Peptidyl-prolyl cis-trans isomerase ROF2 modulates intracellular pH homeostasis in Arabidopsis

[EN] Intracellular pH must be kept close to neutrality to be compatible with cellular functions, but the mechanisms of pH homeostasis and the responses to intracellular acidification are mostly unknown. In the plant Arabidopsis thaliana, we found that intracellular acid stress generated by weak organic acids at normal external pH induces expression of several chaperone genes, including ROF2, which encodes a peptidyl-prolyl cis-trans isomerase of the FK506-binding protein class. Loss of function of ROF2, and especially double mutation of ROF2 and the closely related gene ROF1, results in acid sensitivity. Over-expression of ROF2 confers tolerance to intracellular acidification by increasing proton extrusion from cells. The activation of the plasma membrane proton pump (H+-ATPase) is indirect: over-expression of ROF2 activates K+ uptake, causing depolarization of the plasma membrane, which activates the electrogenic H+ pump. The depolarization of ROF2 over-expressing plants explains their tolerance to toxic cations such as lithium, norspermidine and hygromycin B, whose uptake is driven by the membrane potential. As ROF2 induction and intracellular acidification are common consequences of many stresses, this mechanism of pH homeostasis may be of general importance for stress tolerance.This work was supported by grants BFU2008-00604 from the Ministerio de Ciencia e Innovacion (Madrid, Spain) and PROMETEO/2010/ 038 of the 'Conselleria de Educacion' (Valencia, Spain). We thank Dr Eugenio Grau (Sequencing Service, Instituto de Biologia Molecular y Celular de Plantas, Valencia, Spain) for sequencing of the various genes, and Dr Vicente Fornes (Instituto de Tecnologia Quimica, Valencia, Spain) for assistance with atomic absorption spectrophotometry. None of the authors has a conflict of interest to declare.Bissoli, G.; Niñoles Rodenes, R.; Fresquet Corrales, S.; Palombieri, S.; Bueso Ródenas, E.; Rubio, L.; Garcia-Sanchez, MJ.... (2012). Peptidyl-prolyl cis-trans isomerase ROF2 modulates intracellular pH homeostasis in Arabidopsis. Plant Journal. 70(4):704-716. https://doi.org/10.1111/j.1365-313X.2012.04921.xS70471670

PRX2 and PRX25, peroxidases regulated by COG1, are involved in seed longevity in Arabidopsis

[EN] Permeability is a crucial trait that affects seed longevity and is regulated by different polymers including proanthocyanidins, suberin, cutin and lignin located in the seed coat. By testing mutants in suberin transport and biosynthesis, we demonstrate the importance of this biopolymer to cope with seed deterioration. Transcriptomic analysis of cog1-2D, a gain-of-function mutant with increased seed longevity, revealed the upregulation of several peroxidase genes. Reverse genetics analysing seed longevity uncovered redundancy within the seed coat peroxidase gene family; however, after controlled deterioration treatment, seeds from the prx2 prx25 double and prx2 prx25 prx71 triple mutant plants presented lower germination than wild-type plants. Transmission electron microscopy analysis of the seed coat of these mutants showed a thinner palisade layer, but no changes were observed in proanthocyanidin accumulation or in the cuticle layer. Spectrophotometric quantification of acetyl bromide-soluble lignin components indicated changes in the amount of total polyphenolics derived from suberin and/or lignin in the mutant seeds. Finally, the increased seed coat permeability to tetrazolium salts observed in the prx2 prx25 and prx2 prx25 prx71 mutant lines suggested that the lower permeability of the seed coats caused by altered polyphenolics is likely to be the main reason explaining their reduced seed longevityRenard, J.; Martínez-Almonacid, I.; Sonntag, A.; Molina, I.; Moya-Cuevas, J.; Bissoli, G.; Muñoz-Bertomeu, J.... (2020). PRX2 and PRX25, peroxidases regulated by COG1, are involved in seed longevity in Arabidopsis. Plant Cell & Environment. 43(2):315-326. https://doi.org/10.1111/pce.13656S315326432Almagro, L., Gómez Ros, L. V., Belchi-Navarro, S., Bru, R., Ros Barceló, A., & Pedreño, M. A. (2008). Class III peroxidases in plant defence reactions. Journal of Experimental Botany, 60(2), 377-390. doi:10.1093/jxb/ern277Bailly, C., El-Maarouf-Bouteau, H., & Corbineau, F. (2008). From intracellular signaling networks to cell death: the dual role of reactive oxygen species in seed physiology. Comptes Rendus Biologies, 331(10), 806-814. doi:10.1016/j.crvi.2008.07.022Beisson, F., Li, Y., Bonaventure, G., Pollard, M., & Ohlrogge, J. B. (2007). The Acyltransferase GPAT5 Is Required for the Synthesis of Suberin in Seed Coat and Root of Arabidopsis. The Plant Cell, 19(1), 351-368. doi:10.1105/tpc.106.048033Belmonte, M. F., Kirkbride, R. C., Stone, S. L., Pelletier, J. M., Bui, A. Q., Yeung, E. C., … Harada, J. J. (2013). Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed. Proceedings of the National Academy of Sciences, 110(5), E435-E444. doi:10.1073/pnas.1222061110Bernards, M. A. (2002). Demystifying suberin. Canadian Journal of Botany, 80(3), 227-240. doi:10.1139/b02-017Bernards, M. A., Summerhurst, D. K., & Razem, F. A. (2004). Oxidases, peroxidases and hydrogen peroxide: The suberin connection. Phytochemistry Reviews, 3(1-2), 113-126. doi:10.1023/b:phyt.0000047810.10706.46Bolger, A. M., Lohse, M., & Usadel, B. (2014). Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics, 30(15), 2114-2120. doi:10.1093/bioinformatics/btu170Bueso, E., Muñoz-Bertomeu, J., Campos, F., Brunaud, V., Martínez, L., Sayas, E., … Serrano, R. (2013). ARABIDOPSIS THALIANA HOMEOBOX25 Uncovers a Role for Gibberellins in Seed Longevity. Plant Physiology, 164(2), 999-1010. doi:10.1104/pp.113.232223Châtelain, E., Satour, P., Laugier, E., Ly Vu, B., Payet, N., Rey, P., & Montrichard, F. (2013). Evidence for participation of the methionine sulfoxide reductase repair system in plant seed longevity. Proceedings of the National Academy of Sciences, 110(9), 3633-3638. doi:10.1073/pnas.1220589110Clerkx, E. J. M., Blankestijn-De Vries, H., Ruys, G. J., Groot, S. P. C., & Koornneef, M. (2004). Genetic differences in seed longevity of various Arabidopsis mutants. Physiologia Plantarum, 121(3), 448-461. doi:10.1111/j.0031-9317.2004.00339.xCosio, C., & Dunand, C. (2009). Specific functions of individual class III peroxidase genes. Journal of Experimental Botany, 60(2), 391-408. doi:10.1093/jxb/ern318Czechowski, T., Stitt, M., Altmann, T., Udvardi, M. K., & Scheible, W.-R. (2005). Genome-Wide Identification and Testing of Superior Reference Genes for Transcript Normalization in Arabidopsis. Plant Physiology, 139(1), 5-17. doi:10.1104/pp.105.063743Debeaujon, I., Léon-Kloosterziel, K. M., & Koornneef, M. (2000). Influence of the Testa on Seed Dormancy, Germination, and Longevity in Arabidopsis. Plant Physiology, 122(2), 403-414. doi:10.1104/pp.122.2.403Duroux, L., & Welinder, K. G. (2003). The Peroxidase Gene Family in Plants: A Phylogenetic Overview. Journal of Molecular Evolution, 57(4), 397-407. doi:10.1007/s00239-003-2489-3Fedi, F., O’Neill, C. 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Current Opinion in Plant Biology, 10(3), 252-259. doi:10.1016/j.pbi.2007.04.004GoffL TrapnellC&KelleyD(2014)CummeRbund: Analysis exploration manipulation and visualization of Cufflinks high‐throughput sequencing data. R package version 2.22.0.Gou, M., Hou, G., Yang, H., Zhang, X., Cai, Y., Kai, G., & Liu, C.-J. (2016). The MYB107 Transcription Factor Positively Regulates Suberin Biosynthesis. Plant Physiology, 173(2), 1045-1058. doi:10.1104/pp.16.01614Graça, J. (2015). Suberin: the biopolyester at the frontier of plants. Frontiers in Chemistry, 3. doi:10.3389/fchem.2015.00062Haughn, G., & Chaudhury, A. (2005). Genetic analysis of seed coat development in Arabidopsis. Trends in Plant Science, 10(10), 472-477. doi:10.1016/j.tplants.2005.08.005Herrero, J., Fernández-Pérez, F., Yebra, T., Novo-Uzal, E., Pomar, F., Pedreño, M. Á., … Zapata, J. M. (2013). Bioinformatic and functional characterization of the basic peroxidase 72 from Arabidopsis thaliana involved in lignin biosynthesis. 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Involvement of AtLAC15 in lignin synthesis in seeds and in root elongation of Arabidopsis. Planta, 224(5), 1185-1196. doi:10.1007/s00425-006-0300-6Li-Beisson, Y., Shorrosh, B., Beisson, F., Andersson, M. X., Arondel, V., Bates, P. D., … Ohlrogge, J. (2013). Acyl-Lipid Metabolism. The Arabidopsis Book, 11, e0161. doi:10.1199/tab.0161Mandel, T., Candela, H., Landau, U., Asis, L., Zilinger, E., Carles, C. C., & Williams, L. E. (2016). Differential regulation of meristem size, morphology and organization by the ERECTA, CLAVATA and class III HD-ZIP pathways. Development. doi:10.1242/dev.129973Milne, I., Stephen, G., Bayer, M., Cock, P. J. A., Pritchard, L., Cardle, L., … Marshall, D. (2012). Using Tablet for visual exploration of second-generation sequencing data. Briefings in Bioinformatics, 14(2), 193-202. doi:10.1093/bib/bbs012Molina, I., Bonaventure, G., Ohlrogge, J., & Pollard, M. (2006). The lipid polyester composition of Arabidopsis thaliana and Brassica napus seeds. Phytochemistry, 67(23), 2597-2610. doi:10.1016/j.phytochem.2006.09.011Molina, I., Ohlrogge, J. B., & Pollard, M. (2007). Deposition and localization of lipid polyester in developing seeds of Brassica napus and Arabidopsis thaliana. The Plant Journal, 53(3), 437-449. doi:10.1111/j.1365-313x.2007.03348.xMoreira‐Vilar F C. Siqueira‐Soares R deC Finger‐Teixeira A. Oliveira de D. M. Ferro AP Rocha daG J. Ferrarese M deLL Santos dosW. D. Ferrarese‐Filho O(2014).The Acetyl Bromide Method Is Faster Simpler and Presents Best Recovery of Lignin in Different Herbaceous Tissues than Klason and Thioglycolic Acid Methods. PLoS ONE 9:e110000.https://doi.org/10.1371/journal.pone.0110000Oñate-Sánchez, L., & Vicente-Carbajosa, J. (2008). DNA-free RNA isolation protocols for Arabidopsis thaliana, including seeds and siliques. BMC Research Notes, 1(1), 93. doi:10.1186/1756-0500-1-93Østergaard, L., Teilum, K., Mirza, O., Mattsson, O., Petersen, M., Welinder, K. G., … Henriksen, A. (2000). Plant Molecular Biology, 44(2), 231-243. doi:10.1023/a:1006442618860Pfaffl, M. W. (2001). A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research, 29(9), 45e-45. doi:10.1093/nar/29.9.e45Passardi, F., Longet, D., Penel, C., & Dunand, C. (2004). The class III peroxidase multigenic family in rice and its evolution in land plants☆☆☆. Phytochemistry, 65(13), 1879-1893. doi:10.1016/j.phytochem.2004.06.023Pedreira, J., Herrera, M. T., Zarra, I., & Revilla, G. (2010). The overexpression of AtPrx37, an apoplastic peroxidase, reduces growth in Arabidopsis. Physiologia Plantarum, 141(2), 177-187. doi:10.1111/j.1399-3054.2010.01427.xPollard, M., Beisson, F., Li, Y., & Ohlrogge, J. B. (2008). Building lipid barriers: biosynthesis of cutin and suberin. Trends in Plant Science, 13(5), 236-246. doi:10.1016/j.tplants.2008.03.003Quiroga, M., Guerrero, C., Botella, M. A., Barceló, A., Amaya, I., Medina, M. I., … Valpuesta, V. (2000). A Tomato Peroxidase Involved in the Synthesis of Lignin and Suberin. Plant Physiology, 122(4), 1119-1128. doi:10.1104/pp.122.4.1119Rains, M. K., Gardiyehewa de Silva, N. D., & Molina, I. (2017). Reconstructing the suberin pathway in poplar by chemical and transcriptomic analysis of bark tissues. Tree Physiology, 38(3), 340-361. doi:10.1093/treephys/tpx060Russell, W. R., Burkitt, M. J., Scobbie, L., & Chesson, A. (2005). EPR Investigation into the Effects of Substrate Structure on Peroxidase-Catalyzed Phenylpropanoid Oxidation. Biomacromolecules, 7(1), 268-273. doi:10.1021/bm050636oSano, N., Rajjou, L., North, H. M., Debeaujon, I., Marion-Poll, A., & Seo, M. (2015). Staying Alive: Molecular Aspects of Seed Longevity. Plant and Cell Physiology, 57(4), 660-674. doi:10.1093/pcp/pcv186Shigeto, J., Itoh, Y., Hirao, S., Ohira, K., Fujita, K., & Tsutsumi, Y. (2015). Simultaneously disrupting AtPrx2 , AtPrx25 and AtPrx71 alters lignin content and structure in Arabidopsis stem. 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Identification of novel seed longevity genes related to oxidative stress and seed coat by genome wide association studies and reverse genetics

[EN] Seed longevity is a polygenic trait of relevance for agriculture and for understanding the effect of environment on the ageing of biological systems. In order to identify novel longevity genes, we have phenotyped the natural variation of 270 ecotypes of the model plant,Arabidopsis thaliana, for natural ageing and for three accelerated ageing methods. Genome-wide analysis, using publicly available single-nucleotide polymorphisms (SNPs) data sets, identified multiple genomic regions associated with variation in seed longevity. Reverse genetics of 20 candidate genes in Columbia ecotype resulted in seven genes positive for seed longevity (PSAD1,SSLEA,SSTPR,DHAR1,CYP86A8,MYB47andSPCH) and five negative ones (RBOHD,RBOHE,RBOHF,KNAT7andSEP3). In this uniform genetic background, natural and accelerated ageing methods provided similar results for seed-longevity in knock-out mutants. The NADPH oxidases (RBOHs), the dehydroascorbate reductase (DHAR1) and the photosystem I subunit (PSAD1) highlight the important role of oxidative stress on seed ageing. The cytochrome P-450 hydroxylase, CYP86A8, and the transcription factors, MYB47, KNAT7 and SEP3, support the protecting role of the seed coat during seed ageing.Ministerio de Ciencia, Innovacion y Universidades, Grant/Award Number: BIO2017-88898-PRenard, J.; Niñoles Rodenes, R.; Martínez-Almonacid, I.; Gayubas, B.; Mateos-Fernández, R.; Bissoli, G.; Bueso Rodenas, E.... (2020). Identification of novel seed longevity genes related to oxidative stress and seed coat by genome wide association studies and reverse genetics. Plant Cell & Environment. 43(10):2523-2539. https://doi.org/10.1111/pce.13822S25232539431

A mechanism of growth inhibition by abscisic acid in germinating seeds of Arabidopsis thaliana based on inhibition of plasma membrane H+-ATPase and decreased cytosolic pH, K+, and anions

[EN] The stress hormone abscisic acid (ABA) induces expression of defence genes in many organs, modulates ion homeostasis and metabolism in guard cells, and inhibits germination and seedling growth. Concerning the latter effect, several mutants of Arabidopsis thaliana with improved capability for H+ efflux (wat1-1D, overexpression of AKT1 and ost2-1D) are less sensitive to inhibition by ABA than the wild type. This suggested that ABA could inhibit H+ efflux (H+ -ATPase) and induce cytosolic acidification as a mechanism of growth inhibition. Measurements to test this hypothesis could not be done in germinating seeds and we used roots as the most convenient system. ABA inhibited the root plasma-membrane H+ -ATPase measured in vitro (ATP hydrolysis by isolated vesicles) and in vivo (H+ efflux from seedling roots). This inhibition involved the core ABA signalling elements: PYR/PYL/RCAR ABA receptors, ABA-inhibited protein phosphatases (HAB1), and ABA-activated protein kinases (SnRK2.2 and SnRK2.3). Electrophysiological measurements in root epidermal cells indicated that ABA, acting through the PYR/PYL/RCAR receptors, induced membrane hyperpolarization (due to K+ efflux through the GORK channel) and cytosolic acidification. This acidification was not observed in the wat1-1D mutant. The mechanism of inhibition of the H+ -ATPase by ABA and its effects on cytosolic pH and membrane potential in roots were different from those in guard cells. ABA did not affect the in vivo phosphorylation level of the known activating site (penultimate threonine) of H+ -ATPase in roots, and SnRK2.2 phosphorylated in vitro the C-terminal regulatory domain of H+ -ATPase while the guard-cell kinase SnRK2.6/OST1 did not.This work was funded by grants BFU2011-22526 (to RS) and BIO2011-23446 (to PLR) of the Spanish 'Ministerio de Economia y Competitividad', Madrid, Spain, and grant PROMETEO/2010/038 (to RS) of the 'Generalitat Valenciana', Valencia, Spain. MGG was funded by a JAE-DOC contract of the Spanish 'Consejo Superior de Investigaciones Cientificas', Madrid, Spain. We thank Dr Toshinori Kinoshita (Nagoya University, Nagoya, Japan) for the rabbit antibody against the last 9 aa of AHA2 H+-ATPase with the penultimate Thr947 phosphorylated. We also thank the Proteomics Facility of the 'Centro Nacional de Biotecnologia', Madrid, Spain, for the attempts to identify the phosphorylation site of the H+-ATPase.Planes Ferrer, MD.; Niñoles Rodenes, R.; Rubio, L.; Bissoli, G.; Bueso Ródenas, E.; Garcia-Sanchez, MJ.; Alejandro Martínez, S.... (2015). A mechanism of growth inhibition by abscisic acid in germinating seeds of Arabidopsis thaliana based on inhibition of plasma membrane H+-ATPase and decreased cytosolic pH, K+, and anions. Journal of Experimental Botany. 66(3):813-825. https://doi.org/10.1093/jxb/eru442S81382566

Prolyl isomerases are important determinants of intracellular pH homeostasis in Arabidopsis thaliana

Our previous work in yeast has demonstrated that overexpression of FPR1, and others FKBP immunophilins, conferred tolerance to weak organic acids such as acetic and sorbic acid. FK506 binding proteins (FKBP) where originally identified as the cellular targets of the immunosuppressant drugs rapamycin an FK506. FKBPs are peptidyl-prolyl cis-trans isomerases (PPase EC 5.1.2.8) that catalize the isomerization of peptidyl prolyl bonds between cis and trans configuration. FKBP are ubiquitous proteins that can be found either as a single catalytic proteins or being part of more complex proteins. To assess the implication of FKBP proteins in weak acid tolerance in plants we have generated lines of Arabidopsis thaliana overexpressing two different proteins: yeast FPR1 an Arabidopsis FKBP65 (ROF2). We isolated knock-out mutant rof2 and rof1 from T-DNA mutant seeds collection of Salk institute. Finally we crossed the single mutants to get the double mutant rof1 x rof2. In presence of acetic acid transgenic lines overexpressing any of these genes grew better than wild type plant. On the other hand an AtFKBP65 loss-of-function mutant line showed weak acid sensitivity. We Observed a similar behavior in presence of toxic cations (Norspermidine, Hygromycim B) suggesting a role in K+ transport and we have got the confirmation with the growth al low levels of K+. We excluded the direct participation of plasma membrane ATPase because its activity in rof2 knock out mutant is higher. Furthermore ROF2 overexpression lines show a lower activity than wild-type. With 35S:: ROF2-GFP construction it was possible to see a cytosolic and nuclear cellular distribution that in presence of weak acid condition change: the ROF"-GFP leave the nuclear region. In absence of stress we have observed a gain of apical dominance in 35S::AtFKBP65 mutants and its loss in FKBP65 knock-out line. The roots of AtFKBP65 knock-out mutants have reduced number of lateral roots and exogenous application of IAA was ablBissoli, G. (2013). Prolyl isomerases are important determinants of intracellular pH homeostasis in Arabidopsis thaliana [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/27596Palanci

Salt-tolerance ofPhaseolus vulgarisL. is a function of the potentiationextent of antioxidant enzymes and the expression profiles of polyamine encoding genes

[EN] Selection and improvement of crop salt tolerance constitutes an urgent need for increasing agricultural and food production to feed the growing human population. Two bean (Phaseolus vulgaris L.) genotypes, namely Tema and Djadida varying in their tolerance to NaCl, were selected to investigate the determinant physiolog-ical and molecular mechanisms underlying salt tolerance. At the physiological level, salinity resulted in a sig-nificant decrease in growth, water status, stomatal conductance and photosynthesis for both genotypes. The potassium content was higher in the salt-tolerant genotype Tema under normal and saline conditions. Both genotypes had a significant increase in proline, glucose, fructose and sucrose concentrations, but glucose and fructose were differentially higher in the salt-tolerant genotype. While putrescine and spermidine concentra-tions decreased, spermine concentration significantly enhanced under salt stress along with antioxidant enzymes activities namely catalase, glutathione reductase and ascorbate peroxidase. The increase was higher in the salt-tolerant genotype. The expression levels of genes encoding enzymes involved in spermine biosyn-thesis revealed an upregulation of spermine synthase (SPMS) and a downregulation of polyamine oxidase (PAO4). Overall, the difference between genotypes is more quantitative than qualitative; the salt-tolerant genotype Tema displayed a better physiological and molecular response under salinity. By measuring these molecules in salt-stressed plants, several genotypes could be screened for breeding programmes leading to improve salt tolerance in beans. (c) 2021 SAAB. Published by Elsevier B.V. All rights reserved.Taïbi, K.; Abderrahima, LA.; Boussaid, M.; Bissoli, G.; Taïbi, F.; Achir, M.; Souana, K.... (2021). Salt-tolerance ofPhaseolus vulgarisL. is a function of the potentiationextent of antioxidant enzymes and the expression profiles of polyamine encoding genes. South African Journal of Botany. 140:114-122. https://doi.org/10.1016/j.sajb.2021.03.045S11412214

Endosperm Persistence in Arabidopsis Results in Seed Coat Fractures and Loss of Seed Longevity

Seeds are specialized plant organs that carry, nurture, and protect plant offspring. Developmental coordination between the three genetically distinct seed tissues (the embryo, endosperm, and seed coat) is crucial for seed viability. In this study, we explore the relationship between the TFs AtHB25 and ICE1. Previous results identified ICE1 as a target gene of AtHB25. In seeds, a lack of ICE1 (ice1-2) suppresses the enhanced seed longevity and impermeability of the overexpressing mutant athb25-1D, but surprisingly, seed coat lipid polyester deposition is not affected, as shown by the double-mutant athb25-1D ice1-2 seeds. zou-4, another mutant lacking the transcriptional program for proper endosperm maturation and for which the endosperm persists, also presents a high sensitivity to seed aging. Analysis of gso1, gso2, and tws1-4 mutants revealed that a loss of embryo cuticle integrity does not underlie the seed-aging sensitivity of ice1-2 and zou-4. However, scanning electron microscopy revealed the presence of multiple fractures in the seed coats of the ice1 and zou mutants. Thus, this study highlights the importance of both seed coat composition and integrity in ensuring longevity and demonstrates that these parameters depend on multiple factors

Silicon carbide thin films for EUV and soft X-ray applications

We have deposited SiC thin films using two different deposition techniques, Pulsed Electron Deposition (PED) and Pulsed Laser Deposition (PLD). The PED technique is a relatively new technique where a pulsed electron flux with high energy directly hits the target surface producing a plasma in a very similar way to PLD, where, instead, a pulsed laser beam is used. These two techniques can give very dense thin films with stoichiometry much closer to the target one\u2019s compared to others physical vapour deposition techniques (sputtering, e-beam). The major drawback of PED is that the thin film surface is affected by the presence of particulate, due to the impact of the high energy electron beam with the target. In the PLD system we used a magnetic field to curve the plasma path in the vacuum and we placed the substrate at 90\ub0 with respect to the target allowing the deposition of particulate-free samples. The characterization of the films has been made by measuring the optical reflectance vs angle of incidence in the EUV region (from 121.6nm down to 40.7 nm), taking measurements at different time from deposition. X-ray photoemission measurements have been also carried out to show stoichiometry and the presence of contaminants. Other measurements such as X-ray diffraction, atomic force microscopy and profiling were also carried out to check crystalline domains and surface roughness

An Arabidopsis Mutant Over-Expressing Subtilase SBT4.13 Uncovers the Role of Oxidative Stress in the Inhibition of Growth by Intracellular Acidification

Intracellular acid stress inhibits plant growth by unknown mechanisms and it occurs in acidic soils and as consequence of other stresses. In order to identify mechanisms of acid toxicity, we screened activation-tagging lines of Arabidopsis thaliana for tolerance to intracellular acidification induced by organic acids. A dominant mutant, sbt4.13-1D, was isolated twice and shown to over-express subtilase SBT4.13, a protease secreted into endoplasmic reticulum. Activity measurements and immuno-detection indicate that the mutant contains less plasma membrane H+-ATPase (PMA) than wild type, explaining the small size, electrical depolarization and decreased cytosolic pH of the mutant but not organic acid tolerance. Addition of acetic acid to wild-type plantlets induces production of ROS (Reactive Oxygen Species) measured by dichlorodihydrofluorescein diacetate. Acid-induced ROS production is greatly decreased in sbt4.13-1D and atrboh-D,F mutants. The latter is deficient in two major NADPH oxidases (NOXs) and is tolerant to organic acids. These results suggest that intracellular acidification activates NOXs and the resulting oxidative stress is important for inhibition of growth. The inhibition of acid-activated NOXs in the sbt4.13-1D mutant compensates inhibition of PMA to increase acid tolerance

Seed coat lignification level is crucial in Capsicum spp seed longevity

[EN] Capsicum (pepper) is known for its poor seed germination, particularly seed longevity is usually much shorter than other Solanaceae. However, the molecular mechanisms involved are mostly unknown in these species. The present study examines the differences in seed longevity among Capsicum species and varietal types. Feral or less domesticated species, such as Capsicum chinense and particularly Capsicum frutescens, showed higher germination rates than the more domesticated Capsicum annuum after accelerated seed aging treatments. In addition, variability was detected in the expression of genes involved in the response to seed deterioration. The differences observed in ASPG1 expression led us to study the seed protein profile in dry and germinating seeds. Seed storage protein mobilization during germination was faster in seed aging-resistant genotypes. Similarly, the transcriptional change observed for the orthologous gene of the trans-species regulator AtHB25 prompted us to study the structure and molecular components of the seed coat in peppers. All the Capsicum pepper accessions analyzed presented very lignified testa and we observed a positive correlation between the amount of lignin and seed viability. Our results provide essential information to explain the poor germination observed in pepper seeds and provide an experimental framework for future improvements in this important characterThe authors want to thank Marisol Gascon of the microscope service at the IBMCP. This work has been partially financed by the project PID2019-110221RR-C32, FEDER/ERDF funds.Bissoli, G.; Bono, M.; Martinez-Almonacid, I.; Moreno Peris, E.; Renard, J.; Espinosa, A.; Naranjo, MA.... (2022). Seed coat lignification level is crucial in Capsicum spp seed longevity. Physiologia Plantarum. 174(1):1-9. https://doi.org/10.1111/ppl.13600S19174