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Publikation

Abukhalaf, M.; Proksch, C.; Thieme, D.; Ziegler, J.; Hoehenwarter, W.; Changing turn-over rates regulate abundance of tryptophan, GS biosynthesis, IAA transport and photosynthesis proteins in Arabidopsis growth defense transitions BMC Biol. 21, 249, (2023) DOI: 10.1186/s12915-023-01739-3

Background Shifts in dynamic equilibria of the abundance of cellular molecules in plant-pathogen interactions need further exploration. We induced PTI in optimally growing Arabidopsis thaliana seedlings for 16 h, returning them to growth conditions for another 16 h. Methods Turn-over and abundance of 99 flg22 responding proteins were measured chronologically using a stable heavy nitrogen isotope partial labeling strategy and targeted liquid chromatography coupled to mass spectrometry (PRM LC–MS). These experiments were complemented by measurements of mRNA and phytohormone levels. Results Changes in synthesis and degradation rate constants (Ks and Kd) regulated tryptophane and glucosinolate, IAA transport, and photosynthesis-associated protein (PAP) homeostasis in growth/PTI transitions independently of mRNA levels. Ks values increased after elicitation while protein and mRNA levels became uncorrelated. mRNA returned to pre-elicitation levels, yet protein abundance remained at PTI levels even 16 h after media exchange, indicating protein levels were robust and unresponsive to transition back to growth. The abundance of 23 PAPs including FERREDOXIN-NADP( +)-OXIDOREDUCTASE (FNR1) decreased 16 h after PAMP exposure, their depletion was nearly abolished in the myc234 mutant. FNR1 Kd increased as mRNA levels decreased early in PTI, its Ks decreased in prolonged PTI. FNR1 Kd was lower in myc234, mRNA levels decreased as in wild type. Conclusions Protein Kd and Ks values change in response to flg22 exposure and constitute an additional layer of protein abundance regulation in growth defense transitions next to changes in mRNA levels. Our results suggest photosystem remodeling in PTI to direct electron flow away from the photosynthetic carbon reaction towards ROS production as an active defense mechanism controlled post-transcriptionally and by MYC2 and homologs. Target proteins accumulated later and PAP and auxin/IAA depletion was repressed in myc234 indicating a positive effect of the transcription factors in the establishment of PTI.
Publikation

Naumann, C.; Heisters, M.; Brandt, W.; Janitza, P.; Alfs, C.; Tang, N.; Toto Nienguesso, A.; Ziegler, J.; Imre, R.; Mechtler, K.; Dagdas, Y.; Hoehenwarter, W.; Sawers, G.; Quint, M.; Abel, S.; Bacterial-type ferroxidase tunes iron-dependent phosphate sensing during Arabidopsis root development Curr. Biol. 32, 2189-2205, (2022) DOI: 10.1016/j.cub.2022.04.005

Access to inorganic phosphate (Pi), a principal intermediate of energy and nucleotide metabolism, profoundly affects cellular activities and plant performance. In most soils, antagonistic Pi-metal interactions restrict Pi bioavailability, which guides local root development to maximize Pi interception. Growing root tips scout the essential but immobile mineral nutrient; however, the mechanisms monitoring external Pi sta-tus are unknown. Here, we show that Arabidopsis LOW PHOSPHATE ROOT 1 (LPR1), one key determinant of Fe-dependent Pi sensing in root meristems, encodes a novel ferroxidase of high substrate specificity and affinity (apparent KM ∼2 μmM Fe2+). LPR1 typifies an ancient, Fe-oxidizing multicopper protein family that evolved early upon bacterial land colonization. The ancestor of streptophyte algae and embryophytes (land plants) acquired LPR1-type ferroxidase from soil bacteria via horizontal gene transfer, a hypothesis supported by phylogenomics, homology modeling, and biochemistry. Our molecular and kinetic data on LPR1 regulation indicate that Pi-dependent Fe substrate availability determines LPR1 activity and function. Guided by the metabolic lifestyle of extant sister bacterial genera, we propose that Arabidopsis LPR1 monitors subtle concentration differentials of external Fe availability as a Pi-dependent cue to adjust root meristem maintenance via Fe redox signaling and cell wall modification. We further hypothesize that the acquisition of bacterial LPR1-type ferroxidase by embryophyte progenitors facilitated the evolution of local Pi sensing and acquisition during plant terrestrialization.
Publikation

Niemeyer, M.; Moreno Castillo, E.; Ihling, C. H.; Iacobucci, C.; Wilde, V.; Hellmuth, A.; Hoehenwarter, W.; Samodelov, S. L.; Zurbriggen, M. D.; Kastritis, P. L.; Sinz, A.; Calderón Villalobos, L. I. A.; Flexibility of intrinsically disordered degrons in AUX/IAA proteins reinforces auxin co-receptor assemblies Nat. Commun. 11, 2277, (2020) DOI: 10.1038/s41467-020-16147-2

Cullin RING-type E3 ubiquitin ligases SCFTIR1/AFB1-5 and their AUX/IAA targets perceive the phytohormone auxin. The F-box protein TIR1 binds a surface-exposed degron in AUX/IAAs promoting their ubiquitylation and rapid auxin-regulated proteasomal degradation. Here, by adopting biochemical, structural proteomics and in vivo approaches we unveil how flexibility in AUX/IAAs and regions in TIR1 affect their conformational ensemble allowing surface accessibility of degrons. We resolve TIR1·auxin·IAA7 and TIR1·auxin·IAA12 complex topology, and show that flexible intrinsically disordered regions (IDRs) in the degron’s vicinity, cooperatively position AUX/IAAs on TIR1. We identify essential residues at the TIR1 N- and C-termini, which provide non-native interaction interfaces with IDRs and the folded PB1 domain of AUX/IAAs. We thereby establish a role for IDRs in modulating auxin receptor assemblies. By securing AUX/IAAs on two opposite surfaces of TIR1, IDR diversity supports locally tailored positioning for targeted ubiquitylation, and might provide conformational flexibility for a multiplicity of functional states.
Publikation

Bassal, M.; Abukhalaf, M.; Majovsky, P.; Thieme, D.; Herr, T.; Ayash, M.; Tabassum, N.; Al Shweiki, M. R.; Proksch, C.; Hmedat, A.; Ziegler, J.; Lee, J.; Neumann, S.; Hoehenwarter, W.; Reshaping of the Arabidopsis thaliana Proteome Landscape and Co-regulation of Proteins in Development and Immunity Mol. Plant 13, 1709-1732, (2020) DOI: 10.1016/j.molp.2020.09.024

Proteome remodeling is a fundamental adaptive response, and proteins in complexes and functionally related proteins are often co-expressed. Using a deep sampling strategy we define core proteomes of Arabidopsis thaliana tissues with around 10 000 proteins per tissue, and absolutely quantify (copy numbers per cell) nearly 16 000 proteins throughout the plant lifecycle. A proteome-wide survey of global post-translational modification revealed amino acid exchanges pointing to potential conservation of translational infidelity in eukaryotes. Correlation analysis of protein abundance uncovered potentially new tissue- and age-specific roles of entire signaling modules regulating transcription in photosynthesis, seed development, and senescence and abscission. Among others, the data suggest a potential function of RD26 and other NAC transcription factors in seed development related to desiccation tolerance as well as a possible function of cysteine-rich receptor-like kinases (CRKs) as ROS sensors in senescence. All of the components of ribosome biogenesis factor (RBF) complexes were found to be co-expressed in a tissue- and age-specific manner, indicating functional promiscuity in the assembly of these less-studied protein complexes in Arabidopsis. Furthermore, we characterized detailed proteome remodeling in basal immunity by treating Arabidopsis seeldings with flg22. Through simultaneously monitoring phytohormone and transcript changes upon flg22 treatment, we obtained strong evidence of suppression of jasmonate (JA) and JA-isoleucine (JA-Ile) levels by deconjugation and hydroxylation by IAA-ALA RESISTANT3 (IAR3) and JASMONATE-INDUCED OXYGENASE 2 (JOX2), respectively, under the control of JASMONATE INSENSITIVE 1 (MYC2), suggesting an unrecognized role of a new JA regulatory switch in pattern-triggered immunity. Taken together, the datasets generated in this study present extensive coverage of the Arabidopsis proteome in various biological scenarios, providing a rich resource available to the whole plant science community.
Publikation

Ibañez, C.; Delker, C.; Martinez, C.; Bürstenbinder, K.; Janitza, P.; Lippmann, R.; Ludwig, W.; Sun, H.; James, G. V.; Klecker, M.; Grossjohann, A.; Schneeberger, K.; Prat, S.; Quint, M.; Brassinosteroids Dominate Hormonal Regulation of Plant Thermomorphogenesis via BZR1 Curr. Biol. 28, 303-310.e3, (2018) DOI: 10.1016/j.cub.2017.11.077

Thermomorphogenesis is defined as the suite of morphological changes that together are likely to contribute to adaptive growth acclimation to usually elevated ambient temperature [1, 2]. While many details of warmth-induced signal transduction are still elusive, parallels to light signaling recently became obvious (reviewed in [3]). It involves photoreceptors that can also sense changes in ambient temperature [3, 4, 5] and act, for example, by repressing protein activity of the central integrator of temperature information PHYTOCHROME-INTERACTING FACTOR 4 (PIF4 [6]). In addition, PIF4 transcript accumulation is tightly controlled by the evening complex member EARLY FLOWERING 3 [7, 8]. According to the current understanding, PIF4 activates growth-promoting genes directly but also via inducing auxin biosynthesis and signaling, resulting in cell elongation. Based on a mutagenesis screen in the model plant Arabidopsis thaliana for mutants with defects in temperature-induced hypocotyl elongation, we show here that both PIF4 and auxin function depend on brassinosteroids. Genetic and pharmacological analyses place brassinosteroids downstream of PIF4 and auxin. We found that brassinosteroids act via the transcription factor BRASSINAZOLE RESISTANT 1 (BZR1), which accumulates in the nucleus at high temperature, where it induces expression of growth-promoting genes. Furthermore, we show that at elevated temperature BZR1 binds to the promoter of PIF4, inducing its expression. These findings suggest that BZR1 functions in an amplifying feedforward loop involved in PIF4 activation. Although numerous negative regulators of PIF4 have been described, we identify BZR1 here as a true temperature-dependent positive regulator of PIF4, acting as a major growth coordinator.
Publikation

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.
Publikation

Winkler, M.; Niemeyer, M.; Hellmuth, A.; Janitza, P.; Christ, G.; Samodelov, S. L.; Wilde, V.; Majovsky, P.; Trujillo, M.; Zurbriggen, M. D.; Hoehenwarter, W.; Quint, M.; Calderón Villalobos, L. I. A.; Variation in auxin sensing guides AUX/IAA transcriptional repressor ubiquitylation and destruction Nat. Commun. 8, 15706, (2017) DOI: 10.1038/ncomms15706

Auxin is a small molecule morphogen that bridges SCFTIR1/AFB-AUX/IAA co-receptor interactions leading to ubiquitylation and proteasome-dependent degradation of AUX/IAA transcriptional repressors. Here, we systematically dissect auxin sensing by SCFTIR1-IAA6 and SCFTIR1-IAA19 co-receptor complexes, and assess IAA6/IAA19 ubiquitylation in vitro and IAA6/IAA19 degradation in vivo. We show that TIR1-IAA19 and TIR1-IAA6 have distinct auxin affinities that correlate with ubiquitylation and turnover dynamics of the AUX/IAA. We establish a system to track AUX/IAA ubiquitylation in IAA6 and IAA19 in vitro and show that it occurs in flexible hotspots in degron-flanking regions adorned with specific Lys residues. We propose that this signature is exploited during auxin-mediated SCFTIR1-AUX/IAA interactions. We present evidence for an evolving AUX/IAA repertoire, typified by the IAA6/IAA19 ohnologues, that discriminates the range of auxin concentrations found in plants. We postulate that the intrinsic flexibility of AUX/IAAs might bias their ubiquitylation and destruction kinetics enabling specific auxin responses.
Publikation

Hoehenwarter, W.; Mönchgesang, S.; Neumann, S.; Majovsky, P.; Abel, S.; Müller, J.; Comparative expression profiling reveals a role of the root apoplast in local phosphate response BMC Plant Biol. 16, 106, (2016) DOI: 10.1186/s12870-016-0790-8

BackgroundPlant adaptation to limited phosphate availability comprises a wide range of responses to conserve and remobilize internal phosphate sources and to enhance phosphate acquisition. Vigorous restructuring of root system architecture provides a developmental strategy for topsoil exploration and phosphate scavenging. Changes in external phosphate availability are locally sensed at root tips and adjust root growth by modulating cell expansion and cell division. The functionally interacting Arabidopsis genes, LOW PHOSPHATE RESPONSE 1 and 2 (LPR1/LPR2) and PHOSPHATE DEFICIENCY RESPONSE 2 (PDR2), are key components of root phosphate sensing. We recently demonstrated that the LOW PHOSPHATE RESPONSE 1 - PHOSPHATE DEFICIENCY RESPONSE 2 (LPR1-PDR2) module mediates apoplastic deposition of ferric iron (Fe3+) in the growing root tip during phosphate limitation. Iron deposition coincides with sites of reactive oxygen species generation and triggers cell wall thickening and callose accumulation, which interfere with cell-to-cell communication and inhibit root growth.ResultsWe took advantage of the opposite phosphate-conditional root phenotype of the phosphate deficiency response 2 mutant (hypersensitive) and low phosphate response 1 and 2 double mutant (insensitive) to investigate the phosphate dependent regulation of gene and protein expression in roots using genome-wide transcriptome and proteome analysis. We observed an overrepresentation of genes and proteins that are involved in the regulation of iron homeostasis, cell wall remodeling and reactive oxygen species formation, and we highlight a number of candidate genes with a potential function in root adaptation to limited phosphate availability. Our experiments reveal that FERRIC REDUCTASE DEFECTIVE 3 mediated, apoplastic iron redistribution, but not intracellular iron uptake and iron storage, triggers phosphate-dependent root growth modulation. We further highlight expressional changes of several cell wall-modifying enzymes and provide evidence for adjustment of the pectin network at sites of iron accumulation in the root.ConclusionOur study reveals new aspects of the elaborate interplay between phosphate starvation responses and changes in iron homeostasis. The results emphasize the importance of apoplastic iron redistribution to mediate phosphate-dependent root growth adjustment and suggest an important role for citrate in phosphate-dependent apoplastic iron transport. We further demonstrate that root growth modulation correlates with an altered expression of cell wall modifying enzymes and changes in the pectin network of the phosphate-deprived root tip, supporting the hypothesis that pectins are involved in iron binding and/or phosphate mobilization.
Publikation

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.
Publikation

Delker, C.; Sonntag, L.; James, G.; Janitza, P.; Ibañez, C.; Ziermann, H.; Peterson, T.; Denk, K.; Mull, S.; Ziegler, J.; Davis, S.; 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.
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