Auxin biosynthetic intermediate genes and their role in developmental growth and plasticity in higher plants

Abstract

Auxins are a group of phytohormones that regulate several aspects of plant growth and development. Indole-3-acetic acid (IAA) is the predominant form of auxin in plants and several IAA biosynthetic pathways have been previously proposed but remain genetically uncharacterized. One of the proposed pathways is the indole-3-pyruvic acid (IPyA) pathway, which is inferred to regulate key developmental processes such as apical hook formation and shade avoidance. Recent molecular evidence suggests the existence of the pathway in higher plants but remains unverified due to the elusive nature of IPyA in vitro. Extending on these recent advances, this research was aimed at investigating aspects of IPyA-dependent auxin biology in Pisum sativum (pea) using reverse genetics, expression profiling, and analytical techniques. As a result the genes PsTAR2, PsTAR 5g Mt 80, and PsTAR 5g Mt 90, which are inferred to encode key enzymes in the IPyA pathway, were cloned. On expression analysis PsTAR2 was found to be slightly heightened in response to IPyA-inducing conditions (shade) while IAA levels remained unaltered contrary to previous reports. Moreover, the inferred homologs PsTAR 5g Mt 80 and PsTAR 5g Mt 90 appeared down-regulated in the same conditions suggesting functional divergence in the gene family. Thus, PsTAR2 was thought to be solely responsible for regulating IPyA-dependent auxin synthesis. Consequently, using a reverse genetic approach, called TILLING, the PsTAR2 gene was mutated in order to study the down-stream effects of IPyA deficiency. The procedure is currently underway and in the process of isolating two novel pstar2 (IPyA) mutant lines consisting of a missense mutation (pstar2 4280) and a highly desired knockout mutation (pstar2 918). On completion the novel mutants are anticipated to be indispensable to future IPyA-auxin investigations in higher plants. In light of the unstable nature of IPyA, a protocol has been formulated using UPLC for fractioning followed by MS/MS analysis. This technique appears to be very promising as a robust IPyA detection protocol in plant extracts.