Altered expression of the<Emphasis Type="Italic"> Arabidopsis</Emphasis> ortholog of<Emphasis Type="Italic"> DCL</Emphasis> affects normal plant development

The DCL (defective chloroplasts and leaves) gene of tomato (Lycopersicon esculentum Mill.) is required for chloroplast development, palisade cell morphogenesis, and embryogenesis. Previous work suggested that DCL protein is involved in 4.5S rRNA processing. The Arabidopsis thaliana (L.) Heynh. genome contains five sequences encoding for DCL-related proteins. In this paper, we investigate the function of AtDCL protein, which shows the highest amino acid sequence similarity with tomato DCL. AtDCL mRNA was expressed in all tissues examined and a fusion between AtDCL and green fluorescent protein (GFP) was sufficient to target GFP to plastids in vivo, consistent with the localization of AtDCL to chloroplasts. In an effort to clarify the function of AtDCL, transgenic plants with altered expression of this gene were constructed. Deregulation of AtDCL gene expression caused multiple phenotypes such as chlorosis, sterile flowers and abnormal cotyledon development, suggesting that this gene is required in different organs. The processing of the 4.5S rRNA was significantly altered in these transgenic plants, indicating that AtDCL is involved in plastid rRNA maturation. These results suggest that AtDCL is the Arabidopsis ortholog of tomato DCL, and indicate that plastid function is required for normal plant development.Abbreviations DCL Defective chloroplasts and leaves - GFP Green fluorescent protein     

Cloning and expression of <Emphasis Type="Italic">mel</Emphasis> gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain.     

Molecular analysis of the<Emphasis Type="Italic"> CRINKLY4</Emphasis> gene family in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

The maize (Zea mays L.) CRINKLY4 (CR4) gene encodes a serine/threonine receptor-like kinase that controls an array of developmental processes in the plant and endosperm. The Arabidopsis thaliana (L.) Heynh. genome encodes an ortholog of CR4, ACR4, and four CRINKLY4-RELATED (CRR) proteins: AtCRR1, AtCRR2, AtCRR3 and AtCRK1. The available genome sequence of rice (Oryza sativa L.) encodes a CR4 ortholog, OsCR4, and four CRR proteins: OsCRR1, OsCRR2, OsCRR3 and OsCRR4, not necessarily orthologous to the Arabidopsis CRRs. A phylogenetic study showed that AtCRR1 and AtCRR2 form a clade closest to the CR4 group while all the other CRRs form a separate cluster. The five Arabidopsis genes are differentially expressed in various tissues. A construct formed by fusion of the ACR4 promoter and the GUS reporter, ACR4::GUS, is expressed primarily in developing tissues of the shoot. The ACR4 cytoplasmic domain functions in vitro as a serine/threonine kinase, while the AtCRR1 and AtCRR2 kinases are not active. The ability of ACR4 to phosphorylate AtCRR2 suggests that they might function in the same signal transduction pathway. T-DNA insertions were obtained in ACR4, AtCRR1, AtCRR2, AtCRR3 and AtCRK1. Mutations in acr4 show a phenotype restricted to the integuments and seed coat, suggesting that Arabidopsis might contain a redundant function that is lacking in maize. The lack of obvious mutant phenotypes in the crr mutants indicates they are not required for the hypothetical redundant function.     

Distribution of similar self-incompatibility (<Emphasis Type="Italic">S</Emphasis>) haplotypes in different genera,<Emphasis Type="Italic"> Raphanus</Emphasis> and<Emphasis Type="Italic"> Brassica</Emphasis>

The nucleotide sequences of ten SP11 and nine SRK alleles in Raphanus sativus were determined, and deduced amino acid sequences were compared with those of Brassica SP11 and SRK. The amino acid sequence identity of class-I SP11s in R. sativus was about 30% on average, the highest being 52.2%, while that of the S domain of class-I SRK was 77.0% on average and ranged from 70.8% to 83.9%. These values were comparable to those of SP11 and SRK in Brassica oleracea and B. rapa. SP11 of R. sativus S-21 was found to be highly similar to SP11 of B. rapa S-9 (89.5% amino acid identity), and SRK of R. sativus S-21 was similar to SRK of B. rapa S-9 (91.0%). SP11 and SRK of R. sativus S-19 were also similar to SP11 and SRK of B. oleracea S-20, respectively. These similarities of both SP11 and SRK alleles between R. sativus and Brassica suggest that these S haplotype pairs originated from the same ancestral S haplotypes.     

Novel<Emphasis Type="Italic"> eceriferum</Emphasis> mutants in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

We conducted a novel non-visual screen for cuticular wax mutants in Arabidopsis thaliana (L.) Heynh. Using gas chromatography we screened over 1,200 ethyl methane sulfonate (EMS)-mutagenized lines for alterations in the major A. thaliana wild-type stem cuticular chemicals. Five lines showed distinct differences from the wild type and were further analyzed by gas chromatography and scanning electron microscopy. The five mutants were mapped to specific chromosome locations and tested for allelism with other wax mutant loci mapping to the same region. Toward this end, the mapping of the cuticular wax (cer) mutants cer10 to cer20 was conducted to allow more efficient allelism tests with newly identified lines. From these five lines, we have identified three mutants defining novel genes that have been designated CER22, CER23, and CER24. Detailed stem and leaf chemistry has allowed us to place these novel mutants in specific steps of the cuticular wax biosynthetic pathway and to make hypotheses about the function of their gene products.Abbreviations EMS Ethyl methane sulfonate - SEM Scanning electron microscopy - SSLP Simple sequence length polymorphism - WT Wild type     

In vivo evidence for a regulatory role of the kinase activity of the <Emphasis Type="Italic">linotte</Emphasis>/<Emphasis Type="Italic">derailed</Emphasis> receptor tyrosine kinase,a <Emphasis Type="Italic">Drosophila</Emphasis> Ryk ortholog

The RYK subfamily of receptor tyrosine kinases is characterised by unusual, but highly conserved, amino acid substitutions in the kinase domain. The linotte/derailed gene encodes a Drosophila RYK subfamily member involved in embryonic and adult central nervous system development. Previous studies have shown that the kinase activity of this receptor is not required in vivo for its embryonic function. In this study, we have investigated the role of the cytoplasmic domain and the kinase activity of the linotte/derailed receptor tyrosine kinase in adult brain development. Our results indicate that these domains are not essential for adult brain development but they are required for the proper regulation of the activity of this receptor. This sheds light on a regulatory role for the kinase activity of a RYK subfamily member.Edited by C DesplanEmmanuel Taillebourg and Caroline Moreau-Fauvarque contributed equally to this work     

Cloning and expression of the sucrose phosphorylase gene from <Emphasis Type="Italic">Leuconostoc </Emphasis><Emphasis Type="Italic">mesenteroides</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The gene encoding sucrose phosphorylase (742sp) in Leuconostoc mesenteroides NRRL B-742 was cloned and expressed in Escherichia coli. The nucleotide sequence of the transformed 742sp comprised an ORF of 1,458 bp giving a protein with calculated molecular mass of 55.3 kDa. 742SPase contains a C-terminal amino acid sequence that is significantly different from those of other Leu. mesenteroides SPases. The purified 742SPase had a specific activity of 1.8 U/mg with a K _m of 3 mM with sucrose as a substrate; optimum activity was at 37°C and pH 6.7. The purified 742SPase transferred the glucosyl moiety of sucrose to cytosine monophosphate (CMP). Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Members of the<Emphasis Type="Italic"> S</Emphasis>-receptor kinase multigene family in<Emphasis Type="Italic"> Senecio squalidus</Emphasis> L. (Asteraceae), a species with sporophytic self-incompatibility

While the molecular basis of sporophytic self-incompatibility (SSI) has been investigated extensively in the Brassicaceae, almost nothing is known about the molecular regulation of SSI in other families, such as the Asteraceae. In species of Brassica and in Arabidopsis lyrata, a stigma-specific serine-threonine receptor kinase (SRK) and its cognate ligand, a pollen coating-borne cysteine-rich protein (SCR/SP11), determine the female and male sides of the SSI response, respectively. Here we have used RT-PCR with degenerate primers to conserved regions of SRK to amplify three SRK-like gene fragments expressed in stigmas of Senecio squalidus (Asteraceae). The Senecio S-receptor-like kinase (SSRLK) sequences share ~43% amino acid sequence identity with Brassica SRK₃ but higher amino acid sequence identity (~50%) with two Solanum bulbocastanum receptor-like kinase genes of unknown function. Despite expression in stigmas, all three SSRLKs were expressed at varying levels in floral and vegetative tissues. No allelic polymorphism was detected for the three SSRLKs in two S homozygous lines of S. squalidus or three other lines of S. squalidus carrying different S alleles. A full-length cDNA clone was obtained for SSRLK1 and its predicted amino acid sequence revealed significant structural differences to Brassica SRKs, most notably a major N-terminal truncation of 169 amino acids and the presence of just 8 conserved cysteine residues within the putative receptor domain instead of 12. Together, the sequence characteristics and expression characteristics of SSRLKs suggest that they are unlikely to be directly involved in the SSI response of S. squalidus. These findings are discussed in terms of the evolution of the SRK multigene family and the molecular basis of SSI in S. squalidus and the Asteraceae.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Phylogeny and divergence of basal angiosperms inferred from<Emphasis Type="Italic"> APETALA3</Emphasis>- and<Emphasis Type="Italic"> PISTILLATA</Emphasis>-like MADS-box genes

The B-class MADS-box genes composed of APETALA3 (AP3) and PISTILLATA (PI) lineages play an important role in petal and stamen identity in previously studied flowering plants. We investigated the diversification of the AP3-like and PI-like MADS-box genes of eight species in five basal angiosperm families: Amborella trichopoda (Amborellaceae); Brasenia schreberi and Cabomba caroliniana (Cabombaceae); Euryale ferox, Nuphar japonicum, and Nymphaea tetragona (Nymphaeaceae); Illicium anisatum (Illiciaceae); and Kadsura japonica (Schisandraceae). Sequence analysis showed that a four amino acid deletion in the K domain, which was found in all previously reported angiosperm PI genes, exists in a PI homologue of Schisandraceae, but not in six PI homologues of the Amborellaceae, Cabombaceae, and Nymphaeaceae, suggesting that the Amborellaceae, Cabombaceae, and Nymphaeaceae are basalmost lineages in angiosperms. The results of molecular phylogenetic analyses were not inconsistent with this hypothesis. The AP3 and PI homologues from Amborella share a sequence of five amino acids in the 5 region of exon 7. Using the linearized tree and likelihood methods, the divergence time between the AP3 and PI lineages was estimated as somewhere between immediately after to several tens of millions of years after the split between angiosperms and extant gymnosperms. Estimates of the age of the most recent common ancestor of all extant angiosperms range from ~140–210 Ma, depending on the trees used and assumptions made.     

Identification and characterization of two <Emphasis Type="Italic">uvrA</Emphasis> genes of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> pathovar citri

Two uvrA-like genes, designated uvrA1 and uvrA2, that may be involved in nucleotide excision repair in Xanthomonas axonopodis pv. citri (X. a. pv. citri) strain XW47 were characterized. The uvrA1 gene was found to be 2,964 bp in length capable of encoding a protein of 987 amino acids. The uvrA2 gene was determined to be 2,529 bp with a coding potential of 842 amino acids. These two proteins share 71 and 39% identity, respectively, in amino acid sequence with the UvrA protein of Escherichia coli. Analyses of the deduced amino acid sequence revealed that UvrA1 and UvrA2 have structures characteristic of UvrA proteins, including the Walker A and Walker B motifs, zinc finger DNA binding domains, and helix-turn-helix motif with a polyglycine hinge region. The uvrA1 or uvrA2 mutant, constructed by gene replacement, was more sensitive to DNA-damaging agents methylmethane sulfonate (MMS), mitomycin C (MMC), or ultraviolet (UV) than the wild type. The uvrA1 mutant was four orders of magnitude more sensitive to UV irradiation and two orders of magnitude more sensitive to MMS than the uvrA2 mutant. The uvrA1uvrA2 double mutant was one order of magnitude more sensitive to MMS, MMC, or UV than the uvrA1 single mutant. These results suggest that UvrA1 plays a more important role than UvrA2 in DNA repair in X. a. pv. citri. Both uvrA1 and uvrA2 genes were found to be constitutively expressed in the wild type and lexA1 or lexA2 mutant of X. a. pv. citri, and treatment of these cells with sublethal dose of MMC did not alter the expression of these two genes. Results of electrophoresis mobility shift assays revealed that LexA1 or LexA2 does not bind to either the uvrA1 or the uvrA2 promoter. These results suggest that uvrA expression in X. a. pv. citri is not regulated by the SOS response system.     

IS<Emphasis Type="Italic">Rm31</Emphasis>, a new insertion sequence of the IS<Emphasis Type="Italic">66</Emphasis> family in<Emphasis Type="Italic"> Sinorhizobium meliloti</Emphasis>

Sinorhizobium meliloti natural populations show a high level of genetic polymorphism possibly due to the presence of mobile genetic elements such as insertion sequences (IS), transposons, and bacterial mobile introns. The analysis of the DNA sequence polymorphism of the nod region of S. meliloti pSymA megaplasmid in an Italian isolate led to the discovery of a new insertion sequence, ISRm31. ISRm31 is 2,803 bp long and has 22-bp-long terminal inverted repeat sequences, 8-bp direct repeat sequences generated by transposition, and three ORFs (A, B, C) coding for proteins of 124, 115, and 541 amino acids, respectively. ORF A and ORF C are significantly similar to members of the transposase family. Amino acid and nucleotide sequences indicate that ISRm31 is a member of the IS66 family. ISRm31 sequences were found in 30.5% of the Italian strains analyzed, and were also present in several collection strains of the Rhizobiaceae family, including S. meliloti strain 1021. Alignment of targets sites in the genome of strains carrying ISRm31 suggested that ISRm31 inserts randomly into S. meliloti genomes. Moreover, analysis of ISRm31 insertion sites revealed DNA sequences not present in the recently sequenced S. meliloti strain 1021 genome. In fact, ISRm31 was in some cases linked to DNA fragments homologous to sequences found in other rhizobia species.     

Molecular cloning and characterization of crustin from mud crab<Emphasis Type="Italic"> Scylla paramamosain</Emphasis>

Antimicrobial peptides (AMPs) are important components of the host innate immune response against microbial invasion. In the present study, we report the identification and characterization of a crustin (CrusSp) from the hemocyte of mud crab, Scylla paramamosain using an expressed sequence tag (EST) and rapid amplification cDNA end (RACE) approaches. Analysis of the nucleotide sequence revealed seven different variances of the CrusSp cDNA in mud crab. The open reading frame encodes a protein of 111 amino acids with 21 residues signal sequence. The predicted molecular mass of the mature protein (90 amino acids) is 10.27 kDa with an estimated pI of 8.54. Analysis of the protein domain features indicated typical conserved cysteine residues containing a single whey acidic protein (WAP) domain at the C-terminus. A neighbour-joining tree showed that S. paramamosain crustin is closely related to other crustin homologues, and displays the highest similarity to crustin antimicrobial peptide in shore crab Carcinus maenas. Four exons and three introns were identified within the 999 bp genomic DNA sequence of CrusSp. Tissue distribution analysis showed that CrusSp was highly expressed in hemocytes, gills, intestines and muscle but it was not expressed in hepatopancreas and eyestalks. To gain insight into the in vitro antimicrobial activities of CrusSp, the mature peptide coding region was cloned into E. coli for heterologous expression. The recombinant CrusSp could inhibit the growth of gram-positive bacteria but had no inhibition activity against gram-negative bacteria. These results indicated the involvement of CrusSp in the innate immunity of S. paramamosain.     

Ectopic Expression of an <Emphasis Type="Italic">FT</Emphasis> Homolog from <Emphasis Type="Italic">Citrus</Emphasis> Confers an Early Flowering Phenotype on Trifoliate Orange (<Emphasis Type="Italic">Poncirus trifoliata</Emphasis> L. Raf.)

Citrus FT (CiFT) cDNA, which promoted the transition from the vegetative to the reproductive phase in Arabidopsis thaliana, when constitutively expressed was introduced into trifoliate orange (Poncirus trifoliata L. Raf.). The transgenic plants in which CiFT was expressed constitutively showed early flowering, fruiting, and characteristic morphological changes. They started to flower as early as 12 weeks after transfer to a greenhouse, whereas wild-type plants usually have a long juvenile period of several years. Most of the transgenic flowers developed on leafy inflorescences, apparently in place of thorns; however, wild-type adult trifoliate orange usually develops solitary flowers in the axils of leaves. All of the transgenic lines accumulated CiFT mRNA in their shoots, but there were variations in the accumulation level. The transgenic lines showed variation in phenotypes, such as time to first flowering and tree shape. In F₁ progeny obtained by crossing ‘Kiyomi’ tangor (C. unshiu × sinensis) with the pollen of one transgenic line, extremely early flowering immediately after germination was observed. The transgene segregated in F₁ progeny in a Mendelian fashion, with complete co-segregation of the transgene and the early flowering phenotype. These results showed that constitutive expression of CiFT can reduce the generation time in trifoliate orange.     

Structural and transcriptional analysis of<Emphasis Type="Italic"> S</Emphasis>-locus F-box genes in<Emphasis Type="Italic"> Antirrhinum</Emphasis>

A class of ribonucleases termed S-RNases, which control the pistil expression of self-incompatibility, represents the only known functional products encoded by the S locus in species from the Solanaceae, Scrophulariaceae and Rosaceae. Previously, we identified a pollen-specific F-box gene, AhSLF (S locus F-box)-S₂, very similar to S₂-RNase in Antirrhinum, a member of the Scrophulariaceae. In addition, AhSLF-S₂ also detected the presence of its homologous DNA fragments. To identify these fragments, we constructed two genomic DNA libraries from Antirrhinum self-incompatible lines carrying alleles S₁S₅ and S₂S₄, respectively, using a transformation-competent artificial chromosome (TAC) vector. With AhSLF-S₂-specific primers, TAC clones containing both AhSLF-S₂ and its homologs were subsequently identified (S₂TAC, S₅TACa, S₄TAC, and S₁TACa). DNA blot hybridization, sequencing and segregation analyses revealed that they are organized as single allelic copies (AhSLF-S₂, -S₁, -S₄ and -S₅) tightly linked to the S-RNases. Furthermore, clusters of F-box genes similar to AhSLF-S₂ were identified. In total, three F-box genes (AhSLF-S₂, -S₂A and -S₂C) in S₂TAC (51 kb), three (AhSLF-S₄, -S₄A and -S₄D) in S₄TAC (75 kb), two (AhSLF-S₅ and -S₅A) in S₅TACa (55 kb), and two (AhSLF-S₁ and -S₁E) in S₁TACa (71 kb), respectively, were identified. Paralogous copies of these genes show 38–54% identity, with allelic copies sharing 90% amino acid identity. Among these genes, three (AhSLF-S₂C, -S₄D and -S₁E) were specifically expressed in pollen, similar to AhSLF-S₂, implying that they likely play important roles in pollen, whereas three AhSLF-SA alleles showed no detectable expression. In addition, several types of retroelements and transposons were identified in the sequenced regions, revealing some detailed information on the structural diversity of the S locus region. Taken together, these results indicate that both single allelic and tandemly duplicated genes are associated with the S locus in Antirrhinum. The implications of these findings in evolution and possible roles of allelic AhSLF-S genes in the self-incompatible reaction are discussed in species like Antirrhinum.Sequence data from this article have been deposited with the EMBL/GenBank databases under accession numbers AJ300474, AJ515534, AJ515536 and AJ515535