Betaine Aldehyde Dehydrogenase Research Articles

The Effect of Salinity Pretreatment on Cd Accumulation and Cd-Induced Stress in BADH -Transgenic and Nontransgenic Rice Seedlings

The influence of betaine aldehyde dehydrogenase (BADH) and salinity pretreatment on oxidative stress under cadmium (Cd) toxicity was investigated in rice cv. Xiushui 11 and its BADH-transgenic line Bxiushui 11. The results showed that plants previously treated with 4.25 and 8.5 mM NaCl, respectively, for 5 days each had higher Cd concentrations in both roots and shoots of the two rice genotypes compared with the controls. Malondialdehyde (MDA) content in both leaves and roots was increased by salinity pretreatment and was significantly lower in the salinity-pretreatment plants than in the controls when the plants were consequently exposed to Cd stress. Salinity pretreatment also increased proline content and the activities of superoxide dismutase (SOD) and peroxidase (POD) in both leaves and roots. It can be assumed that salinity pretreatment enhances the defensive ability of plants against oxidative stress through increasing activities of antioxidative enzymes. The BADH-transgenic line (Bxiushui 11) had lower Cd and MDA content, higher SOD and POD activities, and higher proline content than its wild type (Xiushui 11). The current results suggest that betaine, a product of BADH expression, improves the tolerance of rice plants to Cd stress through increasing the activities of antioxidative enzymes and osmoprotectant content.

Osmotic Regulation of Betaine Content in Leymus chinensis Under Saline-alkali Stress and Cloning and Expression of Betaine Aldehyde Dehydrogenase(BADH) Gene

Abstract The potted Leymus chinensis seedlings were treated with saline-alkali solution of six different(from I to VI) concentrations. The results demonstrate that the betaine content and Betaine-aldehyde dehydrogenase( BADH: EC 1.2.1.8) activities have a direct relation with increased stressing time in the same treatment; both exhibit a single peak with increasing the concentration of saline-alkali solution, and number V shows the highest value. The BADH gene of Leymus chinensis was cloned by RT-PCR and RACE technology and was designated as LcBADH . The cDNA sequence of LcBADH was 1774bp including the open reading frame(ORF) of 1521bp(coding 506 amino acids). The vector of prokaryotic expression was constructed by inserting the LcBADH gene fragment into pET30a(+) and transformed into E. coli BL21 (DE3). The result of SDS-PAGE shows that the idio-protein with a molecular mass of 56.78 kDa was effectively expressed in the recombinant bacteria induced by isopropyl β-D -thiogalactoside(IPTG).

Discovery of a new fragrance allele and the development of functional markers for the breeding of fragrant rice varieties

The recessive fgr gene on chromosome 8 is associated with rice fragrance. It has been reported that this gene is a non-functional badh2 allele and that the functional Badh2 allele encoding putative betaine aldehyde dehydrogenase (BADH2) could render rice non-fragrant. Here we report the discovery of a new badh2 allele and the development of functional markers for the badh2 locus. A total of 24 fragrant and ten non-fragrant rice varieties were studied and sequenced for their Badh2/badh2 loci. Of the 24 fragrant rice varieties, 12 were found to have the known badh2 allele (badh2-E7), which has an 8-bp deletion and three single nucleotide polymorphisms (SNPs) in exon 7; the others had a novel null badh2 allele (badh2-E2), which has a sequence identical to that of the Badh2 allele in exon 7, but with a 7-bp deletion in exon 2. Both null badh2 alleles are responsible for rice fragrance. Based on sequence divergence amongst the functional Badh2 and two null badh2 alleles, we developed functional markers which can be easily used to distinguish non-fragrant from fragrant rice and to differentiate between two kinds of fragrant rice. These functional markers will find their usefulness in breeding for fragrant rice varieties via marker-assisted selection.

A novel betaine aldehyde dehydrogenase gene from Jatropha curcas, encoding an enzyme implicated in adaptation to environmental stress

A novel betaine aldehyde dehydrogenase gene, named JcBD1, was cloned by RT-PCR and RACE techniques from Jatropha curcas L., an important plant species as its seed is the raw material for biodiesels. The cDNA was 1769 bp in length with a complete open reading frame of 1509 bp, encoding 503 amino acids with a predicted molecular mass of 54.585 kD and a p I of 5.32. The deduced amino acid sequence showed substantial similarities to BADH enzymes present in other plants, including the absolute conservative region in the possible active site. Southern blot analysis indicated that the JcBD1 gene belongs to a multigene family. Semi-quantitative RT-PCR analyses revealed that JcBD1 gene was expressed in roots, stems, leaves, flowers, and young seeds, but that the expression level was highest in leaves and stems. And the JcBD1 gene was increasedly expressed in leaves undergoing environmental stress like drought (30% PEG), heat (50 °C), and salt (300 mM NaCl). The JcBD1 protein was functionally expressed in Escherichia coli ( E. coli). E. coli cells transformed with the JcBD1 gene obviously grew better than control cells. The enzyme activity of expressed JcBD1 protein was detected. Over-expression of JcBD1 enzyme in E. coli conferred its resistance to abiotic stressors like salt.

Screening of genes regulated by cold shock in Shewanella piezotolerans WP3 and time course expression of cold-regulated genes

The differential gene transcription of a deep-sea bacterium Shewanella piezotolerans WP3 in response to cold shock was analyzed by RNA arbitrarily primed PCR. Ninety primer sets were used to scan two different RNA pools derived from the culture of cold shock and its control (culture at its optimal grown temperature). Ninety-four putative differentially expressed fragments were identified and cloned. Six out of the 94 fragments were confirmed to be truly differentially transcribed in terms of cold shock by reverse Northern dot blot and then sequenced. Sequence blast analysis showed that the six differentially transcribed genes are putative genes for zonular occludens toxin, chaperon GroEL, efflux transporter, Sua5/YciO/YrdC/YwlC family protein, betaine-aldehyde dehydrogenase, and DEAD box RNA helicase, respectively. The time course expression profiles of these six genes from 0 to 90 min upon cold shock were quantified by real-time PCR. Deletion mutation of the highest induced gene--RNA helicase gene, had no significant impact on the growth of the strain no matter upon cold shock or under permanent low temperature. It is suggested that one or more additional DEAD box RNA helicase genes compensate for the loss of the function of the mutated gene.

Genetic engineering of the biosynthesis of glycinebetaine leads to increased tolerance of photosynthesis to salt stress in transgenic tobacco plants

Genetically engineered tobacco (Nicotiana tabacum L.) with the ability to synthesis glycinebetaine (GB) in chloroplasts was established by introducing the BADH gene for betaine aldehyde dehydrogenase from spinach (Spinacia oleracea L.). The genetic engineering resulted in enhanced tolerance of growth of young seedlings to salt stress. This increased tolerance was not due to improved water status, since there were no significant differences in accumulation of sodium and chloride, leaf water potential, and relative water content between wild type and transgenic plants under salt stress. Salt stress resulted in a decrease in CO2 assimilation and such a decrease was much greater in wild type plants than in transgenic plants. Though salt stress showed no damage to PSII, there were a decrease in the maximal PSII electron transport rate in vivo and an increase in non-photochemical quenching (NPQ) and these changes were greater in wild type plants than in transgenic plants. In addition, salt stress inhibited the activities of ribulose 1,5-bisphosphate carboxylase/oxygenase, chloroplastic fructose-1,6-bisphosphatase, fructose-1,6-bisphosphate aldolase, and phosphoribulokinase and such a decrease was also greater in wild type plants than in transgenic plants, suggesting that GB protects these enzymes against salt stress. However, there were no significant changes in the activities of phosphoglycerate kinase, triose phosphate isomerase, ribulose-5-phosphate isomerase, transketolase, and sedoheptulose-1,7-bisphosphatase in both wild type and transgenic plants. The results in this study suggest that enhanced tolerance of CO2 assimilation to salt stress may be one of physiological bases for increased tolerance of growth of transgenic plants to salt stress.

Co-ordinate expression of glycine betaine synthesis genes linked by the FMDV 2A region in a single open reading frame in Pichia pastoris

The genes encoding the two enzymes choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) of glycine betaine synthesis in Suaeda salsa were cloned and fused with the 2A region of foot-and-mouth disease virus in a single open reading frame. The fused genes were placed under the control of the alcohol oxidase (AOX1) promoter in pPIC3B and transformed into P. pastoris GS115. The expression of the fused genes in P. pastoris and the ability of recombinant yeasts to tolerate environmental stresses were studied. The results showed that induced with 0.5% methanol for 96 h, the maximal activities of CMO and BADH in the tested recombinant yeasts were 45- and 44-fold higher than those in the control yeast transformed empty vector only, respectively; the content of glycine betaine in the recombinant yeasts was 28- to 35-fold higher than that in the control. The fused genes linked by 2A region of foot-and-mouth disease virus were expressed in P. pastoris successfully and the polyprotein was 'cleaved' to each functional protein. The yeasts transformed the fused genes, which were more resistant to salt, methanol, and high temperature stresses than the control as result of glycine betaine synthesis genes introduced.

TaqMan based real time PCR method for quantitative detection of basmati rice adulteration with non-basmati rice

A TaqMan based real time PCR method is developed for the first time to check the adulteration in basmati rice. Betaine aldehyde dehydrogenase 2 (BAD2) gene specific primers and probes were used to detect adulterant non-basmati rice content in basmati samples. The results obtained with market samples and validation samples (basmati rice spiked with non-basmati rice in different proportions) clearly revealed that this technique is useful to detect the adulteration very accurately with 1% detection limit.

Functional analysis of BADH gene promoter from Suaeda liaotungensis K.

A 1,993 bp region upstream of the gene encoding the betaine aldehyde dehydrogenase (BADH) was isolated from Suaeda liaotungensis K., and the analysis of the promoter sequence has revealed the existence of several putative cis-elements by the PLACE database. In this study, according to the characteristic of the BADH promoter, five chimeric constructs varied in the length of promoter fragments from -1,993, -1,466, -1,084, -573 and -300 to +62 bp relative to the transcriptional start site were placed to the upstream of the beta-glucuronidase (GUS) coding region and transferred to Nicotiana tabacum L.cv.89 by Agrobacterium tumefaciens-mediated leaf-disc transformation. The functional properties of each promoter fragment were examined by GUS histochemical staining and fluorescence quantitative analyses in the transgenic tobacco leaves treated with different NaCl concentrations for 48 h. The results show that healthy transgenic plants had decreased GUS activity in leaves, whereas a higher GUS activity was observed when the transgenic plants were challenged with sodium chloride (NaCl). Induction levels were proportional to the concentration of NaCl treatment, allowing fine-tuning of protein expression. GUS enzyme activity was enhanced 6.3-fold in transgenic tobacco leaves containing -300 bp promoter fragment in the presence of 400 mmol/l NaCl compared to the noninductive leaves. This suggests that the smallest promoter fragment (-300 to +62 bp) possesses all the essential cis-acting elements and is sufficient for NaCl induction.

Complex, unusual conformational changes in kidney betaine aldehyde dehydrogenase suggested by chemical modification with disulfiram

The NAD +-dependent animal betaine aldehyde dehydrogenases participate in the biosynthesis of glycine betaine and carnitine, as well as in polyamines catabolism. We studied the kinetics of inactivation of the porcine kidney enzyme (pkBADH) by the drug disulfiram, a thiol-reagent, with the double aim of exploring the enzyme dynamics and investigating whether it could be an in vivo target of disulfiram. Both inactivation by disulfiram and reactivation by reductants were biphasic processes with equal limiting amplitudes. Under certain conditions half of the enzyme activity became resistant to disulfiram inactivation. NAD + protected almost 100% at 10 μM but only 50% at 5 mM, and vice versa if the enzyme was pre-incubated with NAD + before the chemical modification. NADH, betaine aldehyde, and glycine betaine also afforded greater protection after pre-incubation with the enzyme than without pre-incubation. Together, these findings suggest two kinds of active sites in this seemingly homotetrameric enzyme, and complex, unusual ligand-induced conformational changes. In addition, they indicate that, in vivo, pkBADH is most likely protected against disulfiram inactivation.

The Suaeda liaotungensis kitag betaine aldehyde dehydrogenase gene improves salt tolerance of transgenic maize mediated with minimum linear length of DNA fragment

In this research we established a particular vector-free and marker-free plant transformation system of maize to overcome the obstacles of biosafety limits. The BADH gene was introduced into maize by pollen-tube pathway, using the principle of minimum linear length of the transformation element, which was composed of only the BADH gene, expression regulatory sequence (35S CAMV promoter, NOS terminator), and T-DNA border sequence at both sides. Twenty-seven of 2076 transformed samples were positive in PCR amplification and the PCR positive rate of T1 generation was 1.3%. Further Southern blotting results indicated that the BADH gene was integrated into maize genome. Transgenic lines of progeny were examined for tolerance to NaCl by induced salt stress with 250 mM NaCl Hoagland solution. After 15 days of treatment, 73.9–100% of the transgenic seedlings survived and grew well, whereas most wild-type seedlings wilted and showed loss of chlorophyll. Only 8.9% of the wild-type plants survived but gradually died after salt stress. The electrical conductivity of the transgenic line of progeny after salt stress was lower than wild type. The transgenic progeny had higher glycinebetaine and Chlorophyll content than wild type after salt stress.

Functional defect at the rice choline monooxygenase locus from an unusual post‐transcriptional processing is associated with the sequence elements of short‐direct repeats

Glycine betaine (GB), a quaternary ammonium solute, plays a crucial role in developing osmotic tolerance. Rice contains a choline monooxygenase (CMO) and two betaine aldehyde dehydrogenase homologues that are required for GB synthesis, but usually no GB is accumulated in rice (Oryza sativa). To elucidate the molecular processes that underlie the GB deficiency in rice, an experiment involving rice and spinach (Spinacia oleracea) was conducted to analyze the products transcribed from CMO genes. Reverse transcription-polymerase chain reaction (RT-PCR) was used to obtain CMO transcripts and a sequencing approach was employed to analyze the structural composition of various CMO transcripts. The results showed that most rice CMO transcripts were processed incorrectly, retaining introns or deleted of coding sequences; the unusual deletion events occurred at sequence elements of the short-direct repeats. In conclusion, the production of incorrect CMO transcripts results in a deficiency of the full-length CMO protein and probably reduces GB accumulation considerably in rice plants. Sequence comparison results also implied that the unusual deletion-site selection might be mediated by the short-direct repeats in response to stress conditions.

Proteomic analyses indicate induction of hepatic carbonyl reductase/20 β-hydroxysteroid dehydrogenase B in rainbow trout exposed to sewage effluent

Proteomic analyses were performed to identify regulated liver proteins in rainbow trout ( Oncorhynchus mykiss) caged upstream and downstream from a sewage treatment works (STW). Two-dimensional gel electrophoresis, image analysis and FT-ICR mass-spectrometry revealed four regulated protein spots. The three down-regulated spots contained betaine aldehyde dehydrogenase, lactate dehydrogenase and an unidentified protein respectively. The only up-regulated spot consisted of both mitochondrial ATP synthase alpha-subunit and carbonyl reductase/20 β-hydroxysteroid dehydrogenase (CR/20 β-HSD). Further studies using quantitative PCR revealed a 13.5-fold induction of CR/20 β-HSD B mRNA following STW effluent exposure. The CR/20 β-HSD B gene was not regulated by 17 α-ethinylestradiol, suggesting that its induction downstream from the STW is due to other factors than exposure to estrogens. Image analysis was initially performed on four gels from each group. These analyses suggested 15 regulated spots. However, validation of the 15 spots by increasing the number of replicates confirmed only four regulated spots. Hence, the present study also demonstrates the need for sufficient biological/technical replication in the interpretation of proteomic data.

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

AbstractThe 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 betaine), 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 a 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% NaCl. Cell membrane of the wild type was seaerely damaged as determined by higher EL under salinity stress. K+and Ca2+ contents of all tested lines decreased under salt stress, but the transgenic plants showed a significantly higher accumulation of K+ and Ca2+ than the wild type. In contrast, the wild type had significantly higher Cl and Na+ 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.(Handling editor: Jin‐Zhong Cui)

Glycinebetaine Accumulation, Physiological Characterizations and Growth Efficiency in Salt‐tolerant and Salt‐sensitive Lines of Indica Rice (Oryza sativaL. ssp.indica) in Response to Salt Stress

AbstractThe aim of this research was to investigate betaine aldehyde dehydrogenase (BADH) and glycinebetaine (Glybet) biosynthesis in photoautotrophic rice seedlings. The role of Glybet on physiological and growth responses to salt stress in both salt‐tolerant and salt‐sensitive lines is to be investigated. The BADH activity in salt‐tolerant seedlings cultured under extreme salt stress (342 mmNaCl) progressively increased during the first few hours until it peaked after 72 h. This was about 2.5 times greater than in salt‐sensitive plants. Similarly, the amount of Glybet detected in salt‐tolerant lines was 1.3 times more than in salt‐sensitive lines at 96 h salt exposure. The BADH activities were positively related to Glybet accumulation in both salt‐tolerant and salt‐sensitive lines. The accumulation of Glybet in salt‐tolerant lines was directly correlated with pigment stabilization. Relative water content in the salt‐tolerant lines was closely related to water oxidation in photosystem II (PSII), defined by maximum quantum yield of PSII (Fv/Fm). In addition, a high concentration of total chlorophyll is more efficient in capturing light energy, defined by photochemical quenching. The concentrations of chlorophyllaand total carotenoid were positively related to the quantum efficiency of PSII (ΦPSII) and non‐photochemical quenching, respectively, resulting in a high net‐photosynthetic rate (NPR) and the promotion of growth. The high level of Glybet in salt‐tolerant lines plays a role as a salt defensive response mechanism in terms of pigment stabilization and water oxidation in PSII, resulting in high NPR and growth efficiency.

An Unusual Posttranscriptional Processing in Two Betaine Aldehyde Dehydrogenase Loci of Cereal Crops Directed by Short, Direct Repeats in Response to Stress Conditions

Various abilities to synthesize and accumulate glycine betaine (GB) are crucial for angiosperms to develop salt and drought tolerances. In higher plants, GB is synthesized by a two-step oxidation of choline via an intermediate form of betaine aldehyde, and catalyzed by choline monooxygenase and betaine aldehyde dehydrogenase (BADH). In this study, numerous truncated and/or recombinant transcripts of two BADH homologs resulting from an unusual posttranscriptional processing were detected in rice (Oryza sativa) and other cereal crops, including maize (Zea mays), wheat (Triticum aestivum), and barley (Hordeum vulgare). The observed events took place at the 5' exonic region, and led to the insertion of exogenous gene sequences and a variety of deletions that resulted in the removal of translation initiation codon, loss of functional domain, and frame-shifts with premature termination by introducing stop codon. By contrast, the BADH transcripts from dicotyledonous species, such as spinach (Spinacia oleracea), Arabidopsis (Arabidopsis thaliana), and tomato (Solanum lycopersicum), had correctly processed mRNA. This suggests the differentiation of posttranscriptional processing in BADH genes potentially contributes to the variation of GB-synthesizing capacities among various plant species. In addition, comprehensive sequence analyses demonstrated that extensive sequence similarities (named as short, direct repeats) are of paired presence surrounding the junctions of both the deletion and/or insertion sites in the unusual BADH transcripts. The site selection for the deletion/insertion was altered in response to the stress conditions. This indicates that the sequence elements of short, direct repeats are probably required for the recognition of the deletion/insertion sites.

Salinity induced changes in proline and betaine contents and synthesis in two aquatic macrophytes differing in salt tolerance

The present work investigated the effect of NaCl and seawater salinity (SWS) on accumulation of proline and glycinebetaine (betaine) and expression of two key enzymes of their biosynthetic pathway, betainealdehyde dehydrogenase (BADH) and Δ1-pyrroline-5-carboxylate synthetase (P5CS) in two aquatic macrophytes, salt-tolerant Najas gramenia and moderately-salt-tolerant Najas indica. N. gramenia did not show any accumulation of betaine or expression of BADH in response to NaCl or SWS treatments. The content of proline increased significantly in response to the salt treatments. High expression of P5CS was also observed, however, the transcript abundance did not correlate with the amount of proline. N. indica, exhibited significantly greater accumulation of proline in response to SWS, which contained Ca2+ in addition to NaCl, than in response to NaCl alone.

The accumulation and synthesis of betaine in winter skate (Leucoraja ocellata)

The present study investigated aspects of betaine metabolism in an elasmobranch fish, the winter skate (Leucoraja ocellata). Based on the level of choline dehydrogenase (ChoDH) activity, the liver and kidney appear to be the major sites of betaine synthesis and the mitochondrial localization of ChoDH and betaine aldehyde dehydrogenase (BADH) indicates that the metabolic organization of betaine synthesis in winter skate is similar to other vertebrates. Food deprivation did not affect white muscle betaine content, and prolonged starvation (70 days) appeared to decrease the total hepatic betaine synthetic capacity. There was no decrease in ChoDH or BADH activity at the mitochondrial level with starvation, suggesting any decrease is due to catabolism of hepatic reserves rather than downregulation of betaine synthesis. Skates fed a high betaine diet (frozen squid approximately 55 micromol g(-1)) had elevated white muscle betaine content compared to those fed a low betaine diet (frozen herring <2 micromol g(-1)); however, high dietary betaine intake did not affect the activity of betaine synthesizing enzymes in liver. Acclimation to elevated salinity (120 and 130% seawater) did not result in an increase in white muscle betaine content. Taken as a whole, the present data suggest that diet is a major determinant of muscle betaine in the winter skate and that betaine is of marginal importance as an intracellular osmolyte in this species.

Substrate‐induced inactivation of the chloroplastic betaine aldehyde dehydrogenase

Substrate‐induced inactivation of the chloroplastic betaine aldehyde dehydrogenase

Ethanol attenuates Aldh9 mRNA expression in Japanese medaka ( Oryzias latipes) embryogenesis

The mechanisms of teratogenic effects of ethanol in Japanese medaka embryogenesis were investigated by testing the hypothesis that ethanol or its metabolite ameliorates the expression of ethanol metabolizing enzymes. We have previously demonstrated that ethanol is unable to alter the expression pattern of alcohol dehydrogenase (ADH) mRNA, the first enzyme of ethanol metabolism, in medaka embryos during development. We, therefore, extended our investigation to aldehyde dehydrogenase (ALDH) system, the next enzyme of alcohol metabolic pathway. As the first step towards studying the regulation of Aldh mRNA expression by ethanol, we have cloned a cDNA by reverse transcriptase polymerase chain reaction (RT-PCR) from adult Japanese medaka ( Oryzias latipes) liver representing the medaka ALDH9 gene product, with a coding region of 1515 nucleotides. The deduced amino acid sequences share 81.2% identity with cod liver betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8), and 71.1% identity with human ALDH9A1 sequences. RT-PCR analysis further showed that in adults Aldh9 mRNA is constitutively expressed in all organs tested (brain, eye, gill, GI, heart, liver, kidney, muscle, skin, testis and ovary). Using semi-quantitative (rRT-PCR) and quantitative real time RT-PCR (qRT-PCR), we detected Aldh9 mRNA at all time points of development and the expression was lowest between ∼ 1 and 8 h post-fertilization (hpf). Treatment of the embryos with ethanol for 48 h post-fertilization (hpf) attenuates (delayed) the expression of Aldh9 mRNA. This delayed expression of Aldh9 mRNA by ethanol may enhance acetaldehyde concentration in the embryo and induce teratogenesis during development.