Selenium (Se) is an essential element for many organisms but is toxic at higher levels. CpNifS is a chloroplastic NifS-like protein in Arabidopsis (Arabidopsis thaliana) that can catalyze the conversion of cysteine into alanine and elemental sulfur (S 0 ) and of selenocysteine into alanine and elemental Se (Se 0 ). We overexpressed CpNifS to investigate the effects on Se metabolism in plants. CpNifS overexpression significantly enhanced selenate tolerance (1.9-fold) and Se accumulation (2.2-fold). CpNifS overexpressors showed significantly reduced Se incorporation into protein, which may explain their higher Se tolerance. Also, sulfur accumulation was enhanced by approximately 30% in CpNifS overexpressors, both on media with and without selenate. Root transcriptome changes in response to selenate mimicked the effects observed under sulfur starvation. There were only a few transcriptome differences between CpNifS-overexpressing plants and wild type, besides the 25-to 40-fold increase in CpNifS levels. Judged from x-ray analysis of near edge spectrum, both CpNifS overexpressors and wild type accumulated mostly selenate (Se VI ). In conclusion, overexpression of this plant NifS-like protein had a pronounced effect on plant Se metabolism. The observed enhanced Se accumulation and tolerance of CpNifS overexpressors show promise for use in phytoremediation.

NifS-like proteins provide the sulfur (S) for the formation of iron-sulfur (Fe-S) clusters, an ancient and essential type of cofactor found in all three domains of life. Plants are known to contain two distinct NifS-like proteins, localized in the mitochondria (MtNifS) and the chloroplast (CpNifS). In the chloroplast, five different Fe-S cluster types are required in various proteins. These plastid Fe-S proteins are involved in a variety of biochemical pathways including photosynthetic electron transport and nitrogen and sulfur assimilation. In vitro, the chloroplastic cysteine desulfurase CpNifS can release elemental sulfur from cysteine for Fe-S cluster biogenesis in ferredoxin. However, because of the lack of a suitable mutant allele, the role of CpNifS has not been studied thus far in planta. To study the role of CpNifS in Fe-S cluster biogenesis in vivo, the gene was silenced by using an inducible RNAi (interference) approach. Plants with reduced CpNifS expression exhibited chlorosis, a disorganized chloroplast structure, and stunted growth and eventually became necrotic and died before seed set. Photosynthetic electron transport and carbon dioxide assimilation were severely impaired in the silenced plant lines. The silencing of CpNifS decreased the abundance of all chloroplastic Fe-S proteins tested, representing all five Fe-S cluster types. Mitochondrial Fe-S proteins and respiration were not affected, suggesting that mitochondrial and chloroplastic Fe-S assembly operate independently. These findings indicate that CpNifS is necessary for the maturation of all plastidic Fe-S proteins and, thus, essential for plant growth.Fe-S proteins ͉ inducible RNAi ͉ photosynthesis ͉ Arabidopsis thaliana

Early embryonic cell cycles in Drosophila consist of rapidly alternating S and M phases. Three genes, pan gu (png), plutonium (plu), and giant nuclei (gnu) coordinate these early S-M cycles by ensuring adequate Cyclin B protein levels. Mutations in any of these genes result in unregulated DNA replication and a lack of mitosis ("giant nuclei" phenotype). png encodes a serine/threonine protein kinase, and plu and gnu encode small, novel proteins. We show that PNG, PLU, and GNU constitute a novel protein kinase complex that specifically regulates S-M cell cycles. All three proteins are required for PNG kinase activity and are phosphorylated by PNG in vitro. Yeast two-hybrid screening revealed a direct interaction between PNG and PLU, and their co-expression is required for physical association and activation of PNG kinase. Artificial dimerization of PLU via fusion to either GST or FK506 binding protein (in the presence of dimerizing agent) abrogates the requirement for GNU to activate PNG kinase. We propose a model in which GNU normally regulates embryonic cell cycles by promoting transient dimerization of a core PNG/PLU complex, thereby stimulating PNG kinase activity. Protein kinases play crucial regulatory roles in the cell cycle. CDK/cyclin complexes control transitions throughout the cell cycle, and their proper regulation is essential for ensuring the orderly progression of DNA replication and mitosis (for review, see Murray and Hunt 1993). Association of a cyclin subunit with a CDK subunit is needed both for kinase activity and to confer substrate specificity. Thus, one mechanism for control of these kinase complexes involves the accumulation of threshold levels of cyclins via transcription and regulated degradation of cyclin proteins.Modified cell cycles are used to achieve particular developmental goals. Organisms that must undergo rapid embryogenesis, such as marine invertebrates, amphibians, and insects, utilize a streamlined cell cycle in which DNA replication (S phase) and mitosis (M phase) alternate without intervening gaps. The S-M cycles are driven by maternally provided stockpiles of protein and RNA, eliminating a need for gap phases for gene expression or growth (Foe et al. 1993). Zygotic transcription has not yet begun, so the S-M cell cycles differ from the archetypal cell cycle in that they are regulated solely by posttranscriptional mechanisms. In Drosophila embryos, an additional distinction is that the nuclei divide synchronously in a common cytoplasm (syncytium) during the S-M cycles.During the first seven cell cycles of Drosophila embryogenesis, levels of the mitotic Cyclins A and B as well as their partner kinase CDK1 are high due to maternal stockpiling, and no detectable fluctuations in their levels or CDK1 activity occur (Edgar et al. 1994). However, localized degradation of Cyclin B during these early cycles has been reported (Huang and Raff 1999), and injection of a stabilized form of Cyclin B into early embryos causes mitotic arrest (Su et al. 1998). Thus, localized oscillations in ...

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