Aconitate isomerase from maize leaves: Light-dependent expression and kinetic properties.

Aconitate isomerase (EC 5.3.3.7) interconverts cis- and trans-isomers of aconitic acid. Expression of the gene encoding this enzyme was studied in maize (Zea mays L.) leaves depending on light regime. Aconitate isomerase was induced by white and by red light indicating the involvement of phytochrome in the regulation of gene expression. The enzyme was partially purified from maize leaves. The value of Km was 0.75 mM with cis-aconitate and 0.92 mM with trans-aconitate, pH optimum was 8.0-8.2 with both substrates, citrate and malate suppressed its activity. It is concluded that aconitate isomerase actively participates in the interconversion of cis- and trans-aconitate in the light providing a possibility of using the pool of trans-aconitate for the regulation of the tricarboxylic acid cycle activity and mediating citrate/isocitrate supply for the biosynthetic and signaling purposes in photosynthetic cells.

Regulation of expression of the mitochondrial and peroxisomal forms of citrate synthase in maize during germination and in response to light.

Expression of genes encoding the mitochondrial and peroxisomal forms of citrate synthase (EC 2.3.3.1) was studied in maize (Zea mays L.) in scutella during germination and in leaves depending on light regime. During germination, citrate synthase activity increased in scutella both in mitochondria and in fatty-acid metabolizing peroxisomes (glyoxysomes) by day 6 and then declined. This was preceded by the peak of expression of the genes encoding the mitochondrial (Csy1) and peroxisomal (Csy2) forms of citrate synthase occurring on the day 3 of germination, after which the expression of Csy1 gradually and of Csy2 sharply declined. The decrease of expression of both genes was followed by the increase of promoter methylation which was more intensive for the gene encoding the mitochondrial form. In leaves, the activity of the mitochondrial form was much higher than that of the peroxisomal form and increased in darkness, while the peroxisomal form was almost undetectable in darkness and increased in the light. The mitochondrial form was inhibited by white and red light while the peroxisomal form was induced by white, red and blue light indicating the involvement of phytochrome and cryptochrome. The mechanism of light regulation of citrate synthase involved promoter methylation leading to the inhibition of corresponding genes and exhibiting opposite patterns for Csy1 and Csy2. Citrate synthase was purified from mitochondria and glyoxysomes of maize scutellum. The mitochondrial form had higher optimum pH as compared to the glyoxysomal form and possessed higher affinity to oxaloacetate and acetyl-CoA. It is concluded that expression of citrate synthase during germination and in response to light is regulated by methylation of promoters of corresponding genes.

Expression and promoter methylation of succinate dehydrogenase and fumarase genes in maize under anoxic conditions.

Succinate dehydrogenase (SDH) and fumarase enzyme activity and expression of genes encoding the SDH subunits A (Sdh1-2), B (Sdh2-3), C (Sdh3), D (Sdh4) and the mitochondrial (Fum1) and cytosolic (Fum2) isoforms of fumarase were quantified in maize (Zea mays L.) seedlings exposed to atmospheres of air (control), N2, and CO2. The catalytic activity of SDH gradually declined in plants exposed to N2 atmospheres, with ∼40% activity remaining after 24h. In seedlings incubated in CO2, the suppression was even more pronounced. Fumarase activity was more stable, decreasing by one third after 24h of anoxia. The level of Sdh1-2 transcripts in seedlings declined significantly under N2 and even more rapidly upon exposure to CO2, with a concomitant increase in methylation of the corresponding promoters. The level of Sdh2-3 and Sdh3 transcripts also decreased under N2 and CO2, but the changes in promoter methylation were less pronounced, whereas the changes in the level of Sdh4 expression and promoter methylation were minor. Expression of Fum1 and Fum2 was affected by N2 and CO2 atmospheres, however without changes in corresponding promoter methylation. It is concluded that, under conditions of oxygen deficiency, succinate accumulates mainly due to downregulation of SDH gene expression and reduction of enzyme activity, and to a lesser extent due to the decrease of fumarase gene expression.