Integrated metabolomic and transcriptomic analysis revealed the flavonoid biosynthesis and regulation in Areca catechu.

INTRODUCTION: Flavonoids are active substances in many herbal medicines, and Areca catechu fruit (AF), an important component in traditional Chinese medicine (TCM), is rich in flavonoids. Different parts of AF, Pericarpium Arecae (PA) and Semen Arecae (SA), have different medicinal effects in prescription of TCM. OBJECTIVE: To understand flavonoid biosynthesis and regulation in AF. METHODOLOGY: The metabolomic based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the transcriptome based on high-throughput sequencing technology were combined to comprehensively analyse PA and SA. RESULTS: From the metabolite dataset, we found that 148 flavonoids showed significant differences between PA and SA. From the transcriptomic dataset, we identified 30 genes related to the flavonoid biosynthesis pathway which were differentially expressed genes in PA and SA. The genes encoding the key enzymes in the flavonoid biosynthesis pathway, chalcone synthase and chalcone isomerase (AcCHS4/6/7 and AcCHI1/2/3), were significantly higher expressed in SA than in PA, reflecting the high flavonoid concentration in SA. CONCLUSIONS: Taken together, our research acquired the key genes, including AcCHS4/6/7 and AcCHI1/2/3, which regulated the accumulation of flavonol in AF. This new evidence may reveal different medicinal effects of PA and SA. This study lays a foundation for investigating the biosynthesis and regulation of flavonoid biosynthesis in areca and provides the reference for the production and consumption of betel nut.

Genome-Wide Association Analysis of Fruit Shape-Related Traits in Areca catechu.

The areca palm (Areca catechu L.) is one of the most economically important palm trees in tropical areas. To inform areca breeding programs, it is critical to characterize the genetic bases of the mechanisms that regulate areca fruit shape and to identify candidate genes related to fruit-shape traits. However, few previous studies have mined candidate genes associated with areca fruit shape. Here, the fruits produced by 137 areca germplasms were divided into three categories (spherical, oval, and columnar) based on the fruit shape index. A total of 45,094 high-quality single-nucleotide polymorphisms (SNPs) were identified across the 137 areca cultivars. Phylogenetic analysis clustered the areca cultivars into four subgroups. A genome-wide association study that used a mixed linear model identified the 200 loci that were the most significantly associated with fruit-shape traits in the germplasms. In addition, 86 candidate genes associated with areca fruit-shape traits were further mined. Among the proteins encoded by these candidate genes were UDP-glucosyltransferase 85A2, the ABA-responsive element binding factor GBF4, E3 ubiquitin-protein ligase SIAH1, and LRR receptor-like serine/threonine-protein kinase ERECTA. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that the gene that encoded UDP-glycosyltransferase, UGT85A2, was significantly upregulated in columnar fruits as compared to spherical and oval fruits. The identification of molecular markers that are closely related to fruit-shape traits not only provides genetic data for areca breeding, but it also provides new insights into the shape formation mechanisms of drupes.

Cryopreservation of Arecanut (Areca catechu L.) Pollen.

BACKGROUND: Cryopreservation opens new avenues in the field of genetic resource conservation, especially in recalcitrant seeded palms such as arecanut for which field genebanks are exposed to pest and disease attacks and natural calamities. It is only through cryopreservation that the safety of the conserved germplasm can be assured at a relatively low cost for extended periods. OBJECTIVE: The objective of this work was to standardize various aspects of arecanut pollen cryopreservation, viz. collection and desiccation of pollen, in vitro germination, viability and fecundity studies. MATERIALS AND METHODS: Pollens of three arecanut genotypes (Sumangala, Hirehalli Dwarf and Hirehalli Dwarf x Sumangala) were collected in December 2013-February 2014. In vitro viability tests were conducted using fresh and desiccated pollen. Desiccated pollen was cryopreserved by direct immersion in liquid nitrogen and cryostored for different durations (24 hours to 2 years). Viability and fertility studies were conducted using cryopreserved pollen. RESULTS: Pollen extraction was achieved from fully opened male flowers by desiccation at room temperature (33-34 degree C). A medium containing 2.5 g/L sucrose was found to be best for in vitro germination at room temperature. There was no significant difference in germination between desiccated and cryopreserved pollen whereas pollen tube length decreased significantly after cryopreservation. Fertility studies using HD x Sumangala pollen cryostored for various durations (1 month, 1 year and 2 years) showed the setting of 70, 43 and 62%, respectively. Normal nut set was observed using cryopreserved pollen. CONCLUSION: Pollen cryopreservation is a viable option for germplasm conservation and hybridization programmes in arecanut.

Genetic variation detected by RAPD markers in natural populations of babassu palm (Attalea speciosa Mart.).

The purpose of this study was to analyze the effects of management on the genetic structure of natural populations of Attalea speciosa in the State of Piauí, Brazil, using random-amplified polymorphic DNA (RAPD) markers. Three babassu populations under different management systems were selected. Polymerase chain reactions were performed for 20 RAPD primers. A total of 146 bands were generated, 141 of which were polymorphic (96.58%), with a variation of 4 and 12 loci and an average of 7 bands per primer. A dendrogram revealed a clear separation between the three populations (0.57). Data reliability and node consistency were verified by bootstrap values and the cophenetic correlation coefficient (88.15%). Coefficients of similarity between pairs of genotypes ranged from 0.26 to 0.86, with a mean of 0.57. Nei's genetic diversity index (HE) value of the population sampled in Teresina was 0.212, of Esperantina it was 0.195, and of José de Freitas it was 0.207. After the HE was decomposed, the complete diversity was found to be 0.3213. Genetic differentiation between populations was 0.362, and the estimation of gene flow between populations was low (0.879). Analysis of molecular variance revealed that 59.52% of the variation was contained within populations, and 40.48% was between populations. RAPD markers were effective for genetic diversity analysis within and between natural babassu populations, and exhibited a high level of polymorphism. Genetic diversity was the highest within populations; variability was lower in the managed populations than in the undisturbed populations.

Gene co-expression network analysis in areca floral organ and the potential role of the AcMADS17 and AcMADS23 in transgenic Arabidopsis.

Areca catechu L., a monocot belonging to the palm family, is monoecious, with female and male flowers separately distributed on the same inflorescence. To discover the molecular mechanism of flower development in Areca, we sequenced different floral samples to generate tissue-specific transcriptomic profiles. We conducted a comparative analysis of the transcriptomic profiles of apical sections of the inflorescence with male flowers and the basal section of the inflorescence with female flowers. Based on the RNA sequencing dataset, we applied weighted gene co-expression network analysis (WGCNA) to identify sepal, petal, stamen, stigma and other specific modules as well as hub genes involved in specific floral organ development. The syntenic and expression patterns of AcMADS-box genes were analyzed in detail. Furthermore, we analyzed the open chromatin regions and transcription factor PI binding sites in male and female flowers by assay for transposase-accessible chromatin sequencing (ATAC-seq) assay. Heterologous expression revealed the important role of AcMADS17 and AcMADS23 in floral organ development. Our results provide a valuable genomic resource for the functional analysis of floral organ development in Areca.

Molecular Signature of Long Non-Coding RNA Associated with Areca Nut-Induced Head and Neck Cancer.

The areca nut is a high-risk carcinogen for head and neck cancer (HNC) patients in Southeast Asia. The underlying molecular mechanism of areca nut-induced HNC remains unclear, especially regarding the role of long non-coding RNA (lncRNA). This study employed a systemic strategy to identify lncRNA signatures related to areca nut-induced HNC. In total, 84 cancer-related lncRNAs were identified. Using a PCR array method, 28 lncRNAs were identified as being dysregulated in HNC cells treated with areca nut (17 upregulated and 11 downregulated). Using bioinformatics analysis of The Cancer Genome Atlas Head-Neck Squamous Cell Carcinoma (TCGA-HNSC) dataset, 45 lncRNAs were differentially expressed in tumor tissues from HNC patients (39 over- and 6 under-expressions). The integrated evaluation showed 10 lncRNAs dysregulated by the areca nut and altered expression in patients, suggesting that these panel molecules participate in areca nut-induced HNC. Five oncogenic (LUCAT1, MIR31HG, UCA1, HIF1A-AS2, and SUMO1P3) and tumor-suppressive (LINC00312) lncRNAs were independently validated, and three key molecules were further examined. Pathway prediction revealed that LUCAT1, UCA1, and MIR31HG modulate multiple oncogenic mechanisms, including stress response and cellular motility. Clinical assessment showed that these lncRNAs exhibited biomarker potentials in diagnosis (area under the curve = 0.815 for LUCAT1) and a worse prognosis (both p < 0.05, survival analysis). Cellular studies further demonstrated that MIR31HG facilitates areca nut-induced cancer progression, as silencing this molecule attenuated arecoline-induced invasion ability in HNC cells. This study identified lncRNA signatures that play a role in areca nut-induced HNC. These molecules may be further applied in risk assessment, diagnosis, prognosis, and therapeutics for areca nut-associated malignancies.

Genome skims analysis of betel palms (Areca spp., Arecaceae) and development of a profiling method to assess their plastome diversity.

The betel nut (Areca catechu L., Arecaceae) is a monoecious cultivated palm tree that is widespread in tropical regions. It is mainly cultivated for producing areca nuts, from which seeds are extracted and chewed by local populations principally in the Indo-Pacific region. Seeds contain alkaloids which are central nervous system stimulants and are highly addictive. Wild relatives of the betel nut are distributed in South Asia and Australasia, with ca. 40-50 Areca species currently recognized. The geographic origin(s) of the betel nut and its subsequent diffusion and diversification remains poorly documented. Here, a genome skimming approach was applied to screen nucleotidic variation in the most abundant genomic regions. Low coverage sequencing data allowed us to assemble full plastomes, mitochondrial regions (either full mitogenomes or the full set of mitochondrial genes) and the nuclear ribosomal DNA cluster for nine representatives of the Areca genus collected in the field and herbarium collections (including a 182-years old specimen collected during the Dumont d'Urville's expedition). These three genomic compartments provided similar phylogenetic signals, and revealed very low genomic diversity in our sample of cultivated betel nut. We finally developed a genotyping method targeting 34 plastid DNA microsatellites. This plastome profiling approach is useful for tracing the spread of matrilineages, and in combination with nuclear genomic data, can resolve the history of the betel nut. Our method also proves to be efficient for analyzing herbarium specimens, even those collected >100 years ago.