Arabidopsis peroxisomal malate dehydrogenase functions in β‐oxidation but not in the glyoxylate cycle

Abstract

SummaryThe aim was to determine the function of peroxisomal NAD + -malate dehydrogenase (PMDH) in fatty acid boxidation and the glyoxylate cycle in Arabidopsis. Seeds in which both PMDH genes are disrupted by T-DNA insertions germinate, but seedling establishment is dependent on exogenous sugar. Mutant seedlings mobilize their triacylglycerol very slowly and growth is insensitive to 2,4-dichlorophenoxybutyric acid. Thus mutant seedlings are severely impaired in b-oxidation, even though microarray analysis shows that b-oxidation genes are expressed normally. The mutant phenotype was complemented by expression of a cDNA encoding PMDH with either its native peroxisome targeting signal-2 (PTS2) targeting sequence or a heterologous PTS1 sequence. In contrast to the block in b-oxidation in mutant seedlings, [14 C]acetate is readily metabolized into sugars and organic acids, thereby demonstrating normal activity of the glyoxylate cycle. We conclude that PMDH serves to reoxidize NADH produced from fatty acid b-oxidation and does not participate directly in the glyoxylate cycle.

Disrupting Autophagy Restores Peroxisome Function to anArabidopsis lon2Mutant and Reveals a Role for the LON2 Protease in Peroxisomal Matrix Protein Degradation

“…Membranes were blocked in 8% milk solution (or 5% BSA for the anti-ATG3 antibody) in 20 mM Tris, pH 7.5, 150 mM NaCl, and 0.1% Tween 20 and subsequently incubated overnight at 4°C with primary antibodies in blocking solution. Rabbit antibodies against ATG3 (1:10,000; Phillips et al, 2008), ATG7 (1:1000; Doelling et al, 2002), ICL (1:1000; Maeshima et al, 1988), HPR (1:10,000; Agrisera AS11 1797), MLS (1:25,000; Olsen et al, 1993), the PED1 isoform of thiolase (1:2500; Lingard et al, 2009), PEX5 (1:100;Zolman and Bartel, 2004), PEX7 (1:800; Ramón and Bartel, 2010), PEX14 (1:10,000; Agrisera AS08 372), and PMDH2 (1:2000; Pracharoenwattana et al, 2007) were diluted as indicated. Mouse antibodies against HSC70 (1:20,000 or 1:50,000; StressGen Bioreagents SPA-817) were used.…”

Autophagy-Related Proteins Are Required for Degradation of Peroxisomes inArabidopsisHypocotyls during Seedling Growth

“…Immunoblots were incubated in a blocking solution (PBS containing 0.1% [v/v] Triton X-100 and either 5% [w/v] nonfat dry milk or 3% [w/v] BSA). Primary antibodies were diluted in blocking solution as follows: 1:2000 for anti-ICL (Ettinger and Harada, 1990) and 1:1000 for anti-GFP (Roche Applied Science), anti-MLS (Ettinger and Harada, 1990), anti-pHPR (Agrisera) (Kleczkowski et al, 1986), anti-peroxisomal malate dehydrogenase (Pracharoenwattana et al, 2007), and anti-histone H3 (Abcam). Chemiluminescence was detected using horseradish peroxidase-conjugated secondary antibodies and SuperSignal West Pico Chemiluminescent Kit (Thermo Scientific).…”

Peroxisomal Malate Dehydrogenase Is Not Essential for Photorespiration in Arabidopsis But Its Absence Causes an Increase in the Stoichiometry of Photorespiratory CO2 Release

“…The HPR mutant showed reduced levels of the NADHdependent peroxisomal HPR activity, but rates of photosynthesis were only reduced by 25% compared with wild-type plants when measured under ambient CO 2 concentrations (Murray et al, 1989). Related mutants lacking PMDH have also been reported in Arabidopsis (Arabidopsis thaliana) as growing slower in air, but surviving (Pracharoenwattana et al, 2007). It was proposed that an alternative pathway may metabolize hydroxypyruvate during photorespiration in these mutants, such as a cytosolic HPR or being linked to simultaneous b-oxidation in the peroxisome (Murray et al, 1989;Kleczkowski et al, 1990;Pracharoenwattana et al, 2007).…”