Molecular genetic and biochemical analysis of gibberellic acid involvement in the stages of soybean (Glycine max L.) nodule development

Abstract

This thesis aimed to characterise four genes identified to be involved in early nodulation in Glycine max (soybean). The first gene studied was a GmTIR-NBS-LRR gene, a Toll/Interleukin 1 receptor - Nucleotide Binding Site - Leucine-Rich Repeat (TIR-NBS-LRR) suspected of being involved in host specificity in regards to rhizobia. Over-expression, silencing and histochemical promoter studies of this gene saw no phenotypic changes. This is due to the previously unknown existence of a transcript variant (TV) of the gene, as well as the likely existence of alternative promoter(s) (AP). Future characterisation work will focus on understanding and identifying the TVs and possible APs of GmTIR-NBS-LRR. The remaining three early nodulation genes are involved in GA biosynthesis, a plant hormone whose role in nodulation is still unclear. The genes GmGA20ox a, GmGA2ox and GmGA3ox 1a were analysed through silencing and histochemical promoter studies, over-expression and phytogenic analysis and histochemical promoter studies, respectably. Additionally, measurement of endogenous GAs in G. max roots which had yet to be reported was carried out. No phenotypic changes were observed following either silencing or over-expression for any of the GA biosynthesis genes. The histochemical promoter studies of GmGA20ox a highlighted a likely role in facilitating infection thread formation and early nodule development in the cortical cells. GmGA3ox 1a appeared to be more general, but still NF dependent in its expression, through its widespread presence in the phloem during early nodulation. Successful measurement of endogenous GA showed that, independent of nodulation, GA3 is the more abundant bioactive GA in soybean compared to GA1. This differs from Pisum sativum (pea) roots where GA3 was not detected and GA1 is most abundant. The recent availability of G. max mutants of many of these genes through Soybase (http://soybase.org), coupled with the new method for measuring endogenous GA in G. max roots, opens up many pathways for further effective characterisation of these nodulation specific genes and thus, a better understanding of nodulation as a whole.