Preparation Of Monoclonal Antibody Against Indole-3-acetic Acid And Analysis Of Ethylene Action On Auxin In Regulating Root Gravitropism

Gravitropism is very important for plant to take up the water, mineral ions and to harvest light, which are necessary for growth and development. Recent advance in molecular biology, associated with the adoption of the mutants have allowed major breakthroughs in our understanding of both root and shoot gravitropism. Now, it is confident that auxin plays the central role in regulating the gravitropism in plants.It has been discovered that the gravitropism had changed in some mutants on ETH biosynthesis or signaling. So extremely probably, ETH affects the gravitropism via auxin. In this case, such mutants might be used as the ideal model plants for the study of the closs-talk between ETH and auxin.In order to study ETH's action on auxin in the mutants, it is necessary to establish an efficient quantitative assay of IAA in plants. Firstly, we produced a monoclonal antibody to IAA. The antibody showed high specificity to MelAA but little cross-reactivity with some compounds structurally similar to IAA and some intermediates in IAA metabolism. And then, based on this antibody, an indirect enzyme-linked immunosorbent assay (ELISA), with high specificity, sensitivity and precision, was developed for quantification of IAA in plants.The materials in this study were Arabidopsis thaliana.(WT) and its mutants including ethylene over producer mutant (eto1-1), ethylene insensitive root mutant (eir1-1) and ethylene insensitive mutant (etr1-3).From the phenotypes of 12-d-old seedlings of Arabidopsis WT and mutants, the root gravitropism was obviously enhanced in etol-1, but diminished in eir1-1 and etr1-3, compared with WT. The shoot negative gravitropism was also diminished in etiolated seedlings of etr1-3.The IAA contents in shoots and roots of 12-d-old seedlings of Arabidopsis WT andmutants were assayed. High ETH level (in eto1-1) showed the promotion of IAA synthesis or inhibition of the IAA catabolism, thus improves the root gravitropism. Insensitivity to ETH (in eir1-1 root and etr1-3) led to the impossibility of IAA transportion from shoot tip to shoot bottom or root, so that result in the diminishment of root gravitropism. It is concluded that ETH perform its action on gravitropism through the regulation of both IAA contents and IAA polar transportation.The above results were attested by exogenous application of IAA and TIBA. IAA (1 10-7mol/L) succeeded in restoring the root gravitropism in eir1-1 and etr1-3, while TIBA (1 10-5mol/L), the commonly used inhibitor of IAA polar transportation, diminished the root gravitropism in eto1-1 and WT.