In vivo effects of <Emphasis Type="Italic">NbSiR</Emphasis> silencing on chloroplast development in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>

Sulfite reductase (SiR) performs dual functions, acting as a sulfur assimilation enzyme and as a chloroplast (cp-) nucleoid binding protein. In this study, we examined the in vivo effects of SiR deficiency on chloroplast development in Nicotiana benthamiana. Virus-induced gene silencing of NbSiR resulted in leaf yellowing and growth retardation phenotypes, which were not rescued by cysteine supplementation. NbSiR:GFP fusion protein was targeted to chloroplasts and colocalized with cp-nucleoids. Recombinant full-length NbSiR protein and the C-terminal half of NbSiR possessed cp-DNA compaction activities in vitro, and expression of full-length NbSiR in E. coli caused condensation of genomic DNA. NbSiR silencing differentially affected expression of plastid-encoded genes, inhibiting expression of several genes more severely than others. In the later stages, depletion of NbSiR resulted in chloroplast ablation. In NbSiR-silenced plants, enlarged cp-nucleoids containing an increased amount of cp-DNA were observed in the middle of the abnormal chloroplasts, and the cp-DNAs were predominantly of subgenomic sizes based on pulse field gel electrophoresis. The abnormal chloroplasts developed prolamellar body-like cubic lipid structures in the light without accumulating NADPH:protochlorophyllide oxidoreductase proteins. Our results suggest that NbSiR plays a role in cp-nucleoid metabolism, plastid gene expression, and thylakoid membrane development.     

The chloroplast ribosomal protein L21 gene is essential for plastid development and embryogenesis in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Embryogenesis in higher plants is controlled by a complex gene network. Identification and characterization of genes essential for embryogenesis will provide insights into the early events in embryo development. In this study, a novel mutant with aborted seed development (asd) was identified in Arabidopsis. The asd mutant produced about 25% of albino seeds at the early stage of silique development. The segregation of normal and albino seeds was inherited as a single recessive embryo-lethal trait. The gene disrupted in the asd mutant was isolated through map-based cloning. The mutated gene contains a single base change (A to C) in the coding region of RPL21C (At1g35680) that is predicted to encode the chloroplast 50S ribosomal protein L21. Allele test with other two T-DNA insertion lines in RPL21C and a complementation test demonstrated that the mutation in RPL21C was responsible for the asd phenotype. RPL21C exhibits higher expression in leaves and flowers compared with expression levels in roots and developing seeds. The RPL21C–GFP fusion protein was localized in chloroplasts. Cytological observations showed that the asd embryo development was arrested at the globular stage. There were no plastids with normal thylakoids and as a result no normal chloroplasts formed in mutant cells, indicating an indispensable role of the ASD gene in chloroplasts biogenesis. Our studies suggest that the chloroplast ribosomal protein L21 gene is required for chloroplast development and embryogenesis in Arabidopsis.     

The complete chloroplast genome sequence of <Emphasis Type="Italic">Pseudoroegneria libanotica</Emphasis>, genomic features,and phylogenetic relationship with <Emphasis Type="Italic">Triticeae</Emphasis> species

Pseudoroegneria libanotica is an important herbage diploid species possessing the St genome. The St genome participates in the formation of nine perennial genera in Triticeae (Poaceae). The whole chloroplast (cp) genome of P. libanotica is 135 026 bp in length. The typical quadripartite structure consists of one large single copy of 80 634 bp, one small single copy of 12 766 bp and a pair of inverted regions (20 813 bp each). The cp genome contains 76 coding genes, four ribosomal RNA and 30 transfer RNA genes. Comparative sequence analysis suggested that: 1) the 737 bp deletion in the cp of P. libanotica was specific in Triticeae species and might transfer into its nuclear genome; 2) hot-spot regions, indels in intergenic regions and protein coding sequences mainly led to the length variation in Triticeae; 3) highly divergence regions combined with negative selection in rpl2, rps12, ccsA, rps8, ndhH, petD, ndhK, psbM, rps3, rps18, and ndhA were identified as effective molecular markers and could be considered in future phylogenetic studies of Triticeae species; and 4) ycf3 gene with rich cpSSRs was suitable for phylogeny analysis or could be used for DNA barcoding at low taxonomic levels. The cpSSRs distribution in the coding regions of diploid Triticeae species was shown for the first time and provided a valuable source for developing primers to study specific simple sequence repeat loci.     

Complete Chloroplast Genome Sequence of <Emphasis Type="Italic">Glycine max</Emphasis> and Comparative Analyses with other Legume Genomes

Lack of complete chloroplast genome sequences is still one of the major limitations to extending chloroplast genetic engineering technology to useful crops. Therefore, we sequenced the soybean chloroplast genome and compared it to the other completely sequenced legumes, Lotus and Medicago. The chloroplast genome of Glycine is 152,218 basepairs (bp) in length, including a pair of inverted repeats of 25,574 bp of identical sequence separated by a small single copy region of 17,895 bp and a large single copy region of 83,175 bp. The genome contains 111 unique genes, and 19 of these are duplicated in the inverted repeat (IR). Comparisons of Glycine, Lotus and Medicago confirm the organization of legume chloroplast genomes based on previous studies. Gene content of the three legumes is nearly identical. The rpl22 gene is missing from all three legumes, and Medicago is missing rps16 and one copy of the IR. Gene order in Glycine, Lotus, and Medicago differs from the usual gene order for angiosperm chloroplast genomes by the presence of a single, large inversion of 51 kilobases (kb). Detailed analyses of repeated sequences indicate that many of the Glycine repeats that are located in the intergenic spacer regions and introns occur in the same location in the other legumes and in Arabidopsis, suggesting that they may play some functional role. The presence of small repeats of psbA and rbcL in legumes that have lost one copy of the IR indicate that this loss has only occurred once during the evolutionary history of legumes.     

The chloroplast genome from a lycophyte (microphyllophyte), <Emphasis Type="Italic">Selaginella uncinata</Emphasis>, has a unique inversion,transpositions and many gene losses

We determined the complete nucleotide sequence of the chloroplast genome of Selaginella uncinata, a lycophyte belonging to the basal lineage of the vascular plants. The circular double-stranded DNA is 144,170 bp, with an inverted repeat of 25,578 bp separated by a large single copy region (LSC) of 77,706 bp and a small single copy region (SSC) of 40,886 bp. We assigned 81 protein-coding genes including four pseudogenes, four rRNA genes and only 12 tRNA genes. Four genes, rps15, rps16, rpl32 and ycf10, found in most chloroplast genomes in land plants were not present in S. uncinata. While gene order and arrangement of the chloroplast genome of another lycophyte, Hupertzia lucidula, are almost the same as those of bryophytes, those of S. uncinata differ considerably from the typical structure of bryophytes with respect to the presence of a unique 20 kb inversion within the LSC, transposition of two segments from the LSC to the SSC and many gene losses. Thus, the organization of the S. uncinata chloroplast genome provides a new insight into the evolution of lycophytes, which were separated from euphyllophytes approximately 400 million years ago. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

PRBP plays a role in plastid ribosomal RNA maturation and chloroplast biogenesis in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>

In the present study, we investigated protein characteristics and physiological functions of PRBP (plastid RNA-binding protein) in Nicotiana benthamiana. PRBP fused to green fluorescent protein (GFP) localized to the chloroplasts. Recombinant PRBP proteins bind to single-stranded RNA in vitro, but not to DNA in a double- or a single-stranded form. Virus-induced gene silencing (VIGS) of PRBP resulted in leaf yellowing in N. benthamiana. At the cellular level, PRBP depletion disrupted chloroplast biogenesis: chloroplast number and size were reduced, and the thylakoid membrane was poorly developed. In PRBP-silenced leaves, protein levels of plastid-encoded genes were significantly reduced, whereas their mRNA levels were normal regardless of their promoter types indicating that PRBP deficiency primarily affects translational or post-translational processes. Depletion of PRBP impaired processing of the plastid-encoded 4.5S ribosomal RNA, resulting in accumulation of the larger precursor rRNAs in the chloroplasts. In addition, PRBP-deficient chloroplasts contained significantly reduced levels of mature 4.5S and 5S rRNAs in the polysomal fractions, indicating decreased chloroplast translation. These results suggest that PRBP plays a role in chloroplast rRNA processing and chloroplast development in higher plants.     

Photosynthesis in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Mutants with Reduced Chloroplast Number

We have used a class of Arabidopsis mutants altered in the accumulation and replication of chloroplasts (arc mutants) to investigate the effect of reduced chloroplast number on the photosynthetic competence of leaves. Each of the arc mutants examined (arc3, arc5, and arc6) accumulate only a few (2–15) large chloroplasts per mesophyll cell [K.A. Pyke and R.M. Leech (1992) Plant Physiology 99: 1005–1008]. The increased plastid size maintains a constant plastid to mesophyll cell volume, which has been suggested to compensate for the lower chloroplast number. In fact, we find that reduced chloroplast number has an effect on both the composition and structure of the photosynthetic apparatus, and that each arc mutant has an altered photosynthetic capacity, and we conclude that photosynthetic competence is dependent on proper chloroplast division and development.     

The cellulose synthase (<Emphasis Type="Italic">CESA</Emphasis>) gene superfamily of the moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

The CESA gene superfamily of Arabidopsis and other seed plants comprises the CESA family, which encodes the catalytic subunits of cellulose synthase, and eight families of CESA-like (CSL) genes whose functions are largely unknown. The CSL genes have been proposed to encode processive β-glycosyl transferases that synthesize noncellulosic cell wall polysaccharides. BLAST searches of EST and shotgun genomic sequences from the moss Physcomitrella patens (Hedw.) B.S.G. were used to identify genes with high similarity to vascular plant CESAs, CSLAs, CSLCs, and CSLDs. However, searches using Arabidopsis CSLBs, CSLEs, and CSLGs or rice CSLFs or CSLHs as queries identified no additional CESA superfamily members in P. patens, indicating that this moss lacks representatives of these families. Intron insertion sites are highly conserved between Arabidopsis and P. patens in all four shared gene families. However, phylogenetic analysis strongly supports independent diversification of the shared families in mosses and vascular plants. The lack of orthologs of vascular plant CESAs in the P. patens genome indicates that the divergence of mosses and vascular plants predated divergence and specialization of CESAs for primary and secondary cell wall syntheses and for distinct roles within the rosette terminal complexes. In contrast to Arabidopsis, the CSLD family is highly represented among P. patens ESTs. This is consistent with the proposed function of CSLDs in tip growth and the central role of tip growth in the development of the moss protonema. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. Accession numbers: DQ417756, DQ417757, DQ898284–6, DQ898147–54, DQ902545–51.     

<Emphasis Type="Italic">Anthocyaninless1</Emphasis> gene of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> encodes a UDP-glucose:flavonoid-3-<Emphasis Type="Italic">O</Emphasis>-glucosyltransferase

We isolated several mutants of Arabidopsis thaliana (L.) Heynh. that accumulated less anthocyanin in the plant tissues, but had seeds with a brown color similar to the wild-type. These mutants were allelic with the anthocyaninless1 (anl1) mutant that has been mapped at 15.0 cM of chromosome 5. We performed fine mapping of the anl1 locus and determined that ANL1 is located between the nga106 marker and a marker corresponding to the MKP11 clone. About 70 genes are located between these two markers, including three UDP-glucose:flavonoid-3-O-glucosyltransferase-like genes and a glutathione transferase gene (TT19). A mutant of one of the glucosyltransferase genes (At5g17050) was unable to complement the anl1 phenotype, showing that the ANL1 gene encodes UDP-glucose:flavonoid-3-O-glucosyltransferase. ANL1 was expressed in all tissues examined, including rosette leaves, stems, flower buds and roots. ANL1 was not regulated by TTG1.     

Study of tauopathies by comparing <Emphasis Type="Italic">Drosophila</Emphasis> and human <Emphasis Type="Italic">tau</Emphasis> in <Emphasis Type="Italic">Drosophila</Emphasis>

The microtubule-binding protein tau has been investigated for its contribution to various neurodegenerative disorders. However, the findings from transgenic studies, using the same tau transgene, vary widely among different laboratories. Here, we have investigated the potential mechanisms underlying tauopathies by comparing Drosophila (d-tau) and human (h-tau) tau in a Drosophila model. Overexpression of a single copy of either tau isoform in the retina results in a similar rough eye phenotype. However, co-expression of Par-1 with d-tau leads to lethality, whereas co-expression of Par-1 with h-tau has little effect on the rough eye phenotype. We have found analogous results by comparing larval proteomes. Through genetic screening and proteomic analysis, we have identified some important potential modifiers and tau-associated proteins. These results suggest that the two tau genes differ significantly. This comparison between species-specific isoforms may help to clarify whether the homologous tau genes are conserved. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This study was supported by the National Science Foundation of China (30270341; 30630028), the Multidisciplinary Program (Brain and Mind) of the Chinese Academy of Sciences, the Major State Basic Research Program (“973 program”; G2000077800; G2006CB806600; 2006CB911003), the Precedent Project of Important Intersectional Disciplines in the Knowledge Innovation Engineering of the Chinese Academy of Sciences (KJCX1-09-03).     

Identification of Pathogenicity-Related Genes in the Vascular Wilt Fungus <Emphasis Type="Italic">Verticillium dahliae</Emphasis> by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-Mediated T-DNA Insertional Mutagenesis

Verticillium dahliae is the causal agent of vascular wilt in many economically important crops worldwide. Identification of genes that control pathogenicity or virulence may suggest targets for alternative control methods for this fungus. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was applied for insertional mutagenesis of V. dahliae conidia. Southern blot analysis indicated that T-DNAs were inserted randomly into the V. dahliae genome and that 69% of the transformants were the result of single copy T-DNA insertion. DNA sequences flanking T-DNA insertion were isolated through inverse PCR (iPCR), and these sequences were aligned to the genome sequence to identify the genomic position of insertion. V. dahliae mutants of particular interest selected based on culture phenotypes included those that had lost the ability to form microsclerotia and subsequently used for virulence assay. Based on the virulence assay of 181 transformants, we identified several mutant strains of V. dahliae that did not cause symptoms on lettuce plants. Among these mutants, T-DNA was inserted in genes encoding an endoglucanase 1 (VdEg-1), a hydroxyl-methyl glutaryl-CoA synthase (VdHMGS), a major facilitator superfamily 1 (VdMFS1), and a glycosylphosphatidylinositol (GPI) mannosyltransferase 3 (VdGPIM3). These results suggest that ATMT can effectively be used to identify genes associated with pathogenicity and other functions in V. dahliae.     

Common genomic features of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> subsp. <Emphasis Type="Italic">doylei</Emphasis> strains distinguish them from <Emphasis Type="Italic">C. jejuni</Emphasis> subsp. <Emphasis Type="Italic">jejuni</Emphasis>

Background  

Campylobacter jejuni has been divided into two subspecies: C. jejuni subsp. jejuni (Cjj) and C. jejuni subsp. doylei (Cjd). Nearly all of the C. jejuni strains isolated are Cjj; nevertheless, although Cjd strains are isolated infrequently, they differ from Cjj in two key aspects: they are obtained primarily from human clinical samples and are associated often with bacteremia, in addition to gastroenteritis. In this study, we utilized multilocus sequence typing (MLST) and a DNA microarray-based comparative genomic indexing (CGI) approach to examine the genomic diversity and gene content of Cjd strains.     

Complete chloroplast genome sequence of <Emphasis Type="Italic">Capsicum</Emphasis><Emphasis Type="Italic">baccatum</Emphasis> var. <Emphasis Type="Italic">baccatum</Emphasis>

Molecular markers derived from the complete chloroplast genome can provide effective tools for species identification and phylogenetic resolution. Complete chloroplast (cp) genome sequences of Capsicum species have been reported. We herein report the complete chloroplast genome sequence of Capsicum baccatum var. baccatum, a wild Capsicum species. The total length of the chloroplast genome is 157,145 bp with 37.7 % overall GC content. One pair of inverted repeats, 25,910 bp in length, was separated by a small single-copy region (17,974 bp) and large single-copy region (87,351 bp). This region contains 86 protein-coding genes, 30 tRNA genes, 4 rRNA genes, and 11 genes contain one or two introns. Pair-wise alignments of chloroplast genome were performed for genome-wide comparison. Analysis revealed a total of 134 simple sequence repeat (SSR) motifs and 282 insertions or deletions variants in the C. baccatum var. baccatum cp genome. The types and abundances of repeat units in Capsicum species were relatively conserved, and these loci could be used in future studies to investigate and conserve the genetic diversity of the Capsicum species.