Biology subjects

Zubieta, C.

Publications and source records attributed to Zubieta, C..

14 recordsLinked to original sources

bioRxiv2026

SEPALLATA MADS transcription factors act as key regulators in fertilization efficiency, ovule outer integument growth and mucilage secretory cell differentiation in Arabidopsis

In angiosperms, ovule development requires the activity of the C, D and E classes of MADS genes, which encode key transcriptional regulators of reproductive development. The SEPALLATA (SEP) MADS transcription factors (MTFs), which belong to the E class, act as organizing hubs of MADS heterotetrameric complexes and play an essential role in the development of flower organs. However, the role of the SEP genes in ovule and seed development has been difficult to determine due to redundancy in the subclade, the lack of observable phenotypes in single and double sep1 sep2 mutants and the homeotic conversion of the carpel into sepal or leaf in higher order sep mutants. Here, we engineered a version of SEP3 (SEP3{Delta}M) that encodes a protein lacking the DNA-binding MADS-domain but retains the oligomerization domains needed for MADS protein heterotetramerization. In vitro experiments demonstrated the ability of SEP3{Delta}M to interact with the C and D classes of MTF, reducing the capability of such MADS complex to efficiently bind DNA. sep3{Delta}M plants showed a delay in flower opening and organ maturation and a reduced fertility. The ovules exhibited reduced outer integument growth, and the few seeds that developed showed impaired mucilage secretion upon imbibition. RNA-seq analysis of sep3{Delta}M demonstrated misregulation of genes involved in outer integument and seed coat development. Taken together, these data indicate the key role of SEP3-containing MADS complexes in proper ovule outer integument growth and seed coat development.

plant biology↗

bioRxiv2026

Structural Evolution of LEAFY Reveals DNA-Mediated Cooperativity and Dimerization Shifts at the Water-to-Land Transition

The evolution of transcription factor (TF) DNA-binding specificity is a major driver of gene regulatory innovation. Unlike most TFs, which diversify through gene duplication and neofunctionalization, the plant-specific LEAFY (LFY) TF evolved novel binding specificities without extensive duplication. Here, we combine experimental structural determination and biochemical assays to reveal how LFYs dimerization and DNA-binding preferences shifted during the water-to-land transition. We present crystal structures of the LFY DNA-binding domain (DBD) from the hornwort Nothoceros aenigmaticus and the alga Interfilum paradoxum bound to DNA, demonstrating two distinct dimerization mechanisms: one mediated by direct protein-protein interactions and another driven by DNA-mediated cooperativity. In the ancestral state, LFY likely bound DNA as a dimer through DNA-mediated cooperativity, with protein-protein dimerization emerging later, enforcing new DNA-binding preferences. Our findings support a revised evolutionary scenario for LFY, highlighting the dynamic interplay between protein-DNA and protein-protein interactions as key drivers of TF binding specificity. This work deepens our understanding of how structural adaptations in TFs underpin evolutionary transitions in gene regulation.

plant biology↗

bioRxiv2026

EARLY FLOWERING 3 (ELF3): a novel role in integrating environmental stimuli with root stem cell niche maintenance

Maintaining stem cell niche (SCN) homeostasis in the root apical meristem (RAM) is essential for proper root growth and thus for overall plant development. In Arabidopsis thaliana, a group of slowly dividing cells at the SCN center known as the quiescent center (QC) maintain the surrounding stem cells, including the distally located columella stem cells (CSCs) which give rise to the differentiated columella cells. Many actors, including the PLETHORA (PLT) family transcription factors, regulate the QC quiescence and CSC fate. However, little is known about the integration of external and/or internal cues into regulating SCN homeostasis. In this study, we report for the first time the interaction between PLT3 and the thermosensor and circadian clock related transcriptional regulator, EARLY FLOWERING 3 (ELF3), which are both expressed in the root SCN. We show that ELF3 localizes, similar to PLT3, to subcellular condensates and sustains the QC and CSC fate. We demonstrate that ELF3 forms condensates in vitro and in vivo, in the cytoplasm, as well as in the nucleus, where it then co-localizes with PLT3. We reveal that the interaction of ELF3 and PLT3 is mediated by their intrinsically disordered prion-like domains (PrDs). Furthermore, transient expression in human epithelial cells (HEp-2) cells and in Nicotiana benthamiana shows that PHYTOCHROME INTERACTING FACTORS 3 and 4 (PIF3/4) function as nuclear shuttles for ELF3, recruiting it to nuclear condensates, where it co-localizes with PLT3, PIF3, and PIF4. Accordingly, we propose a model where the co-localization and interactions of ELF3, PLT3, PIF3, and PIF4 represent a dynamic mechanism to integrate environmental signals into SCN maintenance and cell fate decisions.

plant biology↗

bioRxiv2024

Metal ion activation and DNA recognition by the Deinococcus radiodurans manganese sensor DR2539

The accumulation of manganese ions is crucial for scavenging reactive oxygen species (ROS) and protecting the proteome of Deinococcus radiodurans (Dr). However, metal homeostasis still needs to be tightly regulated to avoid toxicity. DR2539, a dimeric transcription regulator, plays a key role in Dr manganese homeostasis. Despite comprising three well-conserved domains: a DNA binding domain, a dimerization domain, and an ancillary domain, both the metal ion activation mechanism and the DNA recognition mechanism remain elusive. In this study, we present biophysical analyses and the structure of the dimerization and DNA binding domains of DR2539 in its holo form and in complex with the 21 bp pseudo-palindromic repeat of the dr1709 promotor region. These findings shed light into the activation and recognition mechanisms. The dimer presents eight manganese binding sites that induce structural conformations essential for DNA binding. The analysis of the protein-DNA interfaces elucidates the significance of Tyr59 and helix H3 sequence in the interaction with the DNA. Finally, the structure in solution as determined by small angle X-ray scattering experiments and supported by AlphaFold modelling provides a model illustrating the conformational changes induced upon metal binding.

biophysics↗

bioRxiv2023

The ALOG domain defines a new family of plant-specific transcription factors acting during Arabidopsis flower development

The ALOGs (Arabidopsis LIGHT-DEPENDENT SHORT HYPOCOTYLS 1 and Oryza G1) are Transcription Factors (TFs) from an evolutionarily conserved plant-specific family shown to play critical roles in meristem identity, inflorescence architecture and organ boundaries in diverse species from mosses to higher flowering plants. However, the DNA binding-specificity and molecular determinants of protein-DNA interactions of this family were uncharacterized. Using in vitro genome-wide studies, we identified the conserved DNA motif bound by ALOG proteins from the liverwort Marchantia polymorpha and the flowering plants Arabidopsis, tomato and rice. In order to determine the amino acids responsible for DNA-binding specificity, we solved the 2.1[A] structure of the ALOG DNA binding domain in complex with its cognate DNA. The ALOG DBD is an all-alpha helical domain with a structural zinc ribbon insertion and an N-terminal disordered NLS. The NLS sequence forms an integral part of the DNA binding domain and contributes to direct base read-out. To define the function of a group of redundant ALOG proteins in the model plant Arabidopsis thaliana, we generated a series of alog mutants and uncovered their participation in a gene regulatory network involving the other floral regulators LEAFY, BLADE-ON-PETIOLE and PUCHI, all active in defining boundary regions between reproductive meristems and repressing bracts development. Taken together, this work provides the biochemical and structural basis for DNA-binding specificity of an evolutionarily conserved TF family and reveals its role as a key player in defining organ boundaries in Arabidopsis. Significance StatementTranscription Factors (TFs) are key proteins that bind specific regions in the genome and regulate the expression of associated genes. Not all organisms possess the same set of TFs and some, like the ALOGs, are specific to the plant kingdom. These TFs have been shown to play important roles from mosses to flowering plants. However, it was not known what DNA motif they recognize and how they bind DNA. Here we identify this motif, we show it is widely conserved in evolution and we understand how this new type of DNA binding domain works at the structural level. In addition, we also show that several ALOG genes from Arabidopsis share a redundant function within the genetic network underlying correct floral meristem development.

plant biology↗

bioRxiv2023

SEPALLATA-driven MADS transcription factor tetramerization is required for inner whorl floral organ development

MADS genes encode transcription factors that act as master regulators of plant reproduction and flower development. The SEPALLATA (SEP) subfamily is required for the development of floral organs and plays roles in inflorescence architecture and development of the floral meristem. The SEPALLTAs act as organizers of MADS complexes, forming both heterodimers and heterotetramers in vitro. To date, the MADS complexes characterized in angiosperm floral organ development contain at least one SEPALLATA protein. Whether DNA-binding by SEPALLATA-containing dimeric MADS complexes are sufficient for launching floral organ identity programs, however, is not clear as only defects in floral meristem determinacy were observed in tetramerization impaired SEPALLATA mutants. Here, we used a combination of genome-wide binding studies, high resolution structural studies of the SEP3/AGAMOUS tetramerization domain, structure-based mutagenesis and complementation experiments in sep1 sep2 sep3 and sep1 sep2 sep3 ag-4 plants transformed with versions of SEP3 encoding tetramerization mutants. We demonstrate that while SEP3 heterodimers are able to bind DNA both in vitro and in vivo and recognize the majority of SEP3 wild type binding sites genome-wide, tetramerization is not only required for floral meristem determinacy, but also absolutely required for floral organ identity in the second, third and fourth whorls.

plant biology↗

bioRxiv2023

Phase separation and molecular ordering of the prion-like domain of the thermosensory protein EARLY FLOWERING 3

Liquid-liquid phase separation (LLPS) is an important mechanism enabling the dynamic compartmentalisation of macromolecules, including complex polymers such as proteins and nucleic acids, and occurs as a function of the physicochemical environment. In the model plant, Arabidopsis thaliana, LLPS by the protein EARLY FLOWERING3 (ELF3) occurs in a temperature sensitive manner and controls thermoresponsive growth. ELF3 contains a largely unstructured prion-like domain (PrLD) that acts as a driver of LLPS in vivo and in vitro. The PrLD contains a poly-glutamine (polyQ) tract, whose length varies across natural Arabidopsis accessions. Here, we use a combination of biochemical, biophysical and structural techniques to investigate the dilute and condensed phases of the ELF3 PrLD with varying polyQ lengths. We demonstrate that the dilute phase of the ELF3 PrLD forms a monodisperse higher order oligomer that does not depend on the presence of the polyQ sequence. This species undergoes LLPS in a pH and temperature-sensitive manner and the polyQ region of the protein tunes the initial stages of phase separation. The liquid phase rapidly undergoes aging and forms a hydrogel as shown by fluorescence and atomic force microscopies. Furthermore, we demonstrate that the hydrogel assumes a semi-ordered structure as determined by small angle X-ray scattering, electron microscopy and X-ray diffraction. These experiments demonstrate a rich structural landscape for a PrLD protein and provide a framework to describe the structural and biophysical properties of biomolecular condensates.

biophysics↗

bioRxiv2022

The F-box cofactor UFO redirects the LEAFY floral regulator to novel cis-elements

In angiosperms, flower patterning requires the localized expression of the APETALA3 (AP3) floral homeotic gene involved in petal and stamen development. AP3 is synergistically induced by the master transcription factor (TF) LEAFY (LFY) and the F-box protein UNUSUAL FLORAL ORGANS (UFO), but the molecular mechanism underlying this synergy has remained unknown. Here we show that the connection to ubiquitination pathways suggested by the F-box domain of UFO is mostly dispensable for its function and that UFO instead acts by forming a transcriptional complex with LFY and binds to newly discovered regulatory elements. Cryo-electron microscopy explains how a LFY-UFO complex forms on these novel DNA sites due to direct interaction of UFO with LFY and DNA. Finally, we show that this complex has a deep evolutionary origin, largely predating flowering plants. This work reveals a novel mechanism of an F-box protein in directly modulating the DNA-binding specificity of a master TF.

plant biology↗

bioRxiv2021

Cell layer-specific expression of the B-class MADS-box gene PhDEF drives petal tube or limb development in petunia flowers

Floral homeotic MADS-box transcription factors ensure the correct morphogenesis of floral organs, which are organized in different cell layers deriving from the meristematic L1, L2 and L3 layers. How cells from these distinct layers acquire their respective identity and coordinate their growth to ensure normal floral organ morphogenesis is unresolved. Here, we study petunia petals that form a limb and tube through congenital fusion, a complex morphology that coevolved with pollinators. We have identified petunia mutants expressing the B-class MADS-box gene PhDEF in the epidermis or in the mesophyll of the petal only, called wico and star respectively. Strikingly, wico flowers form a strongly reduced tube while their limbs are almost normal, while star flowers form a normal tube but very reduced and unpigmented limbs, showing that petunia petal morphogenesis is highly modular. Comparative transcriptome analysis of star, wico and wild-type petals revealed a strong down-regulation of the anthocyanin production pathway in star petals including its major regulator ANTHOCYANIN2 (AN2). We found that PhDEF directly binds to AN2 regulatory sequence in vitro by gel shift assay, and in vivo by chromatin immunoprecipitation, suggesting that PhDEF directly activates the petal pigmentation pathway by activating AN2. Altogether, we show that cell-layer specific homeotic activity in petunia petals differently impacts tube and limb development, revealing the relative importance of the different cell layers in the modular architecture of petunia petals.

plant biology↗

bioRxiv2021

The Intervening Domain Is Required For DNA-binding and Functional Identity of Plant MADS Transcription Factors

The MADS transcription factors (TF) are an ancient protein family with a high degree of sequence identity that bind almost identical DNA sequences across all eukaryotic kingdoms of life, yet fulfill dramatically different physiological roles. In plants, the family is divided into two main lineages, type I and II, based on sequence conservation of the DNA-binding MADS-box domain (M domain) with yeast and animal M domains. Here, we demonstrate that DNA binding in both lineages absolutely requires a short amino acid sequence C-terminal to the M domain called the Intervening domain (I domain) in type II MADS. Structural elucidation of the MI domains from the floral regulator, SEPALLATA3 (SEP3), shows a highly conserved MADS-box fold with the I domain forming an alpha helix and acting to stabilize the M domain. Based on secondary structure prediction, sequences fulfilling the same function as the SEP3 I domain can be found in both lineages of plant MADS TFs, suggesting the I domain is a conserved and required part of the DNA-binding domain. Using the floral organ identity MADS TFs, SEP3, APETALA1 (AP1) and AGAMOUS (AG), domain swapping demonstrate that the I domain alters DNA-binding specificity based on seq-DAP-seq experiments. Yeast 2-hybrid experiments further revealed the role of the I domain in dimerization specificity. Surprisingly, introducing AG carrying the I domain of AP1 in the Arabidopsis ap1 mutant, resulted in a high degree of complementation and restoration of first and second whorl organs. Taken together, these data demonstrate that the I domain acts both as an integral part of the DNA-binding domain and strongly contributes to the functional identity of the MADS TF.

plant biology↗

bioRxiv2021

Unraveling the role of MADS transcription factor complexes in apple tree dormancy using sequential DAP-seq

BackgroundThe effect of global warming on dormancy and flowering patterns of crop trees threatens world-wide fruit production and food security. In Rosaceous tree species, it is believed that a group of genes encoding MADS transcription factors (TFs) controls temperature-mediated dormancy cycle. These genes are similar to SHORT VEGETATIVE PHASE (SVP) from Arabidopsis thaliana and referred as DORMANCY-ASSOCIATED MADS-BOX (DAM) genes. ResultsBy making use of apple tree (Malus x domestica) as a model for Rosaceous species, we have investigated the function of MADS TFs during the dormancy cycle. We found that MdDAM and other dormancy related MADS TFs form multimeric complexes with MdSVPa, and that MdSVPa is essential for the transcriptional complex activity. Then, for the first time in non-model plant species, we performed sequential DNA Affinity Purification sequencing (seq-DAP-seq) to define the genome-wide binding sites of these MADS TF complexes. Target genes associated with the binding sites were identified by combining seq-DAP-seq data with transcriptomics datasets obtained by the inducible glucocorticoid receptor expression system, and reanalyzing preexisting data related to dormancy cycle in apple trees. ConclusionWe have determined a gene regulatory network formed by MdSVPa-containing complexes that regulate the dormancy cycle in apple trees in response to environmental cues. Key genes identified with our genomic approach and the elucidated regulatory relationships provide leads for breeding fruit trees better adapted to changing climate conditions. Moreover, we provide novel molecular evidence on the evolutionary functional segregation between DAM and SVP proteins in the Rosaceae family.

plant biology↗

bioRxiv2021

The LEAFY floral regulator displays pioneer transcription factor properties

Pioneer transcription factors (TFs) are a special category of TFs with the capacity to bind to closed chromatin regions in which DNA is wrapped around histones and often highly methylated. Subsequently, they are able to modify the chromatin state to initiate gene expression. In plants, LEAFY (LFY) is a master floral regulator and has been suggested to act as a pioneer TF in Arabidopsis. Here, we demonstrate that LFY is able to bind both methylated and non-methylated DNA using a combination of in vitro genome-wide binding experiments and structural modeling. Comparisons between regions bound by LFY in vivo and chromatin accessibility data suggest that LFY binds a subset of regions occupied by nucleosomes. We confirm that LFY is able to bind nucleosomal DNA in vitro using reconstituted nucleosomes. Finally, we show that constitutive LFY expression in seedling tissues is sufficient to induce chromatin accessibility in the LFY direct target genes, APETALA1 and AGAMOUS. Taken together, our study suggests that LFY possesses key pioneer TF features that contribute to launch the floral gene expression program.

plant biology↗

bioRxiv2019

Molecular mechanisms of Evening Complex activity in Arabidopsis

The Evening Complex (EC), composed of the DNA-binding protein LUX ARRHYTHMO (LUX) and two additional proteins, EARLY FLOWERING 3 (ELF3) and ELF4, is a transcriptional repressor complex and a core component of the plant circadian clock. In addition to maintaining oscillations in clock gene expression, the EC also participates in temperature and light entrainment and regulates important clock output genes such as PHYTOCHROME INTERACTING FACTOR 4 (PIF4), a key transcription factor involved in temperature dependent plant growth. These properties make the EC an attractive target for altering plant development through targeted mutations to the complex. However, the molecular basis for EC function was not known. Here we show that binding of the EC requires all three proteins and that ELF3 decreases the ability of LUX to bind DNA whereas the presence of ELF4 restores interaction with DNA. To be able to manipulate this complex, we solved the structure of the DNA-binding domain of LUX bound to DNA. Using structure-based design, a LUX variant was constructed that showed decreased in vitro binding affinity but retained specificity for its cognate sequences. This designed LUX allele modulates hypocotyl elongation and flowering. These results demonstrate that modifying the DNA-binding affinity of LUX can be used to titrate the repressive activity of the entire EC, tuning growth and development in a predictable manner.\n\nSignificance StatementCircadian gene expression oscillates over a 24 hr. period and regulates many genes critical for growth and development. In plants, the Evening Complex (EC), a three-protein repressive complex made up of LUX ARRYTHMO, EARLY FLOWERING 3 and EARLY FLOWERING 4, acts as a key component of the circadian clock and is a regulator of thermomorphogenic growth. However, the molecular mechanisms of complex formation and DNA-binding have not been identified. Here we determine the roles of each protein in the complex and present the structure of the LUX DNA-binding domain in complex with DNA. Based on these data, we used structure-based protein engineering to produce a version of the EC with altered in vitro and in vivo activity. These results demonstrate that the EC can be modified to alter plant growth and development at different temperatures in a predictable manner.

plant biology↗

bioRxiv2018

Cryo-EM structure of adenovirus type 3 fibre with desmoglein 2 shows a novel mode of receptor engagement

Attachment of adenovirus (HAd) to host cell is a critical step of infection. This work reports the cryo-electron microscopy (cryo-EM) structure of a non-symmetrical complex smaller than 100kDa formed by the trimeric human adenovirus of type 3 fibre knob (HAd3K) and human desmoglein 2 (DSG2). The structure reveals a unique stoichiometry, shedding light to new adenovirus infection strategies and providing new insights for adenoviral vector development.

microbiology↗