Journal of Experimental Botany, Vol. 52, No. 356, pp. 577-590,
April 2001
© 2001 Oxford University Press
Compartmentation of photosynthesis in cells and tissues of C4 plants
1 School of Biological Sciences, Washington State University, Pullman, WA 99164-4236, USA
2 Department of Anatomy and Morphology, V. L. Komarov Botanical Institute of Russian Academy of Sciences, Prof. Popov Street 2, 197376 St Petersburg, Russia
3 Department of Plant Physiology, Ural State University, Lenin Avenue 51, 620083 Ekaterinburg, Russia
4 Centro de Estudios Fotosintéticos y Bioquímicos, (CEFOBI), Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina
Received 31 March 2000; Accepted 24 November 2000
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Abstract |
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 Top Abstract C4 pathway of photosynthesisInitial studies on the...Mechanical isolation of...Enzymatic isolation of mesophyll...In situ methods for...Distribution of C4-specific mRNAImmunolocalization of C4...Cell- and organ-specific...SummaryReferences |
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Critical to defining photosynthesis in C4 plants is understanding the intercellular and intracellular compartmentation of enzymes between mesophyll and bundle sheath cells in the leaf. This includes enzymes of the C4 cycle (including three subtypes), the C3 pathway and photorespiration. The current state of knowledge of this compartmentation is a consequence of the development and application of different techniques over the past three decades. Initial studies led to some alternative hypotheses on the mechanism of C4 photosynthesis, and some controversy over the compartmentation of enzymes. The development of methods for separating mesophyll and bundle sheath cells provided convincing evidence on intercellular compartmentation of the key components of the C4 pathway. Studies on the intracellular compartmentation of enzymes between organelles and the cytosol were facilitated by the isolation of mesophyll and bundle sheath protoplasts, which can be fractionated gently while maintaining organelle integrity. Now, the ability to determine localization of photosynthetic enzymes conclusively, through in situ immunolocalization by confocal light microscopy and transmission electron microscopy, is providing further insight into the mechanism of C4 photosynthesis and its evolution. Currently, immunological, ultrastructural and cytochemical studies are revealing relationships between anatomical arrangements and photosynthetic mechanisms which are probably related to environmental factors associated with evolution of these plants. This includes interesting variations in the C4 syndrome in leaves and cotyledons of species in the tribe Salsoleae of the family Chenopodiaceae, in relation to evolution and ecology. Thus, analysis of structure–function relationships using modern techniques is a very powerful approach to understanding evolution and regulation of the photosynthetic carbon reduction mechanisms.
Key words: Anatomy, C4 plants, chloroplasts, gene expression, immunolocalization, photosynthetic enzymes, ultrastructure.
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C4 pathway of photosynthesis |
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TopAbstractC4 pathway of photosynthesisInitial studies on the...Mechanical isolation of...Enzymatic isolation of mesophyll...In situ methods for...Distribution of C4-specific mRNAImmunolocalization of C4...Cell- and organ-specific...SummaryReferences |
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In the 1960s, it was recognized that some plants have a unique pathway of assimilating atmospheric CO2 (see the historical account in Hatch, 1999
, including the early work of Karpilov, Kortschack et al., and Hatch and Slack). As what is now termed the C4 pathway was being identified in various species, it was also recognized that it was associated with a special leaf anatomy. Large, distinctive bundle sheath cells, with prominent chloroplasts, surround the vascular tissue, which, in turn, are surrounded by a layer of chloroplast-containing mesophyll cells; more than a century ago Haberlandt called this Kranz anatomy (Edwards and Walker, 1983).
Once the association between Kranz anatomy and the fixation of atmospheric CO2 into C4 acids was made, there were immediate questions about the biochemical mechanism of carbon assimilation and the role of the two photosynthetic cell types. It became clear that C4 plants have very high levels of phosphoenolpyruvate carboxylase (PEPC) compared to C3 plants, where ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco) is the primary carboxylase, which results in the fixation of atmospheric CO2 via the C3 pathway. Subsequently, three C4 acid decarboxylases were identified: NADP-malic enzyme (NADP-ME), NAD-malic enzyme (NAD-ME), and phosphoenolpyruvate carboxykinase (PEP-CK), in that order, which were key to developing an understanding of the C4 mechanism (i.e. C4 plants fix atmospheric CO2 into C4 acids via the C4 cycle, C4 acids are decarboxylated and the CO2 donated to Rubisco of the C3 pathway) (see the historical summary by Hatch, 1999). Since the 1960s, there have been parallel studies on the biochemistry of C4 photosynthesis and the compartmentation of metabolic processes of carbon assimilation between mesophyll and bundle sheath cells. These include enzymology of the C4 cycle, the C3 pathway of photosynthesis and photorespiration. More recently, research on the C4 mechanism has been focused on the molecular evolution of C4-specific genes and their differential expression among various organs and between the two photosynthetic cell types (Ku et al., 1996; Sheen, 1999). This paper discusses the development and application of techniques for studying compartmentation in C4 photosynthesis which have been critical in elucidating this metabolic process, and which continue to play a key role in advancing current understanding of the evolution and diversity of the C4 syndrome in plants.
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Initial studies on the compartmentation of key enzymes, metabolites and understanding of C4 photosynthesis |
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TopAbstractC4 pathway of photosynthesisInitial studies on the...Mechanical isolation of...Enzymatic isolation of mesophyll...In situ methods for...Distribution of C4-specific mRNAImmunolocalization of C4...Cell- and organ-specific...SummaryReferences |
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When Hatch and Slack found, by 14CO2–12CO2 pulse–chase experiments, the initial incorporation of label into the C4-carboxyl carbon of oxaloacetate, malate and aspartate, followed by labelling in the C-1 carboxyl of phosphoglyceric acid (PGA) and then hexose-P in sugarcane, they developed a working model for photosynthesis in C4 plants (Hatch and Slack, 1966
). The model included a ‘C4 cycle’, with fixation of atmospheric CO2 by pyruvate or phosphoenolpyruvate (PEP) to generate a C4 acid, transfer of the C4-carboxyl to an acceptor to form the C-1 carboxyl of 3-phosphoglyceric acid (PGA), and then use of the 3-carbon product as the substrate for fixing another CO2. In this initial model of C4 photosynthesis, a transcarboxylase reaction was suggested (direct transfer of carbon from a C4 acid to an acceptor to form PGA). It was logical not to postulate C4 acid decarboxylation, CO2 release and refixation by Rubisco, since in initial studies Rubisco activities from whole leaf extracts were relatively low, and the efficiency by which a C4 acid decarboxylase and refixation of CO2 by Rubisco could function was questionable (exposure of leaves to 5% 12CO2 during the chase gave a similar labelling pattern). At that time, the relationship between Kranz anatomy and the biochemical pathway had not been established, and the mechanism of photorespiration via ribulose 1,5-bisphosphate (RuBP) oxygenase in C3 plants under limiting CO2 had not been discovered. After the connection was made between plants fixing atmospheric CO2 into C4 acids and Kranz anatomy, it was obviously important to determine the function of the two chlorenchyma cell types, mesophyll and bundle sheath cells. Already, a number of hypotheses had been put forth about the mechanism of photosynthesis in plants having Kranz type anatomy, prior to any knowledge of the biochemistry involved. Even Haberlandt (Haberlandt, 1896), in his descriptions of plants with Kranz anatomy in the late 1800s, suggested there might be some co-operative function of the two cell types in photosynthesis.
From the 1940s to the early 1960s, there was speculation that the mesophyll cells may assimilate atmospheric CO2 and that the bundle sheath chloroplasts may only function like amyloplasts to store starch, or possibly to fix CO2 generated by respiration in vascular tissue (see Rhoades and Carvalho, 1944, and other references in Slack, 1969).
Subsequent to their 14CO2–12CO2 pulse-chase isotope results, from 1967–1969 Hatch and Slack searched for enzymes which might be involved in C4 photosynthesis and found key enzymes, including PEPC, NADP-ME, pyruvate,Pi dikinase (PPDK), and Rubisco (the latter at low levels), along with evidence for other enzymes of the C3 cycle. Recognition that sugarcane and other plants with this pattern of photosynthesis have two types of chlorenchyma cells (mesophyll and bundle sheath), led to studies on compartmentation of the enzymes.
The earliest applications of techniques to study cell-specific compartmentation of enzymes in C4 plants were the non-aqueous fractionation methods used by Slack (Slack, 1969; Slack et al., 1969) and differential grinding (Björkman and Gauhl, 1969). Application of non-aqueous density fractionation (Slack et al., 1969; Slack, 1969) provided important insight into the compartmentation of some of the key enzymes of photosynthesis between mesophyll and bundle sheath chloroplasts of maize and amaranth. Using freeze-dried, macerated leaf tissue, a step-wise fractionation was made in different densities of hexane-carbon tetrachloride (1.30, 1.33, 1.36, and 1.40 g ml-1). The procedure employed gave pelleted samples having densities of <1.30, 1.30–1.33, 1.33–1.36, 1.36–1.40, and >1.40 g ml-1. Earlier work with the non-aqueous technique (Stocking, 1959) had shown that de-starched chloroplasts of tobacco (C3) are lighter than most other cell constituents. Slack et al. showed that the low density fraction,<1.30 g ml-1, was rich in the starchless, grana-containing mesophyll chloroplasts, while the high density fractions, 1.36–1.40 and >1.40 g ml-1, were rich in the agranal, starch-containing bundle sheath chloroplasts (Slack et al., 1969). It was concluded that PPDK, NADP-malate dehydrogenase (NADP-MDH) and glycerate kinase occur in mesophyll chloroplasts and that Rubisco and NADP-ME occur in bundle sheath chloroplasts. NADP-triose-P dehydrogenase and PGA-kinase were found in both mesophyll and bundle sheath chloroplast fractions. The non-aqueous method gave variable results with PEPC, which, in some preparations, was more associated with the lower density mesophyll chloroplast fraction, while in other preparations it appeared at high densities. Although it was suggested that the enzyme may be associated with the bounding membrane of mesophyll chloroplasts, its compartmentation (cell type and intracellular localization) was not clear. Subsequently, it became clear that this enzyme occurs in the cytosol of mesophyll cells, based on the fractionation of mesophyll protoplasts and in situ immunolocalization studies (Gutierrez et al., 1974b; Voznesenskaya et al., 1999; as discussed later). The reasons for the variation with the non-aqueous technique, and whether the enzyme may have some association with the outer chloroplast envelope in situ, are unknown.
Slack et al. also fed 14CO2 to maize leaves for 25 s, and then analysed the distribution of metabolites with the non-aqueous density fractionation (Slack et al., 1969). The major labelling of malate, aspartate, and PGA occurred in the mesophyll chloroplast fraction while the major labelling of fructose phosphates and ribulose phosphates was in the bundle sheath chloroplast fraction. At this time, two alternative hypotheses on C4 photosynthesis were proposed: (1) mesophyll chloroplasts fix atmospheric CO2 through a C4 acid cycle and a transcarboxylase reaction (e.g. with glycerate as a product in the latter reaction, and its conversion to 3-PGA via glycerate kinase, which would be consistent with labelling of PGA in the mesophyll chloroplast), and bundle sheath chloroplasts fix mainly respired CO2 through RuBP carboxylase, or (2) mesophyll chloroplasts fix atmospheric CO2 via PEPC into C4 acids, and C4 acids donate CO2 to Rubisco in bundle sheath cells via NADP-ME.
There are limitations with the non-aqueous method which can complicate interpretations of the results: it does not give a complete separation of any one cellular compartment into one density fraction, adsorption of cytosolic material to organelles may influence their density, and the yield of bundle sheath chloroplasts may be low, in part by loss of starch from bundle sheath chloroplasts, resulting in their partitioning to a lighter fraction. For a review of the non-aqueous method see Stitt et al. (Stitt et al., 1989), and for its use in studying the distribution of metabolites between the chloroplasts and extracellular compartments in maize mesophyll and bundle sheath cells, see Weiner and Heldt (Weiner and Heldt, 1992). In current applications of the non-aqueous technique, tetrachlorethylene instead of carbon tetrachloride is used, due to the higher toxicity of the latter, and an iterative mathematical approach can be used more accurately to calculate the distribution between compartments (Moore et al., 1997).
Other differential separation techniques were used early on which added to the information provided by the non-aqueous separation techniques. Björkman and Gauhl showed that C4 plants have substantial Rubisco activity, suggesting its function in photosynthesis in these species (Björkman and Gauhl, 1969). They also employed a sequential grinding procedure with Atriplex rosea (C4). Using increasing force to break bundle sheath cells, they found PEPC was high in the initial extract and low in the terminal extract, while Rubisco was three times higher in the terminal extract. On this basis, they suggested PEPC was predominantly in mesophyll cells and that Rubisco was highest in bundle sheath cells. Adapting this differential grinding technique to maize and Gomphrena globosa (C4), Berry et al. suggested NADP-ME as well as Rubisco occurs in bundle sheath cells and that PEPC occurs in mesophyll cells (Berry et al., 1970). The substantial activities of Rubisco and NADP-ME in the initial extract (30–40% of Rubisco on a soluble protein basis) left it uncertain whether the C3 pathway also functioned in mesophyll cells. These approaches were limited by the lack of strict isolation of individual mesophyll or bundle sheath cells.
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Mechanical isolation of mesophyll cells and bundle sheath cells/strands |
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TopAbstractC4 pathway of photosynthesisInitial studies on the...Mechanical isolation of...Enzymatic isolation of mesophyll...In situ methods for...Distribution of C4-specific mRNAImmunolocalization of C4...Cell- and organ-specific...SummaryReferences |
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Separation of intact mesophyll and bundle sheath cells of a C4 plant provided a direct means of studying intercellular compartmentation of enzymes between both cell types and testing their functions in photosynthetic metabolism (Edwards et al., 1970
; Edwards and Black, 1971). A procedure combining gentle grinding in a mortar and filtration through a series of nylon mesh filters with precise porosity, allowed for rapid isolation and purification (c. 30 min) of individual mesophyll and bundle sheath cells (the latter from bundle sheath strands) (Edwards et al., 1970; Edwards and Black, 1971). This was first accomplished with the C4 monocot Digitaria sanguinalis, an NADP-ME type species. Species of this genera are particularly amenable to isolation of both cell types, whereas in C4 species of other genera tested, mesophyll cells are largely or completely broken during mechanical treatment. From a series of studies with D. sanguinalis, it became clear that enzymes of the carboxylation phase of the C4 pathway were in mesophyll cells, and that light-dependent fixation of CO2 into C4 acids occurred when the cells were provided with pyruvate and 14CO2. Bundle sheath cells showed the capacity for light-dependent fixation of CO2 via Rubisco into PGA. Subsequently, bundle sheath strands isolated mechanically or enzymatically, were used in many studies on C4 photosynthesis. These included studies on the capacity for the Hill reaction with the addition of Hill oxidants, on light-dependent CO2 fixation, on metabolism of C4 acids by the three different C4 subtypes, and on the capacity for photorespiration via Rubisco under CO2-limited conditions (Edwards and Huber, 1981; Edwards and Walker, 1983; Hatch, 1999; Kanai and Edwards, 1999). In C4 plants, there are numerous plasmodesmata connections between mesophyll and bundle sheath cells. When cells are isolated, these connections are severed and apparently remain unsealed, and metabolites such as malate and pyruvate can be taken into the cells through the open plasmodesmata. This interesting feature extends the types of experiments that can be done with the isolated cells. It made possible studies to test the ability of these cells to metabolize various suspected intermediate compounds in the light (e.g. pyruvate and 3-PGA by mesophyll cells; ribose-5-P and C4 acids by bundle sheath cells), and to evaluate the effects of addition of these substances on 14CO2 fixation or photosynthetic O2 evolution.
While mesophyll and bundle sheath cells isolated by mechanical force were useful for studies on photosynthetic metabolism of these cell types, they also had their limitations. First, preparations of the two cell types were always cross-contaminated (particularly in bundle sheath, e.g. 5–10% contamination by mesophyll cells), making precise determination of compartmentation of any enzymes between the two cell types difficult. Second, due to potential physical damage of the isolated cells, enzymes associated with the cytosol may be lost and thus underestimated. Third, both cell types could only be isolated effectively from Digitaria species by this mechanical means, such that comparative studies were limited to this genera. Fourth, both mesophyll and bundle sheath cells are very difficult to break, such that it was impossible to isolate intact organelles to study intracellular compartmentation. As a consequence, methods to overcome these shortfalls for separation of intact cells or organelles needed to be developed.
In summary, by 1970, application of non-aqueous and mechanical isolation techniques produced a view of the C4 photosynthetic mechanism in NADP-ME species like sugarcane, sorghum, and D. sanguinalis (Slack et al., with non-aqueous fractionation, Björkman and Gauhl, and Berry et al., with bundle sheath strands, and Edwards et al., with mesophyll and bundle sheath cells). It formed the basis of the pathway for NADP-ME species as it is known today (Hatch et al., 1971). However, there was controversy over compartmentation and function of mesophyll and bundle sheath cells in C4 photosynthesis for several years. For example, there was evidence that chloroplasts isolated from young maize leaves were capable of photosynthesis by the Calvin cycle, and when leaves of maize were incubated in the absence of CO2, PEP was depleted, which was unexpected if atmospheric CO2 was fixed by PEPC (see results of Gibbs and colleagues in Hatch et al., 1971, and later evidence and explanation for this effect, Usuda, 1987; Leegood and von Caemmerer, 1988). Application of differential grinding techniques resulted in different proposals about compartmentation of PEPC and Rubisco. This included a radically different scheme of C4 photosynthesis based on results from differential grinding techniques with NADP-ME species like sugarcane and Pennisetum purpureum (Coombs and Baldry, 1972). It was suggested that CO2 was fixed by PEPC in epidermal cells, that malate donated CO2 to Rubisco in mesophyll cells for fixation through the C3 pathway, and that the bundle sheath chloroplasts only functioned to store starch (consistent with earlier speculations on the role of bundle sheath chloroplasts by Rhoades and Carvalho, 1944). Soon, a new technical approach, the enzymatic isolation and separation of mesophyll protoplasts and bundle sheath cells, firmly established the co-operative function of these cell types in C4 photosynthesis; not only in the NADP-ME type species, but also in other C4 subgroups as well.
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Enzymatic isolation of mesophyll and bundle sheath preparations |
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TopAbstractC4 pathway of photosynthesisInitial studies on the...Mechanical isolation of...Enzymatic isolation of mesophyll...In situ methods for...Distribution of C4-specific mRNAImmunolocalization of C4...Cell- and organ-specific...SummaryReferences |
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Enzymatic isolation of mesophyll protoplasts and bundle sheath strands from C4 plants (free of epidermal tissue), using a mixture of fungal cellulase-pectinase (Kanai and Edwards, 1973
a, b), was a technical advancement which allowed the properties of these cells to be explored in various C4 species. The method was especially successful with C4 monocots, including species of all three subgroups. There was a clear separation of the two cell types with very little cross contamination, and use of the dye Evans Blue (which is excluded from mesophyll protoplasts retaining their selective permeability) showed a high degree of intactness (Kanai and Edwards, 1973b). Significantly, this technique allowed studies on the intercellular distribution of enzymes between the cell types (e.g. photosynthetic, photorespiratory, glycolytic, and enzymes of nitrogen and sulphate assimilation) (Edwards and Walker, 1983). With respect to C4 photosynthesis, these results showed that enzymes of the carboxylation phase of the C4 cycle, including PEPC, are in mesophyll cells, while C4 acid decarboxylases, along with phosphoribulokinase and Rubisco, are in bundle sheath cells (Kanai and Edwards, 1973c; Ku et al., 1974). This included species used in the studies of Baldry and Coombs that specifically addressed their proposal. Research on these preparations included studies on the photochemical properties (relative levels of PSI and PSII, and capacity for photochemistry), and on the effects of addition of various metabolites on photosynthetic metabolism (Edwards and Walker, 1983).
Isolated bundle sheath strands have been used for many years to study their photosynthetic metabolism (e.g. fixation of CO2 and utilization of C4 acids as donors of CO2 to the C3 pathway). Methods include enzymatic isolation, as described above, mechanical isolation or combining enzymatic treatment with subsequent mechanical isolation. With some species, the latter method has the advantage of providing a purer bundle sheath preparation and a shorter preparation time. Methods of isolation and photosynthetic studies with bundle sheath strands have been described earlier (Edwards and Huber, 1981; Hatch, 1987; Furbank et al., 1990; Meister et al., 1996).
Mesophyll protoplasts can be broken gently while maintaining the integrity of chloroplasts (intactness 90% or greater) and other organelles (e.g. mitochondria and peroxisomes) by passing them several times through a 20 micron nylon mesh. Mesophyll protoplast extracts (containing functional chloroplasts and cytosolic PEPC) were used to study the energetics of pyruvate conversion to PEP and reduction of oxaloacetate to malate (Edwards and Huber, 1979). These results showed that mesophyll chloroplasts are capable of generating ATP not only by linear electron flow to NADP, but also by PSI-dependent cyclic electron flow and by the O2-dependent Mehler peroxidase reaction. Mesophyll protoplasts were valuable for studying the intercellular compartmentation of photosynthetic enzymes in species representing the three C4 subgroups, and for characterizing certain chloroplast transporters associated with the C4 pathway (Edwards and Huber, 1979, 1981; Kanai and Edwards, 1999).
A number of photosynthetic enzymes in C4 plants are regulated by light–dark transitions, including several enzymes of the C4 cycle. Mesophyll and bundle sheath preparations have been of value in studying the mechanism of light activation of several enzymes (Usuda et al., 1984; Nakamoto and Edwards, 1986). In this regard, mesophyll protoplasts of sorghum and D. sanguinalis have been used to study the signal transduction pathway controlling phosphorylation/dephosphorylation of PEPC (Pierre et al., 1992; Giglioli-Guivarc'h et al., 1996).
In 1984, Jenkins and Russ developed a mechanical procedure for isolating functional mesophyll chloroplasts from maize and several other C4 species (Jenkins and Russ, 1984). The procedure (preparation time of about 20 min by maceration of maize leaves in a Sorvall blender, and purification by centrifugation of the intact chloroplasts through a 32% Percoll gradient) provided good yields of chloroplasts (80–90% intactness), with negligible contamination by bundle sheath chloroplasts.
In 1979, bundle sheath and mesophyll protoplasts were isolated from Panicum miliaceum, an NAD-ME type monocot, and functional chloroplasts were isolated from both protoplast types (Edwards et al., 1979). Subsequently, both mesophyll and bundle sheath protoplasts were isolated from Portulaca grandiflora, a succulent NADP-ME C4 dicot (Ku et al., 1981), Flaveria trinervia, an NADP-ME type dicot and Atriplex spongiosa, an NAD-ME type dicot (Moore et al., 1984), and several PEP-CK monocots (Ku et al., 1980; Chapman and Hatch, 1983; Watanabe et al., 1984). The bundle sheath protoplasts are larger and more dense than the mesophyll protoplasts and can be separated by density gradient centrifugation. In these studies, bundle sheath protoplasts have been used to investigate the intracellular compartmentation of various enzymes associated with pathways of C4 acid decarboxylation, CO2 fixation, and photorespiration. As noted earlier, it is difficult to isolate intact and functional chloroplasts and other organelles from bundle sheath strands; but there has been some success with Flaveria bidentis (Meister et al., 1996) and maize (Kanai and Edwards, 1999). As protoplasts were being used for studies on C4 photosynthesis, they were also employed to resolve questions on compartmentation of enzymes and metabolites, and the mechanism of photosynthesis in CAM and C3 species (Robinson and Walker, 1979; Edwards and Walker, 1983; Gardeström and Wigge, 1988; Ku et al., 1980; Stitt et al., 1989; Winter et al., 1982).
Mesophyll and bundle sheath preparations enzymatically isolated from greening maize seedlings have been used extensively by Sheen as a means of investigating gene regulation and signal transduction (Sheen, 1995). Her studies with maize mesophyll protoplasts, as a single-cell transient expression system, have provided novel information about sugar sensing and feedback inhibition of transcription of photosynthetic genes. This simple technique is also useful for rapid identification of promoter enhancer or suppresser elements for gene transcription (Sheen, 1991; Imaizumi et al., 1997) and for isolation of cell-specific transcriptional factors regulating the expression of C4 photosynthesis genes. Mesophyll protoplasts isolated from greening, etiolated maize seedlings are very active and have high transcriptional activity. After introduction of the gene into isolated protoplasts by electroporation, for high levels of expression of the inserted gene it is essential to maintain viability of the protoplasts during the following 24 h incubation under low light. Increasing the pH of the incubation medium from 5.8, a pH traditionally used for tissue culture, to 7.0 or 8.0, greatly enhanced the level of gene expression by 10- or 20-fold, respectively (M Ku, M Taniguchi, M Matsuoka, T Sugiyama, unpublished data). In addition, inclusion of 10 mM KCl and 10 mM NaHCO3 at pH 7.0 in the incubation medium further stimulated gene expression by more than 60-fold. Thus, maintenance of ion homeostasis and photosynthetic viability by the isolated mesophyll protoplasts is important for the cells to express the introduced gene. The major limitation of this technique for promoter analysis is that development- and tissue-specific, and to some extent cell-specific, regulation of gene transcription cannot be examined. Stable transformants will be required for these analyses.
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In situ methods for studying compartmentation of mRNA and protein in C4 photosynthesis |
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TopAbstractC4 pathway of photosynthesisInitial studies on the...Mechanical isolation of...Enzymatic isolation of mesophyll...In situ methods for...Distribution of C4-specific mRNAImmunolocalization of C4...Cell- and organ-specific...SummaryReferences |
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While study of cellular compartmentation of photosynthetic enzymes in C4 leaves can be facilitated by separation and isolation of relatively pure cell types or organelles from mature tissues, this approach is time-consuming and, in some cases, it is not applicable to very young leaves or other tissues. In addition, the specific intracellular localization of a given enzyme among the many compartments within a cell type cannot be determined with certainty. There are two cytological approaches to solve this problem in studying the expression and compartmentation of photosynthetic enzymes in C4 plants. In situ hybridization and immunolabelling have been developed to detect the site of expression of a specific mRNA or protein, respectively, directly in the tissues of interest. These complementary techniques are particularly useful for detecting distribution of specific transcripts and proteins directly in developing and mature leaves and cotyledons of C4 plants.
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Distribution of C4-specific mRNA |
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TopAbstractC4 pathway of photosynthesisInitial studies on the...Mechanical isolation of...Enzymatic isolation of mesophyll...In situ methods for...Distribution of C4-specific mRNAImmunolocalization of C4...Cell- and organ-specific...SummaryReferences |
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Initially, Sheen and colleagues (Sheen, 1999
) first extracted total RNA from isolated mesophyll protoplasts and bundle sheath strands and examined the cell-specific differential expression of various C4 photosynthesis genes. Subsequently, in situ hybridization was employed directly on leaf sections to detect specific mRNA.
For in situ hybridization of mRNA, plasmids (DNA template) have been used to generate sense and antisense RNA probes for Rubisco LSU and SSU, PEPC and PPDK. The sense strand probe is a critical control for this technique. Both radioactive and stable-labelled probes can be generated. For obvious reasons, stable-labelled probes, such as those tagged with digoxigenin, are easier to work with and have found considerable use for studies on C3 and C4 species. During in vitro synthesis of probes, digoxigenin-11-UTP is added to generate ‘dig-labelled probes’ which can later be detected by a secondary reaction. Paraffin sections are prepared and hybridized with labelled transcripts under carefully controlled conditions. Hybridized transcripts are detected using anti-digoxigenin antisera conjugated to alkaline phosphatase (other probes can also be used) in combination with a colour detection system (Wang et al., 1992, 1993). In more recent investigations, rhodamine-conjugated secondary antibodies were used, and sections were analysed using a confocal imaging system (Ramsperger et al., 1996). Localization of mRNAs for enzymes of the C4 pathway and Rubisco of the C3 pathway has been studied in several C4 species, with the most detailed studies conducted on maize and Amaranthus hypochondriacus (Wang et al., 1992, 1993; Long and Berry, 1996; Ramsperger et al., 1996; Langdale et al., 1988; Sheen, 1999; Dengler and Nelson, 1999).
One of the most interesting problems in understanding the developmental aspect of C4 photosynthesis is elucidating the initial C4 gene expression patterns and post-transcriptional regulation in developing organs. In mature leaves, it has been demonstrated that mRNAs for the small and large subunit of Rubisco, and the NADP-ME (maize) and NAD-ME (amaranth) are expressed exclusively in bundle sheath cells, while PPDK, PEPC and NADP-MDH are expressed in mesophyll cells. However, there is only partial information on the control of expression of mRNA and synthesis of these proteins during development. There are differences in the environmentally- and developmentally-dependent signals controlling the expression of these genes in the few species studied, and in situ hybridization may help us to understand the regulation of C3/C4 gene development under different conditions further. In situ hybridization is a powerful technique for tissue and cell-specific localization of gene expression. However, it must be remembered that the intensity of the signal seen does not necessarily translate into differences in protein accumulation due to translational regulation, and it does not give information on subcellular distribution of the protein encoded by the transcript. This is particularly illustrated by observations of macromolecular trafficking of both protein and mRNA between highly differentiated cells such as companion cells and sieve elements (Kuhn et al., 1997; Xonocostle-Cazares et al., 1999). A different in situ technique, immunolocalization, can be used to clarify such relationships.
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Immunolocalization of C4 photosynthesis proteins |
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TopAbstractC4 pathway of photosynthesisInitial studies on the...Mechanical isolation of...Enzymatic isolation of mesophyll...In situ methods for...Distribution of C4-specific mRNAImmunolocalization of C4...Cell- and organ-specific...SummaryReferences |
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As discussed earlier, following the discovery of a C4 pathway and Kranz anatomy, several in vitro isolation techniques were employed to determine the intercellular and intracellular compartmentation of enzymes of C4 photosynthesis. However, a leaf is a complex organ with many tissue types and considerable variation in size of veins and relative distribution of cell types. Thus, isolation techniques tend to give a limited picture of compartmentation.
Immunocytochemistry, a technique that was developed to take advantage of antibodies as very specific ‘stains’ for probing tissue and cellular structure and function, was soon employed to confirm and expand the cell isolation results. This technique combines the high chemical specificity of antibodies and the high spatial resolution of microscopy. In 1977, Hattersley et al. used immunofluorescent labelling to study localization of Rubisco in plants. Antibodies to Rubisco were used to locate the enzyme in leaves of 42 species (Hattersley et al., 1977). They used an indirect labelling approach, whereby leaf segments were fixed in ethanol; then, hand-cut leaf blade sections were rinsed briefly in buffered saline and incubated for 1 h with antiserum. After rinsing, the leaf blade sections were incubated in the dark in fluorescein isothiocyanate (FITC)-labelled sheep anti-rabbit immunoglobulin. Rinsed sections were mounted in 50% glycerol (aqueous) containing 1% thymol and then sections were observed with a Zeiss Photomicroscope III set up for epifluorescence, and photographed within 24 h of preparation. All 29 species having a distinct Kranz anatomy showed high levels of fluorescence for bundle sheath cells when sections were treated with Rubisco antibodies and limited fluorescence from mesophyll cells. Thus, evidence for high Rubisco protein was demonstrated in BSC across various C4 species by this in situ method indicating this is a consistent feature in evolution of C4 plants.
Direct in situ methods have obvious advantages over in vitro methods, where uncertainties may exist about stability of proteins during isolation, and degree of purity of the fractions obtained. In addition, there are many variations in Kranz anatomy, and not all species are amenable to cell isolation. For example, among the Poaceae (the family with the largest number of C4 species) there are three C4 subtypes which have ‘classical’ anatomical and structural properties, including NADP-ME species having a single bundle sheath layer, and NAD-ME and PEP-CK types having a double bundle sheath layer with the C3 cycle in the outer layer (Gutierrez et al., 1974a; Dengler and Nelson, 1999). There are other C4 species of Poaceae which have ‘non-classical’ variants in the type of Kranz anatomy, including the aristidoid type which has a double chlorenchyma sheath. In the Chenopodiaceae (which has the largest number of C4 species among dicot families) there are four variants of Kranz anatomy including atriplicoid, kochioid, salsoloid, and suaedoid types (Carolin et al., 1975). Thus, in situ immunolocalization is a valuable tool for studying compartmentation of enzymes across taxonomic groups where C4 photosynthesis has evolved multiple times resulting in variations in anatomy and biochemistry.
With subsequent studies on immunolocalization of enzymes, a consistent feature across the different types of C4 plants analysed is the selective, high level of PEPC in mesophyll cells, and localization of Rubisco in bundle sheath cells, and both malic enzymes, NAD-ME and NADP-ME, in bundle sheath cells (Dengler and Nelson, 1999; Drincovich et al., 1998; Maurino et al., 1997; Sinha and Kellogg, 1996; Madhavan et al., 1996; Voznesenskaya et al., 1999). Initial studies of several C4 monocots on the localization of PPDK, the enzyme which regenerates PEP, showed high activity in mesophyll cells, with little or no activity in bundle sheath cells. However, studies of species among four different lineages of C4 evolution in the grasses show variation in PPDK localization from mesophyll, to bundle sheath, to both cell types (Sinha and Kellogg, 1996). Since two ATP are required per pyruvate converted to PEP via this enzyme, it would be of interest to examine the energetics and function of the C4 cycle between mesophyll and bundle sheath cells of such species.
Immunolocalization techniques for studies of compartmentation in C4 plants have improved significantly since the initial work (Hattersley et al., 1977). While immunofluorescence on unembedded or paraffin-embedded tissue samples is still a very powerful technique, the resolution is somewhat limited due to section thickness and problems with structural preservation at the subcellular level. In addition, autofluorescence of tissues in the absence of antibody can limit interpretations of the absolute compartmentation of proteins of interest.
Initial improvements in immunocytochemistry dealt with changes in fixation protocols and the chemical fixatives used (formaldehyde, paraformaldehyde, glutaraldehyde, and combinations of these), and the testing of various embedding media which allowed better structural preservation compared to free hand sectioning (paraffin, epoxy and acrylic resins). In particular, the use of resin-embedded leaf material for light and electron microscopy has greatly improved the ability to resolve the distribution patterns of various enzymes of the C4 pathway at the cellular and subcellular levels. The development of improved fixation techniques (including freeze-substitution and microwave processing), new resins designed for retention of antigen recognition, availability of improved fluorescent probes, new gold probes and silver enhancing techniques, and laser scanning confocal microscopy, have allowed cellular and subcellular localization of a range of relevant enzymes of the C4 pathway in a large number of species. For the application of immunolocalization techniques in studies with C4 plants see the following studies (Perrot-Rechenmann et al., 1982, 1983; Bauwe, 1984; Reed and Chollet, 1985; Rawsthorne, 1992; Wang et al., 1993; Dengler et al., 1995; Sinha and Kellogg, 1996; Ueno, 1992, 1996, 1998; Maurino et al., 1997; Drincovich et al., 1998; Voznesenskaya et al., 1999).
Currently, one of the most precise methods is the immunocytochemical technique which uses protein (Protein A, G, or IgG)-conjugated gold particles as a secondary probe. This method has been applied successfully to the study of compartmentation of enzymes of carbon assimilation in C3–C4 and C4 plants, and its undoubted advantage is the possibility for its use not only for light microscopy level investigations (using normal light, epipolarization or confocal imaging systems), but also at the electron microscopy level for establishing the intracellular and organellar localization of different enzymes (Rawsthorne, 1992; Ueno, 1992, 1996, 1998; Maurino et al., 1997; Drincovich et al., 1998; Voznesenskaya et al., 1999). Fixation of material with the paraformaldehyde+glutaraldehyde mixture and embedding it in Lowicryl or L.R. White acrylic resin gives good preservation of tissues and organelles, and does not require removing the resin. In particular, the development of gold probes for the immunolocalization allowed for TEM-level studies of the subcellular distribution of the enzymes of the C4 pathway. In combination with silver enhancing procedures, gold probes provide a high resolution localization technique at the light microscope level (Maurino et al., 1997; Drincovich et al., 1998; Voznesenskaya et al., 1999).
An example of the spatial resolution and preciseness of immunolabelling, even at the light microscope level, using gold probes are demonstrated in Fig. 1. Figure 1A and B illustrate immunogold labelling of Rubisco in the C4 plant maize versus the C3 plant rice. It shows Rubisco is exclusively confined to chloroplasts of the bundle sheath cells of mature maize leaves. While rice has a distinct bundle sheath around the vascular tissue, these sheath cells are less developed and have few chloroplasts; the primary site of labelling of Rubisco is seen in the chloroplasts of the upper layer of mesophyll cells. For all immunolocalization studies it is important to run preimmune (or non-immune) controls to be sure there is little, or no, non-specific labelling of the tissue in the absence of the primary antibody, which is illustrated in Fig. 1D for maize. Figure 1C shows the immunolocalization of PEPC in maize. The label is restricted to the mesophyll cells and close examination of these sections shows the label is in the cytoplasm while the chloroplasts are unlabelled. PEPC immunolabelling was also done on rice, but as expected there was very little, or no, labelling, consistent with low PEPC activity in the C3 rice leaf (not shown).
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In studying compartmentation of enzymes of photosynthetic carbon metabolism in different chloroplast-containing tissues of various species, the partitioning of carbon into carbohydrates, including starch, is also of interest. It is thought sucrose is predominantly synthesized in mesophyll cells; evidence with various C4 species shows starch is normally synthesized in bundle sheath cells. However, enzymes for their biosynthesis are found in both cell types (Leegood and Walker, 1999
). Using the PAS procedure for staining polysaccharides, Fig. 2 illustrates the heavy deposition of starch in bundle sheath chloroplasts of maize, the site of Rubisco localization, as well as the appearance of starch in the guard cells, while mesophyll cells are essentially devoid of starch. Such cytochemical techniques combining chemical selectivity and spatial resolution can be of significant value in combination with immuno and in situ techniques. For determining the distribution of metabolites between mesophyll and bundle sheath cells during C4 photosynthesis, different rapid fractionation techniques were developed earlier (Leegood, 1985; Stitt and Heldt, 1985), but these rely on the ability to obtain fractions enriched in the respective cell types, which, as pointed out above, is not possible with many species.
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The current state of these microscope-based methods and their ability to solve some of the intriguing problems of C4 photosynthesis is illustrated in a recent study (Voznesenskaya et al., 1999
) involving several species in the tribe Salsoleae of the family Chenopodiaceae. In a given species, different photosynthetic vegetative organs often have the same type of CO2 fixation (Edwards and Walker, 1983). However, unlike other species previously examined, some chenopods having C4 type photosynthesis in leaves have C3 metabolism in cotyledons. Initially, Butnik discovered non-Kranz cotyledons in some C4 chenopods, for example, in the Salsola genus (Butnik, 1984). Later studies using the ‘pulse–chase’, 14CO2–12CO2, technique and analysis of isolated enzymes provided evidence for C3 and C4 type photosynthesis in cotyledons among species of the tribe Salsoleae having C4 photosynthesis in leaves (Pyankov et al., 1999, 2000). Anatomically, four types of cotyledons were identified, two C3 types, having dorsoventral and isopalisade mesophyll structure, and two Kranz types (atriplicoid and salsoloid). Immunolocalization studies of these tissues are important for evaluating the mechanism of photosynthesis for several reasons. As noted, there is large diversity in anatomy. Among species of the tribe Salsoleae, leaves and cotyledons can have up to four chloroplast-containing tissues: hypoderm, mesophyll, bundle sheath, and water storage. Thus, the role of each cell type in photosynthesis needs to be identified. Cotyledons in some species are very small and are difficult to characterize photosynthetically by most techniques. Also, in some species, like Haloxylon, where the carbon isotope fractionation values in cotyledons are intermediate between C3 and C4 plants (Pyankov et al., 1999), immunolocalization studies can help resolve the mechanism of carbon fixation (Voznesenskaya et al., 1999).
In the study of Voznesenskaya et al. (Voznesenskaya et al., 1999), immunocytochemical localization of four main photosynthetic enzymes (Rubisco, PEPC, NAD-ME, and NADP-ME) was determined in four species from the tribe Salsoleae exhibiting C4 type CO2 fixation of the NAD- or NADP-ME subtype. The species studied have in common salsoloid structure in leaves (or stem in the case of Haloxylon), but with different leaf anatomy in cotyledons: C3 dorsoventral, C3 isopalisade, C4 atriplicoid, and C4 salsoloid. It was shown that, irrespective of the nature of assimilating organs having Kranz anatomy (leaf, cotyledon or stem), Rubisco was strongly localized to bundle sheath cells, and PEPC was localized in mesophyll cells, while malic enzymes were restricted to bundle sheath cells. Both types of cotyledons with C3 anatomy showed ordinary C3 Rubisco localization in all mesophyll cells and the absence of C4 enzyme labelling. Electron microscopy revealed the localization of NAD-ME in mitochondria, while NADP-ME was in chloroplasts of bundle sheath cells in the respective C4 types. Staining for polysaccharides showed sites of starch accumulation, which generally paralleled the localization of Rubisco. It was apparent that in some C4 organs, the hypoderm and water storage tissue also have chloroplasts which contain Rubisco, which store starch, and which, thus, perform C3 photosynthesis.
The immunolocalization approach is also important for addressing the question of whether CAM occurs in succulent species of Chenopodiaceae. A degree of CAM has been suggested, but not proven, in some Chenopodiaceae species because they have water storage tissue with a significant number of chloroplasts (Zalenskii and Glagoleva, 1981; Bil’ et al., 1983). However, little or no PEPC or malic enzyme protein was detected by immunolocalization in water storage cells, suggesting there is no CAM or donation of CO2 from C4 acids to Rubisco in these cells. Rather, the occurrence of Rubisco in water storage tissue of some species suggests a role for refixation of respired CO2 from vascular tissue which is centrally located in the leaf (Voznesenskaya et al., 1999).
At this point, the limiting factor for use of immunocytochemistry in photosynthesis research is not so much the methodology but the availability of antibodies to the enzymes of importance to the pathways being studied. While a number of companies are actively involved in producing antibodies to thousands of proteins for animal and human research purposes, most antibodies for plant biology research are produced by individual researchers and are of limited availability.
The in situ immunolocalization method for analysing compartmentation of enzymes of C4 photosynthesis is also ideal for developmental studies (Maurino et al., 1997) and studies with C3–C4 intermediates (Hattersley et al., 1977; Bauwe, 1984; Reed and Chollet, 1985; Hylton et al., 1988, Drincovich et al., 1998). It is particularly difficult to separate the cell types in the intermediate species because the bundle sheath cells and Kranz anatomy are less developed than in C4 plants. A common feature of intermediates, demonstrated with immunocytochemistry, is localization of Rubisco in both mesophyll and bundle sheath cells. In this case, the Rubisco in bundle sheath is the site of refixation of photorespired CO2 (where glycine decarboxylase of the photorespiratory pathway is specifically localized), and fixation of CO2 delivered to the bundle sheath by a limited C4 pathway in certain C3–C4 intermediates.
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Cell- and organ-specific expression of C4-specific genes—gene promoter analysis in transgenic C4 plants |
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TopAbstractC4 pathway of photosynthesisInitial studies on the...Mechanical isolation of...Enzymatic isolation of mesophyll...In situ methods for...Distribution of C4-specific mRNAImmunolocalization of C4...Cell- and organ-specific...SummaryReferences |
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Recent molecular studies on the C4 mechanism have focused on evolution of C4-specific genes from existing genes in ancestral C3 plants, and regulation of their expression in C4 plants (Ku et al., 1996
; Rosche et al., 1998; Sheen, 1999). Relative to the corresponding genes in C3 plants, the key features of C4-specific genes are high-level expression, and organ- and cell-specific expression. During the course of evolution, C4-specific genes acquired modifications in their promoter regions which regulate the specific patterns of expression. Homologous transgenic C4 plants have been used to identify the promoter elements of C4 genes that are essential for directing organ-specific and cell-specific expression. In this approach, a fragment of the gene promoter region is fused to a reporter gene (e.g. GUS-glucuranidase, LUC-firefly luciferase or GFP-green fluorescent protein) and used for transformation. The transcriptional strength of the promoter in the transgenic plants can be determined by analysing the reporter protein: enzymic activity of GUS and LUC, histochemical staining of GUS, and fluorescence signal by GFP. Histochemical staining of GUS in tissues provides a direct visualization of the site of expression, but does not allow a good quantitative estimation of expression level, especially between different experiments. However, this can be remedied by assaying the enzymic activity of GUS directly in protein preparations extracted from different tissues or isolated cell types (Taniguchi et al., 2000). As shown in Fig. 3, a 0.9 kb promoter sequence from the maize PPDK gene directed GUS expression predominantly in the mesophyll chloroplasts of a transgenic maize leaf. On the other hand, the constitutive 35S cauliflower mosaic virus promoter directed GUS expression in both cell types. The maize PPDK gene promoter sequences apparently contain the necessary cis-acting elements for its cell-specific expression. While cytochemical staining of GUS activity is a simple method for direct, visual determination of the reporter enzyme protein localization, it does not permit resolution at the intracellular level. It is, thus, more useful to determine specificity for expression in particular cell types. After fixation of the tissues, the blue product produced by GUS tends to stick to organelles, especially chloroplasts, even though the protein is located in the cytosol. A concomitant immunolabelling will help resolve this problem. Finally, there is growing interest in transferring C4 genes to C3 plants (Ku et al., 1999), and methods of studying compartmentation of gene expression and protein accumulation will be critical to this work (Sheehy, 2000).
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Summary |
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TopAbstractC4 pathway of photosynthesisInitial studies on the...Mechanical isolation of...Enzymatic isolation of mesophyll...In situ methods for...Distribution of C4-specific mRNAImmunolocalization of C4...Cell- and organ-specific...SummaryReferences |
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The use of various techniques in studying the cellular compartmentation of photosynthetic metabolism in C4 plants has been critical in elucidating the mechanism of C4 photosynthesis. The current understanding has been dependent on improvement of, or development of, new techniques over the past several decades. In the future, cell-specific analysis will be required to further the understanding of C4 photosynthesis, and to analyse the potential of utilizing the genetic information associated with this process to improve crop productivity. This includes studies on the taxonomic-based diversity in the C4 mechanism (anatomy and biochemistry), the further characterization of compartmentation of enzymes, and of specific transporters in organelles which are required for C4 photosynthesis, elucidation of signalling processes which are responsible for the control of the development of Kranz anatomy and the associated biochemistry in C4 plants, and determination of the consequences of transforming C3 plants with genes from C4 plants which are responsible for Kranz anatomy and C4 photosynthesis.
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Acknowledgments |
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This work was partly supported by NSF Grant IBN-9807916 and Civilian Research and Development Foundation Grant RB1-264. Thanks to T Kostman and N Tarlyn for assistance in preparing the figures. The microsocopy was done in the WSU Electron Microscopy Center.
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Notes |
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5 To whom correspondence should be addressed. Fax: +1 509 335 3184. E-mail: edwardsg{at}wsu.edu
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Abbreviations |
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NAD-ME, NAD-malic enzyme; NADP-MDH, NADP-malate dehydrogenase; NADP-ME, NADP-malic enzyme; PEP, phosphoenolpyruvate; PEPC, phosphoenolpyruvate carboxylase; PEP-CK, phosphoenolpyruvate carboxykinase; PPDK, pyruvate,Pi dikinase; PGA, phosphoglyceric acid, Rubisco, ribulose 1,5-bisphosphate carboxylase-oxygenase; RuBP, ribulose 1,5-bisphosphate..
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References |
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TopAbstractC4 pathway of photosynthesisInitial studies on the...Mechanical isolation of...Enzymatic isolation of mesophyll...In situ methods for...Distribution of C4-specific mRNAImmunolocalization of C4...Cell- and organ-specific...SummaryReferences |
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