The importance of flavin monooxygenases (OsYUCCA), tryptophan decarboxylases (OsTDC), nitrilases (OsNIT), and aldehyde oxidases (OsAO) for auxin production in developing rice grains was investigated. Indole-3-acetic acid (IAA) levels in grains increased from approximately 20ng/g FW to 2 μg/g FW during 14 days after flowering (DAF), with the largest increase in IAA (4 and 7 days DAF) correlating with the major gain in grain fresh weight. The rice genome was found to contain 14 OsYUCCA, 7 OsTDC, 4 OsAO, and 2 OsNIT genes. Phylogenetic analysis showed that OsTDC1 has orthologues across the plant kingdom. OsTDC1 was expressed in developing grains at 1, 7, and 21 DAF, however quantitative RT-PCR analysis did not show a clear correlation between OsTDC1 expression and IAA synthesis. Phylogenetic analysis of OsYUCCAs classified OsYUCCAs 9-14, in the same clade as AtYUCCA10 and AtYUCCA11, which are reported to be involved in seed development. A strong correlation between expression of OsYUCCAs 9 & 11 and IAA content suggested these genes are crucial for IAA synthesis in rice grains. Phylogenetic analysis of AOs suggested that major plant groups inherited one AO sequence with isoforms being products of recent gene duplication. This was surprising as AOs have proposed involvement in both abscisic acid and IAA synthesis. Expression analysis and sequencing showed that of four rice AOs, two (OsAO1 and OsAO2) were expressed in grains at 1, 7, and 21 DAF. OsNIT1 and OsNIT2 are part of a conserved clade with members from a diverse group of plants. RT-PCR results as well as on-line microarray data showed expression of OsNITs in grains at 1, 7, and 21 DAF. Despite evidence of expression there was no clear correlation observed between AO or NIT transcripts and IAA content. Data thus provided strong evidence for the involvement of YUCCA in auxin synthesis in developing grains, but only weak evidence for involvement of TDC, AO and NIT. The observed correlation between expression of tryptophan amino transferase (OsTAA1), and OsYUCCA 9 & 11 however suggested that an alternative pathway which involves both YUCCA and TAA should be considered. This would require a different catalytic activity for YUCCA than that previously proposed.

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.