Regulation of reserve carbohydrates in hull-less barley grain

Abstract

(1,3;1,4)-β-Glucans, which have many health benefits, represent the major cell wall component in barley endosperm. There have been a number of studies that have altered the amount of (1,3;1,4)-β-glucan in the grain, however, the effects of modifying (1,3;1,4)-β-glucan on various carbohydrate metabolic pathways and its impact on grain development have not previously been clearly defined. Here, we used transgenic grain with increased (1,3;1,4)-β-glucan and a (1,3;1,4)-β-glucanless (bgl) mutant (OUM125) supplied by Professor K. Sato (Okayama University), to investigate the link between carbohydrate metabolism and grain development in hull-less barley. Hull-less barley was investigated as it is more suitable for food use due to absence of maternal (husk) tissues. High (1,3;1,4)-β-glucan barley grain (cv Torrens) was successfully generated by over-expressing HvCslF6, via agrobacterium-mediated transformation. Transgenic grain had up to 70% more (1,3;1,4)-β-glucan than the wild type. Examination of developing transgenic grain revealed a large fluid filled cavity in the endosperm, which resulted in shrunken grain at maturity. The endosperm transfer region of the developing grain was ruptured by 10 days after pollination (DAP), which became more pronounced through development. Quantitative real-time PCR (QPCR) analysis uncovered genes related to cell wall, starch, sucrose and fructan biosynthesis that were differentially expressed across grain development. Starch metabolic genes were downregulated in the early storage phase and the fructan biosynthetic gene, 6-SFT, was upregulated during the later storage phase. Increased amounts of sucrose and fructan were found in the cavity and endosperm tissue of the transgenic grain. The link between altered sugar homeostasis, the large endosperm cavity and the poorly formed endosperm transfer region in transgenic grain was explored by immuno-histochemical microscopy. Cell walls in the endosperm transfer region were poorly formed and variations occurred in the abundance of mannan polysaccharides. Additionally, the development of both the subaleurone and aleurone layers were altered with regards to cell number, shape and position. To further understand the relationship between cell wall, starch and fructan metabolism in barley, transcript profiles of genes related to their metabolism were examined in the (1,3;1,4)- β-glucanless OUM125 mutant. Inactive CSLF6 synthase activity in OUM125 resulted in upregulation of the CslH1 gene from 19 DAP, resulting in traces of BG1 antibody labelling in the mutant pericarp. Other cell wall-related genes including CslF3, CslF7, CslF10, CesA2, CesA3 and Gsl2 were upregulated from 15 DAP in the mutant grain. Deposition of arabinoxylan, callose and cellulose was altered in the absence of (1,3;1,4)-β-glucan in the mutant endosperm. QPCR analysis identified changes in the expression of starch and fructan biosynthetic genes during the storage phase. At grain maturity, sucrose and fructan contents had increased, while the amount of starch remained unchanged. Research findings from this project provide fundamental knowledge about carbon partitioning in grain across development and suggest that small changes in polysaccharide synthesis and deposition can have significant effects on other metabolic processes important for correct grain development. While decreasing the amount of (1,3;1,4)-β-glucan in the barley endosperm had a low impact on grain morphology and carbon partitioning, significantly increasing the (1,3;1,4)-β-glucan content had major deleterious effects on a number of key processes.