Assessing Radish Health during Space Cultivation by Gene Transcription.

During the Advanced Plant Habitat experiment 2, radish plants were grown in two successive grow-outs on the International Space Station (ISS) for 27 days each. On days 10, 18, and 24, leaf punch (LP) samples were collected and frozen. At harvest, bulb tissue was sampled with oligo-dT functionalized Solid Phase Gene Extraction (SPGE) probes. The space samples were compared with samples from ground controls (GC) grown at the Kennedy Space Center (KSC) under the same conditions as on the ISS, with notably elevated CO2 (about 2500 ppm), and from lab plants grown under atmospheric CO2 but with light and temperature conditions similar to the KSC control. Genes corresponding to peroxidase (RPP), glucosinolate biosynthesis (GIS), protein binding (CBP), myrosinase (RMA), napin (RSN), and ubiquitin (UBQ) were measured by qPCR. LP from day 24 and bulb samples collected at harvest were compared with RNA-seq data from material that was harvested, frozen, and analyzed after return to Earth. The results showed stable transcription in LP samples in GC but decreasing values in ISS samples during both grow-outs, possibly indicative of stress. SPGE results were similar between GC and ISS samples. However, the RNA-seq analyses showed different transcription profiles than SPGE or LP results, possibly related to localized sampling. RNA-seq of leaf samples showed greater variety than LP data, possibly because of different sampling times. RSN and RPP showed the lowest transcription regardless of method. Temporal analyses showed relatively small changes during plant development in space and in ground controls. This is the first study that compares developmental changes in space-grown plants with ground controls based on a comparison between RNA-seq and qPCR analyses.

Preamble to the Special Edition Plants and Microgravity.

The need to study plant systems in space has a long history and space experiments on plants were recognized for their scientific value and as necessity to provide life support for humans and other non-photosynthetic organisms [...].

Changes in endogenous abscisic acid and stomata of the resurrection fern, Pleopeltis polypodioides, in response to de- and rehydration.

PREMISE: While angiosperms respond uniformly to abscisic acid (ABA) by stomatal closure, the response of ferns to ABA is ambiguous. We evaluated the effect of endogenous ABA, hydrogen peroxide (H2 O2 ), nitric oxide (NO), and Ca2+ , low and high light intensities, and blue light (BL) on stomatal opening of Pleopeltis polypodioides. METHODS: Endogenous ABA was quantified using gas chromatography-mass spectrometry; microscopy results and stomatal responses to light and chemical treatments were analyzed with Image J. RESULTS: The ABA content increases during initial dehydration, peaks at 15 h and then decreases to one fourth of the ABA content of hydrated fronds. Following rehydration, ABA content increases within 24 h to the level of hydrated tissue. The stomatal aperture opens under BL and remains open even in the presence of ABA. Closure was strongly affected by BL, NO, and Ca2+ , regardless of ABA, H2 O2 effect was weak. CONCLUSIONS: The decrease in the ABA content during extended dehydration and insensitivity of the stomata to ABA suggests that the drought tolerance mechanism of Pleopeltis polypodioides is independent of ABA.

Lipid Rafts and Plant Gravisensitivity.

The necessity to include plants as a component of a Bioregenerative Life Support System leads to investigations to optimize plant growth facilities as well as a better understanding of the plant cell membrane and its numerous activities in the signaling, transport, and sensing of gravity, drought, and other stressors. The cell membrane participates in numerous processes, including endo- and exocytosis and cell division, and is involved in the response to external stimuli. Variable but stabilized microdomains form in membranes that include specific lipids and proteins that became known as (detergent-resistant) membrane microdomains, or lipid rafts with various subclassifications. The composition, especially the sterol-dependent recruitment of specific proteins affects endo- and exo-membrane domains as well as plasmodesmata. The enhanced saturated fatty acid content in lipid rafts after clinorotation suggests increased rigidity and reduced membrane permeability as a primary response to abiotic and mechanical stress. These results can also be obtained with lipid-sensitive stains. The linkage of the CM to the cytoskeleton via rafts is part of the complex interactions between lipid microdomains, mechanosensitive ion channels, and the organization of the cytoskeleton. These intricately linked structures and functions provide multiple future research directions to elucidate the role of lipid rafts in physiological processes.

Transcription Profile of Auxin Related Genes during Positively Gravitropic Hypocotyl Curvature of Brassica rapa.

Unlike typical negative gravitropic curvature, young hypocotyls of Brassica rapa and other dicots exhibit positive gravitropism. This positive curvature occurs at the base of the hypocotyl and is followed by the typical negative gravity-induced curvature. We investigated the role of auxin in both positive and negative hypocotyl curvature by examining the transcription of PIN1, PIN3, IAA5 and ARG1 in curving tissue. We compared tissue extraction of the convex and concave flank with Solid Phase Gene Extraction (SPGE). Based on Ubiquitin1 (UBQ1) as a reference gene, the log (2) fold change of all examined genes was determined. Transcription of the examined genes varied during the graviresponse suggesting that these genes affect differential elongation. The transcription of all genes was upregulated in the lower flank and downregulated in the upper flank during the initial downward curving period. After 48 h, the transcription profile reversed, suggesting that the ensuing negative gravicurvature is controlled by the same genes as the positive gravicurvature. High-spatial resolution profiling using SPGE revealed that the transcription profile of the examined genes was spatially distinct within the curving tissue. The comparison of the hypocotyl transcription profile with the root tip indicated that the tip tissue is a suitable reference for curving hypocotyls and that root and hypocotyl curvature are controlled by the same physiological processes.

Blueprints for Constructing Microgravity Analogs.

The desire to understand gravitational effects on living things requires the removal of the very factor that determines life on Earth. Unfortunately, the required free-fall conditions that provide such conditions are limited to a few seconds unless earth-orbiting platforms are available. Therefore, attempts have been made to create conditions that simulate reduced gravity or gravity-free conditions ever since the gravity effects have been studied. Such conditions depend mostly on rotating devices (aka clinostats) that alter the gravity vector faster than the biological response time or create conditions that compensate sedimentation by fluid dynamics. Although several sophisticated, commercial instruments are available, they are unaffordable to most individual investigators. This article describes important considerations for the design and construction of low cost but versatile instruments that are sturdy, fully programmable, and affordable. The chapter focuses on detailed construction, programming of microcontrollers, versatility, and reliability of the instrument.

Plant biology for space exploration - Building on the past, preparing for the future.

A review of past insights of space experiments with plants outlines basic space and gravity effects as well as gene expression. Efforts to grow plants in space gradually incorporated basic question on plant productivity, stress response and cultivation. The prospect of extended space missions as well as colonization of the Moon and Mars require better understanding and therefore research efforts on biomass productivity, substrate and water relations, atmospheric composition, pressure and temperature and substrate and volume (growth space) requirements. The essential combination of using plants not only for food production but also for regeneration of waste, and recycling of carbon and oxygen production requires integration of complex biological and engineering aspects. We combine a historical account of plant space research with considerations for future research on plant cultivation, selection, and productivity based on space-related environmental conditions.

Desiccation Mitigates Heat Stress in the Resurrection Fern, Pleopeltis polypodioides.

Although heat and desiccation stresses often coincide, the response to heat especially in desiccation tolerant plants is rarely studied. We subjected hydrated Pleopeltis polypodioides fronds to temperatures up to 50°C and dehydrated fronds up to 65°C for 24 h. The effect of heat stress was evaluated using morphological changes, photosystem (PS) II efficiency, and metabolic indicators. Pinnae of dried fronds exposed to more than 40°C curled tighter and became brittle compared to fronds dried at lower temperatures. Exposure to > 50°C leads to discolored fronds after rehydration. Hydrated fronds turned partially brown at > 35°C. Chlorophyll fluorescence (Ft) and quantum yield (Qy) increased following re-hydration but the recovery process after 40°C treatment lasted longer than at lower temperatures. Similarly, hydrated fronds showed reduced Qy when exposed to > 40°C. Dried and hydrated fronds remained metabolically active up to 40°C. Hydroperoxides and lipid hydroperoxides in dried samples remained high up to 50°C, but decreased in hydrated fronds at > 40°C. Catalase (CAT) and glutathione (GSH) oxidizing activities remained high up to 40°C in dehydrated fronds and up to 35°C in hydrated fronds. Major fatty acids detected in both dehydrated and hydrated fronds included palmitic (C16:0) and stearic (C18:0) acids, oleic (18:1), linoleic (C18:2); and linolenic (C18:3) acids. Linolenic acid was most abundant. In dried fronds, all fatty acids decreased at > 35°C. The combined data indicate that the thermotolerance of dry fronds is about 55°C but is at least 10°C lower for hydrated fronds.

Comparison of Microgravity Analogs to Spaceflight in Studies of Plant Growth and Development.

Life on Earth has evolved under the influence of gravity. This force has played an important role in shaping development and morphology from the molecular level to the whole organism. Although aquatic life experiences reduced gravity effects, land plants have evolved under a 1-g environment. Understanding gravitational effects requires changing the magnitude of this force. One method of eliminating gravity''s influence is to enter into a free-fall orbit around the planet, thereby achieving a balance between centripetal force of gravity and the centrifugal force of the moving object. This balance is often mistakenly referred to as microgravity, but is best described as weightlessness. In addition to actually compensating gravity, instruments such as clinostats, random-positioning machines (RPM), and magnetic levitation devices have been used to eliminate effects of constant gravity on plant growth and development. However, these platforms do not reduce gravity but constantly change its direction. Despite these fundamental differences, there are few studies that have investigated the comparability between these platforms and weightlessness. Here, we provide a review of the strengths and weaknesses of these analogs for the study of plant growth and development compared to spaceflight experiments. We also consider reduced or partial gravity effects via spaceflight and analog methods. While these analogs are useful, the fidelity of the results relative to spaceflight depends on biological parameters and environmental conditions that cannot be simulated in ground-based studies.

Augmentation of root gravitropism by hypocotyl curvature in Brassica rapa seedlings.

Main Conclusion Root gravitropism of primary roots is assisted by curvature of the hypocotyl base. Root gravitropism is typically described as the sequence of signal perception, signal processing, and response that causes differential elongation and the establishment of a new gravitropic set-point angle. We describe two components of the graviresponse of Brassica seedlings that comprise a primary curvature of the root tip and a later onset but stronger curvature that occurs at the base of the hypocotyl. This second curvature is preceded by straightening of the tip region but leads to the completion of the alignment of the root axis. Curvature in both regions require a minimum displacement of 20 deg. The rate of tip curvature is a function of root length. After horizontal reorientation, tip curvature of five mm long roots curved twice as fast as 10 mm long roots (33.6 ±â€¯3.3 vs. 14.3 ±â€¯1.5 deg hr-1). The onset of curvature at the hypocotyl base is correlated with root length, but the rate of this curvature is independent of seedling length. Decapping of roots prevented tip curvature but the curvature at base of hypocotyl was unaffected. Endodermal cells at the root shoot junction show numerous, large and sedimenting amyloplasts, which likely serve as gravity sensors (statoliths). The amyloplasts at the hypocotyl were 3-4 µm in diameter, similar in size to those in the root cap, and twice the size of starch grains in the cortical layers of hypocotyl or elsewhere in the root. These data indicate that the root shoot reorientation of young seedlings is not limited to the root tip but includes more than one gravitropically responsive region.

Irradiation effects of MeV protons on dry and hydrated Brassica rapa seeds.

Although space radiation is a known risk for space travel and eventual colonization of Moon or Mars, relatively few data exist on radiation effects on potential crop plants. We studied Brassica rapa to assess the tolerance of seeds and seedlings to radiation by exposing dry and hydrated B. rapa seeds to 1, 2 and 3 MeV proton ions of various fluences and examined the effect on germination and root growth. Modeling penetration depth with SRIM code indicated that the applied energy was insufficient to penetrate the seeds; therefore, all energy was deposited into the tissue. Subsequent germination varied based on the incident ion energy and fluence (dose). Dry and hydrated seeds germinate after ion fluence (1013 ions cm-2) irradiation, but the germination percentage decreased with increasing fluence for ions that could penetrate the seed coat (> 1 MeV). Despite their greater volume and mass, hydrated seeds were more sensitive to irradiation than dry seeds. Damage of the seed coat after irradiation led to faster germination and initial seedling growth. Our results suggest that the seed coat represents a valuable natural radiation protection and that low energy protons, the prevailing solar radiation, are suitable for studying radiation effects in seeds and plants.

Biochemical responses of the desiccation-tolerant resurrection fern Pleopeltis polypodioides to dehydration and rehydration.

The epiphytic fern Pleopeltis polypodioides can tolerate repeated drying and rehydration events without conspicuous damage. To understand the biochemical principles of drought-tolerance, we analyzed the effect of dehydration and rehydration at 25 °C on hydroperoxide and lipid hydroperoxide, the activities of antioxidative (catalase and glutathione-oxidizing) enzymes and evaluated changes in fatty acid composition and saturation levels. Dehydration increased peroxide concentration and the activity of glutathione oxidases, but reduced catalase activity. During dehydration, the biosynthesis of palmitic (C16:0), linoleic (C18:2), linolenic (C18:3) and stearic acid (C18:0) increased 18, 12, 20, and 8-fold, respectively. In contrast, rehydration lowered levels of peroxides, the activity of glutathione-oxidizing enzymes, and fatty acids but increased catalase activity. The coordinated changes during de- and rehydration suggest that lipids and oxidative and antioxidative enzymes are components of the drought-resistance system.

Tissue accumulation patterns and concentrations of potassium, phosphorus, and carboxyfluorescein translocated from pine seed to the root.

MAIN CONCLUSION: Potassium (K), phosphorous (P), and carboxyfluorescein (CF) accumulate in functionally distinct tissues within the pine seedling root cortex. Seedlings of Pinus pinea translocate exogenous CF and endogenous K and P from the female gametophyte/cotyledons to the growing radicle. Following unloading in the root tip, these materials accumulate in characteristic spatial patterns. Transverse sections of root tips show high levels of P in a circular ring of several layers of inner cortical cells. K and CF are minimal in the high P tissue. In contrast, high levels of K and CF accumulate in outer cortical cells, and in the vascular cylinder. These patterns are a property of living tissue because they change after freeze-thaw treatment, which kills the cells and results in uniform distribution of K and P. K concentration can be reduced to undetectable levels by incubation of roots in 100 mM NaCl. Inductively coupled plasma optical emission spectrometry (ICP-OES) analysis and scanning electron microscopy (SEM)/energy-dispersive X-ray spectroscopy (EDS) of root segments both reliably determine K and P concentrations.

The role of peltate scales in desiccation tolerance of Pleopeltis polypodioides.

MAIN CONCLUSION: The extreme drought tolerance of the resurrection fern is in part the result of the dorsal scales that assist in water distribution and controlled desiccation. We studied the effect of peltate scales on water uptake and loss of the desiccation-tolerant epiphytic fern Pleopeltis polypodioides using optical and FTIR microscopy and staining with calcofluor, solophenyl flavine7GFE, and Ruthenium Red. We provide information on structure, property, and function of the scales by measuring water uptake and dehydration, contact angles, and metabolic activity. Peltate scales mainly contain cellulose, xylogalactans, and pectin. Water is absorbed from the center of scales, and the overlapping arrangement of scales facilitates surface spreading of water. Intact fronds hydrated fully within 5 h of imbibition of the apical pinna, without scales water uptake stopped after 1 h. Hydration rates via rhizomes followed a longer time course but also improved in the presence of scales. Fronds with and without scales lost half of their water content in 15 or 4 h, respectively. The overall metabolism of rapidly dehydrated fronds was significantly reduced compared with slowly dehydrated fronds. Thus, water management and metabolism of Pleopeltis are dependent on surface properties determined by peltate scales.

Oxygen dependency of germinating Brassica seeds.

Establishing plants in space, Moon or Mars requires adaptation to altered conditions, including reduced pressure and composition of atmospheres. To determine the oxygen requirements for seed germination, we imbibed Brassica rapa seeds under varying oxygen concentrations and profiled the transcription patterns of genes related to early metabolism such as starch degradation, glycolysis, and fermentation. We also analyzed the activity of lactate dehydrogenase (LDH) and alcohol dehydrogenase (ADH), and measured starch degradation. Partial oxygen pressure (pO2) greater than 10% resulted in normal germination (i.e., protrusion of radicle about 18 hours after imbibition) but lower pO2 delayed and reduced germination. Imbibition in an oxygen-free atmosphere for three days resulted in no germination but subsequent transfer to air initiated germination in 75% of the seeds and the root growth rate was transiently greater than in roots germinated under ambient pO2. In hypoxic seeds soluble sugars degraded faster but the content of starch after 24 h was higher than at ambient oxygen. Transcription of genes related to starch degradation, α-amylase (AMY) and Sucrose Synthase (SUS), was higher under ambient O2 than under hypoxia. Glycolysis and fermentation pathway-related genes, glucose phosphate isomerase (GPI), 6-phosphofructokinase (PFK), fructose 1,6-bisphosphate aldolase (ALD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate decarboxylase (PDC), LDH, and ADH, were induced by low pO2. The activity of LDH and ADH was the highest in anoxic seeds. Germination under low O2 conditions initiated ethanolic fermentation. Therefore, sufficient oxygen availability is important for germination before photosynthesis provides necessary oxygen and the determination of an oxygen carrying capacity is important for uniform growth in space conditions.

Use of high gradient magnetic fields to evaluate gravity perception and response mechanisms in plants and algae.

Magnetic gradients have the valuable property of exerting a repulsive ponderomotive force onto diamagnetic compounds. A carefully designed gradient and proper positioning of biological material can be used to manipulate gravisensing organelles such as amyloplasts of higher plants and other statoliths such as the BaSO4-filled vesicles of Characean algae. This chapter describes the main considerations of magnetic gradients and their application as a localized force field to manipulate (sort) cellular organelles based on their magnetic properties. Many of the inferences from such activities have yet to be investigated.

Profiling Gene Expression in Germinating Brassica Roots.

Based on previously developed solid-phase gene extraction (SPGE) we examined the mRNA profile in primary roots of Brassica rapa seedlings for highly expressed genes like ACT7 (actin7), TUB (tubulin1), UBQ (ubiquitin), and low expressed GLK (glucokinase) during the first day post-germination. The assessment was based on the mRNA load of the SPGE probe of about 2.1 ng. The number of copies of the investigated genes changed spatially along the length of primary roots. The expression level of all genes differed significantly at each sample position. Among the examined genes ACT7 expression was most even along the root. UBQ was highest at the tip and root-shoot junction (RS). TUB and GLK showed a basipetal gradient. The temporal expression of UBQ was highest in the MZ 9 h after primary root emergence and higher than at any other sample position. Expressions of GLK in EZ and RS increased gradually over time. SPGE extraction is the result of oligo-dT and oligo-dA hybridization and the results illustrate that SPGE can be used for gene expression profiling at high spatial and temporal resolution. SPGE needles can be used within two weeks when stored at 4 °C. Our data indicate that gene expression studies that are based on the entire root miss important differences in gene expression that SPGE is able to resolve for example growth adjustments during gravitropism.

Mapping and candidate genes associated with saccharification yield in sorghum.

Sorghum (Sorghum bicolor (L.) Moench) is a high-yielding, stress tolerant energy crop for lignocellulosic-based biofuel production. Saccharification is a process by which hydrolytic enzymes break down lignocellulosic materials to fermentable sugars for biofuel production, and mapping and identifying genes underlying saccharification yield is an important first step to genetically improve the plant for higher biofuel productivity. In this study, we used the ICRISAT sorghum mini core germplasm collection and 14 739 single nucleotide polymorphism markers to map saccharification yield. Seven marker loci were associated with saccharification yield and five of these loci were syntenic with regions in the maize genome that contain quantitative trait loci underlying saccharification yield and cell wall component traits. Candidate genes from the seven loci were identified but must be validated, with the most promising candidates being ß-tubulin, which determines the orientation of cellulose microfibrils in plant secondary cell walls, and NST1, a master transcription factor controlling secondary cell wall biosynthesis in fibers. Other candidate genes underlying the different saccharification loci included genes that play a role in vascular development and suberin deposition in plants. The identified loci and candidate genes provide information into the factors controlling saccharification yield and may facilitate increasing biofuel production in sorghum.

Analysis of magnetic gradients to study gravitropism.

PREMISE OF THE STUDY: Gravitropism typically is generated by dense particles that respond to gravity. Experimental stimulation by high-gradient magnetic fields provides a new approach to selectively manipulate the gravisensing system. METHODS: The movement of corn, wheat, and potato starch grains in suspension was examined with videomicroscopy during parabolic flights that generated 20 to 25 s of weightlessness. During weightlessness, a magnetic gradient was generated by inserting a wedge into a uniform, external magnetic field that caused repulsion of starch grains. The resultant velocity of movement was compared with the velocity of sedimentation under 1 g conditions. RESULTS: The high-gradient magnetic fields repelled the starch grains and generated a force of at least 0.6 g. Different wedge shapes significantly affected starch velocity and directionality of movement. CONCLUSIONS: Magnetic gradients are able to move diamagnetic compounds under weightless or microgravity conditions and serve as directional stimulus during seed germination in low-gravity environments. Further work can determine whether gravity sensing is based on force or contact between amyloplasts and statocyte membrane system.