Biology subjects

Botella, M. A.

Publications and source records attributed to Botella, M. A..

14 recordsLinked to original sources

bioRxiv2026

Synaptotagmin-mediated plasma membrane homeostasis is required to recruit the endocytic machinery during cold stress

Endocytosis depends on plasma membrane (PM) lipid composition, yet how plants sustain endocytic machinery recruitment during stress remains unclear. We investigated whether synaptotagmins SYT1 and SYT3 function in membrane homeostasis and endocytosis in Arabidopsis thaliana. In this work, we revealed that cold stress reversibly blocked FM4-64 internalization in syt1syt3 double mutants by displacing peripheral and core TPC subunits from the PM. Loss of SYT1 reduced brassinosteroid receptor internalization during cold stress, while double mutants showed enhanced brassinosteroid signaling. SYTs and VAP27 genes showed a condition-dependent genetic interaction in endocytosis: the syt1/vap27-1/vap27-3 triple mutant resembles vap27-1/vap27-3 under control conditions and syt1 during cold stress, suggesting overlapping contributions to endocytic regulation. Using molecular dynamics simulations of protein-membrane interactions, phosphoinositide depletion combined with increased diacylglycerol weakened TPC core membrane association. Our findings identify SYT1 and SYT3 as essential components for sustaining endocytosis during abiotic stress and support a model in which lipid homeostasis at endoplasmic reticulum-PM contact sites preserves the membrane composition required for TPC-dependent endocytosis.

plant biology↗

bioRxiv2026

Integration of SYT1 Interactomics and Dual-Localization Proteomics Links ER-PM Contacts to Lignin Deposition

Membrane contact sites (MCSs) are evolutionarily conserved intracellular nanodomains that physically bridge opposing lipid bilayers to facilitate non-vesicular communication and maintain cellular homeostasis. In plants, endoplasmic reticulum-plasma membrane (ER-PM) contact sites play fundamental roles in environmental adaptation, and are populated by specialized proteins which act as tethers such as Synaptotagmin 1 (SYT1). However, a comprehensive view of the molecular machinery governing processes at these junctions is still needed. In this work, we integrate affinity purification mass spectrometry, TurboID proximity labeling, and a dual-localization reanalysis of HyperLOPIT spatial proteomics to functionally map the protein interaction landscape of the ER-PM contact sites protein SYT1. Beyond recovering established ER-PM MCS functions, our analysis identified uncharacterized proteins as bona fide resident components of these junctions, and revealed that these nanodomains act as docking platforms that anchor the monolignol biosynthetic complex. By spatially organizing Membrane Steroid Binding Proteins and cytochrome P450 enzymes, our findings support a model where SYT1-mediated anchoring of this metabolon to ER-PM contact sites optimizes monolignol export required for stress-induced lignification. Ultimately, this proteomic framework expands the functional repertoire of ER-PM contact sites, opening new avenues to uncover hidden roles of MCSs across diverse eukaryotic systems.

molecular biology↗

bioRxiv2026

Molecular mechanisms of E-Syt-mediated stress resistance

Membrane contact sites (MCS) between the endoplasmic reticulum (ER) and the plasma membrane (PM) enable direct intermembrane exchange of signals and metabolites. The Extended Synaptotagmins (E-Syts) are an evolutionary conserved family of ER-PM tethers essential to maintain PM integrity under stress conditions. To investigate the underlying molecular mechanisms, we employed cellular reconstitution experiments in yeast and plants. We show that E-Syt-mediated stress tolerance relies on ER-PM MCS targeting, which requires the E-Syt N-terminal membrane anchor, a minimal set of two C2 domains and an SMP domain. C2 domains are sufficiently conserved that interspecies domains can sustain both PM localization and stress response. The role of the SMP domain in ER-PM localization is also conserved, but SMP function in stress resistance is species-specific. Furthermore, cryo-electron tomography uncovers a scaffolding role for the SMP domain in maintaining ER-PM distance, and in the formation of ER membrane peaks with extreme curvature that appear necessary for stress tolerance. Collectively, our findings reveal the individual and synergistic roles of all E-Syt modules in maintaining cellular homeostasis under stress.

cell biology↗

bioRxiv2025

Novel substrate affinity of FaCCR1 and FaCCR1/FaOCT4 expression control the content of medium-chain esters in strawberry fruit

Fruit flavor and aroma are influenced by the sugars-to-acid ratio and most importantly by the specific profile and abundance of volatile organic compounds (VOCs). While over 550 VOCs have been identified in strawberry fruit, fewer than 20 are believed to be key for aroma, being esters the major components. A major and stable quantitative trait locus (QTL) explaining a large percentage (40%) of phenotypic variation for several medium-chain esters (MCE) was mapped at about 19 Mb in chromosome 6A using both a biparental population and genome-wide association studies (GWAS). Using a combination of genetic mapping, global transcriptomic studies of bulked contrasting lines, and in vivo and in silico functional analyses, we identified the cinnamoyl Co-A reductase FaCCR1(6A) and the organic cation/carnitine transporter FaOCT4(6A) as underlying genes. Subcellular localization experiments indicated that FaCCR1(6A) and FaOCT4(6A) localize at the cytosol and tonoplast membrane respectively. Transient overexpression of FaCCR1(6A) and FaOCT4(6A) in fruits resulted in a significant increase in MCE content. Despite the well-described function of CCRs in the biosynthesis of monolignols, in vitro enzymatic assays and molecular docking revealed that FaCCR1(6A) also presents high affinity for MCE precursors, indicating a previously unidentified function of this protein in volatile biosynthesis. Based in our findings, we have developed a predictive KASP assay in FaOCT4(6A) for selecting superior cultivars with enhanced fruit aroma.

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↗

bioRxiv2024

Unravelling Different Biological Roles Of Plant Synaptotagmins

Endoplasmic Reticulum-Plasma Membrane contact sites (ER-PM CS) are evolutionarily conserved membrane domains found in all eukaryotes, where the endoplasmic reticulum (ER) closely interfaces with the plasma membrane (PM). This short distance, typically 10-30 nm, is achieved in Arabidopsis through the action of tether proteins such as Synaptotagmins (SYTs). Arabidopsis comprises five SYT members (SYT1-SYT5), but whether they possess overlapping or distinct biological functions remains elusive. SYT1 is the best-characterized gene member and plays an essential role in the resistance to abiotic stress. This study reveals that while the functionally redundant SYT1 and SYT3 genes are involved in salt and cold stress resistance, SYT5 is associated with Pseudomonas syringae resistance despite evidence of in vivo interaction between SYT1 and SYT5. Structural phylogenetic analysis indicates that SYT1 and SYT5 clades emerged early in the evolution of land plants. These protein clades exhibit different structural features, rationalizing their distinct biological roles.

plant biology↗

bioRxiv2024

Concerted Transport and Phosphorylation of Diacylglycerol at ER-PM Contacts Sites Regulates Phospholipid Dynamics During Stress

ABSTRACTA universal response of plants to environmental stresses is the activation of plasma membrane (PM) phospholipase C (PLC) that hydrolyzes phosphatidylinositol phosphate (PIP) to produce soluble inositol phosphate (IP) and diacylglycerol (DAG). DAG produced in this way can be either phosphorylated by PM diacylglycerol kinases (DGKs) to produce the second messenger phosphatidic acid (PA) or transferred to the endoplasmic reticulum (ER) by the Synaptotagmin 1 (SYT1) protein at ER-PM Contact Sites (CS). In Arabidopsis, the clearance of DAG at the PM (avoiding deleterious accumulation) by the transfer activity of SYT is essential to maintain PM stability after stress. In this study we identify that DGK1 and DGK2 form a module with SYT1 at ER-PM CS through interaction of their C1 and C2 domains respectively. Global transcriptomic and metabolomic analyses confirms that SYT1 and DGK1/DGK2 are functionally related and lipidomic analysis supports the hypothesis that DGK1 and DGK2 function at the ER by phosphorylating DAG transferred by SYT1 from the PM. DGK1 and DGK2 show structural similarity to human DGK{varepsilon}, the DGK isoform that function at ER-PM CS in the phosphoinositide (PI) cycle. Our data indicate that components of the PI cycle are conserved between animals and plants and provide a novel mechanism leading to an increase in the efficiency of the PI cycle by channeling the transport and hydrolysis of DAG at the ER-PM CS.

plant biology↗

bioRxiv2022

Identification of plant exclusive lipid transfer SMP proteins at membrane contact sites in Arabidopsis and Tomato

Membrane contact sites (MCS) are regions where two membranes of different organelles are close but not fused; they coordinate non-vesicular communication between organelles and are involved in a wide variety of physiological functions, including membrane lipid homeostasis. Amongst proteins localized at MCS are those containing a lipid transport domain known as synaptotagmin-like mitochondrial-lipid binding protein (SMP), being the mammalian Extended Synaptotagmins, the yeast Tricalbins and the plant Synaptotagmin 1 (SYT1) the best SMP proteins characterized so far. They are all localized at endoplasmic reticulum-plasma membrane contact sites (ER-PM CS). We have carried out in-silico genome-wide identification of genes encoding SMP proteins in Arabidopsis and tomato. We have identified the plant exclusive NTMC2T5 proteins as ER-chloroplast CS components which make them extremely interesting as the route for lipid trafficking into and out of chloroplasts remains unknown. Additionally, NTMC2T5 over-expressions caused a significant clustering of chloroplast around nucleus. Moreover, SYT6, NTMC2T6 and TEX2 have been identified as ER-Trans-Golgi Network CS proteins. These proteins associated between them and with the exocytosis related proteins VAMP721 and VAMP727. Since the functional roles of many of these genes are unknown, this gene collection provides a useful resource for future studies. HIGHLIGHTPlant exclusive lipid transport proteins were identified at membrane contact sites. SYT6, TEX2 and NTMC2T6 proteins are localized at ER-TGN. NTMC2T5 proteins are localized at ER-Chloroplast and induced chloroplast-nucleus clustering.

plant biology↗

bioRxiv2022

Peripheral membrane proteins modulate stress tolerance by safeguarding cellulose synthases

Controlled primary cell wall remodeling adapts plant growth under stressful conditions, but how these changes are conveyed to adjust cellulose synthesis is unknown. Here, we identify the Tetratricopeptide Thioredoxin-Like (TTL) proteins as new members of the cellulose synthase complex (CSC) and describe their unique and hitherto unknown dynamic association with the CSC under cellulose-deficient conditions. We find that TTLs are essential for maintaining cellulose synthesis under salinity stress, establishing a stress-resilient cortical microtubule array, and stabilizing CSCs at the plasma membrane. To fulfill these functions, TTLs interact with Cellulose Synthase1 (CESA1) and engage with cortical microtubules to promote their polymerization. We propose that TTLs function as bridges connecting stress perception with dynamic regulation of cellulose biosynthesis at the plasma membrane. One Sentence SummaryTTLs are peripheral membrane proteins that maintain the integrity of the cellulose synthase complex upon adverse conditions.

plant biology↗

bioRxiv2021

The structure and flexibility analysis of the Arabidopsis Synaptotagmin 1 reveal the basis of its regulation at membrane contact sites

Cell function requires the maintenance of membrane lipid homeostasis as changes in cellular environment unbalance this equilibrium. The non-vesicular lipid transfer at endoplasmic reticulum (ER) and plasma membrane (PM) contact sites (CS) is central to restore it. Extended synaptotagmins (E-Syts) are ER proteins that play a central role in this process as they act as molecular tethers with PM and as lipid transfer proteins between these organelles. E-Syts are constitutively anchored to the ER through an N-terminal hydrophobic segment and bind to the PM via C-terminal C2 domains. In plants, synaptotagmins (SYTs) are orthologous of E-Syts and regulate the ER-PM communication by the activity of their two C2 domains in response to abiotic stresses. We have combined macromolecular crystallography, small-angle X-ray scattering, structural bioinformatics and biochemical data to analyze the regulation of plant synaptotagmin 1 (SYT1). Our data show that the binding of SYT1 to the PM is regulated by the interaction of the first C2 domain through a Ca2+-dependent lipid binding site and by a site for phosphorylated forms of phosphatidylinositol in such a way that two different molecular signals are integrated in response to stress. In addition, our data show that SYT1 is highly flexible by virtue of up to three hinge points, including one that connects the two C2 domains. This feature provides conformational freedom to SYT1 to define a large and complementary interaction surface with the PM. This structural plasticity, in turn, may facilitate lipid extraction, protein loading and subsequent transfer between PM and ER. Data DepositionThe atomic coordinates and structure factors have been deposited in the Protein Data Bank, https://www.pdb.org/ [PDB ID codes Ca2+ and Cd2+ complexes of SYT1C2A (7AS6 and 7ATP, respectively)]. The final SAXS models were deposited and are available at SASBDB https://www.sasbdb.org/ [ID codes SASDKG6 for the SMP2C2A construct SASDKJ9 for the C2AB construct and SASDKK9 in presence of Ca2+]

plant biology↗

bioRxiv2020

Synaptotagmins Maintain Diacylglycerol Homeostasis at Endoplasmic Reticulum-Plasma Membrane Contact Sites during Abiotic Stress

Endoplasmic Reticulum-Plasma Membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to WT while the levels of most glycerolipid species remain unchanged. Additionally, SYT1-GFP preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a crucial SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

molecular biology↗

bioRxiv2020

Organization and control of the ascorbate biosynthesis pathway in plants

The enzymatic steps involved in O_SCPLOWLC_SCPLOW-ascorbate biosynthesis in photosynthetic organisms (the Smirnoff-Wheeler, SW pathway) has been well established and here we comprehensively analyze the subcellular localization, potential physical interactions of SW pathway enzymes and assess their role in control of ascorbate synthesis. Transient expression of GFP-fusions in Nicotiana benthamiana and Arabidopsis (Arabidopsis thaliana) mutants complemented with genomic constructs showed that while GME is cytosolic, VTC1, VTC2, VTC4, and O_SCPLOWLC_SCPLOW-GalDH have cytosolic and nuclear localization. While transgenic lines GME-GFP, VTC4-GFP and O_SCPLOWLC_SCPLOW-GalDH-GFP driven by their endogenous promoters accumulated the fusion proteins, the functional VTC2-GFP protein is detected at low level using immunoblot in a complemented vtc2 null mutant. This low amount of VTC2 protein and the extensive analyses using multiple combinations of SW enzymes in N. benthamiana supported the role of VTC2 as the main control point of the pathway on ascorbate biosynthesis. Interaction analysis of SW enzymes using yeast two hybrid did not detect the formation of heterodimers, although VTC1, GME and VTC4 formed homodimers. Further coimmunoprecipitation (CoIP) analysis indicted that consecutive SW enzymes, as well as the first and last enzymes (VTC1 and O_SCPLOWLC_SCPLOW-GalDH), associate thereby adding a new layer of complexity to ascorbate biosynthesis. Finally, metabolic control analysis incorporating known kinetic characteristics, showed that previously reported feedback repression at the VTC2 step confers a high flux control coefficient and rationalizes why manipulation of other enzymes has little effect on ascorbate concentration. One sentence summaryMetabolic engineering, genetic analysis and functional mutant complementation identify GDP-O_SCPLOWLC_SCPLOW-galactose phosphorylase as the main control point in ascorbate biosynthesis in green tissues.

molecular biology↗

bioRxiv2019

A putative ER-PM contact site complex relocalizes in response to Rare Earth Elements-induced endocytosis

In plant cells, environmental stressors induce changes in the cytosolic concentration of calcium ([Ca2+]cyt) that are transduced by Ca2+-sensing proteins. To confer specificity to the stress signaling response, [Ca2+]cyt sensing must be tightly regulated in space and time; the molecular mechanisms that restrict the localization and dynamics of Ca2+ sensors in plants, however, are largely unknown. In this report, we identify a putative Ca2+-sensitive complex containing the synaptotagmins 1 and 5 (SYT1 and SYT5) and the Ca2+-dependent lipid binding protein (CLB1), which is enriched at ER-PM contact sites (EPCS) and relocalizes in response to Rare Earth Elements (REEs)-induced endocytosis. Our results show that endocytosed REEs influence cytosolic Ca2+ signaling, as indicated by the activation of the Ca2+/Calmodulin-based ratiometric sensor GCaMP3, and promote the cytoskeleton-dependent accumulation of ER-PM contact sites at the cell cortex. Based on these results, we propose that the EPCS-localized SYT1/SYT5/CLB1 complex is part of an evolutionarily conserved and spatially regulated Ca2+-responsive mechanism that control cER-PM communication during stress episodes.

plant biology↗

bioRxiv2018

TTL proteins scaffold brassinosteroid signaling components at the plasma membrane to optimize signal transduction in plant cells

Brassinosteroids (BRs) form a group of steroidal hormones essential for plant growth, development and stress responses. Here, we report that plant-specific TETRATRICOPEPTIDE THIOREDOXIN-LIKE (TTL) proteins are positive regulators of BR signaling functioning as scaffold for BR signaling components in Arabidopsis. TTL3 forms a complex with all core components involved in BR signaling, including the receptor kinase BRASSINOSTEROID INSENSITIVE1 (BRI1), the transcription factor BRASSINAZOLE RESISTANT1 (BZR1) and the phosphatase BRI1-SUPPRESSOR1 (BSU1), but excluding the co-receptor BAK1. TTL3 is mainly localized in the cytoplasm, but BR treatment increases its localization at the plasma membrane, where it strengthens the association with BR signaling components. Consistent with a role in BR signaling, mutations in TTL3 and related TTL1 and TTL4 genes cause reduced BR responsiveness. We propose a mechanistic model for BR signaling, in which cytoplasmic/nuclear BR components bound to TTL proteins are recruited to the plasma membrane upon BR perception, which in turn allows the assembly of a BR signaling complex, leading to the de-phosphorylation and nuclear accumulation of the transcription factors BZR1 and BES1.

molecular biology↗