Hemerocallis citrina Baroni (H. citrina) is an important specialty vegetable that is not only edible and medicinal but also has ornamental value. However, much remains unknown about the regulatory mechanisms associated with the growth, development, and flowering rhythm of this plant. CO, as a core regulatory factor in the photoperiod pathway, coordinates light and circadian clock inputs to transmit flowering signals. We identified 18 COL genes (HcCOL1-HcCOL18) in the H. citrina cultivar \'Mengzihua\' and studied their chromosomal distribution, phylogenetic relationships, gene and protein structures, collinearity, and expression levels in the floral organs at four developmental stages. The results indicate that these genes can be classified into three groups based on phylogenetic analysis. The major expansion of the HcCOL gene family occurred via segmental duplication, and the Ka/Ks ratio indicated that the COL genes of Arabidopsis thaliana, Oryza sativa, Phalaenopsis equestris, and H. citrina were under purifying selection. Many cis-elements, including light response elements, abiotic stress elements, and plant hormone-inducible elements, were distributed in the promoter sequences of the HcCOL genes. Expression analysis of HcCOL genes at four floral developmental stages revealed that most of the HcCOL genes were expressed in floral organs and might be involved in the growth, development, and senescence of the floral organs of H. citrina. This study lays a foundation for the further elucidation of the function of the HcCOL gene in H. citrina and provides a theoretical basis for the molecular design breeding of H. citrina.

Salicylic acid (SA) is an essential phytoregulator that is widely used to promote the synthesis of high-value nutraceuticals in plants. However, its application in daylily, an ornamental plant highly valued in traditional Chinese medicine, has not been reported. Herein, we investigated the exogenous SA-induced physiological, transcriptional and biochemical changes in long yellow daylily (LYD). We found that 2 mg/L foliar SA treatment significantly improved LYD plant growth and yield. Transcriptome sequencing and differentially expressed genes (DEGs) analysis revealed that the phenylpropanoid biosynthesis, isoquinoline alkaloid biosynthesis, sulfur metabolism, plant hormone signal transduction and tyrosine metabolism were significantly induced in SA-treated leaves. Many transcription factors and antioxidant system-related DEGs were induced under the SA treatment. Biochemical analyses showed that the leaf contents of soluble sugar, soluble protein (Cpr), ascorbic acid (AsA) and colchicine were significantly increased by 15.15% (from 30.16 ± 1.301 to 34.73 ± 0.861 mg/g), 19.54% (from 60.3 ± 2.227 to 72.08 ± 1.617 mg/g), 30.45% (from 190.1 ± 4.56 to 247.98 ± 11.652 μg/g) and 73.05% (from 3.08 ± 0.157 to 5.33 ± 0.462 μg/g), respectively, under the SA treatment. Furthermore, we identified 15 potential candidate genes for enhancing the growth, production and phytochemical content of LYD. Our results provide support for the bioaccumulation of colchicine in yellow daylily and valuable resources for biotechnological-assisted production of this important nutraceutical in Hemerocallis spp.

BACKGROUND: Hemerocallis citrina Baroni (Huang hua cai in Chinese) is a perennial herbaceous plant grown for its flower buds that are eaten fresh or dried and is known as the vegetarian three treasures. The nuclear genome of H. citrina has been reported, but the intraspecific variation of the plastome (plastid genome) has not yet been studied. Therefore, the panplastome of this species collected from diverse locations is reported here for the first time.
RESULTS: In this study, 65 H. citrina samples were resequenced, de novo assembled, and aligned with the published plastome of H. citrina to resolve the H. citrina panplastome. The sizes of the 65 newly assembled complete plastomes of H. citrina ranged from 156,048 bp to 156,263 bp, and the total GC content ranged from 37.31 to 37.34%. The structure of the complete plastomes showed a typical tetrameric structure, including a large single copy (LSC), a small single copy (SSC), and a pair of inverted repeat regions (IRA and IRB). Many nucleotide variants were identified between plastomes, among which the variants in the intergenic spacer region were the most abundant, with the highest number of variants concentrated in the LSC region. Based on the phylogenetic tree constructed using the ML method, population structure analysis, and principal component analysis (PCA), the panplastome data were subdivided into five genetic clusters. The C5 genetic cluster was mostly represented by samples from Qidong, Hunan Province, while samples from Shanxi and Shaanxi Provinces were classified into the C4 genetic cluster. The greatest genetic diversity was found in the C1 genetic cluster, and the greatest genetic distance between any two clusters was found between the C4 and C5 clusters.
CONCLUSIONS: The resolution of the panplastome and the analysis of the population structure of H. citrina plastomes provide important data for future breeding projects and germplasm preservation.

BACKGROUND: Hemerocallis citrina Baroni is a traditional medical and edible plant. It is rich in flavonoid compounds, which are a kind of important bioactive components with various health benefits and pharmaceutical value. However, the flavonoid metabolomics profile and the comparison of flavonoid compounds from different parts of H. citrina is scarce.
RESULTS: In this study, flavonoid metabolites were investigated from roots, stems, leaves and flowers of H. citrina. A total of 364 flavonoid metabolites were identified by UPLC-MS/MS based widely targeted metabolomics, and the four plant parts showed huge differences at flavonoid metabolic level. Compared to roots, 185, 234, and 119 metabolites accounted for upregulated differential flavonoid metabolites (DFMs) in stems, leaves, and flowers, respectively. Compared to stems, 168 and 29 flavonoid metabolites accounted for upregulated DFMs in leaves and flowers, respectively. Compared to leaves, only 29 flavonoid metabolites accounted for upregulated DFMs in flowers. A number of 35 common flavonoid metabolites were observed among six comparison groups, and each comparison group had its unique differential metabolites. The most abundant flavonoid metabolites in the four parts are flavonols and flavones, followed by flavanones, chalcones, flavanols, flavanonols, anthocyanidins, tannin, and proanthocyanidins. 6,7,8-Tetrahydroxy-5-methoxyflavone, 7,8,3\',4\'-tetrahydroxyflavone, 1-Hydroxy-2,3,8-trimethoxyxanthone, Farrerol-7-O-glucoside, 3\',7-dihydroxy-4\'-methoxyflavone, 3,3\'-O-Dimethylellagic Acid, 5-Hydroxy-6,7-dimethoxyflavone, Nepetin (5,7,3\',4\'-Tetrahydroxy-6-methoxyflavone), (2s)-4,8,10-trihydroxy-2-methoxy-1 h,2 h-furo[3,2-a]xanthen-11-one are dominant in roots. Isorhamnetin-3-O-(6\'\'-malonyl)glucoside-7-O-rhamnoside, 7-Benzyloxy-5-hydroxy-3\',4\'-methylenedioxyflavonoid, 3-Hydroxyphloretin-4\'-O-glucoside are dominant in stems. Chrysoeriol-7-O-glucoside, Epicatechin glucoside, Kaempferol-3-O-rhamnoside (Afzelin)(Kaempferin)*, Azaleatin (5-O-Methylquercetin), Chrysoeriol-5-O-glucoside, Nepetin-7-O-glucoside(Nepitrin), 3,5,7,2\'-Tetrahydroxyflavone; Datiscetin, Procyanidin B2*, Procyanidin B3*, Procyanidin B1, Isorhamnetin-3-O-(6\'\'-acetylglucoside) are dominant in leaves. kaempferol-3-p-coumaroyldiglucoside, Delphinidin-3-O-sophoroside-5-O-glucoside, Limocitrin-3-O-sophoroside, Kaempferol-3-O-rutinoside(Nicotiflorin), Luteolin-7-O-(6\'\'-malonyl)glucoside-5-O-rhamnoside are dominant in flowers.
CONCLUSIONS: There was significant difference in flavonoid metabolites among different parts of H. citrina. Leaves had relative higher metabolites contents than other parts. This study provided biological and chemical evidence for the different uses of various plant parts of H. citrina, and these informations are important theoretical basis for the food industry, and medical treatment.

Hemerocallis citrina is a perennial herbaceous plant that is dedicated to mothers in Chinese culture and is widely distributed across the country. As a popular species with a long history of cultivation and utilization, it is renowned for its remarkable edible and medicinal value. In this study, we integrated Illumina short-read and Oxford Nanopore long-read sequencing to generate a complete mitochondrial genome (mitogenome) assembly of H. citrina. The H. citrina mitogenome has a multiple chromosomal structure consisting of three circular molecules that are 45,607 bp, 239,991 bp, and 182,864 bp long. We correspondingly annotated 66 genes, comprising 45 protein-coding genes (PCGs), 17 tRNA genes, and 4 rRNA genes. Comparative analysis of gene organization indicated that six syntenic gene clusters were conserved in the mitogenomes of the compared plants. The investigation of repeat content revealed repeat-rich nature of the H. citrina mitogenome, for which plentiful dispersed repeats were characterized to correlate with the size of the mitogenome. The codon usage behavior disclosed that Leucine (Leu) and Serine (Ser) were the most preferred amino acids in H. citrina, and nearly all of the codons with relative synonymous codon usage (RSCU) values greater than 1 showed the preference of A or T ending. Moreover, we inferred a total of 679 RNA editing sites in all mitochondrial PCGs, which presented perfect C-to-U types and tended to lead to the alteration of internal codons. Subsequent selective pressure analysis showed that the majority of the PCGs had undergone evolutionary negative selections, with atp9 in particular undergoing strong stabilizing selection, reflecting its indispensable function in mitogenomes. According to the phylogenetic analysis, H. citrina is close to the species Allium cepa (Amaryllidaceae) and Asparagus officinalis (Asparagaceae) in evolutionary terms. Overall, this project presents the first complete mitogenome of H. citrina, which could provide a reference genome for the comprehensive exploration of the Asphodelaceae family and can facilitate further genomic breeding and evolutionary research on this medicine-food homologous plant.

Daylily (Hemerocallis citrina Baroni) is a perennial herb whose flowers are commonly used in traditional Chinese cuisine. It is commercially cultivated in the Loess plateau of Gansu province, China. From July to October 2020, necrotic lesions were observed on the foliage of daylily plants in Huan County, Gansu, China, with an average disease incidence of 90%, and 52 to 86 disease index across four fields (approximate 6 hectares). Lesions were fusiform or nearly fusiform yellowish-brown spots of different sizes and a yellow irregular border. Older lesions were almost dark brown that often coalesced and expanded to cover the entire leaves. Thirty-four samples were collected from plants with typical foliar symptoms. Symptomatic tissues were excised from the margins of the lesions and sterilized with 75% ethanol for 20 s and 0.1% NaClO for 2 min, rinsed with sterilized water four times, dried on sterile paper towels, and cultured on Potato Dextrose Agar medium at 25°C for 7 days. A total of 34 fungal isolates with 100% isolation frequency were obtained and characterized. Colonies were white, becoming pale brown with age, reverse turned grayish black with age and irregular pale yellowish borders on the reverse side. Conidia (n=50) were hyaline, one-celled, subcylindrical with obtuse to slightly rounded ends, of 12-18.5×3.5-6 µm in size, (avg. 15.5×4.8 µm). The isolates were designated as K2010301 (51-54) and deposited in the Microbiological Culture Collection Center at College of Pastoral Agriculture Science and Technology, Lanzhou University (China). For fungal identification to species level, genomic DNA of a representative isolate (isolate MG) was extracted. Internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase-1 (CHS-1) and beta-tubulin (TUB2) were amplified using V9G/ITS4, GDF1/GDR1, CHS-354R/CHS-79F, and T1/Bt-2b primer sets (Damm et al., 2012), respectively, and deposited in GenBank under accession numbers MW811458, MW836582, MW836581, and MW836584. BLASTn showed higher than 99% identity with Colletotrichum siamense (GenBank: KP703350 (ITS), MN884050 (GAPDH), MN894598 (CHS-1), and KX578815 (TUB2)). A Bayesian inference analysis of the four concatenated loci showed that isolate MG grouped in the C. siamense clade. Pathogenicity tests were performed by spraying a spore suspension (1×105 conidia/mL) of a 10-day-old culture of isolate \"MG\" onto 3 healthy and asymptomatic daylily plants. Three control plants were only sprayed with the same volume of sterile distilled water. The inoculated plants were covered with black plastic bags for 2 days to maintain high relative humidity. Anthracnose symptoms resembling those observed in the field developed after 7 days on all inoculated plants, while no symptoms were observed on the control plants. The fungus was reisolated and identified as C. siamense based on morphological features and DNA sequence analysis, fulfilling Koch\'s postulates. It has been demonstrated that C. liliacearum (Zhuang, 2005), C. gloeosporioides, and C. spaethianum (Yang et al., 2012) are anthracnose pathogens of H. citrina. To our knowledge, this is the first report of C. siamense causing daylily anthracnose worldwide. This fungal pathogen represents a severe threat and has the potential to cause yield losses of daylily, so further studies should focus on epidemiology and effective management strategies of this disease.

Hemerocallis citrina Baroni (Liliaceae), a Liliaceae plant, has been widely used in food and traditional medicine. This study investigated the safety of ethanol extracts from Hemerocallis citrina (HCE) after oral treatment (p.o.) and evaluating the anti-inflammatory mechanism of HCE in a lipopolysaccharide (LPS)-induced depressive-like model. First, in an 8-week experimental procedure, blood and tissue samples collected from mice were used for biochemical and histopathological analysis every two weeks. Neither the body weight nor relative organ weights were affected by HCE administration. Only the total cholesterol levels were decreased by HCE administration. Histopathological analysis showed no significant liver and kidney changes caused by HCE. In addition, in an LPS-induced mouse depressive-like model, HCE significantly reversed the reduction of sucrose preference with LPS. The results also indicated that LPS activated the nuclear factor-κB (NF-κB), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in the prefrontal cortex. In contrast, these activations were normalized by HCE pretreatment. In summary, our study provided essential evidence for the safety of Hemerocallis citrina in both food and medicine. The results also demonstrated that HCE exhibited antidepressant-like effects that might be related to inhibition of the NF-κB signaling pathway.

BACKGROUND: Daylily flowers, the flower and bud parts of Hemerocallis citrina or H. fulva, are well known as Wang-You-Cao in Chinese, meaning forget-one\'s sadness plant. However, the major types of active constituents responsible for the neurological effects remain unclear. This study was to examine the protective effects of hydroalcoholic extract and fractions and to identify the active fractions.
METHODS: The extract of daylily flowers was separated with AB-8 resin into different fractions containing non-phenolic compounds, phenolic acid derivatives and flavonoids as determined using UPLC-DAD chromatograms. The neuroprotective activity was measured by evaluating the cell viability and lactate dehydrogenase release using PC12 cell damage models induced by corticosterone and glutamate. The neurological mechanisms were explored by determining their effect on the levels of dopamine (DA), 5-hydroxy tryptamine (5-HT), γ-aminobutyric acid (GABA), noradrenaline (NE) and acetylcholine (ACh) in the cell culture medium measured using an LC-MS/MS method.
RESULTS: Pretreatment of PC12 cells with the extract and phenolic fractions of daylily flowers at concentrations ranging from 0.63 to 5 mg raw material/mL significantly reversed corticosterone- and glutamate-induced neurotoxicity in a dose-dependent manner. The fractions containing phenolic acid derivatives (0.59% w/w in the flowers) and/or flavonoids (0.60% w/w) exerted similar dose-dependent neuroprotective effect whereas the fractions with non-phenolic compounds exhibited no activity. The presence of phenolic acid derivatives in the corticosterone- and glutamate-treated PC12 cells elevated the DA level in the cell culture medium whereas flavonoids resulted in increased ACH and 5-HT levels.
CONCLUSIONS: Phenolic acid derivatives and flavonoids were likely the active constituents of daylily flowers and they conferred a similar extent of neuroprotection, but affected the release of neurotransmitters in a different manner.

BACKGROUND: Depression induce distressed emotional state and cognitive deficits simultaneously, which both should be improved in the treatment. Hemerocallis citrina Baroni (HC) is a traditional herbal medicine in Eastern-Asia areas and the total phenols extract of HC (HCPE) contains the main active ingredients. It has been reported that HC has the emotional improvement effect. But the cognitive effect of HC was seldom researched.
OBJECTIVE: We designed to evaluate the antidepressant and cognitive improvement effect of HCPE using a chronic unpredictable mild stress (CUMS) model, and the potential mechanisms were explored by investigating the corticosterone (CORT), monoamine neurotansmitters, brain-derived neurotropic factor (BDNF) and oxidative stress.
METHODS: The depression rats were induced by CUMS procedures and treated with HCPE (10, 20, 40mg/kg/day, by gastric gavage). The antidepressant effect was evaluated by sucrose preference test, open field test and body weight, while the cognitive improvement was investigated using morris water maze test. Besides, the levels of monoamine neurotransmitters in the hippocampus and frontal cortex were measured by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The serum CORT and BDNF in hippocampus were test using enzyme-linked immunosorbent assay (ELISA) kits. The oxidative stress indicators in frontal cortex were also analyzed.
RESULTS: HCPE (40mg/kg) improved the emotion and cognition related behaviors in depression effectively. Moreover, HCPE increased the neurotransmitters concentration (5-HT, DA and NE) in the hippocampus and frontal cortex compared with CUMS rats. Meanwhile, the CUMS induced changes of serum corticosterone level and the hippocampus BDNF level were reversed. Besides, HCPE reduced malondialdehyde (MDA) in the frontal cortex of model rats.
CONCLUSIONS: It suggested that HCPE could improve the depression-like emotional status and associated cognitive deficits in CUMS rats, which might be mediated by regulation of neurotransmitters and BDNF levels in brain, alleviation of corticosterone level as well as the alleviation of oxidative stress.

A previous study found that the antidepressant-like effects of ethanolic extracts from Hemerocallis citrina are predominantly related to the flavonoid, hesperidin. The study herein aimed to explore the antidepressant-like mechanism of hesperidin in mice induced by chronic mild stress (CMS). The results indicated that hesperidin reversed the reduction of sucrose preference and the elevation of immobility time in mice induced by CMS. In addition, the increase in serum corticosterone levels and decrease in hippocampal extracellular signal-regulated kinase (ERK) phosphorylation and brain-derived neurotrophic factor (BDNF) levels in CMS mice were also ameliorated by hesperidin treatment. In contrast, improvement by hesperidin was suppressed by pretreatment with ERK inhibitor SL327. Taken together, our findings confirmed the antidepressant-like effect of hesperidin and indicated that hesperidin-induced BDNF up-regulation was mediated in an ERK-dependent manner.