Gravity perception and gravitropic response of inflorescence stems in Arabidopsis thaliana.

Shoots of higher plants exhibit negative gravitropism. However, little is known about the site of gravity perception in shoots and the molecular mechanisms of shoot gravitropic responses. Our recent analysis using shoot gravitropism 1(sgr1)/scarecrow(scr) and sgr7/short-root (shr) mutants in Arabidopsis thaliana indicated that the endodermis is essential for shoot gravitropism and strongly suggested that the endodermis functions as the gravity-sensing cell layer in dicotyledonous plant shoots. In this paper, we present our recent analysis and model of gravity perception and gravitropic response of inflorescence stems in Arabidopsis thaliana.     

Red light-induced suppression of gravitropism in moss protonemata.

Moss protonemata are among the few cell types known that both sense and respond to gravity and light. Apical cells of Ceratodon protonemata grow by oriented tip growth which is negatively gravitropic in the dark or positively phototropic in unilateral red light. Phototropism is phytochrome-mediated. To determine whether any gravitropism persists during irradiation, cultures were turned at various angles with respect to gravity and illuminated so that the light and gravity vectors acted either in the same or in different directions. Red light for 24h (> or = l40nmol m-2 s-1) caused the protonemata to be oriented directly towards the light. Similarly, protonemata grew directly towards the light regardless of light position with respect to gravity indicating that all growth is oriented strictly by phototropism, not gravitropism. At light intensities < or = l00nmol m-2 s-1, no phototropism occurs and the mean protonemal tip angle remains above the horizontal, which is the criterion for negative gravitropism. But those protonemata are not as uniformly upright as they would be in the dark indicating that low intensity red light permits gravitropism but also modulates the response. Protonemata of the aphototropic mutant ptr1 that lacks a functional Pfr chromophore, exhibit gravitropism regardless of red light intensity. This indicates that red light acts via Pfr to modulate gravitropism at low intensities and to suppress gravitropism at intensities < or = 140nmol m-2 s-1.     

The role of calcium accumulation and the cytoskeleton in the perception and response of Coprinus cinereus to gravity.

The role of Ca2+ in the gravitropic perception and/or response mechanism of Coprinus cinereus was examined by treating stipes with inhibitors of Ca2+ transport and calmodulin. Inhibitors had no effect on gravity perception but significantly diminished gravitropism. It is concluded that, under the conditions tested, Ca2+ is not involved in gravity perception by Coprinus stipes, but does contribute to transduction of the gravitropic impulse. The results would be consistent with regulation of the gravitropic bending process requiring accumulation of Ca2+ within a membrane-bound compartment. Treatment of stipes with an actin inhibitor caused a significantly delayed response, a result not observed with the Ca2+ inhibitors. This suggests that cytoskeletal elements may be involved directly in perception of gravity by Coprinus stipes while Ca(2+)-mediated signal transduction may be involved in directing growth differentials.     

Spaceflight experiments with Arabidopsis starch-deficient mutants support a statolith-based model for graviperception.

In order to help resolve some of the controversy associated with ground-based research that has supported the starch-statolith theory of gravity perception in plants, we performed spaceflight experiments with Arabidopsis in Biorack during the January 1997 and May 1997 missions of the Space Shuttle. Seedlings of wild-type (WT) Arabidopsis, two reduced-starch strains, and a starchless mutant were grown in microgravity and then were given either a 30, 60, or 90 minute gravity stimulus on a centrifuge. By the 90 min 1-g stimulus, the WT exhibited the greatest magnitude of curvature and the starchless mutant exhibited the smallest curvature while the two reduced starch mutants had an intermediate magnitude of curvature. In addition, space-grown plants had two structural features that distinguished them from the controls: a greater number of root hairs and an anomalous hypocotyl hook structure. However, the morphological changes observed in the flight seedlings are likely to be due to the effects of ethylene present in the spacecraft. (Additional ground-based studies demonstrated that this level of ethylene did not significantly affect gravitropism nor did it affect the relative gravitropic sensitivity among the four strains.) Nevertheless, this experiment on gravitropism was performed the "right way" in that brief gravitational stimuli were provided, and the seedlings were allowed to express the response without further gravity stimuli. Our spaceflight results support previous ground-based studies of these and other mutants since increasing amounts of starch correlated positively with increasing sensitivity to gravity.     

What is the threshold amount of starch necessary for full gravitropic sensitivity?

In preparation for microgravity experiments, we studied the kinetics of gravitropism in seedlings of wild-type (WT) Arabidopsis and three starch-deficient mutants. One of these mutants is starchless (ACG 21) while the other two are intermediate starch mutants (ACG 20 and ACG 27). In root cap cells, ACG 20 and 27 have 51% and 60% of the WT amount of starch, respectively. However, in endodermal cells of the hypocotyl, ACG 20 has a greater amount of starch than ACG 27. WT roots and hypocotyls were much more responsive to gravity than were the respective organs of the starchless mutant, and the intermediate starch mutants exhibited reduced gravitropism but had responses that were close to that of the WT. In roots, ACG 27 (more starch) was more responsive than ACG 20 (less starch), while in hypocotyls, ACG 20 (more starch) had a greater response than ACG 27 (less starch). Taken together, our data are consistent with the starch-statolith hypothesis for gravity perception in that the degree of graviresponsiveness is proportional to the total mass of plastids per cell. These results also suggest that (in roots) 51-60% starch is close to the threshold amount of starch needed for full gravitropism and that the gravity sensing system is "overbuilt."     

Gravitropism and phototropism in protonemata of the moss Pohlia nutans (hedw.) Lindb

The gravitropism of protonemata of Pohlia nutans is described and compared with that of other mosses. In darkness, protonemata showed negative gravitropism. Under uniform illumination they grew radially over the substrate surface, whereas unilateral illumination induced positive phototropic growth. Gravitropism was coupled with starch synthesis and amyloplast formation. Protonematal gravitropic growth is more variable than the strict negative gravitropism of Ceratodon chloronema.     

Circumnutation augmented in clinostatted plants by a tactile stimulus

Dark-grown, 4-day old, $\text{Helianthus annuus}$ seedlings were rotated for 20 hr on horizontal clinostats to minimize the amplitude of circumnutation. Then a Plexiglas sheet was placed gently against the tip of the cotyledons. By time-lapse video imaging (using intermittent IR illumination to which the plants were insensitive) movements of the clinostatted plants were observed before, during, and after the period of mechanical contact. Immediately after the Plexiglas sheet was removed residual nutation increased in amplitude almost three-fold, then declined over the next 7 hr to the prestimulation level. This demonstration of enhancement of circumnutation by mechanical contact is consistent with the model of an endogenous oscillator that can be stimulated by factors other than gravity.     

Agravitropic mutant for the study of hydrotropism in seedling roots

Roots have been shown to respond to a moisture gradient by positive hydrotropism. Agravitropic mutant plants are useful for the study of the hydrotropism in roots because on Earth hydrotropism is obviously altered by the gravity response in the roots of normally gravitropic plants. The roots are able to sense water potential gradient as small as 0.5 MPa mm⁻¹. The root cap includes the sensing apparatus that causes a differential growth at the elongation region of roots. A gradient in apoplastic calcium and calcium influx through plasmamembrane in the root cap is somehow involved in the signal transduction mechanism in hydrotropism, which may cause a differential change in cell wall extensibility at the elongation region. We have isolated an endoxy loglucan transferase (EXGT) gene that is strongly expressed in pea roots and appears to be involved in the differential growth in hydrotropically responding roots. Thus, it is now possible to study hydrotropism in roots by comparing with or separate from gravitropism. These results also imply that microgravity conditions in space are useful for the study of hydrotropism and its interaction with gravitropism.     

Graviresponses in fungi.

Although the orientation of mycelial hyphal growth is usually independent of the gravity vector, individual specialised hyphae can show response to gravity. This is exemplified by the sporangiophore of Phycomyces, but the most striking gravitropic reactions occur in mushroom fruit bodies. During the course of development of a mushroom different tropisms predominate at different times; the young fruit body primordium is positively phototropic, but negative gravitropism later predominates. The switch between tropisms has been associated with meiosis. The spore-bearing tissue is positively gravitropic and responds independently of the stem. Bracket polypores do not show tropisms but exhibit gravimorphogenetic responses: disturbance leads to renewal of growth producing an entirely new fruiting structure. Indications from both clinostat and space flown experiments are that the basic form of the mushroom (overall tissue arrangement of stem, cap, gills, hymenium, veil) is established independently of the gravity vector although maturation, and especially commitment to the meiosis-sporulation pathway, requires the normal gravity vector. The gravity perception mechanism is difficult to identify. The latest results suggest that disturbance of cytoskeletal microfilaments is involved in perception (with nuclei possibly being used as statoliths), and Ca2(+)-mediated signal transduction may be involved in directing growth differentials.     

The promotive effect of latrunculin B on maize root gravitropism is concentration dependent.

The cytoskeleton has been proposed to be a key player in the gravitropic response of higher plants. A major approach to determine the role of the cytoskeleton in gravitropism has been to use inhibitors to disrupt the cytoskeleton and then to observe the effect that such disruption has on organ bending. Several investigators have reported that actin or microtubule inhibitors do not prevent root gravitropism, leading to the conclusion that the cytoskeleton is not involved in this process. However, there are recent reports showing that disruption of the actin cytoskeleton with the actin inhibitor, latrunculin B, promotes the gravitropic response of both roots and shoots. In roots, curvature is sustained during prolonged periods of clinorotation despite short periods of gravistimulation. These results indicate that an early gravity-induced signal continues to persist despite withdrawal of the constant gravity stimulus. To investigate further the mechanisms underlying the promotive effect of actin disruption on root gravitropism, we treated maize roots with varying concentrations of latrunculin B in order to determine the lowest concentration of latrunculin B that has an effect on root bending. After a 10-minute gravistimulus, treated roots were axially rotated on a one rpm clinostat and curvature was measured after 15 hours. Our results show that 100 nM latrunculin B induced the strongest promotive effect on the curvature of maize roots grown on a clinostat. Moreover, continuously gravistimulated roots treated with 100 nM latrunculin B exhibited stronger curvature responses while decapped roots treated with this concentration of latrunculin B did not bend during continuous gravistimulation. The stronger promotive effect of low concentrations of latrunculin B on the curvature of both clinorotated and continuously gravistimulated roots suggests that disruption of the finer, more dynamic component of the actin cytoskeleton could be the cause of the enhanced tropic responses of roots to gravity.     

Laser ablation of root cap cells: implications for models of graviperception.

The initial event of gravity perception by plants is generally thought to occur through sedimentation of amyloplasts in specialized sensory cells. In the root, these cells are the columella which are located toward the center of the root cap. To define more precisely the contribution of columella cells to root gravitropism, we used laser ablation to remove single columella cells or groups of these cells and observed the effect of their removal on gravity sensing and response. Complete removal of the cap or all the columella cells (leaving peripheral cap cells intact) abolishes the gravity response of the root. Removal of stories of columella revealed differences between regions of the columella with respect to gravity sensing (presentation time) versus graviresponse (final tropic growth response of the root). This fine mapping revealed that ablating the central columella located in story 2 had the greatest effect on presentation time whereas ablating columella cells in story 3 had a smaller or no effect. However, when removed by ablation the columella cells in story 3 did inhibit gravitropic bending, suggesting an effect on translocation of the gravitropic signal from the cap rather than initial gravity perception. Mapping the in vivo statolith sedimentation rates in these cells revealed that the amyloplasts of the central columella cells sedimented more rapidly than those on the flanks do. These results show that cells with the most freely mobile amyloplasts generate the largest gravisensing signal consistent with the starch statolith hypothesis of gravity sensing in roots.     

Statolith positioning by microfilaments in Chara rhizoids and protonemata.

The rhizoids of the green alga Chara are tip-growing cells with a precise positive gravitropism. In rhizoids growing downwards the statoliths never sediment upon the cell wall at the very tip but keep a minimal distance of approximately 10 micrometers from the cell vertex. It has been argued that this position is attained by a force acting upon the statoliths in the basal direction and that this force is generated by an interaction between actin microfilaments and myosin on the statolith membrane. This hypothesis received experimental support from (1) effects of the actin-attacking drug cytochalasin, (2) experiments under microgravity conditions, and (3) clinostat experiments. Using video-microscopy it is now shown that this basipetal force also acts on statoliths during sedimentation. As a result, many statoliths in Chara rhizoids do not simply fall along the plumb line while sedimenting during gravistimulation, but move basipetally. This statolith movement is compared to the ones occurring in the unicellular Chara protonemata during gravistimulation. Dark-grown protonemata morphologically closely resemble the rhizoids but respond negatively gravitropic. In contrast to the rhizoids a gravistimulation of the protonemata induces a transport of statoliths towards the tip. This transport is mainly along the cell axis and not parallel to the gravity vector. It is stressed that the sedimentation of statoliths in Chara rhizoids and protonemata as well as in gravity sensing cells in mosses and higher plants is accompanied by statolith movements based on interactions with the cytoskeleton. In tip-growing cells these movements direct the statoliths to a definite region of the cell where they can sediment and elicit a gravitropic curvature. In the statocytes of higher plants the interactions of the statoliths with the cytoskeleton probably do not serve primarily to move the statoliths but to transduce mechanical stresses from the sedimenting statoliths to the plasma membrane.     

Protein crystals in Phycomyces sporangiophores are involved in graviperception.

The sporangiophores of the zygomycete fungus Phycomyces blakesleeanus contain octahedral crystals with diameters of up to 5 micrometers in their vacuole. The crystals are associated with the intracellular membrane system. In tilted or horizontally placed sporangiophores, the crystals sediment to the respective lower face of the vacuole with a velocity of up to 100 micrometers per minute. The sedimentation is completed within about 2 minutes, well within the latency period for the negative gravitropic response of Phycomyces. Crystal-lacking mutant strains display a smaller maximal bending angle and a reduced gravitropic bending rate in comparison to the wild type. We therefore conclude that the crystals serve as statoliths for gravitropism in Phycomyces.     

Genetic analysis of the gravitropic set-point angle in lateral roots of Arabidopsis.

Research on gravity responses in plants has mostly focused on primary roots and shoots, which typically orient to a vertical orientation. However, the distribution of lateral organs and their characteristically non-vertical growth orientation are critical for the determination of plant form. For example, in Arabidopsis, when lateral roots emerge from the primary root, they grow at a nearly horizontal orientation. As they elongate, the roots slowly curve until they eventually reach a vertical orientation. The regulation of this lateral root orientation is an important component affecting overall root system architecture. We found that this change in orientation is not simply due to the onset of gravitropic competence, as non-vertical lateral roots are capable of both positive and negative gravitropism. Thus, the horizontal growth of new lateral roots appears to be determined by what is called the gravitropic set-point angle (GSA). This developmental control of the GSA of lateral roots in Arabidopsis provides a useful system for investigating the components involved in regulating gravitropic responses. Using this system, we have identified several Arabidopsis mutants that have altered lateral root orientations but maintain normal primary root orientation.     

High temperature alters the growth reaction of Pottia protonemata

Under gravistimulation, dark-grown protonemata of Pottia intermedia revealed negative gravitropism with a growth rate of approximately 28 μm·h⁻¹ at room temperature (20 °C). In 7 days, the protonema formed a bundle of vertically oriented filaments. At an elevated temperature (30 °C), bundles of vertically growing filaments were also formed. However, both filament growth rate and amplitude of the gravicurvature were reduced. Red light (RL) irradiation induced a positive phototropism of most apical protonemal cells at 20 °C. In a following period of darkness, approximately two-thirds of such cells began to grow upward again, recovering their negative gravitropism. RL irradiation at the elevated temperature caused a partial increase in the number of protonemal cells with negative phototropism, but the protonemata did not exhibit negative gravitropism after transfer to darkness. The negative gravitropic reaction was renewed only when protonemata were placed at 20 °C. A dramatic decrease in starch amount in protonemal apical cells, which are sensitive to both gravity and light, occurred at the higher temperature. Such a decrease may be one of the reasons for the inhibition of the protonemal gravireaction at the higher temperature. The observation has a bearing on the starch-statolith theory.     

High temperature alters the growth reaction of Pottia protonemata.

Under gravistimulation, dark-grown protonemata of Pottia intermedia revealed negative gravitropism with a growth rate of approximately 28 μm·h⁻¹ at room temperature (20 °C). In 7 days, the protonema formed a bundle of vertically oriented filaments. At an elevated temperature (30 °C), bundles of vertically growing filaments were also formed. However, both filament growth rate and amplitude of the gravicurvature were reduced. Red light (RL) irradiation induced a positive phototropism of most apical protonemal cells at 20 °C. In a following period of darkness, approximately two-thirds of such cells began to grow upward again, recovering their negative gravitropism. RL irradiation at the elevated temperature caused a partial increase in the number of protonemal cells with negative phototropism, but the protonemata did not exhibit negative gravitropism after transfer to darkness. The negative gravitropic reaction was renewed only when protonemata were placed at 20 °C. A dramatic decrease in starch amount in protonemal apical cells, which are sensitive to both gravity and light, occurred at the higher temperature. Such a decrease may be one of the reasons for the inhibition of the protonemal gravireaction at the higher temperature. The observation has a bearing on the starch-statolith theory.     

Gravitropism and phototropism in protonemata of the moss Pohlia nutans (Hedw.) Lindb.

The gravitropism of protonemata of Pohlia nutans is described and compared with that of other mosses. In darkness, protonemata showed negative gravitropism. Under uniform illumination they grew radially over the substrate surface, whereas unilateral illumination induced positive phototropic growth. Gravitropism was coupled with starch synthesis and amyloplast formation. Protonematal gravitropic growth is more variable than the strict negative gravitropism of Ceratodon chloronema.     

Cell biology of plant gravity sensing.

The debate about whether gravity sensing relies upon statoliths (amyloplasts that sediment) has intensified with recent findings of gravitropism in starchless mutants and of claims of hydrostatic gravity sensing. Starch and significant plastid sedimentation are not necessary for reduced sensing in mutant roots, but plastids might function here if there were a specialized receptor for plastid mass e.g. in the ER. Alternatively, components in addition to amyloplasts might provide mass for sensing. The nucleus is dense and its position is regulated, but no direct data exist for its role in sensing. If the weight of the protoplast functioned in sensing, why would there be specific cytological specializations favoring sedimentation rather than cell mass? Gravity has multiple effects on plants in addition to gravitropism. There may be more than one mechanism of gravity sensing.     

The relationship between profiles of plagiogravitropism and morphometry of columella cells during the development of lateral roots of Vigna angularis

There has been no convincing explanation on a mechanism inducing plagiogravitropism of lateral roots. The present work deals with gravitropic features of Vignaangularis lateral roots during the course of their growth and morphometric analysis of root caps, columella cells and amyloplasts. Regardless of the magnitude of deviation of the primary root axis from the gravity vector, the newly emerging lateral roots tended to keep a constant angle to the gravity vector. They modified gravireaction several times during the course of their development: a first horizontal-growth stage when they grow in the cortex of primary roots (stage I), a sloping-down growth stage from their emergence to a length of about 1 mm (stage II), a second horizontal-growth stage from a length of about 1 mm to that of over 4 mm (stage III) and a curving-down stage thereafter (stage IV). The columella cells with amyloplasts large enough to sediment were not fully differentiated in the stage I but the turning point from the stage I to II was associated with the development of amyloplasts which were able to sediment toward the distal part of the cell. Amyloplasts were significantly small in the lateral roots over 10 mm long compared with those in ones 0–10 mm long, suggesting that they rapidly develop immediately after the lateral roots emerge from primary roots and then gradually decrease their size when the lateral roots grow over 10 mm long. This dimensional decrease of amyloplasts may be partially involved in weak gravireaction in the stage III. Evidence was not presented indicating that a switchover from the stage III to IV was connected with the dimension of root caps, the number of columella cells and the development of amyloplasts. Some factors at the molecular level rather than at the cellular and tissue levels are probably dominant to induce the stage IV.     

Correction of B2bot for NeQuick during low solar activity at Hainan station

This work is a continuation of the previous article and it focuses on low solar activity and modeling effort. NeQuick model uses Epstein layer formalism to model each part of the profile. We study the diurnal and seasonal variations of B2_bot, ΔB2 (B2_best − B2_NeQuick2) and R (B2_best/B2_{NeQuick 2}) at Hainan station during low solar activity. The results show it is possible to improve the B2_bot parameter of the NeQuick model at that region during low solar activity. Then, we use a function ?(t) with LT in different seasons to correct the B2_bot formula of NeQuick 2. The correction shows that (1) By the correction formula, the B2_bot of NeQuick is improved. The maximum standard deviation is improved for 9 km. (2) The correction formula is more effective in summer than in equinox and winter and performs better during early morning hours than during the rest of the day.