Auxin Biosynthesis, Signalling and Function During Early Stages of Endosperm Development in Rice (Oryza sativa L.)

Rice endosperm feeds more than half of the world's population. Its development is a complex process with multiple layers of regulation. The molecular events occurring during its early stages of development determine largely the final grain size and weight, which are key components of grain yield. A large and rapid increase in the content of indole-3-acetic acid (IAA), the main in planta auxin, occurs during early endosperm development. However, auxin action in early endosperm is poorly understood. In this study, I aimed to explore spatio-temporal expression of IAA biosynthesis and signalling genes during early stages of endosperm development in rice. My other aim was to assess the effects of IAA on grain fill and to determine the auxin-response genes in early grains. I found that OsYUC12, one of the three grain-specific OsYUCCAs, was expressed in the aleurone, sub-aleurone and embryo at 3-8 days after pollination (DAP), suggesting early aleurone, sub-aleurone and embryo as sites of IAA biosynthesis. The non-canonical OsIAA29 was strongly co-expressed with OsYUC12. Its spatial expression was restricted to dorsal aleurone, where it could be part of an auxin signalling pathway. OsIAA29 has orthologues only in cereal and non-cereal grass species; expression of the orthologues is early grain- or endosperm-specific. This gene may have evolved independently in the grass family (Poaceae) and acquired conserved functions related to aleurone development. Furthermore, a cluster of MYB transcription factors orthologous to maize Myb-Related Protein-1 (ZmMRP-1) may act downstream of the auxin signalling in dorsal aleurone and regulate its differentiation in relation to apoplastic nutrient transfer. Taking my cue from IAA biosynthesis and signalling, I used a chemical approach to determine its function in early rice grains. Application of two IAA biosynthesis inhibitors, Lkynurenine and 4-phenoxyphenylboronic acid (PPBo), to rice spikelets from 3 to 10 DAP reduced significantly grain IAA levels. Genes encoding small cysteine-rich peptides, seed storage proteins and amino acid transporters were down-regulated in auxin-deficient grains at 5 DAP. These genes are expressed specifically in the aleurone, sub-aleurone and embryo. Auxin may also regulate homeostasis of ethylene, cytokinins, gibberellins and abscisic acid in early grains. Inhibitor-treated panicles showed extensive post-fertilisation seed abortion, which consequently led to significant reduction in their total weight at maturity. This suggested a crucial role of auxin in grain retention, probably through regulation of coenocyte cellularisation. Furthermore, I showed short-lived expression of three OsARFs (OsARF13, OsARF14 and OsARF16) and two OsAUX/IAAs (OsIAA8 and OsIAA14) in early grains at 2-6 DAP. OsARF16 is most likely to interact with OsIAA8 and OsIAA14 in early endosperm. Thus, the results from this study suggested auxin action during key events of early endosperm development such as cellularisation of coenocyte nuclei, early aleurone differentiation and initiation of starch and storage protein biosynthesis, which take place at 3-7 DAP. These findings will point towards new areas of research that will further our understanding of the role of auxin in cereal endosperm development