Root Plastids Research Articles

The Effect of Nitrogen Nutrition on the Cellular Localization of Glutamine Synthetase Isoforms in Barley Roots.

Glutamine synthetase (GS) was detected by immunogold localization in the cytosol and plastids of roots of 7-d-old barley (Hordeum vulgare L. cv Klaxon) seedlings grown in the presence or absence of NO3- (15 mM) or NH4+ (30 mM). The number of GS polypeptides changed during root development, and this was affected by N nutrition. There was no evidence of a NO3--inducible root plastid GS.In apical 5- to 10-mm regions of the root the concentration of immunogold labeling of cytosolic GS was higher in the cortical parenchyma than in the vascular cells of the stele, irrespective of N nutrition. This labeling was at least 50% higher in both cell types in N-free compared with N-grown (either NO3- or NH4+) seedlings. In contrast, GS specific activity was highest in roots of NO3--grown seedlings. It is suggested that this indicates the presence of inactive GS in roots grown without N. This study has identified both cell- and development-specific responses of GS to N nutrition.

Pyruvate dehydrogenase complex and acetyl-CoA carboxylase in pea root plastids: their characterization and role in modulating glycolytic carbon flow to fatty acid biosynthesis

The pyruvate dehydrogenase complex (PDC) and acetyl-CoA carboxylase (ACC, EC 6.4.1.2) have been characterized in pea root plastids. PDC activity was optimum in the presence of 1.0 mM pyruvate, 1.5 mM NAD+ 0.1 mM CoA, 0.1 mM TPP, 5 mM MgCl2, 3.0 mM cysteine-HCl, and 0.1 M Tricine (pH 8.0) and represents approximately 47% of the total cellular activity. ACC activity was greatest in the presence of 1.0 mM acetyl-CoA, 4 mM NaHCO3 mM ATP, 10 mM MgCl2, 2.5 mM dithiothreitol, and 100 mM Tricine (pH 8.0). Both enzymes were stimulated by reduced sulphydryl reagents and inhibited by sulphydryl inhibitors. ACC was also inhibited by malonyl-CoA while PDC was inhibited by both malonyl-CoA and NADH. Both enzymes were stimulated by DHAP and UDP-galactose while ACC was also stimulated by PEP and F1,6P. Palmitic acid and oleic acid both inhibited ACC, but had essentially no effect on PDC. Palmitoyl-CoA inhibited both enzymes while PA and Lyso-PA inhibited PDC, but stimulated ACC. The results presented support the hypothesis that PDC and ACC function in a co-ordinated fashion to promote glycolytic carbon flow to fatty acid biosynthesis in pea root plastids.

The Utilization of Glycolytic Intermediates as Precursors for Fatty Acid Biosynthesis by Pea Root Plastids.

Radiolabeled pyruvate, glucose, glucose-6-phosphate, acetate, and malate are all variously utilized for fatty acid and glycerolipid biosynthesis by isolated pea (Pisum sativum L.) root plastids. At the highest concentrations tested (3-5mM), the rates of incorporation of these precursors into fatty acids were 183, 154, 125, 99 and 57 nmol h-1 mg-1 protein, respectively. In all cases, cold pyruvate consistently caused the greatest reduction, whereas cold acetate consistently caused the least reduction, in the amounts of each of the other radioactive precursors utilized for fatty acid biosynthesis. Acetate incorporation into fatty acids was approximately 55% dependent on exogenously supplied reduced nucleotides (NADH and NADPH), whereas the utilization of the remaining precursors was only approximately 10 and 20% dependent on added NAD(P)H. In contrast, the utilization of all precursors was greatly dependent (85-95%) on exogenously supplied ATP. Palmitate, stearate, and oleate were the only fatty acids synthesized from radioactive precursors. Higher concentrations of each precursor caused increased proportions of oleate and decreased proportions of palmitate synthesized. Radioactive fatty acids from all precursors were incorporated into glycerolipids. The data presented indicate that the entire pathway from glucose, including glycolysis, to fatty acids and glycerolipids is operating in pea root plastids. This pathway can supply both carbon and reduced nucleotides required for fatty acid biosynthesis but only a small portion of the ATP required

Dynamics of small heat shock protein distribution within the chloroplasts of higher plants.

Accumulation of the small heat shock proteins (sHSPs) in response to high temperature stress is thought to contribute to the development of thermotolerance in eukaryotic organisms, but the mechanism of action is unknown. We are investigating the chloroplast-localized sHSP, HSP21, with the goal of defining its contribution to the acquisition of thermotolerance in plants. Following an initial heat stress and period of recovery, HSP21 is localized primarily in the soluble fraction of the chloroplast. During an additional stress, HSP21 undergoes a temperature-dependent redistribution from the soluble to the insoluble chloroplast fraction in both isolated organelles and intact plants. The change in HSP21 partitioning is accompanied by depletion of the 10-11 S HSP21-containing complexes from the soluble chloroplast fraction. HSP21 in the insoluble fraction cannot be solubilized by nonionic detergent under conditions that release essentially all the pigments and proteins from the thylakoid membranes, indicating that HSP21 in its insoluble state is not dependent for its insolubility on attachment to an intact membrane. The temperature-dependent redistribution of HSP21 is affected by light intensity but occurs in both leaf and root plastids, suggesting that the function of this activity is not strictly related to the presence of the photosynthetic apparatus. Our study indicates that the chloroplast sHSP has dynamic properties similar to those of cytoplasmic sHSPs from plants and other organisms and suggests that the ability to partition between a soluble and an insoluble state reflects a functionally important property of all sHSPs.

Digestion of Intraplastidic Hexosephosphate Isomerase by Trypsin in Preparations of Root Plastids

Summary The effect of tryptic digestion on hexosephosphate isomerase activity in pea root plastids and cytosol was investigated. Maintenance of organelle integrity conferred substantial protection from proteolysis when compared with broken organelles. Electrophoretic studies indicated the presence of two major isoenzymes of hexosephosphate isomerase in pea roots, one cytosolic the other plastidial. Further studies indicated that the loss of hexosephosphate isomerase activity from organelles during tryptic digestion is due to proteolysis of the enzyme within or released from the organelle and not loss of contaminating cytosolic activity which was not susceptible to trypsin proteolysis.

Porins from plants. Molecular cloning and functional characterization of two new members of the porin family.

Porins are voltage-gated diffusion pores found in all eukaryotic kingdoms. Here we describe, for the first time, the identification and characterization of two cDNAs encoding porins from plants. Peptide sequences obtained from a 30-kDa protein of envelope membranes from pea root plastids allowed the isolation of two cDNA clones from pea and maize. On the protein level, both proteins are homologous by 58%. Sequence comparison against the Swiss-Prot sequence data base revealed a homology of about 25% to mitochondrial porins from fungi and human. Computer-aided predictions of the secondary structure of the plant porins revealed the presence of 16 antiparallel beta-strands that are also found in mitochondrial porins. Porins from non-green plastids and from the outer mitochondrial membrane were reconstituted into planar lipid bilayers. The proteins showed high pore-forming activities and similar single-channel conductances. In vitro translated porin was preferentially imported only into non-green plastids but not into chloroplasts. To our knowledge, this is the first example of selective import of a plastid protein into different types of plastids. This finding is in line with the observation that an immunoreactive 30-kDa band was only found in non-green plastids and mitochondria but not in chloroplasts. We conclude that mitochondria and non-green plastids possess homologous porin proteins, whereas chloroplasts are characterized by a different type of porin.

The role of the triose-phosphate shuttle and glycolytic intermediates in fatty-acid and glycerolipid biosynthesis in pea root plastids

The capacity of the triose-phosphate shuttle and various combinations of glycolytic intermediates to substitute for the ATP requirement for fatty-acid and glycerolipid biosynthesis in pea (Pisum sativum L.) root plastids was assessed. In all cases, ATP gave the greatest rates of fatty-acid and glycerolipid biosynthesis. Rates of up to 66 and 27 nmol·(mg protein)−1·h−1 were observed for the incorporation of acetate and glycerol-3-phosphate into lipids in the presence of ATP. In the absence of exogenously supplied ATP, the triose-phosphate shuttle gave up to 44 and 33% of the ATP-control activity in promoting fatty-acid and glycerolipid biosynthesis from acetate and glycerol-3-phosphate, respectively. The optimum shuttle components were 2 mM dihydroxyacetonephosphate (DHAP), 2 mM oxaloacetic acid and 4 mM inorganic phosphate (referred to as the DHAP shuttle). Glyceraldehyde-3-phosphate, as a shuttle triose, was approximately 82% as effective as DHAP in promoting fatty-acid synthesis while 2-phosphoglycerate, 3-phosphoglycerate, and phosphoenolpyruvate were only 27–37% as effective as DHAP. When glycolytic intermediates were used as energy sources for fatty-acid synthesis, in the absence of both exogenously supplied ATP and the triose-phosphate shuttle, phosphoenolpyruvate, 2-phosphoglycerate, fructose-6-phosphate and glucose-6-phosphate each gave 48%, 17%, 23% and 17%, respectively, of the ATP-control activity. Other triose phosphates tested were much less effective in promoting fatty-acid synthesis. When exogenously supplied ATP was supplemented with the DHAP shuttle or glycolytic intermediates, the complete shuttle increased fatty-acid biosynthesis by 37% while DHAP alone resulted in 24% stimulation. Glucose-6-phosphate, fructose-6-phosphate and glycerol-3-phosphate similarly all improved the rates of fatty-acid synthesis by 20–30%. In contrast, 3-phosphoglycerate, 2-phosphoglycerate and phosphoenolpyruvate all inhibited fatty-acid synthesis by approximately 10% each. The addition of the DHAP shuttle and glycolytic intermediates with or without exogenously supplied ATP caused an increase in the proportion of radioactive oleate and a decrease in the proportion of radioactive palmitate synthesized. The use of these alternative energy sources resulted in higher amounts of free fatty acids and triacylglycerol, and lower amounts of diacylglycerol and phosphatidic acid. The data presented here indicate that ATP is superior in promoting in-vitro fatty-acid biosynthesis in pea root plastids; however, both the triose-phosphate shuttle and glycolytic metabolism can produce some of the ATP required for fatty-acid biosynthesis in these plastids.

Tissue-specific expression of the large ATP synthase gene cluster in spinach plastids

Plastids present in different tissues may vary morphologically and functionally, despite the fact that all plastids within the same plant contain identical genomes. This is achieved by regulation of expression of the plastid genome by tissue-specific factors, the mechanisms of which are not fully understood. The proton translocating ATP synthase/ATPase is a multisubunit complex composed of nine subunits, six encoded in the plastid and three in the nucleus. We have investigated the tissue-specific expression of the large ATP synthase gene cluster in spinach (Spinacia oleracea). This gene cluster encodes four of the six plastid-encoded ATP synthase genes. Transcript abundance, transcriptional activity, and transcript stability were investigated relative to gene dosage in root plastids and in stem, leaf, and flower chloroplasts. All three of these factors display significant tissue-specific variation. It was intriguing to discover that, although transcript abundance normalized to gene dosage varies in each tissue, transcript abundance as a proportion of the entire plastid RNA population in each tissue is not significantly different. Thus it appears that in these tissues the variation in transcription and stability of transcripts derived from the large ATP synthase gene cluster balances to yield an equivalent proportion of these transcripts in the plastid RNA population. Expression of this gene cluster in photosynthetic as well as non-photosynthetic tissues may facilitate the plasticity of structure and function which is characteristic of plastids.

The role of the triose-phosphate shuttle and glycolytic intermediates in fatty-acid and glycerolipid biosynthesis in pea root plastids

The role of the triose-phosphate shuttle and glycolytic intermediates in fatty-acid and glycerolipid biosynthesis in pea root plastids

The Purification and Properties of Ferredoxin-NADP+-Oxidoreductase from Roots of Pisum sativum L.

A ferredoxin-NADP+-oxidoreductase (FNR) was purified to homogeneity from pea root plastids to a specific activity of 200 nkat · mg protein−1, following acetone precipitation and ferredoxin affinity chromatography. The molecular weight of the enzyme was estimated to be 36,000 and 33,800 by SDS-polyacrylamide gel electrophoresis and molecular exclusion chromatography, respectively. The absorption spectrum of the enzyme suggests it contains flavin as a prosthetic group. The enzyme requires NADPH and did not use NADH as an electron donor. The Km values for NADPH and ferredoxin were calculated to be 28 and 5 μM, respectively. The enzyme exhibited optimal activity at pH 8.0. Although resembling the leaf enzyme in most properties, amino terminal sequencing demonstrates clear differences between the leaf and root proteins and suggests closer homology of the pea root enzyme with the enzyme from spinach roots. A polyclonal antibody against the pea root plastid enzyme was raised by the immunization of rabbits. Judging by immunodiffusion only partial identity was observed between the root plastid and chloroplast FNR. The root plastid FNR enzyme activity was precipitated with increasing concentrations of the antibody, in contrast to the chioroplast enzyme which was not inhibited. The potential usefulness of these antibodies is discussed.

ADP/ATP Translocator from Pea Root Plastids (Comparison with Translocators from Spinach Chloroplasts and Pea Leaf Mitochondria).

The kinetic properties of the adenosine 5[prime]-diphosphate/adenosine 5[prime]-triphosphate (ADP/ATP) translocator from pea (Pisum sativum L.) root plastids were determined by silicone oil filtering centrifugation and compared with those of spinach (Spinacia oleracea L.) chloroplasts and pea leaf mitochondria. In addition, the ADP/ATP transporting activities from the above organelles were reconstituted into liposomes. The Km(ATP) value of the pea root ADP/ATP translocator was 10 [mu]M and that for ADP was 46 [mu]M. Corresponding values of the spinach ADP/ATP translocator were 25 [mu]M and 28 [mu]M, respectively. Comparable results were obtained for the reconstituted ATP transport activities. The transport was highly specific for ATP and ADP. Adenosine 5[prime]-monophosphate (AMP) caused only a slight inhibition and phosphoenolpyruvate and inorganic pyrophosphate caused no inhibition of ATP uptake. With pea root plastids and spinach chloroplasts, Km values >1 mM were obtained for ADP-glucose. Since the concentrations of ATP and ADP-glucose in the cytosolic compartment of spinach leaves have been determined as 2.5 and 0.6 mM, respectively, a transport of ADP-glucose by the ADP/ATP translocator does not appear to have any physiological significance in vivo. Although both the plastidial and the mitochondrial ADP/ATP translocators were inhibited to some extent by carboxyatractyloside, no immunological cross-reactivity was detected between the plastidial and the mitochondrial proteins. It seems probable that these proteins derive from different ancestors.

A 330 bp region of the spinach nitrite reductase gene promoter directs nitrate‐inducible tissue‐specific expression in transgenic tobacco

Nitrite reductase is an enzyme in the nitrate assimilatory pathway whose expression is induced upon the addition of nitrate. Furthermore, it is known to be located in chloroplasts in leaves and plastids in roots. A 3.1 kb 5′ upstream region of the spinach nitrite reductase (NiR) gene promoter was shown previously to confer nitrate inducibility on the β‐glucuronidase (GUS) reporter gene expression in both the leaves and the roots of transgenic tobacco plants. In the present study, this 3.1 kb promoter fragment as well as a series of promoter deletion constructs, fused to a GUS gene, were utilized to delineate the region of NiR promoter involved in nitrate regulation of NiR expression by studying the cellular localization of NiR—GUS expression as well as its regulation by nitrate. In plants carrying the longest promoter fragment (−3100 from the transcription start site) and promoter sequences progressively deleted to −330 bp, the expression of GUS was markedly increased in the presence of nitrate, and this expression was found to occur in mesophyll cells in leaves and in the vascular tissues of stem and roots. When nitrate was added to NiR—GUS plants grown in the absence of nitrate, significant levels of GUS activity could be seen in the roots after 2 h and in the leaves after 6 h. Further 5′ deletion of the promoter to −200 bp abolished the nitrate induction of GUS expression, indicating that the 130 bp region of the nitrite reductase promoter located between −330 and −200 is required for full nitrate‐inducible tissue‐specific expression.

Glycolytic enzymes in non-photosynthetic plastids of pea (Pisum sativum L.) roots

The presence of the glycolytic enzymes from hexokinase to pyruvate kinase in plastids of seedling pea (Pisum sativum L.) roots was investigated. The recoveries, latencies and specific activities of each enzyme in different fractions was compared with those of organelle marker enzymes. Tryptic-digestion experiments were performed on each enzyme to determine whether activities were bound within membranes. The results indicate that hexokinase (EC 2.7.1.2) and phosphoglyceromutase (EC 5.4.2.1) are absent from pea root plastids. The possible function of the remaining enzymes is considered.

Induction of ferredoxin‐NADP+ oxidoreductase and ferredoxin synthesis in pea root plastids during nitrate assimilation

SummaryA 14.5 kDa protein with antigenic components in common with pea leaf ferredoxin was detected on transblots of the soluble proteins of pea root plastids. The amount of this protein was found to increase during the induction of nitrate assimilation in pea roots, reaching a maximal level at 8–12 h. Concurrent with this, a fourfold increase in NADPH‐dependent ferredoxin‐NADP+ oxidoreductase (FNR) activity was observed corresponding to an increase in the amount of this protein detected immunologically on transblots using a leaf FNR antibody. These changes were not observed in plastids from roots of plants grown on ammonia or depleted of nitrogen. It is suggested that in addition to the already well reported induction by nitrate of nitrate reductase and nitrite reductase, there is a co‐induction of a plastid located ferredoxin and FNR. Both these proteins are necessary for the transfer of reductant generated by the oxidative pentose phosphate pathway to nitrite reductase.

Localization of [gamma]-Glutamylcysteine Synthetase and Glutathione Synthetase Activity in Maize Seedlings.

Fresh weight, protein, cysteine, [gamma]-glutamylcysteine, glutathione, and the extractable activity of the enzymes of glutathione biosynthesis, [gamma]-glutamylcysteine synthetase (EC 6.3.2.2) and glutathione synthetase (EC 6.3.2.3), were measured in roots, scutella, endosperms, and shoots of 3-, 7-, and 11-d-old maize (Zea mays L. cv LG 9) seedlings. In 3-d-old seedlings, the scutella represented 14% of the seedling fresh weight, containing 43% of total protein and 63 and 55% of the activity of [gamma]-glutamylcysteine synthetase and glutathione synthetase, respectively; in 11-d-old seedlings, the corresponding values were 4.5% for fresh weight, 8.0% for protein content, and 14 and 20% for the enzyme activities. The highest concentrations of thiols were found for cysteine (0.27 mM) in the roots, for glutathione (4.4 mM) in the shoots, and for [gamma]-glutamylcysteine (13 [mu]M) in the scutella of 3-d-old seedlings. The enzyme activities of roots were localized in subcellular fractions after sucrose density gradient centrifugation. Nearly half of the [gamma]-glutamylcysteine synthetase activity was detected in the root proplastids of 4-d-old seedlings, whereas <10% of the glutathione synthetase activity was localized in this organelle. Our results demonstrate the importance of scutella in glutathione synthesis in the early stage of seedling development. Unlike chloroplasts, root plastids show only a small proportion of glutathione synthetase activity.

Studies of the Enzymic Capacities and Transport Properties of Pea Root Plastids.

Plastids have been isolated from pea (Pisum sativum L.) roots with a high degree of purity and intactness. In these plastids, the activity of enzymes involved in carbohydrate metabolism have been analyzed and corrected for cytosolic contamination. The results show that fructose-1,6-bisphosphatase, NAD-glyceraldehyde phosphate dehydrogenase, and phosphoglyceromutase are not present in pea root plastids. Transport measurements revealed that inorganic phosphate, dihydroxyacetone phosphate (DHAP), 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, and glucose-6-phosphate (Glc6p) are transported across the envelope in a counterexchange mode. Transport of glucose-1-phosphate was definitely excluded. The oxidation of Glc6P by intact plastids resulted almost exclusively in the formation of DHAP. The parallel measurement of DHAP formation and NO2- consumption during Glc6P-supported nitrite reduction yielded a ratio of NO2-reduced/DHAP formed of 1.6, which is relatively close to the theoretical value of 2.0. These results show that the oxidation of Glc6P, involving the uptake of Glc6P and the release of DHAP, and the reduction of NO2- are very tightly coupled to each other.

Reductant for glutamate synthase in generated by the oxidative pentose phosphate pathway in non‐photosynthetic root plastids

SummaryPurified pea root plastids were supplied with glutamine, 2‐oxoglutarate and phosphorylated sugars. Formation of glutamate was linear for 75 min and dependent upon the intactness of the organelle. Glucose‐6‐phosphate and ribose‐5‐phosphate were the most effective substrates in supporting glutamate synthesis. Flux through the oxidative pentose phosphate pathway during glutamate synthesis in purified plastids was followed by monitoring the release of 14CO2 from [1‐14C]glucose‐6‐phosphate. 14CO2 evolution from C‐1 was dependent upon the presence of both glutamine and 2‐oxoglutarate and could be inhibited by the application of azaserine. The data are discussed in view of the role of the oxidative pentose phosphate pathway in non‐photosynthetic plastids.

Energy Requirements for Fatty Acid and Glycerolipid Biosynthesis from Acetate by Isolated Pea Root Plastids

Fatty acid and glycerolipid biosynthesis from [(14)C]acetate by isolated pea root plastids is completely dependent on exogenously supplied ATP. CTP, GTP, and UTP are ineffective in supporting fatty acid biosynthesis, all resulting in <3% of the activity obtained with ATP. However, ADP alone or in combination with inorganic phosphate (Pi) or pyrophosphate (PPi) gave up to 28% of the ATP control activity, whereas AMP + PPi, PPi alone, or Pi alone were ineffective in promoting fatty acid biosynthesis. The components of the dihydroxyacetonephosphate (DHAP) shuttle (DHAP, oxaloacetate, and Pi), which promote intraplastidic ATP synthesis, restored 41% of the control ATP activity, whereas the omission of any of the shuttle components abolished this activity. When the DHAP shuttle components were supplemented with ADP, the rate of fatty acid biosynthesis was completely restored to that observed in the presence of ATP. Under the conditions of ADP + DHAP shuttle-driven fatty acid biosynthesis, exogenously supplied ATP gave only a 6% additional stimulation of activity. In general, variations in the energy source had only small effects on the proportions of radioactive fatty acids and glycerolipids synthesized. Most notably, higher amounts of radioactive oleic acid, free fatty acids, and diacylglycerol and lower amounts of phosphatidic acid were observed when ADP and/or the DHAP shuttle were substituted for ATP. The results presented here indicate that, although isolated pea root plastids readily utilize exogenously supplied ATP for fatty acid biosynthesis, these plastids can also synthesize sufficient ATP when provided with the appropriate cofactors.

Characterization of Fatty Acid Biosynthesis in Isolated Pea Root Plastids

Fatty acid biosynthesis from Na[1-(14)C]acetate was characterized in plastids isolated from primary roots of 7-day-old germinating pea (Pisum sativum L.) seeds. Fatty acid synthesis was maximum at 82 nanomoles per hour per milligram protein in the presence of 200 micromolar acetate, 0.5 millimolar each of NADH, NADPH, and coenzyme A, 6 millimolar each of ATP and MgCl(2), 1 millimolar each of MnCl(2) and glycerol-3-phosphate, 15 millimolar KHCO(3), 0.31 molar sucrose, and 0.1 molar Bis-Tris-propane, pH 8.0, incubated at 35 degrees C. At the standard incubation temperature of 25 degrees C, fatty acid synthesis was essentially linear for up to 6 hours with 80 to 120 micrograms per milliliter plastid protein. ATP and coenzyme A were absolute requirements, whereas divalent cations, potassium bicarbonate, and reduced nucleotides all variously improved activity two- to 10-fold. Mg(2+) and NADH were the preferred cation and nucleotide, respectively. Glycerol-3-phosphate had little effect, whereas dithiothreitol and detergents generally inhibited the incorporation of [(14)C]acetate into fatty acids. On the average, the principal radioactive products of fatty acid biosynthesis were approximately 39% palmitic, 9% stearic, and 52% oleic acid. The proportions of these fatty acids synthesized depended on the experimental conditions.

Tissue and Cellular Distribution of Glutamine Synthetase in Roots of Pea (Pisum sativum) Seedlings

The effect of nitrate application on glutamine synthetase activity in roots of pea (Pisum sativum L.) seedlings (2 weeks old) was studied. Separation of organelles from root fragments by sucrose density-gradient centrifugation revealed that both nitrite reductase and glutamine synthetase activities increased in root plastids as a response to nitrate application and that no such response was induced by ammonium application. Glutamine synthetase activity was also found to increase in plastids with distance from apex in nitrate-treated plants, the highest specific activity being located in the fourth 1-centimeter segment. Separation by SDS-PAGE and characterization by Western blotting showed that cytosolic glutamine synthetase contains one subunit polypeptide (28 kilodaltons) and that plastid glutamine synthetase contains both the 38-kilodalton subunit and a heavier subunit. When nitrate was present in the nutrient solution, the heavier subunit increased in abundance in protein fractions obtained from purified root plastids.