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.

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.

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.

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.

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.

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.

Tissue-specific and maturity-dependent distribution of pyridine alkaloids in Areca triandra.

Areca nuts (seeds of Areca catechu L.) are a traditional and popular masticatory in India, Bangladesh, Malaysia, certain parts of China, and some other countries. Four related pyridine alkaloids (arecoline, arecaidine, guvacoline, and guvacine) are considered being the main functional ingredients in areca nut. Until now, A. catechu is the only known species producing these alkaloids in the Arecaceae family. In the present study, we investigated alkaloid contents in 12 Arecaceae species and found that only Areca triandra Roxb. contained these pyridine alkaloids. We further analyzed in more detail tissue-specific and development-related distribution of these alkaloids in leaves, male and female flowers and fruits in different stages of maturity in A. triandra by ultra-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry. Results revealed that the alkaloids were most abundant in young leaves, the pericarp of ripe fruits and the endosperm of unripe fruits in developmental stage 2. Abundance of the 4 different alkaloids in A. triandra fruits varied during maturation. Pericarps of ripe fruits had the highest arecaidine concentration (4.45 mg g-1) and the lowest guvacoline concentration (0.0175 mg g-1), whereas the endosperm of unripe fruits of developmental stage 2 contained the highest guvacoline concentration (3.39 mg g-1) and the lowest guvacine concentration (0.245 mg g-1). We conclude that A. triandra is useful in future as a further valuable source of Areca alkaloids.

The Pentratricopeptide Repeat Protein Pigment-Defective Mutant2 is Involved in the Regulation of Chloroplast Development and Chloroplast Gene Expression in Arabidopsis.

The development of functional chloroplasts, which is assisted by a series of nuclear-encoded auxiliary protein factors, is essential for plant autotrophic growth and development. To understand the molecular mechanisms underlying chloroplast development, we isolated and characterized a pigment-defective mutant, pdm2, and its corresponding variegated RNA interference (RNAi) lines in Arabidopsis. Sequence analysis revealed that PDM2 encodes a pentatricopeptide repeat protein that belongs to the P subgroup. Confocal microscopic analysis and immunoblotting of the chloroplast protein fraction showed that PDM2 was located in the stroma. In RNAi plants, protein-related photosynthesis was severely compromised. Furthermore, analysis of the transcript profile of chloroplast genes revealed that plastid-encoded polymerase-dependent transcript levels were markedly reduced, while nuclear-encoded polymerase-dependent transcript levels were increased, in RNAi plants. In addition, PDM2 affects plastid RNA editing efficiency in most editing sites, apparently by directly interacting with multiple organellar RNA editing factor 2 (MORF2) and MORF9. Thus, our results demonstrate that PDM2 is probably involved in the regulation of plastid gene expression required for normal chloroplast development.

Development of the photosynthetic apparatus of Cunninghamia lanceolata in light and darkness.

Here, we compared the development of dark- and light-grown Chinese fir (Cunninghamia lanceolata) cotyledons, which synthesize chlorophyll in the dark, representing a different phenomenon from angiosperm model plants. We determined that the grana lamellar membranes were well developed in both chloroplasts and etiochloroplasts. The accumulation of thylakoid membrane protein complexes was similar between chloroplasts and etiochloroplasts. Measurement of chlorophyll fluorescence parameters indicated that photosystem II (PSII) had low photosynthetic activities, whereas the photosystem I (PSI)-driven cyclic electron flow (CEF) rate exceeded the rate of PSII-mediated photon harvesting in etiochloroplasts. Analysis of the protein contents in etiochloroplasts indicated that the light-harvesting complex II remained mostly in its monomeric conformation. The ferredoxin NADP+ oxidoreductase and NADH dehydrogenase-like complexes were relatively abundantly expressed in etiochloroplasts for Chinese fir. Our transcriptome analysis contributes a global expression database for Chinese fir cotyledons, providing background information on the regulatory mechanisms of different genes involved in the development of dark- and light-grown cotyledons. In conclusion, we provide a novel description of the early developmental status of the light-dependent and light-independent photosynthetic apparatuses in gymnosperms.

Expression of Root Genes in Arabidopsis Seedlings Grown by Standard and Improved Growing Methods.

Roots of Arabidopsis thaliana seedlings grown in the laboratory using the traditional plant-growing culture system (TPG) were covered to maintain them in darkness. This new method is based on a dark chamber and is named the improved plant-growing method (IPG). We measured the light conditions in dark chambers, and found that the highest light intensity was dramatically reduced deeper in the dark chamber. In the bottom and side parts of dark chambers, roots were almost completely shaded. Using the high-throughput RNA sequencing method on the whole RNA extraction from roots, we compared the global gene expression levels in roots of seedlings from these two conditions and identified 141 differently expressed genes (DEGs) between them. According to the KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment, the flavone and flavonol biosynthesis and flavonoid biosynthesis pathways were most affected among all annotated pathways. Surprisingly, no genes of known plant photoreceptors were identified as DEGs by this method. Considering that the light intensity was decreased in the IPG system, we collected four sections (1.5 cm for each) of Arabidopsis roots grown in TPG and IPG conditions, and the spatial-related differential gene expression levels of plant photoreceptors and polar auxin transporters, including CRY1, CRY2, PHYA, PHYB, PHOT1, PHOT2, and UVR8 were analyzed by qRT-PCR. Using these results, we generated a map of the spatial-related expression patterns of these genes under IPG and TPG conditions. The expression levels of light-related genes in roots is highly sensitive to illumination and it provides a background reference for selecting an improved culture method for laboratory-maintained Arabidopsis seedlings.

Screening and Validation of Housekeeping Genes of the Root and Cotyledon of Cunninghamia lanceolata under Abiotic Stresses by Using Quantitative Real-Time PCR.

Cunninghamia lanceolata (Chinese fir) is a fast-growing and commercially important conifer of the Cupressaceae family. Due to the unavailability of complete genome sequences and relatively poor genetic background information of the Chinese fir, it is necessary to identify and analyze the expression levels of suitable housekeeping genes (HKGs) as internal reference for precise analysis. Based on the results of database analysis and transcriptome sequencing, we have chosen five candidate HKGs (Actin, GAPDH, EF1a, 18S rRNA, and UBQ) with conservative sequences in the Chinese fir and related species for quantitative analysis. The expression levels of these HKGs in roots and cotyledons under five different abiotic stresses in different time intervals were measured by qRT-PCR. The data were statistically analyzed using the following algorithms: NormFinder, BestKeeper, and geNorm. Finally, RankAggreg was applied to merge the sequences generated from three programs and rank these according to consensus sequences. The expression levels of these HKGs showed variable stabilities under different abiotic stresses. Among these, Actin was the most stable internal control in root, and GAPDH was the most stable housekeeping gene in cotyledon. We have also described an experimental procedure for selecting HKGs based on the de novo sequencing database of other non-model plants.

Genome-wide analysis reveals dynamic changes in expression of microRNAs during vascular cambium development in Chinese fir, Cunninghamia lanceolata.

MicroRNAs (miRNAs) are small noncoding regulatory RNAs that play key roles in the process of plant development. To date, extensive studies of miRNAs have been performed in a few model plants, but few efforts have focused on small RNAs (sRNAs) in conifers because of the lack of reference sequences for their enormous genomes. In this study, Solexa sequencing of three sRNA libraries obtained from dormant, reactivating, and active vascular cambium in Chinese fir (Cunninghamia lanceolata) using tangential cryosectioning identified 20 known miRNA families and 18 novel potential miRNAs, of which nine novel miRNA precursors were validated by RT-PCR and sequencing. More than half of these novel miRNAs displayed stage-specific expression patterns in the vascular cambium. Furthermore, analysing the 103 miRNAs and their predicted targets indicated that about 70% appeared to negatively regulate their targets, of which two target genes involved in the regulation of cambial cell division were validated via RNA ligase-mediated rapid amplification of 5'-cDNA ends (RLM 5'-RACE) and transient co-expression in Nicotiana benthamiana leaves. Interestingly, miRNA156 and miRNA172 may regulate the phase transition in vascular cambium from dormancy to active growth. These results provide new insights into the important regulatory functions of miRNAs in vascular cambium development and wood formation in conifers.

The signal transducer NPH3 integrates the phototropin1 photosensor with PIN2-based polar auxin transport in Arabidopsis root phototropism.

Under blue light (BL) illumination, Arabidopsis thaliana roots grow away from the light source, showing a negative phototropic response. However, the mechanism of root phototropism is still unclear. Using a noninvasive microelectrode system, we showed that the BL sensor phototropin1 (phot1), the signal transducer NONPHOTOTROPIC HYPOCOTYL3 (NPH3), and the auxin efflux transporter PIN2 were essential for BL-induced auxin flux in the root apex transition zone. We also found that PIN2-green fluorescent protein (GFP) localized to vacuole-like compartments (VLCs) in dark-grown root epidermal and cortical cells, and phot1/NPH3 mediated a BL-initiated pathway that caused PIN2 redistribution to the plasma membrane. When dark-grown roots were exposed to brefeldin A (BFA), PIN2-GFP remained in VLCs in darkness, and BL caused PIN2-GFP disappearance from VLCs and induced PIN2-GFP-FM4-64 colocalization within enlarged compartments. In the nph3 mutant, both dark and BL BFA treatments caused the disappearance of PIN2-GFP from VLCs. However, in the phot1 mutant, PIN2-GFP remained within VLCs under both dark and BL BFA treatments, suggesting that phot1 and NPH3 play different roles in PIN2 localization. In conclusion, BL-induced root phototropism is based on the phot1/NPH3 signaling pathway, which stimulates the shootward auxin flux by modifying the subcellular targeting of PIN2 in the root apex transition zone.

A membrane microdomain-associated protein, Arabidopsis Flot1, is involved in a clathrin-independent endocytic pathway and is required for seedling development.

Endocytosis is essential for the maintenance of protein and lipid compositions in the plasma membrane and for the acquisition of materials from the extracellular space. Clathrin-dependent and -independent endocytic processes are well established in yeast and animals; however, endocytic pathways involved in cargo internalization and intracellular trafficking remain to be fully elucidated for plants. Here, we used transgenic green fluorescent protein-flotillin1 (GFP-Flot1) Arabidopsis thaliana plants in combination with confocal microscopy analysis and transmission electron microscopy immunogold labeling to study the spatial and dynamic aspects of GFP-Flot1-positive vesicle formation. Vesicle size, as outlined by the gold particles, was ∼100 nm, which is larger than the 30-nm size of clathrin-coated vesicles. GFP-Flot1 also did not colocalize with clathrin light chain-mOrange. Variable-angle total internal reflection fluorescence microscopy also revealed that the dynamic behavior of GFP-Flot1-positive puncta was different from that of clathrin light chain-mOrange puncta. Furthermore, disruption of membrane microdomains caused a significant alteration in the dynamics of Flot1-positive puncta. Analysis of artificial microRNA Flot1 transgenic Arabidopsis lines established that a reduction in Flot1 transcript levels gave rise to a reduction in shoot and root meristem size plus retardation in seedling growth. Taken together, these findings support the hypothesis that, in plant cells, Flot1 is involved in a clathrin-independent endocytic pathway and functions in seedling development.

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.

γ-Aminobutyric acid (GABA) homeostasis regulates pollen germination and polarized growth in Picea wilsonii.

γ-Aminobutyric acid (GABA) is a four-carbon non-protein amino acid found in a wide range of organisms. Recently, GABA accumulation has been shown to play a role in the stress response and cell growth in angiosperms. However, the effect of GABA deficiency on pollen tube development remains unclear. Here, we demonstrated that specific concentrations of exogenous GABA stimulated pollen tube growth in Picea wilsonii, while an overdose suppressed pollen tube elongation. The germination percentage of pollen grains and morphological variations in pollen tubes responded in a dose-dependent manner to treatment with 3-mercaptopropionic acid (3-MP), a glutamate decarboxylase inhibitor, while the inhibitory effects could be recovered in calcium-containing medium supplemented with GABA. Using immunofluorescence labeling, we found that the actin cables were disorganized in 3-MP treated cells, followed by the transition of endo/exocytosis activating sites from the apex to the whole tube shank. In addition, variations in the deposition of cell wall components were detected upon labeling with JIM5, JIM7, and aniline blue. Our results demonstrated that calcium-dependent GABA signaling regulates pollen germination and polarized tube growth in P. wilsonii by affecting actin filament patterns, vesicle trafficking, and the configuration and distribution of cell wall components.

The regulation of cambial activity in Chinese fir (Cunninghamia lanceolata) involves extensive transcriptome remodeling.

Chinese fir (Cunninghamia lanceolata), a commercially important tree for the timber and pulp industry, is widely distributed in southern China and northern Vietnam, but its large and complex genome has hindered the development of genomic resources. Few efforts have focused on analysis of the modulation of transcriptional networks in vascular cambium during the transition from active growth to dormancy in conifers. Here, we used Illumina sequencing to analyze the global transcriptome alterations at the different stages of vascular cambium development in Chinese fir. By analyzing dynamic changes in the transcriptome of vascular cambium based on our RNA sequencing (RNA-Seq) data at the dormant, reactivating and active stages, many potentially interesting genes were identified that encoded putative regulators of cambial activity, cell division, cell expansion and cell wall biosynthesis and modification. In particular, the genes involved in transcriptional regulation and hormone signaling were highlighted to reveal their biological importance in the cambium development and wood formation. Our results reveal the dynamics of transcriptional networks and identify potential key components in the regulation of vascular cambium development in Chinese fir, which will contribute to the in-depth study of cambial differentiation and wood-forming candidate genes in conifers.

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.

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.