Polarity of statocytes in lentil seedling roots grown in space (Spacelab D1 Mission)

A morphometric analysis of root statocytes was performed on seedlings of lentil ( Lens culinaris L., cv. Verte du Puy) in order to determine the effects of microgravity on the polarity of these cells. Seedlings were grown: (1) on the ground, (2) in microgravity, (3) on a 1 g centrifuge in space, (4) first in microgravity and then placed on a 1 g centrifuge for 3 h. Dry seeds were hydrated in space (except for the ground control) for 25 h in darkness at 22°C in the Biorack facility developed by the European Space Agency. At the end of the experiment, the seedlings were photographed and fixed in glutaraldehyde in the Biorack glove box. The average shape of the statocytes and the location of endoplasmic reticulum, amyloplasts and nucleus in the cells were analysed in the four samples. By considering the cell shape, it appears that the morphology of the statocytes on the ground was different from that observed in the space samples. Cell polarity was similar in microgravity and in the centrifuged samples except for the distribution of the amyloplasts. These organelles were not distributed at random in near zero gravity, and they were more numerous in the proximal than in the distal half. Moreover, the statoliths were more voluminous in microgravity than in the centrifuged samples. The nucleus was closer to the cell center in the statocytes of roots grown in microgravity than in statocytes of roots grown in microgravity and then placed on the 1 g centrifuge for 3 h. It is hypothesized that the nucleus is attached to the cell periphery and that its location is dependent upon gravity.     

Sensitivity to gravistimulus of lentil seedling roots grown in space during the IML 1 Mission of Spacelab

The gravitropic curvature of seedlings of lentil ( Lens culinaris L. cv. Verte du Puy) grown in microgravity and stimulated on the 1 g centrifuge for 5 to 60 min was followed by time lapse photography in near weightlessness in the frame of the IML 1 Mission of Spacelab. In microgravity, the root tip could overshoot the direction of the 1 g acceleration after bending, whereas roots stimulated on the ground did not reach the direction of the gravity vector. On earth, there is, therefore, a regulation (inhibition of root curvature), which is gravity dependent. In space, the initial rate of curvature as well as the amplitude of curvature varied as a function of the quantity of stimulation (Q, in g min). For a given quantity of stimulation, the rate of curvature remained constant for 80 min. The bending has thus a certain inertia, which is linked to the mechanism of differential growth. The presentation time (Tp) of the lentil root was calculated by extrapolation to zero curvature of the regression line representing either the initial rate of curvature or the amplitude of curvature at 2 h after the end of the stimulation. Tp was estimated to 27 and 26 s. respectively. These results confirm the values of Tp obtained by clinostats, and they also lead to a reconsideration of the causes of the kinetics of root curvature.     

Densitometric analysis of nuclear DNA content in lentil roots grown in space

Lentil seedlings were grown for 28 h in space, on board Spacelab (IML 1 Mission) and growth of the primary root was analysed. The length of cortical cells was less in near weightlessness than on the 1 g centrifuge (flight control) and mitotic index was lower but there was no apparent perturbation in the mitosis. To further investigate which phase of cell cycle was modified, densitometric analysis of nuclear DNA content in cortical cells was carried out by the mean of an image processing system (SAMBA). In microgravity there was a decrease in DNA synthesis and a promotion of the arrest in the G2 phase of cell cycle. These results, and other ones obtained elsewhere on a slowly rotating clinostat, led us to think that in microgravity the perturbation of the gravisensing cells and/or the absence of convection could account for the modification of cell growth registered in the primary root.     

Cell cycle and cell differentiation in lentil roots grown on a slowly rotating clinostat

Root growth was studied for seedlings of lentil ( Lens culinaris L. cv. Verte du Puy) grown for 27 h either on a slowly rotating clinostat (0.9 rev. min⁻¹) of vertically (controls). Horizontal clinorotation was employed, so that the longitudinal axis of the root was parallel to the axis of the rotation. Morphological (root length and orientation) and cellular (cell proliferation and cell elongation) parameters were studied. The cell cycle was also analysed by flow cytometry. Root length deviation of the roots from the initial orientation was observed on the clinostat; this deviation could be due to spontaneous oscillation. Cell elongation of the clinostat-rotated roots occurred closer to the tip than in the vertical roots, but the mitotic index was not modified. Clinorotation did not change the frequencies of the G1, S and G2 phases of the cell cycle. These results were compared to those obtained during the D1 mission on Spacelab, 1985. The effects of microgravity on root orientation and mitotic index were not simulated by clinorotation.     

Photomorphogenesis and pigment induction in lentil seedling roots exposed to low light conditions

Although roots are normally hidden in soil, they may inadvertently be exposed to low light levels in experiments or in natural conditions through cracks or light transmittance through the soil. Light has been implicated in root morphogenesis. Thus, effects of low light conditions on lentil (Lens culinaris L. cv. Verte du Puy) root morphology and root pigmentation were studied. Lentil seedlings were grown in peat or transparent, nutrient-fortified agar at a 12-h light (PAR 240 μmol · m(-2) · s(-1)), 12-h dark cycle. Roots were exposed to low levels (≈ 1-10 μmol · m(-2) · s(-1)) of broadband white light, either directly or indirectly by aboveground light penetrating the growth medium. Control roots were grown in darkness. In situ spectroscopy was used to measure transmittance and reflectance spectra of intact root tissue by mounting the upper part of the primary root directly in a spectrophotometer equipped with an integrating sphere attachment. The transmittance and reflectance spectra were used to calculate the in situ root absorbance spectrum. Absorbance bands were found in the regions 480-500 nm and 650-680 nm, possibly due to low levels of root-localised carotenoids and chlorophylls, respectively. Low light levels (≈ 1-10 μmol · m(-2) · s(-1) ) transmitted through the growth medium significantly increased root pigment concentration and root biomass, and altered root morphology by enhancing lateral root formation and inhibiting root elongation relative to roots grown in complete darkness. The light-induced changes in root morphogenesis and pigmentation appear to be primarily due to upper root light perception.     

Oscillatory growth movements of roots in weightlessness

The gravitropic curvature of lentil roots ( Lens culinaris L. cv. Verte du Puy), grown in near weightlessness and stimulated on a 1-g centrifuge for 5 to 60 min was followed by time lapse photography. The experiment was carried out in the frame of the IML 1 Mission of Spacelab. Due to the applied acceleration field, the tip of the roots bent and reoriented with respect to the acceleration vector. However, visual inspection of the data could indicate an oscillatory movement superimposed on the gravitropic reorientation.
We applied two signal processing techniques, fast Fourier transform (FFT) and maximum entropy spectral analysis (MESA), to provide quantitative data about the oscillatory movements of the lentil roots under gravity free conditions. In the case with very few data points in the time series the MESA method is superior to the conventional FFT. In the lentil root movements, the Fourier analysis could not extract and resolve the oscillatory signals present in the time series. The MESA approach revealed oscillations with periods around 35 and 50 min for the present lentil roots.
Circumnutations are, therefore, present in roots also in weightlessness.     

Actin filaments responsible for the location of the nucleus in the lentil statocyte are sensitive to gravity

The location of the nucleus in statocytes or lentil roots grown: 1), at 1 g on the ground, 2), on a 1 g centrifuge in space, 3), in simulated microgravity on a slowly rotating clinostat (0.9 rmp) 4), in microgravity in space was investigated and statistically evaluated. In cells differentiated at 1 g on the ground, the nuclear membrane was almost in contact with the plasmalemma lining the proximal cell wall, whereas in statocytes of roots crown on the clinostat there was a distance of 0.47 micrometers (horizontal clinorotation) and or 0.76 micrometers (vertical clinorotation) between these membranes. However, in microgravity the nucleus was the most displaced, 0.87 micrometers from the proximal cell wall. Centrifugation of vertically grown roots in the root-tip direction showed that the threshold of centrifugal force to detach all nuclei from the proximal cell wall was about 40 g. In statocytes developed in the presence of cytochalasin B at 1 g the nuclei were sedimented on the amyloplasts at the distal cell pole, demonstrating that the location of the nucleus depends on actin filaments. The results obtained are in agreement with the hypothesis that gravity causes a tension of actin filaments and that this part of the cytoskeleton undergoes a relaxation in microgravity.     

Genetics of resistance to ascochyta blight (Ascochyta lentis) of lentil and the identification of closely linked RAPD markers

 Foliar resistance to Ascochyta lentis is controlled at a single major locus by a dominant gene (AbR ₁ ) in the lentil accession ILL5588 (cv ‘Northfield’). Flanking RAPD markers that are closely linked to the resistance locus in coupling phase were identified by bulked segregant analysis. Out of 261 decanucleotide primers screened 7 produced a polymorphic marker that segregated with the resistance locus, and all markers were found to exist within a single linkage group. Five of the seven RAPD markers were within 30 cM of the resistance locus. Log likelihood analysis for detecting QTL associated with the foliar resistance revealed that a single narrow peak accounted for almost 90% of the variance of resistance between the bulks. Preliminary mapping in an F₃ population revealed that the closest flanking markers were approximately 6 and 14 centiMorgans (cM) away from the resistance locus. These markers should be useful for the discrimination of resistant germplasm through marker-assisted selection in future breeding programmes and represent the first essential step towards the map-based cloning of this resistance gene. Received: 18 December 1997 / Accepted: 9 June 1998     

Effect of benzylaminopurine on in vitro and in vivo root development in lentil, Lens culinaris Medik

The effect of benzylaminopurine (BAP) on the formation of roots from lentil shoots regenerated on media containing BAP was studied. Seedling shoot tips, first nodes and bractlets, and immature seeds cultured on the initiation media containing 2.25 or 0.225 mg/l of BAP regenerated multiple bud shoots. The regenerated shoots formed roots in percentages ranging from 4.6 to 39.9% on a rooting medium (R medium) containing 2 mg/l of indoleacetic acid. Rooting success on R medium depended upon the cytokinin used in the initiation media, its concentration, and the time elapsed during shoot formation on these media prior to transplanting regenerated shoots to R medium. In vivo study of root growth of lentil seedlings demonstrated the strong inhibitory effect of BAP on root growth reflected in a drastic reduction of the mitotic index of the root meristem. Received: 27 August 1996 / Revision received: 12 December 1996 / Accepted: 15 January 1997     

Oxidation of benzylamine Br-derivatives by lentil seedling copper-amine oxidase

ABSTRACT

Copper amine oxidase was shown to be able to catalyse the oxidative deamination of 2-, 3- and 4-Br-derivatives of benzylamine to the corresponding aldehydes, that all absorb at 250 nm. This change in the absorption spectrum made it possible to follow the enzyme reaction. 2-Br-benzylamine, 3-Br-benzylamine, and 4-Br-benzylamine showed K_m values similar to benzylamine, but 3-Br-benzylamine showed a slower k_c, which allows it to be a catalytically more efficient substrate. Under anaerobic conditions the native enzyme oxidised 1 equivalent of all Br-derivatives and released 1 equivalent of aldehyde per enzyme subunit. These findings demonstrate that, in anaerobic conditions, the enzyme can oxidise substrates with a single incomplete turnover. The possible involvement of the cofactor 6-hydroxydopa quinone and of a negatively charged residue in the oxidation of Br-benzylamines is discussed.     

The role of extracellular free-calcium gradients in gravitropic signalling in maize roots

Gravitropism in roots has been proposed to depend on a downward redistribution of calcium across the root cap. However, because of the many calcium-binding sites in the apoplast, redistribution might not result in a physiologically effective change in the apoplasmic calcium activity. To test whether there is such a change, we measured the effect of gravistimulation on the calcium activity of statocyte cell walls with calcium-specific microelectrodes. Such a measurement must be made on a tissue with gravity sensing cells at the surface. To obtain such a tissue, decapped maize roots (Zea mays L. cv. Golden Cross Bantam) were grown for 31 h to regenerate gravitropic sensitivity, but not root caps. The calcium activity in the apoplasm surrounding the gravity-sensing cells could then be measured. The initial pCa was 2.60 ± 0.28 (approx 2.5 mM). The calcium activity on the upper side of the root tip remained constant for 10 min after gravistimulation, then decreased 1.7-fold. On the lower side, after a similar lag the calcium activity increased 1.6-fold. Control roots, which were decapped but measured before recovering gravisensitivity (19 h), showed no change in calcium activity. To test whether this gradient is necessary for gravitropic curvature, we eliminated the calcium activity gradient during gravitropism by applying a mobile calcium-binding site (di-nitro-BAPTA; 1,2-bis(2-amino-5-nitro-phenoxy)ethane-N,N,N,N-tetraacetic acid) to the root cap; this treatment eliminated gravicurvature. A calcium gradient may be formed by proton-induced calcium desorption if there is a proton gradient. Preventing the formation of apoplastic pH gradients, using 10 and 50 mM 2-(N-morpholino)ethanesulfonic acid (Mes) buffer or 10 mM fusicoccin to stimulate proton excretion maximally, did not inhibit curvature; therefore the calcium gradient is not a secondary effect of a proton gradient. We have found a distinct and rapid differential in the apoplasmic calcium activity between the upper and lower sides of gravistimulated maize root tips which is necessary for gravitropism.Abbreviations BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid - FC fusicoccin - Mes 2-(N-morpholino)ethanesulfonic acid The authors thank Phyllis Woolwine for drawing Fig. 1, Dr. Sarbjit Virk for assistance with total calcium measurements, Dr. Paul Sampson for statistical advice, and Michael Newton for developing the EM algorithm to analyze the time-series data. This work was supported by NASA grant NAGW-1394 and by a NASA Research Associateship to T.B. through NASA grant NAGW-70.     

Automorphogenesis of maize roots under simulated microgravity conditions

Plant seedlings show exaggerated growth responses on a three-dimensional clinostat. Such an automorphogenesis appears to be one of major factors which govern the life cycle of higher plants under a microgravity environment. On the three-dimensional clinostat, maize roots exhibited curvatures in three different portions; 1) the basal region just protruding from the coleorhiza, 2) the region between the mature and the elongation zone, and 3) the elongation zone, several mm from the tip. Even non-clinostatted control roots showed some degree of curvature. The curvature occurred at random without any dorsiventrality. There was no difference in the osmotic concentration of the cell sap between the convex and the concave halves of any region. However, the convex, rapidly expanding side exhibited a higher extensibility of the cell wall in some regions, which appears to be a cause of the curvature. In order to understand the role of gravity in regulation of plant growth and development, we should clarify a series of events by which an automorphogenesis is induced under simulated microgravity conditions.     

Site of graviperception in roots: a re-examination

Two lines of evidence have been cited to support the assertion that the root cap is the sole site of graviperception in the root. The first evidence is based on surgical removal of the cap, which abolishes the response to gravity. This is sufficient to conclude that the cap is involved in gravitropism, but not to conclude that the cap is the site of graviperception. The second is based on the results of centrifugation experiments, in which different parts of the plant are subjected to different centrifugal forces. The data from such experiments have been cited to support the conclusion that the perception of gravity is limited to the rootcap. However, these data actually support the conclusion that gravity is perceived throughout the root tip, and not only in the root cap. We believe that the data support the conclusion that the root cap is involved in root gravitropism, but that there is inadequate evidence to conclude that the cap is the sole site of graviperception.     

Quantification of curvature production in cylindrical organs, such as roots and hypocotyls

Differential growth curvature rate (DGCR), defined as the spatial derivative of the tropic speed, was derived as a measure of curvature production in cylindrical organs. Its relation to usual concepts, such as curvature (kappa), rate of curvature (dkappa/dt) and differential growth profiles, was determined. A root gravitropism model, testing the hypothesis of one and two motors, exemplified its capabilities.DGCR was derived using cylindrical geometry and its meaning was obtained through a curvature conservation equation. The root gravitropism model was solved using a discrete difference method on a computer.DGCR described curvature production independently of growth, and was superior to dkappa/dt, which underestimated production. Moreover, DGCR profiles were able to differ between one and two motors, while profiles of kappa and dkappa/dt were not.The choice of the measure of curvature production has a large impact on experimental results, in particular when spatial and temporal patterns of differential growth need to be determined. DGCR was shown to fulfill the accuracy needed in the quantification of curvature production and should thus serve as a helpful tool for measurements.     

兵豆萌发过程中初生根皮层细胞的细胞周期活动的动态分析

观察了兵豆(Lens culinaris Medic.)初生根原皮层组织的细胞周期在其种子萌发过程中时间和空间上的动态变化.免疫组织化学和细胞学证据表明,原皮层细胞分别在种子吸胀大约13 h和17 h开始DNA复制和细胞分裂.最早进行DNA复制和细胞分裂的细胞位于远基端1 mm附近,但这些分裂细胞的DNA复制是在种子成熟过程中完成的,而不是在萌发后.第一个细胞周期的激活样式表明,这些细胞并不同步激活,而是依次进入细胞周期,且进入的次序与自身在根尖中的相对位置有关.在兵豆初生根原皮层组织中,邻近位置上的细胞的细胞周期同步化程度较高.     

兵豆萌发过程中初生根皮层细胞的细胞周期活动的动态分析

观察了兵豆 (LensculinarisMedic.)初生根原皮层组织的细胞周期在其种子萌发过程中时间和空间上的动态变化。免疫组织化学和细胞学证据表明 ,原皮层细胞分别在种子吸胀大约 13h和 17h开始DNA复制和细胞分裂。最早进行DNA复制和细胞分裂的细胞位于远基端 1mm附近 ,但这些分裂细胞的DNA复制是在种子成熟过程中完成的 ,而不是在萌发后。第一个细胞周期的激活样式表明 ,这些细胞并不同步激活 ,而是依次进入细胞周期 ,且进入的次序与自身在根尖中的相对位置有关。在兵豆初生根原皮层组织中 ,邻近位置上的细胞的细胞周期同步化程度较高。     

Abscisic acid,xanthoxin and violaxanthin in the caps of gravistimulated maize roots

The occurrence and distribution of abscisic acid (ABA), xanthoxin (Xa) and the carotenoid violaxanthin (Va) were investigated in root tips of maize (Zea mays L. cv. Merit). In roots grown in the dark, Va and ABA were present in relatively high amounts in the root cap and in low amounts in the adjacent terminal 1.5 mm of the root. Xanthoxin was present in equal concentrations in both regions. In roots exposed to light, the ABA distribution was reversed, with relatively low levels in the root cap and high levels in the adjacent 1.5-mm segment. Light also caused a decrease in Va in both regions of the root and an increase in Xa, especially in the cap. In the maize cultivar used for this work, light is necessary for gravitropic curving. This response occurs within the same time frame as the light-induced ABA redistribution as well as the changes in the levels of Va and Xa. These data are consistent with a role for ABA in root gravitropism and support the proposal that Xa may arise from the turnover of Va.Abbreviations ABA abscisic acid - GC gas chromatography - HPLC high-performance liquid chromatography - GC-MS gas chromatography-mass spectroscopy - V_a violaxanthin - Xa xanthoxin     

Computer-based video digitizer analysis of surface extension in maize roots

We used a video digitizer system to measure surface extension and curvature in gravistimulated primary roots of maize (Zea mays L.). Downward curvature began about 25 +/- 7 min after gravistimulation and resulted from a combination of enhanced growth along the upper surface and reduced growth along the lower surface relative to growth in vertically oriented controls. The roots curved at a rate of 1.4 +/- 0.5 degrees min-1 but the pattern of curvature varied somewhat. In about 35% of the samples the roots curved steadily downward and the rate of curvature slowed as the root neared 90 degrees. A final angle of about 90 degrees was reached 110 +/- 35 min after the start of gravistimulation. In about 65% of the samples there was a period of backward curvature (partial reversal of curvature) during the response. In some cases (about 15% of those showing a period of reverse bending) this period of backward curvature occurred before the root reached 90 degrees. Following transient backward curvature, downward curvature resumed and the root approached a final angle of about 90 degrees. In about 65% of the roots showing a period of reverse curvature, the roots curved steadily past the vertical, reaching maximum curvature about 205 +/- 65 min after gravistimulation. The direction of curvature then reversed back toward the vertical. After one or two oscillations about the vertical the roots obtained a vertical orientation and the distribution of growth within the root tip became the same as that prior to gravistimulation. The period of transient backward curvature coincided with and was evidently caused by enhancement of growth along the concave and inhibition of growth along the convex side of the curve, a pattern opposite to that prevailing in the earlier stages of downward curvature. There were periods during the gravitropic response when the normally unimodal growth-rate distribution within the elongation zone became bimodal with two peaks of rapid elongation separated by a region of reduced elongation rate. This occurred at different times on the convex and concave sides of the graviresponding root. During the period of steady downward curvature the elongation zone along the convex side extended farther toward the tip than in the vertical control. During the period of reduced rate of curvature, the zone of elongation extended farther toward the tip along the concave side of the root. The data show that the gravitropic response pattern varies with time and involves changes in localized elongation rates as well as changes in the length and position of the elongation zone. Models of root gravitropic curvature based on simple unimodal inhibition of growth along the lower side cannot account for these complex growth patterns.     

Modification of the gravitropic response of seedling roots of raneseed (Brassica napus) transformed by Agrobacterium rhizogenes A4

We have examined the growth and gravitropic response of seedling roots of rapeseed ( Brassica napus . CrGC5–1) transformed by Agrobacterium rhizogenes A4, in order to evaluate if this could constitute a new model system for the study of gravitropism. The transformed clone chosen for study had integrated full-length TL- and TR-DNA from pRi (the root inducing plasmid), and thus included all of the agrobacterial genes potentially involved in the modified phenotype of transformed plants. In the vertical position, the growth rate of transformed roots was higher than controls. During 24 h of continuous stimulation, the optimal angle for gravitropic bending in normal roots was 135° (with respect to the gravity axis), with decreasing response at 90° and 45°. For transformed roots, slight curvature developed at 45° and at 90°, and stronger curvature was observed at 135°, though transformed roots tips never reached the vertical position. The minimum stimulation time necessary to elicit a response (presentation time) was also determined: it was signficantly shorter in normal roots (80 s) than in transformed ones (120 s). The results show that pRi transformed roots are less sensitive to gravity than normal roots.     

Growth and gravitropism of corn roots in solution

Growth and early gravitropic responses of corn roots in solution have been studied using time-lapse photography. Aeration was required for both root growth and gravitropism. The optimum pH for gravitropism was in the range 5 to 6. The bending response seemed to be greater for roots in non-buffered solution than in buffered solution. Fastest growth and maximum curvature occurred with about 0.2 mol m⁻³ Ca²⁺. Under some conditions, the gravitropic response started with apparently negligible time delay after the start of the gravitropic stimulus. This may denote graviperception in or near the elongation zone itself. This mechanism for early but relatively weak gravitropism may help to explain a variety of gravitropic responses such as the ‘early wrong way’ curvature, and the behaviour of roots whose columella cells lack amyloplasts. More rapid bending appears to start at about 20 min, which is consistent with observations on roots in humid air and with the accepted statolith model of perception in the root cap.