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(American Journal of Botany. 2001;88:992-1005.)
© 2001 Botanical Society of America, Inc.

Structure and Development

Ontogenetic derivation and cell differentiation in photosynthetic tissues of C3 and C4 Cyperaceae1

Connie L. Soros2 and Nancy G. Dengler

Department of Botany, University of Toronto, Toronto, Ontario, Canada M5S 1A1

Received for publication May 5, 2000. Accepted for publication July 20, 2000.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Four variants of Kranz anatomy occur in the Cyperaceae. Three of these anatomical types (fimbristyloid, chlorocyperoid, and eleocharoid) are unique among taxa with C4 photosynthesis in that the photosynthetic carbon reduction tissue (PCR, functional equivalent of bundle sheath) is located within the vascular strand and is separated from the primary carbon assimilation tissue (PCA, positional equivalent of mesophyll) by the mestome sheath layer. In the fourth anatomical type, rhynchosporoid, PCR tissue is located in the position of the mestome sheath. In this study, we compared two aspects of development of PCR and PCA tissues in representatives of the C3 and C4 types: (1) ontogenetic derivation and (2) cellular differentiation. Analysis of the planes of cell division associated with procambial strand formation indicated that PCR tissue is always derived from the procambium, while PCA tissue is derived from the ground meristem. These cell lineages remain distinct after the initial organization of vascular strands. Analysis of cell differentiation using accumulation of cell-type-specific photosynthetic enzymes as markers of differentiation indicated that, with one exception, a low level of non-cell-specific enzyme accumulation preceded abundant and cell-specific accumulation of photosynthetic enzymes at the distal end of the leaf elongation zone. Enzyme accumulation coincided spatially (and temporally) with structural aspects of cell differentiation. Previous cladistic analyses have indicated that these anatomical types represent separate evolutionary origins of the C4 pathway, and the differences in developmental pathways observed here reflect these independent origins from C3 ancestors.

Key Words: C4 photosynthesis • cell differentiation • cell lineage • Cyperaceae • Kranz anatomy • PEPCase • RuBPCase


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
The C4 photosynthetic pathway requires the coordinated activity of two photosynthetic cell types: primary carbon assimilation cells (PCA cells, the positional equivalent of mesophyll cells) and photosynthetic carbon reduction cells (PCR cells, usually the positional equivalent of bundle sheath cells). Each cell type has distinct ultrastructure and a unique enzyme complement, reflecting the compartmentalization of the C4 pathway. These two different types of photosynthetic cells were described first in the sedge species, Cyperus longus (Haberlandt, 1882 ). Haberlandt (1882, 1914 ) referred to the pattern of leaf anatomy with radially arranged mesophyll cells and a conspicuous bundle sheath as "Kranz" (German for wreath) anatomy. The term Kranz has since been used to refer to both the entire suite of structural characteristics associated with C4 plants and the enlarged, chloroplast-rich bundle sheath (PCR) cells alone (Johnson and Brown, 1973 ; Brown, 1975 ). Shortly after the discovery of C4 photosynthesis (Hatch and Slack, 1966 ), the association between Kranz anatomy and the C4 biochemical pathway was demonstrated, including the functional significance of tissue arrangement, the division of labor between the two photosynthetic cell types, and the role of a diffusion barrier at the interface between PCA and PCR cells (Hatch, Slack, and Johnson, 1967 ; Laetsch, 1971, 1974 ; Gutierrez, Gracen, and Edwards, 1974 ; Hatch, Kagawa, and Craig, 1975 ).

Kranz anatomy of leaves and photosynthetic culms in C4 Cyperaceae differs from other C4 families in one primary respect: at least one nonphotosynthetic cell layer lies between the PCA and PCR cells. While the anatomical variants found in all other families with C4 taxa meet the "one cell distant" criterion (i.e., PCA and PCR tissues are directly adjacent) (Hattersley and Watson, 1975 ), many C4 taxa of the Cyperaceae break this rule. This unusual separation of PCA and PCR tissue in the Cyperaceae may have implications for both the diffusion of C4 intermediate metabolites (Soros and Dengler, 1998 ) and for developmentally important signalling pathways (Nelson and Langdale, 1992 ; Brutnell and Langdale, 1998 ; Dengler and Nelson, 1999 ; Dengler and Taylor, 2000 ).

Four anatomical subtypes have been recognized in taxa of the Cyperaceae with C4 photosynthesis: the rhynchosporoid, chlorocyperoid, fimbristyloid, and eleocharoid types (Takeda, Ueno, and Agata, 1980 ; Bruhl, Stone, and Hattersley, 1987 ; Bruhl and Perry, 1995 ). The types are defined by: the presence or absence of the parenchymatous bundle sheath layer, the position of the PCR tissue, and, in taxa where the PCR tissue is in the position of inner border parenchyma, whether the PCR layer is interrupted by large metaxylem elements or is continuous. In the rhynchosporoid type, the PCR layer is in the position of the mestome sheath of comparable C3 Cyperaceae, and a parenchymatous bundle sheath is present (Fig. 1B; Takeda, Ueno, and Agata, 1980 ). In the chlorocyperoid and fimbristyloid types, PCR tissue is in the position of inner border parenchyma of the vascular tissue of comparable C3 Cyperaceae and, in large major veins, the PCR layer is interrupted by large metaxylem elements (Fig. 1C, D). A mestome sheath is present in both types. The fimbristyloid type always has at least a partial parenchymatous bundle sheath, whereas this sheath consists of only a few cells or is absent in the chlorocyperoid type (Lerman and Raynal, 1972 ; Raynal, 1973 ; Hattersley and Watson, 1975 ; Carolin, Jacobs, and Vesk, 1977 ). In the eleocharoid anatomical type, the PCR layer is in the position of inner border parenchyma, but this layer is continuous, extending between the large metaxylem vessels and mestome sheath in major veins (Fig. 1E; Bruhl, Stone, and Hattersley, 1987 ; Ueno et al., 1988 ; Bruhl and Perry, 1995 ).



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  		Fig. 1. Diagrams (upper row) and micrographs (lower row) of transverse sections of major vascular bundles in C3 and C4 anatomical types in the Cyperaceae. (A) Cyperus eragrostis (C3). (B) Rhynchospora rubra (C4, rhynchosporoid type). (C) Pycreus polystachyos (C4, chlorocyperoid type). (D) Eleocharis vivipara (C4, fimbristyloid type). (E) Eleocharis retroflexa (C4, eleocharoid type). Scale bar = 25 µm. Figure Abbreviations: IBP, inner border parenchyma; ICS, intercellular space; M, mesophyll; MP, metaphloem; MS, mestome sheath; MX, metaxylem; PBS, parenchymatous bundle sheath; PCA, primary carbon assimilation tissue; PCR, photosynthetic carbon reduction tissue; PP, protophloem; PX, protoxylem; VT, vascular tissue.

 
With relatively few exceptions, each of these anatomical types has evolved in a separate tribe of the Cyperaceae (Soros and Bruhl, 2000 ). When anatomical types were mapped onto phylogenetic trees constructed from a morphological data set (Bruhl, 1995 ) and a combined morphological and rbcL sequence data set (Muasya et al., 2000 ), analyses indicated that the C4 pathway has arisen at least four times in the Cyperaceae. The chlorocyperoid type arose in the tribe Cypereae, the fimbristyloid type in the tribe Abildgaardieae, and the eleocharoid type in the tribe Scirpeae, all subfamily Cyperoideae (Bruhl, 1995 ; Soros and Bruhl, 2000 ). Both the rhynchosporoid and chlorocyperoid types arose in the Rhynchosporeae (subfamily Caricoideae; Bruhl, 1995 ). Each anatomical type has evolved in concert with one of the C4 biochemical subtypes: the rhynchosporoid, chlorocyperoid and fimbristyloid anatomical types are the NADP-malic enzyme biochemical subtype (NADP-ME). The eleocharoid anatomical type (and one anomalous fimbristyloid species, Eleocharis vivipara; Bruhl, Stone, and Hattersley, 1987 ; Ueno et al., 1988 ) is the NAD-malic enzyme (NAD-ME) biochemical type.

In this study, we investigated two aspects of the development of PCA and PCR cells in species of the Cyperaceae that represent these major photosynthetic and anatomical types (C3, rhynchosporoid, chlorocyperoid, and eleocharoid; Table 1). Our goal was to determine whether major clades differed in the developmental pathways used to achieve mature Kranz anatomy. First, we analyzed cell lineage relationships among the precursors of PCR, PCA, and associated tissues in order to determine structural equivalence between tissues in C3 and the C4 anatomical types. Since grass and sedge leaves have distinct longitudinal cell files, the lineages of vascular tissue and associated PCR and PCA tissues can be followed from primary meristematic tissue to maturity. Analysis of serial cross sections makes it possible to relate the position of differentiating tissues above the leaf or culm extension zone with origin of tissue files within procambium and ground meristem (Dengler, Dengler, and Hattersley, 1985 ; Bosabalidis, Evert, and Russin, 1994 ). For example, in the Poaceae, the PCR tissue in the "single sheath" C4 anatomical type is derived from procambium, while in "double sheath" anatomical types PCR tissue is derived from ground meristem (Dengler, Dengler, and Hattersley, 1985 ; Nelson and Dengler, 1992 ). It has been previously hypothesized that PCR tissue of the chlorocyperoid, fimbristyloid, and eleocharoid types originated from the inner border parenchyma of ancestral C3 species and is therefore ontogenetically derived from procambial tissue (Brown, 1975 ; Carolin, Jacobs, and Vesk, 1977 ; Ueno, 1983 ; Ueno and Koyama, 1987 ; Ueno, Samejima, and Koyama, 1989 ; Estelita, 1992 ). Observations of positional relationships in mature tissues support this hypothesis; however, this is the first developmental study to specifically test whether PCR tissue in the C4 anatomical types of the Cyperaceae is derived from procambium.


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  		Table 1.  Sources of plant material, biochemical and anatomical types, and voucher specimens for species sampled

 
The second aspect of C4 development investigated for each of these anatomical types was the pattern of cell differentiation. This was done using photosynthetic enzyme accumulation as a marker of cell differentiation. In order for the C4 pathway to operate, mature PCA and PCR cells must each have specialized anatomy and cell-specific enzyme complements. While PCA cells resemble C3 mesophyll cells anatomically, PCR cells are conspicuously larger and have more numerous and/or larger chloroplasts, an asymmetric distribution of organelles, and modified cell walls (Edwards and Walker, 1983; Dengler and Nelson, 1999 ; Dengler and Taylor, 2000 ). PCR cells accumulate ribulose-1,5-bisphosphate carboxylase (RuBPCase), other enzymes of the C3 Calvin (PCR) cycle, and one or more decarboxylating enzymes (NADP-ME, NAD-ME, or phosphoenol-pyruvate carboxykinase). The PCA cells accumulate carbonic anhydrase and the C4 pathway enzymes phosphoenol-pyruvate carboxylase (PEPCase), pyruvate orthophosphate dikinase, and malate dehydrogenase (Hatch, 1987 ; Kanai and Edwards, 1999 ; Furbank, Hatch, and Jenkins, 2000 ). In the present study, we have characterized the pattern of accumulation of cell-specific enzymes as markers of C4 tissue differentiation, namely RuBPCase in PCR cells and PEPCase in PCA cells.

Previous developmental studies correlating cell-specific enzyme accumulation and/or mRNA expression with structural differentiation of photosynthetic tissues have been carried out for the C4 grass Zea mays (Langdale et al., 1988 ) and for the C4 dicots Atriplex rosea (Liu and Dengler, 1994 ; Dengler et al., 1995 ) and species of Amaranthus (Wang, Klessig, and Berry, 1992 ; Wang et al., 1993a, b ; Ramsperger, Summers, and Berry, 1996 ; McCormac et al., 1997 ). These studies have reached opposing conclusions regarding the coordination of structural and biochemical characteristics and the timing of the unique C4 pattern of RuBPCase expression (upregulated in PCR cells and downregulated in PCA cells). By combining information about ontogenetic derivation with markers for photosynthetic cell differentiation, and by making a phylogenetically meaningful comparison across major photosynthetic types in the Cyperaceae, this study is able to assess the influence of cell position and lineage on the time course of cell differentiation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Plant material and growth conditions
Plants of the C3 species Cyperus eragrostis and three C4 species, Rhynchospora rubra (NADP-ME, rhynchosporoid anatomical type), Pycreus polystachyos (NADP-ME, chlorocyperoid anatomical type), and Eleocharis retroflexa (NAD-ME, eleocharoid anatomical type) were grown in the University of Toronto greenhouses. Locations and collectors of seed or preserved material are listed in Table 1; vouchers are deposited at the Royal Ontario Museum Herbarium, Canada (TRT). In addition to anatomical typing, all species examined in this study were previously biochemically typed by enzyme analysis and/or carbon isotope ratios (Table 1).

Identification of leaf elongation zone
Since leaves and culms of Cyperaceae species grow through the activity of a basal intercalary meristem, it was necessary to identify the location of the leaf (or culm) elongation zone in order to study patterns of cell division and cell differentiation (Soros and Dengler, 1996 ). Total leaf or culm lengths were measured for five replicate plants for each of the four species investigated. Leaves were fixed in formalin–glacial acetic acid–ethanol (70%) and rinsed in 70% ethanol. The lengths of five epidermal cells were measured at 1.0-mm intervals along the length of each expanding leaf using a stage micrometer on a Reichert Polyvar microscope. Since we previously found that epidermal cell elongation could be used as a marker for the distal boundary of the zone of leaf elongation (identified using independent criteria), the distance from the leaf base to the most proximal fully elongated epidermal cells was measured (Soros and Dengler, 1996 ). As an additional verification, the distal boundary of the leaf elongation zone was identified in serial paraffin sections as the basalmost location where protoxylem lacunae of major veins first became evident (Soros and Dengler, 1996 ).

Ontogenetic derivation of photosynthetic tissues
Ontogenetic origins of photosynthetic tissues were studied using vegetative shoot apices from plants that were 4–8 wk old. The apices were fixed in 4% paraformaldehyde and 2% glutaraldehyde, postfixed in 2% OsO4, and embedded in Epon or Spurr's resin (Analychem Corp. Ltd., Markham, Ontario, Canada). Serial transverse sections 3 µm thick were made on a Sorvall MT-2 ultramicrotome (Norwalk, Connecticut, USA) and stained using 0.05% Toluidine Blue O in 0.1% sodium carbonate. Representative sections were photographed on a Polyvar microscope (Reichert-Jung, Vienna, Austria).

Positional equivalence of PCR tissue was inferred from determination of cell lineage relationships of veins and associated tissue. Planes of formative cell divisions in procambium and ground meristem were determined in serial sections, using either visible mitotic figures (metaphase through telophase) or pairs of sister cells, separated by a conspicuously thin (presumed to be recently formed) cell wall (Fig. 2). Division figures were scored as giving rise to daughter cells in either one or two of the following four regions: A, all procambium but the outermost layer; B, the outermost layer of procambium; C, the layer of ground meristem adjacent to the procambium; D, all other ground meristem (Fig. 2; Dengler, Dengler, and Hattersley, 1985 ). The orientations of all formative (longitudinal) divisions occurring within the zone of cell division at the base of an expanding leaf or culm were recorded by scoring between the distal end of the leaf extension zone where basipetally differentiating protoxylem and protophloem first appeared (Soros and Dengler, 1996 ) and the disk of leaf insertion where leaf (or culm) procambial strands extended basipetally to connect with the vascular bundles of the main stem. Counts were made along the length of ten vascular bundles (five major and five minor) for each of three replicate apices per species, with careful attention not to count the same event twice. Reported values are averages for three apices (see Fig. 5). For this study, major bundles were identified as those with protoxylem lacunae and large metaxylem elements, while minor veins were those without protoxylem lacunae and small metaxylem elements. These counts provided quantitative data for otherwise qualitative descriptions of pattern, thus providing additional support for interpretation of positional relationships in mature tissues.



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  		Fig. 2. Photomicrograph (A) and diagram (B) indicating cell layer designations in developing procambium (A and B regions) and ground meristem (C and D regions). See text for explanation. Arrow points to thin, presumed recently formed cell wall. Scale bar = 25 µm

 


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  		Fig. 5. Relative proportions (percentage of total, mean + 1 SE) of various division planes in developing procambium and associated ground meristem in major and minor veins (combined) of leaves and culms of C3 and C4 anatomical types in the Cyperaceae. (A) Cyperus eragrostis (C3). (B) Rhynchospora rubra (C4, rhynchosporoid type). (C) Pycreus polystachyos (C4, chlorocyperoid type). (D) Eleocharis retroflexa (C4, eleocharoid type). A, B, C, D regions as illustrated in Fig. 2 . N, total number of divisions counted (ten veins per apex; three apices)

 
Immunolocalization
Isolation of RuBPCase proteins and production of antibodies were performed as described in Dengler et al. (1995) . Polyclonal antibodies to PEPCase enzyme, prepared as described in Wang, Klessig, and Berry (1992) , were generously provided by Dr. James O. Berry (State University of New York, Buffalo, New York). Three replicate apices, each containing five to ten leaves per species were examined for each enzyme. Shoot apices were fixed in 3 : 1 ethanol : glacial acetic acid overnight at room temperature. Dehydration and embedding methods used were described by Dengler et al. (1995) . Serial transverse sections 7 µm thick were cut on a Spencer AO rotary microtome. Sections were selected at 0.1-mm intervals for the basalmost 1.3 mm, 0.3-mm intervals from 1.3 to 3.1 mm, and 1.0-mm intervals for the remaining portion of each shoot apex and mounted on poly-L-lysine (100 µg/mL) coated slides.

Immunolocalizations were carried out using reagents from the ImmunoselectTM Immunocytochemistry, ELISA, and Immunoblotting System (Gibco BRL, Burlington, Ontario, Canada). The immunolocalization protocol was followed as described in Dengler et al. (1995) . Primary antibodies were diluted 1 : 5000 for RuBPCase and 1 : 2000 for PEPCase. Parallel control slides using preimmune serum in place of primary antibody were run with each RuBPCase experiment, and slides omitting the primary antibody but including all other steps were run as controls for the PEPCase experiments. In some experiments, other species with known immunolocalization patterns (e.g., Stenotaphrum secundatum; Sud and Dengler, 2000 ) were included on the same slides as positive controls.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Identification of the leaf elongation zone
In each of the representative species of the four anatomical types examined, the zone of elongation was more or less constant in extent across leaves of increasing ages, based on the pattern of epidermal cell elongation. In each species, the youngest macroscopically visible leaf or culm was shorter than the elongation zone (Fig. 3; Soros and Dengler, 1996 ). Mature leaves that had ceased expansion still had a zone of shorter epidermal cells at the base, although mature vascular tissue extended across the zone (Soros and Dengler, 1996 ).



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  		Fig. 3. Mean total length (+1 SE) and mean elongation zone length (+1 SE) of macroscopic leaves or culms of C3 and C4 anatomical types in the Cyperaceae. (A) Cyperus eragrostis (C3). (B) Rhynchospora rubra (C4, rhynchosporoid type). (C) Pycreus polystachyos (C4, chlorocyperoid type). (D) Eleocharis retroflexa (C4, eleocharoid type). Length of the elongation zone was inferred from epidermal cell lengths and position of protoxylem lacunae. Solid bar, elongation zone; Open bar, mature tissue; PX, region without protoxylem lacunae

 
In growing leaves of Cyperus eragrostis (C3), the leaf elongation zone comprised the basal 11.2 ± 1.2 mm of total leaf length (based on epidermal cell length), while protoxylem lacunae were evident 9.3 ± 0.7 mm from the leaf base (Fig. 3A). Leaves stopped growing when they reached ~175 mm in length, so that the elongation zone occupied ~6–8% of the mature leaf length. The leaves or culms of the C4 representative species were shorter and had proportionately shorter zones of leaf elongation, but these still occupied ~8% of mature leaf or culm length (Fig. 3B–D). In leaves of Rhynchospora rubra (rhynchosporoid type), the elongation zone extended ~3.0 mm, based on epidermal cell measurements and 2.4 ± 0.2 mm, based on the level of protoxylem lacunae (Fig. 3B). In leaves of Pycreus polystachyos (chlorocyperoid type), the elongation zone extended 5.8 ± 0.5 mm based on epidermal cell lengths and 4.3 ± 0.3 mm based on level of protoxylem lacunae (Fig. 3C). In culms of Eleocharis retroflexa (eleocharoid type), the basal elongation zone extended 2.7 ± 0.3 mm based on epidermal cell lengths and 3.7 ± 0.7 mm based on the level of protoxylem lacunae (Fig. 3D).

Ontogenetic derivation of PCR and associated tissues
In the four representative species examined, longitudinal veins were organized during the first four to five plastochrons of leaf development. Major veins appeared first within the leaf primordium and then extended basipetally into the disk of leaf insertion within the stem. At the earliest stages of major vein development observed, procambium was generally distinguished from ground meristem by smaller cell diameter and absence of vacuoles within the cytoplasm (Fig. 4A). Minor vein procambial strands arose in a similar way, but after major vein procambium was continuous with the stem bundles. Patterns of cell proliferation within minor veins generally mirrored those of major veins, although there were fewer divisions, reflecting smaller vein size. Because of this similarity, data for major and minor veins were combined (Fig. 5).



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  		Fig. 4. Transverse sections of major vein (A–D) and minor vein (E–F) procambial strand formation in Cyperus eragrostis (C3). (A) Pattern of procambial cells indicating derivation from three precursors (arrow). (B) Procambial strand with "A" and "B" regions. (C) Procambial strand with "A/A" division figure (arrow). Recent "C/D" division in ground meristem adjacent to procambium (arrowhead). (D) Differentiation of protophloem and protoxylem from "A" region. (E) Pattern of procambial cells indicating derivation from one precursor (arrow). (F) Procambial strand with "A" and "B" regions. (G) Procambial strand with "A/A" division figure (arrow). (H) Differentiation of protophloem and protoxylem from "A" region. Scale bar = 25 µm. Regions are as in Fig. 2

 
In the C3 species Cyperus eragrostis, procambial strands could be recognized by the reduction in size of vacuoles and by the distinctive planes of cell division (Fig. 4A, E). Major vein procambial strands appeared to be derived from one to three precursor cells (Fig. 4A), while minor vein procambial strands appeared to be derived from one precursor (Fig. 4E). Division patterns in both major and minor procambial strands gave rise to distinct "A" and "B" regions at an early stage of development (Figs. 2, 4B, F). Subsequent divisions within the procambium occurred either within the peripheral "B" layer or within the "A" region, maintaining the distinctness of these two lineages (Fig. 4C, G). During the initial stages of procambial strand formation, longitudinal divisions within the adjacent ground meristem occurred in all planes (Fig. 4A, B, E, F). As the procambial strand grew in width, however, ground meristem divisions oriented tangentially to the surface of the procambial strand gave rise to the "C" layer of ground meristem directly adjacent to the procambium (Figs. 2, 4C, G). After formation of the "C" layer from the rest of the ground meristem ("D" region), it tended to remain distinct from surrounding ground tissue (Fig. 4D). In both major and minor veins, the differentiation of the first protophloem elements within region "A" provided a marker, indicating that region "A" formed the vascular tissue with inner border parenchyma at its periphery, region "B" formed the mestome sheath layer, region "C" formed the parenchymatous bundle sheath layer, and region "D" formed adjacent ground tissue (Figs. 1A, 2, 4D, H).

The quantitative analysis of the planes of cell division associated with leaf vein development in Cyperus eragrostis supported these qualitative observations (Fig. 5A). The greatest proportion of divisions (~40%) gave rise to derivatives within the "A" region (A/A divisions). The proportion of divisions that gave rise to derivatives in both "A" and "B" regions was small (<5%), reflecting the separation of vascular tissue and mestome sheath lineages from an early stage. In contrast, the proportion of divisions that gave rise to two derivatives within the "B" layer was higher (~15%). Within the ground meristem, divisions that gave rise to one derivative in the "C" region and one in the "D" region (C/D) were more frequent than either pattern with both derivatives in the same region (C/C and D/D divisions, Fig. 5A).

The C4 species Rhynchospora rubra (rhynchosporoid type), Pycreus polystachyos (chlorocyperoid type), and Eleocharis retroflexa (eleocharoid type) had similar qualitative and quantitative patterns of cell division plane associated with major and minor vein ontogeny as the C3 species Cyperus eragrostis (Fig. 5B–D). For all four species, there were no instances of divisions occurring across the boundary of procambial and ground meristem lineages (B/C divisions; Fig. 5B–D). Once formed, the lineage restriction between procambium (regions A and B) and ground meristem (regions C and D) was maintained throughout the developmental stages observed. Thus all cells derived from layers "A" and "B" (the vascular tissue, including inner border parenchyma, and mestome sheath) were exclusively procambial in origin, while those from layers "C" and "D" (parenchymatous bundle sheath and mesophyll) were exclusively from ground meristem. The PCR tissue arose from the peripheral layer of the procambium in Rhynchospora rubra and from cells just to the interior of the peripheral layer in Pycreus polystachyos and Eleocharis retroflexa. Metaxylem elements of major veins differentiated within the peripheral procambial layer in Pycreus polystachyos, but to the interior of this layer in Eleocharis retroflexa, giving rise to a continuous layer of inner border parenchyma in this anatomical type.

Cell differentiation
In this study, cell differentiation was assessed by the cell-specific accumulation of the photosynthetic enzymes RuBPCase and PEPCase. In the C3 species Cyperus eragrostis, RuBPCase and PEPCase were not detected within the zone of cell division at ~1.0 mm from the leaf base (Figs. 6A, D, 9A). Low-level non-cell-specific accumulation was evident within the zone of elongation, at 2.3 ± 0.3 mm from the leaf base for RuBPCase and at 2.6 ± 0.7mm from the leaf base for PEPCase (Figs. 6B, E, 9A). High-level and cell-specific accumulation of RuBPCase in the mesophyll cells was apparent at 14.0 ± 1.0 mm from the base of the leaf, just above the distal end of the zone of leaf elongation (Figs. 3A, 6C, 9A). At this stage, accumulation of PEPCase was barely detectable and was non-cell-specific (Figs. 6F, 9A). Changes in enzyme accumulation were correlated with structural aspects of cell differentiation: low-level, nonspecific accumulation of RuBPCase coincided with the first differentiation of protoxylem and protophloem and the initiation of intercellular space formation in the mesophyll (Figs. 6B, E, 9A). High-level, mesophyll cell-specific accumulation of RuBPCase coincided with maturation of metaxylem and metaphloem and formation of large intercellular spaces in the mesophyll (Figs. 6C, F, 9A).



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  		Fig. 6. Accumulation of RuBPCase (A–C) and PEPCase (D–F) in developing leaves of Cyperus eragrostis (C3). (A, D) Enzyme accumulation not detected. (B, E) Non-cell-specific accumulation of enzymes (arrowheads). (C) High-level, cell-specific accumulation of RuBPCase in mesophyll tissue. (F) Low-level, non-cell-specific accumulation of PEPCase (arrowhead). Scale bar = 50 µm

 
The same general pattern was seen in the three representative C4 species examined, but they differed slightly in timing of enzyme accumulation (Fig. 9B–D). In the chlorocyperoid species, Pycreus polystachyos, neither enzyme was detectable within the zone of cell division (~0.2 mm from the leaf base; Figs. 7A, D, 9C). RuBPCase was detectable, but non-cell-specific, ~0.3 ± 0.2 mm from leaf base (Figs. 7B, 9C). PEPCase was not detectable at this level, but appeared ~1.2 ± 0.3 mm above the leaf base (Figs. 7E, 9C). Accumulation of these enzymes preceded both differentiation of protophloem and protoxylem and formation of extensive intercellular airspace in the mesophyll (Fig. 7B, E). High-level, cell-specific accumulation of RuBPCase within PCR cells was correlated with the distal end of the leaf elongation zone (Figs. 3C, 7C, 9C). High-level, cell-specific accumulation of PEPCase in PCA cells occurred distal to the leaf elongation zone (7.7 ± 0.9 mm from the leaf base) (Figs. 7F, 9C). PEPCase was not detectable or detectable only at low levels in ground tissue more than one cell away from the vein (Fig. 7F). Full maturation of metaxylem and metaphloem and formation of large intercellular spaces occurred distal to the level of maximum enzyme accumulation (not shown).



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  		Fig. 9. Patterns of accumulation of RuBPCase and PEPCase (either not detected, or low-level and non-cell-specific, or high-level and cell-specific) plotted in relation to mean lengths (+1 SE) of leaf or culm elongation zones. Elongation zones indicated by levels of the youngest fully elongated epidermal cells and the first appearance of protoxylem lacunae. (A) Cyperus eragrostis (C3). (B) Rhynchospora rubra (C4, rhynchosporoid type). (C) Pycreus polystachyos (C4, chlorocyperoid type). (D) Eleocharis retroflexa (C4, eleocharoid type)

 


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  		Fig. 7. Accumulation of RuBPCase (A–C) and PEPCase (D–F) in developing leaves of Pycreus polystachyos (C4, chlorocyperoid type). (A, D) Enzyme accumulation not detected. (B, E) Non-cell-specific accumulation of enzymes (arrowheads). (C, F) High-level, cell-specific accumulation of enzymes. Scale bar = 50 µm

 
In the eleocharoid species, Eleocharis retroflexa, a similar pattern of enzyme accumulation was observed, with the exception that RuBPCase was detected right to the base of the culm, where it was non-cell-specific (Fig. 9D). In contrast, accumulation of PEPCase occurred later (and initially was not cell-specific), at 0.3 ± 0.2 mm from the base of the culm. Cell-specific accumulation of both RuBPCase and PEPCase was present at 3.3 ± 0.8 and 5.3 ± 1.2 mm, respectively (Figs. 8A, B, 9D). In mature tissues, PEPCase accumulated in cells in the position of C3 parenchymatous bundle sheath, indicating that cells in this position function as PCA tissue (Fig. 8B).



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  		Fig. 8. Distribution of RuBPCase (A) and PEPCase (B) in mature culm tissues of C4 eleocharoid Eleocharis retroflexa. Scale bar = 50 µm

 
The rhynchosporoid species Rhynchospora rubra accumulated RuBPCase in the layer positionally equivalent to the mestome sheath at maturity. Surprisingly, PEPCase never became cell-specific (Fig. 9B), but accumulated in both PCA and PCR cells, typically at higher levels in the PCR (mestome sheath) layer. We observed this result consistently, even on slides where other C4 species provided positive controls. This observation indicates that this Rhynchospora species may have an unusual version of C4 photosynthesis, and additional species of the genus are currently being investigated.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
The spatial arrangement of photosynthetic tissues in three (fimbristyloid, chlorocyperoid, eleocharoid) of the four C4 anatomical types in the Cyperaceae is highly unusual, because PCR tissue is located in the position of the inner border parenchyma of vascular tissue. In all other C4 families, PCR and PCA tissues are in direct contact (or rarely separated by another cell), facilitating the diffusion of C4 metabolites between the two cell types (Hattersley and Watson, 1975 ; Dengler and Nelson, 1999 ; Leegood, 2000 ). In contrast, C4 metabolites diffusing between PCA and PCR tissue must cross at least one extra cell layer, the mestome sheath, in most C4 species of the Cyperaceae. We have previously speculated that this arrangement might represent an evolutionary "solution" to the problem of isolating PCR tissue (the site of the CO2-concentrating mechanism) from the abundant intercellular airspace that is present in this primarily aquatic group of plants. The position of PCR tissue behind the barrier provided by the suberized walls of the mestome sheath potentially enhances CO2 concentration by reducing apoplastic leakage (Soros and Dengler, 1998 ). Whatever the functional reason for this placement of PCR tissue, this unique version of Kranz anatomy raises interesting developmental questions, particularly regarding the ontogenetic derivation of photosynthetic tissues, the temporal pattern of photosynthetic cell differentiation, and the evolution of these developmental patterns within the Cyperaceae.

Ontogenetic derivation of PCR and associated tissues
In this study, we characterized the planes of cell division associated with procambial strand development in developing leaves or photosynthetic culms in taxa representing C3 and three of the C4 anatomical types within the Cyperaceae. Despite the variation in mature anatomical pattern among the types, we found that PCR tissue is always derived from the procambium and PCA is always derived from the ground meristem. Specifically, procambium gives rise to the vascular tissue, including inner border parenchyma (PCR tissue in the chlorocyperoid, fimbristyloid, and eleocharoid types), and the mestome sheath (PCR tissue in the rhynchosporoid type), while adjacent ground meristem forms the mesophyll (PCA tissue in C4 types) and parenchymatous bundle sheath, when present (Fig. 10). This pattern of tissue derivation had been hypothesized by earlier researchers (Brown, 1975 ; Carolin, Jacobs, and Vesk, 1977 ; Takeda, Ueno, and Agata, 1980 ; Ueno, 1983 ; Ueno and Koyama, 1987 ; Ueno and Samejima, 1989 ), but had not previously been tested by developmental analysis.



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  		Fig. 10. Ontogenetic derivation of PCR, PCA, and associated tissues from procambium or ground meristem for C3 and the C4 anatomical types in the Cyperaceae. (A) C3. (B) C4 rhynchosporoid type. (C) C4 chlorocyperoid type. (D) C4 fimbristyloid type. (E) C4 eleocharoid type

 
Previous studies of the early ontogeny of vascular bundles in C4 grasses have led to conflicting conclusions about tissue layer derivation (Esau, 1943, 1965 ; Dengler, Dengler, and Hattersley, 1985 ; Langdale et al., 1989 ; Bosabalidis, Evert, and Russin, 1994 ). Based on cell division patterns associated with major vein development in stems and leaf sheaths in the grass Zea mays (Poaceae), Esau (1943) concluded that the single bundle sheath layer (PCR tissue) surrounding the veins differentiated partly from cells of the outermost layer of procambium and partly from adjacent ground meristem. In contrast, Dengler, Dengler, and Hattersley (1985) used an approach similar to that used in this study to follow the ontogenetic derivation of photosynthetic tissues in leaves of seven grass species representing the major C4 anatomical types and taxonomic groups. They concluded that bundle sheath (PCR) tissue is derived from the outer layer of procambium (the mestome sheath layer) in maize and other C4 grasses with a single bundle sheath, but from the ground meristem (the parenchymatous bundle sheath layer) in grasses with two bundle sheaths (Dengler, Dengler, and Hattersley, 1985 ; Nelson and Dengler, 1992 ; Sud and Dengler, 2000 ). As seen in this study, their results implied that, once procambial strands arose from the ground meristem early in leaf development, the resultant tissue systems were separated developmentally by a lineage restriction.

A different conclusion was reached with a clonal analysis of vein formation in maize. Langdale et al. (1989) found that some clonal sector boundaries bisected a vein, suggesting veins arose from procambial strands that represented mixed cell lineages. In these cases, bundle sheath cells on one side of the vein clearly shared a closer ontogenetic derivation with adjacent mesophyll than with the bundle sheath on the other half of the vein. This apparent contradiction has been partly resolved by more recent anatomical studies. In maize (Bosabalidis, Evert, and Russin, 1994 ), barley (Trivett and Evert, 1998 ), and Stenotaphrum (Sud and Dengler, 2000 ), incipient procambial strands were first recognized in transverse sections as "assemblages"—groups of two to four laterally adjacent cells derived from a common precursor. Less often, procambial strands appeared to arise from two or more cells that do not share an immediate precursor (Bosabalidis, Evert, and Russin, 1994 ). Bosabalidis, Evert, and Russin (1994) recognized that, if a procambial strand arose in a position that straddled a sector boundary in a variegated plant, it would give rise to the "half veins" observed in mature maize leaves by Langdale et al. (1989) .

Taken together, these studies indicate that procambial strand formation occurs through a two-step process during the development of grass and sedge leaves. First, procambial precursors are delimited from the ground meristem during the early plastochrons of leaf development. If the vein is derived from a single file of precursors, formative (longitudinal) divisions give rise to a procambial strand where all cells are more closely related to each other than to adjacent ground-meristem-derived tissue. If two adjacent precursors give rise to a procambial strand during this first phase, the composition of the mature vein will reflect this "mixed" cell lineage and may form visible "half veins" in a variegated leaf (Langdale et al., 1989 ; Sud and Dengler, 2000 ). In the second phase of development, procambial strands are increased in length through additive (horizontal) divisions. This phase is by far the most protracted and perpetuates the clonal make-up of the original procambial strands. In this study of the Cyperaceae, we observed incipient procambial strands that were derived from one to three ground meristem precursors, but we did not begin quantifying the planes of cell division until formative divisions were underway (i.e., "A" and "B" regions were distinguished). Once this occurred, we found strong evidence for a lineage restriction between procambium and ground meristem: PCR tissue differentiates on one side of this putative restriction and PCA tissue on the other.

Ontogenetic derivation of PCR tissue from the procambium may impose a developmental "constraint" that limits aspects of cell differentiation. In the grass family, procambium-derived PCR tissue has lower cell volumes, fewer organelles, and smaller chloroplasts in comparison to ground meristem-derived PCR tissue, even in intrageneric comparisons (Dengler, Dengler, and Hattersley, 1986 ; Dengler et al.,1994 ). Our data indicate that similar constraints may be in operation in the Cyperaceae. Although individual PCR cells in the position of inner border parenchyma are much larger than their C3 counterparts (Soros and Dengler, 1998 ), the total volume of PCR tissue is lower than in the rhynchosporoid type where PCR tissue develops from the mestome sheath layer (Soros, 1999 ). Despite such putative constraints, in three of four independent origins of C4 photosynthesis in the Cyperaceae (fimbrystyloid, chlorocyperoid, and eleocharoid types), it was the inner border parenchyma rather than the mestome sheath that was converted to PCR tissue. This suggests that it may have been "easier" to modify vascular parenchyma of C3 taxa to PCR cells than to convert the achlorophyllous, thick-walled mestome sheath cells.

The differing ontogenetic derivations of PCR and PCA tissues may also have implications for cell-to-cell communication during differentiation. Plasmodesmata, the cytoplasmic channels between adjacent cells, provide a potential route for the intercellular movement of signaling molecules (Lucas, 1995 ; Kragler, Lucas, and Monzer, 1998 ). Primary plasmodesmata are formed during cytokinesis and thus provide cytoplasmic continuity among cells that share a common lineage (Lucas, 1995 ). In all C4 Cyperaceae types, a common procambial cell lineage gives rise to vascular and PCR tissues, while a separate ground meristem lineage gives rise to PCA tissues. Therefore, in early stages of development, plasmodesmatal connections would provide only a limited pathway for the movement of signal molecules between PCR and PCA precursors. As tissues mature, the formation of secondary plasmodesmata across the existing periclinal walls located between the procambium and ground meristem may override the potential limitation of cell lineage on cell–cell communication (Dengler and Nelson, 1999 ). Studies of plasmodesmatal frequencies between various cell–cell interfaces in mature leaves of C4 plants indicate that the highest plasmodesmatal values occur at the PCR–PCA cell interface, providing an extensive symplastic pathway for the diffusion of C4 acids and other metabolites (Botha and Evert, 1988 ; Robinson-Beers and Evert, 1991 ; Evert, Russin, and Bosabalidis, 1996 ; Kragler, Lucas, and Monzer, 1998 ; Leegood, 2000 ). The nature of cell–cell communication between PCR and PCA cells at the time of differentiation is still one of the major unresolved issues of C4 development (Brutnell and Langdale, 1998 ; Dengler and Nelson, 1999 ; Dengler and Taylor, 2000 ).

PCR and PCA cell differentiation
Photosynthetic enzymes accumulate in three distinct stages in the Cyperaceae leaves and culms examined. First, a region that lacks detectable enzyme accumulation (with the exception of RuBPCase in Eleocharis retroflexa) occurs at the base of the leaf or culm, more or less coinciding with the zone of cell division. Second, a region distal to this accumulates low levels of enzymes in a non-cell-specific manner; this corresponds to the basal portion of the zone of cell elongation. Third, a region of high-level, cell-specific RuBPCase (and PEPCase in C4 species) accumulation extends from the distal end of the zone of elongation (as indicated by epidermal cell length measurements and protoxylem differentiation) to the tip of the leaf or culm. Within this general pattern, RuBPCase appears in PCR cells at an earlier stage than PEPCase does in PCA cells in these C4 species.

Two lines of evidence led Nelson and Langdale (1992) to hypothesize that a developmental signal produced in the vascular tissue (or its precursors) diffuses outward, first to PCR and then to PCA cells, inducing cell-specific developmental change. First, the delayed differentiation of PCA cells with respect to PCR cells (as indicated by the delayed accumulation of PEPCase) suggests that prior differentiation of adjacent PCR cells may be prerequisite to PCA development. Second, mesophyll cells that are more distant from the PCR (bundle sheath) tissue may not show the cell-specific accumulation of PEPCase characteristic of nearby functional PCA cells, indicating that the putative signal from the vascular tissue fails to reach or to influence these more distant cells (Langdale and Nelson, 1991 ; Nelson and Langdale, 1992 ). Similar observations have been made for the C4 dicot Atriplex rosea (Dengler et al., 1995 ) and now for three of the C4 anatomical types in the Cyperaceae (this study). These observations support the idea that leaf vascular tissue provides a positional landmark that guides or influences the pattern of cell differentiation within adjacent tissues (Cerioli et al., 1994 ; Freeling and Lane, 1994 ; Nelson and Dengler, 1997 ).

In at least some C4 species, a C3-like "default" stage precedes the C4-specific pattern of enzyme accumulation. For instance, mRNAs for the small subunit of RuBPCase and NADP-malic enzyme accumulated in all ground meristem cells of leaf primordia in maize and only later became specific to the PCR (bundle sheath) tissue with subsequent structural differentiation (Langdale et al., 1988 ). In the C4 dicot amaranth, the mRNAs for PEPCase and PPDK were detected in apical meristems and leaf primordia, but became cell-specific and highly abundant with the structural differentiation of mesophyll cells (Ramsperger, Summers, and Berry, 1996 ). At the same time, mRNAs for both large and small subunits of RuBPCase were expressed throughout young leaves and did not become PCR-specific until a later stage. In amaranth, full anatomical and biochemical expression of the C4 pathway appeared to be coordinated with the transition of leaf metabolism from sink to source (Wang et al., 1993b ). Our immunolocalization data for these three representatives of the C4 anatomical types in the Cyperaceae corroborated this general pattern. In each species, we found low-level, non-cell-specific accumulation of both RuBPCase and PEPCase proteins within the leaf extension zone of young leaves and culms, while the highly specific and abundant enzyme accumulation coincided with structural differentiation at the distal end of the leaf elongation zone.

Although RuBPCase accumulated prior to PEPCase, the timing of PEPCase accumulation in relation to cell structural differentiation differed among the anatomical types. In Pycreus polystachyos (chlorocyperoid type), the accumulation of PEPCase occurred after cell structural differentiation (i.e., in an anatomically mature region of the leaf distal to the zone of leaf elongation). This pattern of cell-specific enzyme accumulation differed in relative timing from previously examined representatives of C4 Poaceae and C4 dicots where RuBPCase and PEPCase specific accumulation preceded final structural differentiation (Langdale et al., 1988 ; Dengler et al., 1995 ). This difference could reflect the presence of the "extra" (mestome sheath) layer that separates the PCA and PCR tissues in Pycreus, thus increasing the length of the putative signaling pathway from vascular tissue to PCA.

Development and evolution of Kranz anatomy in the Cyperaceae
Cladistic analyses indicate that the C4 pathway has arisen four times in the Cyperaceae (Soros and Bruhl, 2000 ). Each of the C4 anatomical types has evolved in a separate tribe, although the chlorocyperoid type has evolved in both the Cypereae (subfamily Cyperoideae) and the Rhynchosporeae (subfamily Caricoideae). Our species sample included representatives of three of the four C4 variants (chlorocyperpoid, eleocharoid, rhynchosporoid) and a C3 Cypereae species. Although all representatives showed a similar pattern of leaf or culm extension and of delineation of procambium in the basal meristem, each anatomical type was characterized by a distinctive pattern of PCR tissue derivation, providing support for the interpretation of structural equivalence of tissues based on mature anatomy alone. Furthermore, while each anatomical type followed the same general pattern of cell differentiation, with high-level and cell-specific enzyme accumulation occurring at the distal end of the leaf elongation zone, there are distinct differences among the types. Accumulation of PEPCase in PCA cells was conspicuously delayed in Pycreus polystachyos (chlorocyperoid type) and, surprisingly, was present in both PCA and PCR tissue in Rhynchospora rubra (rhynchosporoid type). Another interesting finding was that PEPCase accumulated in the PBS layer of Eleocharis retroflexa, indicating that this layer functions as part of the PCA tissue. Therefore we refer to this layer as PCA rather than PBS as in Soros and Dengler (1998) (see Figs. 1, 10). Assuming these specific patterns are characteristic of the anatomical type and tribes represented, then these differences represent the different developmental pathways associated with independent evolutionary origins.

The C4 photosynthetic pathway has evolved at least 31 times within the flowering plants as a whole (Kellogg, 1999 ). In each evolutionary lineage, RuBPCase, other Calvin cycle enzymes, and one or more decarboxylating enzymes are expressed cell-specifically in PCR tissue, PEPCase is up-regulated in PCA tissue, and the basic elements of Kranz anatomy are present (Sinha and Kellogg, 1996 ; Kellogg, 1999 ). Despite these commonalities, other features of the C4 syndrome can show unique patterns of variation among lineages. For instance, in the grass family (Poaceae), lineages differ in the specificity of accumulation of light-harvesting chlorophyll a/b protein and of pyruvate orthophosphate dikinase (Sinha and Kellogg, 1996 ). In this study of three different C4 lineages in the sedge family (Cyperaceae), we examined the protein accumulation pattern of only two enzymes that were used as markers of cell differentiation. Nonetheless, we observed differences in mature accumulation patterns and in temporal aspects of accumulation. These comparisons of related clades emphasize that the C4 syndrome has been assembled anew at each evolutionary event and that, while certain essential requirements must be met for C4 function, each evolutionary origin may represent a slightly different version of the syndrome (Sinha and Kellogg, 1996 ; Kellogg, 1999 ).

Independent evolutionary origins of the C4 syndrome have resulted in different regulatory mechanisms at the molecular level. For instance, different isoforms of PEPCase have been co-opted for C4 photosynthesis in Zea mays and Flaveria trinervia (Hermans and Westhoff, 1992 ; Kellogg, 1999 ). In Flaveria trinervia, the PEPCase promoter sequence is similar to that of the C3 isozyme, but in maize it appears to be totally new (Schaffner and Sheen, 1992 ). Although the developmental regulation of structural aspects of the C4 syndrome is unknown at the molecular level, it is equally possible that different forms of regulators or of promoter sequences are altered and therefore respond to developmental signals in different ways in C4 taxa than in their C3 ancestors. Our results for the C4 Cyperaceae identify specific tissue regions where such altered development would occur. For instance, cells at the periphery of the procambium respond to signals for altered chloroplast replication and growth, mitochondrial replication, and organelle placement in the rhynchosporoid type, while cells positioned more deeply in the procambium give a similar response to these signals in the chlorocyperoid and eleocharoid types.


   FOOTNOTES
 
1 The authors thank Mr. K. Masumira and Dr. Osamu Ueno for providing material of Rhynchospora rubra, Dr. James O. Berry for providing PEPCase antibody, Dr. Ronald E. Dengler for helpful discussion, Ruchira Sud and Julie Kang for comments on the manuscript, and Petra Donnelly for assistance with preparation of illustrations. Back

2 Author for correspondence, current address: Department of Botany, University of Guelph, Guelph, Ontario, Canada N1G 3B2 (e-mail: csoros{at}uoguelph.ca ). Back


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
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