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.

Multi-omics analysis revealed the mechanism underlying flavonol biosynthesis during petal color formation in Camellia Nitidissima.

BACKGROUND: Camellia nitidissima is a rare, prized camellia species with golden-yellow flowers. It has a high ornamental, medicinal, and economic value. Previous studies have shown substantial flavonol accumulation in C. nitidissima petals during flower formation. However, the mechanisms underlying the golden flower formation in C. nitidissima remain largely unknown. RESULTS: We performed an integrative analysis of the transcriptome, proteome, and metabolome of the petals at five flower developmental stages to construct the regulatory network underlying golden flower formation in C. nitidissima. Metabolome analysis revealed the presence of 323 flavonoids, and two flavonols, quercetin glycosides and kaempferol glycosides, were highly accumulated in the golden petals. Transcriptome and proteome sequencing suggested that the flavonol biosynthesis-related genes and proteins upregulated and the anthocyanin and proanthocyanidin biosynthesis-related genes and proteins downregulated in the golden petal stage. Further investigation revealed the involvement of MYBs and bHLHs in flavonoid biosynthesis. Expression analysis showed that flavonol synthase 2 (CnFLS2) was highly expressed in the petals, and its expression positively correlated with flavonol content at all flower developmental stages. Transient overexpression of CnFLS2 in the petals increased flavonol content. Furthermore, correlation analysis showed that the jasmonate (JA) pathways positively correlated with flavonol biosynthesis, and exogenous methyl jasmonate (MeJA) treatment promoted CnFLS2 expression and flavonol accumulation. CONCLUSIONS: Our findings showed that the JA-CnFLS2 module regulates flavonol biosynthesis during golden petal formation in C. nitidissima.

PmHs1pro-1 monitors Bsursaphelenchus xylophilus infection and activates defensive response in resistant Pinus massoniana.

Plant resistance (R) genes play a crucial role in the detection of effector proteins secreted by pathogens, either directly or indirectly, as well as in the subsequent activation of downstream defence mechanisms. However, little is known about how R genes regulate the defence responses of conifers, particularly Pinus massoniana, against the destructive pine wood nematode (PWN; Bursaphelenchus xylophilus). Here, we isolated and characterised PmHs1pro-1, a nematode-resistance gene of P. massoniana, using bioinformatics, molecular biology, histochemistry and transgenesis. Tissue-specific expressional pattern and localisation of PmHs1pro-1 suggested that it was a crucial positive regulator in response to PWN attack in resistant P. massoniana. Meanwhile, overexpression of PmHs1pro-1 was found to activate reactive oxygen species (ROS) metabolism-related enzymes and the expressional level of their key genes, including superoxide dismutase, peroxidase and catalase. In addition, we showed that PmHs1pro-1 directly recognised the effector protein BxSCD1of PWN, and induced the ROS burst responding to PWN invasion in resistant P. massoniana. Our findings illustrated the molecular framework of R genes directly recognising the effector protein of pathology in pine, which offered a novel insight into the plant-pathogen arms race.

Integrative Analysis of Small RNA and Gene Expression Uncovers a Novel Long-Noncoding MicroRNA Targeting a UDP-Transferase in Camellia chekiangoleosa.

Biosynthesis of specific secondary metabolites in plants involves fine regulation of gene expression. Camellia chekiangoleosa has important economic value: the seeds contain high-quality unsaturated fatty acids and the pericarp is rich in tea saponins. As an important posttranscriptional regulator, the role of microRNAs (miRNAs) in controlling secondary metabolism in C. chekiangoleosa is not fully studied. Here, we investigated the role of miRNAs and their targets in the secondary metabolic regulatory network by comprehensively analyzing small RNAs, transcriptomes, and degradomes from different tissues. We identified 168 known miRNAs and 74 novel miRNAs in the C. chekiangoleosa genome and revealed 15 tandem clusters containing 35 miRNAs. By establishing a gene regulatory network containing miRNAs, target genes, and transcription factors, we unravelled the multiplicity of miRNA tissue-specific regulation of gene expression, which may be tightly linked to the synthesis of secondary metabolites. Furthermore, we characterized a novel long-noncoding miRNA gene (cch-miR3633) that targeted a UDP-transferase gene (CchUGT94E5). We showed that, ectopic expression of CchUGT94E5 caused outgrowth of shoot branching and changes in cytokinin contents in Arabidopsis, indicating a potential role of regulating secondary metabolism. This work provides valuable information for the study of miRNA regulation of secondary metabolism.

Genome-Wide Analysis of MADS-Box Gene Family Reveals CjSTK as a Key Regulator of Seed Abortion in Camellia japonica.

The plant MADS-box transcription factor family is a major regulator of plant flower development and reproduction, and the AGAMOUS-LIKE11/SEEDSTICK (AGL11/STK) subfamily plays conserved functions in the seed development of flowering plants. Camellia japonica is a world-famous ornamental flower, and its seed kernels are rich in highly valuable fatty acids. Seed abortion has been found to be common in C. japonica, but little is known about how it is regulated during seed development. In this study, we performed a genome-wide analysis of the MADS-box gene the in C. japonica genome and identified 126 MADS-box genes. Through gene expression profiling in various tissue types, we revealed the C/D-class MADS-box genes were preferentially expressed in seed-related tissues. We identified the AGL11/STK-like gene, CjSTK, and showed that it contained a typical STK motif and exclusively expressed during seed development. We found a significant increase in the CjSTK expression level in aborted seeds compared with normally developing seeds. Furthermore, overexpression of CjSTK in Arabidopsis thaliana caused shorter pods and smaller seeds. Taken together, we concluded that the fine regulation of the CjSTK expression at different stages of seed development is critical for ovule formation and seed abortion in C. japonica. The present study provides evidence revealing the regulation of seed development in Camellia.

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.

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.

IsoSplitter: identification and characterization of alternative splicing sites without a reference genome.

Long-read transcriptome sequencing is designed to sequence full-length RNA molecules and advantageous for identifying alternative splice isoforms; however, in the absence of a reference genome, it is difficult to accurately locate splice sites, because of the diversity of patterns of alternative splicing (AS). Based on long-read transcriptome data we developed a versatile tool, IsoSplitter, to reverse-trace and validate AS gene "split-sites" with the following features: (1) IsoSplitter initially invokes a modified SIM4 program to find transcript split-sites; (2) each split-site is then quantified, to reveal transcript diversity, and putative isoforms are grouped into gene clusters; (3) an optional step for aligning short-reads is provided, to validate split-sites by identifying unique junction reads, and revealing and quantifying tissue-specific alternative splice isoforms. We tested IsoSplitter AS prediction using datasets from multiple model and non-model plant species, and showed that IsoSplitter pipeline is efficient to handle different transcriptomes with high accuracy. Furthermore, we evaluated the IsoSplitter pipeline compared with that of the splice junction identification tools, Program to Assemble Spliced Alignments (PASA-software needs a reference genome for AS identification) and AStrap, using data from the model plant Arabidopsis thaliana. We found that, IsoSplitter determined more than twice as many AS events than AStrap analysis; and 94.13% of the IsoSplitter predicted AS events were also identified by the PASA analysis. Starting from a simple sequence file, IsoSplitter is an assembly-free tool for identification and characterization of AS. IsoSplitter is developed and implemented in Python 3.5 using the Linux platform and is freely available at https://github.com/Hengfu-Yin/IsoSplitter.

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.

Characterizations of a Class-I BASIC PENTACYSTEINE Gene Reveal Conserved Roles in the Transcriptional Repression of Genes Involved in Seed Development.

The developmental regulation of flower organs involves the spatio-temporal regulation of floral homeotic genes. BASIC PENTACYSTEINE genes are plant-specific transcription factors that is involved in many aspects of plant development through gene transcriptional regulation. Although studies have shown that the BPC genes are involved in the developmental regulation of flower organs, little is known about their role in the formation of double-flower due. Here we characterized a Class I BPC gene (CjBPC1) from an ornamental flower-Camellia japonica. We showed that CjBPC1 is highly expressed in the central whorls of flowers in both single and doubled varieties. Overexpression of CjBPC1 in Arabidopsis thaliana caused severe defects in siliques and seeds. We found that genes involved in ovule and seed development, including SEEDSTICK, LEAFY COTYLEDON2, ABSCISIC ACID INSENSITIVE 3 and FUSCA3, were significantly down-regulated in transgenic lines. We showed that the histone 3 lysine 27 methylation levels of these downstream genes were enhanced in the transgenic plants, indicating conserved roles of CjBPC1 in recruiting the Polycomb Repression Complex for gene suppression.

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.

Transcriptomic Analysis Reveals Key Candidate Genes Related to Seed Abortion in Chinese Jujube (Ziziphus jujuba Mill).

Background: Seed abortion is a common phenomenon in Chinese jujube that seriously hinders the process of cross-breeding. However, the molecular mechanisms of seed abortion remain unclear in jujube. Methods: Here, we performed transcriptome sequencing using eight flower and fruit tissues at different developmental stages in Ziziphus jujuba Mill. 'Zhongqiusucui' to identify key genes related to seed abortion. Histological analysis revealed a critical developmental process of embryo abortion after fertilization. Results: Comparisons of gene expression revealed a total of 14,012 differentially expressed genes. Functional enrichment analyses of differentially expressed genes between various sample types uncovered several important biological processes, such as embryo development, cellular metabolism, and stress response, that were potentially involved in the regulation of seed abortion. Furthermore, gene co-expression network analysis revealed a suite of potential key genes related to ovule and seed development. We focused on three types of candidate genes, agamous subfamily genes, plant ATP-binding cassette subfamily G transporters, and metacaspase enzymes, and showed that the expression profiles of some members were associated with embryo abortion. Conclusion: This work generates a comprehensive gene expression data source for unraveling the molecular mechanisms of seed abortion and aids future cross-breeding efforts in jujube.

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.

Alternative Polyadenylation in response to temperature stress contributes to gene regulation in Populus trichocarpa.

BACKGROUND: Genome-wide change of polyadenylation (polyA) sites (also known as alternative polyadenylation, APA) is emerging as an important strategy of gene regulation in response to stress in plants. But little is known in woody perennials that are persistently dealing with multiple abiotic stresses. RESULTS: Here, we performed a genome-wide profiling of polyadenylation sites under heat and cold treatments in Populus trichocarpa. Through a comprehensive analysis of polyA tail sequences, we identified 25,919 polyA-site clusters (PACs), and revealed 3429 and 3139 genes shifted polyA sites under heat and cold stresses respectively. We found that a small proportion of genes possessed APA that affected the open reading frames; and some shifts were commonly identified. Functional analysis of genes displaying shifted polyA tails suggested that pathways related to RNA metabolism were linked to regulate the APA events under both heat and cold stresses. Interestingly, we found that the heat stress induced a significantly more antisense PACs comparing to cold and control conditions. Furthermore, we showed that a unique cis-element (AAAAAA) was predominately enriched downstream of PACs in P. trichocarpa genes; and this sequence signal was only absent in shifted PACs under the heat condition, indicating a distinct APA mechanism responsive to heat tolerance. CONCLUSIONS: This work provides a comprehensive picture of global polyadenylation patterns in response to temperatures stresses in trees. We show that the frequent change of polyA tail is a potential mechanism of gene regulation responsive to stress, which are associated with distinctive sequence signatures.

CcBLH6, a bell-like homeodomain-containing transcription factor, regulates the fruit lignification pattern.

MAIN CONCLUSION: CcBLH6 is a bell-like homeodomain-containing transcription factor that plays an important role of lignin biosynthesis in the control of fruit lignification pattern in Camellia chekiangoleosa. The fruit of Camellia chekiangoleosa has a unique lignification pattern that features with a thick pericarp containing a low level of lignification. Yet the fruit lignification pattern and the regulatory network of responsible gene transcription are poorly understood. Here, we characterized a bell-like homeodomain-containing (BLH) transcription factor from C. chekiangoleosa, CcBLH6, in the control of fruit lignification. CcBLH6 expression was highly correlated with the unique lignification pattern during fruit development. The ectopic expression of CcBLH6 promoted the lignification process of stem and root in Arabidopsis. We found that expression of genes related to lignin biosynthesis and its transcriptional regulation was altered in transgenic lines. In a Camellia callus-transformation system, overexpression of CcBLH6 greatly enhanced the expression of genes related to lignin biosynthesis and its transcriptional regulation was altered in transgenic lines. In the callus-transformation system, overexpression of CcBLH6 greatly enhanced the lignification of parenchymal cells, and the regulation of several genes involved in lignin accumulation was largely consistent between Arabidopsis and Camellia. Our study reveals a positive role of CcBLH6 in the regulation of lignin biosynthesis during fruit lignification in Camellia.

Two terpene synthases in resistant Pinus massoniana contribute to defence against Bursaphelenchus xylophilus.

Pine wood nematode (PWN; Bursaphelenchus xylophilus), a destructive pest of Pinus massoniana, is causing a severe epidemic of pine wilt disease in China. When invaded by PWN, resistant P. massoniana secretes an abundance of oleoresin terpenoids as a defensive strategy. However, regulatory mechanisms of this defence in resistant P. massoniana have yet to be elucidated. Here, we characterized two terpene synthase genes, α-pinene synthase (PmTPS4) and longifolene synthase (PmTPS21), identified in resistant P. massoniana and investigate the contribution of these genes to the oleoresin defence strategy in resistant masson pines. Up-regulation of these two genes in the stem supported their involvement in terpene biosynthesis as part of the defence against PWN. Recombinant protein expression revealed catalytic activity for the two PmTPSs, with PmTPS4 primarily producing α-pinene, while PmTPS21 produced α-pinene and longifolene simultaneously. The major enzymatic products of the two terpene synthases had inhibitory effects on PWN in vitro. We demonstrated that PmTPS4 and PmTPS21 played positive roles in terpene-defence mechanisms against PWN infestation. The major products of these terpene synthases could directly inhibit the survival rate of PWN in vitro. We revealed that PmTPS21 was a novel bifunctional enzyme capable of simultaneous production of both monoterpene and sesquiterpene.

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.

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.

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.