MicroRNA156 (miR156) functions in maintaining the juvenile phase in plants. However, the mobility of this microRNA has not been demonstrated. So far, only three microRNAs, miR399, miR395, and miR172, have been shown to be mobile. We demonstrate here that miR156 is a potential graft-transmissible signal that affects plant architecture and tuberization in potato (Solanum tuberosum). Under tuber-noninductive (long-day) conditions, miR156 shows higher abundance in leaves and stems, whereas an increase in abundance of miR156 has been observed in stolons under tuber-inductive (short-day) conditions, indicative of a photoperiodic control. Detection of miR156 in phloem cells of wild-type plants and mobility assays in heterografts suggest that miR156 is a graft-transmissible signal. This movement was correlated with changes in leaf morphology and longer trichomes in leaves. Overexpression of miR156 in potato caused a drastic phenotype resulting in altered plant architecture and reduced tuber yield. miR156 overexpression plants also exhibited altered levels of cytokinin and strigolactone along with increased levels of LONELY GUY1 and StCyclin D3.1 transcripts as compared with wild-type plants. RNA ligase-mediated rapid amplification of complementary DNA ends analysis validated SQUAMOSA PROMOTER BINDING-LIKE3 (StSPL3), StSPL6, StSPL9, StSPL13, and StLIGULELESS1 as targets of miR156. Gel-shift assays indicate the regulation of miR172 by miR156 through StSPL9. miR156-resistant SPL9 overexpression lines exhibited increased miR172 levels under a short-day photoperiod, supporting miR172 regulation via the miR156-SPL9 module. Overall, our results strongly suggest that miR156 is a phloem-mobile signal regulating potato development.
The carbon skeletons of over 55,000 naturally occurring isoprenoid compounds are constructed from four fundamental coupling reactions: chain elongation, cyclopropanation, branching, and cyclobutanation. Enzymes that catalyze chain elongation and cyclopropanation are well studied, whereas those that catalyze branching and cyclobutanation are unknown. We have catalyzed the four reactions with chimeric proteins generated by replacing segments of a chain-elongation enzyme with corresponding sequences from a cyclopropanation enzyme. Stereochemical and mechanistic considerations suggest that the four coupling enzymes could have evolved from a common ancestor through relatively small changes in the catalytic site.
Farnesyl diphosphate (FPP) synthase catalyzes the consecutive head-to-tail condensations of isopentenyl diphosphate (IPP, C 5 ) with dimethylallyl diphosphate (DMAPP, C 5 ) and geranyl diphosphate (GPP, C 10 ) to give (E,E)-FPP (C 15 ). The enzyme belongs to a genetically distinct family of chain elongation enzymes that install E-double bonds during each addition of a five-carbon isoprene unit. Analysis of the C 10 and C 15 products from incubations with avian FPP synthase reveals that small amounts of neryl diphosphate (Z-C 10 ) and (Z,E)-FPP are formed along with the E-isomers during the C 5 → C 10 and C 10 → C 15 reactions. Similar results were obtained for FPP synthase from Escherichia coli, Artemisia tridentata (sage brush), Pyrococcus furiosus, and Methanobacter thermautotrophicus and for GPP and FPP synthesized in vivo by E. coli FPP synthase. When (R)-[2-2 H]IPP was a substrate for chain elongation, no deuterium was found in the chain elongation products. In contrast, the deuterium in (S)-[2-2 H]IPP was incorporated into all of the products. Thus, the pro-R hydrogen at C2 of IPP is lost when the E-and Z-double bond isomers are formed. The synthesis of Z-double bond isomers by FPP synthase during chain elongation is unexpected for a highly evolved enzyme and probably reflects a compromise between optimizing double bond stereoselectivity and the need to exclude DMAPP from the IPP binding site.Chain elongation is the fundamental building reaction in the isoprenoid pathway. 1 During this process the growing hydrocarbon chain in an allylic isoprenoid diphosphate is added to isopentenyl diphosphate (IPP). These reactions are catalyzed by polyprenyl diphosphate synthases, a group of prenyltransferases that can be further subdivided into two families according to the stereochemistry, E or Z, of the double bond in the newly added isoprene unit. Members of the two families utilize the same chemical mechanism for chain elongation but are genetically unrelated. 1 Subgroups within each family are selective for the size of the allylic substrate selected for chain elongation and the length of the isoprenoid chain in the final product. Thus, the "trunk" of the isoprenoid biosynthetic pathway is in reality a complex set of trunks that varies from organism to organism rather than a set of linear chain elongation reactions. Members of each family share distinctive highly conserved motifs characteristic of proteins that have evolved from a common ancestor. 2,3 Members of the E-family typically synthesize shorter chain isoprenoid diphosphates found early in the pathway, while members of the Z-family synthesize longer chain diphosphates. 1 Farnesyl diphosphate (FPP) synthase is the prototypal representative of enzymes in the Efamily. FPP synthase catalyzes two reactions -the sequential addition of the hydrocarbon moieties of dimethylallyl diphosphate (C 5 ) and geranyl diphosphate (C
Functional characterization and understanding of the intricate signaling mechanisms in stem-like cells is crucial for the development of effective therapies in melanoma. We have studied whether melanoma cells are phenotypically distinct and hierarchically organized according to their tumorigenic nature. We report that melanoma-specific CD133 cancer stem cells exhibit increased tumor-initiating potential, tumor-endothelial cell interaction, and lung metastasis. These cells are able to transdifferentiate into an endothelial-like phenotype when cultured under endothelial differentiation-promoting conditions. Mechanistically, Notch1 upregulates mitogen-activated protein kinase activation through CD133, which ultimately controls vascular endothelial growth factor and matrix metalloproteinase expression in CD133 stem cells leading to melanoma growth, angiogenesis, and lung metastasis. Blockade or genetic ablation of Notch1 and mitogen-activated protein kinase pathways abolishes melanoma cell migration and angiogenesis. Chromatin immunoprecipitation and reporter assays revealed that Notch1 intracellular domain regulates CD133 expression at the transcriptional level. Andrographolide inhibits Notch1 intracellular domain expression, Notch1 intracellular domain-dependent CD133-mediated mitogen-activated protein kinase and activator protein-1 activation, and epithelial to mesenchymal-specific gene expression, ultimately attenuating melanoma growth and lung metastasis. Human malignant melanoma specimen analyses revealed a strong correlation between Notch1 intracellular domain, CD133, and p-p38 mitogen-activated protein kinase expression and malignant melanoma progression. Thus, targeting Notch1 and its regulated signaling network may have potential therapeutic implications for the management of cancer stem cell-mediated melanoma progression.
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