High-Capacity Conductive Nanocellulose Paper Sheets for Electrochemically Controlled Extraction of DNA Oligomers

Highly porous polypyrrole (PPy)-nanocellulose paper sheets have been evaluated as inexpensive and disposable electrochemically controlled three-dimensional solid phase extraction materials. The composites, which had a total anion exchange capacity of about 1.1 mol kg−1, were used for extraction and subsequent release of negatively charged fluorophore tagged DNA oligomers via galvanostatic oxidation and reduction of a 30–50 nm conformal PPy layer on the cellulose substrate. The ion exchange capacity, which was, at least, two orders of magnitude higher than those previously reached in electrochemically controlled extraction, originated from the high surface area (i.e. 80 m2 g−1) of the porous composites and the thin PPy layer which ensured excellent access to the ion exchange material. This enabled the extractions to be carried out faster and with better control of the PPy charge than with previously employed approaches. Experiments in equimolar mixtures of (dT)6, (dT)20, and (dT)40 DNA oligomers showed that all oligomers could be extracted, and that the smallest oligomer was preferentially released with an efficiency of up to 40% during the reduction of the PPy layer. These results indicate that the present material is very promising for the development of inexpensive and efficient electrochemically controlled ion-exchange membranes for batch-wise extraction of biomolecules

Two euAGAMOUS genes control C-function in Medicago truncatula

[EN] C-function MADS-box transcription factors belong to the AGAMOUS (AG) lineage and specify both stamen and carpel identity and floral meristem determinacy. In core eudicots, the AG lineage is further divided into two branches, the euAG and PLE lineages. Functional analyses across flowering plants strongly support the idea that duplicated AG lineage genes have different degrees of subfunctionalization of the C-function. The legume Medicago truncatula contains three C-lineage genes in its genome: two euAG genes (MtAGa and MtAGb) and one PLENA-like gene (MtSHP). This species is therefore a good experimental system to study the effects of gene duplication within the AG subfamily. We have studied the respective functions of each euAG genes in M. truncatula employing expression analyses and reverse genetic approaches. Our results show that the M. truncatula euAG- and PLENA-like genes are an example of subfunctionalization as a result of a change in expression pattern. MtAGa and MtAGb are the only genes showing a full C-function activity, concomitant with their ancestral expression profile, early in the floral meristem, and in the third and fourth floral whorls during floral development. In contrast, MtSHP expression appears late during floral development suggesting it does not contribute significantly to the C-function. Furthermore, the redundant MtAGa and MtAGb paralogs have been retained which provides the overall dosage required to specify the C-function in M. truncatula.This work was funded by grants BIO2009-08134 and BIO2012-39849-C02-01 from the Spanish Ministry of Economy and Competitiveness and the Ramon y Cajal Program (RYC-2007-00627 to CGM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Serwatowska, J.; Roque Mesa, EM.; Gómez Mena, MC.; Constantin, GD.; Wen, J.; Mysore, KS.; Lund, OS.... (2014). Two euAGAMOUS genes control C-function in Medicago truncatula. PLoS ONE. 9(8):103770-1-103770-12. https://doi.org/10.1371/journal.pone.0103770S103770-1103770-1298Prunet, N., & Jack, T. P. (2013). Flower Development in Arabidopsis: There Is More to It Than Learning Your ABCs. Flower Development, 3-33. doi:10.1007/978-1-4614-9408-9_1Causier, B., Schwarz-Sommer, Z., & Davies, B. (2010). Floral organ identity: 20 years of ABCs. Seminars in Cell & Developmental Biology, 21(1), 73-79. doi:10.1016/j.semcdb.2009.10.005Irish, V. F. (2010). The flowering of Arabidopsis flower development. The Plant Journal, 61(6), 1014-1028. doi:10.1111/j.1365-313x.2009.04065.xHeijmans, K., Morel, P., & Vandenbussche, M. (2012). MADS-box Genes and Floral Development: the Dark Side. Journal of Experimental Botany, 63(15), 5397-5404. doi:10.1093/jxb/ers233Bowman, J. L., Smyth, D. R., & Meyerowitz, E. M. (1989). Genes directing flower development in Arabidopsis. The Plant Cell, 1(1), 37-52. doi:10.1105/tpc.1.1.37Yanofsky, M. F., Ma, H., Bowman, J. L., Drews, G. N., Feldmann, K. A., & Meyerowitz, E. M. (1990). The protein encoded by the Arabidopsis homeotic gene agamous resembles transcription factors. Nature, 346(6279), 35-39. doi:10.1038/346035a0Bradley, D., Carpenter, R., Sommer, H., Hartley, N., & Coen, E. (1993). Complementary floral homeotic phenotypes result from opposite orientations of a transposon at the plena locus of antirrhinum. Cell, 72(1), 85-95. doi:10.1016/0092-8674(93)90052-rPinyopich, A., Ditta, G. S., Savidge, B., Liljegren, S. J., Baumann, E., Wisman, E., & Yanofsky, M. F. (2003). Assessing the redundancy of MADS-box genes during carpel and ovule development. Nature, 424(6944), 85-88. doi:10.1038/nature01741Liljegren, S. J., Ditta, G. S., Eshed, Y., Savidge, B., Bowman, J. L., & Yanofsky, M. F. (2000). SHATTERPROOF MADS-box genes control seed dispersal in Arabidopsis. Nature, 404(6779), 766-770. doi:10.1038/35008089Davies, B., Motte, P., Keck, E., Saedler, H., Sommer, H., & Schwarz-Sommer, Z. (1999). PLENA and FARINELLI: redundancy and regulatory interactions between two Antirrhinum MADS-box factors controlling flower development. The EMBO Journal, 18(14), 4023-4034. doi:10.1093/emboj/18.14.4023Kramer, E. M., Jaramillo, M. A., & Di Stilio, V. S. (2004). Patterns of Gene Duplication and Functional Evolution During the Diversification of the AGAMOUS Subfamily of MADS Box Genes in Angiosperms. Genetics, 166(2), 1011-1023. doi:10.1534/genetics.166.2.1011Becker, A. (2003). The major clades of MADS-box genes and their role in the development and evolution of flowering plants. Molecular Phylogenetics and Evolution, 29(3), 464-489. doi:10.1016/s1055-7903(03)00207-0Irish, V. F. (2003). The evolution of floral homeotic gene function. BioEssays, 25(7), 637-646. doi:10.1002/bies.10292Zahn, L. M., Leebens-Mack, J. H., Arrington, J. M., Hu, Y., Landherr, L. L., dePamphilis, C. W., … Ma, H. (2006). Conservation and divergence in the AGAMOUS subfamily of MADS-box genes: evidence of independent sub- and neofunctionalization events. Evolution Development, 8(1), 30-45. doi:10.1111/j.1525-142x.2006.05073.xFerrandiz, C. (2000). Negative Regulation of the SHATTERPROOF Genes by FRUITFULL During Arabidopsis Fruit Development. Science, 289(5478), 436-438. doi:10.1126/science.289.5478.436Ma, H., Yanofsky, M. F., & Meyerowitz, E. M. (1991). AGL1-AGL6, an Arabidopsis gene family with similarity to floral homeotic and transcription factor genes. Genes & Development, 5(3), 484-495. doi:10.1101/gad.5.3.484Savidge, B., Rounsley, S. D., & Yanofsky, M. F. (1995). Temporal relationship between the transcription of two Arabidopsis MADS box genes and the floral organ identity genes. The Plant Cell, 7(6), 721-733. doi:10.1105/tpc.7.6.721Colombo, M., Brambilla, V., Marcheselli, R., Caporali, E., Kater, M. M., & Colombo, L. (2010). A new role for the SHATTERPROOF genes during Arabidopsis gynoecium development. Developmental Biology, 337(2), 294-302. doi:10.1016/j.ydbio.2009.10.043Fourquin, C., & Ferrándiz, C. (2012). Functional analyses of AGAMOUS family members in Nicotiana benthamiana clarify the evolution of early and late roles of C-function genes in eudicots. The Plant Journal, 71(6), 990-1001. doi:10.1111/j.1365-313x.2012.05046.xKapoor, M., Tsuda, S., Tanaka, Y., Mayama, T., Okuyama, Y., Tsuchimoto, S., & Takatsuji, H. (2002). Role of petuniapMADS3in determination of floral organ and meristem identity, as revealed by its loss of function. The Plant Journal, 32(1), 115-127. doi:10.1046/j.1365-313x.2002.01402.xPan, I. L., McQuinn, R., Giovannoni, J. J., & Irish, V. F. (2010). Functional diversification of AGAMOUS lineage genes in regulating tomato flower and fruit development. Journal of Experimental Botany, 61(6), 1795-1806. doi:10.1093/jxb/erq046Pnueli, L., Hareven, D., Rounsley, S. D., Yanofsky, M. F., & Lifschitz, E. (1994). Isolation of the tomato AGAMOUS gene TAG1 and analysis of its homeotic role in transgenic plants. The Plant Cell, 6(2), 163-173. doi:10.1105/tpc.6.2.163Dreni, L., & Kater, M. M. (2013). MADSreloaded: evolution of theAGAMOUSsubfamily genes. New Phytologist, 201(3), 717-732. doi:10.1111/nph.12555Brunner, A. M. (2000). Plant Molecular Biology, 44(5), 619-634. doi:10.1023/a:1026550205851Perl-Treves, R., Kahana, A., Rosenman, N., Xiang, Y., & Silberstein, L. (1998). Expression of Multiple AGAMOUS-Like Genes in Male and Female Flowers of Cucumber (Cucumis sativus L.). Plant and Cell Physiology, 39(7), 701-710. doi:10.1093/oxfordjournals.pcp.a029424Yu, D., Kotilainen, M., Pöllänen, E., Mehto, M., Elomaa, P., Helariutta, Y., … Teeri, T. H. (1999). Organ identity genes and modified patterns of flower development in Gerbera hybrida (Asteraceae). The Plant Journal, 17(1), 51-62. doi:10.1046/j.1365-313x.1999.00351.xDong, Z., Zhao, Z., Liu, C., Luo, J., Yang, J., Huang, W., … Luo, D. (2005). Floral Patterning in Lotus japonicus. Plant Physiology, 137(4), 1272-1282. doi:10.1104/pp.104.054288Hofer, J. M., & Noel Ellis, T. (2014). Developmental specialisations in the legume family. Current Opinion in Plant Biology, 17, 153-158. doi:10.1016/j.pbi.2013.11.014Fourquin, C., del Cerro, C., Victoria, F. C., Vialette-Guiraud, A., de Oliveira, A. C., & Ferrándiz, C. (2013). A Change in SHATTERPROOF Protein Lies at the Origin of a Fruit Morphological Novelty and a New Strategy for Seed Dispersal in Medicago Genus. Plant Physiology, 162(2), 907-917. doi:10.1104/pp.113.217570Hewitt EJ (1966) Sand and Water Culture Methods Used in the Study of Plant Nutrition. Farnham Royal, UK: Commonwealth Agricultural Bureau.Cheng, X., Wang, M., Lee, H.-K., Tadege, M., Ratet, P., Udvardi, M., … Wen, J. (2013). An efficient reverse genetics platform in the model legumeMedicago truncatula. New Phytologist, 201(3), 1065-1076. doi:10.1111/nph.12575D’ Erfurth, I., Cosson, V., Eschstruth, A., Lucas, H., Kondorosi, A., & Ratet, P. (2003). Efficient transposition of theTnt1tobacco retrotransposon in the model legumeMedicago truncatula. The Plant Journal, 34(1), 95-106. doi:10.1046/j.1365-313x.2003.01701.xTadege, M., Ratet, P., & Mysore, K. S. (2005). Insertional mutagenesis: a Swiss Army knife for functional genomics of Medicago truncatula. Trends in Plant Science, 10(5), 229-235. doi:10.1016/j.tplants.2005.03.009Tadege, M., Wen, J., He, J., Tu, H., Kwak, Y., Eschstruth, A., … Mysore, K. S. (2008). Large-scale insertional mutagenesis using the Tnt1 retrotransposon in the model legume Medicago truncatula. The Plant Journal, 54(2), 335-347. doi:10.1111/j.1365-313x.2008.03418.xCheng, X., Wen, J., Tadege, M., Ratet, P., & Mysore, K. S. (2010). Reverse Genetics in Medicago truncatula Using Tnt1 Insertion Mutants. Plant Reverse Genetics, 179-190. doi:10.1007/978-1-60761-682-5_13Benlloch, R., d’ Erfurth, I., Ferrandiz, C., Cosson, V., Beltrán, J. P., Cañas, L. A., … Ratet, P. (2006). Isolation of mtpim Proves Tnt1 a Useful Reverse Genetics Tool in Medicago truncatula and Uncovers New Aspects of AP1-Like Functions in Legumes. Plant Physiology, 142(3), 972-983. doi:10.1104/pp.106.083543Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., … Higgins, D. G. (2007). Clustal W and Clustal X version 2.0. Bioinformatics, 23(21), 2947-2948. doi:10.1093/bioinformatics/btm404Altschul, S. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research, 25(17), 3389-3402. doi:10.1093/nar/25.17.3389Tamura, K., Dudley, J., Nei, M., & Kumar, S. (2007). MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) Software Version 4.0. Molecular Biology and Evolution, 24(8), 1596-1599. doi:10.1093/molbev/msm092Dellaporta, S. L., Wood, J., & Hicks, J. B. (1983). A plant DNA minipreparation: Version II. Plant Molecular Biology Reporter, 1(4), 19-21. doi:10.1007/bf02712670Schmittgen, T. D., & Livak, K. J. (2008). Analyzing real-time PCR data by the comparative CT method. Nature Protocols, 3(6), 1101-1108. doi:10.1038/nprot.2008.73Constantin, G. D., Krath, B. N., MacFarlane, S. A., Nicolaisen, M., Elisabeth Johansen, I., & Lund, O. S. (2004). Virus-induced gene silencing as a tool for functional genomics in a legume species. The Plant Journal, 40(4), 622-631. doi:10.1111/j.1365-313x.2004.02233.xWesley, S. V., Helliwell, C. A., Smith, N. A., Wang, M., Rouse, D. T., Liu, Q., … Waterhouse, P. M. (2001). Construct design for efficient, effective and high-throughput gene silencing in plants. The Plant Journal, 27(6), 581-590. doi:10.1046/j.1365-313x.2001.01105.xGuerineau F, Mullineaux P (1993) Plant transformation and expression vectors. In: Croy R, editor. Plant Molecular Biology. Oxford, UK: Bios Scientific Publishers, Academic Press. pp. 121–147.Clough, S. J., & Bent, A. F. (1998). Floral dip: a simplified method forAgrobacterium-mediated transformation ofArabidopsis thaliana. The Plant Journal, 16(6), 735-743. doi:10.1046/j.1365-313x.1998.00343.xBenlloch, R., Roque, E., Ferrándiz, C., Cosson, V., Caballero, T., Penmetsa, R. V., … Madueño, F. (2009). Analysis of B function in legumes: PISTILLATA proteins do not require the PI motif for floral organ development inMedicago truncatula. The Plant Journal, 60(1), 102-111. doi:10.1111/j.1365-313x.2009.03939.xRoque, E., Serwatowska, J., Cruz Rochina, M., Wen, J., Mysore, K. S., Yenush, L., … Cañas, L. A. (2012). Functional specialization of duplicated AP3-like genes inMedicago truncatula. The Plant Journal, 73(4), 663-675. doi:10.1111/tpj.12068Flanagan, C. A., Hu, Y., & Ma, H. (1996). Specific expression of the AGL1 MADS-box gene suggests regulatory functions in Arabidopsis gynoecium and ovule development. The Plant Journal, 10(2), 343-353. doi:10.1046/j.1365-313x.1996.10020343.xSieburth, L. E., & Meyerowitz, E. M. (1997). Molecular dissection of the AGAMOUS control region shows that cis elements for spatial regulation are located intragenically. The Plant Cell, 9(3), 355-365. doi:10.1105/tpc.9.3.355Busch, M. A. (1999). Activation of a Floral Homeotic Gene in Arabidopsis. Science, 285(5427), 585-587. doi:10.1126/science.285.5427.585Moyroud, E., Minguet, E. G., Ott, F., Yant, L., Posé, D., Monniaux, M., … Parcy, F. (2011). Prediction of Regulatory Interactions from Genome Sequences Using a Biophysical Model for the Arabidopsis LEAFY Transcription Factor. The Plant Cell, 23(4), 1293-1306. doi:10.1105/tpc.111.083329Grønlund, M., Constantin, G., Piednoir, E., Kovacev, J., Johansen, I. E., & Lund, O. S. (2008). Virus-induced gene silencing in Medicago truncatula and Lathyrus odorata. Virus Research, 135(2), 345-349. doi:10.1016/j.virusres.2008.04.005Mandel, M. A., Bowman, J. L., Kempin, S. A., Ma, H., Meyerowitz, E. M., & Yanofsky, M. F. (1992). Manipulation of flower structure in transgenic tobacco. Cell, 71(1), 133-143. doi:10.1016/0092-8674(92)90272-eMizukami, Y., & Ma, H. (1992). Ectopic expression of the floral homeotic gene AGAMOUS in transgenic Arabidopsis plants alters floral organ identity. Cell, 71(1), 119-131. doi:10.1016/0092-8674(92)90271-dCannon, S. B., Sterck, L., Rombauts, S., Sato, S., Cheung, F., Gouzy, J., … Young, N. D. (2006). Legume genome evolution viewed through the Medicago truncatula and Lotus japonicus genomes. Proceedings of the National Academy of Sciences, 103(40), 14959-14964. doi:10.1073/pnas.0603228103Young, N. D., & Bharti, A. K. (2012). Genome-Enabled Insights into Legume Biology. Annual Review of Plant Biology, 63(1), 283-305. doi:10.1146/annurev-arplant-042110-103754Jager, M. (2003). MADS-Box Genes in Ginkgo biloba and the Evolution of the AGAMOUS Family. Molecular Biology and Evolution, 20(5), 842-854. doi:10.1093/molbev/msg089Johansen, B., Pedersen, L. B., Skipper, M., & Frederiksen, S. (2002). MADS-box gene evolution—structure and transcription patterns. Molecular Phylogenetics and Evolution, 23(3), 458-480. doi:10.1016/s1055-7903(02)00032-5Rutledge, R., Regan, S., Nicolas, O., Fobert, P., Côté, C., Bosnich, W., … Stewart, D. (1998). Characterization of an AGAMOUS homologue from the conifer black spruce ( Picea mariana ) that produces floral homeotic conversions when expressed in Arabidopsis. The Plant Journal, 15(5), 625-634. doi:10.1046/j.1365-313x.1998.00250.xParcy, F., Nilsson, O., Busch, M. A., Lee, I., & Weigel, D. (1998). A genetic framework for floral patterning. Nature, 395(6702), 561-566. doi:10.1038/26903Causier, B., Bradley, D., Cook, H., & Davies, B. (2009). Conserved intragenic elements were critical for the evolution of the floral C-function. The Plant Journal, 58(1), 41-52. doi:10.1111/j.1365-313x.2008.03759.xAiroldi, C. A., & Davies, B. (2012). Gene Duplication and the Evolution of Plant MADS-box Transcription Factors. Journal of Genetics and Genomics, 39(4), 157-165. doi:10.1016/j.jgg.2012.02.008Giménez, E., Pineda, B., Capel, J., Antón, M. T., Atarés, A., Pérez-Martín, F., … Lozano, R. (2010). Functional Analysis of the Arlequin Mutant Corroborates the Essential Role of the ARLEQUIN/TAGL1 Gene during Reproductive Development of Tomato. PLoS ONE, 5(12), e14427. doi:10.1371/journal.pone.0014427Kater, M. M., Colombo, L., Franken, J., Busscher, M., Masiero, S., Van Lookeren Campagne, M. M., & Angenent, G. C. (1998). Multiple AGAMOUS Homologs from Cucumber and Petunia Differ in Their Ability to Induce Reproductive Organ Fate. The Plant Cell, 10(2), 171-182. doi:10.1105/tpc.10.2.171Tsuchimoto, S., van der Krol, A. R., & Chua, N. H. (1993). Ectopic expression of pMADS3 in transgenic petunia phenocopies the petunia blind mutant. The Plant Cell, 5(8), 843-853. doi:10.1105/tpc.5.8.843Airoldi, C. A., Bergonzi, S., & Davies, B. (2010). Single amino acid change alters the ability to specify male or female organ identity. Proceedings of the National Academy of Sciences, 107(44), 18898-18902. doi:10.1073/pnas.1009050107Causier, B., Castillo, R., Zhou, J., Ingram, R., Xue, Y., Schwarz-Sommer, Z., & Davies, B. (2005). Evolution in Action: Following Function in Duplicated Floral Homeotic Genes. Current Biology, 15(16), 1508-1512. doi:10.1016/j.cub.2005.07.063Birchler, J. A., & Veitia, R. A. (2007). The Gene Balance Hypothesis: From Classical Genetics to Modern Genomics. The Plant Cell, 19(2), 395-402. doi:10.1105/tpc.106.049338Birchler, J. A., & Veitia, R. A. (2009). The gene balance hypothesis: implications for gene regulation, quantitative traits and evolution. New Phytologist, 186(1), 54-62. doi:10.1111/j.1469-8137.2009.03087.xEdger, P. P., & Pires, J. C. (2009). Gene and genome duplications: the impact of dosage-sensitivity on the fate of nuclear genes. Chromosome Research, 17(5), 699-717. doi:10.1007/s10577-009-9055-9Freeling, M. (2006). Gene-balanced duplications, like tetraploidy, provide predictable drive to increase morphological complexity. Genome Research, 16(7), 805-814. doi:10.1101/gr.368140

The Genetic Basis of Floral Organ Identity and Its Applications in Ornamental Plant Breeding

Chapter 2Petunia hybrida (or garden petunia) is worldwide one of the most popular bedding plants. At the same time, petunia has a decades-long history as a model species for scientific research to study a variety of processes, including floral organ development. Here we explain the genetic basis of floral organ identity in a comprehensible manner and illustrate the potential of floral organ identity mutants for ornamental plant breeding, using petunia as an example. Although the B-and C-floral organ identity functions are well conserved at the molecular level, indicating broad applicability, different species may exhibit significant differences in the degree of redundancy versus subfunctionalization/ specialization among duplicated pairs of the homeotic genes. This is a direct consequence of the complex origin of different plant genomes, which were shaped by whole-genome, large and small-scale duplication events, often leading to (partial) genetic redundancy. Since classical genetic screens only can uncover nonredundant functions, this is probably the main reason why the use of floral organ identity mutants as breeding targets has remained unexplored in many ornamentals. We discuss how different breeding strategies may cope with this phenomenon

Observational determination of surface radiative forcing by CO2 from 2000 to 2010.

The climatic impact of CO2 and other greenhouse gases is usually quantified in terms of radiative forcing, calculated as the difference between estimates of the Earth's radiation field from pre-industrial and present-day concentrations of these gases. Radiative transfer models calculate that the increase in CO2 since 1750 corresponds to a global annual-mean radiative forcing at the tropopause of 1.82 ± 0.19 W m(-2) (ref. 2). However, despite widespread scientific discussion and modelling of the climate impacts of well-mixed greenhouse gases, there is little direct observational evidence of the radiative impact of increasing atmospheric CO2. Here we present observationally based evidence of clear-sky CO2 surface radiative forcing that is directly attributable to the increase, between 2000 and 2010, of 22 parts per million atmospheric CO2. The time series of this forcing at the two locations-the Southern Great Plains and the North Slope of Alaska-are derived from Atmospheric Emitted Radiance Interferometer spectra together with ancillary measurements and thoroughly corroborated radiative transfer calculations. The time series both show statistically significant trends of 0.2 W m(-2) per decade (with respective uncertainties of ±0.06 W m(-2) per decade and ±0.07 W m(-2) per decade) and have seasonal ranges of 0.1-0.2 W m(-2). This is approximately ten per cent of the trend in downwelling longwave radiation. These results confirm theoretical predictions of the atmospheric greenhouse effect due to anthropogenic emissions, and provide empirical evidence of how rising CO2 levels, mediated by temporal variations due to photosynthesis and respiration, are affecting the surface energy balance