Similarity in uptake and retention of trace amounts of 31 silicon and 68 germanium in rat tissues and cell organelles.

Uptake of Si and Ge (chemical analogue of Si) in rat tissues was compared to establish their subcellular localization and determine if Ge could be used to trace Si in situ. 31Si and 68Ge were found in tissues and in cell organelles after injection of 31Si (OH) 4 or 68Ge (OH) 4. Accumulation of 31Si and 68Ge in the tissues increased for 30 minutes, declined rapidly about 30 minutes and then was gradually but steadily depleted. Simultaneous double labelling with 31Si and 68Ge produced concomitant accumulation and release of both isotopes. After a single IP injection, some 68Ge remained in the spleen and kidney for up to 20 days. The 31Si and 68Ge concentration in the tissues and cell organelles increased linearly with the amount administered (31Si, 10-4 to 10-2 g/kg body wt and 68Ge, 10-9 to 10-3 g/kg body wt). Thus, substitution of 68Ge for 31Si can extend the limit of detection of Si by at least 5 orders of magnitude. Both 31Si and 68Ge were water extractable (47%-74% and 38%-89%, respectively) from liver cell organelles; 45%-81% 31Si and 66%-90% 68Ge were extractable in 10% TCA, while only 10%-59% of either isotope were extractable in organic solvents (acetone, chloroform, ethanol). A small percentage of Si may be bound or polymerized.     

Assembly of the subcellular parts of <Emphasis Type="Italic">Bryopsis hypnoides</Emphasis> Lamouroux into new protoplasts

The chloroplasts, mitochondria, and protoplasm devoid of mature chloroplasts (PMC) of Bryopsis hypnoides Lamouroux were isolated by low-speed and sucrose density centrifugation. The PMC aggregated in artificial seawater, and then protoplasts without mature chloroplasts (PtMCs) were formed. Transmission electron microscopy and cytochemical studies indicated that there were mitochondria, nuclei, vesicles, and other small cell organelles in the PtMCs. Scanning electron microscopy showed that there were holes on the surface of 1-h PtMCs and then fewer holes on the surface of 24-h PtMCs, suggesting that a healing process occurred. The plasma membrane was formed over the surface of the PtMCs. However, the cell wall was not regenerated, and the newly formed PtMCs were ruptured and died in 3 days. Light intensity during alga maintenance before use influenced significantly (one-way ANOVA, P < 0.0001) on the number of PtMCs formed; the highest number of PtMCs was formed at 20μmol/(m² s). When isolated chloroplasts were transferred into seawater, there were only two or three chloroplasts aggregated together. However, isolated mitochondria and the mixed six layers of cell organelles (separated by sucrose density centrifugation) could not aggregate in the artificial seawater. This indicates that the conjunction of cell organelles is important for their aggregation. Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 1, pp. 123–130. The text was submitted by the autors in English.     

Germanium-68 as a possible marker for silicon transport in rat brain

Silicon (Si) is an essential trace element normally present in brain and cerebrospinal fluid, although the mechanism by which it enters and distributes in brain is largely unknown. Due to the short radioactive half-life of³¹Si (156 min) we have investigated the use of⁶⁸Germanium (68-Ge, half-life 282 days) as a possible marker for Si transport in rat brain over longer periods than are possible with³¹Si. Adult male anaesthetised rats were given a bolus of⁶⁸Ge I. V. and arterial blood samples taken during experiments that lasted between 5 min and 3 days. At termination, the brain was removed and analysed for radioactivity as were the plasma samples. Data were analyzed by Graphical Analysis which showed that the blood-brain barrier permeability to⁶⁸Ge (K_in10^–4 ml/min/g) is similar to that for many non protein-bound electrolytes in plasma and that⁶⁸Ge fluxes across cerebral capillaries are bidirectional. The autoradiographic distribution of⁶⁸Ge in brain was homogenous. Our results are in agreement with those of previous studies using³¹Si or⁶⁸Ge, which suggest⁶⁸Ge may be a useful marker for Si when investigating the role of this element in conditions such as neurodegenerative diseases.     

Tryptophan aminotransferase and tryptophan dehydrogenase activities in some cell compartments of spinach leaves: The effect of calcium ions on tryptophan dehydrogenase

The content of spinach-leaf cells was compartmented by differential centrifugation. Three fractions were obtained,i.e. chloroplasts, pellet of remaining organelles sedimenting at 97 000g and cytosol. Enzyme activities of L-tryptophan aminotransferase (TAT) as well as L-tryptophan dehydrogenase (TDH) were demonstrated in all cell fractions. The highest activities of both enzymes were found in the pellet of organelles followed by the enzyme activities in the chloroplasts. The cytosol had the lowest enzyme activities. Chloroplasts are characterized by a relatively higher TDH activity, organelles sedimenting at 97 000g were marked by a relatively higher TAT activity. In all fractions both pyridine nucleotide coenzymes catalyzed the TDH activity. Ca²⁺ in a concentration of 0.8 minol l⁻¹ increased markedly the TDH activity in both directions of its activity. An erratum to this article is available at .     

Influence of silicon on cobalt, zinc, and magnesium in baker's yeast, Saccharomyces cerevisiae

Silicon (Si, as silicate) is involved in numerous important structure and function roles in a wide range of organisms, including man. Silicate availability influences metal concentrations within various cell and tissue types, but, as yet, clear mechanisms for such an influence have been discovered only within the diatoms and sponges. In this study, the influence of silicate on the intracellular accumulation of metals was investigated in baker's yeast (Saccharomyces cerevisiae). It was found that at concentrations up to 10 mM, silicate did not influence the growth rate of S. cerevisiae within a standard complete medium. However, an 11% growth inhibition was observed when silicate was present at 100 mM. Intracellular metal concentrations were investigated in yeast cultures grown without added silicate (−Si) or with the addition of 10 mM silicate (+Si). Decreased amounts of Co (52%), Mn (35%), and Fe (20%) were found within +Si-grown yeast cultures as compared to −Si-grown ones, whereas increased amounts of Mo (56%) and Mg (38%) were found. The amounts of Zn and K were apparently unaffected by the presence of silicon. +Si enhanced the yeast growth rate for low-Zn²⁺ medium, but it decreased the growth rate under conditions of a low Mg²⁺ medium and did not alter the growth rates in high Zn²⁺ and Co²⁺ media. +Si doubled the uptake rate of Co²⁺ but did not influence that of Zn²⁺. We propose that a possible explanation for these results is that polysilicate formation at the cell wall changes the cell wall binding capacity for metal ions. The toxicity of silicate was compared to germanium (Ge, as GeO₂), a member of the same group of elements as Si (group 14). Hence, Si and Ge are chemically similar, but silicate starts to polymerize to oligomers above 5 mM, whereas Ge salts remain as monomers at such concentrations. Ge proved to be far more toxic to yeast than Si and no influence of Si on Ge toxicity was found. We propose that these results relate to differences in cellular uptake.     

Studies on the biochemistry and fine structure of silica shell formation in diatoms

Summary The fine structure and pigmentation of an apochlorotic diatom isolated from decaying Macrocystis pyrifera is described. The morphology of acid cleaned shells suggests that the isolate is Nitzschia alba Lewin and Lewin. Light microscope observations indicated a centrally located nucleus and numerous highly refractile bodies which stained differentially with Nile blue and Sudan black B. The stained globules could be correlated in thin-sectioned profiles with either electron dense or lucent areas depending on the fixation technique. In the electron microscope the nucleus, Golgi complex, and mitochondria were similar in appearance to those described for other diatoms. Proplastid-like organelles, delimited by a double membrane, and containing small vesicles were also observed. Neither carotenoids nor chlorophylls could be detected by spectroscopic or spectrofluorometric analysis in vivo or in organic solvent extracts. Deposition of new walls was initiated by formation of silicon deposition-vesicles in the central region of dividing cells. The acentric raphes were deposited last. The genesis and interrelationship of the old plasmalemma, silicalemma, and newly formed plasmalemma are discussed.     

The ratio of germanium to silicon in plant phytoliths: quantification of biological discrimination under controlled experimental conditions

Slight differences in the chemical behavior of germanium (Ge) and silicon (Si) during soil weathering enable Ge/Si ratios to be used as a tracer of Si pathways. Mineral weathering and biogenic silicon cycling are the primary modifiers of Ge/Si ratios, but knowledge of the biogenic cycling component is based on relatively few studies. We conducted two sets of greenhouse experiments in order to better quantify the range and variability in Ge discrimination by plants. Graminoid species commonly found in North American grassland systems, Agropyron smithii, Schizachyrium scoparium, and Andropogon gerardii were grown under controlled hydroponic environmental conditions. Silicon leaf contents were positively correlated with solution Si and ambient temperature but not with nutrient solution pH, electrical conductivity, or species. The Ge/Si ratio incorporated into phytoliths shows a distribution coefficient [(Ge/Si)_phytolith/(Ge/Si)_solution] of about 0.2 and is remarkably invariant between species, photosynthetic pathway, and solution temperature. Ge seems to be discriminated against during the uptake and translocation of Si to the opal deposition sites by about a factor of five. In the second experiment, a wider range of graminoid species (Agropyron smithii, Bouteloua gracilis, Buchloe dactyloides, Oryzopsis hymenoides, Schizachyrium scoparium and Andropogon gerardii) were grown in two different soil mediums. Plant phytoliths showed a distribution factor of about 0.4 for field grown grasses, and 0.6 for potting soil grown grasses with no clear trends among the species. Evidence of the direction and degree of biological Ge discrimination during plant uptake provides a geochemical finger print for plants and improves the utility of Ge/Si ratios in studies of terrestrial weathering and links between Si cycles in terrestrial and marine systems.     

DIATOM MINERALIZATION OK SILICIC ACID. I Si(OH)4 TRANSPORT CHARACTERISTICS IN NAVICULA PELLICULOSA

Silicic acid transport was studied in the photosynthetic diatom Navicula pelliculosa (Bréb.) Hilse using [⁶⁸Ge] germanic acid (⁶⁸Ge(OH)₄) as a tracer of silicic acid (Si(OH)₄). The initial uptake rate of Si(OH)₄ was dependent on cell number, pH, temperature, light and was promoted by certain monovalent cations in the medium. Na⁺ was more effective than K⁺, whereas Li⁺ and NH⁺₄ were ineffective at promoting uptake. Uncouplers and inhibitors of oxidative phosphorylation and of photophosphorylation reduced uptake by 40–99% of control values. Uptake was also especially sensitive to the sulfhydryl blocking agents at 10^?5 M and to the ionophorous compound valinomycin (10^?7 M) which inhibited uptake by 82%. The Si(OH)₄ transport system displayed Michaelis-Menten-type saturation kinetics with kinetic parameters of K_S= 4.4 p. mol Si(OH)₄· 1^?1, V_max= 334 pmol Si(OH)₄· 10⁶ cells^?1· min^?1. Calculations of the acid soluble silicic acid pool size based on 60 s uptake at 20 μM Si(OH)₄ suggested that intracellular levels of Si could reach 20 mM and as much as 5 mM could exist as free silicic acid, representing maintenance of a 250-fold concentration gradient compared with the medium. Efflux from preloaded cells was dependent on temperature and the Si(OH)₄ concentration of the external medium. In the presence of 100 μMM “cold” Si(OH)₄, approximately 30% of the Si(OH)₄ in preloaded cells was exchanged in 20 min. The initial uptake rate of Si(OH)₄ in logarithmic phase cells was constant, but the uptake rate increased in a linear fashion for 6 h in stationary phase cells. These results suggest that the first step in silica mineralization by diatoms is the active transmembrane transport of Si(OH)₄ by an energy dependent, saturable, membrane-carrier mechanism which requires the monovalent cations Na⁺ and K⁺ and is sensitive to sulfhydryl blocking agents. Silicic acid transport activity also appears to be regulated during different growth stages of the diatom.     

Silicon-containing granules of rat liver,kidney and spleen mitochondria

Summary Isolated rat liver mitochondria containing granule aggregates (25–75 nm in diameter) and small (5–10 nm) electron opaque granules were examined by electron probe X-ray microanalysis. The granule aggregates gave an intense Si signal, while the small granules gave both Si and P signals. Isolated mitochondria of rat liver, spleen and kidney, subjected to detergent solubilization and differential centrifugation, produced two granule fractions: (1) a 10,000g fraction consisting predominantly of granule aggregates (25–75 nm) composed of smaller granules (5–10 nm in diameter), and (2) a 10,000–30,000 g fraction of non-aggregated small granules (5–10 nm). Thin sections of isolated granule aggregates gave Si X-ray signals similar to those obtained from in situ granules. In addition S, Cl, Mg, Cr and Fe X-ray signals were observed. Cr occurred only in the large kidney granules, while Fe occurred in both fractions of the spleen and kidney granules. The presence of Si in the granules was confirmed by chemical analysis of the isolated granules and in vivo radiolabeling of the granules with ³¹Si and ⁶⁸Ge. Contamination within the electron microscope was eliminated by a liquid nitrogen anticontamination device.Supported by Grant GM-08229-13-15 from the National Institutes of Health, USPHSWe are grateful to the Perkin-Elmer Corporation and to their Western Branch Manager, Mr. Michael E. Mullen and Microscopist, Mr. Minoru Saito, for use of and assistance with the Hitachi H 500 transmission electron microscope with the scanning attachment, and to the Kevex Corporation for the use of the Kevex X-ray Spectrometer. We also wish to acknowledge Mary Louise Chiappino for her technical assistance in preparing the thin sections, the final micrographs and X-ray spectra photographs and Darlene Lum for technical assistance in the laboratory     

Adaptations by macroalgae to low carbon availability. II. Ultrastructural specializations, related to the function of a photosynthetic buffer system in the Fucaceae

Abstract Among the brown algae, species of the Fucaceae (Pelvetia, Fucus and Ascophyllum) were found to have a ‘photosynthetic buffering’ system, allowing the algae to carry out oxygen production without a concomitant uptake of inorganic carbon. This system was not found in other brown algae examined (e.g. Halidrys, Laminaria and Desmarestia) nor in 16 examined species of red and green algae. Pelvetia, Fucus and Ascophyllum belong to the littoral algae which are periodically emersed. In the Fucaceae, the meristodermal cells were found to have a special organization of organelles. Towards the outer cell wall there was a prominent layer of mitochondria while the chloroplasts were concentrated towards the inner and side walls. Between the mitochondria and the chloroplasts there was a large number of physodes. This arrangement of organelles was not found in the other brown algae examined nor in red or green algae. The significance of this organization of the mitochondria is discussed in connection with the function of the ‘photosynthetic buffering’ system.     

Determination of the sodium,potassium and chloride ion concentrations in the chloroplasts of the halophyteSuaeda maritima by non-aqueous cell fractionation

Summary Leaf material from the halophyteSuaeda maritima L. Dum. grown under both saline and non-saline conditions was fractionated under non-aqueous conditions in order to determine the ion content of various subcellular compartments. Fractions containing cell walls, nuclei and chloroplasts were successfully prepared and contents of DNA, chlorophyll, protein and Na⁺, K⁺, and Cl^– determined. The cell wall fraction was not apparently heavily contaminated by the other fractions and had a low ion content although the nuclear fraction was contaminated by other organelles. The ion contents of chloroplasts were determined and the results discussed in relation to earlier microscopical data.     

Enzymes of nitrogen assimilation in maize roots

The intracellular distribution of enzymes involved in the Crassulacean acid metabolism (CAM) has been studied in Bryophyllum calycinum Salisb. and Crassula lycopodioides Lam. After separation of cell organelles by isopycnic centrifugation, enzymes of the Crassulacean acid metabolism were found in the following cell fractions: Phosphoenolpyruvate carboxylase in the chloroplasts; NAD-dependent malate dehydrogenase in the mitochondria and in the supernatant; NADP-dependent malate dehydrogenase and phosphoenolpyruvate carboxykinase in the chloroplasts; NADP-dependent malic enzyme in the supernatant and to a minor extent in the chloroplasts; NAD-dependent malic enzyme in the supernatant and to some degree in the mitochondria; and pyruvate; orthophosphate dikinase in the chloroplasts. The activity of the NAD-dependent malate dehydrogenase was due to three isoenzymes separated by (NH₄)₂SO₄ gradient solubilization. These isoenzymes represented 17, 78, and 5% of the activity recovered, respectively, in the order of elution. The isoenzyme eluting first was associated with the mitochondria and the second isoenzyme was of cytosolic origin, while the intracellular location of the third isoenzyme was probably the peroxisome. Based on these findings, the metabolic path of Crassulacean acid metabolism within cells of CAM plants is discussed. New address: Institut für Pflanzenphysiologie und Zellbiologie, Freie Universität Berlin, Königin-Luise-Straße 12-16a. D-1000 Berlin 33     

A study of the intracellular transport of calcium in rat heart

The distribution of in vivo injected ⁴⁵Ca⁺⁺ in the subcellular fractions of rat heart has been studied. Most of the radioactivity of the cell was found to be associated with the subcellular organelles; only a small fraction was recovered in the soluble phase. Mitochondria contained the greatest part of the total radioactivity associated with the subcellular organelles. After injection of ⁴⁵Ca⁺⁺ the specific activity of the mitochondrial calcium pool was several times higher than that of the calcium of the sarcoplasmic reticulum. Pentachlorophenol has been administered to rats to uncouple oxidative phosphorylation in heart mitochondria in vivo and its effect on the distribution of ⁴⁵Ca⁺⁺ in the heart studied. Under these conditions, it has been found that mitochondria contained much less ⁴⁵Ca⁺⁺ than the controls; this decrease was paralleled by an increase of the radioactivity associated with the microsomes and with the final supernatant. Experiments in which ⁴⁵Ca⁺⁺ was added to heart homogenates at 0° indicated that ⁴⁵Ca⁺⁺ also became bound to mitochondria and the other subcellular structures at 0°. However, PCP had no effect on the distribution of radioactivity among the subcellular fractions under these conditions. The results suggest that (1) energy-linked movements of Ca⁺⁺ take place in mitochondria of the intact rat heart, (2) a part of the uptake of ⁴⁵Ca⁺⁺ by mitochondria does not depend on metabolism, and, (3) the movements of Ca⁺⁺ in heart mitochondria in vivo are probably more active than those in the sarcoplasmic reticulum.     

Cell organelles from crassulacean-acid-metabolism (CAM) plants

Cell organelles were isolated from the CAM plants Crassula lycopodioides Lam., Bryophyllum calycinum Salisb. and Sedum rubrotinctum R.T. Clausen by isopycnic centrifugation in sucrose gradients. The inclusion of 2.5% Ficoll in the grinding medium proved to be essential for a satisfactory separation of cell organelles during the subsequent centrifugation. Peroxisomes, mitochondria, and whole and broken chloroplasts were at least partially resolved as judged by marker-enzyme-activity profiles. The isolated peroxisomes contained activities of glycollate oxidase, catalase, hydroxypyruvate reductase, glycine aminotransferase, serine-glyoxylate aminotransferase, and aspartate aminotransferase, comparable to activities found in spinach (Spinacia oleracea L.) leaf peroxisomes. In contrast to spinach, however, only little, if any, particulate malate dehydrogenase activity could be attributed to isolated peroxisomes of the three CAM plants.     

Sorting of precursor proteins between isolated spinach leaf mitochondria and chloroplasts

The precursors of the F₁-ATPase -subunits fromNicotiana plumbaginifolia andNeurospora crassa were imported into isolated spinach (Spinacia oleracea L.) leaf mitochondria. Both F₁ precursors were imported and processed to mature size products. No import of the mitochondrial precursor proteins into isolated intact spinach chloroplasts was seen. Moreover, the precursor of the 33 kDa protein of photosynthetic water-splitting enzyme was not imported into the leaf mitochondria. This study provides the first experimental report ofin vitro import of precursor proteins into plant mitochondria isolated from photosynthetic tissue and enables studies of protein sorting between mitochondria and chloroplasts in a system which is homologous with respect to organelles. The results suggest a high organellar specificity in the plant cell for the cytoplasmically synthesized precursor proteins.     

Radioisotopic Method for Measuring Cell Division Rates of Individual Species of Diatoms from Natural Populations

Silicon is an essential element for diatom frustule synthesis and is usually taken up only by dividing cells. With ⁶⁸Ge, a radioactive analog of Si, the cell cycle marker event of frustule formation was identified for individual species of diatom. The frequency of cells within a population undergoing this division event was estimated, and the cell division rate was calculated. In laboratory cultures, these rates of cell division and those calculated from changes in cell numbers were similar. By dual labeling with ⁶⁸Ge(OH)₄ and NaH¹⁴CO₃, rates of cell division and photosynthesis were coincidently measured for diatoms both in laboratory cultures and when isolated from natural populations in estuarine, offshore, and polar environments. These techniques permit the coupling between photosynthesis and cell division to be examined in situ for individual species of diatom.     

Germanium-68 as an adequate tracer for silicon transport in plants. Characterization of silicon uptake in different crop species

A basic problem in silicon (Si) uptake studies in biology is the lack of an appropriate radioactive isotope. Radioactive germanium-68 ((68)Ge) has been used previously as a Si tracer in biological materials, but its suitability for the study of Si transport in higher plants is still untested. In this study, we investigated (68)Ge-traced Si uptake by four crop species differing widely in uptake capacity for Si, including rice (Oryza sativa), barley (Hordeum vulgare), cucumber (Cucumis sativus), and tomato (Lycopersicon esculentum). Maintenance of a (68)Ge:Si molar ratio that was similar in the plant tissues of all four plant species to that supplied in the nutrient solution over a wide range of Si concentrations demonstrated the absence of discrimination between (68)Ge and Si. Further, using the (68)Ge tracer, a typical Michaelis-Menten uptake kinetics for Si was found in rice, barley, and cucumber. Compared to rice, the relative proportion of root-to-shoot translocated Si was lower in barley and cucumber and especially in tomato (only 30%). Uptake and translocation of Si in rice, barley, and cucumber (Si accumulators) were strongly inhibited by 2,4-dinitrophenol and HgCl(2), but in tomato, as a Si-excluding species, both inhibitors produced the opposite effect. In conclusion, our results suggest the use of the (68)Ge tracer method as an appropriate choice for future studies of Si transport in plants. Our findings also indicate that the restriction of Si from symplast to apoplast in the cortex of Si excluders is a metabolically active process.     

Isolation and Properties of Intact Chromoplasts from Tomato Fruits

Intact chromoplasts were isolated from tomato fruits at differentripening stages by Percoll density gradient centrifugation.The isolated chromoplast fractions were contaminated very littleby other organelles, although the fraction from fully ripenedfruits contained some mitochondria and microbodies. As the transformationof chloroplasts to chromoplasts proceeded, the density of theplastids decreased from 1.096 to 1.075 g-cm^-3 and the decreasewas related to a decrease in chlorophyll and an increase inlycopene in the plastids. (Received December 21, 1983; Accepted April 20, 1984)     

Cytoplasmic inheritance of mitochondria and chloroplasts in the anisogamous brown alga Mutimo cylindricus (Phaeophyceae)

Based on the morphology of gametes, sexual reproduction in brown algae is usually classified into three types: isogamy, anisogamy, and oogamy. In isogamy, chloroplasts and chloroplast DNA (chlDNA) in the sporophyte cells are inherited biparentally, while mitochondria (or mitochondrial DNA, mtDNA) is inherited maternally. In oogamy, chloroplasts and mitochondria are inherited maternally. However, the patterns of mitochondrial and chloroplast inheritance in anisogamy have not been clarified. Here, we examined derivation of mtDNA and chlDNA in the zygotes through strain-specific PCR analysis using primers based on single nucleotide polymorphism in the anisogamous brown alga Mutimo cylindricus. In 20-day-old sporophytes after fertilization, mtDNA and chlDNA derived from female gametes were detected, thus confirming the maternal inheritance of both organelles. Additionally, the behavior of mitochondria and chloroplasts in the zygotes was analyzed by examining the consecutive serial sections using transmission electron microscopy. Male mitochondria were isolated or compartmentalized by a double-membrane and then completely digested into a multivesicular structure 2 h after fertilization. Meanwhile, male chloroplasts with eyespots were observed even in 4-day-old, seven-celled sporophytes. The final fate of male chloroplasts could not be traced. Organelle DNA copy number was also examined in female and male gametes. The DNA copy number per chloroplast and mitochondria in male gametes was lower compared with female organelles. The degree of difference is bigger in mtDNA. Thus, changes in different morphology and DNA amount indicate that maternal inheritance of mitochondria and chloroplasts in this species may be based on different processes and timing after fertilization.

     

Isolation of a cDNA encoding mitochondrial citrate synthase from Arabidopsis thaliana

We isolated a cDNA clone from Arabidopsis thaliana encoding the TCA cycle enzyme, citrate synthase. The plant enzyme displays 48% and 44% amino acid residue similarity with the pig, and yeast polypeptides, respectively. Many proteins, including citrate synthase, which are destined to reside in organelles such as mitochondria and chloroplasts, are the products of the nucleocytoplasmic protein synthesizing machinery and are imported post-translationally to the site of function. We present preliminary investigations toward the establishment of an in vitro plant mitochondrial import system allowing for future studies to dissect this process in plants where the cell must differentiate between mitochondria and chloroplast and direct their polypeptides appropriately.