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.     

Effect of salinity and abscisic acid on accumulation of glycinebetaine and betaine aldehyde dehydrogenase mRNA in Sorghum leaves (Sorghum bicolor)

The effects of salt stress and abscisic acid (ABA) on the expression of betaine aldehyde dehydrogenase (BADH) were determined in sorghum (Sorghum bicolor L.) plants. BADH mRNA expression was induced by salinity, and the timing coincided with the observed glycinebetaine (betaine) accumulation. The leaf water potential in the leaves of the sorghum plants was significantly affected by salinity. In response to salinity, betaine, ABA, Na and Cl accumulations increased 6-, 16-, 90-, and 3-fold, respectively. In the leaf disks from unsalinized plants incubated on NaCl, or ABA solution, the BADH mRNA level was lower than in the ABA-treated disks. Exogenous application of the ABA biosynthetic inhibitor fluridone to the NaCl-treated disks reduced the ABA accumulation and BADH mRNA levels compared with NaCl-treated leaves. The results indicate that the salt-induced accumulation of betaine and BADH mRNA coincides with the presence of ABA.     

Physiological and Growth Responses of Tomato Progenies Harboring the Betaine Alhyde Dehydrogenase Gene to Salt Stress

The responses of five transgenic tomato （Lycopersicon esculentum Mill） lines containing the betaine aldehyde dehydrogenase （BADH） gene to salt stress were evaluated. Proline, betaine （N, N, N-trimethylglycine, hereafter betalne）, chlorophyll and ion contents, BADH activity, electrolyte leakage （EL）, and some growth parameters of the plants under 1.0% and 1.5% NaCl treatments were examined. The transgenic tomatoes had enhanced BADH activity and betaine content, compared to the wild type under stress conditions. Salt stress reduced chlorophyll contents to s higher extent in the wild type than in the transgenic plants. The wild type exhibited significantly higher proline content than the transgenic plants at 0.9% and 1.3% NaCh Cell membrane of the wild type was severely damaged as determined by higher EL under salinity stress. K^＋ and Ca^2＋ contents of all tested lines decreased under salt stress, but the transgenic plants showed a significantly higher accumulation of K^＋ and Ca^2＋ than the wild type. In contrast, the wild type had significantly higher CI- and Na^2＋ contents than the transgenic plants under salt stress. Although yield reduction among various lines varied, the wild type had the highest yield reduction. Fruit quality of the transgenic plants was better in comparison with the wild type as shown by a low ratio of blossom end rot fruits. The results show that the transgenic plants have improved salt tolerance over the wild type.     

甜菜碱醛脱氢酶(BADH)基因转化小麦及其表达

Betaine aldehyde dehydrogenase (BADH) cDNA cloned from Atriplex hortensis L. in the plasmid pABH9 containing maize ubiquitin promoter and bar gene was transferred into wheat (Triticum aestivum L.) by microprojectile bombardment with 4.1% of average frequency of transformation. From 300 young embryo calli bombarded with the plasmid, 24 transgenic plants were obtained showing BADH gene integration by both PCR and Southern blotting analysis. Among the 24 transgenic plants, 13 exhibited higher BADH activity than the control. Some transgenic plants grew normally with healthy roots on the medium containing 0.7% NaCl while the control plants had very poor roots and finally died.     

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.     

Seed germination of the halophyteSuaeda japonica under salt stress

Suaeda japonica Makino belonging to the family Chenopodiaceae, is a halophyte and grows at the shore of Ariake sea in Japan. This plant presumably possesses high salt resistant nature, thus, we examined the mechanisms of seed germination under salt stress. The seeds maintained 80% germination rates on the medium containing 0.7 M NaCl. Germination rates varied depending on salt type; the germination rates under NaCl or KCI exhibited relatively lower values than ones under sodium gluconate or potassium gluconate. This different responses for salts seemed to be as a result of the presence of Cl⁻ ions. Although very high levels of betaine (compatible solute), were kept in the seedlings grown under no salt stress, the contents gradually increased as concentration of NaCl increased. Betaine is a factor present in plants that works to alleviate the effects of excessive soil salts. It is synthesized in leaves from betaine aldehyde, and this process is catabolized by betaine aldehyde dehydrogenase (BADH). When the seedlings were cultivated on the medium without NaCl, relatively high level of BADH activity was found. The activity increased 5-fold in the seedlings grown under 0.5 M NaCl stress. Increases in betaine content and BADH activity were found during seed germination. InS. japonica, the salt stress promoted BADH activity, subsequently endogenous betaine contents were increased, and increased betaine seemed to secure seed germination under salt stress.     

甜菜碱醛脱氢酶(BADH)基因转化小麦及其表达

采用基因枪法将山菠菜甜菜碱醛脱氢酶 (BADH)基因导入小麦 (TriticumaestivumL .)品种 ,并且得以表达。该基因由玉米Ubi1启动子控制。在盐胁迫条件下 ,多数转基因植株叶片的BADH活性比受体亲本提高 1～ 3倍 ,部分植株相对电导率比亲本明显低 ,表明转基因植株的细胞膜在胁迫时有受损较轻倾向。PCR和Southern杂交分析证实外源BADH基因已插入小麦基因组 ,平均转化频率为 4.1%。     

Physiological responses to salt stress of T2 alfalfa progenies carrying a transgene for betaine aldehyde dehydrogenase

Glycinebetaine is an important quaternary ammonium compound generated in response to salt and other osmotic stresses in many organisms. Its synthesis requires the catalysis of betaine aldehyde dehydrogenase encoded by a Betaine Aldehyde Dehydrogenase (BADH) gene that converts betaine aldehyde into glycinebetaine in some halotolerant plants. In this study, a BADH gene was over expressed in transgenic alfalfa (Medicago sativa L) plants using Agrobacterium-mediated transformation. Transgenic alfalfa plants grown under 9‰ NaCl grew well; while non-transgenic control plants turned yellowish in color, wilted, and eventually died. Polymerase chain reaction (PCR) and Northern blot hybridization analyses demonstrated that the BADH gene was transferred into the T2 generation and segregated in a Mendelian fashion. Transgenic alfalfa plants expressing BADH showed significantly higher BADH enzyme activity and betaine contents when grown under 6‰ NaCl. Moreover, proline content in T2 lines were higher while electrolyte leakage and malonaldehyde content were lower in T2 lines compared with non-transgenic plants. These findings indicated that transgenic plants expressing BADH transgene exhibited higher salt tolerance than non-transgenic plants.     

Expression of betaine aldehyde dehydrogenase gene and salinity tolerance in rice transgenic plants

Betaine as one of osmolytes plays an important role in osmoregulation of most high plants. Betaine aldehyde dehydrogenase C BADH) is the second enzyme involved in betaine biosynthesis. The BADH gene from a halophite, Atriplex hortensis, was transformed into rice cultivars by bombarment method. Totally 192 transgenic rice plants were obtained and most of them had higher salt tolerance than controls. Among transgenic plants transplanted in the saline pool containing 0.5% NaCl in a greenhouse, 22 survived, 13 of which set seeds, and the frequency of seed setting was very low, only 10% . But the controls could not grow under the same condition. The results of BADH ac-tivity assay and Northern blot showed that the BADH gene was integrated into chromosomes of transgenic plants and expressed.     

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.     

盐生植物盐爪爪甜菜碱醛脱氢酶基因的克隆及在盐胁迫下的BADH 基因的表达

根据已发表的几种藜科植物甜菜碱醛脱氢酶(BADH) 基因的同源保守区设计了一对引物, 采用RT-PCR 方法从盐生植物盐爪爪( Kalidium foliatum) 中扩增出BADH 基因的1 个开放阅读框架, 其核苷酸序列长1 503 bp , 推测的氨基酸序列全长为500 个氨基酸残基。核苷酸序列与藜科几种盐生植物如滨藜、碱蓬、菠菜、山菠菜和甜菜等的同源性为81% , 与甜土植物水稻的同源性为69%。氨基酸序列与以上两类植物(盐生植物和甜土植物) 的同源性比对为80% 和71% , 说明BADH 基因在藜科盐生植物中是一种较高保守的基因。BADH 基因编码的多肽在高等植物中行使重要的功能。用不同浓度的NaCl 胁迫处理盐爪爪植株, BADH mRNA 的表达水平比对照植株高, 说明盐爪爪BADH 基因的表达受盐诱导, 间接说明甜菜碱醛脱氢酶催化合成的甜菜碱作为渗透调节的小分子物质, 它的积累与盐胁迫存在紧密关联, 本研究为进一步从生理和分子水平阐明盐爪爪的耐盐机制提供一定的参考。     

Enhanced drought and salinity tolerance in transgenic potato plants with a BADH gene from spinach

Drought and salinity are the most important abiotic stresses that affect the normal growth and development of plants. Glycine betaine is one of the most important osmolytes present in higher plants that enable them to cope with environmental stresses through osmotic adjustment. In this study, a betaine aldehyde dehydrogenase (BADH) gene from spinach under the control of the stress-induced promoter rd29A from Arabidopsis thaliana was introduced into potato cultivar Gannongshu 2 by the Agrobacterium tumefaciens system. Putative transgenic plants were confirmed by Southern blot analysis. Northern hybridization analysis demonstrated that expression of BADH gene was induced by drought and NaCl stress in the transgenic potato plants. The BADH activity in the transgenic potato plants was between 10.8 and 11.7 U. There was a negative relationship (y = −2.2083x + 43.329, r = 0.9495) between BADH activity and the relative electrical conductivity of the transgenic potato plant leaves. Plant height increased by 0.4–0.9 cm and fresh weight per plant increased by 17–29% for the transgenic potato plants under NaCl and polyethylene glycol stresses compared with the control potato plants. These results indicated that the ability of transgenic plants to tolerate drought and salt was increased when their BADH activity was increased.     

盐生植物盐爪爪甜菜碱醛脱氢酶基因的克隆及在盐胁迫下的BADH基因的表达

根据已发表的几种藜科植物甜菜碱醛脱氢酶(BADH)基因的同源保守区设计了一对引物,采用RT-PCR方法从盐生植物盐爪爪(Kalidium foliatum)中扩增出BADH基因的1个开放阅读框架,其核苷酸序列长1503bp,推测的氨基酸序列全长为500个氨基酸残基。核苷酸序列与藜科几种盐生植物如滨藜、碱蓬、菠菜、山菠菜和甜菜等的同源性为81%,与甜土植物水稻的同源性为69%。氨基酸序列与以上两类植物(盐生植物和甜土植物)的同源性比对为80%和71%,说明BADH基因在藜科盐生植物中是一种较高保守的基因。BADH基因编码的多肽在高等植物中行使重要的功能。用不同浓度的NaCl胁迫处理盐爪爪植株,BADHmRNA的表达水平比对照植株高,说明盐爪爪BADH基因的表达受盐诱导,间接说明甜菜碱醛脱氢酶催化合成的甜菜碱作为渗透调节的小分子物质,它的积累与盐胁迫存在紧密关联,本研究为进一步从生理和分子水平阐明盐爪爪的耐盐机制提供一定的参考。     

Plastid-expressed choline monooxygenase gene improves salt and drought tolerance through accumulation of glycine betaine in tobacco

Glycine betaine (GlyBet), a quaternary ammonium compound, functions as an osmoprotectant in many organisms including plants. Previous research has shown that over-expression of enzymes for GlyBet biosynthesis in transgenic plants improved abiotic stress tolerance, but so far no study on the effects of plastid-expression of choline monooxygenase, the enzyme that catalyzes the conversion of choline into betaine aldehyde, has been reported. In the present study, tobacco (Nicotiana tabacum L. cv Wisconsin 38) plants were transformed with a gene for choline monooxygenase (BvCMO) from beet (Beta vulgaris) via plastid genetic engineering. Transplastomic plants constitutively expressing BvCMO under the control of the ribosomal RNA operon promoter and a synthetic T7 gene G10 leader were able to accumulate GlyBet in leaves, roots and seeds, and exhibited improved tolerance to toxic level of choline and to salt/drought stress when compared to wild type plants. Transplastomic plants also demonstrated higher net photosynthetic rate and apparent quantum yield of photosynthesis in the presence of 150 mM NaCl. Salt stress caused no significant change on the maximal efficiency of PSII photochemistry (Fv/Fm) in both wild type and transplastomic plants, but a decrease in the actual efficiency of PSII (PhiPSII) was observed, and such a decrease was much greater in wild type plants. Our results demonstrate the feasibility of improving salt and drought tolerance in plants through plastid transformation with BvCMO gene.     

转BADH基因紫花苜蓿山苜2号品种的抗盐性鉴定及系统选育

利用转基因技术创造苜蓿新种质已成为牧草新技术育种的重要组成部分。为了有效地从苜蓿转基因植株及其后代中选育出优良品种,深入研究转基因苜蓿的植物学性状及其产量十分重要。以通过农杆菌介导技术获得的T0代转BADH基因苜蓿为试材,利用分子生物学方法对其自交株系的世代群体连续进行抗盐性鉴定筛选和系统选育,首次获得了具有抗盐碱能力的转基因苜蓿稳定株系。同时,通过品种比较实验、区域实验和生产实验,表明在不同盐碱地条件下,转BADH基因的苜蓿植株产草量明显高于对照（未转基因的中苜1号）,生产实验的干草增产率介于13.11%–24.98%之间。上述结果表明,外源目的基因主要特性的遗传稳定,进而从实践上验证了转BADH基因工程操作的实用性。     

Abscisic acid is involved in the water stress-induced betaine accumulation in pear leaves

ABA exogenously applied to the leaves of the whole plants of pear (Pyrus bretschneideri Redh. cv. Suly grafted on Pyrus betulaefolia Rehd.) significantly increased the betaine concentrations in the leaves when the plants were well watered. The plants subjected to 'drought plus ABA' treatment had significantly higher betaine concentrations in their leaves than those given drought treatment alone. The 'drought plus ABA' treatment increased the amount of betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8) and its activity in the leaves more than did the drought treatment alone. The experiments with detached leaves showed that ABA treatment significantly increased the concentration of betaine, activity of BADH and apparent amount of BADH in non-dehydrated leaves, and enhanced the accumulation of betaine, activity of BADH and apparent amount of BADH in dehydrated leaves. These effects of ABA were both time- and dose-dependent. Two ABA isomers, (-)-cis, trans-ABA and 2-trans, 4-trans-ABA, had no effect on the betaine accumulation in the leaves, showing that the ABA-induced effects are specific. These data demonstrate that ABA is involved in the drought-induced betaine accumulation in the pear leaves.     

Accumulation of glycinebetaine in rice plants that overexpress choline monooxygenase from spinach and evaluation of their tolerance to abiotic stress

BACKGROUND AND AIMS: Glycinebetaine (GB), a quaternary ammonium compound, is a very effective compatible solute. In higher plants, GB is synthesized from choline (Cho) via betaine aldehyde (BA). The first and second steps in the biosynthesis of GB are catalysed by choline monooxygenase (CMO) and by betaine aldehyde dehydrogenase (BADH), respectively. Rice (Oryza sativa), which has two genes for BADH, does not accumulate GB because it lacks a functional gene for CMO. Rice plants accumulate GB in the presence of exogenously applied BA, which leads to the development of a significant tolerance to salt, cold and heat stress. The goal in this study was to evaluate and to discuss the effects of endogenously accumulated GB in rice. METHODS: Transgenic rice plants that overexpressed a gene for CMO from spinach (Spinacia oleracea) were produced by Agrobacterium-mediated transformation. After Southern and western blotting analysis, GB in rice leaves was quantified by (1)H-NMR spectroscopy and the tolerance of GB-accumulating plants to abiotic stress was investigated. KEY RESULTS: Transgenic plants that had a single copy of the transgene and expressed spinach CMO accumulated GB at the level of 0.29-0.43 micromol g(-1) d. wt and had enhanced tolerance to salt stress and temperature stress in the seedling stage. CONCLUSIONS: In the CMO-expressing rice plants, the localization of spinach CMO and of endogenous BADHs might be different and/or the catalytic activity of spinach CMO in rice plants might be lower than it is in spinach. These possibilities might explain the low levels of GB in the transgenic rice plants. It was concluded that CMO-expressing rice plants were not effective for accumulation of GB and improvement of productivity.