The Accumulation of Glycine Betaine Is Dependent on Choline Monooxygenase (OsCMO), Not on Phosphoethanolamine N-Methyltransferase (OsPEAMT1), in Rice (Oryza sativa L. ssp. japonica)

Glycine betaine (GB) is an important osmoprotectant, which improves plant tolerance to various abiotic stresses. In higher plants, GB is synthesized through two-step oxidations of choline, catalyzed by choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH), respectively. Choline, the precursor of GB, is synthesized by phosphoethanolamine N-methyltransferase (PEAMT). Rice is known as a typical non-GB-accumulated species. However, the underlying mechanism related to GB accumulation remains elusive. Here, we determined whether the endogenous accumulation of choline is sufficient to GB biosynthesis in rice and whether the rice CMO protein has the function of oxidizing choline to generate betaine aldehyde. The results showed that overexpression of the rice PEAMT1 gene (OsPEAMT1) resulted in increased levels of choline, while GB content remained unchanged in the transgenic rice plants overexpressing OsPEAMT1. However, the intracellular GB level and the tolerance to salt stress of the transgenic lines overexpressing OsCMO were significantly enhanced. Immunoblotting analysis demonstrated that abundant functional OsCMO proteins with correct size were detected in OsCMO-overexpressing transgenic rice plants, but rarely accumulated in the wild type. Collectively, these results implicated that the endogenous accumulation level of choline is not the major factor leading to non-GB accumulation in rice. Instead, the defective expression of OsCMO resulted in non-GB accumulation.     

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.     

Isolation of a choline monooxygenase cDNA clone from Amaranthus tricolor and its expressions under stress conditions

INTRODUCTIONAmaranth is a C4 dicotyledonous mesophytecrop plant. A. tricofor is a major variety for veg-etable and ornamental crops, and is widely culti-vated in the wor1d. Osmoprotectant glycine betaine(GB) was detected in Amaranthaceae, A. HyPochon-driacus L[2] and A. Caudatus L[3, 4]. GB iswidespread and an effective osmoprotectant in manyplants[3]. We studied the photosynthetic adaptationmechanism of A. trico1or under salt stress due to ac-cumulation of GB[5].GB is synthesized …     

Improved tolerance to salinity and low temperature in transgenic tobacco producing glycine betaine

Glycine betaine is an osmoprotectant found in many organisms, including bacteria and higher plants. The bacterium Escherichia coli produces glycine betaine by a two-step pathway where choline dehydrogenase (CDH), encoded by betA, oxidizes choline to betaine aldehyde which is further oxidized to glycine betaine by the same enzyme. The second step, conversion of betaine aldehyde into glycine betaine, can also be performed by the second enzyme in the pathway, betaine aldehyde dehydrogenase (BADH), encoded by betB. Transformation of tobacco (Nicotiana tabacum), a species not accumulating glycine betaine, with the E. coli genes for glycine betaine biosynthesis, resulted in transgenic plants accumulating glycine betaine. Plants producing CDH were found to accumulate glycine betaine as did F1 progeny from crosses between CDH- and BADH-producing lines. Plants producing both CDH and BADH generally accumulated higher amounts of glycine betaine than plants producing CDH alone, as determined by 1H NMR analysis. Transgenic tobacco lines accumulating glycine betaine exhibited increased tolerance to salt stress as measured by biomass production of greenhouse-grown intact plants. Furthermore, experiments conducted with leaf discs from glycine betaine-accumulating plants indicated enhanced recovery from photoinhibition caused by high light and salt stress as well as improved tolerance to photoinhibition under low temperature conditions. In conclusion, introduction of glycine betaine production into tobacco is associated with increased stress tolerance probably partly due to improved protection of the photosynthetic apparatus.     

植物甜菜碱合成酶的分子生物学和基因工程

甜菜碱是一种非毒性的渗透调节剂。多种高等植物在盐碱或缺水的环境下在细胞中积累甜菜碱 ,以维持细胞的正常膨压。甜菜碱的积累使得许多代谢中的重要酶类在渗透胁迫下能保持活性。在植物中甜菜碱由胆碱经两步氧化得到 ,催化第一步反应的酶是胆碱单加氧酶 (CMO) ,催化第二步反应的酶是甜菜碱醛脱氢酶 (BADH)。本文综述了这两种酶的分子生物学及基因工程研究的最新进展 ,讨论了其基因工程研究的意义。     

Isolation and characterization of a novel peroxisomal choline monooxygenase in barley

Glycine betaine (GB) is a compatible solute accumulated by many plants under various abiotic stresses. GB is synthesized in two steps, choline → betaine aldehyde → GB, where a functional choline-oxidizing enzyme has only been reported in Amaranthaceae (a chloroplastic ferredoxin-dependent choline monooxygenase) thus far. Here, we have cloned a cDNA encoding a choline monooxygenase (CMO) from barley (Hordeum vulgare) plants, HvCMO. In barley plants under non-stress condition, GB had accumulated in all the determined organs (leaves, internodes, awn and floret proper), mostly in the leaves. The expression of HvCMO protein was abundant in the leaves, whereas the expression of betaine aldehyde dehydrogenase (BADH) protein was abundant in the awn, floret proper and the youngest internode than in the leaves. The accumulation of HvCMO mRNA was increased by high osmotic and low-temperature environments. Also, the expression of HvCMO protein was increased by the presence of high NaCl. Immunofluorescent labeling of HvCMO protein and subcellular fractionation analysis showed that HvCMO protein was localized to peroxisomes. [¹⁴C]choline was oxidized to betaine aldehyde and GB in spinach (Spinacia oleracea) chloroplasts but not in barley, which indicates that the subcellular localization of choline-oxidizing enzyme is different between two plant species. We investigated the choline-oxidizing reaction using recombinant HvCMO protein expressed in yeast (Saccharomyces cerevisiae). The crude extract of HvCMO-expressing yeast coupled with recombinant BBD2 protein converted [¹⁴C]choline to GB when NADPH was added as a cofactor. These results suggest that choline oxidation in GB synthesis is mediated by a peroxisomal NADPH-dependent choline monooxygenase in barley plants.     

Metabolic engineering of glycine betaine synthesis: plant betaine aldehyde dehydrogenases lacking typical transit peptides are targeted to tobacco chloroplasts where they confer betaine aldehyde resistance

Certain higher plants synthesize and accumulate glycine betaine, a compound with osmoprotectant properties. Biosynthesis of glycine betaine proceeds via the pathway choline betaine aldehyde glycine betaine. Plants such as tobacco (Nicotiana tabacum L.) which do not accumulate glycine betaine lack the enzymes catalyzing both reactions. As a step towards engineering glycine betaine accumulation into a non-accumulator, spinach and sugar beet complementary-DNA sequences encoding the second enzyme of glycine-betaine synthesis (betaine aldehyde dehydrogenase, BADH, EC 1.2.1.8) were expressed in tobacco. Despite the absence of a typical transit peptide, BADH was targeted to the chloroplast in leaves of transgenic plants. Levels of extractable BADH were comparable to those in spinach and sugar beet, and the molecular weight, isoenzyme profile and K _m for betaine aldehyde of the BADH enzymes from transgenic plants were the same as for native spinach or sugar beet BADH. Transgenic plants converted supplied betaine aldehyde to glycine betaine at high rates, demonstrating that they were able to transport betaine aldehyde across both the plasma membrane and the chloroplast envelope. The glycine betaine produced in this way was not further metabolized and reached concentrations similar to those in plants which accumulate glycine betaine naturally. Betaine aldehyde was toxic to non-transformed tobacco tissues whereas transgenic tissues were resistant due to detoxification of betaine aldehyde to glycine betaine. Betaine aldehyded ehydrogenase is therefore of interest as a potential selectable marker, as well as in the metabolic engineering of osmoprotectant biosynthesis.Abbreviations BADH betaine aldehyde dehydrogenase - bp base pairs - FAB-MS fast atom bombardment-mass spectrometry - GAPDH NADP-linked glyceraldehyde-3-phosphate dehydrogenase We thank Dr. G. An for the gift of the vector pGA643 and Mr. Sylvain Lebeurier for help in maintaining plants. This work was supported, in part, by grants from the Natural Sciences and Engineering Research Council of Canada, the Rockefeller Foundation, and the U.S. Department of Agriculture, and by gifts from CIBAGEIGY Biotechnology.     

Marginal impact of cropping BADH transgenic maize BZ-136 on chemical property,enzyme activity,and bacterial community diversity of rhizosphere soil

Plant and Soil - Transgenic betaine aldehyde dehydrogenase (BADH) maize that overaccumulates glycine betaine (GB) is developed to enhance tolerance to salt stress, while the ecological risk of...     

Betaine aldehyde dehydrogenase in plants

Plant betaine aldehyde dehydrogenases (BADHs) have been the target of substantial research, especially during the last 20 years. Initial characterisation of BADH as an enzyme involved in the production of glycine betaine (GB) has led to detailed studies of the role of BADH in the response of plants to abiotic stress in vivo , and the potential for transgenic expression of BADH to improve abiotic stress tolerance. These studies have, in turn, yielded significant information regarding BADH and GB function. Recent research has identified the potential for BADH as an antibiotic-free marker for selection of transgenic plants, and a major role for BADH in 2-acetyl-1-pyrroline-based fragrance associated with jasmine and basmati style aromatic rice varieties.     

Enhanced stress tolerance in Escherichia coli and Nicotiana tabacum expressing a betaine aldehyde dehydrogenase/choline dehydrogenase fusion protein

In Escherichia coli the osmoprotective compound glycine betaine is produced from choline by two enzymes; choline dehydrogenase (CDH) oxidizes choline to betaine aldehyde and then further on to glycine betaine, while betaine aldehyde dehydrogenase (BADH) facilitates the conversion of betaine aldehyde to glycine betaine. To evaluate the importance of BADH, a BADH/CDH fusion enzyme was constructed and expressed in E. coli and in Nicotiana tabacum. The fusion enzyme displayed both enzyme activities, and a coupled reaction could be measured. The enzyme was characterized regarding molecular weight and the dependence of the enzyme activities on environmental factors (salt, pH, and poly(ethylene glycol) addition). At high choline concentrations, E. coli cells expressing BADH/CDH were able to grow to higher final densities and to accumulate more glycine betaine than cells expressing CDH only. The intracellular glycine betaine levels were almost 5-fold higher for BADH/CDH when product concentration was related to CDH activity. Also, after culturing the cells at high NaCl concentrations, more glycine betaine was accumulated. On medium containing 20 mM choline, transgenic tobacco plants expressing BADH/CDH grew considerably faster than vector-transformed control plants.     

盐胁迫下三色苋甜菜碱及有关酶含量的变化

三色苋(Amaranthus tricolor)不同器官中的甜菜碱(GB)含量显著不同.除子叶外,根、茎和叶的GB含量和茎、叶中的胆碱单加氧酶(CMO)含量都因300 mmol/L的NaCl处理而增加.甜菜碱醛脱氢酶(BADH)的表达无论盐处理与否在所有器官中都能检测到,其含量变化不大.当种子发芽时,具备合成GB的能力,CMO含量增加;在此之前未能检测到CMO,也不能合成GB.研究结果表明三色苋响应盐胁迫而合成GB的关键酶是CMO.     

Genetic manipulation of Japonica rice using the <Emphasis Type="Italic">OsBADH1</Emphasis> gene from Indica rice to improve salinity tolerance

Betaine aldehyde dehydrogenase (BADH) is a major oxidative enzyme that converts betaine aldehyde to glycine betaine (GB), an osmoprotectant compound in plants. Japonica rice (salt-sensitive) was genetically engineered to enhance salt tolerance by introducing the OsBADH1 gene from Indica rice (salt-tolerant), which is a GB accumulator. We produced transgenic rice plants overexpressing the modified OsBADH1 gene under the control of the maize ubiquitin promoter. The transgenic rice showed increased OsBADH1 gene expression and OsBADH1 enzyme production, resulting in the accumulation of GB. It also exhibited enhanced salt tolerance in immature and mature transgenic rice seedlings. The adverse effect of salt stress on seed germination, the growth of immature and mature seedlings, water status, and photosynthetic pigments was alleviated in transgenic seedlings.     

Betaine aldehyde dehydrogenase in sorghum.

The ability to synthesize and accumulate glycine betaine is wide-spread among angiosperms and is thought to contribute to salt and drought tolerance. In plants glycine betaine is synthesized by the two-step oxidation of choline via the intermediate betaine aldehyde, catalyzed by choline monooxygenase and betaine aldehyde dehydrogenase (BADH). Two sorghum (Sorghum bicolor) cDNA clones, BADH1 and BADH15, putatively encoding betaine aldehyde dehydrogenase were isolated and characterized. BADH1 is a truncated cDNA of 1391 bp. BADH15 is a full-length cDNA clone, 1812 bp in length, predicted to encode a protein of 53.6 kD. The predicted amino acid sequences of BADH1 and BADH15 share significant homology with other plant BADHs. The effects of water deficit on BADH mRNA expression, leaf water relations, and glycine betaine accumulation were investigated in leaves of preflowering sorghum plants. BADH1 and BADH15 mRNA were both induced by water deficit and their expression coincided with the observed glycine betaine accumulation. During the course of 17 d, the leaf water potential in stressed sorghum plants reached -2.3 MPa. In response to water deficit, glycine betaine levels increased 26-fold and proline levels increased 108-fold. In severely stressed plants, proline accounted for > 60% of the total free amino acid pool. Accumulation of these compatible solutes significantly contributed to osmotic potential and allowed a maximal osmotic adjustment of 0.405 MPa.     

Genetic Engineering of Glycine Betaine Biosynthesis Reduces Heat-Enhanced Photoinhibition by Enhancing Antioxidative Defense and Alleviating Lipid Peroxidation in Tomato

Glycine betaine (GB) is a compatible solute that accumulates rapidly to enhance heat tolerance in many plants grown under heat stress. In this study, a BADH gene (betaine aldehyde dehydrogenase) from spinach was introduced into tomato (Lycopersicon esculentum cv. ‘Moneymaker’) via Agrobacterium-mediated transformation. Transgenic tomato lines expressing BADH exhibited higher capabilities for GB accumulation. Chlorophyll fluorescence analysis of wild type (WT) and transgenic plants exposed to heat treatment (42 °C) showed that transgenic plants exhibited higher photosynthetic capacities than WT plants. This finding suggests that GB accumulation increases tolerance to heat-enhanced photoinhibition. This increased tolerance was associated with an improvement in D1 protein content, which accelerated the repair of photosystem II (PSII) following heat-enhanced photoinhibition. Significant accumulations of hydrogen peroxide (H₂O₂) and superoxide radical (O₂ ^?) were observed in WT plants under heat stress. However, these accumulations were much less for the transgenic plants. An important finding reported herein is that exogenous GB cannot directly reduce the content of reactive oxygen species (ROS). In accordance with a lower relative electrolyte conductivity and malondialdehyde content, the activities of antioxidant enzymes were higher in transgenic lines than in WT plants, indicating that the degree of membrane injury in the transgenic plants was lower compared to the WT plants. These results suggest that GB accumulation in vivo cannot directly eliminate ROS. Rather, higher antioxidant enzyme activities must be maintained to lessen the accumulation of ROS in transgenic plants and to decrease the degree of membrane injury.     

Functional characterization of choline monooxygenase,an enzyme for betaine synthesis in plants

In plants, the first step in betaine synthesis was shown to be catalyzed by a novel Rieske-type iron-sulfur enzyme, choline monooxygenase (CMO). Although CMO so far has been found only in Chenopodiaceae and Amaranthaceae, the recent genome sequence suggests the presence of a CMO-like gene in Arabidopsis, a betaine non-accumulating plant. Here, we examined the functional properties of CMO expressed in Escherichia coli, cyanobacterium, and Arabidopsis thaliana. We found that E. coli cells in which choline dehydrogenase (CDH) was replaced with spinach CMO accumulate betaine and complement the salt-sensitive phenotype of the CDH-deleted E. coli mutant. Changes of Cys-181 in spinach CMO to Ser, Thr, and Ala and His-287 to Gly, Val, and Ala abolished the accumulation of betaine. The Arabidopsis CMO-like gene was transcribed in Arabidopsis, but its protein was not detected. When the Arabidopsis CMO-like gene was expressed in E. coli, the protein was detected but was found not to promote betaine sysnthesis. Overexpression of spinach CMO in E. coli, Synechococcus sp. PCC7942, and Arabidopsis conferred resistance to abiotic stress. These facts clearly indicate that CMO, but not the CMO-like protein, could oxidize choline and that Cys-181 and His-287 are involved in the binding of Fe-S cluster and Fe, respectively.