The effect of nitrogen status and carbon remobilisation on nitrate assimilation and associated cytokinin signalling in shoots and roots of Lolium perenne.
Thesis DisciplineCellular and Molecular Biology
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
The efficiency of nitrogen (N) assimilation is a critical component of N fertiliser use by plants, regrowth following defoliation and forage production in Lolium perenne L., an important pasture species worldwide. The aim of this study was to gain insight into the effect of modifying the internal carbon (C)/N balance on the efficiency of nitrate (NO3-) assimilation and to determine the relevance of the cytokinins to this process.
The physiological and biochemical responses to perturbation of the internal C/N balance by N treatment to plants grown for 10 weeks in unfertilised soil, defoliation, and exposure to contrasting day length and N supply, were characterised over a seven-day period in the leaf, sheath and root of perennial ryegrass. A spatiotemporal analysis of NO3- use efficiency (NUE) was described in terms of growth response, remobilisation of the water-soluble carbohydrates (WSCs) across their degrees of polymerisation, amino acid profile, together with components of cytokinin signal transduction. The expression of the cytokinin response regulator LpRR genes was monitored by RT-qPCR.
The initial response to high levels (5 mM) of KNO3 addition to N-impoverished plants was characterised by a rapid remobilisation of carbon stores from the low-molecular weight WSC, along with an increase in N content and assimilation into free amino acids. Subsequently, the shoot became the main source of C through remobilisation of a large pool of high-molecular weight WSC. Variations in the cytokinin content and LpRR gene expression suggest long- distance root/shoot signalling within a day of KNO3 treatment, and ongoing input from cytokinin over seven days. The expectation that perennial ryegrass possesses a cytokinin- mediated N-signalling/relay system similar to that characterised in model plant species Arabidopsis thaliana was confirmed. The presence of high levels of cis-zeatin-type cytokinins was considered in the context of hormonal homeostasis under the stress of steady- state N-deficiency.
Plants, manipulated to have contrasting internal C/N balance, were obtained by exposure to either continuous light or short days (8 h light: 16 h dark), and watered with modified N-free Hoagland medium containing either high (5 mM) or low (50 µM) NO3- as sole N source. An increase in C demand was experimentally induced by a defoliation of perennial ryegrass plants. Leaf regrowth following defoliation involved remobilisation of the low- and high- molecular weight WSCs. The extent of the remobilisation and the partitioning of the WSC following defoliation were dependant on the initial WSC levels and on the C and N availability to the plant. Cytokinin levels varied in the sheath and root as early as 8 h following defoliation and preceded an overall increase in amino acids at 24 h. Within 168 h after cutting, a negative feedback brought the amino acid response back towards pre- defoliation levels, and was under control of the C/N ratio. Therefore, it is suggested that WSC remobilisation in the leaf is coordinated with N availability to the root via systemic cytokinin signal leading to efficient N assimilation in the leaf and in the sheath tissues and to early leaf regrowth following defoliation.
The effect of exogenous application of a cytokinin-like compound, CPPU, was tested as a potential signal “priming” the remobilisation of WSCs reserves required for NO3- assimilation. Foliar spray of CPPU did not increase the physiological response of perennial ryegrass plants to N treatment and defoliation, as measured by biomass of regrown leaf material, tiller and leaf number, leaf elongation rate, maximum photosynthetic rate or stomatal conductance. Hydroponic treatment with a cytokinin receptor antagonist, referred to as PI-55, influenced the activation of the cytokinin signalling pathway but without physiological consequences.
Based on the results obtained in the present study, the development of a cytokinin-based N fertiliser would not be expected to improve the NUE of perennial ryegrass. However, based on the effect of N treatment and defoliation on the remobilisation of the WSCs, a recommendation is made that a grazing event and N fertiliser application should be separated in time by several days to allow a recovery of some of the WSC reserves.