Takayoshi Shimazaki

The influence of genes regulating transmembrane transport of Na+ on the salt resistance of Aeluropus lagopoides

Plantlets of Aeluropus lagopoides (Linn.) Trin. Ex Thw. were grown at different NaCl concentrations (26, 167, 373 and 747mM) for 3, 7 and 15 days; their growth, osmotic adjustment, gas exchange, ion compartmentalisation and expression of various genes related to Na+ flux was studied. Plantlets showed optimal growth in non-saline (control; 26mM NaCl) solutions, whereas CO2/H2O gas exchange, leaf water concentration and water use efficiency decreased under all salinity treatments, accompanied by increased leaf senescence, root ash, sodium content and leaf osmolality. A decrease in malondialdehyde (MDA) content with time was correlated with Na+ accumulation in the leaf apoplast and a concomitant increase in Na+ secretion rate. A. lagopoides accumulated a higher concentration of Na+ in root than in leaf vacuoles, corresponding with higher expression of V-NHX and lower expression of PM-NHX in root than leaf tissue. It appears that V-ATPase plays a vital role during Na+ transport by producing an electromotive force, driving ion transport. Leaf calcium increased with increasing salinity, with more rapid accumulation at high salinity than at low salinity, indicating a possible involvement of Ca2+ in maintaining K+:Na+ ratio. Our results suggest that A. lagopoides successfully compartmentalised Na+ at salinities up to 373mM NaCl by upregulating the gene expression of membrane linked transport proteins (V-NHX and PM-NHX). At higher salinity (747mM NaCl), a reduction in the expression of V-NHX and PM-NHX in leaves without any change in the rate of salt secretion, is a possible cause of the toxicity of NaCl.

Metabolic Profiling of Transgenic Potato Tubers Expressing Arabidopsis Dehydration Response Element-Binding Protein 1A (DREB1A)

Untargeted metabolome analyses play a critical role in understanding possible metabolic fluctuations of crops under varying environmental conditions. This study reports metabolic profiles of transgenic potato tubers expressing the Arabidopsis DREB1A transcription factor gene, which induces expression of genes involved in environmental stress tolerance. A combination of targeted and untargeted metabolomics demonstrated considerable metabolome differences between the transgenic lines and nontransgenic parent cultivars. In the transgenic lines, stimulation of stress responses was suggested by elevated levels of the glutathione metabolite, γ-aminobutyric acid (GABA), and by the accumulation of β-cyanoalanine, a byproduct of ethylene biosynthesis. These results suggest that the Arabidopsis DREB1A expression might directly or indirectly enhance endogenous potato stress tolerance systems. The results indicate that transgenesis events could alter the metabolic compositions in food crops, and therefore metabolomics analysis could be a most valuable tool to monitor such changes.

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.

Salt and Drought Stress Tolerances in Transgenic Potatoes and Wild Species

Transgenic potatoes were employed to test whether a diverse range of gene actions can induce different stress tolerance(s). To select available transgenic lines, we measured growth profiles under non-stress conditions in our evaluation of transgenic lines and confirmed productivity under stress(es). We performed microarray analyses to clarify the abiotic stress tolerance mechanism controlled by AtDREB1A. Two transgenic lines out of over 200 independent genotypes displayed stable tuber production under high salinity stress conditions. We identified five significant genes that were induced by abiotic stress with AtDREB1A in potato; the reported homologs in Arabidopsis are downstream of the AtDREB1A gene. Our results suggest that the AtDREB1A gene acts as a transcriptional factor against abiotic stresses in potato, and that potato may have mechanisms in abiotic stress tolerance(s) controlled by a native transcriptional factor similar to AtDREB1A. Besides uses of the exotic genes which can enhance abiotic stress tolerance, exploitation of the native genes in cultivated potatoes is also valuable for elucidating the basic knowledge and for orienting towards uses in crop improvement. Further challenges would be comparing the stress responses between cultivated potatoes and wild species in more detail, in order to examine (1) whether alternative tolerance mechanisms are possible, and (2) if so, whether effective germplasm enhancement could be carried out based on wild species to improve drought and salt stress tolerance of cultivated potato.

Allelopathy assessments for the environmental biosafety of the salt-tolerant transgenic Eucalyptus camaldulensis, genotypes codA12-5B, codA 12-5C, and codA 20C.

Allelopathy tests were conducted on salt-tolerant transgenic eucalyptus trees conferring bacterial codA gene in the designated net-house conditions under Type II use (contained use) of the Japanese law on environmental biosafety aiming for Type I (field use) application. Three transgenic and corresponding nontransgenic genotypes were employed for four different tests: (1) sandwich bioassay; (2) soil germination method; (3) gas chromatography (GC) for volatile substances from the plants; and (4) high-performance liquid chromatography (HPLC) on phenolic compounds from fresh leaves, which are the primary allelopathic substances on the species. The simple approaches, the bioassays, indicated no significant difference between the transgenic and nongenetically modified genotypes. There was no qualitative difference between the transgenic and nontransgenic lines by GC or HPLC. Absence of any quantitative difference was suggested by repetitive examination and subsequent analysis of variance assessments with the chromatographic methods and bioassays. Moreover, it was also indicated that bioassays should be the primary assessment method for allelopathy in considering the simplicity, speed, low cost, and reproducibility of these methods. Overall, substantial equivalence was considered on the three transgenic genotypes with codA gene when compared with the nontransgenic Eucalyptus camaldulensis lines. The experiments supported the application to isolated field testing of the transgenic Eucalyptus camaldulensis genotypes as the first case and experience in Japanese regulatory approval processes Type I Use for the deliberate release to the environment.

Evaluation of the yield of abiotic-stress-tolerant AtDREB1A transgenic potato under saline conditions in advance of field trials

Cultivated potato is a drought-, salinity-, and frost-sensitive species. The transgenic approach is one of the methods used to mitigate abiotic stress. The utility of transgenic potatoes that have abiotic stress tolerance should be judged from their yield under stress conditions. In order to establish transgenic potato lines with the AtDREB1A gene that could be used in practical applications, we screened candidate lines in a growth room with growth profiles under non-stress conditions rather than the expression level of transgene. After identifying better transgenic lines (D163 and D164), yield of those lines under stress conditions was evaluated in the special netted-house. Although the yield was lower than the yield under non-stress conditions, two selected transgenic lines were able to maintain their yield under high saline conditions (EC > 10 mS/cm). In this study, fertilizer was not added beyond what was already contained in the soil mix in order to evaluate the yield of the transgenic lines under saline conditions in as simple a manner as possible. In future studies, it will be necessary to evaluate their yield in a farming context in an isolated field after assessing the environmental biosafety of these transgenic potato lines.