Label-Free Quantitative Proteomics of Pelargonium zonale: Tissue-Specific Differences

Abstract

Variegated Pelargonium zonale plants present excellent model for studying metabolite fluxes and photosynthesis-related processes between the photosynthetically active (green, vG) and inactive (white, vW) tissues within the same leaf under the same microenvironmental conditions. We aimed to investigate the proteomic differences between these two metabolically contrasting tissue types, as well as between vG and plain morphs (G) to gain more insight into the evolutionary benefits of variegation. Label-free proteomics was performed using liquid chromatography coupled to tandem mass spectrometry. The data were searched against the newly expanded RNAseq database for P. zonale leaves. Analysis was done with X!Tandem, using 10 ppm precursor mass precision and fragment 0.02 Th mass tolerance. The identified proteins were filtered using X!TandemPipeline, requiring at least two peptides with E-values lower than 0.01 and a protein E-value<10-5. Peptide ions, and their parental proteins, were quantified by integrating signal intensities from extracted ion currents (XIC) using MassChroQ software. A total of 2707 protein groups were identified. After removing dubious data and peptides with missing values, we obtained 2009 protein group. We annotated 564 and 79 differentially abundant proteins in vG vs. vW and vG vs. G, respectively. The differentially abundant proteins were mapped into metabolic pathways using the modified MapMan 3.6.0RC1 software. Proteins related to photosynthesis and carbohydrate metabolism were more abundant in vG compared with vW, while proteins related to oxidative stress and protein degradation were more abundant in vW than in vG. Compared to vG, G tissue contained more proteins involved in energy production and protein synthesis. Briefly, this study has paved the way to uncover the evolutionary advantages of the variegated phenotype.