Betaine Aldehyde Dehydrogenase Research Articles

Use of Allele-Specific Amplification for Rapid Identification of Aromatic and Non-aromatic Rice Germplasms.

In the current context of global climate change trends, threat to food and nutrition security, collection, conservation and management, and characterization and evaluation of crop germplasms especially traditional landraces are gaining momentum more than ever before. Aromatic rice is an elite category of cultivated rice having huge sociocultural heritage value, fetching premium prices globally. Hence, its identification, in situ conservation, and appropriate characterization are likely to augment reliability of this distinctive category of rice, and rice commodity chain actors. badh2.1 is recognised as the major allele responsible for rice 2-acetyl-1-pyrroline aroma production in a vast number of aromatic rice globally. However, most of the previous works on the genetics and biochemical pathways of aroma expression in rice have encompassed mainly Basmati, Sadri, Della, Jasmine, and a few modern hybrids. But apart from these spotlighted varieties, a myriad of indigenous, aromatic rice germplasms exists. Allele-specific amplification, a low-cost, accurate method invented by Bradbury et al. 2005, can be utilized successfully for discriminating the rarely explored aromatic and nonaromatic rice as described.

Multi-omics analysis reveals the genetic basis of rice fragrance mediated by betaine aldehyde dehydrogenase 2

IntroductionFragrance is an important economic and quality trait in rice. The trait is controlled by the recessive gene betaine aldehyde dehydrogenase 2 (BADH2) via the production of 2-acetyl-1-pyrroline (2AP). ObjectivesVariation in BADH2 was evaluated at the population, genetic, transcriptional, and metabolic levels to obtain insights into fragrance regulation in rice. MethodsWhole-genome resequencing of the Korean World Rice Collection of 475 rice accessions, including 421 breeding lines and 54 wild accessions, was performed. Transcriptome analyses of a subset of 279 accessions, proteome analyses of 64 accessions, and volatile profiling of 421 breeding lines were also performed. ResultsWe identified over 3.1 million high-quality single nucleotide polymorphisms (SNPs) in Korean rice collection. Most SNPs were present in intergenic regions (79%), and 190,148 SNPs (6%) were located in the coding sequence, of which 53% were nonsynonymous. In total, 38 haplotypes were identified in the BADH2 coding region, including four novel haplotypes (one in cultivated and three in wild accessions). Tajima's D values suggested that BADH2 was under balancing selection in japonica rice. Furthermore, we identified 316 expression quantitative trait loci (eQTL), including 185 cis-eQTLs and 131 trans-eQTLs, involved in BADH2 regulation. A protein quantitative trait loci (pQTL) analysis revealed the presence of trans-pQTLs; 13 pQTLs were mapped 1 Mbp from the BADH2 region. Based on variable importance in projection (VIP) scores, 15 volatile compounds, including 2AP, discriminated haplotypes and were potential biomarkers for rice fragrance. ConclusionWe generated a catalog of haplotypes based on a resequencing analysis of a large number of rice accessions. eQTLs and pQTLs associated with BADH2 gene expression and protein accumulation are likely involved in the regulation of 2AP variation in fragrant rice. These data improve our understanding of fragrance and provide valuable information for rice breeding.

Transgenic tobacco co-expressing flavodoxin and betaine aldehyde dehydrogenase confers cadmium tolerance through boosting antioxidant capacity.

Excessive heavy metal (HM) levels in soil have become a source of concern due to their adverse effects on human health and the agriculture industry. Soil contamination by HMs leads to an accumulation of reactive oxygen species (ROSs) within the plant cell and disruption of photosynthesis-related proteins. The response of tobacco lines overexpressing flavodoxin (Fld) and betaine aldehyde dehydrogenase (BADH) to cadmium (Cd) toxicity was investigated in this study. PCR results demonstrated the expected amplicon length of each gene in the transgenic lines. Absolute qRT-PCR demonstrates a single copy of T-DNA integration into each transgenic line. Relative qRT-PCR confirmed overexpression of Fld and BADH in transgenic lines. The maximum quantum yield of photosystem II (Fv/Fm) was measured under Cd toxicity stress and revealed that transgenic lines had a higher Fv/Fm than wild-type (WT) plants. Accumulation of proline, glycine betaine (GB), and higher activity of antioxidant enzymes alongside lower levels of malondialdehyde (MDA) and hydrogen peroxide (H2O2) was indicative of a robust antioxidant system in transgenic plants. Therefore, performing a loop in reducing the ROS produced in the photosynthesis electron transport chain and stimulating the ROS scavenger enzyme activity improved the plant tolerance to Cd stress.

Production of aromatic three-line hybrid rice using novel alleles of BADH2.

Aroma is a key grain quality trait that directly influences the market price of rice globally. Loss of function of betaine aldehyde dehydrogenase 2 (OsBADH2) affects the biosynthesis of 2‐acetyl‐1‐pyrroline (2‐AP), which is responsible for aroma in fragrant rice. The current study was aimed at creating new alleles of BADH2 using CRISPR/Cas9 gene editing technology under the genetic background of the japonica Ningjing 1 (NJ1) and indica Huang Huazhan (HHZ) varieties. Sensory evaluation and analysis using headspace solid‐phase microextraction gas chromatography–mass spectrometry (HS‐SPME‐GC‐MS) showed that the grains of the four homozygous T1 lines with new alleles of BADH2 (nj1‐cr BADH2‐1, nj1‐cr BADH2‐2, hhz‐cr BADH2‐1 and hhz‐cr BADH2‐2) produced moderate fragrance and had significantly increased 2‐AP content compared with wild‐types. Moreover, there were no significant differences in the amylose content and gelatinization temperature among the four lines with new alleles of BADH2 to the wild‐types. Thereafter, we crossed the HHZ background new alleles of BADH2 with CMS line Taonong 1A (TN1A) to produce a three‐line hybrid variety B‐Tao‐You‐Xiangzhan (BTYXZ) with increased grain aroma. The 2‐AP content in grains of the improved BTYXZ‐1 and BTYXZ‐2 reached at 26.16 and 18.74 μg/kg, and the gel consistency of BTYXZ‐1 and BTYXZ‐2 increased significantly by 9.1% and 6.5%, respectively, compared with the wild‐type Tao‐You‐Xiangzhan (TYXZ). However, the γ‐aminobutyric acid (GABA) content in the improved three‐line hybrid rice BTYXZ‐1 (5.6 mg/100 g) and BTYXZ‐2 (10.7 mg/100 g) was significantly lower than that of the TYXZ. These results demonstrated that CRISPR/Cas9 gene editing technology could be successfully utilized in improving aroma in non‐fragrant japonica and indica varieties. In addition, the newly developed BADH2 alleles provided important genetic resources for grain aroma improvement in three‐line hybrid rice.

The power and versatility of genome editing tools in crop improvement

The power and versatility of genome editing tools in crop improvement

Genetic engineering of glycinebetaine synthesis enhances cadmium tolerance in BADH-transgenic tobacco plants via reducing cadmium uptake and alleviating cadmium stress damage

Cadmium (Cd) is a toxic non-essential elements to plants, and its accumulation in plants can affect human health through the food chain. Glycinebetaine (GB) is an effective osmotic adjustment substance for plants to resist abiotic stress, and it plays important roles in various stresses. To investigate whether endogenous GB accumulation in vivo can enhance Cd tolerance, we used betaine aldehyde dehydrogenase (BADH) transgenic tobacco plants to explore the important role of GB under Cd stress conditions. The results showed that the tobacco exposed to cadmium stress, lead to growth inhibition and biomass reduction, decreased photosynthesis, decreased the absorption of essential minerals and increased oxidative stress. However, GB accumulation in transgenic tobacco plants significantly increased Cd tolerance, as evidenced by the decreased growth inhibition, enhanced photosynthetic rate and reduced reactive oxygen species accumulation. GB can regulate Cd uptake and transport, which results in reduced Cd accumulation. Moreover, GB also reduced K+ efflux under Cd stress and regulated ion transporter genes to maintain element balance. Our findings provide a novel role of GB in enhancing Cd stress tolerance in transgenic plants and thus propose GB as a potential candidate for relieving Cd toxicity in other crops.

Root colonization and rhizosphericcommunity structure of Arbuscular Mycorrhizal Fungi inBADH transgenic maize BZ-136 and its recipient under salt stress and neutral soil.

Betaine aldehyde dehydrogenase (BADH) transgenic maize has a capability to grow under drought and salt stress; the risk of planting BADH transgenic maizeon symbiotic microorganisms remains problematic, however. A pot experiment was carried out to assess the impact of BADH transgenic maize BZ-136 on arbuscular mycorrhizal fungi (AMF) colonization in root and community structure in rhizosphere soil compared with that of parental maize Zheng58 in neutral and saline-alkaline soil. Microscope observation found that BZ-136 only had a significantly reduced effect on AMF colonization at the elongation stage (9-14%). High-throughput sequencing analysis revealed that the AMF taxonomic composition kept consistency at the genus level between transgenic BZ-136 and non-transgenic parental Zheng58. NMDS analysis verified the slight difference in community structure between BZ-136 and Zheng58 presented an agrotype-dependent pattern. AMF community indices showed that BZ-136 had a higher richness at the flowering stage in saline-alkaline soil and had a higher diversity at the mature stage in neutral soil. Heatmap analysis also illuminated AMF community structure of transgenic maize at species level was similar to that of non-transgenic maize. In summary, cropping transgenic BADH maize has minor or transient effects on AMF colonization and rhizosphericsoil AMF community structure, while agrotype has a stronger effect on AMF community structure.

Haplotype Analysis of BADH1 by Next-Generation Sequencing Reveals Association with Salt Tolerance in Rice during Domestication.

Betaine aldehyde dehydrogenase 1 (BADH1), a paralog of the fragrance gene BADH2, is known to be associated with salt stress through the accumulation of synthesized glycine betaine (GB), which is involved in the response to abiotic stresses. Despite the unclear association between BADH1 and salt stress, we observed the responses of eight phenotypic characteristics (germination percentage (GP), germination energy (GE), germination index (GI), mean germination time (MGT), germination rate (GR), shoot length (SL), root length (RL), and total dry weight (TDW)) to salt stress during the germination stage of 475 rice accessions to investigate their association with BADH1 haplotypes. We found a total of 116 SNPs and 77 InDels in the whole BADH1 gene region, representing 39 haplotypes. Twenty-nine haplotypes representing 27 mutated alleles (two InDels and 25 SNPs) were highly (p < 0.05) associated with salt stress, including the five SNPs that have been previously reported to be associated with salt tolerance. We observed three predominant haplotypes associated with salt tolerance, Hap_2, Hap_18, and Hap_23, which were Indica specific, indicating a comparatively high number of rice accessions among the associated haplotypes. Eight plant parameters (phenotypes) also showed clear responses to salt stress, and except for MGT (mean germination time), all were positively correlated with each other. Different signatures of domestication for BADH1 were detected in cultivated rice by identifying the highest and lowest Tajima’s D values of two major cultivated ecotypes (Temperate Japonica and Indica). Our findings on these significant associations and BADH1 evolution to plant traits can be useful for future research development related to its gene expression.

Spectroscopic analysis of coenzyme binding to betaine aldehyde dehydrogenase dependent on potassium.

Glycine betaine is the main osmolyte synthesized and accumulated in mammalian renal cells. Glycine betaine synthesis is catalyzed by the enzyme betaine aldehyde dehydrogenase (BADH) using NAD+ as the coenzyme. Previous studies have shown that porcine kidney betaine aldehyde dehydrogenase (pkBADH) binds NAD+ with different affinities at each active site and that the binding is K+ dependent. The objective of this work was to analyze the changes in the pkBADH secondary and tertiary structure resulting from variable concentrations of NAD+ and the role played by K+ . Intrinsic fluorescence studies were carried out at fixed-variable concentrations of K+ and titrating the enzyme with varying concentrations of NAD+ . Fluorescence analysis showed a shift of the maximum emission towards red as the concentration of K+ was increased. Changes in the exposure of tryptophan located near the NAD+ binding site were found when the enzyme was titrated with NAD+ in the presence of potassium. Fluorescence data analysis showed that the K+ presence promoted static quenching that facilitated the pkBADH-NAD+ complex formation. DC data analysis showed that binding of K+ to the enzyme caused changes in the α-helix content of 4% and 12% in the presence of 25 mM and 100 mM K+ , respectively. The presence of K+ during NAD+ binding to pkBADH increased the thermal stability of the complex. These results indicated that K+ facilitated the pkBADH-NAD+ complex formation and suggested that K+ caused small changes in secondary and tertiary structures that could influence the active site conformation.

The Determination of Aromatic Character of Several Local Rice Varieties using Phenotypic Analysis and Molecular DNA

The pandan scent in aromatic rice has been known as the result of 8 bp deletions and 3 Single Nucleotide Polymorphisms (SNPS) in BADH2 gene, which produce non-functional betaine aldehyde dehydrogenase (BADH) enzyme. Several DNA markers for aromatic character based on mutation in BADH2 gene have been developed. In our experiment, we analysed the presence of aromatic character in four local rice variety such as Merah Wangi, Pendok, Genjah Arum, and Mentik Wangi Susu using KOH method and DNA molecular method using three DNA markers to detect mutation that responsible for the development of aromatic character. Phenotype analysis using KOH method showed that Merah Wangi, Genjah Arum, and Mentik Wangi Susu produce pandan scents. PCR analysis using Bradbury and Badex7-5, and RM223 markers showed the presence of BADH2 mutation in Merah Wangi and Mentik Wangi Susu, whereas Pendok and Genjah Arum did not show BADH2 mutation using those used three markers. Our results indicate that among four investigated local rice, only Merah Wangi and Mentik Wangi Susu are categorized as aromatic rice whereas Pendok and Genjah Arum are non-aromatic.Keywords: genetic analysis, aromatic rice, specific markers, genetic mutation.

Fragrance in Pandanus amaryllifoliusRoxb. Despite the Presence of a Betaine Aldehyde Dehydrogenase 2.

Pandanus amaryllifolius Roxb. accumulates the highest concentration of the major basmati aroma volatile 2-acetyl-1-pyrroline (2AP) in the plant kingdom. The expression of 2AP is correlated with the presence of a nonfunctional betaine aldehyde dehydrogenase 2(BADH2) in aromatic rice and other plant species. In the present study, a full-length BADH2 sequence was reconstructed from the transcriptome data of leaf tissue from P. amaryllifolius seedlings. Based on this sequence, a 1509 bp coding sequence was defined that encoded a 54 kD PaBADH2 protein. This revealed the presence of a full-length BADH2 protein in P. amaryllifolius. Moreover, quantitative real-time PCR analysis, combined with BADH2 enzyme activity, confirmed the expression and functionality of the PaBADH2 protein. To understand the apparent structural variation, docking analysis was carried out in which protein showed a good affinity with both betaine aldehyde (BAD) and γ-aminobutyraldehyde (GAB-ald) as substrates. Overall, the analysis showed the presence of a functional BADH2, along with substantial 2AP synthesis (4.38 ppm). Therefore, we conclude that unlike all other plants studied to date, 2AP biosynthesis in P. amaryllifolius is not due to the inactivation of BADH2.

Heat Priming of Lentil (Lens culinaris Medik.) Seeds and Foliar Treatment with γ-Aminobutyric Acid (GABA), Confers Protection to Reproductive Function and Yield Traits under High-Temperature Stress Environments.

Gradually increasing temperatures at global and local scales are causing heat stress for cool and summer-season food legumes, such as lentil (Lens culinaris Medik.), which is highly susceptible to heat stress, especially during its reproductive stages of development. Hence, suitable strategies are needed to develop heat tolerance in this legume. In the present study, we tested the effectiveness of heat priming (HPr; 6 h at 35 °C) the lentil seeds and a foliar treatment of γ-aminobutyric acid (GABA; 1 mM; applied twice at different times), singly or in combination (HPr+GABA), under heat stress (32/20 °C) in two heat-tolerant (HT; IG2507, IG3263) and two heat-sensitive (HS; IG2821, IG2849) genotypes to mitigate heat stress. The three treatments significantly reduced heat injury to leaves and flowers, particularly when applied in combination, including leaf damage assessed as membrane injury, cellular oxidizing ability, leaf water status, and stomatal conductance. The combined HPr+GABA treatment significantly improved the photosynthetic function, measured as photosynthetic efficiency, chlorophyll concentration, and sucrose synthesis; and significantly reduced the oxidative damage, which was associated with a marked up-regulation in the activities of enzymatic antioxidants. The combined treatment also facilitated the synthesis of osmolytes, such as proline and glycine betaine, by upregulating the expression of their biosynthesizing enzymes (pyrroline-5-carboxylate synthase; betaine aldehyde dehydrogenase) under heat stress. The HPr+GABA treatment caused a considerable enhancement in endogenous levels of GABA in leaves, more so in the two heat-sensitive genotypes. The reproductive function, measured as germination and viability of pollen grains, receptivity of stigma, and viability of ovules, was significantly improved with combined treatment, resulting in enhanced pod number (21–23% in HT and 35–38% in HS genotypes, compared to heat stress alone) and seed yield per plant (22–24% in HT and 37–40% in HS genotypes, in comparison to heat stress alone). The combined treatment (HPr+GABA) was more effective and pronounced in heat-sensitive than heat-tolerant genotypes for all the traits tested. This study offers a potential solution for tackling and protecting heat stress injury in lentil plants.

Lanthanum (La) improves growth, yield formation and 2-acetyl-1-pyrroline biosynthesis in aromatic rice (Oryza sativa L.)

BackgroundLanthanum (La) is a rare earth element that can influence plant growth and development. However, the effect of La on growth, yield formation and 2-acetyl-1-pyrroline (2-AP, a key compound responsible for the aroma of rice) biosynthesis in aromatic rice (Oryza sativa L. subsp. japonica Kato) has not been reported. Therefore, the present study investigated the effects of La on growth, photosynthesis, yield formation and 2-AP biosynthesis in aromatic rice through three experiments.ResultsTwo pot experiments and a two-year field trial were conducted with different rates of La application (20–120 LaCl3 mg kg−1 and 12 kg ha−1 LaCl3), and treatments without La application were used as controls. The results showed that the application of LaCl3 at 80 and 100 mg kg−1 and at 12 kg ha−1 greatly increased the 2-AP content (by 6.45–43.03%) in aromatic rice seedlings and mature grains compared with the control. The La treatments also increased the chlorophyll content, net photosynthetic rate and total aboveground biomass of rice seedlings. Higher antioxidant enzyme (superoxide, peroxidase, and catalase) activity was detected in the La treatments than in the control. The La treatments also increased the grain yield, grain number per panicle and seed-setting rate of aromatic rice relative to the control. Moreover, the grain proline and γ-aminobutyric acid contents and the activity of betaine aldehyde dehydrogenase significantly decreased under the La treatment. The application of La to soil enhanced the activity of proline dehydrogenase by 20.62–56.95%.ConclusionsLa improved the growth, yield formation and 2-AP content of aromatic rice and enhanced 2-AP biosynthesis by increasing the conversion of proline to 2-AP and decreasing the conversion of GABald to GABA.

Betaine Aldehyde Dehydrogenase (BADH) vs. Flavodoxin (Fld): Two Important Genes for Enhancing Plants Stress Tolerance and Productivity.

Abiotic stresses, mainly salinity and drought, are the most important environmental threats that constrain worldwide food security by hampering plant growth and productivity. Plants cope with the adverse effects of these stresses by implementing a series of morpho-physio-biochemical adaptation mechanisms. Accumulating effective osmo-protectants, such as proline and glycine betaine (GB), is one of the important plant stress tolerance strategies. These osmolytes can trigger plant stress tolerance mechanisms, which include stress signal transduction, activating resistance genes, increasing levels of enzymatic and non-enzymatic antioxidants, protecting cell osmotic pressure, enhancing cell membrane integrity, as well as protecting their photosynthetic apparatus, especially the photosystem II (PSII) complex. Genetic engineering, as one of the most important plant biotechnology methods, helps to expedite the development of stress-tolerant plants by introducing the key tolerance genes involved in the biosynthetic pathways of osmolytes into plants. Betaine aldehyde dehydrogenase (BADH) is one of the important genes involved in the biosynthetic pathway of GB, and its introduction has led to an increased tolerance to a variety of abiotic stresses in different plant species. Replacing down-regulated ferredoxin at the acceptor side of photosystem I (PSI) with its isofunctional counterpart electron carrier (flavodoxin) is another applicable strategy to strengthen the photosynthetic apparatus of plants under stressful conditions. Heterologous expression of microbially-sourced flavodoxin (Fld) in higher plants compensates for the deficiency of ferredoxin expression and enhances their stress tolerance. BADH and Fld are multifunctional transgenes that increase the stress tolerance of different plant species and maintain their production under stressful situations by protecting and enhancing their photosynthetic apparatus. In addition to increasing stress tolerance, both BADH and Fld genes can improve the productivity, symbiotic performance, and longevity of plants. Because of the multigenic and complex nature of abiotic stresses, the concomitant delivery of BADH and Fld transgenes can lead to more satisfying results in desired plants, as these two genes enhance plant stress tolerance through different mechanisms, and their cumulative effect can be much more beneficial than their individual ones. The importance of BADH and Fld genes in enhancing plant productivity under stress conditions has been discussed in detail in the present review.

The toxicity of selenium and mercury in Suaeda salsa after 7-days exposure

Mercury is one of the major pollutants in the ocean, selenium causes toxicity beyond a certain limit, but there are few comparative toxic studies between them in halophytes. The study was to investigate the toxic effects of selenium (Se4+) and mercury (Hg2+) in halophyte Suaeda salsa at the level of genes, proteins and metabolites after exposure for 7 days. By integrating the results of proteomics and metabolomics, the pathway changed under different treatments were revealed. In Se4+-treated group, the changed 3 proteins and 10 metabolites participated in the process of substance metabolism (amino acid, pyrimidine), citrate cycle, pentose phosphate pathway, photosynthesis, energy, and protein biosynthesis. In Hg2+-treated group, the changed 10 proteins and 10 metabolites were related to photosynthesis, glycolysis, substance metabolism (cysteine and methionine, amino acid, pyrimidine), ATP synthesis and binding, tolerance, sugar-phosphatase activity, and citrate cycle. In Se4++ Hg2+-treated group, the changed 5 proteins an 12 metabolites involved in stress defence, iron ion binding, mitochondrial respiratory chain, structural constituent of ribosome, citrate cycle, and amino acid metabolism. Furthermore, the separate and combined selenium and mercury both inhibited growth of S. salsa, enhanced activity of antioxidant enzymes (superoxide dismutase, peroxidase and catalase), and disturbed osmotic regulation through the genes of choline monoxygenase and betaine aldehyde dehydrogenase. Our experiments also showed selenium could induce synergistic effects in S. salsa. In all, we successfully characterized the effects of selenium and mercury in plant which was helpful to evaluate the toxicity and interaction of marine pollutants.

BADH1 is associated with fragrance in sorghum (Sorghum bicolor (L.) Moench) cultivar ‘Ambemohor’

Aroma is an important trait that can enhance the product value in several crops. Pandan-like fragrance resulting from accumulation of 2-acetyl-1-pyrroline (2AP) is one of the pleasant aromas in food crops which is caused by null or missense mutations in betaine aldehyde dehydrogenase 2 (BADH2) gene. In addition, betaine aldehyde aehydrogenase 1 (BADH1) has shown to be associated with aroma in rice. In this study, we investigated the genetics controlling coconut juice-like fragrance in inflorescence of sorghum cultivar 'Ambemohor'. 2AP analysis in seeds revealed that Ambemohor possessed no 2AP. An F2 population developed from the cross between Ambemohor x KU630 (nonfragrant) segregated into a ratio of 3 (fragrant) : 1 (nonfragrant), suggesting that the coconut juice-like fragrance in Ambemohor is controlled by a single dominant gene, designated 'Aro'. Bulked segregant analysis suggested that the gene controlling fragrance in Ambemohor is located on sorghum chromosome 6. Quantitative trait locus (QTL) analysis identified a major QTL, (qAro6.1, for the fragrance located on chromosome 6 between markers SB3567 and SB3570. Bioinformatics analysis revealed that SB3567 and SB3570 were 217.8 kb apart and there were 29 annotated genes in this region including BADH1. Sequence analysis revealed that BADH1 sequences in Ambemohor and KU630 differed in size, but their coding sequences (CDS) were of same size. CDS alignment revealed four single-nucleotide polymorphisms (SNPs) between Ambemohor and KU630 in which two SNPs caused amino change in BADH1 of Ambemohor. These results suggested that BADH1 is a candidate gene for the coconut juice-like fragrance in Ambemohor.

Molecular characterization of glycine betaine biosynthesis genes from deep sea halophilic bacteria, Bacillus atrophaeus NIOT-DSB21

Glycine betaine (N, N, N-trimethylglycine) is an effective compatible solute, which maintains fluidity of membranes and protects the biological structure of the organisms under stress. In this study, betaine aldehyde dehydrogenase ( GbsA ) and betaine alcohol dehydrogenase ( GbsB ) genes encoding for glycine betaine biosynthesis were PCR amplified from genomic DNA of Bacillus atrophaeus NIOT-DSB21 isolated from deep sea sediment of the active volcanic Barren Island, Andaman and Nicobar Islands. The amplified genes were cloned and nucleotide sequences were determined. The sequencing results showed that GbsA and GbsB genes contain ORF of 1473 bp and 1209 bp long, encoding 490 and 402 amino acids respectively (GenBank accession nos. MW045210 and MW045211). In silico sequence analysis revealed that the GbsA and GbsB sequences of B. atrophaeus were conserved in many eubacteria.

Effect of the drug cyclophosphamide on the activity of porcine kidney betaine aldehyde dehydrogenase.

The enzyme betaine aldehyde dehydrogenase (BADH EC 1.2.1.8) catalyzes the synthesis of glycine betaine (GB), an osmolyte and osmoprotectant. Also, it participates in several metabolic pathways in humans. All BADHs known have cysteine in the active site involved in the aldehyde binding, whereas the porcine kidney enzyme (pkBADH) also has a neighborhood cysteine, both sensitive to oxidation. The antineoplastic and immuno-suppressant pre-drug cyclophosphamide (CTX), and its bioactivation products, have two highly oxidating chlorine atoms. This work aimed to analyze the effect of CTX in the activity of porcine kidney betaine aldehyde dehydrogenase. PkBADH was incubated with varying CTX concentration (0 to 2.0 mM) at 25 °C and lost 50 % of its activity with 2.0 mM CTX. The presence of the coenzyme NAD+ (0.5 mM) decreased 95% the activity in 2.0 mM CTX. The substrate betaine aldehyde (0.05 and 0.4 mM, and the products NADH (0.1-0.5 mM) and GB (1 and 10 mM) did not have an effect on the enzyme inactivation by CTX. The reducing agents, dithiothreitol and β-mercaptoethanol, reverted the pkBADH inactivation, but reduced glutathione (GSH) was unable to restore the enzyme activity. Molecular docking showed that CTX could enter at the enzyme active site, where its chlorine atoms may interact with the catalytic and the neighboring cysteines. The results obtained show that CTX inactivates the pkBADH due to oxidation of the catalytic cysteine or because it oxidizes catalytic and neighborhood cysteine, forming a disulfide bridge with a concomitant decrease in the activity of the enzyme.

Tibet Identification of the Fragrance Allele and Development of the Functional Markers for Fragrance in Taiguoxiaoxiangzhan

The fragrant rice breeding has become an important direction for hybrid rice breeding program. The selection and utilization of parent materials with ideal plant type, fine grain quality, strong resistance and other excellent agronomic characteristics with strong fragrance, especially the utilization and cultivation of the fragrant rice restorer lines, is of great significance to further promote the fragrant hybrid rice breeding. Taiguoxiaoxiangzhan is a restorer line with excellent grain quality, strong resistance and fragrance, which has been widely used in fragrant rice breeding in China. In order to trace the fragrance in Taiguoxiaoxiangzhan, we identified the type of the fragrance gene in Taiguoxiaoxiangzhan was&nbsp; badh2-E7&nbsp; which has an 8 bp deletion and three single nucleotide polymorphisms (SNPs) in exon 7, and this gene is a non-functional&nbsp; badh2 &nbsp;allele and that the functional&nbsp; Badh2 &nbsp;allele encoding betaine aldehyde dehydrogenase (BADH2) could render rice non-fragrant. Furthermore, we found that the Taiguoxiaoxiangzhan increased 2-acetyl-1-pyrroline (2-AP) content (0.108 mg/kg) compared with the non-fragrant rice varieties of Nipponbare and 9311 (&lt;0.01 mg/kg) by the GC-MS method. We also proved that FMbadh2-E7 and InDel-E7, two molecular markers reported earlier, can be applied in different genotypes, including&nbsp; Badh2/Badh2 &nbsp;(non-fragrance homozygote),&nbsp; Badh2/bahd2 &nbsp;(non-fragrance heterozygote) and&nbsp; badh2/badh2&nbsp; (fragrance homozygote). In addition, based on sequence divergence amongst the functional&nbsp; Badh2 &nbsp;and null&nbsp; badh2-E7 &nbsp;allele, we developed two functional markers, FMbadh2-E7A and FMbadh2-E7B, which can be easily used to distinguish non-fragrant from fragrant rice. Genetic analysis shows that the segregation of&nbsp; Badh2/Badh2 ,&nbsp; Badh2/badh2&nbsp; and &nbsp;badh2/badh2 &nbsp;in the F 2 &nbsp;population derived from crosses between Taiguoxiaoxiangzhan and different restorer lines fitted a theoretical ratio of 1:2:1, indicating that the fragrance of Taiguoxiaoxiangzhan is controlled by a single nuclear recessive gene ( bahd2 ). In this study, the identification and genetic analysis of the fragrance gene in Taiguoxiaoxiangzhan and the development of functional markers for fragrance have been studied, which provides a theoretical foundation for the further breeding of fragrant hybrid rice with excellent quality, high yield and strong resistance by molecular marker-assisted selection technology.

The JA‐responsive MYC2‐BADH‐like transcriptional regulatory module in Poncirus trifoliata contributes to cold tolerance by modulation of glycine betaine biosynthesis

Glycine betaine (GB) is known to accumulate in plants exposed to cold, but the underlying molecular mechanisms and associated regulatory network remain unclear. Here, we demonstrated that PtrMYC2 of Poncirus trifoliata integrates the jasmonic acid (JA) signal to modulate cold-induced GB accumulation by directly regulating PtrBADH-l, a betaine aldehyde dehydrogenase (BADH)-like gene. PtrBADH-l was identified based on transcriptome and expression analysis in P. trifoliata. Overexpression and VIGS (virus-induced gene silencing)-mediated knockdown showed that PtrBADH-l plays a positive role in cold tolerance and GB synthesis. Yeast one-hybrid library screening using PtrBADH-l promoter as baits unraveled PtrMYC2 as an interacting candidate. PtrMYC2 was confirmed to directly bind to two G-box cis-acting elements within PtrBADH-l promoter and acts as a transcriptional activator. In addition, PtrMYC2 functions positively in cold tolerance through modulation of GB synthesis by regulating PtrBADH-l expression. Interestingly, we found that GB accumulation under cold stress was JA-dependent and that PtrMYC2 orchestrates JA-mediated PtrBADH-l upregulation and GB accumulation. This study sheds new light on the roles of MYC2 homolog in modulating GB synthesis. In particular, wepropose a transcriptional regulatory module PtrMYC2-PtrBADH-l to advance the understanding of molecular mechanisms underlying the GB accumulation under cold stress.