Effects of elevated atmospheric CO2 concentration on leaf dark respiration of Xanthium strumarium in light and in darkness.

Leaf dark respiration (R) is an important component of plant carbon balance, but the effects of rising atmospheric CO(2) on leaf R during illumination are largely unknown. We studied the effects of elevated CO(2) on leaf R in light (R(L)) and in darkness (R(D)) in Xanthium strumarium at different developmental stages. Leaf R(L) was estimated by using the Kok method, whereas leaf R(D) was measured as the rate of CO(2) efflux at zero light. Leaf R(L) and R(D) were significantly higher at elevated than at ambient CO(2) throughout the growing period. Elevated CO(2) increased the ratio of leaf R(L) to net photosynthesis at saturated light (A(max)) when plants were young and also after flowering, but the ratio of leaf R(D) to A(max) was unaffected by CO(2) levels. Leaf R(N) was significantly higher at the beginning but significantly lower at the end of the growing period in elevated CO(2)-grown plants. The ratio of leaf R(L) to R(D) was used to estimate the effect of light on leaf R during the day. We found that light inhibited leaf R at both CO(2) concentrations but to a lesser degree for elevated (17-24%) than for ambient (29-35%) CO(2)-grown plants, presumably because elevated CO(2)-grown plants had a higher demand for energy and carbon skeletons than ambient CO(2)-grown plants in light. Our results suggest that using the CO(2) efflux rate, determined by shading leaves during the day, as a measure for leaf R is likely to underestimate carbon loss from elevated CO(2)-grown plants.

Plant growth in elevated CO2 alters mitochondrial number and chloroplast fine structure.

With increasing interest in the effects of elevated atmospheric CO(2) on plant growth and the global carbon balance, there is a need for greater understanding of how plants respond to variations in atmospheric partial pressure of CO(2). Our research shows that elevated CO(2) produces significant fine structural changes in major cellular organelles that appear to be an important component of the metabolic responses of plants to this global change. Nine species (representing seven plant families) in several experimental facilities with different CO(2)-dosing technologies were examined. Growth in elevated CO(2) increased numbers of mitochondria per unit cell area by 1.3-2.4 times the number in control plants grown in lower CO(2) and produced a statistically significant increase in the amount of chloroplast stroma (nonappressed) thylakoid membranes compared with those in lower CO(2) treatments. There was no observable change in size of the mitochondria. However, in contrast to the CO(2) effect on mitochondrial number, elevated CO(2) promoted a decrease in the rate of mass-based dark respiration. These changes may reflect a major shift in plant metabolism and energy balance that may help to explain enhanced plant productivity in response to elevated atmospheric CO(2) concentrations.

Elevated CO2 increases productivity and invasive species success in an arid ecosystem.

Arid ecosystems, which occupy about 20% of the earth's terrestrial surface area, have been predicted to be one of the most responsive ecosystem types to elevated atmospheric CO2 and associated global climate change. Here we show, using free-air CO2 enrichment (FACE) technology in an intact Mojave Desert ecosystem, that new shoot production of a dominant perennial shrub is doubled by a 50% increase in atmospheric CO2 concentration in a high rainfall year. However, elevated CO2 does not enhance production in a drought year. We also found that above-ground production and seed rain of an invasive annual grass increases more at elevated CO2 than in several species of native annuals. Consequently, elevated CO2 might enhance the long-term success and dominance of exotic annual grasses in the region. This shift in species composition in favour of exotic annual grasses, driven by global change, has the potential to accelerate the fire cycle, reduce biodiversity and alter ecosystem function in the deserts of western North America.

Effects of short- and long-term elevated CO2 on the expression of ribulose-1,5-bisphosphate carboxylase/oxygenase genes and carbohydrate accumulation in leaves of Arabidopsis thaliana (L.) Heynh.

To investigate the proposed molecular characteristics of sugar-mediated repression of photosynthetic genes during plant acclimation to elevated CO2, we examined the relationship between the accumulation and metabolism of nonstructural carbohydrates and changes in ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) gene expression in leaves of Arabidopsis thaliana exposed to elevated CO2. Long-term growth of Arabidopsis at high CO2 (1000 microL L-1) resulted in a 2-fold increase in nonstructural carbohydrates, a large decrease in the expression of Rubisco protein and in the transcript of rbcL, the gene encoding the large subunit of Rubisco (approximately 35-40%), and an even greater decline in mRNA of rbcS, the gene encoding the small subunit (approximately 60%). This differential response of protein and mRNAs suggests that transcriptional/posttranscriptional processes and protein turnover may determine the final amount of leaf Rubisco protein at high CO2. Analysis of mRNA levels of individual rbcS genes indicated that reduction in total rbcS transcripts was caused by decreased expression of all four rbcS genes. Short-term transfer of Arabidopsis plants grown at ambient CO2 to high CO2 resulted in a decrease in total rbcS mRNA by d 6, whereas Rubisco content and rbcL mRNA decreased by d 9. Transfer to high CO2 reduced the maximum expression level of the primary rbcS genes (1A and, particularly, 3B) by limiting their normal pattern of accumulation through the night period. The decreased nighttime levels of rbcS mRNA were associated with a nocturnal increase in leaf hexoses. We suggest that prolonged nighttime hexose metabolism resulting from exposure to elevated CO2 affects rbcS transcript accumulation and, ultimately, the level of Rubisco protein.

Influence of Plant Growth at High CO2 Concentrations on Leaf Content of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Intracellular Distribution of Soluble Carbohydrates in Tobacco, Snapdragon, and Parsley.

We have examined the possible role of leaf cytosolic hexoses and the expression of mannitol metabolism as mechanisms that may affect the repression of photosynthetic capacity when plants are grown at 1000 versus 380 [mu]L L-1 CO2. In plants grown at high CO2, leaf ribulose-1,5-bisphosphate carboxylase/oxygenase content declined by [greater than or equal to]20% in tobacco (Nicotiana sylvestris) but was not affected in the mannitol-producing species snapdragon (Antirrhinum majus) and parsley (Petroselinum hortense). In the three species mesophyll glucose and fructose at midday occurred almost entirely in the vacuole (>99%), irrespective of growth CO2 levels. The estimated cytosolic concentrations of glucose and fructose were [less than or equal to]100 [mu]M. In the three species grown at high CO2, total leaf carbohydrates increased 60 to 100%, but mannitol metabolism did not function as an overflow mechanism for the increased accumulation of carbohydrate. In both snapdragon and parsley grown at ambient or high CO2, mannitol occurred in the chloroplast and cytosol at estimated midday concentrations of 0.1 M or more each. The compartmentation of leaf hexoses and the metabolism of alternate carbohydrates are further considered in relation to photosynthetic acclimation to high levels of CO2.

EcoCELLs: tools for mesocosm scale measurements of gas exchange.

We describe the use of a unique plant growth facility, which has as its centerpiece four 'EcoCELLs', or 5x7 m mesocosms designed as open-flow, mass-balance systems for the measurement of carbon, water and trace gas fluxes. This system is unique in that it was conceived specifically to bridge the gap between measurement scales during long-term experiments examining the function and development of model ecosystems. There are several advantages to using EcoCELLs, including (i) the same theory of operation as leaf level gas exchange systems, but with continuous operation at a much larger scale; (ii) the ability to independently evaluate canopy-level and ecosystem models; (iii) simultaneous manipulation of environmental factors and measurement of system-level responses, and (iv) maximum access to, and manipulation of, a large rooting volume. In addition to discussing the theory, construction and relative merits of EcoCELLs, we describe the calibration and use of the EcoCELLs during a 'proof of concept' experiment. This experiment involved growing soybeans under two ambient CO2 concentrations (approximately 360 and 710 micromoles mol-1). During this experiment, we asked 'How accurate is the simplest model that can be used to scale from leaf-level to canopy-level responses?' in order to illustrate the utility of the EcoCELLs in validating canopy-scale models.

Plants, CO2 and photosynthesis in the 21st century.

Human activity in the last 200 years has led to a marked increase in the level of CO2 in the atmosphere. Plants sense increases in CO2 levels and initially respond with an increase in photosynthetic rate, which may then slow as the plant adapts. This increase in photosynthetic rate may account in part for the 'disappearance' of an estimated 1.8 gigatons of carbon per year.

Intracellular localization of CA1P and CA1P phosphatase activity in leaves of Phaseolus vulgaris L.

CA1P and CA1P phosphatase occur in the chloroplasts of leaf mesophyll cells of many species. However, whether either may occur exclusively in the chloroplast has not yet been established. To examine their intracellular distribution, mature, dark-or light-treated leaves of Phaseolus vulgaris were frozen, lyophilized and then centrifuged in density gradients of heptane and tetrachloroethylene. After gradient fractionation, both CA1P and CA1P phosphatase activity co-segregated with chloroplast material. Distribution analyses using sub-cellular compartment markers indicated that both CA1P and CA1P phosphatase do occur exclusively in leaf chloroplasts.

Evidence That 2-Carboxyarabinitol 1-Phosphate Binds to Ribulose-1,5-Bisphosphate Carboxylase in Vivo.

An important question concerning the role of carboxyarabinitol 1-phosphate (CA1P) metabolism in the light-dependent regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity is the extent to which CA1P is bound to Rubisco in vivo. We report here the development of an extraction procedure using ammonium sulfate that stabilizes CA1P bound to Rubisco. This procedure exploits the ability of sulfate to bind at the catalytic site of Rubisco and to competitively balance the binding and release of CA1P from Rubisco. In darkened bean leaves about 75% of the Rubisco catalytic sites were found to be bound with CA1P. This confirms previous indirect estimates from gas exchange measurements. We have used this extraction procedure to examine CA1P-Rubisco interactions in bean during a natural transition from darkness to light. With increasing light intensity following sunrise, CA1P degradation proceeded in two distinct phases: first, a majority of the unbound CA1P pool was degraded at very low light levels ([less than or equal to]30 [mu]mol quanta m-2 s-1); second, CA1P initially bound to Rubisco was then degraded at increasing light levels (>30 [mu]mol quanta m-2 s-1). These results indicate that there is a low-fluence activation of CA1P phosphatase that can occur prior to CA1P release by Rubisco activase. This activation may be mediated by NADPH. During sunrise in bean, the level of the catalytically competent form of Rubisco was regulated by CA1P metabolism.

Regulation of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activity in Response to Reduced Light Intensity in C4 Plants.

The light-dependent regulation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity was studied in 16 species of C4 plants representing all three biochemical subtypes and a variety of taxonomic groups. Rubisco regulation was assessed by measuring (a) the ratio of initial to total Rubisco activity, which reflects primarily the carbamylation state of the enzyme, and (b) total Rubisco activity per mol of Rubisco catalytic sites, which declines when 2-carboxyarabinitol 1-phosphate (CA1P) binds to carbamylated Rubisco. In all species examined, the activity ratio of Rubisco declined with a reduction in light intensity, although substantial variation was apparent between species in the degree of Rubisco deactivation. No relationship existed between the degree of Rubisco deactivation and C4 subtype. Dicots generally deactivated Rubisco to a greater degree than monocots. The total activity of Rubisco per catalytic site was generally independent of light intensity, indicating that CA1P and other inhibitors are not major contributors to the light-dependent regulation of Rubisco activity in C4 plants. The light response of the activity ratio of Rubisco was measured in detail in Amaranthus retroflexus, Brachiaria texana, and Zea mays. In A. retroflexus and B. texana, the activity ratio declined dramatically below a light intensity of 400 to 500 [mu]mol of photons m-2 s-1. In Z. mays, the activity ratio of Rubisco was relatively insensitive to light intensity compared with the other species. In A. retroflexus, the pool size of ribulose bisphosphate (RuBP) declined with reduced light intensity except between 50 and 500 [mu]mol m-2 s-1, when the activity ratio of Rubisco was light dependent. In Z. mays, by contrast, the pool size of RuBP was light dependent only below 350 [mu]mol m-2 s-1. These results indicate that, in response to changes in light intensity, most C4 species regulate Rubisco by reversible carbamylation of catalytic sites, as commonly observed in C3 plants. In a few species, notably Z. mays, Rubisco is not extensively regulated in response to changes in light intensity, possibly because the activity of the CO2 pump may become limiting for photosynthesis at subsaturating light intensity.

Identification and levels of 2'-carboxyarabinitol in leaves.

2'-Carboxyarabinitol 1-phosphate (CA1P) is a naturally occurring inhibitor of ribulose-1,5 bisphosphate carboxylase/oxygenase activity. A chloroplast phosphatase has previously been identified that degrades CA1P in vitro to carboxyarabinitol (CA) plus phosphate, but CA has not yet been detected in plants. Here, we detail procedures to isolate and assay CA from leaves and utilize mass spectrometry to demonstrate for the first time that CA is present in plants. CA was present in leaves of all 13 species examined, including those of C(3), C(4), and Crassulacean acid metabolism photosynthetic subgroups. CA was present both in species with high levels of CA1P (e.g. Phaseolus vulgaris, Lycopersicon esculentum, Beta vulgaris) as well as in species with low levels of CA1P (e.g. Spinacea oleracea, Triticum aestivum). CA levels in the light were sometimes greater than those in the dark. Bean leaves had the most CA of any species tested, with levels in the light approaching 1 micromole per milligram of chlorophyll. In illuminated bean leaves, about 63% of the CA is located outside the chloroplast. CA is one of only a few branched chain sugar acids to be identified from plants.

Metabolism of 2'-carboxyarabinitol in leaves.

Results presented here indicate that 2'-carboxyarabinitol (CA) is the in vivo precursor and product of 2'-carboxyarabinitol 1-phosphate (CA1P) metabolism in leaves. When [2-(14)C]CA was fed in the light to leaves of five species known to be highly active in CA1P metabolism (Phaseolus vulgaris, Lycopersicon esculentum, Helianthus annuus, Petunia hybrida, and Beta vulgaris), [(14)C]CA1P was formed in the dark. Reillumination of a Phaseolus leaf caused this [(14)C]CA1P to be rapidly metabolized to [(14)C]CA (t((1/2)) = 1 min). The epimer 2'-carboxyribitol could not substitute for CA in the dark synthesis of CA1P, and CA in the anionic form was a better substrate than CA in the lactone form. In leaves of Phaseolus vulgaris, the active CA pool size used in the dark synthesis of CA1P is between about 70 and 110 nanomoles per milligram of chlorophyll. The photosynthetic electron transport inhibitor diuron did not affect the dark synthesis of [(14)C]CA1P, but did greatly reduce the rate of its subsequent light degradation (t((1/2)) = approximately 10 min). Dark synthesis of [(14)C]CA1P was inhibited by dithiothreitol and NaF. From the present data, we suggest that CA1P and CA participate in a metabolic substrate cycle in vivo.

Measurement of 2-carboxyarabinitol 1-phosphate in plant leaves by isotope dilution.

The level of 2-carboxyarabinitol 1-phosphate (CA1P) in leaves of 12 species was determined by an isotope dilution assay. (14)C-labeled standard was synthesized from [2-(14)C]carboxyarabinitol 1,5-bisphosphate using acid phosphatase, and was added at the initial point of leaf extraction. Leaf CA1P was purified and its specific activity determined. CA1P was found in dark-treated leaves of all species examined, including spinach (Spinacea oleracea), wheat (Triticum aestivum), Arabidopsis thaliana, and maize (Zea mays). The highest amounts were found in bean (Phaseolus vulgaris) and petunia (Petunia hybrida), which had 1.5 to 1.8 moles CA1P per mole ribulose 1,5-bisphosphate carboxylase catalytic sites. Most species had intermediate amounts of CA1P (0.2 to 0.8 mole CA1P per mole catalytic sites). Such intermediate to high levels of CA1P support the hypothesis that CA1P functions in many species as a light-dependent regulator of ribulose 1,5-bisphosphate carboxylase activity and whole leaf photosynthetic CO(2) assimilation. However, CA1P levels in spinach, wheat, and A. thaliana were particularly low (less than 0.09 mole CA1P per mole catalytic sites). In such species, CA1P does not likely have a significant role in regulating ribulose 1,5-bisphosphate carboxylase activity, but could have a different physiological role.

Regulation of Ribulose-1,5-Bisphosphate Carboxylase Activity in Response to Light Intensity and CO(2) in the C(3) Annuals Chenopodium album L. and Phaseolus vulgaris L.

The light and CO(2) response of (a) photosynthesis, (b) the activation state and total catalytic efficiency (k(cat)) of ribulose-1,5-bisphosphate carboxylase (rubisco), and (c) the pool sizes of ribulose 1,5-bisphosphate, (RuBP), ATP, and ADP were studied in the C(3) annuals Chenopodium album and Phaseolus vulgaris at 25 degrees C. The initial slope of the photosynthetic CO(2) response curve was dependent on light intensity at reduced light levels only (less than 450 micromoles per square meter per second in C. album and below 200 micromoles per square meter per second in P. vulgaris). Modeled simulations indicated that the initial slope of the CO(2) response of photosynthesis exhibited light dependency when the rate of RuBP regeneration limited photosynthesis, but not when rubisco capacity limited photosynthesis. Measured observations closely matched modeled simulations. The activation state of rubisco was measured at three light intensities in C. album (1750, 550, and 150 micromoles per square meter per second) and at intercellular CO(2) partial pressures (C(1)) between the CO(2) compensation point and 500 microbars. Above a C(1) of 120 microbars, the activation state of rubisco was light dependent. At light intensities of 550 and 1750 micromoles per square meter per second, it was also dependent on C(1), decreasing as the C(1) was elevated above 120 microbars at 550 micromoles per square meter per second and above 300 microbars at 1750 micromoles per square meter per second. The pool size of RuBP was independent of C(1) only under conditions when the activation state of rubisco was dependent on C(1). Otherwise, RuBP pool sizes increased as C(1) was reduced. ATP pools in C. album tended to increase as C(1) was reduced. In P. vulgaris, decreasing C(1) at a subsaturating light intensity of 190 micromoles per square meter per second increased the activation state of rubisco but had little effect on the k(cat). These results support modelled simulations of the rubisco response to light and CO(2), where rubisco is assumed to be down-regulated when photosynthesis is limited by the rate of RuBP regeneration.

Photosynthetic induction state of leaves in a soybean canopy in relation to light regulation of ribulose-1-5-bisphosphate carboxylase and stomatal conductance.

Photosynthetic induction state, stomatal conductance and light regulation of ribulose-1,5-bisphosphate carboxylase (rubisco) were examined for leaves in a mature, closed soybean (Glycine max) canopy (leaf area index approximately 5) with the objective to determine the extent to which these factors may be limiting the capacity to respond to light transients during sunflecks. When sampled along a vertical gradient, leaves near the bottom of the canopy had lower rubisco contents and chlorophyll a/b ratios as compared with upper leaves. Leaves sampled at midcanopy showed a wide variation in photosynthetic induction state (ratio of the photosynthetic rate achieved after 1 minute exposure to high light to the steady-state assimilation rate achieved after 20 minutes exposure). Both photosynthetic induction state and the initial rubisco activity varied in parallel with stomatal conductance. By contrast there was no correlation between total rubisco activity and stomatal conductance. The results indicate that induction state, as determined by the light regulation of both rubisco activity and stomatal conductance, is an important limitation to the ability of leaves in a soybean canopy to respond to light transients that occur during sunflecks.

Salinity Induced Limitations on Photosynthesis in Prunus salicina, a Deciduous Tree Species.

The response of photosynthetic CO(2) assimilation to salinization in 19 year old Prunus salicina was evaluated under field conditions for a 3 year period. The observed decline in CO(2) assimilation capacity was apparently related to increasing leaf chloride (Cl(-)) content, and independent of changes in leaf carbohydrate status. The response of net CO(2) assimilation (A) to leaf intercellular CO(2) partial pressure (C(i)) indicated that the reduction in the capacity for A with Cl(-) was not the result of decreased stomatal conductance but a consequence of nonstomatal inhibition. The nonstomatal limitations to CO(2) assimilation capacity, as determined by the response of A to C(i) and biochemical assay, were related to a decline in the activity of ribulose 1,5-bisphosphate carboxylase (Rubpcase) and the pool size of triose phosphate, ribulose 1,5-bisphosphate (Rubp) and phosphoglycerate with increasing salinity. Lack of agreement between the initial slope of the A to C(i) response curve and Rubpcase activity suggests the occurrence of heterogeneous stomatal apertures with the high salinity treatment (28 millimolar). Prolonged exposure to chloride salts appeared to increase the Rubp or Pi regeneration limitation, decrease Rubpcase activity and reduce leaf chlorophyll content. Observed changes in the biochemical components of CO(2) fixation may, in turn, affect total leaf carbohydrates, which also declined with time and salinity. The reduction in Rubpcase activity was apparently a consequence of a reduced Rubpcase protein level rather than either a regulatory or inhibitory effect.

Metabolism of 2-carboxyarabinitol 1-phosphate and regulation of ribulose-1,5-bisphosphate carboxylase activity.

Metabolism of 2'-carboxy-D-arabinitol 1-phosphate (CA1P) is an important component in the light-dependent regulation of ribulose-1,5-bisphosphate carboxylase (Rubisco) activity and whole leaf photosynthetic CO2 assimilation in many species, and functions as one mechanism for regulating Rubisco activity when photosynthesis is light-limited. Species differ in their capacity to accumulate CA1P, ranging from those which can synthesize levels of this compound approaching or in excess of the Rubisco catalytic site concentration, to those which apparently lack the capacity for CA1P synthesis. CA1P is structurally related to the six carbon transition state intermediate of the carboxylation reaction and binds tightly to the carbamylated catalytic site of Rubisco, making that site unavailable for catalysis. Under steady-state, the concentration of CA1P in the leaf is highest at low photon flux density (PFD) or in the dark. Degradation of CA1P and recovery of Rubisco activity requires light and is stimulated by increasing PFD. The initial degradation reaction is catalyzed by an enzyme located in the chloroplast stroma, CA1P phosphatase, which yields carboxyarabinitol (CA) and inorganic phosphate as its products. The pathway of CA metabolism in the plant remains to be determined. Synthesis of CA1P occurs in the dark, and in Phaseolus vulgaris this process has been shown to be stimulated by low PFD. The pathway of CA1P synthesis and its relationship to the degradative pathway remains unknown at the present time. The discovery of the existence of this previously unknown carbon pathway in photosynthesis indicates that we still have much to learn concerning the regulation of Rubisco activity and photosynthesis.