Quantitative Proteome Analysis of Jatropha curcas L. Genotypes with Contrasting Levels of Phorbol Esters

Abstract

The phorbol esters in the seeds of Jatropha curcas are a major hindrance to the full exploitation of the potential of this oil crop as a source of raw material for the production of biodiesel. Here, various quantitative proteomic strategies are used to establish the proteomes of roots, leaves, and endosperm of two genotypes of J. curcas with contrasting levels of phorbol esters in the seeds. In total 4532, 1775, and 503 proteins are identified respectively in roots, leaves, and endosperm, comprising 5068 unique proteins; of this total, 185 are differentially abundant in roots, 72 in leaves, and 20 in the endosperm. The biosynthetic pathways for flavonoids and terpenoids are well represented in roots, including the complete set of proteins for the mevalonate and non‐mevalonate/Deoxyxylulose 5‐Phosphate pathways, and proteins involved in the branches which lead to the synthesis tricyclic diterpenoids and gibberellins. Also, casbene synthase which catalyzes the first committed step in the biosynthesis of tigliane‐type diterpenes is identified in roots of both genotypes, but not in leaves and endosperm. This dataset will be a valuable resource to explore the biochemical basis of the low toxicity of Jatropha genotypes with low concentration of phorbol esters in the seeds.

Monitoring casbene synthase in Jatropha curcas tissues using targeted proteomics

“…For the SRM and PRM assays, we set as the internal positive control for the root, endosperm, and leaf samples, the proteins peptidyl-prolyl cis–trans isomerase (A0A067LPN3), legumin type B (A0A067K3Z1) and the chloroplast photosystem II 10 kDa (D6BRD6), respectively, by the reason that these proteins were well identified in previous shotgun proteomics experiments [ 25 ]. Additional file 4 : Table S4 shows SRM and PRM transitions to be monitored.…”

“…Although the expression of CS transcripts in different tissues and developmental stages of J. curcas have been reported [ 9 , 11 , 12 , 14 , 26 , 27 ], no corresponding data on the proteins encoded by the transcripts were produced. Only recently a single proteomics study leads to the identification of CS in roots of J. curcas [ 25 ]. Together these results draw attention to the lack of correlation between experiments at the transcript and protein levels and the use of targeted proteomics approaches to validate gene transcription studies.…”

“…Voucher specimens of HPE and LPE genotypes are deposited at the Herbarium Prisco Bezerra, Federal University of Ceará, under numbers EAC62156 and EAC62157. The endosperm, leaves, and roots collection and preparation for protein extraction were performed as described [ 25 ].…”

“…Protein extraction from each tissue is described in [ 25 ] and was performed according to Vasconcelos et al [ 40 ]. Briefly, tissue powder was mixed with polyvinylpolypyrrolidone (PVPP) and pyridine buffer (50 mM pyridine, 10 mM Thiourea, 1% SDS, pH 5,0) in a proportion of 1:2:40 (w/w/v) and stirred for at least 4 h at 4 °C.…”

“…Although together these studies identified more than 4000 proteins, no CS was identified, and these studies did not attempt to find out whether the lack of CS identification was related to the dynamic range of the protein extracts under analysis. Only recently, Farias et al (2020) gathered evidence for the presence of CS in the roots of two genotypes displaying contrasting levels of PE [ 25 ]. On the other hand, the literature abound on reports on the transcription of the CS gene in developing seeds [ 26 , 27 ], mature seeds [ 26 , 27 ], developing fruit [ 11 , 14 , 27 ], mature fruits [ 14 ], developing leaves [ 12 , 26 , 27 ], mature leaves [ 9 , 26 , 27 ]; roots [ 9 , 26 ], flowers [ 9 , 26 ], tegmen [ 12 ], and stem [ 9 ].…”