Mitochondrial respiration releases carbon dioxide that was previously fixed by photosynthesis, so its rate influences the growth, productivity and energy use efficiency of plants. The tri-carboxylic acid (TCA) cycle is a critical intersection between catabolic and anabolic processes and it plays a central role in mitochondrial respiratory metabolism. Mitochondrial malate dehydrogenase (NAD-MDH) (EC 220.127.116.11) is involved in the final step of the TCA cycle, and is responsible for the inter-conversion of malate and oxaloacetate, concomitant with the reduction or oxidation of reducing equivalents. This thesis aimed to gain a better understanding of the role of mitochondrial MDH (mMDH) in respiratory metabolism during growth and development of Arabidopsis thaliana. This was undertaken by transcript, protein and metabolite analysis on different organs (leaf, seed and root) at distinct developmental stages using single (mmdh1-2 and mmdh2-1) and double (mmdh1-2mmdh2-1) T-DNA knockout lines, as well as in a complemented line (mmdh1mmdh2 35S: MMDH1), in the A. thaliana (Col-0) genetic background. The correlation between naturally varied mMDH levels and leaf respiratory metabolism of A. thaliana ecotypes were also investigated. Ecotype Col-0 was used as a reference for data integration between mMDH mutant experiments and ecotype assessments.
Both findings from mMDH mutants and ecotypes supported the hypothesis that mMDH plays a significant role in modulating leaf respiration in plants as lower protein abundance of mMDH was consistently, significantly and inversely correlated with plant respiration rates. MMDH1 (At1g53240) appeared to be the predominant mitochondrial isoform of NAD-MDH when compared to MMDH2 (At3g15020). This was inferred from higher transcript and protein abundances of MMDH1, and a larger impact of single MMDH1 mutation on plant phenotype, MDH expression compensation and metabolism. Functional redundancy between mMDH isoforms was evident in leaf respiratory metabolism as complementation of MMDH1 cDNA in the mMDH double mutant background restored wild-type growth phenotypes. However, the lack of MMDH2 could not be fully substituted by MMDH1 in heterotrophic organs (seeds and roots) and during certain plant developmental stages, suggesting either some unknown specific roles of MMDH2 or the importance of the native promoter in complementation. Transcript analyses from mMDH mutants and ecotypes suggested that NAD-MDH isoforms with a higher degree of functional redundancy (e.g. MMDH1 and MMDH2) and those with relatively greater participation in the biochemical NAD-MDH network (e.g. MMDH1, CMDH1, CHMDH and PMDH2) showed stronger correlation with each other in expression level. These relationships symbolise a cooperative model of the MDH network in executing global cellular redox homeostasis, probably facilitated by malate-oxaloacetate shuttling system across subcellular compartments. In both mMDH mutants and ecotypes, MMDH1 and SDH1 proteins (At5g66760& At2g18450) were significantly correlated with respiration rates, demonstrating negative and positive relationship with respiration rates respectively. It is therefore suggested that MDH is a negative and SDH is a positive marker for plant respiration rates.
Loss of mitochondrial MDH had significant effects on the overall energy status of Arabidopsis plants. The important roles of mMDH in plant carbon partitioning and carbon use efficiency was evident from the delayed growth, significantly reduced biomass of both autotrophic and heterotrophic organs and seed defects in the mmdh1-2mmdh2-1 double mutant, concomitant with elevated respiration rates throughout Arabidopsis developmental stages. Complete loss or decreased mMDH protein levels in Arabidopsis resulted in a marked accumulation of leaf glycine, consistent with a slowed photorespiratory process. Defective nitrogen metabolism was also consistent with lower abundances of leaf 2-oxoglutarate dehydrogenase subunits (At3g55410 and At5g65750) in the mmdh1-2mmdh2-1 double mutant. Free amino acids, particularly branched-chain amino acids, accumulated in leaves of fast-respiring mmdh1-2mmdh2-1 double mutants and also in low-MDH ecotypes. This was observed to an even greater extent in seeds and roots of mmdh1-2mmdh2-1, consistent with increased catabolism of amino acids in response to lower mMDH protein levels. Collectively, the present study has demonstrated that a deficiency of mMDH proteins affects nitrogen and carbon assimilation as well as the efficiency of carbon usage across Arabidopsis organs and their development and that natural variation in MDH levels may be an important factor in defining respiration rates.
|Qualification||Doctor of Philosophy|
|Publication status||Unpublished - 2015|