Targeted Proteomics Allows Quantification of Ethylene Receptors and Reveals SlETR3 Accumulation in Never-Ripe Tomatoes
2019
Chen, Yi | Rofidal, Valerie | Hem, Sonia | Gil, Julie | Nosarzewska, Joanna | Berger, Nathalie | Demolombe, Vincent | Bouzayen, Mondher | Azhar, Beenish J. | Shakeel, Samina N. | Schaller, G. Eric | Binder, Brad M. | Santoni, Veronique | Chervin, Christian | Génomique et Biotechnologie des Fruits (GBF) ; Institut National de la Recherche Agronomique (INRA)-École nationale supérieure agronomique de Toulouse (ENSAT) ; Institut National Polytechnique (Toulouse) (Toulouse INP) ; Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National Polytechnique (Toulouse) (Toulouse INP) ; Université de Toulouse (UT)-Université de Toulouse (UT) | Biochimie et Physiologie Moléculaire des Plantes (BPMP) ; Institut National de la Recherche Agronomique (INRA)-Centre international d'études supérieures en sciences agronomiques (Montpellier SupAgro)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro) | Dept Biochem ; University of Nairobi (UoN) | Dept Biol Sci ; Univ London Imperial Coll Sci Technol & Med | Department of Biochemistry, Cellular, and Molecular Biology ; The University of Tennessee [Knoxville] | CSC; INRA; National Science Foundation (IOS-1456487), (MCB-1517032); International Research Support Initiative Program of Higher Education Commission of Pakistan
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.
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