15-07: A proteomic and bioinformatic approach to identify target genes for genetic engineering biomass energy crops

Identifying the constituents of the plant cell wall proteome is important for genetic engineering of plant cell walls to enhance their digestibility and conversion to simple sugars.  Here, we used CaCl₂-extraction procedure that targets cell wall and apoplastic proteins to isolate proteins from senescent Arabidopsis stems.  The extracted proteins were fractionated by SDS-PAGE, and digested in-gel with trypsin followed by peptide identification by Nano-LC-ESI-MS/MS.  Nearly 650 proteins were identified, which included 189 apoplastic proteins with 93 of them being cell wall proteins based on gene ontology (GO) analysis.  Overall, about half of the identified cell wall proteins have not been detected in recent published proteomes of the Arabidopsis cell wall.  About 26% (24/93) of these cell wall proteins were glycoside hydrolases (GH) and their inhibitors, including GH 1, 3, 16, 17, 20, 27, 28, 31, 32, 35, 38, and 51.  Among the 19 identified oxido-reductases, at least 13 of them are related to H₂O₂ generation and removal, suggesting the likely existence of a network of proteins for the regulation of an extracellular H₂O₂ homeostasis, which may play an essential role in plant response to a combination of abiotic, development, and senescent stresses.  Protein expression pattern analyses suggest that, because of their precise timing for expression (only in the senescent stage for stems) and their precise location (cell wall and apoplast), seven GH proteins and one peroxidase are promising candidate targets to be genetically manipulated in biomass crops to enhance biomass digestibility. This constitutes a new approach for feedstock plant engineering.