RESUMEN
Overexpression of a pine MYB, PtMYB4, in Arabidopsis caused ectopic lignin deposition and allowed the plants to undergo photomorphogenesis even when they were grown in the dark. The phenotype caused by PtMYB4 overexpression was reminiscent of the previously characterised dark-photomorphogenic mutant, de-etiolated 3 (det3); consequently, we tested the hypothesis that MYB misexpression may explain aspects of the det3 phenotype. We show here that AtMYB61, a member of the Arabidopsis R2R3-MYB family, is misexpressed in the det3 mutant. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) experiments suggested that AtMYB61 was misexpressed in a det3 background relative to wild-type plants. Examination of AtMYB61 promoter activity in a det3 background showed that the spatial control of AtMYB61 expression was lost. In order to determine if such misexpression could explain the mutant phenotype, AtMYB61 was overexpressed in wild-type Arabidopsis plants. Transgenic plants that overexpressed AtMYB61 had the same ectopic lignification and dark-photomorphogenic phenotype as that of the det3 mutant. In order to test if AtMYB61 was necessary for these aspects of the det3 phenotype, AtMYB61 expression was downregulated in det3 plants in both antisense and sense suppression experiments. Suppression of AtMYB61 in a det3 mutant background restored all mutant phenotypes of the det3 mutant associated with development in the dark. Taken together, these results suggest that AtMYB61 misexpression was both sufficient and necessary to explain the ectopic lignification and dark-photomorphogenic phenotypes of the det3 mutant.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Genes myb/genética , Lignina/metabolismo , Factores de Transcripción/genética , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/efectos de la radiación , Oscuridad , Regulación de la Expresión Génica de las Plantas , Genes myb/efectos de la radiación , Luz , Lignina/efectos de la radiación , Morfogénesis/genética , Morfogénesis/efectos de la radiación , Mutagénesis , Fenotipo , Pinus , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas/genética , Factores de Transcripción/efectos de la radiación , Tubulina (Proteína)/genéticaRESUMEN
A cDNA encoding a member of the R2R3-MYB family of transcription factors was cloned from a library constructed from differentiating Pinus taeda (loblolly pine) xylem RNA. This MYB family member, Pinus taeda MYB1 (PtMYB1), was most abundantly expressed in differentiating xylem, as assessed by both ribonuclease protection assays, and by northern blot analysis with poly(A)-enriched RNA. Similar to other plant R2R3-MYB family members, recombinant Pt MYB1 protein was able to bind to AC elements in electrophoretic mobility shift assays (EMSAs). AC elements are DNA motifs rich in adenosine and cytosine that have been implicated in the xylem-localised regulation of genes encoding lignin biosynthetic enzymes. Pt MYB1 not only bound to AC elements, but was also able to induce AC-element-dependent shifts in the electrophoretic mobility of a plant promoter that contains three AC elements, the minimal PHENYLALANINE AMMONIA-LYASE 2 (PAL2) promoter from Phaseolus vulgaris. Transcriptional activation assays conducted using yeast showed that Pt MYB1 also activated transcription, and that it did so in an AC-element-dependent fashion. Pt MYB1 also activated transcription from the minimal PAL2 promoter in plant cells in an AC-element-dependent fashion, as revealed by transient transcriptional activation assays with microprojectile-bombarded tobacco NT-1 cells. Taken together, these finding are consistent with the hypothesis that Pt MYB1 may regulate transcription from cis -acting AC elements in pine xylem.
Asunto(s)
Pinus/genética , Proteínas de Plantas/genética , Estructuras de las Plantas/genética , Proteínas Proto-Oncogénicas c-myb/genética , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Secuencia de Bases , Sitios de Unión/genética , Clonación Molecular , Citidina Trifosfato/genética , Citidina Trifosfato/metabolismo , ADN Complementario/química , ADN Complementario/genética , Ensayo de Cambio de Movilidad Electroforética , Datos de Secuencia Molecular , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Filogenia , Proteínas de Plantas/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-myb/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Análisis de Secuencia de ADN , Transcripción Genética/genéticaRESUMEN
A member of the R2R3-MYB family of transcription factors was cloned from a cDNA library constructed from RNA isolated from differentiating pine xylem. This MYB, Pinus taeda MYB4 (PtMYB4), is expressed in cells undergoing lignification, as revealed by in situ RT-PCR. Electrophoretic mobility shift assays (EMSAs) showed that recombinant PtMYB4 protein is able to bind to DNA motifs known as AC elements. AC elements are ubiquitous in the promoters of genes encoding lignin biosynthetic enzymes. Transcriptional activation assays using yeast showed that PtMYB4 could activate transcription in an AC-element-dependent fashion. Overexpression of PtMYB4 in transgenic tobacco plants altered the accumulation of transcripts corresponding to genes encoding lignin biosynthetic enzymes. Lignin deposition increased in transgenic tobacco plants that overexpressed PtMYB4, and extended to cell types that do not normally lignify. Taken together, these findings are consistent with the hypothesis that PtMYB4 is sufficient to induce lignification, and that it may play this role during wood formation in pine.