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The plant Golgi plays a pivotal role in the biosynthesis of cell wall matrix polysaccharides, protein glycosylation, and vesicle trafficking. Golgi-localized proteins have become prospective targets for reengineering cell wall biosynthetic pathways for the efficient production of biofuels from plant cell walls. However, proteomic characterization of the Golgi has so far been limited, owing to the technical challenges inherent in Golgi purification. In this study, a combination of density centrifugation and surface charge separation techniques have allowed the reproducible isolation of Golgi membranes from Arabidopsis (Arabidopsis thaliana) at sufficiently high purity levels for in-depth proteomic analysis. Quantitative proteomic analysis, immunoblotting, enzyme activity assays, and electron microscopy all confirm high purity levels. A composition analysis indicated that approximately 19% of proteins were likely derived from contaminating compartments and ribosomes. The localization of 13 newly assigned proteins to the Golgi using transient fluorescent markers further validated the proteome. A collection of 371 proteins consistently identified in all replicates has been proposed to represent the Golgi proteome, marking an appreciable advancement in numbers of Golgi-localized proteins. A significant proportion of proteins likely involved in matrix polysaccharide biosynthesis were identified. The potential within this proteome for advances in understanding Golgi processes has been demonstrated by the identification and functional characterization of the first plant Golgi-resident nucleoside diphosphatase, using a yeast complementation assay. Overall, these data show key proteins involved in primary cell wall synthesis and include a mixture of well-characterized and unknown proteins whose biological roles and importance as targets for future research can now be realized.

ENHANCED DISEASE RESISTANCE 1 (EDR1) encodes a CTR1-like kinase and was previously reported to function as a negative regulator of disease resistance and ethylene-induced senescence. Here, we report that the edr1 mutant displays enhanced stress responses and spontaneous necrotic lesions under drought conditions in the absence of pathogen, suggesting that EDR1 is also involved in stress response signaling and cell death regulation. Double mutant analysis revealed that these drought-induced phenotypes require salicylic acid but not ethylene signaling pathways. In addition, the edr1-mediated ethylene-induced senescence phenotype was suppressed by mutations in EIN2, but not by mutations in SID2, PAD4, EDS1, or NPR1, suggesting that EDR1 functions at a point of cross talk between ethylene and salicylic acid signaling that impinges on senescence and cell death. Two edr1-associated phenotypes, drought-induced growth inhibition and ethylene-induced senescence, were suppressed by mutations in ORE9, implicating ubiquitin-mediated protein degradation in the regulation of these phenotypes. However, the ore9 mutation did not suppress edr1-mediated enhanced disease resistance to powdery mildew or spontaneous lesions, indicating that these phenotypes are controlled by separate signaling pathways. To investigate the function of the EDR1 kinase domain, we expressed the C-terminal third of EDR1 in wild-type Columbia and edr1 backgrounds under the control of a dexamethasone-inducible promoter. Overexpression of the EDR1 kinase domain in an edr1 background had no obvious effect on edr1-associated phenotypes. However, overexpression of the EDR1 kinase domain in a wild-type Columbia background caused dominant negative phenotypes, including enhanced disease resistance to powdery mildew and enhanced ethylene-induced senescence; thus, the overexpressed EDR1 kinase domain alone does not exert EDR1 function, but rather negatively affects the function of native EDR1 protein.

Loss-of-function mutations in the Arabidopsis (Arabidopsis thaliana) ENHANCED DISEASE RESISTANCE1 (EDR1) gene confer enhanced resistance to infection by powdery mildew (Golovinomyces cichoracearum). EDR1 encodes a protein kinase, but its substrates and the pathways regulated by EDR1 are unknown. To identify components of the EDR1 signal transduction pathway(s), we conducted a forward genetic screen for mutations that suppressed edr1-mediated disease resistance. Genetic mapping and cloning of one of these suppressor mutations revealed a recessive missense mutation in the KEEP ON GOING gene (KEG; At5g13530), which we designated keg-4. KEG encodes a multidomain protein that includes a RING E3 ligase domain, a kinase domain, ankyrin repeats, and HERC2-like repeats. The KEG protein has previously been shown to have ubiquitin ligase activity and to negatively regulate protein levels of the transcription factor ABCISIC ACID INSENSITIVE5. KEG mRNA levels were found to be 3-fold higher in edr1 mutant plants compared to wild type. Loss-of-function mutations in KEG are seedling lethal and are hypersensitive to glucose and abscisic acid (ABA). The keg-4 mutation, in contrast, conferred resistance to 6% glucose and suppressed edr1-mediated hypersensitivity to ABA, suggesting that the keg-4 mutation suppresses ABA signaling by altering KEG function. Several ABA-responsive genes were found to be further up-regulated in the edr1 mutant following ABA treatment, and this up-regulation was suppressed by the keg-4 mutation. We conclude that edr1-mediated resistance to powdery mildew is mediated, in part, by enhanced ABA signaling.