Evaluation of the anticarcinogenic potential of the endophyte, Streptomyces sp. LRE541 isolated from Lilium davidii var. unicolor (Hoog) Cotton

Background Endophytic actinomycetes, as emerging sources of bioactive metabolites, have been paid great attention over the years. Recent reports demonstrated that endophytic streptomycetes could yield compounds with potent anticancer properties that may be developed as chemotherapeutic drugs. Results Here, a total of 15 actinomycete-like isolates were obtained from the root tissues of Lilium davidii var. unicolor (Hoog) Cotton based on their morphological appearance, mycelia coloration and diffusible pigments. The preliminary screening of antagonistic capabilities of the 15 isolates showed that isolate LRE541 displayed antimicrobial activities against all of the seven tested pathogenic microorganisms. Further in vitro cytotoxicity test of the LRE541 extract revealed that this isolate possesses potent anticancer activities with IC50 values of 0.021, 0.2904, 1.484, 4.861, 6.986, 8.106, 10.87, 12.98, and 16.94 μg/mL against cancer cell lines RKO, 7901, HepG2, CAL-27, MCF-7, K562, Hela, SW1990, and A549, respectively. LRE541 was characterized and identified as belonging to the genus Streptomyces based on the 16S rRNA gene sequence analysis. It produced extensively branched red substrate and vivid pink aerial hyphae that changed into amaranth, with elliptic spores sessile to the aerial mycelia. To further explore the mechanism underlying the decrease of cancer cell viability following the LRE541 extract treatment, cell apoptosis and cell cycle arrest assays were conducted in two cancer cell lines, RKO and 7901. The result demonstrated that LRE541 extract inhibited cell proliferation of RKO and 7901 by causing cell cycle arrest both at the S phase and inducing apoptosis in a dose-dependent manner. The chemical profile of LRE541 extract performed by the UHPLC-MS/MS analysis revealed the presence of thirty-nine antitumor compounds in the extract. Further chemical investigation of the LRE541 extract led to the discovery of one prenylated indole diketopiperazine (DKP) alkaloid, elucidated as neoechinulin A, a known antitumor agent firstly detected in Streptomyces; two anthraquinones 4-deoxy-ε-pyrromycinone (1) and epsilon-pyrromycinone (2) both displaying anticancer activities against RKO, SW1990, A549, and HepG2 with IC50 values of 14.96 ± 2.6 − 20.42 ± 4.24 μg/mL for (1); 12.9 ± 2.13, 19.3 ± 4.32, 16.8 ± 0.75, and 18.6 ± 3.03 μg/mL for (2), respectively. Conclusion Our work evaluated the anticarcinogenic potential of the endophyte, Streptomyces sp. LRE541 and obtained one prenylated indole diketopiperazine alkaloid and two anthraquinones. Neoechinulin A, as a known antitumor agent, was identified for the first time in Streptomyces. Though previously found in Streptomyces, epsilon-pyrromycinone and 4-deoxy-ε-pyrromycinone were firstly shown to possess anticancer activities. Graphical Abstract

Characteristics of Cuticular Wax and Analysis of Wax Biosynthesis Related Genes in Lanzhou Lily(Lilium Davidii Var. Unicolor) Under Drought Stress

The full text of this preprint has been withdrawn by the authors due to author disagreement with the posting of the preprint. Therefore, the authors do not wish this work to be cited as a reference. Questions should be directed to the corresponding author.

Effects of Cold Treatments on Seedling Emergence and Growth of Lilium davidii var. unicolor Bulblets

For Lilium davidii var. unicolor bulblets produced by scale propagation, the effects of cold treatments on the sprouting and development of bulblets were studied. The results showed that 5 °C was a more suitable temperature than 2 or 10 °C. Bulblets treated at 5 °C for 3 weeks presented the best uniformity of seedling emergence, and the sprouting rate was 100%. Moreover, the largest bulbs were observed in this treatment after a growing season. It was found that long storage at low temperatures is unfavorable for bulb development. The weight and circumference of bulbs from bulblets that were cold-treated for more than 5 weeks were significantly less than those treated for 1 to 4 weeks. During the first 4 weeks of cold storage, the starch content of bulblets decreased significantly, coinciding with an increase in soluble sugars. The starch and soluble sugar contents in bulblets stored at 2 and 5 °C changed faster than those in bulblets stored at 10 °C. However, the effect of temperature on carbohydrates diminished gradually as the storage time increased. Long storage of bulblets at low temperatures is not good for subsequent growth and development. The results of this study provide important information for accelerating the scale propagation of L. davidii var. unicolor and maximizing bulb yield.

Characterization of Endophytic Fungi, Acremonium sp., from Lilium davidii and Analysis of Its Antifungal and Plant Growth-Promoting Effects

The present study was aimed at isolating endophytic fungi from the Asian culinary and medicinal plant Lilium davidii and analyzing its antifungal and plant growth-promoting effects. In this study, the fungal endophyte Acremonium sp. Ld-03 was isolated from the bulbs of L. davidii and identified through morphological and molecular analysis. The molecular and morphological analysis confirmed the endophytic fungal strain as Acremonium sp. Ld-03. Antifungal effects of Ld-03 were observed against Fusarium oxysporum, Botrytis cinerea, Botryosphaeria dothidea, and Fusarium fujikuroi. The highest growth inhibition, i.e., 78.39 ± 4.21 % , was observed for B. dothidea followed by 56.68 ± 4.38 % , 43.62 ± 3.81 % , and 20.12 ± 2.45 % for B. cinerea, F. fujikuroi, and F. oxysporum, respectively. Analysis of the ethyl acetate fraction through UHPLC-LTQ-IT-MS/MS revealed putative secondary metabolites which included xanthurenic acid, valyl aspartic acid, gancidin W, peptides, and cyclic dipeptides such as valylarginine, cyclo-[L-(4-hydroxy-Pro)-L-leu], cyclo(Pro-Phe), and (3S,6S)-3-benzyl-6-(4-hydroxybenzyl)piperazine-2,5-dione. Other metabolites included (S)-3-(4-hydroxyphenyl)-2-((S)-pyrrolidine-2-carboxamido)propanoic acid, dibutyl phthalate (DBP), 9-octadecenamide, D-erythro-C18-Sphingosine, N-palmitoyl sphinganine, and hydroxypalmitoyl sphinganine. The strain Ld-03 showed indole acetic acid (IAA) production with or without the application of exogenous tryptophan. The IAA ranged from 53.12 ± 3.20 μg ml-1 to 167.71 ± 7.12 μg ml-1 under different tryptophan concentrations. The strain was able to produce siderophore, and its production was significantly decreased with increasing Fe(III) citrate concentrations in the medium. The endophytic fungal strain also showed production of organic acids and phosphate solubilization activity. Plant growth-promoting effects of the strain were evaluated on in vitro seedling growth of Allium tuberosum. Application of 40% culture dilution resulted in a significant increase in root and shoot length, i.e., 24.03 ± 2.71 mm and 37.27 ± 1.86 mm, respectively, compared to nontreated control plants. The fungal endophyte Ld-03 demonstrated the potential of conferring disease resistance and plant growth promotion. Therefore, we conclude that the isolated Acremonium sp. Ld-03 should be further investigated before utilization as a biocontrol agent and plant growth stimulator.

Transcriptome Profiling Reveals Key Genes In Regulation Of The Tepal Trichome Development In Lilium Pumilum D.C.

Trichome is a specialized structure found on the surface of the plant with important function in survival against abiotic and biotic stress. It is also an important economic trait in crop breeding. Extensive research has investigated the foliar trichome in model plants (Arabidopsis and tomato). However, the developmental mechanism of tepal trichome remains elusive. Lilium pumilum is an edible ornamental bulb and a good breeding parent possessing cold and salt-alkali resistance. Here, we found a natural mutant of Lilium pumilum grown on a highland whose tepals are covered by trichomes. Our data indicate that trichomes of this mutant are multicellular and branchless. Notably, stomata are also developed on the tepal of the mutant as well, suggesting there may be a correlated between trichome and stomata regulation. Furthermore, we isolated 27 differentially expressed genes (DEGs) by comparing the transcriptome profiling between the natural mutant and the wild type. These twenty-seven genes belong to four groups: epidermal cell cycle and division, trichome morphogenesis, stress response, and transcription factors. Quantitative real-time PCR in Lilium pumilum (natural mutant and the wild type) and other lily species (Lilium leichtlinii var. maximowiczii/ trichome; Lilium davidii var. willmottiael, trichomeless) confirmed the validation of RNA-seq data and identified several trichome-related genes.

An optimized method to obtain high-quality RNA from different tissues in Lilium davidii var. unicolor

Background: The high quality, high yield and purity RNA samples are essential for subsequent molecular experiments such as RT-PCR, qPCR and RNA-seq. However, harvest high quality RNA samples from different tissues in Lilium davidii var. unicolor is a great challenge due to its polysaccharides, polyphenols and other secondary metabolites. In this study, three RNA extraction methods, namely modified TRIzol method, Kit and CTAB methods were reported to obtain the total RNA from Lilium davidii var. unicolor, and the efficient RNA extraction protocol (modified TRIzol method) was described. Results: A Nano drop spectrophotometer and 1% gel electrophoresis were used to detect the RNA quality and integrity. Compared with Kit and CTAB methods, the higher RNA concentrations from different tissues were obtained and the A260/280 ratios of RNA samples were ranged from1.97 to 2.27 when using the modified TRIzol method. None of intact RNA bands were gained from the modified CTAB method, indicating that the RNA samples isolation were degraded. As a result of the modified TRIzol method, the RNA samples in the different tissues from Lilium presented clear 28S and 18S bands.Conclusions: Here, modified TRIzol method is an easy, efficient, and low-cost method for RNA isolation from Lilium davidii var. unicolor. Thus, modified TRIzol method is sufficient to gain eligible RNA to support further molecular experiments in Lilium davidii var. unicolor.