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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

Strategy for enriching and bioassaying MtCEP1 peptides using root cultures. (A–D) Axenic root cultures containing either the vector (A, C) or MtCEP1ox (B, D). Root cultures were maintained on solidified Fåhraeus medium (A, B) prior to sub-culturing in liquid Fåhraeus medium (C, D) for peptide isolation. (A) and (B) show one week’s growth after adding an equally-sized root segment to the agar plates and (C) and (D) show two week’s growth in the liquid medium after equally sized root segments were initially sub-cultured. (E) The concentrated exudates from vector control and MtCEP1ox root cultures were incorporated into Fåhraeus medium to a final concentration of 1% of that present in the harvested flasks and bioassayed for inhibition of emerged lateral root number on wild type A17 seedlings (measured as LR number/cm) n=12.
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Figure 1: Strategy for enriching and bioassaying MtCEP1 peptides using root cultures. (A–D) Axenic root cultures containing either the vector (A, C) or MtCEP1ox (B, D). Root cultures were maintained on solidified Fåhraeus medium (A, B) prior to sub-culturing in liquid Fåhraeus medium (C, D) for peptide isolation. (A) and (B) show one week’s growth after adding an equally-sized root segment to the agar plates and (C) and (D) show two week’s growth in the liquid medium after equally sized root segments were initially sub-cultured. (E) The concentrated exudates from vector control and MtCEP1ox root cultures were incorporated into Fåhraeus medium to a final concentration of 1% of that present in the harvested flasks and bioassayed for inhibition of emerged lateral root number on wild type A17 seedlings (measured as LR number/cm) n=12.

Mentions: To isolate the MtCEP1 mature peptides, axenic transformed root cultures were established using the same MtCEP1ox and vector control constructs reported in Imin et al. (2013) (Fig. 1A–D). Upon initial agar or liquid subculture, the starting root mass of the vector control and MtCEP1ox transgenic roots was the same. However, upon growth the MtCEP1ox root cultures formed significantly less root branches (~40% of the control; Student’s t-test, P < 0.001) resulting in a reduced root mass (Fig. 1 B, D). The MtCEP1ox cultures showed similar phenotypes to those previously observed in roots transformed with MtCEP1ox (i.e. a reduced number of lateral roots, CCP site formation and reduced root mass). To validate that biologically active secreted peptides were present in the culture medium, the root exudates from MtCEP1ox and vector control liquid cultures were collected, concentrated and added to the growth medium supporting the growth of wild-type M. truncatula seedlings to assay for their biological activities. The MtCEP1ox exudates reduced lateral root number indicating the presence of sufficient biologically active peptide in the liquid culture (Fig. 1E).


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)

Strategy for enriching and bioassaying MtCEP1 peptides using root cultures. (A–D) Axenic root cultures containing either the vector (A, C) or MtCEP1ox (B, D). Root cultures were maintained on solidified Fåhraeus medium (A, B) prior to sub-culturing in liquid Fåhraeus medium (C, D) for peptide isolation. (A) and (B) show one week’s growth after adding an equally-sized root segment to the agar plates and (C) and (D) show two week’s growth in the liquid medium after equally sized root segments were initially sub-cultured. (E) The concentrated exudates from vector control and MtCEP1ox root cultures were incorporated into Fåhraeus medium to a final concentration of 1% of that present in the harvested flasks and bioassayed for inhibition of emerged lateral root number on wild type A17 seedlings (measured as LR number/cm) n=12.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4526912&req=5

Figure 1: Strategy for enriching and bioassaying MtCEP1 peptides using root cultures. (A–D) Axenic root cultures containing either the vector (A, C) or MtCEP1ox (B, D). Root cultures were maintained on solidified Fåhraeus medium (A, B) prior to sub-culturing in liquid Fåhraeus medium (C, D) for peptide isolation. (A) and (B) show one week’s growth after adding an equally-sized root segment to the agar plates and (C) and (D) show two week’s growth in the liquid medium after equally sized root segments were initially sub-cultured. (E) The concentrated exudates from vector control and MtCEP1ox root cultures were incorporated into Fåhraeus medium to a final concentration of 1% of that present in the harvested flasks and bioassayed for inhibition of emerged lateral root number on wild type A17 seedlings (measured as LR number/cm) n=12.
Mentions: To isolate the MtCEP1 mature peptides, axenic transformed root cultures were established using the same MtCEP1ox and vector control constructs reported in Imin et al. (2013) (Fig. 1A–D). Upon initial agar or liquid subculture, the starting root mass of the vector control and MtCEP1ox transgenic roots was the same. However, upon growth the MtCEP1ox root cultures formed significantly less root branches (~40% of the control; Student’s t-test, P < 0.001) resulting in a reduced root mass (Fig. 1 B, D). The MtCEP1ox cultures showed similar phenotypes to those previously observed in roots transformed with MtCEP1ox (i.e. a reduced number of lateral roots, CCP site formation and reduced root mass). To validate that biologically active secreted peptides were present in the culture medium, the root exudates from MtCEP1ox and vector control liquid cultures were collected, concentrated and added to the growth medium supporting the growth of wild-type M. truncatula seedlings to assay for their biological activities. The MtCEP1ox exudates reduced lateral root number indicating the presence of sufficient biologically active peptide in the liquid culture (Fig. 1E).

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