The completion of plant genome sequences and advances in DNA microarray technology have contributed to the accumulation of a vast number of plant gene expression datasets. Co-expression analyses using such datasets can be used to predict the functions of many plant genes. A network approach has been incorporated into co-expression analysis to visualize gene-to-gene functional relatedness (co-expression network analysis). Applying of this analysis to plant gene expression datasets has led to the accumulation of large quantities of information on plant gene function. Plant gene expression datasets and genome-level information obtained using co-expression analyses in plants can be retrieved from various types of databases. Here, we summarize practical approaches for detecting co-expressed genes in plants and review recent progress in plant co-expression analyses.
Plant metabolites are produced through complex processes that include multiple enzymatic steps, branched pathways and regulation by a number of functionally redundant transcription factors. In addition, plants synthesize and accumulate each metabolite, especially secondary metabolites, in specific tissues and cells during development. Therefore, manipulation of both transcription factors that regulate enzymatic steps of a metabolic pathway (metabolic regulators) and/or that regulate cellular differentiation (developmental regulators) would be an effective strategy for controlling plant metabolites, quantitatively and qualitatively. In this review, we describe the advantages of using transcription factors for metabolic engineering in plants. Transcriptional activators and repressors, including the chimeric repressors generated by CRES-T, are useful tools for the genetic engineering of metabolic pathways. In addition, we propose that the use of both developmental regulators and plant tissue culture technology, in combination with metabolic regulators, would be an effective strategy to increase the productivity of metabolites. We summarize the strategies that have been applied for the detection of regulators and enzyme genes involved in metabolic pathways.
Plastid transformation is a powerful tool for the production of useful compounds in higher plants through metabolic engineering, because it has many advantages over conventional nuclear transformation: high-level foreign protein accumulation, no need for a transit peptide, absence of gene silencing, and convenient transgene stacking in an operon. Plastid transformation has recently yielded remarkable results in the production of highly valued biopharmaceutical proteins and in conferring herbicide and insect resistance. Metabolic pathway engineering by plastid transformation has also produced higher levels of useful compounds than nuclear transformation. Furthermore, recent reports have shown the functional regulation of transgene expression from the plastid genome. In this review, we have focused on the progress of plastid transformation in material production from the aspect of biosynthetic pathway engineering, discussing the issues for future expansion of plastid transformation.
Metabolites in primary metabolic pathways, such as glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway, the Calvin cycle and amino acid biosynthetic pathways, are ionic compounds. It is difficult to analyze these metabolites simultaneously by established methods for metabolite profiling of biological samples such as gas chromatography-mass spectrometry or liquid chromatography-mass spectrometry. Capillary electrophoresis-mass spectrometry (CE-MS) is a promising method for the comprehensive and quantitative analysis of ionic metabolites. This technique enables the simultaneous determination of over 1000 charged compounds. This review discusses the principles and practical procedures for conducting CE-MS, describes specific applications of CE-MS for primary metabolite profiling, and enumerates some of the technical problems associated with CE-MS at present.
The 20 amino acids, except for methionine and tryptophan, are coded for by two to six codons called synonymous codons. Synonymous codons are used differently by different organisms. Hence, codon selection should be important to express recombinant proteins in tobacco chloroplasts. We present here the codon usage table from the entire 79 mRNAs from tobacco chloroplasts. As codons function on mRNAs, codon usage tables should be constructed from mRNAs but not from genes. We devised an in vitro assay for translation efficiencies of codons, and measured the translation efficiencies of several synonymous codon groups in tobacco chloroplasts. Our results indicated that translation efficiencies of individual codons are not always correlated with codon usage. Based on these data, we discuss on synonymous codon selection for expression of inserted genes in tobacco chloroplasts.
Chloroplasts are plant-specific organelles that perform photosynthesis and responsible for the world's primary productivity. Using light energy, chloroplasts produce many important products, including starch, amino acids, lipids, pigments and various secondary products. Therefore, chloroplasts are essential to the lives of all plants and animals alike. Chloroplast transformation is a unique technology to produce huge amount of valuable materials in chloroplasts using photosynthetic energy. In order to control chloroplasts at will, we need more information on molecular basis of chloroplast gene expression and communication between the chloroplast and nucleus. Chloroplast sigma factors are key regulators of the chloroplast gene expression and chloroplast differentiation. This review summarized recent findings on roles of chloroplast sigma factors in the chloroplast differentiation and environmental responses.
Natural rubber is one of the most important polymers produced by plants because it is a strategic raw material used in more than 40,000 products. It has unique properties as a polymer owing to its specific structure, its high molecular weight and yet-to-be-defined contributions of minor components in the latex. Among over 2500 rubber-producing plant species, the Para rubber tree (Hevea brasiliensis Muell. Arg.) is presently the only commercial source of natural rubber. The objective of this review is to provide readers with information on the newest trends and market conditions of natural rubber and also explain the historical background and a global view of Hevea breeding and genetics, together with information about alternative resources.
Eucommia ulmoides Oliver is a deciduous tree distributed in China, and is the only species in its genus, and the only genus in its family. It grows in temperate zones, and produces long-chain trans-polyisoprene throughout the entire plant. This trans-polyisoprene (TPI) derived from Eucommia is called Eucommia rubber (EU-rubber), and is a hydrocarbon with a carbon molecular weight of a few hundred thousand. EU-rubber is a hard rubber with thermoplasticity, and has properties similar to plastic. In terms of practical use, these plant-derived polymers are attracting attention as an industrial raw material to substitute for petroleum, and demand is expected to grow in the future. Hydrocarbon is also a fuel which has energy with the same value as petroleum.
Astaxanthin responsible for the red color of red fish and crustacean has beneficial effects on human health as well as its utility as a pigmentation source in aquaculture. Astaxanthin biosynthetic pathway from β-carotene needs two enzymes, a carotenoid 4,4′-ketolase (oxygenase) and a carotenoid 3,3′-hydroxylase. β-Carotene is usually one of dominant carotenoids in higher plants, which lack the former enzyme. This mini-review elucidates in the earlier section the catalytic functions of a series of the ketolase and hydroxylase enzymes that have been isolated up to now. For the last ten years, pathway engineering approaches of plants including staple crops toward astaxanthin production have been undertaken by introducing and expressing in the plants a ketolase gene sometimes along with a hydroxylase gene. Several successful results have been achieved to produce large amounts of astaxanthin together with other ketocarotenoids in plant tissues such as carrot roots and tobacco leaves.
Licorice is the root and stolon of the Glycyrrhiza plant, which belongs to the family Leguminosae. The licorice plant is an important medicinal herb, and the constituent—glycyrrhizin—is widely used as a natural sweetener and also as a pharmaceutical agent because of its anti-inflammatory and hepatoprotective properties. Licorice is also an indispensable ingredient of traditional Japanese Kampo medicines. Furthermore, licorice extracts are used as cosmetics, food additives, tobacco flavors, and confectionery foods. In this article, we review the importance of licorice and its related products.
Forest produces the most abundant biomass resource on earth, and its ecological and commercial importance in maintaining the global environment and human society is irreplaceable. It is an urgent task to establish technological infrastructure to produce forest biomass in a sustainable manner. Forest tree biotechnology plays a pivotal role to achieve this goal. Genus Eucalyptus contains some of the most valuable and the fastest growing tropical tree species in the world. In 2006, the whole genome sequence of poplar, the first woody plant genome, was released. Subsequently, whole genome sequencing projects of two Eucalyptus species have started, and are currently ongoing. Eucalyptus expression-sequence tag (EST) databases are quite useful for identifying Eucalyptus genes which are orthologous to those of poplar and other model plants. In addition, establishing efficient systems for in vitro regeneration of transformed Eucalyptus will be indispensable for genetic improvement of the species. Importantly, these activities must be done to conserve the global environment and benefit the social welfare of local communities. In this review, the current status and “post-genomics” research directions in Eucalyptus biotechnology are outlined.
Glycine betaine (GB) is an important compatible solute that protects plants against the damaging effects of abiotic stresses. A number of plants have been engineered to contain genes of the GB biosynthetic pathway, which confers enhanced tolerance to a range of abiotic stresses during various plant developmental stages. Unlike natural accumulators, the transgenic plants accumulate very low GB concentrations, insignificant in terms of coping with osmotic stress. The GB accumulation in these transgenic plants varies depending upon their capacity for endogenous choline uptake, the type of gene that catalyzes the GB biosynthetic pathway, and the localization of the transgene product in a particular cellular compartment. This review focuses on recent progress in studies of abiotic stress tolerance conferred by GB in transgenic plants.
The number of nucleotide and protein sequences deposited in public databases has rapidly increased in recent times. To provide consistent annotation of orthologous genes across organisms, annotation using Gene Ontology (GO) terms is shared among most of these databases. GO outlines a structured vocabulary used for describing biological properties of gene products. The illustration of GO terms using complete paths to the root term on the basis of a directed acyclic graph (DAG) approach aids in the systematic grasping of the functional information related to a gene product. However, the website provided by the GO Consortium does not present DAGs for two or more GO terms due to difficulties in the depiction of the complex relationships of such GO terms. To overcome these problems, we have constructed a DAG-based browser, termed DAGViz, that shows DAG-based information of multiple GO terms assigned to one or more genes within a single screen (http://www.pgb.kazusa.or.jp/dagviz/). In the current report, we illustrate the advantages of DAGViz in analyzing GO annotation.
A new Arabidopsis transcriptome and microarray (ARTRA) database has been developed that contains transcriptome sequences and gene-specific sequences for a DNA microarray and RNAi knockdown triggers. Probes for the microarray were developed using sequences from the ARTRA database are characterized by long gene-specific fragments and spotting as a single sense strand. As a result, the gene-specificity and sensitivity of this microarray were both high, and therefore the ratio of gene expression from the present array was highly correlated with that from the Affymetrix GeneChip (R2=0.95). Moreover, AGAMOUS gene expression was reduced to about 10% of wild-type in T1 plants transformed with a vector containing an inverted repeat of the gene-specific fragment of AGAMOUS. Flowers of the T1 plants lacked pistils and stamens, which is the phenotype of known AGAMOUS knockout plants. This result showed that gene knockdown is possible in transgenic plants with the inverted repeat structure using a gene-specific fragment. RNAi knockdown is important as an approach for genes without T-DNA tag lines. Access to the ARTRA database is available to any researcher on request from the web site of http://artra.kazusa.or.jp/.
The Dof (DNA-binding with one finger) genes are members of a family of plant-specific transcription factors that have a highly conserved DNA-binding domain, namely, Dof domain. The Dof domain is a particular class of zinc finger domain that has been demonstrated to bind specifically to DNA sequences with a T/AAAAG core. In the Arabidopsis genomic database, 36 Dof genes have been identified, whereas the functions of most of the members still remain to be studied. Therefore, we attempted to comprehensively and systematically investigate functions of Arabidopsis Dof genes. As the first step, we isolated cDNAs of all the Arabidopsis Dof genes based on the coding sequences identified on the genomic database. Then, we selected genes, which are subjected to further functional analysis, through a phylogenetic analysis, and transformed Arabidopsis cultured cells (line T87) using cDNAs corresponding to the selected genes. After that, we examined transcriptional profiles in the Dof gene-overexpressed calli using an Arabidopsis DNA microarray. From the results, possible involvements of the Dof genes in regulation of metabolic pathways are discussed.
Although several biotechnological researchers have reported the production of useful carotenoids in transgenic plants, the acquirement of genetically stable transgenic plants and subsequent examinations for effects of transgenes on the plants require a lot of time and efforts. Here, we show a simple and efficient approach to evaluate key genes involved in carotenoid biosynthesis using Arabidopsis thaliana suspension-cultured cell line T87. A plasmid for the expression of six key-gene candidates for astaxanthin production was constructed and introduced into A. thaliana T87 cells via Agrobacterium-mediated transformation. Five among twenty transgenic cell lines were isolated as lines resistant to a breaching herbicide norflurazon, and the expression of all the six transgenes in the lines was confirmed by quantitative transcriptional analysis. These five transgenic cell lines were shown to accumulate 4.3 to 21.9 μg g−1 fresh weight of astaxanthin, in addition to other ketocarotenoids containing adonirubin, canthaxanthin, echinenone and 3′-hydroxyechinenone. The amount of the ketocarotenoids was estimated to be 36% to 53% of the total carotenoids. The total carotenoid amount was also increased to 4.7 to 13.9 times that of control cells. We further employed microarray analysis to evaluate effects of the transgenes on endogenous gene expression in the transgenic cells. Transcriptional levels of many endogenous carotenogenic genes, e.g., genes for β-carotene hydroxylase, neoxanthin cleavage enzymes, abscisic aldehyde oxidase, 1-deoxy-D-xylulose 5-phosphate synthase, endoplasmic reticulum-targeted and mitochondria-targeted geranyl-geranyl diphosphate synthase were significantly elevated by the expression of the six genes for astaxanthin biosynthesis. These data suggest that transgenic cells adjust their whole cellular isoprenoid-metabolic system to direct the high-level production of the carotenoids including the ketocarotenoids in the plastids.
Salinity is regarded as a major factor impacting the growth of plants such as trees. Transgenic technology is considered an effective approach to minimizing salt damage by enhancing a plant's salt tolerance. Currently, most of the research on transgenic woody plants is still limited to the laboratory level in vitro and in growth chambers. However, the confined laboratory evaluation of salt tolerance condition yields minimal information since various factors that significantly impact growth, such as weather and season, do not come into play. Field level evaluation of salt tolerance is necessary but is limited in Japan. Transgenic plants should be evaluated in semi-confinement before they are moved to open fields. Therefore, an effective evaluation method for salt tolerance in woody plants is needed in semi-confinement. To evaluate the salt tolerance of transgenic Eucalyptus under semi-confined conditions, we initially developed a new evaluation method using non-transgenic E. camaldulensis. The duration of salt treatment was determined to be the point in time when half of the plants started to exhibit wilting. Additionally, the plants were continuously irrigated with fresh water as a final procedure. With the evaluation system in place, the salt tolerance of transgenic E. globulus containing the choline oxidase (codA) gene was measured. The transgenic plants showed higher survival rates than the non-transgenic plants. This result indicated that the salt tolerance of transgenic E. globulus was enhanced by the introduced codA gene and also established that the new evaluation system is valuable for testing transgenic plants in semi-confinement and will contribute to an increase in the useful applications of transgenic plants.
The development of transgenic plants may help alleviate both environmental and food problems. In Japan, transgenic plants cannot be planted in the field without undergoing an environmental biosafety assessment under confined and semi-confined conditions. The main objectives of environmental biosafety assessment studies are to define the properties of the host plant and to evaluate its influence on other organisms. To appraise the influence of a transgenic plant on other plants, the transgenic plant is examined for new compounds that influence plant growth by measuring germinating seeds. Previously, we assessed the allelopathic activity of several transgenic plants using the sandwich method to assay the allelopathic activity of leachate from dried leaf samples. However, because Eucalyptus leaves are difficult to dry, the sandwich method does not allow the evaluation of multiple samples at the same time. Here, we report a new “homogenized method” that relies on homogenizing fleshy leaf samples, instead of drying them. The alleopathic activity of a non-transgenic plant and an Antisense-lim transgenic plant were evaluated using both the sandwich method and the homogenized method to determine whether the homogenized method was available for in biosafety assessments of transgenic plants. The homogenized method may be an effective and useful tool for evaluating differences between non-transgenic and transgenic plants using multiple, concurrent samples.
The modification and enhancement of plant amino acid accumulation is potentially beneficial in terms of the production of suitable crop plants for food or feeds, and also with respect to possible environmental improvement via the effective use of nitrogen nutrition. It is also expected that modified plants will produce beneficial nitrogenous compounds, such as proteins, secondary metabolites, or nucleic acids. The size of amino acid pools in plants is strictly controlled by certain environmental factors such as light and nutrition. In order to gain an understanding of the mechanisms that control amino acid biosynthesis and metabolism, we investigated the diurnal changes in amino acid contents and also how the amino acid contents change with the progression of growth. We observed that almost all the amino acids in plant leaves undergo diurnal changes and that the pattern of these changes was different in young and old stages. We focused on correlation of the asparagine content and the expression of ASN2 asparagine synthetase, since under our experimental conditions their fluctuating patterns were found to be similar. The asparagine contents of ASN2-overexpressing and -underexpressing plants were increased and decreased, respectively, when they were grown under normal light and nutrient conditions. These changes in asparagine content were marked when the plants were grown under conditions where ammonium was the sole nitrogen source. It has been previously reported that ASN2 expression and ammonium metabolism are correlated. Our findings and these previous observations suggest that ASN2 functions as a regulator of asparagine biosynthesis and metabolism and that it mediates the effective use of nitrogen under ammonium-sufficient conditions.
Since 2004, we have been developing a metabolite database concerning species-metabolite relations called KNApSAcK, which currently contains 49,165 species-metabolite relations incorporating 24,847 metabolites. In the present study, we report current status of KNApSAcK database and it's application to metabolomics fields and propose a new algorithm for detecting fragmentation patterns in a complicated mixture such as a plant tissue and a new scheme for analyzing spectral information leading to peak annotation of GC-MS spectra. When considering samples corresponding to a variety of species in addition to model species, KNApSAcK DB has strong potential for contribution to metabolomics research by way of applying it not only to simple metabolite search but also to further metabolomics analysis.
Genome sequence analysis has revealed the presence of almost infinite numbers of cytochrome P450 genes in a variety of organisms. To establish a robust experimental platform from which to explore the catalytic potential of those putative P450 proteins, we have developed a comprehensive metabolic profiling system based on Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR/MS) analysis, together with recombinant enzyme studies. Here, we report the enzymatic properties of CYP78A5, CYP78A7, CYP78A10, and CYP86C3 of Arabidopsis as short-chain fatty acid hydroxylases, with substrates including lauric acid (C12:0), myristic acid (C14:0), palmitic acid (C16:0), and myristoleic acid (C14:1). The fatty acid hydroxylation activity of CYP78A7 was confirmed using FT-ICR/MS metabolic profiling of P450-gene-overexpressing lines of cultured Arabidopsis T87 cells. The practical application of this metabolic profiling scheme is discussed as a P450 functional characterization platform.
Glycyrrhiza uralensis (Leguminosae, Fabaceae) is the most important medicinal plant used as a natural sweetener, in Chinese herbal medicines, and as a tobacco flavoring agent. Here, we report the generation of 56,857 expressed sequence tags (ESTs) derived from two full-length cDNA libraries produced from rhizomes (stolons) of two strains of G. uralensis, together with differential expression data obtained by MegasortTM analysis. The MegasortTM analysis was performed using two plant organs, namely, thickened roots and a rhizome-like organ, which differed in their glycyrrhizin content. After clustering of the 5′-end of ESTs, 5,542 contigs and 4,932 singletons were generated. A total of 10,474 nonredundant sequences were annotated on the basis of the TAIR database, of which 7,905 (75.5%) sequences exhibited homology to the sequences of registered genes. The gene functions of these 7,905 sequences were classified into Biological Process and Molecular Function on the basis of Gene Ontology (GO).
In this study, as a first step to achieve a goal to produce genetically-engineered sterile Eucalyptus, we focused on the AGAMOUS-like genes which are especially involved in stamen formation. Three new distinct cDNA fragments (EgAGL1, EgAGL2 and EgAGL3) and two distinct genomic DNA fragments (EgAGL1 and EgAGL2) encoding MADS-box proteins were isolated from Eucalyptus grandis. In the present study, three genes have 62–74% homology with the Arabidopsis AGAMOUS (AG) gene in deduced amino acid level. EgAGL1, EgAGL2 and EgAGL3 were classified close to the AGAMOUS gene by phylogenic analysis. EgAGL1 and EgAGL2 were strongly expressed in flower buds, and consequently may regulate stamen formation in Eucalyptus.
In animals, yeasts, bacteria, and viruses, L-glutamine D-fructose-6-phosphate amidotransferase (GFAT) is well characterized, but not in plants. This study identified an Arabidopsis GFAT gene by detecting the enzymatic activity of the recombinant protein produced in a wheat germ in vitro translation system. The Arabidopsis gene and a chlorella virus GFAT gene under the CaMV 35S promoter were introduced into tobacco BY-2 cells. Transgenic cells with the viral gene had higher levels of hexosamines than wild type, but no such hexosamine production was observed in transgenic cells with the Arabidopsis gene. These results clarify metabolic engineering of hexosamines in plants.
Since the woody plants are important bio-materials for paper, chemical and energy production, strategies for improvement of their properties are of considerable interest. We examined the expression of Eucalyptus camaldulensis transcription factor (TF) genes in xylem tissues using an oligo-microarray gene analysis technique. As a result, we isolated 21 TF genes related to xylem development. Following a functional investigation of 7 TFs in vitro, we selected the xylem TF gene EcHB1, which encodes a HD-Zip class II, for investigation as a transgene. EcHB1 expression was driven by the CaMV35S promoter in transgenic tobacco plants, which showed greater fiber length (≤20%) and plant height (≤50%) than wild-type plants. In addition, leaf, root and stem growth were significantly enhanced in the transgenic lines, which also had a lower acid-soluble lignin and hemicellulose content than wild-type. Our results indicate that metabolic flexibility might be involved in these improvements to xylem cell wall biosynthesis and that in addition to providing a growth advantage, such modifications may confer long-term structural integrity to the woody perennials.