Dissecting the gene regulatory networks behind carbon allocation in plants

Plants store excess energy and metabolites derived from photosynthesis in a variety of storage compounds with starch, protein and triacyglycerol (oil, TAG) being the most common. As humans, we are completely dependent on these compounds either directly as food or indirectly, as materials for large number of goods or feed for animals. With an increased interest in sustainable production systems and the need to feed and clothe a growing population in a changing environment, the need for a better understanding of how plants allocate carbon for storage is increasingly important. One key transcription factor involved in the accumulation of TAG into seeds is WRINKLED1 (WRI1). Using a transient gene expression system together with promoter-reporter gene constructs in Nicotiana benthamiana leaves, it was found that WRI1 is negatively regulating its own expression. This was further investigated by domain swapping between WRI1 homologs from diverse plant species to reveal that this mechanism was intrinsic to the tandem DNA binding AP2-domains of WRI1. Fluorescent electrophoretic mobility shift assay (fEMSA) was employed using purified WRI1 showing that WRI1 is unable to directly interact with its own upstream region. During seed development, non-endospermic seeds accumulate large amounts of oil in the embryo during later stages of embryo development. This is known to be regulated by several master regulators commonly called the LAFL-network (LEAFY COTYLEDON1 (LEC1), ABSCISSIC ACID INSENSITIVE3 (ABI3), FUSCA3 (FUS3) and LEC2). Using transient expression in N. benthamiana leaves it was discovered that LEC1 significantly influences the transactivation ability of LEC2 and especially ABI3. Not only is LEC1 modulating the ability of ABI3 to induce NbWRI1 expression, but also a large set of key genes involved in embryo morphogenesis. This suggests that LEC1 and ABI3 tightly collaborate to drive the embryo development from middle to late stage and the transition between morphogenesis to the acquisition of storage compounds. With the purpose of investigating the differences in carbon partitioning between oat (Avena sativa) which is unique among the cereals in storing significant amount of oil in the endosperm with wheat (Triticum aestivum) that has basically no endosperm oil AsWRI1 was transformed into wheat. The resulting transformant lines showed a significantly altered seed phenotype with increased oil accumulation, disrupted endosperm development and reduced sink strength