Comparative analysis of thylakoid protein complexes in the mesophyll and bundle sheath cells from C3, C4 and C3–C4 Paniceae grasses

To better understand the coordination between dark and light reactions during the transition from C₃ to C₄ photosynthesis, we optimized a method for separating thylakoids from mesophyll (MC) and bundle sheath cells (BSCs) across different plant species. We grew six Paniceae grasses including representatives from the C₃, C₃–C₄ and C₄ photosynthetic types and all three C₄ biochemical subtypes [nicotinamide adenine dinucleotide phosphate‐dependent malic enzyme (NADP‐ME), nicotinamide adenine dinucleotide‐dependent malic enzyme (NAD‐ME) and phosphoenolpyruvate carboxykinase (PEPCK)] in addition to Zea mays under control conditions (1000 μmol quanta m^?2 s^?1 and 400 ppm of CO₂). Proteomics analysis of thylakoids under native conditions, using blue native polyacrylamide gel electrophoresis followed by liquid chromatography‐mass spectrometry (LC‐MS), demonstrated the presence of subunits of all light‐reaction‐related complexes in all species and cell types. C₄ NADP‐ME species showed a higher photosystems I/II ratio and a clear accumulation of the NADH dehydrogenase‐like complexes in BSCs, while Cytb₆f was more abundant in BSCs of C₄ NAD‐ME species. The C₄ PEPCK species showed no clear differences between cell types. Our study presents, for the first time, a good separation between BSC and MC for a C₃–C₄ intermediate grass which did not show noticeable differences in the distribution of the thylakoid complexes. For the NADP‐ME species Panicum antidotale, growth at glacial CO₂ (180 ppm of CO₂) had no effect on the distribution of the light‐reaction complexes, while growth at low light (200 μmol quanta m^?2 s^?1) promoted the accumulation of light‐harvesting proteins in both cell types. These results add to our understanding of thylakoid distribution across photosynthetic types and subtypes, and introduce thylakoid distribution between the MC and BSC of a C₃–C₄ intermediate species.     

The efficiency of the CO2-concentrating mechanism during single-cell C4 photosynthesis

The photosynthetic efficiency of the CO₂‐concentrating mechanism in two forms of single‐cell C₄ photosynthesis in the family Chenopodiaceae was characterized. The Bienertioid‐type single‐cell C₄ uses peripheral and central cytoplasmic compartments (Bienertia sinuspersici), while the Borszczowioid single‐cell C₄ uses distal and proximal compartments of the cell (Suaeda aralocaspica). C₄ photosynthesis within a single‐cell raises questions about the efficiency of this type of CO₂‐concentrating mechanism compared with the Kranz‐type. We used measurements of leaf CO₂ isotope exchange (Δ¹³C) to compare the efficiency of the single‐cell and Kranz‐type forms of C₄ photosynthesis under various temperature and light conditions. Comparisons were made between the single‐cell C₄ and a sister Kranz form, S. eltonica[NAD malic enzyme (NAD ME) type], and with Flaveria bidentis[NADP malic enzyme (NADP‐ME) type with Kranz Atriplicoid anatomy]. There were similar levels of Δ¹³C discrimination and CO₂ leakiness (?) in the single‐cell species compared with the Kranz‐type. Increasing leaf temperature (25 to 30 °C) and light intensity caused a decrease in Δ¹³C and ? across all C₄ types. Notably, B. sinuspersici had higher Δ¹³C and ? than S. aralocaspica under lower light. These results demonstrate that rates of photosynthesis and efficiency of the CO₂‐concentrating mechanisms in single‐cell C₄ plants are similar to those in Kranz‐type.     

Photosynthetic nitrogen and water use efficiency of acacia and eucalypt seedlings as afforestation species

The ecophysiological traits of acacia and eucalypt are important in assessing their suitability for afforestation. We measured the gas-exchange rate, the leaf dry mass per area (LMA) and the leaf nitrogen content of two acacia and four eucalypt species. Relative to the eucalypts, the acacias had lower leaf net photosynthetic rate (P _N), lower photosynthetic nitrogen-use efficiency (PNUE), higher water-use efficiency (WUE), higher LMA and higher leaf nitrogen per unit area (N _area). No clear differences were observed within or between genera in the maximum rate of carboxylation (V _cmax) or the maximum rate of electron transport (J _max), although these parameters tended to be higher in eucalypts. PNUE and LMA were negatively correlated. We conclude that acacias with higher LMA do not allocate nitrogen efficiently to photosynthetic system, explaining why their P _N and PNUE were lower than in eucalypts.     

Photosynthetic oxygen exchange in C4 grasses: the role of oxygen as electron acceptor

C₄ grasses of the NAD‐ME type (Astrebla lappacea, Eleusine coracana, Eragrostis superba, Leptochloa dubia, Panicum coloratum, Panicum decompositum) and the NADP‐ME type (Bothriochloa bladhii, Cenchrus ciliaris, Dichanthium sericeum, Panicum antidotale, Paspalum notatum, Pennisetum alopecuroides, Sorghum bicolor) were used to investigate the role of O₂ as an electron acceptor during C₄ photosynthesis. Mass spectrometric measurements of gross O₂ evolution and uptake were made concurrently with measurements of net CO₂ uptake and chlorophyll fluorescence at different irradiances and leaf temperatures of 30 and 40 °C. In all C₄ grasses gross O₂ uptake increased with increasing irradiance at very high CO₂ partial pressures (pCO₂) and was on average 18% of gross O₂ evolution. Gross O₂ uptake at high irradiance and high pCO₂ was on average 3.8 times greater than gross O₂ uptake in the dark. Furthermore, gross O₂ uptake in the light increased with O₂ concentration at both high CO₂ and the compensation point, whereas gross O₂ uptake in the dark was insensitive to O₂ concentration. This suggests that a significant amount of O₂ uptake may be associated with the Mehler reaction, and that the Mehler reaction varies with irradiance and O₂ concentration. O₂ exchange characteristics at high pCO₂ were similar for NAD‐ME and NADP‐ME species. NAD‐ME species had significantly greater O₂ uptake and evolution at the compensation point particularly at low irradiance compared to NADP‐ME species, which could be related to different rates of photorespiratory O₂ uptake. There was a good correlation between electron transport rates estimated from chlorophyll fluorescence and gross O₂ evolution at high light and high pCO₂.     

Growth, gas exchange, leaf nitrogen and carbohydrate concentrations in NAD‐ME and NADP‐ME C4 grasses grown in elevated CO2

Plants with the C₄ photosynthetic pathway have predominantly one of three decarboxylation enzymes in their bundle sheath cells. Within the grass family (Poaceae) bundle sheath leakiness to CO₂ is purported to be lowest in the nicotinamide adenine dinucleotide phosphate-malic enzyme (NADP-ME, EC 1.1.1.40) group, highest in the NAD-ME (EC 1.1.1.39) group and intermediate in the phosphoenolpyruvate carboxykinase (PCK, EC 4.1.1.32) group. We investigated the hypothesis that growth and photosynthesis of NAD-ME C₄ grasses would respond more to elevated CO₂ treatment than NADP-ME grasses. Plants were grown in 8-1 pots in growth chambers with ample water and fertilizer for 39 days at a continuous CO₂ concentration of either 350 or 700 µl l^?1. NAD-ME species included Bouteloua gracilis Lag. ex Steud (Blue grama), Buchloe dactyloides (Nutt.) Engelm. (Buffalo grass) and Panicum virgatum L. (Switchgrass) and the NADP-ME species were Andropogon gerardii Vittman (Big bluestem), Schizachyrium scoparium (Michx.) Nash (Little bluestem), and Sorghastrum nutans (L.) Nash (Indian grass). Contrary to our hypothesis, growth of the NADP-ME grasses was generally greater under elevated CO₂ (significant for A. gerardii and S. nutans), while none of the NAD-ME grasses had a significant growth response. Increased leaf total non-structural carbohydrate (TNC) was associated with greater growth responses of NADP-ME grasses. Decreased leaf nitrogen in NADP-ME species grown at elevated CO₂ was found to be an artifact of TNC dilution. Assimilation (A) vs intercellular CO₂ (C_i) curves revealed that leaf photosynthesis was not saturated at 350 µl l^?1 CO₂ in any of these C₄ grasses. Assimilation of elevated CO₂-grown A. gerardii was higher than in plants grown in ambient CO₂. In contrast, B. gracilis grown in elevated CO₂ displayed lower A, a trait more commonly reported in C₃ plants. Photosynthetic acclimation in B. gracilis was not related to leaf TNC or nitrogen concentrations, but A:C_i curves suggest a reduction in activity of both phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39). Some adaptation of stomatal functioning was also seen in B. gracilis and A. gerardii leaves grown in elevated CO₂. Our study shows that C₄ grasses have the capacity for increased growth and photosynthesis under elevated CO₂ even when water and nutrients are non-limiting. While it was the NADP-ME species which had significant responses in the present study, we have previously reported significant growth increases in elevated CO₂ for B. gracilis.     

Drought limitation of photosynthesis differs between C3 and C4 grass species in a comparative experiment

Phylogenetic analyses show that C₄ grasses typically occupy drier habitats than their C₃ relatives, but recent experiments comparing the physiology of closely related C₃ and C₄ species have shown that advantages of C₄ photosynthesis can be lost under drought. We tested the generality of these paradoxical findings in grass species representing the known evolutionary diversity of C₄ NADP‐me and C₃ photosynthetic types. Our experiment investigated the effects of drought on leaf photosynthesis, water potential, nitrogen, chlorophyll content and mortality. C₄ grasses in control treatments were characterized by higher CO₂ assimilation rates and water potential, but lower stomatal conductance and nitrogen content. Under drought, stomatal conductance declined more dramatically in C₃ than C₄ species, and photosynthetic water‐use and nitrogen‐use efficiency advantages held by C₄ species under control conditions were each diminished by 40%. Leaf mortality was slightly higher in C₄ than C₃ grasses, but leaf condition under drought otherwise showed no dependence on photosynthetic‐type. This phylogenetically controlled experiment suggested that a drought‐induced reduction in the photosynthetic performance advantages of C₄ NADP‐me relative to C₃ grasses is a general phenomenon.     

Nitrogen use strategy drives interspecific differences in plant photosynthetic CO2 acclimation

Rising atmospheric CO₂ concentration triggers an emergent phenomenon called plant photosynthetic acclimation to elevated CO₂ (PAC). PAC is often characterized by a reduction in leaf photosynthetic capacity (A_sat), which varies dramatically along the continuum of plant phylogeny. However, it remains unclear whether the mechanisms responsible for PAC are also different across plant phylogeny, especially between gymnosperms and angiosperms. Here, by compiling a dataset of 73 species, we found that although leaf A_sat increased significantly from gymnosperms to angiosperms, there was no phylogenetic signal in the PAC magnitude along the phylogenetic continuum. Physio-morphologically, leaf nitrogen concentration (N_m), photosynthetic nitrogen-use efficiency (PNUE), and leaf mass per area (LMA) dominated PAC for 36, 29, and 8 species, respectively. However, there was no apparent difference in PAC mechanisms across major evolutionary clades, with 75% of gymnosperms and 92% of angiosperms regulated by the combination of N_m and PNUE. There was a trade-off between N_m and PNUE in driving PAC across species, and PNUE dominated the long-term changes and inter-specific differences in A_sat under elevated CO₂. These findings indicate that nitrogen-use strategy drives the acclimation of leaf photosynthetic capacity to elevated CO₂ across terrestrial plant species.     

Form of inorganic carbon involved as a product and as an inhibitor of c(4) Acid decarboxylases operating in c(4) photosynthesis

These studies demonstrated that CO₂ rather than HCO₃⁻ is the inorganic carbon metabolite produced by the C₄ acid decarboxylases involved in C₄ photosynthesis (chloroplast located NADP malic enzyme, mitochondrial NAD malic enzyme, and cytosolic phosphoenolpyruvate [PEP] carboxykinase). The effect of varying CO₂ or HCO₃⁻ as a substrate for the carboxylation reaction catalyzed by these enzymes or as inhibitors of the decarboxylation reaction was also determined. The K_mCO₂ was 1.1 millimolar for NADP malic enzyme and 2.5 millimolar for PEP carboxykinase. For these two enzymes the velocity in the carboxylating direction was substantially less than for the decarboxylating direction even with CO₂ concentrations at the upper end of the range of expected cellular levels. Activity of NAD malic enzyme in the carboxylating direction was undetectable. The decarboxylation reaction of all three enzymes was inhibited by added HCO₃⁻. For NADP malic enzyme CO₂ was shown to be the inhibitory species but PEP carboxykinase and NAD malic enzyme were apparently inhibited about equally by CO₂ and HCO₃⁻.     

The physiological role of malic enzyme in grape ripening

The high specificity of malic enzyme (ME; EC 1.1.1.40) from grape berries (Vitis vinifera L.) for the naturally occurring l-enantiomer of malic acid, its very selective C₄-decarboxylation, and certain allosteric properties, reported previously, favour the conjecture of a regulatory function of ME in fruit malic acid degradation. On the other hand, high ME activity was detected even during the acid-accumulating phase of berry development. Also, the in vitro reversibility of the reaction supports the possibility of malate formation under conditions facilitating carboxylation of pyruvate, notably high CO₂/HCO ₃ ^- and NADPH/NADP ratios. However, a very limited incorporation of ¹⁴C into malate and the uniform labeling pattern of the dicarboxylic acid after administration of [U-¹⁴C] alanine to grape berries before and after the onset of ripening, indicate that the reverse reaction does not contribute essentially to grape malate synthesis. A regulatory mechanism mediating malic acid remetabolization on the basis of cosubstrate availability, comparable to the control of the hexose monophosphate shunt, is discussed.Abbreviation ME Malic enzyme (l-malate: NADP oxidoreductase)     

Spatial patterns of photosynthetic characteristics and leaf physical traits of plants in the Loess Plateau of China

The spatial patterns of photosynthetic characteristics and leaf physical traits of 171 plants belonging to nine life-forms or functional groups (trees, shrubs, herbs, evergreen trees, deciduous trees, C₃ and C₄ herbaceous plants, leguminous and non-leguminous species) and their relationships with environmental factors in seven sites, Yangling, Yongshou, Tongchuan, Fuxian, Ansai, Mizhi and Shenmu, ranging from south to north in the Loess Plateau of China were studied. The results showed that the leaf light-saturated photosynthetic rate (P_max), photosynthetic nitrogen use efficiency (PNUE), chlorophyll content (Chl), and leaf mass per area (LMA) of all the plants in the Loess Plateau varied significantly among three life-form groups, i.e., trees, shrubs and herbs, and two groups, i.e., evergreen trees and deciduous trees, but leaf nitrogen content differed little among different life-form groups. For the 171 plants in the Loess Plateau, leaf P_max was positively correlated with PNUE. The leaf nitrogen content per unit area (N_area) was positively correlated but Chl was negatively correlated with the LMA. When controlling the LMA, the N_area was positively correlated with the Chl (partial r = 0.20, P < 0.05). With regard to relationships between photosynthetic characteristics and leaf physical traits, the P_max was positively correlated with N _area, while the PNUE was positively correlated with the Chl and negatively correlated with the N_area and LMA. For all the species in the Loess Plateau, the PNUE was negatively correlated with the latitude and annual solar radiation (ASR), but positively correlated with the mean annual rainfall (MAR) and mean annual temperature (MAT). With regard to the leaf physical traits, the leaf Chl was negatively correlated with the latitude and ASR, but positively correlated with the MAR and MAT. However, the N_area and LMA were positively correlated with the latitude and ASR, but negatively correlated with the MAR and MAT. In general, leaf N_area and LMA increased, while PNUE and Chl decreased with increases in the latitude and ASR and decreases in MAR and MAT. Electronic supplementary material The online version of this article () contains supplementary material, which is available to authorized users.     

Maintenance of leaf N controls the photosynthetic CO2 response of grassland species exposed to 9 years of free‐air CO2 enrichment

Determining underlying physiological patterns governing plant productivity and diversity in grasslands are critical to evaluate species responses to future environmental conditions of elevated CO₂ and nitrogen (N) deposition. In a 9‐year experiment, N was added to monocultures of seven C₃ grassland species exposed to elevated atmospheric CO₂ (560 μmol CO₂ mol^?1) to evaluate how N addition affects CO₂ responsiveness in species of contrasting functional groups. Functional groups differed in their responses to elevated CO₂ and N treatments. Forb species exhibited strong down‐regulation of leaf N_mass concentrations (?26%) and photosynthetic capacity (?28%) in response to elevated CO₂, especially at high N supply, whereas C₃ grasses did not. Hence, achieved photosynthetic performance was markedly enhanced for C₃ grasses (+68%) in elevated CO₂, but not significantly for forbs. Differences in access to soil resources between forbs and grasses may distinguish their responses to elevated CO₂ and N addition. Forbs had lesser root biomass, a lower distribution of biomass to roots, and lower specific root length than grasses. Maintenance of leaf N, possibly through increased root foraging in this nutrient‐poor grassland, was necessary to sustain stimulation of photosynthesis under long‐term elevated CO₂. Dilution of leaf N and associated photosynthetic down‐regulation in forbs under elevated [CO₂], relative to the C₃ grasses, illustrates the potential for shifts in species composition and diversity in grassland ecosystems that have significant forb and grass components.     

漓江水陆交错带植物叶性状对水淹胁迫的响应及经济谱分析

以漓江水陆交错带为研究区,分两个条带分别量测了适生植物的5个叶性状指标:最大净光合速率(A_(max))、比叶重(LMA)、单位质量叶片全氮含量(N_(mass))、单位质量叶片全磷含量(P_(mass))、单位质量叶片全钾含量(K_(mass))。研究重度淹没带与微度淹没带不同功能型植物叶性状间的差异,分析并讨论重度淹没带叶性状间的关系与全球尺度是否存在差异,探究重度淹没带植物对水淹生境的生理响应机制。结果如下:(1)重度淹没带植物叶片的A_(mass)、N_(mass)、P_(mass)显著高于微度淹没带。(2)乔木、灌木叶片的LMA均显著高于草本植物,而A_(mass)、PPUE均显著低于草本植物。(3)重度淹没带草本叶性状指标的N_(mass)、P_(mass)、PNUE均显著高于微度微度淹没带,而乔木、灌木的叶性状在两个条带的差异则不显著。(4)重度淹没带植物叶性状关系与全球尺度基本一致,其植物叶片具有低LMA,高A_(mass)、Nmas s、P_(mass)。分析可知,重度淹没带植物在出露期提高叶片光合效率及相关营养水平可能是其适应水淹胁迫特殊生境的关键策略之一;不同功能型植物对同一环境的适应能力存在一定的差异,草本对于水淹环境的响应更为积极,适应能力更好;重度淹没带也存在叶经济谱,其植物在经济谱中属于"快速投资-收益"型物种。     

Significance of mesophyll conductance for photosynthetic capacity and water-use efficiency in response to alkaline stress in Populus cathayana seedlings

Cuttings of Populus cathayana were exposed to three different alkaline regimes (0, 75, and 150 mM Na₂CO₃) in a semicontrolled environment. The net photosynthesis rate (P _N), mesophyll conductance (g _m), the relative limitations posed by stomatal conductance (L _s) and by mesophyll conductance (L _m), photosynthetic nitrogen-use efficiency (PNUE), carbon isotope composition (δ¹³C), as well as specific leaf area (SLA) were measured. P _N decreased due to alkaline stress by an average of 25% and g _m decreased by an average of 57%. Alkaline stress caused an increase of L _m but not L _s, with average L _s of 26%, and L _m average of 38% under stress conditions. Our results suggested reduced assimilation rate under alkaline stress through decreased mesophyll conductance in P. cathayana. Moreover, alkaline stress increased significantly δ¹³C and it drew down CO₂ concentration from the substomatal cavities to the sites of carboxylation (C _i-C _c), but decreased PNUE. Furthermore, a relationship was found between PNUE and C _i-C _c. Meanwhile, no correlation was found between δ¹³C and C _i/C _a, but a strong correlation was proved between δ¹³C and C _c/C _a, indicating that mesophyll conductance was also influencing the ¹³C/¹²C ratio of leaf under alkaline stress.     

Novel characteristics of cassava,Manihot esculenta Crantz,a reputed C3-C4 intermediate photosynthesis species

The cassava plant, Manihot esculenta, grows exceptionally well in low fertility and drought prone environments, but the mechanisms that allow this growth are unknown. Earlier, and sometimes contradictory, work speculated about the presence of a C₄-type photosynthesis in cassava leaves. In the present work we found no evidence for a C₄ metabolism in mature attached cassava leaves as indicated i) by the low, 2 to 8%, incorporation of ¹⁴CO₂ into C₄ organic acids in short time periods, 10 s, and the lack of ¹⁴C transfer from C₄ acids to other compounds in ¹²CO₂, ii) by the lack of C₄ enzyme activity changes during leaf development and the inability to detect C₄ acid decarboxylases, and iii) by leaf CO₂ compensation values between 49 and 65 l of CO₂ 1^–1 and by other infrared gas exchange photosynthetic measurements. It is concluded that the leaf biochemistry of cassava follows the C₃ pathway of photosynthesis with no indication of a C₃-C₄ mechanism.However, cassava leaves exhibit several novel characteristics. Attached leaves have the ability to effectively partition carbon into sucrose with nearly 45% of the label in sucrose in about one min of ¹⁴CO₂ photosynthesis, contrasting with 34% in soybean (C₃) and 25% in pigweed (C₄). Cassava leaves displayed a strong preference for the synthesis of sucrose versus starch. Field grown cassava leaves exhibited high rates of photosynthesis and curvilinear responses to increasing sunlight irradiances with a tendency to saturate only at high irradiances, above 1500 mol m^–2 s^–1. Morphologically, the cassava leaf has papillose epidermal cells on its lower mesophyll surface that form fence-like arrangements encircling guard cells. It is proposed that the active synthesis of sugars has osmotic functions in the cassava plant and that the papillose epidermal cells function to maintain a healthy leaf water status in various environments.Abbreviations ADP adenosine diphosphate - Asp aspartate - BSA bovine serum albumin - CoA coenzyme A - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - FBP fructose-1,6-biphosphate - Gly glycine - HEPES N-2-hydroxyethylpiperazine-N-2-ethansulfonic acid - Mal malate - NAD nicotinamide adenine dinucleotide (oxidized form) - NADH nicotinamide adenine dinucleotide (reduced form) - NADP nicotinamide adenine dinucleotide phosphate (oxidized form) - PAR photosynthetic active radiation (400–700 nm) - PEP phosphenolpyruvate carboxylase - p-FBPase plastid fructose-1,6-biphosphatase - PGA 3-phosphoglyceric acid - PMSF phenylmethylsulfonyl fluoride - PVP polyvinylpyrrolidone - Rubisco ribulose-1,5-biphosphate carboxylase/oxygenase - RuBP ribulose-1,5-biphosphate - Ser serine - sugar-P sugar-phosphates     

Influence of High CO2Partial Pressure on Nitrogen Use Efficiency of the C4Grasses Panicum coloratum and Cenchrus ciliaris

Australia's tropical grasslands are dominated by C₄grasses,characterized by their unique biochemistry and anatomy. Twonaturalized C₄grasses (Panicum coloratum and Cenchrus ciliaris)were used to investigate whether high CO₂partial pressure [p(CO₂)] influences photosynthetic nitrogen use efficiency andplant nitrogen use efficiency (PNUE and NUE respectively). Plantswere grown for 30 d with four levels of N at p(CO₂) of 38 or86 Pa. PNUE was calculated from leaf CO₂assimilation rates (A)and leaf N concentrations, and NUE from total leaf N contentand plant dry mass. At each p(CO₂), PNUE and NUE were greaterfor C. ciliaris than for P. coloratum due to higher A and drymass combined with lower leaf N concentrations. Elevatedp (CO₂)increased PNUE of C. ciliaris only. This effect was due to lowerleaf N concentrations (area basis). At high p(CO₂), NUE of C.ciliaris was also greater. This resulted from a 1.6-fold stimulationof dry mass by high p(CO₂). Although dry mass of P. coloratumwas increased 1.2-fold by elevated p(CO₂), its NUE was unaffected.Leaf transpiration rates were halved at elevated p(CO₂), andwe suggest that this factor plays a major role in the growthresponse of C₄grasses to high p(CO₂). Copyright 2001 Annalsof Botany Company Panicum coloratum, Cenchrus ciliaris, nitrogen use efficiency, elevated CO₂, leaf N concentration, growth, photosynthesis     

Strong photosynthetic acclimation and enhanced water‐use efficiency in grassland functional groups persist over 21 years of CO2 enrichment,independent of nitrogen supply

Uncertainty about long‐term leaf‐level responses to atmospheric CO₂ rise is a major knowledge gap that exists because of limited empirical data. Thus, it remains unclear how responses of leaf gas exchange to elevated CO₂ (eCO₂) vary among plant species and functional groups, or across different levels of nutrient supply, and whether they persist over time for long‐lived perennials. Here, we report the effects of eCO₂ on rates of net photosynthesis and stomatal conductance in 14 perennial grassland species from four functional groups over two decades in a Minnesota Free‐Air CO₂ Enrichment experiment, BioCON. Monocultures of species belonging to C₃ grasses, C₄ grasses, forbs, and legumes were exposed to two levels of CO₂ and nitrogen supply in factorial combinations over 21 years. eCO₂ increased photosynthesis by 12.9% on average in C₃ species, substantially less than model predictions of instantaneous responses based on physiological theory and results of other studies, even those spanning multiple years. Acclimation of photosynthesis to eCO₂ was observed beginning in the first year and did not strengthen through time. Yet, contrary to expectations, the response of photosynthesis to eCO₂ was not enhanced by increased nitrogen supply. Differences in responses among herbaceous plant functional groups were modest, with legumes responding the most and C₄ grasses the least as expected, but did not further diverge over time. Leaf‐level water‐use efficiency increased by 50% under eCO₂ primarily because of reduced stomatal conductance. Our results imply that enhanced nitrogen supply will not necessarily diminish photosynthetic acclimation to eCO₂ in nitrogen‐limited systems, and that significant and consistent declines in stomatal conductance and increases in water‐use efficiency under eCO₂ may allow plants to better withstand drought.     

A genetic link between leaf carbon isotope composition and whole‐plant water use efficiency in the C4 grass Setaria

Genetic selection for whole‐plant water use efficiency (yield per transpiration; WUE_plant) in any crop‐breeding programme requires high‐throughput phenotyping of component traits of WUE_plant such as intrinsic water use efficiency (WUE_i; CO₂ assimilation rate per stomatal conductance). Measuring WUE_i by gas exchange measurements is laborious and time consuming and may not reflect an integrated WUE_i over the life of the leaf. Alternatively, leaf carbon stable isotope composition (δ¹³C_leaf) has been suggested as a potential time‐integrated proxy for WUE_i that may provide a tool to screen for WUE_plant. However, a genetic link between δ¹³C_leaf and WUE_plant in a C₄ species has not been well established. Therefore, to determine if there is a genetic relationship in a C₄ plant between δ¹³C_leaf and WUE_plant under well watered and water‐limited growth conditions, a high‐throughput phenotyping facility was used to measure WUE_plant in a recombinant inbred line (RIL) population created between the C₄ grasses Setaria viridis and S. italica. Three quantitative trait loci (QTL) for δ¹³C_leaf were found and co‐localized with transpiration, biomass accumulation, and WUE_plant. Additionally, WUE_plant for each of the δ¹³C_leaf QTL allele classes was negatively correlated with δ¹³C_leaf, as would be predicted when WUE_i influences WUE_plant. These results demonstrate that δ¹³C_leaf is genetically linked to WUE_plant, likely to be through their relationship with WUE_i, and can be used as a high‐throughput proxy to screen for WUE_plant in these C₄ species.     

Elevated CO2 enhances water relations and productivity and affects gas exchange in C3 and C4 grasses of the Colorado shortgrass steppe.

Six open‐top chambers were installed on the shortgrass steppe in north‐eastern Colorado, USA from late March until mid‐October in 1997 and 1998 to evaluate how this grassland will be affected by rising atmospheric CO₂. Three chambers were maintained at current CO₂ concentration (ambient treatment), three at twice ambient CO₂, or approximately 720 μmol mol^?1 (elevated treatment), and three nonchambered plots served as controls. Above‐ground phytomass was measured in summer and autumn during each growing season, soil water was monitored weekly, and leaf photosynthesis, conductance and water potential were measured periodically on important C₃ and C₄ grasses. Mid‐season and seasonal above‐ground productivity were enhanced from 26 to 47% at elevated CO₂, with no differences in the relative responses of C₃/C₄ grasses or forbs. Annual above‐ground phytomass accrual was greater on plots which were defoliated once in mid‐summer compared to plots which were not defoliated during the growing season, but there was no interactive effect of defoliation and CO₂ on growth. Leaf photosynthesis was often greater in Pascopyrum smithii (C₃) and Bouteloua gracilis (C₄) plants in the elevated chambers, due in large part to higher soil water contents and leaf water potentials. Persistent downward photosynthetic acclimation in P. smithii leaves prevented large photosynthetic enhancement for elevated CO₂‐grown plants. Shoot N concentrations tended to be lower in grasses under elevated CO₂, but only Stipa comata (C₃) plants exhibited significant reductions in N under elevated compared to ambient CO₂ chambers. Despite chamber warming of 2.6 °C and apparent drier chamber conditions compared to unchambered controls, above‐ground production in all chambers was always greater than in unchambered plots. Collectively, these results suggest increased productivity of the shortgrass steppe in future warmer, CO₂ enriched environments.     

Capacity for NADPH regeneration in the leaves of two poplar genotypes differing in ozone sensitivity

Cell capacity for cytosolic NADPH regeneration by NADP‐dehydrogenases was investigated in the leaves of two hybrid poplar (Populus deltoides × Populus nigra) genotypes in response to ozone (O₃) treatment (120 ppb for 17 days). Two genotypes with differential O₃ sensitivity were selected, based on visual symptoms and fallen leaves: Robusta (sensitive) and Carpaccio (tolerant). The estimated O₃ flux (POD₀), that entered the leaves, was similar for the two genotypes throughout the treatment. In response to that foliar O₃ flux, CO₂ assimilation was inhibited to the same extent for the two genotypes, which could be explained by a decrease in Rubisco (EC 4.1.1.39) activity. Conversely, an increase in PEPC (EC 4.1.1.31) activity was observed, together with the activation of certain cytosolic NADP‐dehydrogenases above their constitutive level, i.e. NADP‐G6PDH (EC 1.1.1.49), NADP‐ME (malic enzyme) (EC 1.1.1.40) and NADP‐ICDH (NADP‐isocitrate dehydrogenase) (EC1.1.1.42). However, the activity of non‐phosphorylating NADP‐GAPDH (EC 1.2.1.9) remained unchanged. From the 11th fumigation day, NADP‐G6PDH and NADP‐ME profiles made it possible to differentiate between the two genotypes, with a higher activity in Carpaccio than in Robusta. At the same time, Carpaccio was able to maintain high levels of NADPH in the cells, while NADPH levels decreased in Robusta O₃‐treated leaves. All these results support the hypothesis that the capacity for cells to regenerate the reducing power, especially the cytosolic NADPH pool, contributes to improve tolerance to high ozone exposure.