The impact of ozone on juvenile maize (Zea mays L.) plant photosynthesis: effects on vegetative biomass, pigmentation, and carboxylases (PEPc and Rubisco)

The impact of ozone on crops was more studied in C (3) than in C (4) species. In C (3) plants, ozone is known to induce a photosynthesis impairment that can result in significant depressions in biomass and crop yields. To investigate the impact of O (3) on C (4) plant species, maize seedlings ( ZEA MAYS L. cv. Chambord) were exposed to 5 atmospheres in open-top chambers: non-filtered air (NF, 48 nL L (-1) O (3)) and NF supplied with 20 (+ 20), 40 (+ 40), 60 (+ 60), and 80 (+ 80) nL L (-1) ozone. An unchambered plot was also available. Leaf area, vegetative biomass, and leaf dry mass per unit leaf area (LMA) were evaluated 33 days after seedling emergence in OTCs. At the same time, photosynthetic pigments as well as carboxylase (PEPc and Rubisco) activities and amounts were also examined in the 5th leaf. Ozone enhanced visible symptoms characterizing foliar senescence. Across NF, + 20, + 40, and + 60 atmospheres, both chlorophylls and carotenoids were found to be linearly decreased against increasing AOT40 ( CA. - 50 % in + 60). No supplementary decrease was observed between + 60 and + 80. Total above-ground biomass was reduced by 26 % in + 80 atmosphere; leaf dry matter being more depressed by ozone than leaf area. In some cases, LMA index was consistent to reflect low negative effects caused by a moderate increase in ozone concentration. PEPc and Rubisco were less sensitive to ozone than pigments: only the two highest external ozone doses reduced their activities by about 20 - 30 %. These changes might be connected to losses in PEPc and Rubisco proteins that were decreased by about one-third. The underlying mechanisms for these results were discussed with special reference to C (3) species. To conclude, we showed that both light and dark reactions of C (4) photosynthesis can be impaired by realistic ozone doses.     

Differential effects of chilling on the activity of C4 enzymes in two ecotypes of Echinochloa crus-galli from sites of contrasting climates

Five-week-old plants of Echinochloa crusgalli (L.) Beauv. from Mississippi and from Québec grown under controlled conditions were subjected to dark chilling for 10 h at 5°C or light chilling treatments for 14 h at 7°C under hight light (1 000 μmol m⁻² s⁻¹). The activities of four C₄ enzymes of Québec plants, measured 4 h after the completion of the cold treatment, were not affected by the chilling treatment in the dark. The activities of pyruvate, P_i dikinase (PPDK; EC 2.7.9.1) and NADP⁺-malic enzyme (NADP⁺-ME; EC 1.1.1.40), were significantly reduced in dark-chilled Mississippi plants. Chilling under high light conditions elicited significant levels of reduction in the activities of the four enzymes from both ecotypes but the reductions were significantly less severe for Québec plants. The recovery of activities of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) and PPDK for both ecotypes was completed within 36 to 60 hours following the chilling treatment, but NADP⁺-malate dehydro-genase (NADP⁺-MDH; EC 1.1.1.82) and NADP⁺-ME activities of chilled Mississippi plants remained below that of control plants at the end of the 5-day monitoring period. PPDK was inactivated in vitro at 0 and 10°C and the rates of cold inactivation were significantly higher for PPDK extracted from Mississippi plants. The activity of PEPC of Mississippi extracts was slightly, but significantly reduced by a 60 min treatment at 0°C.     

Influence of light intensity during growth on photosynthesis and activity of several key photosynthetic enzymes in a C4 plant (Zea mays)

Maize ( Zea mays L. Hybrid Sweet Corn, Royal Crest), a C₄ plant, was grown under different light regimes, after which the rate of photosynthesis and activities of several photosynthetic enzymes (per unit leaf chlorophyll) were measured at different light intensities. Plants were grown outdoors under direct sunlight or 23% of direct sunlight, and in growth chambers at photosynthetic photon flux densities of about 20% and 8% of direct sunlight. The plants grown under direct sunlight had a higher light compensation point than plants grown under lower light. At a light intensity about 25% of direct sunlight, plants from all growth regimes had a similar rate of photosynthesis. Under saturating levels of light the plants grown under direct sunlight had a substantially higher rate of photosynthesis than plants grown under the lower light regimes. The higher photosynthetic capacity in the plants grown under direct sunlight was accompanied by an increased activity of several photosynthetic enzymes and in the amount of the soluble protein in the leaf. Among five photosynthetic enzymes examined, RuBP carboxylase (EC 4.1.1.39) and pyruvate, Pi dikinase (EC 2.7.9.1) were generally just sufficient to account for rates of photosynthesis under saturating light; thus, these may be rate limiting enzymes in C₄ photosynthesis. Pyruvate, Pi dikinase and NADP-malate dehydrogenase (EC 1.1.1.82) were the only enzymes examined which were light activated and increased in activity with increasing light intensity. In the low light grown plants the activity of pyruvate, Pi dikinase closely paralleled the photosynthetic rate measured under different light levels. With the plants grown under direct sunlight, as light intensity was increased the activation of pyruvate, Pi dikinase and NADP⁺-malate dehydrogenase proceeded more rapidly than photosynthesis.     

Effects of growth light and nitrogen nutrition on the organization of the photosynthetic apparatus in leaves of a C4 plant, Amaranthus cruentus

Properties of C4 photosynthesis were examined in Amaranthus cruentus L. (NAD-malic enzyme (ME) subtype, dicot) grown under different light and nitrogen (N) conditions, from the viewpoint of N investment into their photosynthetic components. In low-light (LL) leaves, chlorophyll content per leaf area was greater and chlorophyll alb ratio was lower than in high-light (HL) leaves. These indicate that LL leaves invest more N into their light-harvesting systems. However, this N investment did not contribute to the increase in the quantum yield of photosynthesis on the incident photon flux density (PFD) basis (Qi) in LL leaves. N allocation to ribulose 1,5-bisphosphate carboxylasel oxygenase (Rubisco) was significantly higher in HL-high N (HN) leaves than in other leaves. On the other hand, N allocation to C4 enzymes [phosphoenolpyruvate carboxylase (PEPC) and pyruvate Pi dikinase (PPDK)] was unaffected by the growth conditions. Maximum photosynthetic rates (Pmax) per Rubisco content were similar irrespective of the growth light treatments. Carbon isotope ratios (delta13 C) in the leaf dry matter were more negative in LL leaves than in HL leaves (LL = -19.3% per hundred, HL = -16.0% per hundred) and independent of leaf N. Vein density was highest in HL-HN leaves, and leaf thickness was unaffected by the growth light treatments. From these results, we conclude that A. cruentus leaves would not acclimate efficiently to low growth light.     

Relationships between quantum yield for CO2 assimilation, activity of key enzymes and CO2 leakiness in Amaranthus cruentus, a C4 dicot, grown in high or low light

In C(4) photosynthesis, a part of CO(2) fixed by phosphoenolpyruvate carboxylase (PEPC) leaks from the bundle-sheath cells. Because the CO(2) leak wastes ATP consumed in the C(4) cycle, the leak may decrease the efficiency of CO(2) assimilation. To examine this possibility, we studied the light dependence of CO(2) leakiness (phi), estimated by the concurrent measurements of gas exchange and carbon isotope discrimination, initial activities of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and pyruvate, orthophosphate dikinase (PPDK), the phosphorylation state of PEPC and the CO(2) assimilation rate using leaves of Amaranthus cruentus (NAD-malic enzyme subtype, dicot) plants grown in high light (HL) and low light (LL). phi was constant at photon flux densities (PFDs) >200 micromol m(-2) s(-1) and was around 0.3. At PFDs <150 micromol m(-2) s(-1), phi increased markedly as PFD decreased. At 40 micromol m(-2) s(-1), phi was 0.76 in HL and 0.55 in LL leaves, indicating that the efficiency of CO(2) assimilation at low PFD was greater in LL leaves. The activities of Rubisco and PPDK, and the phosphorylated state of PEPC all decreased as PFD decreased. Theoretical calculations with a mathematical model clearly showed that the increase in phi with decreasing PFD contributed to the decrease in the CO(2) assimilation rate. It was also shown that the 'conventional' quantum yield of photosynthesis obtained by fitting the straight line to the light response curve of the CO(2) assimilation rate at the low PFD region is seriously overestimated. Ecological implications of the increase in phi in LL are discussed.     

Photosynthesis and the influence of CO2-enrichment on δ13C values in a C3 halophyte

Abstract Shifts in ?¹³C of the graminaceous C₃ halophyte Puccinellia nuttalliana (Schultes) Hitch. can be induced by salinization. To investigate this phenomenon, three approaches were taken: assay of carboxylases, CO₂-enrichment studies, and gas exchange analysis. Although ribulose-1,5-bisphosphate carboxylase activity decreased with salinity, phosphoenolpyruvate carboxylase activity did not increase and its levels were not atypical of C₃ plants. When plants were grown at four NaCl concentrations under atmospheres of 310 and 1300 cm³ m^?3 CO₂, the CO₂-enrichment enhanced the effects of salinity on ?¹³C. This is consistent with a biophysical explanation for salt-induced shifts in ?¹³C, whereby there is a steepening of the CO₂ diffusion gradient into the leaf. Gas exchange analysis indicated that intercellular CO₂ concentrations were depressed in the leaves of salt-affected plants. This resulted from a greatly decreased stomatal conductance coupled with only small effects on intrinsic photosynthetic capacity. Water-use efficiency was enhanced.     

Anisakis simplex: CO(2)-fixing enzymes and development throughout the in vitro cultivation from third larval stage to adult

We studied the effect of CO(2) on the in vitro cultivation of Anisakis simplex, an aquatic parasitic nematode of cetaceans (final hosts) and fish, squid, crustaceans and other invertebrates (intermediate/paratenic hosts), and, occasionally, of man (accidental host). The results showed that a high pCO(2), at a suitable temperature, is vital for the optimum development of these nematodes, at least from the third larval stage (L3) to adult. After 30 days cultivation in air, molting to L4 (fourth larval stage) was reduced to 1/3, while survival was about 1/3 of that when cultivated in air + 5% CO(2). The activity of the CO(2)-fixing enzymes, PEPCK and PEPC, was also studied. Throughout the development of the worms studied, PEPCK activity was much higher than that of PEPC (e.g., 305 vs. 6.8 nmol/min.mg protein, respectively, in L3 collected from the host fish). The activity of these enzymes in the worms cultivated in air + 5% CO(2) was highest during M3, and was also generally higher than that of those cultivated in air only, especially during molting from L3 to L4 (e.g., in recently molted L4, PEPCK activity was 3.7 times greater than that of PEPC 2.9 times greater than when cultivated in air).     

Some effects of 4-chloro-5-(dimethylamino)-2-phenyl-3(2H)-pyridazinone (San 9785) on the development of chloroplast thylakoid membranes in Hordeum vulgare L.

Chloroplast ultrastructural and photochemical features were examined in 6-d-old barley (Hordeum vulgare L. cv. Sundance) plants which had developed in the presence of 4-chloro-5-(dimethylamino)-2-phenyl-3(2H)-pyridazinone (San 9785). In spite of a substantial modification of the fatty-acid composition of thylakoid lipids there were no gross abnormalities in chloroplast morphology, and normal amounts of membrane and chlorophyll were present. Fluorescence kinetics at 77K demonstrated considerable energetic interaction of photosystem (PS)I and PSII chlorophylls within the altered lipid environment. An interference with electron transport was indicated from altered room-temperature fluorescence kinetics at 20°C. Subtle changes in the arrangements of chloroplast membranes were consistently evident and the overall effects of these changes was to increase the proportion of appressed to nonappressed membranes. This correlated with a lower chlorophyll a/b ratio, an increase in the amount of light-harvesting chlorophylls as determined by gel electrophoresis and fluorescence emission spectra, and an increase in excitation-energy transfer from PSII to PSI, as predicted from current ideas on the organisation of photosystems in appressed and non-appressed thylakoid membranes.Abbreviations CP1 P700-chlorophyll a protein - F_o, F_m, F_v minimal, maximal and variable fluorescence yield - LHCP light-harvesting chlorophyll-protein complex - PSI, PSII photosystem I, II - San 9785 4-chloro-5(dimethylamino)-2-phenyl-3(2H)-pyridazinone     

Mitochondrial malic enzyme (ME2) in pancreatic islets of the human, rat and mouse and clonal insulinoma cells: Simple enzyme assay for mitochondrial malic enzyme 2

Despite interest in malic enzyme(ME)s in insulin cells, mitochondrial malic enzyme (ME2) has only been studied with estimates of mRNA or with mRNA knockdown. Because an mRNA’s level does not necessarily reflect the level of its cognate enzyme, we designed a simple spectrophotometric enzyme assay to measure ME2 activity of insulin cells by utilizing the distinct kinetic properties of ME2. Mitochondrial ME2 uses either NAD or NADP as a cofactor, has a high K_m for malate and is allosterically activated by fumarate and inhibited by ATP. Cytosolic ME (ME1) and the other mitochondrial ME (ME3) use only NADP as a cofactor and have lower K_ms for malate. The assay easily showed for the first time that substantial ME2 activity is present in pancreatic islets of humans, rats and mice and INS-1 832/13 cells. ME2’s presence was confirmed with immunoblotting. There was no evidence that ME3 is present in these tissues.     

Effects of elevated CO2, elevated O3 and potassium deficiency on Norway spruce [Picea abies (L) Karst.]: seasonal changes in photosynthesis and non-structural carbohydrate content

Two clones of 5-year-old Norway spruce [Picea abies (L.) Karst.] were exposed to two atmospheric concentrations of CO2 (350 and 750 μmol mol^?1) and O₃ (20 and 75nmolmol^?1) in a phytotron at the GSF-Forschung-szentrum (Munich) over the course of a single season (April to October). The phytotron was programmed to recreate an artificial climate similar to that at a high elevation site in the Inner Bavarian Forest, and trees were grown in large containers of forest soil fertilized to achieve contrasting levels of potassium nutrition, designated well-fertilized or K-deficient. Measurements of the rate of net CO₂ assimilation were made on individual needle year age classes over the course of the season, chlorophyll fluorescence kinetics were recorded after approximately 23 weeks, and seasonal changes in non-structural carbohydrate composition of the current year's foliage were monitored. Ozone was found to have contrasting effects on the rate of net CO₂ assimilation in different needle age classes. After c. 5 months of fumigation, elevated O₃ increased (by 33%) the rate of photosynthesis in the current year's needles. However, O3 depressed (by 30%) the photo-synthetic rate of the previous year's needles throughout the period of exposure. Chlorophyll fluorescence measurements indicated that changes in photosystem II electron transport played no significant role in the effects of O₃ on photosynthesis. The reasons for the contrasting effects of O₃ on needles of different ages are discussed in the light of other recent findings. Although O₃ enhanced the rate at which CO₂ was fixed in the current year's foliage, this was not reflected in increases in the non-structural carbohydrate content of the needles. The transfer of ambient CO₂-grown trees to a CO₂-enriched atmosphere resulted in marked stimulation in the photosynthetic rate of current and previous year's foliage. However, following expansion of the current year's growth, the photosynthetic rate of the previous year's foliage declined. The extent of photosynthetic adjustment in response to prolonged exposure to elevated CO₂ depended upon the clone, providing evidence of intraspecific variation in the long-term response of photosynthesis to elevated CO₂. The increase in photosynthesis induced by CO₂ enrichment was associated with increased foliar concentrations of glucose, fructose and starch (but no change in sucrose) in the new growth. CO₂ enrichment significantly enhanced the photosynthetic rate of K-deficient needles, but there was a strong CO₂soil interaction in the current year's needles, indicating that the long-term response of trees to a high CO₂ environment may depend on soil fertility. Although the rate of photosynthesis and non-structural carbohydrate content of the new needles were increased in O₃-treated plants grown at higher levels of CO₂, there was no evidence that elevated CO₂ provided additional protection against O₃ damage. Simultaneous exposure to elevated O₃ modified the effects of elevated CO₂ on needle photosynthesis and non-structural carbohydrate content, emphasizing the need to take into account not only soil nutrient status but also the impact of concurrent increases in photochemical oxidant pollution in any serious consideration of the effects of climate change on plant production.     

Seasonal effects of sun exposure and emersion on intertidal seaweed physiology: Fluctuations in antioxidant contents, photosynthetic pigments and photosynthetic efficiency in the red alga Porphyra umbilicalis Kützing (Rhodophyta, Bangiales)

There is a great deal of speculation regarding the physiological and biochemical mechanisms that give certain seaweed species the ability to colonize the intertidal zone. Frequent exposure to ambient temperatures and high irradiance levels in addition to dehydration during tidal emersion generates acute physiological stress. The ability of seaweeds like Porphya to overcome these challenges and survive in such a harsh environment has been linked to elevated reactive oxygen metabolism. The current study focused on measuring seasonal changes in antioxidant enzymes plus alterations in pigment contents and photosynthetic efficiency of P. umbilicalis plants found growing in the uppermost intertidal zone.Our results suggest that P. umbilicalis exhibits increased antioxidant metabolism, which could contribute to its success in colonizing such a stressful habitat. Elevated levels of glutathione reductase GTR, catalase and carotenoid contents during emersion suggested heightened protection against reactive oxygen species ROS damage is a necessary attribute for species in the upper intertidal regions. This hypothesis was further strengthened by the finding that the greatest antioxidant increases were observed during summer months when irradiance levels and temperatures were at their peak. Winter emersion did not elicit the same physiological response, as antioxidant levels were similar in submersed and emersed plants.For the most part, photosynthetic pigments were largely affected by sun exposure and less by emersion stress. Shaded blades maintained higher concentrations of photosynthetic pigments compared to sun exposed thalli concurring with established research. Photosynthetic efficiency measurements indicated emersion and not sun exposure was the greater facilitator of photoinhibitory damage and ROS generation at PSII. The findings of this field study strengthen previous assertions that protection via elevated antioxidant metabolism and increased PSII repair are involved in providing relief from the acute environmental stresses in the intertidal zone.     

Studies on the leaf of Amaranthus retroflexus (Amaranthaceae): ultrastructure,plasmodesmatal frequency,and solute concentration in relation to phloem loading

Both the mesophyll and bundle-sheath cells associated with the minor veins in the leaf of Amaranthus retroflexus L. contain abundant tubular endoplasmic reticulum, which is continuous between the two cell types via numerous plasmodesmata in their common walls. In bundle-sheath cells, the tubular endoplasmic reticulum forms an extensive network that permeates the cytoplasm, and is closely associated, if not continuous, with the delimiting membranes of the chloroplasts, mitochondria, and microbodies. Both the number and frequency of plasmodesmata between various cell types decrease markedly from the bundle-sheath — vascular-parenchyma cell interface to the sicve-tube member — companion-cell interface. For plants taken directly from lighted growth chambers, a stronger mannitol solution (1.4 M) was required to plasmolyze the companion cells and sieve-tube members than that (0.6 M) necessary to plasmolyze the mesophyll, bundle-sheath, and vascular-parenchyma cells. Placing plants in the dark for 48 h reduced the solute concentration in all cell types. Judging from the frequency of plasmodesmata between the various cell types of the vascular bundles, and from the solute concentrations of the various cell types, it appears that assimilates are actively accumulated by the sieve-tube — companion-cell complex from the apoplast.     

Seasonal changes in the effects of free-air CO2 enrichment (FACE) on growth, morphology and physiology of rice root at three levels of nitrogen fertilization

Over time, the relative effects of elevated [CO₂] on the aboveground photosynthesis, growth and development of rice (Oryza sativa L.) are likely to be changed with increasing duration of CO₂ exposure, but the resultant effects on rice belowground responses remain to be evaluated. To investigate the impacts of elevated [CO₂] on seasonal changes in root growth, morphology and physiology of rice, a free‐air CO₂ enrichment (FACE) experiment was performed at Wuxi, Jiangsu, China, in 2002–2003. A japonica cultivar with large panicle was exposed to two [CO₂] (ambient [CO₂], 370 μmol mol⁻¹; elevated [CO₂], 570 μmol mol⁻¹) at three levels of nitrogen (N): low (LN, 15 g N m⁻²), medium (MN, 25 g N m⁻²) and high N (HN, 35 g N m⁻²). Elevated [CO₂] increased cumulative root volume, root dry weight, adventitious root length and adventitious root number at all developmental stages by 25–71%, which was mainly associated with increased root growth rate during early growth period (EGP) and lower rate of root senescence during late growth period (LGP), while a slight inhibition of root growth rate occurred during middle growth period (MGP). For individual adventitious roots, elevated [CO₂] increased average length, volume, diameter and dry weight early in the season, but the effects gradually disappeared in subsequent stages. Total surface area and active adsorption area per unit root dry weight reached their maxima 10 days earlier in FACE vs. ambient plants, but both of them together with root oxidation ability per unit root dry weight declined with elevated [CO₂] during MGP and LGP, the decline being larger during MGP than LGP. The CO₂‐induced decreases in specific root activities during MGP and LGP were associated with a larger amount of root accumulation during EGP and lower N concentration and higher C/N ratio in roots during MGP and LGP in FACE vs. ambient plants. The results suggest that most of the CO₂‐induced increases in shoot growth of rice are similarly associated with increased root growth.     

Acetyl glyceryl ether phosphorylcholine (platelet-activating factor) mediates heightened metabolic activity in macrophages: Studies on PGE, TXB2 and O2 production, spreading, and the influence of calmodulin-inhibitor W-7

The phospholipid mediator AGEPC (acetyl glyceryl ether phosphorylcholine) was examined for its effects on guinea pig peritoneal macrophages. At a concentration of 10⁻⁹ -10⁻⁶ M, AGEPC evoked release of prostaglandin E (PGE) and thromboxane B₂ (TXB₂) from albumin-elicited macrophages. It also triggerd generation of O⁻₂ by Corynebacterium parvum-induced cells. Moreover, it caused augmented spreading of macrophages. The calmodulin antagonis W-7 attenuated AGEPC-mediated O⁻₂ production and cell spreading whereas prostanoid synthesis was enhanced. These novel actions of AGEPC on the major cellular component of the inflammatory process attest to its role as a potent mediator of immunoinflammatory responses.     

Molecular and functional characterization of the plastid-localized Phosphoenolpyruvate enolase (ENO1) from Arabidopsis thaliana

The Arabidopsis thaliana gene At1g74030 codes for a putative plastid phosphoenolpyruvate (PEP) enolase (ENO1). The recombinant ENO1 protein exhibited enolase activity and its kinetic properties were determined. ENO1 is localized to plastids and expressed in most heterotrophic tissues including trichomes and non-root-hair cells, but not in the mesophyll of leaves. Two T-DNA insertion eno1 mutants exhibited distorted trichomes and reduced numbers of root hairs as the only visible phenotype. The essential role of ENO1 in PEP provision for anabolic processes within plastids, such as the shikimate pathway, is discussed with respect to plastid transporters, such as the PEP/phosphate translocator.     

Phylogenetic reconstruction at the species and intraspecies levels in the genus Pisum (L.) (peas) using a histone H1 gene

A phylogenetic analysis of the genus Pisum (peas), embracing diverse wild and cultivated forms, which evoke problems with species delimitation, was carried out based on a gene coding for histone H1, a protein that has a long and variable functional C-terminal domain. Phylogenetic trees were reconstructed on the basis of the coding sequence of the gene His5 of H1 subtype 5 in 65 pea accessions. Early separation of a clear-cut wild species Pisum fulvum is well supported, while cultivated species Pisum abyssinicum appears as a small branch within Pisum sativum. Another robust branch within P. sativum includes some wild and almost all cultivated representatives of P. sativum. Other wild representatives form diverse but rather subtle branches. In a subset of accessions, PsbA-trnH chloroplast intergenic spacer was also analysed and found less informative than His5. A number of accessions of cultivated peas from remote regions have a His5 allele of identical sequence, encoding an electrophoretically slow protein product, which earlier attracted attention as likely positively selected in harsh climate conditions. In PsbA-trnH, a 8bp deletion was found, which marks cultivated representatives of P. sativum.