The role of phosphorylatable serine residues in the DNA-binding domain of Arabidopsis bZIP transcription factors.

Reversible phosphorylation plays a crucial role in regulating the activity of enzymes and other proteins in all living organisms. Particularly, the phosphorylation of transcription factors can modulate their capability to regulate downstream target genes. In plants, basic domain-containing leucine-zipper (bZIP) transcription factors have an important function in the regulation of many developmental processes and adaptive responses to the environment. By a comprehensive sequence analysis, we identified a set of highly conserved, potentially phospho-accepting serines within the DNA-binding domain of plant bZIPs. Structural modelling revealed that these serines are in physical contact with the DNA and predicts that their phosphorylation will have a major influence on the DNA-binding activity of plant bZIPs. In support of this, we show, by means of a quantitative in vitro binding assay, that phosphorylation-mimicking substitutions of some of these serines strongly interfere with the DNA binding of two prototypical Arabidopsis bZIPs, namely AtZIP63 and HY5. Our data suggest that the identified serines could serve as in vivo targets for kinases and phosphatases, allowing the fine-tuning of bZIP factor activity at the DNA-protein interaction level.

Expression patterns within the Arabidopsis C/S1 bZIP transcription factor network: availability of heterodimerization partners controls gene expression during stress response and development.

Members of the Arabidopsis group C/S1 basic leucine zipper (bZIP) transcription factor (TF) network are proposed to implement transcriptional reprogramming of plant growth in response to energy deprivation and environmental stresses. The four group C and five group S1 members form specific heterodimers and are, therefore, considered to cooperate functionally. For example, the interplay of C/S1 bZIP TFs in regulating seed maturation genes was analyzed by expression studies and target gene regulation in both protoplasts and transgenic plants. The abundance of the heterodimerization partners significantly affects target gene transcription. Therefore, a detailed analysis of the developmental and stress related expression patterns was performed by comparing promoter: GUS and transcription data. The idea that the C/S1 network plays a role in the allocation of nutrients is supported by the defined and partially overlapping expression patterns in sink leaves, seeds and anthers. Accordingly, metabolic signals strongly affect bZIP expression on the transcriptional and/or post-transcriptional level. Sucrose induced repression of translation (SIRT) was demonstrated for all group S1 bZIPs. In particular, transcription of group S1 genes strongly responds to various abiotic stresses, such as salt (AtbZIP1) or cold (AtbZIP44). In summary, heterodimerization and expression data provide a basic framework to further determine the functional impact of the C/S1 network in regulating the plant energy balance and nutrient allocation.

Bimolecular fluorescence complementation (BiFC) to study protein-protein interactions in living plant cells.

Dynamic networks of protein-protein interactions regulate numerous cellular processes and determine the ability of cells to respond appropriately to environmental stimuli. However, the study of protein complex formation in living plant cells has remained experimentally difficult and time-consuming and requires sophisticated technical equipment. In this report, we describe a bimolecular fluorescence complementation (BiFC) technique for visualization of protein-protein interactions in plant cells. This approach is based on the formation of a fluorescent complex by two non-fluorescent fragments of the yellow fluorescent protein (YFP) brought together by the association of interacting proteins fused to these fragments. We present the BiFC vectors currently available for the transient and stable transformation of plant cells and provide a detailed protocol for the successful use of BiFC in plants.

Photosynthetic redox regulation of the plastocyanin promoter in tobacco.

Persistent light quality gradients in dense plant populations induce imbalances in the distribution of excitation energy between the photosystems. Plants counteract such conditions by re-adjusting the stoichiometry of photosystems, which involves control of photosynthesis gene expression both in chloroplasts and in the nucleus. Decisive control parameters are redox signals from the photosynthetic electron transport chain, one prominent is the plastoquinone (PQ) pool. In a recent study, a plastocyanin (PC)-promoter::beta-glucuronidase reporter gene construct in tobacco demonstrated reversible redox regulation in response to varying light qualities. Here, northern and Western analyses demonstrate that this promoter regulation also accounts for the accumulation of the endogenous tobacco PetE gene transcripts and the protein amounts of the encoded PC. Hence, the reporter gene construct reflects the natural regulation of this nuclear gene in tobacco. In kinetic experiments, the response of the construct to either oxidation or reduction of the PQ pool was tested by defined light quality shifts. The construct displayed upregulation in response to a reduction signal and downregulation in response to an oxidation signal, both with a half-time of about 24 h. The response was finished after 48 h. DCMU application abolished the upregulation in response to the reduction signal, indicating the dependence on thylakoid membrane electron transport. To study the redox-responsive promoter region in more detail, several promoter deletion constructs were tested for their responsiveness. All constructs displayed a reversible response to light-induced oxidation and reduction signals; however, a minimal promoter region localised between -168 to -79 bp upstream of the transcription start site was sufficient to confer this redox regulation. This indicates that photosynthetic redox signals act on distinct regions in the PC promoter in a manner independent from photoreceptors and upstream cis elements conferring high basic expression in the light.

Post-translational regulation of plant bZIP factors.

The post-translational regulation of transcription factors plays an important role in the control of gene expression in eukaryotes. The mechanisms of regulation include not only factor modifications but also regulated protein-protein interaction, protein degradation and intracellular partitioning. In plants, the basic-region leucine zipper (bZIP) transcription factors contribute to many transcriptional response pathways. Despite this, little is known about their post-translational regulation. Recent findings suggest that plant bZIP factors are under the control of various partially signal-induced and reversible post-translational mechanisms that are crucial for the control of their function. However, the fact that, to date, only a few plant bZIPs have been analyzed with respect to post-translational regulation indicates that we have just identified the tip of an iceberg.

bZIP10-LSD1 antagonism modulates basal defense and cell death in Arabidopsis following infection.

Plants use sophisticated strategies to balance responses to oxidative stress. Programmed cell death, including the hypersensitive response (HR) associated with successful pathogen recognition, is one cellular response regulated by reactive oxygen in various cellular contexts. The Arabidopsis basic leucine zipper (bZIP) transcription factor AtbZIP10 shuttles between the nucleus and the cytoplasm and binds consensus G- and C-box DNA sequences. Surprisingly, AtbZIP10 can be retained outside the nucleus by LSD1, a protein that protects Arabidopsis cells from death in the face of oxidative stress signals. We demonstrate that AtbZIP10 is a positive mediator of the uncontrolled cell death observed in lsd1 mutants. AtbZIP10 and LSD1 act antagonistically in both pathogen-induced HR and basal defense responses. LSD1 likely functions as a cellular hub, where its interaction with AtbZIP10 and additional, as yet unidentified, proteins contributes significantly to plant oxidative stress responses.

Combinatorial control of Arabidopsis proline dehydrogenase transcription by specific heterodimerisation of bZIP transcription factors.

Proline metabolism has been implicated in plant responses to abiotic stresses. The Arabidopsis thaliana proline dehydrogenase (ProDH) is catalysing the first step in proline degradation. Transcriptional activation of ProDH by hypo-osmolarity is mediated by an ACTCAT cis element, a typical binding site of basic leucine zipper (bZIP) transcription factors. In this study, we demonstrate by gain-of-function and loss-of-function approaches, as well as chromatin immunoprecipitation (ChIP), that ProDH is a direct target gene of the group-S bZIP factor AtbZIP53. Dimerisation studies making use of yeast and Arabidopsis protoplast-based two-hybrid systems, as well as bimolecular fluorescence complementation (BiFC) reveal that AtbZIP53 does not preferentially form dimers with group-S bZIPs but strongly interacts with members of group-C. In particular, a synergistic interplay of AtbZIP53 and group-C AtbZIP10 was demonstrated by colocalisation studies, strong enhancement of ACTCAT-mediated transcription as well as complementation studies in atbzip53 atbzip10 T-DNA insertion lines. Heterodimer mediated activation of transcription has been found to operate independent of the DNA-binding properties and is described as a crucial mechanism to modulate transcription factor activity and function.

Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation.

Dynamic networks of protein-protein interactions regulate numerous cellular processes and determine the ability to respond appropriately to environmental stimuli. However, the investigation of protein complex formation in living plant cells by methods such as fluorescence resonance energy transfer has remained experimentally difficult, time consuming and requires sophisticated technical equipment. Here, we report the implementation of a bimolecular fluorescence complementation (BiFC) technique for visualization of protein-protein interactions in plant cells. This approach relies on the formation of a fluorescent complex by two non-fluorescent fragments of the yellow fluorescent protein brought together by association of interacting proteins fused to these fragments (Hu et al., 2002). To enable BiFC analyses in plant cells, we generated different complementary sets of expression vectors, which enable protein interaction studies in transiently or stably transformed cells. These vectors were used to investigate and visualize homodimerization of the basic leucine zipper (bZIP) transcription factor bZIP63 and the zinc finger protein lesion simulating disease 1 (LSD1) from Arabidopsis as well as the dimer formation of the tobacco 14-3-3 protein T14-3c. The interaction analyses of these model proteins established the feasibility of BiFC analyses for efficient visualization of structurally distinct proteins in different cellular compartments. Our investigations revealed a remarkable signal fluorescence intensity of interacting protein complexes as well as a high reproducibility and technical simplicity of the method in different plant systems. Consequently, the BiFC approach should significantly facilitate the visualization of the subcellular sites of protein interactions under conditions that closely reflect the normal physiological environment.

Chloroplast redox control of nuclear gene expression--a new class of plastid signals in interorganellar communication.

Chloroplasts are genetically semiautonomous organelles that contain their own subset of 100-120 genes coding for chloroplast proteins, tRNAs, and rRNAs. However, the great majority of the chloroplast proteins are encoded in the nucleus and must be imported into the organelle after their translation in the cytosol. This arrangement requires a high degree of coordination between the gene expression machineries in chloroplasts and nucleus, which is achieved by a permanent exchange of information between both compartments. The existence of such coordinating signals has long been known; however, the underlying molecular mechanisms and signaling routes are not understood. The present data indicate that the expression of nuclear-encoded chloroplast proteins is coupled to the functional state of the chloroplasts. Photosynthesis, which is the major function of chloroplasts, plays a crucial role in this context. Changes in the reduction/oxidation (redox) state of components of the photosynthetic machinery act as signals, which regulate the expression of chloroplast proteins in both chloroplasts and nucleus and help to coordinate the expression both in compartments. Recent advances in understanding chloroplast redox regulation of nuclear gene expression are summarized, and the importance for intracellular signaling is discussed.