Asaph B. Cousins

Carbon dioxide enrichment inhibits nitrate assimilation in wheat and Arabidopsis.

Nitrate for Me, Ammonium for You The interdependence of plant nitrogen uptake and plant responses to carbon dioxide is well established, but the influence of inorganic nitrogen form—i.e., whether nitrate or ammonium—has been largely ignored. Bloom et al. (p. 899) present evidence from five independent methods in both a monocot and dicot species that carbon dioxide inhibition of nitrate assimilation is a major determinant of plant responses to rising atmospheric concentrations of carbon dioxide. This finding explains several phenomena, including carbon dioxide acclimation and decline in food quality. The large variation in these phenomena among species, locations, or years derives from the large variation in the relative dependence of plants on nitrate and ammonium as nitrogen sources among species, locations, or years. The relative importance of ammonium and nitrate for plant N nutrition in future cropping systems will be critical for quantity and quality of food. The inhibition of photorespiration by elevated carbon dioxide is paralleled by a reduction in the assimilation of nitrate. The concentration of carbon dioxide in Earth’s atmosphere may double by the end of the 21st century. The response of higher plants to a carbon dioxide doubling often includes a decline in their nitrogen status, but the reasons for this decline have been uncertain. We used five independent methods with wheat and Arabidopsis to show that atmospheric carbon dioxide enrichment inhibited the assimilation of nitrate into organic nitrogen compounds. This inhibition may be largely responsible for carbon dioxide acclimation, the decrease in photosynthesis and growth of plants conducting C3 carbon fixation after long exposures (days to years) to carbon dioxide enrichment. These results suggest that the relative availability of soil ammonium and nitrate to most plants will become increasingly important in determining their productivity as well as their quality as food.

Peroxisomal malate dehydrogenase is not essential for photorespiration in Arabidopsis but its absence causes an increase in the stoichiometry of photorespiratory CO2 release.

Peroxisomes are important for recycling carbon and nitrogen that would otherwise be lost during photorespiration. The reduction of hydroxypyruvate to glycerate catalyzed by hydroxypyruvate reductase (HPR) in the peroxisomes is thought to be facilitated by the production of NADH by peroxisomal malate dehydrogenase (PMDH). PMDH, which is encoded by two genes in Arabidopsis (Arabidopsis thaliana), reduces NAD+ to NADH via the oxidation of malate supplied from the cytoplasm to oxaloacetate. A double mutant lacking the expression of both PMDH genes was viable in air and had rates of photosynthesis only slightly lower than in the wild type. This is in contrast to other photorespiratory mutants, which have severely reduced rates of photosynthesis and require high CO2 to grow. The pmdh mutant had a higher O2-dependent CO2 compensation point than the wild type, implying that either Rubisco specificity had changed or that the rate of CO2 released per Rubisco oxygenation was increased in the pmdh plants. Rates of gross O2 evolution and uptake were similar in the pmdh and wild-type plants, indicating that chloroplast linear electron transport and photorespiratory O2 uptake were similar between genotypes. The CO2 postillumination burst and the rate of CO2 released during photorespiration were both greater in the pmdh mutant compared with the wild type, suggesting that the ratio of photorespiratory CO2 release to Rubisco oxygenation was altered in the pmdh mutant. Without PMDH in the peroxisome, the CO2 released per Rubisco oxygenation reaction can be increased by over 50%. In summary, PMDH is essential for maintaining optimal rates of photorespiration in air; however, in its absence, significant rates of photorespiration are still possible, indicating that there are additional mechanisms for supplying reductant to the peroxisomal HPR reaction or that the HPR reaction is altogether circumvented.

Carbonic Anhydrase and Its Influence on Carbon Isotope Discrimination during C4 Photosynthesis. Insights from Antisense RNA in Flaveria bidentis

In C4 plants, carbonic anhydrase (CA) facilitates both the chemical and isotopic equilibration of atmospheric CO2 and bicarbonate (HCO3−) in the mesophyll cytoplasm. The CA-catalyzed reaction is essential for C4 photosynthesis, and the model of carbon isotope discrimination (Δ13C) in C4 plants predicts that changes in CA activity will influence Δ13C. However, experimentally, the influence of CA on Δ13C has not been demonstrated in C4 plants. Here, we compared measurements of Δ13C during C4 photosynthesis in Flaveria bidentis wild-type plants with F. bidentis plants with reduced levels of CA due to the expression of antisense constructs targeted to a putative mesophyll cytosolic CA. Plants with reduced CA activity had greater Δ13C, which was also evident in the leaf dry matter carbon isotope composition (δ13C). Contrary to the isotope measurements, photosynthetic rates were not affected until CA activity was less than 20% of wild type. Measurements of Δ13C, δ13C of leaf dry matter, and rates of net CO2 assimilation were all dramatically altered when CA activity was less than 5% of wild type. CA activity in wild-type F. bidentis is sufficient to maintain net CO2 assimilation; however, reducing leaf CA activity has a relatively large influence on Δ13C, often without changes in net CO2 assimilation. Our data indicate that the extent of CA activity in C4 leaves needs to be taken into account when using Δ13C and/or δ13C to model the response of C4 photosynthesis to changing environmental conditions.

CO2 enrichment inhibits shoot nitrate assimilation in C3 but not C4 plants and slows growth under nitrate in C3 plants

The CO2 concentration in Earths atmosphere may double during this century. Plant responses to such an increase depend strongly on their nitrogen status, but the reasons have been uncertain. Here, we assessed shoot nitrate assimilation into amino acids via the shift in shoot CO2 and O2 fluxes when plants received nitrate instead of ammonium as a nitrogen source (deltaAQ). Shoot nitrate assimilation became negligible with increasing CO2 in a taxonomically diverse group of eight C3 plant species, was relatively insensitive to CO2 in three C4 species, and showed an intermediate sensitivity in two C3-C4 intermediate species. We then examined the influence of CO2 level and ammonium vs. nitrate nutrition on growth, assessed in terms of changes in fresh mass, of several C3 species and a Crassulacean acid metabolism (CAM) species. Elevated CO2 (720 micromol CO2/mol of all gases present) stimulated growth or had no effect in the five C3 species tested when they received ammonium as a nitrogen source but inhibited growth or had no effect if they received nitrate. Under nitrate, two C3 species grew faster at sub-ambient (approximately 310 micromol/mol) than elevated CO2. A CAM species grew faster at ambient than elevated or sub-ambient CO2 under either ammonium or nitrate nutrition. This study establishes that CO2 enrichment inhibits shoot nitrate assimilation in a wide variety of C3 plants and that this phenomenon can have a profound effect on their growth. This indicates that shoot nitrate assimilation provides an important contribution to the nitrate assimilation of an entire C3 plant. Thus, rising CO2 and its effects on shoot nitrate assimilation may influence the distribution of C3 plant species.

The Role of Phosphoenolpyruvate Carboxylase during C4 Photosynthetic Isotope Exchange and Stomatal Conductance

Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) plays a key role during C4 photosynthesis and is involved in anaplerotic metabolism, pH regulation, and stomatal opening. Heterozygous (Pp) and homozygous (pp) forms of a PEPC-deficient mutant of the C4 dicot Amaranthus edulis were used to study the effect of reduced PEPC activity on CO2 assimilation rates, stomatal conductance, and 13CO2 (Δ13C) and C18OO (Δ18O) isotope discrimination during leaf gas exchange. PEPC activity was reduced to 42% and 3% and the rates of CO2 assimilation in air dropped to 78% and 10% of the wild-type values in the Pp and pp mutants, respectively. Stomatal conductance in air (531 μbar CO2) was similar in the wild-type and Pp mutant but the pp mutant had only 41% of the wild-type steady-state conductance under white light and the stomata opened more slowly in response to increased light or reduced CO2 partial pressure, suggesting that the C4 PEPC isoform plays an essential role in stomatal opening. There was little difference in Δ13C between the Pp mutant (3.0‰ ± 0.4‰) and wild type (3.3‰ ± 0.4‰), indicating that leakiness (ϕ), the ratio of CO2 leak rate out of the bundle sheath to the rate of CO2 supply by the C4 cycle, a measure of the coordination of C4 photosynthesis, was not affected by a 60% reduction in PEPC activity. In the pp mutant Δ13C was 16‰ ± 3.2‰, indicative of direct CO2 fixation by Rubisco in the bundle sheath at ambient CO2 partial pressure. Δ18O measurements indicated that the extent of isotopic equilibrium between leaf water and the CO2 at the site of oxygen exchange (θ) was low (0.6) in the wild-type and Pp mutant but increased to 0.9 in the pp mutant. We conclude that in vitro carbonic anhydrase activity overestimated θ as compared to values determined from Δ18O in wild-type plants.

A Limited Role for Carbonic Anhydrase in C4 Photosynthesis as Revealed by a ca1ca2 Double Mutant in Maize

Maize plants with significantly reduced carbonic anhydrase activity have impaired growth at subambient CO2, but photosynthesis in these plants is not limited under current atmospheric conditions. Carbonic anhydrase (CA) catalyzes the first biochemical step of the carbon-concentrating mechanism of C4 plants, and in C4 monocots it has been suggested that CA activity is near limiting for photosynthesis. Here, we test this hypothesis through the characterization of transposon-induced mutant alleles of Ca1 and Ca2 in maize (Zea mays). These two isoforms account for more than 85% of the CA transcript pool. A significant change in isotopic discrimination is observed in mutant plants, which have as little as 3% of wild-type CA activity, but surprisingly, photosynthesis is not reduced under current or elevated CO2 partial pressure (pCO2). However, growth and rates of photosynthesis under subambient pCO2 are significantly impaired in the mutants. These findings suggest that, while CA is not limiting for C4 photosynthesis in maize at current pCO2, it likely maintains high rates of photosynthesis when CO2 availability is reduced. Current atmospheric CO2 levels now exceed 400 ppm (approximately 40.53 Pa) and contrast with the low-pCO2 conditions under which C4 plants expanded their range approximately 10 million years ago, when the global atmospheric CO2 was below 300 ppm (approximately 30.4 Pa). Thus, as CO2 levels continue to rise, selective pressures for high levels of CA may be limited to arid climates where stomatal closure reduces CO2 availability to the leaf.

Discrimination in the Dark. Resolving the Interplay between Metabolic and Physical Constraints to Phosphoenolpyruvate Carboxylase Activity during the Crassulacean Acid Metabolism Cycle

A model defining carbon isotope discrimination (Δ13C) for crassulacean acid metabolism (CAM) plants was experimentally validated using Kalanchoe daigremontiana. Simultaneous measurements of gas exchange and instantaneous CO2 discrimination (for 13C and 18O) were made from late photoperiod (phase IV of CAM), throughout the dark period (phase I), and into the light (phase II). Measurements of CO2 response curves throughout the dark period revealed changing phosphoenolpyruvate carboxylase (PEPC) capacity. These systematic changes in PEPC capacity were tracked by net CO2 uptake, stomatal conductance, and online Δ13C signal; all declined at the start of the dark period, then increased to a maximum 2 h before dawn. Measurements of Δ13C were higher than predicted from the ratio of intercellular to external CO2 (pi/pa) and fractionation associated with CO2 hydration and PEPC carboxylations alone, such that the dark period mesophyll conductance, gi, was 0.044 mol m−2 s−1 bar−1. A higher estimate of gi (0.085 mol m−2 s−1 bar−1) was needed to account for the modeled and measured Δ18O discrimination throughout the dark period. The differences in estimates of gi from the two isotope measurements, and an offset of −5.5‰ between the 18O content of source and transpired water, suggest spatial variations in either CO2 diffusion path length and/or carbonic anhydrase activity, either within individual cells or across a succulent leaf. Our measurements support the model predictions to show that internal CO2 diffusion limitations within CAM leaves increase Δ13C discrimination during nighttime CO2 fixation while reducing Δ13C during phase IV. When evaluating the phylogenetic distribution of CAM, carbon isotope composition will reflect these diffusive limitations as well as relative contributions from C3 and C4 biochemistry.

A Transgenic Approach to Understanding the Influence of Carbonic Anhydrase on C18OO Discrimination during C4 Photosynthesis

The oxygen isotope composition of atmospheric CO2 is an important signal that helps distinguish between ecosystem photosynthetic and respiratory processes. In C4 plants the carbonic anhydrase (CA)-catalyzed interconversion of CO2 and bicarbonate (HCO3−) is an essential first reaction for C4 photosynthesis but also plays an important role in the CO2-H2O exchange of oxygen as it enhances the rate of isotopic equilibrium between CO2 and water. The C4 dicot Flaveria bidentis containing genetically reduced levels of leaf CA (CAleaf) has been used to test whether changing leaf CA activity influences online measurements of C18OO discrimination (Δ18O) and the proportion of CO2 in isotopic equilibrium with leaf water at the site of oxygen exchange (θ). The Δ18O in wild-type F. bidentis, which contains high levels of CA relative to the rates of net CO2 assimilation, was less than predicted by models of Δ18O. Additionally, Δ18O was sensitive to small decreases in CAleaf. However, reduced CA activity in F. bidentis had little effect on net CO2 assimilation, transpiration rates (E), and stomatal conductance (gs) until CA levels were less than 20% of wild type. The values of θ determined from measurements of Δ18O and the 18O isotopic composition of leaf water at the site of evaporation (δe) were low in the wild-type F. bidentis and decreased in transgenic plants with reduced levels of CA activity. Measured values of θ were always significantly lower than the values of θ predicted from in vitro CA activity and gas exchange. The data presented here indicates that CA content in a C4 leaf may not represent the CA activity associated with the CO2-H2O oxygen exchange and therefore may not be a good predictor of θ during C4 photosynthesis. Furthermore, uncertainties in the isotopic composition of water at the site of exchange may also limit the ability to accurately predict θ in C4 plants.

The Coordination of C4 Photosynthesis and the CO2-Concentrating Mechanism in Maize and Miscanthus × giganteus in Response to Transient Changes in Light Quality

Light quality coordinates a CO2-concentrating mechanism during C4 photosynthesis. Unequal absorption of photons between photosystems I and II, and between bundle-sheath and mesophyll cells, are likely to affect the efficiency of the CO2-concentrating mechanism in C4 plants. Under steady-state conditions, it is expected that the biochemical distribution of energy (ATP and NADPH) and photosynthetic metabolite concentrations will adjust to maintain the efficiency of C4 photosynthesis through the coordination of the C3 (Calvin-Benson-Bassham) and C4 (CO2 pump) cycles. However, under transient conditions, changes in light quality will likely alter the coordination of the C3 and C4 cycles, influencing rates of CO2 assimilation and decreasing the efficiency of the CO2-concentrating mechanism. To test these hypotheses, we measured leaf gas exchange, leaf discrimination, chlorophyll fluorescence, electrochromatic shift, photosynthetic metabolite pools, and chloroplast movement in maize (Zea mays) and Miscanthus × giganteus following transitional changes in light quality. In both species, the rate of net CO2 assimilation responded quickly to changes in light treatments, with lower rates of net CO2 assimilation under blue light compared with red, green, and blue light, red light, and green light. Under steady state, the efficiency of CO2-concentrating mechanisms was similar; however, transient changes affected the coordination of C3 and C4 cycles in M. giganteus but to a lesser extent in maize. The species differences in the ability to coordinate the activities of C3 and C4 cycles appear to be related to differences in the response of cyclic electron flux around photosystem I and potentially chloroplast rearrangement in response to changes in light quality.

Influence of light and nitrogen on the photosynthetic efficiency in the C4 plant Miscanthus × giganteus

There are numerous studies describing how growth conditions influence the efficiency of C4 photosynthesis. However, it remains unclear how changes in the biochemical capacity versus leaf anatomy drives this acclimation. Therefore, the aim of this study was to determine how growth light and nitrogen availability influence leaf anatomy, biochemistry and the efficiency of the CO2 concentrating mechanism in Miscanthusxa0×xa0giganteus. There was an increase in the mesophyll cell wall surface area but not cell well thickness in the high-light (HL) compared to the low-light (LL) grown plants suggesting a higher mesophyll conductance in the HL plants, which also had greater photosynthetic capacity. Additionally, the HL plants had greater surface area and thickness of bundle-sheath cell walls compared to LL plants, suggesting limited differences in bundle-sheath CO2 conductance because the increased area was offset by thicker cell walls. The gas exchange estimates of phosphoenolpyruvate carboxylase (PEPc) activity were significantly less than the in vitro PEPc activity, suggesting limited substrate availability in the leaf due to low mesophyll CO2 conductance. Finally, leakiness was similar across all growth conditions and generally did not change under the different measurement light conditions. However, differences in the stable isotope composition of leaf material did not correlate with leakiness indicating that dry matter isotope measurements are not a good proxy for leakiness. Taken together, these data suggest that the CO2 concentrating mechanism in Miscanthus is robust under low-light and limited nitrogen growth conditions, and that the observed changes in leaf anatomy and biochemistry likely help to maintain this efficiency.