The Dynamic Assimilation Technique measures photosynthetic CO2 response curves with similar fidelity to steady-state approaches in half the time.

The net CO2 assimilation (A) response to intercellular CO2 concentration (Ci) is a fundamental measurement in photosynthesis and plant physiology research. The conventional A/Ci protocols rely on steady-state measurements and take 15-40 min per measurement, limiting data resolution or biological replication. Additionally, there are several CO2 protocols employed across the literature, without clear consensus as to the optimal protocol or systematic biases in their estimations. We compared the non-steady-state Dynamic Assimilation Technique (DAT) protocol and the three most used CO2 protocols in steady-state measurements, and tested whether different CO2 protocols lead to systematic differences in estimations of the biochemical limitations to photosynthesis. The DAT protocol reduced the measurement time by almost half without compromising estimation accuracy or precision. The monotonic protocol was the fastest steady-state method. Estimations of biochemical limitations to photosynthesis were very consistent across all CO2 protocols, with slight differences in Rubisco carboxylation limitation. The A/Ci curves were not affected by the direction of the change of CO2 concentration but rather the time spent under triose phosphate utilization (TPU)-limited conditions. Our results suggest that the maximum rate of Rubisco carboxylation (Vcmax), linear electron flow for NADPH supply (J), and TPU measured using different protocols within the literature are comparable, or at least not systematically different based on the measurement protocol used.

Differential sensitivities of photosynthetic component processes govern oxidative stress levels and net assimilation rates in virus-infected cotton.

Cotton (Gossypium hirsutum L.) leafroll dwarf virus disease (CLRDD) is a yield-limiting threat to cotton production and can substantially limit net photosynthetic rates (AN). Previous research showed that AN was more sensitive to CLRDD-induced reductions in stomatal conductance than electron transport rate (ETR) through photosystem II (PSII). This observation coupled with leaf reddening symptomology led to the hypothesis that differential sensitivities of photosynthetic component processes to CLRDD would contribute to declines in AN and increases in oxidative stress, stimulating anthocyanin production. Thus, an experiment was conducted to define the relative sensitivity of photosynthetic component processes to CLRDD and to quantify oxidative stress and anthocyanin production in field-grown cotton. Among diffusional limitations to AN, reductions in mesophyll conductance and CO2 concentration in the chloroplast were the greatest constraints to AN under CLRDD. Multiple metabolic processes were also adversely impacted by CLRDD. ETR, RuBP regeneration, and carboxylation were important metabolic (non-diffusional) limitations to AN in symptomatic plants. Photorespiration and dark respiration were less sensitive than photosynthetic processes, contributing to declines in AN in symptomatic plants. Among thylakoid processes, reduction of PSI end electron acceptors was the most sensitive to CLRDD. Oxidative stress indicators (H2O2 production and membrane peroxidation) and anthocyanin contents were substantially higher in symptomatic plants, concomitant with reductions in carotenoid content and no change in energy dissipation by PSII. We conclude that differential sensitivities of photosynthetic processes to CLRDD and limited potential for energy dissipation at PSII increases oxidative stress, stimulating anthocyanin production as an antioxidative mechanism.

Differential sensitivities of photosynthetic processes and carbon loss mechanisms govern N-induced variation in net carbon assimilation rate for field-grown cotton.

Nitrogen (N) deficiency limits the net carbon assimilation rate (AN), but the relative N sensitivities of photosynthetic component processes and carbon loss mechanisms remain relatively unexplored for field-grown cotton. Therefore, the objective of the current study was to define the relative sensitivity of individual physiological processes driving N deficiency-induced declines in AN for field-grown cotton. Among the potential diffusional limitations evaluated, mesophyll conductance was the only parameter substantially reduced by N deficiency, but this did not affect CO2 availability in the chloroplast. A number of metabolic processes were negatively impacted by N deficiency, and these effects were more pronounced at lower leaf positions in the cotton canopy. Ribulose bisphosphate (RuBP) regeneration and carboxylation, AN, and gross photosynthesis were the most sensitive metabolic processes to N deficiency, whereas photosynthetic electron transport processes, electron flux to photorespiration, and dark respiration exhibited intermediate sensitivity to N deficiency. Among thylakoid-specific processes, the quantum yield of PSI end electron acceptor reduction was the most sensitive process to N deficiency. It was concluded that AN is primarily limited by Rubisco carboxylation and RuBP regeneration under N deficiency in field-grown cotton, and the differential N sensitivities of the photosynthetic process and carbon loss mechanisms contributed significantly to photosynthetic declines.

Effect of leaf temperature on the estimation of photosynthetic and other traits of wheat leaves from hyperspectral reflectance.

A growing number of leaf traits can be estimated from hyperspectral reflectance data. These include structural and compositional traits, such as leaf mass per area (LMA) and nitrogen and chlorophyll content, but also physiological traits such a Rubisco carboxylation activity, electron transport rate, and respiration rate. Since physiological traits vary with leaf temperature, how does this impact on predictions made from reflectance measurements? We investigated this with two wheat varieties, by repeatedly measuring each leaf through a sequence of temperatures imposed by varying the air temperature in a growth room. Leaf temperatures ranging from 20 °C to 35 °C did not alter the estimated Rubisco capacity normalized to 25 °C (Vcmax25), or chlorophyll or nitrogen contents per unit leaf area. Models estimating LMA and Vcmax25/N were both slightly influenced by leaf temperature: estimated LMA increased by 0.27% °C-1 and Vcmax25/N increased by 0.46% °C-1. A model estimating Rubisco activity closely followed variation associated with leaf temperature. Reflectance spectra change with leaf temperature and therefore contain a temperature signal.

Revised mechanism of carboxylation of ribulose-1,5-biphosphate by rubisco from large scale quantum chemical calculations.

Here, we describe a computational approach for studying enzymes that catalyze complex multi-step reactions and apply it to Ribulose 1,5-bisphosphate carboxylase-oxygenase (Rubisco), the enzyme that fixes atmospheric carbon dioxide within photosynthesis. In the 5-step carboxylase reaction, the substrate Ribulose-1,5-bisphosphate (RuBP) first binds Rubisco and undergoes enolization before binding the second substrate, CO2 . Hydration of the RuBP.CO2 complex is followed by CC bond scission and stereospecific protonation. However, details of the roles and protonation states of active-site residues, and sources of protons and water, remain highly speculative. Large-scale computations on active-site models provide a means to better understand this complex chemical mechanism. The computational protocol comprises a combination of hybrid semi-empirical quantum mechanics and molecular mechanics within constrained molecular dynamics simulations, together with constrained gradient minimization calculations using density functional theory. Alternative pathways for hydration of the RuBP.CO2 complex and associated active-site protonation networks and proton and water sources were investigated. The main findings from analysis of the resulting energetics advocate major revision to existing mechanisms such that: hydration takes place anti to the CO2 ; both hydration and CC bond scission require early protonation of CO2 in the RuBP.CO2 complex; CC bond scission and stereospecific protonation reactions are concerted and, effectively, there is only one stable intermediate, the C3-gemdiolate complex. Our main conclusions for interpreting enzyme kinetic results are that the gemdiolate may represent the elusive Michaelis-Menten-like complex corresponding to the empirical Km (=Kc ) with turnover to product via bond scission concerted with stereospecific protonation consistent with the observed catalytic rate. © 2018 Wiley Periodicals, Inc.

Informing models through empirical relationships between foliar phosphorus, nitrogen and photosynthesis across diverse woody species in tropical forests of Panama.

Our objective was to analyze and summarize data describing photosynthetic parameters and foliar nutrient concentrations from tropical forests in Panama to inform model representation of phosphorus (P) limitation of tropical forest productivity. Gas exchange and nutrient content data were collected from 144 observations of upper canopy leaves from at least 65 species at two forest sites in Panama, differing in species composition, rainfall and soil fertility. Photosynthetic parameters were derived from analysis of assimilation rate vs internal CO2 concentration curves (A/Ci ), and relationships with foliar nitrogen (N) and P content were developed. The relationships between area-based photosynthetic parameters and nutrients were of similar strength for N and P and robust across diverse species and site conditions. The strongest relationship expressed maximum electron transport rate (Jmax ) as a multivariate function of both N and P, and this relationship was improved with the inclusion of independent data on wood density. Models that estimate photosynthesis from foliar N would be improved only modestly by including additional data on foliar P, but doing so may increase the capability of models to predict future conditions in P-limited tropical forests, especially when combined with data on edaphic conditions and other environmental drivers.

Does long-term cultivation of saplings under elevated CO2 concentration influence their photosynthetic response to temperature?

BACKGROUND AND AIMS: Plants growing under elevated atmospheric CO2 concentrations often have reduced stomatal conductance and subsequently increased leaf temperature. This study therefore tested the hypothesis that under long-term elevated CO2 the temperature optima of photosynthetic processes will shift towards higher temperatures and the thermostability of the photosynthetic apparatus will increase. METHODS: The hypothesis was tested for saplings of broadleaved Fagus sylvatica and coniferous Picea abies exposed for 4-5 years to either ambient (AC; 385 µmol mol(-1)) or elevated (EC; 700 µmol mol(-1)) CO2 concentrations. Temperature response curves of photosynthetic processes were determined by gas-exchange and chlorophyll fluorescence techniques. KEY RESULTS: Initial assumptions of reduced light-saturated stomatal conductance and increased leaf temperatures for EC plants were confirmed. Temperature response curves revealed stimulation of light-saturated rates of CO2 assimilation (Amax) and a decline in photorespiration (RL) as a result of EC within a wide temperature range. However, these effects were negligible or reduced at low and high temperatures. Higher temperature optima (Topt) of Amax, Rubisco carboxylation rates (VCmax) and RL were found for EC saplings compared with AC saplings. However, the shifts in Topt of Amax were instantaneous, and disappeared when measured at identical CO2 concentrations. Higher values of Topt at elevated CO2 were attributed particularly to reduced photorespiration and prevailing limitation of photosynthesis by ribulose-1,5-bisphosphate (RuBP) regeneration. Temperature response curves of fluorescence parameters suggested a negligible effect of EC on enhancement of thermostability of photosystem II photochemistry. CONCLUSIONS: Elevated CO2 instantaneously increases temperature optima of Amax due to reduced photorespiration and limitation of photosynthesis by RuBP regeneration. However, this increase disappears when plants are exposed to identical CO2 concentrations. In addition, increased heat-stress tolerance of primary photochemistry in plants grown at elevated CO2 is unlikely. The hypothesis that long-term cultivation at elevated CO2 leads to acclimation of photosynthesis to higher temperatures is therefore rejected. Nevertheless, incorporating acclimation mechanisms into models simulating carbon flux between the atmosphere and vegetation is necessary.

The effect of elevated CO2 on photosynthesis is modulated by nitrogen supply and reduced water availability in Picea abies.

It is assumed that the stimulatory effects of elevated CO2 concentration ([CO2]) on photosynthesis and growth may be substantially reduced by co-occurring environmental factors and the length of CO2 treatment. Here, we present the study exploring the interactive effects of three manipulated factors ([CO2], nitrogen supply and water availability) on physiological (gas-exchange and chlorophyll fluorescence), morphological and stoichiometric traits of Norway spruce (Picea abies) saplings after 2 and 3 years of the treatment under natural field conditions. Such multifactorial studies, going beyond two-way interactions, have received only limited attention until now. Our findings imply a significant reduction of [CO2]-enhanced rate of CO2 assimilation under reduced water availability which deepens with the severity of water depletion. Similarly, insufficient nitrogen availability leads to a down-regulation of photosynthesis under elevated [CO2] being particularly associated with reduced carboxylation efficiency of the Rubisco enzyme. Such adjustments in the photosynthesis machinery result in the stimulation of water-use efficiency under elevated [CO2] only when it is combined with a high nitrogen supply and reduced water availability. These findings indicate limited effects of elevated [CO2] on carbon uptake in temperate coniferous forests when combined with naturally low nitrogen availability and intensifying droughts during the summer periods. Such interactions have to be incorporated into the mechanistic models predicting changes in terrestrial carbon sequestration and forest growth in the future.

Photosynthetic and Growth Responses in a Pioneer Tree (Japanese White Birch) and Competitive Perennial Weeds (Eupatorium sp.) Grown Under Different Regimes With Limited Water Supply to Waterlogging.

For a successful natural regeneration of Japanese white birch (Betula platyphylla var. japonica), competitive vegetation should be managed. Here, we clarified how soil water condition modifies the competitiveness of Japanese white birch against perennial weeds, Eupatorium species, based on an ecophysiological approach combining a glasshouse experiment and a field survey. We investigated photosynthetic and growth responses to various water regimes from water deficit to waterlogging (two times-a-week irrigation, three times-a-week irrigation, half waterlogging, and full waterlogging) in pot-grown seedlings of Japanese white birch and the competitive weed Eupatorium makinoi. The ratio of seedling height of Japanese white birch to seedling height of E. makinoi showed a decreasing trend from two times-a-week irrigation to full waterlogging, which suggests a lower competitiveness for light resource in Japanese white birch with increasing soil wetness. The maximum rate of Rubisco carboxylation (Vc,max) based on unit N was lower in waterlogging treatments than in two times- and three times-a-week irrigation in Japanese white birch, whereas E. makinoi showed the opposite response. This suggests that N partitioning into Rubisco and/or Rubisco activation might be suppressed in Japanese white birch but enhanced in E. makinoi under waterlogging. The maximum photochemical efficiency of photosystem II (Fv/Fm) was also lower in seedlings of Japanese white birch grown under waterlogging treatments. We further conducted a field survey on the relationship between Fv/Fm and topographic wetness index (TWI) in seedlings of Japanese white birch and E. glehnii (closely related to E. makinoi) naturally grown in a study site 5 years after canopy tree cutting. Lower Fv/Fm was observed in seedlings of Japanese white birch with increasing TWI, whereas no significant trend was observed in E. glehnii, in agreement with the glasshouse experiment. Thus, keeping soils not always humid might be favorable to photosynthetic performance and growth competitive ability of Japanese white birch against Eupatorium species.

The knockdown of chloroplastic ascorbate peroxidases reveals its regulatory role in the photosynthesis and protection under photo-oxidative stress in rice.

The inactivation of the chloroplast ascorbate peroxidases (chlAPXs) has been thought to limit the efficiency of the water-water cycle and photo-oxidative protection under stress conditions. In this study, we have generated double knockdown rice (Oryza sativa L.) plants in both OsAPX7 (sAPX) and OsAPX8 (tAPX) genes, which encode chloroplastic APXs (chlAPXs). By employing an integrated approach involving gene expression, proteomics, biochemical and physiological analyses of photosynthesis, we have assessed the role of chlAPXs in the regulation of the protection of the photosystem II (PSII) activity and CO2 assimilation in rice plants exposed to high light (HL) and methyl violagen (MV). The chlAPX knockdown plants were affected more severely than the non-transformed (NT) plants in the activity and structure of PSII and CO2 assimilation in the presence of MV. Although MV induced significant increases in pigment content in the knockdown plants, the increases were apparently not sufficient for protection. Treatment with HL also caused generalized damage in PSII in both types of plants. The knockdown and NT plants exhibited differences in photosynthetic parameters related to efficiency of utilization of light and CO2. The knockdown plants overexpressed other antioxidant enzymes in response to the stresses and increased the GPX activity in the chloroplast-enriched fraction. Our data suggest that a partial deficiency of chlAPX expression modulate the PSII activity and integrity, reflecting the overall photosynthesis when rice plants are subjected to acute oxidative stress. However, under normal growth conditions, the knockdown plants exhibit normal phenotype, biochemical and physiological performance.

Impaired leaf CO2 diffusion mediates Cd-induced inhibition of photosynthesis in the Zn/Cd hyperaccumulator Picris divaricata.

Mechanisms of cadmium (Cd)-induced inhibition of photosynthesis in the Zn/Cd hyperaccumulator Picris divaricata were investigated using photosynthesis limitation analysis. P. divaricata seedlings were grown in nutrient solution containing 0, 5, 10, 25, 50, or 75 µM Cd for 2 weeks. Total limitations to photosynthesis (TL) increased from 0% at 5 µM Cd to 68.8% at 75 µM Cd. CO2 diffusional limitation (DL) made the largest contribution to TL, accounting for 93-98% of TL in the three highest Cd treatments, compared to just 2-7% of TL attributable to biochemical limitation (BL). Microscopic imaging revealed significantly decreased stomatal density and mesophyll thickness in the three highest Cd treatments. Chlorophyll fluorescence parameters related to photosynthetic biochemistry (Fv/Fm, NPQ, ΦPSII, and qP) were not significantly decreased by increased Cd supply. Our results suggest that increased DL in leaves is the main cause of Cd-induced inhibition of photosynthesis in P. divaricata, possibly due to suppressed function of mesophyll and stomata. Analysis of chlorophyll fluorescence showed that Cd supply had little effect on photochemistry parameters, suggesting that the PSII reaction centers are not a main target of Cd inhibition of photosynthesis in P. divaricata.