Chapter 4 C4 Photosynthesis: Kranz Forms and Single-Cell C4 in Terrestrial Plants

Edwards, Gerald E.; Voznesenskaya, Elena V.

doi:10.1007/978-90-481-9407-0_4

Gerald E. Edwards³ &
Elena V. Voznesenskaya⁴

Part of the book series: Advances in Photosynthesis and Respiration ((AIPH,volume 32))

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Summary

Plants identified as having C₄ photosynthesis have a C₄ metabolic cycle with phosphoenolpyruvate carboxylase as the initial catalyst for fixation of atmospheric CO₂, and a C₄ acid decarboxylase (NADP-malic enzyme, NAD-malic enzyme, or phosphoenolpyruvate carboxykinase), which releases CO₂ for fixation by the C₃ cycle. Effective donation of CO₂ to Rubisco minimizes competition by O₂ and photorespiration, and thus increases photosynthesis under conditions where CO₂ is limiting. To achieve this, fixation of atmospheric CO₂ in the cytosol by phosphoenolpyruvate carboxylase must be separated from the donation of CO₂ to Rubisco by the decarboxylation of C₄ acids. In most documented C₄ plants, this is accomplished through evolution of various forms of Kranz anatomy, with fixation of atmospheric CO₂ in mesophyll cells and donation of CO₂ from C₄ acids to Rubisco in bundle sheath cells. In the family Chenopodiaceae, two alternative means of accomplishing this spatial separation evolved within individual photosynthetic cells, whereby one cytoplasmic compartment specializes in fixation of atmospheric CO₂ in the carboxylation phase of the C₄ cycle, and the other cytoplasmic compartment specializes in donating CO₂ from C₄ acids to Rubisco. In this chapter, biochemical and structural variations of Kranz anatomy in three major C₄-containing families, Poaceae, Cyperaceae, and Chenopodiaceae, as well as other known forms for dicots, are summarized. Then, the phylogeny, biogeography, development, and structure-function relationships of the single-cell C₄ systems are discussed in comparison to Kranz type C₄ plants.

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Significance of C4 Leaf Structure at the Tissue and Cellular Levels

Coleataenia prionitis, a C₄-like species in the Poaceae

Article 03 January 2021

Transition from C₃ to proto-Kranz to C₃–C₄ intermediate type in the genus Chenopodium (Chenopodiaceae)

Article Open access 31 August 2019

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1 I. Introduction

1.1 A. What Does It Take to Be C₄?

This question was posed in a short commentary on the history of C₄ by (Edwards et al., 2001) who noted that the minimum requirements for the CO₂ concentrating mechanism in C₄ photosynthesis are “(a) cell-specific amplification of enzymes of C₄ photosynthesis; i.e. phosphoenolpyruvate carboxylase (PEPC) in mesophyll, and C₄ acid decarboxylases and Rubisco in bundle sheath cells, with complementary adjustments of photosystem and electron transport activities; (b) novel cell-specific organelle metabolite translocators; (c) symplastic connections of the spatially separated sources and sinks of 4C-dicarboxylic acid transport metabolites; and (d) barriers to CO₂ diffusion between the site of CO₂ fixation by PEPC in mesophyll cells and sites of CO₂ release and refixation by Rubisco in bundle-sheath cells”. These requirements have been met through the multiple, independent evolution of C₄ photosynthesis in different groups of terrestrial plants. Until the recent discovery of two alternative means of performing C₄ photosynthesis within individual chlorenchyma cells (single-cell C₄), all terrestrial C₄ plants were presumed to have Kranz anatomy.

1.2 B. Occurrence of C₄ Among Terrestrial Plants

The earliest studies which led to the identification of C₄ plants were on maize and sugarcane, members of family Poaceae (see review by Hatch, 1999). Since then, C₄ plants have been found in 19 families with the largest number of species appearing in families Poaceae, Cyperaceae and Chenopodiaceae. C₄ is estimated to have evolved independently over 50 times (Muhaidat et al., 2007), resulting in three biochemical subtypes (see Chapter 14 by Drincovich et al.) based on the mechanism of C₄ acid decarboxylation: NADP-malic enzyme (NADP-ME), NAD-malic enzyme (NAD-ME), and phosphoenolpyruvate carboxykinase (PEP-CK).

2 II. Structural and Biochemical Diversity in Kranz Type Anatomy

The occurrence of Kranz anatomy (Kranz means wreath in German) has been known since its initial characterization by Haberlandt (1884). In a broad sense, Kranz anatomy can now be functionally defined to accommodate all known structural variants of Kranz type C₄ plants. A double concentric layer of chlorenchyma cells together form the Kranz tissue with the outer layer capturing atmospheric CO₂ in the C₄ cycle, and the inner layer donating CO₂ from C₄ acids to Rubisco in the C₃ cycle. The outer layer is commonly referred to as mesophyll (M) cells (usually consisting of palisade parenchyma) and the inner layer as specialized bundle sheath (BS) cells or Kranz cells. The M cells are always closer to the atmosphere than the BS cells, and the BS cells, as a rule, have limited contact with intercellular air space. The cells of chlorenchymatous M and BS layers are usually adjacent to one another, but in some cases they are separated by an additional layer of cells. The ratio of M/BS cells is lower than in C₃ plants, and most of the M cells are in direct contact with BS cells. There are two common structural forms, a double concentric layer of chlorenchyma around individual veins, and a double concentric layer which surrounds all the vascular tissue in the leaf. Since C₄ evolved multiple times from different C₃ leaf anatomies, there are various structural types. Specific types are described below which represent striking examples of evolutionary convergence on a common suite of anatomical features.

2.1 A. Structural Diversity

Among C₄ plants, there is considerable variation in features relevant to the C₄ mechanism (Carolin et al., 1973, 1975, 1977, 1978; Laetsch, 1974; Brown, 1975, 1977; Ellis, 1977; Edwards and Walker, 1983; Dengler et al., 1985; Voznesenskaya and Gamaley, 1986; Prendergast and Hattersley, 1987; Prendergast et al., 1987; Ueno et al., 1988a; Hattersley and Watson, 1992; Dengler and Nelson, 1999; Sage, 2004; Muhaidat, et al., 2007). While distinct biochemical and anatomical types have been catalogued since C₄ photosynthesis was first described more than three decades ago (see Hatch, 1971), new structural subgroups continue to be discovered, providing further insight into the evolution of the syndrome. Usually, several characteristics are taken into account to distinguish between different structural and biochemical subtypes. The most important among them are: (1) number of BS layers; (2) presence or absence of a mestome sheath (MS) and its positioning in relation to other parenchyma sheaths (notably in grasses); (3) presence or absence of a suberin lamella (SL) in BS cell walls (in grasses); (4) position of BS organelles (mainly chloroplasts); and (5) chloroplast differentiation between M and BS: BS cells in NADP-ME species have grana-deficient chloroplasts with a few, small mitochondria, while NAD-ME species have chloroplasts with well-developed grana and numerous, large mitochondria with specialized cristae. In both subtypes, M chloroplasts have a reversed pattern of grana development to that expressed in the BS, with abundant, large grana in NADP-ME species, and a deficiency of grana in chloroplasts of M cells in NAD-ME species. Some other features, such as the shape (outline) of the outer parenchyma BS, have been mentioned as being useful in characterizing certain subtypes, for example an uneven outline of the outer BS in PEP-CK type grasses; however, this does not seem to be especially important phylogenetically in relation to C₄ photosynthetic subtypes, as there are many exceptions (Prendergast et al., 1987). Only features of organelle differentiation provide an easy means to predict biochemical subtypes, while other structural characters only give additional information for considering evolutionary development of Kranz anatomy. The main structural forms of Kranz that are known to occur among C₄ species are illustrated in Figs. 1, 2 and 3. Historically, different forms of Kranz anatomy have been referred to either by taxonomic names, or by names descriptive of their structure. We have used taxonomic names to be consistent and concise, recognizing that they are used descriptively and do not always imply phylogenetic identity. An exception is description of the three classical forms of Kranz anatomy associated with the three biochemical subtypes in family Poaceae, which accounts for many C₄ grasses.

2.1.1 1. Poaceae

For C₄ grasses, important distinguishing characteristics include the presence or absence of a MS, and, when present, the size of MS cells and thickness of their cell walls, the positioning of chloroplasts in BS cells, the presence or absence of a SL and, when present, its distribution in the Kranz cell walls (Carolin et al., 1973; Brown, 1975, 1977; Ellis, 1977; Hattersley and Browning, 1981; Hattersley, 1992; Hattersley and Watson, 1992). At least nine structural subtypes have been distinguished on the basis of these features, and most of the known C₄ grasses fit into these subtypes (described below and illustrated in Fig. 1 and Table 1). It is suggested from phylogenetic analyses that C₄ evolved from C₃ a minimum of 17 times in family Poaceae (Christin et al., 2008, 2009). Certain forms of Kranz anatomy evolved multiple times in the family (Table 1).

Table 1. Structural and biochemical forms of Kranz anatomy in relation to the number of independent C₄ lineages in family Poaceae. The number of each lineage is according to Christin et al., (2008, 2009)

Full size table

In the Poaceae, C₄ species occur in subfamilies Panicoideae, Chloridoideae, Aristidoideae, and Micrairoideae (Sanchez-Ken et al., 2007; Vicentini et al., 2008; Christin et al., 2008, 2009). While most C₄ species in subfamily Panicoideae are NADP-ME type, NAD-ME and PEP-CK type species also occur in the subfamily as discussed below. In subfamily Chloridoideae, most C₄ species are NAD-ME type, while a few genera have PEP-CK type species. C₄ species identified in subfamilies Aristidoideae and Micrairoideae are NADP-ME type (Table 1). Structural forms of Kranz anatomy among these biochemical subtypes are discussed below.

2.1.1.1 Classical NADP-ME Type Anatomy

This type of anatomy was originally the so-called Panicoid (Carolin et al., 1973). Among the three major biochemical C₄ subgroups first identified in Poaceae (Gutierrez et al., 1974; Hatch et al., 1975; Brown, 1977), classical NADP-ME type species have a single parenchyma BS (the Kranz BS which is derived from provascular tissue and, thus, lacks a MS), with BS chloroplasts in a centrifugal/peripheral position and with a deficiency in their grana development, whereas, M chloroplasts have well-developed grana (Fig. 1) (Voznesenskaya and Gamaley, 1986; Hattersley, 1992; Yoshimura et al., 2004). C₄ species with classical NADP-ME type anatomy in subfamily Panicoideae occur in tribes Paniceae, Arundinelleae, and Andropogoneae (Hattersley and Watson, 1992; Sage et al., 1999; GPWG, 2001; Vicentini et al., 2008). The degree of grana reduction in chloroplasts in the BS cells varies, from having nearly agranal BS chloroplasts (representatives of tribe Andropogoneae), to having numerous, small grana (tribe Paniceae), to having a few rather large grana (Panicum obseptum or Rhynchelytrum repens). This subtype has fewer mitochondria in BS cells than in the NAD-ME and PEP-CK subtype (Yoshimura et al., 2004). The SL is present in the outer tangential wall, and partly in the radial cell wall of the BS cells (Hattersley and Browning, 1981).

2.1.1.2 Classical NAD-ME Type Anatomy

This structural type of C₄ grasses has a double sheath: a MS with thick cell walls and a few plastids, surrounded by a Kranz type chlorenchyma sheath (derived from ground tissue). Bundle sheath chloroplasts are in a centripetal position and have well-developed grana, while M chloroplasts show different degrees of grana reduction according to species (Fig. 1). A SL is usually absent in BS cells; but if present, is only in BS cell walls adjacent to sclerenchyma cells which do not contain chloroplasts (Hattersley and Browning, 1981). This subgroup also has abundant, large specialized mitochondria in BS cells, the site of the C₄ acid decarboxylase (Gutierrez et al., 1974; Hatch et al., 1975; Brown, 1977; Hattersley and Watson, 1992; Yoshimura et al., 2004). This form of anatomy was originally named Eragrostoid (Carolin et al., 1973). It occurs particularly in subfamily Chloridoideae (the core Chloridoideae and Centropodia lineages); but, also in subfamily Panicoideae, tribe Paniceae, evolving once in the Panicum, Urochloa, Setaria clade, Table 1 (Sage et al., 1999; GPWG, 2001; Aliscioni et al., 2003; Christin et al., 2008, 2009).

2.1.1.3 Classical PEP-CK Type Anatomy

The classical PEP-CK type has a double chlorenchyma sheath similar to the NAD-ME type, with an inner MS and outer Kranz chlorenchyma sheath with grana-containing BS chloroplasts in a centrifugal position, or scattered peripherally around the cell, see Fig. 1 (Gutierrez et al., 1974; Brown, 1977; Dengler and Nelson, 1999; Yoshimura et al., 2004). The level of grana development is very similar in BS and M chloroplasts, and the BS mitochondria are quite small (generally comparable in size to M mitochondria) and are usually more numerous than in NADP-ME species, but less abundant than in NAD-ME species (Yoshimura et al., 2004; Voznesenskaya et al., 2006). Suberin lamella is present in the outer tangential BS cell walls and extends approximately to the middle of the radial cell walls (Hattersley and Browning, 1981). This subtype has been found in subfamily Chloridoideae in Bouteloua, Eleusine, Muhlenbergia, Spartina, Sporobolus, and Zoysia, and in subfamily Panicoideae, evolving once in the Panicum/Urochloa/Setaria clade, e.g. in Brachiaria, Chaetium, Eriochloa, Melinis, and Urochloa, see Table 1 (Sage et al., 1999; Guissani et al., 2001; Aliscioni et al., 2003; Christin et al., 2008).

2.1.1.4 Arundinelloid: Biochemical Subtype NADP-ME

This type of anatomy was studied in detail in genus Arundinella (tribe Arundinelleae) subfamily Panicoideae. Like the classical NADP-ME type, species in this genus have NADP-ME type biochemistry, with BS chloroplasts having reduced grana and in the centrifugal position; MS is absent in all vascular bundles. However, the Kranz anatomy in veins is widely spaced, and there is the unusual occurrence of a row, or rows, of Kranz assemblies between the veins, which sometimes are referred to as distinctive cells because these bundle sheath-like cells are not associated with vascular tissue (Tateoka, 1958), see Fig. 1. A SL is usually present and continuous in the distinctive cells, or interrupted in the radial cell walls in BS cells surrounding the vascular tissue (Hattersley and Browning, 1981). Distinctive cells have structural and biochemical characteristics similar to the BS cells (Crookston and Moss, 1973; Dengler et al., 1990, 1996; Dengler and Dengler, 1990; Wakayama et al., 2002, 2003, 2006). Distinctive cells have also been found in genera Arthraxon and Microstegium (tribe Andropogoneae), where they have ultrastructural characteristics similar to those shown for Arundinella (Ueno, 1995), and in genus Garnotia; but, there is no additional biochemical or ultrastructural data for species of this genus (Tateoka, 1958).

2.1.1.5 Aristidoid: Biochemical Subtype NADP-ME

The Kranz anatomy of species in genus Aristida, tribe Aristideae in subfamily Aristidoideae, is unusual in having three distinct layers of chlorenchyma cells surrounding the vascular tissue: an inner BS, an outer BS, and the M cells (Brown, 1958; Johnson, 1964; Bisalputra et al., 1969), see Fig. 1. Aristida species have NADP-ME type biochemistry, based on analyses of several species in the genus (Gutierrez et al., 1974; Hattersley, 1987; Prendergast et al., 1987; Voznesenskaya, et al., 2005b). Mestome sheath is absent; both the inner and outer sheaths are chlorenchymatous. However, in the inner sheath, chloroplasts are nearly agranal, while the outer BS contains chloroplasts with well-developed grana similar to the M chloroplasts. In this type, only the inner sheath functions as Kranz cells, while the outer BS functions mainly for storage of starch and, possibly, for refixation of photorespired CO₂ (Voznesenskaya et al., 2005b). In the inner BS, chloroplasts are scattered around the cell or tend to be centrifugal, while in the outer parenchyma BS, chloroplasts are located in a centripetal position. The SL is absent in cell walls of both types of BS (Hattersley and Browning, 1981).

2.1.1.6 Stipagrostoid: Biochemical Subtype NADP-ME

The genus Stipagrostis belongs to tribe Aristideae in subfamily Aristidoideae. Like Aristida, Stipagrostis species also evolved NADP-ME type photosynthesis, but they have a different type of Kranz anatomy, named Stipagrostoid (Voznesenskaya et al., 2005a). This subtype has an inner MS consisting of enlarged cells with thinner cell walls and few chloroplasts, and an outer layer of Kranz cells with chloroplasts in the centripetal position (Brown, 1975). In the Kranz cells of Stipagrostis, mitochondria are few and small, and the chloroplasts are deficient in grana compared to M chloroplasts, which have well-developed grana. In contrast, the classical NADP-ME subtype grasses lack a MS and they have Kranz cells with chloroplasts in a centrifugal position. Also, the Kranz cells of Stipagrostis lack a SL in the walls, whereas the classical NADP-ME type grasses have SL in the Kranz cells, which are thought to have originated from the MS.

2.1.1.7 Eriachneoid: Biochemical Subtype NADP-ME

This subtype, which occurs in genera Pheidochloa and Eriachne (subfamily Micrairoideae, Christin et al., 2008, 2009), has NADP-ME type biochemistry (Prendergast et al., 1987) and an inner MS with an outer Kranz sheath, like the Stipagrostoid type. However, unlike Stipagrostis, which has chloroplasts in a centripetal position in Kranz cells, in Pheidochloa and in most Eriachne species (18 of 21) the chloroplasts in Kranz cells are in a centrifugal position (Hattersley, 1987; Prendergast and Hattersley, 1987; Prendergast et al., 1987; Taniguchi et al., 2003). The BS chloroplasts have well-developed grana with numerous, long intergranal thylakoids; in Eriachne aristidea, the degree of grana stacking is lower compared to the M chloroplasts (Taniguchi et al., 2003). For other species, the situation is not very clear. Bundle sheath chloroplasts of E. glabrata, E. obtusa and P. gracilis have an unexpectedly large number of grana (up to 21 thylakoids in a stack) for an NADP-ME subtype (Prendergast et al., 1987); however, there is no data about the degree of grana differentiation in M chloroplasts of these species. The SL is absent in cell walls of Kranz cells (Prendergast et al., 1987).

2.1.1.8 Neurachneoid: Biochemical Subtypes NADP-ME and PEP-CK

Species with this type of anatomy have a double parenchymatous sheath and the MS is absent; however, in this case the inner sheath is Kranz and the outer sheath is a non-specialized parenchyma BS containing only a small number of chloroplasts (see Dengler et al., 1985; Hattersley et al., 1986; Prendergast et al., 1987; Ueno and Sentoku, 2006). It was suggested that the inner Kranz BS in all species having this type of anatomy originated from the MS of C₃ grasses (Brown, 1975, 1977; Dengler et al., 1985). Species with this type of anatomy which perform NADP-ME type photosynthesis are Neurachne munroi, Paraneurachne muelleri in the Neurachne clade, and also Panicum petersonii and P. prionitis in section Prionita; all belong to the subfamily Panicoideae. In both N. munroi and P. muelleri, thick cell walls of the Kranz inner sheath have a SL, which is only continuous in the outer tangential walls and outer parts of radial walls. The outer parenchyma sheath has relatively thin cell walls without SL. Chloroplasts in Kranz cells are distributed evenly in N. munroi, but are in a centrifugal position in P. muelleri. Kranz cell chloroplasts have granal stacks which are less pronounced than in the M chloroplasts (Hattersley et al., 1986).

Alloteropsis semialata, subfamily Panicoideae, represents a very unique case, where diversity in the form of photosynthesis occurs among subspecies, with ssp. semialata being C₄ and ssp. eckloniana being C₃ (Frean et al., 1983; Prendergast et al., 1987; Ueno and Sentoku, 2006; Ibrahim et al., 2009). An Australian accession of spp. semialata biochemically is PEP-CK type with Neurachneoid type anatomy (Prendergast et al., 1987). The Kranz sheath, unlike the classical PEP-CK type species, is considered to be derived from the MS sheath of C₃ plants. Most anatomical characteristics are similar to those cited above: cell walls of Kranz cells are thicker than in the parenchymatous BS and they have a SL. There are abundant chloroplasts and mitochondria in Kranz cells which do not have a special orientation, and chloroplasts have well-developed grana like those in the M cells (Ueno and Sentoku, 2006). The ssp. semialata has high PEP-CK activity and variable amounts of NADP-ME which may be influenced by growth conditions (Prendergast et al., 1987; Ueno and Sentoku, 2006).

2.1.1.9 Other Forms of NAD-ME Type Anatomy

There are several NAD-ME C₄ species in family Poaceae having some anatomical features that are not characteristic of the classical NAD-ME type C₄ species. Like the classical NAD-ME and PEP-CK types, these species have a double parenchyma sheath, with an inner MS and outer Kranz chlorenchyma sheath with grana-containing BS chloroplasts. Also, the BS cells have abundant mitochondria characteristic of NAD-ME type species. However, the BS chloroplasts are not arranged in the centripetal position, but are located in a centrifugal or peripheral position like the PEP-CK species. Also, the BS cell walls generally have a SL which is usually absent in the classical NAD-ME type species. This includes some species in genera Eragrostis, Enneapogon, Triraphis, and some Panicum species of the section Dichotomiflora (Ohsugi and Murata, 1980; Ohsugi et al., 1982; Prendergast et al., 1986). Interestingly, different cultivars of one NAD-ME type species, P. coloratum, were found to have different positions of chloroplasts in the Kranz cells, centripetal versus centrifugal (Ohsugi et al., 1982), which further shows that this feature cannot be taken as a criterion for distinguishing between different biochemical types.

A more extreme structural variant of NAD-ME type species is the Triodioid type anatomy. Species with Triodioid anatomy have two BS: an inner, thin-walled MS and an outer, chlorenchymatous Kranz BS which lacks SL in the cell walls. There are two variants of Kranz anatomy in this genus: Kranz BS form (“drape”) extensions between adjacent vascular bundles, as in Triodia pungens (Hattersley and Watson, 1992), or BS extensions towards patches of M cells on both the abaxial and adaxial sides of the leaf, which are not associated with vascular bundles, for example T. irritans and T. scariosa, as illustrated in Fig. 1 (Dengler and Nelson, 1999). The species which have been studied have NAD-ME type biochemistry; the appearance of mitochondria in Kranz cells is typical for NAD-ME species (Craig and Goodchild, 1977). The chloroplasts in Kranz cells have well-developed grana; but, unlike classical NAD-ME species, they, are in a centrifugal position or peripherally scattered around the cytoplasm, as in PEP-CK type species (Craig and Goodchild, 1977; Prendergast et al., 1987).

2.1.2 2. Family Cyperaceae

In family Cyperaceae, there are four types of Kranz anatomy (Fig. 2). As in family Poaceae, C₃ Cyperaceae species have an inner MS and an outer parenchyma sheath around the vascular tissue. In C₄ Cyperaceae species, the Kranz cells are considered to have evolved either internal to the MS (Fimbristyloid, Chlorocyperoid and Eleocharoid) or from the MS (Rhynchosporoid) (Brown, 1975; Carolin et al., 1977; Gilliland and Gordon-Gray, 1978; Bruhl et al., 1987; Bruhl and Perry, 1995; Soros and Dengler, 1998, 2001; Dengler and Nelson, 1999). In the first case, the MS, which is situated between M and Kranz cells, is nonphotosynthetic, thick-walled and generally suberized (Carolin et al., 1977; Ueno et al., 1988b; Ueno and Samejima, 1989; Bruhl and Perry, 1995; Soros and Dengler, 2001). This sheath may contribute to diffusional resistance of gases and help to minimize leakage of CO₂ generated from decarboxylation of C₄ acids in the Kranz cells. Most C₄ representatives in the family are NADP-ME type; NAD-ME species have only been found in genus Eleocharis (Bruhl et al., 1987; Ueno et al., 1988a; Murphy et al., 2007).

2.1.2.1 Fimbristyloid: Biochemical Subtypes NADP-ME and NAD-ME

This type of anatomy was found in C₄ species of the tribe Fimbristylideae, for example in Bulbostylis and Fimbristylis (see Carolin et al., 1977; Ueno et al., 1988a) and more recently in genus Eleocharis (Bruhl et al., 1987; Ueno, 1998a; Murphy et al., 2007). The Kranz cells originated internal to the MS and do not form a continuous wreath; rather, they are interrupted by metaxylem elements. In this type, there are three BS layers around all vascular bundles, even small ones: Kranz BS surrounded by the MS, and parenchymatous BS (external to the MS) having fewer chloroplasts than in M cells. Both NADP-ME biochemical subtype in genera Fimbristylis and Bulbostylis (Ueno et al., 1986; Ueno, 1998a), and NAD-ME subtype in some species of Eleocharis (Ueno, 1998b; Murphy et al., 2007), have been reported to have Fimbristyloid anatomy. The species of Fimbristylis have Kranz cells with chloroplasts that are centrifugally located and nearly agranal having numerous small, short grana; mitochondria are small and few, consistent with NADP-ME type photosynthesis (Carolin et al., 1977; Gilliland and Gordon-Gray, 1978). One population of Eleocharis vivipara (type 1) and E. retroflexa ssp. chaetaria are NAD-ME type C₄ species with Fimbristyloid-like anatomy; the latter has Kranz cell chloroplasts with well-developed grana and large mitochondria, typical of NAD-ME type C₄ species (Ueno and Samejima, 1989; Ueno et al., 1989; Ueno, 1996a). Immunolocalization studies show M and parenchymatous BS cells of E. vivipara type I (Ueno, 1996b) and Fimbristylis dichotoma (Ueno, 1998a) have PEPC and pyruvate, Pi dikinase (PPDK), indicating both cell types function to capture CO₂ by PEPC, with delivery of C₄ acids to the Kranz cells, where Rubisco is located.

2.1.2.2 Chlorocyperoid: Biochemical Subtype NADP-ME

The Chlorocyperoid type, as a rule, has two layers of BS, with the Kranz cells internal to the MS. Chlorenchyma cells external to the MS include a partial parenchymatous chlorenchyma sheath (occurring in large vascular bundles, it is less developed than in the Fimbristyloid, and may be completely absent in some species) and palisade-like M cells, both of which are considered to function in the carboxylation phase of the C₄ cycle. Kranz cells contain few large centrifugally-arranged chloroplasts having mostly single stroma thylakoids, convoluted in loops. The degree of grana reduction varies in different species. The SL is usually discontinuous in the radial cell wall of the MS and absent from Kranz BS (Ueno et al., 1988a, b; Bruhl and Perry, 1995). In some species (as with the Fimbristyloid), both M and parenchymatous BS cells may function to capture CO₂ by PEPC, with delivery of C₄ acids to the Kranz cells, where Rubisco is exclusively localized (Ueno, 1998a). This type of anatomy has been found in genera Cyperus, Kyllinga, Pycreus and Torulinium of the tribe Cypereae and Lipocarpha in the Lipocarpheae (Carolin et al., 1977; Gilliland and Gordon-Gray, 1978; Ueno et al., 1986, 1988a). Representative species having Chlorocyperoid type anatomy have NADP-ME type C₄ photosynthesis (Ueno et al., 1986; Bruhl et al., 1987). Eleocharis baldwinii, which has NAD-ME biochemistry and ultrastructure, has an intermediate type of anatomy called sub-Chlorocyperiod (Ueno and Samejima, 1989; Ueno, 2004).

2.1.2.3 Rhynchosporoid: Biochemical Subtype NADP-ME

Unlike the other forms of Kranz anatomy in Cyperaceae, in Rhynchosporoid type the Kranz cells evolved from the MS (Takeda et al., 1980). This thick-walled sheath is surrounded by an incomplete chlorenchymatous parenchyma sheath and palisade-like M cells which are considered to function in fixation of atmospheric CO₂ into C₄ acids. Both M and outer parenchyma sheath chloroplasts have similar thylakoid structure with large grana. Kranz cells have numerous, centrifugally-arranged agranal chloroplasts but, unlike the convoluted thylakoids in the previous types, here the thylakoid membranes usually have a parallel arrangement (Gilliland and Gordon-Gray, 1978; Ueno et al., 1988a; Bruhl and Perry, 1995). Mitochondria are comparable in size and number in M and BS cells. The SL is mostly continuous around the cell but can be discontinuous in the radial cell walls. Biochemical analysis indicates NADP-ME type photosynthesis (Ueno et al., 1986; Bruhl et al., 1987).

2.1.2.4 Eleocharoid: Biochemical Subtype NAD-ME

The Eleocharoid type anatomy was named after C₄ species of Eleocharis which have three types of BS, with the innermost Kranz cells forming a continuous wreath. As in Chlorocyperoid and Fimbristyloid types, the Kranz cells originate internal to the MS. The outer parenchyma BS contain some chloroplasts, while the middle MS lacks, or contains only a few, chloroplasts filled with starch; Kranz cells contain numerous organelles typical of NAD-ME species, with no strict orientation in the cell (scattered around the periphery) or tending slightly towards centrifugal. Chloroplasts of Kranz cells have well-developed grana and store starch; chloroplasts of parenchyma BS are smaller than those in M cells, but in both types of cells there are well-developed grana. Usually, a SL is present on both the inner and outer tangential cell walls in the MS, but sometimes it is absent on the radial cell walls (Ueno and Samejima, 1989; Bruhl and Perry, 1995). The Kranz cells have abundant and large mitochondria, typical of NAD-ME type C₄ grasses and dicots (Ueno and Samejima, 1989; Bruhl and Perry, 1995; Ueno, 2004; Ueno and Wakayama, 2004). The genus Eleocharis is very diverse in forms of photosynthesis between species (C₃, C₃–C₄, C₄-like). It includes amphibious species which change their mode of photosynthesis between submerged and terrestrial growth; and both Eleocharoid and Fimbristyloid type anatomy with NAD-ME type photosynthesis has been found among its C₄ species (Bruhl et al., 1987; Bruhl and Perry, 1995; Ueno, 2004; Ueno and Wakayama, 2004; Murphy et al., 2007).

2.1.3 3. Dicotyledons

Among dicot families, it is well-established that family Chenopodiaceae has the largest number of C₄ species and also the greatest diversity in leaf anatomy, including C₃, C₄ Kranz and C₄ single-cell types (Carolin et al., 1975; Pyankov et al., 1992; Sage et al., 1999; Edwards et al., 2004; Voznesenskaya et al., 2007). This family has been studied most extensively, resulting in classification of six types of Kranz anatomy (Fig. 3) which has been extended to several other families (Carolin et al., 1975, 1982; Jacobs, 2001). The C₄ types of leaves vary in the structure and arrangement of chlorenchyma tissue, in arrangement of water storage and vascular tissue, and by the presence, or absence, of various specialized hypodermal cells. Within these six main types of Kranz anatomy, additional anatomical differences have been recognized, indicating potential for further subdivision of structural types of Kranz in the family (Kadereit et al., 2003). The C₄ structural types in family Chenopodiaceae are named after the corresponding taxonomic names, as indicated below. These main structural forms were also given descriptive names (Vasilevskaya and Butnik, 1981; Voznesenskaya and Gamaley, 1986) which are also referred to in the descriptions below. In addition to the six Kranz types in the Chenopodiaceae, other forms have been recognized in dicot lineages found in Cleome (Cleomaceae), Isostigma and Glossocardia (Asteraceae) and Portulaca (Portulacaceae).

2.1.3.1 Atriplicoid: Biochemical Subtypes NAD-ME and NADP-ME

In C₃ dicots, all the vascular bundles are surrounded by a parenchyma sheath which is more or less distinguishable from M tissue; this sheath becomes a specialized Kranz BS in C₄ species. In Atriplicoid type of anatomy which occurs in some dicot species having laminate leaves, the Kranz tissue forms a classical wreath-like structure with concentric layers of chlorenchyma around each vascular bundle. The Kranz BS encloses vascular bundles; although it can become disrupted on the phloem side in larger bundles. There is structural diversity and potential for recognition of additional subtypes where Kranz encloses individual veins in C₄ dicots having flattened leaves. In this type, hypodermal tissue when present usually fulfills the role of water storage tissue. For example, Portulaca oleracea has extensively developed water storage hypoderm with variable positioning of the veins between the abaxial and adaxial sides of the leaf. In Atriplicoid type, palisade M cells are usually arranged radially; but this can vary in different species (Rathnam et al., 1976; Dengler and Nelson, 1999; McKown et al., 2005; Muhaidat et al., 2007). For species of families Chenopodiaceae and Amarathaceae, Kadereit et al. (2003) distinguished four different types of anatomy in laminate leaves within the Atriplicoid type, which differ in the presence or absence of hypoderm, the occurrence of parenchyma cells between M cells, or the occurrence of additional layers of spongy parenchyma on the abaxial side of the leaf. Nevertheless, similar features may be found in other taxons. Two biochemical subtypes, NADP-ME and NAD-ME, have been found in species having this leaf structure, each having differences in chloroplast ultrastructure (Laetsch, 1968; Kennedy and Laetsch, 1974; Carolin et al., 1975, 1978; Rathnam et al., 1976; Gamaley and Voznesenskaya, 1986; Voznesenskaya and Gamaley, 1986; Sage et al., 1999; Marshall et al., 2007; Muhaidat et al., 2007; Akhani et al., 2008). In the NADP-ME subtype, BS chloroplasts have reduced grana, as is typical for this subtype. The NADP-ME subtype is present in Acanthaceae, Aizoaceae, Amaranthaceae, Asteraceae, Boraginaceae, Cariophyllaceae, Chenopodiaceae, Euphorbiaceae, Nyctaginaceae, Portulacaceae and Zygophyllaceae. The opposite variant, NAD-ME type, which has well-developed grana in BS chloroplasts and reduced grana in M chloroplasts, is found in Acanthaceae, Aizoaceae, Amaranthaceae, Chenopodiaceae, Cleomaceae, Euphorbiaceae, Gisekiaceae, Molluginaceae and Portulacaceae. As a rule, Kranz BS have thickened cell walls, but the thickness varies; they are usually thinner in NAD-ME species. Organelles are usually arranged centripetally in BS cells, except for Trianthema triquetra (Aizoaceae), which has centrifugal positioning of organelles (Carolin et al., 1975).

2.1.3.2 Kochioid: Biochemical Subtypes NAD-ME and NADP-ME

Kochioid type species, also referred to as Semi-Wreath type, have laminate or semi-terete to terete succulent leaves with water-storage tissue underneath the chlorenchyma. The main vascular bundle is in the center, and the remaining vascular bundles are located in two paradermal planes on the leaf periphery or around the periphery in terete leaves. The chlorenchyma tissue is distributed along the peripheral veins; BS and M cells form arcs above the vascular bundles. Bundle sheath cells have relatively thick cell walls, and organelles are located in the centripetal position. Kadereit et al. (2003) recognized three different types of anatomy with such distribution of chlorenchyma tissues, differing in the presence or absence of hypoderm in two Kochia species, while in Kirilowia species, vascular bundles with arcs of chlorenchyma are distributed in the lateral plane only on the adaxial side of the leaf, with spongy parenchyma on the abaxial side. Species with this type of anatomy have been found to have NADP-ME type biochemistry and chloroplast ultrastructure (reduction in grana in BS cells is highly pronounced, up to having totally agranal chloroplasts) in genera Bassia and Kochia of family Chenopodiaceae (Gutierrez et al., 1974; Carolin et al., 1975; Gamaley, 1985; Voznesenskaya and Gamaley, 1986; Pyankov et al., 2000a; Jacobs, 2001), and NAD-ME type biochemistry in C₄ species of the genus Zygophyllum with respective granal chloroplasts and numerous specialized mitochondria in BS cells (Crookston and Moss, 1972; Muhaidat et al., 2007). In the latter case, the leaf is cylindrical with the main vascular bundle in the center of water storage tissue. Two layers of Kranz tissue form arcs outside of the small peripheral veins.

2.1.3.3 Salsoloid: Biochemical Subtypes NAD-ME and NADP-ME

Species with Salsoloid type anatomy, also referred to as Kranz-Centrical type, have cylindrical or terete leaves (or stems in aphyllous species) with two concentric layers of chlorenchyma, typical of C₄ Kranz anatomy, located around the periphery of assimilating organs. The central part is occupied by water storage tissue with the main vein in the middle. The net of secondary vascular bundles penetrates into the water storage tissue; and the small peripheral veins contacting with BS cells are facing toward the chlorenchyma by their xylem. In some desert species, a scleromorphous variant of this type has been found which has a high volume of sclerenchymatous tissue in the center around the main vein and/or in the peripheral bundles, with only a small amount of water storage tissue, for example, in reduced leaves of Nanophyton erinaceum, in the leaves and stems of Arthrophytum lehmannianum from family Chenopodiaceae, or in some Calligonum species of family Polygonaceae (see Butnik et al., 2001). Kadereit et al. (2003) distinguished five different types of anatomy within this type: Salsola type with or without hypoderm, Nanophyton type with sclerenchyma, Climacoptera type having no contact of peripheral veins with chlorenchyma and Halothamnus auriculus type with flattened leaves and several secondary veins distributed in the water storage parenchyma in lateral plane and the net of small peripheral veins adjacent to BS cells. Two biochemical subtypes, NADP-ME (in genera Salsola, Halothamnus, Haloxylon, Horaninovia and some others in family Chenopodiaceae) and NAD-ME (for example in genera Salsola, Climacoptera, Halocharis in Chenopodiaceae and Calligonum in Polygonaceae), with their respective ultrastructural chloroplast subtypes, have been found in species with this anatomy (Winter et al., 1977; Voznesenskaya and Gamaley, 1986; Pyankov and Vakhrusheva, 1989; Sage et al., 1999; Pyankov et al., 2000c; Muhaidat et al., 2007). Variants occur with or without hypodermal tissue, which, if present, plays the role of additional water storage tissue. Usually BS chloroplasts are in the centripetal position, but species of the genus Halothamnus (previously named Aellenia) have centrifugally-arranged chloroplasts, see Edwards et al. (2004).

2.1.3.4 Salsinoid: Biochemical Subtype NAD-ME

Species with Salsinoid type anatomy (also referred to as Kranz-Isopalisade Circular type) occur in genus Suaeda, section Salsina (Kadereit et al., 2003; Schütze et al., 2003). They have terete leaves with two concentric layers of chlorenchyma, palisade M and Kranz cells, around the leaf periphery, and water storage tissue in the center of the leaf. The vascular tissue forms a network in the lateral longitudinal plane; there are no peripheral vascular bundles and only the lateral veins may have contact with chlorenchyma. Only one biochemical subtype has been found, NAD-ME, and structural characteristics are typical for this subtype: numerous specialized mitochondria and chloroplasts with well-developed grana in BS cells, and reduced grana in M chloroplasts. Unlike other C₄ subtypes, the Kranz cells have a large vacuole with less abundant organelles which occur in a centripetal position in a relatively thin layer of cytoplasm. This type was originally called Kranz-Suaedoid (Carolin et al., 1975; Jacobs, 2001). However, subsequently this form of Kranz was recognized as Salsina type after the section of Suaeda in which it occurs (Schütze et al., 2003), and is called Salsinoid here for consistency in nomenclature. For a description of the structural and functional features, see (Shomer-Ilan et al., 1975, 1979, 1981; Fisher et al., 1997; Voznesenskaya et al., 2007).

2.1.3.5 Schoberioid: Biochemical Subtype NAD-ME

This is another form of Kranz anatomy in genus Suaeda which recently was called Schoberia after the section in which it occurs (Kadereit et al., 2003; Schütze et al., 2003); it is called Schoberioid type here for consistency in nomenclature (also referred to as Kranz-Isopalisade type). Before more recent phylogenetic analyses of the Suaedoideae subfamily, it was referred to as Conospermoid type anatomy by Freitag and Stichler (2000). It is found in semi-terete leaves with positioning of vascular bundles in a lateral plane. This subtype is unique in having the vascular bundles enclosed by two layers of Kranz type chlorenchyma in the central part of the leaf, with continuous BS extensions between the veins. Large hypodermal cells, which are located between the chlorenchyma and epidermis, function as water storage tissue. These are NAD-ME type species with typical ultrastructural features for this subtype: BS cells have granal chloroplasts and specialized mitochondria, and M cells have reduced chloroplasts with less grana development. Unlike the Salsinoid type, Schoberioid type species have BS chloroplasts located in the centrifugal position. A variant of this type of anatomy has been found inSuaeda cochlearifolia, which has only one layer of BS cells between the vascular bundles (Voznesenskaya et al., 2007).

2.1.3.6 Kranz-Tecticornioid: Biochemical Subtype NAD-ME

This unique structural subtype of Kranz anatomy is found in the genus Halosarcia (H. indica, Salicornieae, family Chenopodiaceae) (Carolin et al., 1982; Jacobs, 2001), which is now included in the broadly circumscribed genus Tecticornia (Shepherd and Wilson, 2007). In general appearance, it is similar to the Kranz-Centrical (Salsoloid) type of leaf anatomy, with peripheral distribution of two chlorenchyma layers in cylindrical assimilating stems and with a net of small peripheral vascular bundles adjoining BS cells. However, in the Kranz-Tecticornioid type, these small veins are oriented with the phloem side facing towards the chlorenchyma. A striking feature of this type is the presence of bands of thick-walled colorless parenchyma cells between groups of chlorenchymatous M cells. Also, in the Kranz cells granal chloroplasts tend to be located centrifugally, or occasionally scattered around the periphery of the cell. Western blot analysis for C₄ acid decarboxylases and immunolocalization studies indicate NAD-ME type C₄ photosynthesis. There are numerous mitochondria in the Kranz cells, which compared to other NAD-ME species in the family are smaller, but they have a similar specialized structure (Voznesenskaya et al., 2008). Mesophyll chloroplasts have reduced grana, characteristic of this biochemical subtype.

2.1.3.7 Pilosoid: Biochemical Subtype NADP-ME

An interesting variant of Kranz anatomy occurs in species of the clade Pilosa, genus Portulaca, family Portulaceae (for example, in P. grandiflora, P. pilosa, P. villosa, P. sclerocarpa) which have terete cylindrical leaves with a circular arrangement of the small vascular bundles around the leaf periphery, with main vein and water storage cells in the center. Each peripheral vein is surrounded by BS cells (sometimes less developed on the inner side), with M cells forming a wreath-like structure only on the outer and lateral sides of the vascular bundles as illustrated in Nishioka et al. (1996) and Kim and Fisher (1990). Thus, the structure of the mesophyll-bundle sheath-vascular bundle complex of each vein is similar to one of the variants of Atriplicoid type anatomy, but differs in having a peripheral arrangement of veins around the leaf (Fig. 4). Flattened leaves of P. amilis have similar arrangement of VB but only about four layers of water storage tissue in the middle part of the leaf. The whole leaf anatomy can be considered to represent an intermediate stage of evolution from laminate Atriplicoid anatomy to Kochioid or directly to Salsoloid. NADP-ME type of biochemistry is well known for P. grandiflora (Gutierrez et al., 1974; Guralnick et al., 2002). It was recently also shown for two other species of this clade, P. pilosa and P. amilis (Voznesenskaya et al., 2010); and, all other studied species with this type of anatomy have similar ultrastructural features of BS and M chloroplastscharacteristic of this biochemical subtype. A similar distribution of vascular bundles was found in Zygophyllum simplex; but, with Kranz tissue forming open arcs typical for Kochioid type of anatomy, showing similar evolutionary trends in different families.

2.1.3.8 Portulacelloid: Biochemical Subtype NADP-ME

Species of the clade/section Portulacella, genus Portulaca (Portulacaceae) have vascular bundles surrounded by two concentric layers of Kranz anatomy distributed only on the adaxial side of the leaf; there are 4–5 layers of water storage tissue on the abaxial side (Voznesenskaya et al., 2010). As in Pilosoid type, palisade M cells are better developed on the upper and lateral sides of vascular bundles. It is NADP-ME biochemical type with centripetal position of grana-deficient chloroplasts in bundle sheath cells.

2.1.3.9 Glossocardioid: Biochemical Subtype NAD-ME and NADP-ME

Kranz anatomy similar to Salsoloid type was reported for representatives of family Asteraceae Glossocardia bosvallia (Das and Raghavendra, 1976) and some Isostigma species (Peter and Katinas, 2003), and also for Cleome angustifolia leaf (Cleomaceae); all these species have semi-terete to terete leaves with concentric layers of Kranz type chlorenchyma surrounding the leaf on the periphery. Leaf venation consists of the central main vein with or without lateral secondary veins embedded in the water storage parenchyma, with sclerenchyma tissue being either present around veins or absent depending on the species. Small peripheral bundles are in contact with BS cells with their xylem side, characteristic of Salsoloid type anatomy. The main difference from the classic Salsoloid anatomy is the absence of even distribution of small veins around the leaf periphery. The biochemical subtype of G. bosvallia based on western blot analysis is NADP-ME, while C. angustifolia is NAD-ME, the biochemical subtype for Isostigma peucedanifolium is NADP-ME based on deficiency of grana in BS chloroplasts (Voznesenskaya, Koteyeva and Edwards, unpublished). This type of structure was designated as Eryngiophyllum (= Chrysanthellum) in Peter and Katinas (2003); but it has only been observed in Isostigma and Glossocardia in family Asteraceae. Therefore, according to the genus where it was first described (Das and Raghavendra, 1976), we define this as Glossocardioid type anatomy.

2.1.3.10 Simplicifolioid: Biochemical Subtype NADP-ME

Isostigma simplicifolium (family Asteraceae) has a form of Kranz anatomy in which the chlorenchyma tissue forms a continuous layer around flattened leaves (Fig. 4). The major veins, which run in parallel with rare anastomoses, are enclosed in the well-developed sclerenchyma sheaths, with only very small bundles lacking sclerenchyma tissue. There is some water storage parenchyma between major veins. The biochemical subtype is NADP-ME based on deficiency of grana in BS chloroplasts (E. Voznesenskaya, N. Koteyeva and G. Edwards, unpublished).

2.1.3.11 Isostigmoid: Biochemical Subtype unknown

An unusual form of Kranz anatomy, called Isostigmoid, was reported for several species of the genus Isostigma in family Asteraceae having flattened leaves (Peter and Katinas, 2003). In Isostigmoid type, instead of Kranz anatomy encircling individual veins, as occurs in Atriplicoid, the two chlorenchyma layers surround several veins together (illustrated in Fig. 4). It is considered an intermediate form between Atriplicoid type and anatomical forms having a continuous ring of Kranz anatomy around the leaf. The biochemical subtype is unknown.

2.1.3.12 Angustifolioid: Biochemical Subtype NAD-ME

This form of Kranz anatomy occurs in cotyledons of the C₄ species Cleome angustifolia (family Cleomaceae). Kranz tissue continuously surrounds the central part of the blade with vascular bundles distributed in the lateral longitudinal plane and with the main vein in the center of the leaf. Vascular bundles are separated by water storage parenchyma. Chlorenchyma layers, consisting of palisade M and BS cells, are located under the epiderm on the adaxial side; whereas, on the abaxial side the M cells adjacent to BS are small and rounded, and they are separated from the epiderm by two layers of spongy M cells (Fig. 4). Bundle sheath and M cells have ultrastructural features characteristic of NAD-ME biochemistry with granal BS chloroplasts and numerous mitochondria in BS cells; but, there is no difference in the granality between BS and M chloroplasts. This type of anatomy differs from all other known C₄ types. The concentric layer of Kranz tissue around veins has some features of Salsinoid type, but this type is unusual in having spongy parenchyma on the abaxial side of the leaf. The biochemical subtype of this species is NAD-ME (Voznesenskaya, Koteyeva and Edwards, unpublished).

2.2 B. Biochemical Diversity: C₄ Cycles and Energy Requirements for C₄ Subtypes

2.2.1 1. Chloroplasts and Mitochondria

Despite C₄ photosynthesis having evolved multiple times in different families and subfamilies with structural variations on Kranz anatomy, species belonging to each biochemical subtype tend to have features in common in terms of the structure of chloroplasts in M and BS cells, and the occurrence of mitochondria in BS cells. In NADP-ME type C₄ species, BS chloroplasts are deficient in grana compared to M chloroplasts, whereas in NAD-ME type species, the M chloroplasts tend to be more deficient in grana development (Gamaley, 1985; Voznesenskaya and Gamaley, 1986; Fisher et al., 1997; Voznesenskaya et al., 1999). In PEP-CK type C₄ species, the granal development is similar for M and BS chloroplasts (Yoshimura et al., 2004; Voznesenskaya et al., 2006). This has been quantified in a number of studies on different photosynthetic types by determining the granal index (the length of all appressed thylakoid membranes as a percentage of the total length of all thylakoid membranes in a chloroplast). Early studies indicate that the degree of grana development in BS chloroplasts correlates to the capacity for photosystem II (PSII) activity, linear electron flow and capacity for generation of NADPH, with low grana-containing chloroplasts being richer in photosystem I (PSI)-mediated cyclic electron flow producing ATP (Edwards and Walker, 1983; Anderson, 1999). Thus, differences in grana development are associated with differences between M and BS cells in the need for NADPH relative to ATP to support C₄ photosynthesis.

Mitochondria are most abundant, and often larger, in BS cells of NAD-ME type species (where the decarboxylase is located in mitochondria), and least abundant in BS cells of NADP-ME type species (where the decarboxylase is located in chloroplasts). Bundle sheath mitochondria in PEP-CK species also function to provide ATP to support decarboxylation via PEP-CK in the cytosol (Burnell and Hatch, 1988), and they are generally intermediate in size and number compared to NAD-ME or NADP-ME type species (Yoshimura et al., 2004; Voznesenskaya et al., 2006).

The basal energy required per CO₂ assimilation in C₄ photosynthesis is the sum of energy to support the C₄ and C₃ cycles. Analyses to date on biochemical subtyping (by Western blots, enzyme assays) indicate each species has a major form of delivery of CO₂ to Rubisco through one type of C₄ acid decarboxylase. For each biochemical subtype, the amount of energy required to support the C₄ cycle for delivery of CO₂ and the C₃ cycle for fixation of CO₂ can be calculated. This is shown in Fig. 5, with illustrations of how the provision of energy can be met cooperatively by M and BS chloroplasts. This demonstrates how the photochemical demands for energy can be shared between the cell types, and the differences between the three types of C₄ cycles. However, the exact photochemical demands for energy within each subgroup may vary between species. This is most evident in NADP-ME type C₄ species, as discussed below.

2.2.2 2. Illustration of Energetics for NADP-ME Type Species

Summary

1.
5 ATP, 2 NADPH required per CO₂ assimilated (2 ATP for the C₄ cycle, 3 ATP, 2 NADPH for the C₃ cycle).
2.
The C₄ cycle delivers primarily malate to BS cells (NADP-ME species are mainly malate formers).
3.
In NADP-ME type C₄ species, BS chloroplasts have fewer grana than do M chloroplasts, but there is variation in the degree of deficiency of grana in the BS chloroplasts. The extremes range from the BS chloroplasts being agranal (in sugarcane, sorghum), to BS chloroplasts having granal indices about half that of M chloroplasts, observed in members of family Chenopodiaceae (see Voznesenskaya et al., 1999). The illustration in Fig. 5a is based on the granal index and linear electron flow to generate NADPH being two fold higher in M than in BS chloroplasts, as observed in some NADP-ME type chenopods (Voznesenskaya et al., 1999). The two fold higher use of reductive power in BS cells could vary due to the amount of 3-phosphoglyceric acid (PGA) shuttled from BS to M chloroplasts for reduction (in the current scheme, one sixth of the PGA). Alternatively, this balance in reductive power could be modified in NADP-ME dicots by a partial shuttle of aspartate from M to BS cells through NADP-ME, see Moore et al. (1984) and Meister et al. (1996). Also, the degree of grana development in BS chloroplasts of NADP-ME type species appears to correlate with the development of a secondary aspartate PEP-CK shuttle (Gutierrez et al., 1974; Wingler et al., 1999; Voznesenskaya et al., 2006).

The deficiency in PSII in BS chloroplasts in this subtype is thought to reduce production of O₂ in BS cells and help maintain a high CO₂/O₂ ratio, which is favorable for limiting ribulose 1,5-bisphosphate (RuBP) oxygenase activity and photorespiration. This results in an increased need for photochemically-produced NADPH in M chloroplasts, see (Edwards and Walker, 1983).

2.2.3 3. Illustration of Energetics for NAD-ME Type Species

Summary

1.
5 ATP, 2 NADPH required per CO₂ assimilated (2 ATP for the C₄ cycle, 3 ATP, 2 NADPH for the C₃ cycle).
2.
The C₄ cycle delivers primarily aspartate to BS cells (NAD-ME species are primarily aspartate formers). The aspartate cycle requires only production of ATP (but not reductive power) to drive PEP regeneration from alanine by the M chloroplasts (Edwards and Walker, 1983; Voznesenskaya et al., 1999). This ATP may be provided via PSI cyclic electron flow in the M chloroplasts. Extensive studies of NAD-ME-type species in family Chenopodiaceae have shown that M chloroplasts are deficient in grana compared to BS chloroplasts (Gamaley, 1985; Gamaley and Voznesenskaya, 1986; Voznesenskaya and Gamaley, 1986; Glagoleva et al., 1991).
3.
In the illustration of Fig. 5b, the use and generation of reductive power in BS chloroplasts is twofold higher than in M chloroplasts. This occurs with the granal index and linear electron flow to generate NADPH being two fold higher in BS than M chloroplasts, as observed in some NAD-ME type chenopods (Voznesenskaya et al., 1999). Again, this partitioning of reductive power can be regulated by the amount of PGA shuttled from BS to M chloroplasts for reduction to triose-P (in the current scheme, one third of the PGA is shuttled to M cells).

2.2.4 4. Illustration of Energetics for PEP-CK Type Species

Summary

1.
3.6 ATP, 2.3 NADPH required per CO₂ assimilated (0.6 ATP and 0.3 NADPH per CO₂ for the C₄ cycles; 3 ATP, 2 NADPH for the C₃ cycle). The PEP-CK type requires less ATP, but more NADPH per CO₂ fixed than the malic enzyme type species.
2.
There are two C₄ cycles, one shuttling aspartate, the other shuttling malate (Fig. 5c). Aspartate is utilized in the cytosol to generate oxaloacetate for the PEP-CK reaction. Malate is used by the mitochondria via NAD-ME, which generates CO₂ and NADH; the NADH is then utilized by the mitochondria to generate ATP to support the PEP-CK decarboxylase reaction (Burnell and Hatch, 1988; Walker and Chen, 2002; Voznesenskaya et al., 2006). In this scheme, the aspartate cycle generates about 70%, and the malate cycle about 30%, of the total CO₂ delivered to the BS cells.
3.
In this illustration (Fig. 5c), an equal amount of reductive power is required by M and BS cells. This is consistent with M and BS chloroplasts of PEP-CK species having a similar granal index, suggestive of equivalent capacity for PSII activity for generating NADPH (Voznesenskaya et al., 2006). Again, this partitioning of reductive power can to some extent be regulated by the amount of PGA shuttled from BS to M chloroplasts for reduction (in the scheme, about 40%).

2.2.5 5. Additional Energy Requirements in C₄ Photosynthesis

Besides the basic requirements for energy to support the C₃ and C₄ cycles, additional energy will be consumed by over-cycling of the C₄ cycle (CO₂ leakage from BS cells, that is 2 ATP per CO₂ lost from BS cells), and due to the occurrence of limited photorespiration because of some O₂ reacting with RuBP, see Kanai and Edwards (1999). The cost of photorespiration is illustrated in Fig. 6. The requirement for reductive power per O₂ reacting with RuBP (eq. to 2 NADPH) is the same as for CO₂ reacting with RuBP, and the scheme illustrates how this can be shared equally between M and BS chloroplasts.

3 III. Single-Cell C₄ Photosynthesis in Terrestrial Plants

For decades following the discovery of C₄ photosynthesis in the 1960s, it was considered that the requirements for C₄, as summarized in the Introduction, could only be met in terrestrial plants by the presence of Kranz type anatomy. Thus, it was surprising to find species in family Chenopodiaceae that undergo traditional C₄ photosynthesis, but have a unique anatomy that does not consist of the Kranz dual-cell system. Instead, the single-cell C₄ system functions in individual chlorenchyma cells by means of intracellular biochemical and organelle compartmentation. Two very novel means of accomplishing this evolved in subfamily Suaedoideae. These systems function by spatial development of two cytoplasmic domains in chlorenchyma cells, which contain dimorphic chloroplasts. The arrangement of M and BS cells that so long has defined terrestrial forms of C₄ plants has now been joined by single-cell C₄ systems as functional anatomical alternatives (Voznesenskaya et al., 2001, 2002; Sage, 2002; Edwards et al., 2004; Akhani et al., 2005; Park et al., 2010).

3.1 A. Occurrence (Family and Phylogeny)

In family Chenopodiaceae, which has C₃ and C₄ species, all C₄ genera except for subfamily Chenopodioideae (including genus Atriplex) occur in a succulent clade made up of subfamilies Salicornioideae/Suaedoideae/Salsoloideae. The single-cell C₄ type species occur in subfamily Suaedoideae (Fig. 7), in two species in genus Bienertia (B. cycloptera Bunge ex Boiss., B. sinuspersici Akhani sp. nov.) and in one species in genus Suaeda, S. aralocaspica (Bunge) Freitag & Schütze (Kadereit et al., 2003; Schütze et al., 2003; Akhani et al., 2005; Kapralov et al., 2006). Suaeda aralocaspica, originally named Borszczowia aralocaspica Bunge, was subsequently classified in the monotypic Suaeda section Borszczowia, with a leaf type called Borszczowoid (Freitag and Stichler, 2000; Schütze et al., 2003; Kapralov et al., 2006).

There are four independent origins of C₄ photosynthesis in subfamily Suaedoideae: two parallel origins of Kranz C₄ anatomy (Salsinoid and Schoberioid in genus Suaeda) (see Section II A 3), and two independent origins of single-cell C₄. The single-cell C₄ plant Suaeda aralocaspica is in tribe Suaedeae. In this tribe, the veins are invariably located in one plane, with the primary vein in the center of the leaf and all deviating bundles in lateral positions. However, unlike other Suaeda species, S. aralocaspica has a primary vein in the center with peripheral veins adjacent to the chlorenchyma tissue (Freitag and Stichler, 2000; Schütze et al., 2003). This species is positioned between the C₃ section Schanginia and a C₃ shrubby section Suaeda, which suggests this type of photosynthesis evolved from C₃ ancestors rather than from a C₄ ancestor with Kranz anatomy (Kapralov et al., 2006). The two Bienertia species occur in an isolated tribe, Bienertieae, with a leaf type called Bienertioid, and the species have no known close relatives. We will use the common names Bienertia and Borszczowia to refer to these two types of single-cell C₄ taxa which are classified as C₄ structural forms called Bienertioid and Borszczowoid, respectively.

3.2 B. Biogeography of Single-Cell C₄ Species

The single-cell C₄ species grow in desert conditions. Borszczowia grows in central Asia from northeast of the Caspian lowlands east to Mongolia and western China (Fig. 8). It is a hygro-halophyte that grows in temperate salt deserts with low night temperatures. The habitat consists of a high water table in salt marshes, which can support continuous leaf development and growth (Freitag and Stichler, 2000; Boyd et al., 2007).

Bienertia cycloptera grows from east Anatolia eastward to Turkmenistan and Pakistani Baluchestan. Its leaves are very succulent and sensitive to extended drought, which can cause wilting and leaf drop. It grows on dryer soils and is confronted with drought stress during the summer (Akhani et al., 2003).

Bienertia sinuspersici occurs in hot climates, and at lower latitudes and elevations than B. cycloptera. It is in a natural biogeographic range occurring from the westernmost coasts of Pakistan and extending westward along the coastal areas in southern Iran and countries surrounding the Persian Gulf. It shows an arc-like, latitudinal range that is separated from the range of B. cycloptera populations by the Zagros and Makran Mountains (Fig. 8). Bienertia sinuspersici differs anatomically by having mostly one to two layers of chlorenchyma cells, versus two to three layers in B. cycloptera. Furthermore, B. sinuspersici is distinguished from B. cycloptera in having longer cotyledon leaves and leaves proper, larger seeds, larger flowers, and larger chromosomes, together with a set of micro-morphological features (Akhani et al., 2005).

3.3 C. Overview of Two Types of Single-CellC₄ Photosynthesis in Terrestrial Plants

Two means of partitioning the function of C₄ photosynthesis between two cytoplasmic compartments evolved in family Chenopodiaceae. Borszczowia produces elongated palisade chlorenchyma cells with dimorphic chloroplasts polarized towards opposite ends of the cell. This is somewhat analogous to having Kranz anatomy, with the M and BS arrangement, without the intervening cell walls. Surprisingly, a completely different solution to performing C₄ photosynthesis in a single cell is found in Bienertia. The chlorenchyma cells of the two Bienertia species have a peripheral, chloroplast-containing, thin layer of cytoplasm (peripheral compartment) and a very unusual chloroplast-containing central cytoplasmic compartment, which are proposed to function like M and BS cells, respectively, in Kranz type C₄. In both systems, the partitioning of biochemically distinct organelles into discrete compartments is considered to result in concentration of CO₂ around the Rubisco-containing chloroplasts, causing inhibition of Rubisco oxygenase activity and photorespiration, as occurs in the typical Kranz system. Models of how these systems operate C₄ photosynthesis have been proposed (Fig. 9, Edwards et al., 2004).

3.4 D. Biochemical Evidence for Function of C₄ Photosynthesis in Single-Cell C₄ Plants

3.4.1 1. General Features Characteristic of C₄

3.4.1.1 Western Blots and Analysis of C₄ Enzymes

Analyses of photosynthetic enzymes by Western blots and enzyme assays show that the single-cell C₄ species have high levels of C₄ cycle enzymes PEPC and PPDK, similar to Kranz type Suaeda, and in contrast to very low levels in the C₃ type Suaeda species (Fig. 10, also see Voznesenskaya et al., 2002). Assays for C₄ acid decarboxylases show that these single-cell C₄ species are NAD-ME type (Fig. 10), as are all Kranz type C₄ species which have been examined in subfamily Suaedoideae, see Voznesenskaya et al., 2007. Also, the single-cell C₄ species have a C₄ type PEPC similar to that in Kranz type species in subfamily Suaedoideae (Lara et al., 2006). This includes having high specific activities, a serine residue near the amino-terminus which undergoes phosphorylation/dephosphorylation, and light/dark regulation by phosphorylation with differential sensitivity to malate (Lara et al., 2006).

3.4.1.2 C₄ Type Carbon Isotope Composition

Reports on carbon isotope values in Bienertia (Winter, 1981; Akhani et al., 1997, 2005; Freitag and Stichler, 2002; Voznesenskaya et al., 2002) and Borszczowia (Freitag and Stichler, 2000; Voznesenskaya et al., 2001) indicated that they have C₄/CAM (Crassulacean acid metabolism) type carbon isotope composition. Although succulent, subsequent studies showed no evidence for performance of CAM (Voznesenskaya et al., 2001, 2002, 2003). Various collections of the two Bienertia species and Borszczowia, from natural habitats and from plants grown under controlled conditions in high light, show that they have C₄ type carbon isotope composition. Analysis of the carbon isotope composition during a growing season in Iran showed B. cycloptera performs C₄ photosynthesis during its life cycle in nature similar to Kranz type C₄ species (Akhani et al., 2009). In Bienertia species, more negative values (−16% to −19%) have been observed in young leaves and during growth under low light (100–200 photosynthetic photon flux density) (Freitag and Stichler, 2002; Voznesenskaya et al., 2002, 2005c). In young leaves, C₄ type chlorenchyma have not fully developed (Voznesenskaya et al., 2005c); growth under low light may limit the developmental transition from C₃ to a fully functional C₄ system (possibly due to incomplete development of dimorphic chloroplasts, or ability to concentrate CO₂ around Rubisco).

3.4.1.3 Physiological Response

The single-cell C₄ species and the Kranz type Suaeda species have low sensitivity of photosynthesis to O₂ under atmospheric levels of CO₂, and low CO₂ compensation points, typical of C₄ plants (Voznesenskaya et al., 2001, 2002, 2007; Edwards et al., 2007). Also, the water use efficiency (μmol CO₂ mmol^–1 water) is about two fold higher in the single-cell C₄ species and the Kranz type C₄ species than in representative C₃ species in subfamily Suaedoideae (Edwards et al., 2007).

3.4.1.4 Resistance to CO₂ Loss

For C₄ photosynthesis to function, high efficiency in trapping of the CO₂ generated by the C₄ pump, and in refixation of photorespired CO₂, are required. This means that the diffusive resistance in C₄ plants for CO₂ from Rubisco to the intercellular air space must be substantially higher than that in C₃ plants. Analyses of the diffusive resistance in the single-cell C₄ species show it is about 50-fold higher than that in C₃ species, and that it is of the same order of magnitude of Kranz type C₄ plants (Edwards et al., 2007).

3.4.2 2. Spatial Compartmentation Enabling Function of NAD-ME Type C₄ Photosynthesis

Critical experiments showing how C₄ photosynthesis functions in these single-cell C₄ species were performed by studying the structural organization by microscopy, by immunolocalization of several photosynthetic enzymes, and by localization of starch. In each single-cell C₄ system, the mechanism of photosynthesis and spatial compartmentation of function are analogous to those of NAD-ME type Kranz species (Fig. 9, Voznesenskaya et al., 2001, 2002; Edwards et al., 2004). In the Bienertia species, the peripheral cytoplasm functions analogous to Kranz type C₄ M cells in fixation of atmospheric CO₂ into C₄ acids, while the central cytoplasmic compartment functions analogous to BS cells in donation of CO₂ from C₄ acids to Rubisco (Voznesenskaya et al., 2001, 2002; Edwards et al., 2004). As illustrated in the model in Fig. 9, CO₂ is fixed by PEPC in the peripheral cytoplasm leading to formation of C₄ acids (aspartate and malate) and their transport via cytoplasmic channels to the central compartment and decarboxylation in mitochondria via NAD-ME, with CO₂ donation to Rubisco. C₃ acids formed from decarboxylation are transported to the peripheral compartment and used for regeneration of PEP from pyruvate in the peripheral chloroplasts. In Borszczowia, the carboxylation phase of the C₄ pathway, with fixation of atmospheric CO₂, functions in the distal part of the cell (analogous to M cells in Kranz type), while donation of CO₂ to Rubisco from decarboxylation of C₄ acids occurs in the proximal part of the cell (analogous to BS cells in the Kranz type NAD-ME species).

3.4.2.1 Dimorphic Chloroplasts (Structure, Enzymes and Starch)

In each structural type of single-cell C₄, C₄ photosynthesis is accomplished in part by the partitioning of two biochemically and ultrastructurally distinct chloroplast types into separate compartments within the cell (Voznesenskaya et al., 2001, 2002, 2005c). These chloroplasts are dimorphic in biochemistry of photosynthesis, in ability to store starch, and in ultrastructure. In addition to immunolocalization studies by confocal microscopy, immunolocalization in Bienertia and Borszczowia by transmission electron microscopy shows rather strong selective labeling of PPDK in one chloroplast type, and Rubisco in the other chloroplast type (E. Voznesenskaya, N. Koteyeva, G. Edwards, unpublished results). The outer chloroplasts supporting the carboxylation phase of the C₄ cycle to fix atmospheric CO₂ have PPDK, which generates the substrate for PEPC by converting pyruvate to PEP, they store little or no starch, and they have a deficiency in grana development. The inner chloroplasts, which fix CO₂ generated from decarboxylation of C₄ acids, have Rubisco, they store starch (and have ADPG pyrophosphorylase, the first committed step for starch biosynthesis), and they have well-developed grana. These features are the same as the respective M and BS chloroplasts in related Kranz type NAD-ME species. In C₄ plants, BS chloroplasts usually store larger amounts of starch than M chloroplasts. In NAD-ME type C₄ species, the grana-deficient chloroplasts are thought to be associated with a lower requirement for reductive power to support the C₄ carboxylation phase in this subgroup (see Section B2).

3.4.2.2 Mitochondria and Peroxisomes

In the single-cell C₄ species, the mitochondria are partitioned to the cytoplasmic compartment, where the Rubisco-containing chloroplasts are located (the proximal end of the cell in Borszczowia and in the central cytoplasmic compartment in Bienertia species). The mitochondria perform two important functions relative to C₄ photosynthesis: generation of CO₂ by decarboxylation of C₄ acids via NAD-ME and decarboxylation of glycine as a result of any photorespiration. Also, peroxisomes are predominantly located in the cytoplasmic compartment with Rubisco-containing chloroplasts (based on transmission electron microscopy and immunolocalization of catalase), which are presumably associated with metabolism of glycolate to glycerate in the glycolate pathway (Voznesenskaya et al., 2001, 2002; Chuong et al., 2006). While the C₄ cycle concentrates CO₂ around Rubisco and suppresses photorespiration, some photorespiration does occur. The selective localization of glycine decarboxylase in BS mitochondria makes photorespired CO₂ available for refixation by Rubisco.

3.5 E. Development of Spatial Compartmentation and Dimorphic Chloroplasts

An intriguing aspect of single-cell C₄ photosynthesis is the development of spatial compartmentation of functions and dimorphic chloroplasts. There is evidence that very young chlorenchyma cells have a single type of chloroplast (monomorphic) which is in a C₃ default mode, with all chloroplasts containing low levels of Rubisco without PPDK, and without spatial separation of organelles into two compartments (Voznesenskaya et al., 2005c). Since enzymes of the C₄ cycle like PPDK and the small subunit of Rubisco are nuclear encoded, there must be posttranscriptional regulation for selective expression of certain proteins in chloroplasts (see chapter 12 by Berry et al. for selective expression in Kranz type C₄). In mature chlorenchyma cells that have formed two cytoplasmic compartments and dimorphic chloroplasts, there is intricate development of the cytoskeleton, which consists of actin and microtubules. Cytoskeleton-disrupting drugs show that microtubules are important in maintaining the two cytoplasmic compartments (Chuong et al., 2006).

3.6 F. Form of Photosynthesis in Different Photosynthetic Organs in Single-Cell C₄ Species

Plants are usually characterized by photosynthetic type according to the mechanism of carbon assimilation in the leaf, which is generally the main photosynthetic organ. Among chenopods having C₄ photosynthesis in leaves, there is variation between species as to the type of photosynthesis in cotyledons (Butnik, 1979, 1984, 1991; Pyankov et al., 1999, 2000b, c; Voznesenskaya et al., 1999, 2004; Akhani and Ghasemkhani, 2007). There are C₄ chenopods, for example Salsola richteri (Salsoloid type), which have the same type of Kranz anatomy in leaves and cotyledons. There are also C₄ chenopods having C₄ photosynthesis in leaves and cotyledons, but different types of Kranz anatomy, for example Salsola laricina, which has Salsoloid anatomy in leaves and Atriplicoid type anatomy in cotyledons. Finally, there are dicots which have C₄ photosynthesis in leaves, but C₃ type anatomy and photosynthesis in cotyledons; for example Salsola gemmascens has Salsoloid type anatomy in leaves and C₃ type anatomy in cotyledons (Pyankov et al., 2000c).

Not all C₄ plants have leaves as the primary photosynthetic organ during vegetative growth. For example, in family Chenopodiaceae, C₄ species of Anabasis, Haloxylon, Halosarcia, Hammada and some species of Halothamnus have reduced leaves with stems as the primary photosynthetic organ (Ocallaghan, 1992; Akhani et al., 1997; Pyankov et al., 2000b).

With respect to single-cell C₄ species, Borszczowia and both Bienertia species have green cotyledons, leaves, and flowers. Studies of the respective organs, including structure of chlorenchyma, immunolocalization of photosynthetic enzymes (Rubisco and PEPC), and Western blots of Rubisco and C₄ cycle enzymes, show they all have unique chlorenchyma cells which are structurally and biochemically developed to perform single-celled C₄ photosynthesis (Voznesenskaya et al., 2001, 2002, 2004; Edwards et al., 2004; Akhani et al., 2005; Boyd et al., 2007). Thus, these species perform single-cell C₄ photosynthesis throughout their life cycle. Leaves make up the majority of the green tissue during vegetative growth of these species, although younger branches have green stems. However, the flowers have green tepals which become a major photosynthetic organ during the latter reproductive phase of growth. Photosynthesis in flowers may have adaptive value for survival of these desert plants. In Bienertia, under increasing temperatures as flowers develop, lower leaves wither and senesce and are replaced by smaller floral leaves consisting of green tepals (Boyd et al., 2007). Also, the stems of B. cycloptera have single-cell C₄ type chlorenchyma cells beneath the epidermis. This, combined with the presence of stomata on the stems, starch in stem chlorenchyma, and light-dependent fixation of atmospheric CO₂ by the stems, suggests they contribute to carbon assimilation in Bienertia. In contrast, the stems of Borszczowia have C₃ type chlorenchyma cells scattered throughout the cortical tissue. They likely function to refix respired CO₂ in stems, since light dependent fixation of external CO₂ is only observed under high atmospheric levels (Boyd et al., 2007).

3.7 G. How Did Single-Cell C₄ Evolve?

In considering how single-cell C₄ plants might have evolved, an analogy can be made to the proposed evolutionary development of Kranz type C₄ plants from C₃ plants (see Chapter 6 by Bauwe). In C₃ plants, M cells are the main photosynthetic tissue in the leaf, since the BS cells have few, or no, chloroplasts. It has been suggested that evolution of C₄ has occurred multiple times by a stepwise progression of structural and biochemical changes which were induced by CO₂-limiting conditions (Monson et al., 1984; Edwards and Ku, 1987; Monson and Moore, 1989; Rawsthorne and Bauwe, 1998; Sage, 2004). The occurrence of intermediates between C₃ and C₄ plants, particularly in the genus Flaveria (Asteraceae), has provided a basis for suggesting how C₄ may have evolved, from C₃, to intermediates which reduce photorespiration without a C₄ cycle, to intermediates having a partially-functioning C₄ cycle, to full development of C₄. In the first type of intermediate, normal C₃ type photosynthesis occurs in M cells; however, the release of CO₂ in photorespiration occurs in mitochondria in BS cells (by selective localization of glycine decarboxylase in BS mitochondria), where a degree of refixation by chloroplasts occurs. This increases the efficiency of photosynthesis under limiting CO₂ (von Caemmerer, 1989).

By analogy, under CO₂-limiting conditions a single-cell C₃-C₄ intermediate species may develop by spatial separation of the fixation of atmospheric CO₂ by the C₃ cycle and the refixation of photorespired CO₂ (see illustration in Fig. 11). This would generate a form of CO₂ concentrating mechanism and reduce loss of CO₂ by photorespiration. It could provide the initial spatial separation with chloroplasts in one cytoplasmic compartment fixing atmospheric CO₂, and chloroplasts and mitochondria in another cytoplasmic compartment functioning to minimize CO₂ loss by photorespiration. The spatial separation of chloroplasts and mitochondria in this hypothetical C₃-C₄ intermediate is like that in the single-cell C₄ plants. This would provide the initial spatial separation of organelles, with subsequent differentiation of chloroplasts and expression of C₄ cycle enzymes leading to development of a functional C₄ system.

4 IV. Future Perspectives

The discovery of C₄ plants among terrestrial species, approximately 40 years ago, and their association with Kranz anatomy, has led to numerous studies on the occurrence, photosynthetic mechanism, structural and biochemical diversity, molecular control of development of two photosynthetic cells, and evolution (Edwards and Walker, 1983; Hatch, 1987; Sage and Monson, 1999). With the paradigm that this occurred in land plants via development of Kranz anatomy, the finding that terrestrial species can conduct C₄ photosynthesis within individual chlorenchyma cells provides a very different system to study C₄. This includes stages in evolution of C₄, the genetic control of development of the requisite spatial separation of functions, the mechanism of chloroplast differentiation, biochemical and biophysical requirements for the function of C₄ photosynthesis, and development of strategies for engineering C₄ photosynthesis into selected C₃ crops.

Abbreviations

BS:: Bundle sheath(s);
Kranz cells:: An inner layer of chlorenchyma cells specialized for C₄ photosynthesis, irrespective of whether there is contact with vascular bundles (sometimes referred to as BS cells in C₄ plants);
M:: Mesophyll;
MS:: Mestome sheath(s);
NAD-ME:: NAD-malic enzyme;
NADP-ME:: NADP-malic enzyme;
PEPC:: Phosphoenolpyruvate carboxylase;
PEP-CK:: Phosphoenolpyruvate carboxykinase;
PGA:: 3-Phosphoglyceric acid;
PPDK:: Pyruvate,Pi dikinase;
PSI:: Photosystem I;
PSII:: Photosystem II;
RuBP:: Ribulose 1,5-bisphosphate;
SL:: Suberin lamella

References

Akhani H and Ghasemkhani M (2007) Diversity of photosynthetic organs in Chenopodiaceae from Golestan National Park (NE Iran) based on carbon isotope composition and anatomy of leaves and cotyledons. Nova Hedwigia Suppl 131: 265–277
Google Scholar
Akhani H, Trimborn P and Ziegler H (1997) Photosynthetic pathways in Chenopodiaceae from Africa, Asia and Europe with their ecological, phytogeographical and taxonomical importance. Plant Syst Evol 206: 187–221
Google Scholar
Akhani H, Ghobadnejhad M and Hashemi SMH (2003) Ecology, biogeography and pollen morphology of Bienertia cycloptera Bunge ex Boiss. (Chenopodiaceae), an enigmatic C₄ plant without Kranz anatomy. Plant Biol 5: 167–178
Google Scholar
Akhani H, Barroca J, Koteeva N, Voznesenskaya E, Franceschi V, Edwards G, Ghaffari SM and Ziegler H (2005) Bienertia sinuspersici (Chenopodiaceae): a new species from Southwest Asia and discovery of a third terrestrial C₄ plant without Kranz anatomy. Syst Bot 30: 290–301
Google Scholar
Akhani H, Ghasemkhani M, Chuong SDX and Edwards GE (2008) Occurrence and forms of Kranz anatomy and characterization of NAD-ME subtype C₄ photosynthesis in Blepharis ciliaris (L) B.L. Burtt (Acanthaceae). J Exp Bot 59: 1755–1765
PubMed CAS Google Scholar
Akhani H, Lara MV, Ghasemkhani M, Ziegler H and Edwards GE (2009) Does Bienertia cycloptera with the single-cell system of C₄ photosynthesis exhibit a seasonal pattern of δ¹³C values in nature similar to co-existing C₄ Chenopodiaceae having the dual-cell (Kranz) system? Photosyn Res 99: 23–36
PubMed CAS Google Scholar
Aliscioni SS, Giussani LM, Zuloaga FO and Kellogg EA (2003) A molecular phylogeny of Panicum (Poaceae: Paniceae): Tests of monophyly and phylogenetic placement within the Panicoideae. Am J Bot 90: 796–821
PubMed CAS Google Scholar
Anderson JM (1999) Insights into the consequences of grana stacking of thylakoid membranes in vascular plants: a personal perspective. Aust J Plant Physiol 26: 625–639
CAS Google Scholar
Bisalputra T, Downton WJS and Tregunna EB (1969) The distribution and ultrastructure of chloroplasts in leaves differing in photosynthetic carbon metabolism. I. Wheat, Sorghum and Aristida (Gramineae). Can J Bot 47: 15–21
CAS Google Scholar
Boyd CN, Franceschi VR, Chuong SDX, Akhani H, Kiirats O, Smith M and Edwards GE (2007) Flowers of Bienertia cycloptera and Suaeda aralocaspica (Chenopodiaceae) complete the life cycle performing single-cell C₄ photosynthesis. Funct Plant Biol 34: 268–281
CAS Google Scholar
Brown WV (1958) Leaf anatomy in grass systematics. Bot Gaz 119: 170–178
Google Scholar
Brown WV (1975) Variations in anatomy, associations, and origin of Kranz tissue. Am J Bot 62: 395–402
Google Scholar
Brown WV (1977) The Kranz syndrome and its subtypes in grass systematics. Mem Torrey Bot Club 23: 1–97
CAS Google Scholar
Bruhl JJ and Perry S (1995) Photosynthetic pathway-related ultrastructure of C₃, C₄ and C₃-like C₃-C₄ intermediate sedges (Cyperaceae), with special reference to Eleocharis. Aust J Plant Physiol 22: 521–530
CAS Google Scholar
Bruhl JJ, Stone NE and Hattersley PW (1987) C₄ acid decarboxylation enzymes and anatomy in sedges (Cyperaceae): first record of NAD-malic enzyme species. Aust J Plant Physiol 14: 719–728
CAS Google Scholar
Burnell JN and Hatch MD (1988) Photosynthesis in phosphoenolpyruvate carboxykinase-type C₄ plants: pathways of C₄ acid decarboxylation in bundle sheath cells of Urochloa panicoides. Arch Biochem Biophys 260: 187–199
PubMed CAS Google Scholar
Butnik AA (1979) Types of seedling development of Chenopodiaceae Vent. Bot Zh 64: 834–842 (In Russian)
Google Scholar
Butnik AA (1984) The adaptation of anatomical structure of the family Chenopodiaceae Vent. species to arid conditions. Summary of biological science doctor degree thesis. Academy of Sciences of Uzbek SSR, Tashkent (In Russian)
Google Scholar
Butnik AA (1991) Family Chenopodiaceae. In: AL Tachtadjan (ed) Comparative seed anatomy. Dicotyledonous. Caryophyllidae-Dilleniidae, pp 77–82 (In Russian). Nauka, Leningrad
Google Scholar
Butnik AA, Ashurmetov OA, Nigmatova RN and Paizieva SA (2001) Ecological anatomy of desert plants of Middle Asia. V. 2. Subshrubs, Subshrublets FAN, Tashkent (In Russian)
Google Scholar
Carolin RC, Jacobs SWL and Vesk M (1973) The structure of the cells of the mesophyll and parenchymatous bundle sheath of the Gramineae. Bot J Linn Soc 66: 259–275
Google Scholar
Carolin RC, Jacobs SWL and Vesk M (1975) Leaf structure in Chenopodiaceae. Bot Jahrbuch Syst Pflanzengesch Pflanzengeogr 95: 226–255
Google Scholar
Carolin RC, Jacobs SWL and Vesk M (1977) The ultrastructure of Kranz cells in the family Cyperaceae. Bot Gaz 138: 413–419
Google Scholar
Carolin RC, Jacobs SWL and Vesk M (1978) Kranz cells and mesophyll in the Chenopodiales. Aust J Bot 26: 683–698
Google Scholar
Carolin RC, Jacobs SWL and Vesk M (1982) The chlorenchyma of some members of the Salicornieae (Chenopodiaceae). Aust J Bot 30: 387–392
Google Scholar
Christin P-A, Besnard G, Samaritani E, Duvall MR, Hodkinson TR, Savolainen V and Salamin N (2008) Oligocene CO₂ decline promoted C₄ photosynthesis in grasses. Current Biol 18: 37–43
CAS Google Scholar
Christin P-A, Salamin N, Kellogg EA, Vicentini A and Besnard G (2009) Integrating phylogeny into studies of C₄ variation in the grasses. Plant Physiol 149: 82–87
PubMed CAS Google Scholar
Chuong SDX, Franceschi VR and Edwards GE (2006) The cytoskeleton maintains organelle partitioning required for single-cell C₄ photosynthesis in Chenopodiaceae species. Plant Cell 18: 2207–2223
PubMed CAS Google Scholar
Craig S and Goodchild DJ (1977) Leaf ultrastructure of Triodia irritans: a C₄ grass possessing an unusual arrangement of photosynthetic tissues. Aust J Bot 25: 277–290
CAS Google Scholar
Crookston RK and Moss DN (1972) C-4 and C-3 carboxylation characteristics in the genus Zygophyllum (Zygophyllaceae). Ann MO Bot Gard 59: 465–470
Google Scholar
Crookston RK and Moss DN (1973) A variation of C₄ leaf anatomy in Arundinella hirta (Gramineae). Plant Physiol 52: 397–402
PubMed CAS Google Scholar
Das VSR and Raghavendra AS (1976) C₄ photosynthesis and a unique type of Kranz anatomy in Glossocordia boswallaea (Asteraceae). Proc Indian Acad Sci 84B: 12–19
Google Scholar
Dengler RE and Dengler NG (1990) Leaf vascular architecture in the atypical C₄ NADP-malic enzyme grass Arundinella hirta. Can J Bot 68: 1208–1221
Google Scholar
Dengler NG and Nelson T (1999) Leaf structure and development in C₄ plants. In: Sage RF and Monson RK (eds) C₄ Plant Biology. Physiological Ecology series, pp 133–172. Academic Press, New York
Google Scholar
Dengler NG, Dengler RE and Hattersley PW (1985) Differing ontogenetic origins of PCR (“Kranz”) sheaths in leaf blades of C₄ grasses (Poaceae). Am J Bot 72: 284–302
Google Scholar
Dengler NG, Dengler RE and Drenville DJ (1990) Comparison of photosynthetic carbon reduction (Kranz) cells having different ontogenetic origins in the C₄ NADP-malic enzyme grass Arundinella hirta. Can J Bot 68: 1222–1232
Google Scholar
Dengler NG, Donnelly PM and Dengler RE (1996) Differentiation of bundle sheath, mesophyll, and distinctive cells in the C₄ grass Arundinella hirta (Poaceae). Am J Bot 83: 1391–1405
Google Scholar
Edwards GE and Ku MSB (1987) The biochemistry of C₃-C₄ intermediates. In: Hatch MD and Boardman NK (eds) The Biochemistry of Plants, pp 275–325. Academic Press, New York
Google Scholar
Edwards GE and Walker DA (1983) C₃, C₄: Mechanisms, and Cellular and Environmental Regulation, of Photosynthesis. Blackwell, Oxford
Google Scholar
Edwards GE, Furbank RT, Hatch MD and Osmond CB (2001) What does it take to be C₄? Lessons from the evolution of C₄ photosynthesis. Plant Physiol 125: 46–49
PubMed CAS Google Scholar
Edwards GE, Franceschi VR and Voznesenskaya EV (2004) Single-cell C₄ photosynthesis versus the dual-cell (Kranz) paradigm. Ann Rev Plant Biol 55: 173–196
CAS Google Scholar
Edwards GE, Voznesenskaya EV, Smith M, Koteyeva N, Park Y-I, Park JH, Kiirats O, Okita TW and Chuong SDX (2007) Breaking the Kranz paradigm in terrestrial C₄ plants: Does it hold promise for C₄ rice? In: Sheehy JE, Mitchell PL and Hardy B (eds) Charting New Pathways to C₄ rice, pp 249–273. International Rice Research Institute, World Scientific, Los Banos, Philippines
Google Scholar
Ellis RP (1977) Distribution of the Kranz syndrome in the Southern African Eragrostoideae and Panicoideae according to bundle sheath anatomy and cytology. Agroplantae 9: 73–110
Google Scholar
Fisher DD, Schenk HJ, Thorsch JA and Ferren WR, Jr. (1997) Leaf anatomy and subgeneric affiliation of C₃ and C₄ species of Suaeda (Chenopodiaceae) in North America. Am J Bot 84: 1198–1210
PubMed CAS Google Scholar
Frean ML, Ariovich D and Cresswell CF (1983) C₃ and C₄ photosynthetic and anatomical forms of Alloteropsis semialata (R. Br.) Hitchcock. II. A comparative investigation of leaf ultrastructure and distribution of chlorenchyma in the two forms. Ann Bot 51: 811–821
Google Scholar
Freitag H and Stichler W (2000) A remarkable new leaf type with unusual photosynthetic tissue in a central Asiatic genus of Chenopodiaceae. Plant Biol 2: 154–160
Google Scholar
Freitag H and Stichler W (2002) Bienertia cycloptera Bunge ex Boiss., Chenopodiaceae, another C₄ plant without Kranz tissues. Plant Biol 4: 121–132
Google Scholar
Gamaley YV (1985) The variations of the Kranz-anatomy in Gobi and Karakum plants. Bot Zh 70: 1302–1314 (In Russian)
Google Scholar
Gamaley YV and Voznesenskaya EV (1986) Structural-biochemical types of C₄ plants. Sov Plant Physiol 33: 616–630
Google Scholar
Gilliland MG and Gordon-Gray KD (1978) Kranz and non-kranz cells in Cyperaceae. Proc Electron Microsc Soc Southern Africa 8: 85–86
Google Scholar
Glagoleva TA, Voznesenskaya EV, Kol’chevskii KG, Kocharyan NI, Pakhomova MV, Chulanovskaya MV and Gamalei YV (1991) Structural-functional characteristics of halophytes of the Ararat valley. Sov Plant Physiol 37: 822
Google Scholar
GPWG (2001) Phylogeny and subfamilial classification of the grasses (Poaceae). Ann MO Bot Gard 88: 373–457
Google Scholar
Guissani LM, Cota-Sanches JH, Zuloaga FO and Kellogg EA (2001) A molecular phylogeny of the grass subfamily Panicoideae (Poaceae) shows multiple origins of C₄ photosynthesis. Am J Bot 88: 1993–2012
Google Scholar
Guralnick LJ, Edwards GE, Ku MSB, Hockema B and Franceschi VR (2002) Photosynthetic and anatomical characteristics in the C₄-Crassulacean acid metabolism-cycling plant, Portulaca grandiflora. Funct Plant Biol 29: 763–773
CAS Google Scholar
Gutierrez M, Gracen VE and Edwards GE (1974) Biochemical and cytological relationships in C₄ plants. Planta 119: 279–300
CAS Google Scholar
Haberlandt G (1884) Physiologische Pflanzenanatomie. Engelmann, Leipzig
Google Scholar
Hatch MD (1971) Mechanism and function of C₄ photosynthesis. In: Hatch MD, Osmond CB and Slatyer RO (eds) Photosynthesis and Photorespiration, pp 139–152. Wiley-Interscience, New York
Google Scholar
Hatch MD (1987) C₄ photosynthesis: a unique blend of modified biochemistry, anatomy and ultrastructure. Biochim Biophys Acta 895: 81–106
CAS Google Scholar
Hatch MD (1999) C₄ photosynthesis: a historical overview. In: Sage RF and Monson RK (eds) C₄ plant biology. Physiological Ecology series, pp 17–46. Academic Press, San Diego
Google Scholar
Hatch MD, Kagawa T and Craig S (1975) Subdivision of C₄-pathway species based on differing C₄ acid decarboxylating systems and ultrastructural features. Aust J Plant Physiol 2: 111–128
CAS Google Scholar
Hattersley PW (1987) Variations in photosynthetic pathway. In: Soderstrom, TR Hilu KW, Campbell CS and Barkworth ME (eds) Grass systematics and evolution, pp 49–64. Smithsonian Institution Press, Washington, DC
Google Scholar
Hattersley PW (1992) C₄ photosynthetic pathway variation in grasses (Poaceae): its significance for arid and semi-arid lands. In: Chapman GP (ed) Desertified Grasslands: Their Biology and Management, pp 181–212. Academic Press, London
Google Scholar
Hattersley PW and Browning AJ (1981) Occurrence of the suberized lamella in leaves of grasses of different photosynthetic types. I. In parenchymatous bundle sheaths and PCR (“Kranz”) sheaths. Protoplasma 109: 371–401
Google Scholar
Hattersley PW and Watson L (1992) Diversification of photosynthesis. In: Chapman GP (ed) Grass evolution and domestication, pp 38–116. Cambridge University Press, Cambridge
Google Scholar
Hattersley PW, Wong S-C, Perry S and Roksandic Z (1986) Comparative ultrastructure and gas exchange characteristics of the C₃-C₄ intermediate Neurachne minor S.T. Blake (Poaceae). Plant Cell Environ 9: 217–233
CAS Google Scholar
Ibrahim DG, Burke T, Ripley BS and Osborne CP (2009) A molecular phylogeny of the genus Alloteropsis (Panicoideae, Poaceae) suggests an evolutionary reversion from C₄ to C₃ photosynthesis. Ann Bot 103: 127–136
PubMed CAS Google Scholar
Jacobs SWL (2001) Review of leaf anatomy and ultrastructure in the Chenopodiaceae (Caryophyllales). J Torrey Bot Soc 128: 236–253
Google Scholar
Johnson MSC (1964) An electron microscope study of the photosynthetic apparatus in plants, with special reference to the Gramineae. Ph.D. thesis, The University of Texas.
Google Scholar
Kadereit G, Borsch T, Weising K and Freitag H (2003) Phylogeny of Amaranthaceae and Chenopodiaceae and the evolution of C₄ photosynthesis. Int J Plant Sci 164: 959–986
CAS Google Scholar
Kanai R and Edwards G (1999) The biochemistry of C₄ photosynthesis. In: Sage RF and Monson RK (eds) C₄ Plant Biology. Physiological Ecology series, pp 49–87. Academic Press, San Diego
Google Scholar
Kapralov MV, Akhani H, Voznesenskaya E, Edwards G, Franceschi VR and Roalson EH (2006) Phylogenetic relationships in the Salicornioideae /Suaedoideae /Salsoloideae s.l. (Chenopodiaceae) clade and a clarification of the phylogenetic position of Bienertia and Alexandra using multiple DNA sequence datasets. Syst Bot 31: 571–585
Google Scholar
Kennedy RA and Laetsch WM (1974) Plant species intermediate for C₃, C₄ photosynthesis. Science 184: 1087–1089
PubMed CAS Google Scholar
Kim I and Fisher DG (1990). Structural aspects of the leaves of seven species of Portulaca growing in Hawaii. Can J Bot 68: 1803–1811
Google Scholar
Laetsch WM (1968) Chloroplast specialization in dicotyledons possessing the C₄-dicarboxylic acid pathway of photosynthetic CO₂ fixation. Am J Bot 55: 875–883
CAS Google Scholar
Laetsch WM (1974) The C₄ syndrome: a structural analysis. Annu Rev Plant Physiol 25: 27–52
CAS Google Scholar
Lara MV, Chuong SDX, Akhani H, Andreo CS and Edwards GE (2006) Species having C₄ single-cell-type photosynthesis in the Chenopodiaceae family evolved a photosynthetic phosphoenolpyruvate carboxylase like that of Kranz-type C₄ species. Plant Physiol 142: 673–684
PubMed CAS Google Scholar
Marshall DM, Muhaidat R, Brown NJ, Liu Z, Stanley S, Griffiths H, Sage RF and Hibberd JM (2007) Cleome, a genus closely related to Arabidopsis, contains species spanning a developmental progression from C₃ to C₄ photosynthesis. Plant J 207: 886–896
Google Scholar
McKown AD, Moncalvo J-M and Dengler NG (2005) Phylogeny of Flaveria (Asteraceae) and inference of C₄ photosynthesis evolution. Am J Bot 92: 1911–1928
PubMed CAS Google Scholar
Meister M, Agostino A and Hatch MD (1996) The roles of malate and aspartate in C₄ photosynthetic metabolism of Flaveria bidentis (L.). Planta 199: 262–269
CAS Google Scholar
Monson RK and Moore Bd (1989) On the significance of C₃-C₄ intermediate photosynthesis to the evolution of C₄ photosynthesis. Plant Cell Env 12: 689–699
CAS Google Scholar
Monson RK, Edwards GE and Ku MSB (1984) C₃-C₄ intermediate photosynthesis in plants. BioScience 34: 563–574
CAS Google Scholar
Moore Bd, Ku MSB and Edwards GE (1984) Isolation of leaf bundle sheath protoplasts from C₄ dicot species and intracellular localization of selected enzymes. Plant Sci Lett 35: 127–138
CAS Google Scholar
Muhaidat RM, Sage RF and Dengler NG (2007) Diversity of Kranz anatomy and biochemistry in C₄ eudicots. Am J Bot 94: 362–381
PubMed CAS Google Scholar
Murphy LR, Barroca J, Franceschi VR, Lee R, Roalson EH, Edwards GE and Ku MSB (2007) Diversity and plasticity of C₄ photosynthesis in Eleocharis (Cyperaceae). Funct Plant Biol 34: 571–580
CAS Google Scholar
Nishioka D, Miyake H and Taniguchi T (1996) Suppression of granal development and accumulation of Rubisco in different bundle sheath chloroplasts of the C₄ succulent plant Portulaca grandiflora. Ann Bot 77: 629
CAS Google Scholar
Ocallaghan M (1992) The ecology and identification of the southern African Salicornieae (Chenopodiaceae). South Afr J Bot 58: 430–439
Google Scholar
Ohsugi R and Murata T (1980) Leaf anatomy, post-illumination CO₂ burst and NAD-malic enzyme activity of Panicum dichotomiflorum. Plant Cell Physiol 21: 1329–1333
CAS Google Scholar
Ohsugi R, Murata T and Chonan N (1982) C₄ syndrome of the species in the Dichotomiflora group of the genus Panicum (Gramineae). Bot Mag 95: 339–347
CAS Google Scholar
Park J, Okita TW and Edwards GE (2010) Expression profiling and proteomic analysis of isolated photosynthetic cells of the non-Kranz C4 species Bienertia sinuspersici. Funct Plant Biol 37: 1–13
Google Scholar
Peter G and Katinas L (2003) A new type of Kranz anatomy in Asteraceae. Aust J Bot 51: 217–226
Google Scholar
Prendergast HDV and Hattersley PW (1987) Australian C₄ grasses (Poaceae): leaf blade anatomical features in relation to C₄ acid decarboxylation types. Aust J Bot 35: 355–382
Google Scholar
Prendergast HDV, Hattersley PW, Stone NE and Lazarides M (1986) C4 acid decarboxylation type in Eragrostis (Poaceae): patterns of variation in chloroplast position, ultrastructure, and geographical distribution. Plant Cell Env 9: 333–344
Google Scholar
Prendergast HDV, Hattersley PW and Stone NE (1987) New structural/biochemical associations in leaf blades of C₄ grasses (Poaceae). Aust J Plant Physiol 14: 403–420
CAS Google Scholar
Pyankov VI and Vakhrusheva DV (1989) Pathways of primary CO₂ fixation in C₄ plants of the family Chenopodiaceae from the arid zone of Central Asia. Sov Plant Physiol 36: 178–187
Google Scholar
Pyankov VI, Kuzmin AN, Demidov ED and Maslov AI (1992) Diversity of biochemical pathways of CO₂ fixation in plants of the families Poaceae and Chenopodiaceae from the arid zone of Central Asia. Sov Plant Physiol 39: 411–420
Google Scholar
Pyankov VI, Artyusheva EG and Edwards G (1999) Formation of C₄ syndrome in leaves and cotyledons of Kochia scoparia and Salsola collina (Chenopodiaceae). Russian J Plant Phys 46: 452–466
CAS Google Scholar
Pyankov VI, Gunin PD, Tsoog S and Black CC (2000a)C₄ plants in the vegetation of Mongolia: their natural occurrence and geographical distribution in relation to climate. Oecologia 123: 15–31
Google Scholar
Pyankov VI, Voznesenskaya EV, Kuzmin A, Ku MSB, Black CC and Edwards GE (2000b) Diversity of CO₂ fixation pathways in leaves and cotyledons of Salsola (Chenopodiaceae) plants. Dokl Bot Sci 370: 1–5
Google Scholar
Pyankov VI, Voznesenskaya EV, Kuzmin AN, Ku MSB, Ganko E, Franceschi VR, Black CC, Jr. and Edwards GE (2000c) Occurrence of C₃ and C₄ photosynthesis in cotyledons and leaves of Salsola species (Chenopodiaceae). Photosyn Res 63: 69–84
PubMed CAS Google Scholar
Rathnam CKM, Raghavendra AS and Das VSR (1976) Diversity in the arrangements of mesophyll cells among leaves of certain C₄ dicotyledons in relation to C₄ physiology. Z Pflanzenphysiol 77: 283–291
Google Scholar
Rawsthorne S and Bauwe H (1998) C₃-C₄ intermediate photosynthesis. In: Raghavendra AS (ed) Photosynthesis. A comprehensive treatise, pp 150–162. Cambridge University Press, Cambridge
Google Scholar
Sage RF (2002) C₄ photosynthesis in terrestrial plants does not require Kranz anatomy. Trends Plant Sci 7: 283–285
PubMed CAS Google Scholar
Sage RF (2004) The evolution of C₄ photosynthesis. New Phytol 161: 341–370
CAS Google Scholar
Sage RF and Monson RK (1999) C₄ Plant Biology. Academic Press, San Diego
Google Scholar
Sage RF, Li M and Monson RK (1999) The taxonomic distribution of C₄ photosynthesis. In: RF Sage and RK Monson (eds) C₄ Plant Biology, pp 551–584. Academic Press, New York
Google Scholar
Sanchez-Ken JG, Clark LG, Kellogg EA and Kay EE (2007) Reinstatement and emendation of subfamily Micrairoideae (Poaceae). Syst Bot 32: 71–80
Google Scholar
Schütze P, Freitag H and Weising K (2003) An integrated molecular and morphological study of the subfamily Suaedoideae Ulbr. (Chenopodiaceae). Plant Syst Evol 239: 257–286
Google Scholar
Sede SM, Morrone O, Aliscioni SS, Giussani LM and Zuloaga FO (2009) Oncorachis and Sclerochlamys, two new segregated genera from Streptostachys (Poaceae, Panicoideae, Paniceae): a revision based on molecular, morphological and anatomical characters. Taxon 58: 365–374
Google Scholar
Shepherd KA and Wilson PG (2007) Incorporation of the Australian genera Halosarcia, Pachycornia, Sclerostegia and Tegicornia into Tecticornia (Salicornioideae, Chenopodiaceae). Aust Syst Bot 20: 319–331
Google Scholar
Shomer-Ilan A, Beer S and Waisel Y (1975) Suaeda monoica, a C₄ plant without typical bundle sheaths. Plant Physiol 56: 676–679
PubMed CAS Google Scholar
Shomer-Ilan A, Neumann-Ganmore R and Waisel Y (1979) Biochemical specialization of photosynthetic cell layers and carbon flow paths in Suaeda monoica. Plant Physiol 64: 963–965
PubMed CAS Google Scholar
Shomer-Ilan AS, Nissenbaum A and Waisel Y (1981) Photosynthetic pathways and the ecological distribution of the Chenopodiaceae in Israel. Oecologia 48: 244–248
Google Scholar
Soros CL and Dengler NG (1998) Quantitative leaf anatomy of C₃ and C₄ Cyperaceae and comparisons with the Poaceae. Int J Plant Sci 159: 480–491
Google Scholar
Soros CL and Dengler NG (2001) Ontogenetic derivation and cell differentiation in photosynthetic tissues of C₃ and C₄ Cyperaceae. Am J Bot 88: 992–1005
PubMed CAS Google Scholar
Takeda T, Ueno O and Agata W (1980) The occurrence of C₄ species in the genus Rhynchospora and its significance in Kranz anatomy of the Cyperaceae. Bot Mag 93: 55–65
CAS Google Scholar
Taniguchi Y, Taniguchi M, Kawasaki M and Miyake H (2003) Strictness of the centrifugal location of bundle sheath chloroplasts in different NADP-ME type C₄ grasses. Plant Prod Sci 6: 274–280
Google Scholar
Tateoka T (1958) Notes on some grasses. VIII. On leaf structure of Arundinella and Garnotia. Bot Gaz 120: 101–109
Google Scholar
Ueno O (1995) Occurrence of distinctive cells in leaves of C₄ species in Arthraxon and Microstegium (Andropogoneae-Poaceae) and the structural and immunocytochemical characterization of these cells. Int J Plant Sci 156: 270–289
Google Scholar
Ueno O (1996a) Structural characterization of photosynthetic cells in an amphibious sedge, Eleocharis vivipara, in relation to C₃ and C₄ metabolism. Planta 199: 382–393
CAS Google Scholar
Ueno O (1996b) Immunocytochemical localization of enzymes involved in the C₃ and C₄ pathways in the photosynthetic cells of an amphibious sedge, Eleocharis vivipara. Planta 199: 394–403
CAS Google Scholar
Ueno O (1998a) Immunogold localization of photosynthetic enzymes in leaves of various C₄ plants, with particular reference to pyruvate orthophosphate dikinase. J Exp Bot 49: 1637–1646
CAS Google Scholar
Ueno O (1998b) Induction of Kranz anatomy and C₄-like biochemical characteristics in a submerged amphibious plant by abscisic acid. Plant Cell 10: 571–583
PubMed CAS Google Scholar
Ueno O (2004) Environmental regulation of photosynthetic metabolism in the amphibious sedge Eleocharis baldwinii and comparisons with related species. Plant Cell Env 27: 627–639
CAS Google Scholar
Ueno O and Samejima M (1989) Structural features of NAD-malic enzyme type C₄ Eleocharis: an additional report of C₄ acid decarboxylation types of the Cyperaceae. Bot Mag 102: 393–402
CAS Google Scholar
Ueno O and Sentoku N (2006) Comparison of leaf structure and photosynthetic characteristics of C₃ and C₄ Alloteropsis semialata subspecies. Plant Cell Env 29: 257–268
CAS Google Scholar
Ueno O and Wakayama M (2004) Cellular expression of C₃ and C₄ photosynthetic enzymes in the amphibious sedge Eleocharis retroflexa ssp. chaetaria. J Plant Res 117: 433–441
PubMed CAS Google Scholar
Ueno O, Takeda T and Murata T (1986) C₄ acid decarboxylating enzyme activities of the C₄ species possessing Kranz anatomical types in the Cyperaceae. Photosynthetica 20: 111–116
CAS Google Scholar
Ueno O, Takeda T and Maeda E (1988a) Leaf ultrastructure of C₄ species possessing different Kranz anatomical types in the Cyperaceae. Bot Mag 101: 141–152
Google Scholar
Ueno O, Samejima M and Koyama T (1989). Distribution and evolution of C₄ syndrome in Eleocharis, a sedge group inhabiting wet and aquatic environments, based on culm anatomy and carbon isotope ratios. Ann Bot 64: 425–438
Google Scholar
Ueno O, Samejima M, Muto S and Miyachi S (1988b) Photosynthetic characteristics of an amphibious plant, Eleocharis vivipara: expression of C₄ and C₃ modes in contrasting environments. Proc Natl Acad Sci USA 85: 6733–6737
PubMed CAS Google Scholar
Vasilevskaya VK and Butnik AA (1981) The types of the anatomical structure of the dicotyledon leaves (a contribution to the method of anatomical description). Bot Zh 66: 992–1001 (In Russian)
Google Scholar
Vicentini A, Barber JC, Aliscioni SS, Giussani LM and Kellogg EA (2008) The age of the grasses and clusters of origins of C₄ photosynthesis. Global Change Biology 14: 2963–2977
Google Scholar
von Caemmerer S (1989) A model of photosynthetic CO₂ assimilation and carbon-isotope discrimination in leaves of certain C₃-C₄ intermediates. Planta 178: 463–474
Google Scholar
Voznesenskaya EV and Gamaley YV (1986) The ultrastructural characteristics of leaf types with Kranz-anatomy. Bot Zh 71: 1291–1307 (In Russian)
Google Scholar
Voznesenskaya EV, Franceschi VR, Pyankov VI and Edwards GE (1999) Anatomy, chloroplast structure and compartmentation of enzymes relative to photosynthetic mechanisms in leaves and cotyledons of species in the tribe Salsoleae (Chenopodiaceae). J Exp Bot 50: 1779–1795
CAS Google Scholar
Voznesenskaya EV, Franceschi VR, Kiirats O, Freitag H and Edwards GE (2001) Kranz anatomy is not essential for terrestrial C₄ plant photosynthesis. Nature 414: 543–546
PubMed CAS Google Scholar
Voznesenskaya EV, Franceschi VR, Kiirats O, Artyusheva EG, Freitag H and Edwards GE (2002) Proof of C₄ photosynthesis without Kranz anatomy in Bienertia cycloptera (Chenopodiaceae). Plant J 31: 649–662
PubMed CAS Google Scholar
Voznesenskaya EV, Edwards GE, Kiirats O, Artyusheva EG and Franceschi VR (2003) Development of biochemical specialization and organelle partitioning in the single celled C₄ system in leaves of Borszczowia aralocaspica (Chenopodiaceae). Am J Bot 90: 1669–1680
PubMed CAS Google Scholar
Voznesenskaya EV, Franceschi VR and Edwards GE (2004) Light-dependent development of single cell C₄ photosynthesis in cotyledons of Borszczowia aralocaspica (Chenopodiaceae) during transformation from a storage to a photosynthetic organ. Ann Bot 93: 1–11
Google Scholar
Voznesenskaya EV, Chuong SDX, Kirrats O, Franceschi VR and Edwards GE (2005a) Evidence that C₄ species in genus Stipagrostis, family Poaceae, is NADP-malic enzyme subtype with nonclassical type of Kranz anatomy (Stipagrostoid). Plant Sci 168: 731–739
CAS Google Scholar
Voznesenskaya EV, Chuong SDX, Koteeva NK, Edwards GE and Franceschi VR (2005b) Functional compartmentation of C₄ photosynthesis in the triple-layered chlorenchyma of Aristida (Poaceae). Funct Plant Biol 32: 67–77
CAS Google Scholar
Voznesenskaya EV, Koteyeva NK, Chuong SDX, Edwards GE, Akhani H and Franceschi VR (2005c) Differentiation of cellular and biochemical features of the single-cell C₄ syndrome during leaf development in Bienertia cycloptera (Chenopodiaceae). Am J Bot 92: 1784–1795
PubMed CAS Google Scholar
Voznesenskaya EV, Franceschi VR, Chuong SDX and Edwards GE (2006) Functional characterization of phosphoenolpyruvate carboxykinase type C₄ leaf anatomy: Immuno, cytochemical and ultrastructural analyses. Ann Bot 98: 77–91
PubMed CAS Google Scholar
Voznesenskaya EV, Chuong S, Koteyeva N, Franceschi VR, Freitag H and Edwards GE (2007) Structural, biochemical and physiological characterization of C₄ photosynthesis in species having two vastly different types of Kranz anatomy in genus Suaeda (Chenopodiaceae). Plant Biol 9: 745–757
PubMed CAS Google Scholar
Voznesenskaya EV, Akhani H, Koteyeva NK, Chuong SDX, Roalson EH, Kiirats O, Franceschi VR and Edwards GE (2008) Structural, biochemical and physiological characterization of photosynthesis in two C₄ subspecies of Tecticornia indica and the C₃ species Tecticornia pergranulata (Chenopodiaceae). J Exp Bot 59: 1715–1734
PubMed CAS Google Scholar
Voznesenskaya EV, Koteyeva NK, Edwards GE and Ocampo G (2010) Anatomical and biochemical characterization of photosynthetic types in genus Portulaca L. (Portulacaceae). J Exp Bot 61:3647–3662
Google Scholar
Wakayama M, Ueno O and Ohnishi J (2002) Cellular accumulation of photosynthetic enzymes during leaf development of Arundinella hirta, a C₄ grass with unusual Kranz cells without contact with vascular tissues. Plant Cell Physiol 43: S173–S173
Google Scholar
Wakayama M, Ueno O and Ohnishi J (2003) Photosynthetic enzyme accumulation during leaf development of Arundinella hirta, a C₄ grass having Kranz cells not associated with vascular tissues. Plant Cell Physiol 44: 1330–1340
PubMed CAS Google Scholar
Wakayama M, Ohnishi J and Ueno O (2006) Structure and enzyme expression in photosynthetic organs of the atypical C₄ grass Arundinella hirta. Planta 223: 1243–1255
PubMed CAS Google Scholar
Walker RP and Chen Z-H (2002) Phosphoenolpyruvate carboxykinase: Structure, function and regulation. In: Callow JA (ed) Advances in Botanical Research Incorporating Advances in Plant Pathology, pp 93–189. Academic Press, New York
Google Scholar
Wingler A, Walker RP, Chen Z-H and Leegood RC (1999) Phosphoenolpyruvate carboxykinase is involved in the decarboxylation of aspartate in the bundle sheath of maize. Plant Physiol 120: 539–545
PubMed CAS Google Scholar
Winter K (1981) C₄ plants of high biomass in arid regions of Asia. Occurrence of C₄ photosynthesis in Chenopodiaceae and Polygonaceae from the middle east and USSR. Oecologia 48: 100–106
Google Scholar
Winter K, Kramer D, Troughton JH and Card KA (1977) C₄ pathway of photosynthesis in a member of the Polygonaceae: Calligonum persicum (Boiss. and Buhse) Boiss. Z Pflanzenphysiol 81: 341–346
CAS Google Scholar
Yoshimura Y, Kubota F and Ueno O (2004) Structural and biochemical bases of photorespiration in C₄ plants: quantification of organelles and glycine decarboxylase. Planta 220: 307–317
PubMed CAS Google Scholar

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Acknowledgments

This material is based upon work supported by the National Science Foundation under Grant Nos. IBN-0131098, IBN-0236959 and IBN-0641232, by Civilian Research and Development Foundation grants RB1-2502-ST-03 and RUB1-2829-ST-06, by Russian Foundation of Basic Research grant 05-04-49622 and 08-04-00936,and Bill and Melinda Gates Foundation to IRRI for C₄ Rice Program. We also thank the Franceschi Microscopy and Imaging Center of Washington State University for use of their facilities and staff assistance, and C. Cody for plant growth management. The authors appreciate the helpful discussions with Dr. Nuria Koteyeva and the advice of two reviewers, especially in the sections devoted to grasses and sedges.

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School of Biological Sciences, Washington State University, Pullman, WA, 99164-4236, USA
Gerald E. Edwards
Laboratory of Anatomy and Morphology, V.L. Komarov Botanical Institute of Russian Academy of Sciences, Prof. Popov Street 2, 197376, St. Petersburg, Russia
Elena V. Voznesenskaya

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School of Life Sciences, University of Hyderabad, Hyderabad, 500046, India
Agepati S. Raghavendra
Dept. Botany, University of Toronto, Willcocks Street 25, Toronto, M5S 3B2, Ontario, Canada
Rowan F. Sage

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Edwards, G.E., Voznesenskaya, E.V. (2010). Chapter 4 C₄ Photosynthesis: Kranz Forms and Single-Cell C₄ in Terrestrial Plants. In: Raghavendra, A., Sage, R. (eds) C4 Photosynthesis and Related CO2 Concentrating Mechanisms. Advances in Photosynthesis and Respiration, vol 32. Springer, Dordrecht. https://doi.org/10.1007/978-90-481-9407-0_4

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Chapter 4 C4 Photosynthesis: Kranz Forms and Single-Cell C4 in Terrestrial Plants

Summary

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Significance of C4 Leaf Structure at the Tissue and Cellular Levels

Coleataenia prionitis, a C4-like species in the Poaceae

Transition from C3 to proto-Kranz to C3–C4 intermediate type in the genus Chenopodium (Chenopodiaceae)

Keywords

1 I. Introduction

1.1 A. What Does It Take to Be C4?

1.2 B. Occurrence of C4 Among Terrestrial Plants

2 II. Structural and Biochemical Diversity in Kranz Type Anatomy

2.1 A. Structural Diversity

2.1.1 1. Poaceae

2.1.1.1 Classical NADP-ME Type Anatomy

2.1.1.2 Classical NAD-ME Type Anatomy

2.1.1.3 Classical PEP-CK Type Anatomy

2.1.1.4 Arundinelloid: Biochemical Subtype NADP-ME

2.1.1.5 Aristidoid: Biochemical Subtype NADP-ME

2.1.1.6 Stipagrostoid: Biochemical Subtype NADP-ME

2.1.1.7 Eriachneoid: Biochemical Subtype NADP-ME

2.1.1.8 Neurachneoid: Biochemical Subtypes NADP-ME and PEP-CK

2.1.1.9 Other Forms of NAD-ME Type Anatomy

2.1.2 2. Family Cyperaceae

2.1.2.1 Fimbristyloid: Biochemical Subtypes NADP-ME and NAD-ME

2.1.2.2 Chlorocyperoid: Biochemical Subtype NADP-ME

2.1.2.3 Rhynchosporoid: Biochemical Subtype NADP-ME

2.1.2.4 Eleocharoid: Biochemical Subtype NAD-ME

2.1.3 3. Dicotyledons

2.1.3.1 Atriplicoid: Biochemical Subtypes NAD-ME and NADP-ME

2.1.3.2 Kochioid: Biochemical Subtypes NAD-ME and NADP-ME

2.1.3.3 Salsoloid: Biochemical Subtypes NAD-ME and NADP-ME

2.1.3.4 Salsinoid: Biochemical Subtype NAD-ME

2.1.3.5 Schoberioid: Biochemical Subtype NAD-ME

2.1.3.6 Kranz-Tecticornioid: Biochemical Subtype NAD-ME

2.1.3.7 Pilosoid: Biochemical Subtype NADP-ME

2.1.3.8 Portulacelloid: Biochemical Subtype NADP-ME

2.1.3.9 Glossocardioid: Biochemical Subtype NAD-ME and NADP-ME

2.1.3.10 Simplicifolioid: Biochemical Subtype NADP-ME

2.1.3.11 Isostigmoid: Biochemical Subtype unknown

2.1.3.12 Angustifolioid: Biochemical Subtype NAD-ME

2.2 B. Biochemical Diversity: C4 Cycles and Energy Requirements for C4 Subtypes

2.2.1 1. Chloroplasts and Mitochondria

2.2.2 2. Illustration of Energetics for NADP-ME Type Species

2.2.3 3. Illustration of Energetics for NAD-ME Type Species

2.2.4 4. Illustration of Energetics for PEP-CK Type Species

2.2.5 5. Additional Energy Requirements in C4 Photosynthesis

3 III. Single-Cell C4 Photosynthesis in Terrestrial Plants

3.1 A. Occurrence (Family and Phylogeny)

3.2 B. Biogeography of Single-Cell C4 Species

3.3 C. Overview of Two Types of Single-CellC4 Photosynthesis in Terrestrial Plants

3.4 D. Biochemical Evidence for Function of C4 Photosynthesis in Single-Cell C4 Plants

3.4.1 1. General Features Characteristic of C4

3.4.1.1 Western Blots and Analysis of C4 Enzymes

3.4.1.2 C4 Type Carbon Isotope Composition

3.4.1.3 Physiological Response

3.4.1.4 Resistance to CO2 Loss

3.4.2 2. Spatial Compartmentation Enabling Function of NAD-ME Type C4 Photosynthesis

3.4.2.1 Dimorphic Chloroplasts (Structure, Enzymes and Starch)

3.4.2.2 Mitochondria and Peroxisomes

3.5 E. Development of Spatial Compartmentation and Dimorphic Chloroplasts

3.6 F. Form of Photosynthesis in Different Photosynthetic Organs in Single-Cell C4 Species

3.7 G. How Did Single-Cell C4 Evolve?

4 IV. Future Perspectives

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Chapter 4 C₄ Photosynthesis: Kranz Forms and Single-Cell C₄ in Terrestrial Plants

Coleataenia prionitis, a C₄-like species in the Poaceae

Transition from C₃ to proto-Kranz to C₃–C₄ intermediate type in the genus Chenopodium (Chenopodiaceae)

1.1 A. What Does It Take to Be C₄?

1.2 B. Occurrence of C₄ Among Terrestrial Plants

2.2 B. Biochemical Diversity: C₄ Cycles and Energy Requirements for C₄ Subtypes

2.2.5 5. Additional Energy Requirements in C₄ Photosynthesis

3 III. Single-Cell C₄ Photosynthesis in Terrestrial Plants

3.2 B. Biogeography of Single-Cell C₄ Species

3.3 C. Overview of Two Types of Single-CellC₄ Photosynthesis in Terrestrial Plants

3.4 D. Biochemical Evidence for Function of C₄ Photosynthesis in Single-Cell C₄ Plants

3.4.1 1. General Features Characteristic of C₄

3.4.1.1 Western Blots and Analysis of C₄ Enzymes

3.4.1.2 C₄ Type Carbon Isotope Composition

3.4.1.4 Resistance to CO₂ Loss

3.4.2 2. Spatial Compartmentation Enabling Function of NAD-ME Type C₄ Photosynthesis

3.6 F. Form of Photosynthesis in Different Photosynthetic Organs in Single-Cell C₄ Species

3.7 G. How Did Single-Cell C₄ Evolve?