Identification of Wound-Response Proteins using 2D-Electrophoresis and LC-MS/MS: A Case Study on Agarwood Tree Species, Aquilaria malaccensis Hishamuddin M.S.1 , Lee S.Y 1 , Isa N.M.2 , Lamasudin D.U.2 and Mohamed R.1* 1Department of Forest Management, Faculty of Forestry, Universiti Putra Malaysia 2Department of Cell and Molecular, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia *Corresponding author:
[email protected]
INTRODUCTION Wounding is the main trigger to agarwood (gaharu) production in Aquilaria malaccensis (karas), an endangered tree species native to Malaysia. Agarwood acts as a physical barrier and provides a variety of chemical antagonists to fungal attack. To understand the tree’s response to wound elictors, we applied two proteomic approaches, the gel-based 2D-Electrophoresis and direct sequencing via LC-MS/MS.
MATERIALS AND METHODS
Stem size of the Aquilaria trees was measured before wounding treatment.
A drill machine was used to wound the tree.
Wood tissues were sampled from the wounding area at different time points after treatment.
Extracted protein samples were analyzed via LC-MS/MS.
Bioinformatics analysis.
RESULTS AND DISCUSSION Fig 2: Protein Spots 1902 • Predicted as Malate synthase - Responsible towards the activation of carbohydrate - In Aquilaria, carbohydrate metabolism contributes to the production of agarwood resin. Figure 2: The histogram of protein expression (protein spot 1902)
Figure 1: Master gel of proteome patterns of Aquilaria
Fig 1: Image Master 2D Elite software Expression Analysis • Protein spots 1902 and 5501 - At 6th hour : showed up-regulation - 12th hour to the 48th hour: showed down-regulated - At 48th hour: Expression was relatively lower than the control at 0 hour
Fig 3: Protein Spots 5501 • Predicted as reduced Nicotinamide adenine dinucleotide phosphate (NADPH) quinone oxidoreductase subunit 2 B. - Displayed potential interaction towards agarwood formation during production of sesquiterpenes. Figure 3: The histogram of protein expression (protein spot 5501)
Fig 4: LC-MS/MS ANALYSIS Analysis using Bioinformatics tools • •
Figure 4: Venn Diagram of proteins detected at 0h, 6h and 24h of wounding treatment.
A total of 294 proteins across treatment and control plant were identified. Among them, 128 were specific to wounding, 41 were expressed at 6 h, 87 at 24 h, and another 14 were continuously expressed from 6 h, while 47 were shared. • Example type of identified proteins (predicted): i. Heat Shock and chaperone proteins iv. malate dehydrogenase ii. Protein metabolism v. s-adenosylmethionine synthetase iii. Basic Metabolism vi. glyceraldehyde-3-phosphate dehydrogenase
CONCLUSION It is important to identify wound-response proteins that are related to agarwood formation in Aquilaria tree since agarwood has great pharmaceutical values, broad market prospect and high exploitation potential. Therefore, the proteomic studies towards agarwood formation need to be explored further in order to understand the mechanism behind the agarwood formation.
