Janes, George
(2018)
Factors regulating cortex cell file proliferation under low phosphorus stress in Arabidopsis thaliana roots.
PhD thesis, University of Nottingham.
Abstract
Radial patterning of root hair bearing cells (trichoblasts) in Arabidopsis thaliana (arabidopsis) follows the type 3 root hair patterning mechanism whereby the radial positioning of trichoblasts is coordinated according to the position of underlying cortex cells (Pemberton et al. 2001). Epidermal cells which are positioned over the cleft between two underlying cortex cell files adopt trichoblast identity. Low phosphate stress in arabidopsis roots has been show to result in an increase in the number of cortex cell files from 8 to 12-16, which in turn results in an increase in the number of trichoblast position cells (Zhang et al. 2003, Cederholm and Benfey, 2015). However, little is known about what mechanisms control this proliferation in cortex tissue. Zhang et al. (2003) demonstrate that eir1 (an allele of pinformed2, pin2) mutants are deficient in this response to low P. This finding suggests that PIN2 (an efflux facilitator of directional auxin transport) is required to orchestrate proliferation of cortex cell files under low P. This implies that directional auxin transport and, ultimately, auxin response are required to enable proliferative divisions in cortex tissues under low P. First of all, a deeper anatomical understanding of the cortex cell file number response to low P was developed. That showed that the divisions which lead to the increase in cortex cell file number occur in the first cortex cell next to the quiescent centre. It was also found that the anatomical changes only significantly affect the number of cortex cell and trichoblast cell files, suggesting that it is a cortex specific response to increase radial root hair density. It was further discovered that the root is sensitive to up to 400μM P and responds with increased cortex cell file number within 24 hours. I also showed that this response is independent of iron concentration. It was next hypothesised that the role of directional auxin transport implied that other PIN and AUX/LAX genes may be required as well as activating AUXIN RESPONSE FACTORs (ARFs). These experiments revealed that no other PINs or ARFs play a crucial role in this response. However it was found that PIN2:GFP protein changes its subcellular localisation in response to low P, suggesting that changes in auxin flux direction is required. Based on this, it was hypothesised that PIN2 complementation in the cortex in pin2 would rescue the phenotype. This was indeed the case, but also for PIN2 under the AUX1 promoter, suggesting an additional role for PIN2 in the epidermis and root cap during the low P response. Mathematical modelling suggested that auxin flux out of the cortex, mediated by PIN2, would be required for the response. However, results testing this in vivo with the DII:VENUS auxin reporter were inconclusive. It was suspected that ground tissues patterning regulators, BIRD genes, may also play a role in tissue patterning under low P. jkd (jackdaw) and mgp (magpie) mutants did not show any strong phenotype on low P, but expression analysis using reporter lines and reverse transcription qPCR did suggest changes in regulation. It was then hypothesised that BIRD genes and PIN2 may interact, by crossing pin2 and jkd I found that the two interact to a small degree but it is not evident that they interact directly within the same pathway. It was concluded that this response to low P was controlled by a number of regulatory networks which definitely involves PIN2 mediated auxin transport with a contribution from BIRD genes.
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