Recent progress in the single-cell C₄ photosynthesis in terrestrial plants

Abstract

Currently, single-cell C₄ photosynthesis has been reported in four terrestrial plant species, Bienertia cycloptera, B. sinuspersici, B. kavirense and Suaeda aralocaspica, of family Chenopodiaceae. These species possess novel mechanisms of C₄ photosynthesis through spatial partitioning of organelles and key enzymes in distinct cytoplasmic domains within single chlorenchyma cells. Anatomical and biochemical studies have shown that the three Bienertia species and S. aralocaspica utilize biochemical and organellar compartmentation to achieve the equivalent spatial separation of Kranz anatomy but within a single photosynthetic cell. These discoveries have challenged the paradigm for C₄ photosynthesis in terrestrial plants which had suggested for more than 40 years that the Kranz feature was indispensably required for its C₄ function. In this review, we focus on the recent progress in understanding the cellular and molecular mechanisms that control the spatial relationship of organelles in these unique single-cell C₄ systems. The demonstrated interaction of dimorphic chloroplasts with microtubules and actin filaments has shed light on the importance of these cytoskeleton components in the intracellular partitioning of organelles. Future perspectives on the potential function of the cytoskeleton in targeting gene products to specific subcellular compartments are discussed.

Keywords

C₄ plants / single-cell C₄ photosynthesis / Chenopodiaceae / dimorphic chloroplasts / organelle compartmentation / photosynthetic enzymes / cytoskeleton / protein targeting

Introduction

Photosynthetic organisms assimilate atmospheric CO₂ into organic matter that is essential to all forms of life. Three modes of photosynthesis, C₃, C₄ and crassulacean acid metabolism (CAM), have been identified in terrestrial plants, and each is associated with distinct features of biochemistry, physiology and leaf anatomy (Edwards and Walker, 1983; Winter and Smith, 1996). For example, CAM plants convert atmospheric carbon into organic acids at night through the activity of phosphoenol pyruvate carboxylase (PEPC), and the C₄ product is temporarily stored in large vacuoles in mesophyll cells for later use in the Calvin-Benson cycle during the day (Fig. 1C). These plants are succulent in their anatomy and all the photosynthetically active cells are of similar appearance and biochemical capacity with respect to carbon assimilation. On the other hand, the majority of plants perform C₃ photosynthesis where atmospheric CO₂ is assimilated directly in mesophyll cells by the key enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) into two molecules of C₃ compounds, which are then utilized for the synthesis of other products such as sucrose and starch (Fig. 1A). However, Rubisco is a bifunctional enzyme which catalyzes both carboxylation and oxygenation reactions of the same substrate, ribulose-1,5-bisphosphate (RuBP). Reaction of RuBP with CO₂ results in carbon gain whereas reaction with O₂ results in carbon loss due to a wasteful process known as photorespiration. Thus, C₃ plants generally do not perform well under extreme conditions such as drought, salinity and/or high temperature, where CO₂ becomes a limiting factor for photosynthesis (Hatch and Slack, 1970; Sage, 1999).

Contrary to the C₃ species, some plants evolved a mechanism via the C₄ dicarboxylic acid cycle for concentrating CO₂ and subsequently donating it to the C₃ pathway or Calvin-Benson cycle. These C₄ species have been identified in 19 of ca. 500 families of plants including major agronomic crops such as maize, sugarcane, millets and sorghum (Sage, 2003). Terrestrial C₄ plant species generally exhibit the Kranz-type leaf anatomy consisting of two anatomically and biochemically distinct photosynthetic cell types, the mesophyll and bundle sheath cells, which function cooperatively to concentrate CO₂ at the site of Rubisco and thereby reduce photorespiration (Hatch and Slack, 1970; Edwards and Huber, 1981). In C₄ photosynthesis, atmospheric CO₂ is initially fixed into C₄ acids by PEPC in the mesophyll cells. The C₄ acids are then transported to the bundle sheath cells for the release of CO₂ by C₄ acid decarboxylases [i.e. NAD-/NADP-malic enzyme (ME) or PEP-carboxykinase (CK)] to Rubisco, the key enzyme of the Calvin-Benson cycle (Fig. 1B). The separation of different sets of biochemical reactions among the two cell types allows C₄ plants to outperform C₃ plants under CO₂-limiting conditions, as a result of higher rates of photosynthesis, and greater nitrogen and water use efficiency (Hatch and Slack, 1970; Edwards and Huber, 1981; Sage, 2003). In a Kranz-type C₄ leaf, the mesophyll cells contain two signature enzymes, PEPC for initial CO₂ fixation and pyruvate orthophosphate dikinase (PPDK) for regeneration of the PEPC substrate [i.e. phosphoenolpyruvate (PEP)], while the adjacent bundle sheath cells produce the signature decarboxylases, NAD-/NADP-ME (or PEP-CK in some species), for the liberation of CO₂ from C₄ acids and its refixation via Rubisco of the Calvin-Benson cycle (Fig. 1B).

Discovery of single-cell C₄ photosynthesis in terrestrial plants

For over 40 years, it had been generally accepted that Kranz anatomy was a required feature for C₄ photosynthesis. This paradigm has been challenged with the recent discovery that four members of the Chenopodiaceae family perform C₄ photosynthesis within individual chlorenchyma cells. What has been referred to as “single-cell C₄ photosynthesis” has been found in Bienertia cycloptera (Voznesenskaya et al., 2002, 2005), B. sinuspersici (Akhani et al., 2005; Fig. 2A-C), B. kavirense (Akhani et al., 2012), and Suaeda aralocaspica (previously called Borszczowia aralocaspica;Freitag and Stichler, 2000; Voznesenskaya et al., 2001; Fig. 2D-F). The single-cell C₄ species are annual plants that are distributed in central Asia (S. aralocaspica) and salt deserts in countries surrounding the Persian Gulf (B. cycloptera; B. sinuspersici; B. kavirense). These species exhibit novel C₄ photosynthetic mechanisms through temporal expression of enzymes and spatial partitioning of organelles into two distinct intracellular compartments within individual photosynthetic cells (Fig. 2B, E). As demonstrated by biochemical characterization of the enzymes, carbon isotope discrimination analysis and measurement of leaf gas exchange parameters, these species perform full C₄ photosynthesis in nature or under normal growth conditions. Using immunoelectron microscopy, it has been determined that this remarkable feature of C₄ photosynthesis in a single chlorenchyma cell is accomplished by partitioning of two biochemically and ultrastructurally distinct types of chloroplasts (i.e. dimorphic chloroplasts) in separate cytoplasmic compartments within the cell (Voznesenskaya et al., 2001, 2002, 2005; Chuong et al., 2006). The chlorenchyma cells of the Bienertia species consist of a peripheral cytoplasmic compartment (PCC) with chloroplasts containing PPDK but not Rubisco, and a central cytoplasmic compartment (CCC) with Rubisco-containing chloroplasts surrounding a large number of mitochondria containing NAD-ME (Voznesenskaya et al., 2002; Chuong et al., 2006). The two compartments are connected by thin cytoplasmic channels, which allow for the exchange of metabolic products between the two compartments (Fig. 2C). In an analogy to Kranz anatomy, the end result is equivalent to having a bundle sheath cell in the middle of a mesophyll cell with transvacuolar cytoplasmic channels connecting them.

Surprisingly, S. aralocaspica possesses a different mode of C₄ photosynthesis in a single cell. This species comprises of elongated palisade chlorenchyma cells, in which Rubisco-containing chloroplasts and NAD-ME-containing mitochondria are concentrated at the proximal end of the cell closer to the vascular tissues, and PPDK-containing chloroplasts are uniformly distributed at the distal end of the cell away from the vascular tissues (Voznesenskaya et al., 2001; Chuong et al., 2006; Fig. 2F). This is somewhat analogous to having the Kranz anatomy of the mesophyll and bundle sheath arrangement without the intervening cell walls. However, developmental studies demonstrated that this unique organelle partitioning was not evolved from a typical Kranz arrangement and subsequent cell wall degradation (Voznesenskaya et al., 2003). Rather, distribution of organelles at opposite ends of the chlorenchyma cell and differential expression of enzymes in the organelles at the two regions appear to occur during maturation of the S. aralocaspica chlorenchyma cells. In both single-cell C₄ systems, the partitioning of biochemically distinct organelles into discrete compartments results in higher CO₂ concentration around the Rubisco-containing chloroplasts, which in turn inhibits the oxygenase activity of Rubisco and photorespiration, as commonly occurs in the typical Kranz system. Thus, the partitioning of biochemistry and organelles in the single-cell C₄ systems achieves the equivalent of cellular separation of Kranz anatomy but within a single cell, as illustrated in the proposed models of single-cell C₄ photosynthesis (Edwards et al., 2004; Edwards et al., 2008; Fig. 3).

Although the anatomy and biochemistry of these single-cell C₄ systems are slowly emerging, our overall understanding of their development is still rudimentary. The organization of organelles and biochemistry in these species represents a unique and novel system in higher plants. Furthermore, the peculiar structure and organization of these chlorenchyma cells raise questions about the developmental sequence of the mature chlorenchyma cells, about the cellular and molecular factors controlling the formation of the two intracellular compartments as a result of organelle partitioning, and about the differentiation of the dimorphic chloroplasts involving communication between the nucleus and plastids. As an initial step in further characterizing the development of the single-cell C₄ photosynthesis, anatomical and biochemical experiments were performed to examine the timing and pattern of organelle development and major photosynthesis enzyme expression in both Bienertia species (Voznesenskaya et al., 2005; Chuong et al., 2006; Lara et al., 2008; Park et al., 2009). It was shown that there is one common pool of plastids that partition into different subcellular compartments and then alter their biochemical expression (Voznesenskaya et al., 2005). We have also found that major organelles such as chloroplasts, mitochondria and peroxisomes of the two different genera show strict partitioning into distinct compartments, a feature essential for the function of the single-cell C₄ systems (Chuong et al., 2006; Lung et al., 2011; Fig. 4). This cellular organization appears to be developmentally programmed, as chlorenchyma cells of cotyledons and dark grown leaves also showed the partitioning of organelles (unpublished data). In developing leaves of Bienertia, the accumulation of various proteins and transcripts was determined under different light conditions by using immunoblot and real-time PCR analyses (Lara et al., 2008). It was demonstrated that a substantial amount of Rubisco transcripts accumulates in the early stage of leaf development whereas PEPC and PPDK levels remain low until the late stage. Because the accumulation of transcripts mostly correlates to that of their proteins, it has been proposed that transcriptional control is involved in the developmental regulation of photosynthesis. Overall, these results suggest that the differentiation and compartmentation of organelles and photosynthetic enzymes are a tightly regulated and coordinated process. Recently, considerable progress has been made in the investigation of the development of the single-cell C₄ system by developing several molecular tools for analyzing gene expression. For example, procedures for isolating Bienertia protoplasts with intact cytoplasmic compartments have been developed and optimized allowing transient gene expression experiments to be performed in single-cell C₄ plants (Offermann et al., 2011; Lung et al., 2011; Fig. 4). To better understand the molecular basis of the single-cell C₄ system, transcriptomic and proteomic approaches were also initiated to obtain massive gene and protein profile in isolated chlorenchyma cells (Park et al., 2010). These accomplishments represent initial efforts to better understand the complex cellular organization of the single-cell C₄ species and will serve as an important tool for molecular studies examining gene expression and for the isolation of intact dimorphic chloroplasts that are essential for chloroplast preprotein import assays (Lung et al., 2012).

A role for the cytoskeleton in maintaining organelle compartmentation and possibly targeting of proteins to various subcellular compartments

Previous biochemical, anatomical and molecular studies have demonstrated that the compartmentation of major organelles such as chloroplasts, mitochondria and peroxisomes in chlorenchyma cells of B. sinuspersici and S. aralocaspica is essential for the function of the single-cell C₄ systems (Voznesenskaya et al., 2001, 2002; Chuong et al., 2006; Lara et al., 2008). Immunofluorescence microscopy has further revealed the role of the components of the cytoskeleton in the maintenance of the organelle partitioning in the Bienertia species and S. aralocaspica (Chuong et al., 2006; Park et al., 2009). It has been shown that the spatial distribution of the dimorphic chloroplasts to the CCC of Bienertia involved microtubules as its disruption resulted in the dispersal of chloroplasts (Chuong et al., 2006; Park et al., 2009). Previously, microtubules were also demonstrated to play a role in the anchorage of non-green plastids, the establishment of stromules in the hypocotyls of etiolated tobacco (Kwok and Hanson, 2003), as well as the positioning of mitochondria in the cortical cytoplasm (Van Gestel et al., 2002; Logan et al., 2003). On the other hand, the partitioning of organelles in S. aralocaspica appears to involve both actin microfilaments and microtubules since disruption of its subcellular organization was only observed in the presence of dissociating agents for both cytoskeleton components (Chuong et al. 2006). In fact, it has become apparent that actin microfilaments and microtubules interact during many developmental processes, implying a functional purpose rather than a coincidental occurrence (Chuong et al., 2005; Collings et al., 2006; Petrásek and Schwarzerová,2009; Sampathkumar et al., 2011). This is consistent with studies demonstrating that the photo-movement and positioning of chloroplasts in plant cells are mediated by actin microfilaments and/or microtubules (Sato et al., 2001; Wada et al., 2003; Wada and Suetsugu, 2004). Thus, it is believed that the maintenance of organellar compartmentation in the single-cell C₄ species involves interactions between the dimorphic chloroplasts, with both actin microfilaments and microtubules.

Our observations of direct interaction of dimorphic chloroplasts with actin microfilaments implicate a role for actin in chloroplast movement (Fig. 4A), whereas interactions of the chloroplasts with microtubules may imply anchorage function. In Arabidopsis, actin microfilaments form baskets surrounding the chloroplasts (Kandasamy and Meagher, 1999). In the single-cell C₄ species, microtubules might also participate in the orderly partitioning of chloroplasts. Immunolabeling of the cytoskeleton and transient expression of fluorescent fusion proteins in protoplasts consistently revealed that, in addition to the normal cortical transverse arrangement of microtubules in the chlorenchyma cells, a portion of microtubules encircle the dimorphic chloroplasts (Chuong et al., 2006; Lung et al. 2011; Fig. 4B). Collectively, our recent data suggest that components of the cytoskeleton might mediate and maintain the organelle positioning within distinct cytoplasmic compartments in the remarkable single-cell C₄ systems.

Considering that the spatial partitioning of organelles, enzymes and metabolites is essential for C₄ photosynthesis, the structural and biochemical regulation in Bienertia and S. aralocaspica chlorenchyma cells must be complex as they are happening in the same cell. For instance, the control of organelle movement must operate under a precise and stable cellular process to establish cellular polarity. While earlier studies have indicated that the cytoskeleton, particularly microtubules, plays a crucial role in the spatial compartmentation of organelles (Chuong et al., 2006; Park et al., 2009), little is known about the compartmentation of single-cell C₄ machineries at the biochemical level. To this end, the precise distribution of organelle-specific enzymes must take place for proper functioning of the C₄ cycle. This requirement, however, implies that nuclear-encoded proteins must be specifically targeted to the correct subcellular compartment. Most chloroplast proteins are nuclear-encoded and translated in the cytosol as precursors with targeting sequences (Chen and Schnell, 1999). These preproteins are recognized and imported into the chloroplast by sophisticated proteinaceous apparatus located at the outer and inner membranes (the Tic/Toc complex; Translocon at the Inner/Outer envelope membrane of Chloroplasts) (Hirsch et al., 1994; Schnell et al., 1994). The core of the Toc complex consists of three proteins: Toc75, Toc159 and Toc34. Toc75 forms the protein translocation channel, whereas Toc159 and Toc34 are GTPase receptors for preprotein recognition and regulation of the import events. In Arabidopsis, four Toc159 homologs (i.e. atToc159, atToc132, atToc120 and atToc90) and two Toc34 homologs (i.e. atToc33 and atToc34) have been identified (Kubis et al., 2004). In vivo and biochemical data indicated that the substrate selectivity of the Toc complex is governed by different combinations of the Toc159 and Toc34 isoforms, such that atToc159 preferentially interact with atToc33 for the targeting and import of photosynthetic preproteins, whereas the Toc complex comprising of atToc120/132 and atToc34 is responsible for non-photosynthetic preproteins (Ivanova et al., 2004; Smith et al., 2004). Thus, the protein import function of the Toc complexes is essential for plastid biogenesis and differentiation.

In the single-cell C₄ system, the expression of C₄ genes is under the control of a single nucleus. Thus, during the different stages of leaf development the targeting of nuclear-encoded proteins destined for the dimorphic chloroplasts might involve a selective process to facilitate their proper development and intracellular compartmentation in order to achieve a functional C₄ pathway. As an initial step to understand the molecular mechanism regulating the differential accumulation of key photosynthetic enzymes in dimorphic chloroplasts, we have recently identified the core components of the Toc complex from the single-cell C₄ species (Lung and Chuong, 2012). In B. sinuspersici, the expression profiles of individual Toc159 isoforms appear to correlate with the development and differentiation of the dimorphic chloroplasts. For example, Toc132 was more abundant in meristemic tissues where cells have undifferentiated proplastids, whereas high levels of Toc159 were observed in tissues containing photosynthetically active chloroplasts (Lung and Chuong, 2012). Moreover, immunoelectron microscopic studies using isoform-specific antisera revealed cytosolic and chloroplast envelope-associated localization of both Toc159 and Toc132, suggesting the possibility that these receptors can cycle between different compartments (Lung and Chuong, 2012). The immunolocalization results were further supported by fluorescent protein fusion studies in B. sinuspersici protoplasts as well as by immunoblot analyses of the Toc159 receptors in subcellular fractions (Lung and Chuong, 2012). Collectively, these results suggest that the Toc159 receptors are dynamic and their presence in various subcellular compartments may effectively facilitate their function in preprotein recognition and import. The notion that the Toc159 receptors are able to shuttle between the cytoplasm and chloroplast outer envelope has been previously proposed (Hiltbrunner et al., 2001; Ivanova et al. 2004; Smith, 2006). Further bioinformatics analyses of the Toc159 receptors of the single-cell C₄ system identified a novel transit peptide-like signal at the C terminus (Lung and Chuong, 2012). The function of this sorting signal to target proteins to the outer chloroplast envelope was confirmed using fluorescent fusion proteins in living protoplasts (Lung and Chuong, 2012). Despite the complexity of the subcellular anatomy and the differentiation of dual chloroplast types, our recent success in identifying the active core Toc components indicated that the general plastid protein import model also applies to the unique single-cell C₄ system. The presence of multiple isoforms of the Toc receptors supports the hypothesis that distinct Toc complexes present on each chloroplast type may be responsible for the differential import of proteins into dimorphic chloroplasts leading to their differentiation and establishment of the C₄ pathway in a single cell. With the advent of a novel technique for dimorphic chloroplast isolation (Lung et al., 2012), research is ongoing to test the differential targeting hypothesis using in vitro protein import assays.

The intracellular trafficking of macromolecules must involve the service of one or both components of the cytoskeleton in order to orchestrate their precise distribution. Since the operation of the single-cell C₄ systems requires strict partitioning of organelles and enzymes in distinct subcellular compartments, it is possible that proteins and/or mRNAs must be selectively targeted to the destined region of the cells to ensure the overall functional efficiency of the system. In addition to its role in maintaining the spatial separation of organelles and assisting their movement, it is possible that the cytoskeleton plays a role in the targeting of macromolecules to the proper subcellular compartments for specific cellular function. The involvement of the cytoskeleton in mRNA localization has been well documented in yeast, mammalian and plant cells (Bashirullah et al., 1998; Jansen, 2001; Crofts et al., 2005). The association of mRNAs with the cytoskeleton is important in concentrating the synthesis of proteins close to their site of function. Similarly, the cytoskeleton may participate in the intracellular protein trafficking systems to ensure that proteins are properly delivered to various subcellular compartments. In fact, a recent study reported the interaction between actin microfilaments and the Toc159 receptor (Jouhet and Gray, 2009). However, the role of the cytoskeleton in protein import into chloroplasts remains undetermined. Thus, future studies will focus on identifying and characterizing the molecular factors involved in the intracellular targeting of proteins in the single-cell C₄ systems, with an emphasis on the role of the cytoskeleton components in the process.

Conclusion and future directions

Bienertia and S. aralocaspica constitute an attractive biologic system to examine the molecular and biochemical modifications that underlie the shift from Kranz-type C₄ photosynthesis to single-cell C₄ type. These discoveries have led to a re-examination of the requirements for C₄ photosynthesis and raised questions about evolution of the Kranz-type C₄ system. In addition, they provide excellent examples of the ability of plant cells to establish and maintain biochemically complex compartments based on both partitioning of organelles and differential expression/targeting of gene products within the same cell. The single-cell C₄ systems represent the most complex examples of higher plant cells with respect to polar distribution of enzymes and organelles within a single cell. To further our understanding of these systems, research should be carried out to elucidate the nature of the cellular and molecular signals that trigger the onset of organelle movement leading to the establishment of two intracellular compartments within individual cells, since loss of this partitioning would probably prevent the C₄ cycle to function properly. Current and future studies will focus on providing new information about two key fundamental processes in plants: cellular organization leading to functional domains and photosynthetic carbon assimilation. Understanding the cellular and molecular mechanisms regulating the high-level of photosynthetic gene expression may potentially allow for the introduction of C₄-like characteristics into agriculturally important C₃ crops to increase productivity. This could be particularly important to confer a growth advantage to plants with C₄ or C₄-like features under current and changing climate conditions where increased water stress and reduced CO₂ solubility due to extreme temperatures and salinity.

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