Lipidomic and transcriptomic profiles of glycerophospholipid metabolism during Hemerocallis citrina Baroni flowering.

BACKGROUND: Hemerocallis citrina Baroni (daylily) is a horticultural ornamental plant and vegetable with various applications as a raw material in traditional Chinese medicine and as a flavouring agent. Daylily contains many functional substances and is rich in lecithin, which is mostly composed of glycerophospholipids. To study the comprehensive dynamic changes in glycerophospholipid during daylily flowering and the underlying signalling mechanisms, we performed comprehensive, time-resolved lipidomic and transcriptomic analyses of 'Datong Huanghua 6' daylily. RESULTS: Labelling with PKH67 fluorescent antibodies clearly and effectively helped visualise lipid changes in daylily, while relative conductivity and malonaldehyde content detection revealed that the early stages of flowering were controllable processes; however, differences became non-significant after 18 h, indicating cellular damage. In addition, phospholipase D (PLD) and lipoxygenase (LOX) activities increased throughout the flowering process, suggesting that lipid hydrolysis and oxidation had intensified. Lipidomics identified 558 lipids that changed during flowering, with the most different lipids found 12 h before and 12 h after flowering. Transcriptome analysis identified 13 key functional genes and enzymes in the glycerophospholipid metabolic pathway. The two-way orthogonal partial least squares analysis showed that diacylglycerol diphosphate phosphatase correlated strongly and positively with phosphatidic acid (PA)(22:0/18:2), PA(34:2), PA(34:4), and diacylglycerol(18:2/21:0) but negatively with phospholipase C. In addition, ethanolamine phosphotransferase gene and phospholipid-N-methyltransferase gene correlated positively with phosphatidylethanolamine (PE)(16:0/18:2), PE(16:0/18:3), PE(33:2), and lysophosphatidylcholine (16:0) but negatively with PE(34:1). CONCLUSIONS: Overall, this study elucidated changes in the glycerophospholipid metabolism pathway during the daylily flowering process, as well as characteristic genes, thus providing a basis for future studies of glycerophospholipids and signal transduction in daylilies.

The Transcriptome and Metabolome Reveal Stress Responses in Sulfur-Fumigated Cucumber (Cucumis sativus L.).

Sulfur (S) fumigation is a commonly used sterilization method in horticultural facilities against fungal diseases. S fumigation damaged cucumber leaves, although the response mechanism is unclear. This study analyzes the growth, transcriptome, and metabolomic profiles of young and mature leaves, ovaries, and commercial cucumber fruits to decipher the mechanism of cucumber stress response under S fumigation. S fumigation significantly changed the photosynthetic efficiency and reactive oxygen species (ROS) in leaves, but not fruit development, fruit mass, and peel color. Transcriptome analysis indicated that S fumigation strongly regulated stress defense genes. The weighted gene co-expression network analysis revealed that S fumigation regulated ASPG1, AMC1 defense genes, LECRK3, and PERK1 protein kinase. The abscisic acid (ABA)-mediated model of regulation under S fumigation was constructed. Metabolome analysis showed that S fumigation significantly upregulated or downregulated the contents of amino acids, organic acids, sugars, glycosides, and lipids (VIP > 1 and P-value < 0.05). The opposite Pearson's correlations of these differential metabolites implied that cucumber had different metabolic patterns in short-term and long-term S fumigation. Besides, the elevated levels of proline and triglyceride indicated that stress-responsive mechanisms existed in S-fumigated cucumber. Moreover, the comprehensive analysis indicated that S fumigation elevated secondary S-containing metabolites but decreased sulfate absorption and transportation in cucumber. Overall, our results provided a comprehensive assessment of S fumigation on cucumber, which laid the theoretical foundation for S fumigation in protected cultivation.

Qualitative and Quantitative Analysis on Flavonoid Distribution in Different Floral Parts of 42 Hemerocallis Accessions.

The Hemerocallis accessions is widely consumed as nutritious vegetable and traditional medicine in eastern Asia and used as an ornamental flower worldwide. Compared with most other horticultural products, its flower is richer in polyphenols, flavonoids, carotenoids, and anthocyanins. Therefore, the flower has strong antioxidant activity that inhibits cancer cell proliferation, which could used for health and pharmaceutical purposes. The flavonoids composition and distribution in the flowers, and the content varied between different accssions is still unclear. In this context, eight flavonols, two flavones, and two anthocyanins were determined in Hemerocallis flower by high-performance liquid chromatography (HPLC) coupled with photodiode array and mass spectrometric detectors. Rutin was the most abundant flavonols and cyanidin 3,5-glucoside and cyanidin 3-rutinoside were the major anthocyanins in Hemerocallis tepals, resulting in flower petal coloration, and their content in the petal was higher than that of the sepal. Hierarchical cluster analysis grouped the 42 accessions into four groups, and they were significantly different (p < 0.05) from each other in the ten significant compounds by One-way ANOVA. Overall, the qualitative and quantitative analysis of flavonoid constituents in six floral parts of 42 Hemerocallis accessions were elucidated, which could be helpful for the food and pharmaceutical industries, and lay the foundation for the Hemerocallis flower color research.

Characterization of Hemerocallis citrina Transcriptome and Development of EST-SSR Markers for Evaluation of Genetic Diversity and Population Structure of Hemerocallis Collection.

Hemerocallis spp. commonly known as daylilies and night lilies, are among the most popular ornamental crops worldwide. In Eastern Asia, H. citrina is also widely cultivated as both a vegetable crop and for medicinal use. However, limited genetic and genomic resources are available in Hemerocallis. Knowledge on the genetic diversity and population structure of this species-rich genus is very limited. In this study, we reported transcriptome sequencing of H. citrina cv. 'Datonghuanghua' which is a popular, high-yielding variety in China. We mined the transcriptome data, identified and characterized the microsatellite or simple sequence repeat (SSR) sequences in the expressed genome. From ∼14.15 Gbp clean reads, we assembled 92,107 unigenes, of which 41,796 were annotated for possible functions. From 41,796 unigenes, we identified and characterized 3,430 SSRs with varying motifs. Forty-three SSRs were used to fingerprint 155 Hemerocallis accessions. Clustering and population structure analyses with the genotypic data among the 155 accessions reveal broader genetic variation of daylilies than the night lily accessions which form a subgroup in the phylogenetic tree. The night lily group included accessions from H. citrina, H. lilioasphodelus, and H. minor, the majority of which bloom in the evening/night, whereas the ∼100 daylily accessions bloomed in the early morning suggesting flowering time may be a major force in the selection of night lily. The utility of these SSRs was further exemplified in association analysis of blooming time among these accessions. Twelve SSRs were found to have significant associations with this horticulturally important trait.

Identification and validation of reference genes for quantitative real-time PCR studies in long yellow daylily, Hemerocallis citrina Borani.

Gene expression analysis using reverse transcription quantitative real-time PCR (RT-qPCR) requires the use of reference gene(s) in the target species. The long yellow daylily, Hemerocallis citrina Baroni. is rich in beneficial secondary metabolites and is considered as a functional vegetable. It is widely cultivated and consumed in East Asian countries. However, reference genes for use in RT-qPCR in H. citrina are not available. In the present study, six potential reference genes, actin (ACT), AP-4 complex subunit (AP4), tubulin (TUB), ubiquitin (UBQ), 18S and 60S ribosomal RNA, were selected and their expression stability in different developmental stages, organs and accessions was evaluated using four statistical software packages (geNorm, NormFinder, BestKeeper, and RefFinder). For commercial flower buds of different landraces, the combination of 60S, TUB, and AP4 was appropriate whereas ACT and 60S was suitable for normalization of different organs. In addition, AP4 exhibited the most stable expression in flower buds among different developmental stages. UBQ was less stable than the other reference genes under the experimental conditions except under different organs was 18S. The relative expression levels of two genes, primary-amine oxidase (HcAOC3) and tyrosine aminotransferase (HcTAT) which play important roles in alkaloid biosynthesis were also examined in different organs of the 'Datong' landrace, which further confirmed the results of selected reference genes. This is the first report to evaluate the stability of reference genes in the long yellow daylily that can serve as a foundation for RT-qPCR analysis of gene expression in this species.