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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

The MS/MS spectra of novel MtCEP1 peptide species analysed by Q Exactive Plus Hybrid Quadrupole-Orbitrap MS. (A) Identification of the trihydroxylated D1 peptide. Nano LC-MS/MS spectrum at 15.31min of the [M+2H]2+ for the trihydroxylated D1:HyP4,7,11 (m/z 757.8471) showed three signature ion fragments y5, y9 and y12 indicating that the 15 amino acid MtCEP1 D1 peptide is hydroxylated at all three prolines. (B) Identification of the dihydroxylated and tri-arabinosylated D1 peptide. The MS/MS spectrum at 16.11min of [M+2H]2+ for D1:HyP4,7,Tap11 (m/z 955.9105) showed extra peaks accompanying the three signature ion fragments with a shift of m/z 132 indicating that the peptide is arabinosylated at Pro11 and hydroxylated at Pro4 and Pro7. (C) Identification of the monohydroxylated D2 peptide. The MS/MS spectrum at 10.60min [M+2H]2+ for D2:HyP11 (m/z 804.3817) showed the 15 amino acid MtCEP1 D2 peptide is hydroxylated at Pro11. (This figure is available in colour at JXB online.)
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Figure 3: The MS/MS spectra of novel MtCEP1 peptide species analysed by Q Exactive Plus Hybrid Quadrupole-Orbitrap MS. (A) Identification of the trihydroxylated D1 peptide. Nano LC-MS/MS spectrum at 15.31min of the [M+2H]2+ for the trihydroxylated D1:HyP4,7,11 (m/z 757.8471) showed three signature ion fragments y5, y9 and y12 indicating that the 15 amino acid MtCEP1 D1 peptide is hydroxylated at all three prolines. (B) Identification of the dihydroxylated and tri-arabinosylated D1 peptide. The MS/MS spectrum at 16.11min of [M+2H]2+ for D1:HyP4,7,Tap11 (m/z 955.9105) showed extra peaks accompanying the three signature ion fragments with a shift of m/z 132 indicating that the peptide is arabinosylated at Pro11 and hydroxylated at Pro4 and Pro7. (C) Identification of the monohydroxylated D2 peptide. The MS/MS spectrum at 10.60min [M+2H]2+ for D2:HyP11 (m/z 804.3817) showed the 15 amino acid MtCEP1 D2 peptide is hydroxylated at Pro11. (This figure is available in colour at JXB online.)

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)

The MS/MS spectra of novel MtCEP1 peptide species analysed by Q Exactive Plus Hybrid Quadrupole-Orbitrap MS. (A) Identification of the trihydroxylated D1 peptide. Nano LC-MS/MS spectrum at 15.31min of the [M+2H]2+ for the trihydroxylated D1:HyP4,7,11 (m/z 757.8471) showed three signature ion fragments y5, y9 and y12 indicating that the 15 amino acid MtCEP1 D1 peptide is hydroxylated at all three prolines. (B) Identification of the dihydroxylated and tri-arabinosylated D1 peptide. The MS/MS spectrum at 16.11min of [M+2H]2+ for D1:HyP4,7,Tap11 (m/z 955.9105) showed extra peaks accompanying the three signature ion fragments with a shift of m/z 132 indicating that the peptide is arabinosylated at Pro11 and hydroxylated at Pro4 and Pro7. (C) Identification of the monohydroxylated D2 peptide. The MS/MS spectrum at 10.60min [M+2H]2+ for D2:HyP11 (m/z 804.3817) showed the 15 amino acid MtCEP1 D2 peptide is hydroxylated at Pro11. (This figure is available in colour at JXB online.)
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Related In: Results  -  Collection

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Figure 3: The MS/MS spectra of novel MtCEP1 peptide species analysed by Q Exactive Plus Hybrid Quadrupole-Orbitrap MS. (A) Identification of the trihydroxylated D1 peptide. Nano LC-MS/MS spectrum at 15.31min of the [M+2H]2+ for the trihydroxylated D1:HyP4,7,11 (m/z 757.8471) showed three signature ion fragments y5, y9 and y12 indicating that the 15 amino acid MtCEP1 D1 peptide is hydroxylated at all three prolines. (B) Identification of the dihydroxylated and tri-arabinosylated D1 peptide. The MS/MS spectrum at 16.11min of [M+2H]2+ for D1:HyP4,7,Tap11 (m/z 955.9105) showed extra peaks accompanying the three signature ion fragments with a shift of m/z 132 indicating that the peptide is arabinosylated at Pro11 and hydroxylated at Pro4 and Pro7. (C) Identification of the monohydroxylated D2 peptide. The MS/MS spectrum at 10.60min [M+2H]2+ for D2:HyP11 (m/z 804.3817) showed the 15 amino acid MtCEP1 D2 peptide is hydroxylated at Pro11. (This figure is available in colour at JXB online.)
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