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Auxin induces rapid abundance changes in various signaling proteins, transcriptional regulators, and enzymes such as cell wall modification proteins in roots. Loss of function of 15 top responsive proteins results in altered root phenotypes, demonstrating the power of this approach for reverse genetics screens. Characterization of the auxin responsive protein galacturonosyltransferase 10 demonstrates that this enzyme positively regulates sugar-mediated root meristem maintenance. Novel targeted proteomics assays demonstrate that all six auxin receptors remain stable in response to hormone.

The hormone cytokinin promotes cell differentiation in plant roots by repressing both auxin transport and responses to auxin at the boundary between the meristem and the root elongation zone.

The plant hormones auxin and brassinosteroid promote cell expansion by regulating gene expression. In addition to independent transcriptional responses generated by the two signals, recent microarray analyses indicate that auxin and brassinosteroid also coordinate the expression of a set of shared target genes.

Auxin-regulated gene expression is mediated by two families of transcription factors. The ARF proteins bind to a conserved DNA sequence called the AuxRE and activate transcription. The Aux/IAA proteins repress ARF function, presumably by forming dimers with ARF proteins. Recent genetic studies in Arabidopsis indicate that auxin regulates this system by promoting the ubiquitin-mediated degradation of the Aux/IAA proteins, thus permitting ARF function. Mutations in components of SCF$^TIR1$, a ubiquitin protein ligase (E3) result in stabilization of Aux/IAA proteins and decreased auxin response. Further, recent biochemical experiments indicate that the Aux/IAA proteins bind SCF$^TIR1$ in an auxin-dependent manner.

The SCF (for SKP1, Cullin/CDC53,F-box protein) ubiquitin ligase targets a number of cell cycle regulators, transcription factors, and other proteins for degradation in yeast and mammalian cells. Recent genetic studies demonstrate that plant F-box proteins are involved in auxin responses, jasmonate signaling, flower morphogenesis, photocontrol of circadian clocks, and leaf senescence, implying a large spectrum of functions for the SCF pathway in plant development. Here, we present a molecular and functional characterization of plant cullins. TheArabidopsisgenome contains 11 cullin-related genes. Complementation assays revealed that AtCUL1 but not AtCUL4 can functionally complement the yeast cdc53mutant.Arabidopsismutants containing transfer DNA (T-DNA) insertions in the AtCUL1gene were shown to display an arrest in early embryogenesis. Consistently, both the transcript and the protein of the AtCUL1gene were found to accumulate in embryos. The AtCUL1 protein localized mainly in the nucleus but also weakly in the cytoplasm during interphase and colocalized with the mitotic spindle in metaphase. Our results demonstrate a critical role for the SCF ubiquitin ligase inArabidopsisembryogenesis.

Selective protein degradation by the ubiquitin-proteosome pathway has recently emerged as a powerful regulatory mechanism in a wide variety of cellular processes. Ubiquitin conjugation requires the sequential activity of three enzymes or protein complexes called the ubiquitin-activating enzyme (E1), the ubiquitin-conjugating enzyme (E2), and the ubiquitin-protein ligase (E3). In most eukaryotes, there are a small number of similar E1 isoforms without apparent functional specificity. The specific selection of target proteins is accomplished by the E2 and E3 proteins. One of the best-characterized families of E3s are the SCF complexes. The SCF is composed of a cullin (Cdc53), SKP1, RBX1 and one member of a large family of proteins called F-box proteins. The function of the F-box protein is to interact with target proteins. In some cases, the stability of the F-box protein may regulate activity of the SCF complex. In addition, post-translational modification of the cullin subunit by the ubiquitin-like protein RUB/NEDD8 appears to regulate SCF function. In plants, the SCF has so far been implicated in floral development, circadian clock, and response to the plant growth regulators auxin and jasmonic acid.

The indolic compound auxin regulates virtually every aspect of plant growth and development, but its role in embryogenesis and its molecular mechanism of action are not understood. We describe two mutants of Arabidopsis that define a novel gene called AUXIN-RESISTANT6 (AXR6) which maps to chromosome 4. Embryonic development of the homozygous axr6 mutants is disrupted by aberrant patterns of cell division, leading to defects in the cells of the suspensor, root and hypocotyl precursors, and provasculature. The homozygous axr6 mutants arrest growth soon after germination lacking a root and hypocotyl and with severe vascular pattern defects in their cotyledons. Whereas previously described mutants with similar developmental defects are completely recessive, axr6 heterozygotes display a variety of morphological and physiological alterations that are most consistent with a defect in auxin physiology or response. The AXR6 gene is likely to be important for auxin response throughout the plant, including early development.

Transgenic Arabidopsis thaliana plants constitutively expressing Agrobacterium tumefaciens tryptophan monooxygenase (iaaM) were obtained and characterized. Arabidopsis plants expressing iaaM have up to 4-fold higher levels of free indole-3-acetic acid (IAA) and display increased hypocotyl elongation in the light. This result clearly demonstrates that excess endogenous auxin can promote cell elongation in a whole plant. Interactions of the auxin-overproducing transgenic plants with the phytochrome-deficient hy6-1 and auxin-resistant axrl-3 mutations were also studied. The effects of auxin overproduction on hypocotyl elongation were not additive to the effects of phytochrome deficiency in the hy6-1 mutant, indicating that excess auxin does not counteract factors that limit hypocotyl elongation in hy6-1 seedlings. Auxin-overproducing seedlings are also qualitatively indistinguishable from wild-type controls in their response to red, far-red, and blue light treatments, demonstrating that the effect of excess auxin on hypocotyl elongation is independent of red and blue light-mediated effects. All phenotypic effects of iaaM-mediated auxin overproduction (i.e. increased hypocotyl elongation in the light, severe rosette leaf epinasty, and increased apical dominance) are suppressed by the auxin-resistant axr1-3 mutation. The axr1-3 mutation apparently blocks auxin signal transduction since it does not reduce auxin levels when combined with the auxin-overproducing transgene.

Measurement of dopa decarboxylase (DDC) levels in 109 strains of Drosophila melanogaster isogenic for second chromosomes isolated independently from natural populations was undertaken. One of the most extreme variants, designated Ddc+4, was shown to have about 20% more DDC activity at adult eclosion than a standard laboratory strain used for comparison. The DDC overproduction was shown to segregate with the second chromosome and was mapped to a position within 0.15 map units of the DDC structural gene. The variant was shown to be an underproducer of DDC activity at pupariation and the genetic element responsible for this trait mapped in an identical fashion to that causing overproduction. The temporal phenotype described above was observed in the epidermis but DDC activity levels in neural tissue were normal. Examination of CRM levels at pupariation and eclosion revealed that altered DDC protein levels were responsible for the variant DDC activity levels. Electrophoretic analysis under both denaturing and non-denaturing conditions indicated that the DDC molecules in Ddc+4 and the laboratory strain were indistinguishable. These results suggest that alterations in a genetic element (or elements) lying in close proximity to the structural gene are responsible for the complex temporal phenotype of DDC activity exhibited in the variant Ddc+4.

An activity variant in Drosophila, Ddc+4, which has been isolated from natural populations is shown to affect the level of messenger RNA for dopa decarboxylase (DDC) in a stage specific manner. Newly hatched first instar larvae and newly eclosed adults have 1.5 times the amount of DDC mRNA as the Canton-S laboratory strain. On the other hand, puparia of the variant have only 0.5 times as much DDC mRNA as Canton-S. Genomic Southern analysis revealed that Ddc+4 DNA differed from Canton-S DNA by four small restriction length polymorphisms. To confirm these differences, a library of Ddc+4 was constructed in the ?1059 vector. A clone was recovered spanning the DDC region and compared to cloned Canton-S DNA. Acrylamide gel electrophoresis of restriction fragments revealed that one previously identified insertion really consisted of two smaller ones. One of the other differences identified by the Southern analysis was not confirmed in the cloned DNA of Ddc+4, indicating some divergence had occurred in the variant strain between the time of its isogenization and cloning. The differences between the cloned Ddc+4 DNA and cloned Canton-S DNA consisted of six small restriction length polymorphisms and one restriction site polymorphism. Five of the seven differences lay in the 5' untranslated leader sequence of the DDC mRNA or in the 4.5 kb of DNA upstream of the transcription start site. The existence of these small (<100 bp) insertion/deletion polymorphisms in a strain exhibiting a complex temporal phenotype for DDC activity, suggests natural populations are an excellent source of variation affecting gene expression. Secondly, subtle restriction length polymorphisms near the 5' end of genes may well be an important component of the variation upon which selection might be expected to act.

Three members of a family of highly repeated DNA sequences from Arabidopsis thaliana have been cloned and characterized. The repeat unit has an average length of 180 bp and is tandemly repeated in arrays longer than 50 kb. This family represents more than one percent of the Arabidopsis genome. Sequence comparisons with tandemly repeated DNA sequences from other Cruciferae species show several regions of homology and a similar length of the repeat unit. Homologies are also found to highly repeated sequences from other plant species. When the sequence CCGG occurs in the repeated DNA, the inner cytosine is generally methylated.

We modified a video digitizer system to allow short-term high-resolution measurements of root elongation in intact seedlings of Arabidopsis thaliana (L.) Heynh. We used the system to measure the kinetics of promotion and inhibition of root elongation by applied auxin and to determine the dose-response relationship for auxin action on elongation in roots of wild-type seedlings and seedlings of mutants (axr1, aux1, and axr2) with altered auxin responsiveness. Roots of the mutants showed less inhibition in the presence of inhibitory concentrations of auxin than did roots of the wild type. The latent period preceding the change in elongation rate after auxin application was the same for axr1 and axr2 as for the wild type whereas the latent period for aux1 was about twice as long as for the wild type. Low concentrations (ca. 10-11 M) of auxin induced substantial promotion of root elongation in the wild type and in axr2.

Mutant lines of Arabidopsis thaliana resistant to the artificial auxin 2,4-dichloro phenoxyacetic acid (2,4-D) were isolated by screening for growth of seedlings in the presence of toxic levels of 2,4-D. Genetic analysis of these resistant lines indicated that 2,4-D resistance is due to a recessive mutation at a locus we have designated Axr-1. Mutant seedlings were resistant to approximately 50-fold higher concentrations of 2,4-D than wild-type and were also resistant to 8-fold higher concentrations of indole-3-acetic acid (IAA) than wild-type. Labelling studies with (14C)2,4-D suggest that resistance was not due to changes in uptake or metabolism of 2,4-D. In addition to auxin resistance the mutants have a distinct morphological phenotype including alterations of the roots, leaves, and flowers. Genetic evidence indicates that both auxin resistance and the morphological changes are due to the same mutation. Because of the pleiotropic morphological effects of these mutations the Axr-1 gene may code for a function involved in auxin action in all tissues of the plant.

The axr2 mutation of Arabidopsis thaliana (L.) Heynh. confers resistance to the plant growth hormones auxin, ethylene and abscisic acid. In addition, mutant plants have a pronounced dwarf phenotype and display defects in both shoot and root gravitopism. To further characterize this mutant we have determined the phenotype of both dark- and light-grown mutant seedlings. We find that the height of axr2 hypocotyls is reduced in dark conditions compared with wild-type seedlings and that both dark- and light-grown hypocotyls have a gravitropic defect. In addition, we have examined the cellular anatomy of a variety of wild-type and axr2 tissues using light and scanning electron microscopy. Our results indicate that the axr2 mutation has a dramatic effect on cell length in both the inflorescence and the hypocotyl and a lesser effect on cell number in these tissues. The largest difference was observed in the epidermis of the inflorescence where axr2 cells were approximately eightfold shorter than wild-type cells. We suggest that these reductions in cell length and number are sufficient to explain most aspects of the axr2 phenotype. In addition, we propose that a reduction in auxin-mediated cell elongation is responsible for the gravitropic defect in mutant roots, hypocotyls and floral stems. Finally, we have found that the reduction in epidermal cell size in the mutant inflorescence is accompanied by a fourfold increase in stoma density. The implications of this result for models of stoma development are discussed.

We have screened a large population of M2 seeds ofArabidopsis thaliana for plants which are resistant to exogenously applied indole-acetic acid (IAA). One of the resistant lines identified in this screen carries a dominant mutation which we have namedaxr2. Linkage analysis indicates that theaxr2 gene lies on chromosome 3. Plants carrying theaxr2 mutation are severe dwarfs and display defects in growth orientation of both the shoot and root suggesting that the mutation affects some aspect of gravitropic growth. In addition, the roots ofaxr2 plants lack root hairs. Growth inhibition experiments indicate that the roots ofaxr2 plants are resistant to ethylene and abscisic acid as well as auxin.

Plant genomes encode large numbers of F-box proteins (FBPs), the substrate recognition subunit of SKP1–CULLIN–F-box (SCF) ubiquitin ligases. There are ∼700 FBPs in Arabidopsis, most of which are uncharacterized. TIR1 is among the best-studied plant FBPs and functions as a receptor for the plant hormone auxin. Here we use a yeast two-hybrid system to identify novel TIR1 mutants with altered properties. The analysis of these mutants reveals that TIR1 associates with the CULLIN1 (CUL1) subunit of the SCF through the N-terminal H1 helix of the F-box domain. Mutations that untether TIR1 from CUL1 stabilize the FBP and cause auxin resistance and associated growth defects, probably by protecting TIR1 substrates from degradation. Based on these results we propose that TIR1 is subject to autocatalytic degradation when assembled into an SCF. Further, our results suggest a general method for determining the physiological function of uncharacterized FBPs. Finally, we show that a key amino acid variation in the F-box domain of auxin signalling F-box (AFB1), a closely related FBP, reduces its ability to form an SCF, resulting in an increase in AFB1 levels.

Decades of research have suggested that AUXIN BINDING PROTEIN 1 (ABP1) is an essential membrane-associated auxin receptor, but recent findings directly contradict this view. Here we show that embryonic lethality observed in abp1-1, which has been a cornerstone of ABP1 studies, is caused by the deletion of the neighbouring BELAYA SMERT (BSM) gene, not by disruption of ABP1. On the basis of our results, we conclude that ABP1 is not essential for Arabidopsis development.

Growth is a major factor in plant organ morphogenesis and is influenced by exogenous and endogenous signals including hormones. Although recent studies have identified regulatory pathways for the control of growth during vegetative development, there is little mechanistic understanding of how growth is controlled during the reproductive phase. Using Arabidopsis fruit morphogenesis as a platform for our studies, we show that the microRNA miR172 is critical for fruit growth, as the growth of fruit is blocked when miR172 activity is compromised. Furthermore, our data are consistent with the FRUITFULL (FUL) MADS-domain protein and Auxin Response Factors (ARFs) directly activating the expression of a miR172-encoding gene to promote fruit valve growth. We have also revealed that MADS-domain (such as FUL) and ARF proteins directly associate in planta. This study defines a novel and conserved microRNA-dependent regulatory module integrating developmental and hormone signalling pathways in the control of plant growth.