Nitrogen (N) plays a vital role in plant life, with nitrate (NO3-) and ammonium (NH4+) as the most common inorganic N compounds. Strigolactones (SLs) are carotenoid-derived phytohormones, acting as both endogenous and exogenous signalling molecules to play multiple roles in regulating plant development in response to various environmental stimuli and in concert with many other regulators. Starting from the hypothesis that nitric oxide (NO), auxin and SLs could take part to complex pathway governing the maize root adaptation to different N availabilities, this PhD work mainly investigated the involvement of SLs in the maize root developmental response to both nitrate and ammonium, thanks to a combined physiological and molecular approach. The research was initially focused on studying the effect of the two different N source on SL exudation and biosynthesis. A LC-MS/MS method was applied to identify and quantify the already known SLs in maize root exudates obtained by seedlings grown with different N availabilities. The results indicated a clear inhibitory effect of nitrate on SL production. The expression of genes encoding key SL biosynthesis and transport components was then measured in roots in response to nitrate, ammonium and N-starvation, and a germination bioassay was also performed. The results further confirmed the presence of SLs in the exudates harvested from N-deprived plants, while a massive inhibition of SL exudation resulted as a specific response to nitrate provision. In situ hybridization (ISH) experiments were then performed both in roots and shoot. The effects of 24 h of N-deficiency, nitrate and ammonium supply in the presence of a SL biosynthesis inhibitor and of a synthetic SL analogue on lateral root (LR) density and primary root (PR) length were then evaluated. The results suggested that the stimulation of LR development might be linked to the complete or partial inhibition of SL production observed in response to nitrate and ammonium, respectively. Since our previous results suggest that SLs and auxin might cooperate to regulate the response of maize primary root to nitrate, the hypothesis that the negative effect of nitrate on SL biosynthesis/exudation could depend on auxin was further studied with a SL quantification in planta, gene expression assessment and lateral root density evaluation. Results obtained are in accordance with our previous results on exudates, thus confirming the role of zealactones production as a clear response to N-deprivation. The expression of the auxin-related genes evidenced peculiar trends, thus allowing to select few of them as good candidates to better characterize and deepen the auxinic action involved in the nitrate signalling. LR density was also assessed in seedlings treated as for gene expression analysis. Our preliminary results suggest that SLs and auxin share overlapping and divergent pathways to regulate maize lateral root development in response to nitrate availability. The maize root response to different N provision was then assessed, trying to outline the different signature between nitrate and ammonium. Accordingly, a root transcriptome analysis was assessed to compare gene expression profiles in maize root apex of seedlings exposed to N-depleted solution or supplied with nitrate or ammonium for 24 h. In addition, physiological evaluation of plant development in response to nitrate and ammonium was also performed. The results provided new insight to better characterize how the early sensing of N-deficiency or nitrate/ammonium provision by root could impact on the overall plant growth and physiology.

The maize root response to nitrogen fluctuations: signalling crosstalk with strigolactones, auxin and transcriptional regulation / Ravazzolo, Laura. - (2019 Oct 31).

The maize root response to nitrogen fluctuations: signalling crosstalk with strigolactones, auxin and transcriptional regulation

Ravazzolo, Laura
2019-10-31

Abstract

Nitrogen (N) plays a vital role in plant life, with nitrate (NO3-) and ammonium (NH4+) as the most common inorganic N compounds. Strigolactones (SLs) are carotenoid-derived phytohormones, acting as both endogenous and exogenous signalling molecules to play multiple roles in regulating plant development in response to various environmental stimuli and in concert with many other regulators. Starting from the hypothesis that nitric oxide (NO), auxin and SLs could take part to complex pathway governing the maize root adaptation to different N availabilities, this PhD work mainly investigated the involvement of SLs in the maize root developmental response to both nitrate and ammonium, thanks to a combined physiological and molecular approach. The research was initially focused on studying the effect of the two different N source on SL exudation and biosynthesis. A LC-MS/MS method was applied to identify and quantify the already known SLs in maize root exudates obtained by seedlings grown with different N availabilities. The results indicated a clear inhibitory effect of nitrate on SL production. The expression of genes encoding key SL biosynthesis and transport components was then measured in roots in response to nitrate, ammonium and N-starvation, and a germination bioassay was also performed. The results further confirmed the presence of SLs in the exudates harvested from N-deprived plants, while a massive inhibition of SL exudation resulted as a specific response to nitrate provision. In situ hybridization (ISH) experiments were then performed both in roots and shoot. The effects of 24 h of N-deficiency, nitrate and ammonium supply in the presence of a SL biosynthesis inhibitor and of a synthetic SL analogue on lateral root (LR) density and primary root (PR) length were then evaluated. The results suggested that the stimulation of LR development might be linked to the complete or partial inhibition of SL production observed in response to nitrate and ammonium, respectively. Since our previous results suggest that SLs and auxin might cooperate to regulate the response of maize primary root to nitrate, the hypothesis that the negative effect of nitrate on SL biosynthesis/exudation could depend on auxin was further studied with a SL quantification in planta, gene expression assessment and lateral root density evaluation. Results obtained are in accordance with our previous results on exudates, thus confirming the role of zealactones production as a clear response to N-deprivation. The expression of the auxin-related genes evidenced peculiar trends, thus allowing to select few of them as good candidates to better characterize and deepen the auxinic action involved in the nitrate signalling. LR density was also assessed in seedlings treated as for gene expression analysis. Our preliminary results suggest that SLs and auxin share overlapping and divergent pathways to regulate maize lateral root development in response to nitrate availability. The maize root response to different N provision was then assessed, trying to outline the different signature between nitrate and ammonium. Accordingly, a root transcriptome analysis was assessed to compare gene expression profiles in maize root apex of seedlings exposed to N-depleted solution or supplied with nitrate or ammonium for 24 h. In addition, physiological evaluation of plant development in response to nitrate and ammonium was also performed. The results provided new insight to better characterize how the early sensing of N-deficiency or nitrate/ammonium provision by root could impact on the overall plant growth and physiology.
maize; root; nitrate; ammonium; strigolactones; auxin; transcriptomic approach
The maize root response to nitrogen fluctuations: signalling crosstalk with strigolactones, auxin and transcriptional regulation / Ravazzolo, Laura. - (2019 Oct 31).
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11577/3422232
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