The effect of uni-axial clinostat rotation on germination and root anatomy of Phaseolus vulgaris L.

ABSTRACT

Phaseolus vulgaris L. seed germination and seedling root anatomy were investigated on a slowly rotating clinostat in 1g. Clinostat rotating seeds were oriented as follows: the first group with the longer axis parallel to the rotation pole (horizontal), the other with the longer axis normal to the rotation pole with due attention to the position of the root apex primordium in the dry seeds (vertical). Germination time, percent germination and curvature of developing roots were monitored. Furthermore, the anatomy of the root apex was quantitatively analysed. Seeds placed on the clinostat germinated earlier than controls, and columella cells of roots developed while rotating lost the strict polarity with the nucleus positioned near the proximal periclinal cell wall and amyloplasts sedimented on the distal periclinal wall. Irrespective of seed orientation on the rotation axis, loss of cell polarity occurred as well as a decrease in starch content, modification in cell size, and damage to statocytes whose walls appeared partially digested. Cell size in the elongation zone was also larger in roots rotating on the clinostat than in controls, both in vertically and horizontally placed specimens. Our results demonstrate that prolonged rotation has an effect on the statocyte that continuously perceives gravity from ever-changing directions, although this effect is irrespective of seed position on the rotating axis in P. vulgaris.     

Ultrastructure and movements of cell organelles in the root cap of agravitropic mutants and normal seedlings of Arabidopsis thaliana

The root anatomy and ultrastructure of the agravitropic Arabidopsis thaliana L. mutants Dwf and aux-1 were compared with the gravitropic mutant aux-2 and the wild type (WT) in an attempt to find an explanation for the lack of response to gravity. No differences were found in the organization of the root cap. The central part of the cap (columella) contains 5 storeys of developing, functioning and degenerating statocytes. Their ultrastructure is very similar in all four types of plant. Particular attention was paid to the distribution of rough endoplasmie reticulum (ER). Both in the WT and the mutants the ER is concentrated in the distal part at the "floor" of the cell.
Light micrographs were used to compare the sedimentation rates of movable cell structures in normal and agravitropic root statocytes. A longitudinal movement of amyloplasts and nuclei was observed when the roots were inverted. In WT and aux-2 the rates were on average 6.3 μm h⁻¹ (amyloplasts) and 2.1 μm h⁻¹ (nucleus). In aux-1 the sedimentation rates were significantly lower: 2.4 and 0.6 μm h⁻¹, respectively. Based on magnified electron micrographs of normal and inverted statocytes a morphometrical analysis of the distribution and redistribution of amyloplasts, nuclei, mitochondria, vacuoles and ER was made. The only significant difference was found in the redistribution of amyloplasts between aux-1 and the gravitropical normal types.     

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.     

Ultrastructure and movements of cell structures in normal pea and an ageotropic mutant

The pea mutant (Pisum sativum ageotropum) and the normal pea (P. sativum cv. Sabel) were compared in order to see if there were any differences in root anatomy or submorphology which could explain the presumed ageotropic behaviour of the mutant. In both types the root cap consists of a central core (columella) distinct from the peripheral part. The core contains five to six rows of columella cells, each consisting of 10 to 16 storeys of statocytes. The ultrastructure of the columella cells in the two types is very similar; the main difference is confined to the distribution of rough endoplasmic reticulum (ER), which in the mutant statocytes is evenly distributed throughout the cell, while in the normal pea statocytes it is mainly concentrated in the distal part at the “floor” of the cell. Using light micrographs, the movement of amyloplasts and nuclei have been followed in detail during a 40 min inversion period. The pattern of movement of the amyloplasts is apparently identical in the two types and the distances moved during the inversion period are 39 μm and 44 μm in the normal and mutant statocytes, respectively. The nucleus has not been observed to move in normal pea; a slight rearrangement of the nucleus position can be observed during the period 30 to 40 min after the start of inversion of the mutant. Based on magnified electron micrographs of the statocytes a morphometrical analysis was made of five cell structures – amyloplasts, nuclei, mitochondria, vacuoles and ER – which appeared to be freely movable or redistributable under the influence of the gravitational force.     

Root growth and statocyte polarity in lentil seedling roots grown in microgravity or on a slowly rotating clinostat

The ability of clinostats to simulate microgravity was evaluated by comparing lentil ( Lens culinnrias L. cv. Verte du Puy) seedlings grown in space (Spacelab D1 Mission) with seedlings grown on a slowly rotating elinostat. Seeds were germinated and incubated for 25.5 h at 22°C (1) in microgravity, (2) on a 1g-centrifuge in space. (3) on a slowly rotating elinostat and (4) on the ground. Morphological (root length and orientation) and ultrastructural (distribution of amyloplasts, location of the nucleus in statocytes) parameters were studied. For clinostat experiments, two different configurations were employed: the longitudinal axis of the root was parallel (horizontal elinorotation) or perpendicular (vertical elinorotation) to the axis of rotation. the same configurations were used for the lg-controls. Root length and orientation were similar for roots grown on the clinostat and in microgravity. The amyloplasts were identically distributed in statocytes of horizontally clinorolated roots and in statocytes differentiated in microgravity. However, the location of the nucleus was similar in vertically rotated roots and microgravity samples. Since the involvement of the nucleus in graviperception is not known, it can be concluded that horizontal clinorotation simulates microgravity better than vertical elinorotation.     

ARL2, ARG1 and PIN3 define a gravity signal transduction pathway in root statocytes

ALTERED RESPONSE TO GRAVITY1 (ARG1) and its paralog ARG1-LIKE2 (ARL2) are J-domain proteins that are required for normal root and hypocotyl gravitropism. In this paper, we show that both ARL2 and ARG1 function in a gravity signal transduction pathway with PIN3, an auxin efflux facilitator that is expressed in the statocytes. In gravi-stimulated roots, PIN3 relocalizes to the lower side of statocytes, a process that is thought to, in part, drive the asymmetrical redistribution of auxin toward the lower flank of the root. We show that ARL2 and ARG1 are required for PIN3 relocalization and asymmetrical distribution of auxin upon gravi-stimulation. ARL2 is expressed specifically in the root statocytes, where it localizes to the plasma membrane. Upon ectopic expression, ARL2 is also found at the cell plate of dividing cells during cytokinesis, an area of intense membrane dynamics. Mutations in ARL2 and ARG1 also result in auxin-related expansion of the root cap columella, consistent with a role for ARL2 and ARG1 in regulating auxin flux through the root tip. Together these data suggest that ARL2 and ARG1 functionally link gravity sensation in the statocytes to auxin redistribution through the root cap.     

Early root cap development and graviresponse in white clover (Trifolium repens) grown in space and on a two-axis clinostat.

White clover (Trifolium repens) was germinated and grown in microgravity aboard the Space Shuttle (STS-60, 1994; STS-63, 1995), on Earth in stationary racks and in a slow-rotating two-axis clinostat. The objective of this study was to determine if normal root cap development and early plant gravity responses were dependent on gravitational cues. Seedlings were germinated in space and chemically fixed in orbit after 21, 40, and 72 h. Seedlings 96 h old were returned viable to earth. Germination and total seedling length were not dependent on gravity treatment. In space-flown seedlings, the number of cell stories in the root cap and the geometry of central columella cells did not differ from those of the Earth-grown seedlings. The root cap structure of clinorotated plants appeared similar to that of seedlings from microgravity, with the exception of three-day rotated plants, which displayed significant cellular damage in the columella region. Nuclear polarity did not depend on gravity; however, the positions of amyloplasts in the central columella cells were dependent on both the gravity treatment and the age of the seedlings. Seedlings from space, returned viable to earth, responded to horizontal stimulation as did 1 g controls, but seedlings rotated on the clinostat for the same duration had a reduced curvature response. This study demonstrates that initial root cap development is insensitive to either chronic clinorotation or microgravity. Soon after differentiation, however, clinorotation leads to loss of normal root cap structure and plant graviresponse while microgravity does not.     

Adaptation of Mature Spathiphyllum sp. Plant to Prolonged Simulated Microgravity

Mature Spathiphyllum sp. plants were grown in soil in pots under clinostatic or stationary condition. Photosynthetic rate fell to almost zero after 30 d in clinostat, but recovered to more than 60% of the original on the 62nd day. Grana in chloroplasts remained normal in clinostat. There were few starch grains in chloroplasts after 30 d in clinostat, and almost none on the 62nd day, but numerous globular objects of low electronic density were observed. These were probably related to the adaptative changes of photosynthesis. Cell division in meristematic cells of root cap ceased after 30 d in clinostat, and then resumed to attain a rate even higher than that in the control plant on 62nd day, Further the length of the tap root was more than double that in the control plants. Some of the meristematic cells in the root cap under clinstatic condition transformed into statocyst cells, which senesced and were not transformed into secretory cells. No exocytosis was observed in plant in clinostatic condition, albeit the cell wall was broken from abrasion during root growth, resulting in pseudo-exocytosis, and from which the exudates acted as lubricants for the free extension of the root tip during root growth.     

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

The location of the nucleus in statocytes of lentil roots grown: I), 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 grown on the clinostat there was a distance of 0.47 μm horizontal clinorotation) and of 0.76 μm vertical clinorotation) between these membranes. However, in microgravity the nucleus was the most displaced, 0.87 μm 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.     

The growth and differentiation of root cap columella cells and the proper root grown in the stationary conditions and under clinorotation

The results of light- and electron-microscopic investigations of root apices of Beta vulgaris 3-day-old seedlings grown in the stationary conditions and under clinorotation are presented. It was shown that ultrastructure and topography of organelles in root cap statocytes (graviperceptive cells) and in the cells of distal elongation zone clearly reflected the different direction in their growth and differentiation in space and time in dependence on specialization and functions. Cell growth and genetically determined differentiation occur similarly to control, although certain differences in ultrastructure are evident on metabolism changes.     

银苞芋成熟植株对长时间模拟微重力环境的适应

将成熟的银苞芋（Ｓｐａｔｈｉｐｈｙｌｌｕｍ ｓｐ．）植株连同装有栽培土的花盆置于模拟微重力装置上,旋转３０ ｄ 和６２ ｄ,发现旋转３０ ｄ 的植株光合速率减弱,至６２ ｄ 恢复到６０％ 以上。其主要细胞学变化如下：叶绿体基粒无变化,旋转３０ ｄ 其叶绿体内含极少淀粉颗粒,至６２ ｄ 淀粉颗粒全部消失,但出现多量电子密度低的球形物质,显然在微重力下光合代谢发生了适应性的变化;旋转３０ ｄ 的植株其根冠分生组织细胞暂停分裂,至６２ ｄ 其分裂速度超过对照,主根的长度超过对照的１ 倍以上;旋转植株根冠的分化组织细胞转化为平衡囊细胞,但不能继续转化为分泌细胞,而直接老化,故无胞吐作用。在根伸长时土粒擦破老化细胞壁形成拟胞吐作用,胞吐物成了根尖伸长时的润滑剂     

Adenosine kinase modulates root gravitropism and cap morphogenesis in Arabidopsis

Adenosine kinase (ADK) is a key enzyme that regulates intra- and extracellular levels of adenosine, thereby modulating methyltransferase reactions, production of polyamines and secondary compounds, and cell signaling in animals. Unfortunately, little is known about ADK's contribution to the regulation of plant growth and development. Here, we show that ADK is a modulator of root cap morphogenesis and gravitropism. Upon gravistimulation, soluble ADK levels and activity increase in the root tip. Mutation in one of two Arabidopsis (Arabidopsis thaliana) ADK genes, ADK1, results in cap morphogenesis defects, along with alterations in root sensitivity to gravistimulation and slower kinetics of root gravitropic curvature. The kinetics defect can be partially rescued by adding spermine to the growth medium, whereas the defects in cap morphogenesis and gravitropic sensitivity cannot. The root morphogenesis and gravitropism defects of adk1-1 are accompanied by altered expression of the PIN3 auxin efflux facilitator in the cap and decreased expression of the auxin-responsive DR5-GUS reporter. Furthermore, PIN3 fails to relocalize to the bottom membrane of statocytes upon gravistimulation. Consequently, adk1-1 roots cannot develop a lateral auxin gradient across the cap, necessary for the curvature response. Interestingly, adk1-1 does not affect gravity-induced cytoplasmic alkalinization of the root statocytes, suggesting either that ADK1 functions between cytoplasmic alkalinization and PIN3 relocalization in a linear pathway or that the pH and PIN3-relocalization responses to gravistimulation belong to distinct branches of the pathway. Our data are consistent with a role for ADK and the S-adenosyl-L-methionine pathway in the control of root gravitropism and cap morphogenesis.     

Gravity induced changes in intracellular potentials in statocytes of cress roots

Two glass microelectrodes were inserted from opposite sides of the root cap into statocytes of Lepidium sativum L. immersed in medium with or without cytochalasin D (CD). Intracellular potentials (Eis) of statocytes were measured with reference to an earthed electrode in the bathing solution. In the absence of CD, Ei values were -160 +/- 2 mV (n = 52) in vertical roots. During the recording of Eis, the roots were tilted from the vertical by 45 degrees so that in a tilted root one electrode was on the upper side and the other on the lower side; after 5 min the roots were returned to the vertical. At approximately 64 s after tilting (lasting 5-15 s) there was a transient lowering of Ei (more negative) by an average of 4.7 mV on both the upper and lower sides (n = 52). In some cases, this decrease in Ei was preceded by a transitory increase. Returning the roots to the vertical resulted in a response similar to that obtained by tilting. In roots treated with CD at a concentration of 3 (microM for 1 h, the initial Ei was -145 +/- 2 mV (n = 43), and the lowering of Ei on position change (tilting or returning) was smaller (2.0 mV) in some statocytes (n = 50) and higher (8.1 mV) in others (n = 14) compared to control roots (without and with DMSO). A higher concentration (10 microM) of CD and longer treatment (2 h) further reduced the decrease in Ei (1.1 mV) on position change (n = 26). The observed effects of CD support the hypothesis that statoliths in statocytes are anchored by actin filaments to the plasma membrane and/or to the cortical endoplasmic reticulum. Movement of statoliths during the first step of graviperception may lead to stress changes in actin filaments, affecting the transmembrane potential and also the Ei.     

Gravisensitivity of cress roots: investigations of threshold values under specific conditions of sensor physiology in microgravity

The minimum dose (dose = stimulus x time), one of three threshold values related to gravity, was determined under microgravity conditions for cress roots. Seedlings were cultivated on a 1g centrifuge in orbit and under microgravity, respectively. After continuous stimulation on a threshold centrifuge, minimum doses of 20-30 gs for microgravity roots and 50-60 gs for roots grown on a 1g centrifuge were estimated, which indicated that microgravity roots have a higher sensitivity than 1g roots. These results do not confirm the threshold value of 12gs which was determined for cress roots using the slow rotating clinostat. Following application of intermittent stimuli to microgravity-grown roots, gravitropic responses were observed after two stimuli of 13.5 gs separated by a stimulus-free interval of 118s. Generally, this demonstrates that higher plants are able to 'sum up' stimuli which are below the threshold value. Microscopic investigations of the cellular structure corresponding to stimulations in the range of the threshold value demonstrated a small displacement of statoliths in root statocytes. No significant correlation was observed between gravitropic curvature and statolith displacement. If the statolith theory is accepted, it can be concluded that stimulus transformation must occur in the cytoplasm in the near vicinity of the statoliths and that this transformation system--probably involving cytoskeletal elements--must have been affected during microgravity seedling cultivation.     

Agravitropic behaviour of roots of rapeseed (Brassica napus L.) transformed by Agrobacterium rhizogenes

Transgenic hairy roots of Brassica napus (cv. Omega) have been developed, using Agrobacterium rhizogenes strain AR 25, for use as a model system in the investigation of physiological and morphological differences between transgenic and normal roots. The basic parameters of growth and normal or altered gravitropical behaviour of hairy roots are for the first time presented in this paper together with an ultrastructural and morphological analysis of the root statocytes. The results obtained also represented the basis for the TRANSF0RM-experiment on the IML-2 mission performed onboard the Space Shuttle Columbia. Typical hairy root traits such as hormone-autonomous growth high growth rate, lateral branching, and changed/absence of gravitropism were detected. The transformed nature of the roots was confirmed by Southern blot analyses. The gravitropical behaviour of apices from hairy root cultures of this clone has been compared with root tips from normal seedlings. While the wild type roots curved progressively with increasing stimulation angles, the transformed roots showed no curvature when stimulated at 45 degrees, 90 degrees or 135 degrees on the ground. The morphology and ultrastructure of the root tip regions were examined by light microscopy and transmission electron microscopy. At the ultrastructural level no major differences could be detected between the roots studied. There was, however, a slight reduction in the starch content of most of the amyloplasts of the transgenic root tips, and the root cap was more V-shaped in the transgenic roots than in the wild type. Preliminary results from the Shuttle experiment TRANSFORM show a random distribution of amyloplasts in the root cells of both transformed and wild type root caps after 14 h on a 1xg centrifuge followed by 37 h in microgravity.     

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.     

Growth and differentiation of the root cap columella and root proper under stationary conditions and in clinorotation

Results are presented from light-optical and electron-microscopy studies of the root apices of three-day-old seedlings of table beet (Beta vulgaris) grown under conditions of stationary control and in clinorotation. It is shown that the ultrastructure and topography of the organelles of the root cap statocytes (gravity-perceptive cells) and cells of the distal elongation zone (gravity-receptor cells) of the root proper clearly reflect different directions for their growth and differentiation in space and in time as a function of specialization and function. Growth and genetically determined differentiation of the cells under clinorotation conditions occur as in the control, though certain differences in their ultrastructure attest to changes in metabolism.     

The Role of Starch in the Gravitropic Response of the Lentil Root

It is well accepted that the amyloplasts of the cap are responsible for gravisensing in primary roots. However, roots with starch-depleted plastids are able to respond to gravistimulus, but their curvature is slower than that of roots containing amyloplasts. The goal of our experiment was to analyse the effects of natural variations of statolith starch in the gravitropic response of lentil roots to a stimulation in the horizontal position. In lentil seedlings grown in the vertical position for 26 h, the volume of the amyloplasts in the statocytes differed between individual roots. The amount of starch in the cap was determined parallel to the rate of gravitropic curvature. There was no statistical correlation between the intensity of the gravitropic response and the starch content in the statocytes. Lentil roots were treated with gibberellic acid (GA₃) at 32°C in order to reduce the volume of starch in the statoliths. There was 53% less starch in the cap of GA₃treated roots as compared to the cap of control roots. But there was no relationship between starch content in the cap and the responsiveness of the root to a gravistimulus, except when the amount of starch was small.     

Microtubules in statocytes from roots of cress (Lepidium sativum L.)

Summary Statocytes in root caps ofLepidium sativum L. were examined by means of ultrathin serial sections to evaluate the amount and distribution of cortical microtubules. The microtubules encircle the cell, oriented normal to the root length axis. In the distal cell edges, microtubules form a network, separating the distal complex of endoplasmic reticulum from the plasmalemma. Preprophase bands in meristem cells are observable rarely, structures which can be regarded as nucleating sites for microtubules are lacking. During ageing of the root cap cells, the number of microtubules increases in combination with a decrease of microtubule length. Development of the roots on a horizontal clinostat preserves a younger developmental stage of the microtubule system regarding amount and length of the individual microtubules. Evidence for an involvement of microtubules in graviperception is low, whereas their role in orienting cellulose microfibrils cannot be ruled out. Compression of the distal network of microtubules after centrifugation of the roots indicates that microtubules in statocytes ofLepidium sativum L. roots might function in stabilizing the distal complex of endoplasmic reticulum.     

Analysis and regulation of legume inoculants in Canada: The need for an increase in standards

Herbicide residues may affect seedlings during early stages of their development. We studied this possibility by the use of light and electron microscopy after incubation of germinating seeds ofPisum sativum L. andZea mays L. with different concentrations of chlorsulfuron and metsulfuron-methyl. By in vitro experiments, we have shown that both herbicides caused growth reduction of the very young roots, and severe ultrastructural alterations and injuries of the root caps of both species. Chlorsulfuron caused increase of electron-dense material in the vacuoles, cytoplasmic degeneration even in the inner secretory cell layers of the cap, and disruption of the amyloplast envelopes with release of the statolithic starch grains. In the initial cell complex of the root cap, the herbicides caused the formation of large concentric aggregates of the rough ER and wall disformations in the cells adjacent to this complex. Scanning electron microscopic observations revealed a decrease of the slime layer ensheathing the root cap and the subapical root surface.We conclude that even in early stages of seed germination, both herbicides seriously affect the gravity perception centre (consisting of the statocytes), and the secretory tissue of the root caps, thus probably disturbing the processes of gravitropism and the protective slime secretion of the roots.