Arabidopsis thaliana auxotrophs reveal a tryptophan-independent biosynthetic pathway for indole-3-acetic acid.

Abstract

We used tryptophan auxotrophs of the dicot Arabidopsis thaliana (wall cress) to determine whether tryptophan has the capacity to serve as a precursor to the auxin, indole-3-acetic acid (IAA). Quantitative gas chromatographyselected ion monitoring-mass spectrometry (GC-SIM-MS) The phytohormone auxin plays a central role in plant growth and development as a regulator of numerous biological processes, from cell division, elongation, and differentiation to tropic responses, fruit development, and senescence (1). The biosynthetic route to the synthesis ofthe most abundant plant auxin, indole-3-acetic acid (IAA), has been the subject of controversy for decades. The observation that radiolabeled tryptophan, when applied to watermelon slices, yielded radiolabeled IAA (2) was the first of numerous biochemical studies from which several pathways involving the conversion of tryptophan to IAA have been proposed (Fig. 1). In most of the in vitro studies where radiolabeled tryptophan was converted to IAA, at best only a few percent of the total radioactivity was recovered as IAA. This inefficient conversion of tryptophan to IAA could be accounted for by contaminating epiphytic bacteria (4, 5). Indeed, maize coleoptiles do not convert tryptophan to IAA when incubated in sterile conditions (6). More recently, Baldi et al. (7) showed that feeding tryptophan in high concentration to Lemna gibba (duckweed) increased the pool size of tryptophan but had negligible effects on IAA levels. Additionally, feeding Lemna[15N]tryptophan to the extent that 99% of the tryptophan pool was 15N-labeled did not result in a significant 15N enrichment of IAA. Bandurski et al. (8) showed that maize seedlings grown in the presence of deuterium incorporate label into tryptophan but not IAA. The isolation of tryptophan auxotrophs in both maize and Arabidopsis makes it possible to determine whether tryp-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.tophan is indeed the precursor to IAA. The orange pericarp (orp) mutant of maize does not make tryptophan due to a defect in tryptophan synthase p activity (9), yet it has 50-fold higher levels of IAA (10). We have used tryptophan auxotrophs of the dicot Arabidopsis thaliana (11-13) to determine whether tryptophan is the sole precursor to IAA. In this study we show that there is a tryptophan-independent IAA biosynthetic pathway. Our data indicate that the precursor is likely to be a compound earlier in the tryptophan biosynthetic pathway, between anthranilate and indole. MATERIALS AND METHODSA. thaliana ecotype Columbia carrying mutations trpl-1, trp2-1, and trp3-1 have been described (11-13). Seeds were surface sterilized (11) MA; 98 atom %) was added to a final concentration of50 ,uM. After 13 days the seedlings were weighed, frozen in liquid nitrogen, and stored at -80°C.Isolation of IAA, Indole-3-acetonitrile (IAN), Indole, and Trypt...

Transgenic tobacco plants expressing the Arabidopsis thaliana nitrilase II enzyme

“…Nevertheless, initial observations already pointed to a vital role of nitrilases in early plant development/regeneration. However, both, nitrilase I and II exhibit a high Km (approximately 5 mM) for IAN in vitro (Bartling et al, 1992(Bartling et al, , 1994, which contrasts with low (micromolar) endogenous levels of this precursor (Normanly et al, 1993;Wightman, 1977).…”

Hormonal regulation in green plant lineage families

“…The trend suggests that excised plant tissues, cell cultures or cell free extracts synthesize auxin via the tryptophan-dependent (TD) pathways. The auxin can be produced by tryptophan-independent (TI) pathways also (Wright et al, 1991;Normanly et al, 1993). Depending on the state of growth and rate of cell proliferation, cells can switch from the TI to the TD pathway.…”

CYP83B1, a Cytochrome P450 at the Metabolic Branch Point in Auxin and Indole Glucosinolate Biosynthesis in Arabidopsis

“…This is unexpected for an intermediate in a highly regulated biosynthetic pathway. Also, the observation that the isotope enrichment of IAN is lower than that for IAA when radiolabeled tryptophan is administered to either Arabidopsis roots or shoots (Normanly et al, 1993;Müller et al, 1998) indicates that IAN may not be a direct product of indole-3-acetaldoxime metabolism in IAA biosynthesis. Ectopic overexpression of the nitrilases does not give rise to an apparent IAA phenotype under normal conditions (Normanly et al, 1997;Grsic et al, 1998).…”