ALBERT

Notes: Plant responses to elevated atmospheric CO2 have been characterized generally by stomatal closure and enhanced growth rates. These responses are being increasingly incorporated into global climate models that quantify interactions between the biosphere and atmosphere, altering climate predictions from simpler physically based models. However, current information on CO2 responses has been gathered primarily from studies of crop and temperate forest species. In order to apply responses of vegetation to global predictions, CO2 responses in other commonly occurring biomes must be studied. A Free Air CO2 Enrichment (FACE) study is currently underway to examine plant responses to high CO2 in a natural, undisturbed Mojave Desert ecosystem in Nevada, USA. Here we present findings from this study, and its companion glasshouse experiment, demonstrating that field-grown Ephedra nevadensis and glasshouse-grown Larrea tridentata responded to high CO2 with reductions in the ratio of transpirational surface area to sapwood area (LSR) of 33% and 60%, respectively. Thus, leaf-specific hydraulic conductivity increased and stomatal conductance remained constant or was increased under elevated CO2. Field-grown Larrea did not show a reduced LSR under high CO2, and stomatal conductance was reduced in the high CO2 treatment, although the effect was apparent only under conditions of unusually high soil moisture. Both findings suggest that the common paradigm of 20–50% reductions in stomatal conductance under high CO2 may not be applicable to arid ecosystems under most conditions.

Notes: Hamamelitol is an unusual branched-chain sugar alcohol previously suggested to function as a leaf compatible solute. In this study, we have examined the leaf metabolism and intracelluiar compartmentalization of hamamelitol and other soluble sugars during long-term water stress treatment of Hedera helix (English ivy). Total leaf hamamelitol content was relatively low in greenhouse control plants, but increased 2-fold during water stress treatment to levels approaching those observed in field-grown plants (6–7 μmol g−1 fresh weight). Using density gradient fractionation with non-aqueous solvents, we showed that hamamelitol occurs primarily in the cytoplasm and vacuoles of leaf mesophyll cells. During water stress treatment most of the increase in leaf hamamelitol occurred in the mesophyll cytoplasm, compensating osmotically for a decrease in cytoplasmic sucrose concentration. The maximum concentration of cytoplasmic hamamelitol was 155 mol m−3 and occurred in field-grown plants. Labelling experiments showed that hamamelitol is slowly synthesized from 14CO2 in leaves of H. helix, but is very long-lived (estimated t1/2 of 4 years). Together, these data indicate that hamamelitol probably functions during long-term stress conditions as an osmotically active, compatible solute in plant leaves. We suggest that the signal for enhanced accumulation of hamamelitol during the water stress treatment was initiated by decreased plant growth and increased leaf sucrose hydrolysis.

Notes: Soybean plants (Glycine max (L.) Merr. c.v. Williams) were grown in CO2 controlled, natural-light growth chambers under one of four atmospheric CO2 concentrations ([CO2]): (1) 250 μmol mol–1 24 h d–1[250/250]; (2) 1000 μmol mol–1 24 h d–1[1000/1000]; (3) 250 μmol mol–1 during daylight hours and 1000 μmol mol–1 during night-time hours [250/1000] or (4) 1000 μmol mol–1 during daylight hours and 250 μmol mol–1 during night-time hours [1000/250]. During the vegetative growth phase few physiological differences were observed between plants exposed to a constant 24 h [CO2] (250/250 and 1000/1000) and those that were switched to a higher or lower [CO2] at night (250/1000 and 1000/250), suggesting that the primary physiological responses of plants to growth in elevated [CO2] is apparently a response to daytime [CO2] only. However, by the end of the reproductive growth phase, major differences were observed. Plants grown in the 1000/250 regime, when compared with those in the 1000/1000 regime, had significantly more leaf area and leaf mass, 27% more total plant dry mass, but only 18% of the fruit mass. After 12 weeks of growth these plants also had 19% higher respiration rates and 32% lower photosynthetic rates than the 1000/1000 plants. As a result the ratio of carbon gain to carbon loss was reduced significantly in the plants exposed to the reduced night-time [CO2]. Plants grown in the opposite switching environment, 250/1000 versus 250/250, showed no major differences in biomass accumulation or allocation with the exception of a significant increase in the amount of leaf mass per unit area. Physiologically, those plants exposed to elevated night-time [CO2] had 21% lower respiration rates, 14% lower photosynthetic rates and a significant increase in the ratio of carbon gain to carbon loss, again when compared with the 250/250 plants. Biochemical differences also were found. Ribulose-1,5-bisphosphate carboxylase/ oxygenase concentrations decreased in the 250/ 1000 treatment compared with the 250/250 plants, and phosphoenolpyruvate carboxylase activity decreased in the 1000/250 compared with the 1000/1000 plants. Glucose, fructose and to a lesser extent sucrose concentrations also were reduced in the 1000/250 treatment compared with the 1000/1000 plants. These results indicate that experimental protocols that do not maintain elevated CO2 levels 24 h d–1 can have significant effects on plant biomass, carbon allocation and physiology, at least for fast-growing annual crop plants. Furthermore, the results suggest some plant processes other than photosynthesis are sensitive to [CO2] and under ecologically relevant conditions, such as high night-time [CO2], whole plant carbon balance can be affected.

Notes: Plants grown at elevated CO2 often acclimate such that their photosynthetic capacities are reduced relative to ambient CO2-grown plants. Reductions in synthesis of photosynthetic enzymes could result either from reduced photosynthetic gene expression or from reduced availability of nitrogen-containing substrates for enzyme synthesis. Increased carbohydrate concentrations resulting from increased photosynthetic carbon fixation at elevated CO2 concentrations have been suggested to reduce the expression of photosynthetic genes. However, recent studies have also suggested that nitrogen uptake may be depressed by elevated CO2, or at least that it is not increased enough to keep pace with increased carbohydrate production. This response could induce a nitrogen limitation in elevated-CO2 plants that might account for the reduction in photosynthetic enzyme synthesis. If CO2 acclimation were a response to limited nitrogen uptake, the effects of elevated CO2 and limiting nitrogen supply on photosynthesis and nitrogen allocation should be similar. To test this hypothesis we grew non-nodulating soybeans at two levels each of nitrogen and CO2 concentration and measured leaf nitrogen contents, photosynthetic capacities and Rubisco contents. Both low nitrogen and elevated CO2 reduced nitrogen as a percentage of total leaf dry mass but only low nitrogen supply produced significant decreases in nitrogen as a percentage of leaf structural dry mass. The primary effect of elevated CO2 was to increase non-structural carbohydrate storage rather than to decrease nitrogen content. Both low nitrogen supply and elevated CO2 also decreased carboxylation capacity (Vcmax) and Rubisco content per unit leaf area. However, when Vcmax and Rubisco content were expressed per unit nitrogen, low nitrogen supply generally caused them to increase whereas elevated CO2 generally caused them to decrease. Finally, elevated CO2 significantly increased the ratio of RuBP regeneration capacity to Vcmax whereas neither nitrogen supply nor plant age had a significant effect on this parameter. We conclude that reductions in photosynthetic enzyme synthesis in elevated CO2 appear not to result from limited nitrogen supply but instead may result from feedback inhibition by increased carbohydrate contents.

Notes: Photosynthetic acclimation to elevated CO2 cannot presently be predicted due to our limited understanding of the molecular mechanisms and metabolic signals that regulate photosynthetic gene expression. We have examined acclimation by comparing changes in the leaf content of RuBP carboxylase/oxygenase (Rubisco) with changes in the transcripts of Rubisco subunit genes and with leaf carbohydrate metabolism. When grown at 1000 mm3 dm–3 CO2, 12 of 16 crop species at peak vegetative growth had a 15–44% decrease in leaf Rubisco protein, but with no specific association with changes in transcript levels measured at midday. Species with only modest reductions in Rubisco content (10–20%) often had a large reduction in Rubisco small subunit gene mRNAs (〉 30%), with no reduction in large subunit gene mRNAs. However, species with a very large reduction in Rubisco content generally had only small reductions in transcript mRNAs. Photosynthetic acclimation also was not specifically associated with a change in the level of any particular carbohydrate measured at midday. However, a threshold relationship was found between the reduction in Rubisco content at high CO2 and absolute levels of soluble acid invertase activity measured in plants grown at ambient or high CO2. This relationship was valid for 15 of the 16 species examined. There also occurred a similar, albeit less robust, threshold relationship between the leaf hexose/sucrose ratio at high CO2 and a reduced photosynthetic capacity ≥ 20%. These data indicate that carbohydrate repression of photosynthetic gene expression at elevated CO2 may involve leaf sucrose cycling through acid invertase and hexokinase.

Notes: The photosynthetic induction state under conditions of different lightfleck frequencies or durations, or different shade periods was studied in soybean leaves in order to examine how it might limit utilization of sunflecks in leaf canopies. Induction following an increase in photon flux density (PFD) from strongly limiting to saturating PFDs exhibited two phases; a fast-inducing one, requiring about 1 min and a slow one, requiring up to 60 min for completion. Transfer of fully induced leaves to low light resulted in a rapid decrease in the fast-inducing component, a slower decrease in the slow-inducing component and an even slower decrease in stomatal conductance. Therefore, the decreases in extent of induction appeared to be due to biochemical factors and not to stomatal closure. Under flashing light regimes consisting of 1-s lightflecks given at different frequencies for long periods, a constant induction state was achieved, the measure of induction state increased with the frequency of the lightflecks. This constant induction state also depended on the growth conditions, with shade leaves having a higher value than those grown at high light at any particular lightfleck frequency. The measure of induction state was mostly lower in flashing light as compared to constant light of the same mean PFD, particularly in leaves with a low light saturation point and in short lightflecks. Initial activities of ribulose-1,5-bisphosphate carboxylase (rubisco) were also higher in continuous light and were highly correlated with the measure of induction state. The rapid decrease in extent of induction of soybean leaves during shade periods is an important limitation to the ability of the leaves to respond to light increases similar to those occurring with sunflecks. At least part of the limitation on carbon assimilation during sunflecks due to photosynthetic induction is based on regulation of rubisco activity.

Notes: 
 A max, maximum CO2 assimilation rate
CAB, genes encoding chlorophyll a/b binding proteins
Ci, intercellular CO2 concentration
PGK, the gene encoding 3-phosphoglycerate kinase
PRK, the gene encoding phosphoribulokinase
PSAB, the gene encoding the 83 kDa apoprotein of the PSI reaction centre
PSBA, the gene encoding the D1 protein of photosystem II
RBCS, genes encoding the Rubisco small subunit protein
RBCL, the gene encoding the Rubisco large subunit protein
Rubisco, ribulose-1,5-bisphosphate carboxylase/ oxygenase
SBP, the gene encoding sedoheptulose-1,5-bisphosphatase

There have been many recent exciting advances in our understanding of the cellular processes that underlie photosynthetic acclimation to rising atmospheric CO2 concentration. Of particular interest have been the molecular processes that modulate photosynthetic gene expression in response to elevated CO2 and the biochemical processes that link changes in atmospheric CO2 concentration to the production of a metabolic signal. Central to this acclimation response is a reduction in ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) protein content. Studies indicate that this reduction results from species-dependent variation in the differential use and temporal control of molecular processes. We present a model for the control of Rubisco protein accumulation that emphasizes the role of subunit message translation as well as the abundance of subunit messages as components of the acclimation response. Many studies indicate that photosynthetic acclimation to elevated CO2 results from adjustments in leaf carbohydrate signalling. The repression of photosynthetic gene expression is considered to occur primarily by hexokinase functioning as a hexose flux sensor that ultimately affects transcription. Leaf hexoses may be produced as potential sources of signals primarily by sucrose cycling and secondarily by starch hydrolysis. An increased rate of sucrose cycling is suggested to occur at elevated CO2 by enhanced provision of sucrose to leaf acid invertases. Additionally, sink limitations that accentuate photosynthetic acclimation may result from a relative decrease in the export of leaf sucrose and subsequent increase in cellular sucrose levels and sucrose cycling.

Notes: The reduction of photosynthetic capacity in many plants grown at elevated CO2 is thought to result from a feedback effect of leaf carbohydrates on gene expression. Carbohydrate feedback at elevated CO2 could result from limitations on carbohydrate utilization at many different points, for example export of triose phosphates from the chloroplast, sucrose synthesis and phloem loading, transport in the phloem, unloading of the phloem at the sinks, or utilization for growth of sinks. To determine the relative importance of leaf versus whole plant level limitations on carbohydrate utilization at elevated CO2, and the possible effects on the regulation of photosynthetic capacity, we constructed a treatment system in which we could expose single, attached, soybean leaflets to CO2 concentrations different from those experienced by the rest of the plant. The single leaflet treatments had dramatic effects on the carbohydrate contents of the treated leaflets. However, photosynthetic capacity and rubisco content were unaffected by the individual leaflet treatment and instead were related to the whole plant CO2 environment, despite the fact that the CO2 environment around the rest of the plant had no significant affect on the total non-structural carbohydrate (TNC) contents of the treated leaflets. These results necessitate a re-evaluation of the response mechanisms to CO2 as well as some of the methods used to test these responses. We propose mechanisms by which sink strength could influence leaf physiology independently of changes in carbohydrate accumulation.

Notes: The photosynthetic response of Larrea tridentata Cav., an evergreen Mojave Desert shrub, to elevated atmospheric CO2 and drought was examined to assist in the understanding of how plants from water-limited ecosystems will respond to rising CO2. We hypothesized that photosynthetic down-regulation would disappear during periods of water limitation, and would, therefore, likely be a seasonally transient event. To test this we measured photosynthetic, water relations and fluorescence responses during periods of increased and decreased water availability in two different treatment implementations: (1) from seedlings exposed to 360, 550, and 700 μmol mol–1 CO2 in a glasshouse; and (2) from intact adults exposed to 360 and 550 μmol mol–1 CO2 at the Nevada Desert FACE (Free Air CO2 Enrichment) Facility. FACE and glasshouse well-watered Larrea significantly down-regulated photosynthesis at elevated CO2, reducing maximum photosynthetic rate (Amax), carboxylation efficiency (CE), and Rubisco catalytic sites, whereas droughted Larrea showed a differing response depending on treatment technique. Amax and CE were lower in droughted Larrea compared with well-watered plants, and CO2 had no effect on these reduced photosynthetic parameters. However, Rubisco catalytic sites decreased in droughted Larrea at elevated CO2. Operating Ci increased at elevated CO2 in droughted plants, resulting in greater photosynthetic rates at elevated CO2 as compared with ambient CO2. In well-watered plants, the changes in operating Ci, CE and Amax resulted in similar photosynthetic rates across CO2 treatments. Our results suggest that drought can diminish photosynthetic down-regulation to elevated CO2 in Larrea, resulting in seasonally transient patterns of enhanced carbon gain. These results suggest that water status may ultimately control the photosynthetic response of desert systems to rising CO2.

Notes: 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 (−360 and 710μmol 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.