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Novel MtCEP1 peptides produced in vivo differentially regulate root development in Medicago truncatula.

ABSTRACT: Small, post-translationally modified and secreted peptides regulate diverse plant developmental processes. Due to low natural abundance, it is difficult to isolate and identify these peptides. Using an improved peptide isolation protocol and Orbitrap mass spectrometry, nine 15-amino-acid CEP peptides were identified that corresponded to the two domains encoded by Medicago truncatula CEP1 (MtCEP1). Novel arabinosylated and hydroxylated peptides were identified in root cultures overexpressing MtCEP1. The five most abundant CEP peptides were hydroxylated and these species were detected also in low amounts in vector control samples. Synthetic peptides with different hydroxylation patterns differentially affected root development. Notably, the domain 1 peptide hydroxylated at Pro4 and Pro11 (D1:HyP4,11) imparted the strongest inhibition of lateral root emergence when grown with 5mM KNO3 and stimulated the highest increase in nodule number when grown with 0mM KNO3. Inhibition of lateral root emergence by D1:HyP4,11 was not alleviated by removing peptide exposure. In contrast, the domain 2 peptide hydroxylated at Pro11 (D2:HyP11) increased stage III-IV lateral root primordium numbers by 6-fold (P < 0.001) which failed to emerge. Auxin addition at levels which stimulated lateral root formation in wild-type plants had little or no ameliorating effect on CEP peptide-mediated inhibition of lateral root formation or emergence. Both peptides increased and altered the root staining pattern of the auxin-responsive reporter GH3:GUS suggesting CEPs alter auxin sensitivity or distribution. The results showed that CEP primary sequence and post-translational modifications influence peptide activities and the improved isolation procedure effectively and reproducibly identifies and characterises CEPs.

SUBMITTER: Mohd-Radzman NA

PROVIDER: S-EPMC4526912 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:affy_root-dvt-nitrogen_medicago. The biological question is the study of root development adaptation to external nitrogen availability. One of the typical responses to low N provision consists of an increased root branching which promotes soil exploration. In order to differentiate between genes specifically involved in nitrate response and genes of root development, we had performed experiments on both a wild type and a mutant affected in root architecture, both supplied with two different levels of nitrate. Global gene expression profiling of root cells using microarray analysis will be conducted to identify genes expressed during root branching. First, analysing the differential gene expression between the wild type and the mutant both at low and high level of nitrate, we will identify genes of root development but also genes specifically involved in nitrate response. Then, additional comparisons will concern the modifications of wild type and mutant gene expression between the two levels of nitrate. These genes will be compared to those found to be expressed in the wild type roots under submitted to various nitrogen sources (Ruffel et al., 2008). All together these results will allow us to determine which genes, among the firstly underlined, are specifically involved in root development. The kinetics of expression in different tissues of the most relevant of these genes will be further analysed by qRT-PCR. Three successive experiments in growth chamber were performed on two different genotypes of Medicago truncatula. Each experiment constituted a biological replicate. For each of them, after 4 days of seed cold-treatment followed by 4 days of germination, individual plantlets were transferred onto hydroponic culture tanks containing vigorously aerated nutrient solution. Each tank contained 6 plantlets of J5 and 6 plantlets of the mutant. On one shelf of the growth chamber, nutrient solution in the tanks was supplemented by 1mM of KNO3; on the other shelf, the level of KNO3 in the solution was of 10mM. Nutrient solution was renewed every week. Roots of the two genotypes were harvested separately but at the same time, 10 days after the plantlets transfer onto hydroponic culture tanks. Keywords: dose response, wt vs mutant comparison

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Novel MtCEP1 peptides produced in vivo differentially regulate root development in Medicago truncatula.

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