Elena V. Voznesenskaya

Coordination of Leaf Photosynthesis, Transpiration, and Structural Traits in Rice and Wild Relatives (Genus Oryza)

Linkages of leaf and mesophyll cell traits to CO2 diffusion, photosynthesis, transpiration, and water use efficiency were identified across accessions of the genus Oryza. The genus Oryza, which includes rice (Oryza sativa and Oryza glaberrima) and wild relatives, is a useful genus to study leaf properties in order to identify structural features that control CO2 access to chloroplasts, photosynthesis, water use efficiency, and drought tolerance. Traits, 26 structural and 17 functional, associated with photosynthesis and transpiration were quantified on 24 accessions (representatives of 17 species and eight genomes). Hypotheses of associations within, and between, structure, photosynthesis, and transpiration were tested. Two main clusters of positively interrelated leaf traits were identified: in the first cluster were structural features, leaf thickness (Thickleaf), mesophyll (M) cell surface area exposed to intercellular air space per unit of leaf surface area (Smes), and M cell size; a second group included functional traits, net photosynthetic rate, transpiration rate, M conductance to CO2 diffusion (gm), stomatal conductance to gas diffusion (gs), and the gm/gs ratio. While net photosynthetic rate was positively correlated with gm, neither was significantly linked with any individual structural traits. The results suggest that changes in gm depend on covariations of multiple leaf (Smes) and M cell (including cell wall thickness) structural traits. There was an inverse relationship between Thickleaf and transpiration rate and a significant positive association between Thickleaf and leaf transpiration efficiency. Interestingly, high gm together with high gm/gs and a low Smes/gm ratio (M resistance to CO2 diffusion per unit of cell surface area exposed to intercellular air space) appear to be ideal for supporting leaf photosynthesis while preserving water; in addition, thick M cell walls may be beneficial for plant drought tolerance.

Bienertia sinuspersici (Chenopodiaceae): A New Species from Southwest Asia and Discovery of a Third Terrestrial C4 Plant Without Kranz Anatomy

Abstract Our studies on the enigmatic genus Bienertia (Chenopodiaceae), with its C4 photosynthesis and lack of Kranz anatomy, led us to the discovery of a second species of this previously-supposed monotypic genus. The new species is named Bienertia sinuspersici after its main range around the Persian Gulf countries and the northern side of the Gulf of Oman. Bienertia sinuspersici occurs in hot climates and is a vicariant of Bienertia cycloptera, which is found at higher latitudes and elevations in temperate and cold deserts of the region. Like Bienertia cycloptera, the new species has unique chlorenchyma cells with dimorphic chloroplasts and single cell C4 photosynthesis. However, it differs anatomically by having mostly one to two layers of chlorenchyma cells, versus two to three layers in Bienertia cycloptera. Furthermore, the new species has longer cotyledon leaves, larger seeds, larger flowers, and larger chromosomes, and differs in a set of micro-morphological features. All of this supports our conclusion that this widely distributed, novel plant is an overlooked new species. Bienertia sinuspersici grows well in very hot climates, under conditions which most species can barely tolerate. Its wide distribution indicates that its novel C4 photosynthesis may confer advantages for CO2 fixation in these habitats not found in C4 species having conventional Kranz anatomy.

Phylogenetic Relationships in the Salicornioideae / Suaedoideae / Salsoloideae s.l. (Chenopodiaceae) Clade and a Clarification of the Phylogenetic Position of Bienertia and Alexandra Using Multiple DNA Sequence Datasets

Abstract The Chenopodiaceae includes taxa with both C3 and C4 photosynthesis with diverse kinds of Kranz anatomy and single-celled C4 species without Kranz anatomy; thus, it is of key importance for understanding evolution of C4 photosynthesis. All of the C4 genera except Atriplex, which belongs to Chenopodioideae, are in the Salicornioideae / Suaedoideae / Salsoloideae s.l. (including Camphorosmeae and Sclerolaeneae) clade. Our study focused on the relationships of the main lineages within this clade with an emphasis on the placement of the single cell functioning C4 genus Bienertia using maximum parsimony, maximum likelihood, and Bayesian inference phylogenetic analyses of the nuclear ribosomal ITS and five chloroplast DNA regions (atpB-rbcL, matK, psbB-psbH, rbcL, and trnL-trnF). Further we provide a detailed phylogeny of Alexandra and Suaeda based on ITS, atpB-rbcL, and psbB-psbH. Our molecular data provide strong statistical support for the monophyly of: (1) a Salicornioideae / Suaedoideae / Salsoloideae s.l. clade; (2) a Salicornioideae / Suaedoideae clade; (3) the subfamilies Salicornioideae, Suaedoideae (including Bienertia) and Salsoloideae s.l.; (4) the tribes Suaedeae, Salsoleae, and Camphorosmeae; (5) the Salicornieae if Halopeplideae is included; and (6) Suaeda if Alexandra is included. Alexandra lehmannii is therefore reclassified as Suaeda lehmannii and a new section of Suaeda is created, section Alexandra. There are four independent origins of C4 photosynthesis within the Suaedoideae including two parallel origins of Kranz C4 anatomy (in Suaeda sections Salsina s.l. and Schoberia) and two independent origins of C4 systems without Kranz anatomy (in Bienertia and in Suaeda section Borszczowia).

Analysis of Arabidopsis with highly reduced levels of malate and fumarate sheds light on the role of these organic acids as storage carbon molecules.

While malate and fumarate participate in a multiplicity of pathways in plant metabolism, the function of these organic acids as carbon stores in C3 plants has not been deeply addressed. Here, Arabidopsis (Arabidopsis thaliana) plants overexpressing a maize (Zea mays) plastidic NADP-malic enzyme (MEm plants) were used to analyze the consequences of sustained low malate and fumarate levels on the physiology of this C3 plant. When grown in short days (sd), MEm plants developed a pale-green phenotype with decreased biomass and increased specific leaf area, with thin leaves having lower photosynthetic performance. These features were absent in plants growing in long days. The analysis of metabolite levels of rosettes from transgenic plants indicated similar disturbances in both sd and long days, with very low levels of malate and fumarate. Determinations of the respiratory quotient by the end of the night indicated a shift from carbohydrates to organic acids as the main substrates for respiration in the wild type, while MEm plants use more reduced compounds, like fatty acids and proteins, to fuel respiration. It is concluded that the alterations observed in sd MEm plants are a consequence of impairment in the supply of carbon skeletons during a long dark period. This carbon starvation phenotype observed at the end of the night demonstrates a physiological role of the C4 acids, which may be a constitutive function in plants.

Diversity in leaf anatomy, and stomatal distribution and conductance, between salt marsh and freshwater species in the C4 genus Spartina (Poaceae)

Leaf anatomy, stomatal density, and leaf conductance were studied in 10 species of Spartina (Poaceae) from low versus high salt marsh, and freshwater habitats. Internal structure, external morphology, cuticle structure, and stomatal densities were studied with light and electron microscopy. Functional significance of leaf structure was examined by measures of CO(2) uptake and stomatal distributions. All species have Kranz anatomy and C(4)delta(13)C values. Freshwater species have thin leaves with small ridges on adaxial sides and stomata on both adaxial and abaxial sides. By contrast, salt marsh species have thick leaves with very pronounced ridges on the adaxial side and stomata located almost exclusively on adaxial leaf surfaces. Salt marsh species also have a thicker cuticle on the abaxial than on the adaxial side of leaves, and CO(2) uptake during photosynthesis is restricted to the adaxial leaf surface. Salt marsh species are adapted to controlling water loss by having stomata in leaf furrows on the adaxial side, which increases the boundary layer, and by having large leaf ridges that fit together as the leaf rolls during water stress. Differences in structural-functional features of photosynthesis in Spartina species are suggested to be related to adaptations to saline environments.

Revealing diversity in structural and biochemical forms of C4 photosynthesis and a C3–C4 intermediate in genus Portulaca L. (Portulacaceae)

Portulacaceae is one of 19 families of terrestrial plants in which species having C4 photosynthesis have been found. Representative species from major clades of the genus Portulaca were studied to characterize the forms of photosynthesis structurally and biochemically. The species P. amilis, P. grandiflora, P. molokiniensis, P. oleracea, P. pilosa, and P. umbraticola belong to the subgenus Portulaca and are C4 plants based on leaf carbon isotope values, Kranz anatomy, and expression of key C4 enzymes. Portulaca umbraticola, clade Umbraticola, is NADP-malic enzyme (NADP-ME)-type C4 species, while P. oleracea and P. molokiniensis in clade Oleracea are NAD-ME-type C4 species, all having different forms of Atriplicoid-type leaf anatomy. In clade Pilosa, P. amilis, P. grandiflora, and P. pilosa are NADP-ME-type C4 species. They have Pilosoid-type anatomy in which Kranz tissues enclose peripheral vascular bundles with water storage in the centre of the leaf. Portulaca cf. bicolor, which belongs to subgenus Portulacella, is an NADP-ME C4 species with Portulacelloid-type anatomy; it has well-developed Kranz chlorenchyma surrounding lateral veins distributed in one plane under the adaxial epidermis with water storage cells underneath. Portulaca cryptopetala (clade Oleracea), an endemic species from central South America, was identified as a C3–C4 based on its intermediate CO2 compensation point and selective localization of glycine decarboxylase of the photorespiratory pathway in mitochondria of bundle sheath cells. The C4 Portulaca species which were examined also have cotyledons with Kranz-type anatomy, while the stems of all species have C3-type photosynthetic cells. The results indicate that multiple structural and biochemical forms of C4 photosynthesis evolved in genus Portulaca.

Physiological, anatomical and biochemical characterisation of photosynthetic types in genus Cleome (Cleomaceae)

C4 photosynthesis has evolved many times in 18 different families of land plants with great variation in leaf anatomy, ranging from various forms of Kranz anatomy to C4 photosynthesis occurring within a single type of photosynthetic cell. There has been little research on photosynthetic typing in the family Cleomaceae, in which only one C4 species has been identified, Cleome gynandra L. There is recent interest in selecting and developing a C4 species from the family Cleomaceae as a model C4 system, since it is the most closely related to Arabidopsis, a C3 model system (Brown et al. 2005). From screening more than 230 samples of Cleomaceae species, based on a measure of the carbon isotope composition (δ13C) in leaves, we have identified two additional C4 species, C. angustifolia Forssk. (Africa) and C. oxalidea F.Muell. (Australia). Several other species have δ13C values around -17‰ to -19‰, suggesting they are C4-like or intermediate species. Eight species of Cleome were selected for physiological, anatomical and biochemical analyses. These included C. gynandra, a NAD-malic enzyme (NAD-ME) type C4 species, C. paradoxa R.Br., a C3-C4 intermediate species, and 6 others which were characterised as C3 species. Cleome gynandra has C4 features based on low CO2 compensation point (Γ), C4 type δ13C values, Kranz-type leaf anatomy and bundle sheath (BS) ultrastructure, presence of C4 pathway enzymes, and selective immunolocalisation of Rubisco and phosphoenolpyruvate carboxylase. Cleome paradoxa was identified as a C3-C4 intermediate based on its intermediate Γ (27.5 μmol mol-1), ultrastructural features and selective localisation of glycine decarboxylase of the photorespiratory pathway in mitochondria of BS cells. The other six species are C3 plants based on Γ, δ13C values, non-Kranz leaf anatomy, and levels of C4 pathway enzymes (very low or absent) typical of C3 plants. The results indicate that this is an interesting family for studying the genetic basis for C4 photosynthesis and its evolution from C3 species.

Chapter 4 C4 Photosynthesis: Kranz Forms and Single-Cell C4 in Terrestrial Plants

Plants identified as having C4 photosynthesis have a C4 metabolic cycle with phosphoenolpyruvate carboxylase as the initial catalyst for fixation of atmospheric CO2, and a C4 acid decarboxylase (NADP-malic enzyme, NAD-malic enzyme, or phosphoenolpyruvate carboxykinase), which releases CO2 for fixation by the C3 cycle. Effective donation of CO2 to Rubisco minimizes competition by O2 and photorespiration, and thus increases photosynthesis under conditions where CO2 is limiting. To achieve this, fixation of atmospheric CO2 in the cytosol by phosphoenolpyruvate carboxylase must be separated from the donation of CO2 to Rubisco by the decarboxylation of C4 acids. In most documented C4 plants, this is accomplished through evolution of various forms of Kranz anatomy, with fixation of atmospheric CO2 in mesophyll cells and donation of CO2 from C4 acids to Rubisco in bundle sheath cells. In the family Chenopodiaceae, two alternative means of accomplishing this spatial separation evolved within individual photosynthetic cells, whereby one cytoplasmic compartment specializes in fixation of atmospheric CO2 in the carboxylation phase of the C4 cycle, and the other cytoplasmic compartment specializes in donating CO2 from C4 acids to Rubisco. In this chapter, biochemical and structural variations of Kranz anatomy in three major C4-containing families, Poaceae, Cyperaceae, and Chenopodiaceae, as well as other known forms for dicots, are summarized. Then, the phylogeny, biogeography, development, and structure-function relationships of the single-cell C4 systems are discussed in comparison to Kranz type C4 plants.

Biogeographic patterns of diversification and the origins of C4 in Cleome (Cleomaceae).

Abstract Photosynthetic pathway innovations have had a large impact on patterns of diversification of angiosperm lineages and the biogeographic distribution of ecological assemblages. C4 photosynthesis has been one of the most studied processes in plants with respect to function, structure, occurrence, and response to climatic conditions. One of the most promising areas of research of C4 photosynthesis is in the Cleomaceae. Here we explore the phylogenetic origins of the C4 pathway in the Cleomaceae using maximum parsimony, maximum likelihood, and Bayesian inference analyses of nrDNA ITS sequences. As has been found previously, commonly recognized genera including Buhsia, Cleomella, Dactylaena, Gynandropsis, Isomeris, Oxystylis, Podandrogyne, Polanisia, and Wizlizenia are derived from within a paraphyletic Cleome. The phylogenetic results presented here indicate that there are likely at least five separate origins of carbon concentrating mechanisms in the Cleomaceae, including at least three separate origins of C4 species. Analyses of historical biogeography suggest Cleomaceae originated in central Asia.

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.