Enhanced germination under high-salt conditions of seeds of transgenicArabidopsis with a bacterial gene (codA) for choline oxidase

Arabidopsis thaliana was transformed previously with thecodA gene from the soil bacteriumArthrobacter globiformis. This gene encodes choline oxidase, the enzyme that converts choline to glycinebetaine. Transformation with thecodA gene significantly enhanced the tolerance of transgenic plants to low temperature and high-salt stress. We report here that seeds of transgenic plants that expressed thecodA gene were also more tolerant to salt stress during germination than seeds of non-transformed wild-type plants. Seedlings of transgenic plants grew more rapidly than those of wild-type plants under salt-stress conditions. Furthermore, exogenously applied glycinebetaine was effective in alleviating the harmful effects of salt stress during germination of seeds and growth of young seedlings, a result that suggests that it was glycinebetaine that had enhanced the tolerance of the transgenic plants. These observations indicate that synthesis of glycinebetaine in transgenic plantsin vivo, as a result of the expression of thecodA gene, might be veryuseful in improving the ability of crop plants to tolerate salt stress. The extended abstract of a paper presented at the 13th International Symposium in Conjugation with Award of the International Prize for Biology “Frontier of Plant Biology”     

Transformation of tomato with a bacterial codA gene enhances tolerance to salt and water stresses

Genetically engineered tomato (Lycopersicon esculentum) with the ability to synthesize glycinebetaine was generated by introducing the codA gene encoding choline oxidase from Arthrobacter globiformis. Integration of the codA gene in transgenic tomato plants was verified by PCR analysis and DNA blot hybridization. Transgenic expression of gene was verified by RT-PCR analysis and RNA blot hybridization. The codA-transgenic plants showed higher tolerance to salt stress during seed germination, and subsequent growth of young seedlings than wild-type plants. The codA transgene enhanced the salt tolerance of whole plants and leaves. Mature leaves of codA-transgenic plants revealed higher levels of relative water content, chlorophyll content, and proline content than those of wild-type plants under salt and water stresses. Results from the current study suggest that the expression of the codA gene in transgenic tomato plants induces the synthesis of glycinebetaine and improves the tolerance of plants to salt and water stresses.     

Transformation of Japanese persimmon (Diospyros kaki Thunb.) with a bacterial gene for choline oxidase

This report describes the first successful genetic engineering of tolerance to salt in an agriculturally important species of woody plants by Agrobacterium-mediated transformation with the codA gene of Arthrobacter globiformis. This gene encodes choline oxidase, which catalyzes the oxidation of choline to glycinebetaine. The binary plasmid vector pGC95.091, containing a kanamycin-resistance gene (nptII), a gene for -glucuronidase (gusA) and the codA gene in its T-DNA region, was used with a disarmed strain of Agrobacterium tumefaciens, EHA101, to transform Japanese persimmon (Diospyros kaki Thunb. `Jiro') by the leaf disk transformation method. The pRS95.101 plasmid that included only nptII and gusA in the T-DNA region was used as a control. We selected eight transgenic lines with one or two copies of the T-DNA after transformation with pGC95.091 (PC lines) and three lines after transformation with pRS95.101 (PR lines). The eight PC lines produced choline oxidase and glycinebetaine whereas neither was found in untransformed `Jiro' and in the control PR lines. Transgenic plants grew normally, resembling wild-type plants both in vitro and ex vitro. The activity of photosystem II in leaves of the transgenic Japanese persimmon plants under NaCl stress was determined in terms of the ratio of the variable (F _v) to the maximum (F _m) fluorescence of chlorophyll (F _v/F _m). The rate of decline in (F _v/F _m under NaCl stress was lower in the PC lines than in the control PR lines. These results demonstrated that genetic engineering of Japanese persimmon, which allowed it to accumulate glycinebetaine, enhanced the tolerance to salt stress of this plant.     

Compatibility of glycinebetaine in rice plants: evaluation using transgenic rice plants with a gene for peroxisomal betaine aldehyde dehydrogenase from barley

Glycinebetaine is synthesized in plants by the two‐step oxidation of choline, with betaine aldehyde as the intermediate. The reactions are catalyzed by choline mono‐oxygenase and betaine aldehyde dehydrogenase. Rice plants, which do not accumulate glycinebetaine, possess a gene encoding betaine aldehyde dehydrogenase, whose activity is detectable at low levels. To evaluate the compatibility in rice of glycinebetaine on growth and tolerance to salt, cold and heat, we produced transgenic rice plants by introduction of a cDNA for betaine aldehyde dehydrogenase of barley, which is localized in peroxisomes unlike the chloroplast‐specific localization of betaine aldehyde dehydrogenase in spinach and sugar beet. The transgenic rice plants converted high levels of exogenously applied betaine aldehyde (up to 10 mol m^–3) to glycinebetaine more efficiently than did wild‐type plants. The elevated level of glycinebetaine in transgenic plants conferred significant tolerance to salt, cold and heat stress. However, very high levels of glycinebetaine, resulting from conversion of applied betaine aldehyde to glycinebetaine or from exogenous application, inhibited increases in length of rice plants but not increases in dry weight. Our results suggested that the benefits of accumulation of glycinebetaine by rice plants might be considerable under high light conditions.     

Transformation of Arabidopsis thaliana with the codA gene for choline oxidase; accumulation of glycinebetaine and enhanced tolerance to salt and cold stress

Glycinebetaine is one of the compatible solutes that accumulate in the chloroplasts of certain halotolerant plants when these plants are exposed to salt or cold stress. The codA gene for choline oxidase, the enzyme that converts choline into glycinebetaine, has previously been cloned from a soil bacterium, Arthrobacter globiformis. Transformation of Arabidopsis thaliana with the cloned codA gene under the control of the 35S promoter of cauliflower mosaic virus enabled the plant to accumulate glycinebetaine and enhanced its tolerance to salt and cold stress. At 300 mM NaCl, considerable proportions of seeds of transformed plants germinated well, whereas seeds of wild-type plants failed to germinate. At 100 mM NaCl, transformed plants grew well whereas wild-type plants did not do so. The transformed plants tolerated 200 mM NaCl, which was lethal to wild-type plants. After plants had been incubated with 400 mM NaCl for two days, the photosystem II activity of wild-type plants had almost completely disappeared, whereas that of transformed plants remained at more than 50% of the original level. When exposed to a low temperature in the light, leaves of wild-type plants exhibited symptoms of chlorosis, whereas those of transformed plants did not. These observations demonstrate that the genetic modification of Arabidopsis thaliana that allowed it to accumulate glycinebetaine enhanced its ability to tolerate salt and cold stress.     

Transformation of Arabidopsis with the codA gene for choline oxidase enhances freezing tolerance of plants

Arabidopsis thaliana was transformed with the codA gene from Arthrobacter globiformis, which encodes choline oxidase, the enzyme that synthesizes glycinebetaine from choline. The transformation enabled the plants to accumulate glycinebetaine in chloroplasts, and significantly enhanced the freezing tolerance of plants. Furthermore, the photosynthetic machinery of transformed plants was more tolerant to freezing stress than that of wild-type plants. Exogenous application of glycinebetaine also increased the freezing tolerance of wild-type plants, suggesting that the presence of glycinebetaine in transformed plants had enhanced their ability to tolerate freezing stress. Northern blotting analysis revealed that the enhancement of freezing tolerance was not related to the expression of four cold-regulated genes. These results suggest that engineering of the biosynthesis of glycinebetaine by transformation with the codA gene might be an effective method for enhancing the freezing tolerance of plants.     

Glycinebetaine accumulation is more effective in chloroplasts than in the cytosol for protecting transgenic tomato plants against abiotic stress

Tomato (Lycopersicon esculentum Mill. cv. Moneymaker) plants were transformed with a gene for choline oxidase (codA) from Arthrobacter globiformis. The gene product (CODA) was targeted to the chloroplasts (Chl-codA), cytosol (Cyt-codA) or both compartments simultaneously (ChlCyt-codA). These three transgenic plant types accumulated different amounts and proportions of glycinebetaine (GB) in their chloroplasts and cytosol. Targeting CODA to either the cytosol or both compartments simultaneously increased total GB content by five- to sixfold over that measured from the chloroplast targeted lines. Accumulation of GB in codA transgenic plants was tissue dependent, with the highest levels being recorded in reproductive organs. Despite accumulating, the lowest amounts of GB, Chl-codA plants exhibited equal or higher degrees of enhanced tolerance to various abiotic stresses. This suggests that chloroplastic GB is more effective than cytosolic GB in protecting plant cells against chilling, high salt and oxidative stresses. Chloroplastic GB levels were positively correlated with the degree of oxidative stress tolerance conferred, whereas cytosolic GB showed no such a correlation. Thus, an increase in total GB content does not necessarily lead to enhanced stress tolerance, but additional accumulation of chloroplastic GB is likely to further raise the level of stress tolerance beyond what we have observed.     

Enhanced tolerance to light stress of transgenic Arabidopsis plants that express the codA gene for a bacterial choline oxidase

Arabidopsis thaliana was transformed with the codA gene from Arthrobacter globiformis. This gene encodes choline oxidase, an enzyme that converts choline to glycinebetaine. The photosynthetic activity, monitored in terms of chlorophyll fluorescence, of transformed plants was more tolerant to light stress than that of wild-type plants. This enhanced tolerance to light stress was caused by acceleration of the recovery of the photosystem II (PS II) complex from the photo-inactivated state. The transformed plants synthesized glycinebetaine, but no changes were detected in the relative levels of membrane lipids or in the relative levels of fatty acids in the various membrane lipids. Transformation with the codA gene increased levels of H₂O₂, a by-product of the reaction catalyzed by choline oxidase, by only 50% to 100% under stress or non-stress conditions. The activity of ascorbate peroxidase and, to a lesser extent, that of catalase in transformed plants were significantly higher than in the wild-type plants. These observations suggest that H₂O₂ produced by choline oxidase in the transformed plants might have stimulated the expression of H₂O₂ scavenging enzymes, with resultant maintenance of the level of H₂O₂ within a certain limited range. It appears that glycinebetaine produced in vivo, but not changes in membrane lipids or in the level of H₂O₂, protected the PS II complex in transformed plants from damage due to light stress.     

Genetic engineering of the biosynthesis of glycinebetaine enhances photosynthesis against high temperature stress in transgenic tobacco plants

Genetically engineered tobacco (Nicotiana tabacum) with the ability to synthesis glycinebetaine was established by introducing the BADH gene for betaine aldehyde dehydrogenase from spinach (Spinacia oleracea). The genetic engineering enabled the plants to accumulate glycinebetaine mainly in chloroplasts and resulted in enhanced tolerance to high temperature stress during growth of young seedlings. Moreover, CO2 assimilation of transgenic plants was significantly more tolerant to high temperatures than that of wild-type plants. The analyses of chlorophyll fluorescence and the activation of Rubisco indicated that the enhancement of photosynthesis to high temperatures was not related to the function of photosystem II but to the Rubisco activase-mediated activation of Rubisco. Western-blotting analyses showed that high temperature stress led to the association of Rubisco activase with the thylakoid membranes from the stroma fractions. However, such an association was much more pronounced in wild-type plants than in transgenic plants. The results in this study suggest that under high temperature stress, glycinebetaine maintains the activation of Rubisco by preventing the sequestration of Rubisco activase to the thylakoid membranes from the soluble stroma fractions and thus enhances the tolerance of CO2 assimilation to high temperature stress. The results seem to suggest that engineering of the biosynthesis of glycinebetaine by transformation with the BADH gene might be an effective method for enhancing high temperature tolerance of plants.