We have previously identified the tissue expression patterns of ZmNPF6;6 and ZmNPF6;4 in maize root tips (Wen, Z., et al., The Plant Cell, 2017) and used qPCR as well as custom array cDNA chips to profile gene expression linked to N transport and assimilation in maize roots (Dechorgnat, J., et al., Front Plant Sci, 2018, Dechorgnat, J., et al., BMC Plant Biol., 2019). Although indicative of tissue and / or membrane location, we still know little regarding the transcriptional control of these genes and how tissue type (i.e. crown versus seminal root) changes due to nutritional or developmental cues. A recent study of N associated genes in Arabidopsis roots had used a yeast one-hybrid approach to interrogate known root expressed TFs to define N assimilation regulatory networks . This resource is now available to verify if similar networks exist in agricultural crops and how nitrogen metabolic networks are linked to nutrient-dependent root development processes.
This project will investigate transcriptional regulation of selected transport pathways (NPF6, NRT2, AMT) for evidence of tissue-dependent regulatory controls that define root type and function. The project will dissect the 5’-upstream regions of selected promoter sequences for conserved control motifs and the identification of TFs which guide activity in a root type-dependent manner. A traditional yeast 1-hybrid technology will be employed using libraries developed from primary, crown and seminal root cDNA libraries using contrasting Maize lines (F44 and B73). Each library will be tested against ~2.0 KB promoter sequences amplified from genomic DNA. Root cDNA libraries will developed from nitrate or ammonium grown primary, crown and seminal Maize roots and cloned directionally into a yeast 1-hybrid cassette. Prey cDNA libraries will be tested against co-transformed bait promoter constructs for potential interacting proteins that bind. Identified cDNAs will be sequenced, verified for binding specificity and then analysed for expression activity across root tissue profiles using qPCR. We will analyse the data in the context of identified conserved genes and networks previously identified in Arabidopsis (Gaudinier, A., et al., Nature, 2018. 563(7730). To verify tissue localization, each complete promoter will be subcloned into a T-DNA vector upstream of a GUS reporter gene or in front of a gene:GFP C-terminal fusion construct. Transgenic lines (minimum 10 independent lines) will be developed in collaboration with Dr Kanwarpal Dhugga at CIMMYT (Mexico) using the particle bombardment technique utilizing Corteva Agriscience’s technologies for rapid transformation of maize embryos. T1 seed will be imported into Australia for growth and analysis using a PC2/QC2 certified glasshouse and PC2 laboratory and microscopy containment facility in Camden NSW. Seeds released from quarantine will be evaluated for gene expression (GUS staining of promoter fusions) and intercellular localization (GFP signal via confocal microscopy). To clarify whether transport pathways and root changes observed in F44 and B73 maize lines are regulated by CLE and CEP signalling peptides, the PhD applicant will work with Dr Nijat Imin (University of Auckland) to conduct experiments that profile nitrogen uptake, root development with the expression of peptide coding genes. This will be combined with transcriptional analyses of transport pathways and identified TFs that are challenged with synthetic peptides to delineate the underlying regulatory network.
Additional supervisor for this project is Prof Dabing Zhang (University of Adelaide), Dr Nijat Imin (University of Auckland and Dr Kanwarpal Dhugga. This project will be located in Camden Campus (Centre for Carbon Water and Food).
1) Develop primary, seminal and crown root cDNA libraries for use in yeast one-hybrid assays to identify interacting proteins to differentially expressed nutrient transporters in roots.
2) Development of B73 and F44 lines with NPF6 and AMT1 promoter GUS and promoter:geneGFP constructs for analysis of expression patterns and intercellular expression profiles in roots.
3) Verify the role of peptide signaling networks in regulating root development in response to N supply
HDR Inherent Requirements
In addition to the academic requirements set out in the Science Postgraduate Handbook, you may be required to satisfy a number of inherent requirements to complete this degree. Example of inherent requirement may include:
The opportunity ID for this research opportunity is 2920