Radiotracers for examining biological functions of plants and microbes

Abstract

Tracers are used for qualitative and quantitative investigation of a system. Radiotracers have a radionuclide to observe chemical or biological processes by detection of the radionuclide's decay energy. They are non-disruptive and non-destructive to living systems and can be quantified, imaged, and measured in real time, adding value. This work focuses on radiochemistry and radiotracer techniques to understand maize uptake and localization of micronutrients and the impact of Azospirillum brasilense microbial interactions on these processes. Further, it explored how such interactions can influence stress responses in maize. Finally, it examined how the natural biological functions of A. brasilense bacteria respond to light stimulus conducted through the plant tissues. In this dissertation, the efficacy of using 4-fluorophenylboronic acid (FPBA) as a boton (B) imaging agent, which is a derivative of the B deficiency mimic phenylboronic acid (PBA), was explored. It is shown that radioactively labelled [18F]FPBA (t [subscript 1/2] = 110 m) accumulates at the root tip, the root elongation zone and at lateral root initiation sites in maize roots, and also translocates to the shoot where it accumulates along leaf edges. This is the first time a radiotracer has been utilized to image B in plant systems. Nutritional iron (Fe) content was explored in Azospirillum brasilense associated maize. 59Fe (t [subscript 1/2] = 44.5 d) was used to trace iron uptake kinetics and allocation to leaf. In the presence of functional mutants of this bacteria, iron uptake and allocation to leaf was enhanced in maize seedlings. Maize were grown to maturity and plants associated with the bacteria had greater crop yield (kernels cob-1) and enhanced iron and protein ferritin- the bioavailable form of iron to humans- seed content. Similar studies were completed using zinc ([superscript 65]Zn, t[subscript 1/2]= 244 d), where it was noted that the presence of the low-auxin producing and nitrogen-fixing bacteria strain, ipdC, enhanced zinc uptake but had no ncement effect on allocation or zinc seed filling. Carbon metabolism in response to stresses and microbial interaction was also investigated in maize with [11C]CO2 (t [subscript 1/2] = 20.4 m) radiotracer. In association with A. brasilense, maize fixed more carbon dioxide, allocated more 11C-photosynthates to the roots, and produced more 11C-exudates than control maize. Metabolic differences were studied via radio-HPLC and radio-TLC to reveal association enhanced 11C flow into hydrophobic structural components and amino acids. When nitrogen stressed, non-inoculated maize exhibited a decrease in carbon dioxide fixation, root allocation of 11C-photosynthates, and decreased 11Cexudation compared to control maize. They also saw increased 11C flow into hydrophobic structural components and sugars. When inoculated with A. brasilense and subjected to nitrogen stress, the same enhancements occurred- but fixation, allocation, and exudation recovered to near control maize levels, suggesting these bacteria ameliorate some abiotic stresses. Finally, 59Fe and [11C]CO2 radiotracers were applied to the functional mutants of A. brasilense to uncover how various biological functions were impacted by light exposure. First, light transmittance from shoot to root tissues, called light piping, in maize was shown using a DSLR camera and image intensifier. Studies showed the functional mutants with biological nitrogen fixation (BNF) capacity had enhanced assimilation of 59Fe when exposed to light relative to dark treatments and greater activity of the nitrogenase enzyme as measured by acetylene reduction assay in light, with a greater response noted for red than blue light wavelengths. Carbon assimilation as [11C]CO2 and subsequent metabolism in these bacteria were also impacted by light stimulus.