A Phosphoproteomics Study of the Soybean root necrosis 1 Mutant Revealed Type II Metacaspases Involved in Cell Death Pathway

Abstract

The soybean root necrosis 1 (rn1) mutation causes progressive browning of the roots soon after germination and provides increased tolerance to the soil-borne oomycete pathogen Phytophthora sojae in soybean. Toward understanding the molecular basis of the rn1 mutant phenotypes, we conducted tandem mass tag (TMT)-labeling proteomics and phosphoproteomics analyses of the root tissues of the rn1 mutant and progenitor T322 line to identify potential proteins involved in manifestation of the mutant phenotype. We identified 3,160 proteins. When the p-value was set at ≤0.05 and the fold change of protein accumulation between rn1 and T322 at ≥1.5 or ≤0.67, we detected 118 proteins that showed increased levels and 32 proteins decreased levels in rn1 as compared to that in T322. The differentially accumulated proteins (DAPs) are involved in several pathways including cellular processes for processing environmental and genetic information, metabolism and organismal systems. Five pathogenesis-related proteins were accumulated to higher levels in the mutant as compared to that in T322. Several of the DAPs are involved in hormone signaling, redox reaction, signal transduction, and cell wall modification processes activated in plant-pathogen interactions. The phosphoproteomics analysis identified 22 phosphopeptides, the levels of phosphorylation of which were significantly different between rn1 and T322 lines. The phosphorylation levels of two type II metacaspases were reduced in rn1 as compared to T322. Type II metacaspase has been shown to be a negative regulator of hypersensitive cell death. In absence of the functional Rn1 protein, two type II metacaspases exhibited reduced phosphorylation levels and failed to show negative regulatory cell death function in the soybean rn1 mutant. We hypothesize that Rn1 directly or indirectly phosphorylates type II metacaspases to negatively regulate the cell death process in soybean roots.

Keywords: cell death; metacaspases; phosphoproteomics; proteomics; root necrosis; soybean.

FIGURE 1

Experimental design and schematic diagram of the workflow for quantitative proteomics of the soybean rn1 mutant and T322 lines. The T322 is the wild-type progenitor of the rn1 mutant line. Soybean root samples of the rn1 mutant and T322 lines in triplicate were analyzed in parallel by TMT 6-plex based quantitative proteomics and phosphoproteomics analyses. TMT labeled peptides were divided to three aliquots and used by high pH fractionation for global proteomics, TiO₂ enrichment and IMAC-TiO₂ sequential enrichment for phosphoproteomics, respectively. About 16% of total labeled peptides were used for global proteomics and 84% used for phosphoproteomics study. The 10 high-pH RPLC fractions and 2 enriched samples were subjected to nanoLC-MS/MS analysis and subsequent database search using Mascot. After statistical analyses of the global proteomics data and quantitative phosphopeptides, the differentially accumulated proteins, and phosphoproteins with significantly changed phosphorylated peptides/sites were conducted to identify differentially regulated proteins and phosphoproteins by the Rn1 protein for bioinformatics analysis.

FIGURE 2

GO annotation of the 150 DAPs between necrotic (rn1) and healthy (T322) root tissues. The DAPs were classified into three categories: biological process, cellular component, and molecular function, based on GO terms.

FIGURE 3

KEGG pathway enrichment analysis of the DAPs. (A) Based on the KEGG database pathway information, the DAPs were classified into five known categories; viz., cellular process, environmental information processing, genetic information processing, metabolism, and organismal systems. Note that 15.79% of the proteins have no known functions. (B) Specific pathways and number of proteins in each pathway classified under each of the five categories of the DAPs with known functions shown in (A) are presented. The five categories are shown on the right side of (B). The number of proteins among the pathways is presented at the end of each bar.

FIGURE 4

DAPs involved in pathways induced by biotic and abiotic stresses. The DAPs involved in plant biotic and abiotic stresses were mapped using MapMan software. Proteins involved into the biotic and abiotic stresses are presented in red font. The scale represented the fold changes of DAPs, which is presented in the upper left corner of the figure.

FIGURE 5

GO and KEGG analysis for 22 phosphoproteins that were detectible (Table 2). (A) Based on GO terms, the phosphoproteins were classified into three categories: biological process, cellular component, and molecular function. (B) Specific pathways and number of phosphoproteins in each pathway classified under each of the two categories. Some phosphoproteins are significantly involved in immune system process, oxidative phosphorylation and pentose phosphate pathway, as indicated by “*”.

FIGURE 6

MapMan analysis of 22 PhosPs listed in Table 2. Seven phosphoproteins were induced by biotic and abiotic stresses are shown on both sides of the map.

FIGURE 7

MS/MS spectra show confident identifications of three representative phosphopeptides/sites that were labeled by TMT6-plex from three Metacaspase proteins. (A) An MS/MS spectrum of a triply charged ion at m/z 768.689³⁺ confidently identifying a TMT-labeled tryptic peptide with S174 phosphorylation site from Metacaspase 5 protein (Glyma.08G233300) between necrotic and healthy roots; (B) an MS/MS spectrum of a triply charged ion at m/z 756.327³⁺ confidently identifying a TMT-labeled tryptic peptide with S171 phosphorylation site from Metacaspase 4 protein (Glyma.08G233500), and (C) an MS/MS spectrum of a triply charged ion at m/z 766.330³⁺ confidently identifying a TMT-labeled tryptic peptide with S171 phosphorylation from Metacaspase 4 protein (Glyma.15G219100) between necrotic and healthy roots. The inset of each panel shows the expanded view of relative intensity of 6 TMT reporter ions for determining the abundance changes of the S174, S171, and S171 phosphopeptides in necrotic root triplicate (126, 127, and 128) versus 3 healthy roots (129,130, and 131).