The case for improving Phosphorus-Use Efficiency in crops is widely recognised. Although much is known about the underlying molecular and regulatory mechanisms, improvements have been hampered by the extreme complexity of phosphorus (P) dynamics in soil and plants (across all physical scales), including its soil chemistry and uptake, distribution and deficiency responses in plants. The urgency and direction of phosphate research is also being driven by the availability of finite P stocks to farmers and reducing environmental hazards. Thus, systems approaches become essential to identify the most potent (combinations of) target genes for improving phosphate uptake and utilisation in crops. This study has applied these approaches with the aim of increasing understanding of the regulation of phosphate uptake at three separate spatial scales, primarily in rice, but also in Arabidopsis. The first and major part of this study has focused on the cell scale, wherein novel mathematical models for molecular regulation of phosphate acquisition have been developed. Owing to the sparsity of the data, advanced techniques for parameter fitting were employed, which resulted in an original model, which accurately reflected the profiles of all the genes apart from PHO2. It was clear that miR399-mediated degradation was insufficient to explain the apparent early reduction in PHO2 mRNA levels. Five hypotheses were explored mathematically, of which the most plausible is that there is a phosphate-sensitive transcriptional repressor (PsTR) of PHO2 mRNA synthesis. To support this hypothesis, mRNA was extracted from phosphate-starved and untreated roots over a short, 12-hour time course. Quantitative Polymerase Chain Reactions (qPCR) of PHO2 mRNA both confirmed the early decline predicted by the hypothesis and also revealed a temporary restoration of mRNA levels, which points to PHO2 (a type-2 ubiquitin ligase) regulating its own transcript levels. Sensitivity analysis of these models indicates that the utilisation rate of cytosolic phosphate is the biggest influence on this system. Output from simulations with the original and PsTR models qualitatively reproduced the phenotypes of various published phosphate-research papers, with the exception of RNASEQ data in which phosphate-starved rice roots were resupplied with phosphate. In this instance, the observed rapid drop in mRNA levels for PHO2 and IPS1 were incorrectly predicted, pointing to one or more other regulatory mechanisms not represented in these models. The IPS1 gene encodes a long non-coding RNA that has a poly-A tail. Its RNA also binds to miR399 and accumulates to extremely high levels in plant roots during phosphate stress. A sudden loss of IPS1 would release the bound miR399 causing the observed rapid loss of PHO2 mRNA. The observed IPS1 profile can be explained by either the gene having a “super-promotor” that is capable of extremely high RNA synthesis under low phosphate conditions, or the transcript being protected from degradation by phosphate-sensitive RNA-binding proteins. Informatics analyses favour the latter and a revised model incorporating RNA protection was found to have parameters for IPS1 synthesis that are similar to those normally used in modelling gene regulation. The analyses also point to PUMILIO proteins playing this role. The second part of this work has explored the role of tissue geometry in determining root phosphate levels and flux. Multi-cellular vertex-based models of published Arabidopsis and rice root cross-sections were produced using CellSeT, into which equations for phosphate uptake, flux and utilistion were embedded using OpenAlea. Simulations suggest that Arabidopsis trichoblasts have lower cytosolic phosphate levels than neighbouring epidermal cells, because they have a larger area through which phosphate flows into the inner tissues. This implies that trichoblasts are more sensitive to phosphate stress and reduced phosphate levels could therefore be part of the trigger for initiating root-hair growth. Adding root hairs of varying lengths into this geometry shows that a hair does not have to grow much before the phosphate levels in this trichoblast exceeds those in the neighbouring cells and that phosphate flows to them. This potentially suppresses root-hair formation in nearby trichoblasts. The rice simulations show that aerenchyma dramatically reduces cytosolic phosphate in surrounding cells and point to a role for lacunae in rapid uptake of phosphate, without the need for large water fluxes. Alongside aerenchyma, a higher proportion of fluid-filled lacunae could be a desirable trait for improving nutrient-uptake efficiency. At the whole-plant scale for the third part of this work, a time-course dataset has been generated to record the effect of phosphate starvation over 21 days on the uptake dynamics of eleven other macro- and micro-nutrients. This dataset will be of use in future systems studies of nutrient uptake and interactions.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:689803 |
| Date | January 2016 |
| Creators | Ajmera, Ishan |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://eprints.nottingham.ac.uk/32470/ |
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