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A salt-regulated peptide derived from the CAP superfamily protein negatively regulates salt-stress tolerance in Arabidopsis.

Chien PS, Nam HG, Chen YR - J. Exp. Bot. (2015)

Bottom Line: This peptide was found by searching homologues in Arabidopsis using the precursor of a tomato CAP-derived peptide (CAPE) that was initially identified as an immune signal.In searching for a CAPE involved in salt responses, we screened CAPE precursor genes that showed salt-responsive expression and found that the PROAtCAPE1 (AT4G33730) gene was regulated by salinity.We confirmed the endogenous Arabidopsis CAP-derived peptide 1 (AtCAPE1) by mass spectrometry and found that a key amino acid residue in PROAtCAPE1 is critical for AtCAPE1 production.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, Academia Sinica, Taipei 115, Taiwan Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung 402, Taiwan.

No MeSH data available.


Related in: MedlinePlus

Identification of AtCAPE1 in Arabidopsis. (A) Deduced amino acid sequence of the PROAtCAPE1 product. The predicted secretion signal peptide is underlined. The CAP domain is shaded in grey. The putative AtCAPE1 peptide is shaded in red. The cleavage site predicted to produce AtCAPE1 is indicated with an arrowhead. The putative cleavage signal motif is double-underlined. (B) LC-MS/MS spectrum of the synthetic AtCAPE1. The y-ion is the C-terminal fragments after peptide bond cleavage while the b-ion is the N-terminal fragments. (C) LC-MS/MS spectrum of the identified AtCAPE1 in Arabidopsis. (D) Schema representing the construct used for constitutive overexpression of enhanced yellow fluorescent protein (eYFP)-tagged PROAtCAPE1. The green box shows the CNYx motif. The putative CAPE is shown in red. The numbers indicate the predicted molecular weight of precursor protein tagged with eYFP (45.7kDa) and the cleaved precursor tagged with eYFP (26.3kDa). (E) Production of the precursor PROAtCAPE1 and the cleaved PROAtCAPE1 in CAPE1oxCNYD and CAPE1oxCNAD transgenic plants, where eYFP was fused to PROAtCAPE1 containing wild type (CNYD) and the mutated (CNAD) junction sequence, respectively. T3 seedlings derived from independent transgenic lines were sampled for western blotting with anti-GFP antibody. Coomassie blue staining was used for protein loading control. The lower panel shows the presence of the T-DNA insertions in the transgenic plants by genomic DNA PCR with the primer pair 35S-F’ and eYFP-R’ shown in (D).
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Figure 1: Identification of AtCAPE1 in Arabidopsis. (A) Deduced amino acid sequence of the PROAtCAPE1 product. The predicted secretion signal peptide is underlined. The CAP domain is shaded in grey. The putative AtCAPE1 peptide is shaded in red. The cleavage site predicted to produce AtCAPE1 is indicated with an arrowhead. The putative cleavage signal motif is double-underlined. (B) LC-MS/MS spectrum of the synthetic AtCAPE1. The y-ion is the C-terminal fragments after peptide bond cleavage while the b-ion is the N-terminal fragments. (C) LC-MS/MS spectrum of the identified AtCAPE1 in Arabidopsis. (D) Schema representing the construct used for constitutive overexpression of enhanced yellow fluorescent protein (eYFP)-tagged PROAtCAPE1. The green box shows the CNYx motif. The putative CAPE is shown in red. The numbers indicate the predicted molecular weight of precursor protein tagged with eYFP (45.7kDa) and the cleaved precursor tagged with eYFP (26.3kDa). (E) Production of the precursor PROAtCAPE1 and the cleaved PROAtCAPE1 in CAPE1oxCNYD and CAPE1oxCNAD transgenic plants, where eYFP was fused to PROAtCAPE1 containing wild type (CNYD) and the mutated (CNAD) junction sequence, respectively. T3 seedlings derived from independent transgenic lines were sampled for western blotting with anti-GFP antibody. Coomassie blue staining was used for protein loading control. The lower panel shows the presence of the T-DNA insertions in the transgenic plants by genomic DNA PCR with the primer pair 35S-F’ and eYFP-R’ shown in (D).

Mentions: The deduced coding region of the PROAtCAPE1 product was 172 aa (Fig. 1A). A predicted signal peptide was found at the N terminus with a cleavage site between aa 27 and 28 (SignalP 4.1 Server, http://www.cbs.dtu.dk/services/SignalP/), suggesting that PROAtCAPE1 is a secretory protein that is synthesized in the endoplasmic reticulum (ER) and secreted into the extracellular space. The CAP domain (Gibbs et al., 2008) conserved among CAPs from various species was located in the middle of the sequence (aa 43–160), while the putative AtCAPE1 was at the C-terminal end (Fig. 1A).


A salt-regulated peptide derived from the CAP superfamily protein negatively regulates salt-stress tolerance in Arabidopsis.

Chien PS, Nam HG, Chen YR - J. Exp. Bot. (2015)

Identification of AtCAPE1 in Arabidopsis. (A) Deduced amino acid sequence of the PROAtCAPE1 product. The predicted secretion signal peptide is underlined. The CAP domain is shaded in grey. The putative AtCAPE1 peptide is shaded in red. The cleavage site predicted to produce AtCAPE1 is indicated with an arrowhead. The putative cleavage signal motif is double-underlined. (B) LC-MS/MS spectrum of the synthetic AtCAPE1. The y-ion is the C-terminal fragments after peptide bond cleavage while the b-ion is the N-terminal fragments. (C) LC-MS/MS spectrum of the identified AtCAPE1 in Arabidopsis. (D) Schema representing the construct used for constitutive overexpression of enhanced yellow fluorescent protein (eYFP)-tagged PROAtCAPE1. The green box shows the CNYx motif. The putative CAPE is shown in red. The numbers indicate the predicted molecular weight of precursor protein tagged with eYFP (45.7kDa) and the cleaved precursor tagged with eYFP (26.3kDa). (E) Production of the precursor PROAtCAPE1 and the cleaved PROAtCAPE1 in CAPE1oxCNYD and CAPE1oxCNAD transgenic plants, where eYFP was fused to PROAtCAPE1 containing wild type (CNYD) and the mutated (CNAD) junction sequence, respectively. T3 seedlings derived from independent transgenic lines were sampled for western blotting with anti-GFP antibody. Coomassie blue staining was used for protein loading control. The lower panel shows the presence of the T-DNA insertions in the transgenic plants by genomic DNA PCR with the primer pair 35S-F’ and eYFP-R’ shown in (D).
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Related In: Results  -  Collection

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Figure 1: Identification of AtCAPE1 in Arabidopsis. (A) Deduced amino acid sequence of the PROAtCAPE1 product. The predicted secretion signal peptide is underlined. The CAP domain is shaded in grey. The putative AtCAPE1 peptide is shaded in red. The cleavage site predicted to produce AtCAPE1 is indicated with an arrowhead. The putative cleavage signal motif is double-underlined. (B) LC-MS/MS spectrum of the synthetic AtCAPE1. The y-ion is the C-terminal fragments after peptide bond cleavage while the b-ion is the N-terminal fragments. (C) LC-MS/MS spectrum of the identified AtCAPE1 in Arabidopsis. (D) Schema representing the construct used for constitutive overexpression of enhanced yellow fluorescent protein (eYFP)-tagged PROAtCAPE1. The green box shows the CNYx motif. The putative CAPE is shown in red. The numbers indicate the predicted molecular weight of precursor protein tagged with eYFP (45.7kDa) and the cleaved precursor tagged with eYFP (26.3kDa). (E) Production of the precursor PROAtCAPE1 and the cleaved PROAtCAPE1 in CAPE1oxCNYD and CAPE1oxCNAD transgenic plants, where eYFP was fused to PROAtCAPE1 containing wild type (CNYD) and the mutated (CNAD) junction sequence, respectively. T3 seedlings derived from independent transgenic lines were sampled for western blotting with anti-GFP antibody. Coomassie blue staining was used for protein loading control. The lower panel shows the presence of the T-DNA insertions in the transgenic plants by genomic DNA PCR with the primer pair 35S-F’ and eYFP-R’ shown in (D).
Mentions: The deduced coding region of the PROAtCAPE1 product was 172 aa (Fig. 1A). A predicted signal peptide was found at the N terminus with a cleavage site between aa 27 and 28 (SignalP 4.1 Server, http://www.cbs.dtu.dk/services/SignalP/), suggesting that PROAtCAPE1 is a secretory protein that is synthesized in the endoplasmic reticulum (ER) and secreted into the extracellular space. The CAP domain (Gibbs et al., 2008) conserved among CAPs from various species was located in the middle of the sequence (aa 43–160), while the putative AtCAPE1 was at the C-terminal end (Fig. 1A).

Bottom Line: This peptide was found by searching homologues in Arabidopsis using the precursor of a tomato CAP-derived peptide (CAPE) that was initially identified as an immune signal.In searching for a CAPE involved in salt responses, we screened CAPE precursor genes that showed salt-responsive expression and found that the PROAtCAPE1 (AT4G33730) gene was regulated by salinity.We confirmed the endogenous Arabidopsis CAP-derived peptide 1 (AtCAPE1) by mass spectrometry and found that a key amino acid residue in PROAtCAPE1 is critical for AtCAPE1 production.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, Academia Sinica, Taipei 115, Taiwan Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung 402, Taiwan.

No MeSH data available.


Related in: MedlinePlus