The cytoskeleton maintains organelle partitioning required for single-cell C4 photosynthesis in Chenopodiaceae species

Recently, three Chenopodiaceae species, Bienertia cycloptera, Bienertia sinuspersici, and Suaeda aralocaspica, were shown to possess novel C(4) photosynthesis mechanisms through the compartmentalization of organelles and photosynthetic enzymes into two distinct regions within a single chlorenchyma cell. Bienertia has peripheral and central compartments, whereas S. aralocaspica has distal and proximal compartments. This compartmentalization achieves the equivalent of spatial separation of Kranz anatomy, including dimorphic chloroplasts, but within a single cell. To characterize the mechanisms of organelle compartmentalization, the distribution of the major organelles relative to the cytoskeleton was examined. Examination of the distribution of the cytoskeleton using immunofluorescence studies and transient expression of green fluorescent protein-tagged cytoskeleton markers revealed a highly organized network of actin filaments and microtubules associating with the chloroplasts and showed that the two compartments in each cell had different cytoskeletal arrangements. Experiments using cytoskeleton-disrupting drugs showed in Bienertia and S. aralocaspica that microtubules are critical for the polarized positioning of chloroplasts and other organelles. Compartmentalization of the organelles in these species represents a unique system in higher plants and illustrates the degree of control the plant cell has over the organization and integration of multiorganellar processes within its cytoplasm.     

Differentiation of cellular and biochemical features of the single-cell C4 syndrome during leaf development in Bienertia cycloptera (Chenopodiaceae)

The terrestrial plant Bienertia cycloptera has been shown to accomplish C(4) photosynthesis within individual chlorenchyma cells by spatially separating the phases of carbon assimilation into distinct peripheral and central compartments. In this study, anatomical, physiological, and biochemical techniques were used to determine how this unique compartmentation develops. Western blots show ribulose-1,5-bisphosphate carboxylase (Rubisco) (chloroplastic) is present in the youngest leaves and increases during development, while levels of C(4) enzymes-pyruvate,Pi dikinase (chloroplastic), phosphoenolpyruvate carboxylase (PEPC) (cytosol), and NAD-malic enzyme (mitochondrial)-increase later in development. Immunolocalization confirmed this for Rubisco and PEPC. The youngest chlorenchyma cells have a central nucleus surrounded by monomorphic granal chloroplasts containing Rubisco. Later stages show progressive development of a central cytoplasmic compartment enriched with chloroplasts and mitochondria and of a peripheral cytoplasm with chloroplasts. A complex reticulum of connections between the compartments also developed and was characterized. δ(13)C isotope analyses show mature leaves have distinct C(4)-type isotope composition, while the composition in younger leaves is "C(4)-like." Based on the results, this form of single-cell C(4) photosynthesis develops from a common pool of organelles through partitioning to separate compartments, and the development of biochemically and ultrastructurally dimorphic chloroplasts.     

Bienertia cycloptera Bunge ex Boiss., Chenopodiaceae, another C4 Plant without Kranz Tissues286

Abstract: Following up an earlier study on Borszczowia aralocaspica, a second C₄ leaf type without Kranz tissues is described from Bienertia cycloptera Bunge ex Boiss. The species belongs also to tribe Suaedeae and grows in similar temporarily wet saline habitats in southwestern C Asia. Like Borszczowia, the species has the ability to perform C₄ photosynthesis in single chlorenchyma cells but the cytological compartmentation differs. Evidence is cited from anatomy (cytoplasmic compartmentation, starch formation), from carbon isotope composition and from indirect sources. It is documented by micrographs and δ¹³C values (mean ‐ 14.7 %). Variations of the δ¹³C values subsequently found in newly formed leaves of the same greenhouse plants (‐ 15.5 to ‐ 21.1 %) support the hypothesis that Bienertia is a facultative C₄/C₃ species. The new described bienertioid leaf type differs from the leaves of C₃ species in Suaeda by strict separation of the mesophyll into a central aqueous tissue without chloroplasts and a peripheral 1 ‐ 3 layered chlorenchyma with elaborate cytoplasmic compartmentation. Each cell of the latter contains a peripheral cytoplasmic layer with scattered chloroplasts and a large globular cytoplasmic body located in the central vacuole which is densely packed with starch producing chloroplasts, and joined by the nucleus. Comparative studies on different leaves of the same individuals of ordinary C₄ species demonstrated a remarkable range of variation, from 1.0 % in Salsola tragus up to 2.5 % in Petrosimonia sibirica. This corresponds well with the variance in data from different individuals and populations. For explaining the larger interspecific variation from about 23 other species of Salsoloideae, the hypothesis is developed that in species or groups of taxa specific leakages of their C₄ systems do occur. By three characters unique in tribe Suaedeae and detected during the study, Bienertia is confirmed as well separated from Suaeda and as a monotypic genus.     

Structural, biochemical, and physiological characterization of C4 photosynthesis in species having two vastly different types of kranz anatomy in genus Suaeda (Chenopodiaceae)

C (4) species of family Chenopodiaceae, subfamily Suaedoideae have two types of Kranz anatomy in genus Suaeda, sections Salsina and Schoberia, both of which have an outer (palisade mesophyll) and an inner (Kranz) layer of chlorenchyma cells in usually semi-terete leaves. Features of Salsina (S. AEGYPTIACA, S. arcuata, S. taxifolia) and Schoberia type (S. acuminata, S. Eltonica, S. cochlearifoliA) were compared to C (3) type S. Heterophylla. In Salsina type, two layers of chlorenchyma at the leaf periphery surround water-storage tissue in which the vascular bundles are embedded. In leaves of the Schoberia type, enlarged water-storage hypodermal cells surround two layers of chlorenchyma tissue, with the latter surrounding the vascular bundles. The chloroplasts in Kranz cells are located in the centripetal position in Salsina type and in the centrifugal position in the Schoberia type. Western blots on C (4) acid decarboxylases show that both Kranz forms are NAD-malic enzyme (NAD-ME) type C (4) species. Transmission electron microscopy shows that mesophyll cells have chloroplasts with reduced grana, while Kranz cells have chloroplasts with well-developed grana and large, specialized mitochondria, characteristic of NAD-ME type C (4) chenopods. In both C (4) types, phosphoenolpyruvate carboxylase is localized in the palisade mesophyll, and Rubisco and mitochondrial NAD-ME are localized in Kranz cells, where starch is mainly stored. The C (3) species S. heterophylla has Brezia type isolateral leaf structure, with several layers of Rubisco-containing chlorenchyma. Photosynthetic response curves to varying CO (2) and light in the Schoberia Type and Salsina type species were similar, and typical of C (4) plants. The results indicate that two structural forms of Kranz anatomy evolved in parallel in species of subfamily Suaedoideae having NAD-ME type C (4) photosynthesis.     

Occurrence and forms of Kranz anatomy in photosynthetic organs and characterization of NAD-ME subtype C4 photosynthesis in Blepharis ciliaris (L.) B. L. Burtt (Acanthaceae)

Blepharis (Acanthaceae) is an Afroasiatic genus comprising 129 species which occur in arid and semi-arid habitats. This is the only genus in the family which is reported to have some C(4) species. Blepharis ciliaris (L.) B. L. Burtt. is a semi-desert species with distribution in Iran, Oman, and Pakistan. Its form of photosynthesis was investigated by studying different organs. C(4)-type carbon isotope composition, the presence of atriplicoid type Kranz anatomy, and compartmentation of starch all indicate performance of C(4) photosynthesis in cotyledons, leaves, and the lamina part of bracts. A continuous layer of distinctive bundle sheath cells (Kranz cells) encircle the vascular bundles in cotyledons and the lateral vascular bundles in leaves. In older leaves, there is extensive development of ground tissue in the midrib and the Kranz tissue becomes interrupted on the abaxial side, and then becomes completely absent in the mature leaf base. Cotyledons have 5-6 layers, and leaves 2-3 layers, of spongy chlorenchyma beneath the veins near the adaxial side of the leaf, indicating bifacial organization of chlorenchyma. As the plant matures, bracts and spines develop and contribute to carbon assimilation through an unusual arrangement of Kranz anatomy which depends on morphology and exposure to light. Stems do not contribute to carbon assimilation, as they lack chlorenchyma tissue and Kranz anatomy. Analysis of C(4) acid decarboxylases by western blot indicates B. ciliaris is an NAD-malic enzyme type C(4) species, which is consistent with the Kranz cells having chloroplasts with well-developed grana and abundant mitochondria.     

Resolving the compartmentation and function of C4 photosynthesis in the single-cell C4 species Bienertia sinuspersici

Bienertia sinuspersici is a land plant known to perform C(4) photosynthesis through the location of dimorphic chloroplasts in separate cytoplasmic domains within a single photosynthetic cell. A protocol was developed with isolated protoplasts to obtain peripheral chloroplasts (P-CP), a central compartment (CC), and chloroplasts from the CC (C-CP) to study the subcellular localization of photosynthetic functions. Analyses of these preparations established intracellular compartmentation of processes to support a NAD-malic enzyme (ME)-type C(4) cycle. Western-blot analyses indicated that the CC has Rubisco from the C(3) cycle, the C(4) decarboxylase NAD-ME, a mitochondrial isoform of aspartate aminotransferase, and photorespiratory markers, while the C-CP and P-CP have high levels of Rubisco and pyruvate, Pidikinase, respectively. Other enzymes for supporting a NAD-ME cycle via an aspartate-alanine shuttle, carbonic anhydrase, phosophoenolpyruvate carboxylase, alanine, and an isoform of aspartate aminotransferase are localized in the cytosol. Functional characterization by photosynthetic oxygen evolution revealed that only the C-CP have a fully operational C(3) cycle, while both chloroplast types have the capacity to photoreduce 3-phosphoglycerate. The P-CP were enriched in a putative pyruvate transporter and showed light-dependent conversion of pyruvate to phosphoenolpyruvate. There is a larger investment in chloroplasts in the central domain than in the peripheral domain (6-fold more chloroplasts and 4-fold more chlorophyll). The implications of this uneven distribution for the energetics of the C(4) and C(3) cycles are discussed. The results indicate that peripheral and central compartment chloroplasts in the single-cell C(4) species B. sinuspersici function analogous to mesophyll and bundle sheath chloroplasts of Kranz-type C(4) species.     

Structural changes in the vacuole and cytoskeleton are key to development of the two cytoplasmic domains supporting single-cell C<Subscript>4</Subscript> photosynthesis in <Emphasis Type="Italic">Bienertia sinuspersici</Emphasis>

Bienertia sinuspersici Akhani has an unusual mechanism of C₄ photosynthesis which occurs within individual chlorenchyma cells. To perform C₄, the mature cells have two cytoplasmic compartments consisting of a central (CCC) and a peripheral (PCC) domain containing dimorphic chloroplasts which are interconnected by cytoplasmic channels. Based on leaf development studies, young chlorenchyma cells have not developed the two cytoplasmic compartments and dimorphic chloroplasts. Fluorescent dyes which are targeted to membranes or to specific organelles were used to follow changes in cell structure and organelle distribution during formation of C₄-type chlorenchyma. Chlorenchyma cell development was divided into four stages: 1—the nucleus and chloroplasts occupy much of the cytoplasmic space and only small vacuoles are formed; 2—development of larger vacuoles, formation of a pre-CCC with some scattered chloroplasts; 3—the vacuole expands, cells have directional growth; 4—mature stage, cells have become elongated, with a distinctive CCC and PCC joined by interconnecting cytoplasmic channels. By staining vacuoles with a fluorescent dye and constructing 3D images of chloroplasts, and by microinjecting a fluorescence dye into the vacuole of living cells, it was demonstrated that the mature cell has only one vacuole, which is traversed by cytoplasmic channels connecting the CCC with the PCC. Immunofluorescent studies on isolated chlorenchyma cells treated with cytoskeleton disrupting drugs suspended in different levels of osmoticum showed that both microtubules and actin filaments are important in maintaining the cytoplasmic domains. With prolonged exposure of plants to dim light, the cytoskeleton undergoes changes and there is a dramatic shift of the CCC from the center toward the distal end of the cell.     

Comparison of leaf structure and photosynthetic characteristics of C3 and C4 Alloteropsis semialata subspecies

Alloteropsis semialata (R. Br.) Hitchcock includes both C3 and C4 subspecies: the C3 subspecies eckloniana and the C4 subspecies semialata. We examined the leaf structural and photosynthetic characteristics of these plants. A. semialata ssp. semialata showed high activities of photosynthetic enzymes involved in phosphoenolpyruvate carboxykinase-type C4 photosynthesis and an anomalous Kranz anatomy. Phosphoenolpyruvate carboxylase; pyruvate, Pi dikinase and glycine decarboxylase (GDC) were compartmentalized between the mesophyll (M) and inner bundle sheath cells, whereas ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) occurred in both cells. A. semialata ssp. eckloniana also showed an anomalous non-Kranz anatomy, in which the mestome sheath cells included abundant chloroplasts and mitochondria. Rubisco and GDC accumulated densely in the M and mestome sheath cells, whereas the levels of C4 enzymes were low. The activity levels of photo-respiratory enzymes in both subspecies were intermediate between those in typical C3 and C4 plants. The values of CO2 compensation points in A. semialata ssp. semialata were within the C4 range, whereas those in A. semialata ssp. eckloniana were somewhat lower than the C3 range. These data suggest that the plants are C3-like and C4-like but not typical C3 and C4, and when integrated with previous findings, point to important variability in the expression of C4 physiology in this species complex. A. semialata is therefore an intriguing grass species with which to study the evolutionary linkage between C3 and C4 plants.     

The occurrence of C4 species in the genusRhynchospora and its significance in kranz anatomy of the cyperaceae

Rhynchospora rubra was found to have a low CO₂ compensation point, high δ¹³C value, Kranz leaf anatomy, starch present in the bundle sheath cells and narrow interveinal distance. These observations suggest thatR. rubra is a C₄ plant. A further anatomical survey revealed seven otherRhynchospora species presumably having the C₄ photosynthetic pathway. In the family Cypraceae C₄ plants therefore occur in the tribe Rhynchosporeae as well as in the Scirpeae and Cypereae. The C₄ species ofRhynchospora have a normal Kranz type of leaf anatomy, although the C₄ species ofCyperus andFimbristylis presently known have an abnormal one in which the mestome sheath without chloroplasts is interposed between the Kranz tissue and the rest of the chlorenchyma. Thus inRhynchospora the Kranz tissue is in direct contact with the rest of the chlorenchyma, and it is suggested that the Kranz tissue may be homologous with the mestome sheath.     

Functional characterization of phosphoenolpyruvate carboxykinase-type C4 leaf anatomy: immuno-, cytochemical and ultrastructural analyses

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

Species having C4 single-cell-type photosynthesis in the Chenopodiaceae family evolved a photosynthetic phosphoenolpyruvate carboxylase like that of Kranz-type C4 species

Spatial and temporal regulation of phosphoenolpyruvate carboxylase (PEPC) is critical to the function of C(4) photosynthesis. The photosynthetic isoform of PEPC in the cytosol of mesophyll cells in Kranz-type C(4) photosynthesis has distinctive kinetic and regulatory properties. Some species in the Chenopodiaceae family perform C(4) photosynthesis without Kranz anatomy by spatial separation of initial fixation of atmospheric CO(2) via PEPC from C(4) acid decarboxylation and CO(2) donation to Rubisco within individual chlorenchyma cells. We studied molecular and functional features of PEPC in two single-cell functioning C(4) species (Bienertia sinuspersici, Suaeda aralocaspica) as compared to Kranz type (Haloxylon persicum, Salsola richteri, Suaeda eltonica) and C(3) (Suaeda linifolia) chenopods. It was found that PEPC from both types of C(4) chenopods displays higher specific activity than that of the C(3) species and shows kinetic and regulatory characteristics similar to those of C(4) species in other families in that they are subject to light/dark regulation by phosphorylation and display differential malate sensitivity. Also, the deduced amino acid sequence from leaf cDNA indicates that the single-cell functioning C(4) species possesses a Kranz-type C(4) isoform with a Ser in the amino terminal. A phylogeny of PEPC shows that isoforms in the two single-cell functioning C(4) species are in a clade with the C(3) and Kranz C(4) Suaeda spp. with high sequence homology. Overall, this study indicates that B. sinuspersici and S. aralocaspica have a C(4)-type PEPC similar to that in Kranz C(4) plants, which likely is required for effective function of C(4) photosynthesis.     

A Remarkable New Leaf Type With Unusual Photosynthetic Tissue in a Central Asiatic Genus of Chenopodiaceae

Abstract: From the hygrohalophyte Borszczowia aralocaspica Bunge (Chenopodiaceae), a new leaf type with 1-layered chlorenchyma is described as "borszczovoid" and compared with other leaf types in subfamily Salsoloideae. The chlorenchyma is suspected to represent a unique C₄ type. Evidence is cited from anatomical studies and documented by micrographs and Carbon isotope determinations (ä¹³C values). The 1-layered photosynthetic tissue combines all essential anatomical characters of a 2-layered chlorenchyma of regular C₄ plants and is in intimate contact with concentrically arranged peripheral bundles. The ä¹³C values are − 13.03 ‰ from young stems and − 13.78 ‰ from leaves. The results are discussed in the anatomical, physiological and taxonomic framework. In addition, from distantly-related Suaeda species of section Conosperma the conospermoid leaf type is re-described. It is characterized by typical palisade and Kranz layers and differs from the C₄ suaedoid type by an external water-storaging hypodermis and an arrangement of Kranz cells reminiscent of the atriplicoid type from subfamily Chenopodioideae. From eight other species of Chenopodiaceae ä¹³C values are given for the first time.     

Induction of kranz anatomy and C4-like biochemical characteristics in a submerged amphibious plant by abscisic acid

The amphibious leafless sedge Eleocharis vivipara develops C4-like traits as well as Kranz anatomy under terrestrial conditions, but it develops C3-like traits without Kranz anatomy under submerged conditions. When submerged plants are exposed to aerial conditions, they rapidly produce new photosynthetic tissues with C4-like traits. In this study, experiments were performed to determine whether abscisic acid (ABA), a plant stress hormone, could induce the formation of photosynthetic tissues with Kranz anatomy and C4-like biochemical traits under water in the submerged form. When the submerged plants were grown in water containing 5 &mgr;M ABA, they developed new photosynthetic tissues with Kranz anatomy, forming well-developed Kranz (bundle sheath) cells that contained many organelles. The ABA-induced tissues accumulated large amounts of phosphoenolpyruvate carboxylase, pyruvate orthophosphate dikinase, and NAD-malic enzyme at the appropriate cellular sites. The tissues had 3.4 to 3.8 times more C4 enzyme activity than did tissues of the untreated submerged plants. Carbon-14 pulse and carbon-12 chase experiments revealed that the ABA-induced tissues fixed higher amounts of carbon-14 into C4 compounds and lower amounts of carbon-14 into C3 compounds as initial products than did the submerged plants and that they exhibited a C4-like pattern of carbon fixation under aqueous conditions of low carbon, indicating enhanced C4 capacity in the tissues. This report provides an example of the hormonal control of the differentiation of the structural and functional traits required for the C4 pathway.     

Immunocytochemical localization of enzymes involved in the C3 and C4 pathways in the photosynthetic cells of an amphibious sedge, Eleocharis vivipara

Eleocharis vivipara link, an amphibious leafless sedge, develops traits of C₄ photosynthesis and Kranz anatomy in the terrestrial form but develops C₃-like traits with non-Kranz anatomy when submerged. The cellular localization of C₃ and C₄ enzymes in the photosynthetic cells of the two forms was investigated by immunogold labeling and electron microscopy. The terrestrial form has mesophyll cells and three kinds of bundle sheath cell, namely, parenchyma sheath cells, non-chlorophyllous mestome sheath cells, and Kranz cells. Phosphoenol-pyruvate carboxylase (PEPCase) was present in the cytosol of both the mesophyll cells and the parenchyma sheath cells, with higher-density labeling in the latter, but not in the Kranz cells. Pyruvate, Pi dikinase (PPDK) was found at high levels in the chloroplasts of both the mesophyll cells and the parenchyma sheath cells with some-what stronger labeling in the latter. This enzyme was also absent from the Kranz cells. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was found in the chloroplasts of all types of photosynthetic cell, but labeling was significantly less intense in the parenchyma sheath cells than in other types of cell. The submerged form also has three types of photosynthetic cell, as well as non-chlorophyllous mestome sheath cells, but it lacks the traits of Kranz anatomy as a consequence of modification of the cells. Rubisco was densely distributed in the chloroplasts of all the photosynthetic cells. However, PEPCase and PPDK were found in both the mesophyll cells and the parenchyma sheath cells but at lower levels than in the terrestrial form. These data reveal that the terrestrial form has a unique pattern of cellular localization of C₃ and C₄ enzymes, and they suggest that this pattern and the changes in the extent of accumulation of the various enzymes are the main factors responsible for the difference in photosynthetic traits between the two forms.Abbreviations CAM crassulacean acid metabolism - MC meso phyll cell - PSC parenchyma sheath cell - KC Kranz cell - PEP-Case phosphoenolpyruvate carboxylase - PPDK pyruvate, Pi dikinase - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - LS large subunit - RuBP ribulose-1,5-bisphosphate This study was supported by Grants-in-Aid from the Ministry of Agriculture, Forestry and Fisheries of Japan (Integrated Research Program for the Use of Biotechnological Procedures for Plant Breeding) and from the Science and Technology Agency of Japan (Enhancement of Center-of-Excellence, the Special Coordination Funds for Promoting Science and Technology). The author is grateful to Drs M. Matsuoka and S. Muto for providing the antisera and Dr. M. Samejima for his advice at the early stages of this study.     

Structural characterization of photosynthetic cells in an amphibious sedge, Eleocharis vivipara, in relation to C3 and C4 metabolism

Eleocharis vivipara Link is a unique amphibious leafless sedge. The terrestrial form has Kranz anatomy and the biochemical traits of C₄ plants while the submerged form develops structural and biochemical traits similar to those of C₃ plants. The structural features of the culms, which are the photosynthetic organs, of the two forms were examined and compared. The culms of the terrestrial form have mesophyll cells and three bundle sheaths which consist of three kinds of cell, namely, the innermost Kranz cells that contain large numbers of organelles, the middle mestome sheath cells that lack chloroplasts, and the outermost parenchyma sheath cells that contain chloroplasts. The culms of the submerged form had a tendency towards reduction in numbers and size of Kranz cells and vascular bundles, as compared to the terrestrial form, and they had spherical mesophyll cells that were tightly packed without intercellular spaces inside the epidermis. The submerged form had a higher ratio of cross-sectional area of mesophyll cells plus parenchyma sheath cells to that of Kranz cells than the terrestrial form. The difference was mainly due to a decrease in the number and the size of the Kranz cells and to a marked increase in the size of the mesophyll cells and the parenchyma sheath cells in the submerged form, as compared to the terrestrial form. The Kranz cells of the terrestrial form had basically the structural characteristics of plants of the NAD-malic enzyme type, with the exception of the intracellular location of organelles. The Kranz cells of the submerged form included only a few organelles, and the percentage of organelles partitioned to the Kranz cells was significantly smaller in the submerged form than in the terrestrial form. In addition, the size of chloroplasts of the Kranz cells was 60–70% of that of the terrestrial form. These structural differences between the two forms may be related to the functional differences in their mechanisms of photosynthesis.Abbreviations KC Kranz cell - MC mesophyll cell - PSC parenchyma sheath cell - NAD-ME NAD-malic enzyme - VB vascular bundle This study was supported by Grants-in-Aid from the Ministry of Agriculture, Forestry and Fisheries of Japan (Integrated Research Program for the Use of Biotechnological Procedures for Plant Breeding) and from the Science and Technology Agency of Japan (Enhancement of Center-of-Excellence, the Special Coordination Funds for Promoting Science and Technology).     

Protoplast isolation and transient gene expression in the single-cell C4 species, Bienertia sinuspersici

Although transient gene expression using reporters such as green fluorescent protein is a versatile tool for examining gene functions and intracellular protein trafficking, the establishment of a highly efficient gene manipulation method remains a challenge in many plant species. A reliable transformation protocol has not yet been established for the three single-cell C₄ species, despite their potential of serving as model systems for their extraordinary C₄ photosynthetic metabolism. We report the first protocol optimized for isolating a large-scale and homogenous population of protoplasts from chlorenchyma cells of the single-cell C₄ species Bienertia sinuspersici. Cytochemical staining confirmed the preservation of the unusual subcellular compartmentation of organelles in chlorenchyma cells after cell wall digestion. Approximately 84% of isolated protoplasts expressed the reporter fluorescent protein following our optimized polyethylene glycol-mediated transfection procedures. Fluorescent fusion protein tagged with various intracellular sorting signals demonstrated potential use of the transient gene expression system in subcellular protein localization and organelle dynamics studies. Further applications of the current protoplast isolation and transfection techniques in understanding the novel single-cell C₄ photosynthetic mechanism are discussed.     

Anatomy, chloroplast structure and compartmentation of enzymes relative to photosynthetic mechanisms in leaves and cotyledons of species in the tribe Salsoleae (Chenopodiaceae)

Certain members of the family Chenopodiaceae are the dominant species of the deserts of Central Asia; many of them are succulent halophytes which exhibit C4-type CO2 fixation of the NAD- or NADP-ME (malic enzyme) subgroup. In four C4 species of the tribe Salsoleae, the Salsoloid-type Kranz anatomy in leaves or stems was studied in relation to the diversity in anatomy which was found in cotyledons. Halocharis gossypina, has C4 NAD-ME Salsoloid-type photosynthesis in leaves and C3 photosynthesis in dorsoventral non-Kranz cotyledons; Salsola laricina has C4 NAD-ME Salsoloid-type leaves and C4 NAD-ME Atriplicoid-type cotyledons; Haloxylon persicum, has C4 NADP-ME Salsoloid-type green stems and C3 isopalisade non-Kranz cotyledons; and S. richteri has C4 NADP-ME Salsoloid-type leaves and cotyledons. Immunolocalization studies on Rubisco showed strong labelling in bundle sheath cells of leaves and cotyledons of organs having Kranz anatomy. The C4 pathway enzyme phosphoenolpyruvate carboxylase was localized in mesophyll cells, while the malic enzymes were localized in bundle sheath cells of Kranz-type tissue. Immunolocalization by electron microscopy showed NAD-ME is in mitochondria while NADP-ME is in chloroplasts of bundle sheath cells in the respective C4 types. In some C4 organs, it was apparent that subepidermal cells and water storage cells also contain some chloroplasts which have Rubisco, store starch, and thus perform C3 photosynthesis. In non-Kranz cotyledons of Halocharis gossypina and Haloxylon persicum, Rubisco was found in chloroplasts of both palisade and spongy mesophyll cells with the heaviest labelling in the layers of palisade cells, whereas C4 pathway proteins were low or undetectable. The pattern of starch accumulation correlated with the localization of Rubisco, being highest in the bundle sheath cells and lowest in the mesophyll cells of organs having Kranz anatomy. In NAD-ME-type Kranz organs (leaves and cotyledons of S. laricina and leaves of H. gossypina the granal index (length of appressed membranes as a percentage of total length of all membranes) of bundle sheath chloroplasts is 1.5 to 2.5 times higher than that of mesophyll chloroplasts. In contrast, in the NADP-ME-type Kranz organs (S. richteri leaves and cotyledons and H. persicum stems) the granal index of mesophyll chloroplasts is 1.5 to 2.2 times that of the bundle sheath chloroplasts. The mechanism of photosynthesis in these species is discussed in relation to structural differences.     

C(4) photosynthesis in terrestrial plants does not require Kranz anatomy

C(4) photosynthesis in terrestrial plants was thought to require Kranz anatomy because the cell wall between mesophyll and bundle sheath cells restricts leakage of CO(2). Recent work with the central Asian chenopods Borszczowia aralocaspica and Bienertia cycloptera show that C(4) photosynthesis functions efficiently in individual cells containing both the C(4) and C(3) cycles. These discoveries provide new inspiration for efforts to convert C(3) crops into C(4) plants because the anatomical changes required for C(4) photosynthesis might be less stringent than previously thought.