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.

SummaryThe tolerance responses of plants to many abiotic stresses are conjectured to be controlled by complex gene networks. In the frame of the AtGenExpress project a comprehensive Arabidopsis thaliana genome transcript expression study was performed using the Affymetrix ATH1 microarray in order to understand these regulatory networks in detail. In contrast to earlier studies, we subjected, side-by-side and in a high-resolution kinetic series, Arabidopsis plants, of identical genotype grown under identical conditions, to different environmental stresses comprising heat, cold, drought, salt, high osmolarity, UV-B light and wounding. Furthermore, the harvesting of tissue and RNA isolation were performed in parallel at the same location using identical experimental protocols. Here we describe the technical performance of the experiments. We also present a general overview of environmental abiotic stress-induced gene expression patterns and the results of a model bioinformatics analysis of gene expression in response to UV-B light, drought and cold stress. Our results suggest that the initial transcriptional stress reaction of Arabidopsis might comprise a set of core environmental stress response genes which, by adjustment of the energy balance, could have a crucial function in various stress responses. In addition, there are indications that systemic signals generated by the tissue exposed to stress play a major role in the coordination and execution of stress responses. In summary, the information reported provides a prime reference point and source for the subsequent exploitation of this important resource for research into plant abiotic stress.

The plant hormone indoleacetic acid (IAA) transcriptionally activates early genes in plants. The Aux͞IAA family of early genes encodes proteins that are short-lived and nuclear-localized. They also contain a putative prokaryotic ␤␣␣ DNA binding motif whose formation requires protein dimerization. Here, we show that the pea PS-IAA4 and Arabidopsis IAA1 and IAA2 proteins perform homo-and heterotypic interactions in yeast using the two-hybrid system. Gel-filtration chromatography and chemical cross-linking experiments demonstrate that the PS-IAA4 and IAA1 proteins interact to form homodimers in vitro. Deletion analysis of PS-IAA4 indicates that the ␤␣␣ containing acidic C terminus of the protein is necessary for homotypic interactions in the yeast two-hybrid system. Screening an Arabidopsis -ACT cDNA library using IAA1 as a bait reveals heterotypic interactions of IAA1 with known and newly discovered members of the Arabidopsis Aux͞IAA gene family. The new member IAA24 has similarity to ARF1, a transcription factor that binds to an auxin response element. Combinatorial interactions among the various members of the Aux͞IAA gene family may regulate a variety of late genes as well as serve as autoregulators of early auxin-regulated gene expression. These interactions provide a molecular basis for the developmental and tissue-specific manner of auxin action.

The Arabidopsis calcineurin B-like calcium sensor proteins (AtCBLs) interact with a group of serinethreonine protein kinases (AtCIPKs) in a calciumdependent manner. Here we identify a 24 amino acid domain (NAF domain) unique to these kinases as being required and suf®cient for interaction with all known AtCBLs. Mutation of conserved residues either abolished or signi®cantly diminished the af®nity of AtCIPK1 for AtCBL2. Comprehensive two-hybrid screens with various AtCBLs identi®ed 15 CIPKs as potential targets of CBL proteins. Database analyses revealed additional kinases from Arabidopsis and other plant species harbouring the NAF interaction module. Several of these kinases have been implicated in various signalling pathways mediating responses to stress, hormones and environmental cues. Full-length CIPKs show preferential interaction with distinct CBLs in yeast and in vitro assays. Our ®ndings suggest differential interaction af®nity as one of the mechanisms generating the temporal and spatial speci®city of calcium signals within plant cells and that different combinations of CBL±CIPK proteins contribute to the complex network that connects various extracellular signals to de®ned cellular responses.

SummaryEthylene is a plant hormone that regulates many aspects of growth and development. Despite the well-known association between ethylene and stress signalling, its effects on stomatal movements are largely unexplored. Here, genetic and physiological data are provided that position ethylene into the Arabidopsis guard cell signalling network, and demonstrate a functional link between ethylene and hydrogen peroxide (H 2 O 2 ). In wild-type leaves, ethylene induces stomatal closure that is dependent on H 2 O 2 production in guard cells, generated by the nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase AtrbohF. Ethyleneinduced closure is inhibited by the ethylene antagonists 1-MCP and silver. The ethylene receptor mutants etr1-1 and etr1-3 are insensitive to ethylene in terms of stomatal closure and H 2 O 2 production. Stomata of the ethylene signalling ein2-1 and arr2 mutants do not close in response to either ethylene or H 2 O 2 but do generate H 2 O 2 following ethylene challenge. Thus, the data indicate that ethylene and H 2 O 2 signalling in guard cells are mediated by ETR1 via EIN2 and ARR2-dependent pathway(s), and identify AtrbohF as a key mediator of stomatal responses to ethylene.