Salt-inducible betaine aldehyde dehydrogenase from sugar beet: cDNA cloning and expression

Members of the Chenopodiaceae, such as sugar beet and spinach, accumulate glycine betaine in response to salinity or drought stress. The last enzyme in the glycine betaine biosynthetic pathway is betaine aldehyde dehydrogenase (BADH). In sugar beet the activity of BADH was found to increase two- to four-fold in both leaves and roots as the NaCl level in the irrigation solution was raised from 0 to 500 mM. This increase in BADH activity was paralleled by an increase in level of translatable BADH mRNA. Several cDNAs encoding BADH were cloned from a gt10 libary representing poly(A)⁺ RNA from salinized leaves of sugar beet plants, by hybridization with a spinach BADH cDNA. Three nearly full-length cDNA clones were confirmed to encode BADH by their nucleotide and deduced amino acid sequence identity to spinach BADH; these clones showed minor nucleotide sequence differences consistent with their being of two different BADH alleles. The clones averaged 1.7 kb and contained an open reading frame predicting a polypeptide of 500 amino acids with 83% identity to spinach BADH. RNA gel blot analysis of total RNA showed that salinization to 500 mM NaCl increased BADH mRNA levels four-fold in leaves and three-fold in the taproot. DNA gel blot analyses indicated the presence of at least two copies of BADH in the haploid sugar beet genome.     

菠菜甜菜碱醛脱氢酶基因的克隆和序列分析

以耐盐的菠菜mRNA为模板,经反转录合成甜菜碱醛脱氢酶(BADH)基因第一链cDNA。在人工合成的两端引物引导下,通过多聚酶链式反应(PCR),扩增获得双链cDNA。把重组有BADH基因的pUC19转化至E.coli DH5α菌株,亚克隆后测定了基因的全序列。所得到的BADH基因全长序列为1491bp,编码497个氨基酸。与文献报道的相比较,核苷酸序列同源性99.8％,氨基酸序列同源性达99.6％。在此基础上,构建了BADH基因的高等植物表达载体。     

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.     

Overproduction of spinach betaine aldehyde dehydrogenase in Escherichia coli. Structural and functional properties of wild-type, mutants and E. coli enzymes.

Betaine aldehyde dehydrogenase (BADH) catalyzes the last step in the synthesis of the osmoprotectant glycine betaine from choline. Although betaine aldehyde has been thought to be a specific substrate for BADH, recent studies have shown that human and sugar beet BADHs also catalyze the oxidation of omega-aminoaldehydes. To characterize the kinetic and stability properties of spinach BADH, five kinds of expression vectors encoding full length, mature, E103Q, E103K, and chimera BADHs were constructed. These enzymes together with Escherichia coli BADH were expressed in E. coli and purified. The affinities for betaine aldehyde were similar in the spinach and E. coli BADHs, whereas those for omega-aminoaldehydes were higher in spinach BADH than in E. coli BADH. A chimera BADH in which part of the Rossmann type fold in the spinach BADH was replaced with that of E. coli BADH, showed properties which resembled spinach BADH more than E. coli BADH. The spinach E103K mutant was almost inactive, whereas the E103Q mutant showed a similar activity for the oxidation of betaine aldehyde to that of wild type BADH, but a lower affinity for omega-aminoaldehydes. All spinach BADHs were dimers whereas E. coli BADH was a tetramer. E. coli BADH was more stable at high temperature than spinach BADHs. The E103Q mutant was most labile to high temperature. These properties are discussed in relation to the structure of spinach BADH.     

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。     

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.     

盐胁迫下朝鲜碱茅的甜菜碱醛脱氢酶活性变化及其基因保守区的克隆

文章探讨了朝鲜碱茅甜菜碱醛脱氢酶活性在盐胁迫下的变化,用简并引物扩增了甜菜碱醛脱氢酶基因保守区序列的结果表明,该保守区段长438 bp,推测编码145个氨基酸,包括醛脱氢酶高度保守序列V[T/S]LELGGKSP和其后29位与酶功能有关的Cys。此序列Genbank登录号为EF095710。     

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。     

梭梭甜菜碱醛脱氢酶基因克隆及序列分析

采用RT-PCR、RACE等方法从超旱生、耐盐植物梭梭(Haloxylon ammodendron)中扩增出BADH基因的cDNA序列(命名为HaBADH),其开放阅读框为1 503 bp,推测的氨基酸序列全长为500个氨基酸残基,并含有醛脱氢酶所具有的高度保守的十肽(VTLELGGKSP)以及与酶功能有关的半胱氨酸残基(C).其核苷酸序列与藜科几种盐生植物如盐爪爪(Kalidium foliatum)、中亚滨藜(Atriplex centralasiatica)、三角叶滨藜(Atriplex triangularis)、菠菜(Spinacia oleracea)、山菠菜(Atriplex hortensis)和甜菜(Beta vulgaris)等的相似性均在85%以上,推导编码蛋白的氨基酸序列一致性均在87%以上,表明BADH基因在藜科植物中是一种比较保守的基因.研究结果为进一步从分子水平探明梭梭的抗旱、耐盐机制,挖掘并利用植物抗逆基因奠定基础.     

Oxidation of Betaine Aldehyde by Betaine Dehydrogenase from Spinach Leaves

Betaine content in leaves of fifteen plant species was determined. The results showed higher betaine levels in those salt-, drought-, and chilling-resistant species. Betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8 ) was isolated and partially purified from spinach leaves. Some properties of this enzyme were studied. BADH was precipitated by 60% saturation of (NH4)2SO4. Its activity was not detected in 70% saturation of (NH4)2SO4. BADH has two isoenzymes. The activity of BADH was quite stable below –80℃. It was inhibited by 0.125–1.0 mol/L NaG1 or KC1 but not by Mn2+ and Mo6+, and slightly increased by Mg2+.     

Transgenically Expressed Betaine Aldehyde Dehydrogenase Efficiently Catalyzes Oxidation of Dimethylsulfoniopropionaldehyde and [omega]-Aminoaldehydes

Tobacco (Nicotianum tabacum L.) plants engineered to express a sugar beet (Beta vulgaris L.) betaine aldehyde dehydrogenase (BADH) cDNA acquired not only BADH activity, but also three other aldehyde dehydrogenase activities (those measured with 3-dimethylsulfoniopropionaldehyde, 3-aminopropionaldehyde, and 4-aminobutyraldehyde, all of which are natural products). This shows that BADH is not, as believed up to now, a substrate-specific enzyme and that its role may not be limited to glycine betaine synthesis.     

CoA-dependent methylmalonate-semialdehyde dehydrogenase, a unique member of the aldehyde dehydrogenase superfamily. cDNA cloning, evolutionary relationships, and tissue distribution.

Three overlapping cDNA clones encoding methylmalonate-semialdehyde dehydrogenase (MMSDH; 2-methyl-3-oxopropanoate:NAD+ oxidoreductase (CoA-propanoylating); EC 1.2.1.27) have been isolated by screening a rat liver lambda gt 11 library with nondegenerate oligonucleotide probes synthesized according to polymerase chain reaction-amplified portions coding for the N-terminal amino acid sequence of rat liver MMSDH. The three clones cover a total of 1942 base pairs of cDNA, with an open reading frame of 1569 base pairs. The authenticity of the composite cDNA was confirmed by a perfect match of 43 amino acids known from protein sequencing. The composite cDNA predicts a 503 amino acid mature protein with M(r) = 55,330, consistent with previous estimates. Polymerase chain reaction was used to obtain the sequence of the 32 amino acids corresponding to the mitochondrial entry peptide. Northern blot analysis of total RNA from several rat tissues showed a single mRNA band of 3.8 kilobases. Relative mRNA levels were: kidney greater than liver greater than heart greater than muscle greater than brain, which differed somewhat from relative MMSDH protein levels determined by Western blot analysis: liver = kidney greater than heart greater than muscle greater than brain. A 1423-base pair cDNA clone encoding human MMSDH was isolated from a human liver lambda gt 11 library. The human MMSDH cDNA contains an open reading frame of 1293 base pairs that encodes the protein from Leu-74 to the C terminus. Human and rat MMSDH share 89.6 and 97.7% identity in nucleotide and protein sequence, respectively. MMSDH clearly belongs to a superfamily of aldehyde dehydrogenases and is closely related to betaine aldehyde dehydrogenase, 2-hydroxymuconic semialdehyde dehydrogenase, and class 1 and 2 aldehyde dehydrogenases.     

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.     

Influence of silver nitrate and copper sulfate on somatic embryogenesis,shoot morphogenesis,multiplication, and associated physiological biochemical changes in Gladiolus hybridus L.

Plant Cell, Tissue and Organ Culture (PCTOC) - We conducted a comparative analysis of the effects of two key stress-tolerance genes, spinach betaine aldehyde dehydrogenase (BADH) and cyanobacterial...     

Betaine aldehyde oxidation by spinach chloroplasts

Chenopods synthesize betaine by a two-step oxidation of choline: choline → betaine aldehyde → betaine. Both oxidation reactions are carried out by isolated spinach (Spinacia oleracea L.) chloroplasts in darkness and are promoted by light. The mechanism of betaine aldehyde oxidation was investigated with subcellular fractions from spinach leaf protoplasts. The chloroplast stromal fraction contained a specific pyridine nucleotide-dependent betaine aldehyde dehydrogenase (about 150 to 250 nanomoles per milligram chlorophyll per hour) which migrated as one isozyme on native polyacrylamide gels stained for enzyme activity. The cytosol fraction contained a minor isozyme of betaine aldehyde dehydrogenase. Leaves of pea (Pisum sativum L.), a species that lacks betaine, had no betaine aldehyde dehydrogenase isozymes. The specific activity of betaine aldehyde dehydrogenase rose three-fold in spinach plants grown at 300 millimolar NaCl; both isozymes contributed to the increase. Stimulation of betaine aldehyde oxidation in illuminated spinach chloroplasts was due to a thylakoid activity which was sensitive to catalase; this activity occurred in pea as well as spinach, and so appears to be artifactual. We conclude that in vivo, betaine aldehyde is oxidized in both darkness and light by the dehydrogenase isozymes, although some flux via a light-dependent, H₂O₂-mediated reaction cannot be ruled out.     

Study of a Hydridoma Cell Line Producing Monoclonal Antibodies Against Spinach Betaine Aldehyde Dehydrogenase and Its Application

By fusion of mouse myeloma cells (SP2/O-Ag14) and spleen cells derived from BALB/c mice immunized with spinach betaine aldehyde dehydrogenase (BADH) protein, a hybridoma cell line secreting monoclonal antibodies was obtained. The antibody titer of the ascites was about 1 : 103. Not only could the monoclonal antibodes cross react with the BADH of spinach and sugar beet, it could also cross react with the leaf and root crude extracts of barley, rice, sorghum, and wheat. These results indicated the occurrence of BADH in both the photosynthetic tissue and the non-photosynthetic tissue of these graminea spicies.