Transcriptomic analysis of hub genes regulating albinism in light- and temperature-sensitive albino tea cultivars 'Zhonghuang 1' and 'Zhonghuang 2'.

Albino tea cultivars have high economic value because their young leaves contain enhanced free amino acids that improve the quality and properties of tea. Zhonghuang 1 (ZH1) and Zhonghuang 2 (ZH2) are two such cultivars widely planted in China; however, the environmental factors and molecular mechanisms regulating their yellow-leaf phenotype remain unclear. In this study, we demonstrated that both ZH1 and ZH2 are light- and temperature-sensitive. Under natural sunlight and low-temperature conditions, their young shoots were yellow with decreased chlorophyll and an abnormal chloroplast ultrastructure. Conversely, young shoots were green with increased chlorophyll and a normal chloroplast ultrastructure under shading and high-temperature conditions. RNA-seq analysis was performed for high light and low light conditions, and pairwise comparisons identified genes exhibiting different light responses between albino and green-leaf cultivars, including transcription factors, cytochrome P450 genes, and heat shock proteins. Weighted gene coexpression network analyses of RNA-seq data identified the modules related to chlorophyll differences between cultivars. Genes involved in chloroplast biogenesis and development, light signaling, and JA biosynthesis and signaling were typically downregulated in albino cultivars, accompanied by a decrease in JA-ILE content in ZH2 during the albino period. Furthermore, we identified the hub genes that may regulate the yellow-leaf phenotype of ZH1 and ZH2, including CsGDC1, CsALB4, CsGUN4, and a TPR gene (TEA010575.1), which were related to chloroplast biogenesis. This study provides new insights into the molecular mechanisms underlying leaf color formation in albino tea cultivars.

CsLHY positively regulates cold tolerance by activating CsSWEET17 in tea plants.

Low temperature is one of the most important environmental factors limiting tea plants' geographic distribution and severely affects spring tea's yield and quality. Circadian components contribute to plant responses to low temperatures; however, comparatively little is known about these components in tea plants. In this study, we identified a core clock component the LATE ELONGATED HYPOCOTYL, CsLHY, which is mainly expressed in tea plants' mature leaves, flowers, and roots. Notably, CsLHY maintained its circadian rhythmicity of expression in summer, but was disrupted in winter and held a high expression level. Meanwhile, we found that CsLHY expression rhythm was not affected by different photoperiods but was quickly broken by cold, and the low temperature induced and kept CsLHY expression at a relatively high level. Yeast one-hybrid and dual-luciferase assays confirmed that CsLHY can bind to the promoter of Sugars Will Eventually be Exported Transporters 17 (CsSWEET17) and function as a transcriptional activator. Furthermore, suppression of CsLHY expression in tea leaves not only reduced CsSWEET17 expression but also impaired the freezing tolerance of leaves compared to the control. Our results demonstrate that CsLHY plays a positive role in the low-temperature response of tea plants by regulating CsSWEET17 when considered together.

EGCG, GCG, TFDG, or TSA Inhibiting Melanin Synthesis by Downregulating MC1R Expression.

Without affecting cell viability, epigallocatechin gallate (EGCG), gallocatechin gallate (GCG), theaflavine-3,3'-digallate (TFDG), or theasinensin A (TSA) have been found to effectively reduce intracellular melanin content and tyrosinase (TYR) activity. However, studies on the anti-melanogenic mechanism of the above samples remain weak, and the activities of these samples in regulating melanogenesis at the molecular level lack comparison. Using B16F10 cells with the α-melanocyte-stimulating hormone (α-MSH) stimulation and without the α-MSH stimulation as models, the effects of EGCG, GCG, TFDG, or TSA on cell phenotypes and expression of key targets related to melanogenesis were studied. The results showed that α-MSH always promoted melanogenesis with or without adding the four samples. Meanwhile, the anti-melanogenic activities of the four samples were not affected by whether the α-MSH was added in the medium or not and the added time of the α-MSH. On this basis, the 100 µg/mL EGCG, GCG, TFDG, or TSA did not affect the TYR catalytic activity but inhibited melanin formation partly through downregulating the melanocortin 1 receptor (MC1R), microphthalmia-associated transcription factor (MITF), and the TYR family. The downregulation abilities of catechins on the TYR family and MITF expression were stronger than those of dimers at both the transcription and translation levels, while the ability of dimers to downregulate the MC1R expression was stronger than that of catechins at both the transcription and translation levels to some extent. The results of molecular docking showed that these four samples could stably bind to MC1R protein. Taken together, this study offered molecular mechanisms for the anti-melanogenic activity of the EGCG, GCG, TFDG, and TSA, as potential effective components against the UV-induced tanning reactions, and a key target (MC1R) was identified.

Transcriptome Analysis Reveals That Ascorbic Acid Treatment Enhances the Cold Tolerance of Tea Plants through Cell Wall Remodeling.

Cold stress is a major environmental factor that adversely affects the growth and productivity of tea plants. Upon cold stress, tea plants accumulate multiple metabolites, including ascorbic acid. However, the role of ascorbic acid in the cold stress response of tea plants is not well understood. Here, we report that exogenous ascorbic acid treatment improves the cold tolerance of tea plants. We show that ascorbic acid treatment reduces lipid peroxidation and increases the Fv/Fm of tea plants under cold stress. Transcriptome analysis indicates that ascorbic acid treatment down-regulates the expression of ascorbic acid biosynthesis genes and ROS-scavenging-related genes, while modulating the expression of cell wall remodeling-related genes. Our findings suggest that ascorbic acid treatment negatively regulates the ROS-scavenging system to maintain ROS homeostasis in the cold stress response of tea plants and that ascorbic acid's protective role in minimizing the harmful effects of cold stress on tea plants may occur through cell wall remodeling. Ascorbic acid can be used as a potential agent to increase the cold tolerance of tea plants with no pesticide residual concerns in tea.

CsCIPK11-Regulated Metalloprotease CsFtsH5 Mediates the Cold Response of Tea Plants.

Photosystem II repair in chloroplasts is a critical process involved in maintaining a plant's photosynthetic activity under cold stress. FtsH (filamentation temperature-sensitive H) is an essential metalloprotease that is required for chloroplast photosystem II repair. However, the role of FtsH in tea plants and its regulatory mechanism under cold stress remains elusive. In this study, we cloned a FtsH homolog gene in tea plants, named CsFtsH5, and found that CsFtsH5 was located in the chloroplast and cytomembrane. RT-qPCR showed that the expression of CsFtsH5 was increased with leaf maturity and was significantly induced by light and cold stress. Transient knockdown CsFtsH5 expression in tea leaves using antisense oligonucleotides resulted in hypersensitivity to cold stress, along with higher relative electrolyte leakage and lower Fv/Fm values. To investigate the molecular mechanism underlying CsFtsH5 involvement in the cold stress, we focused on the calcineurin B-like-interacting protein kinase 11 (CsCIPK11), which had a tissue expression pattern similar to that of CsFtsH5 and was also upregulated by light and cold stress. Yeast two-hybrid and dual luciferase (Luc) complementation assays revealed that CsFtsH5 interacted with CsCIPK11. Furthermore, the Dual-Luc assay showed that CsCIPK11-CsFtsH5 interaction might enhance CsFtsH5 stability. Altogether, our study demonstrates that CsFtsH5 is associated with CsCIPK11 and plays a positive role in maintaining the photosynthetic activity of tea plants in response to low temperatures.

A Tea Plant (Camellia sinensis) FLOWERING LOCUS C-like Gene, CsFLC1, Is Correlated to Bud Dormancy and Triggers Early Flowering in Arabidopsis.

Flowering and bud dormancy are crucial stages in the life cycle of perennial angiosperms in temperate climates. MADS-box family genes are involved in many plant growth and development processes. Here, we identified three MADS-box genes in tea plant belonging to the FLOWERING LOCUS C (CsFLC) family. We monitored CsFLC1 transcription throughout the year and found that CsFLC1 was expressed at a higher level during the winter bud dormancy and flowering phases. To clarify the function of CsFLC1, we developed transgenic Arabidopsis thaliana plants heterologously expressing 35S::CsFLC1. These lines bolted and bloomed earlier than the WT (Col-0), and the seed germination rate was inversely proportional to the increased CsFLC1 expression level. The RNA-seq of 35S::CsFLC1 transgenic Arabidopsis showed that many genes responding to ageing, flower development and leaf senescence were affected, and phytohormone-related pathways were especially enriched. According to the results of hormone content detection and RNA transcript level analysis, CsFLC1 controls flowering time possibly by regulating SOC1, AGL42, SEP3 and AP3 and hormone signaling, accumulation and metabolism. This is the first time a study has identified FLC-like genes and characterized CsFLC1 in tea plant. Our results suggest that CsFLC1 might play dual roles in flowering and winter bud dormancy and provide new insight into the molecular mechanisms of FLC in tea plants as well as other plant species.

Genome-wide identification, characterization, and expression analysis of tea plant autophagy-related genes (CsARGs) demonstrates that they play diverse roles during development and under abiotic stress.

BACKGROUND: Autophagy, meaning 'self-eating', is required for the degradation and recycling of cytoplasmic constituents under stressful and non-stressful conditions, which helps to maintain cellular homeostasis and delay aging and longevity in eukaryotes. To date, the functions of autophagy have been heavily studied in yeast, mammals and model plants, but few studies have focused on economically important crops, especially tea plants (Camellia sinensis). The roles played by autophagy in coping with various environmental stimuli have not been fully elucidated to date. Therefore, investigating the functions of autophagy-related genes in tea plants may help to elucidate the mechanism governing autophagy in response to stresses in woody plants. RESULTS: In this study, we identified 35 C. sinensis autophagy-related genes (CsARGs). Each CsARG is highly conserved with its homologues from other plant species, except for CsATG14. Tissue-specific expression analysis demonstrated that the abundances of CsARGs varied across different tissues, but CsATG8c/i showed a degree of tissue specificity. Under hormone and abiotic stress conditions, most CsARGs were upregulated at different time points during the treatment. In addition, the expression levels of 10 CsARGs were higher in the cold-resistant cultivar 'Longjing43' than in the cold-susceptible cultivar 'Damianbai' during the CA period; however, the expression of CsATG101 showed the opposite tendency. CONCLUSIONS: We performed a comprehensive bioinformatic and physiological analysis of CsARGs in tea plants, and these results may help to establish a foundation for further research investigating the molecular mechanisms governing autophagy in tea plant growth, development and response to stress. Meanwhile, some CsARGs could serve as putative molecular markers for the breeding of cold-resistant tea plants in future research.

Triterpene saponins from tea seed pomace (Camellia oleifera Abel) and their cytotoxic activity on MCF-7 cells in vitro.

Triterpenoid saponins are the main active ingredients extracted from Camellia oleifera Abel. In this study, crude saponins (Tc) was extracted from tea seed pomace and purified to obtain total saponins (T0). We used a COSMOSIL C18-OPN to separate T0 into three fractions-highly polar saponins (T1), moderately polar saponins (T2), and weakly polar saponins (T3). HPLC-ESI-MS analysis revealed that T2 were mainly composed of components with m/z ([M-H]-) of 1201.5617, 1187.5822, 1245.5862, and 1215.5779. Cell cycle analysis showed that both T0 and T2 inhibited proliferation and induced S phase arrest of MCF-7 cells. Further cell invasion assays demonstrated T0 and T2 also significantly reduced the invasive potential of MCF-7 cells. So T2 extracted from tea seed pomace (Camellia oleifera) may have effective antitumor activity.

Transcriptomic and Metabolic Insights into the Distinctive Effects of Exogenous Melatonin and Gibberellin on Terpenoid Synthesis and Plant Hormone Signal Transduction Pathway in Camellia sinensis.

Melatonin and gibberellin are bioactive molecules in plants. In the present study, the role of exogenous melatonin (MT) and gibberellin (GA) in the tea plant was explored by transcriptome and metabolic analysis. Results showed that the growth of tea plant was enhanced by MT treatment. The pathways of terpenoid synthesis and plant-pathogen interaction were significantly strengthened, combined with the upregulation of LRR-RLK and transcription factors which contributed to the growth of tea plant. The internode elongation and leaf enlargement were hastened by GA treatment. Significantly modulated expression occurred in the plant hormonal signal transduction, complemented by the upregulation of phenylpropanoid biosynthesis and expansins to achieve growth acceleration, whereas the flavonoid synthesis was repressed in GA treatment. Therefore, the distinctive effect of MT and GA treatment on tea plant was different. The MT exhibited significant promotion in terpenoid synthesis, especially, TPS14 and TPS1. GA was prominent in coordinated regulation of plant hormonal signal transduction.

Oleiferasaponin A2, a Novel Saponin from Camellia oleifera Abel. Seeds, Inhibits Lipid Accumulation of HepG2 Cells Through Regulating Fatty Acid Metabolism.

A new triterpenoid saponin, named oleiferasaponin A2, was isolated and identified from Camellia oleifera defatted seeds. Oleiferasaponin A2 exhibited anti-hyperlipidemic activity on HepG2 cell lines. Further study of the hypolipidemic mechanism showed that oleiferasaponin A2 inhibited fatty acid synthesis by significantly down-regulating the expression of SREBP-1c, FAS and FAS protein, while dramatically promoting fatty acid ß-oxidation by up-regulating the expression of ACOX-1, CPT-1 and ACOX-1 protein. Our results demonstrate that the oleiferasaponin A2 possesses potential medicinal value for hyperlipidemia treatment.

Cytotoxic and Hypoglycemic Activity of Triterpenoid Saponins from Camellia oleifera Abel. Seed Pomace.

One new and three known triterpenoid saponins were isolated and identified from Camellia oleifera seeds through IR, NMR, HR-ESI-MS and GC-MS spectroscopic methods, namely oleiferasaponin A3, oleiferasaponin A1, camelliasaponin B1, and camelliasaponin B2. The structure of oleiferasaponin A3 was elucidated as 16α-hydroxy-21ß-O-angeloyl-22α-O-cinnamoyl-23α-aldehyde-28-dihydroxymethylene-olean-12-ene-3ß-O-[ß-d-galactopyranosyl-(1→2)]-[ß-d-xylopyranosyl-(1→2)-ß-d-galactopyranosyl-(1→3)]-ß-d-gluco-pyranosiduronic acid. Camelliasaponin B1 and camelliasaponin B2 exhibited potent cytotoxic activity on three human tumour cell lines (human lung tumour cells (A549), human liver tumour cells (HepG2), cervical tumour cells (Hela)). The hypoglycemic activity of oleiferasaponin A1 was testified by protecting pancreatic ß-cell lines from high-glucose damage.