Abstract
Ethylene regulates fruit ripening and several plant functions (germination, plant growth, plant-microbe interactions). Protein quantification of ethylene receptors (ETRs) is essential to study their functions, but is impaired by low resolution tools such as antibodies that are mostly nonspecific, or the lack of sensitivity of shotgun proteomic approaches. We developed a targeted proteomic method, to quantify low-abundance proteins such as ETRs, and coupled this to mRNAs analyses, in two tomato lines: Wild Type (WT) and Never-Ripe (NR) which is insensitive to ethylene because of a gain-of-function mutation in ETR3. We obtained mRNA and protein abundance profiles for each ETR over the fruit development period. Despite a limiting number of replicates, we propose Pearson correlations between mRNA and protein profiles as interesting indicators to discriminate the two genotypes: such correlations are mostly positive in the WT and are affected by the NR mutation. The influence of putative post-transcriptional and post-translational changes are discussed. In NR fruits, the observed accumulation of the mutated ETR3 protein between ripening stages (Mature Green and Breaker + 8 days) may be a cause of NR tomatoes to stay orange. The label-free quantitative proteomics analysis of membrane proteins, concomitant to Parallel Reaction Monitoring analysis, may be a resource to study changes over tomato fruit development. These results could lead to studies about ETR subfunctions and interconnections over fruit development. Variations of RNA-protein correlations may open new fields of research in ETR regulation. Finally, similar approaches may be developed to study ETRs in whole plant development and plant-microorganism interactions.
Highlights
Ethylene is a plant hormone involved in many developmental processes such as seed germination, root initiation, root hair development, flower development, sex determination, fruit ripening, senescence, and responses to biotic and abiotic stresses (Merchante et al, 2013)
Recent advances in large-scale shotgun proteomics have led to identify a large set of proteins including SlETR3 and SlETR4 in green to red ripe tomato fruits using the ITAG 2.3 database (Feb 2013) (Szymanski et al, 2017) and SlETR1, 3 and 4, using the UniProt FASTA database (Dec 2015) in red ripe tomatoes (Mata et al, 2017)
In a large-scale labelfree proteomic study, we identified SlETR1, 4, 6, and 7 using the most recent ITAG 3.2 (June 2017), in pooled skin and flesh tissues of both the Wild Type (WT) and NR genotypes of the MicroTom cultivar, in four development stages from immature green to Breaker + 8 days (Table S2a; Methods S1b)
Summary
Ethylene is a plant hormone involved in many developmental processes such as seed germination, root initiation, root hair development, flower development, sex determination, fruit ripening, senescence, and responses to biotic and abiotic stresses (Merchante et al, 2013). Since the initial description of the first ethylene receptor, AtETR1 from Arabidopsis thaliana (Chang et al, 1993), several studies combining genetics, molecular biology, and biochemistry have led to a model whereby the receptors function as negative regulators and ethylene releases this inhibition (Shakeel et al, 2013; Lacey and Binder, 2014; Ju and Chang, 2015). ETR abundance may be a critical determinant of ethylene signaling. This is supported in tomato where a study showed that the level of insensitivity to ethylene is related to the expression level of an ETR1 gain-of-function (GOF) mutant (Gallie, 2010). The ethylene signaling may be governed by the relative amount of WT ETRs versus mutant ETRs
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