Carboxylation capacity is the main limitation of carbon assimilation in High Arctic shrubs.

Increases in shrub height, biomass and canopy cover are key whole-plant features of warming-induced vegetation change in tundra. We investigated leaf functional traits underlying photosynthetic capacity of Arctic shrub species, particularly its main limiting processes such as mesophyll conductance. In this nutrient-limited ecosystem, we expect leaf nitrogen concentration to be the main limiting factor for photosynthesis. We measured the net photosynthetic rate at saturated light (Asat) in three Salix species throughout a glacial valley in High-Arctic tundra and used a causal approach to test relationships between leaf stomatal and mesophyll conductances (gsc, gm), carboxylation capacity (Vcmax), nitrogen and phosphorus concentration (Narea, Parea) and leaf mass ratio (LMA). Arctic Salix species showed no difference in Asat compared to a global data set, while being characterized by higher Narea, Parea and LMA. Vcmax, gsc and gm independently increased Asat, with Vcmax as its main limitation. We highlighted a nitrogen-influenced pathway for increasing photosynthesis in the two prostrate mesic habitat species. In contrast, the erect wetland habitat Salix richardsonii mainly increased Asat with increasing gsc. Overall, our study revealed high photosynthetic capacities of Arctic Salix species but contrasting regulatory pathways that may influence shrub ability to respond to environmental changes in High Arctic tundra.

Synergetic light and cytokinin treatments mitigate the recombinant protein yield depression induced by high-density cultivation of hydroponically-grown Nicotiana benthamiana.

Plant molecular farming is currently operating a transition from soil-based cultures toward hydroponic systems. In this study, we designed a whole-plant NFT (nutrient film technique) platform for the transient expression of influenza virus-like particles harboring hemagglutinin H1 proteins in Nicotiana benthamiana. In particular, we examined the effects of plant density during the post-infiltration expression phase on plant growth and H1 yield in relation to the daily light integral (DLI) received by the crop and the exogenous application of 6-BAP cytokinin (CK). We expected from previous work that high DLI and CK treatments would stimulate the development of highly productive leaves on axillary (secondary) stems and thereby improve the H1 yield at the whole-plant scale. Increasing plant density from 35.7 to 61 plants m-2 during the post-infiltration phase significantly decreased the proportion of axillary leaf biomass by 30% and H1 yield per plant by 39%, resulting in no additional yield gain on a whole-crop area basis. Adding CK to the recirculated nutrient solution decreased the harvested leaf biomass by 31% and did not enhance the relative proportion of S leaves of the plants as previously reported with foliar CK application. There was a 36% increase in H1 yield when doubling the DLI from 14 to 28 mol m-2 s-1, and up to 71% yield gain when combining such an increase in DLI with the hydroponic CK treatment. Contrary to our expectations, leaves located on the main stem, particularly those from the upper half of the plant (i.e., eighth leaf and above), contributed about 80% of total H1 yield. Our study highlights the significantly different phenotype (~30% less secondary leaf biomass) and divergent responses to light and CK treatments of NFT-grown N. benthamiana plants compared to previous studies conducted on potted plants.

When and where soil is important to modify the carbon and water economy of leaves.

Photosynthetic 'least-cost' theory posits that the optimal trait combination for a given environment is that where the summed costs of photosynthetic water and nutrient acquisition/use are minimised. The effects of soil water and nutrient availability on photosynthesis should be stronger as climate-related costs for both resources increase. Two independent datasets of photosynthetic traits, Globamax (1509 species, 288 sites) and Glob13C (3645 species, 594 sites), were used to quantify biophysical and biochemical limitations of photosynthesis and the key variable Ci /Ca (CO2 drawdown during photosynthesis). Climate and soil variables were associated with both datasets. The biochemical photosynthetic capacity was higher on alkaline soils. This effect was strongest at more arid sites, where water unit-costs are presumably higher. Higher values of soil silt and depth increased Ci /Ca , likely by providing greater H2 O supply, alleviating biophysical photosynthetic limitation when soil water is scarce. Climate is important in controlling the optimal balance of H2 O and N costs for photosynthesis, but soil properties change these costs, both directly and indirectly. In total, soil properties modify the climate-demand driven predictions of Ci /Ca by up to 30% at a global scale.

Thermal acclimation of photosynthesis and respiration of southern and northern white spruce seed sources tested along a regional climatic gradient indicates limited potential to cope with temperature warming.

Background and Aims: Knowledge of thermal acclimation of physiological processes of boreal tree species is necessary to determine their ability to adapt to predicted global warming and reduce the uncertainty around the anticipated feedbacks of forest ecosystems and global carbon cycle to climate change. The objective of this work was to examine the extent of thermal acclimation of net photosynthesis (An) and dark respiration (Rd) of two distant white spruce (Picea glauca) seed sources (from south and north of the commerial forest zone in Québec) in response to latitudinal and seasonal variations in growing conditions. Methods: The temperature responses of An, its biochemical and biophysical limitations, and Rd were measured in 1-year-old needles of seedlings from the seed sources growing in eight forest plantations along a regional thermal gradient of 5.5 °C in Québec, Canada. Key Results: The average optimum temperature (Topt) for An was 19 ± 1.2 °C and was similar among seed sources and plantation sites along the thermal gradient. Net photosynthesis at Topt (Aopt) varied significantly among plantation sites and was quadratically related to the mean July temperature (MJT) of plantation sites. Topt for mesophyll conductance, maximum electron transport rate and maximum rate of carboxylation were 28, 22 and 30 °C, respectively. Basal respiration rate (Rd at 10 °C) was linearly and negatively associated with MJT. Q10 of Rd (the rate of change in Rd with a 10 °C increase in temperature) did not show any significant relationship with MJT and averaged 1.5 ± 0.1. The two seed sources were similar in their thermal responses to latitudinal and seasonal variations in growing conditions. Conclusions: The results showed moderate thermal acclimation of respiration and no evidence for thermal acclimation of photosynthesis or local genetic adaptation for traits related to thermal acclimation. Therefore, growth of local white spruces may decline in future climates.

Persistent negative temperature response of mesophyll conductance in red raspberry (Rubus idaeus L.) leaves under both high and low vapour pressure deficits: a role for abscisic acid?

The temperature dependence of mesophyll conductance (gm ) was measured in well-watered red raspberry (Rubus idaeus L.) plants acclimated to leaf-to-air vapour pressure deficit (VPDL) daytime differentials of contrasting amplitude, keeping a fixed diurnal leaf temperature (Tleaf ) rise from 20 to 35 °C. Contrary to the great majority of gm temperature responses published to date, we found a pronounced reduction of gm with increasing Tleaf irrespective of leaf chamber O2 level and diurnal VPDL regime. Leaf hydraulic conductance was greatly enhanced during the warmer afternoon periods under both low (0.75 to 1.5 kPa) and high (0.75 to 3.5 kPa) diurnal VPDL regimes, unlike stomatal conductance (gs ), which decreased in the afternoon. Consequently, the leaf water status remained largely isohydric throughout the day, and therefore cannot be evoked to explain the diurnal decrease of gm . However, the concerted diurnal reductions of gm and gs were well correlated with increases in leaf abscisic acid (ABA) content, thus suggesting that ABA can induce a significant depression of gm under favourable leaf water status. Our results challenge the view that the temperature dependence of gm can be explained solely from dynamic leaf anatomical adjustments and/or from the known thermodynamic properties of aqueous solutions and lipid membranes.​.

Threshold response of mesophyll CO2 conductance to leaf hydraulics in highly transpiring hybrid poplar clones exposed to soil drying.

Mesophyll conductance (gm) has been shown to impose significant limitations to net CO2 assimilation (A) in various species during water stress. Net CO2 assimilation is also limited by stomatal conductance to water (gsw), both having been shown to co-vary with leaf hydraulic conductance (Kleaf). Lately, several studies have suggested a close functional link between Kleaf, gsw, and gm. However, such relationships could only be circumstantial since a recent study has shown that the response of gm to drought could merely be an artefactual consequence of a reduced intercellular CO2 mole fraction (Ci). Experiments were conducted on 8-week-old hybrid poplar cuttings to determine the relationship between Kleaf, gsw, and g m in clones of contrasting drought tolerance. It was hypothesized that changes in gsw and Kleaf in response to drought would not impact on gm over most of its range. The results show that Kleaf decreased in concert with g sw as drought proceeded, whereas gm measured at a normalized Ci remained relatively constant up to a g sw threshold of ~0.15 mol m(-2) s(-1). This delayed gm response prevented a substantial decline in A at the early stage of the drought, thereby enhancing water use efficiency. Reducing the stomatal limitation of droughted plants by diminishing the ambient CO2 concentration of the air did not modify gm or Kleaf. The relationship between gas exchange and leaf hydraulics was similar in both drought-tolerant and drought-sensitive clones despite their contrasting vulnerability to stem cavitation and stomatal response to soil drying. The results support the hypothesis of a partial hydraulic isolation of the mesophyll from the main transpiration pathway.

Variable mesophyll conductance revisited: theoretical background and experimental implications.

The CO(2) concentration at the site of carboxylation inside the chloroplast stroma depends not only on the stomatal conductance, but also on the conductance of CO(2) between substomatal cavities and the site of CO(2) fixation. This conductance, commonly termed mesophyll conductance (g(m) ), significantly constrains the rate of photosynthesis. Here we show that estimates of g(m) are influenced by the amount of respiratory and photorespiratory CO(2) from the mitochondria diffusing towards the chloroplasts. This results in an apparent CO(2) and oxygen sensitivity of g(m) that does not imply a change in intrinsic diffusion properties of the mesophyll, but depends on the ratio of mitochondrial CO(2) release to chloroplast CO(2) uptake. We show that this effect (1) can bias the estimation of the CO(2) photocompensation point and non-photorespiratory respiration in the light; (2) can affect the estimates of ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) kinetic constants in vivo; and (3) results in an apparent obligatory correlation between stomatal conductance and g(m) . We further show that the amount of photo(respiratory) CO(2) that is refixed by Rubisco can be directly estimated through measurements of g(m) .

Production of Biopharmaceuticals in Nicotiana benthamiana-Axillary Stem Growth as a Key Determinant of Total Protein Yield.

Data are scarce about the influence of basic cultural conditions on growth patterns and overall performance of plants used as heterologous production hosts for protein pharmaceuticals. Higher plants are complex organisms with young, mature, and senescing organs that show distinct metabolic backgrounds and differ in their ability to sustain foreign protein expression and accumulation. Here, we used the transient protein expression host Nicotiana benthamiana as a model to map the accumulation profile of influenza virus hemagglutinin H1, a clinically promising vaccine antigen, at the whole plant scale. Greenhouse-grown plants submitted to different light regimes, submitted to apical bud pruning, or treated with the axillary growth-promoting cytokinin 6-benzylaminopurine were vacuum-infiltrated with agrobacteria harboring a DNA sequence for H1 and allowed to express the viral antigen for 7 days in growth chamber under similar environmental conditions. Our data highlight the importance of young leaves on H1 yield per plant, unlike older leaves which account for a significant part of the plant biomass but contribute little to total antigen titer. Our data also highlight the key contribution of axillary stem leaves, which contribute more than 50% of total yield under certain conditions despite representing only one-third of the total biomass. These findings underline the relevance of both considering main stem leaves and axillary stem leaves while modeling heterologous protein production in N. benthamiana. They also demonstrate the potential of exogenously applied growth-promoting hormones to modulate host plant architecture for improvement of protein yields.

Organic Nitrogen Uptake and Assimilation in Cucumis sativus Using Position-Specific Labeling and Compound-Specific Isotope Analysis.

Organic nitrogen is now considered a significant source of N for plants. Although organic management practices increase soil organic C and N content, the importance of organic N as a source of crop N under organic farming management systems is still poorly understood. While dual-labeled (13C and 15N) molecule methods have been developed to study amino acid uptake by plants, multiple biases may arise from pre-uptake mineralization by microorganisms or post-uptake metabolism by the plant. We propose the combination of different isotopic analysis methods with molecule isotopologues as a novel approach to improve the accuracy of measured amino acid uptake rates in the total N budget of cucumber seedlings and provide a better characterization of post-uptake metabolism. Cucumber seedlings were exposed to solutions containing L-Ala-1-13C,15N or U-L-Ala-13C3,15N, in combination with ammonium nitrate, at total N concentrations ranging from 0 to 15 mM N and at inorganic/organic N ratios from 10:1 to 500:1. Roots and shoots were then subjected to bulk stable isotope analysis (BSIA) by Isotope Ratio Mass Spectrometry (IRMS), and to compound-specific stable isotope analysis (CSIA) of the free amino acids by Gas Chromatography - Combustion - Isotope Ratio Mass Spectrometry (GC-C-IRMS). Plants exposed to a lower inorganic:organic N ratio acquired up to 6.84% of their N from alanine, compared with 0.94% at higher ratio. No 13C from L-Ala-1-13C,15N was found in shoot tissues suggesting that post-uptake metabolism of Ala leads to the loss of the carboxyl-C as CO2. CSIA of the free amino acids in roots confirmed that intact Ala is indeed taken up by the roots, but that it is rapidly metabolized. C atoms other than from the carboxyl group and amino-N from Ala are assimilated in other amino acids, predominantly Glu, Gln, Asp, and Asn. Uptake rates reported by CSIA of the free amino acids are nevertheless much lower (16-64 times) than those reported by BSIA. Combining the use of isotopologues of amino acids with compound-specific isotope analysis helps reduce the bias in the assessment of organic N uptake and improves the understanding of organic N assimilation especially in the context of organic horticulture.

Greater efficiency of water use in poplar clones having a delayed response of mesophyll conductance to drought.

Improvement of water use efficiency is a key objective to improve the sustainability of cultivated plants, especially fast growing species with high water consumption like poplar. It is well known that water use efficiency (WUE) varies considerably among poplar genotypes, and it was recently suggested that the use of the mesophyll-to-stomatal conductance ratio (gm/gs) would be an appropriate trait to improve WUE. The responses of 7-week-old cuttings of four hybrid poplar clones and one native Balsam poplar (Populus balsamifera L.) to a water stress-recovery cycle were examined to evaluate the relation between the gm/gs ratio and transpiration efficiency (TE), a leaf-level component of WUE. A contrasting gs response to water stress was observed among the five clones, from stomatal closure early on during soil drying up to limited closure in Balsam poplar. However in the hybrids, the decline in gm was consistently delayed by a few days compared with gs. Moreover, in the most water use-efficient hybrids, the recovery following rehydration occurred faster for gm than for gs. Thus, the delay in the response of gm to drought and its faster recovery upon rewatering increased the gm/gs of the hybrids and this ratio scaled positively with TE. Our results support the use of the gm/gs ratio to select genotypes with improved WUE, and the notion that breeding strategies focusing mainly on stomatal responses to soil drying should also look for a strong curvilinearity between net carbon assimilation rate and gs, the indication of a significant increase in gm/gs in the earlier stages of stomatal closure.

Construction costs, chemical composition and payback time of high- and low-irradiance leaves.

The effect of irradiance on leaf construction costs, chemical composition, and on the payback time of leaves was investigated. To enable more generalized conclusions, three different systems were studied: top and the most-shaded leaves of 10 adult tree species in a European mixed forest, top leaves of sub-dominant trees of two evergreen species growing in small gaps or below the canopy in an Amazonian rainforest, and plants of six herbaceous and four woody species grown hydroponically at low or high irradiance in growth cabinets. Daily photon irradiance varied 3-6-fold between low- and high-light leaves. Specific leaf area (SLA) was 30-130% higher at low light. Construction costs, on the other hand, were 1-5% lower for low-irradiance leaves, mainly because low-irradiance leaves had lower concentrations of soluble phenolics. Photosynthetic capacity and respiration, expressed per unit leaf mass, were hardly different for the low- and high-light leaves. Estimates of payback times of the high-irradiance leaves ranged from 2-4 d in the growth cabinets, to 15-20 d for the adult tree species in the European forest. Low-irradiance leaves had payback times that were 2-3 times larger, ranging from 4 d in the growth cabinets to 20-80 d at the most shaded part of the canopy of the mixed forest. In all cases, estimated payback times were less than half the life span of the leaves, suggesting that even at time-integrated irradiances lower than 5% of the total seasonal value, investment in leaves is still fruitful from a carbon-economy point of view. A sensitivity analysis showed that increased SLA of low-irradiance leaves was the main factor constraining payback times. Acclimation in the other five factors determining payback time, namely construction costs, photosynthetic capacity per unit leaf mass, respiration per unit leaf mass, apparent quantum yield, and curvature of the photosynthetic light-response-curve, were unimportant when the observed variation in each factor was examined.

Carbon flux and growth in mature deciduous forest trees exposed to elevated CO2.

Whether rising atmospheric carbon dioxide (CO2) concentrations will cause forests to grow faster and store more carbon is an open question. Using free air CO2 release in combination with a canopy crane, we found an immediate and sustained enhancement of carbon flux through 35-meter-tall temperate forest trees when exposed to elevated CO2. However, there was no overall stimulation in stem growth and leaf litter production after 4 years. Photosynthetic capacity was not reduced, leaf chemistry changes were minor, and tree species differed in their responses. Although growing vigorously, these trees did not accrete more biomass carbon in stems in response to elevated CO2, thus challenging projections of growth responses derived from tests with smaller trees.

Web-FACE: a new canopy free-air CO2 enrichment system for tall trees in mature forests.

The long-term responses of forests to atmospheric CO2 enrichment have been difficult to determine experimentally given the large scale and complex structure of their canopy. We have developed a CO2 exposure system that uses the free-air CO2 enrichment (FACE) approach but was designed for tall canopy trees. The system consists of a CO2-release system installed within the crown of adult trees using a 45-m tower crane, a CO2 monitoring system and an automated regulation system. Pure CO2 gas is released from a network of small tubes woven into the forest canopy (web-FACE), and CO2 is emitted from small laser-punched holes. The set point CO2 concentration ([CO2]) of 500 µmol mol(-1) is controlled by a pulse-width modulation routine that adjusts the rate of CO2 injection as a function of measured [CO2] in the canopy. CO2 consumption for the enrichment of 14 tall canopy trees was about 2 tons per day over the whole growing season. The seasonal daytime mean CO2 concentration was 520 µmol mol(-1). One-minute averages of CO2 measurements conducted at canopy height in the center of the CO2-enriched zone were within ±20% and ±10% of the target concentration for 76% and 47% of the exposure time, respectively. Despite the size of the canopy and the windy site conditions, performance values correspond to about 75% of that reported for conventional forest FACE with the added advantage of a much simpler and less intrusive infrastructure. Stable carbon isotope signals captured by 80 Bermuda grass (Cynodon dactylon) seedlings distributed within the canopy of treated and control tree districts showed a clearly delineated area, with some nearby individuals having been exposed to a gradient of [CO2], which is seen as added value. Time-integrated values of [CO2] derived from the C isotope composition of C. dactylon leaves indicated a mean (±SD) concentration of 513±63 µmol mol(-1) in the web-FACE canopy area. In view of the size of the forest and the rough natural canopy, web-FACE is a most promising avenue towards natural forest experiments, which are greatly needed.

Elevated CO2 reduces sap flux in mature deciduous forest trees.

We enriched in CO2 the canopy of 14 broad-leaved trees in a species-rich, ca. 30-m-tall forest in NW Switzerland to test whether elevated CO2 reduces water use in mature forest trees. Measurements of sap flux density (JS) were made prior to CO2 enrichment (summer 2000) and throughout the first whole growing season of CO2 exposure (2001) using the constant heat-flow technique. The short-term responses of sap flux to brief (1.5-3 h) interruptions of CO2 enrichment were also examined. There were no significant a priori differences in morphological and physiological traits between trees which were later exposed to elevated CO2 (n=14) and trees later used as controls (n=19). Over the entire growing season, CO2 enrichment resulted in an average 10.7% reduction in mean daily JS across all species compared to control trees. Responses were most pronounced in Carpinus, Acer, Prunus and Tilia, smaller in Quercus and close to zero in Fagus trees. The JS of treated trees significantly increased by 7% upon transient exposure to ambient CO2 concentrations at noon. Hence, responses of the different species were, in the short term, similar in magnitude to those observed over the whole season (though opposite because of the reversed treatment). The reductions in mean JS of CO2-enriched trees were high (22%) under conditions of low evaporative demand (vapour pressure deficit, VPD <5 hPa) and small (2%) when mean daily VPD was greater than 10 hPa. During a relatively dry period, the effect of elevated CO2 on JS even appeared to be reversed. These results suggest that daily water savings by CO2-enriched trees may have accumulated to a significantly improved water status by the time when control trees were short of soil moisture. Our data indicate that the magnitude of CO2 effects on stand transpiration will depend on rainfall regimes and the relative abundance of the different species, being more pronounced under humid conditions and in stands dominated by species such as Carpinus and negligible in mono-specific Fagus forests.