Principal component analysis and biochemical characterization of protein and starch reveal primary targets for improving sorghum grain

Authors: WONG, JOSHUA - University Of California; MARX, DAVID - University Of Nebraska; Wilson, Jeff; LEMAUX, PEGGY - University Of California; BUCHANAN, BOB - University Of California; Pedersen, Jeffrey

Submitted to: Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/8/2010
Publication Date: 10/29/2010
Citation: Wong, J.H., Marx, D.B., Wilson, J.D., Lemaux, P.G., Buchanan, B.B., Pedersen, J.F. 2010. Principal component analysis and biochemical characterization of protein and starch reveal primary targets for improving sorghum grain. Plant Science. Volume 179 (2010) 598-611.

Interpretive Summary: Large variation in the digestibility sorghum grain is known to exist which affects its value when consumed by humans, or when used as animal feed or an industrial feedstock. Many digestion assays have been developed to assess digestion for specific end-uses. In this study, we applied multiple digestion techniques to 18 sorghum lines differing in digestibility. Principal Component Analysis (PCA), a statistical technique used to describe total variation in a set of samples and to describe the weight attributable to measured traits, was applied to the data. Results of PCA revealed that amylose content, total starch content, and protein digestibility accounted for much of the variation in this set of samples. Selected waxy (low amylose) and non-waxy lines were digested separately for various times using a-amylase (digests starch), pepsin (digests protein), and pancreatin (multiple digestive functions). Patterns of total protein revealed both the matrix of protein surrounding the starch and the composition of the protein body varied among the waxy and non-waxy sorghum cultivars. Pepsin digestion of storage protein showed that various types of kafirins (storage protein) were digested at different rates. The lighter 20 kDa gamma-kafirin with its ability to form short repetitive protein structures was more resistant to digestion than the heavier 28 and 25 kDa alpha-kafirins lacking this ability. The resistance of gamma-kafirin to digestion seems to be associated with protein indigestibility in both waxy and non-waxy sorghum cultivars. It appears that traits contributing heavily to the first three Principal Components: amylose content, amount of total starch, and percent protein digestibility has a differential effect on the overall protein digestion pattern of non-waxy and waxy cultivars. The amount of starch affects digestion patterns of the protein matrix and protein body components more in non-waxy than waxy sorghum. Percent protein digestibility is influenced by the nature of the protein matrix but more significantly by the protein body packaging in both non-waxy and waxy sorghum cultivars. The three analyses identified in this study (amylose content, amount of total starch, and percent protein digestibility) are clear targets for giving a general understanding of sorghum digestibility and how digestibility might be improved.

Technical Abstract: Large variation characterizes the digestibility of protein and starch in sorghum grain. Since the utility of the grain largely depends on its digestibility, numerous in vitro digestion assays have been developed to assess these variations. In this study, we applied multiple techniques to assess the digestibility of grain from 18 sorghum lines differing in digestibility. Amylose content ranged from 5.7 -31.9%, while the content of total starch and protein varied from 47.9-80.9% and 10.1-15.0%, respectively. In vitro starch digestion rate (IVSDR) varied from 0.0022 to 0.012 mg glucose released/min/mL. Disappearance of dry matter after 12-hr in vitro digestibility (IVDMD) and percent protein digestibility (%PD) varied from 19.3-33.0% and from 34.6-62.5%, respectively. Principal Component Analysis (PCA) was applied to describe and explain total variance. Results of PCA revealed that amylose content, total starch content, and protein digestibility accounted for much of the variation in this population of samples. Selected waxy (low amylose) and non-waxy lines were digested separately in a time-dependent manner by applying in vitro pancreatin digestion in parallel with in vitro pepsin and alpha-amylase digestion. By comparing the results of pancreatin treatment with those with pepsin and alpha-amylase, we were able to describe differences in starch and protein digestibility in two or more types of grains. Patterns of total protein revealed both the protein matrix and the composition of alpha-, beta- and gamma-kafirins in the protein body varied among the waxy and non-waxy sorghum cultivars. Gel-based analyses of in vitro pepsin digestion of storage protein showed the various types of kafirins were digested at different rates. The 20 kDa gamma-kafirin with its ability to form oliogomers was more resistant than the respective 28 and 25 kDa alpha-kafirins lacking this ability. The resistance of gamma-kafirin to digestion seems to be associated with protein indigestibility in both waxy and non-waxy sorghum cultivars. It appears that traits contributing heavily to the first three Principal Components: amylose content, amount of total starch, and % PD has a differential effect on the overall protein digestion pattern of non-waxy and waxy cultivars. The amount of starch affects digestion patterns of the protein matrix and protein body components more in non-waxy than waxy sorghum. %PD is influence by the nature of the protein matrix but more significantly by the protein body packaging in both non-waxy and waxy sorghum cultivars. Although each of the digestibility analyses examined was purportedly optimal for a specific application, the three analyses identified in this study (amylose content, amount of total starch, and % PD) are clear targets for giving a general understanding of sorghum digestibility and how this property might be improved.