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.

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.

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.

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.

Transcriptome and Degradome Profiling Reveals a Role of miR530 in the Circadian Regulation of Gene Expression in Kalanchoë marnieriana.

Crassulacean acid metabolism (CAM) is an important photosynthetic pathway for plant adaptation to dry environments. CAM plants feature a coordinated interaction between mesophyll and epidermis functions that involves refined regulations of gene expression. Plant microRNAs (miRNAs) are crucial post-transcription regulators of gene expression, however, their roles underlying the CAM pathway remain poorly investigated. Here, we present a study characterizing the expression of miRNAs in an obligate CAM species Kalanchoë marnieriana. Through sequencing of transcriptome and degradome in mesophyll and epidermal tissues under the drought treatments, we identified differentially expressed miRNAs that were potentially involved in the regulation of CAM. In total, we obtained 84 miRNA genes, and eight of them were determined to be Kalanchoë-specific miRNAs. It is widely accepted that CAM pathway is regulated by circadian clock. We showed that miR530 was substantially downregulated in epidermal peels under drought conditions; miR530 targeted two tandem zinc knuckle/PLU3 domain encoding genes (TZPs) that were potentially involved in light signaling and circadian clock pathways. Our work suggests that the miR530-TZPs module might play a role of regulating CAM-related gene expression in Kalanchoë.

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.

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.

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.

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.

Characterization of the complete chloroplast genome of Camellia yuhsienensis Hu, a resilient shrub with strong floral fragrance.

Camellia yuhsienensis Hu is an economically valuable species in the genus Camellia. It is widely used for breeding ornaments and oil varieties. In this study, the complete chloroplast (cp) genome sequence of C. yuhsienensis is assembled and annotated. The whole cp genome of C. yuhsienensis is 156,912 bp in size, composed of a small single copy (SSC) region of 18,296 bp and a large single copy (LSC) region of 86,560 bp, separated by a pair of inverted repeats (IRs, IRA: 86,561-112,588; IRB: 130,885-156,912). The overall GC content of C. yuhsienensis cp genome is 37.3%, with the base content A (31.08%), T (31.63%), C (19.02%), and G (18.27%). The phylogenetic analysis of 15 Camellia species based on 77 protein-coding genes shows that C. yuhsienensis is evolutionarily close to Camellia taliensis.

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.

A self-assembled graphene/polyurethane sponge for excellent electromagnetic interference shielding performance.

With the rapid development of personal computers and portable electronics, people have to get rid of a lot of unwanted electromagnetic pollution. The development of high performance electromagnetic interference (EMI) shielding materials is of critical importance to address ever-increasing military and civilian demand. Owing to its high electrical conductivity and flexible 3D structure, graphene sponge has great potential for excellent EMI shielding performance. However, its EMI shielding performance suffers from the material's poor elasticity and durability. In this paper, we demonstrate the potential of a self-assembled graphene/polyurethane sponge composite, synthesized via a two-step hydrothermal method, for EMI shielding. This kind of material exhibits a high specific EMI shielding effectiveness of 969-1578 dB cm2 g-1 which is comparable or even superior to traditional graphene/polymer sponges. The excellent EMI shielding performance originates from the superconductivity of graphene and the highly porous structure of the graphene/polyurethane sponge. It is found that the polyurethane sponge works as a robust scaffold for graphene to shape its 3D structure. This work introduces a facile yet efficient two-step hydrothermal approach to prepare a graphene/polyurethane sponge with excellent EMI shielding performance and good durability.

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.

Unraveling the Roles of Regulatory Genes during Domestication of Cultivated Camellia: Evidence and Insights from Comparative and Evolutionary Genomics.

With the increasing power of DNA sequencing, the genomics-based approach is becoming a promising resolution to dissect the molecular mechanism of domestication of complex traits in trees. Genus Camellia possesses rich resources with a substantial value for producing beverage, ornaments, edible oil and more. Currently, a vast number of genetic and genomic research studies in Camellia plants have emerged and provided an unprecedented opportunity to expedite the molecular breeding program. In this paper, we summarize the recent advances of gene expression and genomic resources in Camellia species and focus on identifying genes related to key economic traits such as flower and fruit development and stress tolerances. We investigate the genetic alterations and genomic impacts under different selection programs in closely related species. We discuss future directions of integrating large-scale population and quantitative genetics and multiple omics to identify key candidates to accelerate the breeding process. We propose that future work of exploiting the genomic data can provide insights related to the targets of domestication during breeding and the evolution of natural trait adaptations in genus Camellia.