SUMMARYAutophagic recycling of intracellular plant constituents is maintained at a basal level under normal growth conditions but can be induced in response to nutritional demand, biotic stress, and senescence. One route requires the ubiquitin-fold proteins Autophagy-related (ATG)-8 and ATG12, which become attached to the lipid phosphatidylethanolamine (PE) and the ATG5 protein, respectively, during formation of the engulfing vesicle and delivery of its cargo to the vacuole for breakdown. Here, we genetically analyzed the conjugation machinery required for ATG8/12 modification in Arabidopsis thaliana with a focus on the two loci encoding ATG12. Whereas single atg12a and atg12b mutants lack phenotypic consequences, atg12a atg12b double mutants senesce prematurely, are hypersensitive to nitrogen and fixed carbon starvation, and fail to accumulate autophagic bodies in the vacuole. By combining mutants eliminating ATG12a/b, ATG5, or the ATG10 E2 required for their condensation with a method that unequivocally detects the ATG8-PE adduct, we also show that ATG8 lipidation requires the ATG12-ATG5 conjugate. Unlike ATG8, ATG12 does not associate with autophagic bodies, implying that its role(s) during autophagy is restricted to events before the vacuolar deposition of vesicles. The expression patterns of the ATG12a and ATG12b genes and the effects of single atg12a and atg12b mutants on forming the ATG12-ATG5 conjugate reveal that the ATG12b locus is more important during basal autophagy while the ATG12a locus is more important during induced autophagy. Taken together, we conclude that the formation of the ATG12-ATG5 adduct is essential for ATG8-mediated autophagy in plants by promoting ATG8 lipidation.

Autophagy is an important intracellular recycling system in eukaryotes that utilizes small vesicles to traffic cytosolic proteins and organelles to the vacuole for breakdown. Vesicle formation requires the conjugation of the two ubiquitin-fold polypeptides ATG8 and ATG12 to phosphatidylethanolamine and the ATG5 protein, respectively. Using Arabidopsis thaliana mutants affecting the ATG5 target or the ATG7 E1 required to initiate ligation of both ATG8 and ATG12, we previously showed that the ATG8/12 conjugation pathways together are important when plants encounter nutrient stress and during senescence. To characterize the ATG12 conjugation pathway specifically, we characterized a null mutant eliminating the E2-conjugating enzyme ATG10 that, similar to plants missing ATG5 or ATG7, cannot form the ATG12-ATG5 conjugate. atg10-1 plants are hypersensitive to nitrogen and carbon starvation and initiate senescence and programmed cell death (PCD) more quickly than wild type, as indicated by elevated levels of senescence-and PCD-related mRNAs and proteins during carbon starvation. As detected with a GFP-ATG8a reporter, atg10-1 and atg5-1 mutant plants fail to accumulate autophagic bodies inside the vacuole. These results indicate that ATG10 is essential for ATG12 conjugation and that the ATG12-ATG5 conjugate is necessary to form autophagic vesicles and for the timely progression of senescence and PCD in plants.A S with other eukaryotes, plants have developed sophisticated mechanisms to recycle intracellular proteins. Most selective protein turnover occurs by the ubiquitin (Ub)/26S proteasome pathway, which directs the correct removal of short-lived regulatory and abnormal proteins (Smalle and Vierstra 2004). Conversely, autophagy is a catabolic process that is largely responsible for nonselective bulk turnover of cytosolic components from individual proteins and protein complexes to the removal of whole organelles Bassham 2007). It involves the engulfment of cytoplasm in small vesicles followed by their deposition into the lytic vacuole (lysosome in animals) where the vesicles and cargo are quickly degraded by a cache of vacuolar proteases, peptidases, lipases, and other hydrolytic enzymes.Thus far, primarily using the yeasts Saccharomyces cerevisiae and Pichia pastoris as models, at least two autophagic routes have been identified (for reviews see Ohsumi 2001; Klionsky 2007). Microautophagy proceeds by forming tubular invaginations of cytoplasm into the vacuole, which pinch off and release vesicles called autophagic bodies into the vacuolar lumen. In contrast, macroautophagy involves the de novo formation of small double-membrane-bound vesicles called autophagosomes within the cytoplasm, which sequester cytosolic constituents. These vesicles dock with the vacuole, where the outer membrane fuses with the tonoplast to release the inner compartment into the vacuolar lumen as an autophagic body. In addition, a derivative of macroautophagy called the cytoplasm-

SummaryImprinted gene expression refers to differential transcription of alleles depending on their parental origin. To date, most examples of imprinted gene expression in plants occur in the triploid endosperm tissue. The Arabidopsis gene MEDEA displays an imprinted pattern of gene expression and has homology to the Drosophila Polycomb group (PcG) protein Enhancer-of-zeste (E(z)). We have tested the allele-specific expression patterns of the three maize E(z)-like genes Mez1, Mez2 and Mez3. The expression of Mez2 and Mez3 is not imprinted, with a bi-allelic pattern of transcription for both genes in both the endosperm and embryonic tissue. In contrast, Mez1 displays a bi-allelic expression pattern in the embryonic tissue, and a mono-allelic expression pattern in the developing endosperm tissue. We demonstrate that mono-allelic expression of the maternal Mez1 allele occurs throughout endosperm development. We have identified a 556 bp differentially methylated region (DMR) located approximately 700 bp 5¢ of the Mez1 transcription start site. This region is heavily methylated at CpG and CpNpG nucleotides on the non-expressed paternal allele but has low levels of methylation on the expressed maternal allele. Molecular evolutionary analysis indicates that conserved domains of all three Mez genes are under purifying selection. The common imprinted expression of Mez1 and MEDEA, in concert with their likely evolutionary origins, suggests that there may be a requirement for imprinting of at least one E(z)-like gene in angiosperms.

95616 (J. Chandler, J. Callis) Deubiquitinating enzymes are essential to the ubiquitin (Ub)/26S proteasome system where they release Ub monomers from the primary translation products of poly-Ub and Ub extension genes, recycle Ubs from polyubiquitinated proteins, and reverse the effects of ubiquitination by releasing bound Ubs from individual targets. The Ub-specific proteases (UBPs) are one large family of deubiquitinating enzymes that bear signature cysteine and histidine motifs. Here, we genetically characterize a UBP subfamily in Arabidopsis (Arabidopsis thaliana) encoded by paralogous UBP3 and UBP4 genes. Whereas homozygous ubp3 and ubp4 single mutants do not display obvious phenotypic abnormalities, double-homozygous mutant individuals could not be created due to a defect in pollen development and/or transmission. This pollen defect was rescued with a transgene encoding wild-type UBP3 or UBP4, but not with a transgene encoding an active-site mutant of UBP3, indicating that deubiquitination activity of UBP3/UBP4 is required. Nuclear DNA staining revealed that ubp3 ubp4 pollen often fail to undergo mitosis II, which generates the two sperm cells needed for double fertilization. Substantial changes in vacuolar morphology were also evident in mutant grains at the time of pollen dehiscence, suggesting defects in vacuole and endomembrane organization. Even though some ubp3 ubp4 pollen could germinate in vitro, they failed to fertilize wild-type ovules even in the absence of competing wildtype pollen. These studies provide additional evidence that the Ub/26S proteasome system is important for male gametogenesis in plants and suggest that deubiquitination of one or more targets by UBP3/UBP4 is critical for the development of functional pollen.