Widely-targeted metabolomics and transcriptomics identify metabolites associated with flowering regulation of Choy Sum.

Choy Sum, a stalk vegetable highly valued in East and Southeast Asia, is characterized by its rich flavor and nutritional profile. Metabolite accumulation is a key factor in Choy Sum stalk development; however, no research has focused on metabolic changes during the development of Choy Sum, especially in shoot tip metabolites, and their effects on growth and flowering. Therefore, in the present study, we used a widely targeted metabolomic approach to analyze metabolites in Choy Sum stalks at the seedling (S1), bolting (S3), and flowering (S5) stages. In total, we identified 493 metabolites in 31 chemical categories across all three developmental stages. We found that the levels of most carbohydrates and amino acids increased during stalk development and peaked at S5. Moreover, the accumulation of amino acids and their metabolites was closely related to G6P, whereas the expression of flowering genes was closely related to the content of T6P, which may promote flowering by upregulating the expressions of BcSOC1, BcAP1, and BcSPL5. The results of this study contribute to our understanding of the relationship between the accumulation of stem tip substances during development and flowering and of the regulatory mechanisms of stalk development in Choy Sum and other related species.

Natural Albino Mutant of Daylily (Hemerocallis spp.) Reveals a Link between Drought Sensitivity and Photosynthetic Pigments Metabolism.

BACKGROUND: Mutant analysis remains one of the main genetic tools for characterising unclarified gene functions in plants, especially in non-model plants. Daylily (Hemerocallis spp.) is a popular perennial ornamental plant grown worldwide. Analysis of daylily mutants can enhance understanding of genes regulating the albino phenotype and improve the cultivar quality of daylily. METHODS: The natural albino mutant (Alb-⁣/-) was isolated by screening a self-pollinated progeny of daylily cultivar 'black-eyed stella'. Transmission electron microscopy was used in analysing the structure of plastids between mutant and wild-type seedlings. The content of chlorophyll, carotenoids and chlorophyll precursors in plants was measured by ultraviolet spectrophotometry. RNA sequencing and physiological measurements were performed to explore the association between drought tolerance and mutation. RESULTS: All the seedlings of the daylily albino mutants died spontaneously within fifteen days after germination when grown in soil. The carotenoid and chlorophyll content in the leaves of the mutant plants significantly decreased compared with those of the wild-type control. The mutant plants displayed stunted growth, and their leaves were white or light yellow in color. Abnormal plastids such as those showing endomembrane vesiculation and lacking stacking were discovered in the leaves of mutant plants. Furthermore, genetic analysis revealed that a single recessive nuclear gene mutation led to the albino trait, RNA sequencing and real-time quantitative PCR validation showed extensive differences in gene expression between the mutant plants and the wild-type control, and most of the genes related to chlorophyll metabolism were down-regulated, with foldchange ranging from 0.20-0.49. Additionally, the surviving homozygous plants (Alb+⁣/+), which do not contain this mutation, were also isolated by analysing the phenotype of their self-pollinated progeny. The net photosynthesis rate and light saturation point of Alb+⁣/+ were higher than those of heterozygous (Alb+⁣/-) plants. Additionally, the Alb+⁣/+ plants were more tolerant to drought conditions than the Alb+⁣/- plants, suggesting that a heterozygous Alb- mutation is sufficient to negatively affect photosynthetic efficiency and drought tolerance. CONCLUSIONS: The albino mutation negatively affects photosynthetic efficiency and drought tolerance, and homozygous mutation is required for the characteristic albino phenotype. This work highlights the link between albino mutation, photosynthetic pigment metabolism and drought sensitivity in daylily.

Hemerocallis citrina Baroni ameliorates chronic sleep deprivation-induced cognitive deficits and depressive-like behaviours in mice.

Sleep deprivation (SD) is common during spaceflight. SD is known to cause cognitive deficits and depression, requiring treatment and prevention. Hemerocallis citrina Baroni (Liliaceae) is a perennial herb with antidepressant, antioxidant, antitumor, anti-inflammatory, and neuroprotective effects.The aim of our study was to investigate the effects of H. citrina extract (HCE) on SD-induced cognitive decline and depression-like behavior and possible neuroinflammation-related mechanisms. HCE (2 g/kg/day, i.g.) or vortioxetine (10 mg/kg/day, i.g.) were given to mice by oral gavage for a total of 28 days during the SD process. HCE treatment was found to ameliorate SD-induced impairment of short- and long-term spatial and nonspatial memory, measured using Y-maze, object recognition, and Morris water maze tests, as well as mitigating SD-induced depression-like behaviors, measured by tail suspension and forced swimming tests. HCE also reduced the levels of inflammatory cytokines (IL-1ß, IL-18, and IL-6) in the serum and hippocampus. Furthermore, HCE suppressed SD-induced microglial activation in the prefrontal cortex (PFC) and the CA1 and dentate gyrus (DG) regions of the hippocampus. HCE also inhibited the expression of phosphorylated NF-κB and activation of the NLRP3 inflammasome. In summary, our findings indicated that HCE attenuated SD-induced cognitive impairment and depression-like behavior and that this effect may be mediated by the inhibition of inflammatory progression and microglial activation in the hippocampus, as well as the down-regulation of NF-κB and NLRP3 signaling. The findings of these studies showingTthese results indicate that HCE exerts neuroprotective effects and are consistent with the findings of previous studies, suggesting that HCE is beneficial for the prevention and treatment of cognitive decline and depression in SD.

Freeze-dried powder of daylily bud improves bromocriptine-induced lactation disorder in rats via JAK2/STAT5 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Milk deficiency is a prevalent problem in the world. Daylily (Hemerocallis citrina Borani), called the Chinese mother flower, is a traditional vegetable and is believed to possess a galactagogue effect in China. Flavonoids and phenols are considered as the active ingredients of daylily to promote lactation and improve depression. AIM OF THE STUDY: The aim of this study was to investigate the prolactin effects of freeze-dried powder of flower buds of H. citrina Baroni in rat and its action mechanisms. MATERIALS AND METHODS: The chemical constituents of flower buds of H. citrina Baroni treated by different drying techniques were analyzed by ultrahigh pressure liquid chromatography-mass spectrometry. Sprague-Dawley (SD) rat model induced by bromocriptine was used to evaluate the effect of freeze-dried powder of daylily buds on promoting lactation. Network pharmacology method, ELISA, qPCR, and Western blot were used to clarify the action mechanisms. RESULTS: We detected 657 compounds in daylily buds. The relative contents of total flavonoids and phenols in freeze-dried samples were higher than those in dried ones. Bromocriptine, as a dopamine receptor agonist, can significantly inhibit prolactin in rats. Daylily buds can restore the levels of prolactin, progesterone and estradiol depressed by bromocriptine, effectively improve the milk production of the rat, and promote the repair of rat mammary gland tissue. We analyzed the relationship between the chemical components of daylily buds and the genes related to lactation with network pharmacology method, revealing that flavonoids and phenols may be the active components that promoted milk production via JAK2/STAT5 pathway, which was confirmed by the results of qPCR and Western blot. Daylily buds can increase the mRNA expression of PRLR, CSN2, LALBA and FASN and the protein expression of PRLR, JAK2 and STAT5. CONCLUSION: Daylily buds can improve the insufficient lactation of rats induced by bromocriptine through PRLR/JAK2/STAT5 pathway, and the freeze-dried processing method may better retain the active components of flavonoids and phenols that promote milk in daylily.

Comparing the contents, functions and neonicotinoid take-up between floral and extrafloral nectar within a single species (Hemerocallis citrina Baroni).

BACKGROUND AND AIMS: Many angiosperms can secrete both floral (FN) and extrafloral (EFN) nectar. However, much remains unclear about how EFN and FN differ in secretion, composition and ecological function, especially when both FN and EFN are secreted on flowers of the same species. METHODS: Hemerocallis citrina flowers secrete both FN and EFN. The FN and EFN traits including volume, presentation pattern and temporal rhythms of secretion were compared by field observation. Sugar and amino acid contents were analysed using regular biochemical methods, whereas the proteome was investigated by combined gel-based and gel-free approaches. Animal feeders on FN and EFN were investigated by field observation. Hemerocallis citrina plants were exposed by soil drenching to two systemic insecticides, acetamiprid and imidacloprid, and the concentration of these in FN and EFN was measured by ultra-high performance liquid chromatography coupled with mass spectrometry. KEY RESULTS: Hemerocallis citrina FN was concentrated and sucrose dominant, secreted in the mature flower tube and served as a reward for pollinators. Conversely, EFN was hexose rich, more dilute and less rich in sugar and amino acids. EFN was secreted on the outside of developing floral buds, and was likely to attract predatory animals for defence. EFN had fewer phenolics, but more pathogenesis-related components, such as chitinase and glucanase. A significantly different proteomic profile and enzymatic activities between FN and EFN suggest that they had different biosynthesis mechanisms. Both neonicotinoid insecticides examined became present in both nectar types soon after application, but in greater concentration within EFN; EFN also attracted a wider range of insect species than FN. CONCLUSIONS: Hemerocallis citrina FN and EFN differed in production, composition and ecological function. The EFN pathway could be a significant way for neonicotinoids to enter the wild food chain, and must be considered when evaluating the risks to the environment of other systemic insecticides.

Molecular Characterization and Expression Patterns of the HkSVP Gene Reveal Distinct Roles in Inflorescence Structure and Floral Organ Development in Hemerocallis fulva.

SHORT VEGETATIVE PHASE (SVP) genes are members of the well-known MADS-box gene family that play a key role in regulating vital developmental processes in plants. Hemerocallis are perennial herbs that exhibit continuous flowering development and have been extensively used in landscaping. However, there are few reports on the regulatory mechanism of flowering in Hemerocallis. To better understand the molecular basis of floral formation of Hemerocallis, we identified and characterized the SVP-like gene HkSVP from the Hemerocallis cultivar 'Kanai Sensei'. Quantitative RT-PCR (qRT-PCR) indicated that HkSVP transcript was mainly expressed in the vegetative growth stage and had the highest expression in leaves, low expression in petals, pedicels and fruits, and no expression in pistils. The HkSVP encoded protein was localized in the nucleus of Arabidopsis protoplasts and the nucleus of onion epidermal cells. Yeast two hybrid assay revealed that HKSVP interacted with Hemerocallis AP1 and TFL1. Moreover, overexpression of HkSVP in Arabidopsis resulted in delayed flowering and abnormal phenotypes, including enriched trichomes, increased basal inflorescence branches and inhibition of inflorescence formation. These observations suggest that the HkSVP gene may play an important role in maintaining vegetative growth by participating in the construction of inflorescence structure and the development of flower organs.

The numerical classification and grading standards of daylily (Hemerocallis) flower color.

This study collected 183 Hemerocallis varieties to conduct numerical classification of flower color and provide valuable baseline data and foundational theory for normalization and precision of Hemerocallis. The color CIELab phenotypes were collected via colorimeter (CR-10 Plus), which separately measured three sepal and petal parts (throat, eye and limb). The colors of experimental samples were artificially named by the Royal Horticultural Society Colour Chart (RHSCC). All the data were analyzed using R software. The results showed that the throat was predominantly green-yellow, light yellow and yellow; green-yellow accounted for the largest proportion of sepals (67.76%) and petals (69.40%). The eye was more abundant, and there were significant differences between sepals and petals. The limb was clustered into five color groups (orange, yellow, pink, red and purple); the yellow group had the most varieties for both sepals and petals, containing 57.38% and 55.74%, respectively. Both sepals and petals had significant differences (p<0.0001) in color (△E), redness (a*) and color angle (h) for the throat, eye and limb. However, the difference in CIELab phenotypes between the eye and limb were not significant. According to "Dual Classification", the color classification standard was proposed as a 3-level standard. The color of sepal and petal consistency served as the first standard, and the color of limb was the second standard. The color pattern types of pure, gradual change, watermark and eye spot, served as the third standard. It has been proposed that all the 183 experimental varieties were divided into two categories, five groups and finally four types. This study provides a classification basis and reference for numeric and standardized color phenotype description for Hemerocallis.

iTRAQ-based quantitative proteomic analysis reveals dynamic changes during daylily flower senescence.

MAIN CONCLUSION: Sugar-related metabolic biological processes and metabolic pathways as well as invertase, protease, and ribosomal proteins may be critical regulators controlling the circadian rhythm and ephemeral properties of daylily flowers. Daylily is a familiar perennial flower. The daylily flower opens at dawn and withers away at night. Flower longevity in almost all daylily varieties from opening to fading is less than 24 h. In the past decades, the physiological changes and genetic responses to senescence in daylily flowers have been reported. However, the main metabolic pathways and biological processes involved in daylily flower senescence and the proteins involved in premature senility of daylily flowers are poorly understood. Herein, we identified differences between the proteomes of four developmental stages (s1-s4) of daylily flowers using iTRAQ-based quantitative proteomic methods. A total of 445 proteins (containing at least two unique peptides) were identified, and differentially expressed proteins (upregulation ≥ 1.5 or downregulation ≤ 0.67, P value ≤ 0.05) were detected between these stages in the following numbers: 58 (s2/s1), 59 (s3/s1), 31 (s3/s2), 64 (s4/s1), 52 (s4/s2), and 29 (s4/s3). Protein functions and classifications were analyzed based on GO, KEGG, and COG, and expressive hierarchical cluster analysis and functional enrichment analysis for differentially expressed proteins were carried out. A comparison of the late stages (s3 and s4) with the early stage (s1) revealed that the sugar (hexose, monosaccharide, and glucose) metabolic process GO category was the most enriched, and sugar (galactose, pentose, starch, and sucrose) metabolism pathways constituted the most enriched KEGG category. Finally, the potential research value of invertase, protease, and ribosomal proteins for revealing the mechanism underlying the circadian rhythm and ephemeral properties of daylily flowers are discussed. These data and analyses provide new insight into the senescence mechanism of daylily flowers.

Methane delays the senescence and browning in daylily buds by re-established redox homeostasis.

BACKGROUND: During the postharvest senescence of fruits and vegetables, redox imbalance occurs. Although the release of methane (CH4 ) has been observed for a long time and its antioxidant properties have recently been demonstrated in animals and plants, the corresponding physiological role of CH4 in regulating plant senescence has not yet been elucidated. RESULTS: Our results indicate that the postharvest deterioration of daylily buds during storage is greatly ameliorated by the exogenous application of CH4 , particularly in the inhibition of tissue browning. The results are supported by a decrease in the degree of browning and by the corresponding phenotype, which are correlated with the suppressed polyphenoloxidase activity. CH4 also maintains the re-establishment of redox balance, as indicated by the lower relative leakage rate, lipid peroxidation level, and reactive oxygen species accumulation in daylily buds. Furthermore, the decrease of the unsaturated/saturated fatty acid ratio and energy charge during storage was also attenuated. CONCLUSION: These results clearly suggest that the postharvest treatment with CH4 is an effective means of prolonging the storage life of daylily buds. © 2017 Society of Chemical Industry.

Daylily protein constituents of the pollen and stigma a proteomics approach.

This study was aimed at the identification and quantification of the protein components of the pollen grains in parallel with the distal stigmatic tissue of tetraploid cultivars. Proteomes were analyzed using iTRAQ 4plex labeling, peptides separation by online RP-nano-LC and analysis by ESI-MS/MS. Protein identification and quantification were made using the Asparagales database as a reference. A total of 524,037 MS/MS spectra were produced from pollen and stigma samples. From these, a total of 8368 peptides wereidentified corresponding to 994 unique peptides and 432 protein groups. Among them, 128 differentially expressed proteins were retained for further analysis. In absence of the daylily genome availability, we exploited numerous databases and bioinformatics resources to exploring the putative biological functions of these proteins. The profile of differentially expressed proteins suggests an important representation of functions associated to the signalling and response against endogenous and environmental stresses, including several enzymes implicated in the biosynthesis of antibiotics. The abundance in stigma of several structural proteins of the ribosomal sub-units as well as of the core histones suggest that the translation processes and the regulation of gene expression in stigma is a more active mechanism than in pollen. In addition, pollen prioritizes the synthesis of fructose and glucose as opposed to sucrose in stigma as a source of energy. Finally, the modulated proteins in Hemerocallis point to several pathways that give potential clues concerning the molecular mechanisms underlying the functions of the pollen and the stigmatic fluid in daylily reproduction.

Low intensity ultrasound as a pretreatment to drying of daylilies: Impact on enzyme inactivation, color changes and nutrition quality parameters.

Effects of thermal and low intensity ultrasound combined with heat (LIUH) pretreatment prior to microwave vacuum drying on enzyme inactivation, color changes and nutrition quality properties of dried daylilies were investigated. The peroxidase (POD), ascorbic acid oxidase (AAO) and polyphenoloxidase (PPO) thermal and LIUH (0.2 and 0.4W/cm2) inactivation were determined and compared at 70, 80 and 90°C. Significant reduction in the POD, AAO and PPO activity was seen in daylilies after an ambient LIUH pretreatment than thermal pretreatment. POD, AAO and PPO thermal and LIUH inactivation followed the first order kinetics. LIUH pretreatment had a more positive influence on maintaining color of dried daylilies than thermal pretreatment. Furthermore, LIUH pretreatment resulted in a significant increase in chlorophylls, carotenoids (lutein, zeaxanthin and ß-carotene), and a decrease in degree of browning and 5-hydroxymethylfurfural (HMF) when compared with thermal pretreatment. The antioxidant activity and contents of several nutritional components of dried daylilies pretreated by LIUH were also higher than that of dried daylilies pretreated by thermal pretreatment. This study provides a basis for the design of LIUH conditions to control vegetables browning and color changes prior to drying processing.

Endogenous Hydrogen Sulfide Homeostasis Is Responsible for the Alleviation of Senescence of Postharvest Daylily Flower via Increasing Antioxidant Capacity and Maintained Energy Status.

There are limited data concerning the role of endogenous H2S in prolonging the postharvest of vegetables and fruits. Using a fluorescence microscope with a specific probe, we discovered that, during the senescence of postharvest daylily flower, endogenous H2S homeostasis was impaired. The activities of two important synthetic enzymes of H2S, l- and d-cysteine desulfhydrase, exhibited decreasing tendencies. However, NaHS (a H2S donor) not only blocked the decreased H2S production but also extended the postharvest life of daylilies. These beneficial roles were verified by the alleviation of lipid peroxidation and the increased activities of antioxidant enzymes. Meanwhile, the energy status was sustained, and the respiration rate was decreased. In contrast to NaHS, the addition of an inhibitor of H2S synthesis alone aggravated lipid peroxidation and lowered energy charge. Together, the present study implies that endogenous H2S alleviates senescence of postharvest daylilies via increasing antioxidant capacity and maintained energy status.

Signal regulation involved in sulfur dioxide-induced guard cell apoptosis in Hemerocallis fulva.

Chronic and acute exposure to SO2 is associated with increased risks of various damages to plants. In the present study, epidermal strip experiment was employed to investigate SO2-induced guard cells apoptosis and the signal regulation in Hemerocallis fulva. The results showed that with the increase of treatment concentrate of SO2 derivates (a mixture of sodium sulfite and sodium bisulfite, 3:1, mmol L⁻¹/mmol L⁻¹, 1.0-5.0 mmol L⁻¹), the physiological activity of the guard cells declined and cell death occurred. While the concentration of SO2 derivatives exceeded 2.0 mmol L⁻¹, the percentage of cell death increased significantly (P<0.05). Typical features of apoptosis including nuclear condensation, nuclear elongation, fragmentation etc. were found. Meanwhile, concomitant presence of nitric oxide (NO), reactive oxygen species (ROS) and Ca²âº level increment appeared. However, SO2-induced cell death can be effectively blocked by either of the following substances with their respective optimal concentrations: antioxidant ascorbic acid (Asc; 0.05 mmol L⁻¹) or catalase (CAT; 200 U mL⁻¹), nitric oxide (NO) scavenger 2-(4-carboxyphenyl)-4, 4, 5, 5- tetramethylmidiazoline-1-oxyl-3-oxide (c-PTIO; 0.20 mmol L⁻¹), nitrate reductase inhibitor NaN3 (0.20 mmol L⁻¹), Ca²âº chelating agent EGTA (0.05 mmol L⁻¹) or plasma membrane Ca²âº channel blocker LaCl3 (0.05 mmol L⁻¹). In addition to a significant decrease in cell death rate, a reduction in the levels of reactive oxygen species (ROS), NO and Ca²âº was observed. Further study showed that compared to treatment with SO2 alone, Asc treatment led to a decrease in NO and Ca²âº levels and NaN3 treatment led to a decrease in ROS and Ca²âº levels, but the NO and ROS levels of the LaCl3 treatment changed little. All results suggested that NO, ROS and Ca²âº were involved in the apoptosis induced by SO2 in H. fulva. The process might be related to the burst of NO or ROS, which would activate the plasma Ca²âº channel and result in the increase of intercellular Ca²âº.