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: 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

NTMC2T5 proteins as lipid bridges between plastids and ER during cotyledon development.

The conversion of proplastids to etioplasts is a critical process during seed germination, being essential for the development of cotyledons and the plant itself. Etioplasts play a crucial role in these initial stages as they serve as precursors to chloroplasts. The shift from one type of plastid to another involves massive structural changes. Among these, a key step is the formation of all chloroplastic membranes, which requires the synthesis and delivery of lipids to the plastids. Galactolipids are the most prevalent lipids found in chloroplasts. Notably, during the initial phases of photomorphogenesis, there is a noticeable build up of galactolipids within the plastid membranes, originated from the endoplasmic reticulum (ER). Since vesicular transport between these organelles has not been documented, lipid transport may be mediated by lipid transport proteins (LTP) at membrane contact sites (MCS). LTPs are characterized by unique lipid transport domains, such as the synaptotagmin-like mitochondrial lipid-binding (SMP) domain. However, no specific LTP has been identified as responsible for lipid exchange between the endoplasmic reticulum (ER) and chloroplasts. In our studies, we have identified the NTMC2T5 proteins, as putative lipid chloroplast transfer proteins, as they contain a SMP domain. Our confocal microscopy experiments have demonstrated that these proteins are anchored to the outer envelope membrane of the chloroplast, and BiFC analysis we shown that they interact with the ER, forming ER-Chloroplast MCS. QRT-PCR analysis have revealed that N. benthamiana NTMC2T5 gene is mainly expressed in early stages of seedling development. Further analysis in Nicotiana benthamiana CRISPR/Cas9 knock-out mutants showed (1) delayed greening in cotyledons when germinated in light, (2) an albino-like phenotype when germinated in darkness, (3) clear defect in plastid morphogenesis by transmission electron microscopy and (4) lower content of galactolipids.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

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

SYT6 protein role in Arabidopsis thaliana contact sites.

The SYT6 protein from A. thaliana (AT3G18370) has recently been identified as a lipid transfer protein localized at membrane contact sites (MCS). MCS are regions where membranes of two organelles closely approach without membrane fusing, typically within 10-30 nm [1]. Historically, research has primarily focused on endoplasmic reticulum (ER) and plasma membrane (PM) MCS [2], but recently MCS involving the ER and other organelles have come to light. SYT6 is a plant exclusive protein exhibiting a modular structure shared with mammalian Extended-Synaptotagmins (E-SYTs) and other plant synaptotagmins, such as SYT1. Our ongoing experiments suggest that SYT6 anchors itself to the ER via its transmembrane domain (TM), contains a lipid trafficking domain (named SMP) [3] and attaches to specific trans-Golgi Network (TGN) vesicles through its C2 domains and coiled-coil domain. These observations make SYT6 a particularly intriguing protein, given that its physiological roles remain unclear. Currently, our focus lies in studying SYT6 to uncover its expression, subcellular localization and most importantly, its function. Thanks to confocal imaging, we have confirmed SYT6 attachment to the ER and to big and small vesicles in continuous motion, suggesting its involvement in secretory trafficking. These findings, combined with Co-Immunoprecipitation experiments, have confirmed the interaction between SYT6 and specific TGN proteins linked to the independent Golgi TGN (GI-TGN). Moreover, our preliminary findings have showed a correlation between SYT6, exocytosis and autophagy. Furthermore, it has been observed that syt6 mutant exhibits distinct phenotypic traits compared to the wild-type, notably displaying altered negative gravitropism. Altogether, these findings suggest that SYT6 represents a novel ER-TGN CS protein that may play a role in secretory trafficking.This work has been funded by grant PID2021-127649OB-I00 (by MCIN/AEI/ 10.13039/501100011033 and by the European Union), Ayuda D2 Plan Propio by Universidad de Málaga and Proyecto QUAL21 012 IHSM (Consejería de Universidad, Investigación e Innovación, Junta de Andalucía). Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech