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

Mohd-Radzman NA, Binos S, Truong TT, Imin N, Mariani M, Djordjevic MA - J. Exp. Bot. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Plant Sciences, Research School of Biology, College of Medicine, Biology and Environment, The Australian National University, Canberra ACT 0200, Australia.

No MeSH data available.


Related in: MedlinePlus

Identification of MtCEP1 peptide species in MtCEP1ox root and vector control exudates. (A) The pre-propeptide structure of MtCEP1 showing the two 15 amino acid peptide domains. (B) Eight species of MtCEP1 domain 1 (D1) peptide and one species of the domain 2 (D2) peptide were identified with their respective PTMs. HyP: hydroxylated proline; TaP: tri-arabinosylated proline. (C) The relative concentration of the nine MtCEP1 peptide species found in MtCEP1ox exudate and the five species found in the vector control exudate. Serial dilutions of the synthetic peptide were performed to establish a standard calibration curve (20amol to 200 femtomol) from which the concentration of each peptide was extrapolated. (D) The peptide species in MtCEP1ox samples were analysed using Quadrupole-Orbitrap and Q-TOF mass spectrometers. The ratio of the five peptide species identified correlated well between both nano-LC-ESI-MS systems. (E) The five most abundant peptides in MtCEP1ox sample eluted from the column based on their hydrophobicity as indicated by their retention time in the Quadrapole-Orbitrap. (F) The peptides in the vector control samples were detected in relative minute amounts as shown in the extracted ion chromatogram.
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Figure 2: Identification of MtCEP1 peptide species in MtCEP1ox root and vector control exudates. (A) The pre-propeptide structure of MtCEP1 showing the two 15 amino acid peptide domains. (B) Eight species of MtCEP1 domain 1 (D1) peptide and one species of the domain 2 (D2) peptide were identified with their respective PTMs. HyP: hydroxylated proline; TaP: tri-arabinosylated proline. (C) The relative concentration of the nine MtCEP1 peptide species found in MtCEP1ox exudate and the five species found in the vector control exudate. Serial dilutions of the synthetic peptide were performed to establish a standard calibration curve (20amol to 200 femtomol) from which the concentration of each peptide was extrapolated. (D) The peptide species in MtCEP1ox samples were analysed using Quadrupole-Orbitrap and Q-TOF mass spectrometers. The ratio of the five peptide species identified correlated well between both nano-LC-ESI-MS systems. (E) The five most abundant peptides in MtCEP1ox sample eluted from the column based on their hydrophobicity as indicated by their retention time in the Quadrapole-Orbitrap. (F) The peptides in the vector control samples were detected in relative minute amounts as shown in the extracted ion chromatogram.

Mentions: Thus far, most of the successfully isolated and characterized bioactive peptides were derived from single domain peptide-encoding genes (Amano et al., 2007; Ohyama et al., 2008; Matsuzaki et al., 2010; Meng et al., 2012). However, a considerable number of regulatory peptide-coding genes, including CEPs, encode more than one peptide domain (Oelkers et al., 2009; Delay et al., 2013b; Imin et al., 2013; Roberts et al., 2013; Ogilvie et al., 2014). Using the modified peptide isolation and enrichment protocol, mature 15-amino-acid bioactive peptides corresponding to both putative peptide domains of MtCEP1 (Fig. 2A, B) were isolated and identified from MtCEP1ox samples (Figs 2–3 and Supplementary Figs S1–3). The sequences of the eight domain 1 (D1) and one domain 2 (D2) species were determined (Fig. 2B) and the relative concentrations of each peptide were quantified using Quadrupole-Orbitrap MS. Five of the most abundant peptides identified in the MtCEP1ox sample were also identified in the vector control sample in low amounts (Fig. 2C). This is the first time that CEP peptides have been identified in planta without requiring constitutive or induced amplification of the peptide-encoding gene. The nano-LC-Chip-ESI-Q-TOF approach identified the five most abundant MtCEP1 species in the MtCEP1ox sample only (Supplementary Fig. S1).


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

Mohd-Radzman NA, Binos S, Truong TT, Imin N, Mariani M, Djordjevic MA - J. Exp. Bot. (2015)

Identification of MtCEP1 peptide species in MtCEP1ox root and vector control exudates. (A) The pre-propeptide structure of MtCEP1 showing the two 15 amino acid peptide domains. (B) Eight species of MtCEP1 domain 1 (D1) peptide and one species of the domain 2 (D2) peptide were identified with their respective PTMs. HyP: hydroxylated proline; TaP: tri-arabinosylated proline. (C) The relative concentration of the nine MtCEP1 peptide species found in MtCEP1ox exudate and the five species found in the vector control exudate. Serial dilutions of the synthetic peptide were performed to establish a standard calibration curve (20amol to 200 femtomol) from which the concentration of each peptide was extrapolated. (D) The peptide species in MtCEP1ox samples were analysed using Quadrupole-Orbitrap and Q-TOF mass spectrometers. The ratio of the five peptide species identified correlated well between both nano-LC-ESI-MS systems. (E) The five most abundant peptides in MtCEP1ox sample eluted from the column based on their hydrophobicity as indicated by their retention time in the Quadrapole-Orbitrap. (F) The peptides in the vector control samples were detected in relative minute amounts as shown in the extracted ion chromatogram.
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Related In: Results  -  Collection

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Figure 2: Identification of MtCEP1 peptide species in MtCEP1ox root and vector control exudates. (A) The pre-propeptide structure of MtCEP1 showing the two 15 amino acid peptide domains. (B) Eight species of MtCEP1 domain 1 (D1) peptide and one species of the domain 2 (D2) peptide were identified with their respective PTMs. HyP: hydroxylated proline; TaP: tri-arabinosylated proline. (C) The relative concentration of the nine MtCEP1 peptide species found in MtCEP1ox exudate and the five species found in the vector control exudate. Serial dilutions of the synthetic peptide were performed to establish a standard calibration curve (20amol to 200 femtomol) from which the concentration of each peptide was extrapolated. (D) The peptide species in MtCEP1ox samples were analysed using Quadrupole-Orbitrap and Q-TOF mass spectrometers. The ratio of the five peptide species identified correlated well between both nano-LC-ESI-MS systems. (E) The five most abundant peptides in MtCEP1ox sample eluted from the column based on their hydrophobicity as indicated by their retention time in the Quadrapole-Orbitrap. (F) The peptides in the vector control samples were detected in relative minute amounts as shown in the extracted ion chromatogram.
Mentions: Thus far, most of the successfully isolated and characterized bioactive peptides were derived from single domain peptide-encoding genes (Amano et al., 2007; Ohyama et al., 2008; Matsuzaki et al., 2010; Meng et al., 2012). However, a considerable number of regulatory peptide-coding genes, including CEPs, encode more than one peptide domain (Oelkers et al., 2009; Delay et al., 2013b; Imin et al., 2013; Roberts et al., 2013; Ogilvie et al., 2014). Using the modified peptide isolation and enrichment protocol, mature 15-amino-acid bioactive peptides corresponding to both putative peptide domains of MtCEP1 (Fig. 2A, B) were isolated and identified from MtCEP1ox samples (Figs 2–3 and Supplementary Figs S1–3). The sequences of the eight domain 1 (D1) and one domain 2 (D2) species were determined (Fig. 2B) and the relative concentrations of each peptide were quantified using Quadrupole-Orbitrap MS. Five of the most abundant peptides identified in the MtCEP1ox sample were also identified in the vector control sample in low amounts (Fig. 2C). This is the first time that CEP peptides have been identified in planta without requiring constitutive or induced amplification of the peptide-encoding gene. The nano-LC-Chip-ESI-Q-TOF approach identified the five most abundant MtCEP1 species in the MtCEP1ox sample only (Supplementary Fig. S1).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Plant Sciences, Research School of Biology, College of Medicine, Biology and Environment, The Australian National University, Canberra ACT 0200, Australia.

No MeSH data available.


Related in: MedlinePlus