Keiichi Mochida

Large-scale phosphorylation mapping reveals the extent of tyrosine phosphorylation in Arabidopsis.

Protein phosphorylation regulates a wide range of cellular processes. Here, we report the proteome‐wide 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 tyrosine‐specific 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.

Large-Scale Comparative Phosphoproteomics Identifies Conserved Phosphorylation Sites 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 leucine-rich repeat proteins and identified novel conserved phosphorylation sites that may regulate this class of proteins.

Genome-Wide Survey and Expression Analysis of the Plant-Specific NAC Transcription Factor Family in Soybean During Development and Dehydration Stress

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.

Brachypodium as a model for the grasses: Today and the future

Over the past several years, Brachypodium distachyon (Brachypodium) has emerged as a tractable model system to study biological questions relevant to the grasses. To place its relevance in the larger context of plant biology, we outline here the expanding adoption of Brachypodium as a model grass and compare this to the early history of another plant model, Arabidopsis thaliana. In this context, Brachypodium has followed an accelerated path in which the development of genomic resources, most notably a whole genome sequence, occurred concurrently with the generation of other experimental tools (e.g. highly efficient transformation and large collections of natural accessions). This update provides a snapshot of available and upcoming Brachypodium resources and an overview of the community including the trajectory of Brachypodium as a model grass.

Discrimination of homoeologous gene expression in hexaploid wheat by SNP analysis of contigs grouped from a large number of expressed sequence tags

Single-nucleotide polymorphisms (SNPs) are useful markers for gene diagnosis and mapping of genes on chromosomes. However, polyploidy, which is characteristic of the evolution of higher plants, complicates the analysis of SNPs in the duplicated genes. We have developed a new method for SNP analysis in hexaploid wheat. First, we classified a large number of expressed sequence tags (ESTs) from wheat in silico. Those grouped into contigs were anticipated to correspond to transcripts from homoeologous loci. We then selected relatively abundant ESTs, and assigned these contigs to each of the homoeologous chromosomes using a nullisomic/tetrasomic series of Chinese Spring wheat strains in combination with pyrosequencing. The ninety genes assigned were almost evenly distributed into seven homologous chromosomes. We then created a virtual display of the relative expression of these genes. Expression patterns of genes from the three genomes in hexaploid wheat were classified into two major groups: (1) genes almost equally expressed from all three genomes; and (2) genes expressed with a significant preference, which changed from tissue to tissue, from certain genomes. In 11 cases, one of the three genes in the allopolyploid was found to be silenced. No preference for gene-silencing in particular genomes or chromosomes was observed, suggesting that gene-silencing occurred after polyploidization, and at the gene level, not at the chromosome or genome level. Thus, the use of this SNP method to distinguish the expression profiles of three homoeologous genes may help to elucidate the molecular basis of heterosis in polyploid plants.

Advances in Omics and Bioinformatics Tools for Systems Analyses of Plant Functions

Omics and bioinformatics are essential to understanding the molecular systems that underlie various plant functions. Recent game-changing sequencing technologies have revitalized sequencing approaches in genomics and have produced opportunities for various emerging analytical applications. Driven by technological advances, several new omics layers such as the interactome, epigenome and hormonome have emerged. Furthermore, in several plant species, the development of omics resources has progressed to address particular biological properties of individual species. Integration of knowledge from omics-based research is an emerging issue as researchers seek to identify significance, gain biological insights and promote translational research. From these perspectives, we provide this review of the emerging aspects of plant systems research based on omics and bioinformatics analyses together with their associated resources and technological advances.

A Chloroplastic UDP-Glucose Pyrophosphorylase from Arabidopsis Is the Committed Enzyme for the First Step of Sulfolipid Biosynthesis

Plants synthesize a sulfur-containing lipid, sulfoquinovosyldiacylglycerol, which is one of three nonphosphorus glycerolipids that provide the bulk of the structural lipids in photosynthetic membranes. Here, the identification of a novel gene, UDP-glucose pyrophosphorylase3 (UGP3), required for sulfolipid biosynthesis is described. Transcriptome coexpression analysis demonstrated highly correlated expression of UGP3 with known genes for sulfolipid biosynthesis in Arabidopsis thaliana. Liquid chromatography–mass spectrometry analysis of leaf lipids in two Arabidopsis ugp3 mutants revealed that no sulfolipid was accumulated in these mutants, indicating the participation of UGP3 in sulfolipid biosynthesis. From the deduced amino acid sequence, UGP3 was presumed to be a UDP-glucose pyrophosphorylase (UGPase) involved in the generation of UDP-glucose, serving as the precursor of the polar head of sulfolipid. Recombinant UGP3 was able to catalyze the formation of UDP-glucose from glucose-1-phosphate and UTP. A transient assay using fluorescence fusion proteins and UGPase activity in isolated chloroplasts indicated chloroplastic localization of UGP3. The transcription level of UGP3 was increased by phosphate starvation. A comparative genomics study on UGP3 homologs across different plant species suggested the structural and functional conservation of the proteins and, thus, a committing role for UGP3 in sulfolipid synthesis.

Genomics and Bioinformatics Resources for Crop Improvement

Recent remarkable innovations in platforms for omics-based research and application development provide crucial resources to promote research in model and applied plant species. A combinatorial approach using multiple omics platforms and integration of their outcomes is now an effective strategy for clarifying molecular systems integral to improving plant productivity. Furthermore, promotion of comparative genomics among model and applied plants allows us to grasp the biological properties of each species and to accelerate gene discovery and functional analyses of genes. Bioinformatics platforms and their associated databases are also essential for the effective design of approaches making the best use of genomic resources, including resource integration. We review recent advances in research platforms and resources in plant omics together with related databases and advances in technology.

Genetic and Epigenetic Alteration among Three Homoeologous Genes of a Class E MADS Box Gene in Hexaploid Wheat

Bread wheat (Triticum aestivum) is a hexaploid species with A, B, and D ancestral genomes. Most bread wheat genes are present in the genome as triplicated homoeologous genes (homoeologs) derived from the ancestral species. Here, we report that both genetic and epigenetic alterations have occurred in the homoeologs of a wheat class E MADS box gene. Two class E genes are identified in wheat, wheat SEPALLATA (WSEP) and wheat LEAFY HULL STERILE1 (WLHS1), which are homologs of Os MADS45 and Os MADS1 in rice (Oryza sativa), respectively. The three wheat homoeologs of WSEP showed similar genomic structures and expression profiles. By contrast, the three homoeologs of WLHS1 showed genetic and epigenetic alterations. The A genome WLHS1 homoeolog (WLHS1-A) had a structural alteration that contained a large novel sequence in place of the K domain sequence. A yeast two-hybrid analysis and a transgenic experiment indicated that the WLHS1-A protein had no apparent function. The B and D genome homoeologs, WLHS1-B and WLHS1-D, respectively, had an intact MADS box gene structure, but WLHS1-B was predominantly silenced by cytosine methylation. Consequently, of the three WLHS1 homoeologs, only WLHS1-D functions in hexaploid wheat. This is a situation where three homoeologs are differentially regulated by genetic and epigenetic mechanisms.

TriFLDB: A Database of Clustered Full-Length Coding Sequences from Triticeae with Applications to Comparative Grass Genomics

The Triticeae Full-Length CDS Database (TriFLDB) contains available information regarding full-length coding sequences (CDSs) of the Triticeae crops wheat (Triticum aestivum) and barley (Hordeum vulgare) and includes functional annotations and comparative genomics features. TriFLDB provides a search interface using keywords for gene function and related Gene Ontology terms and a similarity search for DNA and deduced translated amino acid sequences to access annotations of Triticeae full-length CDS (TriFLCDS) entries. Annotations consist of similarity search results against several sequence databases and domain structure predictions by InterProScan. The deduced amino acid sequences in TriFLDB are grouped with the proteome datasets for Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and sorghum (Sorghum bicolor) by hierarchical clustering in stepwise thresholds of sequence identity, providing hierarchical clustering results based on full-length protein sequences. The database also provides sequence similarity results based on comparative mapping of TriFLCDSs onto the rice and sorghum genome sequences, which together with current annotations can be used to predict gene structures for TriFLCDS entries. To provide the possible genetic locations of full-length CDSs, TriFLCDS entries are also assigned to the genetically mapped cDNA sequences of barley and diploid wheat, which are currently accommodated in the Triticeae Mapped EST Database. These relational data are searchable from the search interfaces of both databases. The current TriFLDB contains 15,871 full-length CDSs from barley and wheat and includes putative full-length cDNAs for barley and wheat, which are publicly accessible. This informative content provides an informatics gateway for Triticeae genomics and grass comparative genomics. TriFLDB is publicly available at http://TriFLDB.psc.riken.jp/.