Study of Cysteine and Cystine Transport in Plant Mitochondria

This dissertation, "Study of Cysteine and Cystine Transport in Plant Mitochondria" by Deepika, Verma, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Cysteine biosynthesis plays an important role in plants for sulfur assimilation. Cysteine regulates the detoxification of cyanide that is a respiratory inhibitor generated mainly during ethylene biosynthesis in higher plants. Cyanide detoxification occurs by the -substitution of the sulfhydryl of cysteine with cyanide catalyzed into β-cyanoalanine by the -cyanoalanine synthase (β-CAS) enzyme in the mitochondria. In plants and bacteria two enzymes catalyze the synthesis of cysteine; serine acetyltransferase (SAT) transfer acetyl- CoA to serine, generating oacetylserine (OAS) which further combines with sulfide ion to form cysteine, the latter reaction is catalyzed by O-acetyl serine sulfhydrylase (OASS). SAT and OASS associate to form cysteine synthase complex (CSC). The isoforms of SAT and OASS have been found in the cytosol, plastids, and mitochondria of several dicotyledonous plants. The function of cysteine synthesis at various sites and its transport within the compartments of the cell is ambiguous. Two type of dicot plant material, wild-type (WT) Arabidopsis thaliana and pear fruits that are distinct in nature, family and origin were selected for this research. Pears are pomaceous fruits that produce a large amount of ethylene during fruit ripening. The selection of fruit material was based on its ethylene measurements. WT Arabidopsis was grown on MS medium and 15-20 days old rosettes were used for the experimental purpose. The mitochondria from both types of plants were isolated and purified using continuous percoll gradient. It was used for respirometry, liquid scintillation counting, and liquid chromatography-tandem mass spectroscopy (LC-MS/MS) assays. The analysis of quality of mitochondria was based on respiratory control ratio, coupling, ADP: O ratio and cytochrome C oxidase latency test. According to oxygraph assays, cyanide inhibits the respiration process, but the application of L-cysteine as low as 5μM in pear and 10μM L-cysteine in Arabidopsis mitochondria can protect the rate of respiration and assist in cyanide detoxification. 2.5μM cystine also shows the similar effect as tested for pear mitochondria. Cyanide concentration required for total inhibition of respiration range between 10 μM to 100 μM. Amino-oxyacetic acid assay suggested that β-cyanoalanine synthase signals the mitochondrial transport of cysteine and cystine. Dithiothreitol (DTT) reduction and Nethylmaleimide (NEM) assay inferred that cysteine is a preferred substrate for cyanide detoxification. Respirometry analysis shows that L-glutamic acid competes with both cysteine and cystine, and aspartic acid interferes with transportation of cystine more than that of cysteine. The specific inhibitors of Xag transport system (AβH, i.e., aspartic-β-hydroxamate) and Xc system (quisqualic acid, S-4-Carboxyphenylglycine (CPG)) inhibit the cysteine and cystine transport in pears. These inhibitors were further tested in liquid scintillation counting by using Lcysteine S DEGREES35 radioisotope and LC-MS/MS experiments by estimating β-cyanoalanine synthesis. The inhibitory effect of chemical inhibitors on cysteine import was found partial for pear and minimal in Arabidopsis mitochondria. But the results obtained from glutamic acid competition in respiratory experiments correspond well with the competitive assays carried out for liquid scintillation counting and LC-MS/MS. This analysis supports the hypothesis that the cysteine is impo