Auxin is a key regulator of plant growth, mechanistically controlled by a finely tuned transcriptional system. The auxin signalling pathway consists of the transcription factors, auxin response factors (ARFs), the transcriptional repressors, auxin/indole-3-acetic acid (Aux/IAAs), and the transport inhibitor response 1/auxin signalling F-box (TIR1/AFB) proteins that form a co-receptor complex with the Aux/IAA proteins. These proteins modulate the plant’s response to auxin, mediating a myriad of downstream responses. Auxin is involved in most, or all, developmental processes and in responses to environmental cues. It has been found to be a repressor of ripening in grapes, and a decrease in berry auxin concentration is required to allow the initiation of ripening. To investigate the role of the auxin signalling pathway in grape, 19 VviARF transcription factors, 23 VviIAA repressors and six VviAFB receptor family members were identified in Vitis vinifera sp. The conserved domains were analysed in each group of proteins and their presence or absence related to possible function. Phylogenetic trees demonstrated the relationship of the Vvi genes with apple, Arabidopsis, poplar, and tomato family members, some of which have proven function in fruit development. Expression analysis across a 16 week V. vinifera L. cv. Shiraz berry developmental series suggested that 39 of the 48 auxin signalling transcripts were highly expressed pre-veraison (before the onset of ripening) and were down-regulated from veraison and throughout berry ripening, correlating with the high concentration of auxin pre-veraison. The varied expression patterns of these genes suggest participation in a range of developmental processes at different stages during development. The change in expression of a large proportion (39 of 48) of auxin signalling genes at veraison indicates that it is a key change-point in berry development. Those genes expressed early in development may play roles in cell division and cell expansion. Two VviARF transcripts, VviARF27 and 2b, and five VviIAA transcripts, VviIAA15b, 19, 31, 38 and 40, were highly up-regulated post-veraison suggesting that they may play roles in fruit ripening. ARF proteins have also been found to play roles in fruit ripening through interactions with other proteins such as MYBs and bHLHs. The expression patterns of the 48 auxin signalling genes in a nine stage leaf developmental series could be clustered into 12 groups. Interestingly, very few auxin signalling pathway genes were expressed in leaves with a pattern that correlated to the pattern of IAA or IAA-Asp accumulation. Various transcripts had high transcript expression in flowers, roots and/or tendrils again suggesting a diversity in the roles these genes play. Yeast 2-hybrid and bimolecular fluorescence techniques showed that VviARF4-VviIAA19, VviARF27- VviIAA19 and VviARF27-VviIAA27 protein pairs interact and have nuclear localisation. The ARF activator, VviARF27, and VviIAA19 have overlapping expression patterns, in post-veraison berries and flowers, suggesting the interaction between these two proteins may occur in planta and play a role in flowering and berry ripening. From their expression patterns, VviARF4 and VviIAA19 may interact in tendrils, and VvARF27 and VviIAA27 may interact in flowers. Ex planta berry treatments suggested that auxin and ethylene/abscisic acid (ABA) have antagonistic effects on the auxin signalling pathway in grape berries. Auxin treatment up-regulated VviIAA transcripts pre- and post-veraison whilst the enhancers of ripening, ethylene, in the form of Ethrel, and ABA, down-regulated some auxin signalling transcripts, both pre- and post-veraison. ABA may play an important role in ripening by switching off the vegetative pathways pre-veraison, such as photosynthesis, while ethylene enhances ripening factors post-veraison. A model explaining the role of auxin signalling during berry development is proposed where the majority of VviARF and VviIAA proteins function during pre-veraison berry development, when the levels of IAA are high. These are switched off towards veraison, and allow the transition to ripening. During fruit ripening, a select number of VviARF and VviIAA proteins may interact to affect the ripening process – potentially through interactions with other protein families. ABA may down-regulate the auxin signalling pathway pre-veraison, and ethylene down-regulates some members of the pathway post-veraison. These findings support the existence of a fruit specific, complex hormonal network that works in concert to modulate and ensure grape berry growth and ripening. This research represents the most in-depth analysis of the auxin signalling pathway components in V. vinifera to date and highlights the pleiotropic roles the candidates play throughout plant development. These findings may aid in the development of strategies to manipulate berry ripening and identifies areas for future research.Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 201
