Function of FT in Flowering Induction in Two Camellia Species.

FLOWERING LOCUS T (FT), belonging to the FT/TFL1 gene family, is an important gene regulating the flowering transition and inflorescence architecture during plant development. Given its importance to plant adaptation and crop improvement, FT has been extensively studied in related plant research; however, the specific role and underlying molecular mechanisms of FT in the continuous flowering of perennial plants remains elusive. Here, we isolated and characterized homologous FT genes from two Camellia species with different flowering-period phenotypes: CaFT was isolated from Camellia azalea, a precious species blooming in summer and flowering throughout the year, and CjFT was isolated from C. japonica, which blooms in winter and spring. The major difference in the genes between the two species was an additional five-amino acid repeat sequence in C. japonica. FT showed high expression levels in the leaves in both species from January to August, especially in April for C. japonica and in May for C. azalea. CaFT was expressed throughout the year in C. azalea, whereas CjFT was not expressed from September to December in C. japonica. The expression levels of FT in the floral buds were generally higher than those in the leaves. Overexpression of CaFT and CjFT in Arabidopsis indicated that both genes can activate downstream genes to promote flowering. Transgenic callus tissue was obtained by introducing the two genes into C. azalea through Agrobacterium-mediated transformation. Transcriptome and quantitative real-time polymerase chain reaction analyses indicated that both florigen FT genes promoted the expression of downstream genes such as AP1, FUL, and SEP3, and slightly up-regulated the expression of upstream genes such as CO and GI. The above results indicated that CaFT and CjFT played a role in promoting flowering in both camellia species. The expression pattern of CaFT in leaves suggested that, compared to CjFT, CaFT may be related to the annual flowering of C. azalea.

Molecular Mechanisms of Seasonal Gene Expression in Trees.

In trees, the annual cycling of active and dormant states in buds is closely regulated by environmental factors, which are of primary significance to their productivity and survival. It has been found that the parallel or convergent evolution of molecular pathways that respond to day length or temperature can lead to the establishment of conserved periodic gene expression patterns. In recent years, it has been shown in many woody plants that change in annual rhythmic patterns of gene expression may underpin the adaptive evolution in forest trees. In this review, we summarize the progress on the molecular mechanisms of seasonal regulation on the processes of shoot growth, bud dormancy, and bud break in response to day length and temperature factors. We focus on seasonal expression patterns of genes involved in dormancy and their associated epigenetic modifications; the seasonal changes in the extent of modifications, such as DNA methylation, histone acetylation, and histone methylation, at dormancy-associated loci have been revealed for their actions on gene regulation. In addition, we provide an outlook on the direction of research on the annual cycle of tree growth under climate change.

Genomics insights into flowering and floral pattern formation: regional duplication and seasonal pattern of gene expression in Camellia.

BACKGROUND: The formation and domestication of ornamental traits are influenced by various aspects, such as the recognition of esthetic values and cultural traditions. Camellia japonica is widely appreciated and domesticated around the world mainly due to its rich variations in ornamental traits. Ornamental camellias have a diverse range of resources, including different bud variations from Camellia spp. as well as inter- and intra- specific hybridization. Despite research on the formation of ornamental traits, a basic understanding of their genetics and genomics is still lacking. RESULTS: Here, we report the chromosomal-level reference genome of C. japonica through combining multiple DNA-sequencing technologies and obtain a high-density genetic linkage map of 4255 markers by sequencing 98 interspecific F1 hybrids between C. japonica and C. chekiangoleosa. We identify two whole-genome duplication events in C. japonica: one is a shared ancient γ event, and the other is revealed to be specific to genus Camellia. Based on the micro-collinearity analysis, we find large-scale segmental duplication of chromosome 8, resulting to two copies of the AGAMOUS loci, which may play a key role in the domestication of floral shapes. To explore the regulatory mechanisms of seasonal flowering, we have analyzed year-round gene expression patterns of C. japonica and C. azalea-a sister plant of continuous flowering that has been widely used for cross breeding. Through comparative analyses of gene co-expression networks and annual gene expression patterns, we show that annual expression rhythms of some important regulators of seasonal growth and development, including GIGANTEA and CONSTANS of the photoperiod pathway, have been disrupted in C. azalea. Furthermore, we reveal that the distinctive expression patterns of FLOWERING LOCUS T can be correlated with the seasonal activities of flowering and flushing. We demonstrate that the regulatory module involved in GIGANTEA, CONSTANS, and FLOWERING LOCUS T is central to achieve seasonality. CONCLUSIONS: Through the genomic and comparative genomics characterizations of ornamental Camellia spp., we propose that duplication of chromosomal segments as well as the establishment of gene expression patterns has played a key role in the formation of ornamental traits (e.g., flower shape, flowering time). This work provides a valuable genomic platform for understanding the molecular basis of ornamental traits.

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.

Floral organ transcriptome in Camellia sasanqua provided insight into stamen petaloid.

BACKGROUND: The cultivated Camellia sasanqua forms a divergent double flower pattern, and the stamen petaloid is a vital factor in the phenomenon. However, the regulation mechanism remains largely unclear. RESULTS: Here, a comprehensive comparative transcriptome analysis of the wild-type, "semi-double", "peony double", and "rose double" was performed. The cluster analysis of global gene expression level showed petal and stamen difficulty separable in double flower. The crucial pathway and genes related to double flower patterns regulation were identified by pairwise comparisons and weighted gene coexpression network (WGCNA). Divergent genes expression, such as AUX1 and AHP, are involved in plant hormone signaling and photosynthesis, and secondary metabolites play an important role. Notably, the diversity of a petal-specific model exhibits a similar molecular signature to the stamen, containing extensin protein and PSBO1, supporting the stamen petaloid point. Moreover, the expansion of class A gene activity influenced the double flower formation, showing that the key function of gene expression was probably demolished. CONCLUSIONS: Overall, this work confirmed the ABCE model and provided new insights for elucidating the molecular signature of double formation.

Comparative Transcriptome Analysis of Flower Senescence of Camellia lutchuensis.

Background: Flower senescence is the last stage of flower development and affects the ornamental and economic value of flower plants. There is still less known on flower senescence of the ornamental plant Camellia lutchuensis, a precious species of Camellia with significant commercial application value. Methods: Transcriptome sequencing was used to investigate the flower senescence in five developmental stages of C. lutchuensis. Results: By Illumina HiSeq sequencing, we generated approximately 101.16 Gb clean data and 46649 differentially expressed unigenes. Based on the different expression pattern, differentially expressed unigenes were classified into 10 Sub Class. And Sub Class 9 including 8252 unigenes, was highly expressed in the flower senescent stage, suggesting it had a potential regulatory relationship of flower senescence. First, we found that ethylene biosynthesis genes ACSs, ACOs, receptor ETR genes and signaling genes EINs, ERFs all upregulated during flower senescence, suggesting ethylene might play a key role in the flower senescence of C. lutchuensis. Furthermore, reactive oxygen species (ROS) production related genes peroxidase (POD), lipase (LIP), polyphenoloxidase (PPO), and ROS scavenging related genes glutathione S-transferase (GST), glutathione reductase (GR) and superoxide dismutase (SOD) were induced in senescent stage, suggesting ROS might be involved in the flower senescence. Besides, the expression of monoterpenoid and isoflavonoid biosynthesis genes, transcription factors (WRKY, NAC, MYB and C2H2 ), senescence-associated gene SAG20 also were increased during flower senescence. Conclusion: In C. lutchuensis, ethylene pathway might be the key to regulate flower senescence, and ROS signal might play a role in the flower senescence.

Multi-Approach Analysis Reveals Pathways of Cold Tolerance Divergence in Camellia japonica.

Understanding the molecular mechanism of the cold response is critical to improve horticultural plant cold tolerance. Here, we documented the physiological, transcriptome, proteome, and hormonal dynamics to cold stress in temperate genotype (Tg) and subtropical genotype (Sg) populations of Camellia japonica. Tg C. japonica suffered minimal osmotic and oxidative damage compared to Sg C. japonica under the same cold treatment. Transcriptional and translational differences increased under the cold treatment, indicating that Tg C. japonica was affected by the environment and displayed both conserved and divergent mechanisms. About 60% of the genes responding to cold had similar dynamics in the two populations, but 1,896 transcripts and 455 proteins differentially accumulated in response to the cold between Tg and Sg C. japonica. Co-expression analysis showed that the ribosomal protein and genes related to photosynthesis were upregulated in Tg C. japonica, and tryptophan, phenylpropanoid, and flavonoid metabolism were regulated differently between the two populations under cold stress. The divergence of these genes reflected a difference in cold responsiveness. In addition, the decrease in the abscisic acid (ABA)/gibberellic acid (GA) ratio regulated by biosynthetic signal transduction pathway enhanced cold resistance in Tg C. japonica, suggesting that hormones may regulate the difference in cold responsiveness. These results provide a new understanding of the molecular mechanism of cold stress and will improve cold tolerance in horticultural plants.

The genome of oil-Camellia and population genomics analysis provide insights into seed oil domestication.

BACKGROUND: As a perennial crop, oil-Camellia possesses a long domestication history and produces high-quality seed oil that is beneficial to human health. Camellia oleifera Abel. is a sister species to the tea plant, which is extensively cultivated for edible oil production. However, the molecular mechanism of the domestication of oil-Camellia is still limited due to the lack of sufficient genomic information. RESULTS: To elucidate the genetic and genomic basis of evolution and domestication, here we report a chromosome-scale reference genome of wild oil-Camellia (2.95 Gb), together with transcriptome sequencing data of 221 cultivars. The oil-Camellia genome, assembled by an integrative approach of multiple sequencing technologies, consists of a large proportion of repetitive elements (76.1%) and high heterozygosity (2.52%). We construct a genetic map of high-density corrected markers by sequencing the controlled-pollination hybrids. Genome-wide association studies reveal a subset of artificially selected genes that are involved in the oil biosynthesis and phytohormone pathways. Particularly, we identify the elite alleles of genes encoding sugar-dependent triacylglycerol lipase 1, ß-ketoacyl-acyl carrier protein synthase III, and stearoyl-acyl carrier protein desaturases; these alleles play important roles in enhancing the yield and quality of seed oil during oil-Camellia domestication. CONCLUSIONS: We generate a chromosome-scale reference genome for oil-Camellia plants and demonstrate that the artificial selection of elite alleles of genes involved in oil biosynthesis contributes to oil-Camellia domestication.

Identification of Chalcone Isomerase Family Genes and Roles of CnCHI4 in Flavonoid Metabolism in Camellia nitidissima.

Camellia nitidissima is a woody plant with high ornamental value, and its golden-yellow flowers are rich in a variety of bioactive substances, especially flavonoids, that are beneficial to human health. Chalcone isomerases (CHIs) are key enzymes in the flavonoid biosynthesis pathway; however, there is a scarcity of information regarding the CHI family genes of C. nitidissima. In this study, seven CHI genes of C. nitidissima were identified and divided into three subfamilies by phylogenetic analysis. The results of multiple sequence alignment revealed that, unlike CnCHI1/5/6/7, CnCHI2/3/4 are bona fide CHIs that contain all the active site and critical catalytic residues. Analysis of the expression patterns of CnCHIs and the total flavonoid content of the flowers at different developmental stages revealed that CnCHI4 might play an essential role in the flavonoid biosynthesis pathway of C. nitidissima. CnCHI4 overexpression significantly increased flavonoid production in Nicotiana tabacum and C. nitidissima. The results of the dual-luciferase reporter assay and yeast one-hybrid system revealed that CnMYB7 was the key transcription factor that governed the transcription of CnCHI4. The study provides a comprehensive understanding of the CHI family genes of C. nitidissima and performed a preliminary analysis of their functions and regulatory mechanisms.

The Flavonoid Biosynthesis Network in Plants.

Flavonoids are an important class of secondary metabolites widely found in plants, contributing to plant growth and development and having prominent applications in food and medicine. The biosynthesis of flavonoids has long been the focus of intense research in plant biology. Flavonoids are derived from the phenylpropanoid metabolic pathway, and have a basic structure that comprises a C15 benzene ring structure of C6-C3-C6. Over recent decades, a considerable number of studies have been directed at elucidating the mechanisms involved in flavonoid biosynthesis in plants. In this review, we systematically summarize the flavonoid biosynthetic pathway. We further assemble an exhaustive map of flavonoid biosynthesis in plants comprising eight branches (stilbene, aurone, flavone, isoflavone, flavonol, phlobaphene, proanthocyanidin, and anthocyanin biosynthesis) and four important intermediate metabolites (chalcone, flavanone, dihydroflavonol, and leucoanthocyanidin). This review affords a comprehensive overview of the current knowledge regarding flavonoid biosynthesis, and provides the theoretical basis for further elucidating the pathways involved in the biosynthesis of flavonoids, which will aid in better understanding their functions and potential uses.

Characterization and phylogenetic analysis of the complete chloroplast genome of Camellia chrysanthoides (Theaceae).

Camellia chrysanthoides H.T.Chang 1979 is an economic species for its high ornamental and medicinal values. In the present work, the complete chloroplast (cp) genome sequence of C. chrysanthoides was assembled and characterized using Illumina paired-end sequencing data. The total cp genome of C. chrysanthoides is 157,439 bp in size, consisting of a small single copy (SSC) and a large single copy (LSC) separated by a pair of inverted repeats (IRs) with 18,265 bp, 88,162 bp, and 25,506 bp, respectively. Further annotation revealed the cp genome encoded 124 genes, including 82 protein-coding genes, 34 tRNA genes, and eight rRNA genes, and the overall GC content of the cp genome is 37.31%. Phylogenetic analysis based on 80 protein-coding genes shows that C. chrysanthoidesis closely related to C. azalea in the genus Camellia.

Flavonoid 3'-hydroxylase of Camellia nitidissima Chi. promotes the synthesis of polyphenols better than flavonoids.

Camellia nitidissima Chi. is an ornamental plant of the genus Camellia L. Its flowers contain a lot of flavonoids and polyphenols. Flavonoid 3'-hydroxylase (F3'H) plays an important role in the synthesis of flavonoids, polyphenols and anthocyanins. We used PCR amplification, quantitative PCR, High-performance liquid chromatography, subcellular localization, and agrobacterium-mediated leaf disk method to study the the function of CnF3'H. The full length of CnF3'H was 1859 bp (GenBank code: HQ290518.1), with an open reading frame of 1577 bp, and encoded 518 amino acid. A phylogenetic tree analysis showed that CnF3'H was closely related to Camellia sinensis L. and C. sinensis cultivar Zhonghuang. CnF3'H was expressed in flowers, leaves, fruits, sepals, petals and stamens of C. nitidissima, and during the flowering process the expression level in flower decreased initially and then increased. CnF3'H expression was significantly positive correlated with polyphenol contents in C. nitidissima. A CnF3'H-EGFP expression vector was constructed to do the subcellular localization, we found that CnF3'H was obviously localized in the nuclear envelope and cytomembrane. In transgenic tobacco flowers, the total polyphenol content and various polyphenol constituents were significantly increased with high CnF3'H expression level, while total flavonoid contents and some flavonol constituents were increased slightly. These findings suggest that CnF3'H promotes the synthesis of polyphenols better than flavonoids.

Characterization and phylogenetic significance of the complete chloroplast genome of Camellia Kissii, an economic crop for producing oil.

Camellia kissii is cultivated for a long time as an oil crop for edible and industrial oils, and has the functions of high oil production rate and unique health care. The complete chloroplast (cp) genome sequence of C. kissii is 156,961 bp in length with GC content of 39.29%. It presents a quadrate structure, including a large single-copy (LSC) region (86,640 bp), a small single-copy (SSC) region (18,399 bp), and a pair of inverted repeats (IRs) (25,961 bp). Meanwhile, 15 complete chloroplast genome of Camellia was aligned to explore the phylogenetic significance of Camellia. And the genetic relationship between Camellia kissii and Camellia huana was found to be closest.

Characterization of the complete chloroplast genome of Camellia brevistyla, an oil-rich and evergreen shrub.

Camellia brevistyla is an economic species for its seeds with high oil content and ornamental value, which is cultivated widespreadly across southern China. In this study, the complete chloroplast (cp) genome sequence of C. brevistyla was assembled and annotated in order to future genetic research. The whole cp genome of C. brevistyla is 159,281 bp in size, composed of a small single copy (SSC) region of 15,662 bp and a large single copy (LSC) region of 86,251 bp separated by a pair of inverted repeats (IRs, IRA: 130598: 159281, IRB: 86252: 114935). The overall GC content of C. brevistyla cp genome is 37.19%, with the base content A (31.03%), T (31.78%), C (18.94%), and G (18.25%). Phylogenetic analysis of 20 species based on 74 protein-coding genes shows that C. brevistyla is evolutionarily closest to Camellia danzaiensis.

Integrated Physiological and Transcriptomic Analyses Reveal a Regulatory Network of Anthocyanin Metabolism Contributing to the Ornamental Value in a Novel Hybrid Cultivar of Camellia Japonica.

Camellia japonica is a plant species with great ornamental and gardening values. A novel hybrid cultivar Chunjiang Hongxia (Camellia japonica cv. Chunjiang Hongxia, CH) possesses vivid red leaves from an early growth stage to a prolonged period and is, therefore, commercially valuable. The molecular mechanism underlying this red-leaf phenotype in C. japonica cv. CH is largely unknown. Here, we investigated the leaf coloration process, photosynthetic pigments contents, and different types of anthocyanin compounds in three growth stages of the hybrid cultivar CH and its parental cultivars. The gene co-expression network and differential expression analysis from the transcriptome data indicated that the changes of leaf color were strongly correlated to the anthocyanin metabolic processes in different leaf growth stages. Genes with expression patterns associated with leaf color changes were also discussed. Together, physiological and transcriptomic analyses uncovered the regulatory network of metabolism processes involved in the modulation of the ornamentally valuable red-leaf phenotype and provided the potential candidate genes for future molecular breeding of ornamental plants such as Camellia japonica.

Functional Diversification of the Dihydroflavonol 4-Reductase from Camellia nitidissima Chi. in the Control of Polyphenol Biosynthesis.

Plant secondary metabolism is complex in its diverse chemical composition and dynamic regulation of biosynthesis. How the functional diversification of enzymes contributes to the diversity is largely unknown. In the flavonoids pathway, dihydroflavonol 4-reductase (DFR) is a key enzyme mediating dihydroflavanol into anthocyanins biosynthesis. Here, the DFR homolog was identified from Camellia nitidissima Chi. (CnDFR) which is a unique species of the genus Camellia with golden yellow petals. Sequence analysis showed that CnDFR possessed not only conserved catalytic domains, but also some amino acids peculiar to Camellia species. Gene expression analysis revealed that CnDFR was expressed in all tissues and the expression of CnDFR was positively correlated with polyphenols but negatively with yellow coloration. The subcellular localization of CnDFR by the tobacco infiltration assay showed a likely dual localization in the nucleus and cell membrane. Furthermore, overexpression transgenic lines were generated in tobacco to understand the molecular function of CnDFR. The analyses of metabolites suggested that ectopic expression of CnDFR enhanced the biosynthesis of polyphenols, while no accumulation of anthocyanins was detected. These results indicate a functional diversification of the reductase activities in Camellia plants and provide molecular insights into the regulation of floral color.

Identification of alternatively spliced gene isoforms and novel noncoding RNAs by single-molecule long-read sequencing in Camellia.

Direct single-molecule sequencing of full-length transcripts allows efficient identification of gene isoforms, which is apt to alternative splicing (AS), polyadenylation, and long non-coding RNA analyses. However, the identification of gene isoforms and long non-coding RNAs with novel regulatory functions remains challenging, especially for species without a reference genome. Here, we present a comprehensive analysis of a combined long-read and short-read transcriptome sequencing in Camellia japonica. Through a novel bioinformatic pipeline of reverse-tracing the split-sites, we have uncovered 257,692 AS sites from 61,838 transcripts; and 13,068 AS isoforms have been validated by aligning the short reads. We have identified the tissue-specific AS isoforms along with 6,373 AS events that were found in all tissues. Furthermore, we have analysed the polyadenylation (polyA) patterns of transcripts, and found that the preference for polyA signals was different between the AS and non-AS transcripts. Moreover, we have predicted the phased small interfering RNA (phasiRNA) loci through integrative analyses of transcriptome and small RNA sequencing. We have shown that a newly evolved phasiRNA locus from lipoxygenases generated 12 consecutive 21 bp secondary RNAs, which were responsive to cold and heat stress in Camellia. Our studies of the isoform transcriptome provide insights into gene splicing and functions that may facilitate the mechanistic understanding of plants.

CjPLE, a PLENA-like gene, is a potential regulator of fruit development via activating the FRUITFUL homolog in Camellia.

Fruit patterning involves the cooperation of multiple processes, including metabolic change, cell differentiation, and cell expansion. The FRUITFUL (FUL) and SHATTERPROOF1/2 (SHPs) MADS-box genes are master regulators directing fruit patterning in several eudicots. However, the regulatory mechanisms of the FUL-SHP network in different fruit types remain unclear. Here, we characterized the functions of an ortholog (CjPLE) of SHPs from Camellia japonica. We showed that CjPLE was predominantly expressed in stamen and carpel tissues during the early stage of floral development and that transcripts were abundant in the pericarp tissues during fruit development. The ectopic expression of CjPLE in Arabidopsis caused enhanced development of the carpels, whereas no defects in floral identity were observed. To investigate the downstream targets of CjPLE, overexpression transformants were analysed through a callus transformation system in Camellia azalea. We examined the expression levels of potential downstream target genes and found that two previously identified APETALA1-like genes (CjAPL1/2) were significantly up-regulated. We showed that CjPLE directly bound to the CArG motifs in the promoter region of CjAPL1 (the FUL ortholog). Taken together, our results reveal a possible positive regulation of FUL by SHP in the control of fruit development in Camellia.

The complete chloroplast genome of Camellia vietnamensis, an economic shrub producing edible seed oil.

Camellia vietnamensis is an economic woody plant producing high-value edible oils, which is commonly found and cultivated in south areas of China. To provide genetic information for future genetic research, we have sequenced and assembled the complete chloroplast (cp) genome of C. vietnamensis based on the Illumina Hiseq platform. The total genome size is 161,958 bp in length with 37% GC, which contains a large single copy (LSC, 86,657 bp) region, a small single copy (SSC, 13,347 bp) region, and a pair of inverted repeat (IRs, 30,977 bp) regions. It is comprised of 81 protein-coding genes, 44 transfer RNAs and 4 ribosomal RNAs. To obtain the phylogeny relationship, the cp genome of C. vietnamensis has been compared with other Camellia species; the results indicate that C. vietnamensis is closely related to C. taliensis. This study provides fundamental information of C. vietnamensis cp genome, and it is valuable to the molecular phylogenetic and genetic diversity analyses in future.