A betaine aldehyde dehydrogenase gene in quinoa (<Emphasis Type="Italic">Chenopodium quinoa</Emphasis>): structure,phylogeny, and expression pattern

Betaine aldehyde dehydrogenase (BADH) is widely considered as a key enzyme in glycine betaine metabolism in higher plants. Several paralogous genes encoding different isozymes of BADH have been identified and characterized in some plants; however, until now, only limited information is available about BADH genes in quinoa (Chenopodium quinoa). Here, we report the molecular cloning, structural organization, phylogenetic evolution, and expression profile of a BADH gene (CqBADH1) from quinoa. The translated putative CqBADH1 protein included five conserved features of the ALDH Family 10. Comparisons between the cDNA and genomic sequences revealed that the CqBADH1 gene contained 15 exons and 14 introns. Comparative screening of introns in homologous genes demonstrated that the number and position of the BADH introns were highly conserved among the BADH genes in Amaranthaceae plants and in other more distantly related plant species. A phylogenetic analysis showed that CqBADH1 had the closest relationship with a protein from Atriplex canescens and belonged to the ALDH10 family. Expression profile analyses indicated that CqBADH1 was expressed only in root, and showed time-dependent expression profiles under NaCl-stress condition. Moreover, in quinoa, NaCl stress led to increased levels of CqBADH1 mRNA accompanied by the accumulation of glycine betaine. This is the first study to describe a BADH gene in quinoa.     

Genome-wide analysis and characterization of molecular evolution of the <Emphasis Type="Italic">HCT</Emphasis> gene family in pear (<Emphasis Type="Italic">Pyrus bretschneideri</Emphasis>)

Lignin is a major component of stone cells in pear fruit, which significantly affects fruit quality. Hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT), a recently discovered enzyme in plants, is an important gene that participates in the formation of lignin. Although HCT gene cloning and expression patterns have been studied in several species, including pear, there is still no extensive genome-wide bioinformatics analysis on the whole gene family, and the evolutionary history of HCT gene family is still unknown. A total of 82 HCT genes were identified in pear, most of which have one or two exons, and all with the conserved HXXXD motif and transferase domains. Based on the structural characteristics and phylogenetic analysis of these sequences, the HCT gene family genes could be classified into four main groups. Structural analysis also revealed that 25 % of HCT genes share a MYB binding site. Expansion of the HCT gene family mostly occurred before the divergence between Arabidopsis and Rosaceae, with whole-genome duplication or segmental duplication events playing the most important role in the expansion of the HCT gene family in pear. At the same time, purifying selection also played a critical role in the evolution of HCT genes. Five of the 82 HCT genes were verified by qRT-PCR to correspond to the pattern of stone cell formation during pear fruit development. The genome-wide identification, chromosome localization, gene structures, synteny, and expression analyses of pear HCT genes provide an overall insight into HCT gene family and their potential involvement in growth and development of stone cells.     

MdMYB4 enhances apple callus salt tolerance by increasing <Emphasis Type="Italic">MdNHX1</Emphasis> expression levels

Here, we performed comparative miRNA profiling in wild type and early flowering transgenic Chrysanthemum morifolium with constitutive expression of APETALA1 (AP1)-like gene, HAM92 (Helianthus annuus). Six sRNA libraries constructed from leaves and shoot apexes after the short day photoperiod initiation, as well as from opened inflorescence after anthesis were sequenced and analyzed. A total of 324 members (163 families) of putative conserved miRNAs and 30 candidate novel miRNAs specific for C. morifolium (cmo-miRNAs) were identified. Bioinformatic analysis revealed 427 and 138 potential mRNA targets for conserved and novel cmo-miRNAs, respectively. These genes were described in Gene Ontology terms and found to be implicated in a broad range of signaling pathways. Plant- and tissue-specific expression of 9 highly conserved cmo-miRNAs was compared between wild type and transgenic chrysanthemum lines with ectopic expression of AP1-like genes HAM92 and CDM111 (C. morifolium), using RT-qPCR and cmo-miR162a as a reference miRNA. The results of our study provide a framework for further investigation of miRNA evolution and functions in higher plants, as well as their roles in flowering control.     

The Lactate Dehydrogenase Gene <Emphasis Type="Italic">LDH-C1</Emphasis>, a New Molecular Marker for Phylogenetic Analysis of Salmonid Fishes (Salmoniformes: Salmonidae)

We tested the locus of the nuclear lactate dehydrogenase gene (LDH-C1) as a phylogenetic marker in specimens of 11 salmonid genera (Thymallus, Coregonus, Hucho, Brachymystax, Salmo, Salmothymus, Acantholingua, Parahucho, Salvelinus, Parasalmo, and Oncorhynchus). All the sequences were veraciously clustered according to their taxonomic affiliation at the species and genus levels. It is shown that used complex of characters contains a phylogenetic signal that represents specific information about the phylogenesis process. This allows us to recommend the LDH-C1 locus to specify the phylogeny of salmonids in the combined analysis of several independent nuclear genes and mitochondrial DNA.     

Human cytomegalovirus <Emphasis Type="Italic">UL145</Emphasis> gene is highly conserved among clinical strains

Human cytomegalovirus (HCMV), a ubiquitous human pathogen, is the leading cause of birth defects in newborns. A region (referred to as UL/b′) present in the Toledo strain of HCMV and low-passage clinical isolates) contains 22 additional genes, which are absent in the highly passaged laboratory strain AD169. One of these genes, UL145 open reading frame (ORF), is located between the highly variable genes UL144 and UL146. To assess the structure of the UL145 gene, the UL145 ORF was amplified by PCR and sequenced from 16 low-passage clinical isolates and 15 non-passage strains from suspected congenitally infected infants. Nine UL145 sequences previously published in the GenBank were used for sequence comparison. The identities of the gene and the similarities of its putative protein among all strains were 95.9–100% and 96.6–100%, respectively. The post-translational modification motifs of the UL145 putative protein in clinical strains were conserved, comprising the protein kinase C phosphorylation motif (PKC) and casein kinase II phosphorylation site (CK-II). We conclude that the structure of the UL145 gene and its putative protein are relatively conserved among clinical strains, irrespective of whether the strains come from patients with different manifestations, from different areas of the world, or were passaged or not in human embryonic lung fibroblast (HELF) cells.     

Identification,characterization, and expression of the <Emphasis Type="Italic">SWEET</Emphasis> gene family in <Emphasis Type="Italic">Phalaenopsis equestris</Emphasis> and <Emphasis Type="Italic">Dendrobium officinale</Emphasis>

Sugars are important molecules that function not only as primary metabolites, but also as nutrients and signal molecules in plants. The sugar transport protein genes family SWEET has been recently identified. The availability of the Dendrobium officinale and Phalaenopsis equestris genome sequences offered the opportunity to study the SWEET gene family in this two orchid species. We identified 22 and 16 putative SWEET genes, respectively, in the genomes of D. officinale and P. equestris using comprehensive bioinformatics analysis. Based on phylogenetic comparisons with SWEET proteins from Arabidopsis and rice, the DoSWEET and PeSWEET proteins could be divided into four clades; among these, clade II specifically lacked PeSWEETs and clade IV specifically lacked DoSWEETs, and there were orthologs present between D. officinale and P. equestris. Protein sequence alignments suggest that there is a predicted serine phosphorylation site in each of the highly conserved MtN3/saliva domain regions. Gene expression analysis in four tissues showed that three PeSWEET genes were most highly expressed in the flower, leaf, stem, and root, suggesting that these genes might play important roles in growth and development in P. equestris. Analysis of gene expression in different floral organs showed that five PeSWEET genes were highly expressed in the column (gynostemium), implying their possible involvement in reproductive development in this species. The expression patterns of seven PeSWEETs in response to different abiotic stresses showed that three genes were upregulated significantly in response to high temperature and two genes were differently expressed at low temperature. The results of this study lay the foundation for further functional analysis of SWEET genes in orchids.     

Genetic signatures of plant resistance genes with known function within and between species

Plant disease resistance (R) genes have undergone significant evolutionary divergence to cope with rapid changes in pathogens. These highly variable evolutionary patterns may have contributed to diversity in R gene protein families or structures. Here, the evolutionary patterns of 76 identified R genes and their homologs were investigated within and between plant species. Results demonstrated that nucleotide binding sites and leucine-rich-repeat genes located in loci with complex evolutionary histories tended to evolve rapidly, have high variation in copy numbers, exhibit high levels of nucleotide variation and frequent gene conversion events, and also exhibit high non-synonymous to synonymous substitution ratios in LRR regions. However, non-NBS-LRR R genes are relatively well conserved with constrained variation and are more likely to participate in the basic defense system of hosts. In addition, both conserved and highly divergent evolutionary patterns were observed for the same R genes and were consistent with inter- and intra-specific distributions of some R genes. These results thus indicate either continuous or altered evolutionary patterns between and within species. The present investigation is the first attempt to investigate evolutionary patterns among all clearly functional R genes. The results reported here thus provide a foundation for future plant disease studies.     

Genomic location and expression analysis of expansin gene family reveals the evolutionary and functional significance in <Emphasis Type="Italic">Triticum aestivum</Emphasis>

The plant-specific expansin proteins constitute an ancient and major gene family known to have roles in regulating diverse biological processes in plants. Although the functions of many expansin genes have been identified in wheat and other species, little is known about the evolution and genomic locations of the expansin genes in wheat (Triticum aestivum). In this study, a total of 87 expansin genes were identified in the wheat genome, including 52 EXPAs, 42 EXPBs and 4 EXLAs. The EXLB gene was not found in the wheat genome. Phylogenetic tree and comparative analysis revealed amplification of the EXPBs in rice, maize and wheat. The predicted wheat expansins were distributed across 14 of 21 chromosomes with different densities, 3 tightly co-located clusters and 15 paralogous pairs, indicating that tandem duplication and segmental duplication events also played roles in the evolution of expansins in wheat. In addition, the gene structures and conserved protein domains of wheat expansins suggest high levels of conservation within the phylogenetic subgroups. Analysis of a published microarray database showed that most wheat expansin genes exhibit different expression levels in different tissues and developmental stages. To our knowledge, this is the first report of a genome-wide analysis of the wheat expansin gene family, which should provide valuable information for further elucidating the classification and putative functions of the entire gene family.     

Fish growth hormone genes: Divergence of coding sequences in salmonid fishes

Comparison of coding nucleotide sequences of the paralogous GH1 and GH2 genes, as well as of the growth hormone amino acid sequences, in the species of closely related salmonid genera Salvelinus, Oncorhynchus, and Salmo was performed. It was demonstrated that, in different groups of salmonids, the amino acid substitution rates were considerably different. In some cases, an obvious discrepancy between the divergence of growth hormone genes and phylogenetic schemes based on other methods and approaches was revealed. These findings suggest that the reason may be multidirectional selection at duplicated genes at different stages of evolution.     

Analysis of <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> M145 genes <Emphasis Type="Italic">SCO4164</Emphasis> and <Emphasis Type="Italic">SCO5854</Emphasis> encoding putative rhodaneses

Streptomyces coelicolor genome carries two apparently paralogous genes, SCO4164 and SCO5854, that encode putative thiosulfate sulfurtransferases (rhodaneses). These genes (and their presumed translation products) are highly conserved and widely distributed across actinobacterial genomes. The SCO4164 knockout strain was unable to grow on minimal media with either sulfate or sulfite as the sole sulfur source. The SCO5854 mutant had no growth defects in the presence of various sulfur sources; however, it produced significantly less amounts of actinorhodin. Furthermore, we discuss possible links between basic interconversions of inorganic sulfur species and secondary metabolism in S. coelicolor.     

Adaptive mutation related to cellulose producibility in <Emphasis Type="Italic">Komagataeibacter medellinensis</Emphasis> (<Emphasis Type="Italic">Gluconacetobacter xylinus</Emphasis>) NBRC 3288

Gluconacetobacter xylinus (formerly Acetobacter xylinum and presently Komagataeibacter medellinensis) is known to produce cellulose as a stable pellicle. However, it is also well known to lose this ability very easily. We investigated the on and off mechanisms of cellulose producibility in two independent cellulose-producing strains, R1 and R2. Both these strains were isolated through a repetitive static culture of a non-cellulose-producing K. medellinensis NBRC 3288 parental strain. Two cellulose synthase operons, types I and II, of this strain are truncated by the frameshift mutation in the bcsBI gene and transposon insertion in the bcsCII gene, respectively. The draft genome sequencing of R1 and R2 strains revealed that in both strains the bcsBI gene was restored by deletion of a nucleotide in its C-rich region. This result suggests that the mutations in the bcsBI gene are responsible for the on and off mechanism of cellulose producibility. When we looked at the genomic DNA sequences of other Komagataeibacter species, several non-cellulose-producing strains were found to contain similar defects in the type I and/or type II cellulose synthase operons. Furthermore, the phylogenetic relationship among cellulose synthase genes conserved in other bacterial species was analyzed. We observed that the cellulose genes in the Komagataeibacter shared sequence similarities with the γ-proteobacterial species but not with the α-proteobacteria and that the type I and type II operons could be diverged from a same ancestor in Komagataeibacter.     

LIN7 Cell-Wall Invertase Orthologs in Cultivated and Wild Tomatoes (<Emphasis Type="Italic">Solanum</Emphasis> Section Lycopersicon)

Plant acid invertases are considered to be the key enzymes in sucrose unloading and carbohydrate supply to sink tissues. Acid cell-wall invertases control sucrose transport via the apoplastic pathway during sink initiation and expansion. In this study, we identified 12 LIN7 gene homologs encoding cell-wall invertases in red- and green-fruited tomato accessions (Solanum section Lycopersicon) of self-compatible and self-incompatible species. All genes consisted of six exons and five introns, including highly conserved 9-bp exon II. Identification of 226 exonic single nucleotide polymorphisms as well as extremely high intron variability indicates a significant interspecific divergence among the examined tomato accessions. Computational prediction revealed protein structure typical for the glycosyl hydrolase family 32 and conserved catalytic sites described for other plant cell-wall invertases. LIN7 expression in mature buds and flowers confirms LIN7 role in the development of pollen tubes and grains. The variability in gene and protein sequences and species-specific differences in LIN7 expression patterns may be responsible for putative functional divergence of invertases. Furthermore, we performed phylogenetic analysis of the Solanum section Lycopersicon species based on the LIN7 gene, which clearly divided the analyzed tomato accessions into two main clusters corresponding to self-compatible and self-incompatible species and was in agreement with the separation into red- and green-fruited plants. Given that LIN7 plays an essential role in tomato fertility and fruit ripening, the characterization of protein variability within species of section Lycopersicon may be useful to evaluate the potential application of the encoding genes for tomato breeding programs.     

Location of low copy genes in chromosomes of <Emphasis Type="Italic">Brachiaria</Emphasis> spp.

Repetitive DNA sequences have been widely used in cytogenetic analyses. The use of gene sequences with a low-copy-number, however, is little explored especially in plants. To date, the karyotype details in Brachiaria spp. are limited to the location of rDNA sites. The challenge lies in developing new probes based on incomplete sequencing data for the genus or complete sequencing of related species, since there are no model species with a sequenced genome in Brachiaria spp. The present study aimed at the physical location of conserved genes in chromosomes of Brachiaria ruziziensis, Brachiaria brizantha, and Brachiaria decumbens using RNAseq data, as well as sequences of Setaria italica and Sorghum bicolor through the fluorescent in situ hybridization technique. Five out of approximately 90 selected sequences generated clusters in the chromosomes of the species of Brachiaria studied. We identified genes in synteny with 5S and 45S rDNA sites, which contributed to the identification of chromosome pairs carrying these genes. In some cases, the species of Brachiaria evaluated had syntenic segments conserved across the chromosomes. The use of genomic sequencing data is essential for the enhancement of cytogenetic analyses.     

The kinase MSK1 is required for induction of c-<Emphasis Type="Italic">fos</Emphasis> by lysophosphatidic acid in mouse embryonic stem cells

Background

The regulation of the immediate-early gene c-fos serves as a paradigm for signal-activated gene induction. Lysophosphatidic acid is a potent serum-borne mitogen able to induce c-fos.

Results

Analysing the signalling events following stimulation of mouse embryonic stem cells with serum and lysophosphatidic acid, we show that the extracellular signal-regulated kinase (ERK) pathway is involved in mediating c-fos induction. We demonstrate that the ERK-activated kinase MSK1 is required for full c-fos promoter activation, as well as for the phosphorylation of cAMP-responsive element (CRE) binding proteins. We propose that MSK1 contributes to ERK-mediated c-fos promoter activation by targeting CRE binding proteins.

Conclusion

These results show that MSK1 is an important ERK-activated mediator of mitogen-stimulated c-fos induction. In addition, they indicate that MSK1 could act through CRE binding proteins to achieve c-fos promoter activation. Thus, they further our understanding of the complex regulation of the model immediate-early gene c-fos.

     

Phylogenetic characterization and novelty of organic sulphur metabolizing genes of <Emphasis Type="Italic">Rhodococcus</Emphasis> spp. (Eu-32)

Rhodococcus spp. (Eu-32) has the unique ability to metabolize organic sulphur containing compounds like dibenzothiophene through an extended sulphur specific pathway (Akhtar et al., in FEMS Microbiol Lett 301:95–102, 2009). Efforts were made to isolate and characterize the presumed desulphurizing genes (dszABC) involved in the sulphur specific pathway of isolate Eu-32 by employing standard and degenerate polymerase chain reaction primers. The partial dszA gene sequence of isolate Eu-32 showed 92 % sequence identity with a putative FMNH-2 dependent monooxygenase of Rhodococcus erythropolis PR4. The dszC gene sequence showed 99 % homology with the dibenzothiophene monooxygenase desulphurizing enzyme of another Rhodococcus species. The dszB gene was not unambiguously identified. A phylogenetic analysis by maximum likelihood method of the 16S rRNA gene and deduced DszA and C amino acid sequences suggest that horizontal gene transfer events might have taken place during the evolution of desulphurizing genes of Rhodococcus spp. (Eu-32).     

Identification of medaka magnetoreceptor and cryptochromes

Magnetoreception is a hallmark ability of animals for orientation and migration via sensing and utilizing geomagnetic fields. Magnetoreceptor (MagR) and cryptochromes (Cry) have recently been identified as the basis for magnetoreception in Drosophila. However, it has remained unknown whether MagR and Cry have conserved roles in diverse animals. Here we report the identification and expression of magr and cry genes in the fish medaka (Oryzias latipes). Cloning and sequencing identified a single magr gene, four cry genes and one cry-like gene in medaka. By sequence alignment, chromosomal synteny and gene structure analysis, medaka cry2 and magr were found to be the orthologs of human Cry2 and Magr, with cry1aa and cry1ab being coorthologs of human Cry1. Therefore, magr and cry2 have remained as single copy genes, whereas cry1 has undergone two rounds of gene duplication in medaka. Interestingly, magr and cry genes were detected in various stages throughout embryogenesis and displayed ubiquitous expression in adult organs rather than specific or preferential expression in neural organs such as brain and eye. Importantly, magr knockdown by morpholino did not produce visible abnormality in developing embryos, pointing to the possibility of producing viable magr knockouts in medaka as a vertebrate model for magnet biology.