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The endodermis and shoot gravitropism
trends in plant science reviews
The endodermis and shoot gravitropism Masao Tasaka, Takehide Kato and Hidehiro Fukaki Shoots and roots of higher plants exhibit negative and positive gravitropism, respectively. A variety of gravitropic mutants have recently been isolated from Arabidopsis, the characterization of which demonstrates that the molecular mechanisms of the gravitropic responses in roots, hypocotyls and inflorescence stems are different. The cytological and molecular analysis of two mutants, shoot gravitropism 1 (sgr1), which is allelic to scarecrow (scr), and sgr7, which is allelic to short-root (shr), indicate that the endodermis is the site of gravity perception in shoots. These data suggest a new model for shoot gravitropism.
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any environmental parameters, including light, temperature, water and nutrients, as well as other organisms, affect plant growth. Gravity is one such parameter – horizontally oriented shoots curve upward and roots curve downward because of it. This process is called gravitropism, and can be divided into four sequential steps: • Gravity perception • Signal formation in the gravity perceptive cell • Intracellular and intercellular signal transduction • Asymmetric cell elongation between the upper and lower sides of the responding organs1,2. Gravitropism has been considered one of the great mysteries of plant behavior for almost 190 years3. Most classical experiments regarded the responding organs as ‘black boxes’ and analyzed the relationship between stimulus and response to infer what was inside the boxes, without directly studying the cellular or molecular mechanisms. Recently, a more direct, genetic approach has been adopted using Arabidopsis1,2,4–27. At least three organs of Arabidopsis show a gravitropic response: the root, the hypocotyl and the inflorescence stem. In this review, we mainly focus on recent shoot gravitropism data.
• Class IV – mutants that have defective gravitropism in hypocotyls and roots but not in inflorescence stems20 (Fukaki et al., unpublished data). • Class V – mutants that exhibit only a defective gravitropic phenotype in roots4,8,9,13–15; it includes many auxin-related mutants. This indicates that at least some elements of the gravitropic mechanisms in these three organs are genetically different. It is not surprising that roots and shoots show positive and negative gravitropism, respectively. However, it is interesting that hypocotyls and inflorescence stems use slightly different molecular mechanisms, even though both organs are negatively gravitropic and are anatomically classified as shoots. One possible explanation is that the developmental origins of these organs are different. Inflorescence stems derive from the shoot apical meristem, which originates from the apical portion of the embryo, whereas the hypocotyl differentiates from the middle part of the embryo. The presence of the class IV mutants, in which both hypocotyls and roots are affected, is also interesting, because these organs respond in opposite directions and have different embryonic origins. Such observations indicate that it is important to analyze the molecular mechanisms of gravitropism in each organ, independently.
Gravitropic response of inflorescence stems
When mature Arabidopsis plants are horizontally oriented, their inflorescence stems bend upward in darkness. Gravitropism of inflorescence stems has several characteristics in common with those of other shoots28. For example, decapitated stem segments anchored in an agar block and turned horizontally in the dark, show a similar gravitropic response to intact stems, indicating that the stem segment contains all the essential components for a gravitropic response28. The time course of this response is shown in Fig. 1. The time sequence suggests that there is no ‘master zone’ organizing all the stem parts, and that each part of the elongation zone has an ability to respond to gravity. This is confirmed by many physiological experiments28. For example, when the stem segment is cut at any position in the elongation zone, all the cut fragments respond to gravity28. Arabidopsis mutants with defective gravitropism
To analyze the molecular mechanisms of gravitropism, mutants with defective gravitropism have been isolated and characterized from Arabidopsis4–27. The organs affected by these mutations are summarized in Fig. 2. The mutations are divided into five different classes: • Class I – mutants that show an abnormal gravitropic response in inflorescence stems only16,19. • Class II – mutants that show defective gravitropism in both inflorescence stems and hypocotyls, but normal gravitropism in roots16,19,22. • Class III – mutants that affect roots, hypocotyls and inflorescence stems6,7,10,11.
Fig. 1. The gravitropic response of a decapitated inflorescence stem segment of Arabidopsis. The segment was horizontally inclined in darkness at 238C. The arrow indicates the orientation of gravity (g). A photograph was taken at each time period, and the photographs overlain by a computer-assisted graphical method. After a short initial lag, the stem bends upwards in the most rapidly elongating region (0.5 h), causing the apical end to overshoot the vertical (2 h), so that the stem shows a ‘U’ shape. After 3 h, the apical region returns to the vertical but the basal region continues bending up to its original orientation so that the segment becomes ‘S’ shaped. After 6 h the apical region passes over the vertical again. After 24 h, the stem becomes vertical between the elongation zone and the nonbending basal zone28.
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The endodermis is the gravity perception site in shoots
Plants are exposed to a constant 1 g force; the gravitropic response is triggered by the perception of a change in the relative gravity orientation. How plants perceive the gravity vector is one of the important questions in gravitropism. g According to the starch–statolith hyrhg pothesis, gravity is perceived by organelles Hypocotyl slr called statoliths29,30. Physiological data suggest that the amyloplast, which contains agr/eir1 Root starch granules, is equivalent to the statoaux1 lith in higher plants. Sedimented amyloaxr1 plasts are located in root-cap columella ax4/rgr1 cells and in the starch sheath layer in stems. Mutants that have lost the ability to syntheFig. 2. Genetic regulation of gravitropism in Arabidopsis. Roots, hypocotyls and inflorescence size starch granules in amyloplasts have stems exhibit a gravitropic response. Agravitropic mutants in Arabidopsis are divided into five also been isolated from other plant species31. groups depending on the combination of abnormal gravitropic organs that are affected. Abbreviations: sgr, shoot gravitropism; pgm, phosphoglucomutase deficient; axr, auxin In Arabidopsis, the phosphoglucomutase resistant; rhg, root and hypocotyl gravitropism; slr, solitary root; agr, agravitropic, which deficient (pgm) mutant and a starch-deficient is allelic to eir, ethylene insensitive root; aux, auxin-resistant; axr, auxin-resistant; rgr, mutant are well-characterized mutants in reduced root gravitropism. which phosphoglucomutase activity is absent6,7. These mutants lack starch both in columella cells and in starch sheath cells and show reduced gravitropic responses in all three organs6,7,21,27, Wild type Mutant indicating that starch is necessary for a normal response. This was confirmed using other mutants that synthesize starch partially18,21,27. SCR /SGR1 and SHR /SGR7 scr /sgr1 and shr /sgr7 In roots, the columella cells in the cap are probably the gravitysensing cells29,30. In addition to the data using the starchless muEndodermis formation Loss of endodermis tants, an elegant cytological experiment supports this possibility32. (inflorescence stem, When different layers of columella cells are selectively ablated by hypocotyl and root) laser, the rate of root response is dependent on the number and the layer of remaining cells. Moreover, the importance of each cell layer in the columella for graviperception is correlated with the Starch sheath formation Loss of starch sheath (inflorescence stem and sedimentation rate of amyloplasts in each cell32. hypocotyl) For shoots, there is no direct evidence as to where the gravity perceptive tissue is, except for the presence of sedimented amyloplasts in the starch sheath or endodermis. Inflorescence stems and Normal gravitropism No gravitropism hypocotyls of shoot gravitropism 1 (sgr1) show no gravitropic response16,22. Longitudinal sections of sgr1 inflorescence stems reveal no endodermal cells in the positions where sedimented amyloplasts are found in inflorescence stems of the wild type, and there are no sedimented amyloplasts anywhere in mutant stems22. Hypocotyls of sgr1 have only two irregular cell layers between the epidermis and the stele compared with two layers of cortex and one layer of endodermis in the wild type22. A few amyloplasts have been observed in the abnormal cells in sgr1, but these never sediment. The inflorescence stems and hypocotyls of another mutant, sgr7, also exhibit no gravitropic response, have no endodermal layer in either inflorescence stems or hypocotyls and have no sedimented amyloplasts22. However, both sgr1 and sgr7 have normal sedimented amyloplasts in root columella cells and show a normal gravitropic response in roots22. Genetic analysis indicates that SGR1 is allelic Fig. 3. The endodermis is essential for shoot gravitropism to SCARECROW (SCR) and SGR7 is allelic to SHORT-ROOT in Arabidopsis. SHOOT GRAVITROPISM 1 [which is allelic to 22 (SHR) . Both genes have been identified as affecting the difSCARECROW (SGR1/SCR)] and SGR 7 [which is allelic to ferentiation of the root and the hypocotyl endodermis33,34. The SHORT-ROOT (SHR)] genes are essential for the differentiation SCR/SGR1 gene possibly encodes a transcription factor from a of endodermal cells in inflorescence stems, hypocotyls and roots. Endodermal cells in inflorescence stems and hypocotyls contain new bZIP family34. The above data indicate that the SCR/SGR1 sedimented amyloplasts, which are important for sensing the gravand SHR/SGR7 genes are also essential for endodermal cell difity vector. Mutations in each gene cause the loss of both endoderferentiation in shoots (Fig. 3). When one is mutated, shoots lose mal cell differentiation and a gravitropic response in inflorescence endodermal cells and cannot respond to gravity22, indicating that stems and hypocotyls. endodermal cells are essential for gravitropism. Moreover, because only the endoderm possesses sedimented amyloplasts in Inflorescence stem
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(a)
(b)
Endodermal cell
g
Cortex Endodermis Stele
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Fig. 4. Model for gravity perception in inflorescence stems in Arabidopsis. (a) Stage after apical overshooting in inflorescence stem showing a ‘U’ shape. The apical region is bent up past the vertical to the left. Short black arrows indicate the directions of the subsequent upward bend; red bars indicate the lower sides of the stems that elongate more than the upper sides in the same sections. The stem can be considered to consist of numerous thin sections, each of which recognizes the gravity vector independently, and the lower part of each section elongates further than the upper part, suggesting that the response of the whole stem is the sum of each local response. (b) Cross section of horizontally-oriented stem. Black arrows indicate the orientation of gravity (g); red arrow indicates the radial direction of each endodermal cell.
shoots, it is likely that endodermal cells are the cells which perceive gravity in shoots. The distribution of endodermal cells in inflorescence stems has been analyzed in Arabidopsis2,27. In hypocotyls and inflorescence stems, the endodermal cells surrounds the stele in a cylindrical layer one cell thick. Cells containing sedimented amyloplasts are present throughout the stem elongation zone. Although the distribution pattern of cells containing sedimented amyloplasts in shoots varies in different plant species – in most plants these cells are arranged in a radially symmetrical manner in shoots35.
Stele Endodermis Cortex Epidermis
Endodermis Endodermis differentiation Amyloplast development Perception of gravity vector Production of signal Amyloplast
Model for gravity perception in shoots
There are two important characteristics of stem gravity sensing. The first is that stems can recognize the gravity vector throughout the elongation zone. For example, stem segments cut from any part of the elongation zone are gravitropic28. The cylindrical distribution of endodermal cells in the entire elongation zone is well adapted for this function. In the case of the Arabidopsis inflorescence stem, the stem does not show the same elongation ability throughout the elongation zone28. The apical region elongates faster than the basal region. When the stem segment is placed horizontally, the most actively elongating region starts to bend followed by the remaining regions that are gradually involved in bending (Fig. 1). When the apical region stands vertically,
Elongation zone Differential cell elongation
Signal transduction (auxin)
Amyloplast
Cortex and epidermis Signal transduction Differential cell elongation
g
Root columella cells Amyloplast development Perception of gravity vector Production of signal
Fig. 5. Model of regulation of root and inflorescence stem gravitropism in Arabidopsis. The gravity vector (g) in inflorescence stems is perceived in endodermal cells and the signal (red arrow) is transported to the outer layers. Granules in endodermal cells indicate sediment amyloplasts. In roots, the gravity vector (g) is perceived by the columella cells of the root cap. A signal (red arrow) is transported from the columella cells to the elongation zone.
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trends in plant science reviews basal regions continue to bend to the same orientation, the apical region then bends over the vertical position passively and the stem forms a ‘U’ shape. The apical region then turns back to a different orientation from its original movement because of new gravitropic stimulation, while the basal region bends up continuously to the same orientation of its original movement. As a result, both regions show relatively different gravitropic responses. These movements suggest that the apical and basal regions may recognize the relative direction of the gravity vector independently during the gravitropic response (Fig. 4a). The second important characteristic of gravitropism is that inflorescence stems can recognize the directional change of gravity when they are inclined in any direction because of the radially symmetrical distribution of endodermal cells. But, how does the endodermal tissue recognize the gravity vector between the upper and lower parts of the section as being different when stems are inclined? And how do these cells produce different signals to induce asymmetrical cell elongation between the upper and lower parts? One possible explanation is shown in Fig. 4b. If an endodermal cell is able to recognize its own radial orientation, it could recognize a difference between its own radial direction and the gravity vector. This difference gradually changes from the upper to the lower part of the stem depending on the position of each cell in the section, and each endodermal cell can provide a different strength of signal to control stem cell elongation overall. Recognizing the gravity vector and the response – a future perspective of gravitropism
Amyloplasts are common as a statolith in all three gravi-responsive organs in Arabidopsis. The starch-deficient Arabidopsis mutant shows reduced gravitropism in all these organs18,21,27, suggesting that this organelle may work as a ‘weight’. It is not clear whether the sedimenting movement itself is essential for graviperception. The graviperception mechanism may include not only amyloplasts but also other cellular components that work as a ‘balance’. Such components may also be present in all graviperceiving cells. This mechanism may recognize gravity as a ‘vector’ (orientation of gravity) rather than a ‘scalar’ (strength of gravity) signal. Although there are some models for a graviperception mechanism, there is little molecular evidence29,30. A signal(s) is produced based on the sensing of the gravity vector in root-columella and in shoot-endodermal cells. It is not clear whether the signal(s) itself and the signal-production system are common to all gravitropic organs. Signals produced in sensing cells must be transmitted to effector cells, for example, from columella cells to the elongation zone (Fig. 5). These two regions are separated along the apical–basal axis of the root. By contrast, in inflorescence stems, signals produced in endodermal cells are transported to outer cell layers. This means that the graviperception cell layer and effector cell layers are present in concentric positions in the cross section, perpendicular to the apical–basal axis. This difference in anatomy could account for some of the organ specificity of different gravitropic mutants. Now that the gravity perception sites in roots, hypocotyls and inflorescence stems have become clearer in Arabidopsis, it should be possible to understand gravitropism in each organ at the tissue, cell and molecular levels. Some reduced gravitropic mutants have been characterized, but it is important to continue to isolate more gravitropic mutants and to analyze their physiology, cytology and molecular biology to fully elucidate the gravitropic mechanism. It is not unreasonable to expect that all these genes will be cloned in the near future through the efforts of the Arabidopsis genomesequencing projects. Combining the sequence data with physiological, cytological and molecular data from these mutants should 106
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provide both an outline of gravitropism and a detailed scenario of each step at the molecular level. Gravitropism includes many important and essential plant physiological processes, such as perception of an environmental stimulus, signal formation, intra- and inter-cellular signal transduction, growth control by plant hormones and cell and tissue differentiation. The analysis of gravitropism should advance many other fundamental fields of plant biology, and the outcome of understanding gravitropism might provide new ideas for agriculture and horticulture. Acknowledgements
We thank Dr F.D. Sack (Ohio State University, USA) for critical reading of the manuscript and valuable suggestions. This work was supported, in part, by Grants-in-Aid for General Scientific Research and Scientific Research on a Priority Area from the Ministry of Education, Science and Culture of Japan, and by a grant for ‘Research for the Future’ Program from The Japan Society for the Promotion of Science to M.T. and by a Grantin-Aid to H.F. from the Japan Ministry of Education, Science and Science and Culture. H.F was supported by a fellowship from the Japanese Society for the Promotion of Science. References 1 Fukaki, H., Fujisawa, H. and Tasaka, M. (1996) How do plant shoots bend up? The initial step to elucidate the molecular mechanisms of shoot gravitropism using Arabidopsis thaliana, J. Plant Res. 109, 129–137 2 Fukaki, H. and Tasaka, M. Gravity perception and gravitropic response of inflorescence stems in Arabidopsis thaliana, Adv. Space Res. (in press) 3 Darwin, C. (1881) The Power of Movement in Plants (assisted by Darwin, F.), John Murray, London 4 Mirza, J.I. et al. (1984) The growth and gravitropic responses of wild-type and auxin-resistant mutants of Arabidopsis thaliana, Physiol. Plant. 60, 516–522 5 Caspar, T., Huber, S.C. and Somerville, C.R. (1985) Alterations in growth, photosynthesis, and respiration in a starchless mutant of Arabidopsis thaliana (L.) deficient in chloroplast phosphoglucomutase activity, Plant Physiol. 79, 11–17 6 Caspar, T. and Pickard, B.G. (1989) Gravitropism in a starchless mutant of Arabidopsis, Planta 177, 185–197 7 Kiss, J.Z., Hertel, R. and Sack, F.D. (1989) Amyloplasts are necessary for full gravitropic sensitivity in roots of Arabidopsis thaliana, Planta 117, 198–206 8 Bell, C.J. and Maher, E.P. (1990) Mutants of Arabidopsis thaliana with abnormal gravitropic responses, Mol. Gen. Genet. 220, 289–293 9 Lincoln, C., Britton, J.H. and Estelle, M. (1990) Growth and development of axr1 mutants of Arabidopsis, Plant Cell 2, 1071–1080 10 Wilson, A.K. et al. (1990) A dominant mutation in Arabidopsis confers resistance to auxin, ethylene and abscisic acid, Mol. Gen. Genet. 222, 377–383 11 Timpte, C.S., Wilson, A.K. and Estelle, M. (1992) Effects of the axr2 mutation of Arabidopsis on cell shape in hypocotyl and inflorescence, Planta 188, 271–278 12 Leyser, H.M.O. et al. (1993) Arabidopsis auxin-resistance gene AXR1 encodes a protein related to ubiquitin-activating enzyme E1, Nature 346, 161–164 13 Roman, G. et al. (1995) Genetic analysis of ethylene signal transduction in Arabidopsis thaliana: five novel mutant loci integrated into a stress response pathway, Genetics 139, 1393–1409 14 Simmons, C. et al. (1995) A novel root gravitropism mutant of Arabidopsis thaliana exhibiting altered auxin physiology, Physiol. Plant. 93, 790–798 15 Hobbie, L. and Estelle, M. (1995) The axr4 auxin-resistant mutants of Arabidopsis thaliana define a gene important for root gravitropism and lateral root initiation, Plant J. 7, 211–220 16 Fukaki, H., Fujisawa, H. and Tasaka, M. (1996) SGR1, SGR2 and SGR3: novel genetic loci involved in shoot gravitropism in Arabidopsis thaliana, Plant Physiol. 110, 945–955 17 Bennett, M.J. et al. (1996) Arabidopsis AUX1 gene: a permease-like regulator of root gravitropism, Science 273, 948–950
trends in plant science reviews 18 Kiss, J.Z., Wright, J.B. and Caspar, T. (1996) Gravitropism in roots of intermediate-starch mutants of Arabidopsis, Physiol. Plant. 94, 237–244 19 Yamauchi, Y. et al. (1997) Mutations in the SGR4, SGR5 and SGR6 loci of Arabidopsis thaliana alter the shoot gravitropism, Plant Cell Physiol. 38, 530–535 20 Fukaki, H., Fujisawa, H. and Tasaka, M. (1997) The RHG gene is involved in root and hypocotyl gravitropism in Arabidopsis thaliana, Plant Cell Physiol. 38, 804–810 21 Kiss, J.Z. et al. (1997) Reduced gravitropism in hypocotyls of starch-deficient mutants of Arabidopsis, Plant Cell Physiol. 38, 518–525 22 Fukaki, H. et al. (1998) Genetic evidence that the endodermis is essential for shoot gravitropism in Arabidopsis thaliana, Plant J. 14, 425–430 23 Luschnig, C. et al. (1998) EIR1, a root-specific protein involved in auxin transport, is required for gravitropism in Arabidopsis thaliana, Genes Dev. 12, 2175–2187 24 Utsuno, K. et al. (1998) AGR, an agravitropic locus of Arabidopsis thaliana, encodes a novel membrane-protein family member, Plant Cell Physiol. 39, 1111–1118 25 Müller, A. et al. (1998) AtPIN2 defines a locus of Arabidopsis for root gravitropism control, EMBO J. 17, 6903–6911 26 Rouse, D. et al. (1998) Changes in auxin response from mutations in an AUX/IAA gene, Science 279, 1371–1373 27 Weise, S.E. and Kiss, J.Z. Gravitropism of inflorescence stems in starchdeficient mutants of Arabidopsis, Int. J. Plant Sci. (in press) 28 Fukaki, H., Fujisawa, H. and Tasaka, M. (1996) Gravitropic response of inflorescence stems in Arabidopsis thaliana, Plant Physiol. 110, 933–943
29 Sack, F.D. (1991) Plant gravity sensing, Int. Rev. Cytol. 127, 193–252 30 Sack, D.F. (1997) Plastids and gravitropic sensing, Planta 203, 63–68 31 Kiss, J.Z. and Sack, F.D. (1989) Reduced gravitropic sensitivity in roots of a starch-deficient mutant of Nicotiana sylvestris, Planta 180, 123–130 32 Blancaflor, E.B., Fasano, J.M. and Gilroy, S. (1998) Mapping the functional roles of cap cells in the response of Arabidopsis primary roots to gravity, Plant Physiol. 116, 213–222 33 Scheres, B. et al. (1995) Mutations affecting the radial organization of the Arabidopsis root display specific defects throughout the radial axis, Development 121, 53–62 34 Di Laurenzio, L. et al. (1996) The SCARECROW gene regulates an asymmetric cell division that is essential for generating the radial organization of the Arabidopsis root, Cell 86, 423–433 35 Hawker, L.E. (1932) A quantitative study of the geotropism of seedlings with special reference to the nature and development of their statolith apparatus, Ann. Bot. 66, 121–157
Masao Tasaka*, Takehide Kato and Hidehiro Fukaki are at the Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0101, Japan.
*Author for correspondence (tel 181 743 72 5480;
fax 181 743 72 5489; e-mail [email protected]).
Function of the ubiquitin–proteosome pathway in auxin response J. Carlos del Pozo and Mark Estelle Proteolysis of important regulatory proteins by the ubiquitin–proteosome pathway is a key aspect of cellular regulation in eukaryotes. Genetic studies in Arabidopsis indicate that response to auxin depends on the function of proteins in this pathway. The auxin transport inhibitor resistant 1 (TIR1) protein is part of a ubiquitin–protein–ligase complex (E3), known as SKP1 CDC53 F-boxTIR1 (SCFTIR1), that possibly directs ubiquitin-modification of protein regulators of the auxin response. In yeast, a similar E3 complex, SCF CDC4, is regulated by conjugation of the ubiquitin-related protein Rub1 to the Cdc53 protein. In Arabidopsis, the AUXIN-RESISTANT1 (AXR1) gene encodes a subunit of the RUB1-activating enzyme, the first enzyme in the RUB-conjugation pathway. Loss of AXR1 results in loss of auxin response. These results suggest a model in which RUB1 modification regulates the activity of SCFTIR1, thereby directing the degradation of the repressors of the auxin response.
A
uxin [indole-3-acetic acid; (IAA)] regulates many important aspects of plant growth and development, including apical dominance, tropic responses, lateral root and root hair formation, and vascular tissue differentiation1. In spite of the importance of this hormone, the molecular basis of auxin response is poorly understood. Using Arabidopsis, several groups have adopted genetic approaches to identify a collection of auxin-response mutants2. One of these mutants, auxin-resistant1 (axr1), was isolated as an auxin-resistant mutant that has a pleiotropic phenotype associated with a decreased response to auxin3. In addition, axr1 plants are deficient in auxin-regulated gene expression, suggesting that
AXR1 is required for auxin signal transduction4,5. The AXR1 gene encodes a protein that participates in activation of the ubiquitinrelated protein RUB1 (Refs 6,7). The auxin transport inhibitor resistant 1 (tir1) mutant also exhibits reduced auxin response. TIR1 is a member of the F-box family of proteins. Studies in yeast and animals indicate that F-box proteins are part of an E3 ubiquitin– ligase complex called the SKP1 CDC53 F-box (SCF)8,9. The cloning and characterization of these auxin-response genes suggest that auxin signaling is mediated through the ubiquitin pathway. Recent molecular data on AXR1 and TIR1 function in Arabidopsis are providing new insight into auxin response.
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The endodermis and shoot gravitropism Masao Tasaka, Takehide Kato and Hidehiro Fukaki Shoots and roots of higher plants exhibit negative and positive gravitropism, respectively. A variety of gravitropic mutants have recently been isolated from Arabidopsis, the characterization of which demonstrates that the molecular mechanisms of the gravitropic responses in roots, hypocotyls and inflorescence stems are different. The cytological and molecular analysis of two mutants, shoot gravitropism 1 (sgr1), which is allelic to scarecrow (scr), and sgr7, which is allelic to short-root (shr), indicate that the endodermis is the site of gravity perception in shoots. These data suggest a new model for shoot gravitropism.
M
any environmental parameters, including light, temperature, water and nutrients, as well as other organisms, affect plant growth. Gravity is one such parameter – horizontally oriented shoots curve upward and roots curve downward because of it. This process is called gravitropism, and can be divided into four sequential steps: • Gravity perception • Signal formation in the gravity perceptive cell • Intracellular and intercellular signal transduction • Asymmetric cell elongation between the upper and lower sides of the responding organs1,2. Gravitropism has been considered one of the great mysteries of plant behavior for almost 190 years3. Most classical experiments regarded the responding organs as ‘black boxes’ and analyzed the relationship between stimulus and response to infer what was inside the boxes, without directly studying the cellular or molecular mechanisms. Recently, a more direct, genetic approach has been adopted using Arabidopsis1,2,4–27. At least three organs of Arabidopsis show a gravitropic response: the root, the hypocotyl and the inflorescence stem. In this review, we mainly focus on recent shoot gravitropism data.
• Class IV – mutants that have defective gravitropism in hypocotyls and roots but not in inflorescence stems20 (Fukaki et al., unpublished data). • Class V – mutants that exhibit only a defective gravitropic phenotype in roots4,8,9,13–15; it includes many auxin-related mutants. This indicates that at least some elements of the gravitropic mechanisms in these three organs are genetically different. It is not surprising that roots and shoots show positive and negative gravitropism, respectively. However, it is interesting that hypocotyls and inflorescence stems use slightly different molecular mechanisms, even though both organs are negatively gravitropic and are anatomically classified as shoots. One possible explanation is that the developmental origins of these organs are different. Inflorescence stems derive from the shoot apical meristem, which originates from the apical portion of the embryo, whereas the hypocotyl differentiates from the middle part of the embryo. The presence of the class IV mutants, in which both hypocotyls and roots are affected, is also interesting, because these organs respond in opposite directions and have different embryonic origins. Such observations indicate that it is important to analyze the molecular mechanisms of gravitropism in each organ, independently.
Gravitropic response of inflorescence stems
When mature Arabidopsis plants are horizontally oriented, their inflorescence stems bend upward in darkness. Gravitropism of inflorescence stems has several characteristics in common with those of other shoots28. For example, decapitated stem segments anchored in an agar block and turned horizontally in the dark, show a similar gravitropic response to intact stems, indicating that the stem segment contains all the essential components for a gravitropic response28. The time course of this response is shown in Fig. 1. The time sequence suggests that there is no ‘master zone’ organizing all the stem parts, and that each part of the elongation zone has an ability to respond to gravity. This is confirmed by many physiological experiments28. For example, when the stem segment is cut at any position in the elongation zone, all the cut fragments respond to gravity28. Arabidopsis mutants with defective gravitropism
To analyze the molecular mechanisms of gravitropism, mutants with defective gravitropism have been isolated and characterized from Arabidopsis4–27. The organs affected by these mutations are summarized in Fig. 2. The mutations are divided into five different classes: • Class I – mutants that show an abnormal gravitropic response in inflorescence stems only16,19. • Class II – mutants that show defective gravitropism in both inflorescence stems and hypocotyls, but normal gravitropism in roots16,19,22. • Class III – mutants that affect roots, hypocotyls and inflorescence stems6,7,10,11.
Fig. 1. The gravitropic response of a decapitated inflorescence stem segment of Arabidopsis. The segment was horizontally inclined in darkness at 238C. The arrow indicates the orientation of gravity (g). A photograph was taken at each time period, and the photographs overlain by a computer-assisted graphical method. After a short initial lag, the stem bends upwards in the most rapidly elongating region (0.5 h), causing the apical end to overshoot the vertical (2 h), so that the stem shows a ‘U’ shape. After 3 h, the apical region returns to the vertical but the basal region continues bending up to its original orientation so that the segment becomes ‘S’ shaped. After 6 h the apical region passes over the vertical again. After 24 h, the stem becomes vertical between the elongation zone and the nonbending basal zone28.
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The endodermis is the gravity perception site in shoots
Plants are exposed to a constant 1 g force; the gravitropic response is triggered by the perception of a change in the relative gravity orientation. How plants perceive the gravity vector is one of the important questions in gravitropism. g According to the starch–statolith hyrhg pothesis, gravity is perceived by organelles Hypocotyl slr called statoliths29,30. Physiological data suggest that the amyloplast, which contains agr/eir1 Root starch granules, is equivalent to the statoaux1 lith in higher plants. Sedimented amyloaxr1 plasts are located in root-cap columella ax4/rgr1 cells and in the starch sheath layer in stems. Mutants that have lost the ability to syntheFig. 2. Genetic regulation of gravitropism in Arabidopsis. Roots, hypocotyls and inflorescence size starch granules in amyloplasts have stems exhibit a gravitropic response. Agravitropic mutants in Arabidopsis are divided into five also been isolated from other plant species31. groups depending on the combination of abnormal gravitropic organs that are affected. Abbreviations: sgr, shoot gravitropism; pgm, phosphoglucomutase deficient; axr, auxin In Arabidopsis, the phosphoglucomutase resistant; rhg, root and hypocotyl gravitropism; slr, solitary root; agr, agravitropic, which deficient (pgm) mutant and a starch-deficient is allelic to eir, ethylene insensitive root; aux, auxin-resistant; axr, auxin-resistant; rgr, mutant are well-characterized mutants in reduced root gravitropism. which phosphoglucomutase activity is absent6,7. These mutants lack starch both in columella cells and in starch sheath cells and show reduced gravitropic responses in all three organs6,7,21,27, Wild type Mutant indicating that starch is necessary for a normal response. This was confirmed using other mutants that synthesize starch partially18,21,27. SCR /SGR1 and SHR /SGR7 scr /sgr1 and shr /sgr7 In roots, the columella cells in the cap are probably the gravitysensing cells29,30. In addition to the data using the starchless muEndodermis formation Loss of endodermis tants, an elegant cytological experiment supports this possibility32. (inflorescence stem, When different layers of columella cells are selectively ablated by hypocotyl and root) laser, the rate of root response is dependent on the number and the layer of remaining cells. Moreover, the importance of each cell layer in the columella for graviperception is correlated with the Starch sheath formation Loss of starch sheath (inflorescence stem and sedimentation rate of amyloplasts in each cell32. hypocotyl) For shoots, there is no direct evidence as to where the gravity perceptive tissue is, except for the presence of sedimented amyloplasts in the starch sheath or endodermis. Inflorescence stems and Normal gravitropism No gravitropism hypocotyls of shoot gravitropism 1 (sgr1) show no gravitropic response16,22. Longitudinal sections of sgr1 inflorescence stems reveal no endodermal cells in the positions where sedimented amyloplasts are found in inflorescence stems of the wild type, and there are no sedimented amyloplasts anywhere in mutant stems22. Hypocotyls of sgr1 have only two irregular cell layers between the epidermis and the stele compared with two layers of cortex and one layer of endodermis in the wild type22. A few amyloplasts have been observed in the abnormal cells in sgr1, but these never sediment. The inflorescence stems and hypocotyls of another mutant, sgr7, also exhibit no gravitropic response, have no endodermal layer in either inflorescence stems or hypocotyls and have no sedimented amyloplasts22. However, both sgr1 and sgr7 have normal sedimented amyloplasts in root columella cells and show a normal gravitropic response in roots22. Genetic analysis indicates that SGR1 is allelic Fig. 3. The endodermis is essential for shoot gravitropism to SCARECROW (SCR) and SGR7 is allelic to SHORT-ROOT in Arabidopsis. SHOOT GRAVITROPISM 1 [which is allelic to 22 (SHR) . Both genes have been identified as affecting the difSCARECROW (SGR1/SCR)] and SGR 7 [which is allelic to ferentiation of the root and the hypocotyl endodermis33,34. The SHORT-ROOT (SHR)] genes are essential for the differentiation SCR/SGR1 gene possibly encodes a transcription factor from a of endodermal cells in inflorescence stems, hypocotyls and roots. Endodermal cells in inflorescence stems and hypocotyls contain new bZIP family34. The above data indicate that the SCR/SGR1 sedimented amyloplasts, which are important for sensing the gravand SHR/SGR7 genes are also essential for endodermal cell difity vector. Mutations in each gene cause the loss of both endoderferentiation in shoots (Fig. 3). When one is mutated, shoots lose mal cell differentiation and a gravitropic response in inflorescence endodermal cells and cannot respond to gravity22, indicating that stems and hypocotyls. endodermal cells are essential for gravitropism. Moreover, because only the endoderm possesses sedimented amyloplasts in Inflorescence stem
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sgr3 sgr5 sgr6
sgr1 sgr2 sgr4 sgr7
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(a)
(b)
Endodermal cell
g
Cortex Endodermis Stele
g
Epidermis
Fig. 4. Model for gravity perception in inflorescence stems in Arabidopsis. (a) Stage after apical overshooting in inflorescence stem showing a ‘U’ shape. The apical region is bent up past the vertical to the left. Short black arrows indicate the directions of the subsequent upward bend; red bars indicate the lower sides of the stems that elongate more than the upper sides in the same sections. The stem can be considered to consist of numerous thin sections, each of which recognizes the gravity vector independently, and the lower part of each section elongates further than the upper part, suggesting that the response of the whole stem is the sum of each local response. (b) Cross section of horizontally-oriented stem. Black arrows indicate the orientation of gravity (g); red arrow indicates the radial direction of each endodermal cell.
shoots, it is likely that endodermal cells are the cells which perceive gravity in shoots. The distribution of endodermal cells in inflorescence stems has been analyzed in Arabidopsis2,27. In hypocotyls and inflorescence stems, the endodermal cells surrounds the stele in a cylindrical layer one cell thick. Cells containing sedimented amyloplasts are present throughout the stem elongation zone. Although the distribution pattern of cells containing sedimented amyloplasts in shoots varies in different plant species – in most plants these cells are arranged in a radially symmetrical manner in shoots35.
Stele Endodermis Cortex Epidermis
Endodermis Endodermis differentiation Amyloplast development Perception of gravity vector Production of signal Amyloplast
Model for gravity perception in shoots
There are two important characteristics of stem gravity sensing. The first is that stems can recognize the gravity vector throughout the elongation zone. For example, stem segments cut from any part of the elongation zone are gravitropic28. The cylindrical distribution of endodermal cells in the entire elongation zone is well adapted for this function. In the case of the Arabidopsis inflorescence stem, the stem does not show the same elongation ability throughout the elongation zone28. The apical region elongates faster than the basal region. When the stem segment is placed horizontally, the most actively elongating region starts to bend followed by the remaining regions that are gradually involved in bending (Fig. 1). When the apical region stands vertically,
Elongation zone Differential cell elongation
Signal transduction (auxin)
Amyloplast
Cortex and epidermis Signal transduction Differential cell elongation
g
Root columella cells Amyloplast development Perception of gravity vector Production of signal
Fig. 5. Model of regulation of root and inflorescence stem gravitropism in Arabidopsis. The gravity vector (g) in inflorescence stems is perceived in endodermal cells and the signal (red arrow) is transported to the outer layers. Granules in endodermal cells indicate sediment amyloplasts. In roots, the gravity vector (g) is perceived by the columella cells of the root cap. A signal (red arrow) is transported from the columella cells to the elongation zone.
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trends in plant science reviews basal regions continue to bend to the same orientation, the apical region then bends over the vertical position passively and the stem forms a ‘U’ shape. The apical region then turns back to a different orientation from its original movement because of new gravitropic stimulation, while the basal region bends up continuously to the same orientation of its original movement. As a result, both regions show relatively different gravitropic responses. These movements suggest that the apical and basal regions may recognize the relative direction of the gravity vector independently during the gravitropic response (Fig. 4a). The second important characteristic of gravitropism is that inflorescence stems can recognize the directional change of gravity when they are inclined in any direction because of the radially symmetrical distribution of endodermal cells. But, how does the endodermal tissue recognize the gravity vector between the upper and lower parts of the section as being different when stems are inclined? And how do these cells produce different signals to induce asymmetrical cell elongation between the upper and lower parts? One possible explanation is shown in Fig. 4b. If an endodermal cell is able to recognize its own radial orientation, it could recognize a difference between its own radial direction and the gravity vector. This difference gradually changes from the upper to the lower part of the stem depending on the position of each cell in the section, and each endodermal cell can provide a different strength of signal to control stem cell elongation overall. Recognizing the gravity vector and the response – a future perspective of gravitropism
Amyloplasts are common as a statolith in all three gravi-responsive organs in Arabidopsis. The starch-deficient Arabidopsis mutant shows reduced gravitropism in all these organs18,21,27, suggesting that this organelle may work as a ‘weight’. It is not clear whether the sedimenting movement itself is essential for graviperception. The graviperception mechanism may include not only amyloplasts but also other cellular components that work as a ‘balance’. Such components may also be present in all graviperceiving cells. This mechanism may recognize gravity as a ‘vector’ (orientation of gravity) rather than a ‘scalar’ (strength of gravity) signal. Although there are some models for a graviperception mechanism, there is little molecular evidence29,30. A signal(s) is produced based on the sensing of the gravity vector in root-columella and in shoot-endodermal cells. It is not clear whether the signal(s) itself and the signal-production system are common to all gravitropic organs. Signals produced in sensing cells must be transmitted to effector cells, for example, from columella cells to the elongation zone (Fig. 5). These two regions are separated along the apical–basal axis of the root. By contrast, in inflorescence stems, signals produced in endodermal cells are transported to outer cell layers. This means that the graviperception cell layer and effector cell layers are present in concentric positions in the cross section, perpendicular to the apical–basal axis. This difference in anatomy could account for some of the organ specificity of different gravitropic mutants. Now that the gravity perception sites in roots, hypocotyls and inflorescence stems have become clearer in Arabidopsis, it should be possible to understand gravitropism in each organ at the tissue, cell and molecular levels. Some reduced gravitropic mutants have been characterized, but it is important to continue to isolate more gravitropic mutants and to analyze their physiology, cytology and molecular biology to fully elucidate the gravitropic mechanism. It is not unreasonable to expect that all these genes will be cloned in the near future through the efforts of the Arabidopsis genomesequencing projects. Combining the sequence data with physiological, cytological and molecular data from these mutants should 106
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provide both an outline of gravitropism and a detailed scenario of each step at the molecular level. Gravitropism includes many important and essential plant physiological processes, such as perception of an environmental stimulus, signal formation, intra- and inter-cellular signal transduction, growth control by plant hormones and cell and tissue differentiation. The analysis of gravitropism should advance many other fundamental fields of plant biology, and the outcome of understanding gravitropism might provide new ideas for agriculture and horticulture. Acknowledgements
We thank Dr F.D. Sack (Ohio State University, USA) for critical reading of the manuscript and valuable suggestions. This work was supported, in part, by Grants-in-Aid for General Scientific Research and Scientific Research on a Priority Area from the Ministry of Education, Science and Culture of Japan, and by a grant for ‘Research for the Future’ Program from The Japan Society for the Promotion of Science to M.T. and by a Grantin-Aid to H.F. from the Japan Ministry of Education, Science and Science and Culture. H.F was supported by a fellowship from the Japanese Society for the Promotion of Science. References 1 Fukaki, H., Fujisawa, H. and Tasaka, M. (1996) How do plant shoots bend up? The initial step to elucidate the molecular mechanisms of shoot gravitropism using Arabidopsis thaliana, J. Plant Res. 109, 129–137 2 Fukaki, H. and Tasaka, M. Gravity perception and gravitropic response of inflorescence stems in Arabidopsis thaliana, Adv. Space Res. (in press) 3 Darwin, C. (1881) The Power of Movement in Plants (assisted by Darwin, F.), John Murray, London 4 Mirza, J.I. et al. (1984) The growth and gravitropic responses of wild-type and auxin-resistant mutants of Arabidopsis thaliana, Physiol. Plant. 60, 516–522 5 Caspar, T., Huber, S.C. and Somerville, C.R. (1985) Alterations in growth, photosynthesis, and respiration in a starchless mutant of Arabidopsis thaliana (L.) deficient in chloroplast phosphoglucomutase activity, Plant Physiol. 79, 11–17 6 Caspar, T. and Pickard, B.G. (1989) Gravitropism in a starchless mutant of Arabidopsis, Planta 177, 185–197 7 Kiss, J.Z., Hertel, R. and Sack, F.D. (1989) Amyloplasts are necessary for full gravitropic sensitivity in roots of Arabidopsis thaliana, Planta 117, 198–206 8 Bell, C.J. and Maher, E.P. (1990) Mutants of Arabidopsis thaliana with abnormal gravitropic responses, Mol. Gen. Genet. 220, 289–293 9 Lincoln, C., Britton, J.H. and Estelle, M. (1990) Growth and development of axr1 mutants of Arabidopsis, Plant Cell 2, 1071–1080 10 Wilson, A.K. et al. (1990) A dominant mutation in Arabidopsis confers resistance to auxin, ethylene and abscisic acid, Mol. Gen. Genet. 222, 377–383 11 Timpte, C.S., Wilson, A.K. and Estelle, M. (1992) Effects of the axr2 mutation of Arabidopsis on cell shape in hypocotyl and inflorescence, Planta 188, 271–278 12 Leyser, H.M.O. et al. (1993) Arabidopsis auxin-resistance gene AXR1 encodes a protein related to ubiquitin-activating enzyme E1, Nature 346, 161–164 13 Roman, G. et al. (1995) Genetic analysis of ethylene signal transduction in Arabidopsis thaliana: five novel mutant loci integrated into a stress response pathway, Genetics 139, 1393–1409 14 Simmons, C. et al. (1995) A novel root gravitropism mutant of Arabidopsis thaliana exhibiting altered auxin physiology, Physiol. Plant. 93, 790–798 15 Hobbie, L. and Estelle, M. (1995) The axr4 auxin-resistant mutants of Arabidopsis thaliana define a gene important for root gravitropism and lateral root initiation, Plant J. 7, 211–220 16 Fukaki, H., Fujisawa, H. and Tasaka, M. (1996) SGR1, SGR2 and SGR3: novel genetic loci involved in shoot gravitropism in Arabidopsis thaliana, Plant Physiol. 110, 945–955 17 Bennett, M.J. et al. (1996) Arabidopsis AUX1 gene: a permease-like regulator of root gravitropism, Science 273, 948–950
trends in plant science reviews 18 Kiss, J.Z., Wright, J.B. and Caspar, T. (1996) Gravitropism in roots of intermediate-starch mutants of Arabidopsis, Physiol. Plant. 94, 237–244 19 Yamauchi, Y. et al. (1997) Mutations in the SGR4, SGR5 and SGR6 loci of Arabidopsis thaliana alter the shoot gravitropism, Plant Cell Physiol. 38, 530–535 20 Fukaki, H., Fujisawa, H. and Tasaka, M. (1997) The RHG gene is involved in root and hypocotyl gravitropism in Arabidopsis thaliana, Plant Cell Physiol. 38, 804–810 21 Kiss, J.Z. et al. (1997) Reduced gravitropism in hypocotyls of starch-deficient mutants of Arabidopsis, Plant Cell Physiol. 38, 518–525 22 Fukaki, H. et al. (1998) Genetic evidence that the endodermis is essential for shoot gravitropism in Arabidopsis thaliana, Plant J. 14, 425–430 23 Luschnig, C. et al. (1998) EIR1, a root-specific protein involved in auxin transport, is required for gravitropism in Arabidopsis thaliana, Genes Dev. 12, 2175–2187 24 Utsuno, K. et al. (1998) AGR, an agravitropic locus of Arabidopsis thaliana, encodes a novel membrane-protein family member, Plant Cell Physiol. 39, 1111–1118 25 Müller, A. et al. (1998) AtPIN2 defines a locus of Arabidopsis for root gravitropism control, EMBO J. 17, 6903–6911 26 Rouse, D. et al. (1998) Changes in auxin response from mutations in an AUX/IAA gene, Science 279, 1371–1373 27 Weise, S.E. and Kiss, J.Z. Gravitropism of inflorescence stems in starchdeficient mutants of Arabidopsis, Int. J. Plant Sci. (in press) 28 Fukaki, H., Fujisawa, H. and Tasaka, M. (1996) Gravitropic response of inflorescence stems in Arabidopsis thaliana, Plant Physiol. 110, 933–943
29 Sack, F.D. (1991) Plant gravity sensing, Int. Rev. Cytol. 127, 193–252 30 Sack, D.F. (1997) Plastids and gravitropic sensing, Planta 203, 63–68 31 Kiss, J.Z. and Sack, F.D. (1989) Reduced gravitropic sensitivity in roots of a starch-deficient mutant of Nicotiana sylvestris, Planta 180, 123–130 32 Blancaflor, E.B., Fasano, J.M. and Gilroy, S. (1998) Mapping the functional roles of cap cells in the response of Arabidopsis primary roots to gravity, Plant Physiol. 116, 213–222 33 Scheres, B. et al. (1995) Mutations affecting the radial organization of the Arabidopsis root display specific defects throughout the radial axis, Development 121, 53–62 34 Di Laurenzio, L. et al. (1996) The SCARECROW gene regulates an asymmetric cell division that is essential for generating the radial organization of the Arabidopsis root, Cell 86, 423–433 35 Hawker, L.E. (1932) A quantitative study of the geotropism of seedlings with special reference to the nature and development of their statolith apparatus, Ann. Bot. 66, 121–157
Masao Tasaka*, Takehide Kato and Hidehiro Fukaki are at the Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0101, Japan.
*Author for correspondence (tel 181 743 72 5480;
fax 181 743 72 5489; e-mail [email protected]).
Function of the ubiquitin–proteosome pathway in auxin response J. Carlos del Pozo and Mark Estelle Proteolysis of important regulatory proteins by the ubiquitin–proteosome pathway is a key aspect of cellular regulation in eukaryotes. Genetic studies in Arabidopsis indicate that response to auxin depends on the function of proteins in this pathway. The auxin transport inhibitor resistant 1 (TIR1) protein is part of a ubiquitin–protein–ligase complex (E3), known as SKP1 CDC53 F-boxTIR1 (SCFTIR1), that possibly directs ubiquitin-modification of protein regulators of the auxin response. In yeast, a similar E3 complex, SCF CDC4, is regulated by conjugation of the ubiquitin-related protein Rub1 to the Cdc53 protein. In Arabidopsis, the AUXIN-RESISTANT1 (AXR1) gene encodes a subunit of the RUB1-activating enzyme, the first enzyme in the RUB-conjugation pathway. Loss of AXR1 results in loss of auxin response. These results suggest a model in which RUB1 modification regulates the activity of SCFTIR1, thereby directing the degradation of the repressors of the auxin response.
A
uxin [indole-3-acetic acid; (IAA)] regulates many important aspects of plant growth and development, including apical dominance, tropic responses, lateral root and root hair formation, and vascular tissue differentiation1. In spite of the importance of this hormone, the molecular basis of auxin response is poorly understood. Using Arabidopsis, several groups have adopted genetic approaches to identify a collection of auxin-response mutants2. One of these mutants, auxin-resistant1 (axr1), was isolated as an auxin-resistant mutant that has a pleiotropic phenotype associated with a decreased response to auxin3. In addition, axr1 plants are deficient in auxin-regulated gene expression, suggesting that
AXR1 is required for auxin signal transduction4,5. The AXR1 gene encodes a protein that participates in activation of the ubiquitinrelated protein RUB1 (Refs 6,7). The auxin transport inhibitor resistant 1 (tir1) mutant also exhibits reduced auxin response. TIR1 is a member of the F-box family of proteins. Studies in yeast and animals indicate that F-box proteins are part of an E3 ubiquitin– ligase complex called the SKP1 CDC53 F-box (SCF)8,9. The cloning and characterization of these auxin-response genes suggest that auxin signaling is mediated through the ubiquitin pathway. Recent molecular data on AXR1 and TIR1 function in Arabidopsis are providing new insight into auxin response.
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