Download Function of the ubiquitin–proteosome pathway in auxin response

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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,
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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
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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.
1360 - 1385/99/$ – see front matter © 1999 Elsevier Science. All rights reserved. PII: S1360-1385(99)01382-5
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Ubiquitin–proteosome pathway
In eukaryotic organisms, ubiquitin conjugation to target proteins
and subsequent degradation by the multisubunit proteosome plays
an important role in diverse cellular processes ranging from cellcycle regulation to signal transduction10. The ubiquitin-conjugation
pathway consists of three enzymes10. Ubiquitin is initially activated
in an ATP-dependent reaction by the ubiquitin-activating enzyme
(E1) forming a thiolester bond between a conserved cysteine
within the E1 and the terminal carboxyl group of ubiquitin. The
second step in the pathway involves the transfer of ubiquitin from
the E1 to a cysteine within an enzyme, the ubiquitin-conjugating enzyme (E2). Finally, ubiquitin is covalently attached to target proteins by an isopeptide linkage between the C-terminus of ubiquitin
and the e-amino group of lysine residues within the target. For
some E2s, this reaction can occur in vitro without the participation
of other proteins. However, isopeptide bond formation in vivo
probably requires participation of a third protein or protein complex, the ubiquitin protein ligase (E3).
In most organisms there are one or two E1 genes and a larger
family of E2 genes. The E2 proteins are specialized for various
processes. For example, in yeast, Ubc3 (also known as Cdc34) is
required for the degradation of several proteins that regulate the
cell cycle, while Ubc2 (also known as Rad6) is involved in DNA
repair. The E3s are structurally diverse and can be either a single protein or a complex of proteins. One such complex is called
the SCF and is composed of Cdc53 (or cullin), SKP1, and a third
protein that contains a SKP1-binding domain called the F-box9.
It is thought that the function of the F-box proteins is to recognize and recruit specific substrates for degradation. The bestcharacterized SCF complexes are SCFCdc4, SCFGrr1 and SCFMet30
from budding yeast9. Each SCF recruits several targets for ubiquitination. For example, SCFCdc4 recruits phosphorylated forms of
Sic1p and Far1p – both inhibitors of the cyclin-dependent kinase
– for ubiquitination and subsequent degradation. Similarly, SCFGrr1
is implicated in the ubiquitination and degradation of phosphorylated CLN, while SCFMet30 mediates the degradation of the CDKinhibitory kinase, Swe1 and proteins involved in regulation of
methionine biosynthesis11.
Most of the components of the ubiquitin-conjugation pathway and subunits of the proteosome, have been identified in
plants12. As these proteins are highly conserved between plants,
animals and fungi, it is probable that the pathway has similar
functions in all three kingdoms. However, there is little specific
information on the function of the pathway in plants. The bestcharacterized plant substrate for ubiquitination is phytochrome
A, which is degraded rapidly upon photoconversion to the far
red form12. There is also evidence that the ubiquitin-conjugation
pathway has a role in stress responses, senescence and pollen
maturation13–15.
repeats (LRRs) and an F-box, suggesting that TIR1 might be part
of an SCF complex9. This hypothesis is supported by recent results that demonstrate binding between TIR1 and two SKP1 homologs, Arabidopsis SKP 1-LIKE [ASK1; also known as ATskp1
(Ref. 16)] and ASK2. These interactions were first demonstrated
in a yeast two-hybrid test and later confirmed by immunoprecipitation from plant extracts (W. Gray, and M. Estelle, unpublished).
Although the biochemical function of TIR1 has not been established directly, these results suggest that TIR1 is part of an SCF
complex.
Expression of the TIR1 gene has been analyzed using both promoter-GUS fusion genes and in situ hybridization with RNA
probes. Expression is strongest in the primary root- and lateral
root-meristems, which is consistent with a key role for the protein in root development (W. Gray, L. Walker and M. Estelle,
unpublished). Because the tir1 mutants are deficient in lateral root
formation, it was of interest to determine when TIR1 function
is required during lateral root initiation. This was investigated
utilizing a cyc1At-GUS fusion gene obtained from Peter Doerner
(Salk Institute, San Diego, CA, USA). During formation of a lateral root, the cyc1At gene is expressed in pericycle cells in G2 1
mitosis phases of the cell cycle17. In lines carrying the cyc1AtGUS fusion, GUS staining appears before the first cell division that
marks the initiation of lateral root primordium formation. If cyc1At
is expressed before TIR1 is required in the developing lateral root
primordium, instances of single stained pericycle cells, which represent arrested lateral root primordia, would be expected in tir1
mutant roots. If, however, TIR1 is required for induction of cyc1At
expression in the pericycle cells, such arrested primordia would
not appear. In the tir1 mutant, expression of cyc1At is restricted to
the few lateral roots that develop, indicating that TIR1 is needed
for initial induction of cyc1At expression (W. Gray and M.
Estelle, unpublished). This result suggests that TIR1 is required
for pericycle cells to respond to the inductive signal, presumably
auxin.
Three additional genes related to TIR1 have been identified in
Arabidopsis. Two of these are called LRR F-BOX1 (LRF1) and
LRF2. Although the functions of LRF1 and LRF2 are unknown,
these proteins show 64% and 69% sequence homology to TIR1,
respectively, suggesting that they might have a related function8
(L. Walker and M. Estelle, unpublished). The third member of
this family is the CORONATINE INSENSITIVE 1 (COI1) gene18.
The coi1 mutants are insensitive to jasmonic acid (JA), indicating
that this gene is essential for JA response19. COI1 is 34% identical
to TIR1, and like TIR1, interacts with ASK1 and ASK2 in a yeast
two-hybrid test (J.G. Turner, unpublished). These exciting results
suggest that the SCF and ubiquitin-mediated processes might be a
recurring theme in plant hormone action.
Ubiquitin-like proteins
Auxin response and the ubiquitin pathway
As seedling root growth is inhibited by relatively low concentrations of auxin, it enables screens for auxin-resistant mutants to
be performed on a large scale2. Four of the genes identified by this
approach have now been cloned, and surprisingly, two of these,
AXR1 and TIR1, encode proteins that probably function in a ubiquitin pathway. The tir1 mutants were isolated in a screen for seedlings showing resistance to auxin-transport inhibitors and were
subsequently found to be deficient in auxin response rather than
transport8. The roots of tir1 plants are resistant to growth inhibition by auxin, but the mutant plants are also deficient in many
other auxin-regulated processes, including lateral root formation
and auxin-dependent hypocotyl elongation8. Molecular characterization of the locus indicates that the TIR1 protein has 16 leucine-rich
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March 1999, Vol. 4, No. 3
Eukaryotes possess at least two families of ubiquitin-like proteins
(Table 1). Smt3p (budding yeast) and SUMO-1 (mammals) are in
one family, while Rub1p (yeast and Arabidopsis) and NEDD8
(human)20 proteins are in the second family. Like ubiquitin, these
proteins are covalently attached to specific target proteins by
conjugation pathways involving E1- and E2-like activities. However, unlike ubiquitin-E1, both Smt3p- and Rub1p-E1 enzymes
are heterodimers, with one subunit similar in sequence to the Nterminal half of ubiquitin-E1 and the other subunit similar to the
C-terminal half of ubiquitin-E1. In Saccharomyces cereviseae, the
heterodimeric E1 (Aos1 and Uba2p) and the E2 enzyme (Ubc9)
are responsible for the activation and conjugation of Smt3p
(Ref. 20). Smt3p molecules are attached to nuclear proteins that
have yet to be identified. The mammalian homolog, SUMO-1, is
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Table 1. Ubiquitin-like proteins and their activating enzymes
Auxin response and the
RUB-conjugation pathway
Three RUB genes are known in Arabidopsis28. The products of two of these (RUB1
E1 N-terminal E1 C-terminal Ubiquitin- Targets
Function
Refs
and RUB2) differ by a single amino acid,
related protein related protein like
while the third (RUB3) is 78% identical
to the other two. A bipartite E1 enzyme,
b
Auxin response
6,7
AXR1
ECR1
RUB1
Cullin
consisting of the AXR1 and ECR1 proteins, activates RUB1 and RUB2. This has
Ula1p
Uba3p
Rub1p
Cdc53p
Cell-cycle regulation 25,26
been demonstrated in vitro using purified
proteins and in extracts prepared from
APP-BP1
UBA3
NEDD8
Cullin
Cell-cycle regulation; 20,27
plant tissues7. The complete absence of
possible involvement
RUB1-activation in extracts prepared from
in Alzheimer’s disease
axr1-12 mutant seedlings indicates that
AXR1 is the predominant species in this
Aso1p
Uba2p
Smt3p
ND
ND
20
tissue. AXR1–ECR1 will also activate
ND
ND
SUMO-1
RanGAP1 Nuclear pore complex 21
RUB3 in vitro, but much less efficiently
formation
than RUB1 and RUB2, suggesting that
another E1 enzyme activates RUB3 (J.C.
ND
ND
SUMO-1
PML
Cellular localization 23
del Pozo and M. Estelle, unpublished).
Only one ECR1 gene has been identified in
ND
ND
SUMO-1
IkBa
Inhibition of
24
the Arabidopsis genome, however, a gene
ubiquitination and
closely related to AXR1 has been identidegradation of IkBa
fied, and it is possible that the product of
this gene functions together with ECR1 to
a
Ubiquitin-activating enzyme.
b
activate RUB3 (Ref. 6).
Data unpublished.
As in yeast, the biochemical function of
Abbreviations: ND, not determined; PML, promyelocytic leukemia complex.
RUB modification in plants is unknown.
However, unlike yeast, defects in the
RUB-conjugation pathway have dramatic
conjugated to several proteins (RanGAP1, PML and IkBa), and consequences. Mutations in AXR1 result in diverse defects in
by contrast with ubiquitin signaling, modification with SUMO-1 auxin-regulated processes that include2,3:
appears to affect the subcellular location or biological function of • Apical dominance
its targets rather than their metabolic stability21–24.
• Inflorescence and hypocotyl elongation
Two groups have shown that when the cytoplasmic RanGAP1 • Gravitropism and phototropism
is modified with SUMO-1, it interacts with RanGAP2 and forms • Root and root hair elongation
part of the nuclear-pore complex implicated in controlling pro- • Lateral root formation
tein traffic to the nucleus21,22. SUMO-1 modification of PML • Tissue growth in culture
changes the subcellular location from soluble nucleoplasmic to In addition, the mutants are deficient in auxin-induced transcripnuclear bodies23. Recently, an interesting regulatory system has tion of several families of auxin-regulated genes4,5. This pleiobeen reported involving activation of NF-kB via modification tropic phenotype implies a central role for AXR1 in most, and
with SUMO-1 (Ref. 24). The transcription factor, NF-kB, is indi- perhaps all, auxin-mediated events in the plant. Indeed, immunorectly activated by ubiquitination and degradation of the repres- localization studies show that AXR1 protein accumulates in
sor, IkBa. SUMO-1 is conjugated to IkBa at the same lysine dividing and elongating cells throughout the plant, but is absent in
residue as ubiquitin, suggesting that SUMO-1-modification might mature nongrowing cells7 (Fig. 1). Further, these studies indicate
prevent ubiquitination of IkBa and block signal-induced acti- that the protein is primarily located in the nucleus (Fig. 1), sugvation of NF-kB.
gesting that the targets of the RUB-conjugation pathway are likely
E1-like enzymes also activate RUB1 and related proteins. In to be nuclear7.
yeast it is the heterodimer Enr2p (also known as Ula1p) and
Uba3p, in plants it is AXR1–ECR1, and in humans it is APP- Auxin-response model
BP1–huba3 that are responsible for Rub1p, RUB1 and NEDD8 It is striking that AXR1 is implicated in the regulation of a comactivation7,20. The E2 required for Rub1p or NEDD8 conjugation plex that is proposed to include TIR1. As previously noted, in
is the Ubc12 protein25 (J.C. del Pozo and M. Estelle, unpublished). yeast there is a genetic interaction between mutants in ENR2 (the
At present the only known Rub1-modified protein is the Cdc53 AXR1 homolog) and CDC4, an F-box protein that is found in an
protein in yeast and humans, a protein already mentioned here SCF complex26. Is there a similar genetic interaction in Arabidin the context of the ubiquitin–ligase SCF complex25–27. Rub1p opsis? The answer is yes, because the axr1 and tir1 mutations have
modification does not result in degradation of Cdc53p (Ref. 26). a synergistic interaction8. The level of 2,4-D resistance in doubleDeletion of either RUB1 or ENR2 from the yeast genome and con- mutant seedlings is greater than would be predicted if the effects
sequent loss of Rub1-modified Cdc53p has little effect on pheno- were simply additive, suggesting that these two genes function in
type. However, both deletions enhance the phenotype of mutations the same or overlapping pathways. Because CDC53 (cullin) is a
in the SKP1, CDC4 and CDC53 genes, all of which encode SCF major target of RUB modification in yeast and in humans, it is
components26. These genetic interactions suggest that Rub1p con- reasonable to expect that Arabidopsis cullin is modified by RUB.
jugation to Cdc53p modulates the activity of the SCF complex in Preliminary data suggest that a member of the cullin family in
some way.
Arabidopsis is modified with RUB1 (J.C. del Pozo and M. Estelle,
a
March 1999, Vol. 4, No. 3
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unpublished). This modification could alter the properties of the
SCF complex in some way. One attractive possibility is that RUBmodified cullin favors assembly of SCFTIR1 over other SCF complexes, thereby promoting the degradation of the repressors of
auxin response. For simplicity, this pathway(s) is referred to as the
AXR1–TIR1 pathway (Fig. 2). At present, there is no information
on how the auxin signal influences the pathway. Auxin might
affect the activity of the RUB-conjugation pathway, thereby altering the levels or activity of SCFTIR1. Alternatively, auxin might
promote interaction between the SCF complex and its potential
substrates. For other SCF complexes, potential substrates must be
phosphorylated before they are recognized by the SCF (Refs 9,
10). There are several reports that activity of a MAP kinase pathway affects auxin response29,30. Perhaps the auxin signal is transduced via this pathway, resulting in phosphorylation of the targets
of the AXR1–TIR1 pathway.
What are the targets of the AXR1–TIR1 pathway?
Fig. 1. Immunolocalization of auxin-resistant 1 (AXR1) in thin
sections using antiserum directed against recombinant AXR1.
Sections were prepared and stained as described in Ref. 7. Section
through the apical region of a six-day-old seedling. (a) AXR1 is
present in expanding cotyledons and the newly formed first pair of
leaves. (b) At higher magnification, AXR1 protein is visible only
in the nucleus.
IAA
?
AXR1
E1
ECR1
E2
1
TIR
P1
C5
3
E2
SK
CD
If AXR1 and TIR1 mediate auxin response by regulating the
degradation of specific proteins, the identity of these molecules
is clearly of great interest. One group of candidates is the proteins involved in auxin-regulated gene expression. Auxin stimulates transcription of specific genes within a few minutes of
auxin application31,32. Rapid auxin-induction of these primary
response genes is independent of protein synthesis and is presumably required for the transcription of secondary genes associated
with auxin response. Because cycloheximide alone will induce
expression of many primary auxin-response genes it has been
suggested that their transcription might be repressed by shortlived repressors. One of the best-characterized families of primary response genes is the Aux/IAA gene family31. This family is
large, with .20 members that encode small short-lived nuclear
proteins. Several lines of evidence suggest that the Aux/IAA
proteins function in transcriptional regulation. They share two
domains (domains III and IV) with ARF1, a transcription factor
that binds a canonical auxin-response
element (AuxRE)33. Domains III and IV
act as dimerization domains promoting
formation of Aux/IAA homodimers and
heterodimers as well as formation of Aux/
IAA-ARF1 dimers.
What is the function of these various
dimers?
R
Repressor
P
Auxin-dependent KINASE?
Ubiquitin
Activation of
auxin-regulated
primary response
genes
Proteosome
RUB1
Activation of
auxin-regulated
secondary response
genes
Long-term consequences
Fig. 2. A model for auxin-resistant 1 (AXR1)–ECR1 and auxin transport inhibitor resistant 1
(TIR1) function in auxin response. According to this model, auxin-regulated gene expression is repressed through the action of one or more repressor proteins (R). When a cell is
exposed to auxin, these repressors are phosphorylated by an unknown protein kinase and
ubiquitinated through the action of SCFTIR1. RUB-modification of CDC53 is required for
normal function of SCFTIR1. Auxin might regulate the activity of SCFTIR1, perhaps via the
RUB-modification pathway, or it might stimulate phosphorylation of the repressor proteins.
Abbreviations: IAA, indole-3-acetic acid; E1, ubiquitin-activating enzyme; E2, ubiquitinconjugating enzyme.
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March 1999, Vol. 4, No. 3
Genetic studies suggest strongly that some
members of the Aux/IAA family promote
auxin response, presumably by stimulating
transcription of other genes. The semidominant axr3 mutants have a constitutive
auxin-response phenotype that includes
excessive adventitious rooting, increased
apical dominance, agravitropism and ectopic expression of the auxin-regulated
gene SAUR-AC1 (Ref. 34). The AXR3 gene
was recently cloned and encodes IAA17, a
member of the Aux/IAA family35. Strikingly, all three mutant alleles examined
affect the same two adjacent residues in
conserved domain II. Five intragenic revertants of axr3 were characterized, each
with a phenotype intermediate between
wild type and axr3. Three of these revertants had a single amino acid change
outside of domain II and the other two
affected splice junctions and resulted in
either truncation of the protein or insertion of 11 extra amino acids. The revertants are likely to result in reduction of
protein function. The semi-dominant nature
of axr3 and the recovery of apparent
loss-of-function revertants indicate that the
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original mutations are gain-of-function. Such gain-of-function
could be a consequence of a dominant negative effect such as formation of a non-functional complex with ARF or Aux/IAA proteins35. Alternatively, the mutations might result in increased
stability of IAA17–AXR3, resulting in inappropriate activation of
downstream genes. The direct measurement of IAA17–AXR3
protein stability in axr3 and wild-type plants should help to resolve this issue.
In addition to activation of auxin response, transfection experiments with carrot protoplasts suggest that binding of some
Aux/IAA proteins to ARF1 prevents transactivation of some
auxin-response genes33. Experiments with cycloheximide suggest
that transcription of the Aux/IAA genes is repressed by short-lived
repressors. Conceivably, the Aux/IAA proteins are the short-lived
repressors. Thus, induction of the auxin-response genes might
require a transient increase in the degradation of their protein
products.
How might the AXR1–TIR1 pathway be involved in
these processes?
Although rapid protein turnover appears to be important for function of the Aux/IAA proteins, the story is complicated because on
the one hand, with Aux/IAA as an activator, degradation would
seem to reduce auxin response, while on the other, with Aux/IAA
as a repressor, degradation would promote auxin response. In
addition, the stability of the pea IAA4 or IAA6 proteins is not
affected by auxin, suggesting that if AXR1 and TIR1 modulate
degradation of these proteins, they do so in an auxin-independent
manner36. Again, the importance of AXR1 and TIR1 in determining
levels of Aux/IAA will be established by directly measuring protein turnover in mutant plants. These experiments are under way.
Another approach to identifying targets of the pathway is to
isolate modifiers of the original axr1 and tir1 mutants. The bestcharacterized line is a suppressor of axr1 called sar1 (Ref. 37).
Genetic studies of the sar1 mutants indicate that the gene functions downstream of AXR1, suggesting that the SAR1 protein might
be a target of the pathway. However, recent studies have shown
that sar1 does not suppress the tir1 mutants, indicating that SAR1
is unlikely to be a target of the putative SCFTIR1. Nevertheless,
SAR1 might be a target of one of the TIR1-related proteins or it
might have some other function in the pathway. The answer to this
question awaits the cloning of the SAR1 gene. The analysis of additional modifiers of axr1 and tir1, both suppressors and enhancers,
is currently under way.
Conclusions
The studies outlined in this review suggest that ubiquitin-mediated
events have an important role in auxin action. Based on our understanding of F-box proteins and the SCF complex, it is likely that
auxin response depends on the degradation of one or more proteins. This degradation is facilitated through modification of a
protein, probably a member of the cullin family, by RUB. Clearly
there are several important outstanding issues and questions with
respect to this model. First, RUB-modification of cullin must be
confirmed in vivo, and the biochemical consequences of modification must be established. Second, the targets of the pathway,
presumably repressors of auxin response, need to be determined.
Third and perhaps most important, we need to understand how
auxin affects activity of the AXR1–TIR1 pathway. Ongoing studies of possible auxin receptors and signaling components should
provide insight into this question. Finally, it will be interesting to
see how broadly the paradigm of regulated protein degradation,
and the SCF complex in particular, has been utilized in cell signaling in plants.
Acknowledgements
We thank Bill Gray for helpful discussion and useful comments
on the manuscript. J.C.d.P. is supported by a long-term fellowship
from the Spanish Government. Research in our laboratory is supported by NIH-GM43644 and NSF IBN-9604398 to M.E.
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J. Carlos del Pozo and Mark Estelle* are at the Dept of Biology,
Indiana University, Bloomington, IN 47405, USA.
*Author for correspondence (tel 11 812 855 8535;
fax 11 812 855 6705; e-mail [email protected]).
Dinitroaniline herbicide resistance
and the microtubule cytoskeleton
Richard G. Anthony and Patrick J. Hussey
Dinitroaniline herbicides have been used for pre-emergence weed control for the past
25 years in cotton, soybean, wheat and oilseed crops. Considering their long persistence
and extensive use, resistance to dinitroanilines is fairly rare. However, the most widespread
dinitroaniline-resistant weeds, the highly resistant (R) and the intermediate (I) biotypes of the
invasive goosegrass Eleusine indica, are now infesting more than 1000 cotton fields in the
southern states of the USA. The molecular basis of this resistance has been identified, and
found to be a point mutation in a major microtubule cytoskeletal protein, a-tubulin. These
studies have served both to explain the establishment of resistance and to reveal fundamental
properties of tubulin gene expression and microtubule structure.
T
he cytoskeleton is a highly dynamic
structure that is involved in many
key processes including cell division, organelle movement and formation
of the cell wall. It is composed of three
fibrous elements, the microtubules, actin
filaments and intermediate filaments, but
the term ‘cytoskeleton’ includes a whole
catalogue of other proteins that anchor,
crosslink or regulate the network within
the cell. The microtubule cytoskeleton is
the target site for many drugs, some are
used as antitumour agents (the plant alkaloids colchicine, vinblastine and taxol), as
fungicides (benzimidazoles, griseofulvin),
and as herbicides (dinitroanilines, phosphorothioamidates and N-phenyl carbamates). These anti-microtubule agents
generally have different affinities for tubulins from metazoan, protozoan, plant and
112
March 1999, Vol. 4, No. 3
(a)
(b)
Fig. 1. Effect of dinitroaniline herbicide on seedling roots. Growth of sensitive (S-biotype)
Eleusine indica seedling roots grown (a) in the absence of herbicide and (b) in the presence
of trifluralin (0.03 mg l21). The herbicide-treated seedlings have the characteristic injury
symptoms associated with the dinitroanilines, that is, excessive swelling of the meristematic
root tip (arrow). Scale bar 5 1 mm.
1360 - 1385/99/$ – see front matter © 1999 Elsevier Science. All rights reserved. PII: S1360-1385(99)01378-3