NTMC2T5.1 is involved in chloroplast clustering around nucleus in Nicotiana benthamiana.

Plants have developed mechanisms to protect themselves from pathogens and resist their attacks. Among these, clustering of chloroplasts around the nuclear envelope is a phenomenon that has been proposed as a general response to the perception of pathogens, such as viruses and bacteria [1]. Additionally, it has been hypothesized that chloroplast clustering could serve as an efficient way of transferring signals —such as lipids and reactive oxygen species— between these two organelles in order to induce changes in the expression of pathogen defence-related genes. Synaptotagmin-like mitochondrial-lipid-binding (SMP) domain proteins are evolutionarily conserved in eukaryotes and participate in the formation of membrane contact sites between organelles. In particular, their SMP domains allow the lipid transport between the membranes of different organelles [2]. Our group is focus on studying NTMC2T5 proteins, a group of SMP proteins, with two homologs in Arabidopsis thaliana. By transiently overexpressing AtNTMC2T5.1 and some truncated versions in Nicotiana benthamiana leaf cells, and using confocal microscopy, we have determined the subcellular localization of the encoded protein. This protein is anchored to the chloroplast outer envelope and interacts with the membranes of other organelles, like the ER and the nuclear envelope membrane [3]. Additionally, we have detected that overexpression of NTMC2T5 proteins causes a significant chloroplast clustering around nucleus, that it was not observed when overexpressing other proteins. Thus, we have estimated the contribution of its functional domains in the clustering. Our results suggest that the C-terminal hydrophobic region (HR) of AtNTMC2T5.1 is essential for this process. Moreover, our Arabidopsis ntmc2t5.1/t5.2 knock down plants showed slower growth when treated with flagelin22 and reduced chloroplast clustering when treated with H2O2.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

NTMC2T5: lipid transfer proteins at ER-chloroplast contact sites involved in plant stress.

Chloroplasts are the site of fatty acid synthesis in plants; however, these fatty acids are assembled into glycerolipids at the ER. Later on, some of these ER-assembled glycerolipids will be transferred back to the chloroplasts to be further modified and to form part of the chloroplastic membranes. Previous reports have shown that under some abiotic stresses, these plastid membranes suffer a large lipid remodelling and new precursors massively need to be transported from the ER to the chloroplast or vice versa. It has been suggested that the newly synthetized ER lipids are delivered to chloroplast via a non-vesicular pathway, likely through lipid transport proteins (LTP). These LTP would be localized in membrane contact sites (MCS). Some LTP at MCS contain particular domains, as the synaptotagmin-like mitochondrial lipid-binding (SMP) domain. We have studied the occurrence of SMP proteins in A. thaliana and S. lycopersicum. By using transient expression in N. benthamiana leaves and confocal microscopy, we have identified the NTMC2T5 family with two homologs in A. thaliana and only one in S. lycopersicum. They are anchored to the chloroplast outer membrane, and they interact in trans with the ER (ER-chloroplast MCS). We have observed that clustering of chloroplasts around the nucleus occurred when we overexpressed these proteins and Arabidopsis double knock-out mutant for these proteins showed less chloroplasts attached to nuclei at control conditions. And, we have investigated the NTMC2T5 protein domains involved in this clustering. Moreover, our analysis has demonstrated that Arabidopsis simple mutants show lower germination rates in media supplemented with NaCl and lower rates of expanded cotyledons in media supplemented with ABA. We have also performed biotinylation-based proximity labelling proteomics experiments in order to identify interactors of these proteins. Finally, we have performed lipidomic analysis to understand the role of these proteins.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

NTMC2T5: a newly identified lipid transfer proteins at ER-chloroplast contact sites involved in development and stress response.

In plants, fatty acid synthesis takes place at chloroplasts, and they are assembled into glycerolipids and sphingolipids at the endoplasmic reticulum (ER). Then, the newly ER synthetized lipids are sent back to the chloroplast to form part of their membranes. Since, no vesicular transport has been described between these two organelles, lipid transport might be mediated by lipid transport proteins (LTP) via a non-vesicular pathway.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

SMP-CONTAINING PROTEINS AT MEMBRANE CONTACT SITES: SUBCELLULAR LOCALIZATION AND CHARACTERIZATION.

Membrane contact sites (MCS) are microdomains where two membranes of two different organelles are in close apposition, but they do not fuse. MCS are essential for non-vesicular transport of lipids. This lipid transport is mediated by several families of proteins which all of them contain a lipid transport domain, as the synaptotagmin-like mitochondrial lipid-binding (SMP) domain. The most studied SMP protein is Arabidopsis SYT1 which is known to be involved in tolerance to multiple abiotic stresses. Later studies in other SMP proteins of the same family have shown that SYT1 and homologous such as SYT3 or SYT5 gave similar results. However, little information is available about the role other SMP proteins in plants. We have studied the occurrence of additional SMP proteins in A. thaliana and S. lycopersicum. In order to identify these proteins, SMP sequences from human and yeast were used to identify their remote orthologues in A. thaliana and S. lycopersicum, allowing the identification of several putative encoding SMP domains. We have found that some of the identified proteins are exclusive of plants as they do not have direct orthologs in yeast nor human. Transient expression in N. benthamiana leaves followed by confocal microscopy was used to study the subcellular localization of these proteins. Our results show that some of these proteins are localized at ER-Golgi contact sites and two other proteins at ER-Chloroplast sites. Finally, to determine whether these proteins are involved in abiotic stress tolerance, we have analysed the root growth and seed germination rates of Arabidopsis mutants for these genes under different conditions. Some of these mutants have shown different germination rates in media supplemented with NaCl and different rates of expanded cotyledons in media supplemented with ABA. These results suggest that some these proteins may be implicated in abiotic stress signalling through an ABA-dependent pathway.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. This work is supported by grants from: Ministerio de Ciencia, Innovación y Universidades (grant PGC2018-098789-B-I00), UMA-FEDER (grant UMA18-FEDERJA-154) and Ministerio de Ciencia e Innovación (BIO2017-82609-R)

Localization and characterization of SMP-containing proteins in Membrane Contact Sites

Membrane contact sites (MCS) are discrete regions where two membranes from different organelles are closely apposed (10-30 nm). In those regions, non-vesicular transfer of lipids takes place to ensure proper organelle functioning. Arabidopsis SYT1 is one of the best characterized MCS protein, and it plays a relevant role in tolerance to abiotic stresses. SYT1 is a SMP (synaptotagmin-like mitocondrial lipid binding domain) containing protein localized at ER-PM contact sites. Recent studies suggest that this protein transfer glycerolipids between these two membranes. However, little is known about other SMP-containing proteins in plants, as their localization or their role in abiotic stress. We have focused on studying the rest of SMP-containing proteins in Arabidopsis thaliana and Solanum lycopersicum. To identify them, human E-Syt1 sequence was used to find the remote orthologues in plants. An interesting highlight of those results was that some SMP-containing proteins are exclusive from plants, there are no orthologues in human nor yeast. The subsequent step was the study of their subcellular location, that was carried out in Nicotiana benthamiana by transient expression of the SMP-containing proteins from Arabidopsis and Solanum, followed by confocal microscopy imaging. We have found that those proteins locate in different MCS across the cell: SYT6, NTMC2T6 and Tex2 localise in ER-Golgi contact sites, NTMC2T5 in ER-Chloroplast contact sites, and we have also confirmed that Solanum CLB1 and SYT5 localized at ER-PM contact sites as their Arabidopsis counterparts. Additionally, we have analysed the root growth, seed germination rates and fully expanded cotyledons of Arabidopsis mutants for these genes in media supplemented with salt or ABA, and our results suggest that some of these proteins might be implicated in abiotic stress signalling through an ABA pathway.This work is supported by grants from: Ministerio de Ciencia, Innovación y Universidades (grant PGC2018-098789-B-I00), UMA-FEDER (grant UMA18-FEDERJA-154) and Ministerio de Ciencia e Innovación (BIO2017-82609-R), and meeting assistance was granted by Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

IDENTIFICATION OF SYT1 INTERACTORS CONNECTS CALCIUM SIGNALING, ENDOPLASMIC RETICULUM BENDING, PLASMODESMATA AND MEMBRANE CONTACT SITES

Synaptotagmin1 (SYT1) is an Arabidopsis thaliana protein essential for tolerance to several abiotic stresses (Schapire et al., 2008; Pérez-Sancho et al., 2015; Ruiz-Lopez et al., 2020). SYT1 forms endoplasmic reticulum-plasma membrane contact sites (ER-PM CS), microdomains conserved across eukaryotes where protein tethers maintain the membranes of the ER and the PM in close apposition (∼30 nm) without fusing. The short distance between membranes facilitates processes such as ion and lipid transport (Pérez-Sancho et al., 2016). For example, SYT1 transports diacylglycerol (DAG) from the PM to the ER during abiotic stress to assure PM integrity (Ruiz-Lopez et al., 2020). Usually, protein complexes form the core of contact sites. In particular, SYT1 forms dimers with SYT1, SYT3, SYT5 and CLB1; all members of the Arabidopsis SYT family and EPCS tethers (Lee et al., 2020; Ruiz-Lopez et al., 2020). We found that SYT1 interacts with proteins involved in different cellular processes by non-targeted proteomic approaches (IP-MS and TAP-tag). Thus, SYT1 interacts with reticulons (RTN), ER-resident proteins responsible for ER curvature, which is crucial for ER morphology and lipid transport at ER-PM CS (Collado et al., 2019). Additionally, SYT1 interacts with ECAs, Ca2+-ATPases located at the ER membrane. SYT1 also interacts with sterol methyltransferases (SMTs), key enzymes in the route of sitosterol and stigmasterol biosynthesis. The homeostasis of the sitosterol and stigmasterol is crucial for the tolerance to wound, heat and bacterial stress presumably by affecting PM fluidity. MCTPs (multiple C2 domains and transmembrane region proteins), plasmodesmata-exclusive proteins are also SYT1 interactors (Brault et al., 2019). We are now investigating the role of SYT1 in these processes using biochemical, genetic and cellular biology approaches

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

CLIL y enseñanza de español para extranjeros: una propuesta de trabajo con Webquests

Suplemento del número 13 de marcoELE (julio - diciembre de 2011) Debido a la creciente penetración de las TIC en la sociedad y por lo tanto la enseñanza, este estudio nos conduce hacia una de sus aplicaciones en el aula más motivadoras, la WebQuest. Este modelo pedagógico, que usa los recursos de Internet de forma dirigida, es más que una plantilla de diseño instructivo, promueve el pensamiento de orden superior, usa dinámicas de aprendizaje cooperativo y andamiaje. Tras una introducción sobre sus conceptos esenciales más la aportación de ideas nuevas para su transición a la Web 2.0., analizaremos la conveniencia de interacción del modelo WebQuest con el enfoque CLIL aplicado a la enseñanza/aprendizaje de ELE y haremos una propuesta concreta que tendrá dos ramas temáticas, el arte y la lengua española

NTMC2T5: nueva proteína localizada en puntos de contacto cloroplasto-retículo implicada en estrés biótico.

Las proteínas con un dominio SMP se encuentran evolutivamente conservadas y participan en la formación de puntos de contacto de membrana (MCS, membrane contact sites), unas estructuras donde las membranas de dos orgánulos se encuentran muy cerca pero sin llegar a contactar y que están implicadas en el transporte lipídico entre compartimentos celulares, entre otros procesos. En este trabajo se han estudiado las proteínas NTMC2T5, un nuevo grupo de proteínas SMP que se localizan en cloroplastos e interaccionan con la envoltura nuclear formando MCS. Para su estudio, se han generado líneas knock-out de Nicotiana benthamiana para este gen utilizando CRISPR/Cas9. Se observó que los cotiledones de las plántulas amarilleaban y se realizó un conteo de cloroplastos totales, observándose una reducción significativa del número de cloroplastos con respecto a plantas wild-type. Estos resultados apuntan a una probable implicación de NTMC2T5 en el desarrollo y en la transición de etioplasto a cloroplasto. Recientemente, se ha demostrado que la agrupación de cloroplastos alrededor del núcleo es una respuesta general a la percepción de patógenos. Por ello, hemos estudiado el agrupamiento de cloroplastos al sobreexpresar el gen AtNTMC2T5.1 y versiones truncadas del mismo en hojas de Nicotiana benthamiana, y los resultados demuestran que NTMC2T5.1 interviene en el agrupamiento de cloroplastos y media la formación de MCS entre el núcleo y los cloroplastos. Además, se trataron plantas de Arabidopsis thaliana mutantes para los genes AtNTMC2T5.1 y AtNTMC2T5.2 con flagelina 22, y se observó que los mutantes presentaron una reducción significativa en el peso fresco. Además, cabe destacar que estos mutantes presentaban un menor agrupamiento de cloroplastos con respecto a plantas control. En resumen, los resultados sugieren que el agrupamiento de cloroplastos alrededor del núcleo está mediado por las proteínas NTMC2T5 y promueve la resistencia ante el ataque por patógenos.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

NTMC2T5: newly identified lipid transfer proteins at ER-chloroplast contact sites.

In plants, fatty acid synthesis takes place at chloroplasts, and they are assembled into glycerolipids and sphingolipids at the endoplasmic reticulum (ER). Then, the newly synthetized lipids in the ER are delivered to chloroplast (mostly) via a non-vesicular pathway, likely through lipid transport proteins (LTP). These LTP would be localized in ER-chloroplast membrane contact sites (MCS), which are microdomains where membranes of these two different organelles are closely apposed but not fussing. Synaptotagmin-like mitochondrial-lipid-binding (SMP) domain proteins are evolutionarily conserved LTP in eukaryotes that localize at membrane CS. They are involved in tethering of these MCS through interaction with other proteins/membrane lipids and in transferring of glycerolipids between these two membranes. We have studied the occurrence of SMP proteins in A. thaliana and S. lycopersicum by searching remote orthologs of human E-Syt1 (SMP protein). By using transient expression in N. benthamiana leaves and confocal microscopy, we have identified the NTMC2T5 family with two homologs in A. thaliana and only one in S. lycopersicum that are anchored to the chloroplast outer membrane and are interacting with the ER (at ER-chloroplast CS). Our preliminary data have unequivocally demonstrated that NTMC2T5 proteins are anchored to the outer envelope membrane in chloroplast, and they bind in trans the ER. Additionally, it is predicted that these proteins contain a SMP domain which is a lipid-transfer domain, indicating that these proteins could be responsible for some of the lipid transferring events at ER-chloroplast CS that are still unknown. Our preliminary phenotypic analyses have shown that these proteins are involved in hypocotyl length in darkness and plant growth under Nitrogen deficiency. Interestingly, we have observed that clustering of chloroplasts around the nucleus occurred when we overexpressed these proteins in Nicotiana benthamiana leaves and ArabidopsisUniversidad de Málaga. Campus de Excelencia Internacional Andalucía Tech