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1.
Plant Physiol ; 176(2): 1469-1484, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29203557

RESUMO

8,14-seco-Triterpenoids are characterized by their unusual open C-ring. Their distribution in nature is rare and scattered in taxonomically unrelated plants. The 8,14-seco-triterpenoid α-onocerin is only known from the evolutionarily distant clubmoss genus Lycopodium and the leguminous genus Ononis, which makes the biosynthesis of this seco-triterpenoid intriguing from an evolutionary standpoint. In our experiments with Ononis spinosa, α-onocerin was detected only in the roots. Through transcriptome analysis of the roots, an oxidosqualene cyclase, OsONS1, was identified that produces α-onocerin from squalene-2,3;22,23-dioxide when transiently expressed in Nicotiana bethamiana In contrast, in Lycopodium clavatum, two sequential cyclases, LcLCC and LcLCD, are required to produce α-onocerin in the N. benthamiana transient expression system. Expression of OsONS1 in the lanosterol synthase knockout yeast strain GIL77, which accumulates squalene-2,3;22,23-dioxide, verified the α-onocerin production. A phylogenetic analysis predicts that OsONS1 branches off from specific lupeol synthases and does not group with the known L. clavatum α-onocerin cyclases. Both the biochemical and phylogenetic analyses of OsONS1 suggest convergent evolution of the α-onocerin pathways. When OsONS1 was coexpressed in N. benthamiana leaves with either of the two O. spinosa squalene epoxidases, OsSQE1 or OsSQE2, α-onocerin production was boosted, most likely because the epoxidases produce higher amounts of squalene-2,3;22,23-dioxide. Fluorescence lifetime imaging microscopy analysis demonstrated specific protein-protein interactions between OsONS1 and both O. spinosa squalene epoxidases. Coexpression of OsONS1 with the two OsSQEs suggests that OsSQE2 is the preferred partner of OsONS1 in planta. Our results provide an example of the convergent evolution of plant specialized metabolism.


Assuntos
Transferases Intramoleculares/metabolismo , Lycopodium/enzimologia , Ononis/enzimologia , Triterpenos/metabolismo , Transferases Intramoleculares/genética , Lycopodium/química , Lycopodium/genética , Ononis/química , Ononis/genética , Folhas de Planta/química , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/química , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Nicotiana/química , Nicotiana/enzimologia , Nicotiana/genética
2.
Metab Eng ; 49: 1-12, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30016654

RESUMO

Triterpene cyclases catalyze the first committed step in triterpene biosynthesis, by forming mono- to pentacyclic backbone structures from oxygenated C30 isoprenoid precursors. Squalene epoxidase precedes this cyclization by providing the oxygenated and activated substrate for triterpene biosynthesis. Three squalene epoxidases from Cucurbita pepo (CpSEs) were isolated and shown to have evolved under purifying selection with signs of sites under positive selection in their N- and C-termini. They all localize to the Endoplasmic Reticulum (ER) and produce 2,3-oxidosqualene and 2,3:22,23-dioxidosqualene when expressed in a yeast erg1 (squalene epoxidase) erg7 (lanosterol synthase) double mutant. Co-expression of the CpSEs with four different triterpene cyclases, either transiently in Nicotiana benthamiana or constitutively in yeast, showed that CpSEs boost triterpene production. CpSE2 was the best performing in this regard, which could reflect either increased substrate production or superior channeling of the substrate to the triterpene cyclases. Fluorescence Lifetime Imaging Microscopy (FLIM) analysis with C. pepo cucurbitadienol synthase (CpCPQ) revealed a specific interaction with CpSE2 but not with the other CpSEs. When CpSE2 was transformed into C. pepo hairy root lines, cucurbitacin E production was increased two folds compared to empty vector control lines. This study provides new insight into the importance of SEs in triterpene biosynthesis, suggesting that they may facilitate substrate channeling, and demonstrates that SE overexpression is a new tool for increasing triterpene production in plants and yeast.


Assuntos
Citrullus/genética , Cucurbita/genética , Liases Intramoleculares , Microrganismos Geneticamente Modificados , Nicotiana , Proteínas de Plantas , Plantas Geneticamente Modificadas , Esqualeno Mono-Oxigenase , Triterpenos/metabolismo , Citrullus/enzimologia , Cucurbita/enzimologia , Expressão Gênica , Liases Intramoleculares/biossíntese , Liases Intramoleculares/genética , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Esqualeno Mono-Oxigenase/biossíntese , Esqualeno Mono-Oxigenase/genética , Nicotiana/genética , Nicotiana/metabolismo
3.
Plant J ; 74(2): 280-93, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23452278

RESUMO

The majority of land plants live in symbiosis with arbuscular mycorrhizal fungi from the phylum Glomeromycota. This symbiosis improves acquisition of phosphorus (P) by the host plant in exchange for carbohydrates, especially under low-P availability. The symbiosome, constituted by root cortex cells accommodating arbuscular mycorrhizal fungal hyphae, is the site at which bi-directional exchange of nutrients and metabolites takes place. Uptake of orthophosphate (Pi) in the symbiosome is facilitated by mycorrhiza-specific plant Pi transporters. Modifications of the potato Pi transporter 3 (StPT3) promoter were analysed in transgenic mycorrhizal roots, and it was found that the CTTC cis-regulatory element is necessary and sufficient for a transcriptional response to fungal colonization under low-Pi conditions. Phylogenetic footprinting also revealed binary combination of the CTTC element with the Pi starvation response-associated PHR1-binding site (P1BS) in the promoters of several mycorrhiza-specific Pi transporter genes. Scanning of the Lotus japonicus genome for gene promoters containing both cis-regulatory elements revealed a strong over-representation of genes involved in transport processes. One of these, LjVTI12, encoding a member of the SNARE family of proteins involved in membrane transport, exhibited enhanced transcript levels in Lotus roots colonized with the arbuscular mycorrhizal fungus Glomus intraradices. Down-regulation of LjVTI12 by RNA interference resulted in a mycorrhiza-specific phenotype characterized by distorted arbuscule morphology. The results highlight cooperative cis-regulation which integrates mycorrhiza and Pi starvation signaling with vesicle trafficking in symbiosome development.


Assuntos
Lotus/metabolismo , Micorrizas/fisiologia , Proteínas de Plantas/metabolismo , Lotus/genética , Proteínas de Plantas/genética , Interferência de RNA , Solanum tuberosum/metabolismo , Solanum tuberosum/microbiologia
4.
Plant Physiol ; 156(4): 2141-54, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21705655

RESUMO

Gene networks involved in inorganic phosphate (Pi) acquisition and homeostasis in woody perennial species able to form mycorrhizal symbioses are poorly known. Here, we describe the features of the 12 genes coding for Pi transporters of the Pht1 family in poplar (Populus trichocarpa). Individual Pht1 transporters play distinct roles in acquiring and translocating Pi in different tissues of mycorrhizal and nonmycorrhizal poplar during different growth conditions and developmental stages. Pi starvation triggered the up-regulation of most members of the Pht1 family, especially PtPT9 and PtPT11. PtPT9 and PtPT12 showed a striking up-regulation in ectomycorrhizas and endomycorrhizas, whereas PtPT1 and PtPT11 were strongly down-regulated. PtPT10 transcripts were highly abundant in arbuscular mycorrhiza (AM) roots only. PtPT8 and PtPT10 are phylogenetically associated to the AM-inducible Pht1 subfamily I. The analysis of promoter sequences revealed conserved motifs similar to other AM-inducible orthologs in PtPT10 only. To gain more insight into gene regulatory mechanisms governing the AM symbiosis in woody plant species, the activation of the poplar PtPT10 promoter was investigated and detected in AM of potato (Solanum tuberosum) roots. These results indicated that the regulation of AM-inducible Pi transporter genes is conserved between perennial woody and herbaceous plant species. Moreover, poplar has developed an alternative Pi uptake pathway distinct from AM plants, allowing ectomycorrhizal poplar to recruit PtPT9 and PtPT12 to cope with limiting Pi concentrations in forest soils.


Assuntos
Perfilação da Expressão Gênica , Família Multigênica/genética , Micorrizas/fisiologia , Proteínas de Transporte de Fosfato/química , Proteínas de Transporte de Fosfato/genética , Populus/genética , Populus/microbiologia , Contagem de Colônia Microbiana , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas/genética , Genótipo , Glomeromycota/efeitos dos fármacos , Glomeromycota/crescimento & desenvolvimento , Glomeromycota/fisiologia , Glucuronidase/metabolismo , Anotação de Sequência Molecular , Micorrizas/efeitos dos fármacos , Proteínas de Transporte de Fosfato/metabolismo , Fosfatos/farmacologia , Filogenia , Plantas Geneticamente Modificadas , Populus/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade da Espécie
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