Quantitative evaluation of IAA conjugate pools in Arabidopsis thaliana.

CONCLUSIONS

This work has demonstrated that the major method of estimating the amount of unknown IAA conjugates-base hydrolysis-can be significantly complicated by chemical artifacts such as glucobrassicin or protein degradation. The concept of 'bound auxin' traces its origin back to more than 80 years ago and has driven research on the sources and forms of these plant hormones since. Indeed, analytical studies have demonstrated that the majority of cellular auxin is conjugated to simple sugars, cyclitols, glycans, amino acids, and other biomolecules. A number of studies have confirmed the enzymatic systems responsible for the synthesis and hydrolysis of a number of such conjugates in Arabidopsis thaliana and some of these compounds have been identified in situ. However, the amount of indole-3-acetic acid (IAA) released upon treating Arabidopsis tissue extracts with base, a commonly employed technique for estimating the amount of IAA conjugates, greatly exceeded the summation of all the IAA conjugates known individually to be present in Arabidopsis. This discrepancy has remained as an unsolved question. In this study, however, we found that a significant portion of the IAA found after base treatment could be attributed to chemical conversions other than conjugate hydrolysis. Specifically, we showed that glucobrassicin conversion, previously thought to occur at insignificant levels, actually accounted for the majority of solvent soluble IAA released and that proteinaceous tryptophan degradation accounted for a large portion of solvent insoluble IAA. These studies clearly demonstrated the limits associated with using a harsh technique like base hydrolysis in determining IAA conjugates and support using more direct approaches such as mass spectrometry-based strategies for unambiguous characterizations of the total complement of IAA conjugates in new plant materials under study.