Updated November 1, 2021:
The overarching goal of this project is to increase protein content by reducing the flow of carbon into cell wall polysaccharides and to improve the incorporation of sulfur-containing amino acids into protein at the expense of other uses for sulfur. During this quarter, mutant lines for a candidate gene affecting cysteine accumulation were obtained and planted. Generation of transgenic lines targeting different enzymes in cell wall polysaccharide synthesis and sulfur amino acid metabolism also continued according to schedule. In addition, progress was made in developing improved biochemical methods to measure intermediate compounds in the synthesis of cell wall polysaccharides and sulfur amino acids. These methods will be used to determine the changes in seed metabolism in the mutant and transgenic lines currently growing.
Updated November 1, 2021:
The overarching goal of this project is to increase protein content by reducing the flow of carbon into cell wall polysaccharides and to improve the incorporation of sulfur-containing amino acids into protein at the expense of other uses for sulfur. During this quarter, improved methods to quantify intermediates involved in cell wall polysaccharide synthesis and sulfur amino acid synthesis were developed. Intermediates for sulfur amino acid synthesis were subsequently measured at different stages of seed development in Wm82 wild-type seed. Mutant lines for a candidate gene affecting cysteine accumulation were grown and tissue collection of developing seeds initiated. Generation of transgenic lines targeting different enzymes in cell wall polysaccharide synthesis and sulfur amino acid metabolism also continued according to schedule, with harvesting of transgenic seeds expected in the coming weeks.
Updated November 1, 2021:
The overarching goal of this project is to increase protein content by reducing the flow of carbon into cell wall polysaccharides and to improve the incorporation of sulfur-containing amino acids into protein at the expense of other uses for sulfur. During this quarter, developing and mature seed from mutant lines for a candidate gene affecting cysteine accumulation were harvested. Transcript levels of the target gene and a closely related paralog were quantified to determine whether the paralog is upregulated in response to the mutations. Analysis of seed composition revealed reductions in protein content in the mutant lines relative to wild-type controls. Levels of cysteine and methionine were lower in one line. Generation of transgenic lines targeting different enzymes in cell wall polysaccharide synthesis and sulfur amino acid metabolism continued according to schedule, with T1 seeds collected and germination and selection of T2 plants initiated.
Updated May 31, 2022:
The overarching goal of this project is to increase protein content by reducing the flow of carbon into cell wall polysaccharides and to improve the incorporation of sulfur-containing amino acids into protein at the expense of other uses for sulfur. During this quarter, the amino acid composition of seed from mutant lines for a candidate gene affecting cysteine accumulation was measured. Minor differences in some amino acids were observed, but cysteine levels were not affected. Interestingly, mutants had higher levels of protein and lower levels of oil compared to the wild-type controls. In addition, homozygous transgenic lines targeting different enzymes in cell wall polysaccharide synthesis and sulfur amino acid metabolism were identified using an innovative quantitative PCR method and developing seeds collected for analysis. For lines analysed so far, strong reductions in target gene expression suggest effective RNAi silencing and alterations in amino acids and their intermediates are consistent with an impact on amino acid metabolism. Finally, combined transcriptome data and metabolite levels obtained from the same developing seed samples were analyzed together to identify how changes in gene expression affected the synthesis and turnover of cysteine and methionine at different stages of seed development.