Project description:MicroRNAs (miRNA) are ~21 nucleotide long, small endogenous non-coding RNAs that functioning in regulation of gene expression found in many eukaryotes. In this study, small RNA libraries of opium poppy from four different tissues (leaf, root, capsule, stem) were sequenced using high-throughput next generation Illumina sequencing (Solexa) technology to investigate potential mode of actions of miRNAs in alkaloid biosynthesis. A total of 27 opium poppy miRNAs which have roles in regulation of alkaloid biosynthesis were identified in this study.
Project description:MicroRNAs (miRNA) are ~21 nucleotide long, small endogenous non-coding RNAs that functioning in regulation of gene expression found in many eukaryotes. In this study, small RNA libraries of opium poppy from four different tissues (leaf, root, capsule, stem) were sequenced using high-throughput next generation Illumina sequencing (Solexa) technology to investigate potential mode of actions of miRNAs in alkaloid biosynthesis. A total of 27 opium poppy miRNAs which have roles in regulation of alkaloid biosynthesis were identified in this study. A six chip study using miRNA isolated from four separate tissues (capsule, leaf, stem, root). small RNA libraries of opium poppy tissues were sequenced using high-throughput next generation Illumina sequencing (Solexa) technology to investigate potential mode of actions of miRNAs in alkaloid biosynthesis. Furthermore, the novel opium poppy miRNAs were also confirmed by a direct small RNA cloning strategy. The microarray platform were performed to measure and analyze the mirnome of the different opium poppy tissues.
Project description:The class of fungal indole alkaloids containing the bicyclo[2.2.2]diazaoctane ring is comprised of diverse molecules that display a range of biological activities. While much interest has been garnered due to their therapeutic potential, this class of molecules also displays unique chemical functionality, making them intriguing synthetic targets. Many elegant and intricate total syntheses have been developed to generate these alkaloids, but the selectivity required to produce them in high yield presents great barriers. Alternatively, if we can understand the molecular mechanisms behind how fungi make these complex molecules, we can leverage the power of nature to perform these chemical transformations. Here, we describe the various studies regarding the evolutionary development of enzymes involved in fungal indole alkaloid biosynthesis.
Project description:The tropane alkaloids (TAs) cocaine and hyoscyamine have been used medicinally for thousands of years. To understand the evolutionary origins and trajectories of serial biosynthetic enzymes of TAs and especially the characteristic tropane skeletons, we generated the chromosome-level genome assemblies of cocaine-producing Erythroxylum novogranatense (Erythroxylaceae, rosids clade) and hyoscyamine-producing Anisodus acutangulus (Solanaceae, asterids clade). Comparative genomic and phylogenetic analysis suggested that the lack of spermidine synthase/N-methyltransferase (EnSPMT1) in ancestral asterids species contributed to the divergence of polyamine (spermidine or putrescine) methylation in cocaine and hyoscyamine biosynthesis. Molecular docking analysis and key site mutation experiments suggested that ecgonone synthases CYP81AN15 and CYP82M3 adopt different active-site architectures to biosynthesize the same product ecgonone from the same substrate in Erythroxylaceae and Solanaceae. Further synteny analysis showed different evolutionary origins and trajectories of CYP81AN15 and CYP82M3, particularly the emergence of CYP81AN15 through the neofunctionalization of ancient tandem duplication genes. The combination of structural biology and comparative genomic analysis revealed that ecgonone methyltransferase, which is responsible for the biosynthesis of characteristic 2-substituted carboxymethyl group in cocaine, evolved from the tandem copies of salicylic acid methyltransferase by the mutations of critical E216 and S153 residues. Overall, we provided strong evidence for the independent origins of serial TA biosynthetic enzymes on the genomic and structural level, underlying the chemotypic convergence of TAs in phylogenetically distant species.
Project description:We analyzed the transcriptome of A. acutangulus roots by deep RNA sequencing to dig TAs biosynthetic genes. KOG (Eukaryotic Orthologous Groups) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analyses identified 48 unigenes referring to the tropane, piperidine and pyridine alkaloid biosynthesis, 145 unigenes presumably involved in distribution of arginine to TAs biosynthesis, and 86 unigenes referring to the terpenoid backbone biosynthesis. Furthermore, 82 unigenes annotated as cytochrome P450 family members seemed to be involved in secondary metabolism pathways. Previously unknown TAs biosynthetic genes in A. acutangulus, which encode littorine mutase/monooxygenase (CYP80F1) and diamine oxidase (DAO), were identified by this study.
Project description:Sampada and Sujata are two contrasting genotypes of Papaver somniferum that are contrasting in terms of their latex and alkaloid profiles. The major objective of the present study was to use a small-scale (750 target genes) microarray of P. somniferum for identification of genes that are differentially expressed in the capsule walls of the two contrasting genotypes, Sampada and Sujata. Nidarshana Chaturvedi and Mridula Singh made equal contribution as first authors to this data.
Project description:Plants produce specialized metabolites for their defence. However, specialist herbivores adapt to these compounds and use them for their own benefit. Plants attacked predominantly by specialists may be under selection to reduce or eliminate production of co-opted chemicals: the defence de-escalation hypothesis. We studied the evolution of pyrrolizidine alkaloids (PAs) in Apocynaceae, larval host plants for PA-adapted butterflies (Danainae, milkweed and clearwing butterflies), to test if the evolutionary pattern is consistent with de-escalation. We used the first PA biosynthesis specific enzyme (homospermidine synthase, HSS) as tool for reconstructing PA evolution. We found hss orthologues in diverse Apocynaceae species, not all of them known to produce PAs. The phylogenetic analysis showed a monophyletic origin of the putative hss sequences early in the evolution of one Apocynaceae lineage (the APSA clade). We found an hss pseudogene in Asclepias syriaca, a species known to produce cardiac glycosides but no PAs, and four losses of an HSS amino acid motif. APSA clade species are significantly more likely to be Danainae larval host plants than expected if all Apocynaceae species were equally likely to be exploited. Our findings are consistent with PA de-escalation as an adaptive response to specialist attack.
Project description:Chlorophylls (Chls) are essential cofactors for photosynthesis. One of the least understood steps of Chl biosynthesis is formation of the fifth (E) ring, where the red substrate, magnesium protoporphyrin IX monomethyl ester, is converted to the green product, 3,8-divinyl protochlorophyllide a In oxygenic phototrophs, this reaction is catalyzed by an oxygen-dependent cyclase, consisting of a catalytic subunit (AcsF/CycI) and an auxiliary protein, Ycf54. Deletion of Ycf54 impairs cyclase activity and results in severe Chl deficiency, but its exact role is not clear. Here, we used a Δycf54 mutant of the model cyanobacterium Synechocystis sp. PCC 6803 to generate suppressor mutations that restore normal levels of Chl. Sequencing Δycf54 revertants identified a single D219G amino acid substitution in CycI and frameshifts in slr1916, which encodes a putative esterase. Introduction of these mutations to the original Δycf54 mutant validated the suppressor effect, especially in combination. However, comprehensive analysis of the Δycf54 suppressor strains revealed that the D219G-substituted CycI is only partially active and its accumulation is misregulated, suggesting that Ycf54 controls both the level and activity of CycI. We also show that Slr1916 has Chl dephytylase activity in vitro and its inactivation up-regulates the entire Chl biosynthetic pathway, resulting in improved cyclase activity. Finally, large-scale bioinformatic analysis indicates that our laboratory evolution of Ycf54-independent CycI mimics natural evolution of AcsF in low-light-adapted ecotypes of the oceanic cyanobacteria Prochlorococcus, which lack Ycf54, providing insight into the evolutionary history of the cyclase enzyme.
Project description:To identify the molecular basis of genome-wide transcriptome analysis in induced opium poppy capsule tissues NimbleGen microarray (12X135 K element) analysis was performed. Fungal elicitor methyl jasmonate (MeJa) treatment was applied on capsules of opium poppy during 0, 3, and 12 h. Four sample sets were prepared for array analyses: i) control ii) 0 hour after treatment iii) 3 hour after treatment iv) 12 h after treatment