Phosphorylation regulates cellulose biosynthesis regulation.

Plants comprise over 82% of all biomass on Earth, a third of which is cellulose, making it the most abundant organic compound¹. Cellulose is also essential for plant development and defense against multiple stresses. But, despite its relevance, there remains much to be discovered about its biosynthetic regulation to improve crop’s tolerance to biotic and abiotic stresses. We have described the Tetratricopeptide Thioredoxin‐Like (TTL² proteins as regulators of the cellulose synthase complex (CSC) under cellulose‐deficient conditions³. We found that TTLs are required to maintain cellulose synthesis under salt stress by relocalising from the cytosol to the CSCs, promoting the polymerization of microtubules to form a stress‐resilient cortical microtubule array, and interacting with the CSCs to stabilize them at the plasma membrane. We are currently investigating how TTLs are targeted to the CSCs. We have found that TTL3 is a substrate for a kinase that when mutated show cellulose‐defective phenotypes under stresses that affect cell wall integrity. We are currently analysing how the CSCs and microtubules behave under abiotic stress in mutants for this kinase. This will provide new insights into how changes in phosphorylation status regulate the activity and dynamic localization of these proteins.This work was funded by the Spanish Ministry for Science and Innovation (PID2020-114419RB-I00MCIN/AEI/10.13039/501100011033 ) to MAB. The Andalusian Research Plan co-financed by the European Union (PAIDI 2020- PY20_00084 and UMA20-FEDERJA-023) to MAB. FP was supported by FPU19/02219 fellowships and EMBO Scientific Exchange Grant 10026 and VAS was supported by an Emerging Investigator research project (UMA20-FEDERJA -007) and co-financed by the “Programa Operativo FEDER 2014-2020” and by the “Consejería de Economía y Conocimiento de la Junta de Andalucía”. Funding from the University of Zürich, and the Swiss National Science Foundation grants no. 31003A_182625 and no. 310030_212382 (to CZ). 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)

Overcoming stress: new insights in the regulation of cell wall biosynthesis

In addition to being crucial in plant development and defence, cellulose is the most abundant organic compound of all biomass on Earth1. Therefore, it is essential to elucidate the regulation of its biosynthesis to improve crop's tolerance to biotic and abiotic stresses. We have found the Tetratricopeptide Thioredoxin-Like (TTL)2 proteins as new players in the regulation of the cellulose synthase complex (CSC), identifying its dynamic association with the CSC under cellulose-deficient conditions3. We have found that TTLs are essential to maintain cellulose synthesis under salt stress, mediated by a stress-resilient cortical microtubule array and the stabilization of the CSCs at the plasma membrane. TTLs carry this out by interacting with Cellulose Synthase 1 and promoting the polymerization of microtubules. This dynamic behaviour of TTLs is not specific to salinity stress, and other modifications that cause reduced cellulose content also lead to the re-localization from the cytosol to the CSC. We conclude that TTLs act as intermediates between stress perception and regulation of cellulose biosynthesis to overcome adverse environmental conditions. All TTL proteins contain an Intrinsic Disordered Region at the end terminus, and we are now investigating how changes in phosphorylation regulate the activity and dynamic localization of these proteins.This work was funded by the Spanish Ministry for Science and Innovation (MCIN/AEI/ 10.13039/501100011033) (PGC2018-098789-B-I00) and (PID2019-107657RB-C22) to MAB, NRL and AC respectively. The Andalusian Research Plan co-financed by the European Union (PAIDI 2020-PY20_00084) to MAB and Junta de Andalucía UMA-FEDER project (grant UMA18-FEDERJA-154) to NRL, and the Swiss National foundation to CSR (SNF 31003A_163065/1 to AM). CK was supported by a Peter und Traudl Engelhorn-Stiftung fellowship, an ETH Career Seed Grant (SEED-05 19-2) of the ETH Foundation, an Emerging Investigator grant (NNF20OC0060564) of the Novo Nordisk Foundation, and an Experiment grant (R346-2020-1546) of the Lundbeck foundation. AGM and FP were supported by BES-2015-071256 and FPU19/02219 fellowships respectively, and meeting attendance was supported by Plan Propio de Investigación, Transfe-rencia y Divulgación Científica de la Universidad de Málaga (UMA) Campus de Excelencia Internacional Andalucía Tech. VAS was supported by an Emerging Investigator research project (UMA20-FEDERJA-007) and co-financed by the “Programa Operativo FEDER 2014-2020” and by the “Consejería de Economía y Conocimiento de la Junta de Andalucía”. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Calling for reinforcements: the role of TTL proteins in the regulación of cell wall biosynthesis

Cellulose is the most abundant organic compound of all biomass on Earth1, with highly relevant roles in plant development and defence. Hence, it is essential to understand the regulation of its biosynthesis to improve the crop's tolerance to biotic and abiotic stresses. Tetratricopeptide Thioredoxin-Like (TTL)2 proteins have been identified as new players in regulating the cellulose synthase complex (CSC), uncovering their dynamic association with the CSC under cellulose-deficient conditions3. We show that TTLs are essential to maintain cellulose synthesis under salt stress, mediated by a reinforced cortical microtubule array and the stabilization of the CSCs at the plasma membrane. To perform this, TTLs interact with Cellulose Synthase 1 and promote microtubules polymerization. This dynamic behaviour of TTLs is not specific to salinity stress, and other factors that cause defects in cellulose also cause the re-localization from the cytosol to the CSC. We conclude that TTLs act as intermediates between stress sensing and the regulation of cellulose biosynthesis to overcome adverse environmental conditions. We are now investigating how changes in phosphorylation of the Intrinsic Disordered Region at the end terminus of TTLs regulate their activity and dynamic localization.This work was funded by the Spanish Ministry for Science and Innovation (MCIN/AEI/ 10.13039/501100011033) (PGC2018-098789-B-I00) and (PID2019-107657RB-C22) to MAB, NRL and AC respectively. The Andalusian Research Plan co-financed by the European Union (PAIDI 2020-PY20_00084) to MAB and Junta de Andalucía UMA-FEDER project (grant UMA18-FEDERJA-154, UMA20-FEDERJA-023) to NRL and MAB respectively, and the Swiss National foundation to CSR (SNF 31003A_163065/1 to AM). CK was supported by a Peter und Traudl Engelhorn-Stiftung fellowship, an ETH Career Seed Grant (SEED-05 19-2) of the ETH Foundation, an Emerging Investigator grant (NNF20OC0060564) of the Novo Nordisk Foundation, and an Experiment grant (R346-2020-1546) of the Lundbeck foundation. AGM and FP were supported by BES-2015-071256 and FPU19/02219 fellowships respectively, and meeting attendance was supported by Plan Propio de Investigación, Transferencia y Divulgación Científica de la Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. VAS was supported by an Emerging Investigator research project (UMA20-FEDERJA-007) and co-financed by the “Programa Operativo FEDER 2014-2020” and by the “Consejería de Economía y Conocimiento de la Junta de Andalucía”

Identification of NTMC2T5, a new lipid transfer protein family at ER-chloroplast contact sites involved in stress response

Plants are sessile organisms and they have perfected a complex molecular signalling network to detect and respond to different environmental stresses. In plants, fatty acid synthesis takes place at chloroplasts, and they are assembled into glycerolipids and sphingolipids at the ER. Then, the newly synthetized lipids in the ER are delivered to chloroplast via a non-vesicular pathway, likely through lipid transport proteins. 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. SMP domain proteins are evolutionarily conserved LTP in eukaryotes that localize at MCS. 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 that are anchored to the chloroplast outer membrane and are interacting with the ER (at ER-chloroplast MCS. Our preliminary data have demonstrated that NTMC2T5 proteins are anchored to the 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 MCS that are still unknown. We show the results of the lipidomic analysis we have performed in order to understand the role of these proteins. And our phenotypic analyses have shown that these proteins are involved in salt tolerance. Additionally, we have observed that clustering of chloroplasts occurred when we overexpressed these proteins. And Arabidopsis double knock-out mutant for these proteins showed less chloroplasts attached to the nucleus in epidermal cells, suggesting that these proteins could be involved in these chloroplast signalling events after stress.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

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

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

Peripheral membrane TTL proteins safeguard cellulose synthesis under stress.

Land plants provide around eighty percent of biomass on Earth and roughly one-third corresponds to cellulose (Bar-On et al 2018). Despite its biological and societal importance, many aspects of cellulose biosynthesis and regulation remain elusive. Controlled primary cell wall remodeling allows plant growth under stressful conditions, but how these changes are conveyed to adjust cellulose synthesis is not well understood (Colin et al 2023). In this work, we identify that Tetratricopeptide Thioredoxin-Like (TTL) proteins, which we previous describe as a scaffold of brassinosteroids signalling components, are also new members of the cellulose synthase complex (CSC) and we describe their unique and hitherto unknown dynamic association with the CSC under cellulose-deficient conditions (Amorim-Silva et al 2019 and Kesten, García-Moreno, Amorim-Silva et al 2022). We found out that TTLs are essential for maintaining cellulose synthesis under high salinity conditions, 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. In addition, we are currently working to identify and characterize new components involved in TTLs function and dynamics during cellulose biosynthesis under saline stress conditions. References: Amorim-Silva et al. 2019 The Plant Cell Bar-On et al. 2018 Proc. Natl. Acad. Sci. Colin et al. 2023 The Plant Cell Kesten, García-Moreno, Amorim-Silva et al. 2022 Sci. Adv.Meeting attendance was supported by Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. This work was funded by the Spanish Ministry for Science and Innovation (MCIN/AEI/ 10.13039/501100011033) (PGC2018-098789-B-I00) and (PID2019-107657RB-C22) to MAB, NRL and AC respectively. The Andalusian Research Plan co-financed by the European Union (PAIDI 2020-PY20_00084) to MAB andde Andalucía UMA-FEDER project (grant UMA18-FEDERJA-154) to NRL, and the Swiss National foundation to CSR (SNF 31003A_163065/1 to AM). CK was supported by a Peter und Traudl Engelhorn-Stiftung fellowship, an ETH Career Seed Grant (SEED-05 19-2) of the ETH Foundation, an Emerging Investigator grant (NNF20OC0060564) of the Novo Nordisk Foundation, and an Experiment grant (R346-2020-1546) of the Lundbeck foundation. AGM and FP were supported by BES-2015-071256 and FPU19/02219 fellowships respectively. VAS was supported by an Emerging Investigator research project (UMA20-FEDERJA-007) and co-financed by the “Programa Operativo FEDER 2014-2020” and by the “Consejería de Economía y Conocimiento de la Junta de Andalucía”.

Atravesando fronteras: voces desde Haití hacia Sudamérica; libertad, igualdad y fraternidad

Es una invitación a poder quitarse el velo colonial, poder ver a Haití en sus orígenes como Ayiti. Es conocer y leer, de la mano de pensadores haitianos, el legado de la revolución negra que puso a temblar al mundo occidental, revolución que se oyó a partir de la mano de una mujer negra quien hizo sonar la campana de la libertad

Peripheral membrane proteins modulate stress tolerance by safeguarding cellulose synthases

International audienceControlled primary cell wall remodeling allows plant growth under stressful conditions, but how these changes are conveyed to adjust cellulose synthesis is not understood. 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 high-salinity conditions, establishing a stress-resilient cortical microtubule array, and stabilizing CSCs at the plasma membrane. To fulfill these functions, TTLs interact with CELLULOSE SYNTHASE 1 (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