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.

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.