NaCl dependent production of coniferin in Alluaudiopsis marnieriana suspension cultured cells.

A stable salt-tolerant cell-suspension culture of Alluaudiopsis marnieriana was established, and intracellular compounds that accumulated under salt-stress conditions were investigated. HPLC/MS, and NMR analyses indicated that enhanced accumulation of coniferin was found during the growth phase in medium containing 150 mM NaCl. Coniferin or its derivatives may play an important role in salt-tolerance mechanisms in this plant.

Development and evaluation of novel salt-tolerant Eucalyptus trees by molecular breeding using an RNA-Binding-Protein gene derived from common ice plant (Mesembryanthemum crystallinum L.).

The breeding of plantation forestry trees for the possible afforestation of marginal land would be one approach to addressing global warming issues. Here, we developed novel transgenic Eucalyptus trees (Eucalyptus camaldulensis Dehnh.) harbouring an RNA-Binding-Protein (McRBP) gene derived from a halophyte plant, common ice plant (Mesembryanthemum crystallinum L.). We conducted screened-house trials of the transgenic Eucalyptus using two different stringency salinity stress conditions to evaluate the plants' acute and chronic salt stress tolerances. Treatment with 400 mM NaCl, as the high-stringency salinity stress, resulted in soil electrical conductivity (EC) levels >20 mS/cm within 4 weeks. With the 400 mM NaCl treatment, >70% of the transgenic plants were intact, whereas >40% of the non-transgenic plants were withered. Treatment with 70 mM NaCl, as the moderate-stringency salinity stress, resulted in soil EC levels of approx. 9 mS/cm after 2 months, and these salinity levels were maintained for the next 4 months. All plants regardless of transgenic or non-transgenic status survived the 70 mM NaCl treatment, but after 6-month treatment the transgenic plants showed significantly higher growth and quantum yield of photosynthesis levels compared to the non-transgenic plants. In addition, the salt accumulation in the leaves of the transgenic plants was 30% lower than that of non-transgenic plants after 15-week moderate salt stress treatment. There results suggest that McRBP expression in the transgenic Eucalyptus enhances their salt tolerance both acutely and chronically.

Total synthesis of (-)-thallusin: utilization of enzymatic hydrolysis resolution.

(-)-Thallusin, isolated from a marine bacterium, is the only known natural product to act as an algal morphogenesis inducer. Because (-)-thallusin can only be obtained in exceedingly limited amounts from microbial cultivation, a synthetic supply of this compound is highly desirable. Here, we describe a novel synthetic pathway to (±)-thallusin and the first asymmetric synthesis of (-)-thallusin utilizing the enzymatic hydrolysis resolution with the combination of lipase PS-30 and lipase M Amamo-10.

Differential analysis of protein expression in RNA-binding-protein transgenic and parental rice seeds cultivated under salt stress.

Transgenic plants tolerant to various environmental stresses are being developed to ensure a consistent food supply. We used a transgenic rice cultivar with high saline tolerance by introducing an RNA-binding protein (RBP) from the ice plant (Mesembryanthemum crystallinum); differences in salt-soluble protein expression between nontransgenic (NT) and RBP rice seeds were analyzed by 2D difference gel electrophoresis (2D-DIGE), a gel-based proteomic method. To identify RBP-related changes in protein expression under salt stress, NT and RBP rice were cultured with or without 200 mM sodium chloride. Only two protein spots differed between NT and RBP rice seeds cultured under normal conditions, one of which was identified as a putative abscisic acid-induced protein. In NT rice seeds, 91 spots significantly differed between normal and salt-stress conditions. Two allergenic proteins of NT rice seeds, RAG1 and RAG2, were induced by high salt. In contrast, RBP rice seeds yielded seven spots and no allergen spots with significant differences in protein expression between normal and salt-stress conditions. Therefore, expression of fewer proteins was altered in RBP rice seeds by high salt than those in NT rice seeds.

Assessment of the salt tolerance and environmental biosafety of Eucalyptus camaldulensis harboring a mangrin transgene.

Increasing soil salinization of arable land has a major impact on the global ecosystem. One approach to increase the usable global forest area is to develop transgenic trees with higher tolerance to conditions of salt stress. An allene oxide cyclase homolog, mangrin, contains a core protein domain that enhances the salt tolerance of its host. We utilized this feature to develop improved salt-tolerant eucalyptus trees, by using transgenic Eucalyptus camaldulensis carrying the mangrin gene as a model. Since the Japanese government requires an environmental biosafety assessment for the surrounding biosphere, we performed experiments on trees grown in a special netted-house. This study examined the transgenic E. camaldulensis carrying the mangrin gene to assess the feasibility of using these transformants, and assessed their salt tolerance and environmental biosafety. We found that seven of 36 transgenic genotypes had significantly higher salt tolerance than non-transformants, and more importantly, that these plants had no significant impact on environmental biosafety. These results suggest that introduction of the mangrin gene may be one approach to safely enhance salt tolerance in genetically modified Eucalyptus species, and that the transformants have no apparent risks in terms of environmental biosafety. Thus, this study provides valuable information regarding the use of transgenic trees in situ.

Heterologous expression of a plant RelA-SpoT homologue results in increased stress tolerance in Saccharomyces cerevisiae by accumulation of the bacterial alarmone ppGpp.

The bacterial alarmone ppGpp is present only in bacteria and the chloroplasts of plants, but not in mammalian cells or eukaryotic micro-organisms such as yeasts and fungi. The importance of the ppGpp signalling system in eukaryotes has therefore been largely overlooked. Here, we demonstrated that heterologous expression of a relA-spoT homologue (Sj-RSH) isolated from the halophilic plant Suaeda japonica in the yeast Saccharomyces cerevisiae results in accumulation of ppGpp, accompanied by enhancement of tolerance against various stress stimuli, such as osmotic stress, ethanol, hydrogen peroxide, high temperature and freezing. Unlike bacterial ppGpp accumulation, ppGpp was accumulated in the early growth phase but not in the late growth phase. Moreover, nutritional downshift resulted in a decrease in ppGpp level, suggesting that the observed Sj-RSH activity to synthesize ppGpp is not starvation-dependent, contrary to our expectations based on bacteria. Accumulated ppGpp was found to be present solely in the cytosolic fraction and not in the mitochondrial fraction, perhaps reflecting the ribosome-independent ppGpp synthesis in S. cerevisiae cells. Unlike bacterial inosine monophosphate (IMP) dehydrogenases, the IMP dehydrogenase of S. cerevisiae was insensitive to ppGpp. Microarray analysis showed that ppGpp accumulation gave rise to marked changes in gene expression, with both upregulation and downregulation, including changes in mitochondrial gene expression. The most prominent upregulation (38-fold) was detected in the hypothetical gene YBR072C-A of unknown function, followed by many other known stress-responsive genes. S. cerevisiae may provide new opportunities to uncover and analyse the ppGpp signalling system in eukaryotic cells.

Indoleamine 2,3-dioxygenase promotes peritoneal dissemination of ovarian cancer through inhibition of natural killercell function and angiogenesis promotion.

The purpose of this study was to clarify the relationship between ovarian cancer peritoneal dissemination and indoleamine 2,3-dioxygenase (IDO) expression, and to explore the possibility of IDO-targeting molecular therapy for ovarian cancer. We transfected an IDO expression vector into the IDO-non-expressing human ovarian cancer cell line OMC-1, and established an IDO-expressing cell line (OMC-1/IDO) to examine the relationship between IDO expression and cancer cell growth in vitro and in vivo. IDO expression did not influence cancer cell growth and invasion in vitro, but promoted tumor growth and peritoneal dissemination in vivo. Immunostaining showed that IDO expression inhibited natural killer (NK) cell accumulation in tumors and promoted tumor angiogenesis. In addition, the oral administration of the IDO inhibitor 1-methly-tryptophan inhibited the growth of OMC-1/IDO-derived subcutaneous tumors in mice. These findings indicate that IDO promotes the peritoneal dissemination of ovarian cancer by inhibiting NK cell accumulation in tumors and promoting angiogenesis, supporting the applicability of IDO-targeting molecular therapy in ovarian cancer.

Post-stress metabolism involves umbelliferone production in anthocyanin-producing and nonproducing cells of Glehnia littoralis suspension cultures.

The effects of yeast extract on the accumulation of transcripts of phenylalanine ammonia-lyase (PAL, EC 4.1.3.5) and chalcone synthase (CHS, EC 2.3.1.74), PAL and CHS enzyme activity and furanocoumarin and anthocyanin metabolites over a 48 h period were studied in anthocyanin-producing (Violet) and non-producing (White) cell suspension cultures of Glehnia littoralis. In the course of this period, umbelliferone, which had not been detected earlier, was detected in the culture medium of the Violet as well as White cells. In White cells, the PAL transcript accumulation and an increase in PAL activity were in good agreement with the level of umbelliferone, and was followed by the induction of bergapten. In the case of the Violet cells, the accumulation of PAL and CHS transcripts, and the increases in PAL and CHS enzyme activity as well as the anthocyanin level, all of which were highly expressed in nontreated cells, were temporarily suppressed. However, the suppression of the PAL transcript and PAL activity was not as great as that of the CHS transcript accumulation and CHS activity, in which a sharp transient increase of umbelliferone production soon after elicitation appears to be a factor.

Putative cis-elements in the promoter region of the carrot phenylalanine ammonia-lyase gene induced during anthocyanin synthesis.

Deletion mutants of the carrot phenylalanine ammonia-lyase gene promoter were used to survey cis-elements for their effect on expression of promoter activity by transient expression. Two putative cis-elements were required to give full activity, but a third might be the most important in regulation of the promoter by 2,4-dichlorophenoxyacetic acid.

Plant RelA/SpoT homolog confers salt tolerance in Escherichia coli and Saccharomyces cerevisiae.

To analyze the mechanisms of salt tolerance in the halophyte Suaeda japonica, Escherichia coli was used as a host organism to undertake functional screening of cDNAs encoding proteins that may play an important role for the salt-tolerance mechanisms. A transformant expressing RelA/SpoT homolog (Sj-RSH) was found to have enhanced salt tolerance. In E. coli, RelA/SpoT controlled the amount of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), which are the effectors of the bacterial stringent response. Complementation analysis using the relA mutant of E. coli showed that Sj-RSH conferred the phenotype associated with (p)ppGpp synthesis. Furthermore, expression of Sj-RSH driven by the GAL1 promoter also gave rise to enhanced salt tolerance in yeast. Northern blot analyses of the yeast transformant revealed that the transcriptional levels of stress responsive genes including GPD1, VMA6, BMH1, HYP1 and HOG1 were clearly enhanced in the Sj-RSH transformant when compared with an empty vector transformant under stress-free and 1.5 M NaCl stress conditions. These results suggest that (p)ppGpp synthesis mediated by plant RelA/SpoT homologs plays a critical role for the transcriptional induction of several stress responsive genes, directly or indirectly in yeast, and that the conserved stress-resistance system may exist in higher plants.

The role of plant CCTalpha in salt- and osmotic-stress tolerance.

To find key genes essential for salt tolerance in the mangrove plant, Bruguiera sexangula, functional screening was performed using Escherichia coli as the host organism. A transformant expressing a cytosolic chaperonin-containing TCP-1alpha (CCTalpha) homologue displayed enhanced salt tolerance. Analysis in E. coli of the functional region revealed that a sequence of only 218 amino acids, containing the apical domain, is necessary for osmotolerance. Furthermore, this domain shows chaperone activity in vitro. Therefore, CCTalpha facilitates the folding of proteins without ATP or the cage-like structure, and may play an important role in stress tolerance, at least in B. sexangula.

Expression of mangrove allene oxide cyclase enhances salt tolerance in Escherichia coli, yeast, and tobacco cells.

To analyze the mechanisms of salt tolerance in the mangrove plant, Bruguiera sexangula, functional screening for cDNAs encoding proteins essential for salt tolerance was performed using Escherichia coli as the host organism. A transformant expressing a protein homologous to Lycopersicon (tomato) allene oxide cyclase (AOC) displayed enhanced salt tolerance. However, this unusual trait is not conferred by Lycopersicon AOC or its Arabidopsis homolog. Analysis of the functional region revealed a sequence of only 70 amino acids, which contains an unusual sequence that is essential for the salt-tolerant phenotype. On the basis of its unusual function, the mangrove AOC homolog is designated "mangrin". Furthermore, expression of mangrin driven by the GAL1 promoter and the 35S cauliflower mosaic virus (CaMV) promoter in Saccharomyces cerevisiae and tobacco cell lines, respectively, also gave rise to enhanced salt tolerance. Mangrin transcripts increased in cultured B. sexangula cells in response to salt stress. We propose that mangrin plays an important role in the salt-tolerance mechanism of B. sexangula, and that the biosynthesis of mangrin might be an effective means of enhancing salt tolerance in higher plants.