State of Brassica rapa photosynthetic membranes in microgravity.

The structural characteristics of the photosynthetic apparatus of Brassica rapa plants grown on board the space shuttle Columbia (STS-87) for 15 days were examined using the methods of transmission electron microscopy and statistic programme STAT. Maintaining of the same growth conditions for control plants was realized with great accuracy using the Orbiter Environmental simulator in Kennedy Space Center. A grana number per a medial section 1.8 times decreased in microgravity. Considerable changes were also revealed in the grana structure in microgravity in comparison with th ground control, namely: 1/a greater diversity in the thylakoid length with granae and 2/ lateral shifting of the thylakoids lateral shifting of the thylakoids relative one to another. The previous mentioned pheomenon was found for 64% of the invested granae. Shifting of the thylakoids in the granae in microgravity led to increasing of the grana thylakoid surface exposed to a stroma. In addition, the volume of stromal thylakoids increased. The peculiarities in the photosynthetic apparatus structure in microgravity are supposed to be an evidence of decreasing in the light harvesting complex amount of photosystem II (PSII).

Growth in microgravity increases susceptibility of soybean to a fungal pathogen.

The influence of microgravity on the susceptibility of soybean roots to Phytophthora sojae was studied during the Space Shuttle Mission STS-87. Seedlings of soybean cultivar Williams 82 grown in spaceflight or at unit gravity were untreated or inoculated with the soybean root rot pathogen P. sojae. At 3, 6 and 7 d after launch while still in microgravity, seedlings were photographed and then fixed for subsequent microscopic analysis. Post-landing analysis of the seedlings revealed that at harvest day 7 the length of untreated roots did not differ between flight and ground samples. However, the flight-grown roots infected with P. sojae showed more disease symptoms (percentage of brown and macerated areas) and the root tissues were more extensively colonized relative to the ground controls exposed to the fungus. Ethylene levels were higher in spaceflight when compared to ground samples. These data suggest that soybean seedlings grown in microgravity are more susceptible to colonization by a fungal pathogen relative to ground controls.

Vascular defense responses in rice: peroxidase accumulation in xylem parenchyma cells and xylem wall thickening.

The rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae is a vascular pathogen that elicits a defensive response through interaction with metabolically active rice cells. In leaves of 12-day-old rice seedlings, the exposed pit membrane separating the xylem lumen from the associated parenchyma cells allows contact with bacterial cells. During resistant responses, the xylem secondary walls thicken within 48 h and the pit diameter decreases, effectively reducing the area of pit membrane exposed for access by bacteria. In susceptible interactions and mock-inoculated controls, the xylem walls do not thicken within 48 h. Xylem secondary wall thickening is developmental and, in untreated 65-day-old rice plants, the size of the pit also is reduced. Activity and accumulation of a secreted cationic peroxidase, PO-C1, were previously shown to increase in xylem vessel walls and lumen. Peptide-specific antibodies and immunogold-labeling were used to demonstrate that PO-C1 is produced in the xylem parenchyma and secreted to the xylem lumen and walls. The timing of the accumulation is consistent with vessel secondary wall thickening. The PO-C1 gene is distinct but shares a high level of similarity with previously cloned pathogen-induced peroxidases in rice. PO-C1 gene expression was induced as early as 12 h during resistant interactions and peaked between 18 and 24 h after inoculation. Expression during susceptible interactions was lower than that observed in resistant interactions and was undetectable after infiltration with water, after mechanical wounding, or in mature leaves. These data are consistent with a role for vessel secondary wall thickening and peroxidase PO-C1 accumulation in the defense response in rice to X. oryzae pv. oryzae.

Plants, plant pathogens, and microgravity--a deadly trio.

Plants grown in spaceflight conditions are more susceptible to colonization by plant pathogens. The underlying causes for this enhanced susceptibility are not known. Possibly the formation of structural barriers and the activation of plant defense response components are impaired in spaceflight conditions. Either condition would result from altered gene expression of the plant. Because of the tools available, past studies focused on a few physiological responses or biochemical pathways. With recent advances in genomics research, new tools, including microarray technologies, are available to examine the global impact of growth in the spacecraft on the plant's gene expression profile. In ground-based studies, we have developed cDNA subtraction libraries of rice that are enriched for genes induced during pathogen infection and the defense response. Arrays of these genes are being used to dissect plant defense response pathways in a model system involving wild-type rice plants and lesion mimic mutants. The lesion mimic mutants are ideal experimental tools because they erratically develop defense response-like lesions in the absence of pathogens. The gene expression profiles from these ground-based studies will provide the molecular basis for understanding the biochemical and physiological impacts of spaceflight on plant growth, development and disease defense responses. This, in turn, will allow the development of strategies to manage plant disease for life in the space environment.

DNA content and differentiation of root apical cells of Brassica rapa plants grown in microgravity.

Root cap is proposed to be a graviperceptive tissue in the plant root, and it is composed of several cell types. One such cell type, the columella cells, are thought to initiate the gravity-induced signal transduction cascade, and these cells arise from the activity of the meristematic zone of the root cap. There is, in fact, a continuum of cells in the central column of the root cap representing the meristematic cells, developing columella cells, mature cells, and those that will soon be sloughed off into the soil. In order to study the functional roles of the root cap cells in gravity-sensing, we compared the ultrastructural organization, differentiation, and DNA content in the meristematic, elongating, and differentiating cells of root tips in Brassica rapa plants grown in space microgravity and at 1g. The experiments were also designed to determine the reactions of root cap cells in both main roots (in which the original root cap was present in an embryonic form within the seed) and lateral roots (in which the root cap formed completely in space after seed germination on orbit) to the space microgravity. This study (ROOTS) was performed in collaboration with the B-PAC experiment on the Space shuttle "Columbia" mission STS-87 (Collaborative US/Ukrainian Experiment (CUE) during November 19-December 5, 1997.

Some characteristics of photosynthetic apparatus under conditions of spaceflight.

During colonization of space by humans, the bioregenerative life-support systems on board the space ships will require the plants with a highly efficient photosynthesis, a process producing food and O2 and removing CO2-Therefore, in recent years the scientists increasingly focus the their attention to study on photosynthetic apparatus of plants grown in space. Although the available data are quite scanty and, at times, controversial, it is Considered that the space grown plants differ from around control plants by growth and development, many structural and functional indices and metabolism. Data exist showing changes in the chlorophyll (Chl) content, structure and number of chloroplasts in the cell, swelling of thylakoids and decrease in the number and size of starch grains in the chloroplasts (for reviews, see Halstead and Dutcher, 1987; Kordyum, 1997). The decrease of shoot fresh weight and reduction of CO2-saturated photosynthetic rate at saturating light intensities in space grown wheat plants in comparison with ground control have been reported by Tripathy et al. (1996). The thylakoids isolated from space grown plants displayed lower rates of electron transport through photosystems I and II (PSI and PSII) and in a whole chain. This study aimed to examine the electron transport rates through PSI and PSII in the isolated thylakoids, to elucidate if there are any differences in accumulation of thylakoid membranes between space grown Brassica rapa plants and ground control plants (based on Chl quantity) and to measure the Chl a/b ratio in isolated thylakoids. These studies were part of the Collaborative US/Ukrainian program during the STS-87 mission (1997).

Ultrastructural observation of altered chloroplast morphology in space-grown Brassica rapa cotyledons.

Photosynthesis will be indispensable in a bioregenerative life-support systems for long space missions. It is critical understand the effects of space on this complex process, especially the loss of gravity. Past has noted changes in plant growth and development; differences about cell size, shape, division, and differentiation; and plastid distribution and structure alterations. The amyloplast-containing columelar cells in root tips were carefully examined since they are likely gravity-sensing sites. Changes on photosynthetic physiology and chloroplast structure have been reported. Both increases and decreases of chlorophyll and carotenoid contents were reported. Structural changes of thylakoid membranes in chloroplasts were observed in pea and Arabidopsis grown in space or clinorotation. Recently, a decrease of CO2 assimilation rate and of electron transport rate of both PSI and PSII on thylakoid membranes were reported in space-grown wheat. These imply an overall decrease of photosynthetic activities, and implicate thylakoid-old structural changes. For example, PSI activity, and its reaction center subunits (PsaA, PsaB, and PsaC) and the LHCIs, were decreased under microgravity. Here, we further examined cellular morphology and ultrastructural features of the chloroplast and its thylakoid membranes by electron microscopy and in situ immunolocalization.

Spaceflight effects on structural and some biochemical parameters of Brassica rapa photosynthetic apparatus.

Chloroplasts play a crucial role in sustaining life on Earth by their dual property in performing the primary fixation of carbon and also in releasing oxygen for use in respiration. Collection of light and its transformation into chemical energy occurs in a thylakoid membrane which is one of the most remarkable transducing systems in the biological world. In order for the light-dependent reactions could take place, a high degree of molecular organization of its constituents is needed. Some results obtained in the framework of the Collaborative Ukrainian Experiment mission (STS-87) which was performed on board of the space shuttle "Columbia" are presented in the given paper. A goal of the study was to obtain data on some parameters of photosynthetic apparatus, namely the chloroplast structure, pigment content and lipid composition of Brassica rapa plants grown in microgravity.

Long-term exposure to spaceflight conditions affects bacterial response to antibiotics.

Bacteria exposed to the spaceflight environment have been shown to have an increased growth rate and an increased resistance to antibiotics. The mechanism of resistance has not yet been identified, as the resistance is quickly lost upon return to Earth. To more fully characterize the spaceflight-induced resistance to antibiotics, 4 species of bacteria were exposed to microgravity for 4 months on the Space Station MIR. Upon return to Earth, these cultures were challenged with a suite of 12 antibiotics of varying modes of action. In contrast to reports from short-term space flights, we find that long-term exposure to microgravity causes bacteria to become more susceptible to most, but not all, antibiotics. Each species responds differently to the suite of antibiotics, frequently becoming less resistant, but occasionally more resistant to the antibiotic. A pattern enabling prediction of response is not yet discernible. While contradicting the results from short-term pure culture research, this experiment confirms results from astronaut and cosmonaut skin flora samples.

Plastid distribution in columella cells of a starchless Arabidopsis mutant grown in microgravity.

Wild-type and starchless Arabidopsis thaliana mutant seedlings (TC7) were grown and fixed in the microgravity environment of a U.S. Space Shuttle spaceflight. Computer image analysis of longitudinal sections from columella cells suggest a different plastid positioning mechanism for mutant and wild-type in the absence of gravity.

Clinorotation affects morphology and ethylene production in soybean seedlings.

The microgravity environment of spaceflight influences growth, morphology and metabolism in etiolated germinating soybean. To determine if clinorotation will similarly impact these processes, we conducted ground-based studies in conjunction with two space experiment opportunities. Soybean (Glycine max [L.] Merr.) seeds were planted within BRIC (Biological Research In Canister) canisters and grown for seven days at 20 degrees C under clinorotation (1 rpm) conditions or in a stationary upright mode. Gas samples were taken daily and plants were harvested after seven days for measurement of growth and morphology. Compared to the stationary upright controls, plants exposed to clinorotation exhibited increased root length (125% greater) and fresh weight (42% greater), whereas shoot length and fresh weight decreased by 33% and 16% respectively. Plants grown under clinorotation produced twice as much ethylene as the stationary controls. Seedlings treated with triiodo benzoic acid (TIBA), an auxin transport inhibitor, under clinorotation produced 50% less ethylene than the untreated control subjected to the same gravity treatment, whereas a treatment with 2,4-D increased ethylene by five-fold in the clinorotated plants. These data suggest that slow clinorotation influences biomass partitioning and ethylene production in etiolated soybean plants.

Organization of photosystem I polypeptides examined by chemical cross-linking.

Photosystem I from the cyanobacterium Synechocystis sp. PCC 6803 was examined using the chemical cross-linkers glutaraldehyde and N-ethyl-1-3-[3-(dimethylamino)propyl]carbodiimide to investigate the organization of the polypeptide subunits. Thylakoid membranes and photosystem I, which was isolated by Triton X-100 fractionation, were treated with cross-linking reagents and were resolved using a Tricine/urea low-molecular-weight resolution gel system. Subunit-specific antibodies and western blotting analysis were used to identify the components of cross-linked species. These analyses identified glutaraldehyde-dependent cross-linking products composed of small amounts of PsaD and PsaC, PsaC and PsaE, and PsaE and PsaF. The novel cross-link between PsaE and PsaF was also observed following treatment with N-ethyl-1-3-[3-(dimethylamino)propyl]carbodiimide. These cross-linking results suggest a structural interaction between PsaE and PsaF and predict a transmembrane topology for PsaF.

Cortical microtubules in sweet clover columella cells developed in microgravity.

Electron micrographs of columella cells from sweet clover seedlings grown and fixed in microgravity revealed longitudinal and cross sectioned cortical microtubules. This is the first report demonstrating the presence and stability of this network in plants in microgravity.

Effects of clinorotation and microgravity on sweet clover columella cells treated with cytochalasin D.

The cytoskeleton of columella cells is believed to be involved in maintaining the developmental polarity of cells observed as a reproducible positioning of cellular organelles. It is also implicated in the transduction of gravitropic signals. Roots of sweet clover (Melilotus alba L.) seedlings were treated with a microfilament disrupter, cytochalasin D, on a slowly rotating horizontal clinostat (2 rpm). Electron micrographs of treated columella cells revealed several ultrastructural effects including repositioning of the nucleus and the amyloplasts and the formation of endoplasmic reticulum (ER) whorls. However, experiments performed during fast clinorotation (55 rpm) showed an accumulation (but no whorling) of a disorganized ER network at the proximal and distal pole and a random distribution of the amyloplasts. Therefore, formation of whorls depends upon the speed of clinorotation, and the overall impact of cytochalasin D suggests the necessity of microfilaments in organelle positioning. Interestingly, a similar drug treatment performed in microgravity aboard the US Space Shuttle Endeavour (STS-54, January 1993) caused a displacement of ER membranes and amyloplasts away from the distal plasma membrane. In the present study, we discuss the role of microfilaments in maintaining columella cell polarity and the utility of clinostats to simulate microgravity.

Mutational analysis of photosystem I polypeptides in the cyanobacterium Synechocystis sp. PCC 6803. Targeted inactivation of psaI reveals the function of psaI in the structural organization of psaL.

We cloned, characterized, and inactivated the psaI gene encoding a 4-kDa hydrophobic subunit of photosystem I from the cyanobacterium Synechocystis sp. PCC 6803. The psaI gene is located 90 base pairs downstream from psaL, and is transcribed on 0.94- and 0.32-kilobase transcripts. To identify the function of PsaI, we generated a cyanobacterial strain in which psaI has been interrupted by a gene for chloramphenicol resistance. The wild-type and the mutant cells showed comparable rates of photoautotrophic growth at 25 degrees C. However, the mutant cells grew slower and contained less chlorophyll than the wild-type cells, when grown at 40 degrees C. The PsaI-less membranes from cells grown at either temperature showed a small decrease in NADP+ photoreduction rate when compared to the wild-type membranes. Inactivation of psaI led to an 80% decrease in the PsaL level in the photosynthetic membranes and to a complete loss of PsaL in the purified photosystem I preparations, but had little effect on the accumulation of other photosystem I subunits. Upon solubilization with nonionic detergents, photosystem I trimers could be obtained from the wild-type, but not from the PsaI-less membranes. The PsaI-less photosystem I monomers did not contain detectable levels of PsaL. Therefore, a structural interaction between PsaL and PsaI may stabilize the association of PsaL with the photosystem I core. PsaL in the wild-type and PsaI-less membranes showed equal resistance to removal by chaotropic agents. However, PsaL in the PsaI-less strain exhibited an increased susceptibility to proteolysis. From these data, we conclude that PsaI has a crucial role in aiding normal structural organization of PsaL within the photosystem I complex and the absence of PsaI alters PsaL organization, leading to a small, but physiologically significant, defect in photosystem I function.

Microgravity and clinorotation cause redistribution of free calcium in sweet clover columella cells.

In higher plants, calcium redistribution is believed to be crucial for the root to respond to a change in the direction of the gravity vector. To test the effects of clinorotation and microgravity on calcium localization in higher plant roots, sweet clover (Melilotus alba L.) seedlings were germinated and grown for two days on a slow rotating clinostat or in microgravity on the US Space Shuttle flight STS-60. Subsequently, the tissue was treated with a fixative containing antimonate (a calcium precipitating agent) during clinorotation or in microgravity and processed for electron microscopy. In root columella cells of clinorotated plants, antimonate precipitates were localized adjacent to the cell wall in a unilateral manner. Columella cells exposed to microgravity were characterized by precipitates mostly located adjacent to the proximal and lateral cell wall. In all treatments some punctate precipitates were associated with vacuoles, amyloplasts, mitochondria, and euchromatin of the nucleus. A quantitative study revealed a decreased number of precipitates associated with the nucleus and the amyloplasts in columella cells exposed to microgravity as compared to ground controls. These data suggest that roots perceive a change in the gravitational field, as produced by clinorotation or space flights, and respond respectively differently by a redistribution of free calcium.

Rice cationic peroxidase accumulates in xylem vessels during incompatible interactions with Xanthomonas oryzae pv oryzae.

A cationic peroxidase, PO-C1 (molecular mass 42 kD, isoelectric point 8.6), which is induced in incompatible interactions between the vascular pathogen Xanthomonas oryzae pv oryzae and rice (Oryza sativa L.), was purified. Amino acid sequences from chemically cleaved fragments of PO-C1 exhibited a high percentage of identity with deduced sequences of peroxidases from rice, barley, and wheat. Polyclonal antibodies were raised to an 11-amino acid oligopeptide (POC1a) that was derived from a domain where the sequence of the cationic peroxidase diverged from other known peroxidases. The anti-POC1a antibodies reacted only with a protein of the same mobility as PO-C1 in extracellular and guttation fluids from plants undergoing incompatible responses collected at 24 h after infection. In the compatible responses, the antibodies did not detect PO-C1 until 48 h after infection. Immunoelectron microscopy was used to demonstrate that PO-C1 accumulated within the apoplast of mesophyll cells and within the cell walls and vessel lumen of xylem elements of plants undergoing incompatible interactions.

A homolog of an Escherichia coli phosphate-binding protein gene from Xanthomonas oryzae pv. oryzae.

A Xanthomonas oryzae pv. oryzae gene with sequence similarity to an Escherichia coli phosphate-binding protein gene (phoS) produces a periplasmic protein of apparent M(r) 35,000 when expressed in E. coli. Amino terminal sequencing revealed that a signal peptide is removed during transport to the periplasm in E. coli.

Clinorotation affects soybean seedling morphology.

Although spaceflight does not appear to significantly affect seed germination, it can influence subsequent plant growth. On STS-3 and SL-2, decreased growth (measured as plant length, fresh weight and dry weight) was noted for pine, oat and mung bean. In the CHROMEX-01 and -02 experiments with Haplopappus and in the CHROMEX-03 experiment with Arabidopsis, enhanced root growth was noted in the space-grown plants. In the CHROMEX-04 experiment with wheat, both leaf fresh weight and leaf area were diminished in the space-grown plants but there was no difference in total plant height (CS Brown, HG Levine, and AD Krikorian, unpublished data). These data suggest that microgravity impacts growth by whole plant partitioning of assimilates. The objective of the present study was to determine the influence of clinorotation on the growth and morphology of soybean seedlings grown in the BRIC (Biological Research In Canister) flight hardware. This experiment provided baseline data for a spaceflight experiment (BRIC-03) flown on STS-63 (Feb. 3-11, 1995).