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Keywords:

  • C4 evolution;
  • CO2 gas exchange;
  • Kranz leaf anatomy;
  • mestome sheath;
  • photorespiration;
  • photosynthetic enzyme localization

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Alloteropsis semialata (R. Br.) Hitchcock includes both C3 and C4 subspecies: the C3 subspecies eckloniana and the C4 subspecies semialata. We examined the leaf structural and photosynthetic characteristics of these plants. A. semialata ssp. semialata showed high activities of photosynthetic enzymes involved in phosphoenolpyruvate carboxykinase-type C4 photosynthesis and an anomalous Kranz anatomy. Phosphoenolpyruvate carboxylase; pyruvate, Pi dikinase and glycine decarboxylase (GDC) were compartmentalized between the mesophyll (M) and inner bundle sheath cells, whereas ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) occurred in both cells. A. semialata ssp. eckloniana also showed an anomalous non-Kranz anatomy, in which the mestome sheath cells included abundant chloroplasts and mitochondria. Rubisco and GDC accumulated densely in the M and mestome sheath cells, whereas the levels of C4 enzymes were low. The activity levels of photorespiratory enzymes in both subspecies were intermediate between those in typical C3 and C4 plants. The values of CO2 compensation points in A. semialata ssp. semialata were within the C4 range, whereas those in A. semialata ssp. eckloniana were somewhat lower than the C3 range. These data suggest that the plants are C3-like and C4-like but not typical C3 and C4, and when integrated with previous findings, point to important variability in the expression of C4 physiology in this species complex. A. semialata is therefore an intriguing grass species with which to study the evolutionary linkage between C3 and C4 plants.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Plants adapt to various environments via particular biochemical and physiological functions. It is well known that different modes of photosynthetic metabolism, such as C3, C4 and crassulacean acid metabolism (CAM), have evolved in plants (Ehleringer & Monson 1993). It is generally thought that C4 plants evolved from C3 plants primarily in response to the reduction of atmospheric CO2 concentration during geological time (Ehleringer, Cerling & Helliker 1997). In C3 photosynthesis, the atmospheric CO2 is fixed by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Photorespiration inevitably follows, owing to the oxygenase reaction of Rubisco. C4 plants use a particular mechanism to concentrate CO2 at the reaction site of Rubisco and thereby suppress photorespiration. In general, the leaves of C4 plants exhibit a Kranz-type anatomy, in which two types of photosynthetic cells, the mesophyll (M) cells and the bundle sheath (BS) cells, are differentiated. In the M cells, atmospheric CO2 is primarily fixed into C4 acids by phosphoenolpyruvate carboxylase (PEPC). The acids are transferred into the BS cells, in which they are decarboxylated. The released CO2 is re-fixed by Rubisco and assimilated into the C3 cycle. The C3 compound generated during decarboxylation is used in the M cells to regenerate phosphoenolpyruvate (PEP) by the work of pyruvate, Pi dikinase (PPDK) (Hatch 1987; Leegood 2002). C4 plants photosynthesize more efficiently than C3 plants under conditions of high light intensity and temperature, and low CO2 conductance (Ehleringer & Monson 1993).

C4 plants occur in various taxonomic groups of monocot and dicot plants (Sage, Li & Monson 1999). The grass family (Poaceae) includes a large number of C4 species of multiple origins in the subfamilies Chloridoideae, Panicoideae and Arundinoideae (Brown 1977; Hattersley & Watson 1992). C4 plants are divided into three C4 biochemical sub-types depending on the process of decarboxylation of C4 acids: (1) nicotinamide adenine dinucleotide phosphate (NADP)–malic enzyme (ME) type, (2) nicotinamide adenine dinucleotide (NAD)–ME type and (3) phosphoenolpyruvate carboxykinase (PCK) type (Hatch 1987). The grass family includes all three C4 sub-types; the PCK-type species occur only in this family (Sage et al. 1999). Current work is investigating the phylogenetic relationships of C4 taxon with C3 taxon at the molecular level (Duvall, Noll & Minn 2001; Giussani et al. 2001; Duvall et al. 2003).

Alloteropsis semialata is a unique grass, including both Kranz and non-Kranz leaf anatomies, and therefore C3 and C4 photosynthetic modes, within this one species (Ellis 1974a,b). Gibbs Russell (1983) re-examined the taxonomic position of A. semialata by using vegetative characters and classified the Kranz and the non-Kranz forms into the subspecies semialata and eckloniana, respectively. Previous studies on the photosynthetic characteristics of the two subspecies have demonstrated that the respective C4 and C3 characteristics correspond to the activities of photosynthetic enzymes, carbon isotope ratios and CO2 gas exchange (Brown 1977; Barrett, Frean & Cresswell 1983; Frean, Ariovich & Cresswell 1983a; Frean et al. 1983b; Prendergast, Hattersley & Stone 1987). A. semialata ssp. eckloniana ranges from southern to tropical Africa; A. semialata ssp. semialata is widely distributed from southern and tropical Africa to India, southeast Asia and Australia. In South Africa, A. semialata ssp. eckloniana grows mainly in cool montane grassland with rainfall of 750 mm or more, whereas A. semialata ssp. semialata thrives in semidry hot grassland at low altitude with rainfall of 775 mm or less, surrounding the main distribution range of A. semialata ssp. eckloniana (Ellis 1974a). Liebenberg & Fossey (2001) reported that in South Africa the distributions of the two subspecies overlap and they are growing in mixed populations. Although some authors doubt that the two photosynthetic forms constitute the same species (Liebenberg & Fossey 2001), it seems certain that they are taxonomically close (Gibbs Russell 1983).

The genus Alloteropsis also represents a unique taxonomic group within the grass family. This genus belongs to the Paniceae tribe of subfamily Panicoideae and consists of five species (Hattersley & Watson 1992). Except for A. semialata, all other species of this genus are C4 grasses (Brown 1977; Hattersley & Watson 1992). A. semialata ssp. semialata is a PCK-type plant with Neurachneae-type leaf anatomy (Prendergast et al. 1987), but three of the C4 species of the genus Alloteropsis are predicted to be of the NAD–ME type (Hattersley & Watson 1992). Thus, it seems that this genus includes different C4 biochemical subtypes with the C3 form. It is not yet known how this photosynthetic diversity came about within the genus. A. semialata affords a unique opportunity to study the evolutionary relationships of C3 and C4 plants.

We examined the structural, biochemical and physiological characteristics of leaves in plants of the two subspecies of A. semialata, placing special emphasis on the immunocytochemical localization of photosynthetic and photorespiratory enzymes. The results demonstrate that the plants we examined can not be classified into either typical C3 or C4 types, but that rather they have C3-like and C4-like photosynthetic characteristics.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Plant materials

Seeds of the two subspecies of A. semialata were provided by Dr J. Lynch (Pennsylvania State University, University Park, PA, USA), who collected them from plants that grew from seeds provided by the Grassland Research Centre, Genebank, South Africa (Halsted & Lynch 1996). Tillers of plants grown from the seeds were transplanted into 4.5 L pots filled with a 1:1 mixture of field soil and a commercial soil mix for vegetables (ISEKI, Tokyo, Japan). The plants were grown in a growth chamber maintained at 27 °C during the light period (14 h) and at 20 °C during the dark period (10 h) for about 3 months. The photon irradiance was provided by metal halide lamps at a photon flux density of 400 µmol m−2 s−1 (wavelength, 400–700 nm) at plant height. The plants were watered daily. Fully expanded leaves were used for the experiments. For enzyme assays and measurements of gas exchange characteristics, several other C3 and C4 species and a C3–C4 intermediate species (Moricandia arvensis) were used as control plants. They were grown from seeds under the same conditions as those for A. semialata.

Determination of chromosome number

The somatic chromosomes in the meristematic cells of root tips were examined. The root tips were pretreated in 2 m m 8-hydroxyquinoline for 2 h at room temperature. They were fixed in acetic alcohol (1:1) for 1 h at room temperature, macerated in a mixture (pH 4.0) of 4% cellulase Onozuka RS, 1% pectolyase Y-23, 7.5 m m KCl and 7.5 m m ethylenediaminetetraacetic acid (EDTA) for 1 h at 37 °C, and mounted on a glass slide. The squashed root tips on the glass slide were refixed in acetic alcohol (1:3) then dried. These were stained with Giemsa's solution for 30 min and examined under a light microscope to determine the chromosome number.

Anatomical and ultrastructural observations

Leaf samples were collected 2–3 h after the start of the light period. Small segments taken from the middle portion of leaf blades were fixed in 3% glutaraldehyde in 50 m m sodium phosphate buffer (pH 6.8) at room temperature for 2 h. They were then washed with phosphate buffer and postfixed in 2% OsO4 in phosphate buffer for 2 h. They were then dehydrated through an acetone series and embedded in Spurr's resin (Spurr 1969). Transverse ultra-thin sections of leaves were stained with uranyl acetate and lead citrate, mounted on copper grids and viewed under a transmission electron microscope (Hitachi H-7000, Hitachi, Tokyo, Japan) at 75 kV. The sections (about 1 µm) of leaves on the glass slides were stained with toluidine blue O.

Antisera

The following antisera were used for immunogold electron microscopy: anti-pea leaf Rubisco large subunit (LS) antiserum (courtesy of S. Muto, Nagoya University, Nagoya, Japan), anti-pea leaf glycine decarboxylase (GDC) P-protein antiserum (courtesy of D. J. Oliver, University of Idaho, Moscow, ID, USA) and anti-maize leaf PEPC and PPDK antisera (courtesy of M. Matsuoka, Nagoya University). These antisera were the same as those used in previous studies of C3 and C4 species (e.g. Ueno 1992, 1998). The antisera were used at a dilution of 1:1000 for Rubisco LS and GDC P-protein and 1:500 for PEPC and PPDK.

Protein A–immunogold electron microscopy

Small segments of the leaf blades were fixed in 3% (v/v) glutaraldehyde in 50 m m sodium phosphate (pH 6.8), dehydrated through an ethanol series and embedded in Lowicryl K4M resin (Chemische Werke Lowi GmbH, Waldkraiburg, Germany) as previously described (Ueno 1992). Ultra-thin sections were collected on nickel grids coated with Formvar (polyvinylformal). The sections on grids were incubated in 0.5% (w/v) bovine serum albumin (BSA) in phosphate-buffered saline (PBS) containing 10 m m sodium phosphate (pH 7.2), 150 m m NaCl and 0.1% (v/v) Tween 20 for 30 min and then in antiserum diluted with 0.5% BSA in PBS. For the control leaf blade sections, the antiserum was replaced with a non-immune serum. The grids were washed several times with PBS and incubated in a 1:40 dilution of a suspension of 15 nm protein A–colloidal gold particles (EY Laboratories, San Mateo, CA, USA) for 30 min. After several washing with PBS and distilled water, the sections were stained with uranyl acetate and lead citrate. The density of labelling was determined by counting the gold particles on electron micrographs at 25 000 × magnification and by calculating the number per unit area (µm−2). Between 5 and 12 individual cells were examined on several immuno-labelled sections.

Enzyme assays

The leaf blades (0.25 g) were ground with 0.5 g of sea sand, 25 mg of polyvinylpyrrolidone and 1 mL of grinding medium on ice. The grinding medium contained 50 m mN-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (Hepes)–KOH (pH 7.5), 0.2 m m EDTA, 2.5 m m MgCl2, 2.5 m m MnCl2, 5 m m dithiothreitol (DTT) and 0.7% (w/v) BSA. Homogenates were filtered through a gauze, and the filtrates were centrifuged at 10 000 g for 5 min at 4 °C. For the NAD–ME assay, the filtrates were treated with 0.5% (v/v) Triton X-100 at room temperature for 5 min before centrifugation. The supernatants were used for the assay of enzyme activity. For the Rubisco assay, the enzyme was pre-incubated in the presence of 10 m m NaHCO3 and 10 m m MgCl2 at 25 °C for 10 min to obtain maximum activation. All enzymes were assayed spectrophotometrically in 1 mL reaction mixtures at 25 °C. The reaction mixtures for each enzyme were as follows:

  • 1
    PEPC[enzyme class (EC) 4.1.1.31]: 50 m m tris (hydroxymethyl)aminomethane (Tris)–HCl (pH 8.0), 4 m m DTT, 10 m m MgCl2, 10 m m NaHCO3, 0.2 m m reduced nicotinamide adenine dinucleotide (NADH), 5 U malate dehydrogenase, 2.5 m m PEP and 10 µL enzyme extract;
  • 2
    NADP–ME (EC 1.1.1.40): 25 m m Tris–HCl (pH 8.0), 5 m m DTT, 0.2 m m EDTA, 5 m m malate, 0.5 m m NADP, 10 m m MgCl2 and 10 µL enzyme extract;
  • 3
    NAD–ME (EC 1.1.1.39): 25 m m Hepes–KOH (pH 7.2), 5 m m DTT, 0.2 m m EDTA, 5 m m malate, 2 m m NAD, 25 µm NADH, 1 U malate dehydrogenase, 0.15 m m coenzyme A (CoA), 4 m m MnCl2 and 10 µL enzyme extract;
  • 4
    PCK (EC 4.1.1.49): 50 m m Hepes–KOH (pH 7.0), 5 m m DTT, 0.2 m m adenosine 5′-triphosphate (ATP), 1.2 m m oxaloacetate, 4 U pyruvate kinase, 2.5 m m MgCl2, 2.5 m m MnCl2 and 10 µL enzyme extract;
  • 5
    NADP–malate dehydrogenase (EC 1.1.1.82): 25 m m Tris–HCl (pH 8.0), 1 m m EDTA, 0.5 m m oxaloacetate, 0.2 m m reduced nicotinamide adenine dinucleotide phosphate (NADPH) and 10 µL enzyme extract;
  • 6
    NAD–malate dehydrogenase (EC 1.1.1.37): 25 m m Tris–HCl (pH 8.0), 1 m m EDTA, 0.5 m m oxaloacetate, 0.2 m m NADH and 10 µL enzyme extract;
  • 7
    Aspartate aminotransferase (EC 2.6.1.1): 50 m m Hepes–KOH (pH 8.0), 2 m m EDTA, 0.03 m m pyridoxial phosphate, 2.5 m m aspartate, 2.5 m mα-ketoglutarate, 0.2 m m NADH, 4 U malate dehydrogenase and 10 µL enzyme extract;
  • 8
    Alanine aminotransferase (EC 2.6.1.2): 50 m m Hepes–KOH (pH 8.0), 2 m m EDTA, 0.03 m m pyridoxial phosphate, 10 m m alanine, 2.5 m mα-ketoglutarate, 0.2 m m NADH, 4 U lactate dehydrogenase and 10 µL enzyme extract;
  • 9
    Rubisco (EC 4.1.1.39): 50 m m Hepes–KOH (pH 8.0), 2.5 m m DTT, 1 m m EDTA, 5 m m ATP, 5 m m phosphocreatine, 0.16 m m NADH, 2 U phosphoglyceric phosphokinase, 2 U creatine phosphokinase, 2 U glyceraldehyde-3-phosphate dehydrogenase, 0.25 m m NaHCO3, 20 m m MgCl2, 0.6 m m ribulose-1,5-bisphosphate and 10 µL enzyme extract;
  • 10
    Glycolate oxidase (EC 1.1.3.1): 33 m m Tris–HCl (pH 7.8), 2.7 m m EDTA, 0.008% (v/v) Triton X-100, 3.3 m m phenylhydrazine, 0.67 m m oxidized glutathione–Na, 0.2 m m flavin mononucleotide, 5 m m glycolic acid and 20 µL enzyme extract (Feierabend & Beevers 1972);
  • 11
    Hydroxypyruvate reductase (EC 1.1.1.29): 25 m m sodium phosphate buffer (pH 5.8), 0.001% (v/v) Triton X-100, 0.2 m m NADH, 1 m m DTT, 20 m m sodium glyoxylate and 10 µL enzyme extract (Feierabend & Beevers 1972); and
  • 12
    Catalase (EC 1.11.1.6): 50 m m potassium phosphate buffer (pH 7.2), 10 m m H2O2 and 10 µL enzyme extract.

For the kinetic properties of PEPC, the supernatants were desalted by passage through a Sephadex G-25 column (Neal & Florini 1973). The column was previously equilibrated with a buffer containing 50 m m Hepes–KOH (pH 7.5), 0.2 m m EDTA and 5 m m DTT. The reaction mixture contained 50 m m Tris–HCl (pH 8.0), 4 m m DTT, 10 m m MgCl2, 10 m m NaHCO3, 0.2 m m NADH, 5 U malate dehydrogenase, 10 µL enzyme extract and various concentrations of PEP. The kinetic constants were calculated from Lineweaver–Burke plots.

The levels of chlorophyll (Chl) were determined by the method of Arnon (1949).

Measurements of CO2 gas exchange

The net photosynthetic rate was measured with an LI-6400 portable photosynthesis system (Li-Cor, Lincoln, NE, USA). Measurements were made at a photosynthetic photon flux density (PPFD) of 1000 µmol m−2 s−1, a leaf temperature of 25 °C and a CO2 concentration of 350 µL L−1. Light within the chamber was provided by a 6400-02 light-emitting diode (LED) light source (Li-Cor). The value of the CO2 compensation point (Γ) was determined by changing the CO2 concentration in the chamber. Γ was measured at a PPFD of 300 and 1000 µmol m−2 s−1. The leaf temperature was maintained at 25 °C. Although it is known that a problem of leakage at the chamber occurs in this photosynthesis system, correction of data was not made.

Statistical analysis

Using one-way analysis of variance (anova) with the Tukey–Kramer Hamilton Standard Division (HSD) test, we tested the significance of any differences in labelling densities of photosynthetic and photorespiratory enzymes between cell types, at a probability of P < 0.05. We tested the significance of any differences in enzyme activities between the two subspecies using the Student's t-test.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Chromosome number

Previous cytological studies have indicated that A. semialata ssp. eckloniana is diploid (2n = 2x = 18), whereas A. semialata ssp. semialata can be hexaploid (2n = 6x = 54), octoploid (2n = 8x = 72) or dodecaploid (2n = 12x = 108) (Frean & Marks 1988; Liebenberg & Fossey 2001). From the count of chromosome number in root tip preparations, A. semialata ssp. eckloniana was found to have 2n = 18 chromosomes, whereas A. semialata ssp. semialata had 2n = 54 chromosomes.

Inner leaf structure

The leaf blades of A. semialata ssp. semialata showed an anomalous Kranz-type anatomy (Fig. 1a,c), called ‘Neurachneae type’ (Hattersley & Watson 1992). Two types of BS surrounded the vascular bundles. The M cells did not show the distinct radial arrangement that is generally seen in the M cells of C4 plants (Dengler & Nelson 1999). The inner BS cells were filled with abundant chloroplasts and mitochondria (Fig. 2b). These organelles did not show a constant intracellular location. The chloroplasts had well-developed grana (Fig. 2b). The cell walls of the inner BS cells were thicker than those of the M and outer BS cells (Fig. 2a), and suberized lamellae occurred in the outer tangential walls and the outer half of radial walls (Fig. 2b). The outer BS cells were smaller than the inner BS cells, and the quantity of organelles was less than that in the inner BS cells (Fig. 1a,c). The chloroplasts also had well-developed grana (Fig. 2a,b). No suberized lamellae was observed in the cell walls of the outer BS cells (Fig. 2a). The M cells included chloroplasts with well-developed grana and smaller mitochondria than in the inner BS cells (Fig. 2a,b).

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Figure 1. Leaf anatomy of the two subspecies of Alloteropsis semialata: (a) A. semialata ssp. semialata. (b) A. semialata ssp. eckloniana. (c) A small and a large vascular bundle of A. semialata ssp. semialata. (d) A small vascular bundle of A. semialata ssp. eckloniana. (e) A large vascular bundle of A. semialata ssp. eckloniana. (a,b) Cross-sections from samples embedded in Spurr's resin. (c–e) Hand-cut sections. M, mesophyll cell; OBS, outer bundle sheath cell; IBS, inner bundle sheath cell. Scale bar = 100 µm.

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image

Figure 2. Ultrastructure leaf anatomy of the two subspecies of Alloteropsis semialata: (a) A mesophyll (M) cell and outer bundle sheath (OBS) cells of A. semialata ssp. semialata. Part of an inner bundle sheath (IBS) cell is also seen on the right margin. (b) IBS cells and an OBS cell of A. semialata ssp. semialata. (c) An M cell of A. semialata ssp. eckloniana. (d) IBS cells of A. semialata ssp. eckloniana. (e) IBS and OBS cells of A. semialata ssp. eckloniana. c, chloroplast; mt, mitochondrion; P, peroxisome; s, starch grain; SL, suberized lamella. Scale bars for (a–d) = 2 µm, (e) = 4 µm.

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The leaf blades of A. semialata ssp. eckloniana showed an anomalous non-Kranz-type anatomy (Fig. 1b,d,e). In this subspecies also two types of BS surrounded the vascular bundle, but the inner BS cells were smaller than the outer. The outer and inner BS cells are apparently similar to the parenchyma sheath and the mestome sheath, respectively, in leaves of C3 grasses. However, the inner BS cells differed from typical mestome sheath cells of C3 grasses by including considerable amounts of photosynthetic organelles. In hand-cut sections, the inner BS cells clearly included chloroplasts (Fig. 1d,e). The chloroplasts in the inner BS cells were smaller than those in the M cells, but were almost the same in size as those of the outer BS cells (Fig. 2c–e). The mitochondria were somewhat larger than those in the outer BS cells (Fig. 2e). In the inner BS cells, suberized lamellae occurred in the cell walls in a similar manner as that seen in the inner BS cells of A. semialata ssp. semialata (Fig. 2d). The outer BS cells included small numbers of organelles (Fig. 2e), a general feature of the parenchyma sheath in C3 grass leaves (Yoshimura, Kubota & Ueno 2004). The M cells included chloroplasts with well-developed grana (Fig. 2c). The M cells neighbouring the outer BS cells, which showed a stick-like shape in the cross-section, were radially arranged.

The leaf blades of A. semialata ssp. eckloniana had wider interveinal distances [mean ± standard deviation (SD), 231.0 ± 41.5 µm, n = 30] than those of A. semialata ssp. semialata (87.0 ± 20.5 µm, n = 30), as seen in comparisons of most C3 and C4 leaves (Dengler & Nelson 1999). The value in A. semialata ssp. eckloniana fell in the lower range of C3 grasses (mean, 303.8 µm), whereas the value of A. semialata ssp. semialata corresponded more closely to NADP–ME grasses (89.2 µm) than to NAD–ME and PCK grasses (142.0 µm) (Kawamitsu et al. 1985).

Immunolocalization of photosynthetic and photorespiratory enzymes

In the leaf blades of A. semialata ssp. semialata, the labelling for PEPC was found in the cytosol of the M and outer BS cells (Fig. 3a,b), but not in those of the inner BS cells (Fig. 3c). The density of labelling was somewhat higher in the cytosol of the outer BS cells than in that of the M cells (Table 1). The labelling for PPDK occurred in the chloroplasts of the M and outer BS cells (Fig. 3d,e), but not in those of the inner BS cells (Fig. 3f). The density of labelling was clearly higher in the chloroplasts of the outer BS cells than in those of the M cells (Table 1). The labelling for Rubisco LS was found in all chloroplasts in the three types of photosynthetic cells (Fig. 3g–i; Table 1). The labelling for the P-protein of GDC, which is the key enzyme of the glycolate pathway and is involved in the decarboxylation of glycine, was found in the mitochondria of the inner BS cells (Fig. 3l), but none or only weak labelling was found in those of the M and outer BS cells (Fig. 3j,k; Table 1).

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Figure 3. Immunogold labelling of photosynthetic and photorespiratory enzymes in the leaves of Alloteropsis semialata ssp. semialata. Phosphoenolpyruvate carboxylase (PEPC) in (a) a mesophyll (M) cell, (b) an outer bundle sheath (OBS) cell and (c) an inner bundle sheath (IBS) cell. (d–f) Pyruvate, Pi dikinase (PPDK). (g–i) Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS). (j–l) Glycine decarboxylase (GDC) P-protein. c, chloroplast; s, starch grain; mt, mitochondrion. Scale bars = 0.5 µm.

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Table 1.  Immunogold labelling of photosynthetic and photorespiratory enzymes in the photosynthetic cells of the two subspecies of Alloteropsis semialata
Subspecies and enzymeCell fractionNumber of gold particles (µm−2)
M cellsOBS cellsIBS cells
  1. The number of gold particles per square micrometre is given as mean ± standard deviation. The number of organelles or cell profiles examined is given in parentheses. Within the same row, the different letters indicate a significant difference at P < 0.05.

  2. M, mesophyll; OBS, outer bundle sheath; IBS, inner bundle sheath; PEPC, phosphoenolpyruvate carboxylase; PPDK, pyruvate, Pi dikinase; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase; LS, large subunit; GDC, glycine decarboxylase.

Alloteropsis semialata ssp. semialata
 PEPCCytosol 60.9 ± 13.5 (9)a 80.7 ± 9.8 (7)b  1.7 ± 0.8 (10)c
Organelles  2.5 ± 0.8 (8)  0.5 ± 0.6 (5)  0.7 ± 0.2 (10)
 PPDKChloroplasts 54.5 ± 11.7 (12)a106.0 ± 15.5 (9)b  1.4 ± 0.8 (13)c
Cytosol + other organelles  0.3 ± 0.5 (8)  0.5 ± 0.3 (9)  1.1 ± 0.4 (5)
 Rubisco LSChloroplasts 88.2 ± 13.9 (11)a 95.3 ± 13.2 (13)a 99.8 ± 16.6 (13)a
Cytosol + other organelles  0.4 ± 0.4 (9)  0.3 ± 0.3 (12)  0.8 ± 0.4 (6)
 GDC P-proteinMitochondria 13.3 ± 11.4 (37)a 10.2 ± 10.7 (23)a 50.9 ± 20.9 (42)b
Cytosol + other organelles  0.6 ± 0.4 (12)  1.5 ± 0.8 (9)  1.2 ± 0.8 (10)
Alloteropsis semialata ssp. eckloniana
 Rubisco LSChloroplasts116.2 ± 7.7 (12)a104.4 ± 29.8 (11)a184.8 ± 34.7 (12)b
Cytosol + other organelles  1.7 ± 2.1 (7)  1.6 ± 2.0 (7)  1.1 ± 0.7 (11)
 GDC P-proteinMitochondria106.5 ± 25.3 (53)a 28.2 ± 10.9 (19)b222.0 ± 48.8 (38)c
Cytosol + other organelles  0.3 ± 0.2 (12)  1.4 ± 1.2 (9)  0.6 ± 0.4 (11)

In the leaf blades of A. semialata ssp. eckloniana, no significant labelling for PEPC or PPDK was found in the three types of photosynthetic cells (Fig. 4a–f). However, the dense labelling for Rubisco LS occurred in all chloroplasts in these cells (Fig. 4g–i). The density of labelling was higher in the chloroplasts of the inner BS cells than in those of the M and outer BS cells (Table 1). The labelling for the P-protein of GDC was found in the mitochondria of the three types of photosynthetic cells (Fig. 4j–l), but the density of labelling was variable among the mitochondria of these cells (Table 1). The density was highest in the inner BS cells and lowest in the outer BS cells, where only weak labelling was found.

image

Figure 4. Immunogold labelling of photosynthetic and photorespiratory enzymes in the leaves of Alloteropsis semialata ssp. eckloniana. Phosphoenolpyruvate carboxylase (PEPC) in (a) a mesophyll (M) cell, (b) an outer bundle sheath (OBS) cell and (c) an inner bundle sheath (IBS) cell. (d–f) Pyruvate, Pi dikinase (PPDK). (g–i) Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS). (j–l) Glycine decarboxylase (GDC) P-protein. P, peroxisome; c, chloroplast; mt, mitochondrion; s, starch grain. Scale bars = 0.5 µm.

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Activities of photosynthetic and photorespiratory enzymes

As would be expected, a high activity of PEPC was found in the leaf blades of A. semialata ssp. semialata(Table 2). Of the C4 acid decarboxylating enzymes, PCK showed the highest activity. The activity level of NADP–malate dehydrogenase was very low, whereas those of NAD–malate dehydrogenase, alanine aminotransferase and aspartate aminotransferase were high. These data indicate that A. semialata ssp. semialata belongs with PCK-type C4 plants, although NADP–ME showed higher activity than in the PCK C4 control plant, Panicum maximum.

Table 2.  Activities of photosynthetic and photorespiratory enzymes in the leaves of the two subspecies of Alloteropsis semialata and the control C3 and C4 grasses
EnzymeA. semialata ssp. semialata (µmol [mg Chl]−1 h−1)A. semialata ssp. eckloniana (µmol [mg Chl]−1 h−1)Panicum maximum (C4) (µmol [mg Chl]−1 h−1)Triticum aestivum (C3) (µmol [mg Chl]−1 h−1)
  1. The values are given as the means ± standard deviation of three to five plants. The different letters indicate significant difference at P < 0.05 between the two subspecies.

  2. Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase; PEPC, phosphoenolpyruvate carboxylase; NADP, nicotinamide adenine dinucleotide phosphate; ME, malic enzyme; PCK, phosphoenolpyruvate carboxykinase; NAD, nicotinamide adenine dinucleotide; ND, not determined.

Rubisco   221 ± 61a   204 ± 63a   292 ± 8   243 ± 19
PEPC 1 471 ± 390a   118 ± 32b 1 086 ± 53    58 ± 16
NADP–ME    93 ± 24a    58 ± 9b     6 ± 1    21 ± 3
NAD–ME    45 ± 12a     9 ± 2b    61 ± 8    13 ± 4
PCK   432 ± 37near 0   679 ± 56ND
NADP–malate dehydrogenase    16 ± 7a    14 ± 2aNDND
NAD–malate dehydrogenase 9 641 ± 1504a 2 918 ± 362b 5 093 ± 537  3 776 ± 722
Alanine aminotransferase   534 ± 16 a   455 ± 89a   344 ± 62    200 ± 49
Aspartate aminotransferase 2 327 ± 437 a   165 ± 30b 2 355 ± 170     43 ± 12
Glycolate oxidase    45 ± 19 a    49 ± 4 a    11 ± 1     85 ± 8
Hydroxypyruvate reductase   482 ± 92 a   348 ± 9b   241 ± 35    574 ± 124
Catalase72 200 ± 19 500a96 600 ± 17 100a27 300 ± 2400119 800 ± 12 900

In the leaf blades of A. semialata ssp. eckloniana, the activity level of the PEPC was lower than in A. semialata ssp. semialata, whereas the activity level of Rubisco was almost the same as that in A. semialata ssp. semialata (Table 2). No activity of PCK was detected, and activity levels of other C4 enzymes were generally lower than those in A. semialata ssp. semialata. However, there were no significant differences in activity levels of NADP–malate dehydrogenase and alanine aminotransferase between the two subspecies. The activity levels of PEPC and NADP–ME were higher than those in the C3 control plant, wheat.

We also examined the activities of three photorespiratory enzymes – glycolate oxidase, hydroxypyruvate reductase and catalase – which are localized in peroxisomes (Ps) (Table 2). There were no significant differences in the activity levels of glycolate oxidase and catalase between A. semialata ssp. semialata and eckloniana. However, the activity levels of these enzymes were intermediate between those in the C3 and C4 control plants.

Michaelis constant (Km) PEP values of PEPC

We measured the Km PEP values of PEPC in A. semialata ssp. semialata and eckloniana. The Km value in A. semialata ssp. semialata was 1.21 ± 0.01 m m (mean ± SD, n = 3), whereas that in A. semialata ssp. eckloniana was 0.22 ± 0.04 m m (n = 3). These values are within the respective ranges of Km (PEP) values of PEPC in C4 and C3 plants (Ting & Osmond 1973; Engelmann et al. 2003).

Gas exchange characteristics

Under both PPFDs, A. semialata ssp. semialata showed near-zero values of Γ, which is typical of C4 plants, as seen in the Γ-values of the C4 control species (Table 3) (Brown & Morgan 1980). Under a high PPFD, A. semialata ssp. eckloniana showed a somewhat lower Γ-value than the values of the C3 control species, but a higher value than that of the C3–C4 intermediate, M. arvensis. The Γ-value of C3–C4 intermediate plants increases as PPFD is reduced, but that of C3 and C4 plants is unaffected (Brown & Morgan 1980; Holaday, Harrison & Chollet 1982; Ueno et al. 2003). No significant difference was found in the Γ-values of A. semialata ssp. eckloniana at different PPFDs, although the value in M. arvensis was increased under a low PPFD (Table 3).

Table 3.  Photosynthetic rate and effect of PPFD on the CO2 compensation point (Γ) in the leaves of the two subspecies of Alloteropsis semialata and the control C3 and C4 plants
SpeciesPhotosynthetic rate (µmol m−2 s−1)Γ (µmol mol−1)Ratio of high to low PPFD
High PPFDLow PPFD
  1. The values are given as the means ± SD of four measurements. High and low PPFDs are 1000 and 300 µmol m−2 s−1, respectively.

  2. PPFD, photosynthetic photon flux density; ND, not determined.

A. semialata ssp. semialata12.0 ± .8near 0near 00
A. semialata ssp. eckloniana13.6 ± 1.735.1 ± 1.136.6 ± 1.10.96
Arundinella hirta (C4)21.7 ± 1.7 2.8 ± 2.0NDND
Sorghum bicolor (C4)22.0 ± 1.1 1.0 ± 0.7near 00
Moricandia arvensis (C3–C4)22.0 ± 0.518.9 ± 4.232.7 ± 5.10.58
Avena sativa (C3)14.6 ± 1.843.1 ± 3.240.3 ± 0.61.07
Glycine max (C3)11.7 ± 1.647.2 ± 1.646.6 ± 2.11.01

The photosynthetic rate was almost the same in the two subspecies (Table 3). That in A. semialata ssp. semialata was lower than those in the C4 control species and the C3–C4 intermediate. That in A. semialata ssp. eckloniana was almost the same as those in the C3 control species. These data corresponded with those reported by Halsted & Lynch (1996) but differed from those by Barrett et al. (1983), in which A. semialata ssp. semialata showed higher photosynthetic rate than A. semialata ssp. eckloniana.

DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Photosynthetic mode of A. semialata ssp. semialata

A. semialata ssp. semialata showed an anomalous Kranz-type leaf anatomy. The structural features of the inner BS cells were typical of the BS cells of PCK-type C4 grasses. However, the inner BS cells were filled with numerous organelles without a constant intracellular location (Frean et al. 1983a). These features clearly differ from that of the typical PCK grasses, in which the chloroplasts are centrifugally located (Hattersley & Browning 1981; Dengler & Nelson 1999; Yoshimura et al. 2004).

Our immunocytochemical study revealed that PEPC, PPDK and GDC P-protein showed the cellular patterns of expression typical of C4 plants (Hatch 1987; Yoshimura et al. 2004). However, Rubisco occurred in all types of photosynthetic cells. This cellular pattern differs from that of typical C4 plants, in which Rubisco is restricted to the BS cells (Hatch 1987; Leegood 2002). It is evident that the labelling of Rubisco in the M and outer BS cells is not an artefact, because in our previous immunocytochemical study with the same antiserum for Rubisco LS we confirmed Rubisco expression specific to the BS cells in many C4 species (Ueno 1998). In addition, we confirmed by Western blot analysis that the antiserum specifically recognizes Rubisco LS of A. semialata ssp. semialata leaves. The cellular patterns of the C3 and C4 enzymes in A. semialata ssp. semialata were similar to those reported in the C4-like terrestrial forms of the amphibious Eleocharis species (Ueno 2001, 2004). There was also a tendency for PEPC and PPDK to be more strongly expressed in the cells (the outer BS cells) neighbouring the vascular bundles than in distant cells (the M cells), as found in the Eleocharis species (Ueno 2004). The inner leaf structure of A. semialata ssp. semialata is somewhat similar to that of Aristida species having M cells and double chlorenchymatous BS (Ueno 1992). However, the intercellular localization of photosynthetic enzymes in A. semialata ssp. semialata differed from that of Aristida species in which the PEPC and Rubisco are compartmentalized between the M cells and the double BS (Ueno 1992).

The data on enzymatic activities suggested that A. semialata ssp. semialata could be classified into the PCK type, which corresponded with the result from a previous study (Prendergast et al. 1987). Frean et al. (1983b) reported that the decarboxylation mode of A. semialata ssp. semialata may change depending on growth temperature: plants grown at a high temperature inside a greenhouse (28/10 °C) showed a high activity of NADP–ME, but plants grown at a low temperature outside the greenhouse (16/4 °C) had a high activity of PCK. It is interesting that the A. semialata ssp. semialata plants we examined have a higher activity of NADP–ME than the PCK grasses previously examined. Prendergast et al. (1987) reported the NADP–ME activities of 12.0 ± 7.2 µmol [mg Chl]−1 h−1 in PCK grasses (mean ± SD  of  15  species)  and  16.2 ± 5.2 µmol [mg  Chl]−1  h−1 in A. semialata ssp. semialata from Australia. A. semialata ssp. semialata had the BS cells in the mestome sheath position and dense vasculature, which are characteristic of leaves in the NADP–ME grasses (Brown 1977; Kawamitsu et al. 1985). It is unknown whether a relatively high activity of NADP–ME found in A. semialata ssp. semialata is related to the possession of such leaf structural features.

A. semialata ssp. semialata displayed higher activities of the photorespiratory enzymes glycolate oxidase and catalase than those reported in 28 species of C4 grasses (12.9 ± 5.5 and 32 500 ± 13 000 µmol [mg Chl]−1 h−1, respectively) (Ueno, Yoshimura & Sentoku 2005). This may reflect that A. semialata ssp. semialata has C4-like characteristics but not complete C4 characteristics. At present, it is unknown whether the Rubisco protein in the M and outer BS cells is active. The Γ of A. semialata ssp. semialata was very low, irrespective of light intensity. This would be due to the localization of GDC P-protein in the inner BS cells and the tightness of cell walls to CO2 leakage, in addition to the operation of the C4 cycle. Any CO2 leaking from the inner BS cells would be refixed by the PEPC of the M and outer BS cells. In the C3–C4 intermediate grass Neurachne minor, C4-like values of Γ have been reported, but there is no evidence of the operation of the C4 cycle, as demonstrated by C3-like δ13C values (Hattersley et al. 1986). The leaf anatomical features of N. minor are similar to those in A. semialata ssp. semialata. Presumably, such structural features and the localization of GDC P-protein to the BS cells cause the reduced values of Γ without high PEPC activity in N. minor (Hattersley et al. 1986; Brown & Hattersley 1989). As far as we know (Sage et al. 1999), this is the first report of a C4-like plant in the grass family. Note that A. semialata ssp. semialata is the first C4-like plant with PCK biochemistry, because C4-like plants of the genus Flaveria have an NADP–ME biochemistry (Cheng et al. 1989) and those in Eleocharis have an NAD–ME biochemistry (Ueno 2001, 2004).

Photosynthetic mode of A. semialata ssp. eckloniana

The anatomical structure of the leaves of A. semialata ssp. eckloniana was also anomalous and differed from that of typical C3 grasses. The outer BS cells showed typical features of the C3-type parenchyma sheath, whereas the inner BS cells included abundant chloroplasts and mitochondria. The features of the inner BS cells are somewhat similar to those found in the BS cells of C3–C4 intermediate plants (Edwards & Ku 1987; Brown & Hattersley 1989). The inner BS cells were somewhat larger than the mestome sheath cells of typical C3 grasses and included the suberized lamellae in the cell walls, as in the C3-type mestome sheath cells (Hattersley & Browning 1981). A comparison of the anatomical features of the leaves of A. semialata ssp. semialata and eckloniana with those of C3 grasses suggests that the inner BS cells of A. semialata ssp. semialata and eckloniana may have originated from the mestome sheath cells of C3 grasses, as proposed by Ellis (1974a) and Brown (1977). It seems that the inner BS cells of A. semialata ssp. eckloniana represent an advanced condition of C3-type mestome sheath cells. Frean et al. (1983a) reported that A. semialata ssp. eckloniana showed non-Kranz leaf anatomy and was almost devoid of chloroplasts in the inner BS (mestome sheath) cells, differing from the result of A. semialata ssp. eckloniana plants that we examined. In addition, the M cells of A. semialata ssp. eckloniana were radially arranged, surrounding the vascular bundle, also differing from the arrangement of M cells in typical C3 grasses.

The chloroplasts of the inner BS cells accumulated much Rubisco, as did the chloroplasts of the M cells. More interestingly, the mitochondria of the inner BS cells accumulated much GDC P-protein, with higher labelling density rather than in the M mitochondria, although the M accumulated more GDC P-protein than the inner BS because of the higher volume ratio of the M to the inner BS. The cellular distribution pattern of GDC P-protein differed from that found in C3–C4 intermediate plants, in which the protein is localized only in the BS mitochondria (Hylton et al. 1988). However, it is similar to the cellular pattern that has recently been reported in hybrid plants between a C3–C4 intermediate and a C3 species of the Brassicaceae (Ueno et al. 2003). The activities of photosynthetic enzymes lay in the ranges of C3 plants, although the activities of PEPC and NADP–ME were slightly higher than those in typical C3 plants. The Km PEP values of the PEPC were within the range of those in C3 plants (Ting & Osmond 1973; Engelmann et al. 2003). A. semialata ssp. eckloniana had lower activities of glycolate oxidase and catalase than those reported in seven species of C3 grasses (87.5 ± 29.4 and 130 000 ± 36 300 µmol [mg Chl]−1 h−1, respectively) (Ueno et al. 2005).

Typically, C3 plants have Γ-values > 40 µmol mol−1, whereas C4 plants have Γ-values < 5 µmol mol−1 (Edwards & Ku 1987). Barrett et al. (1983) reported typical C3Γ-values (about 50 µmol mol−1) for A. semialata ssp. eckloniana. Thus, the Γ-value of A. semialata ssp. eckloniana we examined was lower than those in typical C3 plants and in the A. semialata ssp. eckloniana examined by Barrett et al (1983). However, the Γ-value of A. semialata ssp. eckloniana was independent of light intensity. Altogether, we conclude that the A. semialata ssp. eckloniana we examined is a C3-like plant with weak C3–C4 intermediate characteristics.

A.semialata is a unique grass that evolutionarily links C3 and C4 plants

The present study showed that the two subspecies of A. semialata can be classified into neither true C3 nor true C4 types. An immunohistochemical study on A. semialata ssp. semialata plants growing in Australia has indicated that Rubisco is localized in the BS cells but not in the M and outer BS cells (Hattersley, Watson & Osmond 1977), suggesting that this A. semialata ssp. semialata plant is a true C4 plant. This result contrasts with our immunogold results, which suggest that A. semialata ssp. semialata includes both C4-like and true C4 types. Previous studies (Ellis 1974a; Brown 1977; Hattersley & Watson 1992) reported that in A. semialata plants growing in Africa, there are several intermediates between A. semialata ssp. eckloniana and semialata with respect to leaf anatomy and carbon isotope ratios. At present, it is unknown whether these intermediate plants represent transitional conditions of evolution of photosynthetic mode or originate from natural hybridization between A. semialata ssp. eckloniana and semialata (Hattersley & Watson 1992).

The plants we examined were confirmed to be diploid (2n = 2x = 18) for A. semialata ssp. eckloniana, and hexaploid (2n = 6x = 54) for A. semialata ssp. semialata. It seems unlikely that the plants originated from natural hybridization. This and previous studies suggest that A. semialata may include plants with different photosynthetic modes, namely C3, C3-like, C3-C4 intermediate, C4-like and C4 modes. Such variability in the photosynthetic modes in A. semialata may be comparable with the species-level variation in the photosynthetic modes found in the genus Flaveria (Edwards & Ku 1987). More strict taxonomic studies will be required to determine whether the forms of A. semialata constitute a single species. Nevertheless, A. semialata represents a unique grass that evolutionarily links C3 and C4 plants.

Plasticity between C3 and C4 modes has been reported in only a few plants, although CAM plants are well known to switch photosynthetic modes depending on environmental conditions (Ehleringer & Monson 1993). The amphibious leafless species of Eleocharis differentiate between C4-like mode under terrestrial conditions and C3 or C3–C4 intermediate mode under water (Ueno 2001, 2004). In this case, changes in the water environment induce the conversion of photosynthetic mode. In the C3–C4 intermediate species Mollugo verticillata (Syre & Kennedy 1977) and Flaveria linearis (Teese 1995), significant fluctuations of Γ-values within the C3–C4 intermediate range have been found among populations from different regions and within populations, respectively. These fluctuations may depend on both temperature and water availability in M. verticillata and temperature in F. linearis. Ueno, Samejima & Koyama (1989) reported that the New World leafless land sedge Eleocharis spegazzinii includes both plants with intermediate culm anatomy and C3 carbon isotope ratios and plants with C4-like culm anatomy and C4-like carbon isotope ratios. Thus, the cases of Mollugo, Flaveria and E. spegazzinii might partially resemble the variation in photosynthetic modes seen in A. semialata, although it is unknown whether a single plant of A. semialata modifies its photosynthetic mode according to environmental changes.

A. semialata is an interesting plant with which to study the evolutionary relationships from C3 to C4 plants and vice versa. The situation of the photosynthetic modes seen in A. semialata may represent the path of evolution from C3 to C4 plants. Alternatively, it may represent the reversion from C4 to C3 plants. It is thought that C4 photosynthesis evolved from C3 photosynthesis with addition of the C4 decarboxylic acid pathway and with differentiation of the two photosynthetic cell types (Ehleringer & Monson 1993; Sage 2004). At present, there is no clear evidence for the reversion from C4 to C3 plants. However, recent molecular phylogenetic studies have suggested that the reversion of C4 plants to C3 plants might have occurred in several lineages of the Poaceae (Duvall et al. 2001; Giussani et al. 2001; Duvall et al. 2003) and the Chenopodiaceae (Pyankov et al. 2001). A molecular phylogenetic analysis of the genus Alloteropsis will be required to elucidate the evolutionary sequence of photosynthetic modes, whereas further extensive cytological, anatomical, biochemical and physiological studies of A. semialata plants obtained from various localities will give important clues to the evolution of C4 plants.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

We thank Dr J. Lynch for generously supplying seeds of A. semialata, and Dr Y. Yoshimura for technical assistance in the gas exchange measurement. This study was partly supported by a grant-in-aid from the Ministry of Agriculture, Forestry and Fisheries of Japan (BIØDESIGN Project) to O. Ueno.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  • Arnon D.I. (1949) Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiology 24, 115.
  • Barrett D.R., Frean M.L. & Cresswell C.F. (1983) C3 and C4 photosynthetic and anatomical forms of Alloteropsis semialata (R. Br.) Hitchcock. 1. Variability in photosynthetic characteristics, water utilization efficiency and leaf anatomy. Annals of Botany 51, 801809.
  • Brown R.H. & Hattersley P.W. (1989) Leaf anatomy of C3–C4 species as related to evolution of C4 photosynthesis. Plant Physiology 91, 15431550.
  • Brown R.H. & Morgan J.A. (1980) Photosynthesis of grass species differing in carbon dioxide fixation pathways. VI. Differential effects of temperature and light intensity on photorespiration in C3, C4, and intermediate species. Plant Physiology 66, 541544.
  • Brown W.V. (1977) The Kranz syndrome and its subtypes in grass systematics. Memoirs of the Torrey Botanical Club 23, 197.
  • Cheng S.-H., Moore B.D., Wu J., Edwards G.E. & Ku M.S.B. (1989) Photosynthetic plasticity in Flaveria brownii. Growth irradiance and the expression of C4 photosynthesis. Plant Physiology 89, 11291135.
  • Dengler N.G. & Nelson T. (1999) Leaf structure and development in C4 plants. In C4 Plant Biology (eds R.F.Sage & R.K.Monson), pp. 133172. Academic Press, San Diego, CA.
  • Duvall M.R., Noll J.D. & Minn A.H. (2001) Phylogenetics of Paniceae (Poaceae). American Journal of Botany 88, 19881992.
  • Duvall M.R., Saar D.E., Grayburn W.S. & Holbrook G.P. (2003) Complex transitions between C3 and C4 photosynthesis during the evolution of Paniceae: a phylogenetic case study emphasizing the position of Steinchisma hians (Poaceae), a C3–C4 intermediate. International Journal of Plant Sciences 164, 949958.
  • Edwards G.E. & Ku M.S.B. (1987) Biochemistry of C3–C4 intermediates. In The Biochemistry of Plants, Vol. 10, Photosynthesis (eds M.D.Hatch & N.K.Boardman), pp. 275325. Academic Press, San Diego, CA.
  • Ehleringer J.R. & Monson R.K. (1993) Evolutionary and ecological aspects of photosynthetic pathway variation. Annual Review of Ecology and Systematics 24, 411439.
  • Ehleringer J.R., Cerling T.E. & Helliker B.R. (1997) C4 photosynthesis, atmospheric CO2, and climate. Oecologia 112, 285299.
  • Ellis R.P. (1974a) The significance of the occurrence of both Kranz and non-Kranz leaf anatomy in the grass species Alloteropsis semialata. South African Journal of Science 70, 169173.
  • Ellis R.P. (1974b) Anomalous vascular bundle sheath structure in Alloteropsis semialata leaf blades. Bothalia 11, 273275.
  • Engelmann S., Blasing O.E., Gowik U., Svensson P. & Westhoff P. (2003) Molecular evolution of C4 phosphoenolpyruvate carboxylase in the genus Flaveria: a gradual increase from C3 to C4 characteristics. Planta 217, 717725.
  • Feierabend J. & Beevers H. (1972) Developmental studies on microbodies in wheat leaves. I. Conditions influencing enzyme development. Plant Physiology 49, 2832.
  • Frean M.L. & Marks E. (1988) Chromosome numbers of C3 and C4 variants within the species Alloteropsis semialata (R. Br.) Hitchc. (Poaceae). Botanical Journal of the Linnean Society 97, 255259.
  • Frean M.L., Ariovich D. & Cresswell C.F. (1983a) C3 and C4 photosynthetic and anatomical forms of Alloteropsis semialata (R. Br.) Hitchcock. 2. A comparative investigation of leaf ultrastructure and distribution of chlorenchyma in the two forms. Annals of Botany 51, 811821.
  • Frean M.L., Barrett D.R., Ariovich D., Wolfson M. & Cresswell C.F. (1983b) Intraspecific variability in Alloteropsis semialata (R. Br.) Hitchc. Bothalia 14, 901913.
  • Gibbs Russell G.E. (1983) The taxonomic position of C3 and C4Alloteropsis semialata (Poaceae) in Southern Africa. Bothalia 14, 205213.
  • Giussani L.M., Cota-Sanchez J.H., Zuloaga F.O. & Kellogg E.A. (2001) A molecular phylogeny of the grass subfamily Panicoideae (Poaceae) shows multiple origins of C4 photosynthesis. American Journal of Botany 88, 19932012.
  • Halsted M. & Lynch J. (1996) Phosphorus responses of C3 and C4 species. Journal of Experimental Botany 47, 497505.
  • Hatch M.D. (1987) C4 photosynthesis: a unique blend of modified biochemistry, anatomy and ultrastructure. Biochimica et Biophysica Acta 895, 81106.
  • Hattersley P.W. & Browning A.J. (1981) Occurrence of the suberized lamella in leaves of grasses of different photosynthetic types. I. In parenchymatous bundle sheaths and PCR (Kranz) sheaths. Protoplasma 109, 371401.
  • Hattersley P.W. & Watson L. (1992) Diversification of photosynthesis. In Grass Evolution and Domestication (ed. G.P.Chapman), pp. 38116. Cambridge University Press, Cambridge, UK.
  • Hattersley P.W., Watson L. & Osmond C.B. (1977) In situ immunofluorescent labeling of ribulose-1,5-bisphosphate carboxylase in leaves of C3 and C4 plants. Australian Journal of Plant Physiology 4, 523539.
  • Hattersley P.W., Wong S.-H., Perry S. & Roksandic Z. (1986) Comparative ultrastructure and gas exchange characteristics of the C3–C4 intermediate Neurachne minor S. T. Blake (Poaceae). Plant, Cell and Environment 9, 217233.
  • Holaday A.S., Harrison A.T. & Chollet R. (1982) Photosynthetic/Photorespiratory CO2 exchange characteristics of the C3–C4 intermediate species Moricandia arvensis. Plant Science Letters 27, 181189.
  • Hylton C.M., Rawsthorne S., Smith A.M., Jones D.A. & Woolhouse H.W. (1988) Glycine decarboxylase is confined to the bundle-sheath cells of leaves of C3–C4 intermediate species. Planta 175, 452459.
  • Kawamitsu Y., Hakoyama S., Agata W. & Takeda T. (1985) Leaf interveinal distances corresponding to anatomical types in grasses. Plant and Cell Physiology 26, 589593.
  • Leegood R.C. (2002) C4 photosynthesis: principles of CO2 concentration and prospects for its introduction into C3 plants. Journal of Experimental Botany 53, 581590.
  • Liebenberg E.J.L. & Fossey A. (2001) Comparative cytogenetic investigation of the two subspecies of the grass Alloteropsis semialata (Poaceae). Botanical Journal of the Linnean Society 137, 243248.
  • Neal M.W. & Florini J.R. (1973) A rapid method for desalting small volumes of solution. Analytical Biochemistry 55, 627639.
  • Prendergast H.D.V., Hattersley P.W. & Stone N.E. (1987) New structural/biochemical associations in leaf blades of C4 grasses (Poaceae). Australian Journal of Plant Physiology 14, 403420.
  • Pyankov V.I., Artyusgeva E.G., Edwards G.E., Black C.C. Jr & Soltis P.S. (2001) Phylogenetic analysis of tribe Salsoleae (Chenopodiaceae) based on ribosomal ITS sequences: implications for the evolution of photosynthetic types. American Journal of Botany 88, 11891198.
  • Sage R.F. (2004) Evolution of C4 photosynthesis. New Phytologist 161, 341370.
  • Sage R.F., Li M. & Monson R.K. (1999) The taxonomic distribution of C4 photosynthesis. In C4 Plant Biology (eds R.F.Sage & R.K.Monson), pp. 551584. Academic Press, San Diego, CA.
  • Spurr A.R. (1969) A low viscosity epoxy resin embedding medium for electron microscopy. Journal of Ultrastructural Research 26, 3143.
  • Syre R.T. & Kennedy R.A. (1977) Ecotypic differences in the C3 and C4 photosynthetic activity in Mollugo verticillata, a C3–C4 intermediate. Planta 134, 257262.
  • Teese P. (1995) Intraspecific variation for CO2 compensation point and different growth among variants in a C3–C4 intermediate plant. Oecologia 102, 371376.
  • Ting I.P. & Osmond C.B. (1973) Photosynthetic phosphoenolpyruvate carboxylases. Characteristics of alloenzymes from leaves of C3 and C4 plants. Plant Physiology 51, 439447.
  • Ueno O. (1992) Immunogold localization of photosynthetic enzymes in leaves of Aristida latifolia, a unique C4 grass with a double chlorenchymatous bundle sheath. Physiologia Plantarum 85, 189196.
  • Ueno O. (1998) Immunogold localization of photosynthetic enzymes in leaves of various C4 plants, with particular reference to pyruvate orthophosphate dikinase. Journal of Experimental Botany 49, 16371646.
  • Ueno O. (2001) Environmental regulation of C3 and C4 differentiation in the amphibious sedge Eleocharis vivipara. Plant Physiology 127, 15241532.
  • Ueno O. (2004) Environmental regulation of photosynthetic metabolism in the amphibious sedge Eleocharis baldwinii and comparisons with related species. Plant, Cell and Environment 27, 627639.
  • Ueno O., Samejima M. & Koyama T. (1989) Distribution and evolution of C4 syndrome in Eleocharis, a sedge group inhabiting wet and aquatic environments, based on culm anatomy and carbon isotope ratios. Annals of Botany 64, 425438.
  • Ueno O., Bang S.W., Wada Y., Kondo A., Ishihara K., Kaneko Y. & Matsuzawa Y. (2003) Structural and biochemical dissection of photorespiration in hybrids differing in genome constitution between Diplotaxis tenuifolia (C3–C4) and radish (C3). Plant Physiology 132, 15501559.
  • Ueno O., Yoshimura Y. & Sentoku N. (2005) Variation in the activity of some enzymes of photorespiratory metabolism in C4 grasses. Annals of Botany 95 , ( in press).
  • Yoshimura Y., Kubota F. & Ueno O. (2004) Structural and biochemical bases of photorespiration in C4 plants: quantification of organelles and glycine decarboxylase. Planta 220, 307317.