Discussion
In this study, the enzymatic activity of SDH in B. oleracea was evaluated using the indirect measurement of DCIP concentration by qualitative spectrophotometric assay. DCIP was used as an artificial electron acceptor, which picked up electrons from the reduced succinate, as the ETC was inhibited by the addition of sodium azide. The activity of SDH was assayed in the presence of malonate, a known competitive inhibitor of SDH. Malonate is known to cause an increase in ROS production (Fernandez-Gomez et al., 2004). The objectives of this study were to analyze the rate at which SDH oxidizes succinate, and the impacts of malonate on the rate of SDH activity. The major results noted were the slight decrease in DCIP concentration in the MFF, and the sharp decreases in the MF and MF-M samples. The DCIP concentration decreased in the MFF with an overall rate of 0.015 µM/minute. This unexpected decrease may be a result of the inherent inconsistencies in differential centrifugation therefore some mitochondria may have remained in the sample. As expected, the MF samples showed a strong decrease in DCIP concentration occurred with a maximum rate of 0.13 µM/minute. Surprisingly, the MF-M samples showed the most rapid and significant decrease in DCIP concentration with a maximum rate of 0.22 µM/minute. This result may have occurred due to the mitochondrial membrane collapse caused by the addition of malonate. The addition of malonate was expected to cause little to no change in DCIP concentration because malonate is a competitive inhibitor of SDH.
Other relevant research in this field focused similarly on SDH, yet they alternatively looked at the production of ROS and the effects of malonate on mitochondrial membrane potential. In their research, Fernandez-Gomez et al. (2004) evaluated the effects of malonate as a competitive inhibitor of complex II (SDH) using a Percoll gradient to isolate mitochondria from the brain of Sprague-Dawley rats, and autofluorescence to detect NAD(P)H as a quantification of SDH activity instead of DCIP. Fernandez-Gomez et al. (2004) measured cell viability to determine the effects of malonate through quantification of lactate dehydrogenase activity. It was concluded that malonate caused a mitochondrial membrane potential collapse as shown by the release of tetramethylrhodamine ethyl ester, a membrame-permeant fluorescent probe in a concentration-dependent manner of malonate (Fernandez-Gomez et al., 2004). The results of Fernandez-Gomez et al. (2004) indicated the relation between the addition of malonate and a mitochondrial membrane collapse due to the rapid release of ROS. Our study demonstrated similar results indicated by the rapid decrease in DCIP concentration in the MF-M samples. Our results demonstrate that malonate causes a mitochondrial membrane potential collapse implied by the rapid reduction of DCIP concentration following the addition of malonate. The methods used in the study conducted by Fernandez-Gomez et al. (2004) were more far more complex and precise than the methods used to conduct our study, although the results of our study appear to be conclusive with their results. While they did use an animal model, mitochondria function is generally conserved between eukaryotic organisms, although it may have affected the ability to compare the results of our study to theirs.
In the research conducted by Jardim-Messeder et al. (2015), the production of ROS by SDH was analyzed in Arabidopsis thaliana and Oryza sativa, and the effects of malonate on ROS production were studied. ROS production was assessed using the effects of competitive and noncompetitive inhibitors of SDH. Quantification of SDH activity was also measured using DCIP and spectrophotometric measurements, although they utilized the molar absorption coefficient of reduced DCIP to calculate the rate of SDH activity rather than using a standard curve of DCIP as used in our study. The plant species utilized in their study may have affected the results due to the presence of chlorophyll in the cells, which may have caused disproportionate data dependent on which region of the plant was used. Jardim-Messeder et al. (2015) also used several competitive and noncompetitive inhibitors of SDH, including malonate to conduct their study while we only used malonate. The results of our study provide conclusive results that are in agreement with the results of Jardim-Messeder et al. (2015), due to the observed decrease in DCIP concentration in the MF samples. From their results, Jardim-Messeder et al. (2015) concluded that SDH is a site of ROS production. The implications of ROS production by SDH on the regulation of plant growth and stress responses may be significant. The results of their study indicated that the inhibition of SDH may cause some decreases in plant growth.
The results of our study may have been effected by several factors, such as the temperature at which our experiment was conducted, the concentration of mitochondria in the MFF and MF samples, and the health of the cells collected from B. oleracea. The temperature of our experiment was not carefully regulated as it was conducted on ice, thus some cellular degradation may have occurred due to a higher temperature than optimal for the B. oleracea. The concentrations of mitochondria present in the fractions may not been absolutely ideal, due to the inherent impurities of differential centrifugation, thus causing the slight decrease in DCIP concentration that occurred in the MFF samples due to some residual mitochondria in the MFF. The viability of the B. oleracea cells was not calculated before testing which have had some effect on the observed impact of malonate, as healthy cells may not have been affected as greatly by the addition of malonate due to a greater abundance of antioxidant systems such as FAD.
In this study, the activity of SDH was analyzed in varying sample conditions of MFF, MF and MF-M to assess the rate of SDH through the reduction of succinate to fumarate. The MF-M sample was used to test the effects of malonate on SDH activity as a competitive inhibitor. The results collected demonstrate that malonate causes a mitochondrial membrane potential collapse indicated by the rapid decrease in DCIP concentration from the release of ROS. The DCIP concentration in the MF-M fraction decreased overall at a rate of 0.089 µM/minute with the most drastic decrease occurring between time zero minutes and five minutes with a rate of 0.22 µM/minute. The MF samples showed a significant decrease as well with an overall and maximum rate of 0.13 µM/minute and 0.073 µM/minute, respectively, this decrease was not as rapid as the one observed in the MF-M. A slight decrease in DCIP concentration occurred in the MFF, decreasing with an overall rate of 0.015 µM/minute. The inhibitory effects of malonate on SDH could be further studied using varying concentrations to assess when it becomes toxic to the cell. The varying concentrations of malonate would allow further understanding of malonate concentration decreases SDH activity and the correlation between the two. An understanding of the prevalence of malonate in plant cells is currently lacking and could contribute to the research on regulation of plant growth and responses to environmental stress. Research into how the rate of SDH activity is determined by health of cells may be useful in determining the optimal rates of ROS production and cellular respiration in healthy cells.