Biomedical subjects

Kazuo Shinozaki

Publications and source records attributed to Kazuo Shinozaki.

At least 19 recordsLinked to original sources

PubMed2007

Curated genome annotation of Oryza sativa ssp. japonica and comparative genome analysis with Arabidopsis thaliana.

We present here the annotation of the complete genome of rice Oryza sativa L. ssp. japonica cultivar Nipponbare. All functional annotations for proteins and non-protein-coding RNA (npRNA) candidates were manually curated. Functions were identified or inferred in 19,969 (70%) of the proteins, and 131 possible npRNAs (including 58 antisense transcripts) were found. Almost 5000 annotated protein-coding genes were found to be disrupted in insertional mutant lines, which will accelerate future experimental validation of the annotations. The rice loci were determined by using cDNA sequences obtained from rice and other representative cereals. Our conservative estimate based on these loci and an extrapolation suggested that the gene number of rice is approximately 32,000, which is smaller than previous estimates. We conducted comparative analyses between rice and Arabidopsis thaliana and found that both genomes possessed several lineage-specific genes, which might account for the observed differences between these species, while they had similar sets of predicted functional domains among the protein sequences. A system to control translational efficiency seems to be conserved across large evolutionary distances. Moreover, the evolutionary process of protein-coding genes was examined. Our results suggest that natural selection may have played a role for duplicated genes in both species, so that duplication was suppressed or favored in a manner that depended on the function of a gene.

Arabidopsis↗

PubMed2006

Cell-free synthesis of zinc-binding proteins.

Cell-free protein synthesis has become one of the standard methods for protein expression. The cell-free method is suitable for the synthesis of a protein that requires a ligand for its enzymatic activity and/or structure formation and stabilization, since it is an open system, which allows us to add the proper ligand to the reaction mixture. A large number of proteins that require zinc for their function are involved in diverse cellular processes, including transcription, DNA replication, metabolism, and cell signaling. In this study, we analyzed the effects of zinc on the cell-free synthesis of plant-specific zinc-binding transcription factors. The solubility and/or stability of the proteins were significantly increased in the presence of the proper concentration of zinc during the cell-free reaction. NMR analyses confirmed that correctly folded proteins were synthesized by the cell-free method. These results indicate that the cell-free method can be used to synthesize correctly folded and functional zinc-binding proteins.

Arabidopsis Proteins↗

PubMed2006

Gene networks involved in drought stress response and tolerance.

Plants respond to survive under water-deficit conditions via a series of physiological, cellular, and molecular processes culminating in stress tolerance. Many drought-inducible genes with various functions have been identified by molecular and genomic analyses in Arabidopsis, rice, and other plants, including a number of transcription factors that regulate stress-inducible gene expression. The products of stress-inducible genes function both in the initial stress response and in establishing plant stress tolerance. In this short review, recent progress resulting from analysis of gene expression during the drought-stress response in plants as well as in elucidating the functions of genes implicated in the stress response and/or stress tolerance are summarized. A description is also provided of how various genes involved in stress tolerance were applied in genetic engineering of dehydration stress tolerance in transgenic Arabidopsis plants.

Abscisic Acid↗

PubMed2006

Dual function of an Arabidopsis transcription factor DREB2A in water-stress-responsive and heat-stress-responsive gene expression.

Transcription factor DREB2A interacts with a cis-acting dehydration-responsive element (DRE) sequence and activates expression of downstream genes involved in drought- and salt-stress response in Arabidopsis thaliana. Intact DREB2A expression does not activate downstream genes under normal growth conditions. A negative regulatory domain exists in the central region of DREB2A, and deletion of this region transforms DREB2A to a constitutive active form (DREB2A CA). We carried out microarray analysis of transgenic Arabidopsis-overexpressing DREB2A CA and found that the overexpression of DREB2A CA induces not only drought- and salt-responsive genes but also heat-shock (HS)-related genes. Moreover, we found that transient induction of the DREB2A occurs rapidly by HS stress, and that the sGFP-DREB2A protein accumulates in nuclei of HS-stressed cells. DREB2A up-regulated genes were classified into three groups based on their expression patterns: genes induced by HS, genes induced by drought stress, and genes induced by both HS and drought stress. DREB2A up-regulated genes were down-regulated in DREB2A knockout mutants under stress conditions. Thermotolerance was significantly increased in plants overexpressing DREB2A CA and decreased in DREB2A knockout plants. Collectively, these results indicate that DREB2A functions in both water and HS-stress responses.

Arabidopsis↗

PubMed2006

MEKK1 is required for MPK4 activation and regulates tissue-specific and temperature-dependent cell death in Arabidopsis.

Innate immunity signaling pathways in both animals and plants are regulated by mitogen-activated protein kinase (MAPK) cascades. An Arabidopsis MAPK cascade (MEKK1, MKK4/MKK5, and MPK3/MPK6) has been proposed to function downstream of the flagellin receptor FLS2 based on biochemical assays using transient overexpression of candidate components. To genetically test this model, we characterized two mekk1 mutants. We show here that MEKK1 is not required for flagellin-triggered activation of MPK3 and MPK6. Instead, MEKK1 is essential for activation of MPK4, a MAPK that negatively regulates systemic acquired resistance. We also showed that MEKK1 negatively regulates temperature-sensitive and tissue-specific cell death and H(2)O(2) accumulation that are partly dependent on both RAR1, a key component in resistance protein function, and SID2, an isochorismate synthase required for salicylic acid production upon pathogen infection.

Arabidopsis↗

PubMed2006

An Arabidopsis chloroplast-targeted Hsp101 homologue, APG6, has an essential role in chloroplast development as well as heat-stress response.

Analysis of albino or pale-green (apg) mutants is important for identifying nuclear genes responsible for chloroplast development and pigment synthesis. We have identified 38 apg mutants by screening 11 000 Arabidopsis Ds-tagged lines. One mutant, apg6, contains a Ds insertion in a gene encoding APG6 (ClpB3), a homologue of the heat-shock protein Hsp101 (ClpB1). We isolated somatic revertants and identified two Ds-tagged and one T-DNA-tagged mutant alleles of apg6. All three alleles gave the same pale-green phenotype. These results suggest that APG6 is important for chloroplast development. The APG6 protein contains a transit peptide and is localized in chloroplasts. The plastids of apg6 pale-green cells were smaller than those of the wild type, and contained undeveloped thylakoid membranes. APG6 mRNA accumulated in response to heat shock in various organs, but not in response to other abiotic stresses. Under normal conditions, APG6 is constitutively expressed in the root tips, the organ boundary region, the reproductive tissues of mature plants where plastids exist as proplastids, and slightly in the stems and leaves. In addition, constitutive overexpression of APG6 in transgenic plants inhibited chloroplast development and resulted in a mild pale-green phenotype. The amounts of chloroplast proteins related to photosynthesis were markedly decreased in apg6 mutants. These results suggest that APG6 functions as a molecular chaperone involved in plastid differentiation mediating internal thylakoid membrane formation and conferring thermotolerance to chloroplasts during heat stress. The APG6 protein is not only involved in heat-stress response in chloroplasts, but is also essential for chloroplast development.

Alleles↗

PubMed2006

Loss of Necrotic Spotted Lesions 1 associates with cell death and defense responses in Arabidopsis thaliana.

We isolated a lesion mimic mutant, necrotic spotted lesions 1 (nsl1), from Ds-tagged Arabidopsis thaliana accession No-0. The nsl1 mutant exhibits a growth retardation phenotype and develops spotted necrotic lesions on its rosette and cauline leaves. These phenotypes occur in the absence of pathogens indicating that nsl1 mutants may constitutively express defense responses. Consistent with this idea, nsl1 accumulates high levels of callose and autofluorescent phenolic compounds localized to the necrotic lesions. Furthermore RNA gel blot analysis revealed that genes associated with disease resistance activation are upregulated in the nsl1 mutants and these plants contain elevated levels of salicylic acid (SA). Crossing nsl1 with an SA deficient mutant, eds16-1, revealed that the nsl1 lesions and growth retardation are dependent upon SA. The nsl1 phenotypes are not suppressed under either the rar1-10 or sgt1b-1 genetic background. NSL1 encodes a novel 612aa protein which contains a membrane-attack complex/perforin (MACPF) domain, which is conserved in bacteria, fungi, mammals and plants. The possible modes of action of NSL1 protein in negative regulation of cell death programs and defense responses are discussed.

Amino Acid Sequence↗

PubMed2006

A trial of phenome analysis using 4000 Ds-insertional mutants in gene-coding regions of Arabidopsis.

Mutant lines covering all Arabidopsis genes allow us to pursue systematic functional genomics. A comprehensive phenotype description, called a phenome, is highly sought after in the profiling of -omics data. We selected 4000 transposon-insertional lines with transposon insertions in their gene-coding regions, and systematically observed the visible phenotype of each line. For the first 3 weeks after germination, plants were grown on agar plates and the juvenile phenotypes were recorded. Then the plants were transferred to soil and their phenotypes were recorded at each growth stage. About 140 lines showed clear and reproducible visible phenotypes, including novel phenotypic mutants as well as previously reported ones. All descriptions of the mutants showing visible phenotypes were classified into eight primary categories (seedling, leaves, flowering and growth, stems, branching, flowers, siliques and seed yield) and 43 secondary categories of morphological phenotypes. Phenotypic images have been entered into a searchable database (http://rarge.gsc.riken.jp/phenome/). One example investigated through the use of plural alleles was a mutant of a novel gene related to glycerolipid biosynthesis, with a unique visible phenotype of sepal opening. Our results suggest that we can find more novel visible phenotypes and their corresponding genes, and that phenotypic mutants of gene knockouts are not exhausted yet. This study provides basic data on large-scale phenotyping of gene knockout lines in plants, and will contribute to the completion of an international effort to develop a phenome database of all the functional genes in Arabidopsis.

Arabidopsis↗

PubMed2006

Crosstalk between abiotic and biotic stress responses: a current view from the points of convergence in the stress signaling networks.

Plants have evolved a wide range of mechanisms to cope with biotic and abiotic stresses. To date, the molecular mechanisms that are involved in each stress has been revealed comparatively independently, and so our understanding of convergence points between biotic and abiotic stress signaling pathways remain rudimentary. However, recent studies have revealed several molecules, including transcription factors and kinases, as promising candidates for common players that are involved in crosstalk between stress signaling pathways. Emerging evidence suggests that hormone signaling pathways regulated by abscisic acid, salicylic acid, jasmonic acid and ethylene, as well as ROS signaling pathways, play key roles in the crosstalk between biotic and abiotic stress signaling.

Plant Diseases↗

PubMed2006

A 9-cis-epoxycarotenoid dioxygenase inhibitor for use in the elucidation of abscisic acid action mechanisms.

The plant hormone abscisic acid (ABA) accumulates in response to drought stress and confers stress tolerance to plants. 9-cis-Epoxycarotenoid dioxygenase (NCED), the key regulatory enzyme in the ABA biosynthesis pathway, plays an important role in ABA accumulation. Treatment of plants with abamine, the first NCED inhibitor identified, inhibits ABA accumulation. On the basis of structure-activity relationship studies of abamine, we identified an inhibitor of ABA accumulation more potent than abamine and named it abamineSG. An important structural feature of abamineSG is a three-carbon linker between the methyl ester and the nitrogen atom. Treatment of osmotically stressed plants with 100 microM abamineSG inhibited ABA accumulation by 77% as compared to the control, whereas abamine inhibited the accumulation by 35%. The expression of AB A-responsive genes and ABA catabolic genes was strongly inhibited in abamineSG-treated plants under osmotic stress. AbamineSG is a competitive inhibitor of the enzyme NCED, with a K(i) of 18.5 microM. Although the growth of Arabidopsis seedlings was inhibited by abamine at high concentrations (>50 microM), an effect that was unrelated to the inhibition of ABA biosynthesis, seedling growth was not affected by 100 microM abamineSG. These results suggest that abamineSG is a more potent and specific inhibitor of ABA biosynthesis than abamine.

Abscisic Acid↗

PubMed2006

Genes for the peptidoglycan synthesis pathway are essential for chloroplast division in moss.

The general consensus is that a cyanobacterium phagocytosed by a host cell evolved into the plastids of red and green algae, land plants, and glaucophytes. In contrast to the plastids of glaucophytes, which retain a cyanobacterial-type peptidoglycan layer, no wall-like structures have been detected in plastids from other sources. Although the genome of Arabidopsis thaliana contains five genes that are essential for peptidoglycan synthesis, MurE, MurG, two genes for D-Ala-D-Ala ligase (Ddl), and the gene for translocase I (MraY), their functions have not been determined. We report that the moss Physcomitrella patens has nine homologous genes related to peptidoglycan biosynthesis: MurA, B, C, D, E, and F, Ddl, genes for the penicillin-binding protein Pbp, and dd-carboxypeptidase (Dac). Corroborating a computer prediction, analysis of the GFP fusion proteins with the N terminus of PpMurE or of PpPbp suggests that these proteins are located in the chloroplasts. Gene disruption of the PpMurE gene in P. patens resulted in the appearance of macrochloroplasts both in protonema and in leaf cells. Moreover, gene knockout of the P. patens Pbp gene showed inhibition of chloroplast division in this moss; however, no Pbp gene was found in A. thaliana.

Arabidopsis↗

PubMed2006

A plant locus essential for phylloquinone (vitamin K1) biosynthesis originated from a fusion of four eubacterial genes.

Phylloquinone is a compound present in all photosynthetic plants serving as cofactor for Photosystem I-mediated electron transport. Newly identified seedling-lethal Arabidopsis thaliana mutants impaired in the biosynthesis of phylloquinone possess reduced Photosystem I activity. The affected gene, called PHYLLO, consists of a fusion of four previously individual eubacterial genes, menF, menD, menC, and menH, required for the biosynthesis of phylloquinone in photosynthetic cyanobacteria and the respiratory menaquinone in eubacteria. The fact that homologous men genes reside as polycistronic units in eubacterial chromosomes and in plastomes of red algae strongly suggests that PHYLLO derived from a plastid operon during endosymbiosis. The principle architecture of the fused PHYLLO locus is conserved in the nuclear genomes of plants, green algae, and the diatom alga Thalassiosira pseudonana. The latter arose from secondary endosymbiosis of a red algae and a eukaryotic host indicating selective driving forces for maintenance and/or independent generation of the composite gene cluster within the nuclear genomes. Besides, individual menF genes, encoding active isochorismate synthases (ICS), have been established followed by splitting of the essential 3' region of the menF module of PHYLLO only in genomes of higher plants. This resulted in inactivation of the ICS activity encoded by PHYLLO and enabled a metabolic branch from the phylloquinone biosynthetic route to independently regulate the synthesis of salicylic acid required for plant defense. Therefore, gene fusion, duplication, and fission events adapted a eubacterial multienzymatic system to the metabolic requirements of plants.

Algal Proteins↗

PubMed2006

Functional analysis of an Arabidopsis transcription factor, DREB2A, involved in drought-responsive gene expression.

Transcription factors DREB1A/CBF3 and DREB2A specifically interact with cis-acting dehydration-responsive element/C-repeat (DRE/CRT) involved in cold and drought stress-responsive gene expression in Arabidopsis thaliana. Intact DREB2A expression does not activate downstream genes under normal growth conditions, suggesting that DREB2A requires posttranslational modification for activation, but the activation mechanism has not been clarified. DREB2A domain analysis using Arabidopsis protoplasts identified a transcriptional activation domain between residues 254 and 335, and deletion of a region between residues 136 and 165 transforms DREB2A to a constitutive active form. Overexpression of constitutive active DREB2A resulted in significant drought stress tolerance but only slight freezing tolerance in transgenic Arabidopsis plants. Microarray and RNA gel blot analyses revealed that DREB2A regulates expression of many water stress-inducible genes. However, some genes downstream of DREB2A are not downstream of DREB1A, which also recognizes DRE/CRT but functions in cold stress-responsive gene expression. Synthetic green fluorescent protein gave a strong signal in the nucleus under unstressed control conditions when fused to constitutive active DREB2A but only a weak signal when fused to full-length DREB2A. The region between DREB2A residues 136 and 165 plays a role in the stability of this protein in the nucleus, which is important for protein activation.

Amino Acid Sequence↗

PubMed2006

An Arabidopsis SBP-domain fragment with a disrupted C-terminal zinc-binding site retains its tertiary structure.

SQUAMOSA promoter-binding proteins (SBPs) form a major family of plant-specific transcription factors, mainly related to flower development. SBPs share a highly conserved DNA-binding domain of approximately 80 amino acids (SBP domain), which contains two non-interleaved zinc-binding sites formed by eight conserved Cys or His residues. In the present study, an Arabidopsis SPL12 SBP-domain fragment that lacks a Cys residue involved in the C-terminal zinc-binding pocket was found to retain a folded structure, even though only a single Zn2+ ion binds to the fragment. Solution structure of this fragment determined by NMR is very similar to the previously determined structures of the full SBP domains of Arabidopsis SPL4 and SPL7. Considering the previous observations that chelating all the Zn2+ ions of SBPs resulted in the complete unfolding of the structure and that a mutation of the Cys residue equivalent to that described above impaired the DNA-binding activity, we propose that the Zn2+ ion at the N-terminal site is necessary to maintain the overall tertiary structure, while the Zn2+ ion at the C-terminal site is necessary for the DNA binding, mainly by guiding the basic C-terminal loop to correctly fit into the DNA groove.

Amino Acid Sequence↗

PubMed2006

A flexible representation of omic knowledge for thorough analysis of microarray data.

BACKGROUND: In order to understand microarray data reasonably in the context of other existing biological knowledge, it is necessary to conduct a thorough examination of the data utilizing every aspect of available omic knowledge libraries. So far, a number of bioinformatics tools have been developed. However, each of them is restricted to deal with one type of omic knowledge, e.g., pathways, interactions or gene ontology. Now that the varieties of omic knowledge are expanding, analysis tools need a way to deal with any type of omic knowledge. Hence, we have designed the Omic Space Markup Language (OSML) that can represent a wide range of omic knowledge, and also, we have developed a tool named GSCope3, which can statistically analyze microarray data in comparison with the OSML-formatted omic knowledge data. RESULTS: In order to test the applicability of OSML to represent a variety of omic knowledge specifically useful for analysis of Arabidopsis thaliana microarray data, we have constructed a Biological Knowledge Library (BiKLi) by converting eight different types of omic knowledge into OSML-formatted datasets. We applied GSCope3 and BiKLi to previously reported A. thaliana microarray data, so as to extract any additional insights from the data. As a result, we have discovered a new insight that lignin formation resists drought stress and activates transcription of many water channel genes to oppose drought stress; and most of the 20S proteasome subunit genes show similar expression profiles under drought stress. In addition to this novel discovery, similar findings previously reported were also quickly confirmed using GSCope3 and BiKLi. CONCLUSION: GSCope3 can statistically analyze microarray data in the context of any OSML-represented omic knowledge. OSML is not restricted to a specific data type structure, but it can represent a wide range of omic knowledge. It allows us to convert new types of omic knowledge into datasets that can be used for microarray data analysis with GSCope3. In addition to BiKLi, by collecting various types of omic knowledge as OSML libraries, it becomes possible for us to conduct detailed thorough analysis from various biological viewpoints. GSCope3 and BiKLi are available for academic users at our web site http://omicspace.riken.jp.

Journal Article↗

PubMed2006

Engineering drought tolerance in plants: discovering and tailoring genes to unlock the future.

The ability of plants to tolerate drought conditions is crucial for agricultural production worldwide. Recent progress has been made in our understanding of gene expression, transcriptional regulation and signal transduction in plant responses to drought. Molecular and genomic analyses have facilitated gene discovery and enabled genetic engineering using several functional or regulatory genes to activate specific or broad pathways related to drought tolerance in plants. Several lines of evidence have indicated that molecular tailoring of genes has the potential to overcome a number of limitations in creating drought-tolerant transgenic plants. Recent studies have increased our understanding of the regulatory networks controlling the drought stress response and have led to practical approaches for engineering drought tolerance in plants.

Biotechnology↗

PubMed2006

Monitoring expression profiles of Arabidopsis genes during cold acclimation and deacclimation using DNA microarrays.

A comparative analysis of gene expression profiles during cold acclimation and deacclimation is necessary to elucidate the molecular mechanisms of cold stress responses in higher plants. We analyzed gene expression profiles in the process of cold acclimation and deacclimation (recovery from cold stress) using two microarray systems, the 7K RAFL cDNA microarray and the Agilent 22K oligonucleotide array. By both microarray analyses, we identified 292 genes up-regulated and 320 genes down-regulated during deacclimation, and 445 cold up-regulated genes and 341 cold down-regulated genes during cold acclimation. Many genes up-regulated during deacclimation were found to be down-regulated during cold acclimation, and vice versa. The genes up-regulated during deacclimation were classified into (1) regulatory proteins involved in further regulation of signal transduction and gene expression and (2) functional proteins involved in the recovery process from cold-stress-induced damages and plant growth. We also applied expression profiling studies to identify the key genes involved in the biosynthesis of carbohydrates and amino acids that are known to play important roles in cold acclimation. We compared genes that are regulated during deacclimation with those regulated during rehydration after dehydration to discuss the similarity and difference of each recovery process.

Acclimatization↗

PubMed2006

Abscisic acid-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1.

bZIP-type transcription factors AREBs/ABFs bind an abscisic acid (ABA)-responsive cis-acting element named ABRE and transactivate downstream gene expression in Arabidopsis. Because AREB1 overexpression could not induce downstream gene expression, activation of AREB1 requires ABA-dependent posttranscriptional modification. We confirmed that ABA activated 42-kDa kinase activity, which, in turn, phosphorylated Ser/Thr residues of R-X-X-S/T sites in the conserved regions of AREB1. Amino acid substitutions of R-X-X-S/T sites to Ala suppressed transactivation activity, and multiple substitution of these sites resulted in almost complete suppression of transactivation activity in transient assays. In contrast, substitution of the Ser/Thr residues to Asp resulted in high transactivation activity without exogenous ABA application. A phosphorylated, transcriptionally active form was achieved by substitution of Ser/Thr in all conserved R-X-X-S/T sites to Asp. Transgenic plants overexpressing the phosphorylated active form of AREB1 expressed many ABA-inducible genes, such as RD29B, without ABA treatment. These results indicate that the ABA-dependent multisite phosphorylation of AREB1 regulates its own activation in plants.

Abscisic Acid↗