Diversity of Kranz anatomy and biochemistry in C4 eudicots

C(4) photosynthesis and Kranz anatomy occur in 16 eudicot families, a striking example of convergent evolution. Biochemical subtyping for 13 previously undiagnosed C(4) eudicot species indicated that 10 were NADP-malic enzyme (ME) and three were NAD-ME. A total of 33 C(4) species, encompassing four Kranz anatomical types (atriplicoid, kochioid, salsoloid, and suaedioid), and 21 closely related C(3) species were included in a quantitative anatomical study in which we found that, unlike similar studies in grasses and sedges, anatomical type had no predictive value for the biochemical subtype. In a multivariate canonical discriminant analysis, C(4) species were distinguished from C(3) species by the mesophyll to bundle sheath ratio and exposure of the bundle sheath surface to intercellular space. Discrimination between NADP-ME and NAD-ME was not significant, although in a Mantel test grouping by biochemical subtype was significant, while grouping by family was not. This comprehensive survey of C(4) anatomy and biochemistry unequivocally demonstrated that atriplicoid anatomy and NADP-ME biochemistry predominate in many evolutionary lineages. In addition to a main decarboxylating enzyme, high activity of a second decarboxylating enzyme was often observed. Notably, PEP-carboxykinase activity was significant in a number of species, demonstrating that this enzyme could also serve as a secondary pathway for C(4) metabolism in eudicots.     

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.     

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.     

Photochemical Characteristics of Mesophyll and Bundle Sheath Chloroplasts from C4 Plants

Mesophyll and bundle sheath chloroplasts were isolated by differential grinding from the leaves of two NADP-ME C₄ plants, Setaria italica Beauv. cv. H-1, Pennisetum typhoides S & H. cv. AKP-2, and a NAD-ME C₄ species Amaranthus paniculatus L. The mesophyll chloroplasts of C₄ plants possessed slightly lower K_m for ADP and Pi than those of bundle sheath chloroplasts. The Hill reaction activities and noncyclic photophosphorylation rates of the bundle sheath chloropiasts from S. italica and P. typhoides were less than one-fifth of those by the mesophyll chloroplasts. But the bundle sheath chloroplasts of A. paniculatus exhibited high rates of Hill reaction, cyclic as well as noncyclic photophosphorylation. The pigment- and eyiochrome composition suggested a relative enrichment of PS 1 in bundle sheath chloroplasts of S. italica and P. typhoides. The chain exists in both mesophyll and bundle sheath chloroplasts. As much as 35–52% of leaf chlorophyll was located in the bundle sheath chloroplasts. The photochemical activities of bundle sheath chloroplasts are significant though a major part of leaf photochemical potential is associated with the mesophyll chloroplasts.     

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.     

Growth Analysis of Soybean Seedlings During the Lifespan of the Cotyledons

Patterns of seedling growth of Glycine max in light and darknesswere compared during the period from germination to cotyledonabscission. Fitted growth curves and the derived functions,relative growth rate, unit leaf rate, leaf area ratio and specificleaf area, were used to assess the relative importance in seedlinggrowth of cotyledon storage reserves, cotyledon photosynthesisand leaf photosynthesis. The cotyledons are of an intermediatetype with a predominant storage and a minimal photosyntheticfunction. Cotyledon reserves support seedling growth until theprimary leaves expand, after which growth depends on leaf photosynthesis. Glycine max (L.) Merr., soybean, cotyledons, growth analysis, seedling development     

Developmental and Environmental Effects on the Expression of the C3-C4 Intermediate Phenotype in Moricandia arvensis

Cellular anatomy and expression of glycine decarboxylase (GDC) protein were studied during leaf development of the C₃-C₄ intermediate species Moricandia arvensis. Leaf anatomy was initially C₃-like and the number and profile area of mitochondria in the bundle-sheath cells were the same as those in adjacent mesophyll cells. Between a leaf length of 6 and 12 mm there was a bundle-sheath-specific, 4-fold increase in the number of mitochondrial profiles, followed by a doubling of their individual profile areas as the leaves expanded further. Subunits of GDC were present in whole-leaf extracts before the anatomical development of bundle-sheath cells. Whereas the GDC H-protein content of leaves increased steadily throughout development, the increase in GDC P-protein was synchronous with the development of mitochondria in the bundle sheath. The P-protein was confined to bundle-sheath mitochondria throughout leaf development, and its content in individual mitochondria increased before the anatomical development of the bundle sheath. Anatomical and biochemical attributes of the C₃-C₄ character were present in the cotyledons and sepals but not in other photosynthetic organs/tissues. In leaves and cotyledons that developed in the dark, the expression of the P-protein and the organellar development were reduced but the bundle-sheath cell specificity was retained.     

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.     

C4‐type gene expression is not directly dependent on Kranz anatomy in an amphibious sedge Eleocharis vivipara Link

Eleocharis vivipara Link alters its photosynthetic mode depending on the growth environment. It utilizes C4 photosynthesis when grown under terrestrial conditions (terrestrial form) and C3 photosynthesis when grown under submerged conditions (submerged form). The photosynthetic organ (the mature internodal region of the culm) of the terrestrial form shows typical Kranz anatomy with well-developed bundle sheath cells, while the bundle sheath cells of the submerged form are not developed. In the mature internodal region of the terrestrial form, expression of the genes encoding two carboxylases, the small subunit of ribulose 1,5-bisphosphate carboxylase (RbcS) and phosphoenolpyruvate carboxylase (Ppc), occurred mainly in bundle sheath cells and in mesophyll cells, respectively, as seen in a typical C4 leaf. In the submerged form, RbcS was expressed in both bundle sheath cells and mesophyll cells, and no expression of Ppc was observed. In the immature internodal region with undeveloped bundle sheath cells, both life forms showed the same expression pattern as in C3 plants: RbcS expression was localized in mesophyll cells and no Ppc expression was observed. The C4-type expression pattern was established concomitantly with the development of bundle sheath cells during tissue maturation in the terrestrial internode. In contrast to the terrestrial form, the submerged form maintains C3-type gene expression during tissue maturation. When the terrestrial culm was submerged, a region of transition from the terrestrial form to the submerged form was established in newly sprouting culms. In this transitional region, C4-type expression of the two carboxylase genes was still maintained even though the development of bundle sheath cells was repressed. This observation suggests that the C4-type cell-specific gene expression pattern does not depend on the formation of Kranz anatomy.     

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     

Different functions of vicilin and legumin are reflected in the histopattern of globulin mobilization during germination of vetch (Vicia sativa L.)

The temporal and spatial patterns of storage-globulin mobilization were immunohistochemically pursued in the embryonic axis and cotyledons of vetch seed (Vicia sativa L.) during germination and early seedling growth. Embryonic axes as well as cotyledons of mature seeds contain protein bodies with stored globulins. Prevascular strands of axes and cotyledons, the radicle and epidermal layers of axis organs were nearly exclusively stained by vicilin antibodies whereas the cotyledonous storage mesophyll gave similar staining for vicilin and legumin. Globulin breakdown started locally where growth and differentiation commenced in the axis. There, vicilin mobilization preceded legumin mobilization. Thus vicilin represents the initial source of amino acids for early growth and differentiation processes in vetch. Legumin presumably only serves as a bulk amino acid source for subsequent seedling growth during postgerminative globulin degradation. During the first 2–3 d after the start of imbibition the axis was depleted of globulins whereas no decrease in immunostainability was detected in the cotyledons except in their vascular strands where immunostainability was almost completely lost at this time. Continuous vascular strands were established at the third day when globulin breakdown was finished in the axis but had just started in the cotyledon mesophyll. Protein mobilization proceeded in a small zone from the epidermis towards the vascular strands in the center of the cotyledons. In this zone the storage cells, which initially appeared densely packed with starch grains and protein bodies, concomitantly transformed into cells with a large central vacuole and only a thin cytoplasmic layer attached to the cell wall. These results agree well with the hypothesis that during the first 2 d after imbibition the axis is autonomous in amino acid provision. After the endogenous reserves of the axis are depleted and the conductive tissue has differentiated, globulins are mobilized in the cotyledons, suggesting that then the amino acid supply is taken over by the cotyledons. For comparison with other degradation patterns we used garden bean (Phaseolus vulgaris L) and rape (Brassica napus L.) as reference plants. Received: 3 August 1999 / Accepted: 11 December 1999     

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.     

A Comparative Study of the Role of the Cotyledon in Seedling Development

Seedling development was studied in four species in which thecotyledons had different capacities for expansion and CO²-flxation. The highest growth-rates were shown by the two species in whichthe cotyledons showed the most expansion and also the greatest¹⁴CO²-fixation capacity. Cotyledons of these species becamevery leaf-like and retained a high proportion of the radiocarbonfixation products during their growth in contrast to the hypogealrunner-bean cotyledon which had a high percentage export valuealthough total fixation was low. Seedlings with leaf-like cotyledons which had the dual roleof storage and subsequent provision of photosynthetic organshad delayed leaf development with early growth concentratedin cotyledon, hypocotyl, and root whereas species with cotyledonsless well adapted for photosynthesis produced leaves at an earlierstage. Thus, the pattern of early seedling development was closelyrelated to the degree of photosynthetic adaptation shown bythe cotyledons. The evidence suggests that in the species studied, cotyledonsadapted for the dual role appear to provide a more efficientsystem for early seedling development.     

A comparative anatomical and biochemical analysis in salsola (Chenopodiaceae) species with and without a Kranz type leaf anatomy: a possible reversion of C4 to C3 photosynthesis

Leaf anatomy was studied by light and electron microscopy and the leaf activities of RUBP carboxylase, PEP carboxylase, and malic enzyme were assayed in: Salsola australis and S. oreophila grown on the West Pamirs at 1800 m altitude; in S. australis grown on the East Pamirs at 3860 m; and in S. arbusculiformis grown in the Kisil-Kum desert in Middle Asia near 500 m. Carbon isotope fractionation ratio values also were measured on whole leaf tissue for 18 Salsola species field collected in these and other regions of the former USSR. S. australis leaves are cylindrical and in cross section exhibit a peripheral ring of mesophyll and then an inner ring of bundle sheath type cells; and its biochemical characteristics and deltaC values are typical of a C4 species of the NADP-malic enzyme malate-forming group. These traits were expressed independent of the plant growth altitude up to 4000 m. C4 type deltaC values were obtained in 14 of the Salsola species. Anatomical, structural, and biochemical features typical of the C4 syndrome were absent in S. oreophila and S. arbusculiformis. Four Salsola species, including these two, had C3-type deltaC values. Their cylindrical leaves in cross section exhibited two to three peripheral rings as layers of palisade parenchyma. Although their vascular bundles were surrounded by green bundle sheath cells, their organelle numbers were comparable to those in mesophyll cells. Neither bundle sheath cell wall thickenings nor dimorphic chloroplasts in two leaf cell types were observed. In S. oreophila, there was a high activity of RuBP carboxylase, but a low activity of C4 cycle enzymes. Interpretation of these data lends evidence to the hypothesis that a small group of C3 Salsola species, including S. oreophila, S. arbusculiformis, S. montana, and S. pachyphylla, arose as the result of a reversion of a C4 to a C3 type of photosynthetic CO2 fixation in the cooler climates of Middle Asia.     

Early stages of leaf development in <Emphasis Type="Italic">has</Emphasis> mutant of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The elucidation of molecular mechanisms underlying the leaf development can be facilitated by the detailed anatomical study of leaf development mutants. We present an analysis of leaf anatomy and morphogenesis during early developmental stages in has mutant of Arabidopsis thaliana. The recessive has mutation affects a number of aspects in plant development, including the shape and size of both cotyledons and leaves. The earliest developmental observations suggest almost synchronous growth of the first two leaf primordia of has mutant. No significant disruption of the cell division pattern in the internal tissue is observed at the earliest stages of development, with the major anatomical difference compared to wild type primordia being the untimely maturation of mesophyll tissue cells in has mutant. At the stage of leaf blade formation, structure disruption becomes clearly evident, by irregular arrangement of the cell layers and the lack of polarity in juvenile has leaves. One distinguishing feature of the mutant leaf anatomy is the absence of mesophyll tissue differentiation. Altered has mutant leaf morphology could be at least partially accounted for by the ectopic STM activity that was found at the base of leaf primordia during early stages of leaf development in has plants.     

COMPARATIVE BUNDLE SHEATH AND MESOPHYLL DIFFERENTIATION IN THE LEAVES OF THE C4 GRASSES PANICUM EFFUSUM AND P. BULBOSUM

The two C₄ Panicum species examined differ in C₄ acid decarboxylation type and in developmental origin of bundle sheaths in major veins of their leaf blades. In Panicum effusum R.Br. (NAD-malic enzyme type) both mesophyll (PCA) and chlorenchymatous bundle sheath (PCR) cells are derived from ground meristem. In contrast, in Panicum bulbosum H.B.K. (NADP-malic enzyme type), bundle sheath cells are derived from procambium, while mesophyll develops from ground meristem. To test the hypothesis that the developmental divergence of bundle sheath and mesophyll cells would occur earlier when these two tissues had different ontogenetic origins (in P. bulbosum) than when these tissues had the same origin (in P. effusum), the development of major veins in each species was investigated. We measured cell length and cross sectional area, plastid and mitochondrial number, plastid area, vacuole area fraction, wall thickness and fraction adjacent to intercellular space using direct and digitizer measurements of transmission electron micrographs of leaf cross sections of successive developmental stages. Many of the statistically significant changes in the structural parameters measured occurred late in development of both species. The magnitude of developmental change in P. effusum PCR cells was sometimes more dramatic, viz. changes in cell and PCR plastid area, and in mitochondrion number per cell. However, earlier divergence of PCR and PCA length and volume, and wall fraction adjacent to intercellular space in P. bulbosum than in P. effusum indicates that ontogenetic origin of PCR cells from procambium could determine the timing of at least some developmental events.     

Modulation of the venation pattern of cotyledons of transgenic tobacco for the tumorigenic <Emphasis Type="Italic">6b</Emphasis> gene of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> AKE10

Neoplastic plant-tissue formation, termed crown gall disease, is induced on infection with Agrobacterium tumefaciens. The tumorous tissues develop an extensive vascular system, with a venation pattern distinct from that of native host plants. We report here that the plant-tumorigenic 6b gene of the A. tumefaciens strain AKE10 is capable of inducing extensive vein formation in transgenic tobacco seedlings with distinct pattern formation. Unlike the wild-type cotyledons, transgenic cotyledons had wavy and striate veins depending on the extent of severity of leaf morphology. Graph analysis of the transgenic cotyledonous vein patterns revealed an increase in the number of branch points of veins, end-points of veins, and areas surrounded by the veins. Histological analysis showed abnormal tissue growth on the abaxial side of the cotyledon blades and continual formation of adventitious veins. These adventitiously formed veins included inverted dorso-ventrality and formation of a radial axis.     

Leaf and inflorescence peduncle anatomy: a contribution to the taxonomy of Rapateaceae

The anatomy of leaves and inflorescence peduncles was studied in species of Monotrema (4), Stegolepis (1) and Saxofridericia (1), aiming to contribute to the taxonomy of Rapateaceae. The form and structure of leaf blade midrib and the form of the inflorescence peduncle are diagnostic characteristics for the studied species. Monotrema is distinguished by: epidermal and vascular bundle outer sheath cells containing phenolic compounds in both organs; leaf blade with palisade and spongy chlorenchyma, arm-parenchyma, and air canals between the vascular bundles; leaf sheath with phenolic idioblasts in the mesophyll; inflorescence peduncle with tabular epidermal cells and air canals in the cortex and pith. Such characteristics support the recognition of Monotremoideae, which includes Monotrema. Stegolepis guianensis is distinguished by thick-walled epidermal cells and a plicate chlorenchyma in both organs; leaf blade with subepidermal fiber strands in abaxial surface and a heterogeneous mesophyll; inflorescence peduncle with rounded epidermal cells, a hypodermis with slightly thick-walled cells, and a pith with isodiametric cells and vascular bundles. Saxofridericia aculeata is distinguished by papillate epidermal cells in both organs; unifacial leaf blade with subepidermal fiber strands in both surfaces and a regular chlorenchyma; leaf sheath with a hypodermis in both surfaces and fiber bundles in the mesophyll; inflorescence peduncle with an undefined cortex and a hypodermis with thick-walled cells. S. guianensis shares few characteristics with S. aculeata, supporting their placement in different tribes.     

VARIATION IN THE ORGANIZATION OF THE STOMATAL COMPLEX IN THE LEAF EPIDERMIS OF MONOCOTYLEDONS AND ITS BEARING ON THEIR PHYLOGENY

Stebbins , G. L., and G. S. Khush . (U. California, Davis.) Variation in the organization of the stomatal complex in the leaf epidermis of monocotyledons and its bearing on their phylogeny. Amer. Jour. Bot. 48(1): 51–59. Illus. 1961.—Using macerated pieces of epidermal tissue from living plants and herbarium specimens, stomatal complexes of 192 species belonging to 49 families of monocotyledons were studied. Four categories are recognized, 2 with 4 or more subsidiary cells surrounding the guard cells, 1 with 2 subsidiaries, and 1 with none. Development of the 2-subsidiary type, studied in acetocarmine preparations of Juncus effusus and Sagittaria montevidensis, resembles that in Gramineae previously described. No correlation was found between type of stomatal complex and either leaf shape or type of xylem vessel, but some correlation exists between this character and type of seed germination, vascular anatomy of seedling, growth habit of mature plant, and geographic distribution. Types with 4 or more subsidiaries are most commonly phanerophytes with tropical distribution, many vascular bundles in the cotyledon, and hypogeal germination. Complexes with 2 subsidiaries occur in many families of a diverse nature, but the most primitive plants with this type are hydrophytes or helophytes with tropical or temperate distribution, 1 vascular bundle in the cotyledon, and epigeal germination. Stomatal complexes without subsidiaries are almost confined to the Liliales and their more specialized derivatives. These plants are predominantly geophytes with temperate or tropical distribution, 2 bundles in the cotyledon, and epigeal germination. Reasons are advanced for suggesting that the type with many subsidiaries is the most primitive and the other 2 types have been derived from it independently by reduction of the number of subsidiary cells.