Uncovering salt tolerance mechanisms in pepper plants: a physiological and transcriptomic approach.

[EN] Background Pepper is one of the most cultivated crops worldwide, but is sensitive to salinity. This sensitivity is dependent on varieties and our knowledge about how they can face such stress is limited, mainly according to a molecular point of view. This is the main reason why we decided to develop this transcriptomic analysis. Tolerant and sensitive accessions, respectively called A25 and A6, were grown for 14 days under control conditions and irrigated with 70 mM of NaCl. Biomass, different physiological parameters and differentially expressed genes were analysed to give response to differential salinity mechanisms between both accessions. Results The genetic changes found between the accessions under both control and stress conditions could explain the physiological behaviour in A25 by the decrease of osmotic potential that could be due mainly to an increase in potassium and proline accumulation, improved growth (e.g. expansins), more efficient starch accumulation (e.g. BAM1), ion homeostasis (e.g. CBL9, HAI3, BASS1), photosynthetic protection (e.g. FIB1A, TIL, JAR1) and antioxidant activity (e.g. PSDS3, SnRK2.10). In addition, misregulation of ABA signalling (e.g. HAB1, ERD4, HAI3) and other stress signalling genes (e.g. JAR1) would appear crucial to explain the different sensitivity to NaCl in both accessions. Conclusions After analysing the physiological behaviour and transcriptomic results, we have concluded that A25 accession utilizes different strategies to cope better salt stress, being ABA-signalling a pivotal point of regulation. However, other strategies, such as the decrease in osmotic potential to preserve water status in leaves seem to be important to explain the defence response to salinity in pepper A25 plants.This work was financed by the INIA (Spain) and the Ministerio de Ciencia, Innovacion y Universidades (RTA2017-00030-C02-00) and the European Regional Development Fund (ERDF). Lidia Lopez-Serrano is a beneficiary of a doctoral fellowship (FPI-INIA).Lopez-Serrano, L.; Calatayud, Á.; López Galarza, SV.; Serrano Salom, R.; Bueso Rodenas, E. (2021). Uncovering salt tolerance mechanisms in pepper plants: a physiological and transcriptomic approach. BMC Plant Biology. 21(1):1-17. https://doi.org/10.1186/s12870-021-02938-2S11721

Seed tolerance to deterioration in arabidopsis is affected by virus infection

[EN] Seed longevity is the period during which the plant seed is able to germinate. This property is strongly influenced by environment conditions experienced by seeds during their formation and storage. In the present study we have analyzed how the biotic stress derived from the infection of Cauliflower mosaic virus (CaMV), Turnip mosaic virus (TuMV), Cucumber mosaic virus (CMV) and Alfalfa mosaic virus (AMV) affects seed tolerance to deterioration measuring germination rates after an accelerated aging treatment. Arabidopsis wild type plants infected with AMV and CMV rendered seeds with improved tolerance to deterioration when compared to the non -inoculated plants. On the other hand, CaMV infection generated seeds more sensitive to deterioration. No seeds were obtained from TuMV infected plants. Similar pattern of viral effects was observed in the double mutant athb22 athb25, which is more sensitive to accelerated seed aging than wild type. However, we observed a significant reduction of the seed germination for CMV (65% vs 55%) and healthy (50% vs 30%) plants in these mutants. The seed quality differences were overcomed using the A. thaliana athb25-1D dominant mutant, which over accumulated gibberellic acid (GA), except for TuMV which generated some siliques with low seed tolerance to deterioration. For AMV and TuMV (in athb25-1D), the seed quality correlated with the accumulation of the messengers of the gibberellin 3-oxidase family, the mucilage of the seed and the GA1. For CMV and CaMV it was not a good correlation suggesting that other factors are affecting seed viability. (C) 2017 Elsevier Masson SAS. All rights reserved.We thank L. Corachan and I. Martinez for their excellent technical assistance. This work was supported by grant BI02014-54862-R from the Spanish Direccion General de Investigacion Cientifica y Tecnica (DGICYT) and the Prometeo Program GV2014/010 from the Generalitat Valenciana.Bueso Rodenas, E.; Serrano Salom, R.; Pallas, V.; Sanchez Navarro, JA. (2017). Seed tolerance to deterioration in arabidopsis is affected by virus infection. Plant Physiology and Biochemistry. 116:1-8. https://doi.org/10.1016/j.plaphy.2017.04.020S1811

BvCOLD1: A novel aquaporin from sugar beet (Beta vulgaris L.) involved in boron homeostasis and abiotic stress

[EN] In this report we have identified BvCOLD1, a novel aquaporin from sugar beet (Beta vulgaris) which is only conserved in the Chenopodioideae family. BvCOLD1 is expressed in all plant organs investigated and located in the endoplasmic reticulum. Transport experiments in yeast indicated that BvCOLD1 is able to transport glycerol and boron, the most limiting oligoelement for sugar beet cultivation. Overexpression of BvCOLD1 in Arabidopsis thaliana plants conferred tolerance to cold, to different abiotic stresses and the ability to grow under boron limiting conditions, therefore this novel aquaporin may be an important target to design new crops with enhanced boron homeostasis and abiotic stress tolerance.Ministerio de Economia y Competitividad, Grant/Award Number: BIO2016-77776-P; Secretaria de Estado de Investigacion, Desarrollo e Innovacion, Grant/Award Number: AGL2013-47886-R; Direccion General Investigacion Cientifica; MINECO, Grant/Award Numbers: BIO2014-61826 and BIO2016-77776-P; Universitat Politecnica de Valencia, Grant/Award Number: PAID-06-10-1496Porcel, R.; Bustamante-González, AJ.; Ros, R.; Serrano Salom, R.; Mulet, JM. (2018). BvCOLD1: A novel aquaporin from sugar beet (Beta vulgaris L.) involved in boron homeostasis and abiotic stress. Plant Cell & Environment. 41(12):2844-2857. https://doi.org/10.1111/pce.13416284428574112Aroca, R., Amodeo, G., Fernández-Illescas, S., Herman, E. M., Chaumont, F., & Chrispeels, M. J. (2004). The Role of Aquaporins and Membrane Damage in Chilling and Hydrogen Peroxide Induced Changes in the Hydraulic Conductance of Maize Roots. Plant Physiology, 137(1), 341-353. doi:10.1104/pp.104.051045Biancardi, E. (2005). Genetics and Breeding of Sugar Beet. doi:10.1201/9781482280296Bienert, G. P., Møller, A. L. B., Kristiansen, K. A., Schulz, A., Møller, I. M., Schjoerring, J. K., & Jahn, T. P. (2006). Specific Aquaporins Facilitate the Diffusion of Hydrogen Peroxide across Membranes. Journal of Biological Chemistry, 282(2), 1183-1192. doi:10.1074/jbc.m603761200Bissoli, G., Niñoles, R., Fresquet, S., Palombieri, S., Bueso, E., Rubio, L., … Serrano, R. (2012). Peptidyl-prolyl cis-trans isomerase ROF2 modulates intracellular pH homeostasis in Arabidopsis. 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Early gene expression events in the laminar abscission zone of abscission-promoted citrus leaves after a cycleof water stress/rehydration: involvement of CitbHLH1

[EN] Leaf abscission is a common response of plants to drought stress. Some species, such as citrus, have evolved a specific behaviour in this respect, keeping their leaves attached to the plant body during water stress until this is released by irrigation or rain. This study successfully reproduced this phenomenon under controlled conditions (24h of water stress followed by 24h of rehydration) and used it to construct a suppression subtractive hybridization cDNA library enriched in genes involved in the early stages of rehydration-promoted leaf abscission after water stress. Sequencing of the library yielded 314 unigenes, which were spotted onto nylon membranes. Membrane hybridization with petiole (Pet)- and laminar abscission zone (LAZ)-enriched RNA samples corresponding to early steps in leaf abscission revealed an almost exclusive preferential gene expression programme in the LAZ. The data identified major processes such as protein metabolism, cell-wall modification, signalling, control of transcription and vesicle production, and transport as the main biological processes activated in LAZs during the early steps of rehydration-promoted leaf abscission after water stress. Based on these findings, a model for the early steps of citrus leaf abscission is proposed. In addition, it is suggested that CitbHLH1, the putative citrus orthologue of Arabidopsis BIGPETAL, may play major roles in the control of abscission-related events in citrus abscission zonesWork at the Centre de Genomica was supported by INIA grant RTA08-00065-00-00 and Ministerio de Ciencia e Innovacion-FEDER grants AGL2007-65437-C04-01, PSG-06-0000-2009-8, IPT-01-0000-2010-43, and AGL2011-30240. J.A. and P. M. were recipients of INIA predoctoral fellowships and M. C. and A. C. of INIA/CCAA and 'Ramon y Cajal' postdoctoral contracts, respectively. The help and expertise of E. Blazquez, I. Sanchis, and A. Boix are gratefully acknowledged.Agustí, J.; Gimeno, J.; Merelo, P.; Serrano Salom, R.; Cercós, M.; Conesa, A.; Talón, M.... (2012). Early gene expression events in the laminar abscission zone of abscission-promoted citrus leaves after a cycleof water stress/rehydration: involvement of CitbHLH1. Journal of Experimental Botany. 63:6079-6091. https://doi.org/10.1093/jxb/ers270S607960916

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

Role of Arabidopsis UV RESISTANCE LOCUS 8 in plant growth reduction under osmotic stress and low levels of UV-B

In high-light environments, plants are exposed to different types of stresses, such as an excess of UV-B, but also drought stress which triggers a common morphogenic adaptive response resulting in a general reduction of plant growth. Here, we report that the Arabidopsis thaliana UV RESISTANCE LOCUS 8 (UVR8) gene, a known regulator of the UV-B morphogenic response, was able to complement a Saccharomyces cerevisiae osmo-sensitive mutant and its expression was induced after osmotic or salt stress in Arabidopsis plants. Under low levels of UV-B, plants overexpressing UVR8 are dwarfed with a reduced root development and accumulate more flavonoids compared to control plants. The growth defects are mainly due to the inhibition of cell expansion. The growth inhibition triggered by UVR8 overexpression in plants under low levels of UV-B was exacerbated by mannitol-induced osmotic stress, but it was not significantly affected by ionic stress. In contrast, uvr8-6 mutant plants do not differ from wild-type plants under standard conditions, but they show an increased shoot growth under high-salt stress. Our data suggest that UVR8-mediated accumulation of flavonoid and possibly changes in auxin homeostasis are the underlying mechanism of the observed growth phenotypes and that UVR8 might have an important role for integrating plant growth and stress signals.This work was supported by the Interuniversity Attraction Poles Programme (IUAP P7/29 'MARS') initiated by the Belgian Science Policy Office, by grants from Ghent University (Bijzonder Onderzoeksfonds Methusalem project no. BOF08/01M00408), and by grants to the MIUR project FIRB Plant-STRESS.Fasano, R.; Gonzalez, N.; Tosco, A.; Dal Piaz, F.; Docimo, T.; Serrano Salom, R.; Grillo, S.... (2014). Role of Arabidopsis UV RESISTANCE LOCUS 8 in plant growth reduction under osmotic stress and low levels of UV-B. Molecular Plant. 7(5):773-791. https://doi.org/10.1093/mp/ssu002S7737917

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. 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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

The insecticide DDT targets the OSCP and subunit D of the Apis mellifera ATP synthase

1, 1-bis (p-Chlorophenyl) -2, 2, 2-trichloroethane (DDT) has been used for control of malaria mosquitoes and other insect vectors of human diseases since 1945. Its use poses an environmental dilemma and efforts to replace it have been hampered by lack of information about its molecular target. This work identifies the 23 kDa band responsible for the DDT sensitivity in bees, as the OSCP and subunit "d" of the ATP synthase. The OSCP of the bee's ATP synthase contained 207 amino acids compared to 190 in bovine, which is insensitive to DDT, and the identities were only 47%. Subunit "d" of the bees had no counterpart in the bovine. Whether DDT is interacting only with OSCP, only with subunit "d", or with both subunits, remains to be assessed. Identification of the molecular target of DDT will lead the way to new target based insecticides aimed to protect plant, combat malaria and other insect transmitted diseases.This work was supported by grant Dnr nr 348-2004-4977 from the Swedish Research Council.Younis, HM.; Serrano Salom, R.; Abdel Razik, RK.; Rydström, J. (2011). The insecticide DDT targets the OSCP and subunit D of the Apis mellifera ATP synthase. Journal of Bioenergetics and Biomembranes. 43:457-463. https://doi.org/10.1007/s10863-011-9378-zS45746343Berenbaum M (2005) http://www.wasshingtonpost.com/wp-dyn/content/article/2005/06/04/AR2005060400130.htmlBowman MC, Acree F Jr, Corbett MK (1960) J Agr Food Chem 8:406–408Fenn JB, Mann M, Meng CK, Wong SF, Whitehouse CM (1989) Science 246:64–71Gribble GW (1994) Environ Sci Technol 28:310A–318AHayes WJ Jr (1975) Toxicology of pesticides. Williams & Wilkins, Baltimore, pp 11–14Higuti T, Kuroiwa K, Miyazaki S, Toda H, Kakuno T, Sakiyama F (1993) J Biochem 114:714–717Hong S, Pedersen PL (2008) Microbiol Mol Biol Rev 72:590–641Jukes TH (1977) Trends Biochem Sci 2:38–39Laemmli UK (1970) Nature (London) 227:680–685Lauger P, Martin H, Muller P (1944) Helv Chim Acta 27:892Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) J Biol Chem 193:265–275Mayer B, Witting I, Trifilieff E, Karas M, Schagger H (2007) Mol Cell Proteomics 6.10:1690–1699Metcalf RL (1972) In pest control: strategies for the future. Natl Acad Sci Washington DC, pp 137–156Metcalf RL (1973) J Agric Food Chem 21:511–519Metcalf RL, Kapoor IP, Hirwe AS (1971) Bull World Health Organ 44:363–374Mitchell P (1966) Biol Rev 41:455–502Nedergaard J, Cannon B (1979) Methods Enzymol 55:3–28Pullman ME, Penefsky HS, Datta A, Racker E (1960) J Biol Chem 235:3322–3329Racker E, Chien TF, Kandrach A (1975) FEBS Lett 57:14–18Rees DM, Leslie AGW, Walker JE (2009) Proc Natl Acad Sci USA 106:21597–21601Roberts DR, Laughlin LL, Hsheih P, Legters LJ (1997) Emerg Infect Dis 3(3):1–11Serrano R, Kanner BI, Racker E (1976) J Biol Chem 251:2453–2461Shevchenko A, Wilm M, Vorm O, Mann M (1996) Anal Chem 68:850–858Smith AL (1967) Methods Enzymol 10:81–86Steen H, Mann M (2004) Nat Rev Mol Cell Biol 5:699–711UNEP (2007) Future plans for work on DDT elimination. A Stockholm Convention, Secretariat Position Paper. November 2007Younis HM, Abo-El-Saad MM, Abdel-Razik RK, Abo-Seda SA (2002) Biotechnol Appl Biochem 35:9–17Younis HM, Telford JN, Koch RB (1978) Pestic Biochem Physiol 8:271–27

A novel role for protein kinase Gcn2 in yeast tolerance to intracellular acid stress

Intracellular pH conditions many cellular systems, but its mechanisms of regulation and perception are mostly unknown. We have identified two yeast genes important for tolerance to intracellular acidification caused by weak permeable acids. One corresponded to LEU2 and functions by removing the dependency of the leu2 mutant host strain on uptake of extracellular leucine. Leucine transport is inhibited by intracellular acidification, and either leucine oversupplementation or overexpression of the transporter gene BAP2 improved acid growth. Another acid-tolerance gene is GCN2, encoding a protein kinase activated by uncharged tRNAs during amino acid starvation. Gcn2 phosphorylates eIF2¿ (eukaryotic initiation factor 2¿) (Sui2) at Ser51 and this inhibits general translation, but activates that of Gcn4, a transcription factor for amino acid biosynthetic genes. Intracellular acidification activates Gcn2 probably by inhibition of aminoacyl-tRNA synthetases because we observed accumulation of uncharged tRNAleu without leucine depletion. Gcn2 is required for leucine transport and a gcn2-null mutant is sensitive to acid stress if auxotrophic for leucine. Gcn4 is required for neither leucine transport nor acid tolerance, but a S51A sui2 mutant is acid-sensitive. This suggests that Gcn2, by phosphorylating eIF2¿, may activate translation of an unknown regulator of amino acid transporters different from Gcn4.This work was funded by grants from the Spanish Ministerio de Ciencia e Innovacion (Madrid) [grant number BFU2008-00604] and the Generalitat Valenciana (Valencia) [grant number Prometeo/2010/038].Hueso Lorente, G.; Aparicio Sanchis, R.; Montesinos De Lago, C.; Lorenz, S.; Murguía Ibáñez, JR.; Serrano Salom, R. (2012). A novel role for protein kinase Gcn2 in yeast tolerance to intracellular acid stress. Biochemical Journal. 441(1):255-264. doi:10.1042/BJ20111264S255264441