Conserved Amino Acid Sequences Research Articles

Advancing evolution: Bacteria break down gene silencer to express horizontally acquired genes.

Horizontal gene transfer advances bacterial evolution. To benefit from horizontally acquired genes, enteric bacteria must overcome silencing caused when the widespread heat-stable nucleoid structuring (H-NS) protein binds to AT-rich horizontally acquired genes. This ability had previously been ascribed to both anti-silencing proteins outcompeting H-NS for binding to AT-rich DNA and RNA polymerase initiating transcription from alternative promoters. However, we now know that pathogenic Salmonella enterica serovar Typhimurium and commensal Escherichia coli break down H-NS when this silencer is not bound to DNA. Curiously, both species use the same protease - Lon - to destroy H-NS in distinct environments. Anti-silencing proteins promote the expression of horizontally acquired genes without binding to them by displacing H-NS from AT-rich DNA, thus leaving H-NS susceptible to proteolysis and decreasing H-NS amounts overall. Conserved amino acid sequences in the Lon protease and H-NS cleavage site suggest that diverse bacteria degrade H-NS to exploit horizontally acquired genes.

Purification and Characterization of the Lecithin-Dependent Thermolabile Hemolysin Vhe1 from the Vibrio sp. Strain MA3 Associated with Mass Mortality of Pearl Oyster (Pinctada fucata).

In a previous study, we isolated a Vibrio sp. strain MA3 and its virulence factor, a hemolysin encoded by vhe1. This strain is associated with mass mortalities of the pearl oyster Pinctada fucata. In the present study, the vhe1 gene from strain MA3 was cloned and its encoded product was purified and characterized. Our results show that the vhe1 gene encodes a protein of 417 amino acids with an estimated molecular mass of 47.2kDa and a pI of 5.14. The deduced protein, Vhe1, was found to contain the conserved amino acid sequence (GDSL motif) of the hydrolase/esterase superfamily and five conserved blocks characteristic of SGNH hydrolases. A BLAST homology search indicated that Vhe1 belongs the lecithin-dependent hemolysin/thermolabile hemolysin (LDH/TLH) family. In activity analyses, the optimal temperature for both the hemolytic and phospholipase activities of Vhe1 was 50°C. Vhe1 hemolytic activity and phospholipase activity were highest at pH 8.5 and pH 8.0, respectively. However, both enzymatic activities sharply decreased at high temperature (> 50°C) and pH < 7.0. Compared with previously reported hemolysins, Vhe1 appeared to be more thermal- and pH-labile. Both its hemolytic activity and phospholipase activity were significantly inhibited by CuCl2, CdCl2, ZnCl2, and NiCl2, and slightly inhibited by MnCl2 and CoCl2. Vhe1 showed higher phospholipase activity toward medium-chain fatty acids (C8-C12) than toward shorter- and longer-chain fatty acids. These results accumulate knowledge about the LDH/TLH of V. alginolyticus, which detailed characterization has not been reported, and contribute to solving of the mass mortality of pearl oyster.

Expression of tissue factor pathway inhibitor 2 in the follicles of chicken ovaries and its regulatory mechanism in cultured granulosa cells1

Tissue factor pathway inhibitor 2 (TFPI2) plays a key role in female reproduction. However, its expression and function in chickens are still unclear. In the present study, ovarian tissues from 30 w and 15 w laying chickens were analyzed using RNA-seq to identify the differentially expressed gene TFPI2. The full-length cDNA of TFPI2 was obtained from adult chicken ovaries by rapid-amplification of cDNA ends (RACE), and the putative TFPI2 protein was found to share a highly conserved amino acid sequence with known bird homologs. In addition, TFPI2 was widely expressed in the tissues of adult chicken follicles according to quantitative real-time PCR (qRT–PCR) and Western blotting. Immunohistochemistry suggested that the TFPI2 protein existed in chicken ovary follicles at different developmental states, such as primordial follicles, the ovarian stroma, and the granulosa and theca layers of prehierarchical follicles (6-8 mm) and preovulatory follicles (F1). In vitro, Follicle stimulating hormone or Luteinizing hormone (FSH/LH) stimulated the expression of TFPI2 in chicken granulosa cells. FSH-/LH-induced TFPI2 mRNA expression was mediated by signaling pathways such as the PKA, PKC, PI3K, and mTOR pathways. Functionally, TFPI2 promoted the proliferation and viability of cultured granulosa cells and decreased the secretion of Progesterone (P4) and Estrogen (E2) and the mRNA abundance of key steroidogenic enzymes (STAR, Cyp17a1, Cyp19a1 and 3B-HSD) as well as MMPs (MMP2, 7, 9 and 11). Mechanistically, TFPI2 inhibited the expression of MMP7 via the Wnt signaling pathway. These findings indicate that TFPI2 may play an important role in regulating chicken follicular development and ovulation and suggest the molecular regulation mechanisms.

Human disease-associated calmodulin mutations alter calcineurin function through multiple mechanisms

Calmodulin (CaM) is a ubiquitous, calcium-sensing protein that regulates a multitude of processes throughout the body. In response to changes in [Ca2+], CaM modifies, activates, and deactivates enzymes and ion channels, as well as many other cellular processes. The importance of CaM is highlighted by the conservation of an identical amino acid sequence in all mammals. Alterations to CaM amino acid sequence were once thought to be incompatible with life. During the last decade modifications to the CaM protein sequence have been observed in patients suffering from life-threatening heart disease (calmodulinopathy). Thus far, inadequate or untimely interaction between mutant CaM and several proteins (LTCC, RyR2, and CaMKII) have been identified as mechanisms underlying calmodulinopathy. Given the extensive number of CaM interactions in the body, there are likely many consequences for altering CaM protein sequence. Here, we demonstrate that disease-associated CaM mutations alter the sensitivity and activity of the Ca2+-CaM-enhanced serine/threonine phosphatase calcineurin (CaN). Biophysical characterization by circular dichroism, solution NMR spectroscopy, stopped-flow kinetic measurements, and MD simulations provide mechanistic insight into mutation dysfunction as well as highlight important aspects of CaM Ca2+ signal transduction. We find that individual CaM point mutations (N53I, F89L, D129G, and F141L) impair CaN function, however, the mechanisms are not the same. Specifically, individual point mutations can influence or modify the following properties: CaM binding, Ca2+ binding, and/or Ca2+kinetics. Moreover, structural aspects of the CaNCaM complex can be altered in manners that indicate changes to allosteric transmission of CaM binding to the enzyme active site. Given that loss of CaN function can be fatal, as well as evidence that CaN modifies ion channels already associated with calmodulinopathy, our results raise the possibility that altered CaN function contributes to calmodulinopathy.

Genome-wide identification of Alfin Like (AL) transcription factors and their regulatory role in abiotic stress responses in Poplar (Populus trichocarpa)

Poplar is a valuable natural resource that is extensively utilized to manufacture many types of paper and wood products. It has considerable economic and ecological importance. However, forest dieback caused by environmental stressors has been increasing in recent years. Abiotic stress is managed by the Alfin-like (AL) gene in plants. The highly conserved DUF3594 domain and PHD-finger motif, which belong to the family Alfin-like transcription factors (AL-TFs), are essential for the metabolic and physiological reactions of plants to abiotic stress. Although the environmental stress sensitivity of AL-TFs has not yet been elucidated, these TFs play a vital role in regulating plant growth. We performed a database search and discovered 9 AL TFs. Using the AL-protein sequences from a variety of plant species, gene structures as well as conserved amino acid sequences, cis-elements of promoter regions, and events in the evolution of genes were examined. A phylogenetic tree was constructed and divided into six distinct groups. The expression of PtAL genes was specific to certain organs, and their gene mapping reveled that these genes are situated on seven out of the nineteen chromosomes present in Populus trichocarpa. Moreover, the majority of PtAL genes were strongly expressed in response to abiotic conditions such as low temperature (4 °C), high temperature (39 °C), salt (100 mM NaCl solution), and drought (Water holding). Two PtAL genes (PtAL4 and PtAL6) were sensitive to all abiotic stressors evaluated. The highly expressed PtAL genes (PtAL5 and PtAL7) discovered in this study could be employed in the Populus breeding program to generate stress-resistant cultivars. Our research shows that PtAL genes are very important for plant growth and development and for their ability to cope with environmental stress. This discovery offers valuable insights for improving plant stress tolerance.

The transcription factor Ste12-like increases the mycelial abiotic stress tolerance and regulates the fruiting body development of Flammulina filiformis.

Flammulina filiformis is one of the most commercially important edible fungi worldwide, with its nutritional value and medicinal properties. It becomes a good model species to study the tolerance of abiotic stress during mycelia growth in edible mushroom cultivation. Transcription factor Ste12 has been reported to be involved in the regulation of stress tolerance and sexual reproduction in fungi. In this study, identification and phylogenetic analysis of ste12-like was performed by bioinformatics methods. Four ste12-like overexpression transformants of F. filiformis were constructed by Agrobacterium tumefaciens-mediated transformation. Phylogenetic analysis showed that Ste12-like contained conserved amino acid sequences. All the overexpression transformants were more tolerant to salt stress, cold stress and oxidative stress than wild-type strains. In the fruiting experiment, the number of fruiting bodies of overexpression transformants increased compared with wild-type strains, but the growth rate of stipes slowed down. It suggested that gene ste12-like was involved in the regulation of abiotic stress tolerance and fruiting body development in F. filiformis.

Combining In Vitro and In Silico New Approach Methods to Investigate Type 3 Iodothyronine Deiodinase Chemical Inhibition Across Species.

New approach methodologies (NAMs) are being developed to reduce and replace vertebrate animal testing in support of ecotoxicology and risk assessment. The US Environmental Protection Agency's Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) bioinformatic tool was used to evaluate amino acid sequence conservation of the type 3 iodothyronine deiodinase (DIO3) enzyme across species to demonstrate NAM applications for understanding effects of chemical interactions with a specific protein target. Existing literature was used to identify critical amino acids for thyroid hormone binding and interaction with a reducing cofactor. The SeqAPASS tool identifies whether known critical amino acids involved in ligand binding are exact, partial, or not matches across species compared with a template species based on molecular weight and side chain classification. This evaluation guided the design of variant proteins representing critical amino acid substitutions found in various species. Site-directed mutagenesis of the wild-type (WT) human DIO3 gene sequence was used to create six variant proteins expressed in cell culture, which were then tested in vitro for chemical inhibition. Significant differences in in vitro median inhibitory concentration results were observed among variants for potential competitive inhibitors. A molecular model representing the WT human DIO3 was constructed using Molecular Operating Environment (MOE) software and mutated in silico to create the six variants. The MOE Site Finder tool identified the proposed catalytic and cofactor sites and potential alternative binding sites. Virtual docking did not provide affinity scores with sufficient resolution to rank the potency of the chemical inhibitors. Chemical characteristics, function and location of substituted amino acids, and complexities of the protein target are important considerations in developing NAMs to evaluate chemical susceptibility across species. Environ Toxicol Chem 2023;42:1032-1048. © 2023 University of Wisconsin-Madison. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Rotary properties of hybrid F1-ATPases consisting of subunits from different species

F1-ATPase (F1) is an ATP-driven rotary motor protein ubiquitously found in many species as the catalytic portion of FoF1-ATP synthase. Despite the highly conserved amino acid sequence of the catalytic core subunits: α and β, F1 shows diversity in the maximum catalytic turnover rate Vmax and the number of rotary steps per turn. To study the design principle of F1, we prepared eight hybrid F1s composed of subunits from two of three genuine F1s: thermophilic Bacillus PS3 (TF1), bovine mitochondria (bMF1), and Paracoccus denitrificans (PdF1), differing in the Vmax and the number of rotary steps. The Vmax of the hybrids can be well fitted by a quadratic model highlighting the dominant roles of β and the couplings between α-β. Although there exist no simple rules on which subunit dominantly determines the number of steps, our findings show that the stepping behavior is characterized by the combination of all subunits.

Fusarium mindanaoense sp. nov., a New Fusarium Wilt Pathogen of Cavendish Banana from the Philippines Belonging to the F. fujikuroi Species Complex.

The pathogen causing Fusarium wilt in banana is reported to be Fusarium oxysporum f. sp. cubense (FOC). In 2019, wilt symptoms in banana plants (cultivar: Cavendish) in the Philippines were detected, i.e., the yellowing of the leaves and discoloration of the pseudostem and vascular tissue. The fungus isolated from the vascular tissue was found to be pathogenic to Cavendish bananas and was identified as a new species, F. mindanaoense, belonging to the F. fujikuroi species complex (FFSC); species classification was assessed using molecular phylogenetic analyses based on the tef1, tub2, cmdA, rpb1, and rpb2 genes and morphological analyses. A reciprocal blast search using genomic data revealed that this fungus exclusively included the Secreted in Xylem 6 (SIX6) gene among the SIX homologs related to pathogenicity; it exhibited a highly conserved amino acid sequence compared with that of species in the FFSC, but not with that of FOC. This was the first report of Fusarium wilt in Cavendish bananas caused by a species of the genus Fusarium other than those in the F. oxysporum species complex.

Upstream open reading frame-mediated upregulation of ANAC082 expression in response to nucleolar stress in Arabidopsis.

Perturbations in ribosome biogenesis cause a type of cellular stress called nucleolar or ribosomal stress, which triggers adaptive responses in both animal and plant cells. The Arabidopsis ANAC082 transcription factor has been identified as a key mediator of the plant nucleolar stress response. The 5'-untranslated region (5'-UTR) of ANAC082 mRNA contains an upstream ORF (uORF) encoding an evolutionarily conserved amino acid sequence. Here, we report that this uORF mediates the upregulation of ANAC082 expression in response to nucleolar stress. When transgenic Arabidopsis plants containing a luciferase reporter gene under the control of the ANAC082 promoter and 5'-UTR were treated with reagents that induced nucleolar stress, expression of the reporter gene was enhanced in a uORF sequence-dependent manner. Additionally, we examined the effect of an endoplasmic reticulum (ER) stress-inducing reagent on reporter gene expression because the closest homolog of ANAC082 in Arabidopsis, ANAC103, is involved in the ER stress response. However, the ANAC082 uORF did not respond to ER stress. Interestingly, although ANAC103 has a uORF with an amino acid sequence similar to that of the ANAC082 uORF, the C-terminal sequence critical for regulation is not well conserved among ANAC103 homologs in Brassicaceae. Transient expression assays revealed that unlike the ANAC082 uORF, the ANAC103 uORF does not exert a sequence-dependent repressive effect. Altogether, our findings suggest that the ANAC082 uORF is important for the nucleolar stress response but not for the ER stress response, and that for this reason, the uORF sequence-dependent regulation was lost in ANAC103 during evolution.

Evolutionary conservation of sequence motifs at sites of protein modification

Gene duplications are common in biology and are likely to be an important source of functional diversification and specialization. The yeast Saccharomyces cerevisiae underwent a whole-genome duplication event early in evolution, and a substantial number of duplicated genes have been retained. We identified more than 3500 instances where only one of two paralogous proteins undergoes posttranslational modification despite having retained the same amino acid residue in both. We also developed a web-based search algorithm (CoSMoS.c.) that scores conservation of amino acid sequences based on 1011 wild and domesticated yeast isolates and used it to compare differentially modified pairs of paralogous proteins. We found that the most common modifications-phosphorylation, ubiquitylation, and acylation but not N-glycosylation-occur in regions of high sequence conservation. Such conservation is evident even for ubiquitylation and succinylation, where there is no established 'consensus site' for modification. Differences in phosphorylation were not associated with predicted secondary structure or solvent accessibility but did mirror known differences in kinase-substrate interactions. Thus, differences in posttranslational modification likely result from differences in adjoining amino acids and their interactions with modifying enzymes. By integrating data from large-scale proteomics and genomics analysis, in a system with such substantial genetic diversity, we obtained a more comprehensive understanding of the functional basis for genetic redundancies that have persisted for 100 million years.

Unraveling the Diverse Roles of Neglected Genes Containing Domains of Unknown Function (DUFs): Progress and Perspective.

Domain of unknown function (DUF) is a general term for many uncharacterized domains with two distinct features: relatively conservative amino acid sequence and unknown function of the domain. In the Pfam 35.0 database, 4795 (24%) gene families belong to the DUF type, yet, their functions remain to be explored. This review summarizes the characteristics of the DUF protein families and their functions in regulating plant growth and development, generating responses to biotic and abiotic stress, and other regulatory roles in plant life. Though very limited information is available about these proteins yet, by taking advantage of emerging omics and bioinformatic tools, functional studies of DUF proteins could be utilized in future molecular studies.

The protein family of pyruvate:quinone oxidoreductases: Amino acid sequence conservation and taxonomic distribution

Pyruvate:quinone oxidoreductases (PQOs) catalyse the oxidative decarboxylation of pyruvate to acetate and concomitant reduction of quinone to quinol with the release of CO2. They are thiamine pyrophosphate (TPP) and flavin-adenine dinucleotide (FAD) containing enzymes, which interact with the membrane in a monotopic way. PQOs are considered as part of alternatives to most recognized pyruvate catabolizing pathways, and little is known about their taxonomic distribution and structural/functional relationship.In this bioinformatics work we tackled these gaps in PQO knowledge. We used the KEGG database to identify PQO coding genes, performed a multiple sequence analysis which allowed us to study the amino acid conservation on these enzymes, and looked at their possible cellular function. We observed that PQOS are enzymes exclusively present in prokaryotes with most of the sequences identified in bacteria. Regarding the amino acid sequence conservation, we found that 75 amino acid residues (out of 570, on average) have a conservation over 90 %, and that the most conserved regions in the protein are observed around the TPP and FAD binding sites. We systematized the presence of conserved features involved in Mg2+, TPP and FAD binding, as well as residues directly linked to the catalytic mechanism. We also established the presence of a new motif named “HEH lock”, possibly involved in the dimerization process. The results here obtained for the PQO protein family contribute to a better understanding of the biochemistry of these respiratory enzymes.

Generation of an RNA aptamer against LipL32 of Leptospira isolated by Tripartite-hybrid SELEX coupled with in-house Python-aided unbiased data sorting

Leptospirosis is a potentially life-threatening zoonosis caused by pathogenic Leptospira. The major hurdle of the diagnosis of Leptospirosis lies in the issues associated with current methods of detection, which are time-consuming, tedious and the need for sophisticated, special equipments. Restrategizing the diagnostics of Leptospirosis may involve considerations of the direct detection of the outer membrane protein, which can be faster, cost-saving and require fewer equipments. One such promising marker is LipL32, which is an antigen with high amino acid sequence conservation among all the pathogenic strains. In this study, we endeavored to isolate an aptamer against LipL32 protein via a modified SELEX strategy known as tripartite-hybrid SELEX, based on 3 different partitioning strategies. In this study, we also demonstrated the deconvolution of the candidate aptamers by using in-house Python-aided unbiased data sorting in examining multiple parameters to isolate potent aptamers. We have successfully generated an RNA aptamer against LipL32 of Leptospira, LepRapt-11, which is applicable in a simple direct ELASA for the detection of LipL32. LepRapt-11 can be a promising molecular recognition element for the diagnosis of leptospirosis by targeting LipL32.

VLF: An R package for the analysis of very low frequency variants in DNA sequences.

Here, we introduce VLF, an R package to determine the distribution of very low frequency variants (VLFs) in nucleotide and amino acid sequences for the analysis of errors in DNA sequence records. The package allows users to assess VLFs in aligned and trimmed protein-coding sequences by automatically calculating the frequency of nucleotides or amino acids in each sequence position and outputting those that occur under a user-specified frequency (default of p = 0.001). These results can then be used to explore fundamental population genetic and phylogeographic patterns, mechanisms and processes at the microevolutionary level, such as nucleotide and amino acid sequence conservation. Our package extends earlier work pertaining to an implementation of VLF analysis in Microsoft Excel, which was found to be both computationally slow and error prone. We compare those results to our own herein. Results between the two implementations are found to be highly consistent for a large DNA barcode dataset of bird species. Differences in results are readily explained by both manual human error and inadequate Linnean taxonomy (specifically, species synonymy). Here, VLF is also applied to a subset of avian barcodes to assess the extent of biological artifacts at the species level for Canada goose (Branta canadensis), as well as within a large dataset of DNA barcodes for fishes of forensic and regulatory importance. The novelty of VLF and its benefit over the previous implementation include its high level of automation, speed, scalability and ease-of-use, each desirable characteristics which will be extremely valuable as more sequence data are rapidly accumulated in popular reference databases, such as BOLD and GenBank.

Potential for mercury methylation by Asgard archaea in mangrove sediments.

Methylmercury (MeHg) is a potent neurotoxin that bioaccumulates along food chains. The conversion of MeHg from mercury (Hg) is mediated by a variety of anaerobic microorganisms carrying hgcAB genes. Mangrove sediments are potential hotspots of microbial Hg methylation; however, the microorganisms responsible for Hg methylation are poorly understood. Here, we conducted metagenomic and metatranscriptomic analyses to investigate the diversity and distribution of putative microbial Hg-methylators in mangrove ecosystems. The highest hgcA abundance and expression occurred in surface sediments in Shenzhen, where the highest MeHg concentration was also observed. We reconstructed 157 metagenome-assembled genomes (MAGs) carrying hgcA and identified several putative novel Hg-methylators, including one Asgard archaea (Lokiarchaeota). Further analysis of MAGs revealed that Deltaproteobacteria, Euryarchaeota, Bacteroidetes, Chloroflexi, and Lokiarchaeota were the most abundant and active Hg-methylating groups, implying their crucial role in MeHg production. By screening publicly available MAGs, 104 additional Asgard MAGs carrying hgcA genes were identified from a wide range of coast, marine, permafrost, and lake sediments. Protein homology modelling predicts that Lokiarchaeota HgcAB proteins contained the highly conserved amino acid sequences and folding structures required for Hg methylation. Phylogenetic tree revealed that hgcA genes from Asgard clustered with fused hgcAB genes, indicating a transitional stage of Asgard hgcA genes. Our findings thus suggest that Asgard archaea are potential novel Hg-methylating microorganisms and play an important role in hgcA evolution.

AepG is a glucuronosyltransferase involved in acidic exopolysaccharide synthesis and contributes to environmental adaptation of Haloarcula hispanica

The attachment of a sugar to a hydrophobic lipid carrier is the first step in the biosynthesis of many glycoconjugates. In the halophilic archaeon Haloarcula hispanica, HAH_1206, renamed AepG, is a predicted glycosyltransferase belonging to the CAZy Group 2 family that shares a conserved amino acid sequence with dolichol phosphate mannose synthases. In this study, the function of AepG was investigated by genetic and biochemical approaches. We found that aepG deletion led to the disappearance of dolichol phosphate-glucuronic acid. Our biochemical assays revealed that recombinant cellulose-binding, domain-tagged AepG could catalyze the formation of dolichol phosphate-glucuronic acid in time- and dose-dependent manners. Based on the invivo and invitro analyses, AepG was confirmed to be a dolichol phosphate glucuronosyltransferase involved in the synthesis of the acidic exopolysaccharide produced by H.hispanica. Furthermore, lack of aepG resulted in hindered growth and cell aggregation in high salt medium, indicating that AepG is vital for the adaptation of H.hispanica to a high salt environment. In conclusion, AepG is the first dolichol phosphate glucuronosyltransferase identified in any of the three domains of life and, moreover, offers a starting point for further investigation into the diverse pathways used for extracellular polysaccharide biosynthesis in archaea.

A non-synonymous SNP in homolog of BADH2 gene is associated with fresh pod fragrance in dolichos bean (Lablab purpureus var. lignosus (Prain) Kumari).

Fresh pods are harvestable and marketable economic product in dolichos bean. Fresh pod fragrance is one of the 'farmers' and 'consumers' preferred traits in dolichos bean varieties. The pods with high fragrance fetch a premium price in the market. In breeding programmes, pod fragrance is routinely assessed by organoleptic (sensory) means, which is highly relative and subjective. Identification of linked DNA markers not only offer an objective means but also enable selection of fragment genotypes at seedling stage itself. Betaine aldehyde dehydrogenase (BADH) is known to be the key gene responsible for fragrance in other legumes such as vegetable soybean and mung bean. Taking cues from highly conserved domains in proteins coded by BADH genes, we isolated dolichos bean homolog (LpBADH2) of soybean GmBADH2 gene using reported degenerate primers designed to conserved domains. Analysis of the translated amino acid sequence of LpBADH2 showed high degree of similarity (97.30%) with those of soybean homolog (GmBADH2). Conserved amino acid sequence of aldehyde dehydrogenase-super family were also identified in LpBADH2. Multiple sequence alignment of nucleotide sequences of LpBADH2 with those of related legumes using "ClustalW" revealed the presence of a single non-synonymous single nucleotide polymorphic (SNPs) and three synonymous SNP sites in LpBADH2. The substitution of the amino acid tyrosine in (fragrant genotypes) with phenyl alanine (non-fragrant genotypes) in protein coded by LpBADH2 appeared to be the cause for switch over from fragrance to non-fragrance in dolichos bean. These results are discussed in relation to strategies to breed dolichos bean cultivars with desired level of pod fragrance.

Genome-wide characterization of Alfin-like (AL) genes in apple and functional identification of MdAL4 in response to drought stress.

Eleven Alfin-like (AL) genes were obtained from apple and MdAL4 was selected for improving drought stress tolerance of transgenic apple callus and Arabidopsis. Drought is an important environmental factor affecting plant growth all over the world. Alfin-like (AL) have well-documented functions in abiotic stress response, but their drought stress tolerance in apple (Malus domestica) are poorly understood. According to the transcriptome data, 11 MdAL genes containing conserved Alfin and PHD-finger domain were identified in apple and divided into three subgroups with a total of 35 members from different species. Subsequently, gene structures, conserved amino acid sequences, promoter cis-acting elements, and gene evolution events were analyzed. Based on differential expression of MdALs in response to abiotic stresses, MdAL4, which was highly expressed under drought, was further cloned and investigated. MdAL4 encoding nuclear-localized protein conferred enhanced drought tolerance in overexpressing transgenic calli of apple 'Orin'. Moreover, the ectopic expression of MdAL4 improved the drought tolerance of transgenic Arabidopsis, as judged from remarkably decreased malonaldehyde (MDA) content and electrolyte leakage in MdAL4 overexpressing plants relative to WT. Furthermore, MdAL4 possibly could bind to promoter regions of ROS-scavenging and stress-related genes to improve drought tolerance. Additionally, we found in silico evidence that three proteins containing the WD40 domain that interact with MdAL4. Based on these results, MdAL4 was identified as a positive regulator for improving drought stress of apple.

Membrane Potential-requiring Succinate Dehydrogenase Constitutes the Key to Propionate Oxidation and Is Unique to Syntrophic Propionate-oxidizing Bacteria

Propionate oxidation in Pelotomaculum thermopropionicum is performed under a thermodynamic limit. The most energetically unfavorable reaction in the propionate oxidation pathway is succinate oxidation. Based on previous genomic and transcriptomic ana-lyses, succinate oxidation in P. thermopropionicum under propionate-oxidizing conditions is conducted by the membrane-bound forms of two succinate dehydrogenases (SDHs). We herein examined the activity of SDH, the mechanisms underlying the succinate oxidation reaction in P. thermopropionicum, and the importance of the protein sequences of related genes. SDH activity was highly localized to the membrane fraction. An ana-lysis of the soluble fraction revealed that fumarate reductase received electrons from NADH, suggesting the involvement of membrane-bound SDH in propionate oxidation. We utilized an uncoupler and inhibitors of adenosine triphosphate (ATP) synthase and membrane-bound SDH to investigate whether the membrane potential of P. thermopropionicum supports propionate oxidation alongside hydrogen production. These chemicals inhibited hydrogen production, indicating that membrane-bound SDH requires a membrane potential for succinate oxidation, and this membrane potential is maintained by ATP synthase. In addition, the phylogenetic distribution of the flavin adenine dinucleotide-binding subunit and conserved amino acid sequences of the cytochrome b subunit of SDHs in propionate-oxidizing bacteria suggests that membrane-bound SDHs possess specific conserved amino acid residues that are strongly associated with efficient succinate oxidation in syntrophic propionate-oxidizing bacteria.