The role of mitochondria in three CAM species

dehydrogenase in K. daigremontiana mitochondria. For this reason, it is possible that in the cooperative oxidation of NADH and NADPH, only NADH was faster oxidized, and NADPH was not, leading to the total respiration rate in these cooperative oxidation was not higher than that of individual NADH oxidation. Further experiments must be carried out to define the mechanism of competition during cooperative NADH and NADPH oxidation in K. daigremontiana mitochondria.

6.2.5. Effect of low oxygen concentration on mitochondrial substrate oxidations

interaction between mitochondria, cytosol and other organelles, which involves the transfer of NAD(P)H reducing equivalent across the inner mitochondrial membrane (Raghavendra et al., 1994; Krömer, 1995; Pastore et al., 2003). Our study also showed that during CAM phase III, substrate metabolism in mitochondria related to the cytosol and chloroplast among three investigated CAM species.

Actually during CAM phase III, malate decarboxylation in cytosol of two Kalanchoë species mainly via cytosolic NAD(P)-ME would release NAD(P)H, while malate oxidation in cytosol of A. comosus mainly via cytosolic NAD-MDH to produce OAA. Under these conditions, NADPH concentration can increase in cytosol of two Kalanchoë but not with cytosol of A. comosus. Relatively, we found that mitochondria of two Kalanchoë species easily oxidized NAD(P)H while A. comosus mitochondria only oxidized NADPH in the presences of high Ca²⁺. The characteristic may also relate to required ATP during CAM phase III in cytosol of two Kalanchoë species because that pyruvate orthophosphate dikinase (PPDK) was also accumulated in cytosol of K.

daigremontiana and K. pinnata mesophyll cells (Kondo et al., 1998). It is unclear that if the role of cytosolic PPDK was similarly as chloroplast PPDK, which metabolism would support enough the energy source for their activity. In this study, we found that mitochondria of Kalanchoë species easily oxidized external NADH and NADPH at significant rates (Table 2.2 and Table 3.3), suggesting that cytosolic NAD(P)H oxidation by external NAD(P)H dehydrogenases in mitochondria of Kalanchoë species might supply energy for cytosolic PPDK activity. Furthermore in these species, mitochondrial malate decarboxylation mainly via ME produced pyruvate and CO2. Pyruvate was further converted in TCA cycle or was exported out of mitochondria. The exported pyruvate could directly phosphorylate to PEP by cytosol PPDK or could transport into

the chloroplasts and further phosphorylate to PEP by chloroplasts PPDK (Fig. 6B).

In other hand, the occurrence of very high MDH in both the cytosol and mitochondria and the mitochondrial permeability to both malate and OAA allowed the operation of malate-OAA shuttle in A. comosus. As discussed in chapter 4, this shuttle might operate as a supporting system for the mitochondrion and the cytosol in controlling and regulating malate metabolism in order to supply OAA for PCK activity during the decarboxylation phase of the PCK-CAM plant. In other words, the MDH on either side of the mitochondrial membrane are linked by this shuttle in the daytime conversion of malate to OAA during the decarboxylation phase. In addition, it seems that A. comosus mitochondria not only support ATP for cytosolic PCK activity, but also contribute in supplying the substrate OAA for PCK activity of the decarboxylation phase during the day and for Asp synthesis in the cytosol (Fig. 6A).

Plants grow using light energy to photosynthetically convert atmospheric CO2

into carbon-rich compounds in the chloroplasts. These compounds are then respired in the cytosol and mitochondria to generate the energy and carbon intermediates necessary for biosynthesis (Hoefnagel et al., 1998). This study suggests that CO2 produced during malate metabolites in both cytosolic and mitochondria of three CAM species could also import into chloroplast to further convert for starch synthesis (Fig. 6A and 6B).

It has been indicated that malate release from the vacuole during the day must be close geared to metabolism in CAM plant so that it matches the rate of malate decarboxylation in the cytoplasm and the flux carbon in gluconeogenesis. To operate in phase III of CAM, this gluconeogenic pathway requires ATP (Edward et al., 1982). Our study suggest that mitochondria of three CAM species may contribute their roles not only in supplying the energy (ATP) and NAD(P)H reducing equivalents but also in

supplying pyruvate, OAA as carbon skeletons for metabolite exchange in their cytosol and chloroplast during CAM Phase III (Fig. 6A and 6B).

This study showed that the oxidations of two substrates increased respiration rate and the Alt respiration while the reduction of oxygen concentrations caused a decrease the respiratory property and the Alt respiration in mitochondria of three CAM species. The details of these phenomenons and their role were discussed in chapter 3 and chapter 5, however it required to further study in order to find our the physiological functions of these phenomenons during phase III of these CAM species. Other interesting finding in this study was that external NADH oxidation in A. comosus mitochondria was more engaged on Cyt pathway than on Alt pathway and it could be produce much more ATP than two Kalanchoë species. By this metabolism, A. comosus mitochondria could produce enough ATP for cytosolic PCK activity in daytime.

Future Prospects

Our study contributes to draw a different picture of respiratory property in mitochondria isolated from two Kalanchoë species and A. comosus, especially their malate metabolite in both cytosol and mitochondria. Furthermore, the results in this study indicated that the interactions among the mitochondria, cytosol and chloroplasts in three investigated CAM species. Although main aspects of the characteristics and functions of mitochondria in these species have clearly identified, some remain questions during mitochondrial substrate oxidations are expanded, becoming worth topic for future study

At first, the results in chapter 2 and 3 indicated that during CAM Phase III, the respiration in mitochondria of two Kalanchoë species may support enough ATP for cytosolic PPDK activity. Nevertheless, the physiological role and activity of PPDK in cytosol in two these ME-CAM are unknown and these questions are necessary for future study.

In our study, the NADPH oxidation in mitochondria of two Kalanchoë species was rather differed from that of A. comosus mitochondria. The NADPH oxidation in A.

comosus mitochondria more depended on Ca²⁺ more than that of two Kalanchoë species.

This result may be due to the activity of NADPH dehydrogenase in A. comosus mitochondria required more Ca²⁺ than that of two Kalanchoë species. However, the relationship between Ca²⁺ and activity of NADPH dehydrogenase as well as the role of Ca²⁺ in mitochondrial NADPH oxidations of these CAM species apparently need or are remain to be elucidated.

The malate metabolisms in mitochondria of three investigated CAM species are clearly identified in this study, but the metabolites to be produced by these reactions

such as OAA or pyruvate and the contribution of these products to another metabolism in cytosol or chloroplast were not investigted in this study. Actually, the question that OAA produced during malate oxidation could be converted into TCA cycle or contribute their role in amino acid synthesis in the matrix of A. comosus mitochondria need to be elucidated.

Mitochondrial ETC of three investigated CAM species were connected to Alt and Cyt pathways, and they were not saturated with single substrate. However, the metabolism to control these processes in mitochondria of these CAM species is still not fully understood. This point and the physiological role for the Alt pathway in phase III metabolism should study to fulfill the role of Alt respiration during malate decarboxylation in phase III of these CAM species.

The OAA has been found to rapidly traverse the inner membrane of all the plants mitochondria studied so far. However for the present, the details of OAA transport in plant mitochondria remain unknown, but it is thought that the carrier is specific for OAA. In fact, the locatization of MDH in both cytosol and mitochondria, and the mitochondrial permeability to both malate and OAA could allow the operation of the malate-OAA shuttle in most of the plants mitochondria (Pastore et al., 2003, Hanning et al., 1999). Although Day and Wiskich (1984) indicated that in pea leaf mitochondria, the malate efflux and OAA influx occur on separate carriers, and Zoglowek et al. (1988) have provide evident that a malate-OAA shuttle across the inner mitochondrial membrane is catalyzed by an electrogenic uniport of malate and of OAA linked to a counter exchange, the question whether both malate and OAA are transported by a single transport protein or by two different ones cannot be answered at present (Douce and Neuburger, 1997). This study showed firstly for CAM plants that

the existence of malate-OAA shuttle in A. comosus mitochondrial membrane and by using this shuttle malate and OAA could be easily import in and export out the inner membrane. However, the details for regulation of this shuttle and the carriers and details of transport of OAA and malate in A. comosus are unknown, and these problems are required to study in the future.

The mitochondrial malate metabolism and the malate-OAA shuttle in A.

comosus are very interesting. These findings are complete new and it is the first time to be detected in A. comosus - one typical PCK-CAM species. Especially these finding indicated that the malate metabolism via the shuttle in A. comosus mitochondria plays the suitable roles for total malate metabolism during phase III of PCK-CAM. This shuttle could form the biochemical basis of the interaction between photosynthesis and respiration in A. comosus mitochondria in CAM phase III. However, we really don’t know this metabolism is common for all PCK-CAM plants or it is only a special pathway for A. comosus. So, our results suggest that it is necessary to further investigate mitochondrial malate metabolism with some other PCK-CAM species such as Hoya carnosa, Aloe arborescens, Orchid and others to clearly understand malate metabolism in mitochondria of PCK-CAM plants. If the malate metabolism in mitochondria of these species is also operated similarly as A. comosus via the malate-OAA shuttle, our research would be a common theory. It would be contribute to complete a different picture on total cycle of malate metabolism in both cytosol and mitochondrion during day time between ME-CAM and PCK-CAM plants.

Summary

This study has been performing to obtain a better insight understanding of the mitochondrial respiration and the role of mitochondria during CAM phase III in two ME-CAM species of K. daigremontiana and K. pinnata and one PCK-CAM species of A. comosus. Another purpose of this study is to identify the interactions among the mitochondria, cytosol and chloroplast in three CAM species

In this study, enzyme activities, respiratory properties, and general characteristics of individual and simultaneous substrates oxidations were rather similar in mitochondria of two Kalanchoë species. Mitochondria of three CAM species readily oxidized succinate and NADH with the high rates similarly to mitochondria of other CAM species. However, NADPH and malate in Kalanchoë mitochondria were oxidized via different way from A. comosus mitochondria. Kalanchoë mitochondria easily oxidized NADPH without Ca²⁺ whereas A. comosus mitochondria only oxidized NADPH in the presence of Ca²⁺ at high concentration. I have also found that similarly as other malic enzyme (ME)-CAM species as Sedum praealtum (Arron et al., 1979), K.

blosssfeldiana (Rustin and Queiroz-Claret, 1985) and K. fedtschenkoi (Cook et al., 1995), in K. daigremontiana and K. pinnata, NAD-ME played an important role in mitochondrial malate metabolism in which malate was mainly oxidized by NAD-ME to produce pyruvate and CO2. This result contributed the further experimental evidence to confirm that malate metabolism in mitochondria of ME-CAM plants mainly via active NAD-ME to produce pyruvate and CO2. The main important finding of my research was that A. comosus mitochondria oxidized malate in a different way from mitochondria of ME-CAM species. It was strongly suggested that A. comosus mitochondria oxidized malate via the malate-oxaloacetate (OAA) shuttle. In A. comosus, OAA played a

significant role in the mitochondrial malate metabolism, in which malate was mainly oxidized by malate dehydrogenase (MDH) to produce OAA. The OAA could be exported to outside the mitochondria via a malate-OAA shuttle. The shuttle might to operate as a supporting system for mitochondrion and cytosol in controlling and regulating malate metabolism in order to supply OAA and ATP for PCK activity during decarboxylation phase of the PCK-CAM plants.

The activities of the electron transport chain (ETC) in mitochondria of Kalanchoë and A. comosus were significant differed. Although mitochondrial ETC of these species connected to both alternative (Alt) and cytochrome (Cyt) pathways, their capacities were varied depending on the substrates and species. External NADH oxidation in A. comosus mitochondria was much engaged on Cyt pathway and it could be produced much more ATP than in two Kalanchoë species. This capacity might be one of the metabolisms of A. comosus to help mitochondria function to produce sufficient required ATP for cytosolic PCK activity in daytime. The combination of two or three substrates not only dramatically enhanced respiration rate but also increased the Alt respiration in mitochondria of these CAM species. These results provided further evidence for previous reports that mitochondrial respiration and Alt pathway were not fully saturated with single substrate in mitochondria of these CAM species. In addition, the reduction of low oxygen concentrations caused a decrease not only in the respiratory property but also in the Alt respiration in mitochondria of Kalanchoë and A. comosus.

From this study, it is possible to suggest that during CAM Phase III mitochondria of three CAM species may contribute not only to supply CO2 for chloroplast in the carbon fixation but also to supply ATP and NAD(P)H for malate decarboxylation itself and metabolism after the event.

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