Coordination of the cell-specific distribution of the four subunits of glycine decarboxylase and of serine hydroxymethyltransferase in leaves of C3-C4 intermediate species from different genera

The cell-specific distribution of the four subunit proteins (P, L, T and H) of glycine decarboxylase (GDC) and of serine hydroxymethyltransferase (SHMT) has been studied in the leaves of C₃-C₄ intermediate and C₄ species of three genera (Flaveria, Moricandia and Panicum) using immunogold localization. Antibodies raised against these proteins from pea leaf mitochondria were used to probe Western blots of total leaf proteins of F. linearis Lag., M. arvensis (L.) DC and P. milioides Nees ex Trin. (C₃-C₄), and F. trinervia (Spring.) Mohr and P. miliaceum (L.) (C₄). For all species, each antibody recognised specifically a protein of similar molecular weight to that in pea leaves. In leaves of M. arvensis the P protein was present in the mitochondria of the bundle-sheath cells but was undetectable in those of the mesophyll, whereas the L, T and H proteins and SHMT were present in both cell types. The density of immunogold labelling of SHMT on the mitochondria of mesophyll cells was less than that on those of the bundle-sheath cells, which correlates with the relative activities of SHMT in these cell types. These data reveal that the lack of functional GDC in the mesophyll cells of M. arvensis, which is the principal biochemical reason for reduced photorespiration in this species, is due to the loss of a single subunit protein. This lack of coordinate expression of the subunit proteins of GDC within a photosynthetic cell represents a clear difference between M. arvensis and other C₃ and C₃-C₄ species. None of the GDC proteins was detectable in the mesophyll cells of the C₃-C₄ and C₄ Flaveria and Panicum species but all were present in the bundle-sheath cells. The differences in the distribution of the GDC proteins in leaves of the C₃-C₄ species studied are discussed in relation to the evolution of photosynthetic mechanisms.     

Cellular expression pattern of the glycine decarboxylase P protein in leaves of an intergeneric hybrid between the C3-C4 intermediate species Moricandia nitens and the C3 species Brassica napus

 An intergeneric hybrid plant was produced between the C₃-C₄ intermediate species Moricandia nitens and the C₃ species Brassica napus by sexual hybridization and in vitro embryo rescue. The hybrid nature of the plant was apparent in its morphology and flower pigmentation and was confirmed by leaf isozyme patterns. The overall plant morphology and the shape and thickness of leaves of the hybrid plant were intermediate between those of the parent species. However, the bundle-sheath cells of the hybrid resembled those of the C₃ parent and lacked the organelle development of the C₃-C₄ intermediate parent. Immunogold labelling for the presence of the P subunit of the mitochondrial glycine decarboxylase complex revealed a very similar labelling density on mitochondria in bundle-sheath and mesophyll cells in B. napus, while in  M. nitens the P subunit was only detectable in bundle sheath cells. In the hybrid the labelling density on mesophyll cell mitochondria was almost half of that on the bundle-sheath mitochondria. The CO₂ compensation point of the hybrid was significantly less than that of the C₃ parent but was not as low, nor as responsive to changes in light intensity, as for the C₃-C₄ parent. Received: 23 October 1997 / Accepted: 28 November 1997     

Structural and biochemical bases of photorespiration in C<Subscript>4</Subscript> plants: quantification of organelles and glycine decarboxylase

In C₄ plants, photorespiration is decreased relative to C₃ plants. However, it remains unclear how much photorespiratory capacity C₄ leaf tissues actually have. We thoroughly investigated the quantitative distribution of photorespiratory organelles and the immunogold localization of the P protein of glycine decarboxylase (GDC) in mesophyll (M) and bundle sheath (BS) cells of various C₄ grass species. Specific differences occurred in the proportions of mitochondria and peroxisomes in the BS cells (relative to the M cells) in photosynthetic tissues surrounding a vein: lower in the NADP-malic enzyme (NADP-ME) species having poorly formed grana in the BS chloroplasts, and higher in the NAD-malic enzyme (NAD-ME) and phosphoenolpyruvate carboxykinase (PCK) species having well developed grana. In all C₄ species, GDC was localized mainly in the BS mitochondria. When the total amounts of GDC in the BS mitochondria per unit leaf width were estimated from the immunogold labeling density and the quantity of mitochondria, the BSs of NADP-ME species contained less GDC than those of NAD-ME or PCK species. This trend was also verified by immunoblot analysis of leaf soluble protein. There was a high positive correlation between the degree of granal development (granal index) in the BS chloroplasts and the total amount of GDC in the BS mitochondria. The variations in the structural and biochemical features involved in photorespiration found among C₄ species might reflect differences in the O₂/CO₂ partial pressure and in the potential photorespiratory capacity of the BS cells.Abbreviations BS Bundle sheath - GDC Glycine decarboxylase - M Mesophyll - NAD-ME NAD-malic enzyme - NADP-ME NADP-malic enzyme - PCK Phosphoenolpyruvate carboxykinase     

Predominant localization of mitochondria enriched with glycine-decarboxylating enzymes in bundle sheath cells of Alternanthera tenella, a C3–C4 intermediate species

Mesophyll protoplasts and bundle sheath cells were prepared by enzymatic digestion of leaves of Alternanthera tenella, a C₃-C₄ intermediate species. The intercellular distribution of selected photosynthetic, photorespiratory and respiratory (mitochondrial) enzymes in these meso-phyll and bundle sheath cells was studied. The activity levels of photosynthetic enzymes such as PEP carboxylase (EC 4.1.1.31) or NAD-malic enzyme (EC 1.1.1.39) and photorespiratory enzymes such as glycolate oxidase (EC 1.1.3.1) or NADH-hydroxypyruvate reductase (EC 1.1.1.29) were similar in the two cell types. The activity levels of mitochondrial TCA cycle enzymes such as citrate synthase (EC 4.1.3.7) or fumarase (EC 4.2.1.2) were 2- to 3-fold higher in bundle sheath cells. On the other hand, the activity levels of mitochondrial photorespiratory enzymes, namely glycine decarboxylase (EC 2.1.2.10) and serine hydroxymethyltransferase (EC 2.1.2.1), were 6-9-fold higher in bundle sheath cells than in mesophyll protoplasts. Such preferential localization of mitochondria enriched with the glycine-decarboxylating system in the inner bundle sheath cells would result in efficient refixa-tion of CO₂ from not only photorespiration but also dark respiration before its exit from the leaf. We propose that predominant localization of mitochondria specialized in glycine decarboxylation in bundle sheath cells may form the basis of reduced photorespiration in this C₃-C₄ intermediate species.     

Photosynthesis of Grass Species Differing in Carbon Dioxide Fixation Pathways : VIII. Ultrastructural Characteristics of Panicum Species in the Laxa Group

Ultrastructural studies of leaves of seven Panicum species in or closely related to the Laxa group and classified as C₃, C₄ or C₃-C₄ intermediate were undertaken to examine features associated with C₃ and C₄ photosynthesis. The C₃ species Panicum rivulare Trin. had few organelles in bundle sheath cell profiles (2 chloroplasts, 1.1 mitochondria, and 0.3 peroxisomes per cell section) compared to an average of 10.6 chloroplasts, 17.7 mitochondria, and 3.2 peroxisomes per bundle sheath cell profile for three C₃-C₄ species, Panicum milioides Nees ex Trin., Panicum decipiens Nees ex Trin. and Panicum schenckii Hack. However, two other C₃ species, Panicum laxum Sw. and Panicum hylaeicum Mez, contained about 0.7, 0.5, and 0.3 as many chloroplasts, mitochondria, and peroxisomes, respectively, as in bundle sheath cell profiles of the C₃-C₄ species. Chloroplasts and mitochondria in bundle sheath cells were larger than those in mesophyll cells for the C₄ species Panicum prionitis Griseb. and the C₃-C₄ species, but in C₃ species the organelles were similar in size or were smaller in the bundle sheath cells. The C₃-C₄ species and P. laxum and P. hylaeicum exhibited an unusually close association of organelles in bundle sheath cells with mitochondria frequently surrounded in profile by chloroplasts. The high concentrations in bundle sheath cells of somewhat larger organelles than in mesophyll cells correlates with the reduced photorespiration of the C₃-C₄ species.     

Origin and evolution of C4 photosynthesis in the tribe Salsoleae (Chenopodiaceae) based on anatomical and biochemical types in leaves and cotyledons

 The Chenopodiaceae genus Salsola contains a large number of species with C₄ photosynthesis. Along with derivative genera they have a prominent position among the desert vegetation of Asia and Africa. About 130 species from Asia and Africa were investigated to determine the occurrence of C₃ versus C₄ syndrome in leaves and cotyledons, and to study specific anatomical and biochemical features of photosynthesis in both photosynthetic organs. The species studied belong to all six previously identified sections of the tribe Salsoleae based on morphological characters. Types of photosynthesis were identified using carbon ¹³C/¹²C isotope fractionation. The representatives of all systematic groups were investigated for mesophyll anatomy and biochemical subtypes by determination of enzyme activity (RUBPC, PEPC, NAD- and NADP-ME and AAT) and primary photosynthetic products. Two photosynthetic types (C₃ and C₄) and two biochemical subtypes (NAD- and NADP-ME) were identified in both leaves and cotyledons. Both Kranz and non-Kranz type anatomy were found in leaves and cotyledons, but cotyledons had more diversity in anatomical structure. Strong relationships between anatomical types and biochemical subtypes in leaves and cotyledons were shown. We found convincing evidence for a similar pattern of structural and biochemical features of photosynthesis in leaves and cotyledons within systematic groups, and evaluated their relevance at the evolutionary level. We identified six groups in tribe Salsoleae with respect to photosynthetic types and mesophyll structure in leaves and cotyledons. Two separate lineages of biochemical and anatomical evolution within Salsoleae were demonstrated based on studies of leaves and cotyledons. The sections Caroxylon, Malpighipila, Cardiandra and Belanthera have no C₃ species and only the NAD-ME C₄ subtype has been found in leaves. We suggest the C₄ species in the NADP-ME lineage evolved in Coccosalsola and Salsola sections, and originated in the subsection Arbuscula. Coccosalsola contains many species with C₃ and/or C₃-C₄ intermediate photosynthesis. Within these main evolutionary lineages, species of different taxonomic groups (sections and subsections) had differences in anatomical or/and biochemical features in leaves and cotyledons. We conclude that structural and biochemical changes in the photosynthetic apparatus in species of the tribe Salsoleae were a key factor in their evolution and broad distribution in extreme desert environments. Received January 25, 2001 Accepted July 17, 2001     

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.     

STUDIES ON THE LEAF OF POPULUS DELTOIDES (SALICACEAE): ULTRASTRUCTURE,PLASMODESMATAL FREQUENCY,AND SOLUTE CONCENTRATIONS

The minor veins and contiguous tissues of mature leaves of Populus deltoides Bartr. ex Marsh. were examined with the electron microscope to determine the ultrastructural characteristics of the component cells and to determine the structure, distribution, and frequency of plasmodesmata between the various cell types. In addition, plasmolytic studies were carried out to determine the solute concentrations of the various cell types of the minor veins and contiguous tissues. The cells comprising the mesophyll and bundle sheath contain all the components typical of photosynthetic cells. Paraveinal mesophyll cells and bundle-sheath cells have fewer microbodies and smaller chloroplasts than do palisade parenchyma cells. Vascular parenchyma and companion cells tend to intergrade with one another structurally but can be distinguished from one another by their characteristic plastids. The mature, enucleate sieve-tube member is lined by a parietal layer of cytoplasm consisting of plasmalemma, endoplasmic reticulum, mitochondria, plastids, and P-protein. Plasmodesmata occur along all possible routes from the palisade parenchyma cells to the sieve tubes of the minor veins, and their frequency increases with increasing proximity to the sieve-tube members. Plasmolytic studies revealed that the paraveinal mesophyll cells had a higher C₅₀ (estimated mannitol concentration plasmolyzing, on the average, 50% of a given cell type) than any other cell type of the leaf. Concentration gradients existed along the palisade cell/bundle-sheath cell/companion cell (or vascular parenchyma cell) route as well as along the paraveinal mesophyll cell/bundle-sheath cell/companion cell (or vascular parenchyma cell) route. Considering the frequency of plasmodesmata along these routes, it is conceivable that photosynthate diffuses from palisade cells to the companion cells along concentration gradients. Within the minor veins, the C₅₀ was higher for sieve-tube members than for either companion cells or vascular parenchyma cells, indicating that loading of the sieve tubes is an active, energy-dependent process.     

Photosynthesizing Tissue Development in C4 Cotyledons of Two Salsola Species (Chenopodiaceae)

The quantitative anatomy of developing cotyledons of NAD-malic enzyme species Salsola incanescens and NADP-malic enzyme species S. paulsenii (Chenopodiaceae) was studied. S. incanescens belongs to the group of species with foliar type of seedling development characterized by slowly growing cotyledons and a rosette form at juvenility. The rosette is the consequence of fast leaf formation, which was correlated with a low rate of leaf growth. S. paulsenii belongs to the group with the cotyledonous type of seedling development. A high growth rate of cotyledons, slow leaf formation, and absence of the rosette characterize this type. Slow leaf formation was correlated with a high rate of leaf growth. The Kranz–anatomy in cotyledons of S. incanescens (atriplicoid type) and S. paulsenii (salsoloid type) determines the duration of cotyledon development proceeding for 15 days after seed germination. The rate of growth changes during the developmental period was correlated with the type of seedling development. Cotyledons of a foliar species S. incanescens exhibit 2 to 5 times slower growth changes in cotyledon area, width, thickness, volume of mesophyll and bundle sheath cells, and number of chloroplasts per bundle sheath cell than the cotyledons of a cotyledonous species S. paulsenii. During cotyledon development in both species, the number of chloroplasts per mesophyll cell remained unchanged, and developmental changes in the bundle sheath occurred at higher rate than in mesophyll cells. Thus, these two indices seem to be independent of the type of Kranz–anatomy. The presence of atriplicoid type cotyledons in the species with salsoloid structure of true leaves might indicate a close genetic relationship between these two patterns of Kranz-anatomy.     

Spatial and Temporal Influences on the Cell-Specific Distribution of Glycine Decarboxylase in Leaves of Wheat (Triticum aestivum L.) and Pea (Pisum sativum L.)

The distribution of glycine decarboxylase (GDC) in leaves of pea (Pisum sativum L.) and wheat (Triticum aestivum L.) has been investigated using immunogold labeling of the P-protein subunit of the GDC complex. Mitochondria in photosynthetic mesophyll cells were densely labeled, whereas those in nonphotosynthetic vascular parenchyma and epidermal cells were only weakly labeled. In pea leaves the density of immunogold labeling on mitochondria in the chloroplast-containing bundle sheath and stomatal guard cells was intermediate between that in mesophyll and epidermal cells. In both species the density of labeling on mitochondria in a cell appeared to reflect the photosynthetic capacity of the cell. This relationship was further examined in wheat where a natural developmental gradient exists along the lamina such that cell maturity increases with distance from the basal meristem. In this case the density of labeling on mesophyll cell mitochondria increased with photosynthetic development and with increasing maturity of the cell. Vascular cell mitochondria, however, became less densely labeled as the cells matured. The results indicate a close, positive correlation between the concentration of GDC in the mitochondria and the photosynthetic status of the host cell. This relationship is maintained effectively under the influence of both spatial (i.e. cellular differentiation across the lamina) and temporal (i.e. cellular development along the lamina) constraints.     

Distribution of Photosynthetic Enzymes between Mesophyll, Specialized Parenchyma and Bundle Sheath Cells of Arundinella hirta

Arundinella hirta L. is a C₄ plant having an unusual C₄ leaf anatomy. Besides mesophyll and bundle sheath cells, A. hirta leaves have specialized parenchyma cells which look morphologically like bundle sheath cells but which lack vascular connections and are located between veins, running parallel to them. Activities of phosphoenolpyruvate and ribulose-1,5-bisphosphate carboxylases and phosphoenolpyruvate carboxykinase, NADP-and NAD-malic enzymes were determined for whole leaf extracts and isolated mesophyll protoplasts, specialized parenchyma cells, and bundle sheath cells. The data indicate that A. hirta is a NADP-malic enzyme type C₄ species. In addition, specialized parenchyma cells and bundle sheath cells are enzymatically alike. Compartmentation of enzymes followed the C₄ pattern with phosphoenolpyruvate carboxylase being restricted to mesophyll cells while ribulose-1,5-bisphosphate carboxylase and decarboxylating enzymes were restricted to bundle sheath and specialized parenchyma cells.     

Leaf anatomy of c(3)-c(4) species as related to evolution of c(4) photosynthesis

This study was undertaken to examine the degree of Kranz anatomy development in the species intermediate to C₃ and C₄ types (C₃-C₄) in Panicum, Neurachne, Flaveria, and Moricandia. In each genus, C₃ and/or C₄ species were used for comparison. Leaf transections from each species were examined by light and transmission electron microscopy. The percentages of leaf photosynthetic cell profiles partitioned to bundle sheaths were higher in C₄ than in C₃ species, while C₃-C₄ species tended to be in between. However, percentages for C₃-C₄ species in Moricandia and some C₃-C₄Flaveria species were not greater than C₃. When expressed on a cell profile area basis, C₃-C₄ species partitioned more photosynthetic tissue to bundle sheaths than C₃ species in Moricandia, but not in Flaveria. Neurachne minor S. T. Blake (C₃-C₄) partitioned a very small portion of cell profile area to the inner bundle sheaths (5%) compared to Neurachne munroi F. Muell (C₄) (21%). The percentage of organelles partitioned to bundle sheaths was much greater in C₃-C₄ than in C₃ species. The average C₃ percentages for mitochondria plus peroxisomes were 19, 8, and 19.5% for Neurachne, Flaveria, and Moricandia, respectively, compared to 41, 29, and 46.5% for the C₃-C₄ species. The CO₂ compensation concentration was negatively related to the partitioning of tissue to bundle sheaths and to the percentage of organelles in bundle sheaths. It is concluded that all of the C₃-C₄ species examined have developed some degree of Kranz anatomy and that this altered anatomy is involved in their reduced apparent photorespiration.     

Salsola arbusculiformis, a C3-C4Intermediate in Salsoleae (Chenopodiaceae)

Salsola arbusculiformis is identified as a C₃–C₄intermediatespecies based on anatomical, biochemical and physiological characteristics.This is the first report of a naturally occurring intermediatespecies in the Chenopodiaceae, the family with the largest numberof C₄species amongst the dicots. In the genus Salsola, mostspecies have Salsoloid anatomy with Kranz type bundle sheathcells and C₄photosynthesis, while a few species have Sympegmoidanatomy and were found to have non-Kranz type bundle sheathcells and C₃photosynthesis. In the cylindrical leaves of C₄Salsolawith Salsoloid type anatomy, there is a continuous layer ofdistinct, chlorenchymatous Kranz type bundle sheath cells surroundedby a single layer of mesophyll cells; whereas species with Sympegmoidtype anatomy have an indistinct bundle sheath with few chloroplastsand multiple layers of chlorenchymatous mesophyll cells. However,S. arbusculiformis has intermediate anatomical features. Whileit has two-to-three layers of mesophyll cells, characteristicof Sympegmoid anatomy, it has distinctive, Kranz-like bundlesheath cells with numerous chloroplasts and mitochondria. Measurementsof its CO₂compensation point and CO₂response of photosynthesisshow S. arbusculiformis functions as an intermediate specieswith reduced levels of photorespiration. The primary means ofreducing photorespiration is suggested to be by refixing photorespiredCO₂in bundle sheath cells, since analysis of photosyntheticenzymes (activity and immunolocalization) and¹⁴CO₂labellingof initial fixation products suggests minimal operation of aC₄cycle. Copyright 2001 Annals of Botany Company Immunolocalization, photosynthetic enzymes, C₃–C₄intermediate, C₄-plants, leaf anatomy, Chenopodiaceae, Salsola arbusculiformis     

Characterization of C₃--C₄ intermediate species in the genus Heliotropium L. (Boraginaceae): anatomy, ultrastructure and enzyme activity

Photosynthetic pathway characteristics were studied in nine species of Heliotropium (sensu lato, including Euploca), using assessments of leaf anatomy and ultrastructure, activities of PEP carboxylase and C₄ acid decarboxylases, and immunolocalization of ribulose 1·5‐bisphosphate carboxylase/oxygenase (Rubisco) and the P‐subunit of glycine decarboxylase (GDC). Heliotropium europaeum, Heliotropium calcicola and Heliotropium tenellum are C₃ plants, while Heliotropium texanum and Heliotropium polyphyllum are C₄ species. Heliotropium procumbens and Heliotropium karwinskyi are functionally C₃, but exhibit ‘proto‐Kranz’ anatomy where bundle sheath (BS) cells are enlarged and mitochondria primarily occur along the centripetal (inner) wall of the BS cells; GDC is present throughout the leaf. Heliotropium convolvulaceum and Heliotropium greggii are C₃–C₄ intermediates, with Kranz‐like enlargement of the BS cells, localization of mitochondria along the inner BS wall and a loss of GDC in the mesophyll (M) tissue. These C₃–C₄ species of Heliotropium probably shuttle photorespiratory glycine from the M to the BS tissue for decarboxylation. Heliotropium represents an important new model for studying C₄ evolution. Where existing models such as Flaveria emphasize diversification of C₃–C₄ intermediates, Heliotropium has numerous C₃ species expressing proto‐Kranz traits that could represent a critical initial phase in the evolutionary origin of C₄ photosynthesis.     

Photosynthetic Characteristics of Portulaca grandiflora, a Succulent C(4) Dicot : CELLULAR COMPARTMENTATION OF ENZYMES AND ACID METABOLISM

The succulent, cylindrical leaves of the C₄ dicot Portulaca grandiflora possess three distinct green cell types: bundle sheath cells (BSC) in radial arrangement around the vascular bundles; mesophyll cells (MC) in an outer layer adjacent to the BSC; and water storage cells (WSC) in the leaf center. Unlike typical Kranz leaf anatomy, the MC do not surround the bundle sheath tissue but occur only in the area between the bundle sheath and the epidermis. Intercellular localization of photosynthetic enzymes was characterized using protoplasts isolated enzymatically from all three green cell types.     

Distribution of photorespiratory enzymes between bundle-sheath and mesophyll cells in leaves of the C3−C4 intermediate species Moricandia arvensis (L.) DC

In order to study the location of enzymes of photorespiration in leaves of the C₃–C₄ intermediate species Moricandia arvensis (L.). DC, protoplast fractions enriched in mesophyll or bundlesheath cells have been prepared by a combination of mechanical and enzymic techniques. The activities of the mitochondrial enzymes fumarase (EC 4.2.1.2) and glycine decarboxylase (EC 2.1.2.10) were enriched by 3.0- and 7.5-fold, respectively, in the bundle-sheath relative to the mesophyll fraction. Enrichment of fumarase is consistent with the larger number of mitochondria in bundle-sheath cells relative to mesophyll cells. The greater enrichment of glycine decarboxylase indicates that the activity is considerably higher on a mitochondrial basis in bundle-sheath than in mesophyll cells. Serine hydroxymethyltransferase (EC 2.1.2.1) activity was enriched by 5.3-fold and glutamate-dependent glyoxylate-aminotransferase (EC 2.6.1.4) activity by 2.6-fold in the bundle-sheath relative to the mesophyll fraction. Activities of serine- and alanine-dependent glyoxylate aminotransferase (EC 2.6.1.45 and EC 2.6.1.4), glycollate oxidase (EC 1.1.3.1), hydroxypyruvate reductase (EC 1.1.1.81), glutamine synthetase (EC 6.3.1.2) and phosphoribulokinase (EC 2.7.1.19) were not significantly different in the two fractions. These data provide further independent evidence to complement earlier immunocytochemical studies of the distribution of photorespiratory enzymes in the leaves of this species, and indicate that while mesophyll cells of M. arvensis have the capacity to synthesize glycine during photorespiration, they have only a low capacity to metabolize it. We suggest that glycine produced by photorespiratory metabolism in the mesophyll is decarboxylated predominantly by the mitochondria in the bundle sheath.Abbreviation RuBP ribulose 1,5-bisphosphate     

Evolution of C4 Photosynthesis in Flaveria Species : Isoforms of NADP-Malic Enzyme

NADP-malic enzyme (NADP-ME, EC 1.1.1.40), a key enzyme in C₄ photosynthesis, provides CO₂ to the bundle-sheath chloroplasts, where it is fixed by ribulose-1,5-bisphosphate carboxylase/oxygenase. We characterized the isoform pattern of NADP-ME in different photosynthetic species of Flaveria (C₃, C₃-C₄ intermediate, C₄-like, C₄) based on sucrose density gradient centrifugation and isoelectric focusing of the native protein, western-blot analysis of the denatured protein, and in situ immunolocalization with antibody against the 62-kD C₄ isoform of maize. A 72-kD isoform, present to varying degrees in all species examined, is predominant in leaves of C₃ Flaveria spp. and is also present in stem and root tissue. By immunolabeling, NADP-ME was found to be mostly localized in the upper palisade mesophyll chloroplasts of C₃ photosynthetic tissue. Two other isoforms of the enzyme, with molecular masses of 62 and 64 kD, occur in leaves of certain intermediates having C₄ cycle activity. The 62-kD isoform, which is the predominant highly active form in the C₄ species, is localized in bundle-sheath chloroplasts. Among Flaveria spp. there is a 72-kD constitutive form, a 64-kD form that may have appeared during evolution of C₄ metabolism, and a 62-kD form that is necessary for the complete functioning of C₄ photosynthesis.     

Carbon isotope discrimination in C3-C4 intermediates

Carbon isotope discrimination in C₃–C₄ intermediates is determined by fractionations during diffusion and the biochemical fractionations occurring during CO₂ fixation. These biochemical fractionations in turn depend on the fractionation by Rubisco in the mesophyll, the amount of CO₂ fixation. These biochemical fractionations in turn depend on the fractionation by Rubisco in the mesophyll, the amount of CO₂ fixation occurring in the bundle sheath, the extent of bundle-sheath leakiness and the contribution which C₄-cycle activity makes to the CO₂ pool there. In most instances, carbon isotope discrimination in C₃–C₄ intermediates is C₃-like because only a small fraction of the total carbon fixed is fixed in the bundle sheath. In particular, this must be the case for Flaveria intermediates which initially fix substantial amounts of CO₂ into C₄-acids. In C₃–C₄ intermediates that refix photorespiratory CO₂ alone, it is possible for carbon isotope discrimination to be greater than in C₃-species, particularly at low CO₂ pressures or at high leaf temperatures. Short-term measurements of carbon isotope discrimination and gas exchange of leaves can be used to study the photosynthetic pathways of C₃-C₄ intermediates and their hybrids as has recently been done for C₃ and C₄ species.     

Leaf hydraulic conductance varies with vein anatomy across Arabidopsis thaliana wild‐type and leaf vein mutants

Leaf venation is diverse across plant species and has practical applications from paleobotany to modern agriculture. However, the impact of vein traits on plant performance has not yet been tested in a model system such as Arabidopsis thaliana. Previous studies analysed cotyledons of A. thaliana vein mutants and identified visible differences in their vein systems from the wild type (WT). We measured leaf hydraulic conductance (K_leaf), vein traits, and xylem and mesophyll anatomy for A. thaliana WT (Col‐0) and four vein mutants (dot3‐111 and dot3‐134, and cvp1‐3 and cvp2‐1). Mutant true leaves did not possess the qualitative venation anomalies previously shown in the cotyledons, but varied quantitatively in vein traits and leaf anatomy across genotypes. The WT had significantly higher mean K_leaf. Across all genotypes, there was a strong correlation of K_leaf with traits related to hydraulic conductance across the bundle sheath, as influenced by the number and radial diameter of bundle sheath cells and vein length per area. These findings support the hypothesis that vein traits influence K_leaf, indicating the usefulness of this mutant system for testing theory that was primarily established comparatively across species, and supports a strong role for the bundle sheath in influencing K_leaf.     

Correlations among anatomical,morphological, chemical and agronomic characteristics of leaf blades in Panicum maximum genotypes

Tropical forage grasses present high growth rates and biomass yields, partly due to its C₄ photosynthetic pathway. Considering this, the anatomy of the grasses related to morphology and chemical composition of leaf blades may influence consumption and digestibility, thus interfering in the forage quality. Agronomic, morphological, anatomical and chemical characteristics of leaves of nine Panicum maximum genotypes were evaluated in Brazil, to verify if these characteristics and the associations among them may influence the quality of the leaf blades. A randomized complete blocks design was used with nine treatments and three replications. Three evaluation harvests were done in the rainy season and one in the dry season to evaluate forage yields and quality. Quality was determined through NIRS – Near Infrared Spectrometer. One day prior to each harvest, four leaf blades per plot were harvested for morphological and anatomical evaluations. Results were subjected to analysis of variance and mean comparison by Tukey test, and to simple linear and canonical correlations by SAS. Leaf width was positively correlated with mesophyll. The parenchyma bundle sheath was associated with leaf area and specific leaf area. Neutral detergent fibre was positively correlated with the parenchyma bundle sheath area and specific leaf area. The specific leaf area was negatively correlated with in vitro organic matter digestibility. Morphological differences among P. maximum genotypes did not interfere in biomass accumulation. Considering this, leaf width may be a supplementary tool, that may be used in the early phases of the process of genotype selection, for discriminating qualitatively promising high yielding materials.