Switchgrass proteomics for improved next generation biofuel crops

If advanced biofuels are to replace standard fuels on a gallon-for-gallon basis at competitive pricing, there is a requirement for a new generation of biofuel crops – plants designed specifically to serve as feedstocks for biofuel production. Switchgrass, the US native praire grass, is one of the target crops for biofuel production. In this pilot study we confirmed the capability of proteomics to identify enzymes involved in plant cell wall and sugar biosynthesis using genome resources generated by next generation sequencing approaches.

HFSP Long-Term Fellow Benjamin Schwessinger and colleagues
authored on Mon, 23 March 2015

Plant cell walls are highly structured matrices consisting of the sugar polymers cellulose, hemicellulose and pectin. These plant sugar polymers are the energy supply for the production of advanced biofuels. The polysaccharide cellulose is a concatenation of glucose molecules and is synthesized at the plasma membrane. The matrix polysaccharides (hemicellose and pectin) are biosynthesized within the Golgi apparatus from a wide range of C5 and C6 sugars and deposited to the cell wall via the plant secretory system. In this pilot study, we aimed to identify enzymes associated with cell wall biosynthesis localized to the secretory system of the emerging biofuel feedstock, switchgrass (Panicum virgatum).

Benjamin Schwessinger (left) and Jeemeng Lao. Photo: the Lawrence Berkeley National Laboratory © 2010 The Regents of the University of California, through the Lawrence Berkeley National Laboratory.

We enriched the secretory system from 10-day old dark grown switchgrass shoots using biochemical fractionation techniques and profiled the resultant proteins by shotgun proteomics. We identified over 2500 unique proteins many of which were associated with GO (gene ontology) terms linked to the secretary system and organelles, which supported our enrichment method. The number of identified proteins in the current study is comparable to previous reports investigating the model plant Arabidopsis thaliana for which a high quality reference genome is available. Therefore our proteomic analysis validates the quality and applicability of the current genome assembly of the highly heterogzygous tetraploid switchgrass which was recently generated by next generation sequencing techniques.

In the case of polyploid and heterozygous genomes like switchgrass, proteomics has the potential to correctly identify the functional alleles of biosynthetic enzymes. In our dataset we identified over 60 candidate enzymes involved in cell wall biosynthesis. For example, we uniquely identified five out of ten of the biosynthetic enzymes responsible for producing the precursor sugars for xylan, an important cell wall sugar polymer. We found that for these enzymes both homeologs (orthologs produced by polyploidy) were expressed without any apparent preferential expression of either chromosomal version. If this holds true for other homeologs in switchgrass, the future genetic manipulation of switchgrass will require the simultaneous targeting of both homeologs to achieve desired trait alterations.

Reference

Proteome profile of the endomembrane of developing coleoptiles from switchgrass (Panicum virgatum). Lao J, Sharma M, Sharma R, Fernández-Niño SMG, Schmutz J, Ronald PC, Heazlewood JL and Schwessinger B. Proteomics. 2015 Feb 1.  DOI: 10.1002/pmic.201400487.

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