Figure 16.

Tentative Model Explaining Redirection of Metabolism in the Absence of MDH2 during Photoautotrophic N Deprivation.

FA degradation starts with hydrolysis of TAGs and membrane lipids by lipases. FAs released enter the peroxisomes via an ABC transporter and subsequently are degraded to acetyl-CoAs by the core FA β-oxidation spiral, which consists of four enzymatic activities (ACX, MFP-hydratase, MFP-DH, and KAT). MDH2 plays a role in oxidation of NADH, which is generated by hydroxylacyl-CoA dehydrogenase (MFP-DH) at the third step of β-oxidation spiral. In the absence of MDH2, NADH is likely accumulated, thereby increasing the reduction state of peroxisome. This would result in two metabolic changes in peroxisome: a decrease in malate export and an increase in H₂O₂ level. These two metabolites would in turn significantly alter photosynthesis and chloroplast metabolism. Activated pathways in the mutant are indicated by blue arrows, whereas downregulated pathways are indicated by red arrows. Based on results obtained from this study and current literature, we propose a cascade of events leading to the observed phenotypes in mdh2 mutants: “High oil” phenotype (events 1, 2, and 5): A block in FA β-oxidation combined with increased de novo FA synthesis contributes to higher TAG accumulation. “High starch” phenotype (events 2 to 4): During N starvation, the more active CO₂ fixation and photosynthesis in the mdh2-1 mutant provides more NADPH and carbon precursors for starch synthesis. The increased level of NADPH in chloroplast activates AGPase for starch synthesis and also activates several starch-degrading enzymes; therefore, it results in an increased carbon flux into and out of the starch route. Higher amounts of starch indicate high sink capacity, therefore sustaining photosynthesis. As a consequence, more NADPH is produced by photochemical reactions, and this further activates AGPase, together resulting in the 100 to 300% increase in starch content. “High H₂O₂” phenotype (event 3): The higher level of H₂O₂ in the mutant is supported by the 2-fold increase in the H₂O₂-generating reaction catalyzed by ACX activity. Once it is transmitted to chloroplast, H₂O₂ activates the starch synthesis pathway, as supported further by the observation of starch overaccumulation in wild-type cells supplemented with exogenous H₂O₂. “Sustained photosynthesis” (events 1 and 2): A blockage in FA β-oxidation at the step of MDH2 will result in less malate being produced in the peroxisome. Malate, a recognized electron carrier, can be transported to other compartments via the dicarboxylate transporter. When malate level is high, less NADP⁺ is available as electron acceptor at PSI, therefore downregulating photosynthesis. Conversely, in the absence of MDH2, the malate level is decreased, and more NADP⁺ is available to accept electrons from PSI. ACX, acyl-CoA oxidase; CAT, catalase; CBC, Calvin-Benson-Bassham cycle; CTS1, comatose 1; DH, dehydrogenase; FAD, flavin adenine dinucleotide; FFA, free fatty acid; Fdx, ferredoxin; FNR, ferredoxin-NADP⁺ reductase; KAT, ketoacyl-CoA thiolase; LHC, light-harvesting complex; Mal, malate; MFP, multifunctional protein; NTRC, NADP:thioredoxin reductase C; OAA, oxaloacetate; PQ, plastoquinone; SDP1, sugar dependent 1; SOD, superoxide dismutase.