In plants, basic region/leucine zipper motif (bZIP) transcription factors regulate processes including pathogen defence, light and stress signalling, seed maturation and flower development. The Arabidopsis genome sequence contains 75 distinct members of the bZIP family, of which approximately 50 are not described in the literature. Using common domains, the AtbZIP family can be subdivided into ten groups. Here, we review the available data on bZIP functions in the context of subgroup membership and discuss the interacting proteins. This integration is essential for a complete functional characterization of bZIP transcription factors in plants, and to identify functional redundancies among AtbZIP factors.
Since years, research on SnRK1, the major cellular energy sensor in plants, has tried to define its role in energy signalling. However, these attempts were notoriously hampered by the lethality of a complete knockout of SnRK1. Therefore, we generated an inducible amiRNA::SnRK1α2 in a snrk1α1 knock out background (snrk1α1/α2) to abolish SnRK1 activity to understand major systemic functions of SnRK1 signalling under energy deprivation triggered by extended night treatment. We analysed the in vivo phosphoproteome, proteome and metabolome and found that activation of SnRK1 is essential for repression of high energy demanding cell processes such as protein synthesis. The most abundant effect was the constitutively high phosphorylation of ribosomal protein S6 (RPS6) in the snrk1α1/α2 mutant. RPS6 is a major target of TOR signalling and its phosphorylation correlates with translation. Further evidence for an antagonistic SnRK1 and TOR crosstalk comparable to the animal system was demonstrated by the in vivo interaction of SnRK1α1 and RAPTOR1B in the cytosol and by phosphorylation of RAPTOR1B by SnRK1α1 in kinase assays. Moreover, changed levels of phosphorylation states of several chloroplastic proteins in the snrk1α1/α2 mutant indicated an unexpected link to regulation of photosynthesis, the main energy source in plants.
SummaryIn vivo protein-protein interactions are frequently studied by means of yeast two-hybrid analysis. However, interactions detected in yeast might differ considerably in the plant system. Based on GAL4 DNA-binding (BD) and activation domains (AD) we established an Arabidopsis protoplast two-hybrid (P2H) system. The use of Gateway Ò -compatible vectors enables the high-throughput screening of protein-protein interactions in plant cells. The efficiency of the system was tested by examining the homo-and heterodimerization properties of basic leucine zipper (bZIP) transcription factors. A comprehensive heterodimerization matrix of Arabidopsis thaliana group C and group S bZIP transcription factors was generated by comparing the results of yeast and protoplast two-hybrid experiments. Surprisingly, almost no homodimerization but rather specific and selective heterodimerization was detected. Heterodimers were preferentially formed between group C members (AtbZIP9, -10, -25, -63) and members of group S1 (AtbZIP1, -2, -11, -44, -53). In addition, significant but low-affinity interactions were detected inside group S1, S2 or C AtbZIPs, respectively. As a quantitative approach, P2H identified weak heterodimerization events which were not detected in the yeast system. Thus, in addition to cell biological techniques, P2H is a valuable tool for studying protein-protein interaction in living plant cells.
Control of energy homeostasis is crucial for plant survival, particularly under biotic or abiotic stress conditions. Energy deprivation induces dramatic reprogramming of transcription, facilitating metabolic adjustment. An in-depth knowledge of the corresponding regulatory networks would provide opportunities for the development of biotechnological strategies. Low energy stress activates the Arabidopsis thaliana group S1 basic leucine zipper transcription factors bZIP1 and bZIP53 by transcriptional and posttranscriptional mechanisms. Gain-of-function approaches define these bZIPs as crucial transcriptional regulators in Pro, Asn, and branched-chain amino acid metabolism. Whereas chromatin immunoprecipitation analyses confirm the direct binding of bZIP1 and bZIP53 to promoters of key metabolic genes, such as ASPARAGINE SYNTHETASE1 and PROLINE DEHYDROGENASE, the G-box, C-box, or ACT motifs (ACTCAT) have been defined as regulatory cis-elements in the starvation response. bZIP1 and bZIP53 were shown to specifically heterodimerize with group C bZIPs. Although single loss-of-function mutants did not affect starvation-induced transcription, quadruple mutants of group S1 and C bZIPs displayed a significant impairment. We therefore propose that bZIP1 and bZIP53 transduce low energy signals by heterodimerization with members of the partially redundant C/S1 bZIP factor network to reprogram primary metabolism in the starvation response.
Metabolic adjustment to changing environmental conditions, particularly balancing of growth and defense responses, is crucial for all organisms to survive. The evolutionary conserved AMPK/Snf1/SnRK1 kinases are well-known metabolic master regulators in the low-energy response in animals, yeast and plants. They act at two different levels: by modulating the activity of key metabolic enzymes, and by massive transcriptional reprogramming. While the first part is well established, the latter function is only partially understood in animals and not at all in plants. Here we identified the Arabidopsis transcription factor bZIP63 as key regulator of the starvation response and direct target of the SnRK1 kinase. Phosphorylation of bZIP63 by SnRK1 changed its dimerization preference, thereby affecting target gene expression and ultimately primary metabolism. A bzip63 knock-out mutant exhibited starvation-related phenotypes, which could be functionally complemented by wild type bZIP63, but not by a version harboring point mutations in the identified SnRK1 target sites.DOI: http://dx.doi.org/10.7554/eLife.05828.001
Transcription of Arabidopsis thaliana seed maturation (MAT) genes is controlled by members of several transcription factor families, such as basic leucine zippers (bZIPs), B3s, MYBs, and DOFs. In this work, we identify Arabidopsis bZIP53 as a novel transcriptional regulator of MAT genes. bZIP53 expression in developing seeds precedes and overlaps that of its target genes. Gain-and loss-of-function approaches indicate a correlation between the amount of bZIP53 protein and MAT gene expression. Specific in vivo and in vitro binding of bZIP53 protein to a G-box element in the albumin 2S2 promoter is demonstrated. Importantly, heterodimerization with bZIP10 or bZIP25, previously described bZIP regulators of MAT gene expression, significantly enhances DNA binding activity and produces a synergistic increase in target gene activation. Fulllevel target gene activation is strongly correlated with the ratio of the correspondent bZIP heterodimerization partners. Whereas bZIP53 does not interact with ABI3, a crucial transcriptional regulator in Arabidopsis seeds, ternary complex formation between the bZIP heterodimers and ABI3 increases the expression of MAT genes in planta. We therefore propose that heterodimers containing bZIP53 participate in enhanceosome formation to produce a dramatic increase in MAT gene transcription.
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.
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