Sequencing technology has been rapidly advancing. Giga-sequencers, which produce several gigabases of fragmented sequences per run, are attractive for decoding genomes and expressed sequence tags (ESTs). A variety of plant genomes and ESTs have been sequenced since the decoding of the genome of Arabidopsis thaliana, the model plant. ESTs are useful for functional analyses of genes and proteins and as biomarkers, which are used to identify particular tissues and conditions due to the specificity of their expression. Sequenced plant genomes and ESTs have been entered into public databases, where they are freely downloadable. Sequences representative of particular functions or structures have been collected from public databases to curate smaller databases useful for studying protein function. Here, we discuss the uses of the currently available plant EST datasets. We also demonstrate the use of network module analysis to perform more stable (or irrespective of the difference of performance in each analyzing PC) homology searches and to provide more information on molecular functions of plant ESTs and proteins.
Suppression of lachrymatory factor synthase (LFS) activity in onion (Allium cepa L.) is expected not only to reduce lachrymatory factor released from the disrupted tissues, but also to increase thiosulfinate and their derivatives associated with the characteristic flavor and various health effects. To test this hypothesis, we used transgenic methods to obtain non-lachrymatory onions in which the LFS gene was silenced by RNA interference (RNAi). In transformation experiments, we found that onion calli generated after preconditioning of the suspension-cultured calli on a solid medium were amenable to Agrobacterium-mediated transformation. These calli were observed without or with low levels of yellowish autofluorescence under the fluorescence microscope, and were easily distinguished from the recalcitrant calli having a characteristic yellowish autofluorescence. The frequency of transient green fluorescent protein (GFP) expression significantly increased when these amenable calli were infected with Agrobacterium harboring the plasmid carrying a GFP expression cassette. Southern blot analysis of adaptor ligation PCR products revealed that 4 independent transgenic lines were obtained. The successful transfer of the RNAi construct was confirmed by using PCR. Analysis of the 4 transgenic lines confirmed that the levels of LFS gene transcript, LFS protein, and LFS enzyme activity were reduced to 3.2–11.0%, 0.09–20.7%, and 0.3–10.1%, respectively, of the wild-type control. The thiosulfinate assay suggested that a significant increase in thiosulfinate formation could be expected in the onion bulb extracts in which LFS enzyme activity was suppressed to approximately 1% or less than that of the wild-type control.
Glutathione-dependent formaldehyde dehydrogenase (FALDH) is an enzyme involved in formaldehyde metabolism in eukaryotes. FALDH cDNA was cloned from golden pothos, which is reported to effectively purify gaseous formaldehyde from enclosed room atmosphere. FALDH cDNAs from Arabidopsis, rice and golden pothos were overexpressed in transgenic Arabidopsis, and the enzyme activity was compared to determine the one most suitable for the molecular breeding of formaldehyde-detoxifying plants. The transgenic lines exhibited modified levels of the FALDH transcript, i.e. 10–800% compared to the endogenous transcript, due to either an overexpression or a cosuppression phenotype. The enzyme activity in the crude leaf extract was not proportionate, but did correlate with the transcription levels with certain exceptions. The FALDHs from the three plant species indicated similar enzymatic activity on average. The capacity to detoxify exogenous formaldehyde in the transformants with the FALDHs was determined at the whole plant level. Plants overexpressing FALDH from the three plant species displayed up to a 40% increase in their efficiency to take up exogenous formaldehyde as compared with the wild-type plants. On the other hand, no difference in the survival rate was observed among the transformants and wild type plants on formaldehyde-containing agar medium. These results show the FALDH from golden pothos to be similarly or more effective for detoxifying formaldehyde in transgenic plants compared with Arabidopsis and rice, and that this cDNA is applicable to the molecular breeding of formaldehyde detoxifying plants.
We studied the biosynthetic role of a cytochrome P450, CYP81F4, in Arabidopsis. The CYP81F4 gene was found within a gene expression network containing the transcription factor gene MYB34/ATR1 involved in tryptophan metabolisms. Root metabolic profiles in null cyp81f4 mutant lines were analyzed using an in-house metabolomics scheme based on Fourier transform ion cyclotron resonance mass spectrometry. The cyp81f4 mutant plants exhibited a build-up of indole-3-yl-methyl glucosinolate with concomitant loss of 1-methoxy-indole-3-yl-methyl glucosinolate. A pathway prediction supported by a detailed glucosinolate analysis indicated that CYP81F4, together with an unidentified methyltransferase, is involved in the formation of the methoxy group in the indole ring to yield 1-methoxy-indole-3-yl-methyl glucosinolate in Arabidopsis roots. Current results suggest that 1-methoxy-indole-3-yl-methyl glucosinolate is produced by the activity of CYP81F4 in Arabidopsis roots.
Abstract High target protein concentrations in source materials are important for achieving high purification efficiency. We sought to produce the taste-modifying protein, miraculin, in transgenic tomato fruits and then to extract and purify this protein. In order to improve the efficiency of recombinant miraculin purification from transgenic tomatoes, we tested a salt-stress cultivation technique aimed at increasing miraculin concentration in the tomatoes. Two lines of transgenic tomatoes, 56B and 5B, were grown under salt-stress conditions. There was a higher miraculin content per gram of fresh weight in transgenic tomatoes cultivated under salt stress than in tomatoes grown under non-stressed conditions. The observed increase in miraculin concentration was due to an enrichment effect caused by the miniaturization of tomatoes cultivated under salt stress. When the miraculin-enriched tomatoes were used for miraculin purification, the recovery rate of miraculin was higher.
The effects of different light wavelengths on in vitro asymbiotic germination of mature seeds and seedling growth of Bletilla ochracea Schltr. were examined using five wavelengths from light emitting diodes with peak wavelengths of 470 nm (blue), 525 nm (green), 590 nm (orange), 625 nm (red), and from white light emitting diodes at 40 μmol m−2 s−1. Gellan gum solidified New Dogashima medium without plant growth regulators was used as germination medium. Cultures were maintained at 25°C under 24 h continuous lighting or darkness. The frequencies of seed germination three weeks after sowing reached more than 60% in all light conditions examined including continuous darkness. The highest frequencies of seed germination of 74% were achieved using lighting both with green and orange light emitting diodes, though strong inhibition of seed germination by the specific wavelength was not recognized. Seedling growth of this species was greatly inhibited by darkness. The most effective wavelength of light for rhizoid formation was revealed to be in the range of 590 nm (orange light) and 625 nm (red light), and almost no rhizoid was formed in the darkness. After 3 months of culture, leave width was expanded under white and blue light emitting diodes and was narrower under green, orange and red light. Seedlings grown under white and blue light emitting diodes resulted in thicker pseudobulbs.
Root development is highly affected by nutrient supply under various environmental conditions. A high concentration of nitrate is known to inhibit lateral root growth after emergence from the parent root, implying that cell proliferation ceases at a specific stage in lateral root development. However, the mechanism by which external nitrate availability modulates the cell cycle remains to be elucidated. In this study, we analyzed cell cycle regulation during high-nitrate-mediated inhibition of lateral root growth in Arabidopsis. The expression of mitotic reporter genes, such as those for CDKB2;1 and CYCB1;1, was suppressed in emerged lateral root primordium of seedlings grown under high nitrate conditions, which temporally arrested the outgrowth of the primordium for the first 3 days. In contrast, the expression of CDKA;1, which encodes an ortholog of yeast Cdc2/Cdc28p, was not affected by external nitrate availability. These results indicate that the cell cycle in emerged lateral root primordia is possibly arrested at G1/S under high nitrate conditions but that the primordia retain the competence for cell division. The expression of an auxin response marker was reduced in stunted lateral root primordia under high nitrate conditions, but exogenous auxin application could not suppress growth inhibition. This suggests that reduced auxin accumulation and/or signaling are not the primary cause of high-nitrate-mediated inhibition of lateral root growth.
Genetically transformed lombardy poplar (Populus nigra L. var. italica Koehne) were regenerated after the co-cultivation of stem segments with Agrobacterium tumefaciens strain LBA4404, which harbored a binary vector that included an antisense DNA for ozone-inducible 1-aminocyclopropane-1-carboxylate (ACC) synthase from poplar leaves. Lower rates of ozone-induced ethylene production were observed in transgenic plants than in wild-type plants. Ozone-induced visible damage was attenuated in these lines, and the extent of damage was positively related to the level of ozone-induced ethylene production. In one of these ozone-tolerant lines, the levels of transcripts for ozone-inducible endogenous ACC synthases were suppressed compared with those in wild-type plants, demonstrating that ozone-inducible ACC synthases have a key role in the expression of leaf damage by ozone exposure. Thus, transgenic trees with air pollution tolerance were developed for the first time among woody plants.
Transgenic chrysanthemum plants were constructed to simultaneously express three N-methyltransferases involved in caffeine biosynthetic pathways. Resulting plants produced caffeine at approximately 3 μg g−1 fresh tissue, and were tested for herbivore repellence. When starved second-instar caterpillars of beet armyworms (Spodoptera exigu) were allowed to feed, they ate up to 4.4 mm2 of leaf discs from the wild type plants, while less than 1.5 mm2 of those from the transgenic plants. When third-instars of cotton aphid (Aphis gossypii) were subjected to a choice-test, 27 gathered on wild type leaves, and 6 on transgenic leaves. These results indicate that caffeine-producing chrysanthemum is resistant against herbivores, lepidoptera caterpillars and aphids, both being one of the most serious pests in agriculture. We propose that the method can be practically applied to a variety of important plant species to confer resistance against biotic stresses.
Cryopreservation using an aluminum cryo-plate was successfully applied to in vitro-grown carnation (Dianthus caryophyllus L.) shoot tips. The shoot tips (1–1.5 mm×1 mm) were dissected from the shoot and precultured at 25°C for 2 days on MS medium containing 0.3 M sucrose. The precultured shoot tips were placed on the aluminum cryo-plate containing ten wells embedded with alginate gel. Osmoprotection was performed by immersing the cryo-plates in loading solution (2 M glycerol and 1.4 M sucrose) for 90 min at 25°C. Then, dehydration was performed by immersing the cryo-plates in PVS2 for 25 min at 25°C. After storage in liquid nitrogen, shoot tips attached to cryoplate, were directly immersed into 2 ml 1 M sucrose solution for regeneration. Using this procedure, the average regrowth level of vitrified shoot tips of 4 carnation cultivars reached 95%. This new method has many advantages and will facilitate the cryostorage of reference cultivars of carnation.
Reporter genes are often used to monitor gene expression in various organisms. To investigate the use of a dual-color luciferase reporter assay (DCRA) for monitoring expression of multiple genes in higher plants, we characterized the bioluminescence and the spectra of click beetle luciferases in planta. The DCRA consists of click beetle green luciferase (CBG68luc) and click beetle red luciferase (CBRluc). The green and the red bioluminescence from CBG68luc and CBRluc, respectively, are suitable for nondestructive in vivo assays because luminescence signals can be monitored selectively using optical filters. Using transgenic plants and a transient expression system, we introduced and expressed CBG68luc and CBRluc reporter genes in plant cells and obtained luminescence spectra by spectrometry. The spectral patterns from the transgenic plant cells were similar to those from E. coli transformed with the same reporter genes. Although plant tissues contain pigments and autofluorescent materials, our data suggest that the presence of these do not interfere with gene expression monitoring by DCRA, which is therefore applicable to monitoring the regulation of gene expression in plants.
Of the polyamines (PAs), plants contain at least two kinds of tetraamines, spermine (Spm) and thermospermine (T-Spm). They seem to have different functions in plants because the stem growth defect shown by the loss-of-function mutant plant of ACL5 (encoding T-Spm synthase) was partially complemented by T-Spm but not by Spm in Arabidopsis thaliana. Here we examined the localization of Spm synthase (SPMS) gene promoter activity using the SPMS promoter-β-glucronidase (GUS) gene expressed in transgenic Arabidopsis plants and compared it with that of ACL5 promoter-GUS expression. SPMS promoter activity was detected in almost all organs at all developmental growth stages, while ACL5 promoter activity was only detected in the vascular systems. Upon high salt stress, SPMS promoter activity was highly enhanced in all organs except cotyledons whereas the ACL5 promoter activity was reduced which is consistent with the reduced levels of ACL5 transcripts. The result indicates that SPMS expression is different from that of ACL5 in respect to tissue specificity and stress response, suggesting corresponding differences in functions of Spm and T-Spm.