Determination of Compartmented Metabolite Pools by a Combination of Rapid Fractionation of Oat Mesophyll Protoplasts and Enzymic Cycling

Abstract

ABSTRACIIn vivo pool sizes of a range of metabolites have been determined in subcellular fractions of darkened and illuminated mesophyll protoplasts of Avena sativa L. These estimations were made by combining a method of rapid protoplast fractionation with enzymic cycling techniques. Results are given for reduced and oxidized pyridine nucleotides, triose phosphates, 3-phosphoglycerate, inorganic phosphate, aspartate, malate, oxaloacetate, glutamate, 2-oxoglutarate, and citrate, from chloroplasts, mitochondria, and a fraction representing the remainder of the protoplast. The results indicate distinct differences of compartmented levels of certain metabolites between darkened and illuminated protoplasts.Cells of green tissue have two major systems for energy production, viz. that in chloroplasts and that in mitochondria. In the dark, ATP formed by oxidative phosphorylation is exported from mitochondria to energy consuming sites in the cell, while a back flow of ADP maintains phosphorylation (15). Under illumination there is additional production of ATP by chloroplasts: the latter exceeds considerably that by mitochondria (26).From metabolite determinations on nonaqueously fractionated leaf tissue, Heber and Santarius (12) suggested that mitochondrial respiration of green tissue possibly is inhibited in the light. This hypothesis was deduced from an observed increase in the extrachloroplast ATP/ADP ratio after the onset of illumination, and invoked to the operation of the TP2/PGA shuttle across the inner chloroplast membrane (10,12). Such an increase of the ATP/ADP ratio also has been observed by other authors (8,22). In addition, Goller et al. (6) showed that the incubation of oat mesophyll protoplasts with permeable and specific inhibitors of the light-dependent electron transport is almost without effect on the cytosolic adenylate ratio in the light. These results were taken as evidence that light has no direct effect on oxidative phosphorylation and that, potentially, mitochondria are equally active under darkness and light.Recent experiments with isolated animal and plant mitochondria (5,27) ratio is not the primary factor in the control of respiration in isolated mitochondria. Therefore, Dry and Wiskich (5) concluded that, if leaf mitochondrial respiration is inhibited in the light, the ATP/ADP ratio would be of minor importance in regulation, i.e. respiration may be controlled in more than one way (27). Information on in vivo pool sizes of metabolites in various compartments is limited and mainly has been derived from nonaqueous fractionations of leaf tissue into chloroplast and extrachloroplast material (e.g. Heber and coworkers [11][12][13]29]). A better insight into mechanisms regulating mitochondrial respiration is only possible, however, when pool sizes of a wider range of metabolites within different cellular compartments are known. Importantly, knowledge of how environmental perturbations affect the pool sizes is necessary.In the present report we have combined our method of rapid fractionation of m...

O2-Insensitive Photosynthesis in C3 Plants

“…However, these measurements were made after only 2 min of light during which time photosynthesis probably had not achieved its maximum rate. Chloroplasts from 'rapidly' fractionated oat protoplasts had 3 mm Pi in the experiments of Hampp et al (10). Because of the Pi sensitivity of starch synthesis and enzyme activation (12,13), I believe that the Pi concentration inside the chloroplast can often be as low as 1 (22).…”

“…This limitation occurs in water-stressed leaves, leaves at low temperature, and leaves of plants grown at high CO2. Reports of 02 insensitivity have been published in the past (3,5,10,14,15,18,25,29,31), but no concensus of the possible explanation has yet emerged.…”

Characterization of AtSTOP1 Orthologous Genes in Tobacco and Other Plant Species

“…Each plant was incubated for 24 h in 2 mL of 1/50 MGRL solution containing 1% Suc on a six-well plate in the presence or absence of Al (20 mM Hampp et al (1984) with minor modifications as described by Kihara et al (2003). Oxalate in the medium was quantified calorimetrically using the Enzytec kit for oxalate (R-Biopharm) according to the manufacturer's protocol.…”

Respiration of Mitochondria Isolated from Leaves and Protoplasts of Avena sativa

“…The absence of a cell wall allows ready access to the plasma membrane (22) and facilitates rapid cell fractionation (14). Protoplasts additionally provide the convenience of a homogeneous suspension (19).…”

“…Protoplasts additionally provide the convenience of a homogeneous suspension (19). Oat protoplasts are being used to assess intracellular distribution of metabolites (14), regulation of ion and amino acid flux (19,22), biosynthetic responses to pathogens (29), and the photosynthetic capacity of single cells (15). Consequently, the extent and nature of metabolic differences between protoplasts and cells must be assessed.…”