FOXO1, a member of the FOXO forkhead type transcription factors, is markedly up-regulated in skeletal muscle in energy-deprived states such as fasting and severe diabetes, but its functions in skeletal muscle have remained poorly understood. In this study, we created transgenic mice specifically overexpressing FOXO1 in skeletal muscle. These mice weighed less than the wildtype control mice, had a reduced skeletal muscle mass, and the muscle was paler in color. Microarray analysis revealed that the expression of many genes related to the structural proteins of type I muscles (slow twitch, red muscle) was decreased. Histological analyses showed a marked decrease in size of both type I and type II fibers and a significant decrease in the number of type I fibers in the skeletal muscle of FOXO1 mice. Enhanced gene expression of a lysosomal proteinase, cathepsin L, which is known to be up-regulated during skeletal muscle atrophy, suggested increased protein degradation in the skeletal muscle of FOXO1 mice. Running wheel activity (spontaneous locomotive activity) was significantly reduced in FOXO1 mice compared with control mice. Moreover, the FOXO1 mice showed impaired glycemic control after oral glucose and intraperitoneal insulin administration. These results suggest that FOXO1 negatively regulates skeletal muscle mass and type I fiber gene expression and leads to impaired skeletal muscle function. Activation of FOXO1 may be involved in the pathogenesis of sarcopenia, the age-related decline in muscle mass in humans, which leads to obesity and diabetes.

Protein phosphorylation regulates a wide range of cellular processes. Here, we report the proteomewide mapping of in vivo phosphorylation sites in Arabidopsis by using complementary phosphopeptide enrichment techniques coupled with high-accuracy mass spectrometry. Using unfractionated whole cell lysates of Arabidopsis, we identified 2597 phosphopeptides with 2172 high-confidence, unique phosphorylation sites from 1346 proteins. The distribution of phosphoserine, phosphothreonine, and phosphotyrosine sites was 85.0, 10.7, and 4.3%. Although typical tyrosine-specific protein kinases are absent in Arabidopsis, the proportion of phosphotyrosines among the phospho-residues in Arabidopsis is similar to that in humans, where over 90 tyrosinespecific protein kinases have been identified. In addition, the tyrosine phosphoproteome shows features distinct from those of the serine and threonine phosphoproteomes. Taken together, we highlight the extent and contribution of tyrosine phosphorylation in plants.

Knowledge of phosphorylation events and their regulation is crucial to understand the functional biology of plants. Here, we report a large-scale phosphoproteome analysis in the model monocot rice (Oryza sativa japonica 'Nipponbare'), an economically important crop. Using unfractionated whole-cell lysates of rice cells, we identified 6,919 phosphopeptides from 3,393 proteins. To investigate the conservation of phosphoproteomes between plant species, we developed a novel phosphorylation-site evaluation method and performed a comparative analysis of rice and Arabidopsis (Arabidopsis thaliana). The ratio of tyrosine phosphorylation in the phosphoresidues of rice was equivalent to those in Arabidopsis and human. Furthermore, despite the phylogenetic distance and the use of different cell types, more than 50% of the phosphoproteins identified in rice and Arabidopsis, which possessed ortholog(s), had an orthologous phosphoprotein in the other species. Moreover, nearly half of the phosphorylated orthologous pairs were phosphorylated at equivalent sites. Further comparative analyses against the Medicago phosphoproteome also showed similar results. These data provide direct evidence for conserved regulatory mechanisms based on phosphorylation in plants. We also assessed the phosphorylation sites on nucleotide-binding leucinerich repeat proteins and identified novel conserved phosphorylation sites that may regulate this class of proteins.

Genetic diversity is key to crop improvement. Owing to pervasive genomic structural variation, a single reference genome assembly cannot capture the full complement of sequence diversity of a crop species (known as the ‘pan-genome’1). Multiple high-quality sequence assemblies are an indispensable component of a pan-genome infrastructure. Barley (Hordeum vulgare L.) is an important cereal crop with a long history of cultivation that is adapted to a wide range of agro-climatic conditions2. Here we report the construction of chromosome-scale sequence assemblies for the genotypes of 20 varieties of barley—comprising landraces, cultivars and a wild barley—that were selected as representatives of global barley diversity. We catalogued genomic presence/absence variants and explored the use of structural variants for quantitative genetic analysis through whole-genome shotgun sequencing of 300 gene bank accessions. We discovered abundant large inversion polymorphisms and analysed in detail two inversions that are frequently found in current elite barley germplasm; one is probably the product of mutation breeding and the other is tightly linked to a locus that is involved in the expansion of geographical range. This first-generation barley pan-genome makes previously hidden genetic variation accessible to genetic studies and breeding.

Cloning mammals by means of somatic cell nuclear transfer (SCNT) is highly inefficient because of erroneous reprogramming of the donor genome. Reprogramming errors appear to arise randomly, but the nature of nonrandom, SCNT-specific errors remains elusive. We found that Xist, a noncoding RNA that inactivates one of the two X chromosomes in females, was ectopically expressed from the active X (Xa) chromosome in cloned mouse embryos of both sexes. Deletion of Xist on Xa showed normal global gene expression and resulted in about an eight- to ninefold increase in cloning efficiency. We also identified an Xist-independent mechanism that specifically down-regulated a subset of X-linked genes through somatic-type repressive histone blocks. Thus, we have identified nonrandom reprogramming errors in mouse cloning that can be altered to improve the efficiency of SCNT methods.

Plant-specific NAC transcription factors (TFs) play important roles in regulating diverse biological processes, including development, senescence, growth, cell division and responses to environmental stress stimuli. Within the soybean genome, we identified 152 full-length GmNAC TFs, including 11 membrane-bound members. In silico analysis of the GmNACs, together with their Arabidopsis and rice counterparts, revealed similar NAC architecture. Next, we explored the soybean Affymetrix array and Illumina transcriptome sequence data to analyse tissue-specific expression profiles of GmNAC genes. Phylogenetic analysis using stress-related NAC TFs from Arabidopsis and rice as seeding sequences identified 58 of the 152 GmNACs as putative stress-responsive genes, including eight previously reported dehydration-responsive GmNACs. We could design gene-specific primers for quantitative real-time PCR verification of 38 out of 50 newly predicted stress-related genes. Twenty-five and six GmNACs were found to be induced and repressed 2-fold or more, respectively, in soybean roots and/or shoots in response to dehydration. GmNAC085, whose amino acid sequence was 39%; identical to that of well-known SNAC1/ONAC2, was the most induced gene upon dehydration, showing 390-fold and 20-fold induction in shoots and roots, respectively. Our systematic analysis has identified excellent tissue-specific and/or dehydration-responsive candidate GmNAC genes for in-depth characterization and future development of improved drought-tolerant transgenic soybeans.

Model systems not only allow scientists to investigate complex processes that are difficult to study in nonmodel organisms but also serve to focus community efforts and resources, significantly advancing research. Arabidopsis (Arabidopsis thaliana) has served as a plant model system for almost 30 years and is widely considered the preeminent model plant. The success of Arabidopsis-related research has been driven not only by key features common to any model organism but also by the collaborative environment built by the Arabidopsis community. A decade after the Arabidopsis genome sequence was published, the development of model plants follows a different trajectory. In the past, the development of extensive resources and a large user community happened first and then sequencing the genome followed. Today, however, an organism is selected as a potential model and genome sequencing occurs prior to or concurrent with the development of experimental tools and a user community. Arabidopsis research has provided many scientific breakthroughs (Flavell, 2009). However, its utility as a model is limited to a certain extent when investigating monocot-specific processes.Within the monocots, grasses provide the vast majority of human calories and are increasingly utilized as a sustainable source of energy. Traits including cell wall composition, plant architecture, grain properties,

Ser acetyltransferase (SERAT), which catalyzes O-acetyl-Ser (OAS) formation, plays a key role in sulfur assimilation and Cys synthesis. Despite several studies on SERATs from various plant species, the in vivo function of multiple SERAT genes in plant cells remains unaddressed. Comparative genomics studies with the five genes of the SERAT gene family in Arabidopsis thaliana indicated that all three Arabidopsis SERAT subfamilies are conserved across five plant species with available genome sequences. Single and multiple knockout mutants of all Arabidopsis SERAT gene family members were analyzed. All five quadruple mutants with a single gene survived, with three mutants showing dwarfism. However, the quintuple mutant lacking all SERAT genes was embryo-lethal. Thus, all five isoforms show functional redundancy in vivo. The developmental and compartment-specific roles of each SERAT isoform were also demonstrated. Mitochondrial SERAT2;2 plays a predominant role in cellular OAS formation, while plastidic SERAT2;1 contributes less to OAS formation and subsequent Cys synthesis. Three cytosolic isoforms, SERAT1;1, SERAT3;1, and SERAT3;2, may play a major role during seed development. Thus, the evolutionally conserved SERAT gene family is essential in cellular processes, and the substrates and products of SERAT must be exchangeable between the cytosol and organelles. INTRODUCTIONSulfur plays a pivotal role in plant metabolism and development. Cys, as the first organic-reduced sulfur compound, contributes not only to life as building blocks in proteins, but it also serves as a precursor for the syntheses of Met, glutathione (GSH), cofactors, essential vitamins, sulfur esters, and other sulfur derivatives (Beinert, 2000;Leustek et al., 2000;Marquet, 2001;Gerber and Lill, 2002;Mendel and Hä nsch, 2002;Noctor et al., 2002;Saito, 2004). Ser acetyltransferase (SERAT; EC 2.3.1.30), which catalyzes the formation of O-acetyl-Ser (OAS) from Ser and acetylCoA, links Ser metabolism to Cys biosynthesis. Subsequently, Cys is formed by the condensation of sulfide and OAS, catalyzed by OAS (thiol) lyase (OASTL; EC 4.2.99.8), which is classified within the b-substituted Ala synthase (BSAS) family (Leustek and Saito, 1999;Hatzfeld et al., 2000;Saito, 2004). In contrast with sulfate reduction that occurs exclusively in the plastid, both SERAT and OASTL were found to be associated not only with plastids but also with the cytosol and mitochondria of plant cells (Lunn et al., 1990;Saito, 2000;Hell et al., 2002;Droux, 2003). The reason for the localization of SERAT and OASTL in three compartments where protein synthesis occurs has been conceived to be that the transport of Cys between cellular compartments may be limited (Lunn et al., 1990;Leustek and Saito, 1999;Saito, 2004). However, recent studies on Arabidopsis thaliana T-DNA knockout mutants of OASTL suggested that Cys or its derivatives must be exchangeable between the compartments and that the Cys biosynthesis in plant cells was under compartment-specific regulation (Heeg et al., 2008;Wa...