Isolation and characterization of four novel parsley proteins that interact with the transcriptional regulators CPRF1 and CPRF2.

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.

The DNA binding properties of the parsley bZIP transcription factor CPRF4a are regulated by light.

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. Recent studies have revealed that certain CPRF proteins are regulated in response to light by changes in their expression level and in their intracellular localization. Here, we describe an additional mechanism contributing to the light-dependent regulation of CPRF proteins. We show that the DNA binding activity of the factor CPRF4a is modulated in a phosphorylation-dependent manner and that cytosolic components are involved in the regulation of this process. Moreover, we have identified a cytosolic kinase responsible for CPRF4a phosphorylation. Modification of recombinant CPRF4a by this kinase, however, is insufficient to cause a full activation of the factor, suggesting that additional modifications are required. Furthermore, we demonstrate that the DNA binding activity of the factor is modified upon light treatment. The results of additional irradiation experiments suggest that this photoresponse is controlled by different photoreceptor systems. We discuss the possible role of CPRF4a in light signal transduction as well as the emerging regulatory network controlling CPRF activities in parsley.

Phosphorylation of the parsley bZIP transcription factor CPRF2 is regulated by light.

The analysis of the complex network of signal transduction chains has demonstrated the importance of transcription factor activities for the control of gene expression. To understand how transcription factor activities in plants are regulated in response to light, we analyzed the common plant regulatory factor 2 (CPRF2) from parsley (Petroselinum crispum L.) that interacts with promoter elements of light-regulated genes. Here, we demonstrate that CPRF2 is a phosphoprotein in vivo and that its phosphorylation state is rapidly increased in response to light. Phosphorylation in vitro as well as in vivo occurs primarily within the C-terminal half of the factor, and is caused by a cytosolic 40-kDa protein serine kinase. In contrast to other plant basic leucine-zipper motif factors, phosphorylation of CPRF2 does not alter its DNA binding activity. Therefore, we discuss alternative functions of the light-dependent phosphorylation of CPRF2 including the regulation of its nucleocytoplasmic partitioning.

Nuclear import of the parsley bZIP transcription factor CPRF2 is regulated by phytochrome photoreceptors.

In plants, light perception by photoreceptors leads to differential expression of an enormous number of genes. An important step for differential gene expression is the regulation of transcription factor activities. To understand these processes in light signal transduction we analyzed the three well-known members of the common plant regulatory factor (CPRF) family from parsley (Petroselinum crispum). Here, we demonstrate that these CPRFs, which belong to the basic- region leucine-zipper (bZIP) domain-containing transcription factors, are differentially distributed within parsley cells, indicating different regulatory functions within the regulatory networks of the plant cell. In particular, we show by cell fractionation and immunolocalization approaches that CPRF2 is transported from the cytosol into the nucleus upon irradiation due to action of phytochrome photoreceptors. Two NH2-terminal domains responsible for cytoplasmic localization of CPRF2 in the dark were characterized by deletion analysis using a set of CPRF2-green fluorescent protein (GFP) gene fusion constructs transiently expressed in parsley protoplasts. We suggest that light-induced nuclear import of CPRF2 is an essential step in phytochrome signal transduction.

Cross-linking of chloroplast F0F1-ATPase subunit epsilon to gamma without effect on activity. Epsilon and gamma are parts of the rotor.

Cys residues were directed into positions 17, 28, 41 and 85 of a Cys6-->Ser mutant of subunit epsilon of spinach chloroplast F0F1 ATP synthase. Wild-type and engineered epsilon were expressed in Escherichia coli, purified in the presence of urea, refolded and reassembled with spinach chloroplast F1 lacking the epsilon subunit [F1(-epsilon)]. Cys-containing epsilon variants were modified with a sulfhydryl-reactive photolabile cross-linker. Photocross-linking of epsilon to F1(-epsilon) yielded the same SDS gel pattern of cross-link products independent of the presence or absence of Mg2+ x ADP, phosphate and Mg2+ x ATP. Epsilon (wild type) [Ser6,Cys28]epsilon and [Ser6,Cys41]epsilon were cross-linked with subunit gamma. With chloroplast F0F1 the same cross-link pattern was obtained, except for one extra cross-link, probably between [Ser6,Cys28]epsilon and F0 subunit III. [Ser6,Cys17]epsilon and [Ser6,Cys85]epsilon did not produce cross-links. Cross-linking of epsilon, [Ser6,Cys28]epsilon, [Ser6,Cys41]epsilon to gamma in soluble chloroplast F1 impaired the ability of epsilon to inhibit Ca2+-ATPase activity. The Mg2+-ATPase activity of soluble F1 (measured in the presence of 30% MeOH) was not affected by cross-linking epsilon with gamma. Functional reconstitution of photophosphorylation in F1-depleted thylakoids was observed with F1 in which gamma was cross-linked to [Ser6,Cys28]epsilon or [Ser6,Cys41]epsilon but not with wild-type epsilon. In view of the intersubunit rotation of gamma relative to (alphabeta)3, which is driven by ATP hydrolysis, gamma and epsilon would seem to act concertedly as parts of the 'rotor' relative to the 'stator' (alphabeta)3.