RESUMO
The common plant regulatory factors (CPRFs) from parsley are transcription factors with a basic-leucine-zipper motif that bind to cis-regulatory elements frequently found in promoters of light-regulated genes. Proposed to function in concert with members of other transcription factor families, CPRFs regulate the transcriptional activity of many target genes. Here, we report that, in contrast to CPRF2, which operates as a transcriptional activator, CPRF1 functions as repressor in vivo. Two-hybrid screens using CPRF1 and CPRF2 as "baits" resulted in the isolation of four novel parsley proteins which interact with either CPRF1 or CPRF2 in vivo. Three of these factors represent new parsley bZIP factors, designated CPRF5-CPRF7, whereas the fourth, named CPRF1-interacting protein (CIP), shows no homology to any other known protein. CPRF5 and CIP specifically interact with CPRF1, whilst CPRF6 and CPRF7 exclusively form heterodimers with CPRF2. CPRF5, CPRF6 and CPRF7 are transcription factors that exhibit sequence-specific DNA-binding as well as transactivation abilities, whereas the function of CIP remains elusive. The newly isolated CPRFs and CIP are constitutively localized in the nucleus in parsley protoplasts. Furthermore, mRNA accumulation studies revealed that the expression of these novel bZIP genes and CIP is not altered by exposure to light. We discuss the possible roles of the newly identified proteins in CPRF1- and CPRF2-dependent target gene expression.
Assuntos
Apiaceae/genética , Apiaceae/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/genética , Sequência de Aminoácidos , Apiaceae/efeitos da radiação , Fatores de Transcrição de Zíper de Leucina Básica , Células Cultivadas , Clonagem Molecular , Sequência Conservada , Proteínas de Ligação a DNA/química , Fatores de Ligação G-Box , Zíper de Leucina , Luz , Dados de Sequência Molecular , Proteínas de Plantas/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Raios UltravioletaRESUMO
A family of affinity probes has been generated to detect and purify abscisic-acid (ABA)-binding proteins, by coupling ABA onto carrier proteins (ovalbumin or BSA) through the C1 carboxyl group or the C4' carbonyl group of ABA. ELISA detection showed that these ABA-protein conjugates bound efficiently to the solubilized microsomal protein fraction of Arabidopsis thaliana, but not to the soluble protein fraction. Heat or proteolytic treatments inhibited the binding of the conjugates, indicating the protein nature of these binding sites. After membrane purification of the microsomes, the binding sites were found to be preferentially located in the plasma membrane fraction. The binding of the conjugates was independent of the nature of the carrier protein or the ABA-carrier protein linker, but was competitively inhibited with an anti-ABA mAb. Furthermore, the competitive inhibition of the binding of the conjugates with ABA, but not with the inactive ABA methyl ester analog, demonstrated the specificity of the binding and the saturability of the binding sites. The binding of the conjugates was strictly correlated to the ABA/carrier protein molar coupling ratio, confirming that the affinity of the conjugates to the ABA-binding proteins was enhanced by the increase in the probability of binding events. The experimental approach permits a new insight into the nature of membrane-associated ABA-binding proteins.