Metabolic Activities in Extracts of Mesophyll and Bundle Sheath Cells of Panicum miliaceum (L.) in Relation to the C(4) Dicarboxylic Acid Pathway of Photosynthesis

The activities of certain enzymes related to the carbon assimilation pathway in whole leaves, mesophyll cell extracts, and bundle sheath extracts of the C₄ plant Panicum miliaceum have been measured and compared on a chlorophyll basis. Enzymes of the C₄ dicarboxylic acid pathway—phosphoenolpyruvate carboxylase and NADP-malic dehydrogenase—were localized in mesophyll cells. Carbonic anhydrase was also localized in mesophyll cell extracts. Ribose 5-phosphate isomerase, ribulose 5-phosphate kinase, and ribulose diphosphate carboxylase—enzymes of the reductive pentose phosphate pathway—were predominantly localized in bundle sheath extracts. High activities of aspartate and alanine transaminases and glyceraldehyde-3-P dehydrogenase were found about equally distributed between the photosynthetic cell types. P. miliaceum had low malic enzyme activity in both mesophyll and bundle sheath extracts.     

Distribution of Photosynthetic Enzymes between Mesophyll, Specialized Parenchyma and Bundle Sheath Cells of Arundinella hirta

Arundinella hirta L. is a C₄ plant having an unusual C₄ leaf anatomy. Besides mesophyll and bundle sheath cells, A. hirta leaves have specialized parenchyma cells which look morphologically like bundle sheath cells but which lack vascular connections and are located between veins, running parallel to them. Activities of phosphoenolpyruvate and ribulose-1,5-bisphosphate carboxylases and phosphoenolpyruvate carboxykinase, NADP-and NAD-malic enzymes were determined for whole leaf extracts and isolated mesophyll protoplasts, specialized parenchyma cells, and bundle sheath cells. The data indicate that A. hirta is a NADP-malic enzyme type C₄ species. In addition, specialized parenchyma cells and bundle sheath cells are enzymatically alike. Compartmentation of enzymes followed the C₄ pattern with phosphoenolpyruvate carboxylase being restricted to mesophyll cells while ribulose-1,5-bisphosphate carboxylase and decarboxylating enzymes were restricted to bundle sheath and specialized parenchyma cells.     

Metabolism of epidermal tissues, mesophyll cells, and bundle sheath strands resolved from mature nutsedge leaves

Mesophyll cells and bundle sheath strands were isolated from Cyperus rotundus L. leaf sections infiltrated with a mixture of cellulase and pectinase followed by a gentle mortar and pestle grind. The leaf suspension was filtered through a filter assembly and mesophyll cells and bundle sheath strands were collected on 20-μm and 80-μm nylon nets, respectively. For the isolation of leaf epidermal strips longer leaf cross sections were incubated with the enzymes and gently ground as above. Loosely attached epidermal strips were peeled off with forceps. The upper epidermis, which lacks stomata, could be clearly distinguished from the lower epidermis which contains stomata. Microscopic evidence for identification and assessment of purity is provided for each isolated tissue.Enzymes related to the C₄-dicarboxylic acid cycle such as phosphoenolpyruvate carboxylase, malate dehydrogenase (NADP⁺), pyruvate, P_i dikinase were found to be localized, ≥98%, in mesophyll cells. Enzymes related to operating the reductive pentose phosphate cycle such as RuDP carboxylase, phosphoribulose kinase, and malic enzyme are distributed, ≥99%, in bundle sheath strands. Other photosynthetic enzymes such as aspartate aminotransferase, pyrophosphatase, adenylate kinase, and glyceraldehyde 3-P dehydrogenase (NADP⁺) are quite active in both mesophyll and bundle sheath tissues.Enzymes involved in photorespiration such as RuDP oxygenase, catalase, glycolate oxidase, hydroxypyruvate reductase (NAD⁺), and phosphoglycolate phosphatase are preferentially localized, ≥84%, in bundle sheath strands.Nitrate and nitrite reductase can be found only in mesophyll cells, while glutamate dehydrogenase is present, ≥96%, in bundle sheath strands.Starch- and sucrose-synthesizing enzymes are about equally distributed between the mesophyll and bundle sheath tissues, except that the less active phosphorylase was found mainly in bundle sheath strands. Fructose-1,6-diP aldolase, which is a key enzyme in photosynthesis and glycolysis leading to sucrose and starch synthesis, is localized, ≥90%, in bundle sheath strands. The glycolytic enzymes, phosphoglyceromutase and enolase, have the highest activity in mesophyll cells, while the mitochondrial enzyme, cytochrome c oxidase, is more active in bundle sheath strands.The distribution of total nutsedge leaf chlorophyll, protein, and PEP carboxylase activity, using the resolved leaf components, is presented. ¹⁴CO₂ Fixation experiments with the intact nutsedge leaves and isolated mesophyll and bundle sheath tissues show that complete C₄ photosynthesis is compartmentalized into mesophyll CO₂ fixation via PEP carboxylase and bundle sheath CO₂ fixation via RuDP carboxylase. These results were used to support the proposed pathway of carbon assimilation in C₄-dicarboxylic acid photosynthesis and to discuss the individual metabolic characteristics of intact mesophyll cells, bundle sheath cells, and epidermal tissues.     

Photosynthesis in Flaveria brownii, a C(4)-Like Species: Leaf Anatomy, Characteristics of CO(2) Exchange, Compartmentation of Photosynthetic Enzymes, and Metabolism of CO(2)

Light microscopic examination of leaf cross-sections showed that Flaveria brownii A. M. Powell exhibits Kranz anatomy, in which distinct, chloroplast-containing bundle sheath cells are surrounded by two types of mesophyll cells. Smaller mesophyll cells containing many chloroplasts are arranged around the bundle sheath cells. Larger, spongy mesophyll cells, having fewer chloroplasts, are located between the smaller mesophyll cells and the epidermis. F. brownii has very low CO₂ compensation points at different O₂ levels, which is typical of C₄ plants, yet it does show about 4% inhibition of net photosynthesis by 21% O₂ at 30°C. Protoplasts of the three photosynthetic leaf cell types were isolated according to relative differences in their buoyant densities. On a chlorophyll basis, the activities of phosphoenolpyruvate carboxylase and pyruvate, Pi dikinase (carboxylation phase of C₄ pathway) were highest in the larger mesophyll protoplasts, intermediate in the smaller mesophyll protoplasts, and lowest, but still present, in the bundle sheath protoplasts. In contrast, activities of ribulose 1,5-bisphosphate carboxylase, other C₃ cycle enzymes, and NADP-malic enzyme showed a reverse gradation, although there were significant activities of these enzymes in mesophyll cells. As indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the banding pattern of certain polypeptides of the total soluble proteins from the three cell types also supported the distribution pattern obtained by activity assays of these enzymes. Analysis of initial ¹⁴C products in whole leaves and extrapolation of pulse-labeling curves to zero time indicated that about 80% of the CO₂ is fixed into C₄ acids (malate and aspartate), whereas about 20% of the CO₂ directly enters the C₃ cycle. This is consistent with the high activity of enzymes for CO₂ fixation by the C₄ pathway and the substantial activity of enzymes of the C₃ cycle in the mesophyll cells. Therefore, F. brownii appears to have some capacity for C₃ photosynthesis in the mesophyll cells and should be considered a C₄-like species.     

Carbonic Anhydrase Activity and Localization in Some Plant Species

The aim of this work concerned the study of the differences in the carbonic anhydrase activity and localization between plant species, the photosynthesis of which is carried out according to the C₃ and C₄ pathways respectively. The measurement of enzymatic activity was made with a titrimetric evaluation of the rate of the reaction CO₂+ H₂O ? H⁺+ HCO^?₃. The C₃ plant species showed higher activities than the C₄ species. The localization of carbonic anhydrase was carried out with a histochemical method. The carbonic anhydrase appeared in the chloroplasts both in the mesophyll and the bundle sheath without any difference between C₃ and C₄ plants.     

Lipid peroxidation and the degradation of cytochrome P-450 heme

The enzyme content and functional capacities of mesophyll chloroplasts from Atriplex spongiosa and maize have been investigated. Accompanying evidence from graded sequential blending of leaves confirmed that mesophyll cells contain all of the leaf pyruvate, P_i dikinase, and PEP carboxylase activities and a major part of the adenylate kinase and pyrophosphatase. 3-Phosphoglycerate kinase, NADP glyceraldehyde-3-P-dehydrogenase, and triose-P isomerase activities were about equally distributed between mesophyll and bundle sheath cells but other Calvin cycle enzymes were very largely or solely located in bundle sheath cells. In A. spongiosa extracts of predominantly mesophyll origin the proportion of the released pyruvate, P_i dikinase, adenylate kinase, pyrophosphatase, 3-phosphoglycerate kinase, and NADP glyceraldehyde-3-P dehydrogenase retained in pelleted chloroplasts was similar but varied between 30 and 80% in different preparations. The proportion of these enzymes and NADP malate dehydrogenase recovered in maize chloroplast preparations varied between 15 and 35%. Washed chloroplasts retained most of the activity of these enzymes but ribulose diphosphate carboxylase and other Calvin cycle enzyme activities were undetectable. Among the evidence for the integrity of these chloroplasts was their capacity for light-dependent conversion of pyruvate to phosphoenolpyruvate and O₂ evolution when 3-phosphoglycerate or oxaloacetate were added. These results support our previous conclusions about the function of mesophyll chloroplasts in C₄-pathway photosynthesis and clearly demonstrate that they lack Calvin cycle activity.     

Effects of Polyploidy on Photosynthetic Rates, Photosynthetic Enzymes, Contents of DNA, Chlorophyll, and Sizes and Numbers of Photosynthetic Cells in the C(4) Dicot Atriplex confertifolia

Photosynthetic rates, chlorophyll content, and activities of several photosynthetic enzymes were determined per cell, per unit DNA, and per unit leaf area in five ploidal levels of the C₄ dicot Atriplex confertifolia. Volumes of bundle sheath and mesophyll protoplasts were measured in enzymatic digestions of leaf tissue. Photosynthetic rates per cell, contents of DNA per cell, and activities of the bundle sheath enzymes ribulose 1,5-bisphosphate carboxylase (RuBPC) and NAD-malic enzyme per cell were correlated with ploidal level at 99% or 95% confidence levels, and the results suggested a near proportional relationship between gene dosage and gene products. There was also a high correlation between volume of mesophyll and bundle sheath cells and the ploidal level. Contents of DNA per cell, activity of RuBPC per cell, and volumes of cells were correlated with photosynthetic rate per cell at the 95% confidence level. The mesophyll cells did not respond to changes in ploidy like the bundle sheath cells. In the mesophyll cells the chlorophyll content per cell was constant at different ploidal levels, there was less increase in cell volume than in bundle sheath cells with an increase in ploidy, and there was not a significant correlation (at 95% level) of phosphoenolpyruvate carboxylase activity or content and pyruvate,Pi dikinase activity with increase in ploidy. The number of photosynthetic cells per unit leaf area progressively decreased with increasing ploidy from diploid to hexaploid, but thereafter remained constant in octaploid and decaploid plants. Numbers of cells per leaf area were not correlated with cell volumes. The mean photosynthetic rates per unit leaf area were lowest in the diploid, similar in 4×, 6×, and 8×, and highest in the decaploid. The photosynthetic rate per leaf area was highly correlated with the DNA content per leaf area.     

SPECIALIZATION OF MESOPHYLL MORPHOLOGY IN RELATION TO C4 PHOTOSYNTHESIS IN THE POACEAE

Our current understanding of the photosynthetic process in species utilizing the C₄ photosynthetic pathway suggests that photosynthetic efficiency should be enhanced by: 1) maximizing the conductance of the gas phase transport pathway from the leaf exterior to the mesophyll cell surfaces; 2) maximizing cytoplasmic connections and metabolite transport between bundle sheath and mesophyll parenchyma cells; and 3) minimizing the conductance of the gas phase transport pathway from the bundle sheath cells to the leaf exterior. In this study we have examined several species in the Poaceae with C₄ photosynthesis to determine if there is any evidence for anatomical specialization which would lead to enhanced photosynthetic efficiency by these processes. Observations with light and scanning electron microscopes revealed specializations in mesophyll cell morphology and arrangement which include branched cells forming intercellular channels. These specializations are hypothesized to contribute to photosynthetic efficiency through its influence on the above transport processes.     

Two-Dimensional Electrophoretic Mapping of Proteins of Bundle Sheath and Mesophyll Cells of the C(4) Grass Digitaria sanguinalis (L.) Scop. (Crabgrass)

Two-dimensional electrophoresis was performed on proteins of bundle sheath and mesophyll cells isolated from the C₄ grass Digitaria sanguinalis (L.) Scop. Two-dimensional maps of these proteins were constructed and ribulose-1,5-biphosphate carboxylase and phosphoenolpyruvate carboxylase were identified. Of the total number of proteins found in both cell types, 36% were found only in bundle sheath cells, 17% only in mesophyll cells, and 47% in both cell types. By comparison, the distributions of 48 enzymes assayed in these cell types were 35%, 21%, and 44%, respectively.

Protein patterns were also compared with C₄ plants exhibiting different decarboxylation pathways and, in both bundle sheath and mesophyll cells, proteins were found which were unique to each species. Bundle sheath proteins of one C₄ species were found to be more like bundle sheath proteins of another C₄ species than like mesophyll proteins of the same species.

     

C4 photosynthesis in C3 rice: a theoretical analysis of biochemical and anatomical factors

Engineering C₄ photosynthesis into rice has been considered a promising strategy to increase photosynthesis and yield. A question that remains to be answered is whether expressing a C₄ metabolic cycle into a C₃ leaf structure and without removing the C₃ background metabolism improves photosynthetic efficiency. To explore this question, we developed a 3D reaction diffusion model of bundle‐sheath and connected mesophyll cells in a C₃ rice leaf. Our results show that integrating a C₄ metabolic pathway into rice leaves with a C₃ metabolism and mesophyll structure may lead to an improved photosynthesis under current ambient CO₂ concentration. We analysed a number of physiological factors that influence the CO₂ uptake rate, which include the chloroplast surface area exposed to intercellular air space, bundle‐sheath cell wall thickness, bundle‐sheath chloroplast envelope permeability, Rubisco concentration and the energy partitioning between C₃ and C₄ cycles. Among these, partitioning of energy between C₃ and C₄ photosynthesis and the partitioning of Rubisco between mesophyll and bundle‐sheath cells are decisive factors controlling photosynthetic efficiency in an engineered C₃–C₄ leaf. The implications of the results for the sequence of C₄ evolution are also discussed.     

Microbody enzymes and carboxylases in sequential extracts from c(4) and c(3) leaves

A seven-step sequential grinding procedure was applied to leaves of Atriplex rosea, Sorghum sudanense, and Spinacia oleracea to study the distribution of carboxylases and microbody enzymes. In the extracts from C₄ species there were 7- to 10-fold reciprocal changes in specific activities of ribulose-1, 5-diphosphate carboxylase and phosphoenolpyruvate carboxylase. No such changes occurred in sequential extracts from spinach. No inhibitors of ribulose-1, 5-diphosphate carboxylase were detected when the mesophyll extracts of Sorghum were assayed together with spinach extracts. These results reaffirm the conclusion of others that phosphoenolpyruvate carboxylase is largely confined to the mesophyll in these species and ribulose-1, 5-diphosphate carboxylase to the bundle sheath. The specific activities of glycolate oxidase and hydroxypyruvate reductase in bundle sheath extracts were two to three times those in mesophyll fractions. Catalase behaved similarly in Atriplex rosea but in Sorghum the specific activity was virtually the same in all fractions. From the relative amounts of these enzymes present, and comparison with the data obtained from spinach, it is concluded that typical leaf peroxisomes are present in the bundle sheaths of both C₄ species and in the mesophyll of Atriplex rosea. The relative enzyme activities in the mesophyll of Sorghum suggest that the microbodies there are of the non-specialized type found in many nongreen tissues. The activities of the microbody enzymes in the bundle sheath of Sorghum seem quite inadequate to support photorespiration.     

Intercellular Localization of Assimilatory Sulfate Reduction in Leaves of Zea mays and Triticum aestivum

The intercellular distribution of assimilatory sulfate reduction enzymes between mesophyll and bundle sheath cells was analyzed in maize (Zea mays L.) and wheat (Triticum aestivum L.) leaves. In maize, a C₄ plant, 96 to 100% of adenosine 5′-phosphosulfate sulfotransferase and 92 to 100% of ATP sulfurylase activity (EC 2.7.7.4) was detected in the bundle sheath cells. Sulfite reductase (EC 1.8.7.1) and O-acetyl-l-serine sulfhydrylase (EC 4.2.99.8) were found in both bundle sheath and mesophyll cell types. In wheat, a C₃ species, ATP sulfurylase and adenosine 5′-phosphosulfate sulfotransferase were found at equivalent activities in both mesophyll and bundle sheath cells. Leaves of etiolated maize plants contained appreciable ATP sulfurylase activity but only trace adenosine 5′-phosphosulfate sulfotransferase activity. Both enzyme activities increased in the bundle sheath cells during greening but remained at negligible levels in mesophyll cells. In leaves of maize grown without addition of a sulfur source for 12 d, the specific activity of adenosine 5′-phosphosulfate sulfotransferase and ATP sulfurylase in the bundle sheath cells was higher than in the controls. In the mesophyll cells, however, both enzyme activities remained undetectable. The intercellular distribution of enzymes would indicate that the first two steps of sulfur assimilation are restricted to the bundle sheath cells of C₄ plants, and this restriction is independent of ontogeny and the sulfur nutritional status of the plants.     

Predominant localization of mitochondria enriched with glycine-decarboxylating enzymes in bundle sheath cells of Alternanthera tenella, a C3–C4 intermediate species

Mesophyll protoplasts and bundle sheath cells were prepared by enzymatic digestion of leaves of Alternanthera tenella, a C₃-C₄ intermediate species. The intercellular distribution of selected photosynthetic, photorespiratory and respiratory (mitochondrial) enzymes in these meso-phyll and bundle sheath cells was studied. The activity levels of photosynthetic enzymes such as PEP carboxylase (EC 4.1.1.31) or NAD-malic enzyme (EC 1.1.1.39) and photorespiratory enzymes such as glycolate oxidase (EC 1.1.3.1) or NADH-hydroxypyruvate reductase (EC 1.1.1.29) were similar in the two cell types. The activity levels of mitochondrial TCA cycle enzymes such as citrate synthase (EC 4.1.3.7) or fumarase (EC 4.2.1.2) were 2- to 3-fold higher in bundle sheath cells. On the other hand, the activity levels of mitochondrial photorespiratory enzymes, namely glycine decarboxylase (EC 2.1.2.10) and serine hydroxymethyltransferase (EC 2.1.2.1), were 6-9-fold higher in bundle sheath cells than in mesophyll protoplasts. Such preferential localization of mitochondria enriched with the glycine-decarboxylating system in the inner bundle sheath cells would result in efficient refixa-tion of CO₂ from not only photorespiration but also dark respiration before its exit from the leaf. We propose that predominant localization of mitochondria specialized in glycine decarboxylation in bundle sheath cells may form the basis of reduced photorespiration in this C₃-C₄ intermediate species.     

Photosynthetic Characteristics of Portulaca grandiflora, a Succulent C(4) Dicot : CELLULAR COMPARTMENTATION OF ENZYMES AND ACID METABOLISM

The succulent, cylindrical leaves of the C₄ dicot Portulaca grandiflora possess three distinct green cell types: bundle sheath cells (BSC) in radial arrangement around the vascular bundles; mesophyll cells (MC) in an outer layer adjacent to the BSC; and water storage cells (WSC) in the leaf center. Unlike typical Kranz leaf anatomy, the MC do not surround the bundle sheath tissue but occur only in the area between the bundle sheath and the epidermis. Intercellular localization of photosynthetic enzymes was characterized using protoplasts isolated enzymatically from all three green cell types.     

Mechanism of c(4) photosynthesis: a model describing the inorganic carbon pool in bundle sheath cells

A theoretical model of the composition of the inorganic carbon pool generated in C₄ leaves during steady-state photosynthesis was derived. This model gives the concentrations of CO₂ and O₂ in the bundle sheath cells for any given net photosynthesis rate and inorganic carbon pool size. The model predicts a bundle sheath CO₂ concentration of 70 micromolar during steady state photosynthesis in a typical C₄ plant, and that about 13% of the inorganic carbon generated in bundle sheath cells would leak back to the mesophyll cells, predominantly as CO₂. Under these circumstances the flux of carbon through the C₄ acid cycle would have to exceed the net rate of CO₂ assimilation by 15.5%. With the calculated O₂ concentration of 0.44 millimolar, the potential photorespiratory CO₂ loss in bundle sheath cells would be about 3% of CO₂ assimilation. Among the factors having a critical influence on the above values are the permeability of bundle sheath chloroplasts to HCO₃⁻, the activity of carbonic anhydrase within these chloroplasts, the assumed stromal volume, and the permeability coefficients for CO₂ and O₂ diffusion across the interface between bundle sheath and mesophyll cells. The model suggests that as the net photosynthesis rate changes in C₄ plants, the level and distribution of the components of the inorganic carbon pool change in such a way that C₄ acid overcycling is maintained in an approximately constant ratio with respect to the net photosynthesis rate.     

Distribution of enzymes related to C3 and C4 pathway of photosynthesis between mesophyll and bundle sheath cells of Panicum hians and Panicum milioides

Enzymes of the C₄, C₃ pathway and photorespiration have beenanalyzed for P. hians and P. milioides, which have chlorenchymatousbundle sheath cells in the leaves. On whole leaf extracts thelevels of PEP carboxylase are relatively low compared to C₄species, RuDP carboxylase is typical of C₃ species, and enzymesof photorespiratory metabolism appear somewhat intermediatebetween C₃ and C₄. Substantial levels of PEP carboxylase, RuDPcarboxylase, and photorespiratory enzymes were found in bothmesophyll and bundle sheath cells. Low levels of C₄-acid decarboxylatingenzymes may limit the capacity for C₄ photosynthesis in P. hiansand P. milioides. The results on enzyme activity and distributionbetween mesophyll and bundle sheath cells are consistent withCO₂ fixation via C₃ pathway in these two species. ¹ This research was supported by the College of Agriculturaland Life Sciences, University of Wisconsin, Madison; and bythe University of Wisconsin Research Committee with funds fromthe Wisconsin Alumni Research Foundation; and by the NationalScience Foundation Grant BMS 74-09611. (Received September 16, 1975; )     

Pyruvate, pi dikinase in bundle sheath strands as well as in mesophyll cells in maize leaves

Mesophyll protoplasts and bundle sheath strands were isolated from maize leaves. Light microscopic observation showed the preparations were pure and without cross contamination. Protein blot analysis of mesophyll and bundle sheath cell soluble protein showed that the concentration of pyruvate orthophosphate dikinase (EC 2.7.9.1) is about one-tenth as much in the bundle sheath cells as in mesophyll cells, but about eight times greater than that found in wheat leaves, on the basis of soluble protein. Phosphoenolpyruvate carboxylase (EC 4.1.1.31) was barely detectable in the bundle sheath cells, while ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) and NADP-dependent malic enzyme (EC 1.3.1.37) were exclusively present in the bundle sheath cells and were absent in the mesophyll cells. Whereas pyruvate, Pi dikinase was previously considered localized only in mesophyll cells of C₄ plants, these results clearly demonstrate the presence of appreciable quantities of the enzyme in the bundle sheath cells of the C₄ species maize.     

The distribution of carbonic anhydrase and ribulose diphosphate carboxylase in maize leaves

Extraction of maize (Zea mays) leaves by progressive grinding under suitably protective conditions yields total carbonic anhydrase activities (4800 units per milligram chlorophyll) comparable to the activity in spinach (Spinacia oleracea) leaves. The total ribulose diphosphate carboxylase activity was also equal to or greater than the best literature values for maize. Of the total leaf carbonic anhydrase, 72.5% on a chlorophyll basis was present in the mesophyll cells and 14.2% in the bundle-sheath cells. The distribution of the total leaf ribulose diphosphate carboxylase between the mesophyll and bundle-sheath cells was 42.0 and 48.7% respectively. There was three times as much total chlorophyll in extracts of the mesophyll cells compared with the bundle-sheath cells of maize. Similar results for the above distribution of the two enzymes were found using a differential grinding technique. The possible function of carbonic anhydrase in photosynthesis is discussed. The equal distribution of ribulose diphosphate carboxylase activity between the mesophyll and bundle-sheath cells casts doubt upon the hypothesis that a rigid biochemical compartmentation exists between these cell types in maize.     

Sulfur assimilation in c(4) plants: intercellular compartmentation of adenosine 5'-triphosphate sulfurylase in crabgrass leaves

The activity of adenosine 5′ triphosphate sulfurylase was determined in crabgrass mesophyll cells, bundle sheath strands, and whole leaf extracts. The enzyme was assayed by following molybdate-dependent pyrophosphate release from ATP, ³⁵SO₄²⁻ incorporation into adenosine 5′ phosphosulfate, and ATP synthesis dependent upon adenosine 5′ phosphosulfate and inorganic pyrophosphate. With all assays, greater than 90% of the activity was found in extracts from bundle sheath strands. The activities in whole leaf extracts were consistently intermediate between the activities of mesophyll and bundle sheath extracts and extract-mixing experiments gave no indication of enzyme activation or inhibition in vitro. Whole leaf activities were several hundred-fold less than concurrent measurements of ribulose 1,5-bisphosphate and phosphoenolpyruvate carboxylase activities, which is interpreted as being consistent with the relative amounts of elemental carbon and sulfur found in higher plants. A hypothesis is presented for the intercellular compartmentation of sulfur assimilation in relationship to NO₃⁻ and CO₂ assimilation in leaves of C₄ plants.