A long-term goal of Arabidopsis research is to define the minimal gene set needed to produce a viable plant with a normal phenotype under diverse conditions. This will require both forward and reverse genetics along with novel strategies to characterize multigene families and redundant biochemical pathways. Here we describe an initial dataset of 250 EMB genes required for normal embryo development in Arabidopsis. This represents the first large-scale dataset of essential genes in a flowering plant. When compared with 550 genes with other knockout phenotypes, EMB genes are enriched for basal cellular functions, deficient in transcription factors and signaling components, have fewer paralogs, and are more likely to have counterparts among essential genes of yeast (Saccharomyces cerevisiae) and worm (Caenorhabditis elegans). EMB genes also represent a valuable source of plant-specific proteins with unknown functions required for growth and development. Analyzing such unknowns is a central objective of genomics efforts worldwide. We focus here on 34 confirmed EMB genes with unknown functions, demonstrate that expression of these genes is not embryo-specific, validate a strategy for identifying interacting proteins through complementation with epitope-tagged proteins, and discuss the value of EMB genes in identifying novel proteins associated with important plant processes. Based on sequence comparison with essential genes in other model eukaryotes, we identify 244 candidate EMB genes without paralogs that represent promising targets for reverse genetics. These candidates should facilitate the recovery of additional genes required for seed development.

Manganese (Mn) is an essential metal, but elevated cellular levels are toxic and may lead to the development of an irreversible parkinsonian-like syndrome that has no treatment. Mn-induced parkinsonism generally occurs as a result of exposure to elevated Mn levels in occupational or environmental settings. Additionally, patients with compromised liver function attributable to diseases, such as cirrhosis, fail to excrete Mn and may develop Mn-induced parkinsonism in the absence of exposure to elevated Mn. Recently, a new form of familial parkinsonism was reported to occur as a result of mutations in SLC30A10. The cellular function of SLC30A10 and the mechanisms by which mutations in this protein cause parkinsonism are unclear. Here, using a combination of mechanistic and functional studies in cell culture, Caenorhabditis elegans, and primary midbrain neurons, we show that SLC30A10 is a cell surface-localized Mn efflux transporter that reduces cellular Mn levels and protects against Mn-induced toxicity. Importantly, mutations in SLC30A10 that cause familial parkinsonism blocked the ability of the transporter to traffic to the cell surface and to mediate Mn efflux. Although expression of disease-causing SLC30A10 mutations were not deleterious by themselves, neurons and worms expressing these mutants exhibited enhanced sensitivity to Mn toxicity. Our results provide novel insights into the mechanisms involved in the onset of a familial form of parkinsonism and highlight the possibility of using enhanced Mn efflux as a therapeutic strategy for the potential management of Mn-induced parkinsonism, including that occurring as a result of mutations in SLC30A10.

Glutathione S-transferase Pi (GST ) is a marker protein in many cancers and high levels are linked to drug resistance, even when the selecting drug is not a substrate. S-Glutathionylation of proteins is critical to cellular stress response, but characteristics of the forward reaction are not known. Our results show that GST potentiates S-glutathionylation reactions following oxidative and nitrosative stress in vitro and in vivo. Mutational analysis indicated that the catalytic activity of GST is required. GST is itself redox-regulated. S-Glutathionylation on Cys 47 and Cys 101 autoregulates GST , breaks ligand binding interactions with c-Jun NH 2 -terminal kinase (JNK), and causes GST multimer formation, all critical to stress response. Catalysis of S-glutathionylation at low pK cysteines in proteins is a novel property for GST and may be a cause for its abundance in tumors and cells resistant to a range of mechanistically unrelated anticancer drugs.Glutathione S-transferases (GSTs) 2 are classified as a family of Phase II detoxification enzymes that have classically been described as catalyzing the conjugation of glutathione (GSH) to electrophilic compounds through thioether linkages (1). The Pi class (GST ) is present at high levels in many solid tumors (particularly ovarian, non-small cell lung, breast, liver, pancreas, colon, and lymphomas) and has been indicated in many reports to be overexpressed in drug-resistant tumors (2, 3). Although its increased expression was frequently linked with enhancement of drug detoxification, in most instances the selecting drugs were not substrates of GST . This ambiguity and the high prevalence of GST in tumors have intimated cellular functions for the protein that are unrelated to catalytic detoxification. Recently GST has been identified as an endogenous protein binding partner and regulator of c-Jun NH 2 -terminal kinase (JNK) and peroxiredoxin VI (1-cysPrx) (4 -6). Moreover, oxidative stress causes increased GST expression, the regulation of which has been identified as a downstream event linked to wild-type p53 function (7). Cellular response to oxidative or nitrosative stress includes S-glutathionylation, a post-translational modification characterized by conjugation of glutathione to low pK cysteine sulfhydryl or sulfenic acid moieties in target proteins. This adds a three-amino acid side chain and introduces a net negative charge (as a consequence of glutamic acid) to the protein (8). Consequently, protection from further oxidative damage and/or alteration of protein conformation affecting function and/or cellular localization occurs. Proteins so far identified that are susceptible to S-glutathionylation can be categorized into six distinct clusters: cytoskeletal, glycolysis/energy metabolism, kinases and signaling pathways, calcium homeostasis, antioxidant enzymes, and protein folding (9). Reversibility of S-glutathionylation spontaneously by GSH or catalytically by glutaredoxin or sulfiredoxin (8, 10) 3 provides the cell with a dynamic cycle of regulatory events....