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Publikationen - Molekulare Signalverarbeitung

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Publikation

López-Carrasco, A., Gago-Zachert, S., Mileti, G., Minoia, S., Flores, R. & Delgado, S. The transcription initiation sites of eggplant latent viroid strands map within distinct motifs in their in vivo RNA conformations RNA Biology 13, 83-97, (2016) DOI: 10.1080/15476286.2015.1119365

Eggplant latent viroid (ELVd), like other members of family Avsunviroidae, replicates in plastids through a symmetric rolling-circle mechanism in which elongation of RNA strands is most likely catalyzed by a nuclear-encoded polymerase (NEP) translocated to plastids. Here we have addressed where NEP initiates transcription of viroid strands. Because this step is presumably directed by sequence/structural motifs, we have previously determined the conformation of the monomeric linear (+) and (−) RNAs of ELVd resulting from hammerhead-mediated self-cleavage. In silico predictions with 3 softwares led to similar bifurcated conformations for both ELVd strands. In vitro examination by non-denaturing PAGE showed that they migrate as prominent single bands, with the ELVd (+) RNA displaying a more compact conformation as revealed by its faster electrophoretic mobility. In vitro SHAPE analysis corroborated the ELVd conformations derived from thermodynamics-based predictions in silico. Moreover, sequence analysis of 94 full-length natural ELVd variants disclosed co-variations, and mutations converting canonical into wobble pairs or vice versa, which confirmed in vivo most of the stems predicted in silico and in vitro, and additionally helped to introduce minor structural refinements. Therefore, results from the 3 experimental approaches were essentially consistent among themselves. Application to RNA preparations from ELVd-infected tissue of RNA ligase-mediated rapid amplification of cDNA ends, combined with pretreatments to modify the 5′ ends of viroid strands, mapped the transcription initiation sites of ELVd (+) and (−) strands in vivo at different sequence/structural motifs, in contrast with the situation previously observed in 2 other members of the family Avsunviroidae.

Publikation

Gasperini, D. & Acosta, I. F. and Farmer, E. E. Cotyledon Wounding of Arabidopsis Seedlings. Bio-protocol 6 (2), e1712, (2016)


Publikation

Kowalski, A. M., Gooding, M., Ferrante, A., Slafer, G. A., Orford, S., Gasperini, D. & Griffiths, S. Agronomic assessment of the wheat semi-dwarfing gene Rht8 in contrasting nitrogen treatments and water regimes Field Crop Res 191, 150-160, (2016) DOI: 10.1016/j.fcr.2016.02.026

Reduced height 8 (Rht8) is the main alternative to the GA-insensitive Rht alleles in hot and dry environments where it reduces plant height without yield penalty. The potential of Rht8 in northern-European wheat breeding remains unclear, since the close linkage with the photoperiod-insensitive allele Ppd-D1a is unfavourable in the relatively cool summers. In the present study, two near-isogenic lines (NILs) contrasting for the Rht8/tall allele from Mara in a UK-adapted and photoperiod-sensitive wheat variety were evaluated in trials with varying nitrogen fertiliser (N) treatments and water regimes across sites in the UK and Spain.

The Rht8 introgression was associated with a robust height reduction of 11% regardless of N treatment and water regime and the Rht8 NIL was more resistant to root-lodging at agronomically-relevant N levels than the tall NIL. In the UK with reduced solar radiation over the growing season than the site in Spain, the Rht8 NIL showed a 10% yield penalty at standard agronomic N levels due to concomitant reduction in grain number and spike number whereas grain weight and harvest index were not significantly different to the tall NIL. The yield penalty associated with the Rht8 introgression was overcome at low N and in irrigated conditions in the UK, and in the high-temperature site in Spain. Decreased spike length and constant spikelet number in the Rht8 NIL resulted in spike compaction of 15%, independent of N and water regime. The genetic interval of Rht8 overlaps with the compactum gene on 2DS, raising the possibility of the same causative gene. Further genetic dissection of these loci is required.

Abbreviations

    ANOVA, analysis of variance; Y, yield; HI, harvest index; GN, grain number (m−2); SS, spikelet number (spike−1); SN, spike number (m−2); HD, heading date; AN, anthesis; 12L, length of the second internode from the top; 13L, length of the third internode from the top; PAR, photosynthetically active radiation; R: FR, red: far-red light reflectance ratio; RCBD, randomised complete block design

Publikation

Gasperini, D., Chauvin, A., Acosta, I.F., Kurenda, A., Stolz, S., Chétalat, A., Wolfender J.-L. & Farmer, E.E. Axial and Radial Oxylipin Transport. Plant Physiol. 169, 2244-2254, (2015) DOI: 10.1104/pp.15.01104

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Publikation

Gasperini, D., Chételat, A., Acosta, I.F., Goossens, J., Pauwels, L., Goossens, A., Dreos, R., Alonso, E. & Farmer, E.E. Multilayered Organization of Jasmonate Signalling in the Regulation of Root Growth PLoS Genet. 11 (6), e1005300, (2015) DOI: 10.1371/journal.pgen.1005300

Physical damage can strongly affect plant growth, reducing the biomass of developing organs situated at a distance from wounds. These effects, previously studied in leaves, require the activation of jasmonate (JA) signalling. Using a novel assay involving repetitive cotyledon wounding in Arabidopsis seedlings, we uncovered a function of JA in suppressing cell division and elongation in roots. Regulatory JA signalling components were then manipulated to delineate their relative impacts on root growth. The new transcription factor mutant myc2-322B was isolated. In vitro transcription assays and whole-plant approaches revealed that myc2-322B is a dosage-dependent gain-of-function mutant that can amplify JA growth responses. Moreover, myc2-322B displayed extreme hypersensitivity to JA that totally suppressed root elongation. The mutation weakly reduced root growth in undamaged plants but, when the upstream negative regulator NINJA was genetically removed, myc2-322B powerfully repressed root growth through its effects on cell division and cell elongation. Furthermore, in a JA-deficient mutant background, ninja1 myc2-322B still repressed root elongation, indicating that it is possible to generate JA-responses in the absence of JA. We show that NINJA forms a broadly expressed regulatory layer that is required to inhibit JA signalling in the apex of roots grown under basal conditions. By contrast, MYC2, MYC3 and MYC4 displayed cell layer-specific localisations and MYC3 and MYC4 were expressed in mutually exclusive regions. In nature, growing roots are likely subjected to constant mechanical stress during soil penetration that could lead to JA production and subsequent detrimental effects on growth. Our data reveal how distinct negative regulatory layers, including both NINJA-dependent and -independent mechanisms, restrain JA responses to allow normal root growth. Mechanistic insights from this work underline the importance of mapping JA signalling components to specific cell types in order to understand and potentially engineer the growth reduction that follows physical damage.

Publikation

Farmer, E.E., Gasperini, D. & Acosta, I.F. The squeeze cell hypothesis for the activation of jasmonate synthesis in response to wounding New Phytol. 204, 282-288, (2014) DOI: 10.1111/nph.12897

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Publikation

Acosta, I.F., Gasperini, D., Chételat, A., Stolz, S., Santuari, L. & Farmer, E.E. Role of NINJA in root jasmonate signaling. In: PNAS 110 (38), 15473-15478, (2013) DOI: 10.1073/pnas.1307910110

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Publikation

Gasperini, D., Greenland, A., Hedden, P., Dreos, R., Harwood, W. & Griffiths, S. Genetic and physiological analysis of Rht8 in bread wheat: an alternative source of semi-dwarfism with a reduced sensitivity to brassinosteroids.. In: J. Exp. Bot. 63, 4419-4436, (2012) DOI: 10.1093/jxb/ers138


Publikation

Asquini, E., Gerdol, M., Gasperini, D., Igic, B., Graziosi, G. & Pallavicini A. S-RNase-like Sequences in Styles of Coffea (Rubiaceae). Evidence for S-RNase Based Gametophytic Self-Incompatibility? Tropical Plant Biol. 4, 237-249, (2011) DOI: 10.1007/s12042-011-9085-2

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Publikation

Flores, R., Grubb, C.D., Elleuch, A., Nohales, M.A, Delgado, S. & Gago, S. Rolling-circle replication of viroids, viroid-like satellite RNAs and hepatitis delta virus RNA Biol 8(2), 200-206, (2011)

Viroids and viroid-like satellite RNAs from plants, and the human hepatitis delta virus (HDV) RNA share some properties that include small size, circularity and replication through a rolling-circle mechanism. Replication occurs in different cell compartments (nucleus, chloroplast and membrane-associated cytoplasmatic vesicles) and has three steps: RNA polymerization, cleavage and ligation. The first step generates oligomeric RNAs that result from the reiterative transcription of the circular templates of one or both polarities, and is catalyzed by either the RNA-dependent RNA polymerase of the helper virus on which viroid-like satellite RNAs are functionally dependent, or by host DNA-dependent RNA polymerases that, remarkably, viroids and HDV redirect to transcribe RNA templates. Cleavage is mediated by host enzymes in certain viroids and viroid-like satellite RNAs, while in others and in HDV is mediated by cis-acting ribozymes of three classes. Ligation appears to be catalyzed mainly by host enzymes. Replication most likely also involves many other non-catalytic proteins of host origin and, in HDV, the single virus-encoded protein.

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