Characterization of the Tryptamine Pathway of Auxin Biosynthesis in Developing Rice Grains

Abstract

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.