Michael O. Proudlove

Movement of amino acids into isolated plant mitochondria

Aspartate, glutamate, serine and glycine all permeate the inner membrane of mitochondria isolated from both etiolated and green plant tissues. No significant difference was found in the transport characteristics shown by mitochondria from either tissue. Influx of each amino acid appears diffusional because substrate saturation was not observed and there was no indication of specific inhibition or a requirement for a compensatory or counter ion for uptake. In contrast, uptake of the keto acid pyruvate did appear saturable. Inhibition by α‐cyano‐4‐hydroxycinnamate, mersalyl and FCCP, but not valinomycin, suggests a carrier and a ΔpH mediate pyruvate transport into the matrix.

Evidence for A Ca2+ gradient across the plasma membrane of wheat protoplasts

Fluxes of Ca2+ across the plasma membrane of isolated wheat protoplasts have been measured both as net accumulation and as uptake under steady-state conditions. The ATPase inhibitors, orthovanadate and diethylstibesterol, and the divalent cation ionophore, A23187, were all found to enhance net Ca2+ accumulation by protoplasts. The uptake of Ca2+ under steady-state conditions was also stimulated by A23187 but relatively unaffected by a range of plant hormones or by red or far red light. Light treatments were compared to dark controls with protoplasts isolated from etiolated wheat. The results suggest that plant cells maintain a Ca2+ gradient across their plasma membrane but it appears not to be under phytochrome control.

Pyruvate Transport by Thermogenic Tissue Mitochondria

1. Mitochondria isolated from the thermogenic spadices of Arum maculatum and Sauromatum guttatum plants oxidized external NADH, succinate, citrate, malate, 2-oxoglutarate and pyruvate without the need to add exogenous cofactors. 2. Oxidation of substrates was virtually all via the alternative oxidase, the cytochrome pathway constituting only 10-20% of the total activity, depending on the stage of spadix development. 3. During later stages of spadix development, pyruvate oxidation was enhanced by the addition of aspartate. This was caused by acetyl-CoA condensing with oxaloacetate, produced from pyruvate/aspartate transamination, and so decreasing feedback inhibition of pyruvate dehydrogenase. 4. Pyruvate oxidation was inhibited by the long-chain acid maleimides AM5-11, but not by those with shorter polymethylene side groups, AM1-4. 5. The alpha-cyanocinnamate derivatives UK5099 [alpha-cyano-beta-(1-phenylindol-3-yl)acrylate] and CHCA [alpha-cyano-4-hydroxycinnamate] inhibited pyruvate-dependent O2 consumption and the carrier-mediated uptake of pyruvate across the mitochondrial inner membrane. Characteristics of non-competitive inhibition were observed for CHCA, whereas for UK5099 the results were more complex, suggesting a very low rate of dissociation of the inhibitor-carrier complex. 6. A comparison of the values of Vmax. and Km for oxidation and transport suggested that it was the latter which controls the overall rate of pyruvate oxidation by mitochondria from both tissues.

The Role of Intracellular Organelles in the Regulation of Cytosolic Calcium Levels

Within animal cells, the role of calcium as a second messenger of such diverse activities as cell mobility, elongation and division in addition to its role in neurotransmitter and hormone secretion and its effect on the rate of catabolism is well established (Akerman, 1982; Akerman and Nicholls, 1983). The cytosolic [Ca2+] in animal cells is in the order of 10-7 M (Akerman and Nicholls, 1983) i.e. orders of magnitude lower than its extracellular concentration. Activation by various excitatory or stimulatory signals results in an increase in the internal free Ca 2+ concentration by one or two orders of magnitude. This rise in cytosolic Ca2+ triggers the above mentioned activities via Ca2+-sensitive enzymes located in the cell. Low cytosolic [Ca2+] is maintained by active extrusion driven by a calmodulin-dependent Ca2+ (Ca2+/Mg)-ATPase located in the plasma-membrane. It is buffered by transport systems residing in mitochondria, endoplasmic reticulum, and other organelles Akerman, 1981; Borle, 1981. Whilst the basic principles of cellular Ca2+ regulation as well as Ca2+-dependent enzyme activation have been well documented in animal cells, information on analogous systems in plants is poor. In general, cytosolic [Ca2+] in plants is considered to be low (although no measurements have been reported but see Akerman et al., 1983) and to be controlled both by active extrusion gut of the cell via plasma membrane Ca2+-translocating APTases and by Ca2+ sequestering by mitochondria, vacuoles, endoplasmic reticulum and chloroplasts (Moore and Akerman, 1984).

The Effect of n-polymethylene-carboxymaleimides on Glycine Movement into Pea Leaf Mitochondria

Glycine decarboxylase is a multi-enzyme complex which is located on the inner surface of the inner mitochondrial membrane and catalyses the decarboxylation of glycine to serine (Moore et al., 1977; Sarojini, Oliver, 1983). Considerable controversy still exists as to whether glycine movement into the mitochondria occurs by simple diffusion (Day, Wiskich, 1980; Proudlove, Moore, 1982) or is carrier-mediated (Cavalieri, Huang, 1980; Walker et al., 1982). The latter suggestion is based upon the observations that glycine oxidation and glycine dependent swelling are inhibited by sulphydryl reagents. Direct uptake studies (Day, Wiskich, 1980; Proudlove, Moore, 1982) tended to suggest that glycine enters by simple diffusion, the effect of sulphydryl reagents being probably due to enzyme inhibition. In an attempt to discriminate between these two models we have investigated the effect of a series of N-polymethylenecarboxymaleimides (trivial name, acid maleimides-AM, see Figure 1) on glycine oxidation, decarboxylation and uptake by isolated pea leaf mitochondria. These compounds are a new class of membrane impermeant sulphydryl reagent (Griffiths et el., 1981) that can be used to determine the distribution of the sulphydryl groups in the hydrophobic regions of the membrane.

The Calcium Content of Chloroplast, Mitochondrial and Cytosolic Fractions of Pea Leaf Cells

Evidence that small changes in the cytosolic pCa (from approximately 7 to 6) of animal cells may act as a secondary messenger of hormone action is now widely accepted (Akerman, 1982). For higher plants, however, whilst there are reports that similar, small changes in [Ca2+] can affect the activity of several enzyme-mediated reactions (Moore and Akerman, 1984), there are few data on the Ca2+ levels in the mature cell and the changes which may occur in response to effectors such as light and/or growth regulators (Akerman et al., 1983; Moore and Akerman, 1984). Using isolated protoplasts, which show high rates of CO2-dependent 02 evolution, we have measured both total intra-cellular Ca2+ content and that associated with chloroplastic, mitochondrial and cytosolic/vacuolar fractions of pea leaf cells. Isolating these constituents from protoplasts not only gives maximum yield of intact, active organelles, but also minimizes the time for gross changes in the steady-state [Ca2+] in such cell compartments by using conventional differential centrifugation techniques.

Photorespiratory Development in Wheat. II Chloroplast Dicarboxylate Transport.

The increasing maturity of cells, from base to tip in the primary leaf of wheat, has been used as a model system to study chloroplast biogenesis. Chloroplast area, chlorophyll concentration and protein content and activity, notably RubisCO, all increase dramatically during the early stages of plastid development (1–3). Recent results also show that the activity of glutamine synthetase (GS), a central enzyme in amino acid biosynthesis and photorespiratory nitrogen cycling (4), increases acropetally and that this is due to an increase in the chloroplast isoenzyme (3).

Membrane Potential Measurements in C3 Protoplasts

Membrane potentials have been assessed in isolated organelles using the lipophilic cation methyltriphenyl phosphonium (TPMP+). This distributes across membranes in response to their electrical potential (Scott, Nicholls, 1980). There are few data available, however, on membrane potentials in the intact cell. Microelectrode studies probably reflecting the potential between the vacuole and the external media rather than those generated by energy-conserving cytoplasmic organelles. The present study was undertaken to investigate membrane potentials under various metabolic conditions; in particular, the extent to which ATP generated by the chloroplast in the light may control mitochondrial respiratory activity. Respiration is thought to be under adenylate control (Moore, 1978) so, under photosynthetic conditions, any limitation on mitochondrial electron transport would be reflected by an increased membrane potential in this organelle.

Calcium Fluxes Across the Plasma Membrane of Pea Leaf Protoplasts

Stimulation or excitation of numerous animal cells leads to a rapid increase in the cytosolic free calcium and the activation of certain enzyme systems, either directly or in a calmodulin-dependent fashion. Maintenance of low [Ca2+] is acheived by extrusion across the plasma membrane, by a calmodulin-oependent (Ca2+/Mg)-ATPase and by internal buffering, by mitochondria, endoplasmic reticulum and others (1,2). In plant cells there is now mounting evidence that Ca2+ may play a similar role (see 3 for a recent review). Whilst there is no direct evidence that cytosolic calcium levels change in plant cells, there do appear to be systems available which may be involved in buffering [Ca2+]cyt, again associated with organelles and the plasma membrane (4). In the monocotyledon, wheat- a preliminary study has shown that there is an inwardly directed Ca gradient across the plasma membrane, sustained by a growth regulator/red/far red light-insensitive influx and an ATPase inhibitor-sensitive efflux (5). The susceptibility of chloroplasts of this plant to inhibition by calcium (6), however, prompted us to investigate the kinetics of mesophyll plasma membrane control of cytosolic Ca2+ in the dicotyledon, pea. Chloroplasts from this tissue can accumulate external calcium and the mitochondrial external NADH dehydrogenase is activated by changes in pCa.

Nitrate Reductase Activity in Seeds and Seedlings of Tropical Species

Apart from those plants which have a symbiotic association with nitrogen-fixing bacteria, most plants depend solely on nitrate ions taken in from the soil for their nitrogen requirements. During nitrogen assimilation in plants, this nitrate is reduced to nitrite by the enzyme nitrate reductase (NR). In higher plants, nitrate reductase is extremely unstable and is subject to rapid turn-over, especially at room temperature. The adaptive nature of this enzyme, however, means that it is easily induced, by its substrate nitrate, in roots, stem and leaves of plants1–3.