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.

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.

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.

Zucchini (Cucurbita pepo L.) is one of the main vegetable crops grown under protected cultivation in northern China. Low-temperature (LT) stress severely inhibits the growth of zucchini seedlings, resulting in reductions in yield and quality. Here, using three kinds of different humic acids, including coal-based humic acid (CHA), fulvic acid (FA), and biochemical humic acid (BHA), we investigated the effects of humic acids against LT stress (5 °C) in zucchini seedlings. Treatment with all three kinds of humic acids improves LT stress tolerance by decreasing oxidative damage through increases in antioxidative enzyme activities and the contents of soluble sugar and proline in zucchini seedlings, especially after BHA application. Comparative transcriptomic analysis revealed that a total of 17 differentially expressed genes (DEGs) were commonly induced in the leaves of FA-, CHA-, and BHA-treated zucchini seedlings under LT stress, including calmodulin, ethylene-responsive transcription factors (TFs), peroxidases, and 10 TFs, including two NAC and seven WRKY genes. Altogether, these results indicated that supplementation with humic acids reprograms plant metabolism and modulates the expression of genes involved in ROS scavenging, phytohormone metabolism, or signaling pathways, finally improving LT stress tolerance in zucchini seedlings.

Background: Signal peptides are essential for plant growth and development. In plants, biological processes including cell-cell communication, cellular proliferation and differentiation, cellular determination of self-incompatibility, and defensive responses, all depend heavily on peptide-signaling networks such as CLE (CLAVATA3/Embryo surrounding region-related). The CLEs are indispensable in different periods of plant growth and development, especially in maintaining the balance between proliferation and differentiation of stem cells in various meristematic tissues. The working system of CLE genes in cucumber, an important economical vegetable (Cucumis sativus L.), has not been fully studied yet. The distributional patterns of chromosome-level genome assembly in cucumber provide a fundamental basis for a genome-wide comparative analysis of CLE genes in such plants. Results: A total of 26 individual CLE genes were identified in Chinese long ‘9930’ cucumber, the majority of which belong to unstable short alkaline and hydrophilic peptides. A comparative analysis showed a close relationship in the development of CLE genes among Arabidopsis thaliana, melon, and cucumber. Half of the exon-intron structures of all CsCLEs genes are single-exon genes, and motif 1, a typical CLE domain near the C-terminal functioning in signal pathways, is found in all cucumber CLE proteins but CsCLE9. The analysis of CREs (Cis-Regulatory Elements) in the upstream region of the 26 cucumber CLE genes indicates a possible relationship between CsCLE genes and certain functions of hormone response elements. Cucumber resulted closely related to Arabidopsis and melon, having seven and 15 orthologous CLE genes in Arabidopsis and melon, respectively. Additionally, the calculative analysis of a pair of orthologous genes in cucumber showed that as a part of the evolutionary process, CLE genes are undergoing a positive selection process which leads to functional differentiation. The specific expression of these genes was vigorous at the growth and development period and tissues. Cucumber gene CLV3 was overexpressed in Arabidopsis, more than half of the transformed plants in T1 generation showed the phenomena of obvious weakness of the development of growing point, no bolting, and a decreased ability of plant growth. Only two bolted strains showed that either the pod did not develop or the pod was short, and its development was significantly inferior to that in the wild type. Conclusions: In this study, 26 CLE genes were identified in Chinese long ‘9930’ cucumber genome. The CLE genes were mainly composed of alkaline hydrophilic unstable proteins. The genes of the CLE family were divided into seven classes, and shared close relationships with their homologs in Arabidopsis and melon. The specific expression of these genes was evaluated in different periods of growth and tissue development, and CLV3, which the representative gene of the family, was overexpressed in Arabidopsis, suggesting that it has a role in bolting and fruit bearing in cucumber.

Carbon dioxide (CO₂) is very important for photosynthesis of green plants. CO₂ concentration in the atmosphere is relatively stable, but it drops sharply after sunrise due to the tightness of the greenhouse and the absorption of CO₂ by vegetable crops. Vegetables in greenhouses are chronically CO₂ starved. To investigate the feasibility of using genetic engineering to improve the photosynthesis and yield of greenhouse cucumber in a low CO₂ environment, five genes encoding glyoxylate carboligase (GCL), tartronic semialdehyde reductase (TSR), and glycolate dehydrogenase (GlcDH) in the glycolate catabolic pathway of Escherichia coli were partially or completely introduced into cucumber chloroplast. Both partial pathway by introducing GlcDH and full pathway expressing lines exhibited higher photosynthetic efficiency and biomass yield than wild-type (WT) controls in low CO₂ environments. Expression of partial pathway by introducing GlcDH increased net photosynthesis by 14.9% and biomass yield by 44.9%, whereas the expression of the full pathway increased seed yield by 33.4% and biomass yield by 59.0%. Photosynthesis, fluorescence parameters, and enzymatic measurements confirmed that the introduction of glycolate catabolic pathway increased the activity of photosynthetic carbon assimilation-related enzymes and reduced the activity of photorespiration-related enzymes in cucumber, thereby promoting the operation of Calvin cycle and resulting in higher net photosynthetic rate even in low CO₂ environments. This increase shows an improvement in the efficiency of the operation of the photosynthetic loop. However, the utilization of cucumber of low concentration CO₂ was not alleviated. This study demonstrated the feasibility of introducing the pathway of exogenous glycolate catabolic pathway to improve the photosynthetic and bio-yield of cucumber in a low CO₂ environment. These findings are of great significance for high photosynthetic efficiency breeding of greenhouse cucumber.