Indole-3-acetic acid (IAA) synthesis is required for grain-fill in maize and appears to be regulated by cell-wall invertase (CWIN) activity. OsYUC12 is one of three IAA biosynthesis genes we previously reported as expressed during early rice grain development, correlating with a large increase in IAA content of the grain. This work aimed to investigate further the role of OsYUC12 and its relationship to CWIN activity and invertase inhibitors (INVINH). The analysis shows a brief peak of OsYUC12 expression early in endosperm development. Meta-analysis of microarray data, confirmed by quantitative expression analysis, revealed that OsYUC12 is coexpressed with OsIAA29, which encodes an unusual AUX/IAA transcription factor previously reported as poorly expressed. Maximum expression of OsYUC12 and OsIAA29 coincided with maximum CWIN activity, but also with a peak in INVINH expression. Unlike ZmYUC1, OsYUC12 expression is not reduced in the rice CWIN mutant, gif1. Several reports have investigated CWIN expression in rice grains but none has reported on expression of INVINH in this species. We show that rice has 54 genes encoding putative invertase/pectin methylesterase inhibitors, seven of which are expressed exclusively during grain development. Our results suggest a more complex relationship between IAA, CWIN, and INVINH than previously proposed.

The indole-3-acetic acid (IAA) content of developing grains of 'Oryza sativa' subsp. 'japonica' was measured by combined liquid chromatography, tandem mass spectrometry in multiple-reaction-monitoring mode. The increase from 50 ng g⁻¹ fresh weight to 2.9 µg g⁻¹ fresh weight from 1 to 14 days after pollination was much larger than that previously reported by enzyme-linked immunoassay methods. The largest increase in IAA content coincided with the start of the major starch deposition phase of grain-fill. The increase in IAA content was strongly correlated with the expression of putative IAA biosynthesis genes, OsYUC9, OsYUC11 and OsTAR1, measured by quantitative reverse transcriptase polymerase chain reaction. These results confirm the importance of the tryptophan aminotransferase/YUCCA pathway in this system. All three genes were expressed in endosperm; expression of OsYUC11 appeared to be confined to endosperm tissue. Phylogenetic analysis indicated that OsYUC11 and AtYUC10 belong to a separate clade of YUCCAs, which do not have orthologues outside the Angiosperms. This clade may have evolved with a specific role in endosperm. Expression of tryptophan decarboxylase in developing rice grains did not correlate with IAA levels, indicating that tryptamine is unlikely to be important for IAA synthesis in this system. In light of these observations, we hypothesize that IAA production in developing rice grains is controlled via expression of OsTAR1, OsYUC9, OsYUC11 and that IAA may be important during starch deposition in addition to its previously suggested role early in grain development.

Indole-3-acetaldoxime (IAOx) is a branch point compound of tryptophan (Trp) metabolism in glucosinolate-producing species such as Arabidopsis, serving as a precursor to indole-glucosinolates (IGs), the defense compound camalexin, indole-3-acetonitrile (IAN) and indole-3-acetic acid (IAA). We synthesized [²H₅] and [¹³C₁ₒ¹⁵N₂]IAOx and [¹³C₆], [²H₅] and [2',2'-²H₂]IAN in order to quantify endogenous IAOx and IAN in Arabidopsis and tobacco, a non-IG producing species. We found that side chain-labeled [2',2'-²H₂]IAN overestimated the amount of IAN by 2-fold compared to when [²H₅]IAN was used as internal standard, presumably due to protium–deuterium exchange within the internal standard during extraction of plant tissue. We also determined that [¹³C₁]IAN underestimated the amount of IAN when the ratio of [¹³C₁]IAN standard to endogenous IAN was greater than five to one, whereas either [²H₅]IAN or [¹³C₆]IAN showed a linear relationship with endogenous IAN over a broader range of concentrations. Transgenic tobacco vector control lines did not have detectable levels of IAOx or IAN (limit of detection 100 pg/g fr. wt), while lines expressing either the IAOx-producing CYP79B2 or CYP79B3 genes from Arabidopsis under CaMV 35S promoter control accumulated IAOx in the range of 1–9 μg/g fr. wt. IAN levels in these lines ranged from 0.6 to 6.7 μg/g fr. wt, and IAA levels were 9–14-fold above levels in control lines. An Arabidopsis line expressing the same CYP79B2 overexpression construct accumulated IAOx in two of three lines measured (200 and 400 ng/g fr. wt) and accumulated IAN in all three lines. IAN is proposed to be a metabolite of IAOx or an enzymatic breakdown product of IGs induced upon tissue damage. Since tobacco does not produce detectable IGs, the tobacco data are consistent with IAN being a metabolite of IAOx. IAOx and IAN were also examined in the Arabidopsis activation tagged yucca mutant, and no accumulation of IAOx was found above the limits of detection but accumulation of IAN (3-fold above wt) occurred. The latter was surprising in light of recent reports that rule out IAOx and IAN as intermediates in YUCCA-mediated IAA synthesis.