Cloning and expression analyses of AtMRP4, a novel MRP-like gene from Arabidopsis thaliana

In all organisms glutathione-conjugate transporters (GS-X pumps) mediate the detoxification of a number of xenobiotics by removing them from the cytosol. In addition, GS-X pumps appear to play a role in the processing of endogenous compounds. We have isolated a novel genomic clone from Arabidopsis thaliana that encodes a putative GS-X pump, AtMRP4, which is part of a recently defined gene family. The derived amino acid sequence shares high levels of similarity (55–63%) with human, yeast, and other Arabidopsis homologues. The expression of the different members of the AtMRP gene family in Arabidopsis cell suspensions after treatment with chemicals that modify glutathione metabolism (compounds that induce different types of stress and that act as herbicide antidotes – safeners – in monocotyledonous species) revealed that the members of this gene family are differentially regulated.     

Rice has two distinct classes of protein kinase genes related to SNF1 of Saccharomyces cerevisiae , which are differently regulated in early seed development

We have isolated five cDNA clones (osk1–5) for protein kinases from rice which are related to SNF1 protein kinase of Saccharomyces cerevisiae. Based on the sequence homology, these cDNAs can be classified into two groups, group 1 (osk1) and group 2 (osk2–5). The products of these genes were demonstrated to be functional SNF1-related protein kinases by in vitro and in vivo experiments. Recombinant proteins expressed from both groups of genes were fully active as protein kinases and could phosphorylate SAMS peptide, a substrate specific for the SNF1/AMPK family, as well as themselves (autophosphorylation). Moreover, expression of osk3 cDNA in yeast snf1 mutants restored SNF1 function. Northern blot analyses showed differential expression of these two gene groups; group 1 is expressed uniformly in growing tissues (young roots, young shoots, flowers, and immature seeds), whereas group 2 is strongly expressed in immature seeds. SNF1-related protein kinases have been reported from different plant species, such as rye, barley, Arabidopsis, tobacco, and potato, while the type of gene strongly expressed in immature seeds is known only in cereals such as rye, barley, and, from our findings, in rice. Expression levels of the group 2 genes were further analyzed in seeds during seed maturation. Expression is transiently increased in the early stages of seed maturation and then decreases. The expression peak precedes those of the sbe1 and waxy genes, which are involved in starch synthesis in rice. Taken together, these findings suggest that group 2 OSK genes play important roles in the early stages of endosperm development in rice seeds. Received: 30 April 1998 / Accepted: 20 August 1998     

Three members of the S multigene family are linked to the S locus of Brassica

Two self-incompatibility genes in Brassica, SLG and SRK (SLG encodes a glycoprotein; SRK encodes a receptor-like kinase), are included in the S multigene family. Products of members of the S multigene family have an SLG-like domain (S domain) in common, which may function as a receptor. In this study, three clustered members of the S multigene family, BcRK1, BcRL1 and BcSL1, were characterized. BcRK1 is a putative functional receptor kinase gene expressed in leaves, flower buds and stigmas, while BcRL1 and BcSL1 are considered to be pseudogenes because deletions causing frameshifts were identified in these sequences. Sequence and expression pattern of BcRK1 were most similar to those of the Arabidopsis receptor-like kinase gene ARK1, indicating that BcRK1 might have a function similar to that of ARK1, in processes such as cell expansion or plant growth. Interestingly, the region containing BcRK1, BcRL1 and BcSL1 is genetically linked to the S locus and the physical distance between SLG, SRK and the three S-related genes was estimated to be less than 610 kb. Thus the genes associated with self-incompatibility exist within a cluster of S-like genes in the genome of Brassica. Received: 15 April 1997 / Accepted: 13 June 1997     

Characterization of Repetitive DNA Elements in Arabidopsis

We have applied computational methods to the available database and identified several families of repetitive DNA elements in the Arabidopsis thaliana genome. While some of the elements have features expected of either miniature inverted-repeat transposable elements (MITEs) or retrotransposons, the most abundant class of repetitive elements, the AthE1 family, is structurally related to neither. The AthE1 family members are defined by conserved 5′ and 3′ sequences, but these terminal sequences do not represent either inverted or direct repeats. AthE1 family members with greater than 98% identity are easily identified on different Arabidopsis chromosomes. Similar to nonautonomous DNA-based transposon families, the AthE1 family contains members in which the conserved terminal domains flank unrelated sequences. The primary utility of characterizing repetitive sequences is in defining, at least in part, the evolutionary architecture of specific Arabidopsis loci. The repetitive elements described here make up approximately 1% of the available Arabidopsis thaliana genomic sequence. Received: 13 October 1998 / Accepted: 30 December 1998     

Induction by alkaloids and phenobarbital of Family 4 Cytochrome P450s in Drosophila : evidence for involvement in host plant utilization

In vertebrates, cytochrome P450s of the CYP2 and CYP3 families play a dominant role in drug metabolism, while in insects members of the CYP6 and CYP28 families have been implicated in metabolism of insecticides and toxic natural plant compounds. A degenerate 3^′ RACE strategy resulted in the identification of fifteen novel P450s from an alkaloid-resistant species of Drosophila. The strong (17.4-fold) and highly specific induction of a single gene (CYP4D10) by the toxic isoquinoline alkaloids of a commonly utilized host-plant (saguaro cactus) provides the first indication that members of the CYP4 family in insects may play an important role in the maintenance of specific insect-host plant relationships. Strong barbiturate inducibility of CYP4D10 and two other D. mettleri P450 sequences of the CYP4 family was also observed, suggesting a pattern of xenobiotic responsiveness more similar to those of several vertebrate drug-metabolizing enzymes than to putative vertebrate CYP4 homologs. Received: 14 August 1997 / Accepted: 24 March 1998     

Growth arrest of Schizosaccharomyces pombe following overexpression of mouse type 1 protein phosphatases

The rice disease resistance gene Xa21, which encodes a receptor-like kinase, is a member of a multigene family. Based on comparisons of genomic␣sequences of seven family members, seventeen transposon-like elements were identified in the 5′ and 3′ flanking regions and introns of these genes. Sequence characterization revealed that these elements are diverse, showing similarity to maize Ds, CACTA and miniature inverted repeat-like elements, as well as novel elements. Only two elements were located in presumed coding regions, indicating that integration of transposable elements at the Xa21 disease resistance locus occurred preferentially in noncoding regions. Received: 17 October 1997 / Accepted: 3 February 1998     

Cloning and characterization of PhPI9 involved in floral development from Phalaenopsis Orchid

In the attempt to discover new genes involved in the floral development in monocotyledonousin species, we have cloned and characterized the homologous PISTALLATA-like (PI-like) gene from Phalaenopsis hybrid cultivar named PhPI9 (Ph alaenopsis PI STILLATA # 9). The cDNA of PhPI9 has a fragment of 834 bp and has 60% identity with the PISTILATA from Arabidopsis. The deduced amino acid sequence of PhPI9 had the typical PI-motif. It also formed a subclade with other monocot PI-type genes in phylogenetic analysis. Southern analysis showed that PhPI9 was present in the Phalaenopsis orchid genome as a single copy. Furthermore, it was expressed only in the lip of the Phalaenopsis flower and no expression was detected in vegetative organs. Thus, as a B-function MADS-box gene, PhPI9 specifies floral organ identity in orchids. __________ Translated from Journal of Fudan University (Natural Science), 2006, 45(3): 277–282 [译自: 复旦学报(自然科学版)]     

Genetic differentiation of Arthrobacter population from heavy metal-contaminated environment

Six samples containing extremely high concentration of Pb, Zn, and Cd were obtained from the layers of 5–10 cm and 25–30 cm three tailing piles, with ages of about 10, 20 and more than 80 years, respectively. Then, 48 bacterial strains were obtained from these samples, and subsequently their phylogenetic positions were determined by analysis on the partial sequence of 16S rRNA gene (fragment length ranging from 474 to 708 bp). These isolates were members of the Arthrobacter genus, phylogenetically close to A. keyseri and A. ureafaciens, with sequence ranging from 99.1% to 100%. Furthermore, genetic variation between subpopulations from different samples was revealed by analysis on their randomly amplified polymorphic DNA profile. Nei genetic distance showed that the greatest differentiation occurred between subpopulation A and C. Notably, either genetic distance between subpopulations from the layers of 5–10 cm and 25–30 cm of each tailing pile or between same layers of different tailing pile increased with the history of tailings. Moreover, correlation analysis showed that soluble Pb has a significantly negative relationship with Nei’ gene diversity of subpopulation. It was assumed that soluble Pb may be responsible for the reduced genetic diversity of the Arthrobacter population. Our data provided evidence that genetic differentiation of microbial populations was consistent with the changes of environmental factors, particularly heavy metals. Translated from Acta Ecologica Sinica, 2005, 25(10): 2569–2573 [译自: 生态学报]     

The gene cluster inlC2DE of Listeria monocytogenes contains additional new internalin genes and is important for virulence in mice

In this work we identified and characterized a gene cluster containing three internalin genes of Listeria monocytogenes EGD. These genes, termed inlG, inlH and inlE, encode proteins of 490, 548 and 499 amino acids, respectively, which belong to the family of large, cell wall-bound internalins. The inlGHE gene cluster is flanked by two listerial house-keeping genes encoding proteins homologous to the 6-phospho-β-glucosidase and the succinyl-diaminopimelate desuccinylase of E. coli. A similar internalin gene cluster, inlC2DE, localised to the same position on the L. monocytogenes EGD chromosome was recently described in a different isolate (Dramsi S, Dehoux P, Lebrun M, Goossens PL, Cossart P (1997) Infect Immun 65: 1615–1625). Sequence comparison of the two inl gene clusters indicates that inlG is a new internalin gene, while inlH was generated by a site-specific recombination, leading to an in-frame deletion which removed the 3′-terminal end of inlC2 and the 5′-terminal part of inlD. The third gene of the inlGHE cluster, inlE, is almost identical to the previously reported inlE gene. Our data show that the inlGHE gene cluster is probably transcribed from a major PrfA- independent promoter located upstream of inlG. PCR analysis revealed the presence of the newly identified inl genes inlG and inlH in most L. monocytogenes isolates tested. A mutant which has lost inlG, inlH and inlE by an in-frame deletion exhibited, after oral infection of mice, a significant loss in virulence and shows drastically reduced numbers of viable bacteria in both liver and spleen when compared to the wild-type strain. Received: 21 April 1998 / Accepted: 5 June 1998     

Ectopic expression of mitochondrial gamma carbonic anhydrase 2 causes male sterility by anther indehiscence

Plant mitochondria include gamma-type carbonic anhydrases (γCAs) of unknown function. In Arabidopsis, the γCAs form a gene family of five members which all are attached to the NADH dehydrogenase complex (complex I) of the respiratory chain. Here we report a functional analysis of gamma carbonic anhydrase 2 (CA2). The gene encoding CA2 is constitutively expressed in all plant organs investigated but it is ten fold induced in flowers, particularly in tapetal tissue. Ectopic expression of CA2 in Arabidopsis causes male sterility in transgenic plants. In normal anther development, secondary thickenings of the endothecial cell wall cause anthers to open upon dehydration. Histological analyses revealed that abnormal secondary thickening prevents anther opening in 35S::CA2 transgenic plants. CA2 abundance in transgenic plants is increased 2–3 fold compared to wild-type plants as revealed by Western blotting analyses. Moreover, abundance of other members of the CA family, termed CA3 and CAL2, is increased in transgenic plants. Oxygen uptake measurements revealed that respiration in transgenic plants is mainly based on NADH reduction by the alternative NADH dehydrogenases present in plant mitochondria. Furthermore, the formation of reactive oxygen species (ROS) is very low in transgenic plants. We propose that reduction in ROS inhibits H₂O₂ dependent lignin polymerization in CA2 over-expressing plants, thereby causing male sterility. Gene bank accession number: AY085025 (At1g47260).     

At UBP3 and At UBP4 are two closely related Arabidopsis thaliana ubiquitin-specific proteases present in the nucleus

The ubiquitin-specific proteases (UBPs) are a class of enzymes vital to the ubiquitin pathway. These enzymes cleave ubiquitin at its C-terminus from two types of substrates containing (i) ubiquitin in an α-amino linkage, as found in the primary ubiquitin translation products, polyubiquitin and ubiquitin-ribosomal fusion proteins, or (ii) ubiquitin in an ɛ-amino linkage, as found in multiubiquitin chains either unattached or conjugated to cellular proteins. We have isolated cDNAs for two Arabidopsis thaliana genes, AtUBP3 and AtUBP4, which encode UBPs that are 93% identical. These two cDNAs represent the only two members of this subgroup and encode the smallest UBPs described to date in any organism. Using in vivo assays in Escherichia coli that allow the coexpression of a UBP with a putative substrate, we have shown that AtUBP3 and AtUBP4 can specifically deubiquitinate the artificial substrate Ub-X-β-gal but cannot act upon the natural α-amino-linked ubiquitin fusions Arabidopsis Ub-CEP52 and Arabidopsis polyubiquitin. Affinity-purified antibody prepared against AtUBP3 expressed in E. coli recognizes both AtUBP3 and AtUBP4. AtUBP3 and/or AtUBP4 are present in all Arabidopsis organs examined and at multiple developmental stages. Subcellular localization studies show that AtUBP3 and/or AtUBP4 are present in nuclear extracts. Possible physiological roles for these UBPs are discussed. Received: 14 November 1996 / Accepted: 6 February 1997     

Isolation and characterization of homologues of plant blue-light photoreceptor (cryptochrome) genes from the fern Adiantum capillus-veneris

Many blue-light mediated physiological responses have been studied in the fern Adiantum capillus-veneris. We have isolated genomic clones encoding sequences similar to those encoding blue-light photoreceptors (cryptochromes) in higher plants using the Arabidopsis CRY1 cDNA as a probe, and these positive clones fall into five independent groups. Using RACE procedures, we obtained full-length cDNA sequences for three of these five groups. The deduced amino acid sequences include the photolyase-homologous domain in the N-terminal half, and they also contain a C-terminal extension of about 200 amino acids in length. These structural features indicate that the genes indeed encode Adiantum cryptochromes and represent a small gene family having at least three members. Received: 16 February 1998 / Accepted: 26 April 1998     

Rates of Nucleotide Substitution in Angiosperm Mitochondrial DNA Sequences and Dates of Divergence Between Brassica and Other Angiosperm Lineages

We obtained 16 nucleotide sequences (∼1400 bp each) of the first intron of the mitochondrial (mt) gene for NADH subunit 4 (nad4) from 10 species of Brassicaceae. Using these new sequences and five published sequences from GenBank, we constructed a phylogenetic tree of the Brassicaceae species under study and showed that the rate of nucleotide substitution in the first intron of nad4 is very low, about 0.16–0.23 × 10⁻⁹ substitution per site per year, which is about half of the silent rate in exons of nad4. The ratios of substitution rates in this intron, ITS, and IGS are approximately 1:23:73, where ITS is the nuclear intergenic spacer between 18S and 25S rRNA genes and IGS is the intergenic spacer of 5S rRNA genes. A segment (335 bp) in the first intron of nad4 in Brassicaceae species that is absent in wheat was considered as a nonfunctional sequence and used to estimate the neutral rate (the rate of mutation) in mtDNA to be 0.5–0.7 × 10⁻⁹ substitution per site per year, which is about three times higher than the substitution rate in the rest of the first intron of nad4. We estimated that the dates of divergence are 170–235 million years (Myr) for the monocot–dicot split, 112–156 Myr for the Brassicaceae–Lettuce split, 14.5–20.4 Myr for the Brassica–Arabidopsis split, and 14.5–20.4 Myr for the Arabidopsis–Arabideae split. Received: 14 July 1998 / Accepted: 1 October 1998     

The pea ( Pisum sativum L.) genes sym33 and sym40 control infection thread formation and root nodule function

Two novel non-allelic mutants that were unable to fix nitrogen (Fix⁻) were obtained after EMS (ethyl methyl sulfonate) mutagenesis of pea (Pisum sativum L.). Both mutants, SGEFix⁻–1 and SGEFix⁻–2, form two types of nodules: SGEFix⁻–1 forms numerous white and some pink nodules, while mutant SGEFix⁻–2 forms white nodules with a dark pit at the distal end and also some pinkish nodules. Both mutations are monogenic and recessive. In both lines the manifestation of the mutant phenotype is associated with the root genotype. White nodules of SGEFix⁻–1 are characterised by hypertrophied infection threads and infection droplets, mass endocytosis of bacteria, abnormal morphological differentiation of bacteroids, and premature degradation of nodule symbiotic structures. The structure of the pink nodules of SGEFix⁻–1 does not differ from that of the parental line, SGE. White nodules of SGEFix⁻–2 are characterised by “locked” infection threads surrounded with abnormally thick plant cell walls. In these nodules there is no endocytosis of bacteria into host-cell cytoplasm. The pinkish nodules of SGEFix⁻–2 are characterised by virtually undifferentiated bacteroids and premature degradation of nodule tissues. Thus, the novel pea symbiotic genes, sym40 and sym33, identified after complementation analysis in SGEFix⁻–1 and SGEFix⁻–2 lines, respectively, control early nodule developmental stages connected with infection thread formation and function. Received: 12 June 1998 / Accepted: 25 June 1998     

Phenotypical and structural characterization of the Arabidopsis mutant involved in shoot apical meristem

An Arabidopsis mutant induced by T-DNA insertion was studied with respect to its phenotype, microstructure of shoot apical meristem (SAM) and histochemical localization of the GUS gene in comparison with the wild type. Phenotypical observation found that the mutant exhibited a dwarf phenotype with smaller organs (such as smaller leaves, shorter petioles), and slower development and flowering time compared to the wild type. Optical microscopic analysis of the mutant showed that it had a smaller and more flattened SAM, with reduced cell layers and a shortened distance between two leaf primordia compared with the wild type. In addition, analysis of the histo-chemical localization of the GUS gene revealed that it was specifically expressed in the SAM and the vascular tissue of the mutant, which suggests that the gene trapped by T-DNA may function, in the SAM, and T-DNA insertion could influence the functional activity of the related gene in the mutant, leading to alterations in the SAM and a series of phenotypes in the mutant. __________ Translated from Acta Botanica Boreali-Occidentalia Sinica, 2007, 27(2): 228–232 [译自: 西北植物学报]     

Fine mapping of an Arabidopsis thaliana male sterile mutant EC2-157

An Arabidopsis thaliana male sterile mutant EC2-157 has been isolated using an EMS mutagenesis strategy. Genetic analysis indicated that it was controlled by a single recessive gene called ms157. No pollen grains have been observed in mutant anthers. ms157 Has been mapped to a region of 74kb located in BAC clone T6K22 on chromosome IV using a map-based cloning strategy. As no male sterile genes have been reported in this region, ms157 could be a novel gene related to fertility. The further molecular cloning and functional analysis on this gene should facilitate our understanding of A. thaliana another development. __________ Translated from Journal of Shanghai Normal University (Natural Sciences), 2005, 34(1): 58–63 [译自: 上海师范大学学报 (自然科学版), 2005, 34(1): 58–63]