We have isolated cDNAs from maize (ZGB1) and Arabidopsis (AGB1) encoding proteins homologous to P subunits of guanine nucleotide-binding protein (G protein (9,10) or the transmission of red and blue light-induced signals (11,12). Furthermore, the cloning ofArabidopsis gene GPAI encoding a G protein a subunit designated GPal and its tomato homologue has provided molecular evidence for the existence of G protein-mediated signaling pathway(s) in plants (13, 14). The reports of G protein involvement in a number of different cellular functions raise the possibility that GPal may be involved in more than one signaling pathway. It is possible that G protein f3 subunit(s) might also play a direct role in signaling in plants.In this report, we describe the cloning of the maize ZGB1 and Arabidopsis AGB1 cDNAs encoding proteins that share >41% identity with animal G protein f3 subunits. § This level of homology is greater than that between a known yeast G protein (3subunit and animal ones (15). ZGB1 and AGB1 may represent an additional type of G protein f subunit that is conserved in flowering plants and expressed in roots, leaves, and flowers. MATERIALS AND METHODSMaize Subtracted Library. A maize tassel cDNA library in pCDNAII (Invitrogen) and a maize ear shoot cDNA library in A-Uni-ZAP (Stratagene) were provided by M. Albertsen and G. Huffman, respectively, of Pioneer Hi-Bred International. Biotinylated RNA was generated in vitro from the ear shoot library by using the Gemini Riboprobe system (Promega) with the manufacturer's protocol modified to include 1 mM biotin-11 rUTP (Enzo Diagnostics, instead of photobiotin rUTP) and an increased rUTP concentration (1 mM) in the transcription reaction. A subtracted tassel cDNA library was prepared by hybridization of the biotinylated RNAs with single-stranded DNA from the tassel library as described (16). The second strand of the nonsubtracted DNA molecules was synthesized with Klenow as described (17). One of the obtained clones was designated pPHP2541.S' Rapid Amplification of cDNA Ends (RACE) for PCR. 5' RACE primer extension was performed by using the 5' RACE system (GIBCO/BRL) with leaf and tassel poly(A)+ RNA and the oligonucleotide 5'-GATATCCACAGCCTA-CAGTTG-3' derived from the sequence of the pPHP2541 cDNA insert. The pPHP2541-derived nested primer 5'-GTATTTGATGAGTTGATGGAC-3' and the provided anchor primer were used for PCR amplification with Taq I polymerase (Perkin-Elmer). A clone containing a 0.6-kb PCR product was named pPHP3573.Library Screening, Subcloning, and Sequence Analysis. A A-Uni-ZAP maize tassel cDNA library was screened for a full-length cDNA by using standard conditions and a labeled 0.9-kb insert from pPHP2541 as a probe. A AYES cDNA library from Arabidopsis thaliana (ref. 18; a gift from J. Mulligan) was screened with 32P-labeled 0.9-kb and 0.6-kb maize cDNAs from pPHP2541 and pPHP5373, respectively. cDNAs were subcloned into the Promega vector pGEM7Zf(+) for sequencing of both strands.Southern and Northern Blot Hybridizations. Genomic DNA isol...

The Arabidopsis AGL3 gene was previously identified on the basis of sequence similarity to the floral homeotic gene AGAMOUS (AG), which encodes a protein with a conserved MADS domain that is also found in human and yeast transcription factors (SRF and MCM1, respectively). Analysis of newly isolated full-length cDNA clones as well as genomic clones indicates that AGL3 is indeed a MADS-box gene with a general intron-exon structure similar to other plant MADS-box genes. However, unlike the others, which are expressed specifically in flowers, AGL3 is expressed in all above-ground vegetative organs, as well as in flowers, but not in roots. Furthermore, since AGL3 is MADS-domain protein, it is likely that it is also a DNA-binding protein regulating transcription. To characterize AGL3 as a DNA-binding protein in vitro, we expressed the AGL3 protein in Escherichia coli, and characterized its DNA-binding properties. We show that AGL3 binds to sequences which resemble the target sequences of SRF and MCM1, and have determined the consensus sequence to which AGL3 binds using random oligonucleotides. These results suggest that AGL3 is a widely distributed DNA-binding protein, which may be involved the transcriptional regulation of genes in many cells.

Towards the elucidation of the cellular function(s) of GPal, we have characterized its subcellular localization using immunofluorescence and cell fractionation. GPal is not present in nuclei or chloroplasts. It is a membrane-bound protein, and analysis of isolated endoplasmic and plasma membranes indicates a good correlation between GPal in both the plasma membrane and the ER compartment. Interestingly, these results may suggest more different functions for GPal: it might be involved in transmission of extracellular signals across the plasma membrane and in the cytoplasm, and/or it may also be involved in regulating some aspects of the ER functions or membrane trafficking between both membranes.

Besides the systemic acquired resistance (SAR) induced in response to microbial stimulation, host plants may also acquire resistance to pathogens in response to endogenous stimuli associated with their own development. In tobacco (Nicotiana tabacum), the vegetative-to-flowering transition comes along with a susceptibility-to-resistance transition to the causal agent of black shank disease, the oomycete Phytophthora parasitica. This resistance affects infection effectiveness and hyphal expansion and is associated with extracellular accumulation of a cytotoxic activity that provokes in vitro cell death of P. parasitica zoospores. As a strategy to determine the extracellular events important for restriction of pathogen growth, we screened the tobacco genome for genes encoding secreted or membrane-bound proteins expressed in leaves of flowering plants. Using a signal sequence trap approach in yeast (Saccharomyces cerevisiae), 298 clones were selected that appear to encode for apoplastic, cell wall, or membrane-bound proteins involved in stress response, in plant defense, or in cell wall modifications. Microarray and northern-blot analyses revealed that, at late developmental stages, leaves were characterized by the coordinate up-regulation of genes involved in SAR and in peroxidative cross-linking of structural proteins to cell wall. This suggests the potential involvement of these genes in extracellular events that govern the expression of developmental resistance. The analysis of the influence of salicylic acid on mRNA accumulation also indicates a more complex network for regulation of gene expression at a later stage of tobacco development than during SAR. Further characterization of these genes will permit the formulation of hypotheses to explain resistance and to establish the connection with development.Establishment of acquired resistance to pathogens may be induced by physiological and/or developmental changes taking place in growing plants. The occurrence of a transition from susceptibility to resistance during development is a widely reported phenomenon in monocotyledons and dicotyledons in the case of viruses (Leisner et al., 1992(Leisner et al., , 1993, bacteria (Century et al., 1999;Kus et al., 2002), fungi (Moose and Sisco, 1994; Abedon and Tracy, 1996), and oomycetes (Reuveni et al., 1986;Wyatt et al., 1991: Hugot et al., 1999. Although this phenomenon, age-related resistance (ARR), is well documented from a pathological point of view, few studies have dealt with the genetic and molecular bases of disease control during plant development.On the contrary, numerous studies have investigated defense mechanisms activated in response to pathogen infection and associated with plant disease resistance (Hammond-Kosack and Parker, 2003). These studies have underlined the key role of the host extracellular space as sorting compartment of plant-pathogen interactions. At the early step of infection, the host plant recognizes some pathogensecreted molecules that elicit the coordinate activation of defense reactio...