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.

Canopy photosynthesis and transpiration in microgravity: gas exchange measurements aboard Mir.

The SVET Greenhouse on-board the Orbital Station Mir was used to measure canopy photosynthesis and transpiration rates for the first time in space. During the Greenhouse IIB experiment on Mir (June-January 1997), carbon and water vapor fluxes from two wheat (cv. Superdwarf) canopies were measured using the US developed Gas Exchange Measurement System (GEMS). Gas analyzers capable of resolving CO2 concentration differences of 5 micromoles mol-1 against a background of 0.9% CO2, are necessary to measure photosynthetic and respiratory rates on Mir. The ability of the GEMS gas analyzers to measure these CO2 concentration differences was determined during extensive ground calibrations. Similarly, the sensitivity of the analyzers to water vapor was sufficient to accurately measure canopy evapotranspiration. Evapotranspiration, which accounted for over 90% of the water added to the root zone, was estimated using gas exchange and used to estimate substrate moisture content. This paper presents canopy photosynthesis and transpiration data during the peak vegetative phase of development in microgravity.

Evaluation and modification of commercial infra-red transducers for leaf temperature measurement.

Accurate measurement of the leaf to air temperature gradient is crucial for the determination of stomatal conductance and other plant responses in both single leaves and in plant canopies. This gradient is often less than 1 degree C, which means that leaf temperature must be known to within about +/- 0.1 degree C. This is a challenging task, but new, miniature infra-red transducers from Exergen Corporation (Newton, MA) and Everest Interscience (Tucson, AZ) can be modified and calibrated to achieve this accuracy. The sensors must be modified to add thermal mass and the Exergen sensor requires a measurement of sensor body temperature. Significant error is caused by the discharge of a capacitor in the standard Exergen sensor, but we tested it without the capacitor. The sensors respond rapidly to changes in target temperature, but require 2 to 10 minutes to respond to changes in sensor body temperature, which is often the largest source of error. A new, sensitive method for measuring field of view indicates substantial peripheral vision for both sensors and a wider field of view than specified by the manufacturers. Here we describe sensor output as a function of target and sensor body temperatures, and provide a generic (sensor independent) equation that can be used to achieve +/- 0.2 C accuracy with Exergen sensors. The equation was developed and verified using two black body calibrators.

Adaptation to high CO2 concentration in an optimal environment: radiation capture, canopy quantum yield and carbon use efficiency.

The effect of elevated [CO2] on wheat (Triticum aestivum L. Veery 10) productivity was examined by analysing radiation capture, canopy quantum yield, canopy carbon use efficiency, harvest index and daily C gain. Canopies were grown at either 330 or 1200 micromoles mol-1 [CO2] in controlled environments, where root and shoot C fluxes were monitored continuously from emergence to harvest. A rapidly circulating hydroponic solution supplied nutrients, water and root zone oxygen. At harvest, dry mass predicted from gas exchange data was 102.8 +/- 4.7% of the observed dry mass in six trials. Neither radiation capture efficiency nor carbon use efficiency were affected by elevated [CO2], but yield increased by 13% due to a sustained increase in canopy quantum yield. CO2 enrichment increased root mass, tiller number and seed mass. Harvest index and chlorophyll concentration were unchanged, but CO2 enrichment increased average life cycle net photosynthesis (13%, P < 0.05) and root respiration (24%, P < 0.05). These data indicate that plant communities adapt to CO2 enrichment through changes in C allocation. Elevated [CO2] increases sink strength in optimal environments, resulting in sustained increases in photosynthetic capacity, canopy quantum yield and daily C gain throughout the life cycle.

Characterizing photosynthesis and transpiration of plant communities in controlled environments.

CO2 and water vapor fluxes of hydroponically grown wheat and soybean canopies were measured continuously in several environments with an open gas exchange system. Canopy CO2 fluxes reflect the photosynthetic efficiency of a plant community, and provide a record of plant growth and health. There were significant diurnal fluctuations in root and shoot CO2 fluxes, and in shoot water vapor fluxes. Canopy stomatal conductance (Gc) to water vapor was calculated from simultaneous measurements of canopy temperature (Tcan) and transpiration rates (Tr). Tr in the dark was substantial, and there were large diurnal fluctuations in both Gc and Tr. Canopy net Photosynthesis (Pnet), Tr, and Gc increased with increasing net radiation. Gc increased with Tr, suggesting that the stomata of plants in controlled environments (CEs) behave differently from field-grown plants. A transpiration model based on measurements of Gc was developed for CEs. The model accurately predicted Tr from a soybean canopy.

Quantifying energy and mass transfer in crop canopies: sensors for measurement of temperature and air velocity.

Here we report on the in situ performance of inexpensive, miniature sensors that have increased our ability to measure mass and energy fluxes from plant canopies in controlled environments: 1. Surface temperature. Canopy temperature measurements indicate changes in stomatal aperture and thus latent and sensible heat fluxes. Infrared transducers from two manufacturers (Exergen Corporation, Newton, MA; and Everest Interscience, Tucson, AZ, USA) have recently become available. Transducer accuracy matched that of a more expensive hand-held infrared thermometer. 2. Air velocity varies above and within plant canopies and is an important component in mass and energy transfer models. We tested commercially-available needle, heat-transfer anemometers (1 x 50 mm cylinder) that consist of a fine-wire thermocouple and a heater inside a hypodermic needle. The needle is heated and wind speed determined from the temperature rise above ambient. These sensors are particularly useful in measuring the low wind speeds found within plant canopies. 3. Accurate measurements of air temperature adjacent to plant leaves facilitates transport phenomena modeling. We quantified the effect of radiation and air velocity on temperature rise in thermocouples from 10 to 500 micrometers. At high radiation loads and low wind speeds, temperature errors were as large as 7 degrees C above air temperature.

CO2 crop growth enhancement and toxicity in wheat and rice.

The effects of elevated CO2 on plant growth are reviewed and the implications for crop yields in regenerative systems are discussed. There is considerable theoretical and experimental evidence indicating that the beneficial effects of CO2 are saturated at about 0.12% CO2 in air. However, CO2 can easily rise above 1% of the total gas in a closed system, and we have thus studied continuous exposure to CO2 levels as high as 2%. Elevating CO2 from 340 to 1200 micromoles mol-1 can increase the seed yield of wheat and rice by 30 to 40%; unfortunately, further CO2 elevation to 2500 micromoles mol-1 (0.25%) has consistently reduced yield by 25% compared to plants grown at 1200 micromoles mol-1; fortunately, there was only an additional 10% decrease in yield as the CO2 level was further elevated to 2% (20,000 micromoles mol-1). Yield increases in both rice and wheat were primarily the result of increased number of heads per m2, with minor effects on seed number per head and seed size. Yield increases were greatest in the highest photosynthetic photon flux. We used photosynthetic gas exchange to analyze CO2 effects on radiation interception, canopy quantum yield, and canopy carbon use efficiency. We were surprised to find that radiation interception during early growth was not improved by elevated CO2. As expected, CO2 increased quantum yield, but there was also a small increase in carbon use efficiency. Super-optimal CO2 levels did not reduce vegetative growth, but decreased seed set and thus yield. The reduced seed set is not visually apparent until final yield is measured. The physiological mechanism underlying CO2 toxicity is not yet known, but elevated CO2 levels (0.1 to 1% CO2) increase ethylene synthesis in some plants and ethylene is a potent inhibitor of seed set in wheat.

The limits of crop productivity: validating theoretical estimates and determining the factors that limit crop yields in optimal environments.

Plant scientists have sought to maximize the yield of food crops since the beginning of agriculture. There are numerous reports of record food and biomass yields (per unit area) in all major crop plants, but many of the record yield reports are in error because they exceed the maximal theoretical rates of the component processes. In this article, we review the component processes that govern yield limits and describe how each process can be individually measured. This procedure has helped us validate theoretical estimates and determine what factors limit yields in optimal environments.

Inherent limitations of nondestructive chlorophyll meters: a comparison of two types of meters.

Two types of nondestructive chlorophyll meters were compared with a standard, destructive chlorophyll measurement technique. The nondestructive chlorophyll meters were 1) a custom built, single-wavelength meter, and 2) the recently introduced, dual-wavelengh, chlorophyll meter from Minolta (model SPAD-502). Data from both meters were closely correlated with destructive measurements of chlorophyll (r2 = 0.90 and 0.93; respectively) for leaves with chlorophyll concentrations ranging from 100 to 600 mg m-2, but both meters consistently overestimated chlorophyll outside this range. Although the dual-wavelength meter was slightly more accurate than the single-wavelength meter (higher r2), the light-scattering properties of leaf cells and the nonhomogeneous distribution of chlorophyll in leaves appear to limit the ability of all meters to estimate in vivo chlorophyll concentration.