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Cell Biology |
2Laboratory of Anatomy and Morphology, V. L. Komarov Botanical Institute of Russian Academy of Sciences, Prof. Popov Street 2, 197376, St. Petersburg, Russia; 3School of Biological Sciences, Washington State University, Pullman, Washington 99164-4236 USA; 4Department of Biology, Faculty of Science, University of Tehran, P.O. Box 14155-6455, Tehran, Iran
Received for publication May 19, 2005. Accepted for publication July 27, 2005.
ABSTRACT
The terrestrial plant Bienertia cycloptera has been shown to accomplish C4 photosynthesis within individual chlorenchyma cells by spatially separating the phases of carbon assimilation into distinct peripheral and central compartments. In this study, anatomical, physiological, and biochemical techniques were used to determine how this unique compartmentation develops. Western blots show ribulose-1,5-bisphosphate carboxylase (Rubisco) (chloroplastic) is present in the youngest leaves and increases during development, while levels of C4 enzymes—pyruvate,Pi dikinase (chloroplastic), phosphoenolpyruvate carboxylase (PEPC) (cytosol), and NAD-malic enzyme (mitochondrial)—increase later in development. Immunolocalization confirmed this for Rubisco and PEPC. The youngest chlorenchyma cells have a central nucleus surrounded by monomorphic granal chloroplasts containing Rubisco. Later stages show progressive development of a central cytoplasmic compartment enriched with chloroplasts and mitochondria and of a peripheral cytoplasm with chloroplasts. A complex reticulum of connections between the compartments also developed and was characterized. 
13C isotope analyses show mature leaves have distinct C4-type isotope composition, while the composition in younger leaves is "C4-like." Based on the results, this form of single-cell C4 photosynthesis develops from a common pool of organelles through partitioning to separate compartments, and the development of biochemically and ultrastructurally dimorphic chloroplasts.
Key Words: anatomy • Bienertia cycloptera • C4 photosynthesis • C4 plants • development • immunolocalization • ultrastructure
It is well known that C4 photosynthesis in terrestrial plants is associated with the presence of Kranz type leaf anatomy, which involves biochemical cooperation of two layers of chlorenchymatous cells. In all examined plants having various forms of Kranz anatomy, the carboxylation phase of the C4 cycle takes place in mesophyll cells, whereas the decarboxylation phase occurs in Kranz (or bundle sheath) cells (Edwards and Walker, 1983
; Kanai and Edwards, 1999
; Edwards et al., 2001
). This anatomical separation of biochemical events has been considered the paradigm for C4 photosynthesis. C4 species are of interest because of the specialized anatomy, questions about evolution of this type of photosynthesis, and also because it is considered that the syndrome would be especially effective for agricultural applications since only a few of the major crop species are C4.
Among dicotyledons, the family Chenopodiaceae has the largest number of C4 species, with a high diversity in the Irano-Turanian region, including central and southwest Asia (Akhani et al., 1997
). This family provides a particularly interesting group to study the evolution and anatomy of the C4 photosynthesis syndrome, not only because of the large number of C4 species, but also because of the multiple variations of Kranz anatomy within this family. The most unexpected recent finding was the occurrence of chenopods in the genera Bienertia and Borszczowia, which perform C4 photosynthesis within a single chlorenchyma cell (Edwards et al., 2004
; Akhani et al., 2005
). They have distinct anatomy, named Borszczowoid and Bienertioid (Freitag and Stichler, 2000
, 2002
; Schütze et al., 2003
) and a unique means of spatial compartmentation of the phases of C4 photosynthesis within the individual chlorenchyma cells of the leaf (Voznesenskaya et al., 2001
, 2002
; Edwards et al., 2004
). Three species with these unique features are currently known: Borszczowia aralocaspica, Bienertia cycloptera, and B. sinuspersici.
Bienertia cycloptera, which is distributed in the temperate desert of Iran and a few other localities around Iran (Akhani et al., 2003
) has the most unusual chlorenchyma cells and mechanism of C4 photosynthesis yet found in angiosperm plants (Voznesenskaya et al., 2002
). In this species, the spatial separation of the two stages of C4 photosynthesis occurs in two cellular compartments inside one chlorenchyma cell, a peripheral compartment and a central cytoplasmic compartment (CCC) (Voznesenskaya et al., 2002
). Fixation of atmospheric CO2 takes place in the peripheral cytoplasm by the activity of phosphoenolpyruvate carboxylase (PEPC). This compartment also has grana-deficient chloroplasts containing pyruvate,Pi dikinase (PPDK), which is responsible for regeneration of PEP used by PEPC for CO2 fixation. The CCC contains specialized mitochondria with the decarboxylating NAD-malic enzyme (NAD-ME) and glycine decarboxylase, and granal chloroplasts with Rubisco but little or no PPDK. Thus, the second phase of C4 photosynthesis in B. cycloptera takes place in the CCC (see Voznesenskaya et al., 2002
). The system is analogous to having a Kranz bundle sheath cell inserted in the middle of a mesophyll cell, but without cell walls separating them. The two cytoplasmic compartments appeared to be interconnected by some sort of cytoplasmic channels, which have not been characterized. The formation of two separate, but interconnected and stable, cytoplasmic compartments with completely different organelles is unique in plant cells. Characterization of how this system is formed will help our understanding of regulation of organelle distribution in cells and of differential biochemical expression in cell organelles and will be of interest to those considering the potential for engineering C4 pathways into C3 crop plants.
The purpose of this study was to analyze the development of the single-cell C4 system in B. cycloptera. Leaf anatomy is characterized with a special emphasis on development of the two compartments and their organelles during chlorenchyma cell maturation. The developmental features are correlated with accumulation of key enzymes of the C4 cycle and with full operation of the cycle as determined by carbon isotope discrimination. The results provide new and important insight into this fascinating C4 system.
MATERIALS AND METHODS
Plant material
Seeds of Bienertia cycloptera were collected in central Iran (Tehran Province) and later stored in a freezer at –18°C until the beginning of experimentation. Seeds were germinated on moist paper in petri dishes at room temperature and first maintained at room temperature in petri dishes for 2–3 d at a photosynthetic photon flux density (PPFD) of 20 µmol quanta · m2 · s–1. Seedlings were then transplanted to 10 cm pots containing a soil mixture of two parts commercial potting soil, five parts clay soil, one part sand, and 10 g dolomite powder, then grown for 3 d under 30 PPFD and 30/25°C day/night temperature regime. Beginning on day 4, plants were grown in a growth chamber (model GC-16; Enconair Ecological Chambers, Winnipeg, Manitoba, Canada) under ca. 400 PPFD with a 14 h/10 h light/ dark photoperiod and 25/15°C day/night temperature regime. Leaf samples were taken from plants that were 2–3 mo old.
Scanning electron microscopy (SEM)
Leaf samples were fixed at 4°C in 2% (v/v) paraformaldehyde and 2% (v/v) glutaraldehyde in 0.1 M phosphate buffer (pH 7.2), postfixed in 2% (w/v) OsO4, and then dehydrated in an ethanol series to 100% ethanol, cryofractured in liquid nitrogen, critical-point dried, attached to SEM mounts, sputter-coated with gold and observed with a Hitachi S570 SEM (Hitachi Scientific Instruments, Mountain View, California, USA).
Light and transmission electron microscopy
Samples were taken for general microscopy, immunolocalization, and starch analysis. Samples for ultrastructural study were fixed as for SEM and after acetone dehydration, embedded in Spurr's resin. Cross sections were made on a Reichert Ultracut R ultramicrotome (Reichert-Jung GmbH, Heidelburg, Germany). For light microscopy, semi-thin sections were stained with 1% (w/v) toluidine blue O in 1% (w/v) Na2B4O7; ultra-thin sections were stained for transmission electron microscopy with 2% (w/v) lead citrate or 1 : 2 dilution of 1% (w/v) KMnO4/ 4% (w/v) uranyl acetate. Observations were made with Hitachi H-600 (Hitachi Scientific Instruments) and JEOL-1200 EX transmission electron microscopes (JEOL USA, Inc., Massachusetts, USA).
Staining for polysaccharides
Sections, 0.8–1 µm thick, were made from the same samples, dried onto gelatin-coated slides, incubated in periodic acid (1% w/v) for 30 min, washed and then incubated with Schiff's reagent (Sigma, St. Louis, Missouri, USA) for 1 h. After rinsing, the sections were ready for observation with light microscopy.
Nuclear staining
Isolated, live chlorenchyma cells were used for the visualization of nuclei. For the preparation of chlorenchyma cells, the epidermis was removed by peeling with a fine-tip forceps, and chlorenchyma cells were collected in PME buffer (0.1 M PIPES, 5 mM MgSO4, 5 mM EGTA) supplemented with 0.3 M mannitol and 0.15 M NaCl. Nuclei were stained for 30 min with 10 µg/mL acridine orange (Sigma) in PME buffer at room temperature followed by three 10-min washes in PME. Confocal microscopy was performed on a Bio-Rad MRC 1024 laser scanning confocal microscope with Nikon Eclipse TE 300 inverted microscope (Bio-Rad, Hercules, California, USA). Images were acquired through a x40 Zeiss (Carl Zeiss, Thornwood, New York, USA) Plan-Apochromat oil-immersion. Fluorescence of acridine orange was excited at 488 nm, and emission was detected between 500 and 550 nm. Chlorophyll fluorescence was excited at 594 nm, and emission was detected between 600 and 700 nm. Image processing was performed using Photoshop 6.0 (Adobe, San Jose, California, USA).
In situ immunolocalization
Leaf samples were fixed at 4°C in 2% (v/v) paraformaldehyde and 1.25% (v/v) glutaraldehyde in 0.05 M PIPES buffer, pH 7.2. The samples were dehydrated with a graded ethanol series and embedded in London Resin White (LR White, Electron Microscopy Sciences, Fort Washington, Pennsylvania, USA) acrylic resin. Antibodies used (all raised in rabbit) were anti-spinach Rubisco (LSU) IgG (courtesy of B. McFadden), commercially available anti-maize PEPC IgG (Chemicon, Temecula, California, USA), and anti-Zea mays PPDK IgG (courtesy of T. Sugiyama). For light microscopy, cross sections, 0.8–1 µm thick, were placed onto gelatin-coated slides and blocked for 1 h with TBST + BSA (10 mM Tris-HCl, 0.15 M NaCl, 0.1% v/v Tween 20, 1% w/v bovine serum albumin, pH 7.2). They were then incubated for 3 h with the preimmune serum diluted in TBST + BSA (1 : 100), anti-Rubisco (1 : 500 dilution), anti-PEPC (1 : 200 dilution), and anti-PPDK (1 : 100) antibodies. The slides were washed with TBST + BSA and then treated for 1 h with protein A-gold (10-nm particles diluted 1 : 100 with TBST + BSA). After washing, the sections were exposed to a silver enhancement reagent (Amersham, Arlington Heights, Illinois, USA) for 20 min according to the manufacturer's directions, stained with 0.5% (w/ v) safranin O, and imaged in a reflected/transmitted mode using a Bio-Rad 1024 confocal system with a Nikon Eclipse TE 300 inverted microscope and LaserSharp imaging program (Bio-Rad). The background labeling with preimmune serum was very low, although some infrequent labeling occurred where sections were wrinkled due to trapping of antibodies/label (results not shown, but see Edwards et al., 2001
).
Chlorophyll and protein assay
Chlorophyll was extracted in 80% acetone and analyzed as described (Porra et al., 1989
). Absorbance was measured at 646.6 and 663.6 nm on a Perkin-Elmer (Norwalk, Connecticut, USA) 552A spectrophotometer with three replicates per sample. For protein assay, leaf material was ground in a Ten-Broeck homogenizer with 1 mL of 50 mM HEPES buffer (N-[2-hydroxyethyl]piperazine-N'-[ethanesulfonic acid]) (pH 7.5) and 1% polyvinylpyrrolidone. After centrifugation at 16 000 x g, the supernatant assayed for protein by measuring absorbance at 595 nm using the method of Bradford (1976)
with BSA as a standard.
Western blot analysis
Total proteins were extracted from leaf samples by homogenizing 0.2 g of tissue in 500 µL of extraction buffer (0.1 M Tris-HCl [pH 7.5], 5 mM MgSO4, 10 mM dithiothreitol, 5 mM EDTA, 0.5% [w/v] SDS, 2% [v/v] ß-mercaptoethanol, 10% [v/v] glycerol, 1 mM phenylmethylsulfonyl fluoride and 2.5 µg/mL each of aprotinin, leupeptin, and pepstatin). After centrifugation at 16 000 g for 3 min in a microcentrifuge, the supernatant was collected and protein concentration was determined with the Bradford assay as described before. Protein samples (10 µg) were separated by 12% SDS-PAGE, transferred to nitrocellulose, blocked in phosphate-buffered saline containing 1% BSA and probed with antisera raised against NAD-ME (1 : 5000), PEPC (1 : 60 000), PPDK (1 :10 000), and Rubisco (LSU; 1 : 40 000) overnight. Goat anti-rabbit IgG-alkaline phosphatase conjugate (Bio-Rad) at a dilution of 1 : 50 000 was used as the secondary antibody for detection of the enzymes. Blots were developed with 350 µg/mL nitroblue tetrazolium and 175 µg/mL 5-bromo-4-chloro-3-indolyl phosphate in detection buffer (100 mM Tris-HCl, pH 9.5, 100 mM NaCl, and 5 mM MgCl2). Rubisco and PEPC antibodies were as described before and anti-Amaranthus hypochondriacus L. mitochondrial NAD-ME IgG (65 kDa 
subunit) and anti-Zea mays L. PPDK IgG were courtesy of J. Berry and T. Sugiyama, respectively.
13C carbon isotope determination
Carbon isotope fractionation values were determined on leaf samples taken from plants using a standard procedure relative to PDB (Pee Dee Belemnite) limestone as the carbon isotope standard (Bender et al., 1973
).
RESULTS
Bienertia cycloptera, growing in a natural habitat (Fig. 1A and B) and under our controlled growth conditions (Fig. 1C), has succulent, terete leaves up to 2.5–3.5 cm long. In this study, the youngest (0.08–0.1 cm long, Fig. 1E and F), young (0.2–0.3 cm, Fig. 1D and F), intermediate (0.5–0.6 cm, Fig. 1D), and mature (about 3 cm) leaves (Fig. 1C) were sampled from ca. 1-mo-old plants. Subsequent images (from cross sections in the middle of the leaf) refer to these stages of development.
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The young leaves (ca. 0.2–0.3 cm in length) are also covered by numerous well-differentiated trichomes, mostly located at the adaxial side (Fig. 2B). By this stage of leaf development, the incipient chlorenchyma layer has divided to produce 2–3 layers of chlorenchyma (Figs. 2F, 3D, E). Most of the chlorenchyma cells are still tightly packed at this stage, but intercellular airspaces are beginning to form (Fig. 3D, E). In some chlorenchyma cells, the onset of cellular differentiation, as indicated by the initial development of the CCC, is observed. However, even when the pre-CCC appears as a well-defined ball, it is still positioned adjacent to one or more cell walls with the nucleus located on the periphery (Figs. 2F, 3D, F). All tissues layers are delineated at this stage, water storage cells are expanded with peripheral cytoplasm and large vacuoles, and some starch has accumulated in developing chlorenchyma cells (Fig. 3E).
With leaves at the intermediate stage (ca. 0.5–0.6 cm in length), other than in size, all cells appear similar in disposition and cellular contents to mature leaves. Chlorenchyma cells are oval in profile, have a well-formed CCC (Figs. 2C, G, 3G, I), and starch is restricted to the CCC (Fig. 3H). At this stage of development, nuclei are located on the periphery of the CCC, adjacent to the tangential cell wall (Fig. 3I).
In mature leaves (ca. 3 cm long) of this species there are 2–3 layers of chlorenchyma cells (Figs. 2D, 3J, K). The CCCs are well-delineated in the central region of the cell, with thin cytoplasmic channels connecting them with the peripheral part of the cell (Figs. 2H, 3J). Nuclei are positioned adjacent to the CCC, mostly close to the radial cell wall (Fig. 3L). Starch is abundant in chloroplasts in the CCC of the cell and practically absent in the peripheral chloroplasts and all other tissues (Fig. 3K). It is interesting to note that, in this species, the general outline of the leaf does not change much throughout the developmental stages (compare Fig. 2A–D).
Because the interconnection between the peripheral compartment and CCC is critical to the operation of the C4 cycle, these interconnections were further analyzed by SEM (Fig. 4). Different planes of section or fracture through the mature chlorenchyma cells clearly demonstrate that the connections occur as thin, reticulated sheets rather than as strands (Fig. 4B–F). These cytoplasmic channels occur primarily in the medial longitudinal plane of the cells (Fig. 4B), where they connect the two compartments via the shortest path length. These sheaths are penetrated by numerous apertures of various size (Fig. 4C), which provide continuity of the vacuolar space. The sheets also have junctions or branches, so that when viewed from the perspective of the outside of the axial wall of the cell, they have a honeycomb appearance (Fig. 4D–F). Chloroplasts are sometimes seen within the channels (Fig. 4D).
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In intermediate leaves, approximately 0.5–0.6 cm in length, practically all cells have a well-delineated CCC in the center of the cells, though some CCCs are adjacent to one of the radial cell walls (Fig. 5G). This is probably due to the small size of the cell, as observed with light microscopy (compare with Fig. 3G, I). There is no clear structural differentiation between the peripheral and central chloroplasts at this stage of leaf development, because the internal structure of all chloroplasts contains numerous small grana consisting of 2–7 thylakoids interconnected by intergranal thylakoids (Fig. 5H–J). Large starch grains are found in most chloroplasts (Fig. 5G, H, J). Mitochondria are well developed and are located in the central part of the CCC, surrounded by chloroplasts (Fig. 5H, I), with essentially no, or only a few, mitochondria observed in the peripheral compartment.
The chlorenchyma cells of the mature leaves have expanded considerably, and the CCC is well delineated, suspended in the center of the cell, and interconnected with a peripheral layer of cytoplasm by thin channels (Fig. 5K). The CCC contains chloroplasts having numerous grana with 3–10 thylakoids in stacks and short intergranal thylakoids (Fig. 5L, M), while peripheral chloroplasts have some grana interconnected by numerous, very long intergranal thylakoids (Fig. 5N). Some areas in peripheral chloroplasts do not contain grana at all (data not shown). The cytoplasmic channels between the two cellular compartments occasionally contain some chloroplasts (Fig. 5K). Starch grains are abundant in the CCC, with the largest starch grains located in chloroplasts on the periphery of the ball, and small, or no, grains appear in chloroplasts located in the cytoplasmic channels (Fig. 5K). Starch is rare, or occurs as very small particles, in the chloroplasts of the peripheral compartment. Numerous large mitochondria, having a dense system of tubular and lamellated cristae, are located in the center of the CCC surrounded by chloroplasts. The size of the mitochondria is about 0.8 µm, which is more than twice that in young leaves.
Pattern of accumulation of photosynthetic enzymes
To determine the accumulation of Rubisco and C4 enzymes in the various developmental stages of B. cycloptera leaf tissues, the total soluble protein was extracted, separated by SDS-PAGE, and the levels of specific proteins were examined by immunoblot analysis using antisera raised against the Rubisco large subunit, PEPC, NAD-ME, and PPDK polypeptides. In the youngest leaves, Rubisco and a small amount of PEPC are present, while the other C4 pathway proteins are low or undetectable (Fig. 6). Rubisco and PEPC polypeptides steadily accumulate with age. In contrast, only very small amounts of PPDK and NAD-ME polypeptides are seen in young and intermediate stage leaves, and only in the mature leaf are these proteins at relatively high levels (Fig. 6).
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In the mature leaf, practically all labeling for Rubisco (Fig. 7G) is concentrated in the chloroplasts of the CCC of the chlorenchyma cell; occasionally, low labeling is observed in peripheral chloroplasts of some chlorenchyma cells and in water-storage tissue. PEPC labeling is very heavy in the chlorenchyma cells of mature leaves, and it occurs throughout the cytoplasm both in the peripheral compartment, in channels, and in the CCC (Fig. 7H).
There is no specific labeling for PPDK in the youngest and young leaves, while there was some weak labeling in intermediate leaves in the peripheral chloroplasts and in the CCC (data not shown). In mature leaves, the labeling for PPDK is much higher in the chloroplasts of the peripheral part of the chlorenchyma cells, with lower labeling in the CCC (Voznesenskaya et al., 2002
).
Chlorophyll and protein assays
Table 1 shows changes in the total soluble protein and chlorophyll content during leaf development. The results show that the protein content decreases from young to mature stages of leaf development, both on a fresh and dry mass basis, being nearly three times lower in mature leaves. The content of water in leaves of different ages comprises about 87% of the fresh mass. The total chlorophyll (a + b) content on a fresh and dry mass basis remains about the same during leaf development, while the ratio of chlorophyll a/b is more than two times higher in mature than in the youngest leaves.
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13C carbon isotope determination13C values for leaves during early development (youngest to intermediate) were more negative (ca. –17.6
) than in more mature leaves (ca. –15.7). DISCUSSION
Investigations of leaf development in B. cycloptera characterized the pattern and timing of the development of the dual cytoplasmic compartments in the single-cell C4 system, and correlated this with accumulation of key enzymes required for the biochemical aspects of the system. As with most leaves with two or three palisade chlorenchyma layers, a single "incipient chlorenchyma" layer in the earliest stages undergoes anticlinal and periclinal divisions to develop 2–3 layers of palisade-like chlorenchyma cells in a complete circumferential pattern around the terete leaf. While organelles begin to be partitioned toward one side of the nucleus early on, the CCC only becomes organized after the cells begin to expand, fairly late in development of the leaf. Only in mature leaves does the CCC become located in the center of the cell, while in the earlier stages it is found adjacent to the radial cell walls. Because the CCC is a large structure, cell expansion appears to be required to accommodate its full differentiation within the cell. For the system to be fully functional, a pathway between the CCC and the peripheral compartment must be provided and it must be stable. This study shows that the pathway is achieved by formation of a complex of sheet-like cytoplasmic channels traversing the medial longitudinal regions of the cell between the CCC and the periphery. These channels are extensive, interconnected, and form a honeycomb-like structure as viewed by SEM. This structure may help stabilize the channels, as well as provide an adequate pathway for exchange of substrates and products between the two compartments, as is required in the Kranz system also, but in that case achieved by plasmodesmata. Further analysis of the volume of the connections will be required for complete modeling of the diffusion path for exchange between the two compartments. As the leaf develops, assembly of the two compartments requires a significant change in the distribution of the organelles in a common cytoplasm, as well as quite distinct biochemical and ultrastructural development of chloroplasts in the compartments. The observations presented here demonstrate that the chloroplasts are derived from a common pool of plastids distributed evenly throughout the cytoplasm in the young, incipient chlorenchyma cells. In leaves longer than 0.1 cm, we observed the initial stages of increasing chloroplast and mitochondria numbers around the nucleus. The distribution of these organelles to the perinuclear region may ensure unbiased organelle inheritance during cell division, while allowing efficient mobilization of their functions. Furthermore, development of plastids and mitochondria are strictly regulated by the nucleus (Leon et al., 1998
), and perhaps this proximity mediates rapid development required for further differentiation of organelles. In young leaves, ca. 0.2–0.3 cm in length, the cells are beginning to expand and the first stage of CCC formation is observed as an accumulation of chloroplasts and mitochondria toward one side of the nucleus. At this stage, the "central" vacuole has not fully developed in chlorenchyma cells and this "pre-CCC" in most chlorenchyma cells is adjacent to one of the cell walls. In intermediate leaves, the cells have expanded considerably, and there is clear development of the CCC in the central part of the cell, along with the first appearance of the peripheral cytoplasmic layer with chloroplasts. During this transition, most of the mitochondria have been incorporated into the CCC and few, if any, can be found in the peripheral compartment. There is no differentiation in chloroplast structure at this stage of leaf development, and the central compartment is often still adjacent to at least a small region of cell wall. In mature cells, the compartmentation is complete and differentiation of chloroplasts is now expressed, with less grana development in the peripheral chloroplasts compared to those in the CCC (Voznesenskaya et al., 2002
). There is an increase in the chlorophyll a/b ratio during development (to 4.1–4.4), which may be related to the establishment of the photosystems and to the final differentiation to form the two types of chloroplasts. This is consistent with malic enzyme type C4 plants having chlorophyll a/b ratios in this range, which is higher than that of C3 plants (Ku et al., 1974
; Edwards and Walker, 1983
). The young leaves have high soluble protein on both a fresh and dry mass basis, decreasing about threefold as leaves mature, whereas the chlorophyll content is similar. The reason for this change in protein is uncertain, although some tissues may be more active and have higher protein content when young than when mature, e.g., epidermal and water storage tissue. Since the percentage water content of the tissue remains about the same at various leaf stages (Table 1), the decrease in protein content during development may be compensated for by an increase in other macromolecules (e.g., starch, Fig. 3).
Partitioning of almost all mitochondria to the CCC during leaf development can be seen as a requirement for operation of the C4 cycle in a single cell, because the decarboxylase used, NAD-ME, is a mitochondrial protein (Voznesenskaya et al., 2002
). In mature chlorenchyma cells of this species, both the chloroplasts and mitochondria in the CCC have structural characteristics like those in bundle sheath cells of Kranz- type C4 NAD-ME chenopods, or in the proximal part of chlorenchyma cells of Borszczowia aralocaspica, subfamily Suaedoideae, which is another C4 chenopod with photosynthesis functioning in a single cell (Voznesenskaya et al., 2001
, 2003a
). The mitochondria become larger during leaf development, as was also observed in B. aralocaspica (Voznesenskaya et al., 2003a
). Their structure changes from having a translucent matrix in the center at young stages to densely packed tubular and lamellated cristae in mature leaves. The latter may simply represent a longitudinal section of elongated cristae, a characteristic common for mitochondria of many NAD-ME species in the Chenopodiaceae family (Laetsch, 1968
; Downton, 1971
; Voznesenskaya, 1976
; Shomer-Ilan et al., 1979
; Voznesenskaya and Gamaley, 1986
). While the partitioning of mitochondria into the CCC is a relatively early event, accumulation of the decarboxylase occurs after the CCC and the cell are essentially in their mature state. This likely prevents significant futile cycling of carbon fixation and decarboxylation because PEPC is expressed relatively early during development.
During leaf development of B. cycloptera, there is a transition from the presence of one ultrastructural type of chloroplast without any special orientation in the cell (young leaves) to two structural and biochemical types of chloroplasts located in separate cytoplasmic compartments (mature leaves). An intermediate stage of leaf development occurs in leaves ca. 0.5 cm long, when chloroplasts are located in two different compartments but are not yet structurally differentiated. This suggests that positional influences are important to the ultimate type of chloroplast that develops, rather than the subsequent separation of two, already differentiated chloroplast types. The patterns of structural differentiation in leaves are very similar to those observed in Borszczowia aralocaspica (Voznesenskaya et al., 2003a
), except that the organelles of the two species are located in different places in the cell. The accumulation of Rubisco occurs fairly early during leaf development and in all chloroplasts, regardless of location in the cell and before ultrastructural dimorphism occurs. In contrast, in B. aralocaspica there is increased accumulation of Rubisco in the proximally located chloroplasts in chlorenchyma cells at an intermediate stage of leaf development, and biochemical dimorphism occurs prior to structural dimorphism of chloroplasts. As the leaves of B. cycloptera reach an intermediate stage of development, there is substantial Rubisco in peripheral chloroplasts, but it is lower than that in the CCC chloroplasts; in the mature state, peripheral chloroplasts have little, or no Rubisco, as indicated by immunolabeling. This again points to the importance of intracellular location in determining patterns of development in the organelles within a single cell. In B. cycloptera, as in all previous studies with C4 chenopod species having different types of leaf anatomy (Voznesenskaya et al., 1999
, 2003b
), starch is stored in chloroplasts containing Rubisco. In intermediate leaves, starch is abundant in all chloroplasts of chlorenchyma cells, as is Rubisco. Large amounts of starch accumulates in the CCC of mature chlorenchyma cells but is low, or lacking, in the peripheral chloroplasts (Freitag and Stichler, 2000
; Voznesenskaya et al., 2001
). During leaf development in B. cycloptera, the presence of large starch granules in water-storage cells around the vascular bundle in young leaves may be the result of carbohydrate import from mature leaves (Turgeon, 1989
).
Western blot analysis gave biochemical data that supported our developmental and immunolabeling results. Very low levels of Rubisco could be detected at the youngest stage of development. Likewise, immunolabeling of sections was very low and scattered, even though chloroplasts are present around the nucleus (Fig. 5A–C) and the chlorophyll content on a fresh mass basis is similar to other stages of development (Table 1). Western blots show increased labeling of Rubisco at the young leaf stage, while immunolocalization shows all chloroplasts are labeled. Levels of Rubisco reach a maximum at the intermediate stage and remain about the same at the mature stage, when Rubisco selectively accumulates in the CCC. PEPC, a cytosolic enzyme, is very low in the youngest leaves and progressively accumulates during development in a pattern similar to Rubisco. However, the other key enzymes required for the C4 pathway, PPDK and NAD-ME, are only at low levels until maturity is reached, when there is high accumulation. Previous studies showed PPDK is located in peripheral chloroplasts and NAD-ME in mitochondria of the CCC of mature leaves (Voznesenskaya et al., 2002
). While mitochondria are clearly present in chlorenchyma cells in early stages of development (Fig. 5), the accumulation of NAD-ME in mitochondria occurs after the CCC develops. Thus, the entire pathway is not fully expressed until organelles are completely partitioned into the peripheral and central cytoplasmic compartments and organelle ultrastructural differentiation is essentially complete. This late development of C4 biochemistry in leaves indicates leaves will only function in full C4 mode later in development. If atmospheric CO2 were fixed directly by Rubisco early in development, this would discriminate against fixing 13CO2 and give C3-type carbon isotope composition. Yet, the youngest to intermediate leaves have a C4-like carbon isotope composition. This could occur if development of new leaves is largely supported by import of carbon from C4 photosynthesis in mature leaves. Tissue of B. cycloptera collected from the field has C4-type isotope composition (values ranged from –12.4 to –15.6 with a mean 13C value of –13.9 from 16 collections [Akhani et al., 2005
]), indicating its photosynthesis occurs by development of this C4 system.
This study demonstrates that the single-cell C4 syndrome in B. cycloptera involves a complex program that must coordinate organelle partitioning into two discrete pools, two different ultrastructural differentiation programs for chloroplasts in the same cell, and differential expression of enzymes both spatially and temporally. While similar events occur in the Kranz-type C4 leaf development, each cell of the dual cell system is under the direction of its own nucleus and expresses a program for only one part of the system. Studies on development of C4 photosynthesis in a few Kranz-type C4 species such as maize, amaranth, and Salsola richteri indicate that a transition occurs from monomorphic chloroplasts in the two cell types (similar in structure and in biochemistry with respect to Rubisco, so-called C3 default) followed by development of dimorphic chloroplasts and C4 biochemistry (Berry et al., 1997
; Sheen, 1999
; Voznesenskaya et al., 2003b
). The situation in Borszczowia aralacapsica (Voznesenskaya et al., 2003a
) and in B. cycloptera is complicated by the requirement for control of differential protein and ultrastructural expression within a common cytoplasm. These fascinating single-cell C4 systems will provide excellent models to study mechanisms regulating cellular polarization, both structural and biochemical. Our further studies are exploring regulation of expression, mechanism of partitioning of organelles into the two compartments, how the mature structure is maintained, and the biophysical parameters of operation of C4 photosynthesis in a single cell. These developmental studies provide important information that will aid in the design of experiments to further characterize these unusual cells.
FOOTNOTES
1 Support was received from NSF Grant IBN-0131098, NSF Grant IBN-0236959, NSF Isotope Facility Grant DBI-0116203, Civilian Research and Development Foundation Grant RB1-2502-ST-03 and the Research Council University of Tehran for "Geobotanical studies in different parts of Iran" (H.A.). We also thank the Electron Microscopy Center of Washington State University for use of facilities and for staff assistance and Alexandra Ivanova (Komarov Botanical Institute) for technical assistance. 
5 Author for correspondence (e-mail: edwardsg{at}wsu.edu
)
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