Ludwig-Müller, J.; Denk, K.; Cohen, J. D.; Quint, M. An Inhibitor of Tryptophan-Dependent Biosynthesis of Indole-3-Acetic Acid Alters Seedling Development in Arabidopsis J Plant Growth Regul 29, 242-248, (2010) DOI: 10.1007/s00344-009-9128-1
Although polar transport and the TIR1-dependent signaling pathway of the plant hormone auxin/indole-3-acetic acid (IAA) are well characterized, understanding of the biosynthetic pathway(s) leading to the production of IAA is still limited. Genetic dissection of IAA biosynthetic pathways has been complicated by the metabolic redundancy caused by the apparent existence of several parallel biosynthetic routes leading to IAA production. Valuable complementary tools for genetic as well as biochemical analysis of auxin biosynthesis would be molecular inhibitors capable of acting in vivo on specific or general components of the pathway(s), which unfortunately have been lacking. Several indole derivatives have been previously identified to inhibit tryptophan-dependent IAA biosynthesis in an in vitro system from maize endosperm. We examined the effect of one of them, 6-fluoroindole, on seedling development of Arabidopsis thaliana and tested its ability to inhibit IAA biosynthesis in feeding experiments in vivo. We demonstrated a correlation of severe developmental defects or growth retardation caused by 6-fluoroindole with significant downregulation of de novo synthesized IAA levels, derived from the stable isotope-labeled tryptophan pool, upon treatment. Hence, 6-fluoroindole shows important features of an inhibitor of tryptophan-dependent IAA biosynthesis both in vitro and in vivo and thus may find use as a promising molecular tool for the identification of novel components of the auxin biosynthetic pathway(s).
Raschke, A.; Ibañez, C.; Ullrich, K. K.; Anwer, M. U.; Becker, S.; Glöckner, A.; Trenner, J.; Denk, K.; Saal, B.; Sun, X.; Ni, M.; Davis, S. J.; Delker, C.; Quint, M. Natural variants of ELF3 affect thermomorphogenesis by transcriptionally modulating PIF4-dependent auxin response genes BMC Plant Biol. 15, 197, (2015) DOI: 10.1186/s12870-015-0566-6
BackgroundPerception and transduction of temperature changes result in altered growth enabling plants to adapt to increased ambient temperature. While PHYTOCHROME-INTERACTING FACTOR4 (PIF4) has been identified as a major ambient temperature signaling hub, its upstream regulation seems complex and is poorly understood. Here, we exploited natural variation for thermo-responsive growth in Arabidopsis thaliana using quantitative trait locus (QTL) analysis.ResultsWe identified GIRAFFE2.1, a major QTL explaining ~18 % of the phenotypic variation for temperature-induced hypocotyl elongation in the Bay-0 x Sha recombinant inbred line population. Transgenic complementation demonstrated that allelic variation in the circadian clock regulator EARLY FLOWERING3 (ELF3) is underlying this QTL. The source of variation could be allocated to a single nucleotide polymorphism in the ELF3 coding region, resulting in differential expression of PIF4 and its target genes, likely causing the observed natural variation in thermo-responsive growth.ConclusionsIn combination with other recent studies, this work establishes the role of ELF3 in the ambient temperature signaling network. Natural variation of ELF3-mediated gating of PIF4 expression during nightly growing periods seems to be affected by a coding sequence quantitative trait nucleotide that confers a selective advantage in certain environments. In addition, natural ELF3 alleles seem to differentially integrate temperature and photoperiod information to induce architectural changes. Thus, ELF3 emerges as an essential coordinator of growth and development in response to diverse environmental cues and implicates ELF3 as an important target of adaptation.
Ibañez, C.; Poeschl, Y.; Peterson, T.; Bellstädt, J.; Denk, K.; Gogol-Döring, A.; Quint, M.; Delker, C. Ambient temperature and genotype differentially affect developmental and phenotypic plasticity in Arabidopsis thaliana BMC Plant Biol 17, 114, (2017) DOI: 10.1186/s12870-017-1068-5
BackgroundGlobal increase in ambient temperatures
constitute a significant challenge to wild and cultivated plant species.
Forward genetic analyses of individual temperature-responsive traits
have resulted in the identification of several signaling and response
components. However, a comprehensive knowledge about temperature
sensitivity of different developmental stages and the contribution of
natural variation is still scarce and fragmented at best.ResultsHere, we
systematically analyze thermomorphogenesis throughout a complete life
cycle in ten natural Arabidopsis thaliana accessions grown under long
day conditions in four different temperatures ranging from 16 to 28 °C.
We used Q10, GxE, phenotypic divergence and correlation analyses to
assess temperature sensitivity and genotype effects of more than 30
morphometric and developmental traits representing five phenotype
classes. We found that genotype and temperature differentially affected
plant growth and development with variing strengths. Furthermore,
overall correlations among phenotypic temperature responses was
relatively low which seems to be caused by differential capacities for
temperature adaptations of individual
accessions.ConclusionGenotype-specific temperature responses may be
attractive targets for future forward genetic approaches and
accession-specific thermomorphogenesis maps may aid the assessment of
functional relevance of known and novel regulatory components.
Delker, C.; Sonntag, L.; Geo, V. J.; Janitza, P.; Ibañez, C.; Ziermann, H.; Peterson, T.; Denk, K.; Mull, S.; Ziegler, J.; Davis, S. J.; Schneeberger, K.; Quint, M. The DET1-COP1-HY5 Pathway Constitutes a Multipurpose Signaling Module Regulating Plant Photomorphogenesis and Thermomorphogenesis Cell Rep 9, 1983–1989, (2014) DOI: 10.1016/j.celrep.2014.11.043
Developmental plasticity enables plants to respond to elevated ambient temperatures by adapting their shoot architecture. On the cellular level, the basic-helix-loop-helix (bHLH) transcription factor PHYTOCHROME INTERACTING FACTOR 4 (PIF4) coordinates this response by activating hormonal modules that in turn regulate growth. In addition to an unknown temperature-sensing mechanism, it is currently not understood how temperature regulates PIF4 activity. Using a forward genetic approach in Arabidopsis thaliana, we present extensive genetic evidence demonstrating that the DE-ETIOLATED 1 (DET1)-CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1)-ELONGATED HYPOCOTYL 5 (HY5)-dependent photomorphogenesis pathway transcriptionally regulates PIF4 to coordinate seedling growth in response to elevated temperature. Our findings demonstrate that two of the most prevalent environmental cues, light and temperature, share a much larger set of signaling components than previously assumed. Similar to the toolbox concept in animal embryonic patterning, multipurpose signaling modules might have evolved in plants to translate various environmental stimuli into adaptational growth processes
Quint, M.; Ito, H.; Zhang, W.; Gray, W.M. Characterization of a novel temperature-sensitive allele of the CUL1/AXR6 subunit of SCF ubiquitin-ligases Plant J 43, 371-383, (2005)
Selective protein degradation by the ubiquitin-proteasome pathway has emerged as a key regulatory mechanism in a wide variety of cellular processes. The selective components of this pathway are the E3 ubiquitin-ligases which act downstream of the ubiquitin-activating and -conjugating enzymes to identify specific substrates for ubiquitinylation. SCF-type ubiquitin-ligases are the most abundant class of E3 enzymes in Arabidopsis. In a genetic screen for enhancers of the tir1-1 auxin response defect, we identified eta1/axr6-3, a recessive and temperature-sensitive mutation in the CUL1 core component of the SCFTIR1 complex. The axr6-3 mutation interferes with Skp1 binding, thus preventing SCF complex assembly. axr6-3 displays a pleiotropic phenotype with defects in numerous SCF-regulated pathways including auxin signaling, jasmonate signaling, flower development, and photomorphogenesis. We used axr6-3 as a tool for identifying pathways likely to be regulated by SCF-mediated proteolysis and propose new roles for SCF regulation of the far-red light/phyA and sugar signaling pathways. The recessive inheritance and the temperature-sensitive nature of the pleiotropically acting axr6-3 mutation opens promising possibilities for the identification and investigation of SCF-regulated pathways in Arabidopsis.
Parry, G.; Calderón Villalobos, L.I.; Prigge, M.; Peret, B.; Dharmasiri, S.; Itoh, H.; Lechner, E.; Gray, W.M.; Bennett, M.; Estelle, M. Complex regulation of the TIR/AFB family of auxin receptors Proc Natl Acad Sci USA 106(52), 22540-22545, (2009) DOI: 10.1073/pnas.0911967106
Auxin regulates most aspects of plant growth and development. The hormone is perceived by the TIR1/AFB family of F-box proteins acting in concert with the Aux/IAA transcriptional repressors. Arabidopsis plants that lack members of the TIR1/AFB family are auxin resistant and display a variety of growth defects. However, little is known about the functional differences between individual members of the family. Phylogenetic studies reveal that the TIR1/AFB proteins are conserved across land plant lineages and fall into four clades. Three of these subgroups emerged before separation of angiosperms and gymnosperms whereas the last emerged before the monocot-eudicot split. This evolutionary history suggests that the members of each clade have distinct functions. To explore this possibility in Arabidopsis, we have analyzed a range of mutant genotypes, generated promoter swap transgenic lines, and performed in vitro binding assays between individual TIR1/AFB and Aux/IAA proteins. Our results indicate that the TIR1/AFB proteins have distinct biochemical activities and that TIR1 and AFB2 are the dominant auxin receptors in the seedling root. Further, we demonstrate that TIR1, AFB2, and AFB3, but not AFB1 exhibit significant posttranscriptional regulation. The microRNA miR393 is expressed in a pattern complementary to that of the auxin receptors and appears to regulate TIR1/AFB expression. However our data suggest that this regulation is complex. Our results suggest that differences between members of the auxin receptor family may contribute to the complexity of auxin response.
Quint, M.; Barkawi, L.S.; Fan, K.T.; Cohen, J.D.; Gray, W.M. Arabidopsis IAR4 modulates auxin response by regulating auxin homeostasis Plant Physiol 150, 748-758, (2009) DOI: 10.1104/pp.109.136671
In a screen for enhancers of tir1-1 auxin resistance, we identified two novel alleles of the putative mitochondrial pyruvate dehydrogenase E1α-subunit, IAA-Alanine Resistant4 (IAR4). In addition to enhancing the auxin response defects of tir1-1, iar4 single mutants exhibit numerous auxin-related phenotypes including auxin-resistant root growth and reduced lateral root development, as well as defects in primary root growth, root hair initiation, and root hair elongation. Remarkably, all of these iar4 mutant phenotypes were rescued when endogenous indole-3-acetic acid (IAA) levels were increased by growth at high temperature or overexpression of the YUCCA1 IAA biosynthetic enzyme, suggesting that iar4 mutations may alter IAA homeostasis rather than auxin response. Consistent with this possibility, iar4 mutants exhibit increased Aux/IAA stability compared to wild type under basal conditions, but not in response to an auxin treatment. Measurements of free IAA levels detected no significant difference between iar4-3 and wild-type controls. However, we consistently observed significantly higher levels of IAA-amino acid conjugates in the iar4-3 mutant. Furthermore, using stable isotope-labeled IAA precursors, we observed a significant increase in the relative utilization of the Trp-independent IAA biosynthetic pathway in iar4-3. We therefore suggest that the auxin phenotypes of iar4 mutants are the result of altered IAA homeostasis.
Zhang, W.; Ito, H.; Quint, M.; Huang, H.; Noël, L.D.; Gray, W.M. Genetic analysis of CAND1-CUL1 interactions in Arabidopsis supports a role for CAND1-mediated cycling of the SCFTIR1 complex Proc Natl Acad Sci 105, 8470-8475, (2008) DOI: 10.1073/pnas.0804144105
SKP1-Cullin1-F-box protein (SCF) ubiquitin-ligases regulate numerous aspects of eukaryotic growth and development. Cullin-Associated and Neddylation-Dissociated (CAND1) modulates SCF function through its interactions with the CUL1 subunit. Although biochemical studies with human CAND1 suggested that CAND1 plays a negative regulatory role by sequestering CUL1 and preventing SCF complex assembly, genetic studies in Arabidopsis have shown that cand1 mutants exhibit reduced SCF activity, demonstrating that CAND1 is required for optimal SCF function in vivo. Together, these genetic and biochemical studies have suggested a model of CAND1-mediated cycles of SCF complex assembly and disassembly. Here, using the SCFTIR1 complex of the Arabidopsis auxin response pathway, we test the SCF cycling model with Arabidopsis mutant derivatives of CAND1 and CUL1 that have opposing effects on the CAND1CUL1 interaction. We find that the disruption of the CAND1CUL1 interaction results in an increased abundance of assembled SCFTIR1 complex. In contrast, stabilization of the CAND1CUL1 interaction diminishes SCFTIR1 complex abundance. The fact that both decreased and increased CAND1CUL1 interactions result in reduced SCFTIR1 activity in vivo strongly supports the hypothesis that CAND1-mediated cycling is required for optimal SCF function.
Auxin regulates a host of plant developmental and physiological processes, including embryogenesis, vascular differentiation, organogenesis, tropic growth, and root and shoot architecture. Genetic and biochemical studies carried out over the past decade have revealed that much of this regulation involves the SCFTIR1/AFB-mediated proteolysis of the Aux/IAA family of transcriptional regulators. With the recent finding that the TRANSPORT INHIBITOR RESPONSE1 (TIR1)/AUXIN SIGNALING F-BOX (AFB) proteins also function as auxin receptors, a potentially complete, and surprisingly simple, signaling pathway from perception to transcriptional response is now before us. However, understanding how this seemingly simple pathway controls the myriad of specific auxin responses remains a daunting challenge, and compelling evidence exists for SCFTIR1/AFB-independent auxin signaling pathways.
Raschke, A.; Ibañez, C.; Ullrich, K. K.; Anwer, M. U.; Becker, S.; Glöckner, A.; Trenner, J.; Denk, K.; Saal, B.; Sun, X.; Ni, M.; Davis, S. J.; Delker, C.; Quint, M. Natural Variants of ELF3 Affect Thermomorphogenesis by Transcriptionally Modulating PIF4-Dependent Auxin Response Genes bioRxiv (2015) DOI: 10.1101/015305
Perception and transduction of temperature changes result in altered
growth enabling plants to adapt to increased ambient temperature. While
PHYTOCHROME-INTERACTING FACTOR4 (PIF4) has been identified as a major
ambient temperature signaling hub, its upstream regulation seems complex
and is poorly understood. Here, we exploited natural variation for
thermo-responsive growth in Arabidopsis thaliana using quantitative
trait locus (QTL) analysis. We identified GIRAFFE2.1, a major QTL
explaining ~18% of the phenotypic variation for temperature-induced
hypocotyl elongation in the Bay-0 x Sha recombinant inbred line
population. Transgenic complementation demonstrated that allelic
variation in the circadian clock regulator EARLY FLOWERING3 (ELF3) is
underlying this QTL. The source of variation could be allocated to a
single nucleotide polymorphism in the ELF3 coding region, resulting in
differential expression of PIF4 and its target genes, likely causing the
observed natural variation in thermo-responsive growth. In combination
with other recent studies, this work establishes the role of ELF3 in the
ambient temperature signaling network. Natural variation of
ELF3-mediated gating of PIF4 expression during nightly growing periods
seems to be affected by a coding sequence quantitative trait nucleotide
that confers a selective advantage in certain environments. In
addition, natural ELF3 alleles seem to differentially integrate
temperature and photoperiod cues to induce architectural changes. Thus,
ELF3 emerges as an essential coordinator of growth and development in
response to diverse environmental cues and implicates ELF3 as an
important target of adaptation.