Proteomic Analysis of Near-Isogenic Sunflower Varieties Differing in Seed Oil Traits

Near-isogenic sunflower lines containing 25% (inbred RHA280) and 48% (RHA801) oil by seed dry mass
were comparatively analyzed in biological triplicate at 18 days after flowering using two-dimensional
(both pI 3−10 and 4−7) Difference Gel Electrophoresis. Additionally, two inbred lines varying in oleic
acid content, HA89 (18% oleic) and HA341 (89% oleic), were also analyzed in the same manner. Statistical
analyses of these sunflower lines was performed beginning with fitting a mixed effects linear model
to the log-transformed optical volume of each spot to account for gel variation, followed by testing the
significance between varieties for mean transformed optical spot volumes. The p-values from the spot
analysis procedures were then used to find the cutoff point for differential expression using a 10%
false-discovery rate (FDR). Comparison of the oil content and oleic acid composition lines revealed 77
and 42 protein spots below the 10% FDR cutoff, respectively, and were therefore declared differentially
expressed. Liquid chromatography−tandem mass spectrometry analysis of each of these protein spots
resulted in assignments for 44 and 17 spots, respectively. Fructokinase, plastid phosphoglycerate kinase,
and enolase proteins were determined to be up-regulated in the high oil line, while phosphofructokinase,
cytosolic phosphoglucomutase, and cytsolic phosphoglycerate kinase were up-regulated in the low oil
variety. Additionally, four activities involved in amino acid synthesis were up-regulated in the low oil
variety in addition to 12S storage proteins and a protein similar to legumin storage protein. Interestingly,
two 2-DE spots identified as 14-3-3 proteins were found to be up-regulated in high oleic acid variety.
Alteration of glycolytic and amino acid biosynthetic enzymes, as well as storage protein levels, suggests
seed oil content is tightly linked to carbohydrate metabolism and protein synthesis in a complex manner.
Keywords: oilseeds • seed oil content • DIGE • quantitative proteomics • statistical proteomics