Cytotoxic lignans from the barks of <i>Juglans mandshurica</i>

<p>Phytochemical investigation of the barks of <i>Juglans mandshurica</i> Maxim led to the isolation, purification, and identification of one new lignan named Juglansol A (<b>1</b>), along with nine known compounds (<b>2–10</b>). Their structures were determined by the results of UV, IR, CD, HRESIMS, 1D, and 2D NMR spectroscopic analysis. Compounds <b>1–10</b> were evaluated for their cytotoxicities against A549, HepG2, Hep3B, Bcap-37, and MCF-7 cell lines. The results showed that compound <b>2</b> possessed stronger cytotoxicities against the tested tumor cell lines compared with positive control 5-fluorouracil.</p

A New Mutation, <i>hap1-2</i>, Reveals a C Terminal Domain Function in AtMago Protein and Its Biological Effects in Male Gametophyte Development in <i>Arabidopsis thaliana</i>

<div><p>The exon-exon junction complex (EJC) is a conserved eukaryotic multiprotein complex that examines the quality of and determines the availability of messenger RNAs (mRNAs) posttranscriptionally. Four proteins, MAGO, Y14, eIF4AIII and BTZ, function as core components of the EJC. The mechanisms of their interactions and the biological indications of these interactions are still poorly understood in plants. A new mutation, <i>hap1-2</i>. leads to premature pollen death and a reduced seed production in Arabidopsis. This mutation introduces a viable truncated transcript <i>AtMagoΔC</i>. This truncation abolishes the interaction between AtMago and AtY14 <i>in vitro</i>, but not the interaction between AtMago and AteIF4AIII. In addition to a strong nuclear presence of AtMago, both AtMago and AtMagoΔC exhibit processing-body (P-body) localization. This indicates that AtMagoΔC may replace AtMago in the EJC when aberrant transcripts are to be degraded. When introducing an NMD mutation, <i>upf3-1</i>, into the existing <i>HAP1/hap1-2</i> mutant, plants showed a severely reduced fertility. However, the change of splicing pattern of a subset of SR protein transcripts is mostly correlated with the <i>sr45-1</i> and <i>upf3-1</i> mutations, not the <i>hap1-2</i> mutation. These results imply that the C terminal domain (CTD) of AtMago is required for the AtMago-AtY14 heterodimerization during EJC assembly, UPF3-mediated NMD pathway and the AtMago-AtY14 heterodimerization work synergistically to regulate male gametophyte development in plants.</p></div

Defects in early pollen development in the <i>HAP1/hap1-2</i> mutant.

<p>A–D: Pollen stained by DAPI. (A): <i>qrt</i> mature pollen grains; (B): <i>qrt;HAP1/hap1-2</i> in unicellular stage; (C): <i>qrt;HAP1/hap1-2</i> in bicellular stage; and (D): <i>qrt;HAP1/hap1-2</i> mature pollen grain. E—H: HTR12GFP showing the distribution of centromere histone H3 in <i>qrt;HAP1/hap1-2</i>. Bright dots show detected HTR12GFP representing condensed chromosomes in microspores (E-F), bicellular stage (G) and the mature pollen grain (H) in the <i>qrt;HAP1/hap1-2</i> mutant. Recognizable mutant haploids are indicated by white arrow. Examples of micronuclei are indicated by red arrow. Scale bar = 10 μm.</p

Morphology of flower and anther from different EJC mutants, and their seed yield.

<p>Stage 15 flowers from <i>qrt</i> (A), <i>qrt;HAP1/hap1-2</i> (B), <i>qrt;upf3-1</i> (C), <i>qrt;sr45-1</i> (D), <i>qrt;sr45-1; HAP1/hap1-2</i> (E), <i>qrt;upf3-1;HAP1/hap1-2</i> (F) mutant plants. Small inserts show a close view of representative anthers. Pollen formation within the anther from <i>qrt</i> (G), <i>qrt;HAP1/hap1-2</i> (H), <i>qrt;upf3-1</i> (I), <i>qrt;upf3-1;HAP1/hap1-2</i> (J). Scale bar = 100 μm. (K) Seed yield as a percentage to WT (<i>qrt</i>) was calculated from 16 siliques. Student t-test with Bonferroni correction was used for statistical analysis. Statistical significance (<i>p</i><0.001) is shown as a-d.</p

Both AtMago and AtMagoΔC are detected in P-bodies.

<p>Scale bar = 50 μm. A zoom-in view is shown for each image with a scale bar of 10 μm.</p

Alternative splicing pattern of SR protein genes in WT, <i>HAP1/hap1-2</i>, <i>upf3-1</i>, <i>sr45-1</i>, <i>upf3-1;HAP1/hap1-2</i> (<i>uh</i>) and <i>sr45-1;HAP1/hap1-2</i> (<i>sh</i>) mutant inflorescence tissues.

<p>(A) Alternative splicing pattern was examined on <i>RS40</i>, <i>RS41</i>, <i>RS31</i>, <i>SCL33</i>, <i>SR30</i>, <i>SCL30a</i>, <i>RS31a</i>. <i>GAPDH</i> was used as control. Each band was corresponding to the each splicing isoform. The UTRs were presented by open boxes; the exons were presented by black boxes; the alternatively spliced exons were presented gray boxes, the introns were presented by lines. The position of gene-specific primers was shown by black arrowhead and the stop codons were shown as *. (B) The alternative splicing pattern of selective SR protein gene transcripts was confirmed by qPCR. The ratio of different splicing isoforms (AS) of each genotype was compared to WT (<i>qrt</i>) for student t-test (<i>n</i> = 3). * represents <i>p</i><0.05 and ** represents <i>p</i><0.01.</p

CTD of AtMago is required for AtMago-AtY14 interaction, but not for AtMago-AteIF4AIII.

<p>Western blots showing results from <i>in vitro</i> pull-down assay for AtMagoΔC-AtY14 (A), AtMagoΔC-AteIF4AIII (B), and various AtMago C terminal truncations-AtY14 (C).</p

Characterization and complementation of the <i>HAP1/hap1-2</i> mutant.

<p>(A) <i>HAP1</i> gene structure. Exons were shown as colored boxes; introns were shown as straight lines; UTRs were shown as gray boxes. The T-DNA insertion in <i>hap1-1</i> and <i>hap1-2</i> was indicated by red arrow. (B) rtPCR results showed the expression of a full-length transcript (<i>AtMago</i>), a truncated transcript (<i>AtMagoΔC</i>) and <i>GAPDH</i> in <i>qrt</i>, <i>qrt;HAP1/hap1-2</i>, <i>qrt;sr45-1</i> and <i>qrt;upf3-1</i> mutant plants. Total RNAs were extracted from the inflorescence tissue. The exon composition of the transcript and the position of used primers were shown next to the corresponding rtPCR results. <b>(C)</b> GUS stained pollen grains from <i>qrt</i>, <i>qrt;HAP1/hap1-1</i> and <i>qrt;HAP1/hap1-2</i> showing the identity of mutant pollen grains. Scale bar = 20 μm. (D) Seed number per silique in <i>qrt</i> and <i>qrt;HAP1/hap1-2</i> plants. Scale bar = 1 mm. The quantification was done with sixteen siliques. Student t-test was used for statistical analysis. Error bars represent standard deviations. (E) <i>HAP1</i> gene complemented <i>hap1-2</i> mutant phenotype. The mature plants, GUS-stained pollen grains and seed yield per plant were shown in <i>qrt</i>, <i>qrt;HAP1/hap1-2</i>, <i>HAP1;qrt; hap1-2</i>. (F) qPCR results showing that the <i>AtMago</i> gene expression level was fully recovered in the transgenic plant. <i>GAPDH</i> was used as control. Three biological replicates were used in the analysis. Error bar showed standard deviation. Statistical significance compared to <i>qrt</i> was measured by student t-test (<i>p</i> <0.001).</p

Metabolomics Analysis Reveals that Ethylene and Methyl Jasmonate Regulate Different Branch Pathways to Promote the Accumulation of Terpenoid Indole Alkaloids in <i>Catharanthus roseus</i>

The medicinal plant <i>Catharanthus roseus</i> accumulates large numbers of terpenoid indole alkaloids (TIAs), including the pharmaceutically important vinblastine, vincristine, ajmalicine, and serpentine. The phytohormone ethylene or methyl jasmonate (MeJA) can markedly enhance alkaloid accumulation. The interaction between ethylene or MeJA in the regulation of TIA biosynthesis in <i>C. roseus</i> is unknown. Here, a metabolomics platform is reported that is based on liquid chromatography (LC) coupled with time-of-flight mass spectrometry to study candidate components for TIA biosynthesis, which is controlled by ethylene or MeJA in <i>C. roseus</i>. Multivariate analysis identified 16 potential metabolites mostly associated with TIA metabolic pathways and seven targeted metabolites, outlining the TIA biosynthesis metabolic networks controlled by ethylene or MeJA. Interestingly, ethylene and MeJA regulate the 2-<i>C</i>-methyl-d-erythritol 4-phosphate (MEP) and acetate-mevalonate (MVA) pathways through <i>AACT</i> and <i>HMGS</i> and through <i>DXS</i>, respectively, to induce TIA biosynthesis in <i>C. roseus</i>. Overall, both nontargeted and targeted metabolomics, as well as transcript analysis, were used to reveal that MeJA and ethylene control different metabolic networks to induce TIA biosynthesis

Additional file 7: Figure S4. of Transcriptome analyses reveal SR45 to be a neutral splicing regulator and a suppressor of innate immunity in Arabidopsis thaliana

A flowchart showing the pipeline used to identify alternative splicing events based on AtRTD2. (TIFF 258 kb