Biology subjects

Amorim-Silva, V.

Publications and source records attributed to Amorim-Silva, V..

11 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

Feedback between PI4P signaling and ER-PM contact sites orchestrates polarized root hair growth

Eukaryotic cells are composed of different organelles that communicate with one another through direct contacts, which are necessary for a host of cellular reactions and for responding to different developmental and environmental changes. Plasma membrane (PM) forms extensive contacts with the endoplasmic reticulum (ER) at specific sites named ER-PM contact sites. These contacts play crucial functions in lipid homeostasis, Ca2+ regulation and signaling in all eukaryotes. However, the mechanisms by which plant ER-PM contact site proteins tether to the PM, as well as the dynamics of these contact sites, remain poorly understood. Here, we investigate the importance of phosphoinositides in the establishment and dynamics of ER-PM contact site proteins in plants. We found that phosphatidylinositol-4-phosphate (PI4P), rather than phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), is required for the association of ER-PM contact site proteins with the PM. Furthermore, we identified a PI4P phosphatase, SUPPRESSOR-OF-ACTIN7 (SAC7), that associates with the ER-PM contact site protein SYNAPTOTAGMIN1 (SYT1) and regulates its dynamic association with the PM. In particular, we found that in growing root hairs, a highly polarized cell type, SAC7 removes SYT1-containing contact sites at the growing tip. Consistently, optogenetic induction of ER-PM tethering reduced root hair elongation within minutes of blue light induction. Altogether, we propose a link between SAC7-mediated regulation of PI4P, dynamic ER-PM contact site establishment and polarized cell growth in plants.

Plant Biology↗

bioRxiv2026

Phosphorylation of TTL3 by BIK1 Functions as a Molecular Switch to Control Cellulose Biosynthesis under Salt Stress

Cellulose, a central structural component of plant cell walls, is produced by cellulose synthase complexes (CSCs) at the plasma membrane. Salinity stress is particularly damaging to cellulose biosynthesis, and therefore, plants have developed adaptive mechanisms to cope with these conditions. TETRATRICOPEPTIDE THIOREDOXIN-LIKE (TTL) proteins are essential for growth under salt stress and show a salt-dependent association with CSCs through an as-yet unknown mechanism. Here, we identify a phosphorylation-dependent regulatory mechanism linking salt stress signaling to cellulose biosynthesis through the coordinated action of TTL3 and the receptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE 1 (BIK1). Phosphorylation of Serine 93 in the N-terminal intrinsically disordered region of TTL3 controls its localization, retaining it in the cytosol, while dephosphorylation promotes association with CSCs at the plasma membrane. Biochemical and genetic analysis identified BIK1 as the kinase responsible for TTL3-S93 phosphorylation, with bik1 mutants phenocopying the phosphoablative TTL3S93A in vivo. Transcriptomic analyses reveal a strong overlap of differentially expressed genes between bik1 and a cellulose-deficient mutant, supporting a broader role for BIK1 in cell wall regulation. Notably, TTL proteins do not appear to be involved in the assayed canonical immune responses, suggesting pathway specificity downstream of BIK1. Together, these findings define a signaling module that connects salt stress perception to CSCs regulation and establish BIK1-dependent TTL3 phosphorylation as a molecular switch for maintaining cell wall integrity under abiotic stress.

Plant 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

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

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↗

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↗

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↗