Ground Control Plants Research Articles

Spaceflight impacts xyloglucan oligosaccharide abundance in Arabidopsis thaliana root cell walls

Over the course of more than a decade, space biology investigations have consistently indicated that cell wall remodeling occurs in a variety of spaceflight-grown plants. Here, we describe a mass spectrometric method to study the fundamental composition of xyloglucan, the most abundant hemicellulose in dicot cell walls, in space-grown plants. Four representative Arabidopsis root samples, from a previously conducted spaceflight experiment - Advanced Plant EXperiment - 04 (APEX-04), were used to investigate changes in xyloglucan oligosaccharides abundances in spaceflight-grown plants compared to ground controls. In situ localized enzymatic digestions and surface sampling mass spectrometry analysis provided spatial resolution of the changes in xyloglucan oligosaccharides abundances. Overall, the results showed that oligosaccharide XXLG/XLXG and XXFG branching patterns were more abundant in the lateral roots of spaceflight-grown plants, while XXXG, XLFG, and XLFG/XLFG were more abundant in the lateral roots of ground control plants. In the primary roots, XXFG had a higher abundance in ground controls than in spaceflight plants. This methodology of analyzing the basic components of the cell wall in this paper highlights two important findings. First, that are differences in the composition of xyloglucan oligosaccharides in spaceflight root cell walls compared to ground controls and, second, most of these differences are observed in the lateral roots. Thus, the methodology described in this paper provides insights into spaceflight cell wall modifications for future investigations.

Photosynthetic Performance of Rice Seedlings Originated from Seeds Exposed to Spaceflight Conditions.

The mechanism of the regulation on photosynthesis after spaceflight has not been fully understood. To learn more information about this, we conducted a series of experiments of photosystem, including photosynthetic physiological characteristics (fluorescence parameters, pigment contents), gene expression and proteomic change. We want to examine the response of rice (Oryza sativaDN416), whose seeds were placed in Bio-Radiation Boxon the ShiJian-10(SJ-10) recoverable satellite. Our results demonstrated that the photosynthesis capacity of plants after spaceflight declined, compared to ground control plants. Specifically, Fv/Fm is significantly reduced for 7.5%. Chlorophyll content decreased in the three growth stages of rice, trefoil, tillering and mature stages. To further analyze changes under spaceflight environment, quantitative real-time PCR technology and isobaric tags for relative and absolute quantization (iTRAQ) labeling technology were deployed. We found that the gene expression of important subunits of key enzymes and important structures had been decreased after spaceflight. As for the results of changes in proteins, we discovered that the content of proteins related to electron transport and photosynthesis key enzyme declined. Our experiments can provide reference for further research to learn more about the effects of spaceflight on photosynthesis.

Comparative transcriptomics indicate changes in cell wall organization and stress response in seedlings during spaceflight.

Plants will play an important role in the future of space exploration as part of bioregenerative life support. Thus, it is important to understand the effects of microgravity and spaceflight on gene expression in plant development. We analyzed the transcriptome of Arabidopsis thaliana using the Biological Research in Canisters (BRIC) hardware during Space Shuttle mission STS-131. The bioinformatics methods used included RMA (robust multi-array average), MAS5 (Microarray Suite 5.0), and PLIER (probe logarithmic intensity error estimation). Glycome profiling was used to analyze cell wall composition in the samples. In addition, our results were compared to those of two other groups using the same hardware on the same mission (BRIC-16). In our BRIC-16 experiments, we noted expression changes in genes involved in hypoxia and heat shock responses, DNA repair, and cell wall structure between spaceflight samples compared to the ground controls. In addition, glycome profiling supported our expression analyses in that there was a difference in cell wall components between ground control and spaceflight-grown plants. Comparing our studies to those of the other BRIC-16 experiments demonstrated that, even with the same hardware and similar biological materials, differences in results in gene expression were found among these spaceflight experiments. A common theme from our BRIC-16 space experiments and those of the other two groups was the downregulation of water stress response genes in spaceflight. In addition, all three studies found differential regulation of genes associated with cell wall remodeling and stress responses between spaceflight-grown and ground control plants.

The effect of spaceflight on the gravity-sensing auxin gradient of roots: GFP reporter gene microscopy on orbit.

Our primary aim was to determine whether gravity has a direct role in establishing the auxin-mediated gravity-sensing system in primary roots. Major plant architectures have long been thought to be guided by gravity, including the directional growth of the primary root via auxin gradients that are then disturbed when roots deviate from the vertical as a gravity sensor. However, experiments on the International Space Station (ISS) now allow physical clarity with regard to any assumptions regarding the role of gravity in establishing fundamental root auxin distributions. We examined the spaceflight green fluorescent protein (GFP)-reporter gene expression in roots of transgenic lines of Arabidopsis thaliana: pDR5r::GFP, pTAA1::TAA1–GFP, pSCR::SCR–GFP to monitor auxin and pARR5::GFP to monitor cytokinin. Plants on the ISS were imaged live with the Light Microscopy Module (LMM), and compared with control plants imaged on the ground. Preserved spaceflight and ground control plants were examined post flight with confocal microscopy. Plants on orbit, growing in the absence of any physical reference to the terrestrial gravity vector, displayed typically “vertical” distribution of auxin in the primary root. This confirms that the establishment of the auxin-gradient system, the primary guide for gravity signaling in the root, is gravity independent. The cytokinin distribution in the root tip differs between spaceflight and the ground controls, suggesting spaceflight-induced features of root growth may be cytokinin related. The distribution of auxin in the gravity-sensing portion of the root is not dependent on gravity. Spaceflight appears benign to auxin and its role in the development of the primary root tip, whereas spaceflight may influence cytokinin-associated processes.

Plant growth strategies are remodeled by spaceflight

BackgroundArabidopsis plants were grown on the International Space Station within specialized hardware that combined a plant growth habitat with a camera system that can capture images at regular intervals of growth. The Imaging hardware delivers telemetric data from the ISS, specifically images received in real-time from experiments on orbit, providing science without sample return. Comparable Ground Controls were grown in a sister unit that is maintained in the Orbital Environment Simulator at Kennedy Space Center. One of many types of biological data that can be analyzed in this fashion is root morphology. Arabidopsis seeds were geminated on orbit on nutrient gel Petri plates in a configuration that encouraged growth along the surface of the gel. Photos were taken every six hours for the 15 days of the experiment.ResultsIn the absence of gravity, but the presence of directional light, spaceflight roots remained strongly negatively phototropic and grew in the opposite direction of the shoot growth; however, cultivars WS and Col-0 displayed two distinct, marked differences in their growth patterns. First, cultivar WS skewed strongly to the right on orbit, while cultivar Col-0 grew with little deviation away from the light source. Second, the Spaceflight environment also impacted the rate of growth in Arabidopsis. The size of the Flight plants (as measured by primary root and hypocotyl length) was uniformly smaller than comparably aged Ground Control plants in both cultivars.ConclusionsSkewing and waving, thought to be gravity dependent phenomena, occur in spaceflight plants. In the presence of an orienting light source, phenotypic trends in skewing are gravity independent, and the general patterns of directional root growth typified by a given genotype in unit gravity are recapitulated on orbit, although overall growth patterns on orbit are less uniform. Skewing appears independent of axial orientation on the ISS – suggesting that other tropisms (such as for oxygen and temperature) do not influence skewing. An aspect of the spaceflight environment also retards the rate of early Arabidopsis growth.

Spaceflight effects on consecutive generations of peas grown onboard the Russian segment of the International Space Station

In the period from March 2003 to April 2005 we fulfilled five experimental cultivations of genetically marked dwarf pea species in greenhouse Lada installed in the Russian segment (RS) of the International Space Station (ISS). The purpose of this series of experiments was to make morphologic and genetic analysis of pea plants grown in successive generations. According to our results, pea growth and development over the full cycle of ontogenesis (from seed to seed) taking place in space greenhouse Lada were not different as compared with the ground control plants. In addition, four successive pea crops gathered in space flight did not loose their reproductive functions and formed viable seeds. Genetic analysis of the plants grown from the “space” and “ground” seeds produced by the first to fourth successive crops was performed using the methods of chromosomal aberrations count and Random Amplified Polymorphic DNA (molecular method). No genetic polymorphism was found either in the experimental or control crops. This can serve as a sound argument for the supposition that the genetic apparatus of plants is not impacted by exposure of several successive generations to the conditions of space flight.

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.

Dynamics of Storage Reserve Deposition during Brassica rapa L. Pollen and Seed Development in Microgravity

Pollen and seeds share a developmental sequence characterized by intense metabolic activity during reserve deposition before drying to a cryptobiotic form. Neither pollen nor seed development has been well studied in the absence of gravity, despite the importance of these structures in supporting future long-duration manned habitation away from Earth. Using immature seeds (3-15 d postpollination) of Brassica rapa L. cv. Astroplants produced on the STS-87 flight of the space shuttle Columbia, we compared the progress of storage reserve deposition in cotyledon cells during early stages of seed development. Brassica pollen development was studied in flowers produced on plants grown entirely in microgravity on the Mir space station and fixed while on orbit. Cytochemical localization of storage reserves showed differences in starch accumulation between spaceflight and ground control plants in interior layers of the developing seed coat as early as 9 d after pollination. At this age, the embryo is in the cotyledon elongation stage, and there are numerous starch grains in the cotyledon cells in both flight and ground control seeds. In the spaceflight seeds, starch was retained after this stage, while starch grains decreased in size in the ground control seeds. Large and well-developed protein bodies were observed in cotyledon cells of ground control seeds at 15 d postpollination, but their development was delayed in the seeds produced during spaceflight. Like the developing cotyledonary tissues, cells of the anther wall and filaments from the spaceflight plants contained numerous large starch grains, while these were rarely seen in the ground controls. The tapetum remained swollen and persisted to a later developmental stage in the spaceflight plants than in the ground controls, even though most pollen grains appeared normal. These developmental markers indicate that Brassica seeds and pollen produced in microgravity were physiologically younger than those produced in 1 g. We hypothesize that microgravity limits mixing of the gaseous microenvironments inside the closed tissues and that the resulting gas composition surrounding the seeds and pollen retards their development.

Arabidopsis gene expression patterns are altered during spaceflight

The exposure of Arabidopsis thaliana (Arabidopsis) plants to spaceflight environments results in differential gene expression. A 5-day mission on orbiter Columbia in 1999 (STS-93) carried transgenic Arabidopsis plants engineered with a transgene composed of the alcohol dehydrogenase (Adh) gene promoter linked to the β-Glucuronidase (GUS) reporter gene. The plants were used to evaluate the effects of spaceflight on gene expression patterns initially by using the Adh/GUS transgene to address specifically the possibility that spaceflight induces a hypoxic stress response (Paul, A.L., Daugherty, C.J., Bihn, E.A., Chapman, D.K., Norwood, K.L., Ferl, R.J., 2001. Transgene expression patterns indicate that spaceflight affects stress signal perception and transduction in arabidopsis, Plant Physiol. 126, 613–621). As a follow-on to the reporter gene analysis, we report here the evaluation of genome-wide patterns of native gene expression within Arabidopsis shoots utilizing the Agilent DNA array of 21,000 Arabidopsis genes. As a control for the veracity of the array analyses, a selection of genes was further characterized with quantitative Real-Time RT PCR (ABI - Taqman®). Comparison of the patterns of expression for arrays probed with RNA isolated from plants exposed to spaceflight compared to RNA isolated from ground control plants revealed 182 genes that were differentially expressed in response to the spaceflight mission by more than 4-fold, and of those only 50 genes were expressed at levels chosen to support a conservative change call. None of the genes that are hallmarks of hypoxic stress were induced to this level. However, genes related to heat shock were dramatically induced – but in a pattern and under growth conditions that are not easily explained by elevated temperatures. These gene expression data are discussed in light of current models for plant responses to the spaceflight environment and with regard to potential future spaceflight experiment opportunities.

Gravity independence of seed-to-seed cycling in Brassica rapa.

Growth of higher plants in the microgravity environment of orbital platforms has been problematic. Plants typically developed more slowly in space and often failed at the reproductive phase. Short-duration experiments on the Space Shuttle showed that early stages in the reproductive process could occur normally in microgravity, so we sought a long-duration opportunity to test gravity's role throughout the complete life cycle. During a 122-d opportunity on the Mir space station, full life cycles were completed in microgravity with Brassica rapa L. in a series of three experiments in the Svet greenhouse. Plant material was preserved in space by chemical fixation, freezing, and drying, and then compared to material preserved in the same way during a high-fidelity ground control. At sampling times 13 d after planting, plants on Mir were the same size and had the same number of flower buds as ground control plants. Following hand-pollination of the flowers by the astronaut, siliques formed. In microgravity, siliques ripened basipetally and contained smaller seeds with less than 20% of the cotyledon cells found in the seeds harvested from the ground control. Cytochemical localization of storage reserves in the mature embryos showed that starch was retained in the spaceflight material, whereas protein and lipid were the primary storage reserves in the ground control seeds. While these successful seed-to-seed cycles show that gravity is not absolutely required for any step in the plant life cycle, seed quality in Brassica is compromised by development in microgravity.

Plant reproduction during spaceflight: importance of the gaseous environment.

Plant reproduction is a complex developmental process likely to be disrupted by the unusual environmental conditions in orbital spacecraft. Previous results, reviewed herein, indicated difficulties in obtaining successful seen production in orbit, often relating to delayed plant development during the long-term growth necessary for a complete plant life cycle. Using short-duration exposure to spaceflight, we studied plant reproduction in Arabidopsis thaliana (L.) Heynh, during three flight experiments: CHROMEX-03 on STS-54 (6 d), CHROMEX-04 on STS-51 (10 d), and CHROMEX-05 on STS-68 (11 d). Plants were 13 - 14 d old (rosettes) at time of launch and initiated flowering shoots while in orbit. Plants were retrieved from the orbiters 2 - 3 h after landing and reproductive material was immediately processed for in-vivo observations of pollen viability, pollen tube growth, and esterase activity in the stigma, or fixed for later microscopy. Plants produced equal numbers of flowers to those controls growing on the ground but required special environmental conditions to permit fertilization and early seed development during spaceflight. In CHROMEX-03, plants were grown in closed plant growth chambers (PGCs), and male and female gametophyte development aborted at an early stage in the flight material. In CHROMEX-04, carbon dioxide enrichment was provided to the closed PGCs and reproductive development proceeded normally until the pollination stage, when there was an obstacle to pollen transfer in the spaceflight material. In CHROMEX-05, an air-exchange system was used to provide a slow purging of the PGCs with filtered cabin air. Under these conditions, the spaceflight plants apparently had reproductive development comparable to the ground controls, and immature seeds were produced. In every aspect examined, these seeds are similar to those produced by the ground control plants. The results suggest that if the physical environment around the plant under spaceflight conditions meets the physiological demands of the plant, then reproductive development can proceed normally on orbit.

HISTOCHEMICAL ASSESSMENT OF GROUND-BASED AND MIR-GROWN WHEAT

Our objectives were to ascertain whether wheat (Triticum aestivum L. cv. SuperDwarf) plants, grown aboard the Russian space station Mir in the microgravity of space, exhibited any plant structure and histochemical changes compared with those ground-based plants grown in Moscow, Russia, and Logan, Utah. Plants were harvested at stages of ontogeny corresponding to day 6, 14, 25, 35, and 55 post-emergence and placed in 4% formaldehyde: 1% glutaraldehyde (4F: 1G) fixative, adjusted to pH 7.2, and stored in Aclam plastic bags. Upon return to earth, samples were dehydrated and embedded in Spurr's resin. Use of differential chromophores on semi-thin sections (1 μm) suggests no major artifacts in cellular structure. Enzyme localizations for lignin, carbohydrate, starch, alkaline and acid phosphatase indicate that plants grown aboard Mir appeared to have less lignin than ground control plants. (Supported by NASA Grant NCC 2-831 and the Utah Agr. Expt. Station.)

Cytochemical Localization of Reserves during Seed Development in Arabidopsis thalianaunder Spaceflight Conditions

Successful development of seeds under spaceflight conditions has been an elusive goal of numerous long-duration experiments with plants on orbital spacecraft. Because carbohydrate metabolism undergoes changes when plants are grown in microgravity, developing seed storage reserves might be detrimentally affected during spaceflight. Seed development in Arabidopsis thalianaplants that flowered during 11 d in space on shuttle mission STS-68 has been investigated in this study. Plants were grown to the rosette stage (13 d) on a nutrient agar medium on the ground and loaded into the Plant Growth Unit flight hardware 18 h prior to lift-off. Plants were retrieved 3 h after landing and siliques were immediately removed from plants. Young seeds were fixed and processed for microscopic observation. Seeds in both the ground control and flight plants are similar in their morphology and size. The oldest seeds from these plants contain completely developed embryos and seed coats. These embryos developed radicle, hypocotyl, meristematic apical tissue, and differentiated cotyledons. Protoderm, procambium, and primary ground tissue had differentiated. Reserves such as starch and protein were deposited in the embryos during tissue differentiation. The aleurone layer contains a large quantity of storage protein and starch grains. A seed coat developed from integuments of the ovule with gradual change in cell composition and cell material deposition. Carbohydrates were deposited in outer integument cells especially in the outside cell walls. Starch grains decreased in number per cell in the integument during seed coat development. All these characteristics during seed development represent normal features in the ground control plants and show that the spaceflight environment does not prevent normal development of seeds in Arabidopsis.

Growth and Photosynthetic Responses of Wheat Plants Grown in Space

Growth and photosynthesis of wheat (Triticum aestivum L. cv Super Dwarf) plants grown onboard the space shuttle Discovery for 10 d were examined. Compared to ground control plants, the shoot fresh weight of space-grown seedlings decreased by 25%. Postflight measurements of the O2 evolution/photosynthetic photon flux density response curves of leaf samples revealed that the CO2-saturated photosynthetic rate at saturating light intensities in space-grown plants declined 25% relative to the rate in ground control plants. The relative quantum yield of CO2-saturated photosynthetic O2 evolution measured at limiting light intensities was not significantly affected. In space-grown plants, the light compensation point of the leaves increased by 33%, which likely was due to an increase (27%) in leaf dark-respiration rates. Related experiments with thylakoids isolated from space-grown plants showed that the light-saturated photosynthetic electron transport rate from H2O through photosystems II and I was reduced by 28%. These results demonstrate that photosynthetic functions are affected by the microgravity environment.

Cytological and ultrastructural studies on root tissues.

The anatomy and fine structure of roots from oat and mung bean seedlings, grown under microgravity conditions for 8 days aboard the Space Shuttle, was examined and compared to that of roots from ground control plants grown under similar conditions. Roots from both sets of oat seedlings exhibited characteristic monocotyledonous tissue organization and normal ultrastructural features, except for cortex cell mitochondria, which exhibited a 'swollen' morphology. Various stages of cell division were observed in the meristematic tissues of oat roots. Ground control and flight-grown mung bean roots also showed normal tissue organization, but root cap cells in the flight-grown roots were collapsed and degraded in appearance, especially at the cap periphery. At the ultrastructural level, these cells exhibited a loss of organelle integrity and a highly-condensed cytoplasm. This latter observation perhaps suggests a differing tissue sensitivity for the two species to growth conditions employed in space flight. The basis for abnormal root cap cell development is not understood, but the loss of these putative gravity-sensing cells holds potential significance for long term plant growth orientation during space flight.