Recently, high-throughput technologies for comprehensive genomic, transcriptomic and proteomic analyses have been developed. Large-scale ‘omics data’ obtained from such experimental methods have rapidly accumulated and been stored into web databases. In addition, the number of web databases for molecular biology has rapidly increased, since most new projects construct and maintain new web databases for their own large-scale omics data. This wealth of comprehensive online resources and web databases allow us to extract essential new biological information beyond a dataset obtained from an individual study. For effective and efficient handling of such large sets of data, various kinds of bioinformatics tools are being developed. Here, we review the current state of web databases and bioinformatics tools for plant biosciences and systems biology.
Eukaryotes have a mechanism of RNA-guided regulation of gene expression in which double-stranded RNA inhibits the expression of genes with complementary nucleotide sequences. This mechanism plays a crucial role in many processes including development, stability of the genome, and responses against invading genetic materials. These RNA-guided pathways that control gene expression, collectively termed RNA silencing, are thought to have evolved as a form of innate immunity against viruses. RNA silencing provides a mechanism for downregulating gene expression and a tool that is suitable for analyzing gene function and engineering novel traits in organisms. The phenomenon of RNA silencing was discovered in transgenic petunia plants that had altered patterns of flower color as a consequence of overexpression of the chalcone synthase-A gene responsible for an essential enzyme to biosynthesize anthocyanins. After the “visual” discovery of RNA silencing in petunia, visible phenotypes have played an important role in monitoring the silenced state of a gene in various RNA silencing systems. In particular, a photobleached phenotype in leaf tissues is useful in optimizing a virus-induced gene silencing system. Loss of pigmentation in plant tissues has also led to the detection of naturally occurring RNA-silencing phenomena. Visual changes conferred by endogenous reporter genes provide highly informative assessments of RNA silencing that can be applied to a wide spectrum of plant biotechnology.
We isolated a calcineurin B-like protein (CBL)-interacting protein kinase (EC 126.96.36.199) homolog (VuCIPK1) from cowpea (Vigna unguiculata (L.) Walp.) with significant similarity to AtCIPK3 (86% amino acid identity), which is involved in K+ transport. VuCIPK1 mRNA was detected in the whole plant by RT-PCR. Any significant change in VuCIPK1 mRNA levels was not detected when cowpea was subjected to various environmental stresses. Immunoblot assay with an anti-VuCIPK1 specific antibody and an anti-CBL antibody showed that immunologically CIPK- and CBL-related polypeptides were preferentially associated with membrane fractions of cowpea. The anti-VuCIPK1 antibody cross-reacted to two different CIPK-related polypeptides with relative molecular masses of 51 kDa in root and 55 kDa in mature leaf. Immunoprecipitation assay by anti-VuCIPK1 antibody using cowpea leaf extracts showed that endogenous VuCIPK1 was autophosphorylated on the threonine residues in response to salt stress. The recombinant VuCIPK1 phosphorylated casein in the presence of Mn2+. These observations suggest that membrane-associated VuCIPK1 is involved osmotic stress-activated kinase regulated by its phosphorylation status.
The VERNALIZATION INDEPENDENCE 6 (VIP6)/EARLY FLOWERING 8 (ELF8) gene of Arabidopsis thaliana encodes a protein homologous to CYCLIN THREE REQUIRING 9 (CTR9) component of the yeast transcriptional complex, RNA polymerase II-associated factor 1 (PAF1). It has been demonstrated that mutation alleles in the VIP6/ELF8 show early flowering phenotype, as well as other pleiotropic developmental defects. Here, we provide evidence that seeds with vip6/elf8 homozygous mutations were rarely obtained in all three independent lines of T-DNA insertion. Although viable seeds with homozygous mutant alleles were rarely obtained, they showed pleiotropic phenotype like mutants of other PAF1-related genes, and were sterile. These results suggest that the VIP6/ELF8 gene is essential for plant development.
Carrot (Daucus carota) somatic embryos have been extensively used as an experimental system for studying plant embryogenesis. In maturing zygotic embryos, the phytohormone abscisic acid (ABA) is involved in induction of dormancy and desiccation tolerance. Somatic embryos contain only low levels of endogenous ABA and lack desiccation tolerance and dormancy, but both tolerance and dormancy can be induced by application of exogenous ABA. We analyzed the effects of the optical isomers of ABA, (S)-(+)-2-cis-ABA [(+)-ABA] and (R)-(−)-2-cis-ABA [(−)-ABA], and a racemic mixture of these two isomers (racemic ABA) on the carrot system. Somatic embryos treated with (+)-ABA, (−)-ABA, or racemic ABA showed the same levels of growth inhibition and desiccation tolerance. Similarly, each ABA isomer and racemic ABA had the same effect on accumulation of transcripts for ABA-inducible genes. The results indicate that both (+)-ABA and (−)-ABA cause the same amount of activity in these physiological phenomena. Our findings suggest that strict steric requirements of the ABA signaling pathways are absent in carrot somatic embryos, and we propose that commercially available racemic ABA is as effective as the natural isomer (+)-ABA in induction of dormancy and desiccation tolerance in carrot somatic embryos.
A low glutelin-content mutant, LGC-1, has a mutant gene (lgc1) comprising a tail-to-tail inverted repeat of two highly homologous glutelin B genes (gluB4 and δ-gluB5), resulting in the production of double-stranded glutelin B RNA. Hybrid rice lines between LGC-1 and a transgenic line with the glutelin A1-collagen peptide fusion gene (gluA1-coll) were produced, and F2 hybrid lines homozygous for each or both of the gluA1-coll and lgc1 genes were selected. The expression of gluA1-coll in F2 lines with lgc1 was far lower than that of F2 lines without lgc1 at both the mRNA and protein levels as measured by semi-quantitative RT-PCR and immunoblotting using anti-collagen peptide antibody. The gluA1 mRNA sequence alignment identical to the potential double-stranded RNA formed by gluB4 sense and δ-gluB5 antisense RNAs consisted of 14 nucleotides at most, and was not more than 23 even when one mismatch was allowed within the sequence. Thus, such shorter double-stranded glutelin B RNAs were suggested to suppress the expression of not only endogenous gluA genes as reported previously (Kusaba et al. 2003) but also the exogenous gluA1-coll in the hybrid lines.
The expression of the phenylalanine ammonia-lyase gene (PAL) was induced in anthocynanin-producing suspension-cultured carrot cells. In our previous study, we isolated two PAL genes, DcPAL3 and DcPAL4, from a carrot genomic library, the nucleotide sequences of which are highly similar. Here, the complete nucleotide sequence of DcPAL4 was determined and compared with that of DcPAL3, revealing two miniature inverted repeat transposable elements (MITEs), here designated MITE1 and MITE2, inserted into the proximal promoter region of DcPAL3, but not into that of DcPAL4. The expression of DcPAL4 was not detectable by RT–PCR in cultured carrot cells or the tissues of carrot plants, whereas DcPAL3 expression was detected. Transient expression experiment in carrot protoplasts showed that the DcPAL4 promoter actively produces transcripts, as does the DcPAL3 promoter. To characterize the roles of MITE1 and MITE2 (MITE1/2) in the transgenes of stable transformants, they were inserted into a plant transformation vector containing the 35S–β-glucuronidase (GUS) reporter gene and selectable antibiotic-resistance marker genes, and then introduced into tobacco BY-2 cells using an Agrobacterium method. More regenerated calli carrying the MITE1/2 construct survived on medium containing kanamycin than calli lacking MITE1/2. Regenerated calli containing MITE1/2 were more numerous having stronger GUS activity than those without MITE1/2. These results suggest that MITE1/2 enhances the expression of adjacent transgenes introduced into stable transformants and possibly into the endogenous DcPAL3 gene.
Five non-allelic dull (du) mutants have been isolated and characterized as low amylose mutants in rice (Oryza sativa) endosperm. Synthesis of amylose is controlled by Waxy the gene that encodes granule-bound starch synthase. We previously showed that the du1 and du2 proteins may encode general splicing regulators which affect the expression level and splicing efficiency of Waxyb (Wxb), an allele that has a single-base mutation in the 5′ splice site of intron 1. Here we report that the du3 mutant has also reduced the splicing efficiency of Wxb pre-mRNA. Furthermore, map-based cloning revealed that Du3 encodes the rice homolog of a cap-binding protein 20 kD subunit (CBP20), a component of the heterodimeric nuclear cap-binding complex (CBC) playing a role in pre-mRNA splicing, RNA nuclear export, and nonsense-mediated decay. Transient expression of green fluorescent protein (GFP)-tagged Du3 proteins in rice protoplasts resulted in localization to the nucleus showing a speckled pattern similar to the pattern displayed by other splicing factors.
The effects of erythritol, a safe and nontoxic sweetener, on organisms other than mammals have not been clarified. We investigated the effects of erythritol on plants and microorganisms. The supplementation of erythritol to white radish (Raphanus sativus L.), garlic (Allium sativum), and arabidopsis (Arabidopsis thaliana) enhanced the growth of their seedlings and roots, and shortened germination time. The supplementation of erythritol to a mushroom (Lentinula edodes) markedly enhanced the development of fruiting bodies. These results would suggest that erythritol has the attractive potential as a safe and useful growth accelerator for plants and microorganisms.
A novel and simple DNA extraction method was developed, which was called “the filter-inserted tip method”. This method used glass fiber filters inserted within micropipette tips. It was conducted by drawing and expelling fluid by pipetting using a multichannel pipette. This allowed us to manipulate a large number of samples at the same time for DNA adsorption, fixing, washing and extraction. The 3 mm×3 mm size of glass fiber filter was suitable for this method. To examine the reproducibility of the method, DNA extraction experiment was carried out using rice leaves, and the yielded DNA samples were used for PCR reactions. As a result, PCR amplifications were recognized for 234 out of 236 samples examined, showing high reproducibility of the method. It took approximately 20 min to conduct DNA extractions from 16 samples. These results suggest that this method is suitable for rapid DNA extractions from a large number of samples for PCR reactions, such as marker-assisted selection with simple manipulations, low cost, and without using expensive equipment.
We isolated a cDNA clone encoding a New Zealand spinach (Tetragonia tetragonioides) alkaline α-galactosidase that we designated TtAGA1. The clone was found by monitoring an up-regulated transcripts accumulation with the differential display reverse transcription–polymerase chain reaction method during a treatment of New Zealand spinach shoots with drought. Deduced amino acid sequence suggests that the gene belongs to a family of plant-specific alkaline α-galactosidases. When detached shoots of New Zealand spinach were treated with abiotic stresses such as drought, administration of mannitol (150 mM), wounding, salinity (200 mM and 400 mM sodium chloride), dark, and cold (4°C), drought most enhanced the accumulation of the TtAGA1 transcripts. Treatments with mannitol, wounding, salinity, and dark increased the transcripts accumulation, but cold did not. It is supposed that the degradation of galactosyl saccharides may correlate with drought responses in New Zealand spinach growing in seashore areas.
Biotic and abiotic-induced wounding is one of the severest stresses that plants suffer throughout the growing period. Upon injury, plants rapidly activates a set of genes, which encode diverse proteins to cope with damages. Screening such genes that were transcriptionally activated within 30 min after mechanical wounding in tobacco plants, we identified a particular clone encoding a multfunctional protein, and designated as NtMFP (Nicotiana tabacummultifunctional protein). The deduced polypeptide is constituted of 668 amino acids with an apparent molecular mass of 72.3 kDa, and bacterially expressed protein exhibited a clear β-oxidation activity. Fusion proteins with GFP were observed in cytosol, when expressed in onion epidermal cell layers. In addition to wounding, NtMFP transcripts were induced by tobacco mosaic virus infection, and by jasmonic acid treatments. When NtMFP was suppressed by the RNAi, transgenic tobacco showed dwarfism, early senescence and reduced expresstion of jasmonic acid-responsive genes. Multifunctional protein is generally considered to catalyze multiple steps of the fatty acid β-oxidation. It is also proposed to be involved in the β-oxidation step of jasmonic acid biosynthesis. The present results suggest the possibility that NtMFP commonly functions not only in fatty acid catabolism but also in jasmonic acid biosynthesis pathway.