Strategies for engineering C4 photosynthesis

C₃ photosynthesis is an inefficient process, because the enzyme that lies at the heart of the Benson–Calvin cycle, ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco) is itself a very inefficient enzyme. The oxygenase activity of Rubisco is an unavoidable side reaction that is a consequence of its reaction mechanism. The product of oxygenation, glycollate 2-P, has to be retrieved by photorespiration, a process which results in the loss of a quarter of the carbon that was originally present in glycollate 2-P. Photorespiration therefore reduces carbon gain. Purely in terms of carbon economy, there is, therefore, a strong selection pressure on plants to reduce the rate of photorespiration so as to increase carbon gain, but it also improves water- and nitrogen-use efficiency. Possibilities for the manipulation of plants to decrease the amount of photorespiration include the introduction of improved Rubisco from other species, reconfiguring photorespiration, or introducing carbon-concentrating mechanisms, such as inorganic carbon transporters, carboxysomes or pyrenoids, or engineering a full C₄ Kranz pathway using the existing evolutionary progression in C₃–C₄ intermediates as a blueprint. Possible routes and progress to suppressing photorespiration by introducing C₄ photosynthesis in C₃ crop plants will be discussed, including whether single cell C₄ photosynthesis is feasible, how the evolution of C₃–C₄ intermediates can be used as a blueprint for engineering C₄ photosynthesis, which pathway for the C₄ cycle might be introduced and the extent to which processes and structures in C₃ plant might require optimisation.     

Tissue Sites of Uptake ofC-Labeled C60

This paper describes thein vivobehavior and potential metabolism of C₆₀and a more water-soluble quaternary ammonium salt-derivatized C₆₀. In both cases, a¹⁴C-labeled fullerene core was utilized for the target molecules that were intravenously injected into female Sprague–Dawley rats. The¹⁴C-labeled C₆₀(*C₆₀) was rapidly (within 1 min) cleared from the circulation and the majority of the *C₆₀accumulated in the liver (90–95%). *C₆₀was not eliminated from the liver over the 120-h period of this study. Our results also suggest that C₆₀is not metabolized by the typical oxidative patterns characteristic of other polycyclic aromatics. Therefore, although not acutely toxic, use of C₆₀, or its derivatives that could be cleaved back to the parent C₆₀in vivo, would likely lead to long-term fullerene accumulation in the liver. The uptake of *C₆₀and¹⁴C-labeled ammonium salt-derivatized C₆₀(1)by human keratinocytesin vitroshowed that while both *C₆₀and1are readily taken up by cells,1accumulates more slowly. Additionally, while C₆₀, at rather high concentrations (2.0 μM) and over extended periods of time (8 days), is able to inhibit the growth of human keratinocytes by about 50%, this effect showed little, if any, photoinducability.     

Relationship between allocation of absorbed light energy in PSII and photosynthetic rates of C3 and C4 plants

Two C₃ dicotyledonous crops and five C₄ monocotyledons treated with three levels of nitrogen were used to evaluate quantitatively the relationship between the allocation of absorbed light energy in PSII and photosynthetic rates (P _N) in a warm condition (25–26°C) at four to five levels [200, 400, 800, 1,200 (both C₃ and C₄) and 2,000 (C₄ only) μmol m⁻² s⁻¹] of photosynthetic photon flux density (PPFD). For plants of the same type (C₃ or C₄), there was a linear positive correlation between the fraction of absorbed light energy that was utilized in PSII photochemistry (P) and P _N, regardless of the broad range of their photosynthetic rates due to species-specific effect and/or nitrogen application; meanwhile, the fraction of absorbed light energy that was dissipated through non-photochemical quenching (D) showed a negative linear regression with P _N for each level of PPFD. The intercept of regression lines between P and P _N of C₃ and C₄ plants decreased, and that between D and P _N increased with increasing PPFD. With P and D as the main components of energy dissipation and complementary to each other, the fraction of excess absorbed light energy (E) was unchanged by P _N under the same level of PPFD. At the same level of P _N, C₄ plants had lower P and higher D than C₃ plants, due to the fact that C₄ plants with little or no photorespiration is considered a limited energy sink for electrons. Nevertheless there was a significant negative linear correlation between D and P when data from both C₃ and C₄ plants at varied PPFD levels was merged. The slope of regression lines between P and D was 0.85, indicating that in plants of both types, most of the unnecessary absorbed energy (ca. 85%) could dissipate through non-photochemical quenching, when P was inhibited by low P _N due to species-specific effect and nitrogen limitation at all levels of illumination used in the experiment.     

Characterization of 14 different putative protein kinase cDNA clones of the C4 plantSorghum bicolor

C₄ photosynthesis is functionally dependent on metabolic interactions between mesophyll- and bundle-sheath cells. Although the C₄ cycle is biochemically well understood, many aspects of the regulation of enzyme activities, gene expression and cell differentiation are elusive. Protein kinases are likely involved in these regulatory processes, providing links to hormonal, metabolic and developmental signal-transduction pathways. Here we describe the cloning and characterization of 14 different putative protein kinase leaf cDNA clones from the C₄ plant Sorghum bicolor. These genes belong to three different protein kinase subfamilies: ribosomal protein S6 kinases, SNF1-like protein kinases, and receptor-like protein kinases. We report the partial cDNA sequences, mesophyll/bundle-sheath steady-state mRNA ratios, mesophyll/etiolated leaf steady-state mRNA ratios, and the positions of 14 protein kinase genes on the genetic map of S. bicolor. Only three of the protein kinase genes described here are expressed preferentially in mesophyll cells as compared with the bundle-sheath. Received: 16 January 1998 / Accepted: 3 April 1998     

Leukotriene A4: Enzymatic conversion to leukotriene C4

An unstable epoxide, leukotriene A₄ (5(S)-$\text{trans}$-5,6-oxido-7,9-$\text{trans}$-11,14-$\text{cis}$-eicosatetraenoic acid), was earlier proposed to be an intermediate in the conversion of arachidonic acid into the slow reacting substance (SRS), leukotriene C₄. In the present work synthetic leukotriene A₄ was incubated with human leukocytes or murine mastocytoma cells. A lipoxygenase inhibitor, BW755C, was added in order to prevent leukotriene formation from endogenous substrate. Leukotriene C₄ and 11-$\text{trans}$-leukotriene C₄ were the main products with SRS activity. It was not established whether the 11-$\text{trans}$-compound was formed by isomerization at the leukotriene A₄ or C₄ stage.     

Rat glioma cells (C6) cultured in serum-free defined medium

Rat glioma C₆ cells were adapted to and maintained in serum-free medium for 11 months. Morphological differentiation with extended dendrite processes was observed. This phenomenon is reversible if serum is re-supplemented and is protein or RNA synthesis dependent. The formed cytoplasmic processes rapidly retract when colchicine or vinblastine sulfate is added. db-cAMP is found able to stimulate the extension of cytoplasmic processes of cells cultured in medium containing serum, but no further stimulation was observed in cells adapted to serum-free medium. The serum-free adapted cells retain the ability to synthesize the acidic S-100 protein and the production rate is 25% higher than the cells cultured in serum-supplemented medium. The serum-free adapted cells have a longer population doubling time but the metaphase chromosomes show the same karyotype and modal number as that of C₆ cells continuously cultured in serum-supplemented medium.     

Differential sensitivity of C3 and C4 turfgrass species to increasing atmospheric vapor pressure deficit

Temperature and vapor pressure deficit (VPD) effects on turfgrass growth are almost always confounded in experiments because VPD commonly is substantially increased in elevated-temperature treatments. The objective of this study as to examine specifically the influence of VPD on transpiration response of four ‘warm-season’ (C₄) and four ‘cool-season’ (C₃) turfgrasses to increasing VPD at a stable temperature (29.3 ± 1.5 °C). Although transpiration rates were noticeably lower in C₄ grasses, transpiration rates increased linearly in response to increasing VPD across the range of 0.8–3.0 kPa. In contrast, transpiration rates of C₃ increased sharply with increasing VPD across the range of low VPDs, but became constrained at higher VPDs (>1.35 kPa). Restricted transpiration rate at elevated VPD was most evident in Agrostis palustris and Lolium perenne. Assuming restricted transpiration rates reflect a limitation on leaf CO₂ uptake, these results indicate that the commonly observed decline in growth of C₃ (and success of C₄) grasses at elevated temperature may include a sensitivity to elevated VPD.     

Manipulation by 25-azacycloartanol of the relative percentage of C10, C9 and C8 side-chain sterols in suspension cultures of bramble cells

The addition of 25-azacycloartanol to the medium of suspension cultures of bramble cells resulted, after 6 weeks of growth, in a large decrease in the percentage of C₁₀ side-chain sterols, sitosterol and isofucosterol (83 % of the total in the control, 9 % in the treated cells), and in a spectacular increase in the percentage of C₈ side-chain sterols, cycloartenol, desmosterol and cholesterol (less than 1 % in the control, 53 % in the treated cells). In addition the relative percentage of C₉ side-chain sterols, mainly 24-methylene cholesterol increased significantly (from 16 to 37 %). A secondary effect of 25-azacycloartanol consisted in an increase of the percentage of Δ²⁴ sterols and in a decrease of the percentage of sterols with a saturated side chain. These results are in agreement with an inhibition by 25-azacycloartanol of the C-24 and C-28 methyltransferases and of the Δ²⁴ reductase.     

Liquid chromatographic determination of carotenoids in human serum using an engineered C30 and a C18 stationary phase

A C₃₀ stationary phase was specifically engineered for carotenoid separations, and carotenoid measurements using this column are compared with those obtained using a somewhat more conventional C₁₈ column. Both methods were used to contribute measurements for the certification of carotenoids in Standard Reference Material 968b, Fat-Soluble Vitamins and Cholesterol in Human Serum. Analytes were extracted from the serum into hexane. Measurements on the C₁₈ column were made using a gradient of acetonitrile, methanol, and ethyl acetate, which is described in detail elsewhere. Measurements on the C₃₀ column were made using a gradient of water, methanol, and methyl tert.-butyl ether.     

Infection of maize leaves with Ustilago maydis prevents establishment of C4 photosynthesis

The Basidiomycete fungus Ustilago maydis is the common agent of corn smut and is capable of inducing gall growth on infected tissue of the C₄ plant maize (Zea mays). While U. maydis is very well characterized on the genetic level, the physiological changes in the host plant in response to U. maydis infection have not been studied in detail, yet.Therefore, we examined the influence of U. maydis infection on photosynthetic performance and carbon metabolism in maize leaf galls.At all stages of development, U. maydis-induced leaf galls exhibited carbon dioxide response curves, CO₂ compensation points and enzymatic activities that are characteristic of C₃ photosynthesis, demonstrating that the establishment of C₄ metabolism is prevented in infected tissue. Hexose contents and hexose/sucrose ratio of leaf galls remained high at 6 days post infection, while a shift in free sugar metabolism was observed in the uninfected controls at that time point. Concomitantly, transitory starch production and sucrose accumulation during the light period remained low in leaf galls. Given that U. maydis is infectious on young developing tissue, the observed changes in carbohydrate metabolism suggest that the pathogen manipulates the developing leaf tissue to arrest sink-to-source transition in favor of maintaining sink metabolism in the host cells.Furthermore, evidence is presented that carbohydrate supply during the biotrophic phase of the pathogen is assured by a fungal invertase.     

Functional properties of diapophytoene and related desaturases of C30 and C40 carotenoid biosynthetic pathways

The desaturation reactions of C₃₀ carotenoids from diapophytoene to diaponeurosporene was investigated in vitro and by complementation in Escherichia coli. The expressed diapophytoene desaturase from Staphylococcus aureus inserts three double bonds in an FAD-dependent reaction. The enzyme is inhibited by diphenylamine. In the complementation experiment diapophytoene desaturase was able to convert C₄₀ phytoene to some extend but exhibited a high affinity to ζ-carotene. Comparison to the reaction of a phytoene desaturase from Rhodobacter capsulatus catalyzing a parallel three-step desaturation sequence with the corresponding C₄₀ carotenes revealed that this desaturase can also convert C₃₀ diapophytoene. Other homologous bacterial C₄₀ carotene desaturases could also utilize C₃₀ substrates, including one type of ζ-carotene desaturase which converted diaponeurosporene to diapolycopene. Further complementation experiments including the diapophytoene synthase gene from S. aureus revealed that the C₃₀ carotenogenic pathway is determined by this initial enzyme which is highly homologous to C₄₀ phytoene synthases.     

Root responses along a subambient to elevated CO2 gradient in a C3–C4 grassland

Atmospheric CO₂ (C_a) concentration has increased significantly during the last 20 000 years, and is projected to double this century. Despite the importance of belowground processes in the global carbon cycle, community‐level and single species root responses to rising C_a are not well understood. We measured net community root biomass over 3 years using ingrowth cores in a natural C₃–C₄ grassland exposed to a gradient of C_a from preglacial to future levels (230–550 μmol mol^?1). Root windows and minirhizotron tubes were installed below naturally occurring stands of the C₄ perennial grass Bothriochloa ischaemum and its roots were measured for respiration, carbohydrate concentration, specific root length (SRL), production, and lifespan over 2 years. Community root biomass increased significantly (P<0.05) with C_a over initial conditions, with linear or curvilinear responses depending on sample date. In contrast, B. ischaemum produced significantly more roots at subambient than elevated C_a in minirhizotrons. The lifespan of roots with five or more neighboring roots in minirhizotron windows decreased significantly at high C_a, suggesting that after dense root growth depletes soil resource patches, plants with carbon surpluses readily shed these roots. Root respiration in B. ischaemum showed a curvilinear response to C_a under moist conditions in June 2000, with the lowest rates at C_a<300 μmol mol^?1 and peak activity at 450 μmol mol^?1 in a quadratic model. B. ischaemum roots at subambient C_a had higher SRLs and slightly higher carbohydrate concentrations than those at higher C_a, which may be related to drier soils at low C_a. Our data emphasize that belowground responses of plant communities to C_a can be quite different from those of the individual species, and suggest that complex interactions between and among roots and their immediate soil environment influence the responses of root physiology and lifespan to changing C_a.     

Synthesis, crystal structure and vibrational spectroscopy of a novel mixed ligands Ni(II) pentaborate: [Ni(C4H10N2)(C2H8N2)2][B5O6(OH)4]2

A novel organic-inorganic hybrid pentaborate [Ni(C₄H₁₀N₂)(C₂H₈N₂)₂][B₅O₆(OH)₄]₂ has been synthesized by hydrothermal reaction and characterized by FT-IR, Raman spectroscopy, elemental analyses and DTA-TGA. Its crystal structure was determined from single crystal X-ray diffraction. The structure consists of isolated polyborate anion [B₅O₆(OH)₄]⁻ and nickel complex cation of [Ni(C₄H₁₀N₂)(C₂H₈N₂)₂]²⁺, in which the two kinds of ligands come from the decomposition of triethylenetriamine material. The [B₅O₆(OH)₄]⁻ units are connected to one another through hydrogen bonds, forming a three-dimensional framework with large channel along the a and c axes, in which the templating [Ni(C₄H₁₀N₂)(C₂H₈N₂)₂]²⁺ cations are located. The assignments of the record FT-IR absorption frequencies and Raman shifts were given.     

Synthesis and structure of an organosamarium aryloxide complex, (C5 Me5)2 Sm(OC6HMe4-2,3,5,6)

Bis(pentamethylcyclopentadienyl)samarium bis- (tetrahydrofuranate), (C₅Me₅)₂Sm(THF)₂, reacts with 2,3,5,6-tetramethylphenol in toluene to yield (C₅Me₅)₂Sm(OC₆HMe₄-2,3,5,6). The compound crystallizes in the space group P2₁/c with a = 8.725(3) Å, b=18.821(6) Å, c=18.461(6) Å, β= 111.17(2)°, V = 2827(2) ų and D_c=1.340 g cm⁻³ for Z = 4. Molecules of the aryloxide complex are monomeric and exhibit a bent metallocene structure with a nearly linear Sm---O---C(aryloxide) linkage: Sm---O = 2.13(1) Å, O---C = 1.29(2) Å, and Sm---O---C = 172.3(13)°. Reaction of the samarium complex with phenyllithium produces the previously- characterized species (C₅Me₅)₂Sm(C₆H₅)(THF).     

The promoters of two carboxylases in a C4 plant (maize) direct cell-specific, light-regulated expression in a C3 plant (rice)

C₄ plants have two carboxylases which function in photosynthesis. One, phosphoenolpyruvate carboxylase (PEPC) is localized in mesophyll cells, and the other, ribulose bisphosphate carboxylase (RuBPC) is found in bundle sheath cells. In contrast, C₃ plants have only one photosynthetic carboxylase, RuBPC, which is localized in mesophyll cells. The expression of PEPC in C₃ mesophyll cells is quite low relative to PEPC expression in C₄ mesophyll cells. Two chimeric genes have been constructed consisting of the structural gene encoding β-glucuronidase (GUS) controlled by two promoters from C₄ (maize) photosynthetic genes: (i) the PEPC gene (pepc) and (ii) the small subunit of RuBPC (rbcS). These constructs were introduced into a C₃ cereal, rice. Both chimeric genes were expressed almost exclusively in mesophyll cells in the leaf blades and leaf sheaths at high levels, and no or very little activity was observed in other cells. The expression of both genes was also regulated by light. These observations indicate that the regulation systems which direct cell-specific and light-inducible expression of pepc and rbcS in C₄ plants are also present in C₃ plants. Nevertheless, expression of endogenous pepc in C₃ plants is very low in C₃ mesophyll cells, and the cell specificity of rbcS expression in C₃ plants differs from that in C₄ plants. Rice nuclear extracts were assayed for DNA-binding protein(s) which interact with a cis-regulatory element in the pepc promoter. Gel-retardation assays indicate that a nuclear protein with similar DNA-binding specificity to a maize nuclear protein is present in rice. The possibility that differences in pepc expression in a C₃ plant (rice) and C₄ plant (maize) may be the result of changes in cis-acting elements between pepc in rice and maize is discussed. It also appears that differences in the cellular localization of rbcS expression are probably due to changes in a trans-acting factor(s) required for rbcS expression.     

Protein synthesis during oxygen conformance and severe hypoxia in the mouse muscle cell line C2C12

Oxygen conformance can be described as the ability to reduce energy demand, and hence oxygen consumption, in response to a decline in oxygen availability without a decrease in the concentration of ATP. It has been proposed that oxygen conformance may enhance cellular survival at low oxygen concentrations. We demonstrate that non-contracting C₂C₁₂ cells, a mouse skeletal muscle cell line, are capable of oxygen conformance. Typically, we found oxygen consumption to decline by 30–40% as the concentration of oxygen was reduced from 100 μM to 10 μM. Unexpectedly, the rate of protein synthesis, a major energy consumer in the cell, did not decrease significantly during oxygen conformance. Unlike oxygen conformance, severe hypoxia (<0.5 μM) caused a 36% decline in the concentration of PCr, and under these conditions of energy stress, the rate of protein synthesis declined by 43%. We conclude that there are two distinct metabolic responses to declines in oxygen concentration in non-contracting C₂C₁₂ cells.     

Stomatal responses of C3, C3-C4 and C4Flaveria species to light and intercellular CO2 concentration: implications for the evolution of stomatal behaviour

Stomatal function mediates physiological trade‐offs associated with maintaining a favourable H₂O balance in leaf tissues while acquiring CO₂ as a photosynthetic substrate. The C₃ and C₄ species appear to have different patterns of stomatal response to changing light conditions, and variation in this behaviour may have played a role in the functional diversification of the different photosynthetic pathways. In the current study, we used gain analysis theory to characterize the stomatal conductance response to light intensity in nine different C₃, C₄ and C₃‐C₄ intermediate species Flaveria species. The response of stomatal conductance (g_s) to a change in light intensity represents both a direct (related to a change in incident light intensity, I) and indirect (related to a change in intercellular CO₂ concentration, C_i) response. The slope of the line relating the change in g_s to C_i was steeper in C₄ species, compared with C₃ species, with C₃‐C₄ species having an intermediate response. This response reflects the greater relative contribution of the indirect versus direct component of the g_s versus I response in the C₄ species. The C₃‐C₄ species, Flaveria floridana, exhibited a C₄‐like response whereas the C₃‐C₄ species, Flaveria sonorensis and Flaveria chloraefolia, exhibited C₃‐like responses, similar to their hypothesized position along the evolutionary trajectory of the development of C₄ photosynthesis. There was a positive correlation between the relative contribution of the indirect component of the g_s versus I response and water use efficiency when evaluated across all species. Assuming that the C₃‐C₄ intermediate species reflect an evolutionary progression from fully expressed C₃ ancestors, the results of the current study demonstrate an increase in the contribution of the indirect component of the g_s versus I response as taxa evolve toward the C₄ extreme. The greater relative contribution of the indirect component of the stomatal response occurs through both increases in the indirect stomatal components and through decreases in the direct. Increases in the magnitude of the indirect component may be related to the maintenance of higher water use efficiencies in the intermediate evolutionary stages, before the appearance of fully integrated C₄ photosynthesis.     

δ 13C variation of C3 and C4 plants across an Asian monsoon rainfall gradient in arid northwestern China

We have investigated carbon isotopic compositions of four plant genus/species, Bothriochloa ischaemum (C₄), Stipa bungeana (C₃), Lespedeza sp. (C₃) and Heteropappus less (C₃), along a precipitation gradient in northwest China in order to assess the impact of water availability on the carbon isotopic discrimination against ¹³C during carbon assimilation in this area. This information is necessary for reconstruction of paleovegetation, particularly paleo‐C₃/C₄ plant ratios using δ¹³C value of organic matter in loess and paleosols in the Chinese Loess Plateau. The δ¹³C of C₃ plants, as a group, exhibits a negative correlation with the annual precipitation amount with a total change and sensitivity of 5‰ and ?1.1‰/100 mm, respectively, for the precipitation range from 200 to 700 mm. The C₄ grass, B. ischaemum responds to aridity by decreasing 1.7‰ for over the precipitation range from 350 to 700 mm; the plant δ¹³C is significantly correlated with annual precipitation with a slope ?0.61‰/100 mm. This result implies that without considering the effect of water availability on the plant δ¹³C values, reconstruction of percent C₄ vegetation during the last glaciation can be overestimated by about a factor of two.     

C4 photosynthesis in a single C3 cell is theoretically inefficient but may ameliorate internal CO2 diffusion limitations of C3 leaves

Attempts are being made to introduce C₄ photosynthetic characteristics into C₃ crop plants by genetic manipulation. This research has focused on engineering single‐celled C₄‐type CO₂ concentrating mechanisms into C₃ plants such as rice. Herein the pros and cons of such approaches are discussed with a focus on CO₂ diffusion, utilizing a mathematical model of single‐cell C₄ photosynthesis. It is shown that a high bundle sheath resistance to CO₂ diffusion is an essential feature of energy‐efficient C₄ photosynthesis. The large chloroplast surface area appressed to the intercellular airspace in C₃ leaves generates low internal resistance to CO₂ diffusion, thereby limiting the energy efficiency of a single‐cell C₄ concentrating mechanism, which relies on concentrating CO₂ within chloroplasts of C₃ leaves. Nevertheless the model demonstrates that the drop in CO₂ partial pressure, pCO₂, that exists between intercellular airspace and chloroplasts in C₃ leaves at high photosynthetic rates, can be reversed under high irradiance when energy is not limiting. The model shows that this is particularly effective at lower intercellular pCO₂. Such a system may therefore be of benefit in water‐limited conditions when stomata are closed and low intercellular pCO₂ increases photorespiration.