Integrative metabolome and transcriptome analyses reveal the coloration mechanism in Camellia oleifera petals with different color.

BACKGROUND: Camellia olelfera petals are colorful, and have high ornamental value. However, the color formation mechanism of C. olelfera petals with different color is still unclear. In our study, WGCNA method was applied to integrate metabolites and transcriptomes to investigate the coloration mechanism of four C. olelfera cultivars with different petal colors. RESULTS: Here, a total of 372 flavonoids were identified (including 27 anthocyanins), and 13 anthocyanins were significantly differentially accumulated in C. olelfera petals. Among them, cyanidin-3-O-(6''-O-p-Coumaroyl) glucoside was the main color constituent in pink petals, cyanidin-3-O-glucoside, cyanidin-3-O-galactoside, cyanidin-3-O-rutinoside, and cyanidin-3-O-(6''-O-malonyl) glucoside were the main contributors to candy pink petals, and peonidin-3-O-glucoside was the important color substance responsible for the red petals of C. oleifera. Furthermore, six structural genes (Co4CL1, CoF3H1, CoF3'H, CoANS, CoUGT75C1-4, and CoUGT75C1-5), three MYBs (CoMYB1, CoMYB4, and CoMYB44-3), three bHLHs (CobHLH30, CobHLH 77, and CobHLH 79-1), and two WRKYs (CoWRKY7 and CoWRKY22) could be identified candidate genes related to anthocyanins biosynthesis and accumulation, and lead to the pink and red phenotypes. The regulatory network of differentially accumulated anthocyanins and the anthocyanins related genes in C. olelfera petals were established. CONCLUSIONS: These findings elucidate the molecular basis of the coloration mechanisms of pink and red color in C. olelfera petals, and provided valuable target genes for future improvement of petals color in C. olelfera.

Coloration differences in three Camellia reticulata Lindl. cultivars: 'Tongzimian', 'Shizitou' and 'Damanao'.

Camellia reticulata Lindl., also known as Yunnan Camellia, is an important ornamental plant in China, especially for its large and stunning flowers. A comprehensive understanding of their coloration mechanisms can aid breeders in developing new cultivars and improving their ornamental value; however, it is still unclear in Yunnan Camellia, especially in mixed-color flowers. In this study, we conducted metabolic and transcriptomic comparison analyses to investigate the coloration differences in three Yunnan Camellia cultivars: C. reticulata 'Shizitou' (SZT), C. reticulata 'Damanao' (MN), and C. reticulata 'Tongzimian' (TZM). Our results revealed that the initial flowering stage may play a critical role in the color change of MN. Metabolome analysis demonstrated that cyanidin was the primary anthocyanin in SZT and MN's red region, while its content was low in TZM and MN's white region. According to the transcriptome analysis, the anthocyanins biosynthesis pathway was reconstructed in Yunnan Camellia, and the low expression of CHS was detected in TZM and MN's white region, while ANR maintained a high expression level, which may lead to the low content of cyanidin in them. Transcription factors MYBs, bHLH, and bZIP may play a key role in regulating anthocyanin-structural genes. The co-expression analysis showed that the meristem tissue may play a crucial role in the formation of the mixed white-red color in MN. Our study enriched the genetic basis of flower coloration differences in Yunnan Camellia which will be a valuable genomic resource to understanding the biology of coloration formation and for breeding the Camellia cultivars.

Integrated metabolomics and transcriptomic analysis reveals metabolic changes of flavor compounds of Camellia assamica host plant after parasitized by Viscumarticulatum.

Tea is one of the most popular beverages, it has many health benefits and flavor properties due to the presence of numerous secondary metabolites. Camellia assamica is also a main source of tea, which is mainly planted in the regions of southwest China. In this study, a non-targeted and targeted metabolomics analysis and sensory evaluation on tea leaves with and without mistletoe (Viscum articulatum) was carried out using liquid chromatography-mass spectrometry. RNA-seq-based transcriptomic analysis was conducted in parallel on the same samples, subsequently gene expression and metabolic differentiation were also investigated. Tea leaves with mistletoe presented much lower contents of (-)-catechin, (-)-epicatechin, (-)-gallocatechin gallate and (-)-epicatechin gallate, but significantly higher levels of free amino acids including Arg, Asp, GABA and Gln than that without mistletoe. Transcriptomic analysis also confirmed the main differentially expressed genes (DEGs) containing phenylpropanoid and flavonoid biosynthesis were down-regulated, but genes of amino acid biosynthesis were up-regulated. qRT-PCR analysis further revealed that the relative expression of CsCHS, CsC4H, CsANS, CsLAR, and CsF3H was hindered, while CsglyA and CsilvE expression was increased.

Small RNA profiling reveals that an ovule-specific microRNA, cja-miR5179, targets a B-class MADS-box gene in Camellia japonica.

BACKGROUND AND AIMS: The functional specialization of microRNA and its target genes is often an important factor in the establishment of spatiotemporal patterns of gene expression that are essential to plant development and growth. In different plant lineages, understanding the functional conservation and divergence of microRNAs remains to be explored. METHODS: To identify small regulatory RNAs underlying floral patterning, we performed a tissue-specific profiling of small RNAs in various floral organs from single and double flower varieties (flowers characterized by multiple layers of petals) in Camellia japonica. We identified cja-miR5179, which belongs to a deeply conserved microRNA family that is conserved between angiosperms and basal plants but frequently lost in eudicots. We characterized the molecular function of cja-miR5179 and its target - a B-function MADS-box gene - through gene expression analysis and transient expression assays. KEY RESULTS: We showed that cja-miR5179 is exclusively expressed in ovule tissues at the early stage of floral development. We found that cja-miR5179 targets the coding sequences of a DEFICIENS-like B-class gene (CjDEF) mRNA, which is located in the K motif of the MADS-box domain; and the target sites of miR5179/MADS-box were consistent in Camellia and orchids. Furthermore, through a petal transient-expression assay, we showed that the BASIC PENTACYSTEINE proteins bind to the GA-rich motifs in the cja-miR5179 promoter region and suppresses its expression. CONCLUSIONS: We propose that the regulation between miR5179 and a B-class MADS-box gene in C. japonica has a deep evolutionary origin before the separation of monocots and dicots. During floral development of C. japonica, cja-miR5179 is specifically expressed in the ovule, which may be required for the inhibition of CjDEF function. This work highlights the evolutionary conservation as well as functional divergence of small RNAs in floral development.

Ectopic expression of Camellia oleifera Abel. gibberellin 20-oxidase gene increased plant height and promoted secondary cell walls deposition in Arabidopsis.

MAIN CONCLUSION: Ectopic expression of Camellia oleifera Abel. gibberellin 20-oxidase 1 caused a taller phenotype, promoted secondary cell wall deposition, leaf enlargement, and early flowering, and reduced chlorophyll and anthocyanin accumulation and seed enlargement phenotype in Arabidopsis. Plant height and secondary cell wall (SCW) deposition are important plant traits. Gibberellins (GAs) play important roles in regulating plant height and SCWs deposition. Gibberellin 20-oxidase (GA20ox) is an important enzyme involved in GA biosynthesis. In the present study, we identified a GA synthesis gene in Camellia oleifera. The total length of the CoGA20ox1 gene sequence was 1146 bp, encoding 381 amino acids. Transgenic plants with CoGA20ox1 had a taller phenotype; a seed enlargement phenotype; promoted SCWs deposition, leaf enlargement, and early flowering; and reduced chlorophyll and anthocyanin accumulation. Genetic analysis showed that the mutant ga20ox1-3 Arabidopsis partially rescued the phenotype of CoGA20ox1 overexpression plants. The results showed that CoGA20ox1 participates in the growth and development of C. oleifera. The morphological changes in CoGA20ox1 overexpressed plants provide a theoretical basis for further exploration of GA biosynthesis and analysis of the molecular mechanism in C. oleifera.

Genome-Wide Detection of SPX Family and Profiling of CoSPX-MFS3 in Regulating Low-Phosphate Stress in Tea-Oil Camellia.

Camellia oleifera a member of the family Theaceae, is a phosphorus (P) tolerator native to southern China. The SPX gene family critically regulates plant growth and development and maintains phosphate (Pi) homeostasis. However, the involvement of SPX genes in Pi signaling in Tea-Oil Camellia remains unknown. In this work, 20 SPX genes were identified and categorized into four subgroups. Conserved domains, motifs, gene structure, chromosomal location and gene duplication events were also investigated in the SPX gene family. Defense and stress responsiveness cis-elements were identified in the SPX gene promoters, which participated in low-Pi stress responses. Based on transcriptome data and qRT-PCR results, nine CoSPX genes had similar expression patterns and eight genes (except CoPHO1H3) were up-regulated at 30 days after exposure to low-Pi stress. CoSPX-MFS3 was selected as a key candidate gene by WGCNA analysis. CoSPX-MFS3 was a tonoplast protein. Overexpression of CoSPX-MFS3 in Arabidopsis promoted the accumulation of total P content and decreased the anthocyanin content. Overexpression of CoSPX-MFS3 could enhance low-Pi tolerance by increased biomass and organic acid contents in transgenic Arabidopsis lines. Furthermore, the expression patterns of seven phosphate starvation genes were higher in transgenic Arabidopsis than those in the wild type. These results highlight novel physiological roles of the SPX family genes in C. oleifera under low-Pi stress, and lays the foundation for a deeper knowledge of the response mechanism of C. oleifera to low-Pi stress.

Integrated Analysis of Transcriptome and Metabolome Provides Insight into Camellia oleifera Oil Alleviating Fat Accumulation in High-Fat Caenorhabditis elegans.

Camellia oil (CO) is a high medicinal and nutritional value edible oil. However, its ability to alleviate fat accumulation in high-fat Caenorhabditis elegans has not been well elucidated. Therefore, this study aimed to investigate the effect of CO on fat accumulation in high-fat C. elegans via transcriptome and metabolome analysis. The results showed that CO significantly reduced fat accumulation in high-fat C. elegans by 10.34% (Oil Red O method) and 11.54% (TG content method), respectively. Furthermore, CO primarily altered the transcription levels of genes involved in longevity regulating pathway. Specifically, CO decreased lipid storage in high-fat C. elegans by inhibiting fat synthesis. In addition, CO supplementation modulated the abundance of metabolic biomarkers related to pyrimidine metabolism and riboflavin metabolism. The integrated transcriptome and metabolome analyses indicated that CO supplementation could alleviate fat accumulation in high-fat C. elegans by regulating retinol metabolism, drug metabolism-cytochrome P450, metabolism of xenobiotics by cytochrome P450, ascorbate and aldarate metabolism, and pentose and glucuronate interconversions. Overall, these findings highlight the potential health benefits of CO that could potentially be used as a functional edible oil.

In-depth potential mechanism of combined demulsification pretreatments (isopropanol ultrasonic pretreatments and Ca2+ flow additions) during aqueous enzymatic extractions of Camellia oils.

Emulsification is the practical limitation of aqueous enzymatic extractions of Camellia oils. This study aimed to investigate the influence and demulsification mechanisms of isopropanol ultrasonic pretreatments and Ca2+ additions on aqueous enzymatic extractions of Camellia oils. Combining isopropanol ultrasonic pretreatments with Ca2+ flow additions obtained the highest free oil recovery (78.03 %) and lowest emulsion content (1.5 %). Results indicated that the superior demulsification performance originated from the decrease in emulsion stabilities and formations. First, demulsification pretreatments reduced the oil (14.69 %) and solid (13.21 %) fractions in emulsions to decrease the stability of as-formed emulsions. Meanwhile, isopropanol ultrasonic pretreatments extracted tea saponins (0.38 mg/mL) and polysaccharides (0.23 mg/mL), while Ca2+ combined with protein isolates (5.82 mg/mL), tea saponins (7.48 mg/mL) and polysaccharides (0.78 mg/mL) to form precipitates and reduce emulsion formation. This work could promote the practical application of aqueous enzymatic extractions of Camellia oils and enlighten the rise of advanced demulsification pretreatments.

Novel insight into anthocyanin metabolism and molecular characterization of its key regulators in Camellia sasanqua.

Flower color is a trait that affects the ornamental value of a plant. Camellia sasanqua is a horticultural plant with rich flower color, but little is known about the regulatory mechanism of color diversity in this plant. Here, the anthocyanin profile of 20 C. sasanqua cultivars revealed and quantified 11 anthocyanin derivatives (five delphinidin-based and six cyanidin-based anthocyanins) for the first time. Cyanidin-3-O-(6-O-(E)-p-coumaroyl)-glucoside was the main contributor to flower base color, and the accumulation of cyanidin and delphinidin derivatives differed in the petals. To further explore the molecular mechanism of color divergence, a transcriptome analysis was performed using C. sasanqua cultivars 'YingYueYe', 'WanXia', 'XueYueHua', and'XiaoMeiGui'. The co-expression network related to differences in delphinidin and cyanidin derivatives accumulation was identified. Eleven candidate genes encoding key enzymes (e.g., F3H, F3'H, and ANS) were involved in anthocyanin biosynthesis. Moreover, 27 transcription factors were screened as regulators of the two types of accumulating anthocyanins. The association was suggested by correlation analysis between the expression levels of the candidate genes and the different camellia cultivars. We concluded that cyanidin and delphinidin derivatives are the major drivers of color diversity in C. sasanqua. This finding provides valuable resources for the study of flower color in C. sasanqua and lays a foundation for genetic modification of anthocyanin biosynthesis.

Modulating interfacial structure and lipid digestion of natural Camellia oil body by roasting and boiling processes.

Oil body (OB) is the lipid-storage organelle in oilseed, and its stability is crucial for oilseed processing. Herein, effects of roasting and boiling on the structure, stability, and in vitro lipid digestion of Camellia OB were studied. The interfacial structure and physical stability of the extracted OB were investigated by electrophoresis, confocal-Raman spectroscopy, zeta-potential, and surface hydrophobicity, etc. Boiling caused protein loss on the OB surfaces, forming a stable phospholipid interface, which resulted in coalescence of the droplets (d > 100 µm) and negative ζ-potential (-3 âˆ¼ -8 mV) values at a pH of 2.0. However, roasting partially denatured the proteins in the seeds, which were adsorbed on the OB surfaces. The random coil structure of interfacial protein increased to ∼20 % after thermal treatment. Besides, heating decreased the surface hydrophobicity of OB and improved lipid digestion. After boiling 60 min, the extent of lipolysis increased from 41.7 % (raw) to 57.4 %.

Transcriptome Analysis Reveals Putative Induction of Floral Initiation by Old Leaves in Tea-Oil Tree (Camellia oleifera 'changlin53').

Floral initiation is a major phase change in the spermatophyte, where developmental programs switch from vegetative growth to reproductive growth. It is a key phase of flowering in tea-oil trees that can affect flowering time and yield, but very little is known about the molecular mechanism of floral initiation in tea-oil trees. A 12-year-old Camellia oleifera (cultivar 'changlin53') was the source of experimental materials in the current study. Scanning electron microscopy was used to identify the key stage of floral initiation, and transcriptome analysis was used to reveal the transcriptional regulatory network in old leaves involved in floral initiation. We mined 5 DEGs related to energy and 55 DEGs related to plant hormone signal transduction, and we found floral initiation induction required a high level of energy metabolism, and the phytohormones signals in the old leaves regulate floral initiation, which occurred at stage I and II. Twenty-seven rhythm-related DEGs and 107 genes associated with flowering were also identified, and the circadian rhythm interacted with photoperiod pathways to induce floral initiation. Unigene0017292 (PSEUDO-RESPONSE REGULATOR), Unigene0046809 (LATE ELONGATED HYPOCOTYL), Unigene0009932 (GIGANTEA), Unigene0001842 (CONSTANS), and Unigene0084708 (FLOWER LOCUS T) were the key genes in the circadian rhythm-photoperiod regulatory network. In conjunction with morphological observations and transcriptomic analysis, we concluded that the induction of floral initiation by old leaves in C. oleifera 'changlin53' mainly occurred during stages I and II, floral initiation was completed during stage III, and rhythm-photoperiod interactions may be the source of the main signals in floral initiation induced by old leaves.

Physiological and Transcriptional Analysis Reveals the Response Mechanism of Camellia vietnamensis Huang to Drought Stress.

Drought stress is considered the main obstacle restricting Camellia vietnamensis Huang (C. vietnamensis) yield. Hainan is the southernmost distribution region of C. vietnamensis in China and experiences a drought period annually. To study the drought-stress-response mechanism of C. vietnamensis, we treated seedlings of drought-tolerant (HD1) and drought-sensitive (WH1) cultivars with PEG-6000 (PEG) to simulate drought stress and compared the physiology and transcriptome of their leaves at 0 d, 3 d and 6 d posttreatment. Under drought stress, the growth of C. vietnamensis was inhibited, the relative water content (RWC) of leaves decreased and the contents of malondialdehyde (MDA), antioxidant enzyme activities, osmotic regulatory substances and secondary metabolites increased. Compared with those of WH1, the leaf RWC, osmotic-regulation substance content (proline, soluble protein and soluble sugar) and antioxidant enzyme activity (superoxide dismutase, peroxidase and catalase) of HD1 were significantly increased, while the relative electrical conductivity and MDA content were significantly decreased. Compared with WH1, 2812, 2070 and 919, differentially expressed genes (DEGs) were detected in HD1 0 d, 3 d and 6 d posttreatment, respectively, and the number of DEGs increased with increasing treatment time. The detected DEGs are involved in the drought stress response of C. vietnamensis mainly through plant-hormone signal transduction and lignin and flavonoid biosynthesis pathways. Drought stress significantly activated the expression of several lignin and flavonoid biosynthesis genes in HD1. Moreover, total flavonoid and total polyphenol contents in HD1 were significantly increased, suggesting that the accumulation of flavonoids may be a key factor in the drought stress response of C. vietnamensis. Additionally, 191 DEGs were associated with coding transcription factors (TFs). This study provides insight into the molecular mechanism of the drought stress response of C. vietnamensis and provides a theoretical basis for the development and cultivation of new drought-resistant cultivars.

Photosynthetic carbon and nitrogen metabolism of Camellia oleifera Abel during acclimation to low light conditions.

Tea-oil tree (Camellia oleifera Abel) is an important woody oil crop with high economic value. However, it has low photosynthetic production considering the low light intensity of its growth environment. To understand the acclimation mechanism of tea-oil trees to low light conditions, three light intensity treatments were conducted: high light (450-500 µmol. m-2. s-1), medium light (180-200 µmol. m-2. s-1), and low light (45-50 µmol. m-2. s-1). The carbon (C) and nitrogen (N) metabolism network were constructed by investigating the leaf anatomy, photosynthetic characteristics, N partitioning, transcriptome and metabolome. Results demonstrated that a larger proportion light energy was used for photochemical reactions in an environment with lower light intensity, which resulted in an increase in photosystem II photochemical efficiency and instantaneous light use efficiency (LUE) at the leaf level. As the light intensity increased, decreased electron transfer and carboxylation efficiencies, photorespiration and dark respiration rates, LUE at plant level, and N use efficiency (PNUE) were observed. Leaves trended to harvest more light using higher expression levels of light-harvesting protein genes, higher chlorophyll content, more granum and more tightly stacked granum lamella under lower light intensity. At transcriptional and metabolic levels, the TCA cycle, and the synthesis of starch and saccharides were weakened as light intensity decreased, while the Calvin cycle did not show the regularity between different treatments. Less N was distributed in Rubisco, respiration, and cell wall proteins as light decreased. Storage N was prominently accumulated in forms of amino acids (especially L-arginine) and amino acid derivatives as under medium and low light environments, to make up for C deficiency. Therefore, tea-oil trees actively improve light-harvesting capacity and enlarges the storage N pool to adapt to a low light environment, at the cost of a decrease of photosynthetic C assimilation and PNUE.

Identification of long non-coding RNAs and microRNAs involved in anther development in the tropical Camellia oleifera.

BACKGROUND: Explored the molecular science of anther development is important for improving productivity and overall yield of crops. Although the role of regulatory RNAs, including long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), in regulating anther development has been established, their identities and functions in Camellia oleifera, an important industrial crop, have yet not been clearly explored. Here, we report the identification and characterization of genes, lncRNAs and miRNAs during three stages of the tropical C. oleifera anther development by single-molecule real-time sequencing, RNA sequencing and small RNA sequencing, respectively. RESULTS: These stages, viz. the pollen mother cells stage, tetrad stage and uninucleate pollen stage, were identified by analyzing paraffin sections of floral buds during rapid expansion periods. A total of 18,393 transcripts, 414 putative lncRNAs and 372 miRNAs were identified, of which 5,324 genes, 115 lncRNAs, and 44 miRNAs were differentially accumulated across three developmental stages. Of these, 44 and 92 genes were predicted be regulated by 37 and 30 differentially accumulated lncRNAs and miRNAs, respectively. Additionally, 42 differentially accumulated lncRNAs were predicted as targets of 27 miRNAs. Gene ontology enrichment indicated that potential target genes of lncRNAs were enriched in photosystem II, regulation of autophagy and carbohydrate phosphatase activity, which are essential for anther development. Functional annotation of genes targeted by miRNAs indicated that they are relevant to transcription and metabolic processes that play important roles in microspore development. An interaction network was built with 2 lncRNAs, 6 miRNAs and 10 mRNAs. Among these, miR396 and miR156 family were up-regulated, while their targets, genes (GROWTH REGULATING FACTORS and SQUAMOSA PROMOTER BINDING PROTEIN-LIKE genes) and lncRNAs, were down-regulated. Further, the trans-regulated targets of these lncRNAs, like wall-associated kinase2 and phosphomannose isomerase1, are involved in pollen wall formation during anther development. CONCLUSIONS: This study unravels lncRNAs, miRNAs and miRNA-lncRNA-mRNA networks involved in development of anthers of the tropical C. oleifera lays a theoretical foundation for further elucidation of regulatory roles of lncRNAs and miRNAs in anther development.

Whole-Genome Identification and Analysis of Multiple Gene Families Reveal Candidate Genes for Theasaponin Biosynthesis in Camellia oleifera.

Camellia oleifera is an economically important oilseed tree. Seed meals of C. oleifera have a long history of use as biocontrol agents in shrimp farming and as cleaning agents in peoples' daily lives due to the presence of theasaponins, the triterpene saponins from the genus Camellia. To characterize the biosynthetic pathway of theasaponins in C. oleifera, members of gene families involved in triterpenoid biosynthetic pathways were identified and subjected to phylogenetic analysis with corresponding members in Arabidopsis thaliana, Camellia sinensis, Actinidia chinensis, Panax ginseng, and Medicago truncatula. In total, 143 triterpenoid backbone biosynthetic genes, 1169 CYP450s, and 1019 UGTs were identified in C. oleifera. The expression profiles of triterpenoid backbone biosynthetic genes were analyzed in different tissue and seed developmental stages of C. oleifera. The results suggested that MVA is the main pathway for triterpenoid backbone biosynthesis. Moreover, the candidate genes for theasaponin biosynthesis were identified by WGCNA and qRT-PCR analysis; these included 11 CYP450s, 14 UGTs, and eight transcription factors. Our results provide valuable information for further research investigating the biosynthetic and regulatory network of theasaponins.

De novo transcriptome assembly of the cotyledon of Camellia oleifera for discovery of genes regulating seed germination.

BACKGROUND: Camellia oleifera (C.oleifera) is one of the most important wood oil species in the world. C.oleifera was propagated by nurse seedling grafting. Since the morphology of rootstocks has a significant impact on grafting efficiency and seedling quality, it is necessary to understand the molecular mechanism of morphogenesis for cultivating high-quality and controllable rootstocks. However, the genomic resource for this species is relatively limited, which hinders us from fully understanding the molecular mechanisms of seed germination in C.oleifera. RESULTS: In this paper, using transcriptome sequencing, we measured the gene expression in the C.oleifera cotyledon in different stages of development and the global gene expression profiles. Approximately 45.4 gigabases (GB) of paired-end clean reads were assembled into 113,582 unigenes with an average length of 396 bp. Six public protein databases annotate 61.5% (68,217) of unigenes. We identified 11,391 differentially expressed genes (DEGs) throughout different stages of germination. Enrichment analysis revealed that DEGs were mainly involved in hormone signal transduction and starch sucrose metabolism pathways. The gravitropism regulator UNE10, the meristem regulators STM, KNAT1, PLT2, and root-specific transcription factor WOX11 all have higher gene expression levels in the CAM2 stage (seed soaking), which indicates that the cotyledon-regulated program for germination had initiated when the seeds were imbibition. Our data showed differentially reprogrammed to multiple hormone-related genes in cotyledons during C.oleifera seed germination. CONCLUSION: Cotyledons play vital roles, both as the main nutrient provider and as one primary instructor for seed germination and seedling growth. Together, our study will significantly enrich the genomic resources of Camellia and help us understand the molecular mechanisms of the development in the seed germination and seedling growth of C.oleifera. It is helpful to culture standard and superior quality rootstock for C.oleifera breeding.

Oxidosqualene Cyclases Involved in the Biosynthesis of Diverse Triterpenes in Camellia sasanqua.

Camellia sasanqua is an important economic plant that is rich in lipophilic triterpenols with pharmacological activities including antiallergic, anti-inflammatory, and anticancer activities. However, the key enzymes related to triterpene biosynthesis have seldom been studied in C. sasanqua. Oxidosqualene cyclases (OSCs) are the rate-limiting enzymes related to triterpene biosynthesis. In this study, seven putative OSC genes (CsOSC1-7) were mined from the C. sasanqua transcriptome. Six CsOSCs were characterized for the biosynthesis of diverse triterpene skeletons, including α-amyrin, ß-amyrin, δ-amyrin, dammarenediol-II, ψ-taraxasterol, taraxasterol, and cycloartenol by the heterologous expression system. CsOSC3 was a multiple functional α-amyrin synthase. Three key residues, Trp260, Tyr262, and Phe415, are critical to the catalytic performance of CsOSC3 judging from the results of molecular docking and site-directed mutagenesis. These findings provide important insights into the biosynthesis pathway of triterpenes in C. sasanqua.

Comparative transcriptomic analysis unveils the deep phylogeny and secondary metabolite evolution of 116 Camellia plants.

Camellia plants include more than 200 species of great diversity and immense economic, ornamental, and cultural values. We sequenced the transcriptomes of 116 Camellia plants from almost all sections of the genus Camellia. We constructed a pan-transcriptome of Camellia plants with 89 394 gene families and then resolved the phylogeny of genus Camellia based on 405 high-quality low-copy core genes. Most of the inferred relationships are well supported by multiple nuclear gene trees and morphological traits. We provide strong evidence that Camellia plants shared a recent whole genome duplication event, followed by large expansions of transcription factor families associated with stress resistance and secondary metabolism. Secondary metabolites, particularly those associated with tea quality such as catechins and caffeine, were preferentially heavily accumulated in the Camellia plants from section Thea. We thoroughly examined the expression patterns of hundreds of genes associated with tea quality, and found that some of them exhibited significantly high expression and correlations with secondary metabolite accumulations in Thea species. We also released a web-accessible database for efficient retrieval of Camellia transcriptomes. The reported transcriptome sequences and obtained novel findings will facilitate the efficient conservation and utilization of Camellia germplasm towards a breeding program for cultivated tea, camellia, and oil-tea plants.

Integrative Physiological and Transcriptomic Analysis Reveals the Transition Mechanism of Sugar Phloem Unloading Route in Camellia oleifera Fruit.

Sucrose phloem unloading plays a vital role in photoassimilate distribution and storage in sink organs such as fruits and seeds. In most plants, the phloem unloading route was reported to shift between an apoplasmic and a symplasmic pattern with fruit development. However, the molecular transition mechanisms of the phloem unloading pathway still remain largely unknown. In this study, we applied RNA sequencing to profile the specific gene expression patterns for sucrose unloading in C. oleifera fruits in the apo- and symplasmic pathways that were discerned by CF fluoresce labelling. Several key structural genes were identified that participate in phloem unloading, such as PDBG11, PDBG14, SUT8, CWIN4, and CALS10. In particular, the key genes controlling the process were involved in callose metabolism, which was confirmed by callose staining. Based on the co-expression network analysis with key structural genes, a number of transcription factors belonging to the MYB, C2C2, NAC, WRKY, and AP2/ERF families were identified to be candidate regulators for the operation and transition of phloem unloading. KEGG enrichment analysis showed that some important metabolism pathways such as plant hormone metabolism, starch, and sucrose metabolism altered with the change of the sugar unloading pattern. Our study provides innovative insights into the different mechanisms responsible for apo- and symplasmic phloem unloading in oil tea fruit and represents an important step towards the omics delineation of sucrose phloem unloading transition in crops.

Hair growth-promoting effects of Camellia seed cake extract in human dermal papilla cells and C57BL/6 mice.

OBJECTIVES: Camellia seed cake is a by-product of Camellia oleifera Abel seed after oil extraction. Washing hair with Camellia seed cake extract is a traditional Chinese custom that has lasted for over one thousand years. However, the hair growth-promoting effects of Camellia seed cake extract were not investigated so far. This work examined the effects of de-saponinated Camellia seed cake extracts (DS-CSE) on hair growth, using in vitro and in vivo models. METHODS: The studies on cell proliferation, cell cycle regulation, and K+ channels activation effects of DS-CSE were performed on human dermal papilla cells (DPCs). Relative expression of insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and transforming growth factor-ß (TGF-ß1) in DPCs was determined by RT-PCR. Relative expression of ERK and AKT was determined by Western blot analysis. Hair growth-promoting effects were also measured in C57BL/6J mice model. RESULTS: DS-CSE treatment significantly proliferated DPCs, relating to the increased proportion of DPCs in S and G2 /M phases, the activation of potassium channels and the promoted phosphorylation of ERK and AKT in DPCs. DS-CSE treatment also significantly upregulated the mRNA levels of HGF, VEGF and IGF-1, and downregulated the mRNA level of TGF-ß1. Topical application of DS-CSE promoted hair growth on shaven back mice and also upregulated the expression of VEGF in mice. CONCLUSION: Our results demonstrated that DS-CSE exerts a hair growth-promoting effect in vitro and in vivo by proliferating DPCs through the ERK and AKT signaling pathways and regulating the expression of growth factors.