Circumnutation of rice coleoptiles: its relationships with gravitropism and absence in lazy mutants

Although circumnutation occurs widely in higher plants, its mechanism is little understood. The idea that circumnutation is based on gravitropism has long been investigated, but the reported results have been controversial. We used dark-grown coleoptiles of rice (Oryza sativa L.) to re-investigate this issue. The following results supported the existence of a close relationship between gravitropism and circumnutation: (1) circumnutation disappears on a horizontal clinostat; (2) circumnutation is interrupted by a gravitropic response and re-initiated at a definable phase after gravitropic curvature; (3) circumnutation can be re-established by submergence and a brief gravitropic stimulation in the coleoptiles that have stopped nutating in response to red light; and (4) lazy mutants show no circumnutation. In spite of these results, however, there were cases in which gravitropism and circumnutation could be separated. Firstly, the non-circumnutating lazy coleoptile showed nearly a wild-type level of gravitropic responsiveness in its upper half, although this part was an active site of both gravitropism and circumnutation in wild-type coleoptiles. Secondly, coleoptiles could nutate without overshooting the vertical when developing phototropic curvature. It is concluded that gravitropism influences, but it is not directly involved in the process of circumnutation. It is further suggested that a gravity signal, shared with gravitropism, contributes to the maintenance of circumnutation.     

Circumnutational movement in rice coleoptiles involves the gravitropic response: analysis of an agravitropic mutant and space‐grown seedlings

Plants exhibit helical growth movements known as circumnutation in growing organs. Some studies indicate that circumnutation involves the gravitropic response, but this notion is a matter of debate. Here, using the agravitropic rice mutant lazy1 and space‐grown rice seedlings, we found that circumnutation was reduced or lost during agravitropic growth in coleoptiles. Coleoptiles of wild‐type rice exhibited circumnutation in the dark, with vigorous oscillatory movements during their growth. The gravitropic responses in lazy1 coleoptiles differed depending on the growth stage, with gravitropic responses detected during early growth and agravitropism during later growth. The nutation‐like movements observed in lazy1 coleoptiles at the early stage of growth were no longer detected with the disappearance of the gravitropic response. To verify the relationship between circumnutation and gravitropic responses in rice coleoptiles, we conducted spaceflight experiments in plants under microgravity conditions on the International Space Station. Wild‐type rice seeds were germinated, and the resulting seedlings were grown under microgravity or a centrifuge‐generated 1 g environment in space. We began filming the seedlings 2 days after seed imbibition and obtained images of seedling growth every 15 min. The seed germination rate in space was 92–100% under both microgravity and 1 g conditions. LED‐synchronized flashlight photography induced an attenuation of coleoptile growth and circumnutational movement due to cumulative light exposure. Nevertheless, wild‐type rice coleoptiles still showed circumnutational oscillations under 1 g but not microgravity conditions. These results support the idea that the gravitropic response is involved in plant circumnutation.     

Time course of phytochrome-mediated changes in growth gradients on coleoptiles of Triticum aestivum L.

The length of parenchyma cells along the axis of dark-grown coleoptiles of Triticum aestivum L. and the pattern of competence for red-light-(R-) induced stimulation or inhibition of cell elongation in the course of coleoptile development were determined by microscopic measurements in a file of 240 cells from the tip to the base. On the basis of these measurements distinct zones (responding in different ways to R) were selected for studying the early time course of phytochrome-mediated growth-rate changes in intact coleoptiles by use of a sensitive transducer system. Between 2 d and 4 d after sowing dark-grown coleoptiles showed a graded incline in cell growth activity from the apex to the base (growth gradient). Whereas cell elongation in the coleoptile base ceased 4 d after sowing, cell elongation speeded up in the tip and middle region at that time. Those cells that grew slowly in darkness (tip and middle region between 2d and 3 d after sowing) were stimulated in growth by R-pulse irradiation (1 min R, 660 nm, 1000 J · m^–2). In contrast, the growth of fast-growing cells (base between 2 d and 4 d after sowing, tip and middle region between 4 d and 5 d after sowing) was inhibited by R. However, the starting time for R-induced growth changes was different for different coleoptile zones. The respective data point to the storage of a phytochrome-mediated signal in the cells of the middle region, until these cells become competent to respond to it; alternatively, Pfr, the far-red-light-absorbing form of phytochrome, may be stored in a stable form. Continuous recordings on the effect of R, far-red (FR) and R/FR on the zonal growth responses were made on intact coleoptiles, selected 3 d after sowing. During a 5-h investigation period the R-induced changes in growth rate could be divided into two phases: (i) A transient growth inhibition which started approx. 15 min after R. This response was qualitatively the same in all coleoptile zones investigated (tip, middle region, base). (ii) Zonal-specific growth responses which became measurable approx. 2.5 h after R, i.e. growth promotion in the tip, growth inhibition in the base and an adaptation of growth rate to the dark control level in the middle region. The R-induced growth rate changes were reversible by FR for both phases. Additional growth experiments on excised coleoptile segments under R and auxin application indicated that the zonal-specific growth promotion or inhibition may be not mediated by an influence of R on the auxin level.Abbreviations FR far-red light - Pfr far-red-light-absorbing form of phytochrome - R red light The technical assistance of Mrs. B. Liebe is gratefully acknowledged.     

Identification of the gravitropism-related rice gene LAZY1 and elucidation of LAZY1-dependent and -independent gravity signaling pathways

We identified the gene responsible for three allelic lazy1 mutations of Japonica rice (Oryza sativa L.) by map-based cloning, complementation and RNA interference. Sequence analysis and database searches indicated that the wild-type gene (LAZY1) encodes a novel and unique protein (LAZY1) and that rice has no homologous gene. Two lazy1 mutants were LAZY1 null. Confirming and advancing the previously reported results on lazy1 mutants, we found the following. (i) Gravitropism is impaired, but only partially, in lazy1 coleoptiles. (ii) Circumnutation, observed in dark-grown coleoptiles, is totally absent from lazy1 coleoptiles. (iii) Primary roots of lazy1 mutants show normal gravitropism and circumnutation. (iv) LAZY1 is expressed in a tissue-specific manner in gravity-sensitive shoot tissues (i.e. coleoptiles, leaf sheath pulvini and lamina joints) and is little expressed in roots. (v) The gravitropic response of lazy1 coleoptiles is kinetically separable from that absent from lazy1 coleoptiles. (vi) Gravity-induced lateral translocation of auxin, found in wild-type coleoptiles, does not occur in lazy1 coleoptiles. Based on the genetic and physiological evidence obtained, it is concluded that LAZY1 is specifically involved in shoot gravitropism and that LAZY1-dependent and -independent signaling pathways occur in coleoptiles. It is further concluded that, in coleoptiles, only the LAZY1-dependent gravity signaling involves asymmetric distribution of auxin between the two lateral halves and is required for circumnutation.     

PHYTOCHROME KINASE SUBSTRATE1 regulates root phototropism and gravitropism

Light promotes the expression of PHYTOCHROME KINASE SUBSTRATE1 (PKS1) in the root of Arabidopsis thaliana, but the function of PKS1 in this organ is unknown. Unilateral blue light induced a negative root phototropic response mediated by phototropin 1 in wild-type seedlings. This response was absent in pks1 mutants. In the wild type, unilateral blue light enhanced PKS1 expression in the subapical region of the root several hours before bending was detectable. The negative phototropism and the enhanced PKS1 expression in response to blue light required phytochrome A (phyA). In addition, the pks1 mutation enhanced the root gravitropic response when vertically oriented seedlings were placed horizontally. The negative regulation of gravitropism by PKS1 occurred even in dark-grown seedlings and did not require phyA. Blue light also failed to induce negative phototropism in pks1 under reduced gravitational stimulation, indicating that the effect of pks1 on phototropism is not simply the consequence of the counteracting effect of enhanced gravitropism. We propose a model where the background level of PKS1 reduces gravitropism. After a phyA-dependent increase in its expression, PKS1 positively affects root phototropism and both effects contribute to negative curvature in response to unilateral blue light.     

Interaction of light and gravitropism with nutation of hypocotyls of Arabidopsis thaliana seedlings

Etiolated seedlings of Arabidopsis thaliana nutated under conditions of physiological darkness while about ten percent of monitored individuals exhibited regular elliptical nutation, circumnutation. Pre-irradiation with red light prevented occurrence of circumnutation without having an effect on the average rate of the nutational movement. Phototropic response of seedlings to unilateral blue light appeared to be superimposed over nutation. Throughout gravitropism, some seedlings continued to exhibit nutation suggesting that these two processes are independently controlled. Based on these results, we suggest that nutation in Arabidopsis probably is not controlled by the mechanism predicted by the theory of gravitropic overshoots.     

Hypocotyl growth orientation in blue light is determined by phytochrome A inhibition of gravitropism and phototropin promotion of phototropism

How developing seedlings integrate gravitropic and phototropic stimuli to determine their direction of growth is poorly understood. In this study we tested whether blue light influences hypocotyl gravitropism in Arabidopsis. Phototropin1 (phot1) triggers phototropism under low fluence rates of blue light but, at least in the dark, has no effect on gravitropism. By analyzing the growth orientation of phototropism-deficient seedlings in response to gravitropic and phototropic stimulations we show that blue light not only triggers phototropism but also represses hypocotyl gravitropism. At low fluence rates of blue light phot1 mutants were agravitropic. In contrast, phyAphot1 double mutants grew exclusively according to gravity demonstrating that phytochrome A (phyA) is necessary to inhibit gravitropism. Analyses of phot1cry1cry2 triple mutants indicate that cryptochromes play a minor role in this response. Thus the optimal growth orientation of hypocotyls is determined by the action of phyA-suppressing gravitropism and the phototropin-triggering phototropism. It has long been known that phytochromes promote phototropism but the mechanism involved is still unknown. Our data show that by inhibiting gravitropism phyA acts as a positive regulator of phototropism.     

Metabolic Adaptations of Plant Respiration to Nutritional Phosphate Deprivation

Shoots of the lazy-2 (lz-2) gravitropic mutant of tomato (Lycopersicon esculentum Mill.) have a normal gravitropic response when grown in the dark, but grow downward in response to gravity when grown in the light. Experiments were undertaken to investigate the nature of the light induction of the downward growth of lz-2 shoots. Red light was effective at causing downward growth of hypocotyls of lz-2 seedlings, whereas treatment with blue light did not alter the dark-grown (wild-type) gravity response. Downward growth of lz-2 seedlings is greatest 16 h after a 1-h red light irradiation, after which the seedlings begin to revert to the dark-grown phenotype. lz-2 seedlings irradiated with a far-red light pulse immediately after a red light pulse exhibited no downward growth. However, continuous red or far-red light both resulted in downward growth of lz-2 seedlings. Thus, the light induction of downward growth of lz-2 appears to involve the photoreceptor phytochrome. Fluence-response experiments indicate that the induction of downward growth of lz-2 by red light is a low-fluence phytochrome response, with a possible high-irradiance response component.     

Blue light- and genetically-reversed gravitropic response in protonemata of the moss Ceratodon purpureus

In darkness, protonemal filaments of Ceratodon purpureus (Brid.) grow negatively gravitropically (upwards). Red light induces a positive phototropic response mediated by the photoreceptor phytochrome. A red light treatment also has an inhibitory effect on the gravitropic response, an effect also mediated by phytochrome. In this study the effects of blue light on phototropism and on gravitropism were analysed. Unilateral blue light resulted in only a weak phototropic response, but markedly randomised growth direction. Blue light given together with a gravitropic stimulus reversed the gravitropism, changing it from negative to positive (filaments grow downward). The effect of blue light was also analysed with the mutant ptr116, which is defective in the biosynthesis of the phytochrome chromophore, and in a newly isolated mutant wwr2, which is positively gravitropic in darkness. Blue light induced the same reversal of gravitropism in ptr116 as in the wild type, indicating that phytochrome is not involved in this process. In wwr2 the direction of gravitropism was unaltered by the blue light treatment. Light also affects chlorophyll content and the size of plastids, potential statoliths for gravitropism. Red light induced an increase in plastid size and chlorophyll content in the wild type but not in ptr116. Blue light induced a similar change in wild type plastids. It seems as though light-induced alterations of gravitropism are not simply mediated by alterations in plastid properties, and that red light and blue light evoke fundamentally different responses. Received: 11 July 1997 / Accepted: 30 January 1998     

Genetic analysis of the roles of phytochromes A and B1 in the reversed gravitropic response of the lz-2 tomato mutant

The lz-2 mutation in tomato ( Lycopersicon esculentum ) causes conditional reversal of shoot gravitropism by light. This response is mediated by phytochrome. To further elicit the mechanism by which phytochrome regulates the lz-2 phenotype, phytochrome-deficient lz-2 plants were generated. Introduction of au alleles, which severely block chromophore biosynthesis, eliminated the reversal of hypocotyl gravitropism in continuous red and far-red light. The fri ¹ and tri ¹ alleles were introduced to specifically deplete phytochromes A and B1, respectively. In dark-grown seedlings, phytochrome A was necessary for response to high-irradiance far-red light, a complete response to low fluence red light, and also mediated the effects of blue light in a far-red reversible manner. Loss of phytochrome B1 alone did not significantly affect the behaviour of lz-2 plants under any light treatment tested. However, dark-grown lz-2 plants lacking both phytochrome A and B1 exhibited reduced responses to continuous red and were less responsive to low fluence red light and high fluence blue light than plants that were deficient for phytochrome A alone. In high light, full spectrum greenhouse conditions, lz-2 plants grew downward regardless of the phytochrome deficiency. These results indicate that phytochromes A and B1 play significant roles in mediating the lz-2 phenotype and that at least one additional phytochrome is involved in reversing shoot gravitropism in this mutant.     

Gravitropic microtubule reorientation can be uncoupled from growth

The causal relationship between gravitropic growth responses and microtubule reorientation has been studied. Growth and microtubule reorientation have been uncoupled during the gravitropic response of maize (Zea mays L.) coleoptiles. Microtubule orientation and growth were measured under three different conditions: (i) a gravitropic stimulation where the growth response was allowed to be expressed (intact seedlings were displaced from the vertical position by 90°), (ii) a gravitropic stimulation where the growth response was suppressed (coleoptiles were attached to microscope slides and kept in a horizontal position), (iii) suppression of growth in the absence of gravitropic stimulation (coleoptiles were attached to microscope slides and kept in a vertical position). It was found that (i) gravitropic stimulation can induce a microtubular reorientation from transverse to longitudinal in the upper (slower growing) flank of the coleoptile, and an inhibition of growth; (ii) the reorientation of microtubules precedes the inhibition of growth; (iii) the gravitropic response of microtubules is weaker, not elevated, when the inhibition of growth is artificially enhanced by attaching the coleoptiles to a slide; and (iv) artificial inhibition of growth in the absence of gravitropic stimulation cannot induce a microtubular response. Thus, the extent of microtubule reorientation is not correlated with the extent of growth inhibition. Moreover, these findings demonstrate that microtubules do not reorient passively after growth changes, but actively in response to gravitropic stimulation. Received: 23 November 1999 / Accepted: 10 May 2000     

Control of hypocotyl gravitropism by photochrome in a dicotyledonous seedling (Sesamum indicum L.)

Abstract The present study was prompted by the question as to whether the strong effect of red and far-red light treatments on blue-light-mediated phototropism in the sesame (Sesamum indicum L.) hypocotyl (Woitzik & Mohr, 1988) should be attributed in part to changes initialed by light in the gravitropic counter-response. Light treatments, operating through phytochrome, do indeed strongly affect the gravitropic response. However, the direction of the light effect is the same in gravitropism, as in phototropism. Thus, the gravitropic counter-response leads to an underestimate, rather than an overestimate, of the importance of phytochrome action on phototropic responsiveness. The effect of red and far-red light, operating via phytochrome, on the gravitropic response of the sesame hypocotyl could be studied in the present paper without any interference due to phototropism or light control of longitudinal growth. It was found that the effects of red and far-red pretreatments (given prior to the onset of the stimulus) as well as the action of simultaneously applied red or far-red light (simultaneous to the phototropic or gravitropic stimulus) are very similar in both phototropism and gravitropism. In particular, the seedling is capable of superimposing information about the actual light conditions during bending on the ‘memory’ it has about the light conditions prior to the onset of phototropism or gravitropic stimulation, This striking similarity between the phototropic and gravitropic responses possibly indicates that phytochrome affects the signal-response-chain at a relatively late stage, after the phototropic and the gravitropic signal-response chains have merged. From a teleonomic point of view the action of red and far-red light on phototropic, as well as gravitropic, responsiveness can be conceived as part of a shade escape strategy.     

The light-induced reduction of the gravitropic growth-orientation of seedlings of Arabidopsis thaliana (L.) Heynh. is a photomorphogenic response mediated synergistically by the far-red-absorbing forms of phytochromes A and B

Hypocotyls of dark-grown seedlings of Ara bidosis thaliana exhibit a strong negative gravitropism, which is reduced by red and also by long-wavelength, far-red light treatments. Light treatments using phytochrome A (phyA)- and phytochrome B (phyB)-deficient mutants showed that this response is controlled by phyB in a red/far-red reversible way, and by phyA in a non-reversible, very-low-fluence response. Crosses of the previously analyzed phyB-1 allele (in the ecotype Landsberg erecta background) to the ecotype Nossen wild-type (WT) background resulted in a WT-like negative gravitropism in darkness, indicating that the previously described gravitropic randomization observed with phyB-1 in the dark is likely due to a second mutation independent of that in the PHYB gene.Abbreviations FR long-wavelength far-red light - phyA phytochrome A (holoprotein) - phyB phytochrome B (holoprotein) - Pr red-absorbing form of phytochrome - WT wild type We thank Dr. A. Nagatani (RIKEN Institute, Wako-City, Japan) and Dr. M. Furuya (Hitachi, Hatoyama, Japan) for the phyA-201/phyB-5 double mutant. The work was supported by Deutsche Forschungsgemeinschaft and Human Frontier Science Program grants to E.S.     

Role of Calcium Channels in Phytochrome-Mediated Regulation of Gravitropic Response

The effect of the inhibitors of calcium channels on red-light (R)-mediated inhibition of gravitropic bending was studied in excised wheat (Triticum aestivumL.) coleoptiles. The effect of a short R pulse (2 min) preceding the gravitropic stimulation was completely alleviated by a similar pulse of far-red light (FR), when the latter preceded the gravitropic stimulation and the delay between R and FR pulses did not exceed 20 min. Plant memory of the R pulse lasted up to 40 min. Neither R nor FR exerted any effect on the gravitropic reaction when applied after gravitropic stimulation. Treatment with 1 M of verapamil, LaCl₃, GdCl₃, or ruthenium red before the gravitropic stimulation prevented or released the R-exerted suppression of the gravitropic response (GR). The GR in coleoptiles is apparently regulated by the phytochrome system at the induction phase and involves calcium channels.     

Gravitropic response plays an important role in the nutational movements of the shoots of Pharbitis nil and Arabidopsis thaliana

The shoots of a Japanese strain of morning glory ( Pharbitis nil  ) called 'Shidare-asagao' display agravitropic and weeping growth. It has been shown that this shoot agravitropism may be due to the defective differentiation of endodermal cells that contain statoliths. Roots of the weeping morning glory show normal responsiveness to gravity and the shoots are positively phototropic. Shoots of the morning glory cultivar Violet used as a wild type exhibited distinct circumnutation with circular movements that increase as the plants grow. In weeping morning glory, however, nutation was limited to slight back and forth or side to side movements. To determine whether endodermal cells participate in circumnutation through a function that is independent of their role in gravitropism, the nutational movements of various gravitropic mutants of Arabidopsis thaliana were compared. The inflorescences of wild-type Arabidopsis showed relatively large circular movements. Inflorescences of the pgm-1 mutant, which is defective in starch synthesis, showed reduced nutation. Even more seriously affected were the sgr1-1 / scr-3 and sgr7-1 / shr-2 mutants, which are defective in endodermal cell differentiation, and the auxin-resistant axr2-1 mutant showed no significant nutational movements at all. 1- N -naphthylphthalamic acid (NPA) could inhibit Violet circumnutation, supporting the notion that auxin participates in circumnutation. Thus, the gravitropic response is an essential component in plant shoot circumnutation. Endodermal cells are involved in such circumnutation possibly because of their role in inducing the gravitropic response.     

Gravitropic responses of wild-type and mutant strains of the moss Physcomitrella patens

The gravitropic responses of dark-grown caulonemata and gametophores of wild-type and mutant strains of the moss Physcomitrella patens have been investigated. In the wild-type both caulonemata and gametophores show negative orthogravitropism. No gravitropic response is observed when plants are rotated slowly on a clinostat and the inductive effect of gravity can be replaced by centrifugal force. The gravitropic response of caulonemanta is biphasic, consisting of an initial phase producing a bend of about 20 degrees within 12 h of 90 degrees reorientation and a subsequent slower phase leading to completion of the 90 degrees curvature. No obvious sedimentation of statoliths accompanies this response. Several mutants have been isolated that are either partially or completely impaired in caulonemal gravitropism and one mutant shows a positive gravitropic response. Complementation analysis using somatic hybrids obtained following protoplast fusion indicates that at least three genes can mutate to give an altered gravitropic phenotype. None of these mutants is altered in gametophore gravitropism, suggesting that the gravitropic response of caulonemal filaments may require at least some gene products that are not required for the response of the multicellular gametophores. One class of mutant with impaired caulonemal gravitropism shows a pleiotropic alteration in leaf shape.     

Rapid Suppression of Growth by Blue Light: OCCURRENCE, TIME COURSE, AND GENERAL CHARACTERISTICS

The inhibition of stem elongation in dark-grown seedlings by blue light was studied with marking techniques and with a high-resolution, growth-measuring apparatus. Blue light rapidly suppresses growth in a variety of cultivated species. In some species, the inhibition persists only during the period of irradiation, after which time growth quickly returns to the high dark rate, whereas, in other species, the light response has an additional long-term component which lasts for at least several hours in the dark. The long-term inhibition may be mediated by phytochrome, whereas the rapid, short-term component is specific to a blue-light receptor.     

Novel software for analysis of root gravitropism: comparative response patterns of Arabidopsis wild-type and axr1 seedlings

In an earlier study (Evans, Ishikawa & Estelle 1994, Planta 194, 215-222) we used a video digitizer system to compare the kinetics of auxin action on root elongation in wild-type seedlings and seedlings of auxin response mutants of Arabidopsis thaliana (L.) Heynh. We have since modified the system software to allow determination of elongation on opposite sides of vertical or gravistimulated roots and to allow continuous measurement of the angle of orientation of sequential subsections of the root during the response. We used this technology to compare the patterns of differential growth that generate curvature in roots of the Columbia ecotype and in the mutants axr1-3, axr1-12 and axr2, which show reduced gravitropic responsiveness and reduced sensitivity to inhibition by auxin. The pattern of differential growth during gravitropism differed in roots of wild-type and axr1 seedlings. In wild-type roots, initial curvature resulted from differential inhibition of elongation in the distal elongation zone (DEZ). This was followed by an acceleration of elongation along the top side of the DEZ. In roots of axr1-3, curvature resulted from differential stimulation of elongation whereas in roots of axr1-12 the response was variable. Roots of axr2 did not exhibit gravitropic curvature. The observation that the pattern of differential growth causing curvature is dramatically altered by a change in sensitivity to auxin is consistent with the classical Cholodny-Went theory of gravitropism which maintains that differential growth patterns induced by gravistimulation are mediated primarily by gravi-induced shifts in auxin distribution. The new technology introduced with this report allows automated determination of stimulus response patterns in the small but experimentally popular roots of Arabidopsis.     

Effects of Chlorpromazine on Gravitropism in Avena Coleoptiles

Chlorpromazine (CPZ), an inhibitor of the calcium-activatedform of calmodulin, is readily taken up by the roots of intactoat seedlings but poorly translocated from the roots to thecoleoptile of these plants. However, plants repeatedly rotatedthrough solutions containing low concentrations of CPZ (10^–8–10^–5M)are infiltrated, and under these conditions, CPZ significantlyinhibits the negative gravitropic response of the coleoptilewithout retarding elongation growth. This effect is observablein ‘decapitated’ (apical 1–2 mm removed) coleoptilesections and in intact whole coleoptiles. If exogenous auxinis supplied to the decapitated sections, both their growth ratesand gravitropic responsiveness are increased and, under theseconditions, CPZ can reduce the gravitropic curvature withoutreducing the overall growth rate. These results are discussedin relation to the possible role of calmodulin-dependent calcium-ionpumps in gravitropism. chlorpromazine, gravitropism, calmodulin, calcium, oat, Avena sativa