A novel approach to identify genes that determine grain protein deviation in cereals.

Grain yield and protein content were determined for six wheat cultivars grown over 3 years at multiple sites and at multiple nitrogen (N) fertilizer inputs. Although grain protein content was negatively correlated with yield, some grain samples had higher protein contents than expected based on their yields, a trait referred to as grain protein deviation (GPD). We used novel statistical approaches to identify gene transcripts significantly related to GPD across environments. The yield and protein content were initially adjusted for nitrogen fertilizer inputs and then adjusted for yield (to remove the negative correlation with protein content), resulting in a parameter termed corrected GPD. Significant genetic variation in corrected GPD was observed for six cultivars grown over a range of environmental conditions (a total of 584 samples). Gene transcript profiles were determined in a subset of 161 samples of developing grain to identify transcripts contributing to GPD. Principal component analysis (PCA), analysis of variance (ANOVA) and means of scores regression (MSR) were used to identify individual principal components (PCs) correlating with GPD alone. Scores of the selected PCs, which were significantly related to GPD and protein content but not to the yield and significantly affected by cultivar, were identified as reflecting a multivariate pattern of gene expression related to genetic variation in GPD. Transcripts with consistent variation along the selected PCs were identified by an approach hereby called one-block means of scores regression (one-block MSR).

Fructan biosynthesis in excised leaves of wheat (Triticum aestivum L.): a comparison of de novo synthesis in vivo and in vitro.

Carbohydrate accumulation by excised, continuously illuminated leaves of wheat (Triticum aestivum L.) was followed over a 24 h period. At 0 h, the tissue contained no detectable fructan. In the initial 6 h, only sucrose was accumulated. After 6 h the de novo synthesis of fructans was induced. Fructans accumulated in the sequence 1-kestose, bifurcose, nystose, oligofructans of apparent degree of polymerization (DP) up to 9 and finally, 6-kestose, which was first detected after 22 h. A cell-free protein extract from leaves illuminated for 24 h catalyzed the de novo synthesis of fructan from sucrose. The properties of this fructan synthetic activity (FSA) were characterized. The FSA was stable, exhibiting < 20% loss of activity when stored at 5 °C or 25 °C for 6 h. The FSA exhibited an apparent Km,suc of 114 mM, and an apparent pH optimum at 5.5. The in vitro synthesis of fructan of DP > 3 was not inhibited by sucrose even at 1000 mM. Pyridoxal-hydrochloride at 20 mM did not enhance rates of enzymatic fructan synthesis or significantly inhibit the release of free fructose in the optimized enzymatic reaction. The rate of oligofructan synthesis in the optimized reaction approximated to rates of accumulation in the leaf (1.35 mg g h-1 and 1.18 mg g h-1 respectively). The sequence of oligofructan synthesis in vitro was the same as that observed in the leaf, with the exception that 6-kestose was synthesized early in the time course, in parallel with 1-kestose and bifurcose. Fructans of apparent DP ≤ 8 were detected after 10 h of incubation. When incubated with bifurcose as sole substrate, the cell-free preparation liberated free monosaccharides, without the accumulation of trisaccharide or sucrose as intermediates. The results are discussed with reference to current, conflicting models for the biosynthesis of fructan in cereals.