A shorter road to growth

It is a rare and special treat for scientists when a process turns out to be simpler than expected, rather than more complicated. Nevertheless, this actually appears to be the case for the biosynthetic pathway underlying the production of a plant hormone with an essential role in growth and development.

Indole-3-acetic acid (IAA), a plant hormone belonging to the auxin family, activates a variety of signaling cascades involved with critical functions that include shoot growth, root extension and flowering. Consequently, this molecule has been the subject of intensive investigation for well over 70 years. However, attempts to untangle the mechanisms behind IAA biosynthesis have yielded no clear answers. “Based on the results from biochemical studies conducted from the 1960s to the 1980s, researchers have proposed multiple IAA biosynthesis pathways,” says Hiroyuki Kasahara of the RIKEN Plant Science Center in Yokohama.

Of the four mechanisms proposed to date, two have been the subject of particular interest in recent years; one is mediated by the YUCCA (YUC) protein family, and the other by TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1) and a pair of related enzymes. Both pathways work with the same starting material, the amino acid tryptophan, but prior studies have suggested that YUC and TAA1 otherwise represent components of completely distinct and parallel pathways. According to this model, the former process mediates IAA production via the intermediate tryptamine (TAM), whereas the latter employs indole-3-pyruvic acid (IPA) as an intermediate product.

New findings from Kasahara and colleagues, however, have now overturned this model, revealing that YUC and TAA1 instead work together within a single, unified pathway¹.

To test the relative contributions of these two factors, the researchers generated a variety of genetically modified strains of thale cress (Arabidopsis thaliana), a species commonly used as a model plant. They found that the simultaneous overexpression of genes encoding TAA1 and YUC1 led to increased density of lateral root formation and elevated production of IAA and associated metabolic byproducts, suggesting that these two factors may act synergistically in a common pathway. Experiments in which the researchers overexpressed TAA1 alongside YUC6, a different YUC family member, yielded similar results (Fig. 1).

Given that this synergy is seemingly inconsistent with the hypothesized activities of these two enzyme families, Kasahara and colleagues examined their respective functions more closely. When the researchers knocked out a pair of proteins involved in the TAA pathway, they observed a sharp drop in IPA levels. Likewise, overexpression of TAA1 led to a concurrent increase in IPA production, supporting this protein’s established role in converting tryptophan to IPA as a preliminary step in IAA synthesis.

Their findings with YUC proved more surprising. In vitro experiments have suggested that YUC may facilitate the conversion of TAM intoN-hydroxy-TAM (HTAM), which is in turn modified to yield IAA. As expected, YUC-deficient plants showed clear signs of IAA deficiency (Fig. 2), but did not accumulate TAM as this model would predict. “Our paper shows that this initial characterization of YUC enzyme function was not accurate,” says Kasahara. Instead, these plants exhibited a nearly two-fold increase in the accumulation of IPA, suggesting that YUC enzymes instead act to drive conversion of this compound to IAA.

Isolation of fully functional YUC has posed a major logistical challenge in the past, impairing the direct assessment of these enzymes’ functionin vitro. By systematically testing different cell culture and protein preparation strategies, however, Kasahara and colleagues were able to obtain purified YUC2 and verify their hypothesis, confirming that this enzyme family acts directly downstream from TAA1 and related proteins to convert IPA to IAA. “Plant biologists have assumed that the IAA biosynthesis pathway is very complicated,” says Kasahara, “but we have demonstrated that plants can actually produce IAA in only two steps.”