Showing papers by "Kazuki Saito published in 2007"

Omics-based identification of Arabidopsis Myb transcription factors regulating aliphatic glucosinolate biosynthesis

Abstract: Understanding plant metabolism as an integrated system is essential for metabolic engineering aimed at the effective production of compounds useful to human life and the global environment. The “omics” approach integrates transcriptome and metabolome data into a single data set and can lead to the identification of unknown genes and their regulatory networks involved in metabolic pathways of interest. One of the intriguing, although poorly described metabolic pathways in plants is the biosynthesis of glucosinolates (GSLs), a group of bioactive secondary products derived from amino acids that are found in the family Brassicaceae. Here we report the discovery of two R2R3-Myb transcription factors that positively control the biosynthesis of GSLs in Arabidopsis thaliana by an integrated omics approach. Combined transcriptome coexpression analysis of publicly available, condition-independent data and the condition-specific (i.e., sulfur-deficiency) data identified Myb28 and Myb29 as candidate transcription factor genes specifically involved in the regulation of aliphatic GSL production. Analysis of a knockout mutant and ectopic expression of the gene demonstrated that Myb28 is a positive regulator for basal-level production of aliphatic GSLs. Myb29 presumably plays an accessory function for methyl jasmonate-mediated induction of a set of aliphatic GSL biosynthetic genes. Overexpression of Myb28 in Arabidopsis-cultured suspension cells, which do not normally synthesize GSLs, resulted in the production of large amounts of GSLs, suggesting the possibility of efficient industrial production of GSLs by manipulation of these transcription factors. A working model for regulation of GSL production involving these genes, renamed Production of Methionine-Derived Glucosinolate (PMG) 1 and 2, are postulated.

ATTED-II: a database of co-expressed genes and cis elements for identifying co-regulated gene groups in Arabidopsis

Abstract: Publicly available database of co-expressed gene sets would be a valuable tool for a wide variety of experimental designs, including targeting of genes for functional identification or for regulatory investigation. Here, we report the construction of an Arabidopsis thaliana trans-factor and cis-element prediction database (ATTED-II) that provides co-regulated gene relationships based on co-expressed genes deduced from microarray data and the predicted cis elements. ATTED-II (http://www.atted.bio.titech.ac.jp) includes the following features: (i) lists and networks of co-expressed genes calculated from 58 publicly available experimental series, which are composed of 1388 GeneChip data in A.thaliana; (ii) prediction of cis-regulatory elements in the 200 bp region upstream of the transcription start site to predict co-regulated genes amongst the co-expressed genes; and (iii) visual representation of expression patterns for individual genes. ATTED-II can thus help researchers to clarify the function and regulation of particular genes and gene networks.

Convergent evolution in the BAHD family of acyl transferases: identification and characterization of anthocyanin acyl transferases from Arabidopsis thaliana

Abstract: Members of the BAHD family of plant acyl transferases are very versatile catalytically, and are thought to be able to evolve new substrate specificities rapidly. Acylation of anthocyanins occurs in many plant species and affects anthocyanin stability and light absorption in solution. The versatility of BAHD acyl transferases makes it difficult to identify genes encoding enzymes with defined substrate specificities on the basis of structural homology to genes of known catalytic function alone. Consequently, we have used a modification to standard functional genomics strategies, incorporating co-expression profiling with anthocyanin accumulation, to identify genes encoding three anthocyanin acyl transferases from Arabidopsis thaliana. We show that the activities of these enzymes influence the stability of anthocyanins at neutral pH, and some acylations also affect the anthocyanin absorption maxima. These properties make the BAHD acyl transferases suitable tools for engineering anthocyanins for an improved range of biotechnological applications.

Posttranscriptional Regulation of High-Affinity Sulfate Transporters in Arabidopsis by Sulfur Nutrition

Abstract: High-affinity sulfate transporters SULTR1;1 and SULTR1;2 are expressed at epidermis and cortex of Arabidopsis (Arabidopsis thaliana) roots during sulfur limitation. Here, we report that SULTR1;1 and SULTR1;2 are two essential components of the sulfate uptake system in roots and are regulated at posttranscriptional levels together with the previously reported transcriptional control. Double knockout of SULTR1;1 and SULTR1;2 by T-DNA insertion gene disruption resulted in complete lack of sulfate uptake capacity and severely affected plant growth under low-sulfur conditions. Expression of epitope-tagged proteins SULTR1;1mycHis and SULTR1;2mycHis, under the control of the cauliflower mosaic virus 35S promoter, rescued the uptake of sulfate and the growth of the sultr1;1 sultr1;2 double knockout mutant. The recovery of the double knockout phenotypes was attributable to the posttranscriptional accumulation of sulfate transporter proteins that derive from the epitope-tagged transgenic constructs. Both SULTR1;1mycHis and SUTLR1;2mycHis mRNAs were predominantly found in roots and slightly induced by long-term sulfur limitation. SULTR1;1mycHis and SULTR1;2mycHis proteins were found exclusively in roots, and significantly accumulated by sulfur limitation, correlating with the induction of sulfate uptake activities. In the time course of short-term sulfate starvation treatment, SULTR1;1mycHis and SULTR1;2mycHis proteins were significantly accumulated during the 8- to 72-h period, causing substantial induction of sulfate uptake activities, while their corresponding mRNAs were expressed constantly around the initial levels, except for the transient induction in the first 2 h. This study suggested the importance of root-specific and sulfur deficiency-inducible accumulation of SULTR1;1 and SULTR1;2 sulfate transporter proteins for the acquisition of sulfate from low-sulfur environment.

Unbiased characterization of genotype-dependent metabolic regulations by metabolomic approach in Arabidopsis thaliana

Abstract: Metabolites are not only the catalytic products of enzymatic reactions but also the active regulators or the ultimate phenotype of metabolic homeostasis in highly complex cellular processes. The modes of regulation at the metabolome level can be revealed by metabolic networks. We investigated the metabolic network between wild-type and 2 mutant (methionine-over accumulation 1 [mto1] and transparent testa4 [tt4]) plants regarding the alteration of metabolite accumulation in Arabidopsis thaliana. In the GC-TOF/MS analysis, we acquired quantitative information regarding over 170 metabolites, which has been analyzed by a novel score (ZMC, z-score of metabolite correlation) describing a characteristic metabolite in terms of correlation. Although the 2 mutants revealed no apparent morphological abnormalities, the overall correlation values in mto1 were much lower than those of the wild-type and tt4 plants, indicating the loss of overall network stability due to the uncontrolled accumulation of methionine. In the tt4 mutant, a new correlation between malate and sinapate was observed although the levels of malate, sinapate, and sinapoylmalate remain unchanged, suggesting an adaptive reconfiguration of the network. Gene-expression correlations presumably responsible for these metabolic networks were determined using the metabolite correlations as clues. Two Arabidopsis mutants, mto1 and tt4, exhibited the following changes in entire metabolome networks: the overall loss of metabolic stability (mto1) or the generation of a metabolic network of a backup pathway for the lost physiological functions (tt4). The expansion of metabolite correlation to gene-expression correlation provides detailed insights into the systemic understanding of the plant cellular process regarding metabolome and transcriptome.

Phytochemical genomics in Arabidopsis thaliana: A case study for functional identification of flavonoid biosynthesis genes*

Abstract: The completion of the whole genome sequence of Arabidopsis thaliana has made it possible to explore the phytochemical genomics in this species by determining gene-to- metabolite correlation through the comprehensive analysis of metabolite accumulation and gene expression. In this study, flavonoid profiling of wild-type plants and T-DNA insertion mutants was analyzed using ultra-performance liquid chromatography (UPLC)/photodiode array detection (PDA)/electrospray ionization (ESI)/multiple-stage mass spectrometry (MS n ). Detailed analysis of the metabolite changes in the mutants suggested the functions of genes that have been mutated. In silico coexpression analysis of genes involved in flavonoid metabolism in Arabidopsis was performed using a publicly available transcriptome database of DNA microarrays. We inferred a coexpression framework model of the genes involved in the pathways of flavonol, anthocyanin, and proanthocyanidin synthesis, suggesting specific functions and coregulation of the genes of pathway enzymes and transcription factors. The metabolic profiling of the omt1 mutant lacking a methyltransferase gene narrowed down by the coexpression analysis showed that AtOMT1 (At5g54160) is involved not only in the pro- duction of lignins and sinapoyl esters but also in the methylation of flavonols forming isorhamnetin. These results suggest that the functional genomics approach by detailed target- metabolite profiling with transcriptome coexpression analysis provides an efficient way of identifying novel gene functions involved in plant metabolism.

Camptothecin: Therapeutic Potential and Biotechnology

Abstract: Camptothecin (CPT) and its derivatives have been received considerable attention recently. Two semi-synthetic derivatives, topotecan and irinotecan, are currently prescribed as anticancer drugs. Several more are now in clinical trial. CPT is produced in many plants belonging to unrelated orders of angiosperms. At present, CPT supplied for pharmaceutical use is extracted from the plants, Camptotheca acuminata and Nothapodytes foetida. Several efforts have been made to sustain a stable production of CPT by in vitro cell cultures of C. acuminata, N. foetida and Ophiorrhiza pumila. Recent report showed that plants are not the only sources that produce CPT. CPT was reported to be produced from the endophytic fungus isolated from the inner bark of N. foetida. The hairy root cultures of C. acuminata and O. pumila produce and secrete CPT into the medium in large quantities. These reports suggest the possibility to develop large-scale production of CPT. In addition, recent advance in the cloning and characterization of biosynthetic enzymes involved in CPT biosynthetic pathway provides valuable information for developing genetically engineered CPT-producing plants.

2-Hydroxyisoflavanone Dehydratase is a Critical Determinant of Isoflavone Productivity in Hairy Root Cultures of Lotus japonicus

Abstract: Hairy root cultures of a model legume, Lotus japonicus, were established to characterize two heterologous cDNAs encoding enzymes involved in isoflavone biosynthesis, i.e. licorice 2-hydroxyisoflavanone synthase (IFS) and soybean 2-hydroxyisoflavanone dehydratase (HID) catalyzing sequential reactions to yield isoflavones. While the control and the IFS overexpressor did not accumulate detectable isoflavones, the HID overexpressors did accumulate daidzein and genistein, showing that HID is a critical determinant of isoflavone productivity. Production of coumestrol in all the genotypes and isoliquiritigenin/liquiritigenin in IFS + HID-overexpressing lines was also noted. These results provide insight into the regulatory mechanism that controls isoflavonoid biosynthesis.

Small Antennas for Medical Applications

Abstract: Various types of medical applications of antennas have widely been investigated. Since these antennas are used in and around a human body, the size of the antennas must be small. Particularly, in order to realize minimally invasive treatment, small-size antennas are desired. This paper describes a few antennas for hyperthermia which is one of thermal therapies for cancer. The authors et al. have developed a coaxial-slot antenna, which is one of thin microwave antennas to be employed for interstitial as well as intracavitary microwave hyperthermia. Two different types of coaxial-slot antennas are then introduced for the treatment of brain tumor and bile duct carcinoma.

Predicting state transitions in the transcriptome and metabolome using a linear dynamical system model

Abstract: Modelling of time series data should not be an approximation of input data profiles, but rather be able to detect and evaluate dynamical changes in the time series data. Objective criteria that can be used to evaluate dynamical changes in data are therefore important to filter experimental noise and to enable extraction of unexpected, biologically important information. Here we demonstrate the effectiveness of a Markov model, named the Linear Dynamical System, to simulate the dynamics of a transcript or metabolite time series, and propose a probabilistic index that enables detection of time-sensitive changes. This method was applied to time series datasets from Bacillus subtilis and Arabidopsis thaliana grown under stress conditions; in the former, only gene expression was studied, whereas in the latter, both gene expression and metabolite accumulation. Our method not only identified well-known changes in gene expression and metabolite accumulation, but also detected novel changes that are likely to be responsible for each stress response condition. This general approach can be applied to any time-series data profile from which one wishes to identify elements responsible for state transitions, such as rapid environmental adaptation by an organism.

Oleanane-type Triterpene Glycosides from Glycyrrhiza Uralensis:

Abstract: Chemical investigation of the roots of Glycyrrhiza uralensis resulted in the isolation of six oleanane-type triterpene glycosides (1 – 6), including one new saponin (1) and two (2 and 3) obtained a

Sulfur-responsive promoter of sulfate transporter gene is potentially useful to detect and quantify selenate and chromate

Abstract: Plant-based assays for monitoring contaminated environments provide inexpensive and nontechnical means of environmental analysis. Here we report a model system for monitoring selenium and chromium, which are highly toxic heavy metals for living organisms. The major forms of selenium and chromium in nature are selenate and chromate. As toxic analogs of sulfate, they cause sulfur deficiency in plants by inhibiting the uptake of sulfate from the environment. We used a fusion gene construct consisting of a sulfur-responsive promoter region of the high-affinity sulfate transporter SULTR1;2 from Arabidopsis and green fluorescent protein (GFP; PSULTR1;2-GFP) to quantify the levels of selenate and chromate by GFP accumulation. The PSULTR1;2-GFP transgenic Arabidopsis plants showed drastic increases in GFP with the addition of selenate or chromate to the medium. The increase in GFP was concentration-dependent relative to the amounts of contaminants in the medium, suggesting the potential of PSULTR1;2-GFP plants as indicators in quantifying environmental selenate and chromate.

Metabolic Engineering of Sulfur Assimilation in Plants

Abstract: Sulfur and sulfur-containing products play important roles in plant cells. In order to enhance the ability of the production of sulfur-containing compounds by the metabolic engineering of sulfur assimilation in plants, we constructed transgenic plants over-expressing cysteine synthase (CSase), or serine acetyltransferase (SATase) gene, key enzymes for cysteine biosynthesis in plants. Transgenic plants over-expressing CSase gene were highly tolerant to sulfur dioxide, sulfite, oxidative stress, and cadmium. Besides, transgenic plants over-expressing SATase gene showed the over-accumulation of sulfur-containing compounds, cysteine and glutathione (GSH). These results indicate that the over-expression of CSase or SATase gene is promising for the metabolic engineering of sulfur assimilation in plants