Nuria K. Koteyeva

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.

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.

Structural and physiological analyses in Salsoleae (Chenopodiaceae) indicate multiple transitions among C3, intermediate, and C4 photosynthesis

In subfamily Salsoloideae (family Chenopodiaceae) most species are C4 plants having terete leaves with Salsoloid Kranz anatomy characterized by a continuous dual chlorenchyma layer of Kranz cells (KCs) and mesophyll (M) cells, surrounding water storage and vascular tissue. From section Coccosalsola sensu Botschantzev, leaf structural and photosynthetic features were analysed on selected species of Salsola which are not performing C4 based on leaf carbon isotope composition. The results infer the following progression in distinct functional and structural forms from C3 to intermediate to C4 photosynthesis with increased leaf succulence without changes in vein density: From species performing C3 photosynthesis with Sympegmoid anatomy with two equivalent layers of elongated M cells, with few organelles in a discontinuous layer of bundle sheath (BS) cells (S. genistoides, S. masenderanica, S. webbii) > development of proto-Kranz BS cells having mitochondria in a centripetal position and increased chloroplast number (S. montana) > functional C3–C4 intermediates having intermediate CO2 compensation points with refixation of photorespired CO2, development of Kranz-like anatomy with reduction in the outer M cell layer to hypodermal-like cells, and increased specialization (but not size) of a Kranz-like inner layer of cells with increased cell wall thickness, organelle number, and selective expression of mitochondrial glycine decarboxylase (Kranz-like Sympegmoid, S. arbusculiformis; and Kranz-like Salsoloid, S. divaricata) > selective expression of enzymes between the two cell types for performing C4 with Salsoloid-type anatomy. Phylogenetic analysis of tribe Salsoleae shows the occurrence of C3 and intermediates in several clades, and lineages of interest for studying different forms of anatomy.

Functional analysis of corn husk photosynthesis

The husk surrounding the ear of corn/maize (Zea mays) has widely spaced veins with a number of interveinal mesophyll (M) cells and has been described as operating a partial C3 photosynthetic pathway, in contrast to its leaves, which use the C4 photosynthetic pathway. Here, we characterized photosynthesis in maize husk and leaf by measuring combined gas exchange and carbon isotope discrimination, the oxygen dependence of the CO2 compensation point, and photosynthetic enzyme activity and localization together with anatomy. The CO2 assimilation rate in the husk was less than that in the leaves and did not saturate at high CO2, indicating CO2 diffusion limitations. However, maximal photosynthetic rates were similar between the leaf and husk when expressed on a chlorophyll basis. The CO2 compensation points of the husk were high compared with the leaf but did not vary with oxygen concentration. This and the low carbon isotope discrimination measured concurrently with gas exchange in the husk and leaf suggested C4-like photosynthesis in the husk. However, both Rubisco activity and the ratio of phosphoenolpyruvate carboxylase to Rubisco activity were reduced in the husk. Immunolocalization studies showed that phosphoenolpyruvate carboxylase is specifically localized in the layer of M cells surrounding the bundle sheath cells, while Rubisco and glycine decarboxylase were enriched in bundle sheath cells but also present in M cells. We conclude that maize husk operates C4 photosynthesis dispersed around the widely spaced veins (analogous to leaves) in a diffusion-limited manner due to low M surface area exposed to intercellular air space, with the functional role of Rubisco and glycine decarboxylase in distant M yet to be explained.

Evolution of leaf anatomy and photosynthetic pathways in Portulacaceae.

PREMISE OF THE STUDY Portulacaceae is a family with a remarkable diversity in photosynthetic pathways. This lineage not only has species with different C4 biochemistry (NADP-ME and NAD-ME types) and C3-C4 intermediacy, but also displays different leaf anatomical configurations. Here we addressed the evolutionary history of leaf anatomy and photosynthetic pathways in Portulacaceae. METHODS Photosynthetic pathways were assessed based on leaf anatomy and carbon isotope ratios. Information on the NADP-ME and NAD-ME C4 variants was obtained from the literature. The evolutionary relationships and trait evolution were estimated under a Bayesian framework, and divergence times were calibrated using the ages obtained in a previous study. KEY RESULTS C4 photosynthesis is the main pathway in Portulacaceae. One clade (Cryptopetala), however, includes species that have non-Kranz anatomy and C3 type isotope values, two of which are C3-C4 intermediates. The ancestral leaf anatomy for the family is uncertain. The analysis showed one origin of the C4 pathway, which was lost in the Cryptopetala clade. Nevertheless, when a second analysis was performed taking into account the limited number of species with NAD-ME and NADP-ME data, a secondary gain of the C4 pathway from a C3-C4 intermediate was inferred. CONCLUSIONS The C4 pathway evolved ca. 23 Myr in the Portulacaceae. The number of times that the pathway evolved in the family is uncertain. The diversity of leaf anatomical types and C4 biochemical variants suggest multiple independent origins of C4 photosynthesis. Evidence for a switch from C4 to C3-C4 intermediacy supports the hypothesis that intermediates represent a distinct successful strategy.

Structural, biochemical, and physiological characterization of photosynthesis in two C4 subspecies of Tecticornia indica and the C3 species Tecticornia pergranulata (Chenopodiaceae)

Among dicotyledon families, Chenopodiaceae has the most C(4) species and the greatest diversity in structural forms of C(4). In subfamily Salicornioideae, C(4) photosynthesis has, so far, only been found in the genus Halosarcia which is now included in the broadly circumscribed Tecticornia. Comparative anatomical, cytochemical, and physiological studies on these taxa, which have near-aphyllous photosynthetic shoots, show that T. pergranulata is C(3), and that two subspecies of T. indica (bidens and indica) are C(4) (Kranz-tecticornoid type). In T. pergranulata, the stems have two layers of chlorenchyma cells surrounding the centrally located water storage tissue. The two subspecies of T. indica have Kranz anatomy in reduced leaves and in the fleshy stem cortex. They are NAD-malic enzyme-type C(4) species, with mesophyll chloroplasts having reduced grana, characteristic of this subtype. The Kranz-tecticornoid-type anatomy is unique among C(4) types in the family in having groups of chlorenchymatous cells separated by a network of large colourless cells (which may provide mechanical support or optimize the distribution of radiation in the tissue), and in having peripheral vascular bundles with the phloem side facing the bundle sheath cells. Also, the bundle sheath cells have chloroplasts in a centrifugal position, which is atypical for C(4) dicots. Fluorescence analyses in fresh sections indicate that all non-lignified cell walls have ferulic acid, a cell wall cross-linker. Structural-functional relationships of C(4) photosynthesis in T. indica are discussed. Recent molecular studies show that the C(4) taxa in Tecticornia form a monophyletic group, with incorporation of the Australian endemic genera of Salicornioideae, including Halosarcia, Pachycornia, Sclerostegia, and Tegicornia, into Tecticornia.

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

BACKGROUND AND AIMS Cleomaceae is one of 19 angiosperm families in which C(4) photosynthesis has been reported. The aim of the study was to determine the type, and diversity, of structural and functional forms of C(4) 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 C(4)-type carbon isotope values occurring in separate lineages in the genus (Cleome angustifolia, C. gynandra and C. oxalidea) were shown to have features of C(4) 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 C(4) cycle (compared with the C(3)-C(4) intermediate C. paradoxa and the C(3) 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 C(4) 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, C(3)-C(4) intermediate African species C. paradoxa, suggests adaptation of this lineage to arid environments, which is supported by biogeographical information.

An assessment of the capacity for phosphoenolpyruvate carboxykinase to contribute to C4 photosynthesis

Three C4 acid decarboxylases, phosphoenolpyruvate carboxykinase (PEPCK), NADP-malic enzyme (NADP-ME), and NAD-malic enzyme (NAD-ME) were recruited from C3 plants to support C4 photosynthesis. In Poaceae, there are established lineages having PEPCK type species, and some NADP-ME lineages in which PEPCK contributes to C4. Besides family Poaceae, recently PEPCK has been reported to function in C4 photosynthesis in eudicot species including Cleome gynandra (Cleomaceae), Trianthema portulacastrum and Zaleya pentandra (Aizoaceae). We evaluated PEPCK by enzyme assay and western blots in representatives of Poaceae, Aizoaceae, Cleomaceae, and Chenopodiaceae compared to that in the PEPCK type C4 grass Spartina anglica. Eragrostis nutans was identified as the first NAD-ME type C4 grass having substantial amounts of PEPCK. In the eudicots, including C. gynandra, Cleome angustifolia, T. portulacastrum, Z. pentandra, and nine C4 members of family Chenopodiaceae (which has the most C4 species and diversity in forms among eudicot families), amounts of PEPCK were generally very low (barely detectable up to 4% of that in S. anglica). Based on these results, C4 species can be classified biochemically according to the dominant decarboxylase recruited for C4 function; and, Poaceae remains the only family in which PEPCK is known to have a significant role in C4 photosynthesis.