The two nitrogen mobilisation- and senescence-associated <Emphasis Type="Italic">GS1</Emphasis> and <Emphasis Type="Italic">GDH</Emphasis> genes are controlled by C and N metabolites

In tobacco, the two enzymes of nitrogen metabolism, cytosolic glutamine synthetase (GS1; E.C.6.3.1.2) and glutamate dehydrogenase (GDH; E.C.1.4.1.2), are induced during leaf senescence, whereas the chloroplastic glutamine synthetase (GS2; E.C.6.3.1.2) and nitrate reductase (NR; E.C.1.6.1.1) are repressed in the course of ageing. In this report, we showed in discs of fully expanded Nicotiana tabacum L. cv. Xanthi leaves that sucrose (Suc) and amino acids were involved in the regulation of the expression of GS1 and GDH genes. Suc induced the expression of GS1 and repressed that of GDH. Therefore, we concluded that in response to Suc, GS1 behaved as an early Senescence Associated Gene (SAG), whereas GDH behaved as a late SAG. Moreover, amino acids induced the expression of both genes. Among the amino acids tested as signal molecules, proline (Pro) and glutamate (Glu) were major inducers of GDH and GS1 expression, respectively. Interestingly, an opposite regulation of GS1 and GS2 by Pro and Glu was shown. The contrary effect of Suc on NIA (NR encoding gene) and GDH mRNA accumulation was also emphasized.     

The COBRA gene family in <Emphasis Type="Italic">Populus</Emphasis> and gene expression in vegetative organs and in response to hormones and environmental stresses

COBRA proteins have been shown to be involved in both cell wall expansion and/or cellulose deposition. In this paper, we analyzed all 18 COBRA genes (PtCOBRA) from the completely sequenced Populus trichocarpa genome. The 14-member PtCOBRA subfamily I proteins have high similarities to the Arabidopsis (At) COB subfamily, and members with full length sequences were predicted to possess significant potentials for a GPI-anchor site. The 4-member PtCOBRA subfamily II proteins are 45% longer than subfamily I proteins and lack ω-attachment sites at the C terminus, and are more similar to AtCOBL7 subfamily. The expression of the Populus COBRA family genes were regulated in a tissue-specific manner, and were shown to also respond differentially to inductions of hormones and environmental stimuli which affect plant cell expansion. The high levels of expressions, particularly in shoot tip and young root organs, suggests that at least some Populus COBRA genes are likely involved in regulating cell expansion.     

The ankyrin repeat gene family in rice: genome-wide identification,classification and expression profiling

Ankyrin repeat (ANK) containing proteins comprise a large protein family. Although many members of this family have been implicated in plant growth, development and signal transduction, only a few ANK genes have been reported in rice. In this study, we analyzed the structures, phylogenetic relationship, genome localizations and expression profiles of 175 ankyrin repeat genes identified in rice (OsANK). Domain composition analysis suggested OsANK proteins can be classified into ten subfamilies. Chromosomal localizations of OsANK genes indicated nine segmental duplication events involving 17 genes and 65 OsANK genes were involved in tandem duplications. The expression profiles of 158 OsANK genes were analyzed in 24 tissues covering the whole life cycle of two rice genotypes, Minghui 63 and Zhenshan 97. Sixteen genes showed preferential expression in given tissues compared to all the other tissues in Minghui 63 and Zhenshan 97. Nine genes were preferentially expressed in stamen of 1 day before flowering, suggesting that these genes may play important roles in pollination and fertilization. Expression data of OsANK genes were also obtained with tissues of seedlings subjected to three phytohormone (NAA, GA3 and KT) and light/dark treatments. Eighteen genes showed differential expression with at least one phytohormone treatment while under light/dark treatments, 13 OsANK genes showed differential expression. Our data provided a very useful reference for cloning and functional analysis of members of this gene family in rice. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

NADP‐glutamate dehydrogenase activity in nonstarter lactic acid bacteria: effects of temperature,pH and NaCl on enzyme activity and expression

Aims: To screen the glutamate dehydrogenase (GDH) activity of nonstarter lactic acid bacteria (NSLAB) and to determine the effects of temperature, pH and NaCl values used for cheese ripening on enzyme activity and expression of GDH gene. Methods and Results: A subcellular fractionation protocol and specific enzyme assays were used. The effect of temperature, pH and NaCl on enzyme activity was evaluated. The expression of GDH gene was monitored by real‐time PCR. One selected strain was also used as adjunct starter for cheese making to evaluate the catabolism of free amino acids and the production of volatile organic compounds (VOC) during cheese ripening. The cytoplasm fraction of all strains showed in vitro NADP‐dependent GDH activity. NADP‐GDH activity was markedly strain dependent and varied according to the interactions between temperature, pH and NaCl. Lactobacillus plantarum DPPMA49 showed the highest NADP‐GDH activity under temperature, pH and NaCl values found during cheese ripening. RT‐PCR analysis revealed that GDH expression of Lact. plantarum DPPMA49 was down‐expressed by low temperature (<13°C) and over‐expressed by NaCl (1·87–5·62%). According to NADP‐GDH activity, the highest level of VOC (alcohols, aldehydes, miscellaneous and carboxylic acids) was found in cheeses made with DPPMA49. Conclusions: The results of this study may be considered as an example of the influence of temperature, pH and NaCl on enzyme activity and expression of functional genes, such as GDH, in cheese‐related bacteria. Significance and Impact of the Study: It focuses on the phenotypic and molecular characterization of the NADP‐GDH in lactobacilli under cheese‐ripening conditions. The findings of this study contribute to the knowledge about enzymes involved in the catabolism of amino acids, to be used as an important selection trait for cheese strains.     

Regulation of the <Emphasis Type="Italic">cellulose synthase-like</Emphasis> gene family by light in the maize mesocotyl

The cellulose synthase-like (ZmCSL) gene family of maize was annotated and its expression studied in the maize mesocotyl. A total of 28 full-length CSL genes and another 13 partial sequences were annotated; four are predicted to be pseudogenes. Maize has all of the CSL subfamilies that are present in rice, but the CSLC subfamily is expanded from 6 in rice to 12 in maize, and the CSLH subfamily might be reduced from 3 to 1. Unlike rice, maize has a gene in the CSLG subfamily, based on its sequence similarity to two genes annotated as CSLG in poplar. Light regulation of glycan synthase enzyme activities and CSL gene expression were analyzed in the mesocotyl. A Golgi-localized glucan synthase activity is reduced by ~50% 12 h after exposure to light. β-1,4-Mannan synthase activity is reduced even more strongly (>85%), whereas β-1,4-xylan synthase, callose synthase, and latent IDPase activity respond only slightly, if at all, to light. At least 17 of the CSL genes (42%) are expressed in the mesocotyl, of which four are up-regulated at least twofold, seven are down-regulated at least twofold, and six are not affected by light. The results contribute to our understanding of the structure of the CSL gene family in an important food and biofuel plant, show that a large percentage of the CSL genes are expressed in the specialized tissues of the mesocotyl, and demonstrate that members of the CSL gene family are differentially subject to photobiological regulation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Evolutionary dynamics of the immunoglobulin heavy chain variable region genes in vertebrates

Immunoglobulin heavy chains are polypeptides encoded by four genes: variable (IGHV), joining (IGHJ), diversity (IGHD), and constant (IGHC) region genes. The number of IGHV genes varies from species to species. To understand the evolution of the IGHV multigene family, we identified and analyzed the IGHV sequences from 16 vertebrate species. The results show that the numbers of functional and nonfunctional IGHV genes among different species are positively correlated. The number of IGHV genes is relatively stable in teleosts, but the intragenomic sequence variation is generally higher in teleosts than in tetrapods. The IGHV genes in tetrapods can be classified into three phylogenetic clans (I, II, and III). The clan III and/or II genes are relatively abundant, whereas clan I genes exist in small numbers or are absent in most species. The genomic organization of clan I, II, and III IGHV genes varies considerably among species, but the entire IGHV locus seems to be conserved in the subtelomeric or near-centromeric region of chromosome. The presence or absence of specific IGHV clan members and the lineage-specific expansion and contraction of IGHV genes indicate that the IGHV locus continues to evolve in a species-specific manner. Our results suggest that the evolution of IGHV multigene family is more complex than previously thought and that several factors may act synergistically for the development of antibody repertoire. Electronic supplementary materials The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Expression profile of calcium-dependent protein kinase (CDPKs) genes during the whole lifespan and under phytohormone treatment conditions in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L. ssp. <Emphasis Type="Italic">indica</Emphasis>)

Calcium-dependent protein kinases (CDPKs) control plant development and response to various stress environments through the important roles in the regulation of Ca²⁺ signaling. Thirty-one CDPK genes have been identified in the rice genome by a complete search of the genome based upon HMM profiles. In this study, the expression of this gene family was analyzed using the Affymetrix rice genome array in three rice cultivars: Minghui 63, Zhenshan 97, and their hybrid Shanyou 63 independently. Twenty-seven tissues sampled throughout the entire rice life-span were studied, along with three hormone treatments (GA3, NAA and KT), applied to the seedling at the trefoil stage. All 31 genes were found to be expressed in at least one of the experimental stages studied and revealed diverse expression patterns. We identified differential expression of the OsCPK genes in the stamen (1 day before flowering), the panicle (at the heading stage), the endosperm (days after pollination) and also in callus, in all three cultivars. Eight genes, OsCPK2, OsCPK11, OsCPK14, OsCPK22, OsCPK25, OsCPK26, OsCPK27 and OsCPK29 were found dominantly expressed in the panicle and the stamen, and five genes, OsCPK6, OsCPK7, OsCPK12, OsCPK23 and OsCPK31 were up-regulated in the endosperm stage. The OsCPK genes were also found to be regulated in rice seedlings subjected to different hormone treatment conditions, however their expression were not the same for all varieties. These diverse expression profiles trigger the functional analysis of the CDPK family in rice. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of <Emphasis Type="Italic">Glossy1</Emphasis>-homologous genes in rice involved in leaf wax accumulation and drought resistance

The outermost surfaces of plants are covered with an epicuticular wax layer that provides a primary waterproof barrier and protection against different environmental stresses. Glossy 1 (GL1) is one of the reported genes controlling wax synthesis. This study analyzed GL1-homologous genes in Oryza sativa and characterized the key members of this family involved in wax synthesis and stress resistance. Sequence analysis revealed 11 homologous genes of GL1 in rice, designated OsGL1-1 to  OsGL1-11. OsGL1-1, -2 and -3 are closely related to GL1. OsGL1-4, -5, -6, and -7 are closely related to Arabidopsis CER1 that is involved in cuticular wax biosynthesis. OsGL1-8, -9, -10 and -11 are closely related to SUR2 encoding a putative sterol desaturase also involved in epicuticular wax biosynthesis. These genes showed variable expression levels in different tissues and organs of rice, and most of them were induced by abiotic stresses. Compared to the wild type, the OsGL1-2-over-expression rice exhibited more wax crystallization and a thicker epicuticular layer; while the mutant of this gene showed less wax crystallization and a thinner cuticular layer. Chlorophyll leaching experiment suggested that the cuticular permeability was decreased and increased in the over-expression lines and the mutant, respectively. Quantification analysis of wax composition by GC–MS revealed a significant reduction of total cuticular wax in the mutant and increase of total cuticular wax in the over-expression plants. Compared to the over-expression and wild type plants, the osgl1-2 mutant was more sensitive to drought stress at reproductive stage, suggesting an important role of this gene in drought resistance. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genome-wide analysis of the auxin response factor (ARF) gene family in maize (<Emphasis Type="Italic">Zea mays</Emphasis>)

Auxin response factors (ARFs) are an important family involved in auxin-mediated response through specific binding to auxin response elements (AuxREs). A few members of the ARF family have been functionally characterized in Arabidopsis, rice (Oryza sativa), Poplar (Populous trichocarpa). However, little is known about ARF genes in maize (Zea mays). We performed a comprehensive bioinformatics analysis of the maize ARF gene family including analysis of the genome sequence, conserved domains, chromosomal locations, phylogenetic relationships, gene duplication, and expression profiles. 35 ZmARF genes were identified and categorized into four groups (Class I, II, III, and IV). In addition, a segmental ZmARF duplication event was shown to play an important role in maize ARF gene expansion. 7 ZmARF genes had no expression in specific tissues we obtained, but presented in mixed tissues according to the NCBI EST database, respectively. These studies have laid the theoretical foundation for further functional verification of these ZmARF genes.     

The use of the GDH gene for molecular identification and phylogenetic analysis of the yeast Kluyveromyces marxianus

Summary Our previous work showed that NADP⁺-dependent glutamate dehydrogenase from K. marxianus behaves similarly to its counterpart in S. cerevisiae. It suggested that the ammonia assimilation pathway might be different between K. marxianus and the genetic closed species K. lactis. In the present work, we analyzed the genetic similarity among the GDH gene family in K. marxianus and closed yeasts. Specific primers for GDH genes were designed based on the K. marxianus sequences deposited in the Génolevures Database. One of them, for the KmGDH2 gene, proved to be specific for K. marxianus DNA samples, which confirmed the molecular identification of environmental yeast isolates, and can be proposed for rapid screening of this yeast from environmental samples. The nucleotide sequence revealed that KmGDH2 belongs to the S. cerevisiae GDH1 gene family together with KlGDH gene.     

Molecular analyses of the rice tubby-like protein gene family and their response to bacterial infection

Tubby-like protein family has been identified in various multicellular organisms, indicating its fundamental functions in the organisms. However, the roles of plant tubby-like proteins are unknown. In this study, we have defined the tubby-like protein gene (OsTLP) family with 14 members in rice. Most of the OsTLPs harbor a tubby domain in their carboxyl terminus and an F-box domain in the amino terminus. The expression of all the OsTLPs was induced on infection of Xanthomonas oryzae pv. oryzae, which causes bacterial blight, one of the most devastating diseases of rice worldwide. The maximal expression levels were observed at 2–8 h after infection for all the genes. Eight of the 14 OsTLPs were also responsive to wounding. All the OsTLPs showed differential expression in different tissues at different developmental stages. However, four pairs of the 14 OsTLPs, with each pair having high sequence similarity and distributing on the similar position of different chromosomes, showed similar expression pattern in different tissues, indicating their direct relationship in evolution. These results suggest that the OsTLP family is involved in host–pathogen interaction and it may be also associated with other physiological and developmental activities. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Inventory and Comparative Evolution of the ABC Superfamily in the Genomes of <Emphasis Type="Italic">Phytophthora ramorum</Emphasis> and <Emphasis Type="Italic">Phytophthora sojae</Emphasis>

Automated and manual annotation of the ATP binding cassette (ABC) superfamily in the Phytophthora ramorum and P. sojae genomes has identified 135 and 136 members, respectively, indicating that this family is comparable in size to the Arabidopsis thaliana and rice genomes, and significantly larger than that of two fungal pathogens, Fusarium graminearum and Magnaporthe grisea. The high level of synteny between these oomycete genomes extends to the ABC superfamily, where 108 orthologues were identified by phylogenetic analysis. The largest subfamilies include those most often associated with multidrug resistance. The P. ramorum genome contains 22 multidrug resistance-associated protein (MRP) genes and 49 pleiotropic drug resistance (PDR) genes, while P. sojae contains 20 MRP and 49 PDR genes. Tandem duplication events in the last common ancestor appear to account for much of the expansion of these subfamilies. Recent duplication events in the PDR and ABCG families in both the P. ramorum and the P. sojae genomes indicate that selective expansion of ABC transporters may still be occurring. In other kingdoms, subfamilies define both domain arrangements and proteins having a common phylogenetic origin, but this is not the case for several subfamilies in oomycetes. At least one ABCG type transporter is derived from a PDR transporter, while transporters in the ABCB-half family cluster with transporters from bacterial, plant, and metazoan genomes. Additional examples of transporters that appear to be derived from horizontal transfer events from bacterial genomes include components of transporters associated with iron uptake and DNA repair. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Involvement of NAD(P)+-glutamate dehydrogenase isoenzymes in carbon and nitrogen metabolism in Chlamydomonas reinhardtü

The involvement of three NAD(P)₊-L-glutamate dehydrogenase (GDH; EC 1.4.1.3) isoenzymes, named GDH₁, GDH₂ and GDH₃, in the carbon and nitrogen metabolism of the green alga Chlamydomonas reinhardtü 6145c has been investigated under different environmental and stress conditions. GDH₁ activity decreased, but GDH₂ and GDH₃ activities increased with the age of cultures. When the extracellular ammonium concentration was high, only GDH₁ activity increased with growth whereas GDH₂ and GDH₃ remained unchanged. In the presence of L-methionine-D,L-sulfoximine (MSX), an inhibitor of L-glutamine synthetase (GS), a significant increase of GDH₁ and a slight decrease in GDH₃ activity was observed, whereas GDH₂ did not change. A significant increase in the intracellular 2-oxoglutarate was also found upon addition of azaserine, an inhibitor of L-glutamate synthase (GOGAT) activity. However, no significant changes in GDH isoenzyme activities were observed after addition of azaserine or azaserine plus MSX, except an induction of GDH₃ in the latter case. Moreover, in the presence of ethoxyzolamide (ETZ), an inhibitor of carbonic anhydrase activity, an induction of total GDH activity, mainly due to an increase in GDH₁ and to a minor extent in GDH₂, was observed in cells under low CO₂ (0.03%). In the dark, cells showed an increase in GDH₁ activity, but when acetate was present GDH₁ activity was repressed. All these results taken together suggest a relationship between GDH₁ and nitrogen assimilation, whereas GDH₂ and GDH₃ seem to be involved in the production of 2-oxoglutarate to fuel the tricarboxylic acid (TCA) cycle.     

Evaluation of genes from <Emphasis Type="Italic">eIF4E</Emphasis> and <Emphasis Type="Italic">eIF4G</Emphasis> multigenic families as potential candidates for partial resistance QTLs to <Emphasis Type="Italic">Rice yellow mottle virus</Emphasis> in rice

QTLs for partial resistance to Rice yellow mottle virus (RYMV) in rice were mapped in two populations of doubled-haploid lines (DHLs) and recombinant inbred lines (RILs) derived from the same cross but evaluated for different resistance criteria (virus content and symptom severity). An integrative map was used to compare the two genetic maps and a global analysis of both populations was performed. Most of the QTLs previously identified in DHL population were confirmed with increased significance and precision. As many recent studies evidenced the role of eukaryotic translation initiation factors (eIF) of 4E and 4G families in plant susceptibility to RNA viruses, we checked if these genes co-locate with QTLs of resistance to RYMV. Their systematic in silico identification was carried out on the rice genome and their physical locations were compared to QTL positions on the integrative map. In order to confirm or not the co-locations observed, the analysis was completed by evaluation of near-isogenic lines, QTL fine mapping and sequencing of candidate genes. Three members from eIF4G family could be retained as reliable candidates whereas eIF4E genes, commonly found to govern resistances in other plant/virus interactions, were discarded. Together with the recent identification of an eIF(iso)4G as a major resistance gene, data suggests an important role of genes from eIF4G family in rice resistance to RYMV but does not exclude the contribution of factors different from the translation initiation complex. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.