Viral DNA analysis in <i>N. benthamiana</i> leaf discs and Arabidopsis protoplasts infected with wild-type and mutated BSCTV.

<p>(A) Southern blot analysis of total nucleic acids extracted from leaf discs at 0, 5, 10, 15d after agro-inoculated with wild-type and mutated BSCTV. (B) Southern blot analysis of total nucleic acids extracted from Arabidopsis protoplasts at 0 and 4d after transfection of plasmids containing wild-type and mutated BSCTV.</p

Diagnostic PCR analysis of BSCTV in infected newly emerged leaves of C4 transgenic Arabidopsis.

<p>Genomic DNAs from each of 10 individual plants infected with BSCTV-m1 (A) and BSCTV-m2 (B) were used as templates. P indicates positive control (infected with wild-type BSCTV) and N indicates negative control (not infected with virus). Lane 1 shows DNA from the plants produced symptoms, and lanes 2–10 show DNA from plants without symptoms.</p

The position of two mutations in C4 protein and disease symptoms in <i>N. benthamiana</i> and Arabidopsis infected by BSCTV C4 mutants.

<p>(A) Amino acid sequences of C4 and two C4 mutants created by nucleotide substitution (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011280#s4" target="_blank">materials and methods</a>). Numbers indicate the position of the first and the last amino acid. (B) <i>N. benthamiana</i> plants infected with wild-type and C4 mutated BSCTV two weeks after agro-inoculation. (C) Arabidopsis plants infected with wild-type and C4 mutated BSCTV two weeks after agro-inoculation. a shows 20 plants infected with wild-type, BSCTV-m1 and BSCTV-m2 respectively. b, c, d shows the individual plant infected with wild-type, BSCTV-m1 and BSCTV-m2 respectively.</p

Binding affinity of C4 protein with dsDNA and ssDNA.

<p>(A) Protein-DNA binding assay for C4 binding double strand BSCTV DNA. BSCTV fragments digested by <i>Eco</i>RI were end-labeled with [α- ³²P]dATP and Klenow polynucleotide kinase as a probe. Increasing amounts of C4 proteins (2–5 µg) were incubated with 50 ng probes at 22°C for 30 min. Gels were dried and the migration of labeled DNA was detected by a PhosphorImager. (B) Increasing amounts of C4 protein mixed with 0.5 µg 1 kb linear dsDNA ladder. DNAs were detected by visualization of ethidium bromide stained gels. C) Increasing amounts of C4 protein mixed with 0.2 µg M13 ssDNA. DNAs were detected by visualization of ethidium bromide stained gels. All mixtures were analyzed in 0.7% nondenaturing agarose gels in TBE buffer. The protein/DNA complexes are indicated.</p

Subcellular localization of C4 protein.

<p>A) The fluorescence observation of control GFP protein in Arabidopsis protoplasts. Bar  = 10 µm. (B) The fluorescence observation of GFP-C4 fusion protein in Arabidopsis protoplasts. Bar is common to (A). (C) The fluorescence observation of control GFP protein in <i>N. benthamiana</i> leaves. Bar  = 20 µm. (D) The fluorescence observation of GFP-C4 fusion protein in <i>N. benthamiana</i> leaves. Bar is common to (C). Cells were analyzed by confocol microscopy. F indicates fluorescence, B indicates bright light and M indicates merged. (E) Cell fraction assays of GFP and GFP-C4 fusion protein. Total extract of <i>N. benthamiana</i> leaf cells expressing a GFP control and GFP-C4 fusion proteins were fractionated into soluble (S) and microsomal (M) fractions. GFP and GFP-C4 fusion proteins were detected using a anti-GFP antibody and indicated (top panel). Ponceau S staining of the transferred membrane is displayed as a loading control (bottom panel).</p

Viral DNA analysis in <i>N. benthamiana</i> and Arabidopsis plants agro-inoculated with wild-type and mutated BSCTV.

<p>(A) Southern blot analysis of total nucleic acids extracted from agro-inoculated (0 and 2d) and newly emerged (0, 2, 8, 10, 13, 16 and 20d) leaves of <i>N. benthamiana</i> after agro-inoculation. (B) Southern blot analysis of total nucleic acids extracted from newly emerged leaves of Arabidopsis two weeks after agro-inoculation. Size marker (the first lane on the left) was a mixture of BSCTV fragments digested by <i>Tth</i>111I and <i>Eco</i>RI, and the fragment digested by <i>Eco</i>RI. Fragment sizes are given in kb. The positions of open circle (oc), linear (lin), supercoiled (sc) and single stranded (ss) DNAs, and a population of subgenomic DNA forms are indicated.</p

Integrated Transcriptomic and Metabolomic Analyses Reveal the Molecular and Metabolic Basis of Flavonoids in Areca catechu L.

Areca catechu L., of the Arecaceae family, is widely distributed in tropical Asia. In A. catechu, the extracts and compounds, including flavonoids, have various pharmacological activities. Although there are many studies of flavonoids, the molecular mechanism of their biosynthesis and regulation remains unclear in A. catechu. In this study, 331 metabolites were identified from the root, stem, and leaf of A. catechu using untargeted metabolomics, including 107 flavonoids, 71 lipids, 44 amino acids and derivatives, and 33 alkaloids. The transcriptome analysis identified 6119 differentially expressed genes, and some were enriched in the flavonoid pathway. To analyze the biosynthetic mechanism of the metabolic differences in A. catechu tissues, 36 genes were identified through combined transcriptomic and metabolomic analysis, in which glycosyltransferase genes Acat_15g017010 and Acat_16g013670 were annotated as being involved in the glycosylation of kaempferol and chrysin by their expression and in vitro activities. Flavonoid biosynthesis could be regulated by the transcription factors, AcMYB5 and AcMYB194. This study laid a foundation for further research on the flavonoid biosynthetic pathway of A. catechu

Integrated Transcriptomic and Metabolomic Analyses Reveal the Molecular and Metabolic Basis of Flavonoids in Areca catechu L.

Areca catechu L., of the Arecaceae family, is widely distributed in tropical Asia. In A. catechu, the extracts and compounds, including flavonoids, have various pharmacological activities. Although there are many studies of flavonoids, the molecular mechanism of their biosynthesis and regulation remains unclear in A. catechu. In this study, 331 metabolites were identified from the root, stem, and leaf of A. catechu using untargeted metabolomics, including 107 flavonoids, 71 lipids, 44 amino acids and derivatives, and 33 alkaloids. The transcriptome analysis identified 6119 differentially expressed genes, and some were enriched in the flavonoid pathway. To analyze the biosynthetic mechanism of the metabolic differences in A. catechu tissues, 36 genes were identified through combined transcriptomic and metabolomic analysis, in which glycosyltransferase genes Acat_15g017010 and Acat_16g013670 were annotated as being involved in the glycosylation of kaempferol and chrysin by their expression and in vitro activities. Flavonoid biosynthesis could be regulated by the transcription factors, AcMYB5 and AcMYB194. This study laid a foundation for further research on the flavonoid biosynthetic pathway of A. catechu