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The mechanisms by which plants regulate levels of the phytohormone indole-3-acetic acid (IAA) are complex and not fully understood. One level of regulation appears to be the synthesis and hydrolysis of IAA conjugates, which function in both the permanent inactivation and temporary storage of auxin. Similar to free IAA, certain IAA-amino acid conjugates inhibit root elongation. We have tested the ability of 19 IAA-L-amino acid conjugates to inhibit Arabidopsis seedling root growth. We have also determined the ability of purified glutathione S-transferase (GST) fusions of four Arabidopsis IAAamino acid hydrolases (ILR1, IAR3, ILL1, and ILL2) to release free IAA by cleaving these conjugates. Each hydrolase cleaves a subset of IAA-amino acid conjugates in vitro, and GST-ILR1, GST-IAR3, and GST-ILL2 have K m values that suggest physiological relevance. In vivo inhibition of root elongation correlates with in vitro hydrolysis rates for each conjugate, suggesting that the identified hydrolases generate the bioactivity of the conjugates.

These authors contributed equally to this work. SummaryThe saponins of the model legume Medicago truncatula are glycosides of at least five different triterpene aglycones: soyasapogenol B, soyasapogenol E, medicagenic acid, hederagenin and bayogenin. These aglycones are most likely derived from b-amyrin, a product of the cyclization of 2,3-oxidosqualene. Mining M. truncatula EST data sets led to the identification of sequences putatively encoding three early enzymes of triterpene aglycone formation: squalene synthase (SS), squalene epoxidase (SE), and b-amyrin synthase (b-AS). SS was functionally characterized by expression in Escherichia coli, two forms of SE by complementation of the yeast erg1 mutant, and b-AS by expression in yeast. b-Amyrin was the sole product of the cyclization of squalene epoxide by the recombinant M. truncatula b-AS, as judged by GC-MS and NMR. Transcripts encoding b-AS, SS and one form of SE were strongly and co-ordinately induced, associated with accumulation of triterpenes, upon exposure of M. truncatula cell suspension cultures to methyl jasmonate. Sterol composition remained unaffected by jasmonate treatment. Molecular verification of induction of the triterpene pathway in a cell culture system provides a new tool for saponin pathway gene discovery by DNA array-based approaches.

Most quantum mechanical studies of triterpene synthesis have been done on small models. We calculated mPW1PW91/6-311+G(2d,p)//B3LYP/6-31G* energies for many C30H51O+ intermediates to establish the first comprehensive energy profiles for the cationic cyclization of oxidosqualene to lanosterol, lupeol, and hopen-3beta-ol. Differences among these 3 profiles were attributed to ring strain, steric effects, and proton affinity. Modest activation energy barriers and the ample exothermicity of most annulations indicated that the cationic intermediates rarely need enzymatic stabilization. The course of reaction is guided by hyperconjugation of the carbocationic 2p orbital with parallel C-C and C-H bonds. Hyperconjugation for cations with a horizontal 2p orbital (in the plane of the ABCD ring system) leads to annulation and ring expansion. If the 2p orbital becomes vertical, hyperconjugation fosters 1,2-methyl and hydride shifts. Transition states leading to rings D and E were bridged cyclopropane/carbonium ions, which allow ring expansion/annulation to bypass formation of undesirable anti-Markovnikov cations. Similar bridged species are also involved in many cation rearrangements. Our calculations revealed systematic errors in DFT cyclization energies. A spectacular example was the B3LYP/6-311+G(2d,p)//B3LYP/6-31G* prediction of endothermicity for the strongly exothermic cyclization of squalene to hopene. DFT cyclization energies for the 6-311+G(2d,p) basis set ranged from reasonable accuracy (mPW1PW91, TPSSh with 25% HF exchange) to underestimation (B3LYP, HCTH, TPSS, O3LYP) or overestimation (MP2, MPW1K, PBE1PBE). Despite minor inaccuracies, B3LYP/6-31G* geometries usually gave credible mPW1PW91 single-point energies. Nevertheless, DFT energies should be used cautiously until broadly reliable methods are established.