Genetic dissection of methylcrotonyl CoA carboxylase indicates a complex role for mitochondrial leucine catabolism during seed development and germination

Abstract

SUMMARY3-methylcrotonyl CoA carboxylase (MCCase) is a nuclear-encoded, mitochondrial-localized biotin-containing enzyme. The reaction catalyzed by this enzyme is required for leucine (Leu) catabolism, and it may also play a role in the catabolism of isoprenoids and the mevalonate shunt. In Arabidopsis, two MCCase subunits (the biotinylated MCCA subunit and the non-biotinylated MCCB subunit) are each encoded by single genes (At1g03090 and At4g34030, respectively). A reverse genetic approach was used to assess the physiological role of MCCase in plants. We recovered and characterized T-DNA and transposon-tagged knockout alleles of the MCCA and MCCB genes. Metabolite profiling studies indicate that mutations in either MCCA or MCCB block mitochondrial Leu catabolism, as inferred from the increased accumulation of Leu. Under light deprivation conditions, the hyper-accumulation of Leu, 3-methylcrotonyl CoA and isovaleryl CoA indicates that mitochondrial and peroxisomal Leu catabolism pathways are independently regulated. This biochemical block in mitochondrial Leu catabolism is associated with an impaired reproductive growth phenotype, which includes aberrant flower and silique development and decreased seed germination. The decreased seed germination phenotype is only observed for homozygous mutant seeds collected from a parent plant that is itself homozygous, but not from a parent plant that is heterozygous. These characterizations may shed light on the role of catabolic processes in growth and development, an area of plant biology that is poorly understood.

Methylcrotonyl-CoA Carboxylase Regulates Triacylglycerol Accumulation in the Model Diatom Phaeodactylum tricornutum

“…Consistent with the previous findings in Arabidopsis (Ding et al, 2012), not only the catabolism of Leu, but also that of Val, Ile, Glu, and Thr were disturbed by MCC2 silencing to varying degrees. Furthermore, the existence of redundant cellular Leu may inhibit the absorption of extra nitrogen in culture and lead to incomplete utilization of nitrogen, so carbon utilization by cells might decrease accordingly.…”

“…Furthermore, the existence of redundant cellular Leu may inhibit the absorption of extra nitrogen in culture and lead to incomplete utilization of nitrogen, so carbon utilization by cells might decrease accordingly. In Arabidopsis, knockout of MCCa or MCCb resulted in an impaired reproductive growth phenotype (Ding et al, 2012). Therefore, knockdown of MCC affected lipid synthesis mainly via reducing substrate derived from Leu degradation, so it cannot be excluded that it may also play a minor role in de novo lipid synthesis.…”

Discovery and Characterization of the 3-Hydroxyacyl-ACP Dehydratase Component of the Plant Mitochondrial Fatty Acid Synthase System

“…trans-D 2 -10:1 and trans-D 2 -16:1) were synthesized from a chemical reaction between CoA (EMD Millipore) and trans-D 2 -decenoic acid (TCI Chemicals) or trans-D 2 -dodecenoic acid (Sigma-Aldrich) and purified using an HPLC system (Agilent 1200 HPLC system equipped with a Thermo Scientific Hypersil ODS column [250 mm length, 4 mm i.d., and 5 mm particle size]) as described previously (Förster-Fromme et al, 2008). Structures of these CoA derivatives were determined using a liquid chromatography-mass spectrometry system (Agilent 1100 LC/MSD system) as described previously (Ding et al, 2012). The hydratase activity of mtHD was assayed (for 10 min at 22°C) as described previously (Autio et al, 2008b).…”

The Impact of the Branched-Chain Ketoacid Dehydrogenase Complex on Amino Acid Homeostasis in Arabidopsis

“…Plants synthesize BCAAs and are the main source of these essential nutrients in the diets of humans and agriculturally important animals. In addition to their nutritional value, BCAAs and BCAA-derived metabolites such as glucosinolates, fatty acids, and acyl sugars contribute to plant growth, development, defense, and flavor (Mikkelsen and Halkier, 2003;Taylor et al, 2004;Ishizaki et al, 2005;Slocombe et al, 2008;Araújo et al, 2010;Ding et al, 2012;Kochevenko et al, 2012).…”

“…Plants synthesize BCAAs and are the main source of these essential nutrients in the diets of humans and agriculturally important animals. In addition to their nutritional value, BCAAs and BCAA-derived metabolites such as glucosinolates, fatty acids, and acyl sugars contribute to plant growth, development, defense, and flavor (Mikkelsen and Halkier, 2003;Taylor et al, 2004;Ishizaki et al, 2005;Slocombe et al, 2008;Araújo et al, 2010;Ding et al, 2012;Kochevenko et al, 2012).The BCAA biosynthetic pathway and its regulation have been investigated in Arabidopsis (Arabidopsis thaliana) and other plants for the past two decades, in large part because of the commercial importance of herbicides that inhibit acetohydroxy acid synthase, which is the committing enzyme of BCAA biosynthesis (Singh and Shaner, 1995;Aubert et al, 1997;Singh, 1999;McCourt et al, 2006;Tan et al, 2006;Binder, 2010;Chen et al, 2010;Yu et al, 2010). Strong correlations between the levels of free BCAAs were found in wildtype Arabidopsis seeds and tomato (Solanum lycopersicum) fruits Lu et al, 2008), which suggests coregulation of biosynthesis and/or degradation.…”