38 research outputs found
The Arabidopsis Synaptotagmin1 is enriched in endoplasmic reticulum-plasma membrane contact sites and confers cellular resistance to mechanical stresses
Eukaryotic endoplasmic reticulum (ER)-plasma membrane (PM) contact sites are evolutionarily conserved microdomains that have important roles in specialized metabolic functions such as ER-PM communication, lipid homeostasis, and Ca2+ influx. Despite recent advances in knowledge about ER-PM contact site components and functions in yeast (Saccharomyces cerevisiae) and mammals, relatively little is known about the functional significance of these structures in plants. In this report, we characterize the Arabidopsis (Arabidopsis thaliana) phospholipid binding Synaptotagmin1 (SYT1) as a plant ortholog of the mammal extended synaptotagmins and yeast tricalbins families of ER-PM anchors. We propose that SYT1 functions at ER-PM contact sites because it displays a dual ER-PM localization, it is enriched in microtubule-depleted regions at the cell cortex, and it colocalizes with Vesicle-Associated Protein27-1, a known ER-PM marker. Furthermore, biochemical and physiological analyses indicate that SYT1 might function as an electrostatic phospholipid anchor conferring mechanical stability in plant cells. Together, the subcellular localization and functional characterization of SYT1 highlights a putative role of plant ER-PM contact site components in the cellular adaptation to environmental stresses
Plasma membrane lipid remodeling during cold acclimation is mediated by the ER-PM contact sites-localized synaptotagmins 1 and 3
Cold acclimation is the capacity of certain plants to increase their freezing tolerance in response to a period of low non-freezing temperatures. Cold acclimation involves a series of biochemical and physiological adaptations, including a deep transcriptional reprogramming and drastic changes in the lipid composition of cellular membranes in order to prevent the freeze-induced damage (1). While a profound knowledge has been acquired on the regulation of gene expression triggered by cold-acclimation, very little is known about the mechanisms governing the cold-induced changes in membranes’ lipid composition. In this study we report that in Arabidopsis, the constitutively expressed Synaptotagmin 1 (SYT1) and the cold-induced homolog Synaptotagmin 3 (SYT3) are essential for cold- acclimated freezing tolerance and for the lipid remodelling of the plasma membrane during cold-acclimation. SYT1 and SYT3 are phospholipid-binding proteins located in Endoplasmic Reticulum-Plasma Membrane contact sites (ER-PMcs), conserved structures defined as regions of the cortical ER in close apposition to the PM (2). ER-PMcs facilitate the non-vesicular lipid transport between ER and PM in yeast and mammals, and are essential for lipid homeostasis (3). In contrast to the high and ubiquitous SYT1 expression, SYT3 expression is low and mainly restricted to meristemoids, young stomata, and old primary root. TIRF microscopy analyses show that during cold acclimation there is an increase of SYT1::SYT1:GFP and SYT3::SYT3:GFP signals as spots at the PM. High-resolution lipidome analyses show the over-accumulation of phosphatidylinositols phosphate (PIPs) and glycerolipids in vivo in syt1 and specially syt1/syt3 mutant plants compared to WT in one-week cold-acclimated plants. Interestingly, protein-lipid overlay assays (membrane-strips and PIP-strips) reveal PIPs and glycerolipids as major interactors for both, SYT1 and SYT3. Here we show that 1) Arabidopsis SYT1 and SYT3 are induced by cold, 2) SYT1 and SYT3 localize to ER-PMcs, 3) the specific lipids that directly interact with SYT1 and SYT3 accumulate in syt1/syt3 mutant after cold acclimation, and 4) syt1/syt3 show reduced cold acclimated freezing tolerance. We propose that SYT1 and SYT3 have essential roles in ER-PMcs mediated lipid remodelling during cold acclimation, which in turn leads to freezing tolerance.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech
Characterization of ripe fruit epidermis-specific transcription factors in strawberry
Transcriptome changes during strawberry fruit ripening have been previously reported using either complete fruits or achenes (actual fruits) and receptacles (fleshy part) separately. In order to perform a more detailed study, we have performed a tissue- and stage-specific transcriptome analysis in receptacles of Fragaria vesca fruits, allowing us to infer Gene Regulatory Networks (GRN) in each tissue and stage. In the study, we have focused on the epidermis at the ripe stage, since it plays an important role in defense, as it is the external cell layer in direct contact with the environment, and, in contrast to receptacles of the commercial species, it is the only part of the fruit that accumulates anthocyanins. MapMan analysis of the GRN in ripe epidermis showed that wax and flavonoid biosynthesis were significantly overrepresented functions. Three out of the several TFs found among the main hubs in this GRN were selected to study their biological role, one of them belonging to the MYB family, and two bHLH genes. Protein interaction assays revealed that the MYB protein physically interacts with the two bHLHs, leading to the subcellular relocalization from the cytoplasm to the nucleus in one of them. DAP-seq analyses showed that the bHLH TFs do not bind DNA by themselves, but that genes involved in cuticle formation and flavonoid biosynthesis are among the MYB targets, which were validated by a transactivation assay using the Luciferase/Renilla system. Consistently, MYB-overexpressing stable lines exhibited an upregulation of genes related to cuticle and wax biosynthesis in ripe fruits, and an accumulation of higher amounts of epicuticular waxes in young leaves compared to the WT. We are currently establishing RNAi and CRISPR lines for these three ripe-epidermis specific TFs to further investigate their biological role and performing analyses to understand the effect on gene expression of the interaction between them.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech
Characterization of ripe fruit epidermis-specific transcription factors in strawberry
Transcriptome changes during strawberry fruit ripening have been previously reported using either complete fruits or achenes (actual fruits) and receptacles (fleshy part) separately. In order to perform a more detailed study, we have performed a tissue- and stage-specific transcriptome analysis in receptacles of Fragaria vesca fruits, allowing us to infer Gene Regulatory Networks (GRN) in each tissue and stage. In the study, we have focused on the epidermis at the ripe stage, since it plays an important role in defense, as it is the external cell layer in direct contact with the environment, and, in contrast to receptacles of the commercial species, it is the only part of the fruit that accumulates anthocyanins. MapMan analysis of the GRN in ripe epidermis showed that wax and flavonoid biosynthesis were significantly overrepresented functions. Three out of the several TFs found among the main hubs in this GRN were selected to study their biological role, one of them belonging to the MYB family, and two bHLH genes. Protein interaction assays revealed that the MYB protein physically interacts with the two bHLHs, leading to the subcellular relocalization from the cytoplasm to the nucleus in one of them. DAP-seq analyses showed that the bHLH TFs do not bind DNA by themselves, but that genes involved in cuticle formation and flavonoid biosynthesis are among the MYB targets, which were validated by a transactivation assay using the Luciferase/Renilla system. Consistently, MYB-overexpressing stable lines exhibited an upregulation of genes related to cuticle and wax biosynthesis in ripe fruits, and an accumulation of higher amounts of epicuticular waxes in young leaves compared to the WT. We are currently establishing RNAi and CRISPR lines for these three ripe-epidermis specific TFs to further investigate their biological role and performing analyses to understand the effect on gene expression of the interaction between them.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec
Characterization of ripe fruit epidermis-specific transcription factors in strawberry
The epidermis is the external cell layer in direct contact with the environment, and it plays essential biological roles. Transcriptome analysis (RNA-seq) of Fragaria vesca fruit receptacles at four ripening stages (green, white, turning and red) and of different tissue types of receptacles (pith, vascular bundles, cortex and epidermis) at two ripening stages (green and red) allowed us to infer tissue- and stage-specific Gene Regulatory Networks (GRN). Due to the potential role of the epidermis in defense and in the differential anthocyanin accumulation pattern that shows at the ripe stage of F. vesca fruits (the skin is red, while the inner part is white), we have focused on the GRN of the ripe epidermis. In this study, we aim at the functional characterization of two transcription factors (TFs) that constituted the main hubs of this GRN: a MYB-like gene, and a member of the NAC family of TFs. A MapMan analysis of the genes constituting the GRN in ripe epidermis showed that wax and flavonoid biosynthesis were significantly overrepresented functions in this tissue at the ripe stage. Using the Luciferase/Renilla (Luc/Ren) system, the interaction of the MYB and NAC TFs with their wax-related putative targets was validated. To gain insight into the target genes of these two TFs, we mapped the genome-wide binding sites using DAP-seq analyses. Consistently, MYB bound to a set of genes involved in cuticle formation and flavonoid biosynthesis, while a number of genes involved in solute transport were enriched among the NAC targets. Currently, we are generating CRISPR/Cas9 mutant lines to functionally characterize these two TFs. Furthermore, we are performing protein interaction assays to decipher whether the MYB and NAC TFs interact with each other and with other TFs from the red epidermis GRN.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech
Diacylglycerol transport by Arabidopsis Synaptotagmin 1 at ERplasma membrane contact sites under abiotic stress.
Bulk lipid transport between membranes within cells involves vesicles, however membrane contact sites have recently been discovered as mediators of non-vesicular lipid transfer. ER-PM contact sites are conserved structures defined as regions of the endoplasmic reticulum (ER) that tightly associate with the plasma membrane (PM). Our recent data suggest that the constitutively expressed Arabidopsis Synaptotagmin 1 (SYT1) and the cold-induced homolog AtSYT3 are proteins located in these ER-PM contact sites that are essential for the tolerance various abiotic stresses. Arabidopsis SYTs proteins are integral membrane proteins that contain multiple Ca2+-binding C2 domains and a synaptotagmin-like mitochondrial lipid-binding protein (SMP) domain that contains a hydrophobic groove. In mammals, several SMP proteins are responsible for the inter-organelle transport of glycerophospholipids. Our experiments have demonstrated that there is a recruitment of AtSYT1 and AtSTYT3 to ER-PM contact sites under stress conditions and it requires phosphatidylinositol 4- phosphate, PI(4)P in the PM, in opposition to the recruitment of PI(4,5)P2 in mammals. Moreover, our recent high-resolution lipidome analysis suggest that saturated diacylglycerols (DAGs) are the lipids that AtSYT1 is transferring between the PM and ER. Additionally, we have identified AtDGK2 (diacylglycerol kinase 2) as a key interactor of AtSYT1. Generally, in response to a stress stimulus, a phospholipase C (PLC), hydrolyses PIP2 after the elevation of cytosolic Ca2+, generating DAGs which immediately can be converted to phosphatidic acid (PA) by DGKs.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.
The authors acknowledge the support by the Plan Propio from University of Malaga, Campus de
Excelencia Internacional de Andalucía and by the Redes of Excelencia (BIO2014-56153-REDT) and BIO2017-82609-R, RYC-2016-21172 & PGC2018-098789 of the Ministerio de Economía, Industria y Competitividad
Synaptotagmins at the endoplasmic reticulum-plasma membrane contact sites maintain diacylglycerol homeostasis 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 SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress
Strawberry GRN forever: insights into the transcriptional regulatory network controlling strawberry fruit ripening and quality
Ripening is a critical step for the development of flavor quality in fruits. This character has significantly declined in many fleshy fruits over recent decades. This is particularly significant in strawberry (Fragaria × ananassa), where current cultivars are derived from a narrow germplasm collection. Improving fruit quality requires two important breakthroughs: 1) a precise understanding of the fruit ripening process that will allow the targeting of relevant genes, and 2) the identification of novel alleles responsible for fruit quality traits.
In our project, we aim at the identification and characterization of key transcription factors (TF) involved in fruit ripening regulation and their target genes, in order to infer the Gene Regulatory Network controlling this process. Among them, we have identified two TFs belonging to the NAC (FaRIF) and the BLH9 (FaRPL) family. Functional analyses establishing stable silencing and overexpression lines support that both TFs play a critical role in the regulation of fruit ripening and development. Furthermore, using a stage- and tissue-specific transcriptome analysis, we have identified TFs specifically expressed in the external layer of ripe receptacles of F. vesca fruits, which are involved in the regulation of wax and cuticle formation.
Finally, we have implemented the use of the genome-editing tool CRISPR/Cas9 in the cultivated strawberry, which we expect to open opportunities for engineering this species to improve traits of economic importance
Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)
Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters.
Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs).
Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001).
Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio