Biochemical and chemical biology study of rice OsTAR1 revealed that tryptophan aminotransferase is involved in auxin biosynthesis; identification of a potent OsTAR1 inhibitor, pyruvamine2031

Abstract

IAA, a major form of auxin, is biosynthesized from l-tryptophan via the indole-3-pyruvic acid (IPyA) pathway in Arabidopsis. Tryptophan aminotransferases (TAA1/TARs) catalyze the first step from l-tryptophan to IPyA. In rice, the importance of TAA/TARs or YUC homologs in auxin biosynthesis has been suggested, but the enzymatic activities and involvement of the intermediate IPyA in auxin biosynthesis remain elusive. In this study, we obtained biochemical evidence that the rice tryptophan aminotransferase OsTAR1 converts l-tryptophan to IPyA, and has a Km of 82.02 µM and a Vmax of 10.92 µM min-1 m-1, comparable with those in Arabidopsis. Next, we screened for an effective inhibitor of OsTAR1 from our previously reported inhibitor library for TAA1/TARs, designated pyruvamine (PVM). Differing from previous observations in Arabidopsis, hydroxy-type PVMs, e.g. PVM2031 (previous name KOK2031), had stronger inhibitory effects in rice than the methoxy-type. PVM2031 inhibited recombinant OsTAR1 in vitro. The Ki of PVM2031 was 276 nM. PVM2031 treatment of rice seedlings resulted in morphological changes in vivo, such as reduced lateral root density. Exogenous IAA rescued this growth inhibition, suggesting that the inhibitory effect is auxin specific. Furthermore, rice roots showed reduced IAA levels concomitant with reduced levels of IPyA in the presence of the inhibitors, suggesting that the IPyA pathway is an auxin biosynthesis pathway in rice. Since PVM2031 showed stronger inhibitory effects on rice auxin biosynthesis than known tryptophan aminotransferase inhibitors, we propose that the hydroxy-type PVM2031 is an effective tool for biochemical analysis of the function of auxin biosynthesis in rice roots.

Tillering and small grain 1 dominates the tryptophan aminotransferase family required for local auxin biosynthesis in rice

“…Enzyme activity was exhibited using substrate L‐tryptophan as in MBP‐TSG1. OsTAR1 showed significant aminotransferase activity similar to a previous report (Figure 7A) (Kakei et al 2017); however, both OsTARL1 and OsTARL2 failed to recognize L‐tryptophan and showed no enzyme activity (Figure 7B). In addition, aminotransferase activity of TSG1 was obviously higher than that of OsTAR1 (Figure 7C).…”

“…To further confirm whether mutation of TSG1 in the tsg1 mutant disrupts aminotransferase activity, recombinant MBP‐TSG1 and the mutated version MBP‐TSG1 A447V were expressed in Escherichia coli then the fusion proteins were purified for in vitro assay. In line with previous studies, L‐tryptophan was used to assay the tryptophan aminotransferase activity of TSG1 (Stepanova et al 2008; Tao et al 2008; Kakei et al 2017). The amount of indole‐3‐pyruvic acid‐borate complex produced after each reaction was then determined based on the absorbance at 327 nm, and the findings were used to characterize the enzymatic properties of TSG1 and calculate its kinetic parameters (Figure 3A, B).…”

“…Three homologs of TSG1 aminotransferase were subsequently discovered: OsTAR1 (TRYPTOPHAN AMINOTRANSFERASE RELATED 1) (LOC_Os05g07720), OsTARL1 (TRYPTOPHAN AMINOTRANSFERASE RELATED‐LIKE 1) (LOC_Os01g52010) and OsTARL2 (LOC_Os01g51980). Although OsTAR1 has been previously examined (Abu‐Zaitoon et al 2012; Yoshikawa et al 2014; Kakei et al 2017), the genetic functions of these three homologs and their related phenotypes have yet to be revealed. Whether or not they also show redundancy in auxin metabolism during rice growth also remains unknown.…”

“…Aminotransferase activity assays were performed as previously described (Stepanova et al 2008; Tao et al 2008; Kakei et al 2017) using L‐tryptophan (Sigma‐Aldrich) as a substrate. Aminotransferase activity of TSG1, TSG1 A447V , OsTAR1, OsTARL1, and OsTARL2 at various enzyme concentrations was measured in 100 μL reaction buffer (0.4 mol/L borate buffer (pH 8.5), 10 μmol/L pyridoxal phosphate, 1 mmol/L pyruvic acid) and 1 mmol/L L‐tryptophan.…”

Chemical regulators of plant hormones and their applications in basic research and agriculture*

“…Subsequently, the same group reported pyruvamine PVM1169 (also designated as KOK1169, 3), which is a more specific inhibitor than l-AOPP in Arabidopsis [7]. Kakei et al [8] recently reported that PVM2031 (also designated as KOK2013, 4) outperforms PVM1169 in rice, indicating the species specificity of these inhibitors. A chemical screening aimed at identifying inhibitors of ethylene signaling identified l-kynurenine (l-Kyn, 5), which suppresses ethylene responses in Arabidopsis roots [9].…”