Biology subjects

Alonso, J. M.

Publications and source records attributed to Alonso, J. M..

14 recordsLinked to original sources

bioRxiv2026

An Integrated Spatially Resolved Mechanistic Model of Hierarchical Auxin-Cytokinin-Ethylene Crosstalk Underlying Root Growth Inhibition in Arabidopsis

Decoding how plants integrate multiple hormone signals to coordinate growth requires tools capable of resolving pathway interactions at cellular resolution in living tissue. Here we present ACE (Auxin-Cytokinin-Ethylene) and ACE2, proof-of-concept single-locus reporters to simultaneously capture activity of multiple hormones. Deploying ACE alongside well-established reporters, exogenous hormone treatments, and reverse-genetic perturbations of hormone biosynthesis, signaling, and transport in three-day-old etiolated Arabidopsis seedlings, we dissect the spatiotemporal hierarchy governing primary root elongation and root apical meristem (RAM) size. We demonstrate that both ethylene- and cytokinin-triggered root growth inhibition involve a boost of TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 (TAA1)-mediated auxin biosynthesis and AUXIN RESISTANT1 (AUX1)-dependent auxin redistribution. Two spatially distinct auxin responses underlie the respective root growth effects: ethylene expands TAA1-dependent auxin biosynthesis from the root vasculature into the epidermis and promotes AUX1-mediated auxin import into the transition and elongation zones to inhibit cell elongation, while cytokinin confines ethylene-dependent TAA1-boosted activity to the vasculature and drives auxin accumulation in lateral root cap cells to reduce RAM size. Together, these data establish a reciprocal regulatory loop between these hormones, positioning ethylene as a convergence node in auxin-cytokinin crosstalk, and cytokinin as a modulator of the ethylene-auxin interaction. Critically, the changes in cross-activated reporter patterns described for different genetic backgrounds, alongside quantitative assessment of hormone-specific inhibition of the mutants growth, were consistent with the multi-hormone network established over two decades of research, and added cell-type-resolved spatial detail and a proposed hierarchy for the etiolated seedling root. Finally, a second-generation reporter, ACE2, overcomes key technical limitations of ACE, expanding the platforms capacity toward a higher-order multi-hormone monitoring system. These resources expand the Arabidopsis genetic toolkit and provide a generalizable framework instrumental for dissecting multi-hormone signaling hierarchies at the cellular level.

plant biology↗

bioRxiv2026

Spatiotemporal dynamics of ethylene biosynthesis shape infection and nodule initiation in Medicago truncatula

Ethylene is a well-established negative regulator of nodulation, yet how ethylene biosynthesis and perception are spatially coordinated during early symbiotic signalling remains unresolved. Here, we investigate the dynamics of ethylene responses in Medicago truncatula using transcriptomics, promoter-reporter analyses, loss-of-function approaches and a synthetic reporter. We show that the activity of the ethylene-responsive EBSn reporter shifts from inner root tissues under non-symbiotic conditions to the outer cortex and epidermis following rhizobial inoculation, revealing a spatial reprogramming of ethylene signalling. Among the eight Medicago 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE (ACS) genes, upon rhizobia application MtACS3 is induced in outer root cell layers, while MtACS10 is repressed in the inner cortex and pericycle, mirroring the shift in ethylene perception. Functional analysis demonstrates that MtACS10 restricts nodule initiation, whereas MtACS3 modulates infection thread number, prevents nodule clustering, and contributes to radial positioning of nodule primordia. Rhizobial induced ectopic ACS expression in the root interior counteracts MtACS10 repression and blocks nodulation, highlighting the requirement for spatially confined downregulation of ethylene biosynthesis. Together, these findings establish a framework in which localized shift in ethylene biosynthesis, mediated by distinct Medicago ACS genes, balances infection and organogenesis while co-defining the spatial limits of the root susceptible zone.

plant biology↗

bioRxiv2025

Generative Phenomenology of form perception: Perceptograms and cortical models for amblyopic form distortions

What we see is generated by our cortical neurons processing sensory input. Amblyopia provides a perfect case for studying the neural generation of percepts because abnormal neural development of cortex leads to many amblyopes seeing more complex phantom forms than the viewed stimulus through their amblyopic eye (AE) but not through the fellow eye (FE). Using computer guided dichoptic displays, we showed that 92.6% of phantom percepts generated by single gratings shown to AE were matched exactly by plaids (sums of two gratings) shown to FE. A formal equation between the cortical signals generated by the gratings seen through AE and the signals generated by their matched patterns seen through FE, provided the derivation of AE cortical receptive fields as linear transforms of FE receptive fields. The transformed AE cortical model accurately generated the phantoms, was validated via reverse-correlation, and explained deficits in form detection and spatial localization. This study elucidates the close link between percepts and neuronal receptive fields. TEASERChanges in cortical neurons receptive fields can lead to amblyopes seeing phantom features that are not present in viewed images.

neuroscience↗

bioRxiv2025

The CYP71A, NIT, AMI, and IAMH gene families are dispensable for indole-3-acetaldoxime-mediated auxin biosynthesis in Arabidopsis

Indole-3-acetic acid (IAA) is a crucial auxin governing plant development and environmental responses. While the indole-3-pyruvic acid (IPyA) pathway is the predominant IAA biosynthesis route, other pathways, like the indole-3-acetaldoxime (IAOx) pathway, have been proposed. The IAOx pathway has garnered attention due to its supposed activation in auxin-overproducing mutants (e.g., sur1, sur2, ugt74b1) and the auxin-like responses triggered by exogenous application of its proposed intermediates: IAOx, indole-3-acetonitrile (IAN), and indole-3-acetamide (IAM). However, despite supporting evidence for individual steps, conclusive physiological relevance of the IAOx pathway remains unproven. Using a comprehensive genetic approach combined with metabolic and phenotypic profiling, we demonstrate that mutating gene families proposed to function in the IAOx pathway does not result in prominent auxin-deficient phenotypes, nor are these genes required for high-auxin production in the sur2 mutant. Our findings also challenge the previously postulated linear IAOx pathway. While exogenously provided IAOx, IAN, and IAM can be converted to IAA in vivo, they do not act as precursors for each other. Finally, our findings question the physiological relevance of IAM and IAN as IAA precursors in plants and suggest the existence of a yet uncharacterized auxin biosynthetic route, likely involving IAOx as an intermediate, for the production of IAA in the sur2 mutant. Future identification of the metabolic steps and the corresponding genes in this new pathway may uncover the previously unknown way of synthesizing IAA in plants.

genetics↗

bioRxiv2025

EBSn, a robust synthetic reporter for monitoring ethylene responses in plants

Ethylene is a gaseous plant hormone that controls a wide array of physiologically relevant processes, including plant responses to biotic and abiotic stress, and induces ripening in climacteric fruits. To monitor ethylene in plants, analytical methods, phenotypic assays, gene expression analysis, and transcriptional or translational reporters are typically employed. In the model plant Arabidopsis, two ethylene-sensitive synthetic transcriptional reporters have been described, 5xEBS:GUS and 10x2EBS-S10:GUS. These reporters harbor a different type, arrangement, and number of homotypic cis-elements in their promoters and thus may recruit the ethylene master regulator EIN3 in the context of alternative transcriptional complexes. Accordingly, the patterns of GUS activity in these transgenic lines differ and neither of them encompasses all plant tissues even in the presence of saturating levels of exogenous ethylene. Herein, we set out to develop and test a more sensitive version of the ethylene-inducible promoter that we refer to as EBSnew (abbreviated as EBSn). EBSn leverages a tandem of ten non-identical, natural copies of a novel, dual, everted, 11bp-long EIN3-binding site, 2EBS(-1). We show that in Arabidopsis, EBSn outperforms its predecessors in terms of its ethylene sensitivity, having the capacity to monitor endogenous levels of ethylene and displaying more ubiquitous expression in response to the exogenous hormone. We demonstrate that the EBSn promoter is also functional in tomato, opening new avenues to manipulating ethylene-regulated processes, such as ripening and senescence, in crops.

plant biology↗

bioRxiv2025

Arabidopsis lines with modified ascorbate concentrations reveal a link between ascorbate and auxin biosynthesis

Ascorbate is the most abundant water-soluble antioxidant in plants, and it is an essential molecule for normal plant development. It is present in all green plants, with very different concentrations in different plant species. While ascorbate accumulation is a trait of nutritional, and therefore, agronomical interest, the impact of different concentrations over cellular homeostasis remains elusive. In order to shed light over this question, we leveraged Arabidopsis lines with very low ascorbate (vtc2 mutant with 20% of WT ascorbate levels), and low ascorbate concentration (vtc4 mutant with 65% of WT levels), and we generated a line that accumulates 165% of WT levels (vtc2/OE-VTC2). An 80% reduction of ascorbate increased the expression of genes implicated in defense against pathogens, but repressed genes associated with abiotic stress responses. Unexpectedly, lines with increased (165% of WT) and decreased (65% of WT) ascorbate levels shared 85% of induced transcription factors and the GO terms associated with their transcriptional programs. Among the group of genes whose expression is positively correlated with ascorbate content, we identified TAA1/WEI8, a gene encoding a tryptophan aminotransferase that catalyzes the first step of auxin biosynthesis. Using a combination of genetic and pharmacological approaches in fluorescent and histochemical reporter lines for auxin biosynthesis and signaling activity, we revealed that TAA1- and TAA1-RELATED2 (TAR2)-mediated auxin biosynthesis is necessary for plants to cope with increased ascorbate concentration in a light-dependent manner, revealing a new layer of complexity in the regulatory landscape of redox homeostasis.

plant biology↗

bioRxiv2025

DASH: A versatile and high-capacity gene stacking system for plant synthetic biology

DNA assembly systems based on the Golden Gate method are popular in synthetic biology but have several limitations: small insert size, incompatibility with other cloning platforms, DNA domestication requirement, generation of fusion scars, and lack of post-assembly modification. To address these obstacles, we present the DASH assembly toolset, which combines features of Golden Gate-based cloning, recombineering, and site-specific recombinase systems. We developed (1) a set of donor vectors based on the GoldenBraid platform, (2) an acceptor vector derived from the plant transformation-competent artificial chromosome (TAC) vector, pYLTAC17, and (3) a re-engineered recombineering-ready E. coli strain, CZ105, based on SW105. The initial assembly steps are carried out using the donor vectors following standard GoldenBraid assembly procedures. Importantly, existing parts and transcriptional units created using compatible Golden Gate-based systems can be transferred to the DASH donor vectors using standard single-tube restriction/ligation reactions. The cargo DNA from a DASH donor vector is then efficiently transferred in vivo in E. coli into the acceptor vector by the sequential action of a rhamnose-inducible phage-derived PhiC31 integrase and arabinose-inducible yeast-derived Flippase (FLP) recombinase using CZ105. Furthermore, recombineering-based post-assembly modification, including the removal of undesirable scars, is greatly simplified. To demonstrate the utility of the DASH system, a 116 kb DNA construct harboring a 97 kb cargo consisting of 35 transcriptional units was generated. One of the CDSs in the final assembly was replaced through recombineering, and the in planta functionality of the entire construct was tested in both transient and stable transformants.

synthetic biology↗

bioRxiv2024

Targeted DNA ADP-ribosylation triggers templated repair in bacteria and base mutagenesis in eukaryotes

Base editors create precise genomic edits by directing nucleobase deamination or removal without inducing double-stranded DNA breaks. However, a vast chemical space of other DNA modifications remains to be explored for genome editing. Here, we harness the bacterial anti-phage toxin DarT2 to append ADP-ribosyl moieties to DNA, unlocking distinct editing outcomes in bacteria versus eukaryotes. Fusing an attenuated DarT2 to a Cas9 nickase, we program site-specific ADP-ribosylation of thymines within a target DNA sequence. In tested bacteria, targeting drives efficient homologous recombination in tested bacteria, offering flexible and scar-free genome editing without base replacement nor counterselection. In tested eukaryotes including yeast, plants and human cells, targeting drives substitution of the modified thymine to adenine or a mixture of adenine and cytosine with limited insertions or deletions, offering edits inaccessible to current base editors. Altogether, our approach, called append editing, leverages the addition of a chemical moiety to DNA to expand current modalities for precision gene editing.

synthetic biology↗

bioRxiv2024

Microtubule plus-end dynamics are tightly coupled to the turnover of the MOR1 polymerase

MOR1/XMAP215/Dis1/Stu2 family proteins function as polymerases that promote microtubule assembly through tubulin-interacting N-terminal tumor-overexpressed gene (TOG) domains. It remains unclear how the polymerase activity of these proteins is coordinated with their movement along the growing microtubule lattice. Previously, it was found that a L174F mutation in the N-terminal TOG domain of the 217 kDa Arabidopsis thaliana MICROTUBULE ORGANIZATION 1 (MOR1) protein impaired microtubule growth and shrinkage rates in a temperature-dependent manner. In this study, we used a homologous recombination strategy to generate stable transgenic lines expressing full-length MOR1 and mutant mor1-1L174F proteins tagged with the yellow fluorescent protein YPet. When expressed in mor1 mutant backgrounds, MOR1-3xYPet showed strong association with both the growing and shrinking microtubule plus ends, consistent with its involvement in both polymerization and depolymerization. By contrast, when expressed in a wild-type background, MOR1-3xYPet was redistributed along the microtubule lattice, microtubule growth rates were reduced, and plant growth was stunted. By quantifying fluorescence recovery after photobleaching, we confirmed that MOR1 has greater affinity for the plus end than the lattice. Exploiting the FRAP technique further, we identified a positive and linear correlation between microtubule polymerization rates and MOR1 turnover at the microtubule plus end. When microtubule growth rates were reduced in the mor1-1-YPet line by shifting the temperature from 21 to 30{degrees}C and for MOR1-3xYPet under taxol treatments, MOR1s turnover was delayed. In contrast, microtubule growth rates and MOR1 turnover were hastened at 30{degrees}C or after CRISPR/Cas9-mediated knock out of the CLASP protein. These findings support a new model whereby MOR1 has finite processivity on the microtubule, catalyzing a limited fixed number of tubulin additions before dissociation. HIGHLIGHTSMOR1 has high affinity for both growing and shrinking microtubule plus ends, consistent with a function in both polymerization and depolymerization events. MOR1 has greater affinity for microtubule plus ends than the lattice, and lattice binding associated with overexpression is detrimental to plant growth. MOR1 transiently associates with the microtubule plus end, the duration of which is tightly coupled with its polymerase activity.

cell biology↗

bioRxiv2024

Optical blur affects differently ON and OFF visual pathways

The human eye has a crystalline lens that focuses retinal images at the point of fixation. Outside this fixation region, images are distorted by optical blur, which increases light scatter and reduces the spatial resolution and contrast processed by neuronal pathways. The spectacle lenses that humans use for optical correction also minify or magnify the images, affecting neuronal surround suppression in visual processing. Because light and dark stimuli are processed with ON and OFF pathways that have different spatial resolution, contrast sensitivity and surround suppression, optical blur and image magnification should affect differently the two pathways and the perception of lights and darks. Our results provide support for this prediction in cats and humans. We demonstrate that optical blur expands ON receptive fields while shrinking OFF receptive fields, as expected from the expansion of light stimuli and shrinkage of dark stimuli with light scatter. Spectacle-induced image magnification also shrinks OFF more than ON receptive fields, as expected from the stronger surround suppression in OFF than ON pathways. Optical blur also decreases the population response of OFF more than ON pathways, consistent with the different effects of light scatter on dark and light stimuli and the ON-OFF pathway differences in contrast sensitivity. Based on these results, we conclude that optical blur and image magnification reduce the receptive field sizes and cortical responses of OFF more than ON pathways, making the ON-OFF response balance a reliable signal to optimize the size and quality of the retinal image. HIGHLIGHTSO_LIOptical blur affects ON and OFF pathways differently. C_LIO_LIBlur expands ON but shrinks OFF receptive fields, weakening OFF more than ON signals. C_LIO_LIMagnification from positive blur reduces OFF more than ON signal strength and latency. C_LIO_LION-OFF response balance can signal retinal image quality and guide eye growth. C_LI IN BRIEFPons et al. demonstrate that optical blur affects differently the receptive field properties and responses from ON and OFF visual pathways, revealing a new relation between ON/OFF pathway balance and blurred visual perception. The results highlight a new role of ON and OFF pathways in optimizing retinal image quality that can have implications for the regulation of eye growth. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/618707v1_ufig1.gif" ALT="Figure 1"> View larger version (96K): org.highwire.dtl.DTLVardef@9161eorg.highwire.dtl.DTLVardef@8b11beorg.highwire.dtl.DTLVardef@39e5bdorg.highwire.dtl.DTLVardef@119fd42_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

Development of modular geminivirus-based vectors for high cargo expression and gene targeting in plants

Viral vectors can be useful tools for expressing recombinant proteins as well as delivering gene-editing machinery. Despite their utility, the development and subsequent optimization of these tools is often a difficult and tedious process. Thus, although considerable work has been done to create useful viral vectors for gene editing and protein expression, there is a lack of understanding of how best to design these vectors for specific applications. For instance, it is often unclear whether the inclusion of heterologous promoter sequences or different viral components will improve cargo expression or replicon accumulation. To address some of these hurdles, we designed a GoldenBraid (GB)-compatible viral vector system based on the geminivirus - Beet curly top virus (BCTV). This system allows for simple, modular cloning of a variety of reporter constructs. Making use of this modular cloning strategy, we compared a variety of alternative viral vector architectures. Interestingly, native BCTV promoters outperformed the constitutive 35S promoter, while the removal of the BCTV virion-sense genes promoted reporter expression. Intriguingly, these modifications had no effect on total replicon accumulation. These results show the utility of the new modular BCTV-based viral vectors for protein expression and gene targeting applications, as well as uncover design principles that may inform future geminivirus-based viral vector architectures. We anticipate that the availability of this new modular system will spark the broad adoption of replicon-based strategies in protein expression and gene editing experiments in plants.

synthetic biology↗

bioRxiv2020

A G protein-coupled receptor-like module regulates Cellulose Synthase secretion from the endomembrane system in Arabidopsis

Cellulose synthesis is essential for plant morphology, water transport and defense, and provides raw material for biomaterials and fuels. Cellulose is produced at the plasma membrane by Cellulose Synthase (CESA) protein complexes (CSCs). CSCs are assembled in the endomembrane system and then trafficked from the Golgi apparatus and trans-Golgi Network (TGN) to the plasma membrane. Since CESA enzymes are only active in the plasma membrane, control of CSC secretion is a critical step in the regulation of cellulose synthesis. However, the regulatory framework for CSC secretion is not clarified. In this study, we identify members of a family of seven transmembrane domain-containing proteins (7TMs) as important for cellulose production during cell wall integrity stress. 7TM proteins are often associated with guanine nucleotide-binding protein (G) protein signalling and mutants in several of the canonical G protein complex components phenocopied the 7tm mutant plants. Unexpectedly, the 7TM proteins localized to the Golgi apparatus/TGN where they interacted with the G protein complex. Here, the 7TMs and G proteins regulated CESA trafficking, but did not affect general protein secretion. Furthermore, during cell wall stress, 7TMs localization was biased towards small CESA-containing vesicles, specifically associated with CSC trafficking. Our results thus outline how a G protein-coupled module regulates CESA trafficking and reveal that defects in this process lead to exacerbated responses upon exposure to cell wall integrity stress.

plant biology↗

bioRxiv2020

Structure-function analysis of interallelic complementation in ROOTY transheterozygotes

Auxin is a crucial plant growth regulator. Forward genetic screens for auxin-related mutants have led to the identification of key genes involved in auxin biosynthesis, transport, and signaling. Loss-of-function mutations in the genes involved in indole glucosinolate biosynthesis, a metabolically-related route that produces defense compounds from indolic precursors shared with the auxin pathway, result in auxin overproduction. We identified an allelic series of fertile, hypomorphic mutants for an essential indole glucosinolate route gene ROOTY (RTY) that show a range of high-auxin defects. Genetic characterization of these lines uncovered phenotypic suppression by cyp79b2 b3, wei2, and wei7 mutants and revealed the phenomenon of interallelic complementation in several RTY transheterozygotes. Structural modeling of RTY shed light on the structure-to-function relations in the RTY homo- and heterodimers and unveiled the likely structural basis of interallelic complementation. This work underscores the importance of employing true null mutants in genetic complementation studies.

genetics↗

bioRxiv2019

An improved plant toolset for high-throughput recombineering

Gene functional studies often rely on the expression of a gene of interest as transcriptional and translational fusions with specialized tags. Ideally, this is done in the native chromosomal contexts to avoid potential misexpression artifacts. Although recent improvements in genome editing make it possible to directly modify the target genes in their native chromosomal location, classical transgenesis is still the preferred experimental approach chosen in most gene tagging studies because of its time efficiency and accessibility. We have developed a recombineering-based tagging system that brings together the convenience of the classical transgenic approaches and the high degree of confidence in the obtained results provided by the direct chromosomal tagging achievable by genome editing strategies. These simple and customizable recombineering toolsets and protocols allow for high-throughput generation of a variety of genetic modifications. In addition, a highly efficient recombinase-mediated cassette exchange system has been developed to facilitate the transfer of the desired sequences from a BAC clone to a transformation-compatible binary vector, expanding the use of the recombineering approaches beyond Arabidopsis. The utility of this system is demonstrated by the generation of over 250 whole-gene translational fusions and 123 Arabidopsis transgenic lines corresponding to 62 auxin-related genes, and the characterization of the translational reporter expression patterns for 14 auxin biosynthesis genes.

plant biology↗