Comparison of methods for rooting phylogenetic trees: A case study using Orcuttieae (Poaceae: Chloridoideae)

DNA sequence data (cpDNA trnL intron and nrDNA ITS1 and ITS2) were analyzed to identify relationships within Orcuttieae, a small tribe of endangered grasses endemic to vernal pools in California and Baja California. The tribe includes three genera: Orcuttia, Tuctoria, and Neostapfia. All three genera carry out C₄ photosynthesis but aquatic taxa of Orcuttia lack Kranz anatomy. The unusual habitat preference of the tribe is coupled with the atypical development of C₄ photosynthesis without Kranz anatomy. Furthermore, the tribe has no known close relatives and has been noted to be phylogenetically isolated within the subfamily Chloridoideae. In this study we examine the problem of inferring the root of the tribe in the absence of an identified outgroup, analyze the phylogenetic relationships of the constituent taxa, and evaluate the evolutionary development of C₄ photosynthesis. We compare four methods for inferring the root of the tree: (1) the outgroup method, (2) midpoint rooting, the imposition of a molecular clock for both (3) maximum likelihood (ML) and (4) Bayesian analysis. We examine the consequences of each method for the inferred phylogenetic relationships. Three of the methods (outgroup rooting and the ML and Bayesian molecular clock analyses) suggest that the root of Orcuttieae is between Neostapfia and the Tuctoria/Orcuttia lineage, while midpoint rooting gives a different root. The Bayesian method additionally provides information about probabilities associated with other possible root locations. Assuming that the true root of Orcuttieae is between Neostapfia and the Tuctoria/Orcuttia lineage, our data indicate Neostapfia and Orcuttia are both monophyletic, while Tuctoria is paraphyletic (with no synapomorphies in either dataset) and forming a grade between the other two genera and needs taxonomic revision. Our data support the hypothesis that Orcuttieae was derived from a terrestrial ancestor and evolved specializations to an aquatic environment, including C₄ photosynthesis without Kranz anatomy.     

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.     

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.     

Environmental regulation of photosynthetic metabolism in the amphibious sedge Eleocharis baldwinii and comparisons with related species

The amphibious leafless sedge, Eleocharis baldwinii, expresses C₄ characteristics in the terrestrial form and intermediate characteristics between C₃ and C₄ photosynthesis in the submerged form. This study examined the immunocytochemical localization of C₃ and C₄ enzymes in culms of the two forms to elucidate the regulatory mechanism of photosynthetic metabolism and compared the activities and amounts of C₃ and C₄ enzymes with those in other Eleocharis species, E. vivipara and E. retroflexa, which show C₄ characteristics on land but C₃ and C₄ characteristics under water. The terrestrial form of E. baldwinii exhibited a C₄‐like pattern of enzyme localization. The submerged form exhibited a modified anatomy with well‐developed mesophyll cells and small Kranz cells. The C₄ enzyme levels declined conspicuously in outer mesophyll cells adjacent to the epidermis, whereas Rubisco levels increased throughout the mesophyll in the submerged form. These results suggest that intermediate photosynthesis between C₃ and C₄ photosynthesis in the submerged form results from the predominant operation of the C₃ pathway in the outer mesophyll cells and the C₄ pathway in both the inner mesophyll and Kranz cells. Differences in the degree of C₄ expression in terrestrial forms of Eleocharis species may cause the differences in the expression of photosynthetic modes under water.     

Effects of an inhibitor of phosphoenolpyruvate carboxylase on photosynthesis of the terrestrial forms of amphibious Eleocharis species

The leafless amphibious sedge Eleocharis vivipara develops culms with C₄ traits and Kranz anatomy under terrestrial conditions, but develops culms with C₃ traits and non-Kranz anatomy under submerged conditions. The culms of the terrestrial form have high C₄ enzyme activities, while those of the submerged form have decreased C₄ enzyme activities. The culms accumulate ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the mesophyll cells (MC) and the bundle sheath cells. The Rubisco in the MC may be responsible for the operation of the C₃ pathway in the submerged form. To verify the presence of the C₃ cycle in the MC, we examined the effects of 3,3-dichloro-2-(dihydroxyphosphinoylmethyl) -propenoate (DCDP), an inhibitor of phosphoenolpyruvate carboxylase (PEPC), on photosynthesis in culms of the terrestrial forms of E. vivipara and related amphibious species, E. baldwinii and E. retroflexa ssp. chaetaria. When 1 mM DCDP was fed via the transpiration stream to excised leaves, photosynthesis was inhibited completely in Fimbristylis dichotoma (C₄ control), but by only 20% in potato (C₃ control). In the terrestrial Eleocharis plants, the degree of inhibition of photosynthesis by DCDP was intermediate between those of the C₄ and C₃ plants, at 58–81%. These results suggest that photosynthesis under DCDP treatment in the terrestrial Eleocharis plants is due mainly to fixation of atmospheric CO₂ by Rubisco and probably the C₃ cycle in the MC. These features are reminiscent of those in C₄-like plants. Differential effects of DCDP on photosynthesis of the 3 Eleocharis species are discussed in relation to differences in the degree of Rubisco accumulation and C₃ activity in the MC. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

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).     

SYSTEMATICS OF THE TRIBE ORCUTTIEAE (GRAMINEAE) AND THE DESCRIPTION OF A NEW SEGREGATE GENUS,TUCTORIA

The two genera Neostapfia and Orcuttia, recognized as being closely allied and sharing unusual features, are treated as the sole members of the tribe Orcuttieae. Neostapfia is monotypic; Orcuttia comprises eight taxa, six of which are universally treated as species, and two as varieties. Studies reported here are based on field work throughout the ranges of these two genera and examination of 550 herbarium specimens. Chromosome counts and seed protein analyses for all taxa are presented. These data indicate that each taxon is worthy of specific status and that Orcuttia, as traditionally treated, consists of discordant elements. It is concluded that a more satisfactory taxonomy results when this group is split into two genera. A new genus, Tuctoria, is described, and the combinations T. fragilis (Swallen) J. Reeder, T. greenei (Vasey) J. Reeder, and T. mucronata (Crampton) J. Reeder are proposed.     

The Underwater Life of Secondarily Aquatic Plants: Some Problems and Solutions

The freshwater secondarily aquatic plants, most of which are higher plants, are those returned to the water environment after spending a period of time living on land. The readaptation to living underwater has made it necessary for these plants to put in place morphological and functional strategies to cope with some major problems due to features of the aquatic environment, but also deriving from the specialized organization of their “terrestrial” bodies. The poor O₂ availability underwater accounted for the evolution of wide aerenchyma tissues throughout the plant organs to improve the photosynthetic O₂ flux from the shoot to the roots buried in anoxic sediments and to the neighboring rhizosphere. This favors sediment oxygenation, sustains the aerobic metabolism of roots, and improves the availability and uptake of mineral nutrients, whose delivery to the entire plants, without a transpirational flux, is ensured by an acropetal mass transport depending on root pressure, guttation from hydathodes and channeling by apoplast closure around the vascular tissues. A great expansion of leaf surfaces and an enhanced surface:volume ratio of chloroplast-rich photosynthetic cells help to contact the water medium and to increase the cell/environment exchanges to gain inorganic carbon. Furthermore, different physiological mechanisms operate to cope with the scarce availability of CO₂ and the prevalence of HCO₃ ^? as inorganic carbon form in water. Some of them, like cell wall acidification through H⁺ extrusion by a light-dependent APTase or activation of an apoplastic carbonic anhydrase, operate outside the cells, leading to a conversion of HCO₃ ^? to CO₂, which then diffuses into the cells. Others, on the contrary, act inside the cells to load the active site of Rubisco with CO₂, thus favoring photosynthesis and lowering photorespiration. Aquatic macrophytes with isoetid life form, moreover, can obtain most ot the fixed CO₂ from sediments. In submerged species, in additin to the C₃ cycle, the C₄ and CAM-like photosynthetic metabolisms can also operate, and are modulated by the environmental inorganic carbon availability and the plant photosynthetic demand. Interestingly, in the aquatic plants the C₄ pathway, which can be concomitant with the C₃ one, does not depend on the Kranz anatomy of leaves, but relies on the intracellular compartmentation of carboxylative and decarboxylative enzymes. The CAM-like pathway, defined AAM, which also coexists with the C₃, allows the submerged plants to fix CO₂ in the dark, thus exploiting the higher CO₂ availability in the water medium during the night, and extending to 24?h the period of inorganic carbon assimilation. In almost all the aquatic macrophytes the AAM is only expressed in the submersion state, whereas it is quickly inactivated in emerging leaves in a cell by cell way.     

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.     

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.     

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.     

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.     

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.     

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

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

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.     

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.     

C4 plants in the vegetation of Mongolia: their natural occurrence and geographical distribution in relation to climate

The natural geographical occurrence, carbon assimilation, and structural and biochemical diversity of species with C₄ photosynthesis in the vegetation of Mongolia was studied. The Mongolian flora was screened for C₄ plants by using ¹³C/¹²C isotope fractionation, determining the early products of ¹⁴CO₂ fixation, microscopy of leaf mesophyll cell anatomy, and from reported literature data. Eighty C₄ species were found among eight families: Amaranthaceae, Chenopodiaceae, Euphorbiaceae, Molluginaceae, Poaceae, Polygonaceae, Portulacaceae and Zygophyllaceae. Most of the C4 species were in three families: Chenopodiceae (41 species), Poaceae (25 species) and Polygonaceae, genus Calligonum (6 species). Some new C₄ species in Chenopodiaceae, Poaceae and Polygonaceae were detected. C₄ Chenopodiaceae species make up 45% of the total chenopods and are very important ecologically in saline areas and in cold arid deserts. C₄ grasses make up about 10% of the total Poaceae species and these species naturally concentrate in steppe zones. Naturalized grasses with Kranz anatomy,of genera such as Setaria, Echinochloa, Eragrostis, Panicum and Chloris, were found in almost all the botanical-geographical regions of Mongolia, where they commonly occur in annually disturbed areas and desert oases. We analyzed the relationships between the occurrence of C₄ plants in 16 natural botanical-geographical regions of Mongolia and their major climatic influences. The proportion of C₄ species increases with decreasing geographical latitude and along the north-to-south temperature gradient; however grasses and chenopods differ in their responses to climate. The abundance of Chenopodiaceae species was closely correlated with aridity, but the distribution of the C₄ grasses was more dependent on temperature. Also, we found a unique distribution of different C₄ Chenopodiaceae structural and biochemical subtypes along the aridity gradient. NADP-malic enzyme (NADP-ME) tree-like species with a salsoloid type of Kranz anatomy, such as Haloxylon ammodendron and Iljinia regelii, plus shrubby Salsola and Anabasis species, were the plants most resistant to ecological stress and conditions in highly arid Gobian deserts with less than 100 mm of annual precipitation. Most of the annual C₄ chenopod species were halophytes, succulent, and occurred in saline and arid environments in steppe and desert regions. The relative abundance of C₃ succulent chenopod species also increased along the aridity gradient. Native C₄ grasses were mainly annual and perennial species from the Cynodonteae tribe with NAD-ME and PEP-carboxykinase (PEP-CK) photosynthetic types. They occurred across much of Mongolia, but were most common in steppe zones where they are often dominant in grazing ecosystems. Received: 17 March 1999 / Accepted: 1 November 1999     

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     

Nutrient availability and nutrient use efficiency in plants growing in the transition zone between land and water

The transition zone between terrestrial and freshwater habitats is highly dynamic, with large variability in environmental characteristics. Here, we investigate how these characteristics influence the nutritional status and performance of plant life forms inhabiting this zone. Specifically, we hypothesised that: (i) tissue nutrient content differs among submerged, amphibious and terrestrial species, with higher content in submerged species; and (ii) PNUE gradually increases from submerged over amphibious to terrestrial species, reflecting differences in the availability of N and P relative to inorganic C across the land–water ecotone. We found that tissue nutrient content was generally higher in submerged species and C:N and C:P ratios indicated that content was limiting for growth for ca. 20% of plant individuals, particularly those belonging to amphibious and terrestrial species groups. As predicted, the PNUE increased from submerged over amphibious to terrestrial species. We suggest that this pattern reflects that amphibious and terrestrial species allocate proportionally more nutrients into processes of importance for photosynthesis at saturating CO₂ availability, i.e. enzymes involved in substrate regeneration, compared to submerged species that are acclimated to lower availability of CO₂ in the aquatic environment. Our results indicate that enhanced nutrient loading may affect relative abundance of the three species groups in the land–water ecotone of stream ecosystems. Thus, species of amphibious and terrestrial species groups are likely to benefit more from enhanced nutrient availability in terms of faster growth compared to aquatic species, and that this can be detrimental to aquatic species growing in the land–water ecotone, e.g. Ranunculus and Callitriche.