Genome‐wide transcript analysis of early maize leaf development reveals gene cohorts associated with the differentiation of C4 Kranz anatomy

Photosynthesis underpins the viability of most ecosystems, with C₄ plants that exhibit ‘Kranz’ anatomy being the most efficient primary producers. Kranz anatomy is characterized by closely spaced veins that are encircled by two morphologically distinct photosynthetic cell types. Although Kranz anatomy evolved multiple times, the underlying genetic mechanisms remain largely elusive, with only the maize scarecrow gene so far implicated in Kranz patterning. To provide a broader insight into the regulation of Kranz differentiation, we performed a genome‐wide comparative analysis of developmental trajectories in Kranz (foliar leaf blade) and non‐Kranz (husk leaf sheath) leaves of the C₄ plant maize. Using profile classification of gene expression in early leaf primordia, we identified cohorts of genes associated with procambium initiation and vascular patterning. In addition, we used supervised classification criteria inferred from anatomical and developmental analyses of five developmental stages to identify candidate regulators of cell‐type specification. Our analysis supports the suggestion that Kranz anatomy is patterned, at least in part, by a SCARECROW/SHORTROOT regulatory network, and suggests likely components of that network. Furthermore, the data imply a role for additional pathways in the development of Kranz leaves.     

Cellular expression of C<Subscript>3</Subscript> and C<Subscript>4</Subscript> photosynthetic enzymes in the amphibious sedge<Emphasis Type="Italic"> Eleocharis retroflexa</Emphasis> ssp.<Emphasis Type="Italic"> chaetaria</Emphasis>

The amphibious leafless sedge Eleocharis retroflexa ssp. chaetaria expresses C₄-like biochemical characteristics in both the terrestrial and submerged forms. Culms of the terrestrial form have Kranz anatomy, whereas those of the submerged form have Kranz-like anatomy combined with anatomical features of aquatic plant leaves. We examined the immunolocalization of C₃ and C₄ enzymes in culms of the two forms. In both forms, phosphoenolpyruvate carboxylase; pyruvate, Pi dikinase; and NAD-malic enzyme were compartmentalized between the mesophyll (M) and Kranz cells, but their levels were somewhat reduced in the submerged form. In the terrestrial form, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) occurred mainly in the Kranz cells, and weakly in the M chloroplasts. In the submerged form, the rubisco occurred at higher levels in the M cells than in the terrestrial form. In both forms, the C₄ pattern of enzyme expression was clearer in the M cells adjacent to Kranz cells than in distant M cells. During the transition from terrestrial to submerged conditions, the enzyme expression pattern changed in submerged mature culms that had been formed in air before submergence, and matched that in culms newly developed underwater. It seems that effects of both environmental and developmental factors overlap in the C₄ pattern expression in this plant.     

Structure and enzyme expression in photosynthetic organs of the atypical C4 grass Arundinella hirta

In its leaf blade, Arundinella hirta has unusual Kranz cells that lie distant from the veins (distinctive cells; DCs), in addition to the usual Kranz units composed of concentric layers of mesophyll cells (MCs) and bundle sheath cells (BSCs; usual Kranz cells) surrounding the veins. We examined whether chlorophyllous organs other than leaf blades—namely, the leaf sheath, stem, scale leaf, and constituents of the spike—also have this unique anatomy and the C₄ pattern of expression of photosynthetic enzymes. All the organs developed DCs to varying degrees, as well as BSCs. The stem, rachilla, and pedicel had C₄-type anatomy with frequent occurrence of DCs, as in the leaf blade. The leaf sheath, glume, and scale leaf had a modified C₄ anatomy with MCs more than two cells distant from the Kranz cells; DCs were relatively rare. An immunocytochemical study of C₃ and C₄ enzymes revealed that all the organs exhibited essentially the same C₄ pattern of expression as in the leaf blade. In the scale leaf, however, intense expression of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) occurred in the MCs as well as in the BSCs and DCs. In the leaf sheath, the distant MCs also expressed Rubisco. In Arundinella hirta, it seems that the ratio of MC to Kranz cell volumes, and the distance from the Kranz cells, but not from the veins, affects the cellular expression of photosynthetic enzymes. We suggest that the main role of DCs is to keep a constant quantitative balance between the MCs and Kranz cells, which is a prerequisite for effective C₄ pathway operation.     

Occurrence of C3 and C4 photosynthesis in cotyledons and leaves of Salsola species (Chenopodiaceae)

Most species of the genus Salsola (Chenopodiaceae) that have been examined exhibit C₄ photosynthesis in leaves. Four Salsola species from Central Asia were investigated in this study to determine the structural and functional relationships in photosynthesis of cotyledons compared to leaves, using anatomical (Kranz versus non-Kranz anatomy, chloroplast ultrastructure) and biochemical (activities of photosynthetic enzymes of the C₃ and C₄ pathways, ¹⁴C labeling of primary photosynthesis products and ¹³C/¹²C carbon isotope fractionation) criteria. The species included S. paulsenii from section Salsola, S. richteri from section Coccosalsola, S. laricina from section Caroxylon, and S. gemmascens from section Malpigipila. The results show that all four species have a C₄ type of photosynthesis in leaves with a Salsoloid type Kranz anatomy, whereas both C₃ and C₄ types of photosynthesis were found in cotyledons. S. paulsenii and S. richteri have NADP- (NADP-ME) C₄ type biochemistry with Salsoloid Kranz anatomy in both leaves and cotyledons. In S. laricina, both cotyledons and leaves have NAD-malic enzyme (NAD-ME) C₄ type photosynthesis; however, while the leaves have Salsoloid type Kranz anatomy, cotyledons have Atriplicoid type Kranz anatomy. In S. gemmascens, cotyledons exhibit C₃ type photosynthesis, while leaves perform NAD-ME type photosynthesis. Since the four species studied belong to different Salsola sections, this suggests that differences in photosynthetic types of leaves and cotyledons may be used as a basis or studies of the origin and evolution of C₄ photosynthesis in the family Chenopodiaceae.This revised version was published online in October 2005 with corrections to the Cover Date.     

The efficiency of the CO2-concentrating mechanism during single-cell C4 photosynthesis

The photosynthetic efficiency of the CO₂‐concentrating mechanism in two forms of single‐cell C₄ photosynthesis in the family Chenopodiaceae was characterized. The Bienertioid‐type single‐cell C₄ uses peripheral and central cytoplasmic compartments (Bienertia sinuspersici), while the Borszczowioid single‐cell C₄ uses distal and proximal compartments of the cell (Suaeda aralocaspica). C₄ photosynthesis within a single‐cell raises questions about the efficiency of this type of CO₂‐concentrating mechanism compared with the Kranz‐type. We used measurements of leaf CO₂ isotope exchange (Δ¹³C) to compare the efficiency of the single‐cell and Kranz‐type forms of C₄ photosynthesis under various temperature and light conditions. Comparisons were made between the single‐cell C₄ and a sister Kranz form, S. eltonica[NAD malic enzyme (NAD ME) type], and with Flaveria bidentis[NADP malic enzyme (NADP‐ME) type with Kranz Atriplicoid anatomy]. There were similar levels of Δ¹³C discrimination and CO₂ leakiness (?) in the single‐cell species compared with the Kranz‐type. Increasing leaf temperature (25 to 30 °C) and light intensity caused a decrease in Δ¹³C and ? across all C₄ types. Notably, B. sinuspersici had higher Δ¹³C and ? than S. aralocaspica under lower light. These results demonstrate that rates of photosynthesis and efficiency of the CO₂‐concentrating mechanisms in single‐cell C₄ plants are similar to those in Kranz‐type.     

Does Bienertia cycloptera with the single-cell system of C(4) photosynthesis exhibit a seasonal pattern of delta (13)C values in nature similar to co-existing C (4) Chenopodiaceae having the dual-cell (Kranz) system?

Family Chenopodiaceae is an intriguing lineage, having the largest number of C₄ species among dicots, including a number of anatomical variants of Kranz anatomy and three single-cell C₄ functioning species. In some previous studies, during the culture of Bienertia cycloptera Bunge ex Boiss., carbon isotope values (δ¹³C values) of leaves deviated from C₄ to C₃−C₄ intermediate type, raising questions as to its mode of photosynthesis during growth in natural environments. This species usually co-occurs with several Kranz type C₄ annuals. The development of B. cycloptera morphologically and δ¹³C values derived from plant samples (cotyledons, leaves, bracts, shoots) were analyzed over a complete growing season in a salt flat in north central Iran, along with eight Kranz type C₄ species and one C₃ species. For a number of species, plants were greenhouse-grown from seeds collected from the site, in order to examine leaf anatomy and C₄ biochemical subtype. Among the nine C₄ species, the cotyledons of B. cycloptera, and of the Suaeda spp. have the same respective forms of C₄ anatomy occurring in leaves, while cotyledons of members of tribe Caroxyloneae lack Kranz anatomy, which is reflected in the δ¹³C values found in plants grown in the natural habitat. The nine C₄ species had average seasonal δ¹³C values of −13.9‰ (with a range between species from −11.3 to −15.9‰). The measurements of δ¹³C values over a complete growing season show that B. cycloptera performs C₄ photosynthesis during its life cycle in nature, similar to Kranz type species, with a seasonal average δ¹³C value of −15.2‰. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

C4 photosynthetic modifications in the evolutionary transition from land to water in aquatic grasses

Cladistic analysis supports the conclusion that the Orcuttieae tribe of C₄ grasses reflect evolution from a terrestrial ancestry into seasonal pools. All nine species in the tribe exhibit adaptations to the aquatic environment, evident in the structural characteristics of the juvenile foliage, which persist submerged for 1–3 months prior to metamorphosis to the terrestrial foliage. Aquatic leaves of the least derived or basal genus Neostapfia have few morphological and anatomical characteristics specialized to the aquatic environment and have retained full expression of the C₄ pathway, including Kranz anatomy. Orcuttia species have many derived characteristics and are more specialized to the aquatic environment. These latter species germinate earlier in the season and persist in the submerged stage longer than Neostapfia and evidence from the literature indicates length of submergence is positively correlated with fitness components. Aquatic leaves of Orcuttia species lack Kranz or PCR bundle sheath anatomy, yet ¹⁴C-pulse chase studies indicate >95% malate + aspartate as the initial products of photosynthesis and these products turn over rapidly to phosphorylated sugars, indicating a tight coupling of the C₄ and C₃ cycles. Presence of the C₄ pathway is further supported by enzymological data. Contemporary dogma that Kranz anatomy is a sine qua non for operation of the C₄ pathway is contradicted by the patterns in Orcuttia; however, it is unknown whether the pathway acts as a CO₂ concentrating mechanism in these aquatic plants. Received: 12 June 1997 / Accepted: 10 February 1997     

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.     

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.     

Coleataenia prionitis,a C4-like species in the Poaceae

C₄-like plants represent the penultimate stage of evolution from C₃ to C₄ plants. Although Coleataenia prionitis (formerly Panicum prionitis) has been described as a C₄ plant, its leaf anatomy and gas exchange traits suggest that it may be a C₄-like plant. Here, we reexamined the leaf structure and biochemical and physiological traits of photosynthesis in this grass. The large vascular bundles were surrounded by two layers of bundle sheath (BS): a colorless outer BS and a chloroplast-rich inner BS. Small vascular bundles, which generally had a single BS layer with various vascular structures, also occurred throughout the mesophyll together with BS cells not associated with vascular tissue. The mesophyll cells did not show a radial arrangement typical of Kranz anatomy. These features suggest that the leaf anatomy of C. prionitis is on the evolutionary pathway to a complete C₄ Kranz type. Phosphoenolpyruvate carboxylase (PEPC) and pyruvate, Pi dikinase occurred in the mesophyll and outer BS. Glycine decarboxylase was confined to the inner BS. Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) accumulated in the mesophyll and both BSs. C. prionitis had biochemical traits of NADP-malic enzyme type, whereas its gas exchange traits were close to those of C₄-like intermediate plants rather than C₄ plants. A gas exchange study with a PEPC inhibitor suggested that Rubisco in the mesophyll could fix atmospheric CO₂. These data demonstrate that C. prionitis is not a true C₄ plant but should be considered as a C₄-like plant.

     

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.     

C3 and C4 Photosynthetic and Anatomical forms of Alloteropsis semialata (R.Br.) Hitchcock: I. Variability in Photosynthetic Characteristics, Water Utilization Efficiency and Leaf Anatomy

The grass Alloteropsis semialata (R.Br.) Hitchcock is uniquein that both Kranz and non-Kranz leaf anatomy has been reportedin this species. The present study investigates Kranz formsof A. semialata collected from a single ecological niche. Theseplants exhibit morphological and anatomical differences withrespect to leaf area, stomatal size and stomatal distribution.Carbon dioxide and water exchange measurements in the two formsshow the expected pattern of higher photosynthetic rate andhigher water utilization efficiency associated with Kranz anatomy.No intermediate physiological response or anatomical form wasobserved in this sample. Alloteropsis semialata (R.Br.) Hitchcock, C₃ photosynthetic, C₄ photosynthesis, water utilization, leaf anatomy, Kranz anatomy     

Structural features of NAD-malic enzyme type C4 Eleocharis: An additional report of C4 acid decarboxylation types of the cyperaceae

The genusEleocharis, a blade-less sedge group, has been very recently recorded to include NAD-malic enzyme type C₄ species. The ultrastructural features of culms of two C₄ representatives in the genus were examined in relation to the C₄ acid decarboxylation type. They possessed non-chlorophyllous mestome sheath cells between mesophyll cells and Kranz cells, and were confirmed biochemically to be NAD-malic enzyme type. The oval or lenticular chloroplasts with well-developed grana are scattered in the Kranz cells with abundant large mitochondria, and do not show such centripetal position as is known in the “classical NAD-malic enzyme type”. The suberized lamellae occur in the mestome sheath cells internally surrounding the Kranz sheath and may contribute to maintaining high CO₂ concentration in the Kranz cells. These new structural features of the NAD-malic enzyme type found inEleocharis are added to the structural and functional relationships of the C₄ types in the Cyperaceae reported previously     

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).     

Evidence for a C4 NADP-ME photosynthetic pathway in Vetiveria zizanioides Stapf

ABSTRACT

Leaf anatomy (light and transmission electron microscopy), immunogold localization of Rubisco, photosynthetic enzyme activities, CO₂ assimilation and stomatal conductance were studied in Vetiveria zizanioides Stapf., a graminaceous plant native to tropical and subtropical areas, and cultivated in temperate climates (Northwestern Italy). Leaves possess a NADP-ME Kranz anatomy with bundle sheath cells containing chloroplasts located in a centrifugal position. Dimorphic chloroplasts were also observed; they are agranal and starchy in the bundle sheath and granal starchless in the mesophyll cells. Rubisco immunolocalization studies indicate that this enzyme occurs solely in the bundle sheath chloroplasts. Pyruvate-orthophosphate dikinase, NADP-dependent malate dehydrogenase (NADP-MDH), NADP-dependent malic enzyme (NADP-ME), PEP-carboxykinase and NAD-dependent malic enzyme (NAD-ME) activities were determined. Enzyme activity and some kinetic properties of NADP-ME and NADP-MDH as well as CO₂ compensation point and stomatal conductance values were calculated indicating a NADP-ME C₄ photosynthetic pathway. Biochemical and structural results indicate that V. zizanioides belongs to the C₄ NADP-ME variant. This plant appears to be well adapted to the varying environmental conditions typical of temperate climates, by retaining high enzyme activities and a low CO₂ compensation point.     

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.     

Photosynthetic pathways and ecological distribution ofEuphorbia species in Egypt

Summary The photosynthetic pathways of 42 species of the genusEuphorbia growing wild, naturalized or cultivated in Egypt were investigated. The criteria used included the δ¹³C- and δD-values and Kranz anatomy of the leaves. There is a relationship between the photosynthetic pathway and the ecological conditions in the habitat of a particular species. All 4 CAM species are succulent shrubs, wild or cultivated. The 11 species with C₄ pathways are mainly summer annuals of tropical origin and flourish under the hot summer conditions. The 27 C₃ species are either winter annuals, perennials, perennials flourishing in winter or shrubs; the majority are Mediterranean, European or Saharo-Arabian. Summer annuals with C₃ pathways grow under the shade of the summer crops. Generally, C₃ plants grow under conditions of relatively better water resources and lower temperature than the C₄ plants. The majority of the CAM and C₄ species occur in the southern part of the country, where high temperature is a common feature of the climate.     

Two forward genetic screens for vein density mutants in sorghum converge on a cytochrome P450 gene in the brassinosteroid pathway

The specification of vascular patterning in plants has interested plant biologists for many years. In the last decade a new context has emerged for this interest. Specifically, recent proposals to engineer C₄ traits into C₃ plants such as rice require an understanding of how the distinctive venation pattern in the leaves of C₄ plants is determined. High vein density with Kranz anatomy, whereby photosynthetic cells are arranged in encircling layers around vascular bundles, is one of the major traits that differentiate C₄ species from C₃ species. To identify genetic factors that specify C₄ leaf anatomy, we generated ethyl methanesulfonate‐ and γ‐ray‐mutagenized populations of the C₄ species sorghum (Sorghum bicolor), and screened for lines with reduced vein density. Two mutations were identified that conferred low vein density. Both mutations segregated in backcrossed F₂ populations as homozygous recessive alleles. Bulk segregant analysis using next‐generation sequencing revealed that, in both cases, the mutant phenotype was associated with mutations in the CYP90D2 gene, which encodes an enzyme in the brassinosteroid biosynthesis pathway. Lack of complementation in allelism tests confirmed this result. These data indicate that the brassinosteroid pathway promotes high vein density in the sorghum leaf, and suggest that differences between C₄ and C₃ leaf anatomy may arise in part through differential activity of this pathway in the two leaf types.     

Leaf ultrastructure of C4 species possessing different Kranz anatomical types in the cyperaceae

The leaf ultrastructure of NADP-malic enzyme type C₄ species possessing different anatomical features in the Cyperaceae was examined: types were the Rhynchosporoid type, a normal Kranz type in which mesophyll cells are adjacent to Kranz cells, and Fimbristyloid and Chlorocyperoid types, unusual Kranz types in which nonchlorophyllous mestome sheath intervenes between the two types of green cells. They show structural characteristics basically similar to the NADP-malic enzyme group of C₄ grasses, that is, centrifugally located chloroplasts with reduced grana and no increase of mitochondrial frequency in the Kranz cells. However, the Kranz cell chloroplasts of the Fimbristyloid and Chlorocyperoid types exhibit convoluted thylakoid systems and a trend of extensive development of peripheral reticulum, although those of the Rhynchosporoid type do not possess such particular membrane systems. The suberized lamella, probably a barrier for CO₂ diffusion, is present in the Kranz cell walls of the Rhynchosporoid type and in the mestome sheath cell walls of the other two types, and tightly surrounds the Kranz cells (sheaths) that are the sites of the decarboxylation of C₄ acids. These ultrastructural features are discussed in relation to C₄ photosynthetic function.     

Diversity in forms of C4 in the genus Cleome (Cleomaceae)

Background and Aims

Cleomaceae is one of 19 angiosperm families in which C₄ photosynthesis has been reported. The aim of the study was to determine the type, and diversity, of structural and functional forms of C₄ in genus Cleome.

Methods

Plants of Cleome species were grown from seeds, and leaves were subjected to carbon isotope analysis, light and scanning electron microscopy, western blot analysis of proteins, and in situ immunolocalization for ribulose bisphosphate carboxylase oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPC).

Key Results

Three species with C₄-type carbon isotope values occurring in separate lineages in the genus (Cleome angustifolia, C. gynandra and C. oxalidea) were shown to have features of C₄ photosynthesis in leaves and cotyledons. Immunolocalization studies show that PEPC is localized in mesophyll (M) cells and Rubisco is selectively localized in bundle sheath (BS) cells in leaves and cotyledons, characteristic of species with Kranz anatomy. Analyses of leaves for key photosynthetic enzymes show they have high expression of markers for the C₄ cycle (compared with the C₃–C₄ intermediate C. paradoxa and the C₃ species C. africana). All three are biochemically NAD-malic enzyme sub-type, with higher granal development in BS than in M chloroplasts, characteristic of this biochemical sub-type. Cleome gynandra and C. oxalidea have atriplicoid-type Kranz anatomy with multiple simple Kranz units around individual veins. However, C. angustifolia anatomy is represented by a double layer of concentric chlorenchyma forming a single compound Kranz unit by surrounding all the vascular bundles and water storage cells.

Conclusions

NAD-malic enzyme-type C₄ photosynthesis evolved multiple times in the family Cleomaceae, twice with atriplicoid-type anatomy in compound leaves having flat, broad leaflets in the pantropical species C. gynandra and the Australian species C. oxalidea, and once by forming a single Kranz unit in compound leaves with semi-terete leaflets in the African species C. angustifolia. The leaf morphology of C. angustifolia, which is similar to that of the sister, C₃–C₄ intermediate African species C. paradoxa, suggests adaptation of this lineage to arid environments, which is supported by biogeographical information.