Abstract
Sorghum is an important food staple in rural Asia, India and Africa and a major feedstock in developed countries. Originating in the semi-arid African savannah, sorghum is well adapted to drought prone environments and produces more biomass per unit water than maize. Extensive variability exists within the sorghum gene pool creating a diversity of plant forms with many commercial and industrial applications. Sorghum, provides an alternative to grain crops with greater irrigation and fertiliser requirements, such as maize, however, the grain is generally less digestible, especially upon cooking, due to extensive disulphide cross-linking among sulphur-rich storage proteins in the protein-starch matrix. This reduces the accessibility of proteolytic enzymes to starch, decreasing palatability and nutritional value, and resulting in the need for increased processing. The commercial value of cereals is largely determined by the chemistry of the protein-starch matrix. Located on the periphery of storage protein bodies, cysteine-rich b- and g-kafirins prevent enzymatic access to internally positioned a-kafirins and to starch. The development of mutants is an efficient approach for studying genetic regulation of protein biosynthesis and compartmentalisation and shows how changes in protein profile affect the physical characteristics of the seed. An integrated proteomic approach was employed to characterise 28 sorghum lines with allelic variation at the kafirin loci, including three b-kafirin null mutants, in order to determine the effects of kafirin genetic diversity on the expression of storage proteins. High performance liquid chromatography (HPLC) and capillary electrophoresis were employed to profile water/salt- and alcohol-soluble protein fractions, revealing a wide range of diversity in protein content across the genotypes. Peak profiles were similar among b-kafirin null lines but different to normal lines, with a significant reduction in alcohol-soluble protein and the disappearance of a major set of protein peaks. The relative content of sequentially extracted soluble, insoluble and residual proteins was measured using RP-HPLC to evaluate the degree of cross-linking among storage proteins in the seed. High levels of insoluble and residual protein were associated with reduced digestibility. Gel-based separation of seed proteins and subsequent identification with tandem mass spectrometry identified a range of redox-active proteins affecting storage protein biochemistry. Multiple protein fractions were analysed across the b-kafirin null line QL12 and wild-type line 296B. The redox states of endosperm proteins, of which kafirins are a subset, have been shown to impact on quality traits in addition to the expression of proteins. Thioredoxin, active in the processing of storage proteins at germination, has reported impacts on grain digestibility and was differentially expressed across the genotypes. A range of putative uncharacterised sorghum proteins regulating the folding and disulphide pairing of storage proteins in homologous crop species were detected, as well as a diversity of lysine-rich albumins and globulins, and enzymes involved in starch hydrolysis, such as a-amylase inhibitor. In addition, a high molecular weight g-prolamin homolog was identified in sorghum at the sequence level for the first time. Biochemical factors affecting grain quality were measured across the sample set, including crude protein content, raw and cooked protein digestibility, total starch and flour moisture content. These parameters were evaluated for correlation to the expression of specific classes of proteins, such as the kafirins. Alcohol-soluble RP-HPLC peak distribution profiles showed correlation between a specific peak, likely a g-kafirin, and digestibility. To test the impact of kafirins on the efficiency of ethanol production from sorghum, a subset of ten diverse genetic lines from the sample population, including three b-kafirin null mutants, were evaluated for ethanol yield and fermentation efficiency. Starch content was positively correlated to total ethanol yield (Rs=0.74), and there was a slight positive correlation between protein digestibility and ethanol yield (Rs=0.52). Increases in free amino nitrogen (FAN) content significantly enhanced fermentation efficiency (Rs= 0.65). Multivariate analysis indicated an association between the b-kafirin allele and variation in grain digestibility (P=0.042) and FAN (P=0.036), with subsequent effects on ethanol yield. Sorghum improvement strategies focussed on enhancing digestibility, nutritional value, and fermentation efficiency will increase the usefulness of the crop as a food, feed and biofuel. The most common technique for enhancing cereal protein quality is seed-specific genetic modification of protein biosynthesis for the introduction of genes encoding proteins rich in deficient amino acids or with favourable biochemical characteristics. To achieve this, a comprehensive understanding of storage protein gene function and expression is required for the stable introduction of foreign genes and the manipulation of protein content and structure in the seed. This work contributes to our understanding of sorghum grain protein composition and how it is impacted by diversity in kafirin genetic background. The acquisition of sequence-based information pertaining to the expression of proteins involved in redox activity in the seed, the accumulation of lysine-rich storage proteins and the activity of starch metabolic enzymes, further augments annotation of the sorghum proteome and lends insight to future improvement strategies.
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