Studies on multi-layer aleurone mutants in cereals and their nutritional significance

Abstract

Aleurone cells in the cereal bran fraction store the greatest amount of nutritional components encompassing dietary fibre, phenolic compounds, antioxidants, minerals, phytate, B vitamins and lipid. I observed a substantial enhancement in these nutrients in a novel rice mutant thick aleurone 2 (ta2) possessing a multi-layer aleurone - from about two cells to 12 cells thick. Histological assay of ta2 showed that the aleurone cell structure and tissue organization remained similar to wild-type characteristics. Nutritional analyses of wholegrain flour of field-grown ta2 rice showed concurrent increases in dietary fibre (up 66%), phenolic compounds (up 43%), antioxidants (up 84%), minerals (up 14-23%) and phytate (up 18%), vitamin B3 (up 21%), B6 (up 33%), B9 (up 32%), lipid (up 50%) and simple sugars (up 170%). The field-grown ta2 rice has normal grain morphology, seed size, and weight, suggesting a high potential for being developed as commercially viable bio-fortified rice for consumption. The rice ta2 mutation was mapped to LOC_Os01g11900, corresponding to OsROS1a gene, where a guanine nucleotide was substituted by adenosine at Chr1:6451738. This nucleotide substitution created an alternative RNA splicing site, which resulted in an in-frame insertion of seven amino acids in the OsROS1A protein. Our collaborator in IB-CAS subsequently isolated seven additional multi-layer aleurone rice mutations in OsROS1a by TILLING. All eight mutations were amino acid substitutions; knockout mutations may be lethal. Together these results implied that the thick aleurone trait was achieved through the reduction but not the elimination of OsROS1a activity. However, I still cannot reject the hypothesis that the thick aleurone trait is caused by a gain of function. In the gain of function hypothesis, ta2 mutation can result in in new function in OsROS1a which facilitates the differentiation of endosperm progenitor cells or the trans-differentiation of starchy endosperm cells to aleurone cells during caryopsis development. I attempted to mimic the desired multi-layer aleurone trait to increase the nutritional value in wheat. I identified three wheat homologs of rice OsROS1a, namely TraeROS1a-5A, -5B and -5D. Quantitative PCR and TraeROS1a-5B gene promoter::GUS fusion analyses supported TraeROS1a being expressed in immature endosperm tissues. A suite of wheat transgenics was developed to modulate TraeROS1a gene function in wheat. I used RNAi and artificial microRNA for down-regulation of TraeROS1a. Also, I conducted CRISPR/Cas9 gene editing of the endogenous TraeROS1a gene. Both methods were aimed at knocking down one or two of the three wheat TraeROS1a gene homeologs so as to reduce the total enzyme activity of ROS1A protein. In addition, other genes, CR4 and DEK1, which have previously been shown to affect aleurone development in maize, were modified in wheat. Barley HvCR4 and HvDEK1 genes were ectopically expressed in wheat under starchy endosperm-specific Bx17 or aleurone-specific LTP2 promoters, in further attempts to increase signal perception and induction of multi-layer aleurones. Detailed genotypic and phenotypic analyses have begun on these transgenics, however, completion of this work is beyond the scope of this thesis. Plant transgenics were mainly focused on single nutrient enhancement. This research provides a novel example of multiple nutritional enhancements by the modification of a single gene in cereal crops.