Monitoring casbene synthase in Jatropha curcas tissues using targeted proteomics

Abstract

Background Casbene synthase (CS) is responsible for the first committed step in the biosynthesis of phorbol esters (PE) in the Euphorbiaceae. PE are abundant in the seeds of the biofuel crop Jatropha curcas and its toxicity precludes the use of the protein-rich cake obtained after oil extraction as an animal feed and the toxicity of the fumes derived from burning PE containing biofuel is also a matter of concern. This toxicity is a major hindrance to exploit the potential of this crop as a source of raw material to produce biodiesel. For this reason, the current research on J. curcas is mainly focused on the understanding of the biosynthesis and site of synthesis of PE, as an avenue for the development of genotypes unable to synthesize PE in its seeds. Results Here, we present targeted proteomics assays (SRM and PRM) to detect and quantify CS in leaves, endosperm, and roots of two J. curcas genotypes with contrasting levels of PE. These assays were based on the use of reference isotopic labeled synthetic peptides (ILSP) predicted from 12 gene models of CS from the J. curcas genome. Conclusion Our targeted proteomics methods were able to detect and quantify, for the first time, CS gene products and demonstrate the distribution of CS isoforms only in roots from J. curcas genotypes with a high and low concentration of PE. These methods can be expanded to monitor CS, at the protein level, in different tissues and genotypes of J. curcas.

Untargeted Metabolomic Analysis of Leaves and Roots of Jatropha curcas Genotypes with Contrasting Levels of Phorbol Esters

“…In this work, we applied an untargeted metabolomics approach based on Liquid Chromatography coupled to High‐Resolution Mass Spectrometry (LC‐HRMS) to investigate organic extracts from the leaves and roots of two physic nut genotypes with contrasting PE abundances: Detected Phorbol Ester (DPE) and Not Detected Phorbol Ester (NPE). This research led us to a rich set of metabolite data, with patterns of deposition characteristic of the genotypes and organs analyzed, especially terpenoids, deepening our understanding of the PE biosynthesis and accumulation in physic nut, supporting the recent findings at the proteome level of these genotypes (Pinheiro et al, 2013; Soares et al, 2014; Shah et al, 2015, 2016; Soares et al, 2017; Ramzan et al, 2022; Farias et al, 2020; de Almeida et al, 2021).…”

“…Voucher specimens of the two genotypes were deposited at the Herbarium Prisco Bezerra, Federal University of Ceará, Brazil, under access codes EAC62156 (DPE genotype) and EAC62157 (NPE genotype). The PE concentration in the seed kernel reported in previous work was 0.55 ± 0.06 μg.mg −1 in DPE genotype, whereas these compounds were not detected in NPE (de Almeida et al, 2021).…”

“…In summary, concerning the terpenoid deposition patterns, the diversity of identified diterpenoids in the roots of both genotypes may be related to the expression of the CS enzyme, which is not observed in other tissues (Farias et al, 2020; de Almeida et al, 2021). Furthermore, the notable contrast between the DPE and NPE genotypes mainly concerns the deposition of sesquiterpenoids (farnesylpyrophosphate‐derived metabolites) that are more abundant in the NPE genotype, while diterpenoids and apocarotenoids (geranylgeranylpyrophosphoate‐derived metabolites) were higher in the DPE.…”

“…In summary, concerning the terpenoid deposition patterns, the diversity of identified diterpenoids in the roots of both genotypes may be related to the expression of the CS enzyme, which is not observed in other tissues (Farias et al, 2020;de Almeida et al, 2021).…”

Multivariate Analysis of Seed Chemical Diversity among Jatropha curcas in Botswana

“…The biosynthetic pathway of PE in Jatropha is largely uncharacterized, but the conversion of geranyl diphosphate to casbene by terpene cyclase casbene synthase (CS) is suggested to be a key step in PE biosynthesis [69][70][71]. More than 10 CS gene homologs were found in the Jatropha genome [72,73], and a correlation between the protein abundance of these CS isozymes and PE levels among Jatropha genotypes was reported via targeted proteome analysis [74]. Unraveling the relationship between the variation in PE levels and the CS expression profiles of the Botswanan Jatropha accessions awaits further investigation in the future.…”