Critical Role of COI1-Dependent Jasmonate Pathway in AAL toxin induced PCD in Tomato Revealed by Comparative Proteomics

Abstract

Alternaria alternata f.sp. Lycopersici (AAL) toxin induces programmed cell death (PCD) in susceptible tomato (Solanum lycopersicum) leaves. Jasmonate (JA) promotes AAL toxin induced PCD in a COI1 (coronatine insensitive 1, JA receptor)-dependent manner by enhancement of reactive oxygen species (ROS) production. To further elucidate the underlying mechanisms of this process, we performed a comparative proteomic analysis using tomato jasmonic acid insensitive1 ( jai1), the receptor mutant of JA, and its wild type (WT) after AAL toxin treatment with or without JA treatment. A total of 10367 proteins were identified in tomato leaves using isobaric tags for relative and absolute quantitation (iTRAQ) quantitative proteomics approach. 2670 proteins were determined to be differentially expressed in response to AAL toxin and JA. Comparison between AAL toxin treated jai1 and its WT revealed the COI1-dependent JA pathway regulated proteins, including pathways related to redox response, ceramide synthesis, JA, ethylene (ET), salicylic acid (SA) and abscisic acid (ABA) signaling. Autophagy, PCD and DNA damage related proteins were also identified. Our data suggest that COI1-dependent JA pathway enhances AAL toxin induced PCD through regulating the redox status of the leaves, other phytohormone pathways and/or important PCD components.

Figure 1. Effect of JA pathway on TA induced PCD in detached tomato leaves.

Detached leaflets were incubated under continuous light at 25 °C for 48 h. WT, wild type; jai1, JA receptor mutant; Mock, WT leaves treated with SPB buffer; TA, WT or jai1 leaves treated with TA; TA + JA, WT or jai1 leaves treated with TA and JA. (A) Fully expanded leaflets from nodes between fourth to sixth of 7-week-old plants were treated with 0.2 μM TA with or without 100 μM JA and photographed after 48 h. (B) Leaves from WT and jai1 plants were stained with trypan blue for the degree determination of dead and dying cells after treatment for 48 h. (C) MDA content was detected in WT and jai1 leaflets after co-treated with different concentrations of JA (0, 10, 100, and 500 μM) and 0.2 μM TA for 48 h. Each data point represents the mean of three replicates. Error bars indicate standard deviation of three replicates, asterisks above the bars indicate statistically significant differences between different treatments, as determined by the Student t-tests (**P < 0.01).

Figure 2. Effect of JA pathway on TA induced ROS accumulation in detached tomato leaves.

WT, wild type; jai1, JA receptor mutant; Mock, WT leaves treated with SPB buffer; TA, WT or jai1 leaves treated with TA; TA + JA, or jai1 leaves treated with TA and JA. (A) Leaves of WT and jai1 were stained with DAB for H₂O₂ determination at 48 h after 0.2 μM TA treatment with or without 100 μM JA. (B) Changes of H₂O₂ content in WT and jai1 leaves after 0.2 μM TA treatment for 36, 48 and 72 h. (C) Leaves of WT and jai1 were stained with NBT for O₂^.− determination at 48 h after 0.2 μM TA treatment with or without 100 μM JA. (D) Changes of O₂^.− producing rate in WT and jai1 leaves after 0.2 μM TA treatment for 36, 48 and 72 h. Each data point represents the mean of three replicates. Error bars indicate standard deviation of three replicates, asterisks above the bars indicate statistically significant differences between different treatments, as determined by the Student t-tests (**P < 0.01, ***P < 0.001).

Figure 3. Comparative proteomics identified a large number of proteins involved in TA induced PCD.

(A) Label by iTRAQ reagent. WT refers to the wild type leaves as control; WT + TA, WT leaves treated with TA; WT + TA + JA, WT leaves treated with TA and JA together; jai1 + TA, jai1 leaves treated with TA. Four biological replicates for each treatment and one set of iTRAQ includes two biological replicates. (B) Total protein number identified in two sets with 5% false discovery rate (FDR), set 1 is colored in blue, set 2 is colored in green. (C) Distribution and overlap of differentially expressed (DE) proteins in each comparison. WT + TA was set as the control group for the other groups. Each color represents one contrast. Blue, WT vs WT + TA; Yellow, WT + TA + JA vs WT + TA; Green, jai1 + TA vs WT + TA. (D) GO terms distribution of the overall 10367 proteins categorized in “biological process”. GO-terms were categorized by Blast2Go at level 4 according to their corresponding GO-term (biological process). Each protein may be identified in more than one process.

Figure 4. A putative model showing possible mechanisms of JA regulated AAL toxin response in Solanum Lycopersicum.

The DE proteins caused by JA treatment and COI1 mutation during AAL toxin induced PCD were used to construct the model, and the impairment of COI1 induced protein changes were presented with the contrary change patterns. The identified proteins were assigned to different organelles or groups according to their subcellular localization and molecular functions. Up-regulated proteins are highlighted red and down-regulated proteins are colored green. Arrows and bars represent positive and negative regulation respectively. Red solid lines indicate that the pathways are up-regulated or promoted by JA during TA response, green solid lines with bars or arrows indicate that the pathways are down-regulated by JA during TA response, black solid lines with arrows links the proteins within the same pathway. TA induce PCD by inhibiting the synthesis of ceramide and inducing the overproduction of ROS, COI1-dependent JA pathway may promote this PCD progress by influencing the ROS production and scavenging, other hormone signaling pathways or some possible PCD regulators such as caspase-like proteins, autophagy and DNA repair related proteins. AAO, L-ascorbic acid oxidase; ABI1, ABA insensitive1; ACO1, 1-aminocyclopropane-1-carboxylate oxidase homolog 1-like; AHA, plasma membrane H⁺-ATPase; AOS, allene oxide synthase; ATG11, autophagy related protein 11; CHS, chalcone synthase; CPI, cysteine protease inhibitor; Cyt, Cytochrome; DAP-AT, probable n-succinyldiaminopimelate aminotransferase-like; DDB, DNA damage binding proteins; DDR, DNA damage response; DRA, DNA repair ATPase-related family protein; ER, endoplasmic reticulum; GST, glutathione S-transferase; Golgi, golgi complex; KTI1, kunitz trypsin inhibitor; MMR, DNA mismatch repair; NAT, nucleobase-ascorbate transporter 6-like; Prx, Peroxiredoxin; RNase E, Ribonuclase 2-Like; SBTs, subtilisin-like proteases; TF, transcription factor.