Membrane Proteomics of Arabidopsis Glucosinolate Mutants cyp79B2/B3 and myb28/29

Abstract

Glucosinolates (Gls) constitute a major group of natural metabolites represented by three major classes (aliphatic, indolic and aromatic) of more than 120 chemical structures. In our previous work, soluble proteins and metabolites in Arabidopsis mutants deficient of aliphatic (myb28/29) and indolic Gls (cyp79B2B3) were analyzed. Here we focus on investigating the changes at the level of membrane proteins in these mutants. Our LC/MS-MS analyses of tandem mass tag (TMT) labeled peptides derived from the cyp79B2/B3 and myb28/29 relative to wild type resulted in the identification of 4,673 proteins, from which 2,171 are membrane proteins. Fold changes and statistical analysis showed 64 increased and 74 decreased in cyp79B2/B3, while 28 increased and 17 decreased in myb28/29. As to the shared protein changes between the mutants, one protein was increased and eight were decreased. Bioinformatics analysis of the changed proteins led to the discovery of three cytochromes in glucosinolate molecular network (GMN): cytochrome P450 86A7 (At1g63710), cytochrome P450 71B26 (At3g26290), and probable cytochrome c (At1g22840). CYP86A7 and CYP71B26 may play a role in hydroxyl-indolic Gls production. In addition, flavone 3′-O-methyltransferase 1 represents an interesting finding as it is likely to participate in the methylation process of the hydroxyl-indolic Gls to form methoxy-indolic Gls. The analysis also revealed additional new nodes in the GMN related to stress and defense activity, transport, photosynthesis, and translation processes. Gene expression and protein levels were found to be correlated in the cyp79B2/B3, but not in the myb28/29.

Changes in Whey Proteome between Mediterranean and Murrah Buffalo Colostrum and Mature Milk Reflect Their Pharmaceutical and Medicinal Value

“…The analysis was carried out as previously described by Li et al [ 6 ] and Mostafa et al [ 73 ] with some modifications. Each fraction was subjected to chromatographic separation using the Easy nLC 1200 chromatography system (Thermo Scientific, Waltham, MA, USA) with a nanoliter flow rate.…”

Reconstruction of the Transcriptional Regulatory Network in Arabidopsis thaliana Aliphatic Glucosinolate Biosynthetic Pathway

“…The elongated side chain of amino acid is then subjected to the formation of glucosinolate core structure involving 14 identified aliphatic glucosinolate genes. This phase starts with the conversion of amino acid to aldoxime by cytochrome P450 (CYP79) (Mostafa et al 2017). CYP79F1 metabolizes all the elongated methionederived side chain and CYP79F2 only metabolizes pentahomomethione and hexa-homo-methionine (Redovniković et al 2008).…”

On the Promising Role of Enzyme Activity Assay in Interpreting Comparative Proteomic Data in Plants

“…At present, the validation of proteomic data is mainly done by quantitative RT‐PCR (qRT‐PCR) analysis (measuring the abundance of mRNA), immunoblotting (detection of specific proteins using antibody), and multiple reaction monitoring (MRM) analysis (quantitative comparison of the target proteins) . It has been well documented that transcript levels are not usually in close agreement with protein abundance, because protein abundance depends not only on transcript levels, but also on the rate of protein translation and degradation .…”