Auxin Physiology of the Tomato Mutant diageotropica

The tomato (Lycopersicon esculentum, Mill.) mutant diageotropica (dgt) exhibits biochemical, physiological, and morphological abnormalities that suggest the mutation may have affected a primary site of auxin perception or action. We have compared two aspects of the auxin physiology of dgt and wild-type (VFN8) seedlings: auxin transport and cellular growth parameters. The rates of basipetal indole-3-acetic acid (IAA) polar transport are identical in hypocotyl sections of the two genotypes, but dgt sections have a slightly greater capacity for IAA transport. 2,3,5-Triiodobenzoic acid and ethylene reduce transport in both mutant and wild-type sections. The kinetics of auxin uptake into VFN8 and dgt sections are nearly identical. These results make it unlikely that an altered IAA efflux carrier or IAA uptake symport are responsible for the pleiotropic effects resulting from the dgt mutation. The lack of auxin-induced cell elongation in dgt plants is not due to insufficient turgor, as the osmotic potential of dgt cell sap is less (more negative) than that of VFN8. An auxin-induced increase in wall extensibility, as measured by the Instron technique, only occurs in the VFN8 plants. These data suggest dgt hypocotyls suffer a defect in the sequence of events culminating in auxin-induced cell wall loosening.     

Effect of epibrassinolide on hypocotyl growth of the tomato mutant diageotropica

The effect of epibrassinolide (epiBR) on the growth and gravitropism of hypocotyls was investigated in diageotropica (dgt), a mutant of tomato (Lycopersicon esculentum Mill.). The elongation of (dgt) hypocotyls, which had been reported to be auxin-insensitive, was promoted by increasing concentrations of indole-3-acetic acid (IAA) in the presence of epiBR. α-(p-Chlorophenoxy)isobutyric acid, an inhibitor of auxin action, blocked the enhancement of growth by IAA and epiBR. Time course analysis of IAA-induced dgt hypocotyl elongation in the presence of epiBR revealed typical auxin response kinetics. These results suggest that epiBR restores the auxin responsiveness of dgt hypocotyls with respect to elongation. However, epiBR did not rescue the dgt phenotype with respect to shoot gravitropism. It was therefore concluded that brassinosteroid insensitivity or deficiency is not the primary defect of the dgt mutation. Received: 23 January 1998 / Accepted: 20 June 1998     

The diageotropica mutation alters auxin induction of a subset of the Aux/IAA gene family in tomato

The diageotropica (dgt) mutation has been proposed to affect either auxin perception or responsiveness in tomato plants. It has previously been demonstrated that the expression of one member of the Aux/IAA family of auxin-regulated genes is reduced in dgt plants. Here, we report the cloning of ten new members of the tomato Aux/IAA family by PCR amplification based on conserved protein domains. All of the gene family members except one (LeIAA7) are expressed in etiolated tomato seedlings, although they demonstrate tissue specificity (e.g. increased expression in hypocotyls vs. roots) within the seedling. The wild-type auxin-response characteristics of the expression of these tomato LeIAA genes are similar to those previously described for Aux/IAA family members in Arabidopsis. In dgt seedlings, auxin stimulation of gene expression was reduced in only a subset of LeIAA genes (LeIAA5, 8, 10, and 11), with the greatest reduction associated with those genes with the strongest wild-type response to auxin. The remaining LeIAA genes tested exhibited essentially the same induction levels in response to the hormone in both dgt and wild-type hypocotyls. These results confirm that dgt plants can perceive auxin and suggest that a specific step in early auxin signal transduction is disrupted by the dgt mutation.     

The Diageotropica Gene Differentially Affects Auxin and Cytokinin Responses throughout Development in Tomato

The interactions between the plant hormones auxin and cytokinin throughout plant development are complex, and genetic investigations of the interdependency of auxin and cytokinin signaling have been limited. We have characterized the cytokinin sensitivity of the auxin-resistant diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.) in a range of auxin- and cytokinin-regulated responses. Intact, etiolated dgt seedlings showed cross-resistance to cytokinin with respect to root elongation, but cytokinin effects on hypocotyl growth and ethylene synthesis in these seedlings were not impaired by the dgt mutation. Seven-week-old, green wild-type and dgt plants were also equally sensitive to cytokinin with respect to shoot growth and hypocotyl and internode elongation. The effects of cytokinin and the dgt mutation on these processes appeared additive. In tissue culture organ regeneration from dgt hypocotyl explants showed reduced sensitivity to auxin but normal sensitivity to cytokinin, and the effects of cytokinin and the mutation were again additive. However, although callus induction from dgt hypocotyl explants required auxin and cytokinin, dgt calli did not show the typical concentration-dependent stimulation of growth by either auxin or cytokinin observed in wild-type calli. Cross-resistance of the dgt mutant to cytokinin thus was found to be limited to a small subset of auxin- and cytokinin-regulated growth processes affected by the dgt mutation, indicating that auxin and cytokinin regulate plant growth through both shared and separate signaling pathways.     

The diageotropica gene of tomato encodes a cyclophilin: a novel player in auxin signaling

The single gene, auxin-resistant diageotropica (dgt) mutant of tomato displays a pleiotropic auxin-related phenotype that includes a slow gravitropic response, lack of lateral roots, reduced apical dominance, altered vascular development, and reduced fruit growth. Some auxin responses are unaltered in dgt plants, however, and the levels, metabolism, and transport of auxin appear normal, indicating that the Dgt gene encodes a component of a specific auxin signaling pathway. By combining map-based cloning with comparative microsynteny, we determined that the Dgt gene encodes a cyclophilin (CYP) (LeCYP1; gi:170439) that has not previously been identified as a component of auxin signaling and plant development. Each of the three known dgt alleles contains a unique mutation in the coding sequence of LeCyp1. Alleles dgt ^1-1 and dgt ^1-2 contain single nucleotide point mutations that generate an amino acid change (G₁₃₇R) and a stop codon (W₁₂₈stop), respectively, while dgt ^dp has an amino acid change (W₁₂₈CΔ129–133) preceding a 15 bp deletion. Complementation of dgt plants with the wild-type LeCyp1 gene restored the wild-type phenotype. Each dgt mutation reduced or nullified the peptidyl–prolyl isomerase activity of the GST–LeCYP1 fusion proteins in vitro. RT-PCR and immunoblot analyses indicated that the dgt mutations do not affect the expression of LeCyp1 mRNA, but the accumulation of LeCYP1 protein is greatly reduced for all three mutant alleles. The CYP inhibitor, cyclosporin A, partially mimics the effects of the dgt mutation in inhibiting auxin-induced adventitious root initiation in tomato hypocotyl sections and reducing the auxin-induced expression of the early auxin response genes, LeIAA10 and 11. These observations confirm that the PPIase activity of the tomato CYP, LeCYP1, encoded by the Dgt gene is important for specific aspects of auxin regulation of plant growth, development, and environmental responses.     

Characterization of the growth and auxin physiology of roots of the tomato mutant,diageotropica

Roots of the tomato (Lycopersicon esculentum, Mill.) mutant diageotropica (dgt) exhibit an altered phenotype. These roots are agravitropic and lack lateral roots. Relative to wild-type (VFN8) roots, dgt roots are less sensitive to growth inhibition by exogenously applied IAA and auxin transport inhibitors (phytotropins), and the roots exhibit a reduction in maximal growth inhibition in response to ethylene. However, IAA transport through roots, binding of the phytotropin, tritiated naphthylphthalamic acid ([³H]NPA), to root microsomal membranes, NPA-sensitive IAA uptake by root segments, and uptake of [³H]NPA into root segments are all similar in mutant and wild-type roots. We speculate that the reduced sensitivity of dgt root growth to auxin-transport inhibitors and ethylene is an indirect result of the reduction in sensitivity to auxin in this single gene, recessive mutant. We conclude that dgt roots, like dgt shoots, exhibit abnormalities indicating they have a defect associated with or affecting a primary site of auxin perception or action.Abbreviations BCA bicinchoninic acid - IAA indole 3-acetic acid - dgt diageotropica - IC₅₀ concentration for 50% inhibition of growth - NPA N-1-naphthylphthalamic acid - SCB-1 semicarbazone 1 This research was supported by grants from Sandoz Agro, Inc. (G.K.M), the National Aeronautics and Space Administration (NASA) and the National Science Foundation (T.L.L), and NASA (D.L.R.).     

Insensitivity of the diageotropica tomato mutant to auxin

The sensitivity of excised hypocotyl segments to indoleacetic acid (IAA) in two assays, ethylene production and elongation, was determined in the ethylene-requiring tomato (Lycopersicon esculentum Mill.) mutant, diageotropica (dgt), and its isogenic parent, cv VFN8. Endogenous (uninduced) ethylene synthesis rates were slightly lower in dgt hypocotyls than in VFN8 hypocotyls. Ethylene production was essentially unaffected by IAA in dgt, but was stimulated up to 10-fold by 10 micromolar IAA in VFN8. Elongation of dgt hypocotyls was also insensitive to concentrations of IAA as high as 100 micromolar, as compared to significant elongation of VFN8 hypocotyls in response to 0.1 micromolar IAA. A range of IAA analogs active in VFN8 was also ineffective in stimulating elongation of dgt hypocotyls, suggesting that the differences were not due to rapid metabolism of IAA by dgt tissues. Auxin-induced elongation of VFN8 hypocotyls was unaffected by 2,3,5-triiodobenzoic acid and naphthylphthalamic acid, indicating that polar auxin transport was not a factor in these experiments. Exogenous and auxin-induced ethylene had no effect on the elongation respone of either genotype, nor did exogenous ethylene restore the sensitivity of dgt hypocotyls to IAA. Despite their apparent insensitivity to auxin, dgt hypocotyls elongated dramatically and synthesized ethylene rapidly in response to 1.2 micromolar fusicoccin. These results suggest that the primary effect of the dgt mutation is to reduce the sensitivity of the tissue to auxin. As altered regulation of ethylene synthesis is only one symptom of this fundamental deficiency, dgt should more properly be considered to be the auxin-insensitive tomato mutant.     

Inhibitors of the carrier-mediated influx of auxin in suspension-cultured tobacco cells

 Active auxin transport in plant cells is catalyzed by two carriers working in opposite directions at the plasma membrane, the influx and efflux carriers. A role for the efflux carrier in polar auxin transport (PAT) in plants has been shown from studies using phytotropins. Phytotropins have been invaluable in demonstrating that PAT is essential to ensure polarized and coordinated growth and to provide plants with the capacity to respond to environmental stimuli. However, the function of the influx carrier at the whole-plant level is unknown. Our work aims to identify new auxin-transport inhibitors which could be employed to investigate its function. Thirty-five aryl and aryloxyalkylcarboxylic acids were assayed for their ability to perturb the accumulation of 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene-1-acetic acid (1-NAA) in suspension-cultured tobacco (Nicotiana tabacum L.) cells. As 2,4-D and 1-NAA are preferentially transported by the influx and efflux carriers, respectively, accumulation experiments utilizing synthetic auxins provide independant information on the activities of both carriers. The majority (60%) of compounds half-inhibited the carrier-mediated influx of [¹⁴C]2,4-D at concentrations of less than 10 μM. Most failed to interfere with [³H]NAA efflux, at least in the short term. Even though they increasingly perturbed auxin efflux when given a prolonged treatment, several compounds were much better at discriminating between influx and efflux carrier activities than naphthalene-2-acetic acid which is commonly employed to investigate influx-carrier properties. Structure-activity relationships and factors influencing ligand specificity with regard to auxin carriers are discussed. Received: 28 June 1999 / Accepted: 28 August 1999     

Requirement for the gravity-controlled transport of auxin for a negative gravitropic response of epicotyls in the early growth stage of etiolated pea seedlings

Gravity-controlled transport of auxin was studied for a negative gravitropic response in the early growth stage of etiolated pea (Pisum sativum L. cv. Alaska) seedlings, in which epicotyl bending was observed near the cotyledon nodes of the seedlings grown continuously from seeds germinated in a horizontal or an inclined position. Increased expression of an auxin-inducible gene, PsIAA4/5, was observed in the elongated side of epicotyls grown in a horizontal or an inclined position. Regardless of the conditions of seed germination, polar auxin transport in the proximal side of the first internodes of the seedlings was significantly higher than in the distal side. Polar auxin transport in the proximal side of epicotyls grown in an inclined position was significantly lower than in those grown in a horizontal position. In contrast, lateral auxin distribution from the proximal to distal sides in epicotyls grown in an inclined position was significantly higher than in epicotyls grown in a horizontal position. Accumulation of PsPIN1 mRNA encoding a putative auxin efflux facilitator, which was observed in vascular tissue, cortex and epidermis in the proximal and distal sides of epicotyls, was markedly influenced by gravistimulation. These results strongly suggest that gravistimulation induces changeable polar auxin transport and one-way lateral auxin distribution in epicotyls as well as asymmetric auxin accumulation in the proximal and distal sides of epicotyls, resulting in a negative gravitropic response of epicotyls in the early growth stage of pea seedlings.     

Expression of α-expansin genes in young seedlings of rice (Oryza sativa L.)

 Rice is the only cereal in which germination and coleoptile elongation occur in hypoxia or anoxia. Little is known of the molecular basis directly underlying coleoptile cell extension. In this paper, we describe the expression of α-expansin genes in embryos during seed development and young seedlings grown under various oxygen concentrations. The genes Os-EXP2 and Os-EXP1 were predominantly expressed in the developing seeds, mainly in newly developed leaves, coleoptiles, and seminal roots. These expansins expressed in the developing seeds may give cells the potential to expand after seed imbibition begins. In coleoptiles, Os-EXP4 and Os-EXP2 mRNAs were greatly induced by submergence, while they were weakly detected in aerobic or anoxic conditions. Under submerged soil conditions, the signals hybridized with probes Os-EXP4 and Os-EXP2 in coleoptiles were strongest when coleoptiles elongated in the water layer. These data show that expansin gene expression is highly correlated with coleoptile elongation in response to oxygen concentrations. The Os-EXP4 gene was also expressed in leaves, mesocotyls, and coleorhizas of young seedlings. The growth of these tissues was also correlated with the presence of expansins. Therefore, the evidence derived from this study clearly demonstrates that expansins are indispensable for the growing tissues of rice seedlings. Received: 23 December 1999 / Accepted: 24 February 2000     

Probing the hormonal activity of fractionated molecular humic components in tomato auxin mutants

We progressively reduced the complexity of humic matter by a mild sequential removal of unbound or free components, weakly, and strongly bound molecules. The auxin‐like response of residues from each step was tested using tomato (cv. Micro‐Tom) seedlings expressing DR5 auxin synthetic promoter fused to the β‐glucuronidase (GUS) reporter gene and the low auxin‐sensitivity diageotropica (dgt) mutant. Both exogenous auxin and humic matter promoted lateral root emergence in the control, but failed to induce lateral roots in the dgt mutant. When strongly bound components were removed from humic matter by breaking the ester and ether bonds, the humic residues lost their ability to induce the DR5::GUS activity and lateral root emergence. However, these capacities were retained in the free or weakly bound molecules. These findings confirm that auxin‐like activity in humic matter is associated with complex hydrophobic structures, whose simplification by hydrolysis may release auxin‐like molecules.     

Conditional identification of phosphate-starvation-response mutants in Arabidopsis thaliana

Plants have evolved elaborate metabolic and developmental adaptations to low phosphorus availability. Biochemical responses to phosphate limitation include increased production and secretion of phosphate-acquisition proteins such as nucleases, acid phosphatases, and high-affinity phosphate transporters. However, the signal transduction pathways that sense phosphate availability and integrate the phosphate-starvation response in plants are unknown. We have devised a screen for conditional mutants in Arabidopsis thaliana (L.) Heynh. to dissect signaling of phosphate limitation. Our genetic screen is based on the facultative ability of wild-type Arabidopsis plants to metabolize exogenous DNA when inorganic phosphate is limiting. After screening 50,000 M2 seedlings, we isolated 22 confirmed mutant lines that showed severely impaired growth on medium containing DNA as the only source of phosphorus, but which recovered on medium containing soluble inorganic phosphate. Characterization of nine such mutant lines demonstrated an inability to utilize either DNA or RNA. One mutant line, psr1 (phosphate starvation response), had significantly reduced activities of phosphate-starvation-inducible isoforms of ribonuclease and acid phosphatase under phosphate-limiting conditions. The data suggest that a subset of the selected mutations impairs the expression of more than one phosphate-starvation-inducible enzyme required for utilization of exogenous nucleic acids, and may thus affect regulatory components of a Pi starvation response pathway in higher plants. Received: 23 September 1999 / Accepted: 10 November 1999     

Control of gravimorphogenesis by auxin: accumulation pattern of CS-IAA1 mRNA in cucumber seedlings grown in space and on the ground

Cucumber (Cucumis sativus L.) seedlings grown in microgravity developed a peg on each side of the transition zone between hypocotyl and root, whereas seedlings grown in a horizontal position on the ground developed a peg on the concave side of the gravitropically bending transition zone. The morphological features of the space-grown seedlings were similar to those of seedlings grown in a vertical position on the ground with their radicles pointing down: both became two-pegged seedlings. Morphogenesis of cucumber seedlings is thus inhibited by gravity. Analysis by in-situ hybridization of an auxin-inducible gene, CS-IAA1, showed that its mRNA accumulated to a much greater extent on the lower side of the transition zone in the horizontally placed seedlings on the ground just prior to and during the initiation period of peg formation. On the other hand, when seedlings were grown in microgravity or in a vertical position on the ground, accumulation of CS-IAA1 mRNA occurred all around the transition zone. Accumulation of CS-IAA1 mRNA in horizontally grown seedlings appreciably decreased on the upper side of the transition zone and increased on the lower side upon gravistimulation, compared with the two-pegged seedlings. Application of IAA to seedlings in a horizontal position caused the development of a peg on each side of the transition zone, or a collar-like protuberance, depending on the concentration used. These results suggest that upon gravistimulation the auxin concentration on the upper side of the horizontally placed transition zone is reduced to a level below the threshold value necessary for peg formation. Space-grown seedlings of cucumber might develop two pegs symmetrically because the auxin level in the entire transition zone is maintained above the threshold. This spaceflight experiment verified for the first time that auxin does not redistribute in microgravity. Received: 10 February 2000 / Accepted: 15 March 2000     

Differential expression of four genes encoding 1-aminocyclopropane-1-carboxylate synthase in Lupinus albus during germination, and in response to indole-3-acetic acid and wounding

1-Aminocyclopropane-1-carboxylate (ACC) synthase (ACS; EC 4.4.1.14) is the key regulatory enzyme of the ethylene biosynthetic pathway and is encoded by a multigene family in Arabidopsis thaliana, tomato, mung bean and other plants. Southern blot analysis revealed the existence of at least five ACS genes in white lupin (Lupinus albus L.) genome. Four complete and one partial sequences representing different ACS genes were cloned from the lupin genomic library. The levels of expression of two of the genes, LA-ACS1 and LA-ACS3, were found to increase after hypocotyl wounding. Apparently, these two genes were up-regulated by exogenous IAA treatment of seedlings. The LA-ACS3 mRNA levels were also elevated in the apical part of hypocotyl, which is reported to contain a high endogenous auxin concentration. This gene may be involved in the auxin- and ethylene-controlled apical hook formation. The expression of the LA-ACS4 gene was found to be almost undetectable. This gene may represent a “silent” twin of LA-ACS5 as these two genes share a considerable level of homology in coding and non-coding regions. The LA-ACS5 mRNA is strongly up-regulated in the embryonic axis of germinating seeds at the time of radicle emergence, and was also found in roots and hypocotyls of lupin seedlings. Received: 19 July 1999 / Accepted: 3 March 2000     

Ethylene plays multiple nonprimary roles in modulating the gravitropic response in tomato.

Ethylene is known to interact with auxin in regulating stem growth, and yet evidence for the role of ethylene in tropic responses is contradictory. Our analysis of four mutants of tomato (Lycopersicon esculentum) altered in their response to gravity, auxin, and/or ethylene revealed concentration-dependent modulation of shoot gravitropism by ethylene. Ethylene inhibitors reduce wild-type gravicurvature, and extremely low (0.0005-0.001 microliter L-1) ethylene concentrations can restore the reduced gravitropic response of the auxin-resistant dgt (diageotropica) mutant to wild-type levels. Slightly higher concentrations of ethylene inhibit the gravitropic response of all but the ethylene-insensitive nr (never-ripe) mutant. The gravitropic responses of nr and the constitutive-response mutant epi (epinastic) are slightly and significantly delayed, respectively, but otherwise normal. The reversal of shoot gravicurvature by red light in the lz-2 (lazy-2) mutant is not affected by ethylene. Taken together, these data indicate that, although ethylene does not play a primary role in the gravitropic response of tomato, low levels of ethylene are necessary for a full gravitropic response, and moderate levels of the hormone specifically inhibit gravicurvature in a manner different from ethylene inhibition of overall growth.     

Regulation of auxin transport by aminopeptidases and endogenous flavonoids

 The 1-N-naphthylphthalamic acid (NPA)-binding protein is a putative negative regulator of polar auxin transport that has been shown to block auxin efflux from both whole plant tissues and microsomal membrane vesicles. We previously showed that NPA is hydrolyzed by plasma-membrane amidohydrolases that co-localize with tyrosine, proline, and tryptophan-specific aminopeptidases (APs) in the cotyledonary node, hypocotyl-root transition zone and root distal elongation zone of Arabidopsisthaliana (L.) Heynh. seedlings. Moreover, amino acyl-β-naphthylamide (aa-NA) conjugates resembling NPA in structure have NPA-like inhibitory activity on growth, suggesting a possible role of APs in NPA action. Here we report that the same aa-NA conjugates and the AP inhibitor bestatin also block auxin efflux from seedling tissue. Bestatin and, to a lesser extent, some aa-NA conjugates were more effective inhibitors of low-affinity specific [³H]NPA-binding than were the flavonoids quercetin and kaempferol but had no effect on high-affinity binding. Since the APs are inhibited by flavonoids, we compared the localization of endogenous flavonoids and APs in seedling tissue. A correlation between AP and flavonoid localization was found in 5- to 6-d-old seedlings. Evidence that these flavonoids regulate auxin accumulation in vivo was obtained using the flavonoid-deficient mutant, tt4. In whole-seedling [¹⁴C]indole-3-acetic acid transport studies, the pattern of auxin distribution in the tt4 mutant was shown to be altered. The defect appeared to be in auxin accumulation, as a considerable amount of auxin escaped from the roots. Treatment of the tt4 mutant with the missing intermediate naringenin restored normal auxin distribution and accumulation by the root. These results implicate APs and endogenous flavonoids in the regulation of auxin efflux. Received: 2 December 1999 / Accepted: 16 January 2000     

Morphogenesis in cucumber seedlings is negatively controlled by gravity

 Seedlings of most cucurbitaceous plants develop a peg (protuberance caused by cell outgrowth) on the transition zone between the hypocotyl and root. The peg is necessary for removing the seed coat after germination. In our spaceflight experiments on the STS-95 space shuttle, Discovery, we found that cucumber (Cucumis sativus L.) seedlings grown under microgravity conditions developed two pegs symmetrically at the transition zone. Thus, cucumber seedlings potentially develop two pegs and do not require gravity for peg formation itself, but on the ground the development of one peg is suppressed in response to gravity. This may be considered as negative control of morphogenesis by gravity. Received: 17 August 1999 / Accepted: 4 October 1999     

The<Emphasis Type="Italic"> diageotropica</Emphasis> mutation of tomato disrupts a signalling chain using extracellular auxin binding protein 1 as a receptor

The diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.) is known to lack a number of typical auxin responses. Here we show that rapid auxin-induced growth of seedling hypocotyls is completely abolished by the mutation over the full range of auxin concentrations tested, and also in early phases of the time course. Protoplasts isolated from wild-type hypocotyls respond to auxin by a rapid increase in cell volume, which we measured by image analysis at a high temporal resolution. A similar swelling could be triggered by antibodies directed against a part of the putative auxin-binding domain (box-a) of the auxin-binding protein 1 (ABP1). Induction of swelling both by auxin and by the antibody was not observed in the protoplasts isolated from the dgt mutant. However, dgt protoplasts are able to respond to the stimulator of the H⁺-ATPase, fusicoccin, with normal swelling. We propose that dgt is a signal-transduction mutation interfering with an auxin-signalling pathway that uses ABP1 as a receptor.Abbreviations ABP auxin-binding protein - CCD charge-coupled device - 2,4-D 2,4-dichlorophenoxyacetic acid - dgt diageotropica - FC fusicoccin     

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.