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     

Identification of the cDNA of differentially expressed genes in the transition zone of cucumber seedlings by fluorescent differential display.

Cucumber seedlings have potential to develop two pegs on 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. To understand how the response to gravity suppresses peg formation, we screened cucumber mRNAs to identify the mRNA in the non-peg side that accumulates more than in the peg side. For screening, we determined conditions of fluorescent differential display (FDD). Then, we carried out FDD and found 4 cDNA bands that repeatedly showed stronger intensity in the non-peg side than the peg side. We isolated one of these RT-PCR products. Northern blotting showed the pattern of its mRNA accumulations corresponding to the results of FDD.     

<Emphasis Type="Italic">p</Emphasis>-Chlorophenoxyisobutyric acid impairs auxin response for gravity-regulated peg formation in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis>) seedlings

Cucumber (Cucumis sativus L.) seedlings form a specialized protuberance, the peg, on the transition zone between the hypocotyl and the root. When cucumber seeds germinate in a horizontal position, the seedlings develop a peg on the lower side of the transition zone. To verify the role of auxin action in peg formation, we examined the effect of the anti-auxin, p-chlorophenoxyisobutyric acid (PCIB), on peg formation and mRNA accumulation of auxin-regulated genes. Application of PCIB to cucumber seedlings inhibited peg formation. The application of indole-3-acetic acid (IAA) competed with PCIB and induced peg formation. Furthermore, application of PCIB decreased auxin-inducible CsIAA1 mRNA and increased auxin-repressible CsGRP1 mRNA in the lower side of the transition zone. The differential accumulation of CsIAA1 and CsGRP1 mRNAs in the transition zone of cucumber seedlings grown in a horizontal position was smaller in the PCIB-treated seedlings. These results demonstrate that endogenous auxin redistributes and induces the differential expression of auxin-regulated genes, and ultimately results in the suppression or induction of peg formation in the gravistimulated transition zone of cucumber seedlings.     

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 auxin-resistant diageotropica mutant of tomato responds to gravity via an auxin-mediated pathway

 Hypocotyls of the diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.) do not elongate in response to exogenous auxin, but can respond to gravity. This appears paradoxical in light of the Cholodny-Went hypothesis, which states that shoot gravicurvature results from asymmetric stimulation of elongation by auxin. While light-grown dgt seedlings can achieve correct gravitropic reorientation, the response is slow compared to wild-type seedlings. The sensitivity of dgt seedlings to inhibition of gravicurvature by immersion in auxin or auxin-transport inhibitors is similar to that of wild-type plants, indicating that both an auxin gradient and auxin transport are required for the gravitropic response and that auxin uptake, efflux, and at least one auxin receptor are functional in dgt. Furthermore, dgt gravicurvature is the result of asymmetrically increased elongation as would be expected for an auxin-mediated response. Our results suggest differences between elongation in response to exogenous auxin (absent in dgt) and elongation in response to gravistimulation (present but attenuated in dgt) and confirm the presence of two phases during the gravitropic response, both of which are dependent on functional auxin transport. Received: 16 July 1999 / Accepted: 24 September 1999     

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     

Analysis of peg formation in cucumber seedlings grown on clinostats and in a microgravity (space) environment.

In young cucumber seedlings, the peg is a polar out-growth of tissue that functions by snagging the seed coat, thereby freeing the cotyledons. Previous studies have indicated that peg formation is gravity dependent. In this study we analyzed peg formation in cucumber seedlings (Cucumis sativus L. cv Burpee Hybrid II) grown under conditions of normal gravity, microgravity, and simulated microgravity (clinostat rotation). Seeds were germinated on the ground, in clinostats and on board the space shuttle (STS 95) for 1-2 days, frozen and subsequently examined for their stage of development, degree of hook formation, number of pegs formed, and peg morphology. The frequency of peg formation in space grown seedlings was found to be nearly identical to that of clinostat grown seedlings and to differ from that of seedlings germinated under normal gravity only in a minority of cases; approximately 6% of the seedlings formed two pegs and nearly 2% of the seedlings lacked pegs, whereas such abnormalities did not occur in ground controls. The degree of hook formation was found to be less pronounced for space grown seedlings, compared to clinostat grown seedlings, indicating a greater degree of decoupling between peg formation and hook formation in space. Nonetheless, in all seedlings having single pegs and a hook, the peg was found to be positioned correctly on the inside of the hook, showing that there is coordinate development even in microgravity environments. Peg morphologies were altered in space grown samples, with the pegs having a blunt appearance and many pegs showing alterations in expansion, with the peg extending out over the edges of the seed coat and downwards. These phenotypes were not observed in clinostat or ground grown seedlings.     

Expression of cucumber lipid-body lipoxygenase in transgenic tobacco: lipid-body lipoxygenase is correctly targeted to seed lipid bodies

 A particular isoform of lipoxygenase (LOX, EC 1.13.11.12) localized on lipid bodies has been shown by earlier investigations to play a role during seed germination in initiating the mobilization of triacylglycerols. On lipid bodies of germinating cucumber (Cucumis sativus L.) seedlings, the modification of linoleoyl moieties by this LOX precedes the hydrolysis of the ester bonds. We analyzed the expression and intracellular location of this particular LOX form in leaves and seeds of tobacco (Nicotiana tabacum L.) transformed with one construct coding for cucumber lipid-body LOX and one construct coding for cucumber LOX fused with a hemagglutinin epitope. In both tissues, the amount of lipid-body LOX was clearly detectable. Biochemical analysis revealed that in mature seeds the foreign LOX was targeted to lipid bodies, and the preferred location of the LOX on lipid bodies was verified by immunofluorescence microscopy. Cells of the endosperm and of the embryo exhibited fluorescence based on the immunodecoration of LOX protein whereas very weak fluorescent label was visible in seeds of untransformed control plants. Further cytochemical analysis of transformed plants showed that the LOX protein accumulated in the cytoplasm when green leaves lacking lipid bodies were analyzed. Increased LOX activity was shown in young leaves of transformed plants by an increase in the amounts of endogenous (2E)-hexenal and jasmonic acid. Received: 9 August 1999 / Accepted: 28 September 1999     

Involvement of Ethylene in Gravity-Regulated Peg Development in Cucumber Seedling

Peg development on the lower side of the transition (TR) zoneof the hypocotyl and the root in cucumber seedlings was inhibitedby two inhibitors of ethylene biosynthesis, aminoethoxyvinylglycine and aminooxyacetic acid, and by an inhibitor of ethyleneaction, Ag-thiosulfate. These ethylene inhibitors also inhibitedplumular hook formation of the cucumber hypocotyl. When cucumberseeds were germinated in a vertical position or on a horizontalclinostat, the seedlings exhibited straight growth without formationof a plumular hook and failed to develop a protuberant peg.In the seedlings germinated in a vertical position, exogenousIAA induced a distinct peg-like protuberance, whereas ACC andethylene stimulated overall swelling around the TR zone, whichobviously differed from the normal peg. In horizontally placedseedlings, however, peg development was more pronounced dueto treatment with 5µl/liter of ethylene. These resultsindicate that a high ethylene level in the hook region playssome role in peg development. TIBA, an inhibitor of auxin transport,at 10^–4 M inhibited peg development, as reported previouslyby Witztum and Gersani (1975), but a somewhat lower concentrationof TIBA induced two distinct pegs, on both the lower and uppersides of horizontally grown seedlings. (Received June 12, 1987; Accepted December 11, 1987)     

Auxin-mediated response of cucumber seedlings to gravity]

Gravity regulates peg formation because cucumber seedlings grown in a horizontal position develop a peg on the lower side of the transition zone (TR zone) but not on the upper side. Studies on peg formation have suggested the regulation of peg formation by gravity as follows. Cucumber seedlings potentially develop a peg on both the lower and upper sides of the TR zone. The development of the peg on upper side of the TR zone is suppressed in response to gravity. A phytohormone, auxin, induces peg formation. Upon gravistimulation the auxin concentration on the upper side of the TR zone is reduced to a level below the threshold value necessary for peg formation. The unequally distributed auxin across TR zone is caused by a change in accumulation of auxin influx carrier (CsAUX1) protein and auxin efflux carrier (CsPIN1) protein in response to gravity. In addition, TR zone before peg initiation expresses both CsARF2 (putative activator of auxin response factor) and CsIAA1 (putative repressor of auxin-inducible gene expression), by which TR zone could respond the auxin gradient regulated by gravity.     

A Spaceflight Experiment for the Study of Gravimorphogenesis and Hydrotropism in Cucumber Seedlings

peg , on the transition zone between hypocotyl and root. Our spaceflight experiment verified that the lateral positioning of a peg in cucumber seedlings is modified by gravity. It has been suggested that auxin plays an important role in the gravity-controlled positioning of a peg on the ground. Furthermore, cucumber seedlings grown in microgravity developed a number of the lateral roots that grew towards the water-containing substrate in the culture vessel, whereas on the ground they oriented perpendicular to the primary root growing down. The response of the lateral roots in microgravity was successfully mimicked by clinorotation of cucumber seedlings on the three dimensional clinostat. However, this bending response of the lateral roots was observed only in an aeroponic culture of the seedlings but not in solid medium. We considered the response of the lateral roots in microgravity and on clinostat as positive hydrotropism that could easily be interfered by gravitropism on the ground. This system with cucumber seedlings is thus a useful model of spaceflight experiment for the study of the gravimorphogenesis, root hydrotropism and their interaction. Received 13 September 1999/ Accepted in revised form 12 October 1999     

Analysis of Peg Formation in Cucumber Seedlings Grown on Clinostats and in a Microgravity (Space) Environment

Cucumis sativus L. cv Burpee Hybrid II) grown under conditions of normal gravity, microgravity, and simulated microgravity (clinostat rotation). Seeds were germinated on the ground, in clinostats and on board the space shuttle (STS-95) for 1–2 days, frozen and subsequently examined for their stage of development, degree of hook formation, number of pegs formed, and peg morphology. The frequency of peg formation in space-grown seedlings was found to be nearly identical to that of clinostat-grown seedlings and to differ from that of seedlings germinated under normal gravity only in a minority of cases; ˜6% of the seedlings formed two pegs and nearly 2% of the seedlings lacked pegs, whereas such abnormalities did not occur in ground controls. The degree of hook formation was found to be less pronounced for space-grown seedlings, compared to clinostat-grown seedlings, indicating a greater degree of decoupling between peg formation and hook formation in space. Nonetheless, in all seedlings having single pegs and a hook, the peg was found to be positioned correctly on the inside of the hook, showing that there is coordinate development even in microgravity environments. Peg morphologies were altered in space-grown samples, with the pegs having a blunt appearance and many pegs showing alterations in expansion, with the peg extending out over the edges of the seed coat and downwards. These phenotypes were not observed in clinostat or ground-grown seedlings. Received 12 October 1999/ Accepted in revised form 18 October 1999     

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.     

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.     

Gravity-induced absorbance changes in Phycomyces: a novel method for detecting primary responses of gravitropism

 The negative gravitropism of the sporangiophores of Phycomyces blakesleeanus Burgeff is elicited by different sensory inputs, which include flexure of the growing zone, buoyance of lipid globules and sedimentation of paracrystalline proteins, so-called octahedral crystals (C. Schimek et al., 1999a, Planta 210: 132–142). Gravity-induced absorbance changes (GIACs), which are associated with primary events of gravity sensing, were detected in the growing zones of sporangiophores. After placing sporangiophores horizontally, GIACs were detected after a latency of about 5 min, i.e. 15–25 min prior to gravitropic bending. The spectroscopic properties of the GIACs indicate that gravitropic stimulation could imply the reduction of cytochromes. The GIACs were spectrally distinct from light-induced absorbance changes (LIACs), showing that the primary responses of the light and gravity transduction chains are different. A dual stimulation with gravity and light generated GIAC-LIACs which were distinct from the absorbance changes occurring after the single stimuli and which indicate that light and gravity interact early in the respective transduction chains. Received: 2 September 1999 / Accepted: 9 November 1999     

A spaceflight experiment for the study of gravimorphogenesis and hydrotropism in cucumber seedlings.

Seedlings of Cucurbitaceae plants form a protuberance, termed peg, on the transition zone between hypocotyl and root. Our spaceflight experiment verified that the lateral positioning of a peg in cucumber seedlings is modified by gravity. It has been suggested that auxin plays an important role in the gravity controlled positioning of a peg on the ground. Furthermore, cucumber seedlings grown in microgravity developed a number of the lateral roots that grew towards the water containing substrate in the culture vessel, whereas on the ground they oriented perpendicular to the primary root growing down. The response of the lateral roots in microgravity was successfully mimicked by clinorotation of cucumber seedlings on the three dimensional clinostat. However, this bending response of the lateral roots was observed only in an aeroponic culture of the seedlings but not in solid medium. We considered the response of the lateral roots in microgravity and on clinostat as positive hydrotropism that could easily be interfered by gravitropism on the ground. This system with cucumber seedlings is thus a useful model of spaceflight experiment for the study of the gravimorphogenesis, root hydrotropism and their interaction.     

Differential localization of arabinan and galactan side chains of rhamnogalacturonan 1 in cambial derivatives

 The development of pectin structural features during the differentiation of cambial derivatives was investigated in aspen (Populus tremula L. × P. tremuloides Michx.) using biochemical and immunocytochemical methods. Comparisons were also made between active and resting tissues. Active tissues, in particular cambial cells and phloem derivatives, were characterized by a high pectin content. Use of antibodies raised against arabinan side chains of rhamnogalacturonan 1 (LM6), as well as biochemical analysis, revealed an obvious decrease from the cortex to the differentiating xylem. Galactan side chains, detected with LM5 antibodies, were present mainly in the cambial zone and enlarging xylem cells. In contrast, they were totally absent from sieve-tube cell walls. Image analysis of LM5 immunogold labelling in the cambial zone showed a clustered distribution of galactan epitopes in the radial walls, a distribution which might result from the association of two different periodic processes, namely the exocytosis of galactan and wall expansion. Cessation of cambial activity was characterized by cell wall thickening accompanied by a sharp decrease in the relative amount of pectin and a lowering of the degree of methylesterification. The data provide evidence that the walls of phloem and xylem cells differ in their pectin composition even at a very early stage of commitment. These differences offer useful tools for identifying the initial cells among their immediate neighbours. Received: 12 June 1999 / Accepted: 20 October 1999