Seeds make up over 50% of the world’s agricultural calories, thus discovering genes that regulate seed size is of paramount importance in a world with a rapidly growing population. Several genes that regulate seed size are involved in auxin and cytokinin signalling pathways. Classic experiments by Skoog and Miller demonstrated that auxin and cytokinin in particular ratios could determine cell fate, despite this, no in-depth transcriptome analysis of auxin+cytokinin treated tissues has been performed.
In this work, we identify a new regulator of seed size, ARGONAUTE10 (AGO10), and demonstrate that much of its regulation of seed development is dependent on the overexpression of INDEHISCENT (IND). IND has been linked with both the auxin and cytokinin signalling pathways, thus we performed a transcriptome analysis to investigate the regulation of genes by IND, auxin and cytokinin, and all combinations of those treatments. In this analysis we discover the existence of a set of genes regulated only in the presence of auxin+cytokinin, which we call the Dual Hormone Response (DHR). The DHR contains 518 genes, which is a gene set of comparable size to the gene set regulated by auxin or cytokinin treatment alone. We demonstrate preliminary motif and qRT-PCR data that suggests that the bHLH transcription factor, SPATULA, a binding partner of INDEHISCENT, is involved in regulating the DHR. Further analysis of the transcriptome data revealed that regulation of genes by auxin may be dominant over the regulation of genes by cytokinin, and that this response is dependent on TRANSPORT INHIBITOR RESPONSE 1 (TIR1). Lateral root development in seedlings was more sensitive to auxin than cytokinin, which was abolished in tir1 mutants. Auxin amplified the expression of cytokinin-regulated genes that auxin itself does not regulate in isolation, and vice-versa, and these responses were more sensitive to auxin concentration than cytokinin. Finally, we present preliminary evidence that suggests that cytokinin acts as a direct inhibitor of TIR1 action, diminishing subsequent auxin signalling. Treatment with cytokinin inhibited fluorescence in DII-VENUS root tips within 15 minutes, and also inhibited the auxin-promoted degradation of the Auxin Inducible Degron (AID) in mammalian cells.
This work opens up the field of hormones. Our data suggests that multi-hormone transcriptomes will reveal large sets of genes that are only regulated in the presence of multiple hormones. Such transcriptomes may also reveal the coveted existence of a common set of signalling components to regulate growth. The prioritisation of auxin signalling over cytokinin signalling in regulating genes and lateral root development, suggests the possibility of hormone hierarchies. Finally, the possibility that cytokinin directly inhibits the action of TIR1 represents an important discovery in the interaction between auxin and cytokinin.
