Photosynthetic Enzyme Activities and Localization in Mollugo verticillata Populations Differing in the Levels of C(3) and C(4) Cycle Operation

Ecotypic differences in the photosynthetic carbon metabolism of Mollugo verticillata were studied. Variations in C₃ and C₄ cycle activity are apparently due to differences in the activities of enzymes associated with each pathway. Compared to C₄ plants, the activities of C₄ pathway enzymes were generally lower in M. verticillata, with the exception of the decarboxylase enzyme, NAD malic enzyme. The combined total carboxylase enzyme activity of M. verticillata was greater than that of C₃ plants, possibly accounting for the high photosynthetic rates of this species. Unlike either C₃ or C₄ plants, ribulose bisphosphate carboxylase was present in both mesophyll and bundle sheath cell chloroplasts in M. verticillata. The localization of this enzyme in both cells in this plant, in conjunction with an efficient C₄ acid decarboxylation mechanism most likely localized in bundle sheath cell mitochondria, may account for intermediate photorespiration levels previously observed in this species.     

Geographic distributions and physiological characteristics of co-existing Flaveria species in south-central Mexico

The genus Flaveria consists of 23 species with significant variation in photosynthetic physiologies. We tested whether photosynthetic pathway variation in seven co-existing Flaveria species corresponds to geographic distributions or physiological performance in C₃, C₄, and intermediate species growing under natural conditions in south-central Mexico. We found that Flaveria pringlei (C₃) was the most widely distributed species with multiple growth habits. Numerous populations of Flaveria kochiana (C₄), a recently described species with a previously unknown distribution, were located in the Mixtec region of Oaxaca. Flaveria cronquistii (C₃) and Flaveria ramosissima (C₃-C₄) were only located in the Tehuacán Valley region while Flaveria trinervia (C₄) was widely distributed. Only one population of Flaveria angustifolia (C₃-C₄) and Flaveria vaginata (C₄-like) were located near Izúcar de Matamoros. Midday leaf water potential differed significantly between Flaveria species, but did not vary according to growth habit or photosynthetic pathway. The quantum yield of photosystem II did not vary between species, despite large differences in leaf nitrogen content, leaf shape, plant size and life histories. We did not find a direct relationship between increasing C₄ cycle characteristics and physiological performance in the Flaveria populations examined. Furthermore, C₃ species were not found at higher elevation than C₄ species as expected. Our observations indicate that life history traits and disturbance regime may be the primary controllers of Flaveria distributions in south-central Mexico.     

The requirement for sodium as a micronutrient by species having the c(4) dicarboxylic photosynthetic pathway

Six species having characteristics of plants with the C₄ dicarboxylic photosynthetic pathway, Echinochloa utilis L. Ohwi et Yabuno (Japanese millet), Cynodon dactylon L. (Bermuda grass), Kyllinga brevifolia Rottb., Amaranthus tricolor L. cv. Early splendour, Kochia childsii Hort., and Portulaca grandiflora Hook (rose moss), responded decisively to 0.1 milliequivalent per liter NaCl supplied to their culture solutions initially containing less than 0.08 microequivalent per liter Na. Chlorosis and necrosis occurred in leaves of plants not receiving sodium. Portulaca failed to set flower in the sodium-deficient cultures. Under similar conditions Poa pratensis L. (Kentucky blue grass) having characteristics of the C₃ photosynthetic pathway made normal growth and did not respond to the addition of sodium. It is concluded from these results and previously reported work that sodium is generally essential for species having the C₄ pathway but not for species with the C₃ pathway.     

Changes in Levels of Intermediates of the C(4) Cycle and Reductive Pentose Phosphate Pathway during Induction of Photosynthesis in Maize Leaves

Changes in the level of metabolites of the C₄ cycle and reductive pentose phosphate (RPP) pathway were measured simultaneously with induction of photosynthesis in maize (Zea mays L.) to evaluate what may limit carbon assimilation during induction in a C₄ plant.

After 20 minutes in the dark, there was an immediate rise in photosynthesis during the first 30 seconds of illumination, followed by a gradual rise approaching steady-state rate after 20 minutes of illumination. Among metabolites of the C₄ cycle, there was a net increase in the level of C₃ compounds (the sum of pyruvate, alanine, and phosphoenolpyruvate) during the first 30 seconds of illumination, while there was a net decrease in the level of C₄ acids (malate plus aspartate). The total level of metabolites of the C₄ cycle underwent a sharp increase during this period. At the same time, there was a sharp rise in the level of intermediates of the RPP pathway (ribulose-1,5-bis-phosphate, 3-phosphoglycerate, dihydroxyacetonephosphate, and fructose-1,6-bisphosphate) during the first minute of illumination. The net increase of carbon among intermediates of the C₄ cycle and RPP pathway was far above that of carbon input from CO₂ fixation, and the increase in intermediates of the RPP pathway could not be accounted for by decarboxylation of C₄ acids, suggesting that an endogenous source of carbon supplies the cycles. After 3 minutes of illumination there was a gradual rise in the levels of intermediates of the C₄ cycle and in the total level of metabolites measured in the RPP pathway. This rise in metabolite levels occurs as photosynthesis gradually increases and may be required for carbon assimilation to reach maximum rates in C₄ plants. This latter stage of inductive autocatalysis through the RPP pathway may contribute to the final buildup of these intermediates.

     

Loss of the Transit Peptide and an Increase in Gene Expression of an Ancestral Chloroplastic Carbonic Anhydrase Were Instrumental in the Evolution of the Cytosolic C4 Carbonic Anhydrase in Flaveria

C₄ photosynthesis has evolved multiple times from ancestral C₃ species. Carbonic anhydrase (CA) catalyzes the reversible hydration of CO₂ and is involved in both C₃ and C₄ photosynthesis; however, its roles and the intercellular and intracellular locations of the majority of its activity differ between C₃ and C₄ plants. To understand the molecular changes underlying the evolution of the C₄ pathway, three cDNAs encoding distinct β-CAs (CA1, CA2, and CA3) were isolated from the leaves of the C₃ plant Flaveria pringlei. The phylogenetic relationship of the F. pringlei proteins with other embryophyte β-CAs was reconstructed. Gene expression and protein localization patterns showed that CA1 and CA3 demonstrate high expression in leaves and their products localize to the chloroplast, while CA2 expression is low in all organs examined and encodes a cytosolic enzyme. The roles of the F. pringlei enzymes were considered in light of these results, other angiosperm β-CAs, and Arabidopsis (Arabidopsis thaliana) “omics” data. All three F. pringlei CAs have orthologs in the closely related C₄ plant Flaveria bidentis, and comparisons of ortholog sequences, expression patterns, and intracellular locations of their products indicated that CA1 and CA2 have maintained their ancestral role in C₄ plants, whereas modifications to the C₃ CA3 gene led to the evolution of the CA isoform that catalyzes the first step in the C₄ photosynthetic pathway. These changes included the loss of the chloroplast transit peptide and an increase in gene expression, which resulted in the high levels of CA activity seen in the cytosol of C₄ mesophyll cells.     

Photosynthesis research on yellowtops: Macroevolutionn in progress

The vast majority of angiosperms, including most of the agronomically important crop plants (wheat, etc.), assimilate CO₂ through the inefficient C₃ pathway of photosynthesis. Under ambient conditions these organisms loose about 1/3 of fixed carbon via photorespiration, an energetically wasteful process. Plants with C₄ photosynthesis (such as maize) eliminate photorespiration via a biochemical CO₂-pump and thus have a larger rate of carbon gain. The genus Flaveria (yellowtops, Asteraceae) contains not only C₃ and C₄ species, but also many C₃-C₄ intermediates, which have been interpreted as evolving from C₃ to fully expressed C₄ metabolism. However, the evolutionary significance of C₃-C₄Flaveria-intermediates has long been a matter of debate. A well-resolved phylogeny of nearly all Flaveria species has recently been published. Here, we review pertinent background information and combine this novel phylogeny with physiological data. We conclude that the Flaveria species complex provides a robust model system for the study of the transition from C₃ to C₄ photosynthesis, which is arguably a macroevolutionary event. We conclude with comments relevant to the current Intelligent Design debate.     

Microbial Oxidation of Hydrocarbons and Related Compounds by Whole-Cell Suspensions of the Methane-Oxidizing Bacterium H-2

Previously, a thermophilic obligate methane-oxidizing bacterium, H-2 (type I), was isolated in our laboratory. H-2 is a new type of methylotroph because of the G+C content of DNA; it uses both the ribulose monophosphate pathway and the serine pathway for carbon assimilation and possesses a new quinone. In addition, we found that resting cell suspensions of H-2 had the ability to oxidize a variety of compounds different from the other methane-oxidizing bacteria as follows. (i) C₁ to C₈n-alkanes are hydroxylated and further oxidized, yielding mixtures of the corresponding alcohols, aldehydes, acids, and ketones. Liquid alkanes are transformed through a different oxidative pathway from that of gaseous ones. (ii) Both gaseous (C₂ to C₄) and liquid (C₅, C₆) n-alkenes are oxidized to their corresponding 1,2-epoxides. (iii) Liquid monochloro and dichloro n-alkanes (C₅, C₆) are oxidized, yielding their corresponding acids or haloacids. (iv) Diethyl ether is oxidized to acetic acid; no ethanol and acetaldehyde are detected. (v) Cyclic and aromatic compounds are also oxidized. (vi) Secondary alcohols (C₃ to C₁₀) are oxidized to their corresponding methyl ketones.     

Variations in the Specific Activity of Ribulose-1,5-bisphosphate Carboxylase between Species Utilizing Differing Photosynthetic Pathways

The in vitro specific activity of ribulose-1,5-bisphosphate carboxylase (RuBPCase) (micromoles CO₂ fixed per minute per milligram enzyme) from a number of C₃ and C₄ species and one green alga were measured. RuBPCases from species which utilize the C₄ pathway have a specific activity ~2-fold higher than those from C₃ species. RuBPCase from Chlamydomonas reinhardtii has a specific activity similar to the C₄ enzyme. Higher specific activity forms of RuBPCase are associated with a decreased enzyme affinity for CO₂ (increased K_m[CO₂]). A small but significant difference in the specific activity of RuBPCase from two C₄ decarboxylation types was also observed. The relationship between enzymic properties and the presence or absence of a CO₂ concentrating mechanism is discussed.     

C3 and C4 leaf anatomy types in Camphorosmeae (Camphorosmoideae,Chenopodiaceae)

Complementary to our previous project on the molecular phylogeny of Camphorosmeae, the leaf anatomy of ca. 35 species including all non-Australian and selected Australian species was studied by use of light microscopy. Nine anatomical leaf types were described, compared to previous classifications, and discussed with regard to their putative evolution on the background of phylogenetic trees. Particular emphasis was given to the relationships between the C₃ and C₄ leaf types: Chenolea type (C₃), Eokochia type (C₃), Neokochia type (C₃), Sedobassia type (C₃/C₄ intermediate), Bassia prostrata type (C₄), B. muricata type (C₄), B. eriantha type, B. lasiantha type (C₄), Camphorosma type (C₄). The main results and conclusions were: (1) Two unusual new C₃ leaf types: Chenolea with microfenestrate chlorenchyma, Eokochia with unique complex vascular bundles; (2) Sedobassia interpreted as anatomically C₃/C₄ intermediate by kranz-like bundle sheath cells is the first C₃/C₄ intermediate in Camphorosmeae and found in a derived position; (3) Neokochia type detected as the likely starting point for all four C₄ leaf types and for the C₃/C₄ intermediate; (4) hypodermis of C₄ types originated from outermost chlorenchyma layer of C₃ types and lost multiple times during further evolution; (5) atriplicoid Bassia. lasiantha type without water storage tissue evolved from kochioid B. muricata type; (6) two independent gains of C₄ photosynthesis, one in Bassia and one in Camphorosma; (7) depending on the lineage, leaf architecture remains comparatively stable (Australian Camphorosmeae) or shows an unexpected plasticity (Bassia scoparia group).     

Photosynthetic and photorespiratory characteristics of flaveria species

The genus Flaveria shows evidence of evolution in the mechanism of photosynthesis as its 21 species include C₃, C₃-C₄, C₄-like, and C₄ plants. In this study, several physiological and biochemical parameters of photosynthesis and photorespiration were measured in 18 Flaveria species representing all the photosynthetic types. The 10 species classified as C₃-C₄ intermediates showed an inverse continuum in level of photorespiration and development of the C₄ syndrome. This ranges from F. sonorensis with relatively high apparent photorespiration and lacking C₄ photosynthesis to F. Among the intermediates, the photosynthetic CO₂ compensation points at 30°C and 1150 micromoles quanta per square meter per second varied from 9 to 29 microbars. The values for the three C₄-like species varied from 3 to 6 microbars, similar to those measured for the C₄ species. The activities of the photorespiratory enzymes glycolate oxidase, hydroxypyruvate reductase, and serine hydroxymethyltransferase decreased progressively from C₃ to C₃-C₄ to C₄-like and C₄ species. On the other hand, most intermediates had higher levels of phosphenolpyruvate carboxylase and NADP-malic enzyme than C₃ species, but generally lower activities compared to C₄-like and C₄ species. The levels of these C₄ enzymes are correlated with the degree of C₄ photosynthesis, based on the initial products of photosynthesis. Another indication of development of the C₄ syndrome in C₃-C₄ Flaveria species was their intermediate chlorophyll a/b ratios. The chlorophyll a/b ratios of the various Flaveria species are highly correlated with the degree of C₄ photosynthesis suggesting that the photochemical machinery is progressively altered during evolution in order to meet the specific energy requirements for operating the C₄ pathway. In the progression from C₃ to C₄ species in Flaveria, the CO₂ compensation point decreased more rapidly than did the decrease in O₂ inhibition of photosynthesis or the increase in the degree of C₄ photosynthesis. These results suggest that the reduction in photorespiration during evolution occurred initially by refixation of photorespired CO₂ and prior to substantive reduction in O₂ inhibition and development of the C₄ syndrome. However, further reduction in O₂ inhibition in some intermediates and C₄-like species is considered primarily due to the development of the C₄ syndrome. Thus, the evolution of C₃-C₄ intermediate photosynthesis likely occurred in response to environmental conditions which limit the intercellular CO₂ concentration first via refixation of photorespired CO₂, followed by development of the C₄ syndrome.     

delta-Aminolevulinic acid synthesis in a Cyanidium caldarium mutant unable to make chlorophyll a and phycobiliproteins.

Wild-type cells of the unicellular rhodophyte, Cyanidium caldarium, synthesize chlorophyll a, phycobiliproteins, and heme from δ-aminolevulinic acid during light-dependent chloroplast development but are unable to make photosynthetic pigments in the dark. C. caldarium, mutant GGB-Y, is an obligate heterotroph which, in the light, produces a chloroplast devoid of photosynthetic pigments. The present investigation has shown that δ-aminolevulinic acid is synthesized in cells of mutant GGB-Y incubated with levulinic acid, a competitive inhibitor of δ-aminolevulinic acid dehydrase (the second enzyme in the porphyrin biosynthetic pathway). In vivo, cells of mutant GGB-Y preferentially incorporated C₁ of glutamate and α-ketoglutarate into the C₅ fragment (formaldehyde) of δ-aminolevulinic acid after alkaline periodate degradation. This suggested that δ-aminolevulinic acid arises directly from the carbon skeleton of glutamate and α-ketoglutaric acid. The pattern of incorporation of C₃, C₄, and C₅ of α-ketoglutarate into the C₁–C₄ (succinic acid) fragment of δ-aminolevulinic acid after alkaline periodate degradation was consistent with the origin of δ-aminolevulinic acid from a five-carbon precursor. C₁ and C₂ of glycine and C₂ and C₃ of succinate were incorporated into both the formaldehyde and succinate fragments of δ-aminolevulinic acid in a manner inconsistent with condensation of glycine and succinyl CoA by δ-aminolevulinic acid synthetase, the rate-limiting enzyme in the porphyrin pathway in animals and bacteria. Extracts of the soluble protein from cells of mutant GGB-Y displayed a Soret band at 410 nm indicating the presence of hemoproteins. This shows that mutant GGB-Y cells synthesize heme. The respiration of radiolabeled glutamate, α-ketoglutarate, and glycine to ¹⁴CO₂ is consistent with the existence of mitochondrial cytochromes in cells of mutant GGB-Y and with the ability of the mutant to synthesize δ-aminolevulinic acid. The present results suggest that δ-aminolevulinic acid is synthesized directly from glutamate or α-ketoglutarate and that this is the only process by which the rate-limiting intermediate in the porphyrin pathway is synthesized in C. caldarium. If correct, the rate-limiting, regulative enzyme in the biosynthetic pathway for synthesis of chlorophyll a, bile pigment (phycocyanobilin), and heme must have been completely different in the evolutionary antecedents of modern-day plants and animals.     

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.     

Photosynthetic Characteristics of C(3)-C(4) Intermediate Flaveria Species : III. Reduction of Photorespiration by a Limited C(4) Pathway of Photosynthesis in Flaveria ramosissima

The initial products of photosynthesis by the C₃ species Flaveria cronquistii, the C₄ species F. trinervia, and the C₃-C₄ intermediate species F. ramosissima were determined using a pulse-chase technique with ¹⁴CO₂-¹²CO₂. The intermediate species F. ramosissima incorporated at least 42% of the total soluble ¹⁴C fixed into malate and aspartate after 10 seconds of photosynthesis in ¹⁴CO₂, as compared with 90% for the C₄ species F. trinervia and 5% for the C₃ species F. cronquistii. In both F. ramosissima and F. trinervia, turnover of labeled malate and aspartate occurred during a chase period in ¹²CO₂, although the rate of turnover was slower in the intermediate species. Relative to F. cronquistii, F. ramosissima showed a reduced incorporation of radioactivity into serine and glycine during the pulse period. These results indicate that a functional C₄ pathway of photosynthesis is operating in F. ramosissima which can account for its reduced level of photorespiration, and that this species is a true biochemical intermediate between C₃ and C₄ plants.     

Effect of inland salt-alkaline stress on C4 enzymes, pigments, antioxidant enzymes, and photosynthesis in leaf, bark, and branch chlorenchyma of poplars

The effects of soil salt-alkaline (SA) stress on leaf physiological processes are well studied in the laboratory, but less is known about their effect on leaf, bark and branch chlorenchyma and no reports exist on their effect on C₄ enzymes in field conditions. Our results demonstrated that activities of C₄ enzymes, such as phospholenolpyruvate carboxylase (PEPC), NADP-malic enzyme (NADP-ME), pyruvate orthophosphate dikinase (PPDK), and NADP-dependent malate dehydrogenase (NADP-MDH), could also be regulated by soil salinity/alkalinity in poplar (Populus alba × P. berolinensis) trees, similarly as the already documented changes in activities of antioxidative enzymes, such as superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR), pigment composition, photosynthesis, and respiration. However, compared with 50–90% changes in a leaf and young branch chlorenchyma, much smaller changes in malondialdehyde (MDA), antioxidative enzymes, and C₄ enzymatic activities were observed in bark chlorenchyma, showing that the effect of soil salinity/alkalinity on enzymatic activities was organ-dependent. This suggests that C₄ enzymatic ratios between nonleaf chlorenchyma and leaf (the commonly used parameter to discern the operation of the C₄ photosynthetic pathway in nonleaf chlorenchyma), were dependent on SA stress. Moreover, much smaller enhancement of these ratios was seen in an improved soil contrary to SA soil, when the fresh mass (FM) was used as the unit compared with a calculation on a chlorophyll (Chl) unit. An identification of the C₄ photosynthesis pathway via C₄ enzyme difference between chlorenchyma and leaf should take this environmental regulation and unit-based difference into account.     

Oxygen Requirement and Inhibition of C4 Photosynthesis : An Analysis of C4 Plants Deficient in the C3 and C4 Cycles

The basis for O₂ sensitivity of C₄ photosynthesis was evaluated using a C₄-cycle-limited mutant of Amaranthus edulis (a phosphoenolpyruvate carboxylase-deficient mutant), and a C₃-cycle-limited transformant of Flaveria bidentis (an antisense ribulose-1,5-bisphosphate carboxylase/oxygenase [Rubisco] small subunit transformant). Data obtained with the C₄-cycle-limited mutant showed that atmospheric levels of O₂ (20 kPa) caused increased inhibition of photosynthesis as a result of higher levels of photorespiration. The optimal O₂ partial pressure for photosynthesis was reduced from approximately 5 kPa O₂ to 1 to 2 kPa O₂, becoming similar to that of C₃ plants. Therefore, the higher O₂ requirement for optimal C₄ photosynthesis is specifically associated with the C₄ function. With the Rubisco-limited F. bidentis, there was less inhibition of photosynthesis by supraoptimal levels of O₂ than in the wild type. When CO₂ fixation by Rubisco is limited, an increase in the CO₂ concentration in bundle-sheath cells via the C₄ cycle may further reduce the oxygenase activity of Rubisco and decrease the inhibition of photosynthesis by high partial pressures of O₂ while increasing CO₂ leakage and overcycling of the C₄ pathway. These results indicate that in C₄ plants the investment in the C₃ and C₄ cycles must be balanced for maximum efficiency.     

Glycerolipid synthesis in Chlorella kessleri 11h: I. Existence of a eukaryotic pathway

The fatty acid distributions at the sn-1 and sn-2 positions in major chloroplast lipids of Chlorella kessleri 11h, monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG), were determined to show the coexistence of both C₁₆ and C₁₈ acids at the sn-2 position, i.e. of prokaryotic and eukaryotic types in these galactolipids. For investigation of the biosynthetic pathway for glycerolipids in C. kessleri 11h, cells were fed with [¹⁴C]acetate for 30 min, and then the distribution of the radioactivity among glycerolipids and their constituent fatty acids during the subsequent chase period was determined. MGDG and DGDG were labeled predominantly as the sn-1-C₁₈-sn-2-C₁₆ (C₁₈/C₁₆) species as early as by the start of the chase, which suggested the synthesis of these lipids within chloroplasts via a prokaryotic pathway. On the other hand, the sn-1-C₁₈-sn-2-C₁₈ (C₁₈/C₁₈) species of these galactolipids gradually gained radioactivity at later times, concomitant with a decrease in the radioactivity of the C₁₈/C₁₈ species of phosphatidylcholine (PC). The change at later times can be explained by the conversion of the C₁₈/C₁₈ species of PC into galactolipids through a eukaryotic pathway. The results showed that C. kessleri 11h, distinct from most of other green algal species that were postulated mainly to use a prokaryotic pathway for the synthesis of chloroplast lipids, is similar to a group of higher plants designated as 16:3 plants in terms of the cooperation of prokaryotic and eukaryotic pathways to synthesize chloroplast lipids. We propose that the physiological function of the eukaryotic pathway in C. kessleri 11h is to supply chloroplast membranes with 18:3/18:3-MGDG for their functioning, and that the acquisition of a eukaryotic pathway by green algae was favorable for evolution into land plants.     

Backbone chemical shift assignments of the acyl-acyl carrier protein intermediates of the fatty acid biosynthesis pathway of Plasmodium falciparum

We report the backbone chemical shift assignments of the acyl-acyl carrier protein (ACP) intermediates of the fatty acid biosynthesis pathway of Plasmodium falciparum. The acyl-ACP intermediates butyryl (C₄), -octanoyl (C₈), -decanoyl (C₁₀), -dodecanoyl (C₁₂) and -tetradecanoyl (C₁₄)-ACPs display marked changes in backbone HN, C_α and C_β chemical shifts as a result of acyl chain insertion into the hydrophobic core. Chemical shift changes cast light on the mechanism of expansion of the acyl carrier protein core.