Dataset Information

Control of the polyamine biosynthesis pathway by G2-quadruplexes.

ABSTRACT: G-quadruplexes are naturally-occurring structures found in RNAs and DNAs. Regular RNA G-quadruplexes are highly stable due to stacked planar arrangements connected by short loops. However, reports of irregular quadruplex structures are increasing and recent genome-wide studies suggest that they influence gene expression. We have investigated a grouping of G2-motifs in the UTRs of eight genes involved in polyamine biosynthesis, and concluded that several likely form novel metastable RNA G-quadruplexes. We performed a comprehensive biophysical characterization of their properties, comparing them to a reference G-quadruplex. Using cellular assays, together with polyamine-depleting and quadruplex-stabilizing ligands, we discovered how some of these motifs regulate and sense polyamine levels, creating feedback loops during polyamine biosynthesis. Using high-resolution 1H-NMR spectroscopy, we demonstrated that a long-looped quadruplex in the AZIN1 mRNA co-exists in salt-dependent equilibria with a hairpin structure. This study expands the repertoire of regulatory G-quadruplexes and demonstrates how they act in unison to control metabolite homeostasis.

SUBMITTER: Lightfoot HL

PROVIDER: S-EPMC6067879 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Control of the polyamine biosynthesis pathway by G<sub>2</sub>-quadruplexes.

Lightfoot Helen Louise HL Hagen Timo T Cléry Antoine A Allain Frédéric Hai-Trieu FH Hall Jonathan J

eLife 20180731

G-quadruplexes are naturally-occurring structures found in RNAs and DNAs. Regular RNA G-quadruplexes are highly stable due to stacked planar arrangements connected by short loops. However, reports of irregular quadruplex structures are increasing and recent genome-wide studies suggest that they influence gene expression. We have investigated a grouping of G<sub>2</sub>-motifs in the UTRs of eight genes involved in polyamine biosynthesis, and concluded that several likely form novel metastable RN ...[more]

PMID: 30063205

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Project description:Traditional breeding and molecular approaches have been used to develop tobacco varieties with reduced nicotine and secondary alkaloid levels. However, available low-alkaloid tobacco varieties have impaired leaf quality likely due to the metabolic consequences of nicotine biosynthesis downregulation. Recently, we found evidence that the unbalanced crosstalk between nicotine and polyamine pathways is involved in impaired leaf ripening of a low-alkaloid (LA) Burley 21 line having a mutation at the Nic1 and Nic2 loci, key biosynthetic regulators of nicotine biosynthesis. Since the Nic1 and Nic2 loci are comprised of several genes, all phenotypic changes seen in LA Burley 21 could be due to a mixture of genetics-based responses. Here, we investigated the commercial burley variety TN90 LC and its transgenic versions with only one downregulated gene, either putrescine methyl transferase (PMT-RNAi) or PR50-protein (PR50-RNAi). Nicotine levels of cured lamina of TN90 LC, TN90 PMT-RNAi and TN90 PR50-RNAi, were 70.5 ± 3.8, 2.4 ± 0.5, and 6.0 ± 1.1 mg/g dry weight, respectively. Low-alkaloid transgenic lines showed delayed leaf maturation and impaired leaf quality. We analyzed polyamine contents and ripening markers in wild-type TN90 control plants (WT) and the two transgenic lines. The ripening markers revealed that the PMT-RNAi line showed the most pronounced impaired leaf maturation phenotype at harvest, characterized by higher chlorophyll (19%) and glucose (173%) contents and more leaf mesophyll cells per area (25%), while the ripening markers revealed that maturation of PR50-RNAi plants was intermediate between PMT-RNAi and WT lines. Comparative polyamine analyses showed an increase in free and conjugated polyamines in roots of both transgenic lines, this being most pronounced in the PMT-RNAi plants. For PMT-RNAi plants, there were further perturbations of polyamine content in the leaves, which mirrored the general phenotype, as PR50-RNAi transgenic plants looked more similar to the WT than PMT-RNAi transgenic plants. Activity of ornithine decarboxylase, the enzyme that catalyzes the committing step of polyamine biosynthesis, was significantly higher in roots and mature leaves of PMT-RNAi plants in comparison to WT, while there was no increase observed for arginine decarboxylase. Treatment of both transgenic lines with polyamine biosynthesis inhibitors decreased the polyamine content and ameliorated the phenotype, confirming the intricate interplay of polyamine and nicotine biosynthesis in tobacco and the influence of this interplay on leaf ripening.

Project description:Background and Aims:Polyamines are small metabolites present in all living cells and play fundamental roles in numerous physiological events in plants. The aminopropyltransferases (APTs), spermidine synthase (SPDS), spermine synthase (SPMS) and thermospermine synthase (ACL5), are essential enzymes in the polyamine biosynthesis pathway. In angiosperms, SPMS has evolved from SPDS via gene duplication, whereas in gymnosperms APTs are mostly unexplored and no SPMS gene has been reported. The present study aimed to investigate the functional properties of the SPDS and ACL5 proteins of Scots pine (Pinus sylvestris L.) in order to elucidate the role and evolution of APTs in higher plants. Methods:Germinating Scots pine seeds and seedlings were analysed for polyamines by high-performance liquid chromatography (HPLC) and the expression of PsSPDS and PsACL5 genes by in situ hybridization. Recombinant proteins of PsSPDS and PsACL5 were produced and investigated for functional properties. Also gene structures, promoter regions and phylogenetic relationships of PsSPDS and PsACL5 genes were analysed. Key Results:Scots pine tissues were found to contain spermidine, spermine and thermospermine. PsSPDS enzyme catalysed synthesis of both spermidine and spermine. PsACL5 was found to produce thermospermine, and PsACL5 gene expression was localized in the developing procambium in embryos and tracheary elements in seedlings. Conclusions:Contrary to previous views, our results demonstrate that SPMS activity is not a novel feature developed solely in the angiosperm lineage of seed plants but also exists as a secondary property in the Scots pine SPDS enzyme. The discovery of bifunctional SPDS from an evolutionarily old conifer reveals the missing link in the evolution of the polyamine biosynthesis pathway. The finding emphasizes the importance of pre-existing secondary functions in the evolution of new enzyme activities via gene duplication. Our results also associate PsACL5 with the development of vascular structures in Scots pine.

Dataset Information

Control of the polyamine biosynthesis pathway by G2-quadruplexes.

Publications

Control of the polyamine biosynthesis pathway by G<sub>2</sub>-quadruplexes.

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