this study, Lycoris chinensis bulbs of four developmental stages were compared for starch characteristics. Based on correlation analysis and hierarchical cluster analysis, the relationships among 36 traits were discussed. Compared to commonly consumed starches, L. chinensis starch had higher amylose content (33.4-43.2 %) and weight-average molar mass (36410-82,781 kDa), lower gelatinization temperature (61.8-68.1 °C), gel hardness (19.0-39.5 g) and viscosities. Among developmental stages, starches varied significantly in characteristics. As compared to juvenile stage (S1), mature bulbs (S4) had higher amylose content, lower gelatinization temperature, weight-average molar mass and degree of polymorphism. Correlation analysis revealed that the molecular weight-related traits had significantly positive correlations to gelatinization temperature (Tp, p < 0.05), positive but weak correlations to traits of particle size distribution, significantly negative correlations to AAC and many parameters of viscosity properties (p < 0.05). Based on the results of correlation analysis and hierarchical cluster analysis, the 36 traits of starch characteristics were proposed to be divided into three groups: particle size-related traits, molecular weight-related traits and AAC-related traits. The information presented in the current study are useful for future studies on starches of Lycoris and other bulb species, and instructive for future studies in investigating the "Structure-Function" relationship in starch.

Interplay between Amaryllidaceae alkaloids, the bacteriome and phytopathogens in Lycoris radiata.

Alkaloids are a large group of plant secondary metabolites with various structures and activities. It is important to understand their functions in the interplay between plants and the beneficial and pathogenic microbiota. Amaryllidaceae alkaloids (AAs) are unique secondary metabolites in Amaryllidaceae plants. Here, we studied the interplay between AAs and the bacteriome in Lycoris radiata, a traditional Chinese medicinal plant containing high amounts of AAs. The relationship between AAs and bacterial composition in different tissues of L. radiata was studied. In vitro experiments revealed that AAs have varying levels of antimicrobial activity against endophytic bacteria and pathogenic fungi, indicating the importance of AA synthesis in maintaining a balance between plants and beneficial/pathogenic microbiota. Using bacterial synthetic communities with different compositions, we observed a positive feedback loop between bacteria insensitive to AAs and their ability to increase accumulation of AAs in L. radiata, especially in leaves. This may allow insensitive bacteria to outcompete sensitive ones for plant resources. Moreover, the accumulation of AAs enhanced by insensitive bacteria could benefit plants when challenged with fungal pathogens. This study highlights the functions of alkaloids in plant-microbe interactions, opening new avenues for designing plant microbiomes that could contribute to sustainable agriculture.

Functional Characterization of AP2/ERF Transcription Factors during Flower Development and Anthocyanin Biosynthesis Related Candidate Genes in Lycoris.

The APETALA2/ethylene-responsive transcription factor (AP2/ERF) family has been extensively investigated because of its significant involvement in plant development, growth, fruit ripening, metabolism, and plant stress responses. To date, there has been little investigation into how the AP2/ERF genes influence flower formation and anthocyanin biosynthesis in Lycoris. Herein, 80 putative LrAP2/ERF transcription factors (TFs) with complete open reading frames (ORFs) were retrieved from the Lycoris transcriptome sequence data, which could be divided into five subfamilies dependent on their complete protein sequences. Furthermore, our findings demonstrated that genes belonging to the same subfamily had structural similarities and conserved motifs. LrAP2/ERF genes were analyzed for playing an important role in plant growth, water deprivation, and flower formation by means of gene ontology (GO) enrichment analysis. The expression pattern of the LrAP2/ERF genes differed across tissues and might be important for Lycoris growth and flower development. In response to methyl jasmonate (MeJA) exposure and drought stress, the expression of each LrAP2/ERF gene varied across tissues and time. Moreover, a total of 20 anthocyanin components were characterized using ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) analysis, and pelargonidin-3-O-glucoside-5-O-arabinoside was identified as the major anthocyanin aglycone responsible for the coloration of the red petals in Lycoris. In addition, we mapped the relationships between genes and metabolites and found that LrAP2/ERF16 is strongly linked to pelargonidin accumulation in Lycoris petals. These findings provide the basic conceptual groundwork for future research into the molecular underpinnings and regulation mechanisms of AP2/ERF TFs in anthocyanin accumulation and Lycoris floral development.

SWATH-MS-Based Proteomics Reveals the Regulatory Metabolism of Amaryllidaceae Alkaloids in Three Lycoris Species.

Alkaloids are a class of nitrogen-containing alkaline organic compounds found in nature, with significant biological activity, and are also important active ingredients in Chinese herbal medicine. Amaryllidaceae plants are rich in alkaloids, among which galanthamine, lycorine, and lycoramine are representative. Since the difficulty and high cost of synthesizing alkaloids have been the major obstacles in industrial production, particularly the molecular mechanism underlying alkaloid biosynthesis is largely unknown. Here, we determined the alkaloid content in Lycoris longituba, Lycoris incarnata, and Lycoris sprengeri, and performed a SWATH-MS (sequential window acquisition of all theoretical mass spectra)-based quantitative approach to detect proteome changes in the three Lycoris. A total of 2193 proteins were quantified, of which 720 proteins showed a difference in abundance between Ll and Ls, and 463 proteins showed a difference in abundance between Li and Ls. KEGG enrichment analysis revealed that differentially expressed proteins are distributed in specific biological processes including amino acid metabolism, starch, and sucrose metabolism, implicating a supportive role for Amaryllidaceae alkaloids metabolism in Lycoris. Furthermore, several key genes collectively known as OMT and NMT were identified, which are probably responsible for galanthamine biosynthesis. Interestingly, RNA processing-related proteins were also abundantly detected in alkaloid-rich Ll, suggesting that posttranscriptional regulation such as alternative splicing may contribute to the biosynthesis of Amaryllidaceae alkaloids. Taken together, our SWATH-MS-based proteomic investigation may reveal the differences in alkaloid contents at the protein levels, providing a comprehensive proteome reference for the regulatory metabolism of Amaryllidaceae alkaloids.

Antifungal effects of amaryllidaceous alkaloids from bulbs of Lycoris spp. against Magnaporthe oryzae.

BACKGROUND: Rice blast caused by Magnaporthe oryzae is one of the most devastating diseases of rice, and novel fungicides for controlling rice blast are needed owing to the problem of resistance to commonly used control agents. We previously found that methanol extract of Lycoris radiata (L'Her.) Herb. showed an excellent inhibitory effect on mycelial growth of M. oryzae, indicating its potential for developing control agents against M. oryzae. In this study, we aim to investigate the antifungal effects of different Lycoris spp. against M. oryzae, and clarify the main active components. RESULTS: Extracts from bulbs of seven Lycoris spp. showed excellent inhibitory effects on mycelial growth and spore germination of M. oryzae at 400 mg L-1 . Liquid chromatography-tandem mass spectrometry was employed to analyze the components of the extracts, and heatmap clustering analysis with Mass Profiler Professional software revealed that lycorine and narciclasine may be the main active components. Lycorine and narciclasine, together with three other amaryllidaceous alkaloids (AAs), were then isolated from bulbs of Lycoris spp. Antifungal assays showed that lycorine and narciclasine had good inhibitory activities against M. oryzae in vitro, but the other three AAs showed no antifungal activities under test concentrations. In addition, lycorine and the ethyl acetate part of L. radiata showed good antifungal effects against M. oryzae in vivo, but narciclasine showed phototoxicity on rice when used alone. CONCLUSION: Extracts of test Lycoris spp. and the main active component lycorine have excellent antifungal activities against M. oryzae, and are good candidates for developing control agents against M. oryzae. © 2023 Society of Chemical Industry.

Characterization of the WRKY Gene Family Related to Anthocyanin Biosynthesis and the Regulation Mechanism under Drought Stress and Methyl Jasmonate Treatment in Lycoris radiata.

Lycoris radiata, belonging to the Amaryllidaceae family, is a well-known Chinese traditional medicinal plant and susceptible to many stresses. WRKY proteins are one of the largest families of transcription factors (TFs) in plants and play significant functions in regulating physiological metabolisms and abiotic stress responses. The WRKY TF family has been identified and investigated in many medicinal plants, but its members and functions are not identified in L. radiata. In this study, a total of 31 L. radiata WRKY (LrWRKY) genes were identified based on the transcriptome-sequencing data. Next, the LrWRKYs were divided into three major clades (Group I-III) based on the WRKY domains. A motif analysis showed the members within same group shared a similar motif component, indicating a conservational function. Furthermore, subcellular localization analysis exhibited that most LrWRKYs were localized in the nucleus. The expression pattern of the LrWRKY genes differed across tissues and might be important for Lycoris growth and flower development. There were large differences among the LrWRKYs based on the transcriptional levels under drought stress and MeJA treatments. Moreover, a total of 18 anthocyanin components were characterized using an ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) analysis and pelargonidin-3-O-glucoside-5-O-arabinoside as well as cyanidin-3-O-sambubioside were identified as the major anthocyanin aglycones responsible for the coloration of the red petals in L. radiata. We further established a gene-to-metabolite correlation network and identified LrWRKY3 and LrWRKY27 significant association with the accumulation of pelargonidin-3-O-glucoside-5-O-arabinoside in the Lycoris red petals. These results provide an important theoretical basis for further exploring the molecular basis and regulatory mechanism of WRKY TFs in anthocyanin biosynthesis and in response to drought stress and MeJA treatment.

Identification of Differentially Expressed Genes Related to Floral Bud Differentiation and Flowering Time in Three Populations of Lycoris radiata.

The transition from vegetative to reproductive growth is important for controlling the flowering of Lycoris radiata. However, the genetic control of this complex developmental process remains unclear. In this study, 18 shoot apical meristem (SAM) samples were collected from early-, mid- and late-flowering populations during floral bud differentiation. The histological analysis of paraffin sections showed that the floral bud differentiation could be divided into six stages; the differentiation time of the early group was earlier than that of the middle and late groups, and the late group was the latest. In different populations, some important differential genes affecting the flowering time were identified by transcriptome profiles of floral bud differentiation samples. Weighted gene co-expression network analysis (WGCNA) was performed to enrich the gene co-expression modules of diverse flowering time populations (FT) and floral bud differentiation stages (ST). In the MEyellow module, five core hub genes were identified, including CO14, GI, SPL8, SPL9, and SPL15. The correlation network of hub genes showed that they interact with SPLs, AP2, hormone response factors (auxin, gibberellin, ethylene, and abscisic acid), and several transcription factors (MADS-box transcription factor, bHLH, MYB, and NAC3). It suggests the important role of these genes and the complex molecular mechanism of floral bud differentiation and flowering time in L. radiata. These results can preliminarily explain the molecular mechanism of floral bud differentiation and provide new candidate genes for the flowering regulation of Lycoris.

Comparative transcriptome and metabolome analyses identified the mode of sucrose degradation as a metabolic marker for early vegetative propagation in bulbs of Lycoris.

Vegetative propagation (VP) is an important practice for production in many horticultural plants. Sugar supply constitutes the basis of VP in bulb flowers, but the underlying molecular basis remains elusive. By performing a combined sequencing technologies coupled with ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry approach for metabolic analyses, we compared two Lycoris species with contrasting regeneration rates: high-regeneration Lycoris sprengeri and low-regeneration Lycoris aurea. A comprehensive multi-omics analyses identified both expected processes involving carbohydrate metabolism and transcription factor networks, as well as the metabolic characteristics for each developmental stage. A higher abundance of the differentially expressed genes including those encoding ethylene responsive factors was detected at bulblet initiation stage compared to the late stage of bulblet development. High hexose-to-sucrose ratio correlated to bulblet formation across all the species examined, indicating its role in the VP process in Lycoris bulb. Importantly, a clear difference between cell wall invertase (CWIN)-catalyzed sucrose unloading in high-regeneration species and the sucrose synthase-catalyzed pathway in low-regeneration species was observed at the bulblet initiation stage, which was supported by findings from carboxyfluorescein tracing and quantitative real-time PCR analyses. Collectively, the findings indicate a sugar-mediated model of the regulation of VP in which high CWIN expression or activity may promote bulblet initiation via enhancing apoplasmic unloading of sucrose or sugar signals, whereas the subsequent high ratio of hexose-to-sucrose likely supports cell division characterized in the next phase of bulblet formation.

Transcriptome Analysis of Lycoris chinensis Bulbs Reveals Flowering in the Age-Mediated Pathway.

Lycoris is a summer bulbous flower that commonly needs to go through a long period of vegetative growth for 3 to 5 years before flowering. Plant flowering is regulated by a complex genetic network. Compared with most perennial flowers, knowledge on the molecular mechanism responsible for floral transition in bulbous flowers is lacking, and only a few genes that regulate flowering have been identified with few reports on the floral transition in Lycoris. In this study, we identified many differentially expressed genes (DEGs) and transcription factors (TFs) by RNA-Seq in L. chinensis bulbs of different ages, including one- to four-year-old nonflowering bulbs and four-year-old flowering bulbs. Some DEGs were enriched in Gene Ontology (GO) terms between the three- and four-year-old bulbs, and there most genes were enriched in terms of metabolic process and catalytic activity. In the four-year old bulbs, most of the DEGs that may be involved in flowering were classified under the GO term biological process, which was a totally different result from the vegetative bulbs. Some DEGs between flowering and nonflowering bulbs were enriched in plant hormone signal transduction, including the hormones auxin, cytokinin, abscisic acid, and ethylene, but no DEGs were enriched in the gibberellin pathway. Auxin is the main endogenous phytohormone involved in bulb growth and development, but cytokinin, abscisic acid, and ethylene were shown to increase in flowering bulbs. In addition, energy-metabolism-related genes maintain a high expression level in large bulbs, and some positive regulators (SPL, COL, and AP1) and early flowering genes were also shown to be highly expressed in the meristems of flowering bulbs. It suggested that sugar molecules may be the energy source that regulates the signal transduction of flowering by connecting with phytohormone signaling in Lycoris. A total of 1911 TFs were identified and classified into 89 categories, where the top six families with the largest gene numbers were C2H2, NAC, AP2/ERF-ERF, C3H, MYB-related, and WRKY. Most DEGs were in the AP2/ERF-ERF family, and most of them were downregulated in 4-year-old flowering bulbs. A number of families were reported to be involved in plant flowering, including NAC, AP2/ERF, MYB, WRKY, bZIP, MADS, and NF-Y. These results can act as a genetic resource to aid in the explanation of the genetic mechanism responsible for the flowering of Lycoris and other bulbous flowers.

Early Sucrose Degradation and the Dominant Sucrose Cleavage Pattern Influence Lycoris sprengeri Bulblet Regeneration In Vitro.

Bulblet formation and development determine the quantitative and qualitative traits, respectively, of bulb yield for most flowering bulbs. For Lycoris species, however, the underlying molecular mechanism remains elusive. Here, clonal bulblets of Lycoris sprengeri (Ls) derived from the same probulb were used as explants to establish efficient and inefficient in vitro regeneration systems by adjusting the 6-benzyladenine (BA) concentrations in media. BA application did not change the biological processes among groups but led to earlier decreases in sucrose and total soluble sugar (TSS) contents. Correlation analyses showed that the BA treatments changed the interaction between carbohydrate and endogenous hormone contents during bulblet regeneration. We found that two sucrose degradation enzyme-related genes, cell wall invertase (CWIN) and sucrose synthase, exhibited exactly opposite expression patterns during the competence stage. In addition, the regeneration system that obtained more bulblets showed significantly higher expression of LsCWIN2 than those that obtained fewer bulblets. Our data demonstrate the essential role of BA in accelerating sucrose degradation and the selection of a dominant sucrose cleavage pattern at the competence stage of in vitro bulblet regeneration. We propose that a relatively active CWIN-catalyzed pathway at the competence stage might promote bulblet regeneration, thus influencing bulb yield.

An ATP-Binding Cassette Transporter, LaABCB11, Contributes to Alkaloid Transport in Lycoris aurea.

As a kind of Amaryllidaceae alkaloid which is accumulated in the species of Lycoris plants, lycorine has a range of physiological effects. The biosynthesis pathway of lycorine has been partly revealed, but the transport and accumulation mechanisms of lycorine have rarely been studied. In this study, an ATP-binding cassette (ABC) transporter from Lycoris aurea (L'Hér) Herb., namely LaABCB11, was cloned and functionally characterized. Heterologous expression showed that LaABCB11 transported lycorine in an outward direction, increased the tolerance of yeast cells to lycorine, and caused a lower lycorine accumulation in transformants than control or mutant in yeast. LaABCB11 is associated with the plasma membrane, and in situ hybridization indicated that LaABCB11 was mainly expressed in the phloem of leaves and bulbs, as well as in the cortical cells of roots. These findings suggest that LaABCB11 functions as a lycorine transport and it might be related to the translocation and accumulation of lycorine from the leaves and bulbs to the roots.

Chromosomal variations of Lycoris species revealed by FISH with rDNAs and centromeric histone H3 variant associated DNAs.

Lycoris species have various chromosome numbers and karyotypes, but all have a constant total number of chromosome major arms. In addition to three fundamental types, including metacentric (M-), telocentric (T-), and acrocentric (A-) chromosomes, chromosomes in various morphology and size were also observed in natural populations. Both fusion and fission translocation have been considered as main mechanisms leading to the diverse karyotypes among Lycoris species, which suggests the centromere organization playing a role in such arrangements. We detected several chromosomal structure changes in Lycoris including centric fusion, inversion, gene amplification, and segment deletion by using fluorescence in situ hybridization (FISH) probing with rDNAs. An antibody against centromere specific histone H3 (CENH3) of L. aurea (2n = 14, 8M+6T) was raised and used to obtain CENH3-associated DNA sequences of L. aurea by chromatin immunoprecipitation (ChIP) cloning method. Immunostaining with anti-CENH3 antibody could label the centromeres of M-, T-, and A-type chromosomes. Immunostaining also revealed two centromeres on one T-type chromosome and a centromere on individual mini-chromosome. Among 10,000 ChIP clones, 500 clones which showed abundant in L. aurea genome by dot-blotting analysis were FISH mapped on chromosomes to examine their cytological distribution. Five of these 500 clones could generate intense FISH signals at centromeric region on M-type but not T-type chromosomes. FISH signals of these five clones rarely appeared on A-type chromosomes. The five ChIP clones showed similarity in DNA sequences and could generate similar but not identical distribution patterns of FISH signals on individual chromosomes. Furthermore, the distinct distribution patterns of FISH signals on each chromosome generated by these five ChIP clones allow to identify individual chromosome, which is considered difficult by conventional staining approaches. Our results suggest a different organization of centromeres of the three chromosome types in Lycoris species.

Profiles of Secondary Metabolites (Phenolic Acids, Carotenoids, Anthocyanins, and Galantamine) and Primary Metabolites (Carbohydrates, Amino Acids, and Organic Acids) during Flower Development in Lycoris radiata.

This study aimed to elucidate the variations in primary and secondary metabolites during Lycorisradiata flower development using high performance liquid chromatography (HPLC) and gas chromatography time-of-flight mass spectrometry (GC-TOFMS). The result showed that seven carotenoids, seven phenolic acids, three anthocyanins, and galantamine were identified in the L. radiata flowers. Most secondary metabolite levels gradually decreased according to the flower developmental stages. A total of 51 metabolites, including amines, sugars, sugar intermediates, sugar alcohols, amino acids, organic acids, phenolic acids, and tricarboxylic acid (TCA) cycle intermediates, were identified and quantified using GC-TOFMS. Among the hydrophilic compounds, most amino acids increased during flower development; in contrast, TCA cycle intermediates and sugars decreased. In particular, glutamine, asparagine, glutamic acid, and aspartic acid, which represent the main inter- and intracellular nitrogen carriers, were positively correlated with the other amino acids and were negatively correlated with the TCA cycle intermediates. Furthermore, quantitation data of the 51 hydrophilic compounds were subjected to partial least-squares discriminant analyses (PLS-DA) to assess significant differences in the metabolites of L. radiata flowers from stages 1 to 4. Therefore, this study will serve as the foundation for a biochemical approach to understand both primary and secondary metabolism in L. radiata flower development.

Lycorine hydrochloride suppresses stress-induced premature cellular senescence by stabilizing the genome of human cells.

Lycorine, a natural compound isolated from the traditional Chinese medicinal herb Lycoris radiata, exhibits multiple pharmacological effects, such as anti-inflammatory, antiviral, and anticancer effects. Accumulating evidence also indicates that lycorine might hold the potential to treat age-associated Alzheimer's disease. However, whether lycorine is involved in delaying the onset of cellular senescence and its underlying mechanisms has not been determined. Here, we demonstrate that the salt of lycorine, lycorine hydrochloride, significantly suppressed stress-induced premature cellular senescence (SIPS) by ~2-fold, as determined by senescence-associated beta-galactosidase (SA-ß-gal) staining and the expression of p16 and p21. In addition, pretreating cells with lycorine hydrochloride significantly inhibited the expression of CXCL1 and IL1α, two factors of the senescence-associated secreted phenotype (SASP) in SIPS cells. Further experiments revealed that lycorine hydrochloride promoted both the homologous recombination (HR) and nonhomologous end joining (NHEJ) pathways of DNA double-strand break (DSB) repair. Mechanistic studies suggested that lycorine hydrochloride treatment promoted the transcription of SIRT1 and SIRT6, critical longevity genes positively regulating both HR and NHEJ repair pathways, thereby stimulating DSB repair and stabilizing genomes. Inhibiting SIRT1 enzymatic activity abrogated the protective effect of lycorine hydrochloride on delaying the onset of SIPS, repairing DSBs, and restoring genome integrity. In summary, our work indicates that lycorine hydrochloride might hold therapeutic potential for treating age-associated diseases or promoting healthy aging by stabilizing genomes.