Plant and Cell Physiology Advance Access originally published online on May 27, 2008
Plant and Cell Physiology 2008 49(7):1025-1038; doi:10.1093/pcp/pcn079
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Domain II Mutations in CRANE/IAA18 Suppress Lateral Root Formation and Affect Shoot Development in Arabidopsis thaliana
1 Graduate School of Science and Technology, Kobe University, 1-1 Rokkodai, Kobe, 657-8501 Japan
2 Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, 630-0192 Japan
3 Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodai, Kobe, 657-8501 Japan
*Corresponding author: E-mail, h-fukaki{at}port.kobe-u.ac.jp; Fax, +81-78-803-5721
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Abstract |
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Lateral root formation is an important developmental component of root systems in vascular plants. Several regulatory genes for lateral root formation have been identified from recent studies mainly using Arabidopsis thaliana. In this study, we isolated two dominant mutant alleles, crane-1 and crane-2, which are defective in lateral root formation in Arabidopsis. The crane mutants have dramatically reduced lateral root and auxin-induced lateral root formation, indicating that the crane mutations reduce auxin sensitivity. In addition, the crane mutants have pleiotropic phenotypes in the aerial shoots, including long hypocotyls when grown in the light, up-curled leaves and reduced fertility. The crane mutant phenotypes are caused by a gain-of-function mutation in domain II of IAA18, a member of the Aux/IAA transcriptional repressor family which is expressed in almost all organs. In roots, IAA18 promoter::GUS was expressed in the early stages of lateral root development. In the yeast two-hybrid system, IAA18 interacts with AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19, transcriptional activators that positively regulate lateral root formation. Taken together, our results indicate that CRANE/IAA18 is involved in lateral root formation in Arabidopsis, and suggest that it negatively regulates the activity of ARF7 and ARF19 for lateral root formation.
Keywords: Arabidopsis - Aux/IAA genes - Auxin - IAA18 - Lateral root formation
Abbreviations: AFB, AUXIN RECEPTOR F-BOX PROTEIN; ARF, AUXIN RESPONSE FACTOR; ASL, ASYMMETRIC LEAVES2-LIKE; Aux/IAA, AUXIN/INDOLE-3-ACETIC ACID; AuxRE, auxin response element; CTD, C-terminal domain; EMS, ethyl methanesulfonate; GUS, β-glucuronidase; LBD, LATERAL ORGAN BOUNDARIES-DOMAIN; NAA, naphthaleneacetic acid; NPA, N-1-naphthylphthalamic acid; RT–PCR, reverse transcription–PCR; slr, solitary-root; TIR1, TRANSPORT INHIBITOR RESPONSE1.
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Introduction |
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Lateral root formation is one of the most important events in the development of the root system of vascular plants (Osmont et al. 2007
). Lateral roots are branched secondary roots that are produced de novo from the primary root or parental roots. Formation of lateral roots under adequate regulatory control results in the construction of a root system that supports the plant body and absorbs water and nutrients from soil. Formation of a lateral root primordium is initiated by the highly regulated division of cells in pericycle files of the primary root in most plant species (Barlow et al. 2004). This initiation event is a key step of lateral root formation, but the regulatory mechanisms that control lateral root (primordium) formation are largely unknown.
The plant hormone auxin is known to be an important regulatory agent for plant growth and development, including lateral root formation, embryonic development, tropic responses, apical dominance and vascular development (Woodward and Bartel 2005). In several plant species, exogenously applied auxin promotes pericycle cell division, and causes lateral root formation (Torrey 1950, Blakely et al. 1988, Laskowski et al. 1995), and inhibitors of auxin transport, such as N-1-naphthylphthalamic acid (NPA), suppress the initiation of lateral root primordia (Reed et al. 1998b, Casimiro et al. 2001). Moreover, many Arabidopsis mutants which are impaired in auxin biosynthesis, homeostasis, transport or signaling have fewer lateral roots than the wild type (Fukaki et al. 2007). These observations indicate that auxin is a key regulator of lateral root formation. However, the molecular mechanisms of auxin action in this process are not fully understood.
At the cellular level, auxin regulates the transcription of many genes. Exogenous auxin application rapidly and transiently induces the transcription of early genes such as members of the AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA), SMALL AUXIN-UP RNA (SAUR) and Gretchen Hagen3 (GH3) gene families (Abel and Theologis 1996). The Aux/IAA and AUXIN RESPONSE FACTOR (ARF) transcription regulator families mediate expression of auxin-regulated gene expression (Guilfoyle and Hagen 2007), and 29 Aux/IAA genes have been identified in the Arabidopsis genome (Reed 2001). Expression of each Aux/IAA gene is regulated in a specific temporal and spatial manner, strongly suggesting that Aux/IAA genes have distinct and overlapping functions in auxin signaling (Teale et al. 2006).
Aux/IAA proteins have four highly conserved domains (domains I–IV). Domains III and IV are similar to the C-terminal domains (CTDs) of ARF proteins. ARFs can bind to auxin response elements (AuxREs) in the promoter regions of early auxin response genes and activate or repress their transcription. Domains III and IV mediate homo- or heterodimerization between two Aux/IAA proteins or between Aux/IAA and ARF. Domain I is similar to the conserved domain of the ethylene response factor (ERF) transcriptional repressors, and can inactivate the transcriptional activity of ARFs (Tiwari et al. 2004). Aux/IAAs thus negatively modulate auxin-regulated gene expression as transcriptional repressors via this interaction. Domain II of Aux/IAA contributes to the instability of this protein. Several gain-of-function mutants, which have missense mutations in domain II, have been identified, such as iaa1/axr5 (Yang et al. 2004), iaa3/shy2 (Tian and Reed 1999), iaa7/axr2 (Nagpal et al. 2000), iaa12/bdl (Hamann et al. 2002), iaa14/slr (Fukaki et al. 2002), iaa17/axr3 (Rouse et al. 1998), iaa19/msg2 (Tatematsu et al. 2004) and iaa28 (Rogg et al. 2001). These gain-of-function mutations stabilize the Aux/IAA protein, thereby causing a variety of auxin-related phenotypes (Woodward and Bartel 2005). In the wild type, Aux/IAA proteins are degradated by the ubiquitin–proteasome pathway (Gray et al. 2001). The auxin receptors, TRANSPORT INHIBITOR RESPONSE1 (TIR1) and three TIR1-related AUXIN RECEPTOR F-BOX PROTEINs (AFBs), are subunits of the SCFTIR1/AFBs ubiquitin-ligase complex and act as F-box proteins for recruiting Aux/IAA proteins in Arabidopsis (Dharmasiri et al. 2005, Kepinski and Leyser 2005). The interaction between Aux/IAAs and these F-box proteins is mediated by the Aux/IAA domain II. The missense mutations in domain II inhibit the interaction between Aux/IAA and TIR1/AFBs, thus the mutated Aux/IAA proteins are not degradated, resulting in constitutive inactivation of ARF transcriptional activity.
We previously identified a gain-of-function mutant solitary-root (slr), which has a missense mutation in domain II of IAA14 (Fukaki et al. 2002). The slr mutant has no lateral roots, few root hairs and abnormal gravitropic responses in roots and hypocotyls, strongly suggesting that stabilized mutant IAA14 protein inactivates the transcriptional activity of the ARFs that are responsible for these auxin-mediated developmental processes. The observation that arf 7 arf19 double mutants are very similar to the iaa14/slr mutant in lateral root formation and root gravitropic response (Okushima et al. 2005, Wilmoth et al. 2005) indicates that IAA14, ARF7 and ARF19 probably function in the same genetic pathway of Arabidopsis development. In addition, iaa3/shy2, iaa19/msg2 and iaa28-1 mutants also have reduced numbers of lateral roots, although the phenotypes of these mutants are not as severe as iaa14/slr in terms of lateral root formation. ARF7 and ARF19 interact with several Aux/IAAs including SLR/IAA14, SHY2/IAA3, MSG2/IAA19 and IAA28 in yeasts (Tatematsu et al. 2004, Fukaki et al. 2005, Y. Okushima and H. Fukaki unpublished results). SLR/IAA14 and the other Aux/IAAs thus are likely to be negative regulators of ARF7 and ARF19 through this interaction, thereby negatively regulating lateral root formation. However, it is unknown how many Aux/IAA members regulate lateral root formation because no gain-of-function mutants have been characterized in more than half of the Aux/IAA proteins.
Here, we report the isolation and characterization of two novel Arabidopsis mutant alleles, crane-1 and crane-2, which cause defective lateral root formation. These two mutants are dominant and have almost identical phenotypic characteristics, such as up-curled leaves, dwarfism and low fertility. In crane mutants, lateral root formation is blocked in very early developmental stages. Both crane mutants are due to gain-of-function mutations in IAA18, a member of Aux/IAA gene family. IAA18 is expressed in almost all organs of Arabidopsis, and the promoter activity of IAA18 was strong in the early stages of lateral root formation. In addition, we confirmed that IAA18 protein interacts directly with ARF7 and ARF19 in yeast. Our results indicate that CRANE/IAA18 is involved in lateral root formation and suggest that it functions cooperatively with SLR/IAA14 as a repressor of ARF7 and ARF19.
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Results |
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Isolation of crane mutants
Although the arf 7 arf19 double mutant has an obvious lateral root formation deficiency phenotype (Okushima et al. 2005
), each of the single mutants, arf 7 and arf19, has milder or almost no abnormal phenotype in lateral root formation, presumably because of the functional redundancy between ARF7 and ARF19 (Tatematsu et al. 2004, Okushima et al. 2005, Wilmoth et al. 2005). To identify novel factors that cooperatively regulate lateral root formation with ARF7 and ARF19, we screened ethyl methanesulfonate (EMS)-mutagenized M2 seedlings for enhancers of the arf 7-1 and arf19-4 single mutants. About 150 M2 lines were affected in lateral root formation, and >30 of them were double mutants with a previously known mutation, such as shy2/iaa3, slr/iaa14, tir3, arf 7 (for arf19-4) and arf19 (for arf 7-1), which have been reported to affect lateral root formation (data not shown). Among the remainder, we identified two independent lines from arf 7-1 and arf19-4, respectively, which had almost the same phenotypes as each other, but which did not resemble mutants that had been described previously. We named these two mutants crane-1 and crane-2, respectively, after the origami crane bird. Genetic analysis indicated that the crane-1 and crane-2 mutations are dominant. Even after the backcrossing away from the arf 7-1 or arf19-4 mutation, each crane mutation caused almost identical phenotypes to the original crane-1 arf 7-1 and crane-2 arf19-4 double mutants. Therefore, we analyzed the effects of the single crane mutations on lateral root formation and other developmental events, rather than as a genetic enhancer of arf 7 or arf19.
crane mutants show reduced lateral root formation
The primary roots of 15-day-old light-grown wild-type Col seedlings produced many lateral roots, which are dramatically reduced in crane-1 and crane-2 (Fig. 1A). In 8-day-old seedlings, the length of primary roots was 51.0 ± 8.5 mm (mean ± SD) for Col and 39.5 ± 9.5 mm for crane-2 (Fig. 2A), and the total number of lateral roots was 9.0 ± 2.8 for Col and 2.2 ± 1.8 for crane-2 (Fig. 2B). Lateral root density, which is the number of lateral roots cm–1 primary root, was 1.8 ± 0.6 for Col and 0.6 ± 0.5 for crane-2 (Fig. 2C).
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The aerial parts of these crane mutants also have several distinctive phenotypes (Fig. 1B–H). The cotyledons and rosette leaves of Col are almost flat or slightly down-curled, whereas they are up-curled on the crane mutants (Fig. 1C–E). In addition, crane mutants are dwarfed (Fig. 1B), have longer hypocotyls (Fig. 1C, D, H), shorter stamens (Fig. 1F, G) and lower fertility rates (data not shown) than the wild type. The gravitropic responses of seedling roots and hypocotyls are not dramatically affected by the crane-2 mutation (Supplementary Fig. S1).
The crane mutation blocks the early stages in lateral root formation
In Arabidopsis, the development of a lateral root is described in nine stages (Malamy and Benfey 1997). The formation of a lateral root primordium is initiated by pericycle cell division in a transverse orientation (anticlinal cell division, stage I). Subsequently, some of the divided cells in stage I divide in a longitudinal orientation (periclinal cell division) that produce two layers from a single pericycle cell layer (stage II). After stage II, the lateral root primordium is formed through further cell division and differentiation (stages III and IV). To determine which stages of lateral root development are affected in the crane mutants, we analyzed the expression of a cell cycle marker gene, pCycB1;1::CycB1;1(NT)-GUS, in the crane-2 primary root. In the pCycB1;1::CycB1;1(NT)-GUS line, the chimeric protein containing the destruction box of CycB1;1 fused to β-glucuronidase (GUS) is expressed under the control of the CycB1;1 promoter (Colón-Carmona et al. 1999). As CycB1;1 is expressed only at around the G2–M transition of the cell cycle (Doerner et al. 1996, Fukaki et al. 2002), GUS activity (foci) of 8-day-old light-grown pCycB1;1::CycB1;1(NT)-GUS seedlings was detected in the dividing cells of the root pericycle where lateral root initiation occurs, as well as in the primary root apical meristem (Fig. 3A, B, E). In contrast, the 8-day-old light-grown pCycB1;1::CycB1;1(NT)-GUS/crane-2 seedlings have a smaller number of GUS foci around the root pericycle cells than pCycB1;1::CycB1;1(NT)-GUS/Col seedlings, although foci were detected at the primary root apical meristem (Fig. 3C, E). These observations indicate that the crane mutation reduces the division of pericycle cells in the early stages of lateral root initiation, probably the first anticlinal cell division of the pericycle.
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Auxin sensitivity of crane mutant roots
The crane mutant in lateral root formation and other developmental phenotypes indicates that the crane mutation alters sensitivity to auxin. To investigate this possibility, we examined the effects of exogenous auxin, naphthaleneacetic acid (NAA), on crane-2 root growth. Five-day-old Col and crane seedlings grown on NAA-free medium were transferred to NAA-free or NAA-containing medium and incubated for an additional 4 d. In both Col and crane-2, 0.1 µM NAA reduced the growth of primary roots to a similar extent [32.0 ± 7.1% of control in Col; 43.0 ± 19.1% of control in crane-2 (n = 12); no significant difference was observed between Col and crane-2. (P > 0.05, by Student's t-test)] (Fig. 4A). In contrast, Col seedlings treated with 0.1 µM NAA formed >10 lateral roots per individual (n = 12) whereas the crane-2 seedlings treated with 0.1 µM NAA formed 6.3 lateral roots per individual (n = 12) (P < 0.01, by Student's t-test), indicating that the crane-2 mutant has reduced sensitivity to 0.1 µM NAA in auxin-induced lateral root formation (Fig. 4B). However, 1 µM NAA inhibited primary root growth and induced lateral root formation in the crane-2 mutant similar to Col (Fig. 4A, B). In contrast, as described previously (Fukaki et al. 2002
), slr-1/iaa14 was more resistant to exogenous 0.1 µM NAA in the root growth inhibition assay (Fig. 4A) and produced almost no lateral roots in response to 1 µM NAA (Fig. 4B). These observations indicate that the auxin sensitivity of crane-2 roots is attenuated but not as inhibited as in the slr-1 mutant.
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DR5::GUS is a useful reporter gene for studies on auxin-responsive gene transcription in Arabidopsis (Ulmasov et al. 1997
) and is widely used for visualizing auxin response maxima; it can be useful for following endogenous free auxin distribution (Benková et al. 2003). DR5::GUS expression was observed in the crane-2 background around the distal regions of primary and lateral roots (Fig. 4C–F) and was enhanced by treatment with 1 µM NAA (data not shown) as observed in the wild-type Col.
CRANE encodes IAA18 protein
As described above, some of the pleiotropic phenotypes characteristic of crane mutants are observed among the dominant mutants of Aux/IAA genes such as slr/iaa14 (Liscum and Reed 2002). Among the 29 Aux/IAA genes found in the Arabidopsis genome, the mutants axr5/iaa1 (Yang et al. 2004), shy2/iaa3 (Tian and Reed 1999), bdl/iaa12 (Hamann et al. 2002), slr/iaa14 (Fukaki et al. 2002), axr3/iaa17 (Rouse et al. 1998), msg2/iaa19 (Tatematsu et al. 2004) and iaa28 (Rogg et al. 2001) have been reported previously. All these mutants have an amino acid alteration in domain II of their respective Aux/IAA protein, but no mutants like crane with the phenotypes including long hypocotyls, up-curled leaves and reduced lateral root formation had been reported. The crane locus could not be genetically mapped because not enough F2 seeds were produced due to the very low fertility of F1 plants from a cross between crane and Ler. Therefore, in order to isolate the crane gene, the DNA sequences of IAA2, 4, 5, 8, 9, 10, 11, 13, 15, 16, 18, 26, 27, 29, 31, 32, 33 and 34 were compared in wild-type Col, crane-1 and crane-2 genomic DNA. Both crane-1 and crane-2 have a single-nucleotide missense mutation in IAA18, a member of the Aux/IAA genes (Fig. 5A).
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The IAA18 protein consists of 267 amino acids and has four conserved domains among all of the Aux/IAA proteins (Fig. 5A). The crane-1 and crane-2 mutations cause the Gly99Arg and Gly99Glu substitutions in domain II, respectively (Fig. 5A).
To confirm that CRANE encodes IAA18, we examined whether the 4 kbp genomic fragment containing IAA18 with the crane-2 mutation gives the crane phenotypes in the wild-type background (see Supplementary Fig. S2). Among four independent transgenic lines harboring the 4 kbp fragment, three lines had reduced lateral root formation compared with the non-transgenic controls (Fig. 6A). In addition, these lines had the same developmental abnormalities as crane (Fig. 6A–D and data not shown).
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Previously, a Gly99Glu substitution in domain II (i.e. crane-2) was reported as iaa18-1 in unpublished results by Nagpal and Reed (Reed 2001
). The iaa18-1 allele was not described in detail but defects in cotyledon phyllotaxy, long hypocotyl, short root and up-curled leaves were associated with the mutant (Reed 2001, Liscum and Reed 2002). However, it has not been confirmed whether the iaa18-1 phenotypes are caused by the domain II mutation in the IAA18 gene, but our observation that the two crane mutants have long hypocotyls, short primary roots and up-curled true leaves, as well as fused cotyledons at a very low rate and that the crane phenotypes are caused by the domain II mutation in IAA18 indicates that the iaa18-1 and crane mutants are co-allelic and that they are gain-of-function mutants in IAA18.
To examine the function of the IAA18 gene in the wild type, we analyzed the GABI-Kat line 800H03, which has a T-DNA insertion at the end of the third exon of the IAA18 coding region. No obvious phenotype was observed in this line (data not shown), suggesting that IAA18 functions redundantly with other Aux/IAAs, as is the case with Aux/IAA genes (Overvoorde et al. 2005).
Expression of the IAA18 gene
Quantitative reverse transcription–PCR (RT–PCR) was used to determine the expression pattern of IAA18 in five different organs of Arabidopsis. IAA18 was expressed in the aerial parts and roots of seedlings, rosette leaves, cauline leaves and inflorescences, though expression in roots was lower than in other organs (Fig. 7). This result is consistent with other published microarray data (Zimmermann et al. 2004, Schmid et al. 2005).
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We also examined the IAA18 expression pattern using transgenic plants harboring GUS fused to the 2.0 kb upstream region of IAA18 (pIAA18::GUS, see Supplementary Fig. S2). In the pIAA18::GUS seedlings, GUS expression was observed only in the pericycle where lateral roots are initiated and only during the early stages of lateral root development (Fig. 7B, D, E). No GUS staining was observed in emerged lateral roots or in the primary root meristem (Fig. 7C, F, G). GUS expression in the pericycle was enhanced by 1 µM auxin treatment for 5 h (Fig. 7H), suggesting that IAA18 is expressed in an auxin-dependent manner. In the pIAA18::GUS seedlings, neither cotyledons nor true leaves had apparent GUS staining (data not shown), suggesting that the upstream region used in this experiment may not be sufficient to allow the complete native expression pattern of IAA18, like the case of the pIAA14::GUS line (Fukaki et al. 2002
).
CRANE/IAA18 interacts with ARF7 and ARF19 in yeast
SLR/IAA14 functions cooperatively with ARF7 and ARF19 for initiating lateral root formation, and IAA14 protein interacts directly with ARF7 and ARF19 in a yeast two-hybrid system (Fukaki et al. 2005). IAA18 is co-expressed with ARF7 and ARF19 at the lateral root initiation site (Fig. 7; Okushima et al. 2005), suggesting that IAA18 interacts with ARF7 and ARF19. Yeast two-hybrid experiments between IAA18 and ARF7 or ARF19 were performed to determine if IAA18 protein also interacts with ARF7 and ARF19. The results showed that IAA18 directly interacts with both ARF7 and ARF19 in yeasts (Table 1), suggesting that CRANE/IAA18 functions with ARF7 and ARF19 for lateral root formation in planta.
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A gain-of-function mutation in CRANE/IAA18 blocks lateral root formation
In this study, we identified gain-of-function mutants in CRANE/IAA18, a gene which is involved in Arabidopsis lateral root formation. Phenotypic analysis showed that the crane mutation suppresses the initiation of lateral root formation and attenuates auxin-induced lateral root formation, suggesting that the crane mutation reduces auxin sensitivity in the roots, thereby inhibiting lateral root formation. The crane mutants also have pleiotropic phenotypes in the aerial shoots, including long hypocotyl, up-curled leaves and reduced fertility, indicating the involvement of CRANE in shoot development. Molecular genetic analysis revealed that the crane mutant phenotypes are caused by a missense mutation in domain II of IAA18, a member of the large Aux/IAA protein family, indicating that CRANE encodes IAA18. Previously, Reed (2001
) reported a mutant (iaa18-1) that had the same amino acid substitution in domain II of IAA18 as in crane-2. The iaa18-1 mutant seedlings were briefly described as having long hypocotyls and up-curled leaves (Reed 2001). Although no lateral root phenotype was mentioned, the iaa18-1 mutant phenotypes approximate those of the crane mutants, which lends further support to the supposition that CRANE encodes IAA18.
All of the previously reported gain-of-function mutations of Aux/IAA genes are due to amino acid substitutions within the five amino acids of domain II (GWPPV) (see Fig. 5B). For example, the shy2-1/-2, axr2-1, bodenlos, msg2-4 and iaa28-1 alleles have a mutation in the first proline residue in domain II of IAA3, IAA7, IAA12, IAA19 and IAA28, respectively. Similarly, the shy2-6, axr3-1, slr-1 and msg2-1/3 alleles have mutations in the second proline of IAA3, IAA17, IAA14 and IAA19, respectively. Apparently, substitutions of either of the two proline residues results in severe phenotypes. The glycine to glutamate mutation in IAA3, shy2-3, results in a weak mutant phenotype compared with shy2-2, the proline to serine mutation in IAA3 (Reed et al. 1998a, Tian and Reed 1999). Similarly, the glycine to glutamate mutation in IAA17 domain II, axr3-101, which we found recently, caused milder mutant phenotypes than the axr3-1 and axr3-3 mutants, which have mutations in one of the two proline residues of IAA17 domain II (Supplementary Fig. S3; Leyser et al. 1996, Rouse et al. 1998). The fact that crane-1 and crane-2 have glycine to arginine and glycine to glutamate substitutions in the IAA18 domain II, respectively, suggests that a mutation in either of the two proline residues of the IAA18 domain II would result in a more severe phenotype than crane-1 and crane-2, although such crane alleles have not yet been found.
Recently, the structural basis for the mechanism of interaction of the Aux/IAA protein with TIR1, a component of the auxin receptor, has been reported (Tan et al. 2007). In domain II, tryptophan and the second proline interact with a hydrophobic wall of the TIR1 pocket and pack the auxin molecule into the pocket. The first proline maintains the relative position of these two residues. Glycine is critical for flexibility between the N-terminal region and domain II of the Aux/IAA protein. Both crane mutations may thus affect the flexibility of the IAA18 protein and its degradation by the TIR1-mediated proteolytic pathway.
Aux/IAA proteins have distinct and overlapping functions in plant growth and development
Gain-of-function mutants of SLR/IAA14 (Fukaki et al. 2002), IAA28 (Rogg et al. 2001) and MSG2/IAA19 (Tatematsu et al. 2004) have lateral root formation phenotypes that are similar to crane/iaa18. However, only slr/iaa14 has no lateral roots (Fukaki et al. 2002), but iaa28, msg2/iaa19 and crane/iaa18 initiate a few lateral roots. There are several other phenotypic differences between these mutants. For example, slr/iaa14 has increased apical dominance, but iaa28 has decreased apical dominance (Rogg et al. 2001, Fukaki et al. 2002). In addition, msg2/iaa19 has a unique differential growth of hypocotyls phenotype (Tatematsu et al. 2004) and, as is evident from this work, crane/iaa18 also has unique hypocotyl elongation and leaf growth phenotypes. These phenotypic differences among the four mutants indicate that each Aux/IAA protein has a distinct function in a variety of developmental processes. One of the reasons for this variation is that the expression patterns of most Aux/IAA genes, including IAA14, IAA18, IAA19 and IAA28, are differentially regulated in a tissue- and stage-specific manner (Rogg et al. 2001, Fukaki et al. 2002, Tatematsu et al. 2004, this study). More interestingly, expression of different mutant Aux/IAA proteins, including msg2-1/iaa19, axr2-1/iaa7 and slr-1/iaa14 under the control of the same promoter, resulted in different effects on Arabidopsis development, strongly suggesting that these Aux/IAA proteins have functional differences in repressing ARF activity (Muto et al. 2007). Minor differences in the specificities of their expression patterns and molecular functions would thus result in the phenotypic differences observed among gain-of-function mutants of Aux/IAA genes.
Interactions of Aux/IAAs with ARFs have been investigated using the yeast two-hybrid system (Kim et al. 1997, Ulmasov et al. 1997, Ouellet et al. 2001, Hamann et al. 2002, Hardtke et al. 2004, Tatematsu et al. 2004, Fukaki et al. 2005, Weijers et al. 2005, Weijers et al. 2006), in vitro pull-down assay (Tatematsu et al. 2004, Weijers et al. 2006), co-immunoprecipitation in planta (Weijers et al. 2006) and fluorescence cross-correlation spectroscopy (Muto et al. 2006), but no differential interactions between Aux/IAA proteins and ARFs have been reported in these assays. On the other hand, Aux/IAA genes have the specificities of their expression patterns (Abel and Theologis 1996, Rogg et al. 2001, Fukaki et al. 2002, Hamann et al. 2002, Tatematsu et al. 2004, Weijers et al. 2006, this study), and Aux/IAA proteins have functional differences in repressing ARF activity (Weijers et al. 2006, Muto et al. 2007). These data suggest that Aux/IAA specificity depends on their expression pattern and their functional differences in repressing ARF activity. However, the possibility remains that any binding preference for particular ARFs in planta affects Aux/IAA specificity.
IAA18 expression and function in root and shoot development
Expression analysis by quantitative RT–PCR showed that IAA18 is highly expressed in the aerial organs of Arabidopsis (Fig. 7). This is consistent with the pleiotropic phenotypes observed in the crane mutants (see Fig. 1). Up-curled true leaves were reported in the mutant allele of NPH4/ARF7 (nph4-103) (Nakamoto et al. 2006). Thus, in true leaves, IAA18 might repress NPH4/ARF7 activity. A very similar phenotype to crane was also observed in true leaves of plants with shy2/iaa3 alleles (Tian and Reed 1999) and in a transgenic plant that expresses stabilized mutant IAA26 protein (Padmanabhan et al. 2006). In addition, the arf6 arf8 double mutant has shorter stamens than the wild type (Nagpal et al. 2005), which are also observed in crane mutants (Fig. 1G). This suggests that IAA18 represses ARF6 and ARF8 activity in floral organs. Furthermore, like the crane mutants, the arf8 single mutant grows longer hypocotyls in the light (Tian et al. 2004) (Fig. 1C, D, H), also suggesting that IAA18 represses ARF8 activity in hypocotyls, thereby promoting hypocotyl elongation.
Our analysis using pIAA18::GUS transgenic plants showed that IAA18 is expressed in the early stages of lateral root primordium development (Fig. 7). In Arabidopsis, SLR/IAA14, ARF7 and ARF19 are key transcriptional regulators of lateral root formation (Fukaki et al. 2002, Okushima et al. 2005, Wilmoth et al. 2005). SLR/IAA14 is mainly expressed in the stele throughout the root elongation and mature zones (Fukaki et al. 2002). ARF19 is also expressed throughout root tissues, including the meristematic region (Okushima et al. 2005). On the other hand, expression of ARF7 is restricted to the stele and the early stages of lateral root development (Okushima et al. 2005). Thus, expression of IAA18 overlaps with that of ARF7 and ARF19, in the stele, including in the pericycle where lateral roots are initiated and in the early stages of lateral root primordium. This suggests that IAA18, ARF7 and ARF19 function together to initiate lateral roots. The result from the yeast two-hybrid experiment showing the interaction between CRANE/IAA18 and ARF7 or ARF19 supports this possibility (Table 1).
IAA18 is a negative regulator of lateral root formation in Arabidopsis
In this study, we found that a transcriptional regulator, CRANE/IAA18, is involved in lateral root formation in Arabidopsis. A current model of auxin-mediated lateral root formation in Arabidopsis would first show that in the pericycle cells, where the lateral root is initiated, auxin is perceived by the auxin receptor complex, SCFTIR1/AFBs. Secondly, the ubiquitin–proteasome pathway mediated by the SCFTIR1/AFBs complex and 26S proteasome degrades Aux/IAA proteins including SLR/IAA14, CRANE/IAA18 and the other Aux/IAAs, thereby releasing active ARF7 and ARF19. Then, released ARF transcriptional activators ARF7 and ARF19 induce the expression of genes required for lateral root initiation such as LBD16/ASL18 and LBD29/ASL16 (Okushima et al. 2007).
It is unclear which cell(s) uses CRANE/IAA18-dependent auxin signaling during lateral root formation, and which ARFs are negatively regulated by CRANE/IAA18 in the other auxin-mediated developmental processes, including hypocotyl growth, and shoot organ development in Arabidopsis plants. To answer these questions, further studies using techniques such as real-time detection of the interactions between Aux/IAAs and ARFs in planta will be necessary.
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Plant materials and growth conditions
Arabidopsis thaliana wild-type Columbia (Col) and Landsberg erecta (Ler) accession, slr (Fukaki et al. 2002
), arf 7-1 (SALK_040394; Okushima et al. 2005) and arf19-4 (SALK_009879, obtained from the Arabidopsis Biological Resource Center; Wilmoth et al. 2005) lines were used. pCycB1;1::CycB1;1(NT)-GUS seeds were kindly supplied by Peter Doerner (University of Edinburgh, UK). DR5-GUS seeds were kindly supplied by Tom Guilfoyle (University of Missouri-Columbia, USA). The axr3-1 and axr3-3 mutant seeds were kindly supplied by Ottoline Leyser (University of York, UK). Seeds were surface-sterilized with 0.01% (v/v) Triton X-100 and 10% (v/v) sodium hypochlorite, and plated on Murashige–Skoog medium containing 1.0% (w/v) sucrose solidified with 0.5% (w/v) gelan gum or 1% (w/v) agar. Kanamycin and hygromycin were added to a final concentration of 50 µg ml–1 from an aqueous 50 mg ml–1 stock. Plates and plants transferred to soil were grown at 23°C under constant white light.
Isolation of the crane-1 and crane-2 mutants
About 5,000 each of arf 7-1 and arf19-4 mutant seeds were mutagenized with EMS as described previously (Fukaki et al. 2006). M2 seeds were harvested and sown on the MS plates to screen for mutants with decreased numbers of lateral roots. The crane-1 and crane-2 mutants were isolated from arf 7-1 and arf19-4 M2 populations, respectively. These crane arf double mutants were backcrossed to the wild-type Col, and the resulting F1 generation was crossed with Col to separate the crane mutation from the arf mutation. The arf 7-1 and arf19-4 mutations caused by T-DNA insertion were detected by PCR with the LB-specific primer (5'-GTTGCCCGTCTCACTGGTGAAAAG-3') and gene-specific primers as follows: 5'-TGTTGCACTCCTCTTTGAACC-3' and 5'-TGGACTTTTGCTGACCCACGA-3' for arf 7-1, and 5'-GATGAACAGGAGAGAAGAAGC-3' and 5'-GCGGTTGCATCGAGAAATCCT-3' for arf19-4.
CycB1;1::GUS and DR5::GUS expression in the crane-2 mutant
pCycB1;1::CycB1;1(NT)-GUS (Colón-Carmona et al. 1999) and DR5::GUS (Ulmasov et al. 1997) were crossed with crane-2 by artificial pollination. The resultant F2 seedlings expressing reporter genes were stained and observed as described previously (Malamy and Benfey 1997). For analysis of auxin-induced DR5::GUS expression in crane-2, 7-day-old seedlings of DR5::GUS/crane-2 were transferred to MS plates with or without 1 µM NAA, incubated for 4 h, and stained.
Auxin sensitivity of crane mutants
Five-day-old Col and crane seedlings grown on NAA-free MS plates were transferred to MS plates with or without 1 µM NAA and incubated for an additional 4 d. These plates were photographed with a Nikon COOLPIX2500, and root length and number of lateral roots were tallied using ImageJ software (Abramoff et al. 2004) and NeuronJ plug-in (Meijering et al. 2004).
Cloning of the IAA18 gene
The genomic region containing domain II of the selected Aux/IAA genes (IAA2, 4, 5, 8, 9, 10, 11, 13, 15, 16, 18, 26, 27, 29, 31, 32, 33, 34) was amplified from Col, crane-1 and crane-2 mutant DNA with the primers listed in Supplementary Table S1. PCR products were purified and directly sequenced by standard methods using these PCR primers and BigDye Terminator v3.0 (Applied Biosystems, Lincoln Centre Drive Foster City, CA, USA).
IAA18 was amplified from Col cDNA using the primers: 5'-ATGGAGGGTTATTCAAGAAA-3' and 5'-TCATCTTCTCATTTTCTCTT-3'. The PCR product was cloned into the EcoRV site of the pGreenII (Hellens et al. 2000) and pBluescriptSK+ vectors (Fermentas, Burlington, Ontario, Canada), and confirmed by sequencing using the primers: M13(–20) (5'-GTAAAACGACGGCCAGT-3') and M13RV (5'-AGCGGATAACAATTTCACAC-3').
Transgenic plants expressing the mutated IAA18 gene
To produce the transgenic plants expressing the mutated IAA18 gene, a 3,956 bp genomic region that spans IAA18 was amplified from Col DNA using the primer set: 5'-TATGATGGGATGGTAAATGG-3' and 5'-CCCAAGAGTCCAGACAAAGA-3'. The amplified fragment was cloned into the EcoRV site of pBluescriptSK+ and subcloned into vector pBI301H, a derivative of pBI101 (Clontech, Mountain View, CA, USA) made by the authors, using the EcoRI and SalI sites. This construct was introduced into Agrobacterium tumefaciens strain C58MP90 and transformed into Col plants by the floral dipping method (Clough and Bent 1998).
IAA18 expression
Total RNA was extracted from several Col seedling and adult plant organs using an RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA). cDNAs for each sample were synthesized by reverse transcription using Reverse Transcriptase XL (AMV) (Takara Bio Inc., Otsu, Shiga, Japan). IAA18 expression was analyzed using a Smart Cycler® II System (Cepheid, Sunnyvale, CA, USA) and the primers: 5'-TCGCTGGCAAATACTTCTCTC-3' and 5'-CACTGGACCAGGAGCAGTTC-3'. For an internal control, ACT2 expression was also analyzed using the primers: 5'-TTGTTCCAGCCCTCGTTTGT-3' and 5'-TCATGCTGCTTGGTGCAAGT-3'.
A 2,003 bp fragment including the upstream region and 59 bp of open reading frame was amplified from Col genomic DNA using the primer set: 5'-TATGATGGGATGGTAAATGG-3' and 5'-GGAATCATCAAGTCAAGCAG-3'. The amplified fragment was cloned into the SmaI site of pBI301. This construct was transformed into Col plants as described above. T2 seedlings were stained and observed as described previously (Fukaki et al. 2002).
Interaction between IAA18 protein and ARF7 or ARF19 proteins
Yeast two-hybrid experiments between IAA18 and ARF7 or ARF19 were performed as described previously (Fukaki et al. 2005). IAA18/pAD-GAL4-2.1 was constructed from a XhoI–PstI fragment of IAA18/pBluescriptSK+ and pAD-GAL4-2.1 (Stratagene, La Jolla, CA, USA).
Accession numbers and knockout line
Arabidopsis Genome Initiative locus identifiers for the genes mentioned in this article are as follows: ARF7 (At5g20730), ARF19 (At1g19220), IAA14/SLR (At4g14550), IAA18/CRANE (At1g51950). GABI-Kat line 800H03 was obtained from the Institute for Genome Research and Systems Biology (Rosso et al. 2003).
Supplementary material
Supplementary material mentioned in the article is available to online subscribers at the journal website www.pcp.oxfordjournals.org.
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Funding |
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The Scientific Research on Priority Areas (grants 17027019 and 19060006 to H.F.); Ministry of Education, Culture, Sports, Science and Technology, Japan; the Inamori Foundation (to H.F.); Hyogo Science and Technology Association (to H.F.); the Japan Society for the Promotion of Science (research fellowship for young scientists to Y.O.).
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsFundingAcknowledgmentsReferences |
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We thank Peter Doerner for pCycB1;1::CycB1;1(NT)-GUS, Tom Guilfoyle for DR5::GUS seeds, Ottoline Leyser for the axr3 mutants, and the Arabidopsis Biological Resource Center for the seed stock. We gratefully thank Keiko Uno for her excellent technical assistance.
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(Received March 26, 2008; Accepted May 21, 2008)
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