Journal:

Article Title: Reduced Expression of Aconitase Results in an Enhanced Rate of Photosynthesis and Marked Shifts in Carbon Partitioning in Illuminated Leaves of Wild Species Tomato 1

doi: 10.1104/pp.103.026716

Figure Lengend Snippet: Analysis of the aconitase protein of the tomato genotypes. A, Activity of marker enzymes along a Percoll density gradient used for mitochondrial preparation. Each fraction corresponds to 1 mL of the gradient (from top to base). Each fraction was desalted and assayed for Ppi-dependent phosphofructokinase (PFP) and cytochrome c oxidase (CCO) activities as described in “Materials and Methods”. Fractions 25 to 33 were pooled to form the “mitochondrial fraction.” B, Western-blot analysis of aconitase in cytosolic and mitochondrial fractions. Total protein from the first supernatant and from the pooled mitochondrial fractions (25–33) were analyzed by western blotting, using antiserum raised against pumpkin aconitase that recognizes a protein of approximately 98 kD. C, Zymogram of aconitase activity in leaves of 6-week-old plants from Lp and Aco-1 genotypes. Numbers above the gel indicate the total amount of protein loaded in micrograms. 1 and2, Independent plants. D, Mitochondrial aconitase activity. Aconitase activity was determined in the mitochondrial fractions of Percoll gradients. Data represent the mean ± se of measurements from four plants per genotype. An asterisk indicates those that were determined by the Student's t test to be significantly different between genotypes.

Article Snippet: To test this, we isolated mitochondria from leaves of both Aco-1 and Lp plants using a Percoll gradient purification method ( Millar et al., 2001 ). documents a typical enzyme profile obtained after fractionation of tomato leaf extracts on a Percoll gradient with the profile of pyrophosphate-dependent PFP demarcating the cytoplasm and that of CCO the mitochondria.

Techniques: Activity Assay, Marker, Western Blot