First Report of Fusarium proliferatum Causing Bulb Rot on Lilium davidii var. willmottiae in China.

Lilium davidii var. willmottiae, known as Lanzhou lily, is widely cultivated in China for its edible bulbs. In July 2023, symptoms of bulb rot were observed on Lanzhou lilies harvested from Lanzhou, Gansu Province, during storage at the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences (Beijing, China), at an incidence of nearly 70%. The surface of the lily scales had dark water-stained spots, after the development of which the color gradually darkened, the bulbs became soft, accompanied by a pungent smell. Finally, the whole bulb became ruined and rotten, and there were thick mycelium layers on the bulbs. The infected bulbs were washed with clean water, sterilized with 75% ethanol for 30 s and 2% sodium hypochlorite for 5 min, and then rinsed three times with sterile distilled water. The 5 mm×5 mm tissue pieces from the junction of the diseased part and the healthy part were clipped, placed on potato dextrose agar (PDA) medium and subsequently incubated at 25 °C. Pure cultures were obtained by transferring hyphal tips to new PDA plates. A total of 10 fungal isolates were obtained, all of which exhibited typical Fusarium characteristics. The colonies were white to pink with white to cream-colored aerial mycelia. After 10 to 15 days of incubation, the macroconidia (n = 50) were hyaline, relatively slender with a curve, three to five septate, and 8.73 to 33.24 × 2.16 to 4.19 µm in length. The microconidia (n = 50) were hyaline and pyriform, without septa, and measured 4.04 to 8.48 × 1.24 to 2.65 µm. These morphological characteristics were similar to those described for Fusarium proliferatum (Leslie and Summerell 2006). For molecular identification, a cetyltrimethylammonium bromide (CTAB) protocol was used to extract total genomic DNA (O'Donnell et al., 1998), after which the internal transcribed spacer (ITS), translation elongation factor subunit 1-alpha (TEF1-α) and RNA polymerase Ⅱ subunit 2 (RPB2) genes were amplified using the universal primers ITS1/ITS4, EF1/EF2 and RPB2-5f2/fRPB2-7cr, respectively, and subsequently sequenced (White et al., 1990; O'Donnell et al., 1998; Liu et al., 1999; Reeb et al., 2004; O'Donnell et al., 2007; Jiang et al., 2018). The sequences of a representative isolate (CAAS01) were analyzed and submitted to GenBank under accession numbers OR554007 (ITS), OR594233 (TEF1-α) and OR603932 (RPB2). A BLAST analysis revealed that the sequences of the ITS, TEF1-α, and RPB2 genes shared 100%, 100%, and 100% identity, respectively, with those of Fusarium proliferatum (MT466521.1, MK952792.1, and LT841266.1) in GenBank. In addition, the ITS, TEF1-α and RPB2 sequences shared 100%, 100%, and 100% identity with those of Fusarium annulatum (LC13675, the Fusarium fujikuroi species complex; previously known as the Gibberella fujikuroi species complex) in the Fusarium-ID database. Fusarium proliferatum, whose common synonyms are Gibberella fujikuroi mating population D and Gibberella fujikuroi var. intermedia, is the anamorphic form of the Gibberella fujikuroi complex that belongs to the Nectriaceae family. A phylogenetic tree was constructed based on the combined TEF1-α and RPB2 sequences of CAAS01 and other Fusarium isolates, revealing that CAAS01 was grouped with Fusarium proliferatum. Based on sequence alignment and phylogenetic analysis, the isolate was identified as Fusarium proliferatum. To determine the pathogenicity of the isolated fungi, healthy bulbs were punctured with disposable sterilized needles and soaked in equal amounts of sterile water and conidial suspension (1×107 conidia/mL) for 30 min respectively. The pathogenicity experiment was repeated three times. After 7 days of inoculation at 25 °C and 80% relative humidity, the surface of the inoculated bulbs produced water-stained spots and mycelium layers consistent with the symptoms exhibited by Lilium davidii var. willmottiae bulbs during storage, while the uninoculated lily bulbs remained symptomless. Fusarium proliferatum was reisolated from the infected bulbs and identified based on morphological and molecular characteristics, fulfilling Koch's postulates. To our knowledge, this is the first report of bulb rot on Lilium davidii var. willmottiae caused by Fusarium proliferatum in China. This study will contribute to the development of management strategies for this postharvest disease in Lilium davidii var. willmottiae.

WUSCHEL-related homeobox genes cooperate with cytokinin to promote bulbil formation in Lilium lancifolium.

The bulbil is an important vegetative reproductive organ in triploid tiger lily (Lilium lancifolium). Based on our previously obtained transcriptome data, we screened two WUSCHEL-related homeobox (WOX) genes closely related to bulbil formation, LlWOX9 and LlWOX11. However, the biological functions and regulatory mechanisms of LlWOX9 and LlWOX11 are unclear. In this study, we cloned the full-length coding sequences of LlWOX9 and LlWOX11. Transgenic Arabidopsis (Arabidopsis thaliana) showed increased branch numbers, and the overexpression of LlWOX9 and LlWOX11 in stem segments promoted bulbil formation, while the silencing of LlWOX9 and LlWOX11 inhibited bulbil formation, indicating that LlWOX9 and LlWOX11 are positive regulators of bulbil formation. Cytokinin type-B response regulators could bind to the promoters of LlWOX9 and LlWOX11 and promote their transcription. LlWOX11 could enhance cytokinin pathway signaling by inhibiting the transcription of type-A LlRR9. Our study enriches the understanding of the regulation of plant development by the WOX gene family and lays a foundation for further research on the molecular mechanism of bulbil formation in lily.

First report of Fusarium equiseti causing bulb rot on lily (Lilium 'White planet' ) in China.

Lily (Lilium spp.) is one of the main ornamental plants grown in the world. In addition, bulbs of lily have been extensively used as edible and medicinal herbs in northern and eastern Asia, especially in China (Yu et al. 2015; China Pharmacopoeia Committee 2020; Tang et al. 2021). In August of 2021, a disease of stem and leaf rot was observed on lily cultivar 'White planet' with approximately 25% disease incidence in the greenhouse and fields at the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences (Beijing, China). The bulbs of symptomatic plants were brown and rotten, with sunken lesions. Symptomatic plants showed short, discolored leaves, and eventually lead to stem wilt and death of the whole plants. Infected bulbs were surface sterilized in 75% ethanol for 30 s, then in 2% sodium hypochlorite for 5 min, and rinsed three times with sterile distilled water. A 0.5×0.5 cm2 tissue piece was then placed on potato dextrose agar (PDA) medium and incubated at 25±1℃. After 5 days, the isolate was purified by single spore isolation technique. The singled-spored fungal colony was characterized by fluffy white aerial mycelia, and produced orange pigments with age. After seven days on Spezieller Nahrstoffarmer agar (SNA), conidia produced from simple lateral phialides. Macroconidia have pronounced dorsiventral curvature typical, significantly enlarged in the middle, a tapered whip-liked pointed apical cell and characteristic foot-shaped basal cell, 3 to 6 septate, measuring 18.71 to 43.01×2.89 to 5.56 µm with an average size of 26.98×3.90 µm (n=30). Microconidia were not observed. Typical verrucose thick chlamydospore with rough walls were profuse in chains or clumps, ellipsoidal to subglobose. These morphological characteristics were consistent with Fusarium spp. (Leslie et al. 2006). For molecular identification, the internal transcribed spacer (ITS), translation elongation factor subunit 1-alpha (TEF1-α) and RNA polymeraseⅡsubunit 2 (RPB2) genes were amplified using primers ITS1/ITS4, EF1/EF2 and 5F2/7cR respectively and sequenced (White et al. 1990; Jiang et al. 2018; O'Donnell et al. 2007). Sequences were submitted to GenBank under accession numbers OM078499 (ITS), Accession OM638086 (TEF1-α) and OM638085 (RPB2). BLAST analysis showed that ITS, TEF1-α and RPB2 sequences shared 100%, 99.8%, 99.2% identity to F. equiseti (OM956073, KY081599, MW364892) in GenBank, respectively. In addition, ITS, TEF1-α and RPB2 sequences shared 100%, 99.53%, 100% identity with Fusarium lacertarum (LC7927, Fusarium incarnatum-equiseti species complex) in the Fusarium-ID database. Based on the morphological characteristics and molecular sequences, the isolates were identified as Fusarium equiseti. A pathogenicity test was performed on potted lily ('White planet') under greenhouse conditions (25±1℃ with a 16 h light and 8 h dark cycle). Three healthy lily bulbs were selected and one bulb was planted in each pot filled with sterilized soil. Each pot was inoculated with 5 mL of conidia suspension (1×107 conidia/mL) in te soil around bulbs with a stem length of 3 cm, with an equal amount of sterilized water as a control. This test had three replicates. After 15 days of inoculation, typical symptoms of bulb rotten, like those observed in the greenhouse and fields, developed on the inoculated plants but not on the controls. The same fungus was consistently reisolated from the diseased plants. To our knowledge, this is the first report that F. equiseti caused bulb rot on Lilium in China. Our result should help with future monitoring and control of lily wilt disease.

Development of a multiplex RT-PCR assay for simultaneous detection of Lily symptomless virus, Lily mottle virus, Cucumber mosaic virus, and Plantago asiatica mosaic virus in Lilies.

BACKGROUND: Viral pathogens causing significant economic losses in lilies (Lilium spp. and hybrids) include Lily symptomless virus (LSV), Lily mottle virus (LMoV), Cucumber mosaic virus (CMV), and Plantago asiatica mosaic virus (PlAMV). Rapid and efficient virus detection methods are pivotal to prevent the spread of these viruses. RESULTS: In this study, four specific primer pairs designed from conserved regions of genomic sequences of each virus were used to amplify a 116 bp product for LSV, a 247 bp product for LMoV, a 359 bp product for CMV, and a 525 bp product for PlAMV in a multiplex reverse transcription-polymerase chain reaction (multiplex RT-PCR). The amplified products were clearly separated by 2% agarose gel electrophoresis. The optimal reaction annealing temperature and cycle number were 53.8 °C and 35, respectively. The developed multiplex RT-PCR method was then used to test virus infections from lily samples collected from different regions of China. CONCLUSIONS: An effective multiplex RT-PCR assay was established for the simultaneous detection and differentiation of LSV, LMoV, CMV, and PlAMV in lilies, which offers a useful tool for routine molecular diagnosis and epidemiological studies of these viruses.

Construction of yeast one-hybrid library and screening of transcription factors regulating LhMYBSPLATTER expression in Asiatic hybrid lilies (Lilium spp.).

BACKGROUND: Anthocyanins, which belong to flavonoids, are widely colored among red-purple pigments in the Asiatic hybrid lilies (Lilium spp.). Transcription factor (TF) LhMYBSPLATTER (formerly known as LhMYB12-Lat), identified as the major kernel protein, regulating the anthocyanin biosynthesis pathway in 'Tiny Padhye' of Tango Series cultivars, which the pigmentation density is high in the lower half of tepals and this patterning is of exceptional ornamental value. However, the research on mechanism of regulating the spatial and temporal expression differences of LhMYBSPLATTER, which belongs to the R2R3-MYB subfamily, is still not well established. To explore the molecular mechanism of directly related regulatory proteins of LhMYBSPLATTER in the anthocyanin pigmentation, the yeast one-hybrid (Y1H) cDNA library was constructed and characterized. RESULTS: In this study, we describe a yeast one-hybrid library to screen transcription factors that regulate LhMYBSPLATTER gene expression in Lilium, with the library recombinant efficiency of over 98%. The lengths of inserted fragments ranged from 400 to 2000 bp, and the library capacity reached 1.6 × 106 CFU of cDNA insert, which is suitable to fulfill subsequent screening. Finally, seven prey proteins, including BTF3, MYB4, IAA6-like, ERF4, ARR1, ERF WIN1-like, and ERF061 were screened by the recombinant bait plasmid and verified by interaction with the LhMYBSPLATTER promoter. Among them, ERFs, AUX/IAA, and BTF3 may participate in the negative regulation of the anthocyanin biosynthesis pathway in Lilium. CONCLUSION: A yeast one-hybrid library of lily was successfully constructed in the tepals for the first time. Seven candidate TFs of LhMYBSPLATTER were screened, which may provide a theoretical basis for the study of floral pigmentation.

LhGST is an anthocyanin-related glutathione S-transferase gene in Asiatic hybrid lilies (Lilium spp.).

KEY MESSAGE: LhGST, an anthocyanin-related GST gene, was identified from Asiatic hybrid lilies. Expression and functional analyses demonstrated that LhGST might be involved in anthocyanin sequestration in lily tepals. Anthocyanins are responsible for the pink, red and purple pigmentation of flowers in Asiatic hybrid lilies, synthesized at the cytoplasmic surface of the endoplasmic reticulum (ER) and then transported to the vacuole. To date, the mechanism involved in the intracellular transport of anthocyanins in lilies has not been well elucidated. Here, full-length glutathione S-transferase gene (LhGST) was identified from lilies. Expression analysis revealed that LhGST was positively correlated with anthocyanin accumulation. Phylogenetic tree analysis showed that LhGST clustered with other anthocyanin-related GSTs in the same phi clade. Moreover, functional complementation of an Arabidopsis tt19 mutant demonstrated that LhGST might be involved in anthocyanin accumulation in lily tepals. Additionally, according to phenotype analysis, LhGST was found to be correlated with the transport of anthocyanin in lilies by virus-induced gene silencing (VIGS) assay. In addition, cis-element analysis of the LhGST promoter showed the presence of ABA-, auxin-, MeJA-, gibberellin-, light-, and stress-responsive elements and an MYB recognition site (MRS, CCGTTG). Yeast one-hybrid and dual-luciferase report assays revealed that the promoter of LhGST was activated by LhMYB12-lat, which is a key R2R3-MYB transcription factor that regulates anthocyanin biosynthesis in lilies. In conclusion, our results revealed that LhGST plays a key role in anthocyanin transport and accumulation in the tepals of lilies.

Cytokinin Type-B Response Regulators Promote Bulbil Initiation in Lilium lancifolium.

The bulbil is an important vegetative reproductive organ in triploid Lilium lancifolium whose development is promoted by cytokinins. Type-B response regulators (RRs) are critical regulators that mediate primary cytokinin responses and promote cytokinin-induced gene expression. However, the function of cytokinin type-B Arabidopsis RRs (ARRs) in regulating bulbil formation is unclear. In this study, we identified five type-B LlRRs, LlRR1, LlRR2, LlRR10, LlRR11 and LlRR12, in L. lancifolium for the first time. The five LlRRs encode proteins of 715, 675, 573, 582 and 647 amino acids. All of the regulators belong to the B-I subfamily, whose members typically contain a conserved CheY-homologous receiver (REC) domain and an Myb DNA-binding (MYB) domain at the N-terminus. As transcription factors, all five type-B LlRRs localize at the nucleus and are widely expressed in plant tissues, especially during axillary meristem (AM) formation. Functional analysis showed that type-B LlRRs are involved in bulbil formation in a functionally redundant manner and can activate LlRR9 expression. In summary, our study elucidates the process by which cytokinins regulate bulbil initiation in L. lancifolium through type-B LlRRs and lays a foundation for research on the molecular mechanism of bulbil formation in the lily.

Mechanism of exogenous cytokinins inducing bulbil formation in Lilium lancifolium in vitro.

KEY MESSAGE: The cytokinin pathway promotes the initiation of bulbil formation, and iPA may an important type of cytokinin during bulbil formation in Lilium lancifolium. Bulbils are important vegetative reproductive organs in triploid Lilium lancifolium. We previously showed that cytokinins are involved in bulbil formation, but how cytokinins participate in bulbil formation is not clear. In this study, bulbil formation was divided into three stages on the basis of anatomical and histological observations: the bulbil initiation stage, bulbil primordium-formation stage and bulbil structure-formation stage. The results indicated that iPA was the most critical cytokinin during the bulbil initiation. qRT-PCR revealed that increased iPA content during bulbil initiation was mainly due to increased expression of cytokinin synthesis genes (IPT1/5) and cytokinin activation genes (LOG1/3/5/7) and significantly decreased expression of the cytokinin degradation gene CKX4. Exogenous 6-BA and lovastatin affected the cytokinin pathway and promoted or inhibited bulbil initiation by increasing or decreasing the content of endogenous iPA, respectively. In summary, we demonstrate that cytokinins positively regulate bulbil formation and provide preliminary insight into the regulatory mechanisms by which the cytokinin pathway promotes bulbil initiation.

Transcriptome Analysis Identifies Key Candidate Genes Mediating Purple Ovary Coloration in Asiatic Hybrid Lilies.

Lily tepals have a short lifespan. Once the tepals senesce, the ornamental value of the flower is lost. Some cultivars have attractive purple ovaries and fruits which greatly enhance the ornamental value of Asiatic hybrid lilies. However, little is known about the molecular mechanisms of anthocyanin biosynthesis in Asiatic hybrid lily ovaries. To investigate the transcriptional network that governs purple ovary coloration in Asiatic hybrid lilies, we obtained transcriptome data from green ovaries (S1) and purple ovaries (S2) of Asiatic "Tiny Padhye". Comparative transcriptome analysis revealed 4228 differentially expressed genes. Differential expression analysis revealed that ten unigenes including four CHS genes, one CHI gene, one F3H gene, one F3'H gene, one DFR gene, one UFGT gene, and one 3RT gene were significantly up-regulated in purple ovaries. One MYB gene, LhMYB12-Lat, was identified as a key transcription factor determining the distribution of anthocyanins in Asiatic hybrid lily ovaries. Further qPCR results showed unigenes related to anthocyanin biosynthesis were highly expressed in purple ovaries of three purple-ovaried Asiatic hybrid lilies at stages 2 and 3, while they showed an extremely low level of expression in ovaries of three green-ovaried Asiatic hybrid lilies during all developmental stages. In addition, shading treatment significantly decreased pigment accumulation by suppressing the expression of several unigenes related to anthocyanin biosynthesis in ovaries of Asiatic "Tiny Padhye". Lastly, a total of 15,048 Simple Sequence Repeats (SSRs) were identified in 13,710 sequences, and primer pairs for SSRs were designed. The results could further our understanding of the molecular mechanisms of anthocyanin biosynthesis in Asiatic hybrid lily ovaries.

Lily (Lilium spp.) LhERF4 negatively affects anthocyanin biosynthesis by suppressing LhMYBSPLATTER transcription.

Anthocyanins are among the main pigments involved in the colouration of Asiatic hybrid lily (Lilium spp.). Ethylene, a plant ripening hormone, plays an important role in promoting plant maturation and anthocyanin biosynthesis. However, whether and how ethylene regulates anthocyanin biosynthesis in lily tepals have not been characterized. Using yeast one-hybrid screening, we previously identified an APETALA2 (AP2)/ETHYLENE RESPONSE FACTOR (ERF) named LhERF4 as a potential inhibitor of LhMYBSPLATTER-mediated negative regulation of anthocyanin biosynthesis in lily. Here, transcript and protein analysis of LhERF4, a transcriptional repressor, revealed that LhERF4 directly binds to the promoter of LhMYBSPLATTER. In addition, overexpression of LhERF4 in lily tepals negatively regulates the expression of key structural genes and the total anthocyanin content by suppressing the LhMYBSPLATTER gene. Moreover, the LhERF4 gene inhibits anthocyanin biosynthesis in response to ethylene, affecting anthocyanin accumulation and pigmentation in lily tepals. Collectively, our findings will advance and elucidate a novel regulatory network of anthocyanin biosynthesis in lily, and this research provides new insight into colouration regulation.

Multifaceted roles of LhWRKY44 in promoting anthocyanin accumulation in Asiatic hybrid lilies (Lilium spp.).

The Asiatic hybrid lily (Lilium spp.) is a horticultural crop with high commercial value and diverse anthocyanin pigmentation patterns. However, the regulatory mechanism underlying lily flower color has been largely unexplored. Here, we identified a WRKY transcription factor from lily tepals, LhWRKY44, whose expression was closely associated with anthocyanin accumulation. Functional verification indicated that LhWRKY44 positively regulated anthocyanin accumulation. LhWRKY44 physically interacted with LhMYBSPLATTER and directly bound to the LhMYBSPLATTER promoter, which enhanced the effect of the LhMYBSPLATTER-LhbHLH2 MBW complex activator on anthocyanin accumulation. Moreover, EMSA and dual-luciferase assays revealed that LhWRKY44 activated and bound to the promoters of gene LhF3H and the intracellular anthocyanin-related glutathione S-transferase gene LhGST. Interestingly, our further results showed that LhWRKY44 participated in light and drought-induced anthocyanin accumulation, and improved the drought tolerance in lily via activating stress-related genes. These results generated a multifaceted regulatory mechanism for the LhWRKY44-meditaed enhancement by the environmental signal pathway of anthocyanin accumulation and expanded our understanding of the WRKY-mediated transcriptional regulatory hierarchy modulating anthocyanin accumulation in Asiatic hybrid lilies.

A Na+/H+ antiporter-encoding salt overly sensitive 1 gene, LpSOS1, involved in positively regulating the salt tolerance in Lilium pumilum.

Lilium pumilum has a strong salt tolerance. However, the molecular mechanism underlying its salt tolerance remains unexplored. Here, LpSOS1 was cloned from L. pumilum and found to be significantly enriched at high NaCl concentrations (100 mM). In tobacco epidermal cells, localization analysis showed that the LpSOS1 protein was primarily located in the plasma membrane. Overexpression of LpSOS1 resulted in up-regulation of salt stress tolerance in Arabidopsis, as indicated by reduced malondialdehyde levels and Na+/K+ ratio, and increased activity of antioxidant reductases (including superoxide dismutase, peroxidase, and catalase). Treatment with NaCl resulted in improved growth, as evidenced by increased biomass, root length, and lateral root growth, in both sos1 mutant (atsos1) and wild-type (WT) Arabidopsis plants that overexpressed LpSOS1, Under NaCl treatment,atsos1 and WT Arabidopsis plants overexpressing LpSOS1 exhibited better growth, with higher biomass, root length, and lateral root quantity, whereas in the absence of LpSOS1 overexpression, the plants of both lines were wilted and chlorotic and even died under salt stress. When exposed to salt stress, the expression of stress-related genes was notably upregulated in the LpSOS1 overexpression line of Arabidopsis as compared to the WT. Our findings indicate that LpSOS1 enhances salt tolerance in plants by regulating ion homeostasis, reducing Na+/K+ ratio, thereby protecting the plasma membrane from oxidative damage caused by salt stress, and enhancing the activity of antioxidant enzymes. Therefore, the increased salt tolerance conferred by LpSOS1 in plants makes it a potential bioresource for breeding salt-tolerant crops. Further investigation into the mechanisms underlying lily's resistance to salt stress would be advantageous and could serve as a foundation for future molecular improvements.

Potential Applications of Lilium Plants in Cosmetics: A Comprehensive Review Based on Research Papers and Patents.

The application of cosmetics is indispensable in our current society. In recent years, with an increasing awareness of the long-term health benefits of naturally sourced ingredients, plant-based cosmetic products have gained increasing attention. Lilium belongs to the Liliaceae family, which is one of the main plant families used in cosmetics for skin care treatment. A large number of studies have shown that Lilium plants are rich in components such as phenolic acids, flavonoids, and polysaccharides, with high potential for cosmetic applications. However, the application of lilies in cosmetics has not been systematically reported. This knowledge gap can easily lead to the neglect of its application in cosmetics because lilies are most familiar as ornamental plants. Integrating academic papers and patent publications, we analyzed the potential cosmetic application ingredients in lily, as well as their applications in cosmetics and related efficacy. Patent analysis showed that applications for lily-related cosmetic patents are mainly concentrated in East Asia, including China, Korea, and Japan. The application of lilies involves all aspects of cosmetics, such as sunscreens, facial cleansers, facial masks, conditioners, and so on. Its functions are also rich and diverse, including antiaging, radiation protective, whitening, moisturizing, freckle removal, acne treatment, and hair regeneration promotion. In addition, lilies are compatible with the application of other herbs. Moreover, with a change in people's consumption concepts and the consideration of long-term health benefits, lily-based food and medicine innovation with health care and beautification effects may be a promising direction.

Systematic evaluation on the physicochemical characteristics of a series polysaccharides extracted from different edible lilies by ultrasound and subcritical water.

A series polysaccharide samples extracted from three edible lilies (Lilium davidii var. willmottiae, Lilium brownii var. viridulum, and Lilium lancifolium) by subcritical water and ultrasound-assisted extraction were systematically compared. The results showed that extraction method was a more important factor than lily species. Subcritical water extracted lily polysaccharides (S-LP) with higher yield, molecular weight, neutral glucose and uronic acid content as well as apparent viscosity. Ultrasound-assisted extracted lily polysaccharides (U-LP) with higher reducing sugars and protein content. Moreover, due to the degradation of glycosidic bonds, ultrasonic extraction was easier to obtain lower molecular weight polysaccharides. In addition, the extraction method significantly affected the monosaccharide proportion of polysaccharides, but had no effect on type. Glucose was the main component in S-LP, and glucose and mannose were the main components in U-LP. The micromorphology of different polysaccharide samples was similar, and the scanning electron microscope (SEM) images showed regular/irregular particle clusters with different particle sizes. Overall, the relationships between extraction methods, lily species and polysaccharide properties were preliminarily elucidated, providing a reference for the targeted extraction of specific lily polysaccharides (LP).

The complete plastid genome of Lilium regale E.H.Wilson.

Lilium regale E.H.Wilson is a native lily species in western Sichuan of China and an important resource for lily breeding. In this study, the plastid genome of L. regale was assembled de novo using the next-generation sequencing data. The plastid genome of L. regale was 152,998 bp in length, with a typical quadripartite circle structure consisting of a small single-copy region of 17,529 bp, a large single-copy region of 82,375 bp, and a pair of inverted repeats of 26,547 bp each. A total of 137 different genes were predicted, including 84 protein-coding genes, 38 transfer RNA genes, 8 ribosomal RNA genes, and 7 pseudogenes. The overall GC content of the plastid genome was 36.98%. Phylogenetic analysis revealed that L. regale is most closely related to Lilium leucanthum.

Comprehensive Analysis of Secondary Metabolites in the Extracts from Different Lily Bulbs and Their Antioxidant Ability.

The genus Lilium contains more than 100 wild species and numerous hybrid varieties. Some species of them have been used as medicine and food since ancient times. However, the research on the active components and the medical properties of lilies has only focused on a few species. In this study, the total phenolic acid content (TPC), total flavonoid content (TFC), and antioxidant capacity of 22 representative lilies were systematically investigated. The results showed that the TPC, TFC and antioxidant activity were highly variable among different lilies, but they were significantly positively correlated. Hierarchical cluster analysis indicated that L. henryi and L. regale were arranged in one group characterized by the highest TPC, TFC and antioxidant capacity, followed by Oriental hybrids and Trumpet and Oriental hybrids. The traditional edible and medicinal lilies were clustered in low TPC, TFC and antioxidant capacity group. A total of 577 secondary metabolites, including 201 flavonoids, 153 phenolic acids, were identified in the five species with great differences in antioxidant capacity by extensive targeted metabonomics. Differentially accumulated metabolites (DAMs) analysis reviewed that the DAMs were mainly enriched in secondary metabolic pathways such as isoflavonoid, folate, flavonoid, flavone, flavonol, phenylpropanoid, isoquinoline alkaloid biosynthesis, nicotinate and nicotinamide metabolism and so on. Correlation analysis identified that 64 metabolites were significantly positively correlated with antioxidant capacity (r ≥ 0.9 and p < 0.0001). These results suggested that the genus Lilium has great biodiversity in bioactive components. The data obtained greatly expand our knowledge of the bioactive constituents of Lilium spp. Additionally, it also highlights the potential application of Lilium plants as antioxidants, functional ingredients, cosmetic products and nutraceuticals.

Cloning and Functional Characterization of a Flavonoid Transport-Related MATE Gene in Asiatic Hybrid Lilies (Lilium spp.).

Previous studies have suggested that multidrug and toxic compound extrusion (MATE) proteins might be involved in flavonoid transportation. However, whether MATE proteins are involved in anthocyanin accumulation in Lilium is unclear. Here, a flavonoid transport-related MATE candidate gene, LhDTX35, was cloned from the Asiatic hybrid lily cultivar 'Tiny Padhye' by rapid amplification of 5' and 3' cDNA ends (RACE) and found to encode 507 amino acids. BLASTx results indicated that LhDTX35 showed high homology to the DTX35 genes of other species. Bioinformatics analysis predicted that the protein encoded by LhDTX35 possessed 12 typical transmembrane segments and had functional domains typical of the MATE-like superfamily. Phylogenetic analysis grouped LhDTX35 in the same clade as the DTX35 of other species. Notably, the expression pattern of LhDTX35 was positively correlated with floral anthocyanin accumulation in 'Tiny Padhye'. A subcellular localization assay showed that the protein encoded by LhDTX35 was plasmalemma localized but not nuclear, indicating that the LhDTX35 gene may function as a carrier protein to transport anthocyanins in Lilium. Functional complementation of the ArabidopsisDTX35 gene demonstrated that LhDTX35 could restore silique-infertility and the anthocyaninless phenotype of an ArabidopsisDTX35 mutant. These results indicated that LhDTX35 might be involved in anthocyanin accumulation in Lilium.

Tobacco rattle virus-induced PHYTOENE DESATURASE (PDS) and Mg-chelatase H subunit (ChlH) gene silencing in Solanum pseudocapsicum L.

Virus-induced gene silencing (VIGS) is an attractive tool for determining gene function in plants. The present study constitutes the first application of VIGS in S. pseudocapsicum, which has great ornamental and pharmaceutical value, using tobacco rattle virus (TRV) vectors. Two marker genes, PHYTOENE DESATURASE (PDS) and Mg-chelatase H subunit (ChlH), were used to test the VIGS system in S. pseudocapsicum. The photobleaching and yellow-leaf phenotypes of the silenced plants were shown to significantly correlate with the down-regulation of endogenous SpPDS and SpChlH, respectively (P ≤ 0.05). Moreover, the parameters potentially affecting the efficiency of VIGS in S. pseudocapsicum, including the Agrobacterium strain and the inoculation method (leaf syringe-infiltration, sprout vacuum-infiltration and seed vacuum-infiltration), were compared. The optimized VIGS parameters were the leaf syringe-infiltration method, the Agrobacterium strain GV3101 and the growth of agro-inoculated plants at 25°. With these parameters, the silencing efficiency of SpPDS and SpChlH could reach approximately 50% in S. pseudocapsicum. Additionally, the suitability of various reference genes was screened by RT-qPCR using three candidate genes, and the results demonstrated that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) can serve as a suitable reference for assessing the gene expression levels of VIGS systems in S. pseudocapsicum. The proven application of VIGS in S. pseudocapsicum and the characterization of a suitable reference gene in the present work will expedite the functional characterization of novel genes in S. pseudocapsicum.

Spatiotemporal Transcriptome Analysis Provides Insights into Bicolor Tepal Development in Lilium "Tiny Padhye".

The bicolor Asiatic hybrid lily cultivar "Tiny Padhye" is an attractive variety because of its unique color pattern. During its bicolor tepal development, the upper tepals undergo a rapid color change from green to white, while the tepal bases change from green to purple. However, the molecular mechanisms underlying these changes remain largely uncharacterized. To systematically investigate the dynamics of the lily bicolor tepal transcriptome during development, we generated 15 RNA-seq libraries from the upper tepals (S2-U) and basal tepals (S1-D, S2-D, S3-D, and S4-D) of Lilium "Tiny Padhye." Utilizing the Illumina platform, a total of 295,787 unigenes were obtained from 713.12 million high-quality paired-end reads. A total of 16,182 unigenes were identified as differentially expressed genes during tepal development. Using Kyoto Encyclopedia of Genes and Genomes pathway analysis, candidate genes involved in the anthocyanin biosynthetic pathway (61 unigenes), and chlorophyll metabolic pathway (106 unigenes) were identified. Further analyses showed that most anthocyanin biosynthesis genes were transcribed coordinately in the tepal bases, but not in the upper tepals, suggesting that the bicolor trait of "Tiny Padhye" tepals is caused by the transcriptional regulation of anthocyanin biosynthetic genes. Meanwhile, the high expression level of chlorophyll degradation genes and low expression level of chlorophyll biosynthetic genes resulted in the absence of chlorophylls from "Tiny Padhye" tepals after flowering. Transcription factors putatively involved in the anthocyanin biosynthetic pathway and chlorophyll metabolism in lilies were identified using a weighted gene co-expression network analysis and their possible roles in lily bicolor tepal development were discussed. In conclusion, these extensive transcriptome data provide a platform for elucidating the molecular mechanisms of bicolor tepals in lilies and provide a basis for similar research in other closely related species.

Validation of Reference Genes for Quantitative Real-Time PCR during Bicolor Tepal Development in Asiatic Hybrid Lilies (Lilium spp.).

Quantitative real-time PCR (qRT-PCR) is a reliable and high-throughput technique for gene expression studies, but its accuracy depends on the expression stability of reference genes. To date, several reliable reference gene identifications have been reported in Lilium spp., but none has been obtained for lily tepals at different developmental stages. In this study, ten candidate reference genes were selected and evaluated for their expression stability in Lilium 'Tiny Padhye' during the process of bicolor tepal development. The expression stability of these candidates was evaluated by three software programs (geNorm, NormFinder, and BestKeeper) and the comparative ΔCt method, and comprehensive stability rankings were generated by RefFinder. As a result, TIP41-like family gene (TIP41) and actin (ACT) were the best combination of reference genes for tepals at different developmental stages; TIP41 and F-box family gene (F-box) for tepals under shading treatment; ACT, actin11 (ACT11), and elongation factor 1-α (EF1-α) for different tissues; and ACT, TIP41, and ACT11 for all samples. The selected optimal reference genes were further verified by analyzing the expression levels of flavonoid 3'-hydroxylase (LhF3'H) and anthocyanidin 3-O-glucosyltransfersae (LhUFGT) in tepals at different developmental stages. This study provides useful information for gene expression characterization in lilies under different experimental conditions, and can serve as a basis for similar research in other closely related species.