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

Highlight An 11 aa peptide derived from one of the cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 (CAP) superfamily is salt regulated, conferring salt susceptibility through suppression of salt-tolerance genes.

stored at -80°C until use.
Total RNA was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's recommendations followed by DNase treatments at 37°C for 15 minutes with RQ1 RNase-free DNase (Promega, Madison, WI, USA) to remove the contamination of genomic DNA. Equal volume of Phenol:Chloroform:Isoamyl Alcohol (25:24:1; Sigma, St. Louis, MO, USA) was added to remove the contaminants of protein and DNA, and total RNA was precipitated by ethanol. Five micrograms of total RNA was used for first-strand complementary DNA synthesis in 20 μL reaction with SuperScript III reverse transcriptase (Invitrogen). For RT-PCR, 1 μL of synthesized cDNA was directly used in a total amount of 20 μL reaction to determine the transcriptional level of PROAtCAPE1. For RT-qPCR, 50x dilution of the synthesized cDNA was prepared, and 3 μL of the diluted cDNA was mixed with Fast SYBR Green Master Mix (Applied Biosystems, CA, USA) in a total amount of 10 μL for subsequent RT-qPCR analysis in ABI 7500 Fast Real-Time PCR system (Applied Biosystems).
Gene-specific primers were designed from the 3' untranslated region of Arabidopsis genes by Primer Express 3.0.1 (Applied Biosystems). The transcript of ACTIN2 was used for normalization.

Western Blot Analysis
Ten-day-old CAPE1ox CNYD seedlings were subjected to medium supplemented with and without 125 mM NaCl for various time points. The collected samples were crushed with YSZ Grinding Media (EE-TEC, EZEAG0500) in 2 mL eppendorf tubes by sonication (KURABO,. During the preparation, the samples were submerged in liquid nitrogen for rapid freezing. The samples were then homogenized in extraction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 20 mM EDTA, 1% [w/v] SDS and 10% [v/v] glycerol) at 95°C for 5 min. After centrifuging at 12,000 rpm for 10 min, the supernatant was transferred to a new eppendorf tube. Fifty micrograms of total proteins were loaded on 10% SDS-polyacrylamide gels and analyzed by western blotting. The recombinant PROAtCAPE1 tagged eYFP was detected by anti-GFP antibody (Roche; 11814460001), and horseradish peroxidase-conjugated anti-mouse antibody (Invitrogen; 616520) was used as a secondary antibody. To confirm the equal loading of total proteins, an anti-α-tubulin antibody (Sigma-Aldrich; T5168) was subsequently used to probe the same blot. Table 1. Effect of selected abiotic stresses on expressions of PROAtCAPEs a .
All data used were generated on Affymetrix GeneChip ATH1 22K platform. -, Reduced expression; +, increased expression after treatment; =, no significant effect on expression (defined as P ≥ 0.05 and/or fold-change ≤ 1.5 in expression levels compared with control); ND, PROAtCAPE gene is unavailable in Genevestigator.
a The analysis was done by using Genevestigator 29 microarray database. b Arabidopsis Col-0 were treated with the following stresses and sampled for 0.5, 1, and 3 h: Wounding: punctured with pins; Salt: 150 mM NaCl; Oxidative: 10 μM Methyl Viologen; Osmotic: 300 mM mannitol; Drought: exposed to the air stream for 15 min and returned to the climate chamber; Cold: 4°C. Transcript levels of RD29B were determined. Ten-day old wild-type Ler were treated with 125 mM NaCl for 12 hours while cape1 mutants were treated with 125 mM NaCl supplemented without or with various concentrations of peptides. For the mutants cotreated with salt and peptide, the peptides were pretreated 6 hours before the co-treatments. The relative levels indicate the normalized RD29B transcript from each treatment was compared with the expression of RD29B in wild type at 0 hour. The bars shown here are the average from four biological repeats. Error bars indicate the means ± SE. The different lowercase letters (a-b) indicate significant differences (P ≤ 0.05) between treatments.