Highly variable chloroplast genome from two endangered Papaveraceae lithophytes Corydalis tomentella and Corydalis saxicola

The increasingly wide application of chloroplast (cp) genome super‐barcode in taxonomy and the recent breakthrough in cp genetic engineering make the development of new cp gene resources urgent and significant. Corydalis is recognized as the most genotypes complicated and taxonomically challenging plant taxa in Papaveraceae. However, there currently are few reports about cp genomes of the genus Corydalis. In this study, we sequenced four complete cp genomes of two endangered lithophytes Corydalis saxicola and Corydalis tomentella in Corydalis, conducted a comparison of these cp genomes among each other as well as with others of Papaveraceae. The cp genomes have a large genome size of 189,029–190,247 bp, possessing a quadripartite structure and with two highly expanded inverted repeat (IR) regions (length: 41,955–42,350 bp). Comparison between the cp genomes of C. tomentella, C. saxicola, and Papaveraceae species, five NADH dehydrogenase‐like genes (ndhF, ndhD, ndhL, ndhG, and ndhE) with psaC, rpl32, ccsA, and trnL‐UAG normally located in the SSC region have migrated to IRs, resulting in IR expansion and gene duplication. An up to 9 kb inversion involving five genes (rpl23, ycf2, ycf15, trnI‐CAU, and trnL‐CAA) was found within IR regions. The accD gene was found to be absent and the ycf1 gene has shifted from the IR/SSC border to the SSC region as a single copy. Phylogenetic analysis based on the sequences of common CDS showed that the genus Corydalis is quite distantly related to the other genera of Papaveraceae, it provided a new clue for recent advocacy to establish a separate Fumariaceae family. Our results revealed one special cp genome structure in Papaveraceae, provided a useful resources for classification of the genus Corydalis, and will be valuable for understanding Papaveraceae evolutionary relationships.     

A comparative analysis of complete plastid genomes from Prangos fedtschenkoi and Prangos lipskyi (Apiaceae)

Prangos fedtschenkoi (Regel & Schmalh.) Korovin and P. lipskyi Korovin (Apiaceae) are rare plant species endemic to mountainous regions of Middle Asia. Both are edificators of biotic communities and valuable resource plants. The results of recent phylogenetic analyses place them in Prangos subgen. Koelzella (M. Hiroe) Lyskov & Pimenov and suggest they may possibly represent sister species. To aid in development of molecular markers useful for intraspecific phylogeographic and population‐level genetic studies of these ecologically and economically important plants, we determined their complete plastid genome sequences and compared the results obtained to several previously published plastomes of Apiaceae. The plastomes of P. fedtschenkoi and P. lipskyi are typical of Apiaceae and most other higher plant plastid DNAs in their sizes (153,626 and 154,143 bp, respectively), structural organization, gene arrangement, and gene content (with 113 unique genes). A total of 49 and 48 short sequence repeat (SSR) loci of 10 bp or longer were detected in P. fedtschenkoi and P. lipskyi plastomes, respectively, representing 42–43 mononucleotides and 6 AT dinucleotides. Seven tandem repeats of 30 bp or longer with a sequence identity ≥90% were identified in each plastome. Further comparisons revealed 319 polymorphic sites between the plastomes (IR, 21; LSC, 234; SSC, 64), representing 43.8% transitions (Ts), 56.1% transversions (Tv), and a Ts/Tv ratio of 0.78. Within genic regions, two indel events were observed in rpoA (6 and 51 bp) and ycf1 (3 and 12 bp), and one in ndhF (6 bp). The most variable intergenic spacer region was that of accD/psaI, with 21.1% nucleotide divergence. Each Prangos species possessed one of two separate inversions (either 5 bp in ndhB intron or 9 bp in petB intron), and these were predicted to form hairpin structures with flanking repeat sequences of 18 and 19 bp, respectively. Both species have also incorporated novel DNA in the LSC region adjacent to the LSC/IRa junction, and BLAST searches revealed it had a 100 bp match (86% sequence identity) to noncoding mitochondrial DNA. Prangos‐specific primers were developed for the variable accD/psaI intergenic spacer and preliminary PCR‐surveys suggest that this region will be useful for future phylogeographic and population‐level studies.     

ORGANIZATION OF THE CHLOROPLAST GENOME OF THE FRESHWATER CENTRIC DIATOM CYCLOTELLA MENEGHINIANA1

We constructed a complete physical map and a partial gene map of the chloroplast genome of Cyclotella meneghiniana Kützing clone 1020-1a (Bacillariophyceae). The 128-kb circular molecule contains a 17-kb inverted repeat, which divides the genome into single copy regions of65 kb and 29 kb. This is the largest genome and inverted repeat found in any diatom examined to date. In addition to the 16S and 23S ribosomal RNA genes, the inverted repeat contains both the ndhD gene (as yet unexamined in other diatoms) and the psbA gene (located similarly in one of two other examined diatoms). The Cyclotella chloroplast genome exists as two equimolar populations of inversion isomers that differ in the relative orientation of their single copy sequences. This inversion heterogeneity presumably results from intramolecular recombination within the inverted repeat. For the first time, we map the ndhD, psaC, rpofi, rpoCl, and rpoC2 genes to the chloroplast genome of a chlorophyll c-containing alga. While the Cyclotella chloroplast genome retains some prokaryotic and land plant gene clusters and operons, it contains a highly rearranged gene order in the large and small single copy regions compared to all other examined diatom, algal, and land plant chloroplast genomes.     

A contiguous sequence in spinach nuclear DNA is homologous to three separated sequences in chloroplast DNA

Summary A 3.4-kbp nuclear (n) DNA sequence has greater than 99% sequence homology to three segments of the chloroplast (cp) genes rps2, psbD/C, and psaA respectively. Each of these cpDNA segments is less than 3 kbp in length and appears to be integrated, at least in part, into several (>5) different sites flanked by unique sequences in the nuclear genome. Some of these sites contain longer homologies to the particular genes, while others are only homologous to smaller parts of the cp genes. Both the cpDNA fragments found in the nuclear genome and their flanking nDNA sequences are invested with short repeated A-T rich sequences but, apart from a hexanucleotide sequence and a palindromic sequence identified near each recombination point, there is no obvious structure that can suggest a mechanism of DNA transfer from the chloroplast to the nucleus in spinach.     

Functional loss of all ndh genes in an otherwise relatively unaltered plastid genome of the holoparasitic flowering plant Cuscuta reflexa

We have cloned and sequenced an area of about 9.0 kb of the plastid DNA (ptDNA) from the holoparasitic flowering plant Cuscuta reflexa to investigate the evolutionary response of plastid genes to a reduced selective pressure. The region contains genes for the 16S rRNA, a subunit of a plastid NAD(P)H dehydrogenase (ndhB), three transfer RNAs (trnA, trnI, trnV) as well as the gene coding for the ribosomal protein S7 (rps7). While the other genes are strongly conserved in C. reflexa, the ndhB gene is a pseudogene due to many frameshift mutations. In addition we used heterologous gene probes to identify the other ndh genes encoded by the plastid genome in higher plants. No hybridization signals could be obtained, suggesting that these genes are either lost or strongly altered in the ptDNA of C. reflexa. Together with evidence of deleted genes in the ptDNA of C. reflexa, the plastid genome can be grouped into four classes reflecting a different evolutionary rate in each case. The phylogenetic position of Cuscuta and the significance of ndh genes in the plastid genome of higher plants are discussed.     

Extrinsic polypeptides of spinach photosystem I

By combining Triton X-114 partitioning with alkaline-salt and chaotropic washings of thylakoid membrane vesicles and photosystem I particles, we have studied the protein subunit composition and organization of spinach photosystem I. Upon fractionation of photosystem I particles with Triton X-114, 6 polypeptides of 5.0, 8.2 (psaE), 10.5, 16.6 (psaG), 19.3 and 22.1 kDa (psaD) were considered to be extrinsic membrane proteins. By combining this partitioning with salt washes of thylakoid membranes, the polypeptides of 8.2, 11.6 (psaH), 19.3 and 22.1 kDa were directly shown to be stromally oriented and extrinsic while no extrinsic subunits were identified at the inner thylakoid surface. The 5.0, 8.2, 10.5, 17.2, 19.3 and 22.1 kDa polypeptides appear to have regulatory rather than catalytic functions as their release from photosystem I particles upon high salt-alkali treatment does not affect photosystem I-mediated electron transport.Abbreviations DCIP 2.6-dichlorophenol indophenol - DCMU dichlorophenyl dimethyl urea - LHC light harvesting complex - PVDF polyvinylidene difluoride - SDS sodium dodecyl sulphate - TCA trichloroacetic acid - Tricine (N-tris[Hydroxymethyl]-methylglycine; N-[2-Hydroxy-1,1-bis(hydroxymethyl)-ethyl]glycine) - Tris (tris[Hydroxymethyl]aminomethane)     

玉米ZmPHO2基因家族克隆及其低磷胁迫下的表达

PHO2(编码一个泛素结合酶E2)作为磷高亲和转运体PHT1的负调控子,在维持植物体内磷的动态平衡中发挥重要作用。该研究以拟南芥和水稻中的PHO2为基础,从玉米自交系B73基因组中鉴定出9个ZmPHO2基因家族成员,在系统进化关系上将其分为3类。在玉米自交系178中克隆了上述9个基因的CDS全长序列,保守结构域分析发现,ZmPHO2蛋白质序列中均有1个由约130个氨基酸组成的泛素结合酶E2催化结构域(UBCc),其中包含1个重要的保守氨基酸(半胱氨酸)。实时荧光定量结果表明,低磷胁迫处理后,所有ZmPHO2基因均有表达,并呈现不同的表达模式,主要表现为叶与根之间的组织差异和玉米自交系178与9782之间的基因型差异,而在同一组织多数基因间的表达差异不明显。其中,ZmPHO2;H2在自交系9782的根中持续下调表达,但在叶中持续上调表达,表明ZmPHO2;H2可能参与调控磷素在叶与根之间的运输,以维持地上部分和地下部分磷的平衡。     

The structure and expression of maize genes encoding the major heat shock protein, hsp70

We have isolated and sequenced two maize genomic clones that are homologous to the Drosophila hsp70 gene. One of the maize hsp70 clones contains the entire hsp70 coding region and 81 nucleotides of the 5' nontranslated sequence. The predicted amino acid sequence for this maize protein is 68% homologous to the hsp70 of Drosophila. The second maize hsp70 clone contains only part of the coding sequence and 1.1 kb of the 5' flanking sequence. This 5' flanking sequence contains two sequences homologous to the consensus heat-shock-element sequence. Both maize genes are thermally inducible and each contains an intron in the same position as that of the heat-shock-cognate gene, hsc1, of Drosophila. The presence of an intron in the maize genes is a distinguishing feature in that no other thermally inducible hsp70 genes described to date contain an intron. We have constructed a hybrid hsp70 gene containing the entire hsp70 coding sequence with an intron, and 1.1 kb of the 5' flanking sequence. We demonstrate that this hybrid gene is thermally inducible in a transgenic petunia plant and that the gene is expressed from its own promoter.     

Sequencing of the plastome in the leafless green mycoheterotroph <Emphasis Type="Italic">Cymbidium macrorhizon</Emphasis> helps us to understand an early stage of fully mycoheterotrophic plastome structure

To date, plastome studies of mycoheterotrophic orchids have focused on nongreen mycoheterotrophic or partially mycoheterotrophic species. Cymbidium macrorhizon is a fully mycoheterotrophic orchid that lacks leaves and roots, although its inflorescence rachis is pale green. It has degraded stomata, specific fungal partners, and high concentrations of heavy stable nitrogen and carbon isotopes. Therefore, the plastome of this species is expected to represent an early stage of a fully mycoheterotrophic plastome. In this study, we sequenced the plastomes of C. macrorhizon and closely related species (C. ensifolium, C. kanran, and C. lancifolium). Plastomes of the four Cymbidium species were almost identical structurally, but differed somewhat from those of previously studied species. The genes for the photosynthetic subunits of NADH dehydrogenase, ndhF and ndhH, were absent from all four newly sequenced plastomes, whereas only ndhJ was absent from C. ensifolium. In section Pachyrhizanthe (C. lancifolium and C. macrorhizon), ndhE, ndhI, and ndhJ were pseudogenized. With the exception of ndh and ycf, 64 protein-coding genes in C. macrorhizon were apparently functional. Most of them were highly conserved and under purifying selection. Therefore, no direct evidence is available to suggest that genes related to photosynthesis have lost their functions in C. macrorhizon. This discordance between molecular and physiological features for the trophic status of C. macrorhizon might result from a lag between photosynthetic function loss and relaxed purifying selection.     

The response of electron transport mediated by active NADPH dehydrogenase complexes to heat stress in the cyanobacterium Synechocystis 6803

The electron-transport machinery in photosynthetic membranes is known to be very sensitive to heat. In this study, the rate of electron transport (ETR) driven by photosystem I (PSI) and photosystem II (PSII) during heat stress in the wild-type Synechocystis sp. strain PCC 6803 (WT) and its ndh gene inactiva-tion mutants ΔndhB (M55) and ΔndhD1/ndhD2 (D1/D2) was simultaneously assessed by using the novel Dual-PAM-100 measuring system. The rate of electron transport driven by the photosystems (ETR_PSs) in the WT, M55, and D1/D2 cells incubated at 30℃ and at 55℃ for 10 min was compared. Incubation at 55℃ for 10 min significantly inhibited PSII-driven ETR (ETR^_PSII) in the WT, M55 and D1/D2 cells, and the ex-tent of inhibition in both the M55 and D1/D2 cells was greater than that in the WT cells. Further, PSI-driven ETR (ETR_PSI) was stimulated in both the WT and D1/D2 cells, and this rate was increased to a greater extent in the D1/D2 than in the WT cells. However, ETR_PSI was considerably inhibited in the M55 cells. Analysis of the effect of heat stress on ETR_PSs with regard to the alterations in the 2 active NDH-1 complexes in the WT, M55, and D1/D2 cells indicated that the active NDH-1 supercomplex and medi-umcomplex are essential for alleviating the heat-induced inhibition of ETRPSII and for accelerating the heat-induced stimulation of ETR_PSI, respectively. Further, it is believed that these effects are most likely brought about by the electron transport mediated by each of these 2 active NDH-1 complexes.     

The Mdm2 and p53 genes are conserved in the Arachnids

The p53 protein and its negative regulator the ubiquitin E3 ligase Mdm2 have been shown to be conserved from the Placazoan to man. In common with D.melanogaster and C.elegans, there is a single copy of the p53 gene in T.adhaerens, while in the vertebrates three p53-like genes can be found: p53 , p63 and p73. The Mdm2 gene is not present within the fully sequenced and highly annotated genomes of C.elegans and D.melanogaster. However, it is present in the Placazoan and the presence of multiple distinct p53 genes in the Sea anemone N.vectensis led us to examine the genomes of other phyla for p53 and Mdm2-like genes. We report here the discovery of an Mdm2-like gene and two distinct p53 like genes in the Arachnid Ioxodes scapularis (Northern Deer Tick). The two predicted Deer Tick p53 proteins are much more highly related to the human p53 protein in sequence than are the fruit fly and nematode proteins. One of the Deer tick genes encodes a p53 protein that is initiated within the DNA binding domain of p53 and shows remarkable homology to the newly described N-terminally truncated delta isoforms of human and zebrafish p53.     

Studies on the expression of NDH-H,a subunit of the NAD(P)H-plastoquinone-oxidoreductase of higher-plant chloroplasts

The plastid genomes of higher plants contain eleven reading frames (ndhA-K) that are homologous to genes encoding subunits of the mitochondrial NADH-ubiquinone-oxidoreductase (complex I). The carboxyterminal end of the NDH-H subunit from rice (Oryza sativa L.) was expressed as a fusion protein in Escherichia coli and antibodies against the fusion protein were generated in rabbits. The antibody was used to study the expression of NDH-H, and the following results were obtained: (i) NDH-H is expressed in mono- and dicotyledonous plants, (ii) NDH-H is localized on the stroma lamellae of the thylakoid membrane and (iii) NDH-H is expressed in etioplasts. Together with the finding that two other ndh genes (ndhI and ndhK) are expressed in plastids, these results point to the existence of an NAD(P)H-plastoquinone-oxidoreductase on the thylakoid membrane. The possible function of the enzyme in plastids is discussed and it is suggested that it works in balancing the ATP/ADP and the NADPH/NADP ratios during changing external (i.e. light) or internal (i.e. ATP and NADPH demands of biosynthetic pathways of the plastid) conditions.Abbreviations PVDF polyvinylidene difluoride - SDS sodium dodecyl sulfate We thank Professor M. Sugiura for the rice plastid DNA clone bank, Oliver Buchholz for Sorghum plastid membranes, Pioneer Hi-Bred Inc. for maize and Sorghum seeds and the Deutsche Forschungsgemeinschaft for financial support (SFB189).     

Comparative genomic mapping between a 754 kb region flanking DREB1A in Arabidopsis thaliana and maize

Comparative mapping between model plant species for which the complete genome sequence is known and crop species has been suggested as a new strategy for the isolation of agronomically valuable genes. In this study, we tested whether comparative mapping between Arabidopsisand maize of a small region (754 kb) surrounding the DREB1A gene in Arabidopsis could lead to the identification of an orthologous region in maize containing the DREB1A homologue. The genomic sequence information available for Arabidopsis allowed for the selection of conserved, low-copy genes that were used for the identification of maize homologues in a large EST database. In total, 17 maize homologues were mapped. A second BLAST comparison of these genes to the recently completed Arabidopsis sequence revealed that 15 homologues are likely to be orthologous as the highest similarity score was obtained either with the original Arabidopsis gene or with a highly similar Arabidopsis gene localized on a duplication of the investigated region on chromosome 5. The map position of these genes showed a significant degree of orthology with the Arabidopsis region. Nevertheless, extensive duplications and rearrangements in the Arabidopsisand maize genomes as well as the evolutionary distance between Arabidopsis and maize make it unlikely that orthology and collinearity between these two species are sufficient to aid gene prediction and cloning in maize.