Functional Characterization of Phosphoenolpyruvate Carboxykinase ...

Annals of Botany 98: 77–91, 2006 doi:10.1093/aob/mcl096, available online at www.aob.oxfordjournals.org

Functional Characterization of Phosphoenolpyruvate Carboxykinase-Type C4 Leaf Anatomy: Immuno-, Cytochemical and Ultrastructural Analyses E L E N A V . V O Z N E S E N S K A Y A 1, V I N C E N T R . F R A N C E S C H I 2, S I M O N D . X . C H U O N G 2 and G E R A L D E . E D W A R D S 2,* 1 Laboratory of Anatomy and Morphology, V. L. Komarov Botanical Institute of Russian Academy of Sciences, Prof. Popov Street 2, 197376, St. Petersburg, Russia and 2School of Biological Sciences, Washington State University, Pullman, WA 99164-4236, USA Received: 21 December 2005 Returned for revision: 15 February 2006 Accepted: 22 March 2006 Published electronically: 16 May 2006

Background and Aims Species having C4 photosynthesis belonging to the phosphoenolpyruvate carboxykinase (PEP-CK) subtype, which are found only in family Poaceae, have the most complex biochemistry among the three C4 subtypes. In this study, biochemical (western blots and immunolocalization of some key photosynthetic enzymes) and structural analyses were made on several species to further understand the PEP-CK system. This included PEP-CK-type C4 species Urochloa texana (subfamily Panicoideae), Spartina alterniflora and S. anglica (subfamily Chloridoideae), and an NADP-ME-type C4 species, Echinochloa frumentacea, which has substantial levels of PEP-CK. Key Results Urochloa texana has typical Kranz anatomy with granal chloroplasts scattered around the cytoplasm in bundle sheath (BS) cells, while the Spartina spp. have BS forming long adaxial extensions above the vascular tissue and with chloroplasts in a strictly centrifugal position. Despite some structural and size differences, in all three PEP-CK species the chloroplasts in mesophyll and BS cells have a similar granal index (% appressed thylakoids). Immunolocalization studies show PEP-CK (which catalyses ATP-dependent decarboxylation) is located in the cytosol, and NAD-ME in the mitochondria, in BS cells, and in the BS extensions of Spartina. In the NADP-ME species E. frumentacea, PEP-CK is also located in the cytosol of BS cells, NAD-ME is very low, and the source of ATP to support PEP-CK is not established. Conclusions Representative PEP-CK species from two subfamilies of polyphyletic origin have very similar biochemistry, compartmentation and chloroplast grana structure. Based on the results with PEP-CK species, schemes are presented with mesophyll and BS chloroplasts providing equivalent reductive power which show bioenergetics of carbon assimilation involving C4 cycles (PEP-CK and NAD-ME, the latter functioning to generate ATP to support the PEP-CK reaction), and the consequences of any photorespiration. Key words: C4 grasses, C4 photosynthesis, Kranz anatomy, NADP-ME type, PEP-CK type, Spartina alterniflora, Spartina anglica, Echinocloa frumentacea, Urochloa texana.

INTROD UCTION C4 photosynthesis has evolved a number of times in angiosperm phylogeny and there is considerable variation in the anatomies and biochemistry that are associated with it. Three biochemical subtypes of C4 plants have been identified according to the major means of donation of CO2 from C4 acids to Rubisco in a given species: NAD–malic enzyme (NAD-ME)-, NADP–malic enzyme (NADP-ME)and phosphoenolpyruvate carboxykinase (PEP-CK)-type species (Gutierrez et al., 1974a; Hatch et al., 1975; Hattersley, 1987; Dengler and Nelson, 1999; Kanai and Edwards, 1999). In earlier studies on the three C4 subtypes in family Poaceace they were distinguished by having certain anatomical and ultrastructural features (Gutierrez et al., 1974a; Hatch et al., 1975; Brown, 1977). Later these were defined as ‘classical’ subtypes in the family as a majority of C4 grasses fit these classifications. The classical NADP-ME-type species have a single parenchyma bundle sheath (BS), lack a mestome sheath (MS), and BS chloroplasts are in a centrifugal position and are deficient in grana. This is the so-called panicoid (Carolin et al., 1973) or NADP-ME type of leaf anatomy, belonging to the * For correspondence. E-mail [email protected]

subfamily Panicoideae, tribes Paniceae, Arundinelleae, Andropogoneae and Maydeae (Sage et al., 1999; GPWG, 2001). The classical NAD-ME-type C4 grasses have a double sheath: an MS with thick cell walls and a few plastids, surrounded by a Kranz-type chlorenchyma sheath (cell wall lacking a suberized lamella) with chloroplasts in a centripetal position with well-developed grana. This subtype also has abundant mitochondria which contain the C4 acid decarboxylase, and the anatomy is referred to as eragrostoid (Carolin et al., 1973) or NAD-ME-type leaf anatomy (Hattersley and Watson, 1992). It occurs particularly in tribes Cynodonteae and Eragrostideae in subfamily Chloridoideae; but, also in subfamily Panicoideae, tribe Paniceae (Sage et al., 1999; GPWG, 2001). The classical PEP-CK-type of C4 plant has a double chlorenchyma sheath similar to the NAD-ME type, with an inner MS and outer Kranz chlorenchyma sheath (cell wall having a suberized lamella). However, unlike the classical NAD-ME type, they have grana-containing BS chloroplasts which are in a centrifugal position or scattered around the cell (Gutierrez et al., 1974a; Brown, 1977; Prendergast and Hattersley, 1987; Dengler and Nelson, 1999). The initial report of PEP-CK as a decarboxylase in C4 plants came from studies with Urochloa maxima (Jacq.) R. Webster

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Voznesenskaya et al. — PEP-CK in Grasses

[=Panicum maximum Jacq.], U. texana and Sporobolus poiretii (Edwards et al., 1971). Thus far, PEP-CK-type C4 species have been found in family Poaceae in subfamily Panicoideae (tribe Paniceae) and in subfamily Chloridoideae (tribes Cyanodonteae and Eragrostoideae), based on biochemical analyses (Gutierrez et al., 1974a, 1976; Prendergast et al., 1987; Walker et al., 1997; Sage et al., 1999; Walker and Chen, 2002). To date, no PEP-CK C4 species have been identified among dicots, while NAD-ME and NADP-ME C4 types occur in both monocots and dicots. While leaf anatomy can be an initial indicator of the biochemical type in C4 plants, there is considerable variation in anatomy, giving rise to a number of structural subtypes. In addition to the three classical C4 subtypes, six nonclassical structural C4 subtypes have been identified in family Poaceae (Dengler and Nelson, 1999; Voznesenskaya et al., 2005a). For example, there are non-classical NADME-type C4 species which have anatomical features and positioning of chloroplasts in BS cells like the classical PEP-CK species (Hattersley, 1992). Thus, distinguishing between NAD-ME and PEP-CK subtypes cannot be based on anatomical and ultrastructural features alone. Biochemical analyses of decarboxylases are necessary for definite classification of biochemical C4 subtypes. Among the three classical C4 subtypes, the biochemistry of the PEP-CK-type species is most complex. Complexity arises considering aspartate versus malate shuttles, source of ATP to support PEP-CK in BS cells, and function of the NAD-ME found in PEP-CK species. Two C4 cycles have been proposed in these species, one with the primary flux through an aspartate cycle via PEP-CK, and the other a malate cycle through NAD-ME (Hatch, 1987). The cycle through NAD-ME, besides generating CO2, is proposed to supply ATP in the cytosol of BS cells to support the PEP-CK reaction. In this case, BS mitochondria would also be expected to have a prominent role in C4 photosynthesis, since NAD-ME is normally compartmentalized in mitochondria. Besides the occurrence of PEP-CK-type species among monocots, there is uncertainty over the extent of occurrence of this decarboxylase in other C4 monocots. To date, PEP-CK activity in NAD-ME-type C4 monocots has been found to be low or negligible. Some NADP-ME-type monocots lack PEP-CK (e.g. Sorghum bicolor), while others (e.g. Zea mays, Digitaria sanguinalis) have PEP-CK activity (typically 10–25 % of that in PEP-CK species) (Gutierrez et al., 1974a; Prendergast et al., 1987). PEP-CK has been studied in some NADP-ME species through analysis of activities, regulatory properties, protein content by PEP-CK antibody, and function in isolated BS cells (Walker et al., 1997; Wingler et al., 1999). It was shown in Z. mays, an NADP-ME type where malate is a donor of CO2 through NADP-ME, that aspartate can serve as a donor of CO2 to BS cells through PEP-CK. However, a number of questions remain about PEP-CK and its role in the photosynthetic cycles in the various C4 types. The purpose of this study was to compare the anatomy, ultrastructure and photosynthetic biochemistry of some representative PEP-CK-containing species to

further elucidate the role of this enzyme in carbon fixation of C4 plants. This paper reports on a study of the C4 acid decarboxylase profile (by western blots), immunolocalization of some key photosynthetic enzymes, and the ultrastructure of chloroplasts and mitochondria in PEP-CK species representative of subfamily Chloridoideae (Spartina alterniflora, S. anglica) and subfamily Panicoideae (Urochloa texana). For comparison, also studied were the decarboxylase profile and compartmentalization of PEP-CK in an NADP-ME-type C4 species in subfamily Panicoideae, Echinochloa frumentacea, which has substantial levels of PEP-CK activity (Gutierrez et al., 1974a; Prendergast et al., 1987). Considering the features of PEP-CK species, a scheme is proposed for the biochemistry and energetics in mesophyll (M) and BS cells during C4 photosynthesis.

MATERIA LS A ND METHODS Plant material

Representative species of C4 biochemical subtypes studied include grasses Spartina alterniflora Loisel., S. anglica Hubb., Urochloa texana (Buckl.) R.D. Webster, of PEPCK-type; Zea mays L., Echinochloa crusgalli (L.) Scop., E. frumentacea Link [=E. crusgalli var. frumentacea (Roxb.) W.F. Wight], Digitaria sanguinalis (L.) Scop., Paspalum notatum of NADP-ME-type; Panicum decompositum R.Br. and the dicot Portulaca oleracea L. of the NAD-ME-type. Spartina alterniflora, S. anglica, D. sanguinalis and P. notatum were grown in a greenhouse in 15-cm-diameter pots in commercial potting soil with an approximate 26 C day/18 C night thermoperiod. Maximum midday photon flux density (400–700 nm) was 1200 mmol m–2 s–1 on clear days. Urochloa texana, Z. mays, E. crus-galli, E. frumentacea, P. decompositum and P. oleracea were grown in growth chambers (model GC16; Enconair Ecological Chambers Inc., Winnipeg, Canada) with a step-wise increase in light intensity with maximum midday of approx. 1000 mmoles photosynthetic quanta m 2 s–1 with a 14/10 h light/dark photoperiod and 25 C day/18 C night temperature regime. Fully expanded leaves were sampled from plants grown for 3–5 weeks. Samples were taken from the mid-section of two to three leaves; for microscopy two or three blocks were studied. Western blots

For Western blot analysis, total proteins were extracted from leaves by homogenizing 500 mg of tissue in 05 mL of extraction buffer [100 mM Tris–HCl, pH 75, 5 mM MgSO4, 10 mM dithiotheitol, 5 mM EDTA, 05 % (w/v) SDS, 2 % (v/v) b-mercaptoethanol, 10 % (v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride and 25 mg mL–1 each of aprotinin, leupeptin and pepstatin]. After centrifugation at high speed (14 000 g) for 3 min in a microcentrifuge, the supernatant was collected and protein concentration was determined with Bradford protein assay (Bio-Rad) using bovine serum albumin (BSA) as a standard. Protein samples (10 mg) were separated by 12 % SDS–PAGE, transferred to nitrocellulose membranes, blocked with 1 % (w/v) BSA,

Voznesenskaya et al. — PEP-CK in Grasses and probed overnight with 1 : 5 000 anti-Amaranthus hypochondriacus NAD-ME IgG which was prepared against the 65 kDa a subunit (courtesy of J. Berry: Long et al., 1994), 1 : 5 000 anti-Zea mays NADP-ME IgG prepared against the 62-kDa enzyme (courtesy of C. Andreo; Maurino et al., 1996) or 1 : 10 000 anti-Urochloa maxima PEP-CK IgG (courtesy of R. C. Leegood). The blots were then probed with goat anti-rabbit IgG conjugated to alkaline phosphatase at a dilution of 1 : 50 000 and visualized by a colorimetric detection procedure with 20 mM nitroblue tetrazolium and 75 mM 5-bromo-4-chloro-3-indolyl phosphate in detection buffer (100 mM Tris–HCl, pH 95, 100 mM NaCl and 5 mM MgCl2). Electron microscopy

Samples for ultrastructural studies were fixed at 4 C in 2 % (v/v) paraformaldehyde and 2 % (v/v) glutaraldehyde in 01 M phosphate buffer (pH 72), post-fixed in 4 % (w/v) OsO4, and then after a standard acetone dehydration procedure, embedded in Spurr’s resin. Cross-sections were made on a Reichert Ultracut R ultramicrotome (Reichert-Jung GmbH, Heidelberg, Germany). For electron microscopy, ultra-thin sections were stained with 2 % (w/v) lead citrate or 1 : 2 dilution of 1 % (w/v) KMnO4 : 4 % (w/v) uranyl acetate. Hitachi H-600 and JEM-1200 EX transmission electron microscopes were used for electron microscopy studies. The granal index was calculated as the percentage of the length of all appressed thylakoid membranes to the total length of all thylakoid membranes in a chloroplast. The length of appressed and non-appressed thylakoid membranes (including the length of both intergranal and end granal thylakoid membranes, e.g. all membranes exposed to stroma) were measured with a curvimeter in at least ten micrographs of median chloroplast sections (Lichtenthaler et al., 1982). In-situ immunolocalization

Leaf samples were fixed at 4 C in 2 % (v/v) paraformaldehyde and 125 % (v/v) glutaraldehyde in 005 M phosphate buffer, pH 72. The samples were dehydrated with a graded ethanol series and embedded in London Resin White (LR White, Electron Microscopy Sciences, Fort Washington, PA, USA) acrylic resin. Antibodies used (all raised in rabbit) were anti-spinach Rubisco (LSU) IgG (courtesy of B. McFadden), commercially available anti-maize phosphoenolpyruvate carboxylase (PEPC) IgG (Chemicon, Temecula, CA, USA), anti-Amaranthus hypochondriacus mitochondrial NAD-ME IgG, which was prepared against the 65-kDa a subunit (courtesy of J. Berry; Long et al., 1994), and anti-U. maxima PEPCK IgG (courtesy of R. C. Leegood). Light microscopy

Cross-sections, 08–1 mm thick, were placed onto gelatin coated slides and blocked for 1 h with TBST + BSA (10 mM Tris–HCl, 150 mM NaCl, 01 % v/v Tween 20, 1 % w/v BSA, pH 72). They were then incubated for 3 h with the

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pre-immune serum diluted in TBST + BSA (1 : 100), antiRubisco IgG (1 : 500 dilution), anti-PEPC IgG (1 : 100 dilution), anti-NAD-ME IgG (1 : 100) or anti-PEP-CK IgG (1 : 50). 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 for 20 min according to the manufacturer’s directions (Amersham, Arlington Heights, IL, USA), stained with 05 % (w/v) Safranin O, and imaged in a reflected/ transmitted mode using a BioRad 1024 confocal system with Nikon Eclipse TE 300 inverted microscope and Lasergraph image program 310. The background labelling with pre-immune serum was very low, although some infrequent reflectance of light from thick cell walls, or labelling occurred in areas where the sections were wrinkled due to trapping of protein A–gold particles (results not shown). TEM

Thin sections on Formvar-coated nickel grids were incubated for 1 h in TBST + BSA to block non-specific protein binding on the sections. They were then incubated for 3 h with the preimmune serum diluted in TBST + BSA, or anti-PEP-CK IgG (1 : 50, 1 : 25, 1 : 10). After washing with TBST + BSA, the sections were incubated for 1 h with Protein A–gold (10 nm) diluted 1 : 100 with TBST + BSA. The sections were washed sequentially with TBST + BSA, TBST, and distilled water, and then post-stained with a 1 : 4 dilution of 1 % (w/v) potassium permanganate and 2 % (w/v) uranyl acetate. Images were collected using a JEM-1200 EX transmission electron microscope.

RESULTS Western blot

Figure 1 shows western blot analyses of the C4 acid decarboxylases in the PEP-CK-type species Spartina alterniflora, S. anglica and Urochloa texana, the NADP-ME-type species Echinochloa frumentacea, Zea mays and Sorghum bicolor, and the NAD-ME-type C4 species Portulaca oleracea. When the three PEP-CK species were probed with antibodies to the three decarboxylases, there was a strong immunoreactive band for PEP-CK at 74 kDa and a smaller molecular mass band. PEP-CK has been found to be very susceptible to proteolysis (Walker et al., 1997; Wingler et al., 1999), so the smaller band most likely represents a proteolytic product. The PEP-CK species also had a significant immunoreactive band for NAD-ME, and very faint bands with the NADP-ME antibody at 72 kDa. All three NADP-ME species had a strong immunoreactive band at 62 kDa with the NADP-ME antibody and very little reactivity against NAD-ME antibody. With the PEP-CK antibody, there were distinct reactive bands with the NADP-ME species Z. mays and E. frumentacea but not with S. bicolor. In the NAD-ME species P. oleracea, with antibodies against NAD-ME there was a single high intensity immunoreactive band of 65 kDa and no reaction with PEP-CK or NADP-ME antibodies (Fig. 1).


80

E) -M ) P K AD E) -C ) ) E) -M ea (N D-M EP -CK -CK P P ) ( P A P AD ntac (N ra PE PE ME ea flo ca ( na ( DP- or (N ume i c l li ra a rn fr A lte ang tex s (N bico loa ole a a a h y a a m o c lac tin tin hl ma hu no rtu ar par roc ea org chi o p S P E Z U S S

A 77 50

B

PEP-CK

77 50

NAD-ME

77 50

NADP-ME

C

F I G . 1. Western blot analysis of C4 acid decarboxylases in leaf total protein extracts of several species of differing C4 biochemical types. Spartina alterniflora, S. anglica and Urochloa texana are PEP-CK type; Zea mays, Sorghum bicolor and Echinochloa frumentacea are NADP-ME type, and Portulaca oleracea is NAD-ME type. Equal amounts (10 mg) of total protein were separated by SDS–PAGE, and transferred to nitrocellulose. Blots were probed with antisera raised against (A) PEP-CK, (B) NAD-ME or (C) NADP-ME. Numbers on the left indicate the relative molecular mass standards in kDa.

The immunolabelling for NAD-ME, which is low in all three PEP-CK species examined, is largely confined to BS cells (Fig. 2D and H; low labelling occurs in mesophyll cells of U. texana; Fig. 2D). Electron microscopy observations indicate that labelling for this enzyme is clearly associated with BS mitochondria of both S. anglica (Fig. 5A) and U. texana (Fig. 5B, C), with higher labelling in U. texana (Fig. 5B, C), and absence of labelling in mitochondria of M cells (Fig. 5C). Statistical analysis also shows a high density of gold particles in BS mitochondria for both S. anglica and U. texana (Table 1). In NADP-ME species, the labelling for Rubisco is confined to BS cells (Fig. 3A; results are shown only for E. frumentacea; but all species of this group have similar distribution of labelling), while the labelling for PEPC is in the M cells (Fig. 3B). Bundle sheath cells of E. frumentacea show labelling for PEP-CK (Fig. 3C). Electron microscopy studies demonstrate labelling for this enzyme occurs in the cytosol of BS cells (Fig. 4D, E) and is absent in M cells (Fig. 4F). In other species, such as Zea mays or Digitaria sanguinalis, most labelling of PEP-CK is also found in the cytosol of BS cells, but it is rather low even with the highest concentration of antibodies used in this study (1 : 10) (not shown). In the dicot Portulaca oleracea, an NAD-ME C4 species, there is no immunolabelling for PEP-CK, which is consistent with the western blot analysis.

Immunolabelling

Figures 2 and 3 represent immunolabelling observed by confocal microscopy for selected photosynthetic enzymes in the PEP-CK biochemical type, Urochloa texana and Spartina anglica, and the NADP-ME biochemical type, Echinochloa frumentacea. Among the PEP-CK species studied, U. texana has vascular bundles which are surrounded by an inner MS and an outer chlorenchymatous BS (Fig. 2A–D). In this species, organelles are evenly distributed throughout the BS cell (Fig. 2A). Both S. anglica (Fig. 2) and S. alterniflora (not shown) also have an inner MS. However, as shown with S. anglica, the outer layer of large parenchyma BS not only surrounds the MS and vascular bundle, it also forms long extensions above the xylem part of the bundle (Fig. 2E–G). Organelles in all cells of BS are strictly centrifugally arranged (Fig. 2E–H). In both U. texana and S. anglica, irrespective of the organization of the BS, Rubisco is predominantly compartmentalized in BS chloroplasts (Fig. 2A and E), while labelling for PEPC is restricted to the surrounding M cells (Fig. 2B, F). Reaction with antibody for PEP-CK shows a high level of labelling in BS cells (Fig. 2C, G). Electron microscopy examination of S. anglica shows that this labelling for PEP-CK does not associate with any organelles in BS cells; rather it is located in the cytosol (Fig. 4A, B) and is absent in M cells (Fig. 4C). There is also some apparent infrequent labelling of cell walls (e.g. Fig. 2B, C, F, G) which may be due either to reflected light from thick cell walls or nonspecific binding of antibody. Statistical analysis of the distribution of gold particles (Table 1) confirms that the density of labelling for this antibody is clearly very high in the cytosol in S. anglica with very little labelling in organelles.

Electron microscopy

Comparative electron microscopy of organelle structure in the three PEP-CK C4 species shows that they all have larger chloroplasts in BS than in M cells (Table 2). In U. texana (Fig. 6A–C) the organelles do not have any special orientation in the BS cell (Fig. 6A). Both M and BS chloroplasts have well-developed grana but there is a difference in their size, with larger grana stacks in M chloroplasts (up to 35 thylakoids) while in BS chloroplasts, large grana stacks, up to 25 thylakoids, are rare. The granal index in BS chloroplasts of U. texana is a little higher than in the M chloroplasts (Table 3). While these chloroplasts have nearly the same values of appressed thylakoid density, the main structural difference is in the higher density of non-appressed thylakoids in M chloroplasts, and in the presence of numerous, long intergranal thylakoids between large grana (Fig. 6B). Besides, BS chloroplasts having smaller grana, the length of intergranal thylakoids is also shorter (Fig. 6C). Mesophyll chloroplasts of U. texana also have from one to three layers of vesicular peripheral reticulum (Fig. 6B). In the BS cells of S. anglica, organelles are in a centrifugal position (Fig. 6D). Bundle sheath chloroplasts of S. anglica (Fig. 6E) have significantly larger grana than in U. texana, up to 42 thylakoids in a stack, with numerous intergranal thylakoids. The structure of M chloroplasts of S. anglica (not shown) is similar to BS chloroplasts, and very similar to that in M chloroplasts of U. texana, with large stacks of grana (up to 43 thylakoids) and numerous, long intergranal thylakoids. In S. anglica, the granal indexes in BS cells around the vascular tissue are higher than in M cells, while in BS extensions the granal indexes are

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A

E BSE MC BS

MC

MS

MS

BS

B

F

C

G

D

H

BS MC

MS BS MS

MC

F I G . 2. Reflected/transmitted confocal microscopy imaging of in-situ immunolocalization of photosynthetic enzymes in leaves of grasses of the PEP-CK-type C4 biochemical group. (A–D) Urochloa texana: (A) Rubisco; (B) PEPC; (C) PEP-CK; (D) NAD-ME. (E–H) Spartina anglica: (E) Rubisco; (F) PEPC; (G) PEP-CK; (H) NAD-ME. BS, Bundle sheath; BSE, bundle sheath extension; MC, mesophyll cells; MS, mestome sheath. Scale bars: A–C and E–G = 50 mm; D and H = 20 mm.

similar between the two cell types. The density of both appressed and non-appressed thylakoids is much lower in chloroplasts of BS extensions, where the contact of BS with the vascular bundle is absent (Table 3). The size of

grana in chloroplasts of BS extensions (Fig. 6F) is slightly lower than in BS cells surrounding the vascular bundle which also correlates with a lower density of appressed thylakoids.


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A

B

C

BS MC

F I G . 3. Reflected/transmitted confocal microscopy imaging of in-situ immunolocalization of photosynthetic enzymes in leaves of Echinochloa frumentacea, a member of the NADP-ME-type C4 biochemical group. (A) Rubisco; (B) PEPC; (C) PEP-CK. BS, bundle sheath; MC, mesophyll cells. Scale bars = 50 mm.

A

B

C C

C

M C

M C

D

E

F C

C

C M

M

F I G . 4. Transmission electron microscopy of in situ immunolocalization of PEP-CK in leaves of PEP-CK and NADP-ME-type C4 grasses. (A–C) Spartina anglica: (A, B) PEP-CK localization in cytosol of bundle sheath cells; (C) absence of labelling in mesophyll cells. (D–F) Echinochloa frumentacea: (D–E) immunolocalization of PEP-CK in cytosol of bundle sheath cells; (F) absence of labelling in mesophyll cells. C, Chloroplast; M, mitochondria. Scale bars = 05 mm.

In S. alterniflora, M chloroplasts (Fig. 6G) have a very intensively developed system of large grana. Most of the grana in BS chloroplasts consist of three to eight thylakoids with short intergranal thylakoids between them (Fig. 6H). Both Spartina species have similar granal index values for both chloroplast types (Table 3). However, BS extension

cells have low grana stacking, particularly in S. alterniflora, where most grana consist of only two appressed thylakoids with a few grana having up to five to seven thylakoids (Fig. 6I). In both species of Spartina, the total thylakoid density is higher in M chloroplasts than in BS chloroplasts, with the chloroplasts in BS extensions having the lowest

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T A B L E 1. Immunogold labelling of NAD-ME and PEP-CK enzymes in bundle sheath (BS) cells (number of particles per 1 mm2) NAD-ME in BS cells

PEP-CK in BS cells

Species

Mitochondria

Cytosol + other organelles

Cytosol

Organelles

Spartina anglica Urochloa texana Echinochloa frumentacea

8.40 6 1.32 21.9 6 3.6 n.d.

0.52 6 0.13 0.58 6 0.06 n.d.

45.1 6 8.6 n.d. 9.74 6 1.54

1.76 6 0.40 n.d. 0.24 6 0.06

The number of gold particles is given as mean 6 s.e. The number of partial cell profiles/sections examined for NAD-ME was 16 in Urochloa texana (containing 76 mitochondria profiles) and six in Spartina anglica (containing 22 mitochondria profiles), 10 and 11 for PEP-CK in Spartina anglica and Echinochloa frumentacea, respectively.

A

B

C C

C

M M M C

F I G . 5. Transmission electron microscopy of in-situ immunolocalization of NAD-ME in leaves of PEP-CK-type C4 grasses: (A) labelling for NAD-ME in bundle sheath mitochondria of Spartina anglica; (B) labelling for NAD-ME in bundle sheath mitochondria of Urochloa texana; (C) absence of labelling in mesophyll cell of Urochloa texana. C, Chloroplast; M, mitochondria. Scale bars = 05 mm.

T A B L E 2. Characteristics of mesophyll (M) and bundle sheath (BS) mitochondria and chloroplasts of PEP-CK species Mitochondria, length of axis (mm) Species Urochloa texana Spartina anglica Spartina alterniflora

Axis measured Long Short Long Short Long Short

M 0.6 0.4 0.5 0.3 0.6 0.5

6 6 6 6 6 6

Chloroplasts, length of axis (mm)

BS 0.02 0.01 0.03 0.01 0.02 0.01

0.9 0.6 0.7 0.5 0.7 0.5

6 6 6 6 6 6

0.03 0.02 0.02 0.01 0.04 0.01

M 7.1 1.8 5.0 1.4 6.0 2.1

6 6 6 6 6 6

BS 0.6 0.1 0.1 0.2 0.3 0.1

9.4 3.8 8.1 2.8 8.3 3.4

6 6 6 6 6 6

0.5 0.3 0.2 0.2 0.2 0.1

Numbers represent means 6 s.e. For chloroplasts, n > 20, for mitochondria n = 25–30.

values (Table 3). Also, in both species, M chloroplasts have well-developed vesicular peripheral reticulum, mostly one layer in S. anglica and one to two layers in S. alterniflora. BS chloroplasts may also have one or more layers of peripheral reticulum, or some kind of outgrowth on their ends. A quantitative analysis shows that there are no differences in the structure of chloroplasts in the M cells which surround the vascular bundle and the BS extensions in either Spartina species (Table 3). In U. texana, BS mitochondria are situated mostly in the centre of the cell, between proximal chloroplasts or on the border with the MS. Some mitochondria are also situated

along the outer tangential wall on the border with M cells. In both Spartina species, there is strict centrifugal arrangement of organelles in BS cells irrespective of their position (around the vascular bundle or in the extension). Most of the mitochondria are located internal to the chloroplasts, on the border of the vacuole (Fig. 6D), with a few located in the thin cytoplasmic layer in the centripetal position, especially in cells in the extensions. Among the three PEP-CK species, measurements along the long and short axis show that the mitochondria in M and BS cells are about the same size in S. alterniflora, while the BS mitochondria are larger than M in U. texana and S. anglica. The size of BS mitochondria in


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A

B

C

M

C-M

D

C-BS

E

F

C-BSE

C-BS BS

G

H

I C-BS

C-BSE

C-M

F I G . 6. Transmission electron microscopy of chloroplast structure in leaves of PEP-CK-type C4 grasses. (A–C) Urochloa texana: (A) general view of bundle sheath and mesophyll cell structure; (B) mesophyll chloroplast; (C) bundle sheath chloroplast. (D–F) Spartina anglica: (D) general view of bundle sheath cell with centrifugal chloroplasts; (E) bundle sheath chloroplast from a cell surrounding the vascular bundle; (F) bundle sheath chloroplast from a cell of the bundle sheath extension. (G–I) Spartina alterniflora: (G) mesophyll chloroplast; (H) bundle sheath chloroplast from a cell surrounding the vascular bundle; (I) bundle sheath chloroplast of bundle sheath extension cell. BS, Bundle sheath; C-BS, chloroplasts in bundle sheath; C-BSE, chloroplasts in bundle sheath extensions; C-M, chloroplast in mesophyll cell; M, mesophyll cell. Scale bars: A = 10 mm; D = 5 mm; B, C and E–I = 1 mm.

the PEP-CK species is also larger than mitochondria in BS cells of the NADP-ME species Echinochloa frumentacea (not shown in the table, but see Fig. 4D and E for comparison). The BS mitochondria of U. texana and S. alterniflora have a similar structure with intensively developed tubular cristae, sometimes forming lamellate structures (Fig. 7A, C). In S. anglica the BS mitochondria have numerous calciform or tubular cristae (Fig. 7B). In all three PEP-CK species, the BS cell wall has a suberized lamella with a classical distribution for PEP-CK type, occurring in the outer tangential wall and approximately to

the middle of the radial cell walls. Sometimes they are also present in the corners of the BS cells, and as discontinuous patches in the inner tangential wall in S. anglica. The suberized lamella is thicker in the area of the plasmodesmata and is absent in the MS of the small bundles (results not shown). Plasmodesmata are abundant in the outer tangential cell walls between parenchyma BS and M cells. Some plasmodesmata also occur between BS cells on radial cell walls in U. texana and in both Spartina species, as well as between neighbouring BS cells in extensions (results not shown).

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T A B L E 3. Chloroplast granal index and thylakoid density in palisade mesophyll (M) and bundle sheath (BS) chloroplasts Species

Cell type

Granal index (%)*

Urochloa texana

M BS M (VB) BS (VB) M (Ext) BS (Ext) M (VB) BS (VB) M (Ext) BS (Ext)

42.9 49.2 47.6 58.7 50.8 53.1 51.4 51.4 51.7 46.5

Spartina anglica

Spartina alterniflora

6 6 6 6 6 6 6 6 6 6

2.4 2.4 1.7 1.2 2.1 1.2 1.3 1.3 2.7 3.2

Appressed thylakoid density† 11.9 11.7 24.3 24.9 26.4 14.6 17.1 15.1 20.4 8.4

6 6 6 6 6 6 6 6 6 6

0.7 1.7 1.4 2.4 1.8 3.0 2.1 2.5 1.9 1.6

Non-appressed thylakoid density† 16.1 11.9 26.6 17.4 25.3 12.6 16.0 14.3 18.6 9.3

6 6 6 6 6 6 6 6 6 6

1.3 1.2 1.6 1.6 0.9 1.9 0.3 2.5 1.0 0.6

Total thylakoid density† 28.0 23.5 50.9 42.3 51.6 27.3 33.1 29.3 39.0 17.7

6 6 6 6 6 6 6 6 6 6

1.6 2.7 2.5 3.9 2.0 4.8 2.4 5.0 2.1 2.2

For Spartina anglica and S. alterniflora granal index was measured separately for mesophyll (M) and bundle sheath (BS) chloroplasts located around vascular bundles (VB) and in extensions (Ext). Numbers represent means 6 s.e. The number of chloroplasts measured in each case was ten or more. * The length of all appressed thylakoid membranes as a percentage of the total length of all thylakoid membranes in a chloroplast. † Thylakoid density here represents the length of thylakoid membranes (in mm) per 1 mm2 of chloroplast stroma area (analysed for the total area of the chloroplast excluding starch grains). n.d. = not determined.

DISCUSSION Among C4 species, PEP-CK has only been found to function in C4 photosynthesis in family Poaceae. The classical PEP-CK-type species occur in subfamily Chloridoideae (represented by the two Spartina sp. in this study), where this syndrome arose once in a subclade from NAD-ME subtype (GPWG, 2001; Guissani et al., 2001), and in subfamily Panicoideae (represented by Urochloa texana in this study), where they arose once in a single, well-supported clade with uncertain origin (GPWG, 2001; Guissani et al., 2001). In PEP-CK subtype species, the decarboxylase has a major role in donation of CO2 from C4 acids to Rubisco in BS cells. PEP-CK has also been found among some NADP-ME-type C4 species in subfamily Panicoideae (represented by Echinochloa frumentacea in this study), but generally not among NAD-ME-type species in either subfamily. More research is required to evaluate the occurrence and evolution of C4 species containing PEP-CK, particularly in subfamily Panicoideae. This comparative study of PEP-CK-containing species provides information that is useful for further defining its role in C4 plants. Western blots

Analysis by western blots using antibodies to the three C4 acid decarboxylases showed that the PEP-CK species, Spartina anglica, S. alterniflora and Urochloa texana, have in addition to PEP-CK significant levels of NAD-ME, but little or no NADP-ME. These results are similar to those of Wingler et al. (1999) with the PEP-CK species Chloris gayana, Urochloa panicoides and U. maxima. The western blot data are also generally consistent with reports of enzymatic activities of the decarboxylases in U. texana (Gutierrez et al., 1974a) and S. anglica (Smith et al., 1982) and other PEP-CK species (Gutierrez et al., 1974a; Prendergast et al., 1987). Previously, some activity of PEP-CK was reported in leaf extracts of Portulaca oleracea, an NAD-ME-type C4

species (Gutierrez et al., 1974a), but no immunoreactive protein could be detected by western blots in this species; also, see tests by Wingler et al. (1999) with NAD-ME Amaranthus sp. Thus, currently there is no evidence that PEP-CK is involved in C4 photosynthesis in dicots, but it is interesting to consider why this pathway may not have evolved in dicots. This study further confirms the presence of PEP-CK in some NADP-ME monocots. Western blot analysis demonstrated a strong signal for PEP-CK in the NADPME species, Zea mays and E. frumentacea, and only a very weak signal with Sorghum bicolor. As expected, NADP-ME was high in all three species, while antibody against NAD-ME gave a weak band in all three, similar to earlier results (Wingler et al., 1999). Previous studies by enzyme analysis show that several Echinochloa species, along with several other NADP-ME species, have PEPCK enzymatic activity while some, like S. bicolor, lack PEP-CK activity. While western blots indicate barely detectable levels of NAD-ME in these species, low activity is expected since it is a constitutive enzyme in plants (Artus and Edwards, 1985). NAD-ME enzyme activity is also detectable in NADP-ME species in assays of leaf and BS cell extracts (Gutierrez et al., 1974a; Prendergast et al., 1987). However, it has been suggested that NADP-ME may use NAD partially as substrate and that this could account for some of the activity in crude extracts (Hatch et al., 1982). With the antibody to the 62-kDa NADP-ME purified from Z. mays leaves, western blots gave a major reactive band at 62 kDa with the NADP-ME species. In some cases, a faint reactive band was observed at 72 kDa with the NADP-ME antibody in the NADP-ME and PEP-CK-type species (Fig. 1C). Recently, it was shown that the purified NADP-ME from Z. mays, which was used to make antibodies, co-purifies with a heat shock protein, Hsp 70; which can account for the low intensity 72-kDa reactive band (Lara et al., 2005).


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A

B

C

F I G . 7. Transmission electron microscopy of mitochondrial structure in bundle sheath cells of PEP-CK-type C4 species: (A) Urochloa texana; (B) Spartina anglica; (C) Spartina alterniflora. Scale bars = 05 mm.

Immunolocalization of enzymes

The western blot analyses are supported by the immunolocalization studies. As expected for C4 species, immunolocalization studies for the PEP-CK C4 species U. texana and S. anglica show Rubisco is localized in the BS cells and PEPC in the M cells. Immunolocalization by confocal microscopy shows PEP-CK is localized in BS cells of these PEP-CK-type C4 species. This is consistent with enzymatic analysis with separated M and BS preparations, which show that the enzyme is located in BS cells of Chloris gayana, Brachiaria erucaeformis, Urochloa mosambicensis, U. maxima, U. texana and U. panicoides (Gutierrez et al., 1974b; Ku et al., 1980). The present studies by electron microscopy using the immuno-gold technique showed that PEP-CK is located in the cytosol of BS cells which provides direct in-situ evidence supporting results from fractionation of BS protoplasts of PEP-CK species (Ku et al., 1980; Chapman and Hatch, 1983; Watanabe et al., 1984). Immunolocalization studies by confocal microscopy showed NAD-ME is localized in the BS cells of U. texana and S. anglica, and by electron microscopy in the mitochondria in these cells, which supports in vitro results on compartmentation using isolated BS protoplasts of U. maxima (Chapman and Hatch, 1983), and with isolated mitochondria from BS cells of U. panicoides (see Hatch, 1987). In NADP-ME species, there was previous evidence for PEP-CK in BS cells from analysis of BS extracts, by enzyme activity in Setaria lutescens and Digitaria sanguinalis (Gutierrez et al., 1974b), and protein content by western blots using PEP-CK antibody in U. panicoides (Wingler et al., 1999). Results from immunolocalization on leaf cross-sections with E. frumentacea showed that PEP-CK is located in BS cells; see also results of Walker et al., (1997) with Z. mays and Echinochloa crusgalli. To determine the subcellular compartmentation of PEP-CK in BS cells of NADP-ME-type species, immunolocalization by electron microscopy was performed on E. frumentacea where the enzyme was shown to be located in the cytosol of BS cells. Structure

A common feature of the classical PEP-CK species, including S. anglica, S. alternifolia and U. texana of the present study, is a double chlorenchyma sheath made up of an inner MS and an outer Kranz sheath having a suberized lamella with granal chloroplasts in both M and BS cells.

There is variation in the position of chloroplasts in BS cells as seen in the two Spartina species, in that the chloroplasts are in a centrifugal position, while in U. texana, they are distributed throughout the cell. Also, Spartina has another variation in anatomy with BS extensions which maintain the dual cell-differentiated C4 system in the absence of vascular tissue and an MS. Some PEP-CK-type species have structural dimorphism between M and BS chloroplasts, either by differences in the thylakoid density, grana stacks or size of chloroplasts. The thylakoid density in PEP-CK species studied here was lower in BS than in M chloroplasts, especially in the BS extensions of the Spartina species. All three of the PEP-CK species examined have larger grana in M chloroplasts and much smaller grana stacks in BS chloroplasts, with reduced grana in BS extensions in the two Spartina species. Also, Woo et al. (1971) reported that Chloris gayana has M chloroplasts with greater stacking of thylakoids (17 times) than in BS chloroplasts. Laetsch (1971) reported that Spartina foliosa has larger grana in M than in BS chloroplasts, and that Bouteloua curtipendula has welldeveloped grana in both, but much greater grana stacks in M chloroplasts. On the other hand, based on visual observations, Laetsch (1971) saw no structural dimorphism in grana stacks in Mu¨hlenbergia racemosa. In subsequent studies these species were identified as PEP-CK-type C4 species (Gutierrez et al., 1974a; Sage et al., 1999). In all three PEP-CK species in the present study there was also an obvious structural dimorphism with respect to chloroplast size, in that the BS chloroplasts are larger than M chloroplasts (Table 2). This is also the case in Chloris gayana (Woo et al., 1971), and is apparent for other species from micrographs of Bouteloua curtipendula, Spartina foliosa and Mu¨hlenbergia racemosa (Laetsch, 1971), and from the data of Yoshimura et al. (2004) for C. gayana and U. maxima. While the BS chloroplasts are larger, the proportion of chloroplasts in BS cells is somewhat lower, approx. 45 %, vs. approx. 55 % in M cells from analysis of leaf cross-sections of two PEP-CK species (Yoshimura et al., 2004). In the three PEP-CK species studied, the granal index in M and BS chloroplasts was on average about 50 %, showing little structural dimorphism in this respect. The granal indexes are also similar in M and BS chloroplasts in the PEP-CK species Chloris gayana and Urochloa maxima (Yoshimura et al., 2004). The higher granal index values in that study (68–77) might be due to differences in

Voznesenskaya et al. — PEP-CK in Grasses methodology in measuring the index (see Materials and methods for current analysis following Lichtenthaler et al., 1982). Mitochondria showed structural dimorphism and positional differences between species. It was previously noted from electron microscopy studies (Downton, 1971; Laetsch, 1971) that some C4 species (B. curtipendula, C. gayana, S. foliosa and M. racemosa), which are now known to be PEP-CK type, have large and numerous mitochondria in BS cells. Yoshimura et al. (2004) showed that the BS mitochondria are larger in some PEP-CK species. In the present study, mitochondria in BS were also larger than those in M cells in two species, U. texana and S. anglica, but not in S. alterniflora. Internal mitochondrial structure also differed between the species examined in the present study. In U. texana and S. alterniflora, the BS mitochondria have a similar structure with intensively developed tubular and lamellated cristae, while in S. anglica, they have numerous calciform cristae (Fig. 7) which appear more like the very active mitochondria in C3 species. These data on features of mitochondria in PEP-CK species are in agreement with results of Hatch et al. (1975). Also, they showed that the degree of cristae development is highest in NAD-ME species (together with Chloris gayana of the PEP-CK group), intermediate in other PEP-CK species and lowest in species of the NADP-ME group. There are differences in the position of mitochondria in BS cells of PEP-CK-type C4 species depending on the arrangement of chloroplasts. In S. anglica and S. alterniflora, the mitochondria are largely located internal to the chloroplasts, which are in a centrifugal position; this may enhance the trapping by chloroplasts of CO2 released from mitochondrial NAD-ME. In U. texana, the mitochondria, along with chloroplasts, are scattered around the cytoplasm (also see other reports; Hatch et al., 1975; Hattersley and Browning, 1981). Hattersley and Browning (1981) also noted that mitochondria in some PEP-CK species are usually located between chloroplasts, or sometimes ‘on the outer tangential side of the chloroplasts’, but also they could be found in the zone between BS and M cells. Recently, Yoshimura et al. (2004) calculated that approx. 85 % of the mitochondria are in BS cells vs. approx. 15 % in M cells, based on analysis of leaf crosssections of two PEP-CK species, which is suggestive of a role of BS mitochondria in PEP-CK-type photosynthesis. The classical NADP-ME-type C4 grasses lack an MS as the Kranz cells are thought to be derived from the MS of C3 plants. The NADP-ME species in subfamily Panicoideae which have PEP-CK activity have Kranz anatomy lacking a MS, and BS chloroplasts are deficient in grana. It has been suggested that the occurrence of PEP-CK in NADP-ME species may be correlated with the degree of grana development in BS chloroplasts (Wingler et al., 1999). For example, S. bicolor, which lacks PEP-CK, has agranal BS chloroplasts, whereas Z. mays and other species (e.g. Digitaria sanguinalis, Paspalum notatum) which have PEP-CK, have some grana. The degree of grana stacking has been associated with photosystem II (PSII) activity and production of

87

NADPH (Edwards and Walker, 1983). An aspartate-type C4 cycle through PEP-CK does not transfer reducing power from M to BS cells. Interestingly, an Australian accession of Alloteropsis semialata spp. semialata is an exceptional species which biochemically is PEP-CK-type with neurachneoid-type anatomy. It consists of a doublebundle sheath with a Kranz-type inner sheath with chloroplasts having well-developed grana (Prendergast et al., 1987; Ueno and Sentoku, 2006). This Kranz sheath, like that in the classical NADP-ME-type C4 grasses, is considered to be derived from the MS sheath of C3 plants. Thus, possibly in species where the Kranz sheath originated from the MS in subfamily Panicoideae, there is a range from NADP-ME-type species lacking PEP-CK (e.g. S. bicolor), to those having NADP-ME with PEP-CK (e.g. E. frumentacea), to the extreme case of Alloteropsis semialata, which has high PEP-CK, with lower (Ueno and Sentoku, 2006) or little (Prendergast et al., 1987) NADP-ME activity. Compartmentation of PEP-CK in BS cells and its possible source of ATP

In the present study, immunolocalization shows that PEP-CK is located in the cytosol and NAD-ME in the mitochondria of BS cells of U. texana, S. anglica and S. alterniflora. Extensive studies on isolated BS cells of the PEP-CK species U. panicoides have shown that the ATP required for the cytosolic PEP-CK reaction can be provided by mitochondrial NAD-ME using malate as a substrate (Burnell and Hatch, 1988; Carnal et al., 1993). If NAD-ME strictly functioned to provide ATP to support PEP-CK, then, assuming 25 ATP are produced per NADH generated, NAD-ME would need to function at 40 % of the rate of the PEP-CK reaction. Enzymatic assays show a large variation in the ratio of PEP-CK/NAD-ME activity, including cases of low NAD-ME activity among PEP-CK species grown under different conditions (Prendergast et al., 1987; Hattersley, 1992). If NAD-ME is insufficient, there would need to be an additional means of generating ATP for PEP-CK. Alternatively, at the other extreme, flux through NAD-ME could be greater than 40 % of PEP-CK activity. This could occur through a malate shuttle if the alternative pathway of respiration (non-ATP generating) were engaged in BS mitochondria as observed in Panicum miliaceum, an NAD-ME-type species (Gardestro¨m and Edwards, 1983; Agostino et al., 1996). Thus, it is possible that there is variation in the functional ratio of the two decarboxylases, and in the means of producing ATP to support PEP-CK activity. Thus, a broader survey of the levels (by western blots) and function of decarboxylases in PEP-CK species is needed to determine if PEP-CK activity is obligately linked to a C4 cycle through NAD-ME to meet the ATP requirement. As noted earlier, the present study shows that PEP-CK in the NADP-ME-type species, E. frumentacea, is located in the cytosol in BS cells. Echinachloa frumentacea has very low levels of NAD-ME, similar to constitutive levels in C3 plants, as does other NADP-ME species containing PEP-CK (Walker et al., 1997; Wingler et al., 1999).

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This suggests the ATP requirement for PEP-CK in the cytosol of BS cells is not supported by a malate NADME C4 cycle. Mitochondrial respiration by the TCA cycle, or energy derived from oxidation of some triose phosphate (triose-P) to 3-phosphoglycerate (PGA) in the cytosol (generating ATP and NADPH), could be alternative sources of ATP. Proposed C4 cycle scheme and energetics of photosynthesis in PEP-CK-type species

The leaf anatomy and characteristics of chloroplasts and mitochondria are significant features important to understanding the mechanism of photosynthesis in the different C4 subtypes. The degree of grana development and photochemical features of chloroplasts in M and BS cells are relevant to the respective requirements for ATP and NADPH to support C4 photosynthesis. In C4 photosynthesis, reductive power is required to reduce the PGA generated by Rubisco in BS chloroplasts. In malic enzyme-type species, a logical relationship has been defined between photochemistry of chloroplast types and energy requirements in C4 photosynthesis in the two cell types, while the PEP-CK-type system is more complex. In NADP-MEtype C4 species, BS chloroplasts are deficient in grana and in PSII activity, so M chloroplasts provide most, or all (depending on species), of the reductive power. This is accomplished by a malate shuttle in the C4 cycle which transfers reductive power from M to BS chloroplasts, and by transport of part of the PGA from BS to M chloroplasts for reduction to triose-P. In NAD-ME-type C4 species, an aspartate C4 cycle is predominant in which reductive power is not required, nor transferred, from M to BS cells. Both chloroplasts have grana; although, BS chloroplasts have a higher granal index than M chloroplasts, which have different degrees of grana deficiency depending on the species (Gamaley, 1985; Voznesenskaya et al., 1999). Both chloroplasts have PSII activity (Ku et al., 1974) and share in photochemically generating NADPH for reduction of PGA. The PEP-CK subtype has evolved in two subfamilies (Panicoideae and Chloridoideae) in Poaceae, and studies indicate that they have some features in common, e.g. compartmentalization of photosynthetic enzymes, chloroplast granal index, and abundance of mitochondria in BS cells. In the present study, the granal index was similar in M and BS cells in two species of Spartina (subfamily Chlorodoideae) and in U. texana (subfamily Panicoideae), on average 49 % for M chloroplasts and 52 % for BS chloroplasts. This similar granal index suggests M and BS chloroplasts contribute equally to the generation of reductive power to support carbon assimilation. PEP-CK species have a similar chlorophyll a/b ratio in M and BS chloroplasts (328 vs. 331, average of five species), like that of C3 plants (Mayne et al., 1974). Optical analysis of chloroplasts from PEP-CK-type species indicates both have high levels of PSII (chlorophyll fluorescence at 685 nm/ 735 nm under liquid, delayed light emission from PSII), while the content of the reduced form of reaction centre chlorophyll of photosystem I (PSI) (P700)/mg chlorophyll was about 35 % higher in BS than in M chloroplasts

(Mayne et al., 1974). These results suggest M and BS chloroplasts of PEP-CK species have about equal capacity to generate reductive power. The higher content of P700/mg chlorophyll in BS cells, along with smaller grana stacks, could reflect more PSI cyclic production of ATP in BS chloroplasts. Altogether, from results of this study and the information on PEP-CK species available in the literature, a model of the operation of the PEP-CK system is presented assuming a malate NAD-ME C4 cycle functions to provide ATP to support PEP-CK. Figure 8A shows a scheme of C4 photosynthesis with an NAD-ME C4 cycle with shuttle of malate to BS cells, and a PEP-CK C4 cycle with shuttle of aspartate to BS cells. This scheme indicates how C4 photosynthesis would function in this subtype with equal sharing in the generation of reductive power between M and BS chloroplasts. In the scheme, the C4 cycle with NAD-ME in a shuttle of malate generates ATP, which is utilized in the cytosol by PEP-CK. Two turns of the C4 cycle via NAD-ME generates 5 ATP (assuming 25 ATP are generated per NADH; see Hinkle et al., 1991), through mitochondrial respiration, which supports five turns of the C4 cycle via PEP-CK, with generation of seven CO2 for Rubisco in the Benson–Calvin cycle of BS chloroplasts. Reductive power is generated in BS chloroplasts to support reduction of part of the PGA to triose-P, while reductive power is needed in the M chloroplast to support reduction of part of the PGA from BS chloroplasts, and reduction of oxaloacetate to malate in the NAD-ME cycle. Mesophyll and BS cells in PEP-CK species have similar capacity of reductive phase enzymes, PGA kinase and NADP-triose-P dehydrogenase (Ku and Edwards, 1975). In plant chloroplasts, the currently accepted stochiometry for linear electron transport is production of 26 ATP per 2 NADPH generated per O2 evolved by PSII (Allen, 2003). This is similar to the ratio required in M chloroplasts in the scheme in Fig. 8A, whereas the requirement in the BS chloroplasts is higher (375 ATP/2 NADPH). The higher P700/mg Chl in BS than in M chloroplasts in PEP-CK species, and smaller grana in BS chloroplasts (discussed earlier), may reflect function of cyclic photophosphorylation around PSI to support the additional need for ATP. Partitioning more PGA to M chloroplasts for reduction would result in a further increase in the ATP/NADPH ratio in BS chloroplasts. The CO2 concentrating mechanism in C4 plants is considered to minimize, but not eliminate, ribulose 1,5bisphosphate oxygenase activity in BS chloroplasts. Figure 8B illustrates the consequences of photorespiration in PEP-CK-type species when glycerate, produced in the glycolate pathway, is shuttled to M chloroplasts for reduction (glycerate kinase is located in M cells in C4 plants; Usuda and Edwards, 1980) and ammonia, produced in the glycolate pathway, is reassimilated in BS cells, with M and BS chloroplasts sharing in reduction of PGA. Remarkably, the energy requirements from photochemistry in M and BS chloroplasts per ribulose 1,5-bisphosphate utilized are very similar during net CO2 fixation in C4 photosynthesis (Fig. 8A) and in photorespiration (Fig. 8B).

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Voznesenskaya et al. — PEP-CK in Grasses A

Bundle sheath cell

Mesophyll cell

i. Malate cycle via NAD-ME 2 PEP + 2 CO2 → 2 OAA + 2 Pi 2 OAA + 2 NADPH → 2 Mal + 2 NADP 2 Pyr + 4 ATP → 2 PEP + 4 ADP + 2 Pi ii. Asparate cycle via PEP-Ck 5 PEP + 5 CO2 → 5 OAA + 5 Pi 5 OAA + 5 GLU → 5 Asp + 5 ␣kg iii. CO2 fixation in C3 cycle

2 Mal 2 Pyr

2 MAL 2 Pyr

5 Asp 5 PEP

5 Asp 5 PEP

6 PGA

6 PGA

2 Mal + 2 NAD → 2 Pyr + 2 CO2 + 2 NADH 2 NADH + 5 ADP + 5 Pi → 2 NAD + 5 ATP

5 Asp + 5 αkg → 5 OAA + 5 GLU 5 OAA + 5 ATP → 5 PEP + 5 ADP + 5 CO2

6 PGA + 6 ATP + 6 NADPH→ 6 triose-P + 6 ADP + 6 Pi + 6 NADP

7 CO2 + 7 RuBP → 14 PGA 8 PGA + 8 ATP + 8 NADPH→ 8 triose-P + 8 ADP + 8 Pi + 8 NADP 7 Ribulose-5-P + 7 ATP → 7 RuBP + 7 ADP

Sum of photochemical requirement 10 ATP, 8 NADPH = 2·5 ATP/2 NADPH

Sum of photochemical requirement: 15 ATP, 8 NADPH = 3·75 ATP/2 NADPH

Overall photochemical requirement = 25 ATP, 16 NADPH per 7 CO2 fixed (3·1 ATP/2 NADPH)

B

Mesophyll cell

Bundle sheath cell

6 NADPH, 6 ATP 6 triose-P

6 PGA

3 triose-P

3 glycerate

3 glycerate + 3 CO2 + 3 NH3

3 NADPH, 6 ATP

6 C2

3 ATP, 3 NADPHeq 3 glutamate 6 C3 6 ATP

6 O2 + 3 CO2 + 9 RuBP

6 C3 12 C3

6 NADPH 9 triose-P from mesophyll cell 9 ATP

Total

= 12 ATP, 9 NADPH = 2·7 ATP, 2 NADPH

= 18 ATP, 9 NADPH = 4 ATP, 2 NADPH Total per RuBP 3·3 ATP, 2 NADPH

F I G . 8. Schematic representation of the energetics and biochemical reactions of the PEP-CK-type C4 photosynthesis system, as inferred from this study and information in the literature. (A) Illustration of the energetics in PEP-CK-type C4 photosynthesis with a malate NAD-ME C4 cycle generating ATP to support an aspartate PEP-CK cycle, with equal requirement for reductive power between the two cell types. (B) Proposed consequences of photorespiration, and refixation of photorespired CO2 in bundle sheath cells, on the energy requirements in mesophyll and bundle sheath cells with equal requirement for reductive power between the two cell types. In C4 plants, glycerate kinase, which catalyses phosphorylation of glycerate to PGA, is located in mesophyll chloroplasts (Usuda and Edwards 1980).

In summary, family Poaceae shows substantial diversity in C4 photosynthesis in having three C4 biochemical subtypes and various types of Kranz anatomy. NAD-ME-type C4 species have little or no NADP-ME and PEP-CK; carbon acquisition in this subtype occurs through a C4 cycle via BS mitochondrial NAD-ME. In NADP-MEtype C4 species, a C4 cycle functions through

NADP-ME in BS chloroplasts which have reduced grana. Carbon acquisition is virtually solely through NADP-ME in species like Sorghum bicolor, Saccharum officinale (subfamily Panicoideae), Stipagrostis pennata and Aristida purpurea (subfamily Aristidoideae), which has little or no PEP-CK and little NAD-ME (see the Introduction and Fig. 1; Voznesenskaya et al., 2005a, b).

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It is suggested that an aspartate shuttle via a PEP-CK C4 cycle contributes to carbon acquisition in some NADP-ME species in subfamily Panicoideae; hypothetically, the extent may be correlated with the degree of BS chloroplast grana development. In the classical PEP-CK-type C4 species, the major path of carbon acquisition is through an aspartate PEP-CK C4 cycle which is suggested to be complimented by a malate NAD-ME C4 cycle (Fig. 8). This study provides further insight into the relationships of anatomy, ultrastructural, and biochemical features of PEP-CK-containing C4 species in family Poaceae. ACKNOWLEDGEMENTS We acknowledge support of this work by NSF grants IBN-0131098 and IBN-0236959, and by Civilian Research and Development Foundation grant RB1-2502-ST-03 and Russian Foundation of Basic Research grant 05-04-49622. We also thank the Electron Microscopy Center of Washington State University for use of their facilities and staff assistance, and Dr Nouria Koteyeva for help with calculations.

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