Conservation of dual-targeted proteins in <Emphasis Type="Italic">Arabidopsis</Emphasis> and rice points to a similar pattern of gene-family evolution

Gene duplication followed by acquisition of specific targeting information and dual targeting were evolutionary strategies enabling organelles to cope with overlapping functions. We examined the evolutionary trend of dual-targeted single-gene products in Arabidopsis and rice genomes. The number of paralogous proteins encoded by gene families and the dual-targeted orthologous proteins were analysed. The number of dual-targeted proteins and the corresponding gene-family sizes were similar in Arabidopsis and rice irrespective of genome sizes. We show that dual targeting of methionine aminopeptidase, monodehydroascorbate reductase, glutamyl-tRNA synthetase, and tyrosyl-tRNA synthetase was maintained despite occurrence of whole-genome duplications in Arabidopsis and rice as well as a polyploidization followed by a diploidization event (gene loss) in the latter. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. C. V. Morgante and R. A. O. Rodrigues contributed equally to this work.     

Feasibility of the seed specific cruciferin C promoter in the self excision Cre/<Emphasis Type="Italic">lox</Emphasis>P strategy focused on generation of marker-free transgenic plants

This work is focused on the generation of selectable marker-free transgenic tobacco plants using the self excision Cre/loxP system that is controlled by a strong seed specific Arabidopsis cruciferin C (CRUC) promoter. It involves Agrobacterium-mediated transformation using a binary vector containing the gus reporter gene and one pair of the loxP sites flanking the cre recombinase and selectable nptII marker genes (floxed DNA). Surprisingly, an ectopic activation of CRUC resulting in partial excision of floxed DNA was observed during regeneration of transformed cells already in calli. The regenerated T₀ plants were chimeric, but no ongoing ectopic expression was observed in these one-year-long invitro maintained plants. The process of the nptII removal was expected in the seeds; however, none of the analysed T₀ transgenic lines generated whole progeny sensitive to kanamycin. Detailed analyses of progeny of selected T₀-30 line showed that 10.2% GUS positive plants had completely removed nptII gene while the remaining 86.4% were still chimeras. Repeated activation of the cre gene in T₂ seeds resulted in increased rate of marker-free plants, whereas four out of ten analysed chimeric T₁ plants generated completely marker-free progenies. This work points out the feasibility as well as limits of the CRUC promoter in the Cre/loxP strategy. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Bacterial growth properties at low optical densities

A method for accurate quantification of growth rate and yield of bacterial populations at low densities was developed with a modified version of a stepwise linear model for fitting growth curves based on optical density measurements, and adapted to measurements at low optical densities in 96-well microtiter plates. The method can be used for rapid and precise estimates of growth rate and yield, based on optical density measurements of large numbers of cultures of Escherichia coli. E. coli B lines were serially propagated at low glucose concentration during a long-term evolution experiment. Growth rate and yield of populations sampled from each of 12 lines that evolved for 20,000 generations under these conditions and two ancestral clones was measured. Populations were grown at three different glucose concentrations. Consistent with earlier findings, statistical analysis showed that both exponential growth rate and yield per unit of glucose differed significantly between the three glucose concentrations tested. Significant adaptation of the evolved populations to the nutrient conditions in which they evolved for 20,000 generations was observed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Light-dependent polyploidy control by a CUE protein variant in Arabidopsis

Endoreduplication is a special cell cycle that increases ploidy without cell and nuclear division. In plants endoreduplication is essential for development. We isolated a dominant Arabidopsis mutant from activation tagging lines that had increased polyploidy in darkness. This mutant, ipd1-1D (increased polyploidy level in darkness 1-1D), shows longer hypocotyls and increased ploidy levels only in dark-grown seedlings. The corresponding gene encodes a protein that contains a CUE domain variant. IPD1 is specifically expressed in mitotically dividing cells. Furthermore we show that blue and far-red light can suppress the ploidy increase in ipd1-1D and also suppress the reporter expression in IPD1-promoter β-glucuronidase transgenic plants. These results suggest that IPD1 regulates the endocycle leading to hypocotyl elongation and this function is controlled by blue and far-red light. Electronic Supplementary Material Supplementary material is available for this article at Yuko Tsumoto and Takeshi Yoshizumi contributed equally to this work     

Overexpression of the <Emphasis Type="Italic">Arabidopsis</Emphasis> anaphase promoting complex subunit <Emphasis Type="Italic">CDC27a</Emphasis> increases growth rate and organ size

The Anaphase Promoting Complex (APC) controls CDK activity by targeting the ubiquitin-dependent proteolysis of S-phase and mitosis-promoting cyclins. Here, we report that the ectopic expression of the Arabidopsis CDC27a, an APC subunit, accelerates plant growth and results in plants with increased biomass production. CDC27a overexpression was associated to apical meristem restructuration, protoplasts with higher ³H-thimidine incorporation and altered cell-cycle marker expression. Total protein extracts immunoprecipitated with a CDC27a antibody showed ubiquitin ligase activity, indicating that the Arabidopsis CDC27a gets incorporated into APC complexes. These results indicate a role of AtCDC27a in regulation of plant growth and raise the possibility that the activity of the APC and the rates of plant cell division could be regulated by the concentration of the CDC27a subunit. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Cristian Antonio Rojas and Nubia Barbosa Eloy contributed equally to this work.     

Integrating genes and phenotype: a wheat–Arabidopsis–rice glycosyltransferase database for candidate gene analyses

Glycosyltransferases (GTs) constitute a very large multi-gene superfamily, containing several thousand members identified in sequenced organisms especially in plants. GTs are key enzymes involved in various biological processes such as cell wall formation, storage polysaccharides biosynthesis, and glycosylation of various metabolites. GTs have been identified in rice (Oryza sativa) and Arabidopsis thaliana, but their precise function has been demonstrated biochemically for only a few. In this work we have established a repertoire of virtually all the wheat (Triticum aestivum) GT sequences, using the large publicly available banks of expressed sequences. Based on sequence similarity with Arabidopsis and rice GTs compiled in the carbohydrate active enzyme database (CAZY), we have identified and classified these wheat sequences. The results were used to feed a searchable database available on the web () that can be used for initiating an exhaustive candidate gene survey in wheat applied to a particular biological process. This is illustrated through the identification of GT families which are expressed during cell wall formation in wheat grain maturation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was funded by a grant of the French ministry of research.     

ParA resolvase catalyzes site-specific excision of DNA from the Arabidopsis genome

The small serine resolvase ParA from bacterial plasmids RK2 and RP4 catalyzes the recombination of two identical 133 bp recombination sites known as MRS. Previously, we reported that ParA is active in the fission yeast Schizosaccharomyces pombe. In this work, the parA recombinase gene was placed under the control of the Arabidopsis OXS3 promoter and introduced into Arabidopsis lines harboring a chromosomally integrated MRS-flanked target. The ParA recombinase excised the MRS-flanked DNA and the excision event was detected in subsequent generations in the absence of ParA, indicating germinal transmission of the excision event. The precise site-specific deletion by the ParA recombination system in planta demonstrates that the ParA recombinase can be used to remove transgenic DNA, such as selectable markers or other introduced transgenes that are no longer desired in the final product.     

The role of UDP-glucose:hydroxycinnamate glucosyltransferases in phenylpropanoid metabolism and the response to UV-B radiation in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Arabidopsis harbors four UDP-glycosyltransferases that convert hydroxycinnamates (HCAs) to 1-O-β-glucose esters, UGT84A1 (encoded by At4g15480), UGT84A2 (At3g21560), UGT84A3 (At4g15490), and UGT84A4 (At4g15500). To elucidate the role of the individual UGT84A enzymes in planta we analyzed gene expression, UGT activities and accumulation of phenylpropanoids in Arabidopsis wild type plants, ugt mutants and overexpressing lines. Individual ugt84A null alleles did not significantly reduce the gross metabolic flux to the accumulating compounds sinapoylcholine (sinapine) in seeds and sinapoylmalate in leaves. For the ugt84A2 mutant, LC/MS analysis revealed minor qualitative and quantitative changes of several HCA choline esters and of disinapoylspermidine in seeds. Overexpression of individual UGT84A genes caused increased enzyme activities but failed to produce significant changes in the pattern of accumulating HCA esters. For UGT84A3, our data tentatively suggest an impact on cell wall-associated 4-coumarate. Exposure of plants to enhanced UV-B radiation induced the UGT84A-encoding genes and led to a transient increase in sinapoylglucose and sinapoylmalate concentrations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Promoter methylation and progressive transgene inactivation inArabidopsis

Agrobacterium-transformedArabidopsis plants were generated and the stability of their T-DNA-encoded resistance to kanamycin was examined. Of seven families, each homozygous for a single insertion event, two showed progressive inactivation of resistance over four generations of inbreeding. Loss of resistance was associated with methylation of anSst II site in thenos promoter of the kanamycin resistance gene. Treatment of plant roots from inactive lines with the demethylating agent 5-azacytidine restored the ability of such lines to form callus on kanamycin-containing media. These observations are consistent with the view that methylation is a factor in the progressive inactivation of transgenes inArabidopsis.     

Recombinant near-isogenic lines: a resource for the mendelization of heterotic QTL in maize

Although heterosis is widely exploited in agriculture, a clear understanding of its genetic bases is still elusive. This work describes the development of maize recombinant near-isogenic lines (NILs) for the mendelization of six heterotic QTL previously identified based on a maize (Zea mays L.) RIL population. The efficient and inexpensive strategy adopted to generate sets of NILs starting from QTL-specific residual heterozygous lines (RHLs) is described and validated. In particular, we produced nine pairs of recombinant NILs for all six QTL starting from RHLs F_4:5 originally obtained during the production of the RIL population mentioned above. Whenever possible, two different NIL pairs were generated for each QTL. The efficiency of this procedure was tested by analyzing two segregating populations for two of the selected heterotic QTL for plant height, yield per plant and ears per plant. Both additive and dominant effects were observed, consistently with the presence of the QTL within the introgressed regions. Refinement of QTL detection was consistent with previous observations in terms of effects and position of the considered QTL. The genetic material developed in this work represents the starting point for QTL fine mapping aimed at understanding the genetic bases of hybrid vigor in maize. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular Cloning and Characterization of a Vacuolar H+-pyrophos-phatase Gene, SsVP, from the Halophyte Suaeda salsa and its Overexpression Increases Salt and Drought Tolerance of Arabidopsis

The chenopodiaceae Suaeda salsa L. is a leaf succulent euhalophyte. Shoots of the S. salsa are larger and more succulent when grown in highly saline environments. This increased growth and water uptake has been correlated with a large and specific cellular accumulation of sodium. S. salsa does not have salt glands or salt bladders on its leaves. Thus, this plant must compartmentalize the toxic Na⁺ in the vacuoles. The ability to compartmentalize sodium may result from a stimulation of the proton pumps that provide the driving force for increased sodium transport into the vacuole. In this work, we isolated the cDNA of the vacuolar membrane proton-translocating inorganic pyrophosphatase (H⁺-PPase) from S. salsa. The SsVP cDNA contains an uninterrupted open reading frame of 2292 bp, coding for a polypeptide of 764 amino acids. Northern blotting analysis showed that SsVP was induced in salinity treated leaves. The activities of both the V-ATPase and the V-PPase in Arabidopsis overexpressing SsVP-2 is higher markedly than in wild-type plant under 200 mM NaCl and drought stresses. The Overexpression of SsVP can increase salt and drought tolerance of transgenic Arabidopsis. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users. Shanli Guo, Haibo Yib: These authors contributed equally to this work     

Functional analysis of an Aspergillus ficuum phytase gene in Saccharomyces cerevisiae and its root-specific, secretory expression in transgenic soybean plants

Phytases release inorganic phosphates from phytate in soil. A gene encoding phytase (AfPhyA) was isolated from Aspergillus ficuum and its ability to degrade phytase and release phosphate was demonstrated in Saccharomyces cerevisiae. A promoter from the Arabidopsis Pky10 gene and the carrot extensin signal peptide were used to drive the root-specific and secretory expression of the AfPhyA gene in soybean plants. The phytase activity and inorganic phosphate levels in transgenic soybean root secretions were 4.7 U/mg protein and 439 μM, respectively, compared to 0.8 U/mg protein and 120 μM, respectively, in control soybeans. Our results demonstrated the potential usefulness of the root-specific promoter for the exudation of recombinant phytases and offered a new perspective on the mobilization of phytate in soil to inorganic phosphates for plant uptake. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Guilan Li and Shaohui Yang authors contribute equally to the paper.     

Biosystematic study of the genus <Emphasis Type="Italic">Berberis</Emphasis> L. (Berberidaceae) in Khorassan,NE Iran

Four species of the genus Berberis L. have been reported from different regions, including Khorassan provinces. In this article, a revision of this genus in Khorassan provinces is presented. For better validation of the results, morphological, palynological, chromosomal and molecular studies were conducted on specimens collected from Khorassan provinces and preserved in FUMH and TARI herbaria. Morphological study resulted in recognition of three species of Berberis, from which two species are new records for Khorassan. Four unknown new taxa with new morphological characters were also identified, but left for further analysis because of the high percentage of polyploidy and hybridization in this genus. In order to analyze morphological data, 35 OTUs were provided and scored using the distance method and PCoA. The shape and size of pollen grains were nearly equal in the examined taxa, and there were no obvious differences between them. Chromosomal examination indicated tetraploidy (2n = 4x = 56) in all of the studied taxa. Molecular studies were accomplished by RAPD and sequencing of the ITS region to construct a framework of relationships between the taxa. Molecular studies emphasized the difference in the four unknown taxa from others. The total evidence indicates that Berberis L. shows a high percentage of polyploidy and hybridization.     

Constitutive expression of a meiotic recombination protein gene homolog, OsTOP6A1, from rice confers abiotic stress tolerance in transgenic Arabidopsis plants

Plant productivity is greatly influenced by various environmental stresses, such as high salinity and drought. Earlier, we reported the isolation of topoisomerase 6 homologs from rice and showed that over expression of OsTOP6A3 and OsTOP6B confers abiotic stress tolerance in transgenic Arabidopsis plants. In this study, we have assessed the function of nuclear-localized topoisomerase 6 subunit A homolog, OsTOP6A1, in transgenic Arabidopsis plants. The over expression of OsTOP6A1 in transgenic Arabidopsis plants driven by cauliflower mosaic virus-35S promoter resulted in pleiotropic effects on plant growth and development. The transgenic Arabidopsis plants showed reduced sensitivity to stress hormone, abscisic acid (ABA), and tolerance to high salinity and dehydration at the seed germination; seedling and adult stages as reflected by the percentage of germination, fresh weight of seedlings and leaf senescence assay, respectively. Concomitantly, the expression of many stress-responsive genes was enhanced under various stress conditions in transgenic Arabidopsis plants. Moreover, microarray analysis revealed that the expression of a large number of genes involved in various processes of plant growth and development and stress responses was altered in transgenic plants. Although AtSPO11-1, the homolog of OsTOP6A1 in Arabidopsis, has been implicated in meiotic recombination; the present study demonstrates possible additional role of OsTOP6A1 and provides an effective tool for engineering crop plants for tolerance to different environmental stresses. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genetic transformation of cork oak (Quercus suber L.) for herbicide resistance

The bar gene was introduced into the cork oak genome. Cork oak embryogenic masses were transformed using the Agrobacterium strain AGL1 which carried the plasmid pBINUbiBar. This vector harbours the genes, nptII and bar, the latter under control of the maize ubiquitin promoter. The transgenic embryogenic lines were cryopreserved. Varying activities of phosphinothricin acetyl transferase were detected among the lines, which carried 1–4 copies of the insert. Molecular and biochemical assays confirmed the stability and expression of the transgenes 3 months after thawing the cultures. These results demonstrate genetic engineering of herbicide tolerance in Quercus spp. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Rubén álvarez, Ricardo J. Ordás are contributed equally.     

ps-2, the gene responsible for functional sterility in tomato, due to non-dehiscent anthers, is the result of a mutation in a novel polygalacturonase gene

The recessive mutation ps-2, which appeared spontaneously in tomato, confers functional male sterility due to non-dehiscent anthers. In this study, we isolated and characterized the PS-2 gene. A single nucleotide mutation in a novel tomato polygalacturonase gene is responsible for the ps-2 phenotype. The mutation in ps-2 is responsible for an alternative splicing during maturation of the pre-mRNA, which leads to an aberrant mRNA. Differentiation between ps-2 and wild type (PS-2) anthers only appears in the final developmental stage in which the stomium remains closed in the mutant. To our knowledge, this is the first functional sterility gene isolated in the Solanaceae family. The specific expression of the Arabidopsis homolog of PS-2 in the anther dehiscence zone suggests a conserved mode of action over the plant kingdom, which means that the repression of PS-2 homologs may be a potential way to introduce functional sterility in other species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.