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.     

Complex physiological and molecular processes underlying root gravitropism

Gravitropism allows plant organs to guide their growth in relation to the gravity vector. For most roots, this response to gravity allows downward growth into soil where water and nutrients are available for plant growth and development. The primary site for gravity sensing in roots includes the root cap and appears to involve the sedimentation of amyloplasts within the columella cells. This process triggers a signal transduction pathway that promotes both an acidification of the wall around the columella cells, an alkalinization of the columella cytoplasm, and the development of a lateral polarity across the root cap that allows for the establishment of a lateral auxin gradient. This gradient is then transmitted to the elongation zones where it triggers a differential cellular elongation on opposite flanks of the central elongation zone, responsible for part of the gravitropic curvature. Recent findings also suggest the involvement of a secondary site/mechanism of gravity sensing for gravitropism in roots, and the possibility that the early phases of graviresponse, which involve differential elongation on opposite flanks of the distal elongation zone, might be independent of this auxin gradient. This review discusses our current understanding of the molecular and physiological mechanisms underlying these various phases of the gravitropic response in roots.     

Spatial separation of light perception and growth response in maize root phototropism

Although the effects of gravity on root growth are well known and interactions between light and gravity have been reported, details of root phototropic responses are less documented. We used high-resolution image analysis to study phototropism in primary roots of Zea mays L. Similar to the location of perception in gravitropism, the perception of light was localized in the root cap. Phototropic curvature away from the light, on the other hand, developed in the central elongation zone, more basal than the site of initiation of gravitropic curvature. The phototropic curvature saturated at approximately 10 micromoles m-2 s-1 blue light with a peak curvature of 29 +/- 4 degrees, in part due to induction of positive gravitropism following displacement of the root tip from vertical during negative phototropism. However, at higher fluence rates, development of phototropic curvature is arrested even if gravitropism is avoided by maintaining the root cap vertically using a rotating feedback system. Thus continuous illumination can cause adaptation in the signalling pathway of the phototropic response in roots.     

Cytokinin: perception,signal transduction,and role in plant growth and development

Cytokinins are essential hormones for the proper growth and development of plants. They exert their actions through the phosphorylation of two-component signaling factors. The two-component elements in cytokinin signaling display not only overlapping, but also specific functions throughout a life cycle. These elements regulate the development of shoots, roots, and inflorescence meristems inArabidopsis; shoot meristems in rice; and nodule formation in the lotus. They are also involved in interactions between plants and pathogens. In this review, we examine the mechanism for signaling events initiated by cytokinins inArabidopsis.     

铵减弱拟南芥主根向地性及其相关作用途径

向地性是决定植物根系空间构型的主要因素之一,对植物锚定和水分养分吸收至关重要。除了重力,根系向地性还受土壤环境因子影响。本文采用琼脂培养方法,研究了铵对拟南芥主根向地性反应的影响及相关作用途径。结果表明：短期内,不同浓度（NH4）2SO4均显著抑制主根向地性弯曲,但随着时间的延长,根尖向地性角度逐渐变小。而等（NH4）2SO4浓度的NaCl对主根向地性抑制效应较小,不同浓度的甘露醇不阻碍主根向地性弯曲。纽织化学染色结果显示铵处理12h以内,Col-0根尖没有淀粉体的快速降解过程,并且铵对淀粉体缺失突变体pgm—1主根向地性的影响同Col-0相似。铵处理部分恢复生长素转运载体突变体auxl-22和eir1-1主根向地性缺失。这些结果表明,铵对拟南芥主根向地性的影响独立于根尖淀粉体参与的重力感应途径。     

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.     

Touch modulates gravity sensing to regulate the growth of primary roots of Arabidopsis thaliana

Plants must sense and respond to diverse stimuli to optimize the architecture of their root system for water and nutrient scavenging and anchorage. We have therefore analyzed how information from two of these stimuli, touch and gravity, are integrated to direct root growth. In Arabidopsis thaliana, touch stimulation provided by a glass barrier placed across the direction of growth caused the root to form a step-like growth habit with bends forming in the central and later the distal elongation zones. This response led to the main root axis growing parallel to, but not touching the obstacle, whilst the root cap maintained contact with the barrier. Removal of the graviperceptive columella cells of the root cap using laser ablation reduced the bending response of the distal elongation zone. Similarly, although the roots of the gravisensing impaired pgm1-1 mutant grew along the barrier at the same average angle as wild-type, this angle became more variable with time. These observations imply a constant gravitropic re-setting of the root tip response to touch stimulation from the barrier. In wild-type plants, transient touch stimulation of root cap cells, but not other regions of the root, inhibited both subsequent gravitropic growth and amyloplast sedimentation in the columella. Taken together, these results suggest that the cells of the root cap sense touch stimuli and their subsequent signaling acts on the columella cells to modulate their graviresponse. This interaction of touch and gravity signaling would then direct root growth to avoid obstacles in the soil while generally maintaining downward growth.     

Microsurgical removal of epidermal and cortical cells: evidence that the gravitropic signal moves through the outer cell layers in primary roots of maize

There is general agreement that during root gravitropism some sort of growth-modifying signal moves from the cap to the elongation zone and that this signal ultimately induces the curvature that leads to reorientation of the root. However, there is disagreement regarding both the nature of the signal and the pathway of its movement from the root cap to the elongation zone. We examined the pathway of movement by testing gravitropism in primary roots of maize (Zea mays L.) from which narrow (0.5 mm) rings of epidermal and cortical tissue were surgically removed from various positions within the elongation zone. When roots were girdled in the apical part of the elongation zone gravitropic curvature occurred apical to the girdle but not basal to the girdle. Filling the girdle with agar allowed curvature basal to the girdle to occur. Shallow girdles, in which only two or three cell layers (epidermis plus one or two cortical cell layers) were removed, prevented or greatly delayed gravitropic curvature basal to the girdle. The results indicate that the gravitropic signal moves basipetally through the outermost cell layers, perhaps through the epidermis itself.     

Transport of [3H]IAA label in gravistimulated primary roots of maize

The curvature of roots in response to gravity is attributed to the development of a differential concentration gradient of IAA in the top and bottom of the elongation region of roots. The development of the IAA gradient has been attributed to the redistribution of IAA from the stele to cortical tissues in the elongation region. The gravistimulated redistribution of IAA was investigated by applying [³H]IAA to the cut surface of 5 mm apical primary root segments. The movement of label from the stele-associated [³H]IAA into the root, tip, root cap, and cortical tissues on the top and bottom of the elongation region was determined in vertically growing roots and gravistimulated roots. Label from the stele moved into the region of cell differentiation (root tip) prior to accumulating in the elongation region. Little label was observed in the root cap. Gravistimulation did not increase the amount of label moving from the stele; but gravistimulation did increase the amount of label accumulating in cortical tissues on the lower side of the elongation region, and decreased the amount of label accumulating in cortical tissues on the upper side of the elongation region. Removal of the cap prior to or immediately following gravity stimulation rendered the roots partially insensitive to gravity and also prevented gravity-induced asymmetric redistribution of label. However, removal of the root cap following 30 min of gravistimulation did not alter root curvature or the establishment of an IAA asymmetry across the region of root elongation. These results suggest that a signal originating in the root cap directs auxin redistribution in tissues behind the root cap, leading to the development of an asymmetry of IAA concentration in the elongation region that in turn causes the differential growth rate in the elongation region of a graviresponding root.     

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.     

拟南芥QUA1基因在光信号途径中的表达与功能分析

光信号在植物生长发育过程中具有非常重要的作用。不同的光信号通过调节植物下游基因的表达,进而影响细胞分化、结构和功能的改变,以及组织和器官的形成,参与植物光形态建成。QUA1 (QUASIMODO1)是拟南芥糖基转移酶家族中的一个成员,参与植物细胞壁中果胶的合成。本文以拟南芥qua1-1/cry1以及qua1-1/phyB双突变体为材料,对QUA1基因在光信号途径中的功能进行了分析。结果显示,qua1-1突变体在暗、蓝光、红光以及远红外光培养条件下下胚轴的伸长均受到抑制,QUA1基因的表达同样受到光信号的调节,而且突变体中多种光信号调节基因的表达也受到了影响。通过对qua1-1突变体下胚轴的观察发现,突变体下胚轴表皮细胞长度明显变短。与cry1以及phyB突变体相比,qua1-1/cry1和qua1-1/phyB双突变体下胚轴长度明显变短,而且双突变体中光信号调节基因的表达也有明显变化,表明QUA1可能参与了CRY1以及PHYB介导的蓝光及红光信号传导。以上结果表明QUA1影响了下胚轴细胞的伸长以及光信号调节基因的表达,并参与调控多种光信号传导途径。     

Potassium carrier TRH1 is required for auxin transport in Arabidopsis roots

Disruption of the TRH1 potassium transporter impairs root hair development in Arabidopsis, and also affects root gravitropic behaviour. Rescue of these morphological defects by exogenous auxin indicates a link between TRH1 activity and auxin transport. In agreement with this hypothesis, the rate of auxin translocation from shoots to roots and efflux of [3H]IAA in isolated root segments were reduced in the trh1 mutant, but efflux of radiolabelled auxin was accelerated in yeast cells transformed with the TRH1 gene. In roots, Pro(TRH1):GUS expression was localized to the root cap cells which are known to be the sites of gravity perception and are central for the redistribution of auxin fluxes. Consistent with these findings, auxin-dependent DR5:GUS promoter-reporter construct was misexpressed in the trh1 mutant indicating that partial block of auxin transport through the root cap is associated with upstream accumulation of the phytohormone in protoxylem cells. When [K+] in the medium was reduced from 20 to 0.1 mm, wild type roots showed mild agravitropic phenotype and DR5:GUS misexpression in stelar cells. This pattern of response to low external [K+] was also affected by trh1 mutation. We conclude that the TRH1 carrier is an important part of auxin transport system in Arabidopsis roots.     

植物体内磷脂代谢和信号转导

磷脂不但作为细胞结构的重要组成部分，其代谢产物主料可作为人信号分子，这一推断日益受到人们重视，并为越来越多的实验所证实。本文综述磷脂酶类、磷酸肌醇激酶及其代谢产物参与的植物细胞内的信号转导，阐述它们与植物激素和钙信使系统的关系。     

Secondary messengers and phospholipase A2 in auxin signal transduction

Despite recent progress auxin signal transduction remains largely scetchy and enigmatic. A good body of evidence supports the notion that the ABP1 could be a functional receptor or part of a receptor, respectively, but this is not generally accepted. Evidence for other functional receptors is lacking, as is any clearcut evidence for a function of G proteins. Protons may serve as second messengers in guard cells but the existing evidence for a role of calcium remains to be clearified. Phospholipases C and D seem not to have a function in auxin signal transduction whereas the indications for a role of phospholipase A₂ in auxin signal transduction accumulated recently. Mitogen-activated protein kinase (MAPK) is modulated by auxin and the protein kinase PINOID has a role in auxin transport modulation even though their functional linkage to other signalling molecules is ill-defined. It is hypothesized that signal transduction precedes activation of early genes such as IAA genes and that ubiquitination and the proteasome are a mechanism to integrate signal duration and signal strength in plants and act as major regulators of hormone sensitivity.     

In vivo evidence for the involvement of phospholipase A and protein kinase in the signal transduction pathway for auxin-induced corn coleoptile elongation

Auxin-induced elongation of com coleoptiles is accompanied by cell wall acidification, which depends upon H⁺-pump activity. We tested the hypothesis that phospholipase A and a protein kinase are involved in the pathway of auxin signal transduction leading to H⁺ secretion, and elongation of corn coleoptiles. Initially, the pH of the bath solution at 50–100 μm from the surface of a coleoptile segment (pH_o) ranged between 4.8 and 6.6 when measured with an H⁺-sensitive microelectrode. Twenty or 50 μM lysophosphatidylcholine, 50 μM linolenic acid or 50 μM arachidonic acid induced a decline in pH_o by 0.3 to 2.1 units. The effect was blocked by 1 mM vanadate, suggesting that lysophosphatidylcholine or linolenic acid induced acidification of the apoplast by activating the H⁺-pump. Lysophosphatidylcholine and linolenic acid also accelerated the elongation rate of the coleoptiles. While linolenic acid and arachidonic acid, highly unsaturated fatty acids, promoted pH_o decrease and coleoptile elongation, linoleic acid, oleic acid, and stearic acid, fatty acids with a lesser extent of unsaturation, had no such effects. The effects of lysophosphatidylcholine, linolenic acid, and arachidonic acid on H⁺ secretion were not additive to that of indoleacetic acid (IAA), suggesting that lysophospholipids, fatty acids and auxin use similar pathways for the activation of the H⁺-pump. The phospholipase A₂ inhibitors, aristolochic acid and manoalide, inhibited the IAA-induced pH_o decrease and coleoptile elongation. The general protein kinase inhibitors, H-7 or staurosporine, blocked the IAA- or lysophosphatidylcholine-induced decrease in pH_o. H-7 also inhibited the coleoptile elongation induced by IAA or lysophosphatidylcholine. These results support the hypothesis that phospholipase A is activated by auxin, and that the products of the enzyme, lysophospholipids and fatty acids, induce acidification of the apoplast by activating the H⁺-pump through a mechanism involving a protein kinase, which in turn promotes com coleoptile elongation.     

The Control of Lateral Root Development in Cultured Pea Seedlings. III. Spacing Intervals

The spacing of lateral root primordia in the primary root of Pisum sativum (cv. Alaska) seedlings is influenced by both predetermined lateral root initiation sites in the embryonic radicle and by factors present during seedling growth. When pea seeds were germinated in the presence of the mitotic inhibitor, colchicine, the triarch radicle produced three ranks of primordiomorphs indicating sites of embryonic lateral root primordia. The number of primordiomorphs was not the same along the three xylem strands in the radicle. Normally germinated seedling roots (5 days old) also showed a different number of lateral root primordia associated with the three strands. In both cases, the strand with the greatest number of primordia (or primordiomorphs) was associated with a cotyledonary trace. This indicated a possible role for the cotyledons in setting the pattern of lateral root distribution during radicle development. The spacing of lateral root primordia could be altered by the application of growth regulators. Seedling root tips (2 mm) were removed (? rt) and replaced with indoleacetic acid (+IAA), and in some instances seedlings were also treated with the auxin transport inhibitor, 3,3a-dihydro-2-(p-methoxyphenyl)-8H-pyrazolo[5, 1-α]isoindol-8-one (+DPX). In the growth regulator treatments, primary root elongation was inhibited, a greater number of lateral root primordia were initiated compared to controls, and the spacing intervals between primordia were greatly reduced. The — rt, +IAA, +DPX-treatment resulted in the closest possible spacing intervals (av. 0.4 ? 0.6 mm), but resulted in fused or fasciated laterals. The — rt, + IAA-treatment produced the shortest spacing intervals which resulted in “normal” lateral roots (0.8 ? 1.1 mm).     

Characterization of changes of pain behavior and signal transduction system in food-deprived mice

Fasting in general causes several metabolic changes. In the present study, we examined the possible changes of several types of nociception during the food deprivation were investigated in mice. After the mice were forced into the fasting for 12, 24, or 48?h, the changes of nociception were measured by the tail-flick, writhing, formalin or von-frey tests. We found that the nociceptive behavior induced by intraperitoneally (i.p.) administered acetic acid (writhing response) or intraplantar injection of 5% formalin into the hind-paw were reduced in fasted group. In addition, the tail-flick response and threshold for nociception in mechanical von-frey test were also elevated in fasted group. Moreover, the p-CREB and p-ERK levels in the dorsal root ganglia (DRG) and the spinal cord were reduced in food-deprived group. Furthermore, p-AMPKα₁ expressions in DRG and the spinal cord were up-regulated, whereas p-mTOR in DRG and the spinal cord was down-regulated in food-deprived group. Our results suggest that the chemical, mechanical, and thermal nociceptions appear to be reduced in a food-deprived mouse group. Additionally, reduction of nociception in food-deprived group appears to be closely associated with the expressions of several signal transduction molecules such as ERK, CREB, AMPKα₁ and mTOR proteins in DRG and the spinal cord.     

Calcium-dependent asymmetric movement of 3H-indole-3-acetic acid across gravistimulated isolated root caps of maize

There is evidence that the cap is the initial site of lateral auxin redistribution during the gravitropic response of roots. We tested this further by comparing asymmetric auxin redistribution across the tips of gravistimulated intact roots, decapped roots, isolated root caps and isolated apical sections taken from decapped roots. Gravistimulation caused asymmetric (downward) auxin movement across the tips of intact roots and isolated root caps but not across the tips of decapped roots or across isolated apical root segments. Naphthylphthalamic acid and pyrenoylbenzoic acid, inhibitors of polar auxin transport, inhibited asymmetric auxin redistribution across gravistimulated isolated root caps and across the tips of gravistimulated intact roots. For intact roots there was a positive correlation between the extent of inhibition of assymmetric auxin redistribution by polar auxin transport inhibitors and the extent of inhibition of asymmetric calcium chelating agent, ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid, also caused parallel inhibition of asymmetric auxin redistribution and gravitropic curvature and this effect was reversed by subsequent treatment with calcium. The results support the hypothesis that the cap is a site of early development of auxin asymmetry in gravistimulated roots and that calcium plays an important role in the development of lateral auxin redistribution.     

Organization of cortical microtubules in graviresponding maize roots

Immunofluorescence labeling of cortical microtubules (MTs) was used to investigate the relationship between MT arrangement and changes in growth rate of the upper and lower sides of horizontally placed roots of maize (Zea mays L. cv. Merit). Cap cells and cells of the elongation zone of roots grown vertically in light or darkness showed MT arrangements that were transverse (perpendicular) to the growth direction. Microtubules of cells basal to the elongation zone typically showed oblique orientation. Two hours after horizontal reorientation, cap cells of gravicompetent, light-grown and curving roots contained MTs parallel to the gravity vector. The MT arrangement on the upper side of the elongation zone remained transverse but the MTs of the outer four to five layers of cortical cells along the lower side of the elongation zone showed reorientation parallel to the axis of the root. The MTs of the lower epidermis retained their transverse orientation. Dark-grown roots did not curve and did not show reorientation of MTs in cells of the root cap or elongation zone. The data indicate that MT depolymerization and reorientation is correlated with reduction in growth rate, and that MT reorientation is one of the steps of growth control of graviresponding roots.Abbreviations MT microtubule - QC quiescent center This work was supported by National Science Foundation grant IBN-9118094.     

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.