RESUMO
CaaX-prenyltransferases (CaaX-PTases) catalyse the covalent attachment of isoprenyl groups to conserved cysteine residues located at the C-terminal CaaX motif of a protein substrate. This post-translational modification is required for the function and/or subcellular localization of some transcription factors and components of signal transduction and membrane trafficking machinery. CaaX-PTases, including protein farnesyltransferase (PFT) and type-I protein geranylgeranyltransferase (PGGT-I), are heterodimeric enzymes composed of a common alpha subunit and a specific beta subunit. We have established RNA interference cell lines targeting the beta subunits of PFT and PGGT-I, respectively, in the Catharanthus roseus C20D cell line, which synthesizes monoterpenoid indole alkaloids in response to auxin depletion from the culture medium. In both types of RNAi cell lines, expression of a subset of genes involved in the early stage of monoterpenoid biosynthetic pathway (ESMB genes), including the MEP pathway, is strongly decreased. The role of CaaX-PTases in ESMB gene regulation was confirmed by using the general prenyltransferase inhibitor s-perillyl alcohol (SP) and the specific PFT inhibitor Manumycin A on the wild type line. Furthermore, supplementation of SP inhibited cells with monoterpenoid intermediates downstream of the steps encoded by the ESMB genes restores monoterpenoid indole alkaloids biosynthesis. We conclude that protein targets for both PFT and PGGT-I are required for the expression of ESMB genes and monoterpenoid biosynthesis in C. roseus, this represents a non previously described role for protein prenyltransferase in plants.
Assuntos
Alquil e Aril Transferases/genética , Catharanthus/genética , Regulação da Expressão Gênica de Plantas/genética , Monoterpenos/metabolismo , Alquil e Aril Transferases/antagonistas & inibidores , Alquil e Aril Transferases/metabolismo , Northern Blotting , Catharanthus/citologia , Catharanthus/metabolismo , Técnicas de Cultura de Células , Processos de Crescimento Celular/genética , Regulação para Baixo/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Dados de Sequência Molecular , Monoterpenos/farmacologia , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Prenilação de Proteína/efeitos dos fármacos , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Interferência de RNA/fisiologia , RNA de Plantas/genética , RNA de Plantas/metabolismo , Alcaloides de Triptamina e Secologanina/metabolismo , Fatores de TempoRESUMO
A cDNA encoding a bHLH transcription factor was isolated by the yeast one-hybrid system from a Catharanthus roseus cDNA library using the G-box element of the Strictosidine synthase gene promoter as bait. The corresponding protein (named CrMYC1) was shown to bind specifically to the G-box in yeast. In C. roseus suspension cells CrMYC1 mRNA levels are induced by fungal elicitor and jasmonate suggesting that CrMYC1 may be involved in the regulation of gene expression in response to these signals.
Assuntos
Asteraceae/metabolismo , Carbono-Nitrogênio Liases/genética , Ciclopentanos/farmacologia , Regulação da Expressão Gênica de Plantas/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Asteraceae/efeitos dos fármacos , Asteraceae/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Sequências Hélice-Alça-Hélice , Dados de Sequência Molecular , Oxilipinas , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Especificidade por Substrato , Fatores de Transcrição/metabolismoRESUMO
A novel cDNA (Crubie2) encoding ubiquitin-conjugating enzyme E2 was isolated from a Catharanthus roseus cDNA library. Sequence comparison with Arabidopsis thaliana E2 sequences revealed that CrUBIE2 is a member of a new plant E2 sub-family. Expression of Crubie2 is repressed in developing organs and down-regulated by cytokinin suggesting that a decrease in the ubiquitin-dependent proteolytic pathway may take part in the regulation of alkaloid biosynthesis in C. roseus cell suspensions.