Molecular Characterization of the Calvin Cycle Enzyme Phosphoribulokinase in the Stramenopile Alga Vaucheria litorea and the Plastid Hosting Mollusc Elysia chlorotica

Phosphoribulokinase （PRK）, a nuclear-encoded plastid-localized enzyme unique to the photosynthetic carbon reduction （Calvin） cycle, was cloned and characterized from the stramenopile alga Vaucheria litorea. This alga is the source of plastids for the mollusc （sea slug） Elysia chlorotica which enable the animal to survive for months solely by photoautotrophic CO2 fixation. The 1633-bp V. litorea prk gene was cloned and the coding region, found to be interrupted by four introns, encodes a 405-amino acid protein. This protein contains the typical bipartite target sequence expected of nuclearencoded proteins that are directed to complex （i.e. four membrane-bound） algal plastids. De novo synthesis of PRK and enzyme activity were detected in E. chlorotica in spite of having been starved of V. litorea for several months. Unlike the algal enzyme, PRK in the sea slug did not exhibit redox regulation. Two copies of partial PRK-encoding genes were isolated from both sea slug and aposymbiotic sea slug egg DNA using PCR. Each copy contains the nucleotide region spanning exon 1 and part of exon 2 of V litorea prk, including the bipartite targeting peptide. However, the larger prk fragment also includes intron 1. The exon and intron sequences of prk in E. chlorotica and V/itorea are nearly identical. These data suggest that PRK is differentially regulated in V. litorea and E. chlorotica and at least a portion of the V. litorea nuclear PRK gene is present in sea slugs that have been starved for several months.     

Identification,expression, and characterization of the highly conserved D-xylose isomerase in animals

D-xylose is a necessary sugar for animals. The xylanase from a mollusk, Ampullaria crossean, was previously reported by our laboratory. This xylanase can degrade the xylan into D-xylose. But there is still a gap in our knowledge on its metabolic pathway. The question is how does the xylose enter the pentose pathway？ With the help of genomic databases and bioinformatic tools, we found that some animals, such as bacteria, have a highly conserved D-xylose isomerase （EC 5.3.1.5）. The xyiose isomerase from a sea squirt, Ciona intestinali, was heterogeneously expressed in Escherichia coli and purified to confirm its function. The recombinant enzyme had good thermal stability in the presence of Mg^2＋. At the optimum temperature and optimum pH environment, its specific activity on D-xylose was 0.331 μmol/mg/min. This enzyme exists broadly in many animals, but it disappeared in the genome of Amphibia-like Xenopus laevis. Its sequence was highly conserved. The xylose isomerases from animals are very interesting proteins for the study of evolution.     

Chloroplast Proteomics and the Compartmentation of Plastidial Isoprenoid Biosynthetic Pathways

Recent advances in the proteomic field have allowed high-throughput experiments to be conducted on chloroplast samples. Many proteomic investigations have focused on either whole chloroplast or sub-plastidial fractions. To date, the Plant Protein Database （PPDB, Sun et al., 2009） presents the most exhaustive chloroplast proteome available online. However, the accurate localization of many proteins that were identified in different sub-plastidial compartments remains hypothetical. Ferro et al. （2009） went a step further into the knowledge of Arabidopsis thaliana chloroplast proteins with regards to their accurate localization within the chloroplast by using a semi-quantitative proteomic approach known as spectral counting. Their proteomic strategy was based on the accurate mass and time tags （AMT） database approach and they built up AT_CHLORO, a comprehensive chloroplast proteome database with sub-plastidial localization and curated information on envelope proteins. Comparing these two extensive databases, we focus here on about 100 enzymes involved in the synthesis of chloroplast-specific isoprenoids. Well known pathways （i.e. compartmentation of the methyl erythritol phosphate biosynthetic pathway, of tetrapyrroles and chlorophyll biosynthesis and breakdown within chloroplasts） validate the spectral counting-based strategy. The same strategy was then used to identify the precise localization of the biosynthesis of carotenoids and prenylquinones within chloroplasts （i.e. in envelope membranes, stroma, and/or thylakoids） that remains unclear until now.     

Targeted Gene Knockouts Reveal Overlapping Functions of the Five Physcomitrella patens FtsZ Isoforms in Chloroplast Division,Chloroplast Shaping,Cell Patterning,Plant Development,and Gravity Sensing

Chloroplasts and bacterial cells divide by binary fission. The key protein in this constriction division is FtsZ, a self-assembling GTPase similar to eukaryotic tubulin. In prokaryotes, FtsZ is almost always encoded by a single gene, whereas plants harbor several nuclear-encoded FtsZ homologs. In seed plants, these proteins group in two families and all are exclusively imported into plastids. In contrast, the basal land plant Physcomitrella patens, a moss, encodes a third FtsZ family with one member. This protein is dually targeted to the plastids and to the cytosol. Here, we report on the targeted gene disruption of all ftsZ genes in R patens. Subsequent analysis of single and double knockout mutants revealed a complex interaction of the different FtsZ isoforms not only in plastid division, but also in chloroplast shaping, cell patterning, plant development, and gravity sensing. These results support the concept of a plastoskeleton and its functional integration into the cytoskeleton, at least in the moss R patens.     

Arabidopsis thaliana T-DNA Mutants Implicate GAUT Genes in the Biosynthesis of Pectin and Xylan in Cell Walls and Seed Testa

Galacturonosyltransferase 1 （GAUT1） is an α1,4-D-galacturonosyltransferase that transfers galacturonic acid from uridine 5＇-diphosphogalacturonic acid onto the pectic polysaccharide homogalacturonan （Sterling et al., 2006）. The 25-member Arabidopsis thaliana GAUT1-related gene family encodes 15 GAUT and 10 GAUT-like （GATL） proteins with, respectively, 56-84 and 42-53% amino acid sequence similarity to GAUT1. Previous phylogenetic analyses of AtGAUTs indicated three clades： A through C. A comparative phylogenetic analysis of the Arabidopsis, poplar and rice GAUT families has sub-classified the GAUTs into seven clades： clade A-1 （GAUTs 1 to 3）; A-2 （GAUT4）; A-3 （GAUTs 5 and 6）; A-4 （GAUT7）; B-1 （GAUTs 8 and 9）; B-2 （GAUTs 10 and 11）; and clade C （GAUTs 12 to 15）. The Arabidopsis GAUTs have a distribution comparable to the poplar orthologs, with the exception of GAUT2, which is absent in poplar. Rice, however, has no orthologs of GAUTs 2 and 12 and has multiple apparent orthologs of GAUTs 1, 4, and 7 compared with eitherArabidopsis or poplar. The cell wall glycosyl residue compositions of 26 homozygous T-DNA insertion mutants for 13 of 15 Arabidopsis GAUTgenes reveal significantly and reproducibly different cell walls in specific tissues of gaut mutants 6, 8, 9, 10, 11, 12, 13, and 14 from that of wild-type Arabidopsis walls. Pectin and xylan polysaccharides are affected by the loss of GAUT function, as demonstrated by the altered galacturonic acid, xylose, rhamnose, galactose, and arabinose composition of distinct gaut mutant walls. The wall glycosyl residue compositional phenotypes observed among the gaut mutants suggest that at least six different biosynthetic linkages in pectins and/or xylans are affected by the lesions in these GAUTgenes. Evidence is also presented to support a role for GAUT11 in seed mucilage expansion and in seed wall and mucilage composition.     

Defining the Functional Network of Epigenetic Regulators in Arabidopsis thaliana

Development of ChiP-chip and ChlP-seq technologies has allowed genome-wide high-resolution profiling of chromatin-associated marks and binding sites for epigenetic regulators. However, signals for directing epigenetic modifiers to their target sites are not understood. In this paper, we tested the hypothesis that genome location can affect the involvement of epigenetic regulators using Chromatin Charting （CC） Lines, which have an identical transgene construct inserted at different locations in the Arabidopsis genome. Four CC lines that showed evidence for epigenetic silencing of the luciferase reporter gene were transformed with RNAi vectors individually targeting epigenetic regulators LHP1, MOM1, CMT3, DRD1, DRM2, SUVH2, CLF, and HD1. Involvement of a particular epigenetic regulator in silencing the transgene locus in a CC line was determined by significant alterations in luciferase expression after suppression of the regulator＇s expression. Our results suggest that the targeting of epigenetic regulators can be influenced by genome location as well as sequence context. In addition, the relative importance of an epigenetic regulator can be influenced by tissue identity. We also report a novel approach to predict interactions between epigenetic regulators through clustering analysis of the regulators using alterations in gene expression of putative downstream targets, including endogenous loci and transgenes, in epigenetic mutants or RNAi lines. Our data support the existence of a complex and dynamic network of epigenetic regulators that serves to coordinate and control global gene expression in higher plants.     

Expression,Imprinting, and Evolution of Rice Homologs of the Polycomb Group Genes

Polycomb group proteins （PcG） play important roles in epigenetic regulation of gene expression. Some core PeG proteins, such as Enhancer of Zeste （E（z））, Suppressor of Zeste （12） （Su（z）12）, and Extra Sex Combs （ESC）, are conserved in plants. The rice genome contains two E（z）-Iike genes, OsiEZ1 and OsCLF, two homologs of Su（z）12, OsEMF2a and OsEMF2b, and two ESC-like genes, OsFIE1 and OsFIE2. OsFIE1 is expressed only in endosperm; the maternal copy is expressed while the paternal copy is not active. Other rice PcG genes are expressed in a wide range of tissues and are not imprinted in the endosperm. The two E（z）-Iike genes appear to have duplicated before the separation of the dicots and monocots; the two homologs of Su（z）12 possibly duplicated during the evolution of the Gramineae and the two ESC- like genes are likely to have duplicated in the ancestor of the grasses. No homologs of the Arabidopsis seed-expressed PcG genes MEA and FIS2 were identified in the rice genome. We have isolated T-DNA insertion lines in the rice homologs of three PcG genes. There is no autonomous endosperm development in these T-DNA insertion lines. One line with a T-DNA insertion in OsEMF2b displays pleiotropic phenotypes including altered flowering time and abnormal flower organs, suggesting important roles in rice development for this gene.     

The Earliest Normal Flower from Liaoning Province,China

The early evolution of angiosperms has been a focus of intensive research for more than a century. The Yixian Formation in western Liaoning yields one of the earliest angiosperm macrofioras. Despite multitudes of angiosperm fossils uncovered, including Archaefructus and Sinocarpus, no bona fide normal flower has been dated to 125 Ma （mega-annum） or older. Here we report Callianthus dilae gen. et sp. nov. from the Yixian Formation （Early Cretaceous） in western Liaoning, China as the earliest normal flower known to date. The flower demonstrates a typical floral organization, including tepals, androecium, and gynoecium. The tepals are spatulate with parallel veins. The stamens have a slender filament, a globular anther, bristles at the anther apex, and in situ round-triangular pollen grains. The gynoecium is composed of two stylate carpels enclosed in a fleshy envelope, and develops into a ＂hip＂ when mature. Since the well-accepted history of angiosperms is not much longer than 125 Ma, Callianthus together with Chaoyangia, Archaefructus and Sinocarpus from the Yixian Formation demonstrate a surprisingly high diversity of angiosperms, implying a history of angiosperms much longer than currently accepted.     

Molecular Evolution of VEF-Domain-Containing PcG Genes in Plants

Arabidopsis VERNALIZATION2 （VRN2）, EMBRYONIC FLOWER2 （EMF2）, and FERTILIZATION-INDEPENDENT SEED2 （FIS2） are involved in vernalization-mediated flowering, vegetative development, and seed development, respectively. Together with Arabidopsis VEF-L36, they share a VEF domain that is conserved in plants and animals. To investigate the evolution of VEF-domain-containing genes （VEF genes）, we analyzed sequences related to VEF genes across land plants. To date, 24 full-length sequences from 11 angiosperm families and 54 partial sequences from another nine families were identified. The majority of the full-length sequences identified share greatest sequence similarity with and possess the same major domain structure as Arabidopsis EMF2. EMF2-1ike sequences are not only widespread among angiosperms, but are also found in genomic sequences of gymnosperms, lycophyte, and moss. No FIS2- or VEF-L36-1ike sequences were recovered from plants other than Arabidopsis, including from rice and poplar for which whole genomes have been sequenced. Phylogenetic analysis of the full-length sequences showed a high degree of amino acid sequence conservation in EMF2 homologs of closely related taxa. VRN2 homologs are recovered as a clade nested within the larger EMF2 clade. FIS2 and VEF-L36 are recovered in the VRN2 clade. VRN2 clade may have evolved from an EMF2 duplication event that occurred in the rosids prior to the divergence of the eurosid I and eurosid II lineages. We propose that dynamic changes in genome evolution contribute to the generation of the family of VEF-domain-containing genes, Phylogenetic analysis of the VEF domain alone showed that VEF sequences continue to evolve following EM F2NRN2 divergence in accordance with species relationship. Existence of EMF2-1ike sequences in animals and across land plants suggests that a prototype form of EMF2 was present prior to the divergence of the plant and animal lineages. A proposed sequence of events, based on domain organization and occurrence of intermediate seque     

Effects of Glycerol on the Fluorescence Spectra and Chloroplast Ultrastructure of Phaeodactylum tricornutum （Bacillariophyta）

Responses of the photosynthetic activity of Phaeodactylum tricornutum （Bacillariophyta） to organic carbon glycerol were investigated. The growth rate, photosynthetic pigments, 77 K fluorescence spectra, and chloroplast ultrastructure of P. tricornutum were examined under photoautotrophic, mixotrophic, and photoheterotrophic conditions. The results showed that the specific growth rate was the fastest under mixotrophic conditions. The cell photosynthetic pigment content and values of Chl a/Chl c were reduced under mixotrophic and photoheterotrophic conditions. The value of carotenoid/Chl a was enhanced under mixotrophic conditions, but was decreased under photoheterotrophic conditions. In comparison with photoautotrophic conditions, the fluorescence emission peaks and fluorescence excitation peaks were not shifted. The relative fluorescence of photosystem （PS） Ⅰ and PS Ⅱ and the values of F685/F710 and F685/F738 were decreased. Chloroplast thylakoid pairs were less packed under mixotrophic and photoheterotrophic conditions. There was a strong correlation between degree of chloroplast thylakoid packing and the excitation energy kept in PS Ⅱ. These results suggested that the PS Ⅱ activity was reduced by glycerol under mixotrophic conditions, thereby leading to repression of the photosynthetic activity.     

The Response Difference of Mitochondria in Recalcitrant Antiaris toxicaria Axes and Orthodox Zea mays Embryos to Dehydration Injury

Long-term preservation of recalcitrant seeds is very difficult because the physiological basis on their desiccation sensitivity is poorly understood. Survival of Antiaris toxicaria axes rapidly decreased and that of immature maize embryos very slowly decreased with dehydration. To understand their different responses to dehydration, we examined the changes in mitochondria activity during dehydration. Although activities of cytochrome （Cyt） c oxidase and malate dehydrogenase of the A. toxicaria axis and maize embryo mitochondria decreased with dehydration, the parameters of maize embryo mitochondria were much higher than those of A. toxicaria, showing that the damage was more severe for the A. toxicaria axis mitochondria than for those of maize embryo. The state I and III respiration of the A. toxicaria axis mitochondria were higher than those of maize embryo, the former rapidly decreased, and the latter slowly decreased with dehydration. The proportion of Cyt c pathway to state III respiration for the A. toxicaria axis mitochondria was low and rapidly decreased with dehydration, and the proportion of alternative oxidase pathway was high and slightly increased with dehydration. In contrast, the proportion of Cyt c pathway for maize embryo mitochondria was high, and that of alternative oxidase pathway was low. Both pathways decreased slowly with dehydration.     

Methods for Analysis of Protein Glutathionylation and their Application to Photosynthetic Organisms

Protein S-glutathionylation, the reversible formation of a mixed-disulfide between glutathione and protein thiols, is involved in protection of protein cysteines from irreversible oxidation, but also in protein redox regulation. Recent studies have implicated S-glutathionylation as a cellular response to oxidative/nitrosative stress, likely playing an important role in signaling. Considering the potential importance of glutathionylation, a number of methods have been developed for identifying proteins undergoing glutathionylation. These methods, ranging from analysis of purified proteins in vitro to large-scale proteomic analyses in vivo, allowed identification of nearly 200 targets in mammals. By contrast, the number of known glutathionylated proteins is more limited in photosynthetic organisms, although they are severely exposed to oxidative stress. The aim of this review is to detail the methods available for identification and analysis of glutathionylated proteins in vivo and in vitro. The advantages and drawbacks of each technique will be discussed as well as their application to photosynthetic organisms. Furthermore, an overview of known glutathionylated proteins in photosynthetic organisms is provided and the physiological importance of this post-translational modification is discussed.     

XPA A23G polymorphism is associated with the elevated response to platinum-based chemotherapy in advanced non-small cell lung cancer

DNA repair capacity （DRC） is correlated with sensitivity of cancer cells toward platinum-based chemotherapy. We hypothesize that genetic polymorphisms in DNA repair gene XPA （xeroderma pigmentosum group A） and XPG （xeroderma pigmentosum group G） （ERCC5, excision repair cross-complementation group 5）, which result in inter-individual differences in DNA repair efficiency, may predict clinical response to platinum agents in advanced non-small cell lung cancer （NSCLC） patients. In this study, we find that the A → G change of XPA A23G polymorphism significantly increased response to platinum-based chemotherapy. Polymorphism in XPG His46His was associated with a decreased treatment response, but was not statistically significant.