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.     

Negative gravitropic response of roots directs auxin flow to control root gravitropism

Root tip is capable of sensing and adjusting its growth direction in response to gravity, a phenomenon known as root gravitropism. Previously, we have shown that negative gravitropic response of roots (NGR) is essential for the positive gravitropic response of roots. Here, we show that NGR, a plasma membrane protein specifically expressed in root columella and lateral root cap cells, controls the positive root gravitropic response by regulating auxin efflux carrier localization in columella cells and the direction of lateral auxin flow in response to gravity. Pharmacological and genetic studies show that the negative root gravitropic response of the ngr mutants depends on polar auxin transport in the root elongation zone. Cell biology studies further demonstrate that polar localization of the auxin efflux carrier PIN3 in root columella cells and asymmetric lateral auxin flow in the root tip in response to gravistimulation is reversed in the atngr1;2;3 triple mutant. Furthermore, simultaneous mutations of three PIN genes expressed in root columella cells impaired the negative root gravitropic response of the atngr1;2;3 triple mutant. Our work revealed a critical role of NGR in root gravitropic response and provided an insight of the early events and molecular basis of the positive root gravitropism.     

Early development and gravitropic response of lateral roots in Arabidopsis thaliana

Root system architecture plays an important role in determining nutrient and water acquisition and is modulated by endogenous and environmental factors, resulting in considerable developmental plasticity. The orientation of primary root growth in response to gravity (gravitropism) has been studied extensively, but little is known about the behaviour of lateral roots in response to this signal. Here, we analysed the response of lateral roots to gravity and, consistently with previous observations, we showed that gravitropism was acquired slowly after emergence. Using a lateral root induction system, we studied the kinetics for the appearance of statoliths, phloem connections and auxin transporter gene expression patterns. We found that statoliths could not be detected until 1 day after emergence, whereas the gravitropic curvature of the lateral root started earlier. Auxin transporters modulate auxin distribution in primary root gravitropism. We found differences regarding PIN3 and AUX1 expression patterns between the lateral root and the primary root apices. Especially PIN3, which is involved in primary root gravitropism, was not expressed in the lateral root columella. Our work revealed new developmental transitions occurring in lateral roots after emergence, and auxin transporter expression patterns that might explain the specific response of lateral roots to gravity.     

Nitric oxide mediates gravitropic bending in soybean roots

Plant roots are gravitropic, detecting and responding to changes in orientation via differential growth that results in bending and reestablishment of downward growth. Recent data support the basics of the Cholodny-Went hypothesis, indicating that differential growth is due to redistribution of auxin to the lower sides of gravistimulated roots, but little is known regarding the molecular details of such effects. Here, we investigate auxin and gravity signal transduction by demonstrating that the endogenous signaling molecules nitric oxide (NO) and cGMP mediate responses to gravistimulation in primary roots of soybean (Glycine max). Horizontal orientation of soybean roots caused the accumulation of both NO and cGMP in the primary root tip. Fluorescence confocal microcopy revealed that the accumulation of NO was asymmetric, with NO concentrating in the lower side of the root. Removal of NO with an NO scavenger or inhibition of NO synthesis via NO synthase inhibitors or an inhibitor of nitrate reductase reduced both NO accumulation and gravitropic bending, indicating that NO synthesis was required for the gravitropic responses and that both NO synthase and nitrate reductase may contribute to the synthesis of the NO required. Auxin induced NO accumulation in root protoplasts and asymmetric NO accumulation in root tips. Gravistimulation, NO, and auxin also induced the accumulation of cGMP, a response inhibited by removal of NO or by inhibitors of guanylyl cyclase, compounds that also reduced gravitropic bending. Asymmetric NO accumulation and gravitropic bending were both inhibited by an auxin transport inhibitor, and the inhibition of bending was overcome by treatment with NO or 8-bromo-cGMP, a cell-permeable analog of cGMP. These data indicate that auxin-induced NO and cGMP mediate gravitropic curvature in soybean roots.     

Sending mixed messages: auxin-cytokinin crosstalk in roots

Despite their relatively simple appearance, roots are incredibly complex organs that are highly adapted to differing environments. Many aspects of root development are co-ordinated by subtle spatial differences in the concentrations of the phytohormones auxin and cytokinin. Events from the formation of a root during embryogenesis to the determination of the network of lateral roots are controlled by interactions between these hormones. Recently, interactions have been defined where auxin signaling promotes the expression of cytokinin signaling inhibitors, cytokinin signaling promotes the expression of auxin signaling inhibitors and finally where cytokinin signaling regulates the complex network of auxin transport proteins to position zones of high auxin signaling. We are witnessing a period of discovery in which we are beginning to understand how these hormonal pathways communicate to regulate root formation.     

Gravitropism in lateral roots of Arabidopsis pgm-1 mutants is indistinguishable from that of wild-type

The majority of understanding of root gravity responses comes from the study of primary roots, even though lateral roots make a far greater contribution to root system architecture. The focus of this report is the analysis of gravitropic responses in lateral roots of wild-type background and pgm-1 mutants. Despite the significant reduction in gravitropic response of primary roots of pgm-1 mutants, the lateral roots of this mutant demonstrate wild-type rates of gravitropism, suggesting a significant difference in gravity signal transduction between primary and lateral roots.Key words: gravitropism, lateral roots, pgm-1, root system architecturePlants are extremely sensitive to numerous environmental stimuli, including touch, gravity, light and humidity, among many others. As a pervasive signal on Earth, gravity exerts a persistent influence on plant morphogenesis by directing the primary roots and shoots of most species to align parallel with the gravity vector. The vertical orientations obtained by primary organs has provided for a simple assay of gravitropic responses, and much of our understanding of gravity stimulus perception, signal transduction and differential growth response has been gained by a focus on primary organ systems.With respect to gravity stimulus perception, there is strong evidence that the movement of starch-filled plastids plays a primary role in the detection of a change in the orientation of an organ relative to gravity.¹ Consistent with this evidence, we have recently demonstrated that roots of the starchless mutant of Arabidopsis, pgm-1, respond to gravity at approximately 30% the rate of wild-type roots, and that they lack the wild-type relationship between cap angle and response rate.² Furthermore, pgm-1 roots lack the gravity-induced gradient of auxin reported by DR5-GFP expression, found in wild-type roots, linking plastid sedimentation with the differential auxin transport thought to mediate the differential growth response.³While our understanding of root gravitropism has grown in sophistication and detail, the emerging picture has been compiled almost entirely from observations of primary organ behavior. The degree to which our model of signaling involved in primary root gravitropic responses applies to the behavior of lateral roots is an almost entirely open question, with only a handful of studies investigating lateral root gravitropic responses.^4–6 Toward that end, we have begun to explore the question of lateral root gravitropism in the overall context of root system architecture, and wish to report here on the gravitropic response of lateral roots in wild-type and pgm-1 genetic backgrounds.     

Synergistic action of auxin and cytokinin mediates aluminum‐induced root growth inhibition in Arabidopsis

Auxin acts synergistically with cytokinin to control the shoot stem‐cell niche, while both hormones act antagonistically to maintain the root meristem. In aluminum (Al) stress‐induced root growth inhibition, auxin plays an important role. However, the role of cytokinin in this process is not well understood. In this study, we show that cytokinin enhances root growth inhibition under stress by mediating Al‐induced auxin signaling. Al stress triggers a local cytokinin response in the root‐apex transition zone (TZ) that depends on IPTs, which encode adenosine phosphate isopentenyltransferases and regulate cytokinin biosynthesis. IPTs are up‐regulated specifically in the root‐apex TZ in response to Al stress and promote local cytokinin biosynthesis and inhibition of root growth. The process of root growth inhibition is also controlled by ethylene signaling which acts upstream of auxin. In summary, different from the situation in the root meristem, auxin acts with cytokinin in a synergistic way to mediate aluminum‐induced root growth inhibition in Arabidopsis.     

Role of cytokinin in the regulation of root gravitropism

The models explaining root gravitropism propose that the growth response of plants to gravity is regulated by asymmetric distribution of auxin (indole-3-acetic acid, IAA). Since cytokinin has a negative regulatory role in root growth, we suspected that it might function as an inhibitor of tropic root elongation during gravity response. Therefore, we examined the free-bioactive-cytokinin-dependent ARR5::GUS expression pattern in root tips of transformants of Arabidopsis thaliana (L.) Heynh., visualized high cytokinin concentrations in the root cap with specific monoclonal antibodies, and complemented the analyses by external application of cytokinin. Our findings show that mainly the statocytes of the cap produce cytokinin, which may contribute to the regulation of root gravitropism. The homogenous symmetric expression of the cytokinin-responsive promoter in vertical root caps rapidly changed within less than 30 min of gravistimulation into an asymmetrical activation pattern, visualized as a lateral, distinctly stained, concentrated spot on the new lower root side of the cap cells. This asymmetric cytokinin distribution obviously caused initiation of a downward curvature near the root apex during the early rapid phase of gravity response, by inhibiting elongation at the lower side and promoting growth at the upper side of the distal elongation zone closely behind the root cap. Exogenous cytokinin applied to vertical roots induced root bending towards the application site, confirming the suspected inhibitory effect of cytokinin in root gravitropism. Our results suggest that the early root graviresponse is controlled by cytokinin. We conclude that both cytokinin and auxin are key hormones that regulate root gravitropism.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00425-004-1381-8     

Auxin-dependent regulation of lateral root positioning in the basal meristem of Arabidopsis

In plants, the developmental mechanisms that regulate the positioning of lateral organs along the primary root are currently unknown. We present evidence on how lateral root initiation is controlled in a spatiotemporal manner in the model plant Arabidopsis thaliana. First, lateral roots are spaced along the main axis in a regular left-right alternating pattern that correlates with gravity-induced waving and depends on AUX1, an auxin influx carrier essential for gravitropic response. Second, we found evidence that the priming of pericycle cells for lateral root initiation might take place in the basal meristem, correlating with elevated auxin sensitivity in this part of the root. This local auxin responsiveness oscillates with peaks of expression at regular intervals of 15 hours. Each peak in the auxin-reporter maximum correlates with the formation of a consecutive lateral root. Third, auxin signaling in the basal meristem triggers pericycle cells for lateral root initiation prior to the action of INDOLE-3-ACETIC ACID14 (SOLITARY ROOT).     

Elongation and gravitropic responses of Arabidopsis roots are regulated by brassinolide and IAA

Exogenously applied brassinolide (BL) increased both gravitropic curvature and length of primary roots of Arabidopsis at low concentration (10(-10) M), whereas at higher concentration, BL further increased gravitropic curvature while it inhibited primary root growth. BRI1-GFP plants possessing a high steady-state expression level of a brassinosteroid (BR) receptor kinase rendered the plant's responses to gravity and root growth more sensitive, while BR-insensitive mutants, bri1-301 and bak1, delayed root growth and reduced their response to the gravitropic stimulus. The stimulatory effect of BL on the root gravitropic curvature was also enhanced in auxin transport mutants, aux1-7 and pin2, relative to wild-type plants, and increasing concentration of auxin attenuated BL-induced root sensitivity to gravity. Interestingly, IAA treatment to the roots of bri1-301 and bak1 plants or of plants pretreated with a BL biosynthetic inhibitor, brassinazole, increased their sensitivity to gravity, while these treatments for the BL-hypersensitive transgenic plants, BRI1-GFP and 35S-BAK1, were less effective. Expression of a CYP79B2 gene, encoding an IAA biosynthetic enzyme, was suppressed in BL-hypersensitive plant types and enhanced in BL-insensitive or -deficient plants. In conclusion, our results indicate that BL interacts negatively with IAA in the regulation of plant gravitropic response and root growth, and its regulation is achieved partly by modulating biosynthetic pathways of the counterpart hormone.     

Tomato root growth, gravitropism, and lateral development: correlation with auxin transport.

Tomato (Lycopersicon esculentum, Mill.) roots were analyzed during growth on agar plates. Growth of these roots was inhibited by the auxin transport inhibitors naphthylphthalamic acid (NPA) and semicarbazone derivative I (SCB-1). The effect of auxin transport inhibitors on root gravitropism was analyzed by measurement of the angle of gravitropic curvature after the roots were reoriented 90 degrees from the vertical. NPA and SCB-1 abolished both the response of these roots to gravity and the formation of lateral roots, with SCB-1 being the more effective at inhibition. Auxins also inhibited root growth. Both auxins tested has a slight effect on the gravity response, but this effect is probably indirect, since auxins reduced the growth rate. Auxins also stimulated lateral root growth at concentration where primary root growth was inhibited. When roots were treated with both IAA and NPA simultaneously, a cumulative inhibition of root growth was found. When both compounds were applied together, analysis of gravitropism and lateral root formation indicated that the dominant effect was exerted by auxin transport inhibitors. Together, these data suggest a model for the role of auxin transport in controlling both primary and lateral root growth.     

The effects of auxin on lateral root initiation and root gravitropism in a lateral rootless mutant Lrt1 of rice (Oryza sativa L.)

Auxins control growth and development in plants, including lateral rootinitiation and root gravity response. However, how endogenous auxin regulatesthese processes is poorly understood. In this study, the effects of auxins onlateral root initiation and root gravity response in rice were investigatedusing a lateral rootless mutant Lrt1, which fails to formlateral roots and shows a reduced root gravity response. Exogenous applicationof IBA to the Lrt1 mutant restored both lateral rootinitiation and root gravitropism. However, application of IAA, a major form ofnatural auxin, restored only root gravitropic response but not lateral rootinitiation. These results suggest that IBA is more effective than IAA in lateralroot formation and that IBA also plays an important role in root gravitropicresponse in rice. The application of NAA restored lateral root initiation, butdid not completely restore root gravitropism. Root elongation assays ofLrt1 displayed resistance to 2,4-D, NAA, IBA, and IAA.This result suggests that the reduced sensitivity to exogenous auxins may be due tothe altered auxin activity in the root, thereby affecting root morphology inLrt1.     

Characterization of the glutathione S-transferase (GST) gene family in <Emphasis Type="Italic">Pyrus bretschneideri</Emphasis> and their expression pattern upon superficial scald development

Artemisinin, an antimalarial secondary metabolite produced in Artemisia species, also has been recognized as an allelochemical that inhibits the growth of several plant species. However, the phytotoxicity mechanism of artemisinin is not exhaustively deciphered up to now. In this research, the effects of artemisinin on Arabidopsis thaliana root gravitropic curvature and development were characterized. Exogenously applied artemisinin disturb the root gravitropic responses, inhibited the elongation of primary and lateral roots and root hairs in a concentration-dependent fashion, and prevented the formation of lateral roots and root hairs. Moreover, the number of starch grain and the distribution range of auxin in the root tip was reduced by artemisinin, and the redistribution of auxin was less sensitive to gravity stimulus when treated with artemisinin than that of control. The expression of auxin transporter PIN2 was partially suppressed by artemisinin. Together, the results demonstrated that the effects of artemisinin on root gravitropism and root system development were largely dependent on the reduction of starch grain and auxin levels, as well as the disordered lateral auxin redistribution.     

Gravity signal transduction in primary roots

AIMS: The molecular mechanisms that correlate with gravity perception and signal transduction in the tip of angiosperm primary roots are discussed. SCOPE: Gravity provides a cue for downward orientation of plant roots, allowing anchorage of the plant and uptake of the water and nutrients needed for growth and development. Root gravitropism involves a succession of physiological steps: gravity perception and signal transduction (mainly mediated by the columella cells of the root cap); signal transmission to the elongation zone; and curvature response. Interesting new insights into gravity perception and signal transduction within the root tip have accumulated recently by use of a wide range of experimental approaches in physiology, biochemistry, genetics, genomics, proteomics and cell biology. The data suggest a network of signal transduction pathways leading to a lateral redistribution of auxin across the root cap and a possible involvement of cytokinin in initial phases of gravicurvature. CONCLUSION: These new discoveries illustrate the complexity of a highly redundant gravity-signalling process in roots, and help to elucidate the global mechanisms that govern auxin transport and morphogenetic regulation in roots.     

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.     

Intracellular trafficking and proteolysis of the Arabidopsis auxin-efflux facilitator PIN2 are involved in root gravitropism

Root gravitropism describes the orientation of root growth along the gravity vector and is mediated by differential cell elongation in the root meristem. This response requires the coordinated, asymmetric distribution of the phytohormone auxin within the root meristem, and depends on the concerted activities of PIN proteins and AUX1 - members of the auxin transport pathway. Here, we show that intracellular trafficking and proteasome activity combine to control PIN2 degradation during root gravitropism. Following gravi-stimulation, proteasome-dependent variations in PIN2 localization and degradation at the upper and lower sides of the root result in asymmetric distribution of PIN2. Ubiquitination of PIN2 occurs in a proteasome-dependent manner, indicating that the proteasome is involved in the control of PIN2 turnover. Stabilization of PIN2 affects its abundance and distribution, and leads to defects in auxin distribution and gravitropic responses. We describe the effects of auxin on PIN2 localization and protein levels, indicating that redistribution of auxin during the gravitropic response may be involved in the regulation of PIN2 protein.