We have previously described the lip19 subfamily of basic region-leucine zipper protein (bZIP)-encoding genes from plants. Phylogenetic analysis of bZIP-encoded genes of Arabidopsis thaliana revealed that AtbZIP53 belongs to the lip19 subfamily and that four other members, AtbZIP1, AtbZIP2, AtbZIP11 and AtbZIP44 have close relationships to this subfamily. Here, we further characterized AtbZIP53 and two additional bZIP genes, AtbZIP2 and AtbZIP11. All three gene products are localized to the nucleus and show strong DNA binding activity to hexamer and C/G box hybrid sequences. Transactivation activities of the proteins were examined in plant cells. The expression of the genes upon exposure to various abiotic stresses and to hormone treatments was examined in Arabidopsis. We found that AtbZIP11 and AtbZIP53 were responsive to cytokinin and high salt stress, respectively. Based on these data, the physiological roles are discussed.
We developed a novel RNA virus vector based on the Cucumber mosaic virus (CMV), which is able to efficiently induce gene silencing in plants. We manipulated the RNA 2 of the CMV Y strain, whose genome consists of tripartite components, and introduced restriction sites for cloning a foreign sequence into the vector. To evaluate the vector (designated CMV2-A1) in terms of the ability to induce gene silencing, we cloned portions of the green fluorescent protein (GFP) cDNA or Cauliflower mosaic virus (CaMV) 35S promoter sequences into the vector and inoculated the infectious transcripts into Nicotiana benthamiana plants that express the GFP gene under the control of the CaMV 35S promoter. In both cases, a loss of GFP fluorescence accompanying a reduction in the level of GFP mRNA was induced. The short interfering RNAs (siRNAs) harboring the sequences inserted in the CMV2-A1 vector were detected in the silenced plants. When plants were infected with the virus containing the CaMV 35S promoter sequence, the CaMV 35S promoter sequence in the genomic DNA was heavily methylated. A reduction in the mRNA level of the GFP gene and loss of GFP fluorescence were induced as early as 6 and 12 days post-inoculation, respectively, earlier than the 20 days previously achieved with a Potato virus X vector. These results suggest that the CMV2-A1 vector is suitable for the rapid induction of both transcriptional and post-transcriptional gene silencing.
Tomato plants were transformed with a plasmid that contained a the gene for glutamate decarboxylase (GAD) from Lycopersicon esculentum L. coupled, in the antisense orientation, with the constitutively active 35S promoter from cauliflower mosaic virus. Four independent transformants were obtained. In the fruits of these transgenic plants, the level of expression of GAD mRNA was lower than that in non-transgenic plants. When tomatoes were harvested six weeks after the first flowering, we found that the levels of total free amino acids in transgenic fruits were 1.2 to 3.2 times higher than those in non-transgenic plants. In particular, the level of glutamate in fruits of transgenic plants was about twice that in fruits of non-transgenic plants.
Tobacco plants were transformed with a Nhap type Na+/H+ antiporter gene, SynnhaP1 (slr1595), from a cyanobacterium Synechocystis sp. PCC 6803. Two kinds of promoters, Arabidopsis alcohol dehydrogenase gene promoter (Adh promoter) and CaMV 35S promoter (35S promoter), were used. The transgenic plants driven by Adh promoter accumulated SynNhaP1 proteins only in root whereas the transgenic plants driven by 35S promoter accumulated SynNhaP1 proteins in all tissues. Confocal imaging of SynNhaP1-GFP fusion protein suggests the intracellular localization of SynNhaP1 in plasma membrane. Transgenic plants exhibited higher germination yields, increased biomass during developmental stage, increased seed production, and decreased intracellular Na+ content under salt-stress conditions. The transgenic plants driven by Adh promoter exhibited similar or slightly higher salt tolerance than that by 35S promoter. These results indicate the importance of expression of Na+/H+ antiporter in root for salt tolerance in plant.
Interferon-α (IFN-α) is an important antiviral pharmaceutical. A binary vector containing the first intron of the rice cytosolic SOD gene, the signal sequence of the 10 kDa rice prolamin, the amino-terminal region of β-glucuronidase, a thrombin recognition site, and the mature polypeptide region of human IFN-α was constructed, under the regulation of the cauliflower mosaic virus 35S promoter. Here, we report that transgenic rice cells transformed with this fusion protein vector produced a biologically active IFN-α. The vector was introduced into rice calli by Agrobacterium-mediated methods. Five lines of transgenic calli were obtained. IFN assay demonstrated that these calli expressed fusion proteins bearing biologically active IFN-α. Liquid-cultured cells exhibited stable growth and the production of active IFN-α during 10 successive generations, i.e. in 10 weeks. The expressed proteins were purified by immuno affinity chromatography and reverse-phase HPLC. Repeated selections of cultured cells that had been obtained by dividing calli into small cell aggregates considerably increased the production of IFN-α. Thrombin protease treatment of the fusion protein yielded the intact IFN-α polypeptide. Thus, transgenic suspension rice cells are expected to be useful for the production of large amounts of biologically active proteins at a low cost; moreover, such a system would be easier to employ than animal cell culture systems.
Bisphenol A and benzophenone, diphenyl compounds, were regioselectively hydroxylated, reduced, and glycosylated by the cultured plant cells of Eucalyptus perriniana. Three known biotransformation products and two new products, 2-(3-β-D-glucopyranosyloxy-4-hydroxyphenyl)-2-(4-β-D-glucopyranosyloxyphenyl)propane and 2-(3-β-D-glucopyranosyloxy-4-β-D-glucopyranosyloxyphenyl)-2-(4-hydroxyphenyl)propane, were isolated after seven days' incubation of bisphenol A. This shows that the cultured cells of E. perriniana regioselectively hydroxylate at C-6 and C-12 of bisphenol A and that the glycosides can be formed at the hydroxyl group at C-6, 7, 12, and 13. On the other hand, two known products and two new products, 4-O-[6-O-(α-L-rhamnopyranosyl)-β-D-glucopyranosyl]benzophenone and diphenylmethyl 6-O-(β-D-glucopyranosyl)-β-D-glucopyranoside, were obtained from the biotransformation of benzophenone, showing that the cultured cells of E. perriniana regioselectively hydroxylate at C-4 and reduce the carbonyl group of benzophenone and that glycosylation can occur at the hydroxyl group at C-4 and C-7. Thus, both diphenyl compounds were removed from the culture medium and accumulated in cells as glycosides. The biotransformation with E. perriniana would provide a very useful process for the phytoremediation of endocrine disrupting chemicals such as diphenyl compounds. This procedure using enzymatic reactions is simple and environmentally friendly.
Isorinic acid 3′-hydroxylase (IA3′H) catalyzes a final step in the biosynthetic pathway leading to rosmarinic acid (RA). IA3′H activity was detected in a microsomal fraction prepared from cultured cells of Lithospermum erythrorhizon. Addition of yeast extract or methyl jasmonate to the cell suspension cultures drastically increased IA3′H activity in parallel with increased RA production, whereas addition of salicylic acid enhanced neither IA3′H activity nor RA production. These results are consistent with an integral role for IA3′H in biosynthesis of RA in response to elicitation with yeast extract or methyl jasmonate.
The Arabidopsis ESR1 is thought to be a key gene for commitment to in vitro shoot regeneration in tissue culture. The ESR1 gene encodes a putative transcription factor. We examined the DNA-binding potential for specific sequences. Recombinant ESR1 protein specifically bound to the GCC box, known as the ethylene-responsive element. In addition, ESR1 was localized to the nucleus when expressed in onion epidermal cells. Deletion experiments revealed that the DNA-binding domain or the C-terminal region of ESR1 is essential for ESR1 overexpression to enhance shoot regeneration. The C-terminal region may be involved in transcriptional regulation by ESR1. These results suggest that ESR1 functions as a transcription factor and that its transcriptional regulation is important for the initiation of shoot regeneration.
Risk assessment and management of gene flow via pollen dispersal from genetically modified (GM) plants is critical. To minimize the likelihood of undesirable outcrossing, it is important to better understand the relationship between pollen dispersal distance and outcrossing rate in objective materials under physical containment. This study examined how a special screened greenhouse for Type-2 use, covered by a fine, 1-mm mesh reduced pollen dispersal and affected the outcrossing rate of non-GM yellow maize in the greenhouse and silver maize outside the greenhouse. Results suggest that the mesh-covered greenhouse was effective in reducing the outcrossing rate as a whole, although it may be less effective in reducing the longest distance of pollen dispersal. Further, the isolation distance threshold for the screened greenhouse is discussed in relation to risk assessment.
Beta vulgaris L. accumulates a large amount of glycinebetaine (betaine), a metabolite that is related to salt-tolerance. Production of betaine involves expression of many genes that encode enzymes such as betaine aldehyde dehydrogenase, choline monooxygenase, phosphoethanolamine N-methyltransferase, S-adenosyl-L-methionine synthetase, S-adenosyl-L-homocysteine hydrolase, and methionine synthase. We examined transcriptional regulation of the betaine-related genes in leaf beet (Beta vulgaris L. var cicla, cv. shirogukifudanna). Transcript expression of these betaine-related genes in leaf tissue had the following common features: (1) a similar pattern of transcript induction under salt stress, (2) reduced induction under salt stress in a dark condition, and (3) diurnal rhythms of transcript levels under a photoperiod of 16 h light/8 h darkness. The co-regulation of transcripts may contribute to the effective betaine production without disturbing the biosynthesis of other products in leaf beet.
To develop a screening system for plant activators, which are novel substances that protect plants by enhancing their inherent disease-resistance mechanisms, we utilized a GUS reporter gene system using promoters of the defense-related genes, PR-1 and PR-4. To validate the strategy, we performed subsequent analysis using an Arabidopsis microarray consisting of 1200 full-length cDNA clones representing putative defense-related and regulatory genes. Benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester and 2,6-dichloroisonicotinic acid activated plant defense responses via the salicylic acid (SA)-dependent signaling pathway, and β-aminobutyric acid triggered a primed state in the plant that enables more efficient activation of the SA-, jasmonic acid- and ethylene-signaling pathway. These results suggest that this novel system can be used to screen for candidate plant activators.
On the biotransformation of phenol and monofluorophenols by the cultured cells of Eucalyptus perriniana, phenyl and fluorophenyl β-D-glucosides were isolated after a 1-h incubation. Especially, the yield of 2-fluorophenol is higher than those of the other substrates, i.e., phenol, 3- and 4-fluorophenols. These results indicated that E. perriniana cells selectively glycosylate the hydroxyl group of 2-fluorophenol discriminating phenol, 3-and 4-fluorophenols.
Tobacco BY-2 suspension cells were successfully cryopreserved by a vitrification method combined with an encapsulation technique. Cell cultures cryopreserved using the optimal conditions established in this study could be thawed and grown enough to subculture in fresh medium within 14 days. However, the vitrification method was less effective for cryopreservation of BY-2 than a simplified slow prefreezing method.
Public understanding (PU) of biotechnology and life science is indispensable for developing bio-industry and life science itself. To promote PU, scientific literacy education, especially gene literacy education, should be introduced to the general public. An effective conduit for reaching a broad sector of society is high schools into which more than 95% of young people in Japan enter currently. Gene education involving laboratory activity is effective for obtaining literate students, because practical hands-on activities engage student's attention and deepen their understanding of biotechnology. The text “Recombinant DNA laboratory practice for secondary education” has enabled students to perform transformation experiments with safe host-vector systems in their classroom. Workshops for secondary school teachers have been held in a lot of universities since 2001, and currently gene literacy education involving laboratory activity is available in many high schools and public facilities including museums. To be most successful, we found that gene literacy education requires the collaborative efforts of teachers and scientists, and inter-school or intra-school cooperation status. In the present review, we summarize the current of gene literacy education in Japan.