Isolation of high-quality RNA from gymnosperm and angiosperm trees

Kolosova, Natalia; Miller, Barbara; Ralph, Steven; Ellis, Brian E.; Douglas, Carl J.; Ritland, Kermit; Bohlmann, Jörg

doi:10.2144/04365st06

Cited by 144 publications

(106 citation statements)

References 8 publications

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“…Total RNA was isolated as described previously (44). After cDNA amplification with the SuperScript III reverse transcriptase (Invitrogen), qRT-PCR was performed on a Bio-Rad CFX96 Real-Time system using iTaq SYBR Green Supermix (www.bio-rad.com) and gene-specific primers (Table S2).…”

Section: Methodsmentioning

confidence: 99%

Biosynthesis of the microtubule-destabilizing diterpene pseudolaric acid B from golden larch involves an unusual diterpene synthase

Mafu

Karunanithi

Palazzo

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The diversity of small molecules formed via plant diterpene metabolism offers a rich source of known and potentially new biopharmaceuticals. Among these, the microtubule-destabilizing activity of pseudolaric acid B (PAB) holds promise for new anticancer agents. PAB is found, perhaps uniquely, in the coniferous tree golden larch (Pseudolarix amabilis, Pxa). Here we describe the discovery and mechanistic analysis of golden larch terpene synthase 8 (PxaTPS8), an unusual diterpene synthase (diTPS) that catalyzes the first committed step in PAB biosynthesis. Mining of the golden larch root transcriptome revealed a large TPS family, including the monofunctional class I diTPS PxaTPS8, which converts geranylgeranyl diphosphate into a previously unknown 5,7-fused bicyclic diterpene, coined "pseudolaratriene." Combined NMR and quantum chemical analysis verified the structure of pseudolaratriene, and co-occurrence with PxaTPS8 and PAB in P. amabilis tissues supports the intermediacy of pseudolaratriene in PAB metabolism. Although PxaTPS8 adopts the typical three-domain structure of diTPSs, sequence phylogeny places the enzyme with two-domain TPSs of mono-and sesqui-terpene biosynthesis. Site-directed mutagenesis of PxaTPS8 revealed several catalytic residues that, together with quantum chemical calculations, suggested a substantial divergence of PxaTPS8 from other TPSs leading to a distinct carbocation-driven reaction mechanism en route to the 5,7-trans-fused bicyclic pseudolaratriene scaffold. PxaTPS8 expression in microbial and plant hosts provided proof of concept for metabolic engineering of pseudolaratriene. diterpene biosynthesis | Pseudolarix amabilis | plant natural products | pseudolaric acid | chemotherapeutic drug

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Section: Methodsmentioning

confidence: 99%

Biosynthesis of the microtubule-destabilizing diterpene pseudolaric acid B from golden larch involves an unusual diterpene synthase

Mafu

Karunanithi

Palazzo

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Total RNA was isolated as described previously (Kolosova et al, 2004). First-strand cDNA was synthesized using the SuperScript III First-Strand Synthesis Kit (Invitrogen) and oligo(dT) primers according to the manufacturer's instructions.…”

Section: Quantitative Real-time Pcrmentioning

confidence: 99%

Discovery, Biosynthesis and Stress-Related Accumulation of Dolabradiene-Derived Defenses in Maize

et al. 2018

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“…RNA isolation followed the method of Kolosova et al (2004) with minor modifications. Extractions were scaled down to 50 to 100 mg of tissue; the RNA pellet was washed with 70% ethanol after LiCl precipitation and resuspended in nuclease-free water; RNA integrity was assessed using the RNA 6000 Nano kit (Bioanalyzer 2100; Agilent).…”

Section: Rna Isolationmentioning

confidence: 99%

Evolution of Diterpene Metabolism: Sitka Spruce CYP720B4 Catalyzes Multiple Oxidations in Resin Acid Biosynthesis of Conifer Defense against Insects

et al. 2011

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Diterpene resin acids (DRAs) are specialized (secondary) metabolites of the oleoresin defense of conifers produced by diterpene synthases and cytochrome P450s of the CYP720B family. The evolution of DRA metabolism shares common origins with the biosynthesis of ent-kaurenoic acid, which is highly conserved in general (primary) metabolism of gibberellin biosynthesis. Transcriptome mining in species of spruce (Picea) and pine (Pinus) revealed CYP720Bs of four distinct clades. We cloned a comprehensive set of 12 different Sitka spruce (Picea sitchensis) CYP720Bs as full-length cDNAs. Spatial expression profiles, methyl jasmonate induction, and transcript enrichment in terpenoid-producing resin ducts suggested a role of CYP720B4 in DRA biosynthesis. CYP720B4 was characterized as a multisubstrate, multifunctional enzyme by the formation of oxygenated diterpenoids in metabolically engineered yeast, yeast in vivo transformation of diterpene substrates, in vitro assays with CYP720B4 protein produced in Escherichia coli, and alteration of DRA profiles in RNA interference-suppressed spruce seedlings. CYP720B4 was active with 24 different diterpenoid substrates, catalyzing consecutive C-18 oxidations in the biosynthesis of an array of diterpene alcohols, aldehydes, and acids. CYP720B4 was most active in the formation of dehydroabietic acid, a compound associated with insect resistance of Sitka spruce. We identified patterns of convergent evolution of CYP720B4 in DRA metabolism and ent-kaurene oxidase CYP701 in gibberellin metabolism and revealed differences in the evolution of specialized and general diterpene metabolism in a gymnosperm. The genomic and functional characterization of the gymnosperm CYP720B family highlights that the evolution of specialized metabolism involves substantial diversification relative to conserved, general metabolism.

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Isolation of high-quality RNA from gymnosperm and angiosperm trees

Cited by 144 publications

References 8 publications

Biosynthesis of the microtubule-destabilizing diterpene pseudolaric acid B from golden larch involves an unusual diterpene synthase

Biosynthesis of the microtubule-destabilizing diterpene pseudolaric acid B from golden larch involves an unusual diterpene synthase

Discovery, Biosynthesis and Stress-Related Accumulation of Dolabradiene-Derived Defenses in Maize

Evolution of Diterpene Metabolism: Sitka Spruce CYP720B4 Catalyzes Multiple Oxidations in Resin Acid Biosynthesis of Conifer Defense against Insects

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