Abstract Transcription factors (TFs) play an indispensable role in cell biology as they are responsible for regulating gene activity. TFs act by binding to specific DNA sequences in a gene promoter resulting in either activation or repression of transcription of the target gene. TFs interact with target genes through a DNA binding domain which is often highly conserved and can be used to classify TFs into families. Dof (DNA binding with one finger) TFs are classified by the presence of a highly conserved bi-functional Dof domain. Characterisation of various Dof proteins has identified essential regulatory roles in plant-specific processes. This includes roles in carbon metabolism in maize, seed development and germination in Arabidopsis and cereal crops and circadian responses in Arabidopsis and rice. Despite the important role of Dof proteins in plant growth and development, this family has been studied to a limited extent in wheat with only two Dof proteins reported to date. Therefore, the aim of this thesis is to identify and initiate characterisation of the Dof TF family in wheat (Triticum aestivum) and investigate the potential role of members of this family in wheat productivity-associated physiological processes. Thirty-one Dof genes were identified after extensive searching of available Triticum aestivum ESTs and contig assembly. Phylogenetic analysis grouped these 31 genes into four clades. Extensive gene expression profiling of the TaDof family was undertaken and revealed that the majority of TaDof members were constitutively expressed in major vegetative organs with a few displaying a grain-predominant expression pattern. The TaDof family appears to be enriched with light-responsive or drought down-regulated members, suggesting that the role of this family is predominantly in growth-related processes. To further investigate their role in growth-related physiological processes, two Dof genes, TaDof4 and TaDof5, were selected for more detailed characterisation. TaDof5 was identified to be similar to the Cycling Dof Factors from Arabidopsis and the recently identified rice Dof daily fluctuations genes which are involved in the photoperiodic regulation of flowering time. TaDof5 gene expression was diurnally regulated, had strong expression in the stem and head and a peak in expression level at anthesis. Using publicly available Affymetrix data, correlation analysis suggested co-expression of TaDof5 with a number of circadian-regulated genes associated with flowering. Extensive analysis of the DNA-binding specificity of TaDof5 revealed a preferred binding motif of 5’GAAAAAGTGC. The binding of TaDof5 to DNA requires two (A/T)AAAG(T/C) core motifs in adjacent positions. The binding sequence of TaDof5 was identified in the promoter of one of the TaDof5 co-expressed genes in wheat and subsequent analysis showed that TaDof5 was capable of binding to this promoter region with high affinity. These data suggest that TaDof5 may be involved in photoperiod responses associated with flowering time. TaDof4 was among a large number of previously identified growth-related genes expressed at significantly higher levels in wheat cultivars and progeny lines with high transpiration efficiency (TE) (the amount of biomass produced per unit of water transpired). Field trials were undertaken with the parents and progeny from a cross between Quarrion (high TE) and Genaro 81 (low TE) and demonstrated that the high TE progeny lines had improved early vegetative growth. TaDof4 was therefore characterised further for a role related to biomass production and/or contribution to the TE trait. Expression profiling showed that TaDof4 was consistently expressed at higher levels in the lines with high TE, constitutively expressed in major vegetative organs, drought down-regulated and sucrose up-regulated. Over-expression of TaDof4 identified one line with significantly improved biomass. DNA binding specificity analysis demonstrated that TaDof4 binds to the AAAG(T/C) core target motif essential for Dof DNA binding. These results suggest that TaDof4 is potentially associated with growth-related processes in wheat. In conclusion this study has made the following achievements: (1) identified 31 TaDof family members in Triticum aestivum, (2) used phylogenetic analysis and expression profiling to infer potential functional roles for wheat TaDof family members, (3) further characterised TaDof5 to reveal a potential role in photoperiod responses associated with flowering and elucidated its DNA-binding specificity, (4) further characterised TaDof4 to suggest a potential role in growth-related processes. These results provide fundamental molecular information that increases our understanding of the diverse biological roles of the TaDof family, particularly in growth-related physiological processes in wheat.
| Identifer | oai:union.ndltd.org:ADTP/286522 |
| Creators | Lindsay Shaw |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
Page generated in 0.0023 seconds