Gravity-regulated formation of the peg in developing cucumber seedlings

It has been proposed that peg formation in the vascular transition region (TR zone) between the hypocotyl and the root in Cucurbitaceae seedlings is a gravimorphogenetic phenomenon. Initiation of the peg became visible 36 h after imbibition when cucumber (Cucumis sativus L. cv. Burpee Hybrid II) seeds were germinated in a horizontal position at 24°C in the dark. Simultaneously, sedimented amyloplasts (putative statoliths) were apparent in the sheath cells surrounding the vascular strands, and in the cortical cells immediately adjacent to them, in the TR zone. In contrast, the other cortical cells, some of which were destined to develop into the peg, contained amyloplasts which were not sedimented. These results suggest that the graviperception mechanism for peg formation may be like that of statoliths in shoot gravitropism. By 48 h following imbibition, the cells of the TR zone still had sedimented amyloplasts but had lost their sensitivity to gravity, possibly because of their maturation.     

The effect of a microgravity (space) environment on the expression of expansins from the peg and root tissues of Cucumis sativus

In young cucumber seedlings, the peg is a polar outgrowth of tissue that functions by snagging the seed coat, thereby freeing the cotyledons. The development of the peg is thought to be gravity-dependent and has become a model system for plant-gravity response. Peg development requires rapid cell expansion, a process thought to be catalyzed by α -expansins, and thus was a good system to identify expansins that were regulated by gravity. This study identified 7 new α -expansin cDNAs from cucumber seedlings ( Cucumis sativus L. cv Burpee Hybrid II) and examined their expression patterns. Two α -expansins ( CsExp3 and CsExp4 ) were more highly expressed in the peg and the root. Earlier reports stated that pegs tend not to form in the absence of gravity, so the expression levels were compared in the pegs of seedlings grown in space (STS-95), on a clinostat, and on earth (1 g ). Pegs were observed to form at high frequency on clinostat and space-grown seedlings, yet on clinostats there was more than a 4-fold reduction in the expression of CsExp3 in the pegs of seedlings grown on clinostats vs. those grown at 1 g , while the CsExp4 gene appeared to be turned off (below detection limits). There were no detectable differences in expansin gene expression levels for the pegs of seedlings grown in space or in the orbiter environmental simulator (OES) (1 g ) at NASA. The microgravity environment did not affect the expression of CsExp3 or CsExp4 , and the clinostat did not simulate the microgravity environment well.     

Gravity-induced modification of auxin transport and distribution for peg formation in cucumber seedlings: possible roles for CS-AUX1 and CS-PIN1

Cucurbit seedlings potentially develop a peg on each side of the transition zone between the hypocotyl and root. Seedlings grown in a horizontal position suppress the development of the peg on the upper side of the transition zone in response to gravity. It is suggested that this suppression occurs due to a reduction in auxin levels to below the threshold value. We show in this study that the free indole-3-acetic acid (IAA) content is low, while IAA conjugates are significantly more abundant in the upper side of the transition zone of gravistimulated seedlings, compared to the lower side. A transient increase in mRNA of the auxin-inducible gene, CS-IAA1, was observed in the excised transition zone. The result suggests that the transition zone is a source of auxin. Cucumber seedlings treated with auxin-transport inhibitors exhibited agravitropic growth and developed a peg on each side of the transition zone. Auxin-transport inhibitors additionally caused an increase in CS-IAA1 mRNA accumulation at the transition zone, indicating a rise in intracellular auxin concentrations due to a block of auxin efflux. To study the involvement of the auxin transport system in peg formation, we isolated the cDNAs of a putative auxin influx carrier, CS-AUX1, and putative efflux carrier, CS-PIN1, from cucumber (Cucumis sativus L.) plants. Both genes (CS-AUX1 in particular) were auxin-inducible. Accumulation of CS-AUX1 and CS-PIN1 mRNAs was observed in vascular tissue, cortex and epidermis of the transition zone. A reduced level of CS-AUX1 mRNA was observed in the upper side of the gravistimulated transition zone, compared with the lower side. It is therefore possible that a balance in the activities of auxin influx and efflux carriers controls intracellular auxin concentration at the transition zone, which results in lateral placement of a peg in cucumber seedlings.Abbreviations HFCA 9-hydroxyfluorene-9-carboxylic acid - IAA indole-3-acetic acid - NPA 1-N-naphthylphthalamic acid - TIBA 2,3,5-triiodobenzoic acid     

Differential accumulation of the mRNA of the auxin-repressed gene CsGRP1 and the auxin-induced peg formation during gravimorphogenesis of cucumber seedlings

When cucumber seeds are germinated horizontally, an outgrowth (peg) develops on the lower side of the transition zone between the hypocotyl and the root for pulling the cotyledons and plumule out of the seed coat. We previously suggested that gravistimulation suppresses peg formation on the upper side of the transition zone when placed in a horizontal position. In the gravistimulated transition zone, auxin and the mRNAs of auxin-inducible genes are more abundant in the lower side than in the upper side. Here, using fluorescent differential display, we identified Cucumis sativus glycine-rich protein1(CsGRP1) as a gene whose mRNA accumulated more abundantly on the upper side than on the lower side of the transition zone in response to gravistimulation. Auxin starvation increased CsGRP1 mRNA in segments of the transition zone, and inhibition of polar auxin transport with 2,3,5-triiodobenzoic acid (TIBA) prevented the asymmetric accumulation of CsGRP1 mRNA. These results suggest that gravistimulation increases not only the expression of auxin-inducible genes on the lower side of the transition zone, but also the expression of auxin-repressed genes, such as CsGRP1, on the upper side of cucumber seedlings. In the hypocotyls of 3-day-old seedlings, neither gravistimulation nor changes in auxin level influenced the accumulation of CsGRP1 mRNA. These results suggest that the transition zone responds to gravistimulation in a specific manner by an asymmetric expression of CsGRP1 gene during regulation of peg formation.     

Control of gravimorphogenesis by auxin: accumulation pattern of CS-IAA1 mRNA in cucumber seedlings grown in space and on the ground

Cucumber (Cucumis sativus L.) seedlings grown in microgravity developed a peg on each side of the transition zone between hypocotyl and root, whereas seedlings grown in a horizontal position on the ground developed a peg on the concave side of the gravitropically bending transition zone. The morphological features of the space-grown seedlings were similar to those of seedlings grown in a vertical position on the ground with their radicles pointing down: both became two-pegged seedlings. Morphogenesis of cucumber seedlings is thus inhibited by gravity. Analysis by in-situ hybridization of an auxin-inducible gene, CS-IAA1, showed that its mRNA accumulated to a much greater extent on the lower side of the transition zone in the horizontally placed seedlings on the ground just prior to and during the initiation period of peg formation. On the other hand, when seedlings were grown in microgravity or in a vertical position on the ground, accumulation of CS-IAA1 mRNA occurred all around the transition zone. Accumulation of CS-IAA1 mRNA in horizontally grown seedlings appreciably decreased on the upper side of the transition zone and increased on the lower side upon gravistimulation, compared with the two-pegged seedlings. Application of IAA to seedlings in a horizontal position caused the development of a peg on each side of the transition zone, or a collar-like protuberance, depending on the concentration used. These results suggest that upon gravistimulation the auxin concentration on the upper side of the horizontally placed transition zone is reduced to a level below the threshold value necessary for peg formation. Space-grown seedlings of cucumber might develop two pegs symmetrically because the auxin level in the entire transition zone is maintained above the threshold. This spaceflight experiment verified for the first time that auxin does not redistribute in microgravity. Received: 10 February 2000 / Accepted: 15 March 2000     

Transport and metabolism of h-gibberellin a(1) in dioecious cucumber seedlings

The transport of ³H-GA₁ through hypocotyl segments of cucumber (Cucumis sativus L.) was found to be nonpolar. The transport of ³H-GA₁ was increased by pretreatment with relatively high concentrations of either IAA or Ethephon (2-chloroethylphosphonic acid). Hypocotyl segments from plants of a gynoecious genotype transported more ³H-GA₁ than those of an androecious. The metabolism of ³H-GA₁ in hypocotyl segments was neither related to the sex genotype of the cucumber plant nor influenced by pretreatment with Ethephon. The primary metabolite of GA₁ was suggested to be GA₈. Two other suspected metabolites were not identified. Differences in the endogenous GA of gynoecious and androecious plants could not be accounted for by transport differences.     

Gravitropic responses of the Avena coleoptile in space and on clinostats. IV. The clinostat as a substitute for space experiments

Gravitropic responses of dark grown oat coleoptiles were measured in weightlessness and under clinorotation on earth. The tests in microgravity were conducted in Spacelab during the IML-1 mission and those on clinostats were conducted in laboratories on earth. The same apparatus was used for both kinds of tests. In both cases autotropism and gravitropic responsiveness were determined. This allowed a quantitative comparison between the plants' responses after receiving the same tropistic stimulations either in weightlessness or on clinostats.
Autotropism was observed with oat coleoptiles responding in weightlessness but it did not occur on clinostats. Gravitropic responsiveness was measured as the ratio between the incremental bending response (degrees curvature) and the corresponding incremental g-dose (stimulus intensity times duration for which it was applied). Plants were tested at either of two stages of coleoptile development (i.e. different coleoptile lengths). From a total of six different kinds of critical comparisons that could be made from our tests that provided data for clinorotated vs weightless plants, three showed no significant difference between responses in simulated vs authentic weightlessness. Three other comparisons showed highly significant differences. Therefore, the validity of clinorotation as a general substitute for space flight was not supported by these results.     

Oxygen-mediated cold-acclimation in cucumber (Cucumis sativus) seedlings

Cold acclimation of etiolated cucumber seedlings, consisting of cooling at 12°C for 48 h followed by a warming period at 25°C, led to tolerance to subsequent chilling at 2°C. Tolerance, as evidenced by freedom from chilling injury and continued growth, developed during the warming period in a time-course manner for 12 h but decreased with prolonged warming. A similar increase and subsequent decrease was also observed in the content of palmitic, linoleic and linolenic acids in total lipid fraction from cucumber hypocotyl tissue. During the warming period supra-ambient oxygen stimulated, whereas subambient oxygen inhibited, the increase in fatty acid content as well as development of chilling tolerance. A strong correlation between oxygen-mediated changes in fatty acid content and associated development of cold tolerance suggests that both these processes are interrelated. Cold acclimation, but not cold stress, led to an increase followed by a decrease in CO₂ evolution suggesting that a respiratory upsurge is yet another feature of cold acclimation in cucumbers.     

Physiology of a microgravity environment invited review: microgravity and skeletal muscle.

Spaceflight (SF) has been shown to cause skeletal muscle atrophy; a loss in force and power; and, in the first few weeks, a preferential atrophy of extensors over flexors. The atrophy primarily results from a reduced protein synthesis that is likely triggered by the removal of the antigravity load. Contractile proteins are lost out of proportion to other cellular proteins, and the actin thin filament is lost disproportionately to the myosin thick filament. The decline in contractile protein explains the decrease in force per cross-sectional area, whereas the thin-filament loss may explain the observed postflight increase in the maximal velocity of shortening in the type I and IIa fiber types. Importantly, the microgravity-induced decline in peak power is partially offset by the increased fiber velocity. Muscle velocity is further increased by the microgravity-induced expression of fast-type myosin isozymes in slow fibers (hybrid I/II fibers) and by the increased expression of fast type II fiber types. SF increases the susceptibility of skeletal muscle to damage, with the actual damage elicited during postflight reloading. Evidence in rats indicates that SF increases fatigability and reduces the capacity for fat oxidation in skeletal muscles. Future studies will be required to establish the cellular and molecular mechanisms of the SF-induced muscle atrophy and functional loss and to develop effective exercise countermeasures.     

Reciprocity in the activation of geotropism in oat coleoptiles grown on clinostats

Summary The activation of geotropism in oat coleoptiles, grown on horizonta clinostats, i.e., without the tropic influence of gravity, shows a reciprocal relationship between force and time. Two methods were used to approximate geotropic presentation time. In one, this parameter was estimated by extrapolation to zero response from the linear relationship, response = a+b log stimulation time. In the other, stimulation times of very short as well as longer durations were used; under these conditions, the response curve shows two distinct rates, with the lower rate for stimuli of brief duration. The intersection of the two rate-segments of the response curve was taken as the presentation time. Both methods show reciprocity for the activation of geotropism, but yield significantly different reciprocity constants. The ability of the coleoptile to sense gravity is not affected by gravity compensation. With agar as a growth medium, the magnitude of response to gravity is greater than with sand. However, coleoptiles grown in sand are more sensitive to geotropic activation.Work supported by the U. S. Atomic Energy Commission and the National Aeronautics and Space Administration.     

Differential chilling sensitivity in cucumber (Cucumis sativus) seedlings

Cucumber ( Cucumis sativus L. cv. Poinsett 76) seeds were chilled at 2.5°C in a study of the chilling sensitivity and recovery of radicle tissue. The effect of chilling on radicle growth and the production of carbon dioxide and ethylene was measured. Chilling sensitivity of radicles increased as they grew from 1 to 7 mm in length. The length, not the age of the radicles, determined the level of chilling sensitivity. Apical tissue was most sensitive to chilling and slowest to recover from chilling, followed by subapical and basal tissue. Our data demonstrate that the chilling sensitivity of young seedling radicles differs along their length and that the rapid chilling-induced inhibition of elongation is probably due to an inability of meristematic cells to remain viable and active when chilled.     

Gravitropic responses of the Avena coleoptile in space and on clinostats. I. Gravitropic response thresholds

We conducted a series of gravitropic experiments on Avena coleoptiles in the weightlessness environment of Spacelab. The purpose was to test the threshold stimulus, reciprocity rule and autotropic reactions to a range of g-force stimulations of different intensities and durations The tests avoided the potentially complicating effects of earth's gravity and the interference from clinostat ambiguities. Using slow-speed centrifuges, coleoptiles received transversal accelerations in the hypogravity range between 0.1 and 1.0 g over periods that ranged from 2 to 130 min. All responses that occurred in weightlessness were compared to clinostal experiments on earth using the same apparatus.
Characteristic gravitropistic response patterns of Avena were not substantially different from those observed in ground-based experiments. Gravitropic presentation times were extrapolated. The threshold at 1.0 g was less than 1 min (shortest stimulation time 2 min), in agreement with values obtained on the ground. The least stimulus tested, 0.1 g for 130 min, produced a significant response. Therefore the absolute threshold for a gravitropic response is less than 0.1 g.     

Xylem development and cell wall changes of soybean seedlings grown in space

BACKGROUND AND AIMS: Plants growing in altered gravity conditions encounter changes in vascular development and cell wall deposition. The aim of this study was to investigate xylem anatomy and arrangement of cellulose microfibrils in vessel walls of different organs of soybean seedlings grown in Space. METHODS: Seeds germinated and seedlings grew for 5 d in Space during the Foton-M2 mission. The environmental conditions, other than gravity, of the ground control repeated those experienced in orbit. The seedlings developed in space were compared with those of the control test on the basis of numerous anatomical and ultrastructural parameters such as number of veins, size and shape of vessel lumens, thickness of cell walls and deposition of cellulose microfibrils. KEY RESULTS: Observations made with light, fluorescence and transmission electron microscopy, together with the quantification of the structural features through digital image analysis, showed that the alterations due to microgravity do not occur at the same level in the various organs of soybean seedlings. The modifications induced by microgravity or by the indirect effect of space-flight conditions, became conspicuous only in developing vessels at the ultrastructural level. The results suggested that the orientation of microfibrils and their assembly in developing vessels are perturbed by microgravity at the beginning of wall deposition, while they are still able to orient and arrange in thicker and ordered structures at later stages of secondary wall deposition. CONCLUSIONS: The process of proper cell-wall building, although not prevented, is perturbed in Space at the early stage of development. This would explain the almost unaltered anatomy of mature structures, accompanied by a slower growth observed in seedlings grown in Space than on Earth.