Carbon Sink-to-Source Transition Is Coordinated with Establishment of Cell-Specific Gene Expression in a C4 Plant

Plants that use the highly efficient C4 photosynthetic pathway possess two types of specialized leaf cells, the mesophyll and bundle sheath. In mature C4 leaves, the CO2 fixation enzyme ribulose-1,5-bisphosphate carboxylase (RuBPCase) is specifically compartmentalized to the bundle sheath cells. However, in very young leaves of amaranth, a dicotyledonous C4 plant, genes encoding the large subunit and small subunit of RuBPCase are initially expressed in both photosynthetic cell types. We show here that the RuBPCase mRNAs and proteins become specifically localized to leaf bundle sheath cells during the developmental transition of the leaf from carbon sink to carbon source. Bundle sheath cell-specific expression of RuBPCase genes and the sink-to-source transition began initially at the leaf apex and progressed rapidly and coordinately toward the leaf base. These findings demonstrated that two developmental transitions, the change in photoassimilate transport status and the establishment of bundle sheath cell-specific RuBPCase gene expression, are tightly coordinated during C4 leaf development. This correlation suggests that processes associated with the accumulation and transport of photosynthetic compounds may influence patterns of photosynthetic gene expression in C4 plants.     

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.     

DIFFERING ONTOGENETIC ORIGINS OF PCR (“KRANZ”) SHEATHS IN LEAF BLADES OF C4 GRASSES (POACEAE)

The origin and early development of procambium and associated ground meristem of major and minor veins have been examined in the leaf blades of seven C₄ grass species, representing different taxonomic groups and the three recognized biochemical C₄ types (NAD-ME, PCK, and NADP-ME). Comparisons were made with the C₃ species, Festuca arundinacea. In “double sheath” (XyMS+) species (Panicum effusum, Eleusine coracana, and Sporoboìus elongatus), the procambium of major veins gives rise to xylem, phloem, and a mestome sheath; associated ground meristem differentiates into PCA (“C₄ mesophyll”) tissue and the PCR (“Kranz”) sheath. Development in the C₃ species parallels this pattern, except that associated ground meristem differentiates into mesophyll and a parenchymatous bundle sheath. In contrast, major vein procambium of “single sheath” (XyMS–) species (Panicum bulbosum, Digitaria brownii, and Cymbopogon procerus) differentiates into xylem, phloem and a PCR sheath; associated ground meristem gives rise to PCA tissue. These observations of major vein development support W. V. Brown's hypothesis that the PCR sheaths of “double sheath” (XyMS+) C₄ grasses are homologous with the parenchymatous bundle sheaths of C₃ grasses, while in “single sheath” (XyMS–) C₄ species they are homologous with the mestome sheath. Although there are some similarities in the development of the major and minor vascular bundle procambium in the C₄ species examined, the ontogeny of the smaller minor veins is characterized by a precocious delineation of the PCR sheath layer that may even precede the appearance of the distinctive cytological features of ground meristem and procambium. This contracted development in minor veins appears to be related to their close spacing in mature leaves and to their comparatively late appearance during leaf ontogeny.     

COMPARATIVE BUNDLE SHEATH AND MESOPHYLL DIFFERENTIATION IN THE LEAVES OF THE C4 GRASSES PANICUM EFFUSUM AND P. BULBOSUM

The two C₄ Panicum species examined differ in C₄ acid decarboxylation type and in developmental origin of bundle sheaths in major veins of their leaf blades. In Panicum effusum R.Br. (NAD-malic enzyme type) both mesophyll (PCA) and chlorenchymatous bundle sheath (PCR) cells are derived from ground meristem. In contrast, in Panicum bulbosum H.B.K. (NADP-malic enzyme type), bundle sheath cells are derived from procambium, while mesophyll develops from ground meristem. To test the hypothesis that the developmental divergence of bundle sheath and mesophyll cells would occur earlier when these two tissues had different ontogenetic origins (in P. bulbosum) than when these tissues had the same origin (in P. effusum), the development of major veins in each species was investigated. We measured cell length and cross sectional area, plastid and mitochondrial number, plastid area, vacuole area fraction, wall thickness and fraction adjacent to intercellular space using direct and digitizer measurements of transmission electron micrographs of leaf cross sections of successive developmental stages. Many of the statistically significant changes in the structural parameters measured occurred late in development of both species. The magnitude of developmental change in P. effusum PCR cells was sometimes more dramatic, viz. changes in cell and PCR plastid area, and in mitochondrion number per cell. However, earlier divergence of PCR and PCA length and volume, and wall fraction adjacent to intercellular space in P. bulbosum than in P. effusum indicates that ontogenetic origin of PCR cells from procambium could determine the timing of at least some developmental events.     

Immunocytochemical localization of enzymes involved in the C3 and C4 pathways in the photosynthetic cells of an amphibious sedge, Eleocharis vivipara

Eleocharis vivipara link, an amphibious leafless sedge, develops traits of C₄ photosynthesis and Kranz anatomy in the terrestrial form but develops C₃-like traits with non-Kranz anatomy when submerged. The cellular localization of C₃ and C₄ enzymes in the photosynthetic cells of the two forms was investigated by immunogold labeling and electron microscopy. The terrestrial form has mesophyll cells and three kinds of bundle sheath cell, namely, parenchyma sheath cells, non-chlorophyllous mestome sheath cells, and Kranz cells. Phosphoenol-pyruvate carboxylase (PEPCase) was present in the cytosol of both the mesophyll cells and the parenchyma sheath cells, with higher-density labeling in the latter, but not in the Kranz cells. Pyruvate, Pi dikinase (PPDK) was found at high levels in the chloroplasts of both the mesophyll cells and the parenchyma sheath cells with some-what stronger labeling in the latter. This enzyme was also absent from the Kranz cells. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was found in the chloroplasts of all types of photosynthetic cell, but labeling was significantly less intense in the parenchyma sheath cells than in other types of cell. The submerged form also has three types of photosynthetic cell, as well as non-chlorophyllous mestome sheath cells, but it lacks the traits of Kranz anatomy as a consequence of modification of the cells. Rubisco was densely distributed in the chloroplasts of all the photosynthetic cells. However, PEPCase and PPDK were found in both the mesophyll cells and the parenchyma sheath cells but at lower levels than in the terrestrial form. These data reveal that the terrestrial form has a unique pattern of cellular localization of C₃ and C₄ enzymes, and they suggest that this pattern and the changes in the extent of accumulation of the various enzymes are the main factors responsible for the difference in photosynthetic traits between the two forms.Abbreviations CAM crassulacean acid metabolism - MC meso phyll cell - PSC parenchyma sheath cell - KC Kranz cell - PEP-Case phosphoenolpyruvate carboxylase - PPDK pyruvate, Pi dikinase - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - LS large subunit - RuBP ribulose-1,5-bisphosphate This study was supported by Grants-in-Aid from the Ministry of Agriculture, Forestry and Fisheries of Japan (Integrated Research Program for the Use of Biotechnological Procedures for Plant Breeding) and from the Science and Technology Agency of Japan (Enhancement of Center-of-Excellence, the Special Coordination Funds for Promoting Science and Technology). The author is grateful to Drs M. Matsuoka and S. Muto for providing the antisera and Dr. M. Samejima for his advice at the early stages of this study.     

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.     

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.     

Isolation of Intact and Functional Chloroplasts from Mesophyll and Bundle Sheath Protoplasts of the C(4) Plant Panicum miliaceum

A procedure is described for isolating and purifying mesophyll protoplasts and bundle sheath protoplasts of the C₄ plant Panicum miliaceum. Following enzymic digestion of leaf tissue, mesophyll protoplasts and bundle sheath protoplasts are released and purified by density centrifugation. The lower density of mesophyll protoplasts allowed rapid separation of the two protoplast types. Evidence for separation of mesophyll protoplasts and bundle sheath protoplasts (up to 95% purity) is provided from light microscopy (based on size difference in both chloroplasts and protoplasts), levels of marker enzymes in the preparations (i.e. pyruvate, Pi dikinase and phosphoenolpyruvate carboxylase for mesophyll and ribulose-1,5-bisphosphate carboxylase for bundle sheath), and differences in substrate-dependent O₂ evolution by chloroplasts isolated from protoplasts.     

Distribution of carboxylation and decarboxylation enzymes in isolated mesophyll cells and bundle sheath strands of C 4 plants

Mature leaves of Cyperus rotundus, Cyperus polystachyos, Digitaria decumbens, and Digitaria sanguinalis were separated, using pectinase and cellulase, into pure preparations of mesophyll cells and bundle sheath strands. Assays on these distinct leaf cell types show a clear compartmentation of phosphoenolpyruvate carboxylase, >98%, into mesophyll cells and of ribulose-1, 5-diphosphate carboxylase and malic enzyme, >98%, into the bundle sheath strands. The results clearly establish that the major CO₂ uptake in mesophyll cells is via a β-carboxylation and that both a decarboxylation and a carboxylation reaction occurs in the bundle sheath strands of plants using C₄-dicarboxylic acid photosynthesis.     

An Evaluation of Some Parameters Required for the Enzymatic Isolation of Cells and Protoplasts with CO2 Fixation Capacity from C3 and C4 Grasses

Variable factors affecting the enzymatic isolation of mesophyll protoplasts from Triticum aestivum (wheat), a C₃ gras, and mesophyll protoplasts and bundle sheath strands from Digitaria sanguinalis (crabgrass), a C₄ grass, have been examined with respect to yields and also photosynthetic capacity after isolation. Preparations with high yields and high photosynthetic capacity were obtained when small transverse leaf segments were incubated in enzyme medium in the light at 30°C, without mechanical shaking and without prior vacuum infiltration. Best results were obtained with an enzyme medium that included 0.5 M sorbitol, 1 mM MgCl₂, 1 mM KH₂PO₄, 2% cellulase and 0.1% pectinase at pH 5.5. In gerneral, leaf age and leaf segment size were important factors, with highest yields and photosynthetic capacities obtained from young leaves cut into segments less than 0.8 mm. To facilitate the cutting of such small segments, a mechanical leaf cutter is described that uniformly (± 0.05 mm) cuts leaf tissue into transverse segments of variable size (0.4–2 mm). Isolations that required more than roughly 4 h gave poor yields with reduced photosynthetic capacity; however, using the optimum conditions described, functional preparations could be roughly 2 h. High rates of light dependent CO₂ fixation by the C₄ mesophyll protoplasts required the addition of pyruvate and low levels of oxalacetate, while isolated bundle sheath strands and C₃ mesophyll protoplasts supported CO₂ fixation without added substrates. Rates of CO₂ fixation by isolated wheat protoplasts generally exceeded the reported rates of whole leaf photosynthesis. Wheat mesophyll protoplasts and crabgrass bundle sheath strands were stable when stored at 4°C while C₄ mesophyll protoplasts were stable when stored at 25°C.     

CO2 donation by malate and aspartate reduces photorespiration in Panicum milioides,A C3C4 intermediate species

Oxygen inhibition of leaf slice photosynthesis in Panicum milioides increased from 20% to 30% at 21% O₂ in the presence of maleate, a phosphoenolpyruvate carboxylase inhibitor. The increased O₂ sensitivity was completely reversed by the addition of malate and aspartate, the stable products of the phosphoenolpyruvate carboxylase reaction. The C₄ acids, malate and aspartate, also reduced O₂ inhibition of photosynthesis by isolated bundle sheath strands, but not mesophyll protoplasts. Similarly, only bundle sheath strands exhibited an active C₄ acid-dependent O₂ evolution. Compartmentation of C₄ cycle enzymes, with pyruvate, Pi dikinase in the mesophyll and NAD-malic enzyme in the bundle sheath, was demonstrated. It is concluded that reduced photorespiration in P. milioides is due to a limited potential for C₄ photosynthesis permitting an increase in pCO₂ at the site of bundle sheath ribulosebisphosphate carboxylase.     

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.     

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.     

Parallelism and diversity in multiple origins of c4 photosynthesis in the grass family

C₄ photosynthesis is thought to be an adaptation to warm environments, involving complex changes in the expression of genes governing photosynthesis, intermediary metabolism, and leaf anatomy and histology. Such complexity should be difficult to evolve, yet the pathway has arisen multiple times in the history of the flowering plants and at least four times in the grass family alone. We have used immunolocalization techniques to compare photosynthetic gene expression across all four origins, to determine which genetic changes occur in parallel and which are unique to a particular lineage. The only gene expression patterns common to all origins of the pathway are up-regulation of PEP carboxylase and down-regulation of RuBisCO in mesophyll cells. Both NAD-malic enzyme and NADP-malic enzyme are expressed in bundle sheaths. Expression patterns of light-harvesting chlorophyll a/b binding proteins and pyruvate orthophosphate dikinase appear to be lineage specific, and may be localized to bundle sheaths or to mesophyll or expressed throughout the photosynthetic tissue of the leaf. We suggest that future studies of parallel origin of the C₄ pathway concentrate on regulation of the two carboxylases, as well as the increased density of vascular tissue, which is the only histological characteristic common to all origins of the pathway.     

Differentiation of bundle sheath,mesophyll, and distinctive cells in the C4 grass Arundinella hirta (Poaceae)

The C₄ grass Arundinella hirta is characterized by unusual leaf blade anatomy: veins are widely spaced and files of bundle-sheath-like cells, the distinctive cells, form longitudinal strands that are not associated with vascular tissue. While distinctive cells (DCs) appear to function like bundle sheath cells (BSCs), they differ developmentally in two ways: they are derived from ground meristem rather than procambium and they are formed 1–2 plastochrons later. This study describes ultrastructural features of differentiating of BSCs, DCs, and associated mesophyll cells (MCs) during leaf development. BSCs and DCs differ from adjacent MCs by undergoing earlier cell enlargement, greater rates of chloroplast enlargement, reduction of chloroplast thylakoids at late stages of differentiation, more extensive starch formation, greater wall thickening, and deposition of a suberin lamella. The precocious delimitation of the bundle sheath layer is reflected in earlier BSC enlargement and vacuole growth. Derivation of DCs from ground meristem is correlated with late developmental changes in chloroplast size, wall thickness, and plasmodesmatal density. Despite these differences in timing of events, particularly at early stages, the development of the specialized structural features of BSCs and DCs is essentially similar. Thus, proximity to vascular tissue appears to be nonessential for the coordination and regulation of BSC- and MC-specific developmental events.     

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.