Study On The Interaction Of Light Rare Earths And Rice Mitochondria And Its Mechanism

China enjoys the most abundant resource in rare earth elements. The wide-spread use of these elements would cause inevitable intake by biological systems via absorption as micro-fertiliser, feed additives, exogenous growth hormones, etc. The downstream effects of intake of rare earth elements will render natural food chain contamination, which is the main security issue people have to take into account when utilising these elements. Besides, China is a country that cultivats on rice. Its average rice yield can be increased by8%to20%after application of rare earth elements-containing fertiliser, which might generate decent social and economic effects.At present, understanding of the biological effects of rare earth elements-containing plants has been established on different levels. Despite the huge amount of work and large number of researchers involved in this area, a complete elucidation of the interacting mechanisms between rare earth elements and plant cells and organelles is still on the horizon due to the complexity of plant systems. Among those mechanisms, the well-known Hormesis effect caused by rare earth elements is yet to be clarified. On the other hand, among plant organelles mitochondria is the major concern because of its role in energy generation and in planta metabolism as well as a targeting organelle for toxins. Regarding rice cells, gene clusters of rice mitochondria undergo frequent rearrangement, leading to chimeric gene groups hence infertile male plastid. Given the above information, this thesis aims at applying microcalorimetry, closed chamber respirometry, microscopy and spectroscopy to systematically study the interaction between rice mitochondria and four light rare earth elements as Lanthanum(La), Cerium(Ce), Praseodymium(Pr) and Neodymium(Nd) etc. Based on the experiment results, this paper also tries for the first time to elucidate the underlying mechanism from the perspective of mitochondrial membrane permeability transition (MPT). The author believes that the results will help to build up new theoretical understandings for securing rare earth elements utilisation and high quality rice production.This paper is divided into six parts:In chapter1, we described the biological effects and the putative mechanisms of rare earth elements in planta, structure and functions of plant mitochondria, and the application of microcalorimetry in researches within life sciences. It also presented topic ideas as well as innovative ideas.In chapter2, the bio-chemical effects of lanthanum cation on rice mitochondria was studied. First of all, microcalorimetric experiments were carried out on the influence of La(III) on in vitro metabolism of rice mitochondria. According to the results, La(III) presented obvious Hormesis effect in rice mitochondrial metabolism. Furthermore, experiments were done to investigate the effect of La(III) on mitochondrial MPT. Higher concentrations of La(?) could induce mitochondrial MPT while low concentrations have on effect. Two MPT protecting reagents Cyclosporin A (CsA) and dithiothreitol (DTT) were introduced to characterise the interaction mechanism between high concentration of La(III) and rice mitochondria. It is found out that La(III) could induce CsA-sensitive mitochondrial MPT via either a similar pathway to that of Ca2+or interacting with the thiol-containing protein on mitochondria. Apart from that, La(III) presented in vivo Hormesis effect on the growth of rice seedlings. Compared to control group, La(?)-cultivated breeding group showed higher concentration of La(?) localised in mitochondria. Further investigation into the root apical mitochondrial structure gave the conclusion that treatment of low concentrations of La(?) maintained the entire structure of mitochondria whereas high concentrations of La(III) damaged mitochondrial structure to some extent.In chapter3, the biological effects of cerium cation on rice mitochondria were discussed. First of all, microcalorimetric experiments were carried out on the influence of Ce(?) on metabolism of Xiangzaoxian rice mitochondria in vitro. Different thermogenic curves have been obtained for interaction between various concentrations of Ce(?) and rice mitochondria. Specifically, rice mitochondrial metabolism has been induced under low concentrations of Ce(?)(0?400u?M) whereas inhibited under higher concentrations of Ce(?)(600?1800?M). Apart from microcalorimetry, closed chamber respirometry has been carried out in order to characterise the effect of Ce(?) on mitochondrial State4oxygen consumption. It showed that oxygen consumption rate rose with increasing concentrations of cerium under low concentration regime of Ce(?) while respiration rate decreased with increasing concentration of Ce(?) under higher concentration regime. The results suggested that Ce(?) had induced apparent Hormesis effect on rice mitochondrial respiration and metabolism. In addition, this thesis demonstrated effect on rice mitochondrial structure and functions in presence of Ce(?). Spectrometry and microscopy have been conducted in order to characterise the structural change of rice mitochondria. To be more specific, higher concentrations of Ce(?) induced mitochondrial membrane permeability transition (MPT). Nevertheless, low concentrations barely had any observations. A conclusion that Ce(?) might interact with thiol group and result in rice mitochondrial MPT has been drawn based on the experiments of higher concentrations of Ce(?) with rice mitochondria at presence of three protecting reagents.In chapter4, the biological effects of Praseodymium cation on rice mitochondria was discussed. Isothermal microcalorimeter TAM ? was applied to obtain thermogenic curves of in vitro metabolism of rice mitochondria at the presence of different Pr(?) concentration. The results indicated that Pr(?) could cause Hormesis effect (stimulation under low concentrations whereas inhibition under higher concentrations). UV-visible spectrophotometry and fluorometry were used to study the effect of Pr(?) on rice mitochondrial membrane permeability transition. It is demonstrated that higher concentrations of Pr(?) may induce MPT and the MPT was controllable. Together with rice mitochondrial cytochrome c release, these results suggest that higher concentrations of Pr(?) render mitochondrial dysfunction via MPT. Besides, the effects of different Pr(?) concentrations were also studied on mitochondrial inner membrane H+and K+permeability and mitochondrial membrane lipid peroxidation. It is depicted that Pr(?) could inhibit mitochondrial H+, K+permeability and lipid peroxidation, indicating that Pr(?) can, to some extent, protect mitochondrial oxidative stress and that Pr(?) cause mitochondrial dysfunction via MPT has no relation to oxidative stress.In chapter5, the study was focused on the bio-chemical effects of Neodymium cation on rice mitchondria. Isothermal microcalorimeter TAM ? was applied to obtain therogenic curves of in vitro metabolism of rice mitochondria at presence of different Nd(?) concentration. Specifically, low concentration of Nd(?) stimulated rice mitochondrial metabolism while higher concentration inhibited it. Additionally, Clark electrode was introduced to determine the effect of Nd(?) on rice mitochondrial State4respiration rate. The above two methods indicate that Nd (?)'s influence also obeys Hormesis effect, i.e. stimulation under low concentrations while inhibition under higher concentrations. UV-visible spectrophotometry and fluorometry, together with tunnelling electron microscopy have been used to study the effect of Nd(III) on rice mitochondrial MPT. The release of cytochrome c was also determined. All these pointed to the implication that higher concentrations of Nd(III) could induce rice mitochondrial MPT, leading to structural and functional damage. In addition, the effects of Nd(?) were also studied on mitochondrial inner membrane H+and K+permeability, mitochondrial membrane lipid peroxidation and release of reactive oxygen species (ROS). It is found out that Nd(?) plays a protecting role in mitochondrial oxidative stress, implying that Nd(?)-induced MPT has another responsive pathway rather than oxidative stress. The exact mechanism remains to be elucidated.In chapter6, we summarized previous chapters and outlines the work that has been done. Some further research direction were also presented hereafter.