Genome-wide identification of heavy-metal ATPases genes in Areca catechu: investigating their functionality under heavy metal exposure.

Heavy-metal ATPases (HMAs) play a vital role in plants, helping to transport heavy metal ions across cell membranes.However, insufficient data exists concerning HMAs genes within the Arecaceae family.In this study, 12 AcHMA genes were identified within the genome of Areca catechu, grouped into two main clusters based on their phylogenetic relationships.Genomic distribution analysis reveals that the AcHMA genes were unevenly distributed across six chromosomes. We further analyzed their physicochemical properties, collinearity, and gene structure.Furthermore, RNA-seq data analysis exhibited varied expressions in different tissues of A. catechu and found that AcHMA1, AcHMA2, and AcHMA7 were highly expressed in roots, leaves, pericarp, and male/female flowers. A total of six AcHMA candidate genes were selected based on gene expression patterns, and their expression in the roots and leaves was determined using RT-qPCR under heavy metal stress. Results showed that the expression levels of AcHMA1 and AcHMA3 genes were significantly up-regulated under Cd2 + and Zn2 + stress. Similarly, in response to Cu2+, the AcHMA5 and AcHMA8 revealed the highest expression in roots and leaves, respectively. In conclusion, this study will offer a foundation for exploring the role of the HMAs gene family in dealing with heavy metal stress conditions in A. catechu.

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.

Individual and combined effects of herbicide prometryn and nitrate enrichment at environmentally relevant concentrations on photosynthesis, oxidative stress, and endosymbiont community diversity of coral Acropora hyacinthus.

Nitrogen pollution and pesticides such as photosystem II (PSII) inhibitor herbicides have several detrimental impacts on coral reefs, including breakdown of the symbiosis between host corals and photosynthetic symbionts. Although nitrogen and PSII herbicide pollution separately cause coral bleaching, the combined effects of these stressors at environmentally relevant concentrations on corals have not been assessed. Here, we report the combined effects of nitrate enrichment and PSII herbicide (prometryn) exposure on photosynthesis, oxidative status and endosymbiont community diversity of the reef-building coral Acropora hyacinthus. Coral fragments were exposed in a mesocosm system to nitrate enrichment (9 µmol/L) and two prometryn concentrations (1 and 5 µg/L). The results showed that sustained prometryn exposure in combination with nitrate enrichment stress had significant detrimental impacts on photosynthetic apparatus [the maximum quantum efficiency of photosystem II (Fv/Fm), nonphotochemical quenching (NPQ) and oxidative status in the short term. Nevertheless, the adaptive mechanism of corals allowed the normal physiological state to be recovered following 1 µg/L prometryn and 9 µmol/L nitrate enrichment individual exposure. Moreover, exposure for 9 days was insufficient to trigger a shift in Symbiodiniaceae community. Most importantly, the negative impact of exposure to the combined environmental concentrations of 1 µg/L prometryn and 9 µmol/L nitrate enrichment was found to be significantly greater on the Fv/Fm, quantum yield of non-regulated energy dissipation [Y(NO)], NPQ, and oxidative status of corals compared to the impact of individual stressors. Our results show that interactions between prometryn stress and nitrate enrichment have a synergistic impact on the photosynthetic and oxidative stress responses of corals. This study provides valuable insights into combined effects of nitrate enrichment and PSII herbicides pollution for coral's physiology. Environmental concentrations of PSII herbicides may be more harmful to photosystems and antioxidant systems of corals under nitrate enrichment stress. Thus, future research and management of seawater quality stressors should consider combined impacts on corals rather than just the impacts of individual stressors alone.

A vector-free gene interference system using delaminated Mg-Al-lactate layered double hydroxide nanosheets as molecular carriers to intact plant cells.

BACKGROUND: The Mg-Al-lactate layered double hydroxide nanosheet (LDH-NS) has shown great potential as an optimal nanocarrier for extensive use in plants. However, previous studies in plant sciences have not provided a clear description of the application for the LDH-NSs-based double-stranded RNA (dsRNA) delivery (LDH-dsRNA) system in different tissues of both model and non-model species. RESULTS: LDH-NSs were synthesized by using the co-precipitation method, while the dsRNAs targeting genes of interest were prepared in vitro using T7 RNA polymerase. The LDH-dsRNA bioconjugates with a neutral charge were produced by incubating with the mass ratio of LDH-NSs to dsRNA at 3:1, which were then introduced into intact plant cells using three different approaches, including injection, spray, and soak. The LDH-dsRNA delivery method was optimized by inhibiting the expression of the Arabidopsis thaliana ACTIN2 gene. As a result, soaking A. thaliana seedlings in a medium containing LDH-dsRNA for 30 min led to the silencing of 80% of the target genes. The stability and effectiveness of the LDH-dsRNA system were further confirmed by the high-efficiency knockdown of plant tissue-specific genes, including that encoding phytoene desaturase (PDS), WUSCHEL (WUS), WUSCHEL-related homeobox 5 (WOX5), and ROOT HAIR DEFECTIVE 6 (RHD6). In addition, the LDH-dsRNA system was employed in cassava, where it was found that the expression of the gene encoding nucleotide-binding site and leucine-rich repeat (NBS-LRR) was significantly reduced. As a result, the resistance of cassava leaves to pathogens was weakened. Noteworthy, the injection of LDH-dsRNA into leaves resulted in a significant downregulation of target genes in both stems and flowers, indicating the successful transport of LDH-dsRNA from leaves to other parts of plants. CONCLUSIONS: LDH-NSs have proven to be a highly effective molecular tool for delivering dsRNA into intact plant cells, enabling accurate control of target gene expression.

Systematic Analysis of NRAMP Family Genes in Areca catechu and Its Response to Zn/Fe Deficiency Stress.

Areca catechu is a commercially important medicinal plant widely cultivated in tropical regions. The natural resistance-associated macrophage protein (NRAMP) is widespread in plants and plays critical roles in transporting metal ions, plant growth, and development. However, the information on NRAMPs in A. catechu is quite limited. In this study, we identified 12 NRAMPs genes in the areca genome, which were classified into five groups by phylogenetic analysis. Subcellular localization analysis reveals that, except for NRAMP2, NRAMP3, and NRAMP11, which are localized in chloroplasts, all other NRAMPs are localized on the plasma membrane. Genomic distribution analysis shows that 12 NRAMPs genes are unevenly spread on seven chromosomes. Sequence analysis shows that motif 1 and motif 6 are highly conserved motifs in 12 NRAMPs. Synteny analysis provided deep insight into the evolutionary characteristics of AcNRAMP genes. Among the A. catechu and the other three representative species, we identified a total of 19 syntenic gene pairs. Analysis of Ka/Ks values indicates that AcNRAMP genes are subjected to purifying selection in the evolutionary process. Analysis of cis-acting elements reveals that AcNRAMP genes promoter sequences contain light-responsive elements, defense- and stress-responsive elements, and plant growth/development-responsive elements. Expression profiling confirms distinct expression patterns of AcNRAMP genes in different organs and responses to Zn/Fe deficiency stress in leaves and roots. Taken together, our results lay a foundation for further exploration of the AcNRAMPs regulatory function in areca response to Fe and Zn deficiency.

Transcriptome and targeted metabolomic integrated analysis reveals mechanisms of B vitamin accumulation in Areca catechu nut development.

Areca catechu is well known as a medicinal plant that has high nutritional and medicinal benefits. However, the metabolism and regulatory mechanism of B vitamins during areca nut development remain largely unclear. In this study, we obtained the metabolite profiles of six B vitamins during different areca nut developmental stages by targeted metabolomics. Furthermore, we obtained a panoramic expression profile of genes related to the biosynthetic pathway of B vitamins in areca nuts at different developmental stages using RNA-seq. In total, 88 structural genes related to B vitamin biosynthesis were identified. Furthermore, the integrated analysis of B vitamin metabolism data and RNA-seq data showed the key transcription factors regulating thiamine and riboflavin accumulation in areca nuts, including AcbZIP21, AcMYB84, and AcARF32. These results lay the foundation for understanding metabolite accumulation and the molecular regulatory mechanisms of B vitamins in A. catechu 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.

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.

The genome of Areca catechu provides insights into sex determination of monoecious plants.

The areca palm (Areca catechu) has a monoecious spadix, with male flowers on the apical side and females on the basal side. Here, we applied multiomics analysis to investigate sex determination and floral organ development in areca palms. We generated a chromosome-level reference genome of A. catechu with 16 pseudochromosomes, composed of 2.73 Gb and encoding 31 406 genes. Data from RNA-seq and ATAC-seq (assay for transposase accessible chromatin sequencing) suggested that jasmonic acid (JA) synthesis and signal transduction-related genes were differentially expressed between female and male flowers via epigenetic modifications. JA concentration in female flowers was c. 10 times than that in males on the same inflorescence, while JA concentration in hermaphroditic flowers of abnormal inflorescences was about twice that in male flowers of normal inflorescences. JA promotes the development of female flower organs by decreasing the expression of B-function genes, including AGL16, AP3, PIb and PIc. There is also a region on pseudochromosome 15 harboring sex-related genes, including CYP703, LOG, GPAT, AMS and BiP. Among them, CYP703, AMS and BiP were specifically expressed in male flowers.

AtCPS V326M significantly affect the biosynthesis of gibberellins.

Gibberellins are a class of typical phytohormones, which regulate plant growth and development. The contents of gibberellins dramatically affect the morphology and biomass of plant. The encoding protein of copalyl diphosphate synthase gene (CPS) catalyzes the first-step in the biosynthetic pathway of gibberellins. The mutation in this gene may significantly affect the contents of gibberellins in plants. In this study, we found an EMS-triggered mutant, ga1-168, showing short roots, short hypocotyls, late flowering and dwarf. Map-based cloning revealed that the causal gene of ga1-168 was AtCPS-168, an allele of AtCPS gene. The encoding protein of AtCPS-168 was AtCPS V326M which was resulted from a single-point mutation (guanine to adenine at nucleotide 2768) of AtCPS gene. Protein domain analysis showed that V326 was located in the Terpene_synth domain. The allelism test demonstrated that AtCPS-168 was an allele of AtCPS gene. The transgenic complementation of ga1-168 indicated that AtCPS V326M led to the dwarf and bushy phenotype of ga1-168. The endogenous gibberellins contents analysis suggested that the gibberellins contents of ga1-168 were much lower than that of wild-type. The exogenous GA3 application assay uncovered that application of GA3 can complement the dwarf and bushy phenotype of ga1-168 caused by low endogenous gibberellins contents. Therefore, this study suggested that it is an elegant way to create the ideal plant architecture and height by site-directed mutating the gibberellin biosynthetic genes.

Overexpression of a Gene Encoding Trigonelline Synthase from Areca catechu L. Promotes Drought Resilience in Transgenic Arabidopsis.

Areca catechu L. is a commercially important palm tree widely cultured in tropical and subtropical areas. Its growth and production are severely hindered by the increasing threat of drought. In the present study, we investigated the physiological responses of areca seedlings to drought stress. The results showed that prolonged drought-induced yellowing on the overall area of most leaves significantly altered the chlorophyll fluorescence parameters, including maximum chemical efficiency (Fv/Fm), photochemical efficiency of PSII (Y(II)), photochemical chlorophyll fluorescence quenching (qP) and non-photochemical chlorophyll fluorescence quenching (NPQ). On the 10th day of drought treatment, the contents of proline in the areca leaves and roots increased, respectively, by 12.2 times and 8.4 times compared to normal watering. The trigonelline levels in the leaves rose from 695.35 µg/g to 1125.21 µg/g under 10 days of water shortage, while no significant changes were detected in the content of trigonelline in the roots. We determined the gene encoding areca trigonelline synthase (AcTS) by conducting a bioinformatic search of the areca genome database. Sequence analysis revealed that AcTS is highly homologous to the trigonelline synthases in Coffea arabica (CaTS 1 and CaTS 2) and all possess a conserved S-adenosyl- L-methionine binding motif. The overexpression of AcTS in Arabidopsis thaliana demonstrated that AcTS is responsible for the generation of trigonelline in transgenic Arabidopsis, which in turn improves the drought resilience of transgenic Arabidopsis. This finding enriches our understanding of the molecular regulatory mechanism of the response of areca to water shortage and provides a foundation for improving the drought tolerance of areca seedlings.

The Cassava NBS-LRR Genes Confer Resistance to Cassava Bacterial Blight.

Cassava bacterial blight (CBB) caused by Xanthomonas axonopodis pv. manihotis (Xam) seriously affects cassava yield. Genes encoding nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains are among the most important disease resistance genes in plants that are specifically involved in the response to diverse pathogens. However, the in vivo roles of NBS-LRR remain unclear in cassava (Manihot esculenta). In this study, we isolated four MeLRR genes and assessed their expression under salicylic acid (SA) treatment and Xam inoculation. Four MeLRR genes positively regulate cassava disease general resistance against Xam via virus-induced gene silencing (VIGS) and transient overexpression. During cassava-Xam interaction, MeLRRs positively regulated endogenous SA and reactive oxygen species (ROS) accumulation and pathogenesis-related gene 1 (PR1) transcripts. Additionally, we revealed that MeLRRs positively regulated disease resistance in Arabidopsis. These pathogenic microorganisms include Pseudomonas syringae pv. tomato, Alternaria brassicicola, and Botrytis cinerea. Our findings shed light on the molecular mechanism underlying the regulation of cassava resistance against Xam inoculation.

Optimized synthesis of layered double hydroxide lactate nanosheets and their biological effects on Arabidopsis seedlings.

BACKGROUND: Layered double hydroxide lactate nanosheets (LDH-lactate-NS) are powerful carriers for delivering macro-molecules into intact plant cells. In the past few years, some studies have been carried out on DNA/RNA transformation and plant disease resistance, but little attention has been paid to these factors during LDH-lactate-NS synthesis and delamination, nor has their relationship to the DNA adsorption capacity or transformation efficiency of plant cells been considered. RESULTS: Since the temperature during delamination alters particle sizes and zeta potentials of LDH-lactate-NS products, we compared the LDH-lactate-NS stability, DNA adsorption rate and delivery efficiency of fluorescein isothiocyanate isomer I (FITC) of them, found that the LDH-lactate-NS obtained at 25 °C has the best characters for delivering biomolecules into plant cell. To understand the potential side effects and cytotoxicity of LDH-lactate-NS to plants, we compared the root growth rate between the Arabidopsis thaliana seedlings grown in the culture medium with 1-300 µg/mL LDH-lactate-NS and equivalent raw material, Mg(lactate)2 and Al (lactate)3. Phenotypic analysis showed LDH in a range of 1-300 µg/mL can enhance the root elongation, whereas the same concentration of raw materials dramatically inhibited root elongation, suggesting the nanocrystallization has a dramatical de-toxic effect to Mg(lactate)2 and Al (lactate)3. Since enhancing of root elongation by LDH is an unexpected phenomenon, we further designed experiments to investigate influence of LDH to Arabidopsis seedlings. We further used the gravitropic bending test, qRT-PCR analysis of auxin transport proteins, non-invasive micro-test technology and liquid chromatography-mass spectrometry to investigate the auxin transport and distribution in Arabidopsis root. Results indicated that LDH-lactate-NS affect root growth by increasing the polar auxin transport. CONCLUSIONS: Optimal synthesized LDH-lactate-NS can delivery biomolecules into intact plant cells with high efficiency and low cytotoxity. The working solution of LDH-lactate-NS can promote root elongation via increase the polar auxin transport in Arabidopsis roots.

Genome-Wide Identification of PIN and PILS Gene Families in Areca catechu and the Potential Role of AcPIN6 in Lateral Brace Root Formation.

PIN-FORMED (PIN) and PIN-LIKES (PILS) are two families of auxin transporters that control the directional cell-to-cell transport and intracellular accumulation of auxin, thereby influencing plant growth and development. Most knowledge of PINs and PILSs was obtained from the dicot model plant Arabidopsis thaliana. Here, we focus on the distribution and expression of the PIN and PILS gene families in areca palm (Areca catechu), a monocot tree. The whole genomic dataset of areca palm was used to identify twelve AcPINs and eight AcPILSs, and a phylogenetic tree was constructed of PINS and PILS together with several other palm species, including the date palm (Phoenix dactylifera), oil palm (Elaeis guineensis), and coconut (Cocos nucifera). We further analyzed the expression patterns of AcPIN and AcPILS in areca palm, and found that AcPIN6 displayed an extremely high transcriptional abundance in the brace roots and was extremely stimulated in the lateral root primordium. This result implies that AcPIN6 plays an important role in the growth and formation of brace roots, especially in lateral root initiation. We also overexpressed AcPIN6 and AcPIN6-eGFP in Arabidopsis, and the results revealed that the PIN6 localized on the plasma membrane and affected auxin-related phenomena. Taken together, we analyzed the evolutionary relationships of PINs and PILSs in palm species, and the roles of PIN6 in areca palm root formation. The results will improve the understanding of root system construction in large palm trees.

Effect of static magnetic field on marine mollusc Elysia leucolegnote.

Artificial magnetic fields are unavoidable environment for offshore marine organisms. With the substantially increasing submarine cables, the impact of magnetic field generated by cables on marine organisms has gradually attracted people's attention. However, there are few studies on the effect of magnetic field on molluscs. To explore whether magnetic fields could interfere with the physiological functions of offshore molluscs, here we systematically analyzed the change of metabolism and transcriptome of Elysia leucolegnote exposed to either geomagnetic field or 1.1 T static magnetic field. The blood glucose and lipid levels, as well as the activities of antioxidant enzymes in E. leucolegnote were significantly increased upon the exposure to high static magnetic field for 10 days. Meanwhile, the activities of enzymes related to digestive performance and liver functions were decreased. Possible mechanisms were further revealed through comparative transcriptome analysis. A total of 836 differentially expressed genes were identified, 352 of which were up-regulated and 484 of which were down-regulated after exposure to the high static magnetic field. The up-regulated differential genes were mainly concentrated in lysosomal and apoptotic pathways, and down-regulated differential genes were mainly involved in digestive and immune systems including phagocytosis. This pattern was further confirmed by RT-qPCR analysis. In conclusion, prolonged exposure to a 1.1 T static magnetic field increased oxidative stress and blood glucose and lipid levels, and decreased immunity and physiological conditions in E. leucolegnote. The data we presented here provides a comprehensive view of metabolism change and gene expression pattern of E. leucolegnote exposed to static magnetic field. It may expand our knowledge on the magnetic field effects on offshore mollusc at molecular level, and contribute to clarification of the interaction between marine animals and artificial magnetic fields, which is certainly ecologically important.

Genome-wide investigation of ZINC-IRON PERMEASE (ZIP) genes in Areca catechu and potential roles of ZIPs in Fe and Zn uptake and transport.

Iron (Fe) and Zinc (Zn) are essential nutrient elements for plant growth and development. Here, we observed the effects of Fe and Zn deficiency in seedlings of Areca catechu L. (areca palm), one of the most cultured palm trees in tropic regions. Results revealed that Fe deficiency causes strong chlorosis with the significantly decreased chlorophyll biosynthesis level and photosynthetic activities in the top third young leaf (L3) of seedlings. Zn deficiency caused light chlorosis in all three young leaves which slightly decreased chlorophyll biosynthesis and photosynthetic activities. Analysis of the Fe and Zn concentration in leaves and roots indicated that absorption and distribution of these two ions share cooperative pathways, since Zn deficiency caused Fe increasing, and vice versa. Therefore, we focused on the ZINC-IRON PERMEASE (ZIP) genes in areca trees. From the whole-genome data set we obtained, 6 ZIP genes were classified, and a phylogenetic tree was constructed with other 38 ZIP genes from model plants to find their potential functions. We also analyzed the expression pattern of AcZIP1-6 genes under Zn and Fe deficiency by transcriptomic approaches. With these results, we constructed an expression atlas of AcZIP1-6 genes in leaves and roots of areca seedlings with the dynamic expression levels under Fe and Zn deficient conditions. In conclusion, we provide evidence to understand the absorption and transport of nutrient elements, Fe and Zn, in the tropic agricultural plant A. catechu.

An Antisense Circular RNA Regulates Expression of RuBisCO Small Subunit Genes in Arabidopsis.

Circular RNA (circRNA) is a novel class of endogenous long non-coding RNA (lncRNA) and participates in diverse physiological process in plants. From the dataset obtained by high-throughput RNA sequencing, we identified a circRNA encoded by the sense strand of the exon regions spanning two RuBisCO small subunit genes, RBCS2B and RBCS3B, in Arabidopsis thaliana. We further applied the single specific primer-polymerase chain reaction (PCR) and Sanger sequencing techniques to verify this circRNA and named it ag-circRBCS (antisense and across genic-circular RNA RBCS). Using quantitative real-time PCR (qRT-PCR), we found that ag-circRBCS shares a similar rhythmic expression pattern with other RBCS genes. The expression level of ag-circRBCS is 10-40 times lower than the expression levels of RBCS genes in the photosynthetic organs in Arabidopsis, whereas the Arabidopsis root lacked ag-circRBCS expression. Furthermore, we used the delaminated layered double hydroxide lactate nanosheets (LDH-lactate-NS) to deliver in vitro synthesized ag-circRBCS into Arabidopsis seedlings. Our results indicate that ag-circRBCS could significantly depress the expression of RBCS. Given that ag-circRBCS was expressed at low concentration in vivo, we suggest that ag-circRBCS may represent a fine-tuning mechanism to regulating the expression of RBCS genes and protein content in Arabidopsis.

Identification and expression profiling of genes involved in circadian clock regulation in red dragon fruit (Hylocereus polyrhizus) by full-length transcriptome sequencing.

Crassulacean acid metabolism (CAM) plants fix CO2 at night, exhibiting a reversed regulatory pattern of metabolomic pathways compared with most model plants, which have C3 and C4 pathways. In this study, we used a valuable tropic fruit, red dragon fruit (Hylocereus polyrhizus), as model plant to identify and analyze the circadian regulation genes. Due to the absence of red dragon fruit's whole-genome dataset, we established a full-length transcriptome dataset using single-molecule real-time (SMRT) sequencing method. A 7.66-Gb dataset with 4,552,474 subreads was generated, with an average length of 1,683 bp and an N50 of 2,446 bp. Using this dataset, we identified center oscillator genes: CCA1 (CIRCADIAN CLOCK ASSOCIATED1), ELF3 (EARLY FLOWERING 3), GI (GIGANTEA), LHY (LATE ELONGATED HYPOCOTYL), LNK1 (NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED 1), and TOC1 (TIMING OF CAB EXPRESSION 1); a gene for the input pathway: CRY1 (CRYPTOCHROME); a gene for the output pathway: CO (CONSTANS); and genes related to the CAM pathway: MDH (MALATE DEHYDROGENASE), ME (MALIC ENZYMES), and PPDK (PYRUVATE PHOSPHATE DIKINASE). We further established the 24-h rhythmic expression pattern of these genes and classified these into three groups: HpCCA1, HpELF3, HpLHY, HpLNK1, and HpGI have expression peaks during the day; HpTOC1, HpCO, and HpCRY1 have highest expression levels at night; The genes involved in the CAM pathways, namely, HpMDH, HpME1, and HpPPDK, have double expression peaks in the day and night. Comparison of these expression patterns between red dragon fruit and model plants could provide clues in understanding the circadian clock regulation and the activity of the CAM pathways in cactus plants.

Identifying new compounds with potential pharmaceutical and physiological activity in Areca catechu and Areca triandra via a non-targeted metabolomic approach.

INTRODUCTION: The fruits of Areca catechu, also called areca nuts, are widely used as popular masticatory and traditional herbal medicine in Asia. Besides arecoline and related alkaloids, limited information is available about further primary and secondary metabolites and their potential biological activities. OBJECTIVE: Here we aimed to further enhance our knowledge on phytochemical profiles of A. catechu and Areca triandra fruits. We intended to comprehensively identify metabolites in A. catechu and A. triandra fruits. METHODOLOGY: Metabolites were identified by ultra-performance liquid chromatography triple-quadrupole tandem mass spectrometry (UPLC-MS/MS). The occurrence of 12 selected bioactive compounds in 4 different developmental stages of A. catechu and A. triandra was quantified by LC-MS/MS. RESULTS: A total of 791 metabolites was identified. Of these, 115 metabolites could successfully be mapped to 44 Kyoto Encyclopedia of Genes and Genomes metabolic pathways, and 154 metabolites occurred at significantly different levels in A. catechu compared to A. triandra. Several components with known biological activities were identified for the first time in A. catechu and A. triandra. The abundance of many of these new components was similar in A. catechu and A. triandra, but significantly different between the pericarp and the seeds of A. catechu fruits. CONCLUSIONS: Metabolic profiles indicate that fruits of the Areca species compared here have similar primary and secondary metabolites. Our findings provide new insights into A. catechu and A. triandra as valuable sources for traditional medicine and they pave the way for further studies to potentially improve the underlying pharmaceutical and physiological effects.