Microgravity effects on leaf morphology, cell structure, carbon metabolism and mRNA expression of dwarf wheat.

The use of higher plants as the basis for a biological life support system that regenerates the atmosphere, purifies water, and produces food has been proposed for long duration space missions. The objective of these experiments was to determine what effects microgravity (microg) had on chloroplast development, carbohydrate metabolism and gene expression in developing leaves of Triticum aestivum L. cv. USU Apogee. Gravity naive wheat plants were sampled from a series of seven 21-day experiments conducted during Increment IV of the International Space Station. These samples were fixed in either 3% glutaraldehyde or RNAlater or frozen at -25 degrees C for subsequent analysis. In addition, leaf samples were collected from 24- and 14-day-old plants during the mission that were returned to Earth for analysis. Plants grown under identical light, temperature, relative humidity, photoperiod, CO(2), and planting density were used as ground controls. At the morphological level, there was little difference in the development of cells of wheat under microg conditions. Leaves developed in mug have thinner cross-sectional area than the 1g grown plants. Ultrastructurally, the chloroplasts of microg grown plants were more ovoid than those developed at 1g, and the thylakoid membranes had a trend to greater packing density. No differences were observed in the starch, soluble sugar, or lignin content of the leaves grown in microg or 1g conditions. Furthermore, no differences in gene expression were detected leaf samples collected at microg from 24-day-old leaves, suggesting that the spaceflight environment had minimal impact on wheat metabolism.

Microgravity effects on thylakoid, single leaf, and whole canopy photosynthesis of dwarf wheat.

The concept of using higher plants to maintain a sustainable life support system for humans during long-duration space missions is dependent upon photosynthesis. The effects of extended exposure to microgravity on the development and functioning of photosynthesis at the leaf and stand levels were examined onboard the International Space Station (ISS). The PESTO (Photosynthesis Experiment Systems Testing and Operations) experiment was the first long-term replicated test to obtain direct measurements of canopy photosynthesis from space under well-controlled conditions. The PESTO experiment consisted of a series of 21-24 day growth cycles of Triticum aestivum L. cv. USU Apogee onboard ISS. Single leaf measurements showed no differences in photosynthetic activity at the moderate (up to 600 micromol m(-2) s(-1)) light levels, but reductions in whole chain electron transport, PSII, and PSI activities were measured under saturating light (>2,000 micromol m(-2) s(-1)) and CO(2) (4000 micromol mol(-1)) conditions in the microgravity-grown plants. Canopy level photosynthetic rates of plants developing in microgravity at approximately 280 micromol m(-2) s(-1) were not different from ground controls. The wheat canopy had apparently adapted to the microgravity environment since the CO(2) compensation (121 vs. 118 micromol mol(-1)) and PPF compensation (85 vs. 81 micromol m(-2) s(-1)) of the flight and ground treatments were similar. The reduction in whole chain electron transport (13%), PSII (13%), and PSI (16%) activities observed under saturating light conditions suggests that microgravity-induced responses at the canopy level may occur at higher PPF intensity.

Microgravity does not alter plant stand gas exchange of wheat at moderate light levels and saturating CO2 concentration.

Plant stand gas exchange was measured nondestructively in microgravity during the Photosynthesis Experiment Subsystem Testing and Operations experiment conducted onboard the International Space Station. Rates of evapotranspiration and photosynthesis measured in space were compared with ground controls to determine if microgravity directly affects whole-stand gas exchange of Triticum aestivum. During six 21-day experiment cycles, evapotranspiration was determined continuously from water addition rates to the nutrient delivery system, and photosynthesis was determined from the amount of CO2 added to maintain the chamber CO2 concentration setpoint. Plant stand evapotranspiration, net photosynthesis, and water use efficiency were not altered by microgravity. Although leaf area was significantly reduced in microgravity-grown plants compared to ground control plants, leaf area distribution was not affected enough to cause significant differences in the amounts of light absorbed by the flight and ground control plant stands. Microgravity also did not affect the response of evapotranspiration to changes in chamber vapor pressure difference of 12-day-old wheat plant stands. These results suggest that gravity naïve plants grown at moderate light levels (300 micromol m(-2) s(-1)) behave the same as ground control plants. This implies that future plant-based regenerative life support systems can be sized using 1 g data because water purification and food production rates operate at nearly the same rates as in 1 g at moderate light levels. However, it remains to be verified whether the present results are reproducible in plants grown under stronger light levels.

Induction of hypoxic root metabolism results from physical limitations in O2 bioavailability in microgravity.

Numerous spaceflight experiments have noted changes in the roots that are consistent with hypoxia in the root zone. These observations include general ultrastructure analysis and biochemical measurements to direct measurements of stress specific enzymes. In experiments that have monitored alcohol dehydrogenase (ADH), the data shows this hypoxically responsive gene is induced and is associated with increased ADH activity in microgravity. These changes in ADH could be induced either by spaceflight hypoxia resulting from inhibition of gravity mediated O2 transport, or by a non-specific stress response due to inhibition of gravisensing. We tested these hypotheses in a series of two experiments. The objective of the first experiment was to determine if physical changes in gravity-mediated O2 transport can be directly measured, while the second series of experiments tested whether disruption of gravisensing can induce a non-specific ADH response. To directly measure O2 bioavailability as a function of gravity, we designed a sensor that mimics metabolic oxygen consumption in the rhizosphere. Because of these criteria, the sensor is sensitive to any changes in root O2 bioavailability that may occur in microgravity. In a KC-135 experiment, the sensor was implanted in a moist granular clay media and exposed to microgravity during parabolic flight. The resulting data indicated that root O2 bioavailability decreased in phase with gravity. In experiments that tested for non-specific induction of ADH, we compared the response of transgenic Arabidopsis plants (ADH promoted GUS marker gene) exposed to clinostat, control, and waterlogged conditions. The plants were grown on agar slats in a growth chamber before being exposed to the experimental treatments. The plants were stained for GUS activity localization, and subjected to biochemical tests for ADH, and GUS enzyme activity. These tests showed that the waterlogging treatment induced significant increases in GUS and ADH enzyme activities, while the control and clinostat treatments showed no response. This work demonstrates: (1) the inhibition of gravity-driven convective transport can reduce the O2 bioavailability to the root tip, and (2) the perturbation of gravisensing by clinostat rotation does not induce a nonspecific stress response involving ADH. Together these experiments support the microgravity convection inhibition model for explaining changes in root metabolism during spaceflight.

Farming in space: environmental and biophysical concerns.

The colonization of space will depend on our ability to routinely provide for the metabolic needs (oxygen, water, and food) of a crew with minimal re-supply from Earth. On Earth, these functions are facilitated by the cultivation of plant crops, thus it is important to develop plant-based food production systems to sustain the presence of mankind in space. Farming practices on earth have evolved for thousands of years to meet both the demands of an ever-increasing population and the availability of scarce resources, and now these practices must adapt to accommodate the effects of global warming. Similar challenges are expected when earth-based agricultural practices are adapted for space-based agriculture. A key variable in space is gravity; planets (e.g. Mars, 1/3 g) and moons (e.g. Earth's moon, 1/6 g) differ from spacecraft orbiting the Earth (e.g. Space stations) or orbital transfer vehicles that are subject to microgravity. The movement of heat, water vapor, CO2 and O2 between plant surfaces and their environment is also affected by gravity. In microgravity, these processes may also be affected by reduced mass transport and thicker boundary layers around plant organs caused by the absence of buoyancy dependent convective transport. Future space farmers will have to adapt their practices to accommodate microgravity, high and low extremes in ambient temperatures, reduced atmospheric pressures, atmospheres containing high volatile organic carbon contents, and elevated to super-elevated CO2 concentrations. Farming in space must also be carried out within power-, volume-, and mass-limited life support systems and must share resources with manned crews. Improved lighting and sensor technologies will have to be developed and tested for use in space. These developments should also help make crop production in terrestrial controlled environments (plant growth chambers and greenhouses) more efficient and, therefore, make these alternative agricultural systems more economically feasible food production systems.

Nitrate concentration effects on NO3-N uptake and reduction, growth, and fruit yield in strawberry.

Strawberries (Fragaria xananassa Duch. 'Osogrande') were grown hydroponically with three NO3-N concentrations (3.75, 7.5, or 15.0 mM) to determine effects of varying concentration on NO3-N uptake and reduction rates, and to relate these processes to growth and fruit yield. Plants were grown for 32 weeks, and NO3-N uptake and nitrate reductase (NR) activities in roots and shoots were measured during vegetative and reproductive growth. In general, NO3-N uptake rates increased as NO3-N concentration in the hydroponics system increased. Tissue NO3- concentration also increased as external NO3-N concentration increased, reflecting the differences in uptake rates. There was no effect of external NO3-N concentration on NR activities in leaves or roots during either stage of development. Leaf NR activity averaged approximately 360 nmol NO2 formed/g fresh weight (FW)/h over both developmental stages, while NR activity in roots was much lower, averaging approximately 115 nmol NO2 formed/g FW/h. Vegetative organ FW, dry weight (DW), and total fruit yield were unaffected by NO3-N concentration. These data suggest that the inability of strawberry to increase growth and fruit yield in response to increasing NO3-N concentrations is not due to limitations in NO3-N uptake rates, but rather to limitations in NO3- reduction and/or assimilation in both roots and leaves.

Glycine betaine accumulation, ionic and water relations of red-beet at contrasting levels of sodium supply.

Exposure of plants to sodium (Na) and salinity may increase glycine betaine accumulation in tissues. To study this, red-beet cvs. Scarlet Supreme and Ruby Queen, were grown for 42 days in a growth chamber using a re-circulating nutrient film technique with 0.25 mmol/L K and either 4.75 mmol/L (control) or 54.75 mmol/L (saline) Na (as NaCl). Plants were harvested at weekly intervals and measurements were taken on leaf water relations, leaf photosynthetic rates, chlorophyll fluorescence, chlorophyll levels, glycine betaine levels, and tissue elemental composition. Glycine betaine accumulation increased under salinity and this accumulation correlated with higher tissue levels of Na in both cultivars. Na accounted for 80 to 90% of the total cation uptake under the saline treatment. At final harvest (42 days), K concentrations in laminae ranged from approximately 65-95 micromoles g-1 dry matter (DM), whereas Na in shoot tissue ranged from approximately 3000-4000 micromoles g-1. Leaf sap osmotic potential at full turgor [psi(s100)] increased as lamina Na content increased. Glycine betaine levels of leaf laminae showed a linear relationship with leaf sap [psi(s100)]. Chlorophyll levels, leaf photosynthetic rates, and chlorophyll fluorescence were not affected by Na levels. These results suggest that the metabolic tolerance to high levels of tissue Na in red-beet could be due to its ability to synthesize and regulate glycine betaine production, and to control partitioning of Na and glycine betaine between the vacuole and the cytoplasm.

Low potassium enhances sodium uptake in red-beet under moderate saline conditions.

Due to the discrepancy in metabolic sodium (Na) requirements between plants and animals, cycling of Na between humans and plants is limited and critical to the proper functioning of bio-regenerative life support systems, being considered for long-term human habitats in space (e.g., Martian bases). This study was conducted to determine the effects of limited potassium (K) on growth, Na uptake, photosynthesis, ionic partitioning, and water relations of red-beet (Beta vulgaris L. ssp. vulgaris) under moderate Na-saline conditions. Two cultivars, Klein Bol, and Ruby Queen were grown for 42 days in a growth chamber using a re-circulating nutrient film technique where the supplied K levels were 5.0, 1.25, 0.25, and 0.10 mM in a modified half-strength Hoagland solution salinized with 50 mM NaCl. Reducing K levels from 5.0 to 0.10 mM quadrupled the Na uptake, and lamina Na levels reached -20 g kg-1 dwt. Lamina K levels decreased from -60 g kg-1 dwt at 5.0 mM K to -4.0 g kg-1 dwt at 0.10 mM K. Ruby Queen and Klein Bol responded differently to these changes in Na and K status. Klein Bol showed a linear decline in dry matter production with a decrease in available K, whereas for cv. Ruby Queen, growth was stimulated at 1.25 mM K and relatively insensitive to a further decreases of K down to 0.10 mM. Leaf glycinebetaine levels showed no significant response to the changing K treatments. Leaf relative water content and osmotic potential were significantly higher for both cultivars at low-K treatments. Leaf chlorophyll levels were significantly decreased at low-K treatments, but leaf photosynthetic rates showed no significant difference. No substantial changes were observed in the total cation concentration of plant tissues despite major shifts in the relative Na and K uptake at various K levels. Sodium accounted for 90% of the total cation uptake at the low K levels, and thus Na was likely replacing K in osmotic functions without negatively affecting the plant water status, or growth. Our results also suggest that cv. Ruby Queen can tolerate a much higher Na tissue concentration than cv. Klein Bol before there is any growth reduction. Grant numbers: 12180.

Feasibility of substituting sodium for potassium in crop plants for advanced life support systems.

Recycling of nutrients, air, and water is an integral feature of life support systems designed for long-term space missions. Plants can play a major role in supplying the basic life support requirements, which include providing the crew's food, clean water, and air, and recycling their wastes. The nutrient flux through the plant and human systems needs to be matched in order for nutrients to recycle between humans and plants without an excessive buildup in any one section of the system. Sodium, which is essential at the macronutrient level for human metabolism, has only been shown to be a micronutrient for some plants, with only very limited uptake in most plants. Thus, when Na is added from the outside to meet the human demand in these closed life support systems it will accumulate someplace in the overall system. In simple systems such as these, without a complete biogeological cycle, the buildup of Na could occur in the nutrient solution of the plant system. Various concepts related to the substitution of sodium for potassium in crop plants are currently being investigated by NASA. Results to date suggest that Na concentrations up to 100 g kg-1 dry weight may be achievable in the edible portions of Na-tolerant crops (e.g., red beet and chard). A flow path for nutrient solution high in Na wastes has been suggested for optimizing Na and nitrogen incorporation and utilization from such solutions. Options for further improvements include selecting plant genotypes tolerant to high salinity, which are efficient in Na uptake. This should also be combined with environmental manipulations to maximize Na uptake by crop plants.

Computer model of hydroponics nutrient solution pH control using ammonium.

A computer simulation of a hydroponics-based plant growth chamber using ammonium to control pH was constructed to determine the feasibility of such a system. In nitrate-based recirculating hydroponics systems, the pH will increase as plants release hydroxide ions into the nutrient solution to maintain plant charge balance. Ammonium is an attractive alternative to traditional pH controls in an ALSS, but requires careful monitoring and control to avoid overdosing the plants with ammonium. The primary advantage of using NH4+ for pH control is that it exploits the existing plant nutrient uptake charge balance mechanisms to maintain solution pH. The simulation models growth, nitrogen uptake, and pH of a l-m2 stand of wheat. Simulation results indicated that ammonium-based control of nutrient solution pH is feasible using a proportional integral controller. Use of a 1 mmol/L buffer (Ka = 1.6 x 10(-6)) in the nutrient solution is required.

How far can sodium substitute for potassium in red beet?

Sodium (Na) movement between plants and humans is one of the more critical aspects of bioregenerative systems of life support, which NASA is studying for the establishment of long-term bases on the Lunar or Martian surface. This study was conducted to determine the extent to which Na can replace potassium (K) in red beet (Beta vulgaris L. ssp vulgaris) without adversely affecting metabolic functions such as water relations, photosynthetic rates, and thus growth. Two cultivars, Ruby Queen and Klein Bol, were grown for 42 days at 1200 micromoles mol-1 CO2 in a growth chamber using a re-circulating nutrient film technique with 0%, 75%, 95%, and 98% Na substitution for K in a modified half-strength Hoagland solution. Total biomass of Ruby Queen was greatest at 95% Na substitution and equal at 0% and 98% Na substitution. For Klein Bol, there was a 75% reduction in total biomass at 98% Na substitution. Nearly 95% of the total plant K was replaced with Na at 98% Na substitution in both cultivars. Potassium concentrations in leaves decreased from 120 g kg-1 dwt in 0% Na substitution to 3.5 g kg-1 dwt at 98% Na substitution. Leaf chlorophyll concentration, photosynthetic rate, and osmotic potential were not affected in either cultivar by Na substitution for K. Leaf glycinebetaine levels were doubled at 75% Na substitution in Klein Bol, but decreased at higher levels of Na substitution. For Ruby Queen, glycinebetaine levels in leaf increased with the first increase of Na levels and were maintained at the higher Na levels. These results indicate that in some cultivars of red beet, 95% of the normal tissue K can be replaced by Na without a reduction in growth.

Modeling wheat harvest index as a function of date of anthesis.

A plant growth model developed by Volk and colleagues was modified to partition plant mass production after anthesis into grain and inedible biomass. Using data on wheat (Triticum aestivum) grown in the NASA CELSS Biomass Production Chamber to supply constants for the model, we showed that delaying the date of anthesis 7 days resulted in a 20% decrease in the harvest index. Multiple model components were then assembled to demonstrate the effect of an environmental system failure.

Theoretical and practical considerations of staggered crop production in a BLSS.

A functional Bioregenerative Life Support System (BLSS) will generate oxygen, remove excess carbon dioxide, purify water, and produce food on a continuous basis for long periods of operation. In order to minimize fluctuations in gas exchange, water purification, and yield that are inherent in batch systems, staggered planting and harvesting of the crop is desirable. A 418-day test of staggered production of potato cv. Norland (26-day harvest cycles) using nutrients recovered from inedible biomass was conducted at Kennedy Space Center. The results indicate that staggered production can be sustained without detrimental effects on BLSS life support functions. System yields of H2O, O2 and food were higher in staggered than batch plantings. Plants growing in staggered production or batch production on "aged" solution initiated tubers earlier, and were shorter than plants grown on "fresh" solution. This morphological response required an increase in planting density to maintain full canopy coverage. Plants grown in staggered production used available light more efficiently than the batch planting due to increased side lighting.

Blue light requirements for crop plants used in bioregenerative life support systems.

As part of NASA's Advanced Life Support Program, the Breadboard Project at Kennedy Space Center is investigating the feasibility of using crop plants in bioregenerative life support systems (BLSS) for long-duration space missions. Several types of electric lamps have been tested to provide radiant energy for plants in a BLSS. These lamps vary greatly in terms of spectral quality resulting in differences in growth and morphology of the plants tested. Broad spectrum or "white" light sources (e.g., metal halide and fluorescent lamps) provide an adequate spectrum for normal growth and morphology; however, they are not as electrically efficient as are low-pressure sodium (LPS) or high-pressure sodium (HPS) lamps. Although LPS and HPS, as well as the newly tested red light-emitting diodes (LEDs), have good photosynthetically active radiation (PAR) efficiencies, they are deficient in blue light. Results with several of the crops tested for BLSS (wheat, potato, soybean, lettuce, and radish) have shown a minimum amount of blue light (approximately 30 micromoles m-2 s-1) is necessary for normal growth and development. For example, the lack of sufficient blue light in these lamps has resulted in increased stem elongation and significant reductions in photosynthesis and yield. To avoid problems with blue-deficient lamps and maximize yield, sufficient intensity of HPS or blue light supplementation with red LEDs or LPS lamps is required to meet spectral requirements of crops for BLSS.

A recirculating hydroponic system for studying peanut (Arachis hypogaea L.).

Peanut (Arachis hypogaea L.) plants were grown hydroponically, using continuously recirculating nutrient solution. Two culture tray designs were tested; one tray design used only nutrient solution, while the other used a sphagnum-filled pod development compartment just beneath the cover and above the nutrient solution. Both trays were fitted with slotted covers to allow developing gynophores to reach the root zone. Peanut seed yields averaged 350 gm-2 dry mass, regardless of tray design, suggesting that substrate is not required for hydroponic peanut production.

A modified CROPGRO model for simulating soybean growth in controlled environments.

The CROPGRO crop growth model is adapted in order to analyze experimental data from a soybean (cv. Hoyt) experiment conducted at elevated CO2 levels (1200 micromol mol-1) at Kennedy Space Center, FL. The following adaptations to original CROPGRO produced model agreement with gas-exchange data: the input of square-wave temperature and photosynthetically active radiation (PAR) profiles; the input of the appropriate hydroponic substrate PAR albedo; modified biomass partitioning and developmental parameters; an increased leaf area expansion rate through the fifth vegetative node; a decreased specific leaf area after the fifth vegetative node; and an increased incident diffuse PAR fraction over typical field values. The model demonstrated here suggests that with continued development, modified CROPGRO will be a useful tool in the analysis and eventual optimization of legume production in bioregenerative life support systems.

Use of biologically reclaimed minerals for continuous hydroponic potato production in a CELSS.

Plant-derived nutrients were successfully recycled in a Controlled Ecological Life Support System (CELSS) using biological methods. The majority of the essential nutrients were recovered by microbiologically treating the plant biomass in an aerobic bioreactor. Liquid effluent containing the nutrients was then returned to the biomass production component via a recirculating hydroponic system. Potato (Solanum tuberosum L.) cv. Norland plants were grown on those nutrients in either a batch production mode (same age plants on a nutrient solution) or a staggered production mode (4 different ages of plants on a nutrient solution). The study continued over a period of 418 days, within NASA Breadboard Project's Biomass Production Chamber at the Kennedy Space Center. During this period, four consecutive batch cycles (104-day harvests) and 13 consecutive staggered cycles (26-day harvests) were completed using reclaimed minerals and compared to plants grown with standard nutrient solutions. All nutrient solutions were continually recirculated during the entire 418 day study. In general, tuber yields with reclaimed minerals were within 10% of control solutions. Contaminants, such as sodium and recalcitrant organics tended to increase over time in solutions containing reclaimed minerals, however tuber composition was comparable to tubers grown in the control solutions.

Theoretical and practical considerations for staggered production of crops in a BLSS.

A functional Bioregenerative Life Support System (BLSS) will generate oxygen, remove excess carbon dioxide, purify water, and produce food on a continuous basis for long periods of operation. In order to minimize fluctuations in gas exchange, water purification, and yield that are inherent in batch systems, staggered planting and harvesting of the crop is desirable. A 418-d test of staggered production of potato cv. Norland (26-d harvest cycles) using nutrients recovered from inedible biomass was recently completed at Kennedy Space Center. The results indicate that staggered production can be sustained without detrimental effects on life support functions in a CELSS. System yields of H2O, O2 and food were higher in staggered than batch plantings. Plants growing in staggered production or batch production on "aged" solution initiated tubers earlier, and were shorter than plants grown on "fresh" solution. This morphological response required an increase in planting density to maintain full canopy coverage. Plants grown in staggered production used available light more efficiently than the batch planting due to increased sidelighting.

Accumulation and effect of volatile organic compounds in closed life support systems.

Bioregenerative life support systems (BLSS) being considered for long duration space missions will operate with limited resupply and utilize biological systems to revitalize the atmosphere, purify water, and produce food. The presence of man-made materials, plant and microbial communities, and human activities will result in the production of volatile organic compounds (VOCs). A database of VOC production from potential BLSS crops is being developed by the Breadboard Project at Kennedy Space Center. Most research to date has focused on the development of air revitalization systems that minimize the concentration of atmospheric contaminants in a closed environment. Similar approaches are being pursued in the design of atmospheric revitalization systems in bioregenerative life support systems. in a BLSS one must consider the effect of VOC concentration on the performance of plants being used for water and atmospheric purification processes. In addition to phytotoxic responses, the impact of removing biogenic compounds from the atmosphere on BLSS function needs to be assessed. This paper provides a synopsis of criteria for setting exposure limits, gives an overview of existing information, and discusses production of biogenic compounds from plants grown in the Biomass Production Chamber at Kennedy Space Center.

Effect of elevated carbon dioxide on nutritional quality of tomato.

Tomato (Lycopersicon esculentum Mill.) cvs. Red Robin (RR) and Reimann Philipp (RP) were grown hydroponically for 105 d with a 12 h photoperiod, 26 degrees C/22 degrees C thermoperiod, and 500 micromol m-2 s-1 PPF at either 400, 1200, 5000, or 10,000 micromol mol-1 (0.04, 0.12, 0.50, 1.00 kPa) CO2. Harvested fruits were analyzed for proximate composition, total dietary fiber, nitrate, and elemental composition. No trends were apparent with regard to CO2 effects on proximate composition, with fruit from all treatments and both cultivars averaging 18.9% protein, 3.6% fat, 10.2% ash, and 67.2% carbohydrate. In comparison, average values for field-grown fruit are 16.6% protein, 3.8% fat, 8.1% ash, and 71.5% carbohydrate (Duke and Atchely, 1986). Total dietary fiber was highest at 10,000 micromol mol-1 (28.4% and 22.6% for RR and RP) and lowest at 1000 micromol mol-1 (18.2% and 15.9% for RR and RP), but showed no overall trend in response to CO2. Nitrate values ranged from 0.19% to 0.35% and showed no trend with regard to CO2. K, Mg, and P concentrations showed no trend in response to CO2, but Ca levels increased from 198 and 956 ppm in RR and RP at 400 micromol mol-1, to 2537 and 2825 ppm at 10,000 micromol mol-1. This increase in Ca caused an increase in fruit Ca/P ratios from 0.07 and 0.37 for RR and RP at 400 micromol mol-1 to 0.99 and 1.23 for RR and RP at 10,000 micromol mol-1, suggesting that more dietary Ca should be available from high CO2-grown fruit.