Seed dormancy is a trait of considerable adaptive significance because it maximizes seedling survival by preventing premature germination under unfavorable conditions. Understanding how seeds break dormancy and initiate growth is also of great agricultural and biotechnological interest. Abscisic acid (ABA) plays primary regulatory roles in the initiation and maintenance of seed dormancy. Here we report that the basic leucine zipper transcription factor ABI5 confers an enhanced response to exogenous ABA during germination, and seedling establishment, as well as subsequent vegetative growth. These responses correlate with total ABI5 levels. We show that ABI5 expression defines a narrow developmental window following germination, during which plants monitor the environmental osmotic status before initiating vegetative growth. ABI5 is necessary to maintain germinated embryos in a quiescent state thereby protecting plants from drought. As expected for a key player in ABA-triggered processes, ABI5 protein accumulation, phosphorylation, stability, and activity are highly regulated by ABA during germination and early seedling growth.A bscisic acid (ABA) is a phytohormone regulating the initiation and maintenance of seed dormancy. It also plays an essential role in a plant's response to stress, particularly water deprivation, notably by regulating stomatal aperture (1). So far, ABA-insensitive screens have been widely used to identify molecular genetic components of the ABA signal transduction pathway (2, 3). In these screens, mutagenized Arabidopsis seeds were exposed to ABA concentrations that inhibit germination of wild-type (WT) seeds, and putative mutants that were able to germinate were isolated (2, 3). These screens have allowed the identification of several ABI (ABA-insensitive) genes (4-10) and recent studies have established that ABI1 and ABI3 are key players in vegetative and embryonic ABA responses, respectively (1, 11).Nonetheless, few reports have clarified the physiological role of ABA and mechanisms of action triggered by ABA during germination and early seedling growth. We were led to address these issues by the recent cloning and analysis of ABI5 by two independent groups (4, 5). The abi5 mutation is recessive and ABI5 encodes a putative transcription factor of the basic leucine zipper (bZIP) family (4, 5). The bZIP region of ABI5 shows extensive homology to previously characterized plant (bZIP) transcription factors capable of activating reporter genes containing ABA-responsive DNA elements (ABREs) (12)(13)(14). ABI5 also binds to ABREs in vitro (unpublished results) and dry seeds of abi5 show reduced transcript levels of ABA-responsive and ABRE-containing late embryonic genes such as AtEm1 and AtEm6 (4, 5). Together with the ABA insensitivity of abi5 mutants, these results show that ABI5 is the first bZIP plant factor found to be required in vivo to signal ABA-elicited responses.In the present work, we found that ABA regulates ABI5 accumulation and activity during a limited developmental window. On re...

SummaryThe development of a germinating embryo into an autotrophic seedling is arrested under conditions of water deficit. This ABA-mediated developmental checkpoint requires the bZIP transcription factor ABI5. Here, we used abi3-1, which is also unable to execute this checkpoint, to investigate the relative role of ABI3 and ABI5 in this process. In wild-type Arabidopsis plants, ABI3 expression and activity parallel those described for ABI5 following stratification. During this process, transcript levels of late embryogenesis genes such as AtEm1 and AtEm6 are also re-induced, which might be responsible for the acquired osmotic tolerance in germinated embryos whose growth is arrested. ABI5 expression is greatly reduced in abi3-1 mutants, which has low AtEm1 or AtEm6 expression. Cross complementation experiments showed that 35S-ABI5 could complement abi3-1, whereas 35S-ABI3 cannot complement abi5-4. These results indicate that ABI5 acts downstream of ABI3 to reactivate late embryogenesis programmes and to arrest growth of germinating embryos. Although ABI5 is consistently located in the nucleus, chromosomal immunoprecipitation (ChIP) experiments revealed that ABA increases ABI5 occupancy on the AtEm6 promoter.

Remorins (REMs) are proteins of unknown function specific to vascular plants. We have used imaging and biochemical approaches and in situ labeling to demonstrate that REM clusters at plasmodesmata and in ;70-nm membrane domains, similar to lipid rafts, in the cytosolic leaflet of the plasma membrane. From a manipulation of REM levels in transgenic tomato (Solanum lycopersicum) plants, we show that Potato virus X (PVX) movement is inversely related to REM accumulation. We show that REM can interact physically with the movement protein TRIPLE GENE BLOCK PROTEIN1 from PVX. Based on the localization of REM and its impact on virus macromolecular trafficking, we discuss the potential for lipid rafts to act as functional components in plasmodesmata and the plasma membrane.

Plant immune responses to pathogen attack include the hypersensitive response (HR), a form of programmed cell death occurring at invasion sites. We previously reported on Arabidopsis thaliana MYB30, a transcription factor that acts as a positive regulator of a cell death pathway conditioning the HR. Here, we show by microarray analyses of Arabidopsis plants misexpressing MYB30 that the genes encoding the four enzymes forming the acyl-coA elongase complex are putative MYB30 targets. The acyl-coA elongase complex synthesizes very-long-chain fatty acids (VLCFAs), and the accumulation of extracellular VLCFA-derived metabolites (leaf epidermal wax components) was affected in MYB30 knockout mutant and overexpressing lines. In the same lines, a lipid extraction procedure allowing high recovery of sphingolipids revealed changes in VLCFA contents that were amplified in response to inoculation. Finally, the exacerbated HR phenotype of MYB30-overexpressing lines was altered by the loss of function of the acyl-ACP thioesterase FATB, which causes severe defects in the supply of fatty acids for VLCFA biosynthesis. Based on these findings, we propose a model in which MYB30 modulates HR via VLCFAs by themselves, or VLCFA derivatives, as cell death messengers in plants.

Several Arabidopsis mutants have been isolated that either bypass this ABA-mediated growth arrest or show an enhanced response to ABA (for review, see Finkelstein et al. 2002). The latter category includes mutations in ERA1, which encodes a subunit of a farnesyl transferase (Cutler et al. 1996), and ABH1, which encodes a mRNA cap binding protein (Hugouvieux et al. 2001). Mutants bypassing the ABA-mediated growth inhibition were designated abi1, abi2, abi3, abi4, abi5, abi8 (for ABAinsensitive), cho1, and cho2 (Koornneef et al. 1984;Finkelstein 1994;Lopez-Molina and Chua 2000;Finkelstein et al. 2002;Nambara et al. 2002). The ABI8, CHO1, and CHO2 genes have not yet been cloned Nambara et al. 2002). ABI1 and ABI2 encode two homologous serine/threonine phosphatases of class 2C (Leung et al. 1994(Leung et al. , 1997Meyer et al. 1994;Rodriguez et al. 1998), and both abi1 and abi2 have identical Gly-

Summary A strain‐specific vaccine represents the best possible response to the threat of an influenza pandemic. Rapid delivery of such a vaccine to the world's population before the peak of the first infection wave seems to be an unattainable goal with the current influenza vaccine manufacturing capacity. Plant‐based transient expression is one of the few production systems that can meet the anticipated surge requirement. To assess the capability of plant agroinfiltration to produce an influenza vaccine, we expressed haemagglutinin (HA) from strains A/Indonesia/5/05 (H5N1) and A/New Caledonia/20/99 (H1N1) by agroinfiltration of Nicotiana benthamiana plants. Size distribution analysis of protein content in infiltrated leaves revealed that HA was predominantly assembled into high‐molecular‐weight structures. H5‐containing structures were purified and examination by transmission electron microscopy confirmed virus‐like particle (VLP) assembly. High‐performance thin layer chromatography analysis of VLP lipid composition highlighted polar and neutral lipid contents comparable with those of purified plasma membranes from tobacco plants. Electron microscopy of VLP‐producing cells in N. benthamiana leaves confirmed that VLPs accumulated in apoplastic indentations of the plasma membrane. Finally, immunization of mice with two doses of as little as 0.1 µg of purified influenza H5‐VLPs triggered a strong immune response against the homologous virus, whereas two doses of 0.5 µg of H5‐VLPs conferred complete protection against a lethal challenge with the heterologous A/Vietnam/1194/04 (H5N1) strain. These results show, for the first time, that plants are capable of producing enveloped influenza VLPs budding from the plasma membrane; such VLPs represent very promising candidates for vaccination against influenza pandemic strains.

Remorin proteins have been hypothesized to play important roles during cellular signal transduction processes. Induction of some members of this multigene family has been reported during biotic interactions. However, no roles during host-bacteria interactions have been assigned to remorin proteins until now. We used root nodule symbiosis between Medicago truncatula and Sinorhizobium meliloti to study the roles of a remorin that is specifically induced during nodulation. Here we show that this oligomeric remorin protein attaches to the host plasma membrane surrounding the bacteria and controls infection and release of rhizobia into the host cytoplasm. It interacts with the core set of symbiotic receptors that are essential for perception of bacterial signaling molecules, and thus might represent a plant-specific scaffolding protein.

A cell membrane can be considered a liquid-phase plane in which lipids and proteins theoretically are free to diffuse. Numerous reports, however, describe retarded diffusion of membrane proteins in animal cells. This anomalous diffusion results from a combination of structuring factors including protein-protein interactions, cytoskeleton corralling, and lipid organization into microdomains. In plant cells, plasma-membrane (PM) proteins have been described as relatively immobile, but the control mechanisms that structure the PM have not been studied. Here, we use fluorescence recovery after photobleaching to estimate mobility of a set of minimal PM proteins. These proteins consist only of a PM-anchoring domain fused to a fluorescent protein, but their mobilities remained limited, as is the case for many full-length proteins. Neither the cytoskeleton nor membrane microdomain structure was involved in constraining the diffusion of these proteins. The cell wall, however, was shown to have a crucial role in immobilizing PM proteins. In addition, by single-molecule fluorescence imaging we confirmed that the pattern of cellulose deposition in the cell wall affects the trajectory and speed of PM protein diffusion. Regulation of PM protein dynamics by the plant cell wall can be interpreted as a mechanism for regulating protein interactions in processes such as trafficking and signal transduction.