Root secretion of defense-related proteins is development-dependent and correlated with flowering time

Abstract

Proteins found in the root exudates are thought to play a role in the interactions between plants and soil organisms. To gain a better understanding of protein secretion by roots, we conducted a systematic proteomic analysis of the root exudates of Arabidopsis thaliana at different plant developmental stages. In total, we identified 111 proteins secreted by roots, the majority of which were exuded constitutively during all stages of development. However, defense-related proteins such as chitinases, glucanases, myrosinases, and others showed enhanced secretion during flowering. Defense-impaired mutants npr1-1 and NahG showed lower levels of secretion of defense proteins at flowering compared with the wild type. The flowering-defective mutants fca-1, stm-4, and co-1 showed almost undetectable levels of defense proteins in their root exudates at similar time points. In contrast, root secretions of defense-enhanced cpr5-2 mutants showed higher levels of defense proteins. The proteomics data were positively correlated with enzymatic activity assays for defense proteins and with in silico gene expression analysis of genes specifically expressed in roots of Arabidopsis. In conclusion, our results show a clear correlation between defense-related proteins secreted by roots and flowering time.

FIGURE 1.

2-DE of total A. thaliana Col-0 (WT) root-secreted proteins. Two hundred and fifty micrograms of protein were isoelectrically focused and separated as described under “Experimental Procedures.” A, representative 2-DE gel from root-secreted proteins in WT at day 28 is shown. The molecular masses (kDa) of protein standards are indicated to the left of the gel, and the isoelectric point (pI) is indicated at the top of the gel. The arrows with numbers represent identified proteins that are listed in supplemental Table S1. The gray underlined numbers represent the proteins that are not present in the representative gel but are present in other samples. B, summary of the distribution of root secreted identified protein classes as determined using the protein function data base Pfam and classification schema previously reported for Arabidopsis (91).

FIGURE 2.

Histograms of normalized volume values of the secreted proteins from A. thaliana WT that change throughout the whole 49-day temporal course. The normalized volumes were analyzed by Phoretix 2D Expression software. The spot numbers represent the identified proteins that are listed in supplemental Table S1. Each graph is a subset of proteins grouped together by class/isoforms. The error bars illustrate the S.E. values of three repetitions (n = 3).

FIGURE 3.

Comparative histogram of normalized volume values from each of the 26 spots from 2-DE of A. thaliana WT and the defense-related mutants. The major differences between the root-secreted proteins of A. thaliana ecotype Col-0 (WT), impaired-defense mutant npr1-1, impaired-defense transgenic NahG, and the defense-related mutant cpr5-2 at days 21 and 28 are shown. Two hundred and fifty micrograms of protein for every genotype were analyzed by 2-DE as described under “Experimental Procedures.” The spot numbers represent the identified proteins that are listed in supplemental Table S1. The error bars illustrate the S.E. values of three repetitions (n = 3).

FIGURE 4.

Comparative histogram of normalized volume values from each of the 26 spots from 2-DE of A. thaliana WT and the flowering-related mutants. The major differences between the root-secreted proteins of A. thaliana ecotype Col-0 (WT), the mutant co-1, which flowers later (after day 28) than the WT (day 21) under long day conditions, the mutant fca-1, which has shown late flowering (around day 35), and the mutant stm-4 at days 21 and 28 are shown. Two hundred and fifty micrograms of protein for every genotype were analyzed by 2-DE as described under “Experimental Procedures.” The spot numbers represent the identified proteins that are listed in supplemental Table S1. The error bars illustrate the S.E. values of three repetitions (n = 3).

FIGURE 5.

Enzymatic activity of β-1,3-glucanases, chitinases, and proteases in A. thaliana Col-0 (WT) and defense-related mutants. A, β-1,3-glucanase activity was quantified every 7 days in A. thaliana Col-0 (WT), the impaired defense mutant npr1-1, the impaired defense transgenic NahG, and the defense-related mutant cpr5-2 throughout the whole temporal study; for more details see under “Experimental Procedures.” B, enzymatic activity of chitinases. Chitinolytic activity in root exudates of A. thaliana Col-0 (WT), impaired defense mutant npr1-1, impaired defense transgenic NahG, and the defense-related mutant cpr5-2 was quantified using chitinase azure as a substrate and as described under “Experimental Procedures.” The error bars indicate the S.E. values. The bar with the asterisk represents the days when flowering was observed. The results represent experiments repeated three times with five replicates each.

FIGURE 6.

Enzymatic activity of β-1,3-glucanases, chitinases, and proteases in A. thaliana Col-0 (WT) and flowering-related mutants. A, β-1,3-glucanase activity was quantified every 7 days in A. thaliana Col-0 (WT), the mutant co-1, which flowers later (after day 28) than the WT (day 21) under long day conditions, the mutant fca-1, which has shown late flowering (around day 35), and the mutant stm-4 throughout the whole temporal study; for more details see under “Experimental Procedures.” B, enzymatic activity of chitinases. Chitinolytic activity in root exudates of A. thaliana Col-0 (WT), the mutant co-1, the mutant fca-1, and the mutant stm-4 was quantified using chitinase azure as a substrate and as described under “Experimental Procedures.” The error bars indicate the S.E. values. The results represent experiments repeated three times with five replicates each.

FIGURE 7.

Antibacterial assay from protein root exudates of A. thaliana Col-0 (WT) and the defense-related mutants. Antibacterial activity assays of protein root exudates from the WT, impaired-defense mutant npr1-1, impaired-defense transgenic NahG, and the defense-related mutant cpr5-2 at days 21 and 28. Twenty μg of protein exudates from every genotype were tested against P. syringae pv. lycopersici (DC3000) and P. syringae pv. phaseolicola (Psph 3121). The asterisk indicates the values that are statistically significant at p ≤ 0.05, n = 10. The error bars illustrate the S.E. values. The presented values are from two independent experiments with five biological replicates each.