Preprints
Trenner, J.; Poeschl, Y.; Grau, J.; Gogol-Döring, A.; Quint, M.; Delker, C. Auxin-induced expression divergence between Arabidopsis species likely originates within the TIR1/AFB-AUX/IAA-ARF module bioRxiv (2016) DOI: 10.1101/038422
Auxin is an essential regulator of plant growth and development and
auxin signaling components are conserved among land plants. Yet, a
remarkable degree of natural variation in physiological and
transcriptional auxin responses has been described among Arabidopsis
thaliana accessions. As intra-species comparisons offer only limited
genetic variation, we here inspect the variation of auxin responses
between A. thaliana and A. lyrata. This approach allowed the
identification of conserved auxin response genes including novel genes
with potential relevance for auxin biology. Furthermore, promoter
divergences were analyzed for putative sources of variation. De novo
motif discovery identified novel and variants of known elements with
potential relevance for auxin responses, emphasizing the complex, and
yet elusive, code of element combinations accounting for the diversity
in transcriptional auxin responses. Furthermore, network analysis
revealed correlations of inter-species differences in the expression of
AUX/IAA gene clusters and classic auxin-related genes. We conclude that
variation in general transcriptional and physiological auxin responses
may originate substantially from functional or transcriptional
variations in the TIR1/AFB, AUX/IAA, and ARF signaling network. In that
respect, AUX/IAA gene expression divergence potentially reflects
differences in the manner in which different species transduce identical
auxin signals into gene expression responses.
Preprints
Drost, H.-J.; Gabel, A.; Domazet-Lošo, T.; Quint, M.; Grosse, I. Capturing Evolutionary Signatures in Transcriptomes with myTAI bioRxiv (2016) DOI: 10.1101/051565
Combining transcriptome data of biological processes or response to
stimuli with evolutionary information such as the phylogenetic
conservation of genes or their sequence divergence rates enables the
investigation of evolutionary constraints on these processes or
responses. Such phylotranscriptomic analyses recently unraveled that
mid-developmental transcriptomes of fly, fish, and cress were dominated
by evolutionarily conserved genes and genes under negative selection and
thus recapitulated the developmental hourglass on the transcriptomic
level. Here, we present a protocol for performing phylotranscriptomic
analyses on any biological process of interest. When applying this
protocol, users are capable of detecting different evolutionary
constraints acting on different stages of the biological process of
interest in any species. For each step of the protocol, modular and
easy-to-use open-source software tools are provided, which enable a
broad range of scientists to apply phylotranscriptomic analyses to a
wide spectrum of biological questions.
Preprints
Gantner, J.; Ilse, T.; Ordon, J.; Kretschmer, C.; Gruetzner, R.; Loefke, C.; Dagdas, Y.; Buerstenbinder, K.; Marillonnet, S.; Stuttmann, J. Peripheral infrastructure vectors and an extended set of plant parts for the modular cloning system bioRxiv (2017) DOI: 10.1101/237768
Standardized DNA assembly strategies facilitate the generation of multigene constructs from collections of building blocks in plant synthetic biology. A common syntax for hierarchical DNA assembly following the Golden Gate principle employing Type IIs restriction endonucleases was recently developed, and underlies the Modular Cloning and GoldenBraid systems. In these systems, transcriptional units and/or multigene constructs are assembled from libraries of standardized building blocks, also referred to as phytobricks, in several hierarchical levels and by iterative Golden Gate reactions. This combinatorial assembly strategy meets the increasingly complex demands in biotechnology and bioengineering, and also represents a cost-efficient and versatile alternative to previous molecular cloning techniques. For Modular Cloning, a collection of commonly used Plant Parts was previously released together with the Modular Cloning toolkit itself, which largely facilitated the adoption of this cloning system in the research community. Here, a collection of approximately 80 additional phytobricks is provided. These phytobricks comprise e.g. modules for inducible expression systems, different promoters or epitope tags, which will increase the versatility of Modular Cloning-based DNA assemblies. Furthermore, first instances of a "peripheral infrastructure" around Modular Cloning are presented: While available toolkits are designed for the assembly of plant transformation constructs, vectors were created to also use coding sequence-containing phytobricks directly in yeast two hybrid interaction or bacterial infection assays. Additionally, DNA modules and assembly strategies for connecting Modular Cloning with Gateway Cloning are presented, which may serve as an interface between available resources and newly adopted hierarchical assembly strategies. The presented material will be provided as a toolkit to the plant research community and will further enhance the usefulness and versatility of Modular Cloning.
Preprints
Mitra, D.; Kumari, P.; Quegwer, J.; Klemm, S.; Moeller, B.; Poeschl, Y.; Pflug, P.; Stamm, G.; Abel, S.; Bürstenbinder, K. Microtubule-associated protein IQ67 DOMAIN5 regulates interdigitation of leaf pavement cells in Arabidopsis thaliana bioRxiv (2018) DOI: 10.1101/268466
Plant microtubules form a highly dynamic intracellular network with important roles for regulating cell division, cell proliferation and cell morphology. Its organization and dynamics are coordinated by various microtubule-associated proteins (MAPs) that integrate environmental and developmental stimuli to fine-tune and adjust cytoskeletal arrays. IQ67 DOMAIN (IQD) proteins recently emerged as a class of plant-specific MAPs with largely unknown functions. Here, using a reverse genetics approach, we characterize Arabidopsis IQD5 in terms of its expression domains, subcellular localization and biological functions. We show that IQD5 is expressed mostly in vegetative tissues, where it localizes to cortical microtubule arrays. Our phenotypic analysis of iqd5 loss-of-function lines reveals functions of IQD5 in pavement cell (PC) shape morphogenesis, as indicated by reduced interdigitation of neighboring cells in the leaf epidermis of iqd5 mutants. Histochemical analysis of cell wall composition further suggests reduced rates of cellulose deposition in anticlinal cell walls, which correlate with reduced asymmetric expansion. Lastly, we provide evidence for IQD5-dependent recruitment of calmodulin calcium sensors to cortical microtubule arrays. Our work thus identifies IQD5 as a novel player in PC shape regulation, and, for the first time, links calcium signaling to developmental processes that regulate multi-polar growth in PCs.
Preprints
Anwer, M. U.; Davis, A.; Davis, S. J.; Quint, M. Photoperiod sensing of the circadian clock is controlled by EARLY FLOWERING 3 and GIGANTEA bioRxiv (2018) DOI: 10.1101/321794
ELF3 and GI are two important components of the
Arabidopsis circadian clock. They are not only essential for the
oscillator function but are also pivotal in mediating light inputs to
the oscillator. Lack of either results in a defective oscillator causing
severely compromised output pathways, such as photoperiodic flowering
and hypocotyl elongation. Although single loss of function mutants of
ELF3 and GI have been well-studied, their genetic interaction remains
unclear. We generated an elf3 gi double mutant to study their genetic
relationship in clock-controlled growth and phase transition phenotypes.
We found that ELF3 and GI repress growth differentially during the
night and the day, respectively. Circadian clock assays revealed that
ELF3 and GI are essential Zeitnehmers that enable the oscillator to
synchronize the endogenous cellular mechanisms to external environmental
signals. In their absence, the circadian oscillator fails to
synchronize to the light-dark cycles even under diurnal conditions.
Consequently, clock-mediated photoperiod-responsive growth and
development is completely lost in plants lacking both genes, suggesting
that ELF3 and GI together convey photoperiod sensing to the central
oscillator. Since ELF3 and GI are conserved across flowering plants and
represent important breeding and domestication targets, our data
highlight the possibility of developing photoperiod-insensitive crops by
adjusting the allelic combination of these two key genes.
Preprints
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 receptor assemblies bioRxiv (2019) DOI: 10.1101/787770
Cullin RING-type E3 ubiquitin ligases
SCFTIR1/AFB1-5 and their ubiquitylation targets, AUX/IAAs, sense auxin
concentrations in the nucleus. TIR1 binds a surface-exposed degron in
AUX/IAAs promoting their ubiquitylation and rapid auxin-regulated
proteasomal degradation. Here, we resolved 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. The AUX/IAA PB1 interaction domain also
assists in non-native contacts, affecting AUX/IAA dynamic interaction
states. Our results 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 adopting
a multiplicity of functional states. We postulate IDRs in distinct
members of the AUX/IAA family to be an adaptive signature for protein
interaction and initiation region for proteasome recruitment.
Preprints
Ried, M. K.; Wild, R.; Zhu, J.; Broger, L.; Harmel, R. K.; Hothorn, L. A.; Fiedler, D.; Hothorn, M. Inositol pyrophosphates promote the interaction of
SPX domains with the coiled-coil motif of PHR transcription factors to
regulate plant phosphate homeostasis bioRxiv (2019) DOI: 10.1101/2019.12.13.875393
Phosphorus is an essential nutrient taken up by organisms in the form of
inorganic phosphate (Pi). Eukaryotes have evolved sophisticated Pi
sensing and signalling cascades, enabling them to maintain cellular Pi
concentrations. Pi homeostasis is regulated by inositol pyrophosphate
signalling molecules (PP-InsPs), which are sensed by SPX-domain
containing proteins. In plants, PP-InsP bound SPX receptors inactivate
Myb coiled-coil (MYB-CC) Pi starvation response transcription factors
(PHRs) by an unknown mechanism. Here we report that a InsP8 – SPX
complex targets the plant-unique CC domain of PHRs. Crystal structures
of the CC domain reveal an unusual four-stranded anti-parallel
arrangement. Interface mutations in the CC domain yield monomeric PHR1,
which is no longer able to bind DNA with high affinity. Mutation of
conserved basic residues located at the surface of the CC domain disrupt
interaction with the SPX receptor in vitro and in planta, resulting in
constitutive Pi starvation responses. Together, our findings suggest
that InsP8 regulates plant Pi homeostasis by controlling the oligomeric
state and hence the promoter binding capability of PHRs via their SPX
receptors.
Preprints
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.
Preprints
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 bioRxiv (2017) DOI: 10.1101/017285
Background: Global 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. Results: Here, we systematically analyze thermomorphogenesis throughout a complete life cycle in ten natural Arabidopsis thaliana accessions grown in four different temperatures ranging from 16 to 28 °C. We used Q 10 , 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. Conclusion: Genotype-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.
Preprints
Drost, H.-G.; Bellstädt, J.; Ó'Maoiléidigh, D. S.; Silva, A. T.; Gabel, A.; Weinholdt, C.; Ryan, P. T.; Dekkers, B. J. W.; Bentsink, L.; Hilhorst, H. W. M.; Ligterink, W.; Wellmer, F.; Grosse, I.; Quint, M. Post-embryonic hourglass patterns mark ontogenetic transitions in plant development bioRxiv (2015) DOI: 10.1101/035527
The historic developmental hourglass concept depicts the convergence of
animal embryos to a common form during the phylotypic period. Recently,
it has been shown that a transcriptomic hourglass is associated with
this morphological pattern, consistent with the idea of underlying
selective constraints due to intense molecular interactions during body
plan establishment. Although plants do not exhibit a morphological
hourglass during embryogenesis, a transcriptomic hourglass has
nevertheless been identified in the model plant Arabidopsis thaliana.
Here, we investigated whether plant hourglass patterns are also found
post-embryonically. We found that the two main phase changes during the
life cycle of Arabidopsis, from embryonic to vegetative and from
vegetative to reproductive development, are associated with
transcriptomic hourglass patterns. In contrast, flower development, a
process dominated by organ formation, is not. This suggests that plant
hourglass patterns are decoupled from organogenesis and body plan
establishment. Instead, they may reflect general transitions through
organizational checkpoints.