Transcription factor Sp3 as target for SUMOylation in vivo

A group of sequence-specific DNA-binding proteins related to the transcription factor Sp1 (specificity protein 1) has been implicated in the regulation of many different genes, since binding sites for these transcription factors (GC/GT boxes) are a recurrent motif in regulatory sequences of these genes. In contrast to the transcriptional activators Sp1 and Sp4, the ubiquitously expressed Sp3 protein can both activate and repress transcription. The complex activity of Sp3 depends on two glutamine-rich activation domains, similar to those found in Sp1 and Sp4, and, adjacent to these, on an inhibitory domain unique to Sp3. The critical lysine residue in the Sp3 inhibitory domain lies within a consensus motif (IK551EE) that targets proteins for SUMO modification. SUMO (small ubiquitin-related modifier) is covalently attached to lysine residues in target proteins via an isopeptide linkage in a multi-step process that is analogous to ubiquitination. The present work analyses various aspects of SUMO conjugation to Sp3 in vivo. Studying modification of Sp3 by SUMO is complicated by the existence of a number of Sp3 isoforms. Immunoblot analyses revealed four distinct Sp3 proteins, two slow migrating of more than 100 kDa and two fast migrating species. Seven to eight Sp3 bands appeared, when cells were lysed in denaturing conditions. The additional protein species represent SUMO modified Sp3 isoforms. Currently, it is not known whether the relative distribution of the different Sp3 isoforms is regulated. However, a significant shift towards the long isoforms of Sp3, however, is observed in Sp1-/- ES cells demonstrating that Sp3 isoform expression principally can change in vivo. In addition, this observation suggests that the long isoforms of Sp3 may take over Sp1 functions under Sp1 knockout conditions. When Sp3 is overexpressed along with SUMO1 and SUMO2 in cells in culture, attachment of both SUMO paralogues to Sp3 occurred with almost equal efficiency. Beside lysine 551 within the inhibitory domain, there are two other potential SUMOylation sites in Sp3 (VKQE at position 9 and IKDE at position 120). This study revealed that SUMOylation takes place exclusively at K551, present in all four isoforms. Visualization of endogenous Sp3 by immumofluorescence showed a sponge-like, diffuse appearance, located predominantly in the nucleus. Evolutionally closely related Sp family members Sp1 and Sp2 are also located in the nucleus and the subcellular localization patterns are similar to Sp3. Ectopic expression of SUMO1 fused to GFP (green fluorescent protein) led to the accumulation of this fusion protein within subnuclear “dots” or PODs (promyelocytic leukemia oncogenic domains), whereas endogenous Sp3 remained diffusely distributed throughout nuclei. In addition, the wild-type Sp3 isoforms and the SUMOylation-deficient mutants of Sp3were located in the nucleus exhibiting also a sponge-like, diffuse appearance. Analyzing the Sp3 expression in different cell lines and mouse organs revealed that the relative level of Sp3 modification by SUMO is not cell line or organ dependent. In addition, no variation in Sp3 expression pattern after serum starvation, serum induction and heat shock was observed. Ultraviolet radiation or Tumor Necrosis Factor alpha and Cycloheximide treatment of mammalian cells did not alter the SUMOylation level of Sp3 protein in our experimental conditions. A significant reduction in Sp3 SUMO modification was observed upon treatment with MG-132, a cell-permeable inhibitor of the proteasome. Possibly this proteasome inhibitor prevents proteasome degradation of SUMO specific isopeptidase, which subsequently remove the Sp3-SUMO moiety. PIAS1 (protein inhibitor of activated STAT) was previously cloned in a two-hybrid screen by using the inhibitory domain of Sp3. Moreover, it was shown that PIAS1 strongly enhances SUMO-modification of Sp3 in vitro and thus acts as an SUMO E3 ligase towards Sp3. PIAS1-associated proteins might confer substrate specificity towards Sp3 and other transcription factors and/or regulate PIAS1 activity in vivo. For the purification and identification of PIAS1-associated proteins, a number of C-terminal tagged expression plasmids were constructed for constitutive and inducible expression. The dual-tag affinity purification system established in this thesis work contains a small 15 amino acid artificial tag (BiotinTAG) that becomes biotinylated by the BirA ligase upon co-transfection of an appropriate expression construct. To enhance specificity, a second tag was included in the expression vectors (Calmodulin Binding Peptide or alternatively FLAG or Triple-FLAG). In addition, dual tags expression plasmids for Sp3 were constructed. The establishment of stable cell lines expressing these fusion proteins in an inducible manner was initiated. Such cell lines might be ideal for further analyzes of PIAS1 activities and to purify PIAS1 (Sp3) associated factors

Stress tolerance mechanisms in Juncus: responses to salinity and drought in three Juncus species adapted to different natural environments

[EN] Comparative studies on the responses to salinity and drought were carried out in three Juncus species, two halophytes (Juncus maritimus Lam. and Juncus acutus L.) and one more salt-sensitive (Juncus articulatus L.). Salt tolerance in Juncus depends on the inhibition of transport of toxic ions to the aerial part. In the three taxa studied Na+ and Cl accumulated to the same extent in the roots of salt treated plants; however, ion contents were lower in the shoots and correlated with the relative salt sensitivity of the species, with the lowest levels measured in the halophytes. Activation of K+ transport at high salt concentration could also contribute to salt tolerance in the halophytes. Maintenance of cellular osmotic balance is mostly based on the accumulation of sucrose in the three species. Yet, neither the relative salt-induced increase in sugar content nor the absolute concentrations reached can explain the observed differences in salt tolerance. In contrast, proline increased significantly in the presence of salt only in the salt-tolerant J. maritimus and J. acutus, but not in J. articulatus. Similar patterns of osmolyte accumulation were observed in response to water stress, supporting a functional role of proline in stress tolerance mechanisms in JuncusThis work was partly funded by a grant to O.V. from the Spanish Ministry of Science and Innovation (Project CGL2008-00438/BOS), with contribution by the European Regional Development Fund. Mohamad Al Hassan was a recipient of an Erasmus Mundus pre-doctoral scholarship financed by the European Commission (Welcome Consortium)Al Hassan, M.; López Gresa, MP.; Boscaiu Neagu, MT.; Vicente Meana, Ó. (2016). Stress tolerance mechanisms in Juncus: responses to salinity and drought in three Juncus species adapted to different natural environments. FUNCTIONAL PLANT BIOLOGY. 43:949-960. https://doi.org/10.1071/FP16007S94996043Al Hassan, M., Chaura, J., López-Gresa, M. P., Borsai, O., Daniso, E., Donat-Torres, M. P., … Boscaiu, M. (2016). Native-Invasive Plants vs. Halophytes in Mediterranean Salt Marshes: Stress Tolerance Mechanisms in Two Related Species. Frontiers in Plant Science, 7. doi:10.3389/fpls.2016.00473Albert, R., & Popp, M. (1977). Chemical composition of halophytes from the Neusiedler Lake region in Austria. Oecologia, 27(2), 157-170. doi:10.1007/bf00345820Ashraf, M., & Foolad, M. R. (2007). Roles of glycine betaine and proline in improving plant abiotic stress resistance. Environmental and Experimental Botany, 59(2), 206-216. doi:10.1016/j.envexpbot.2005.12.006Bartels, D., & Sunkar, R. (2005). Drought and Salt Tolerance in Plants. Critical Reviews in Plant Sciences, 24(1), 23-58. doi:10.1080/07352680590910410Bates, L. S., Waldren, R. P., & Teare, I. D. (1973). Rapid determination of free proline for water-stress studies. Plant and Soil, 39(1), 205-207. doi:10.1007/bf00018060Boscaiu, M., Ballesteros, G., Naranjo, M. A., Vicente, O., & Boira, H. (2011). Responses to salt stress in Juncus acutus and J. maritimus during seed germination and vegetative plant growth. Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology, 145(4), 770-777. doi:10.1080/11263504.2011.628446Boscaiu, M., Lull, C., Llinares, J., Vicente, O., & Boira, H. (2012). Proline as a biochemical marker in relation to the ecology of two halophytic Juncus species. Journal of Plant Ecology, 6(2), 177-186. doi:10.1093/jpe/rts017Bose, J., Rodrigo-Moreno, A., & Shabala, S. (2013). ROS homeostasis in halophytes in the context of salinity stress tolerance. Journal of Experimental Botany, 65(5), 1241-1257. doi:10.1093/jxb/ert430Boyer, J. S. (1982). Plant Productivity and Environment. Science, 218(4571), 443-448. doi:10.1126/science.218.4571.443Chen, T. H. H., & Murata, N. (2008). Glycinebetaine: an effective protectant against abiotic stress in plants. Trends in Plant Science, 13(9), 499-505. doi:10.1016/j.tplants.2008.06.007Clarke, L. D., & Hannon, N. J. (1970). The Mangrove Swamp and Salt Marsh Communities of the Sydney District: III. Plant Growth in Relation to Salinity and Waterlogging. The Journal of Ecology, 58(2), 351. doi:10.2307/2258276Drabkova, L., Kirschner, J., & Vlcek, C. (2006). Phylogenetic relationships within Luzula DC. and Juncus L. (Juncaceae): A comparison of phylogenetic signals of trnL-trnF intergenic spacer, trnL intron and rbcL plastome sequence data. Cladistics, 22(2), 132-143. doi:10.1111/j.1096-0031.2006.00095.xDuBois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric Method for Determination of Sugars and Related Substances. Analytical Chemistry, 28(3), 350-356. doi:10.1021/ac60111a017Espinar, J. L., Garcia, L. V., & Clemente, L. (2005). Seed storage conditions change the germination pattern of clonal growth plants in Mediterranean salt marshes. American Journal of Botany, 92(7), 1094-1101. doi:10.3732/ajb.92.7.1094Espinar, J. L., García, L. V., Figuerola, J., Green, A. J., & Clemente, L. (2006). Effects of salinity and ingestion by ducks on germination patterns of Juncus subulatus seeds. Journal of Arid Environments, 66(2), 376-383. doi:10.1016/j.jaridenv.2005.11.001Fita, A., Rodríguez-Burruezo, A., Boscaiu, M., Prohens, J., & Vicente, O. (2015). Breeding and Domesticating Crops Adapted to Drought and Salinity: A New Paradigm for Increasing Food Production. Frontiers in Plant Science, 6. doi:10.3389/fpls.2015.00978Flowers, T. J., & Colmer, T. D. (2008). Salinity tolerance in halophytes*. New Phytologist, 179(4), 945-963. doi:10.1111/j.1469-8137.2008.02531.xFlowers, T. J., Hajibagheri, M. A., & Clipson, N. J. W. (1986). Halophytes. The Quarterly Review of Biology, 61(3), 313-337. doi:10.1086/415032Flowers, T. J., Munns, R., & Colmer, T. D. (2014). Sodium chloride toxicity and the cellular basis of salt tolerance in halophytes. Annals of Botany, 115(3), 419-431. doi:10.1093/aob/mcu217Gagneul, D., Aïnouche, A., Duhazé, C., Lugan, R., Larher, F. R., & Bouchereau, A. (2007). A Reassessment of the Function of the So-Called Compatible Solutes in the Halophytic Plumbaginaceae Limonium latifolium. Plant Physiology, 144(3), 1598-1611. doi:10.1104/pp.107.099820GIL, R., LULL, C., BOSCAIU, M., BAUTISTA, I., LIDÓN, A., & VICENTE, O. (2011). Soluble Carbohydrates as Osmolytes in Several Halophytes from a Mediterranean Salt Marsh. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 39(2), 09. doi:10.15835/nbha3927176Gil, R., Boscaiu, M., Lull, C., Bautista, I., Lidón, A., & Vicente, O. (2013). Are soluble carbohydrates ecologically relevant for salt tolerance in halophytes? Functional Plant Biology, 40(9), 805. doi:10.1071/fp12359Gil, R., Bautista, I., Boscaiu, M., Lidon, A., Wankhade, S., Sanchez, H., … Vicente, O. (2014). Responses of five Mediterranean halophytes to seasonal changes in environmental conditions. AoB PLANTS, 6(0), plu049-plu049. doi:10.1093/aobpla/plu049Glenn, E. (1999). Salt Tolerance and Crop Potential of Halophytes. Critical Reviews in Plant Sciences, 18(2), 227-255. doi:10.1016/s0735-2689(99)00388-3GORHAM, J., HUGHES, L., & WYN JONES, R. G. (2006). Chemical composition of salt-marsh plants from Ynys Môn (Anglesey): the concept of physiotypes. Plant, Cell & Environment, 3(5), 309-318. doi:10.1111/1365-3040.ep11581858Grieve, C. M., & Grattan, S. R. (1983). Rapid assay for determination of water soluble quaternary ammonium compounds. Plant and Soil, 70(2), 303-307. doi:10.1007/bf02374789Gupta, B., & Huang, B. (2014). Mechanism of Salinity Tolerance in Plants: Physiological, Biochemical, and Molecular Characterization. International Journal of Genomics, 2014, 1-18. doi:10.1155/2014/701596Hamamoto, S., Horie, T., Hauser, F., Deinlein, U., Schroeder, J. I., & Uozumi, N. (2015). HKT transporters mediate salt stress resistance in plants: from structure and function to the field. Current Opinion in Biotechnology, 32, 113-120. doi:10.1016/j.copbio.2014.11.025Hariadi, Y., Marandon, K., Tian, Y., Jacobsen, S.-E., & Shabala, S. (2010). Ionic and osmotic relations in quinoa (Chenopodium quinoa Willd.) plants grown at various salinity levels. Journal of Experimental Botany, 62(1), 185-193. doi:10.1093/jxb/erq257Jones, E., Simpson, D., Hodkinson, T., Chase, M., & Parnell, J. (2007). The Juncaceae-Cyperaceae Interface: A Combined Plastid Sequence Analysis. Aliso, 23(1), 55-61. doi:10.5642/aliso.20072301.07Kumari, A., Das, P., Parida, A. K., & Agarwal, P. K. (2015). Proteomics, metabolomics, and ionomics perspectives of salinity tolerance in halophytes. Frontiers in Plant Science, 6. doi:10.3389/fpls.2015.00537Munns, R., & Termaat, A. (1986). Whole-Plant Responses to Salinity. Functional Plant Biology, 13(1), 143. doi:10.1071/pp9860143Munns, R., & Tester, M. (2008). Mechanisms of Salinity Tolerance. Annual Review of Plant Biology, 59(1), 651-681. doi:10.1146/annurev.arplant.59.032607.092911Naidoo, G., & Kift, J. (2006). Responses of the saltmarsh rush Juncus kraussii to salinity and waterlogging. Aquatic Botany, 84(3), 217-225. doi:10.1016/j.aquabot.2005.10.002Niu, X., Bressan, R. A., Hasegawa, P. M., & Pardo, J. M. (1995). Ion Homeostasis in NaCl Stress Environments. Plant Physiology, 109(3), 735-742. doi:10.1104/pp.109.3.735Ozgur, R., Uzilday, B., Sekmen, A. H., & Turkan, I. (2013). Reactive oxygen species regulation and antioxidant defence in halophytes. Functional Plant Biology, 40(9), 832. doi:10.1071/fp12389Pang, Q., Chen, S., Dai, S., Chen, Y., Wang, Y., & Yan, X. (2010). Comparative Proteomics of Salt Tolerance inArabidopsis thalianaandThellungiella halophila. Journal of Proteome Research, 9(5), 2584-2599. doi:10.1021/pr100034fPartridge, T. R., & Wilson, J. B. (1987). Salt tolerance of salt marsh plants of Otago, New Zealand. New Zealand Journal of Botany, 25(4), 559-566. doi:10.1080/0028825x.1987.10410086RAVEN, J. A. (1985). TANSLEY REVIEW No. 2. REGULATION OF PH AND GENERATION OF OSMOLARITY IN VASCULAR PLANTS: A COST-BENEFIT ANALYSIS IN RELATION TO EFFICIENCY OF USE OF ENERGY, NITROGEN AND WATER. New Phytologist, 101(1), 25-77. doi:10.1111/j.1469-8137.1985.tb02816.xRodrı́guez-Navarro, A. (2000). Potassium transport in fungi and plants. Biochimica et Biophysica Acta (BBA) - Reviews on Biomembranes, 1469(1), 1-30. doi:10.1016/s0304-4157(99)00013-1Rozema, J. (1976). An Ecophysiological Study on the Response to Salt of Four Halophytic and Glycophytic Juncus Species. Flora, 165(2), 197-209. doi:10.1016/s0367-2530(17)31845-5Rozema, J. (1991). Growth, water and ion relationships of halophytic monocotyledonae and dicotyledonae; a unified concept. Aquatic Botany, 39(1-2), 17-33. doi:10.1016/0304-3770(91)90019-2Smirnoff, N., & Cumbes, Q. J. (1989). Hydroxyl radical scavenging activity of compatible solutes. Phytochemistry, 28(4), 1057-1060. doi:10.1016/0031-9422(89)80182-7Szabados, L., & Savouré, A. (2010). Proline: a multifunctional amino acid. Trends in Plant Science, 15(2), 89-97. doi:10.1016/j.tplants.2009.11.009Vicente, M. J., Conesa, E., Álvarez-Rogel, J., Franco, J. A., & Martínez-Sánchez, J. J. (2007). Effects of various salts on the germination of three perennial salt marsh species. Aquatic Botany, 87(2), 167-170. doi:10.1016/j.aquabot.2007.04.004Vinocur, B., & Altman, A. (2005). Recent advances in engineering plant tolerance to abiotic stress: achievements and limitations. Current Opinion in Biotechnology, 16(2), 123-132. doi:10.1016/j.copbio.2005.02.001Watson, E. B., & Byrne, R. (2009). Abundance and diversity of tidal marsh plants along the salinity gradient of the San Francisco Estuary: implications for global change ecology. Plant Ecology, 205(1), 113-128. doi:10.1007/s11258-009-9602-7Weimberg, R. (1987). Solute adjustments in leaves of two species of wheat at two different stages of growth in response to salinity. Physiologia Plantarum, 70(3), 381-388. doi:10.1111/j.1399-3054.1987.tb02832.xZhu, J.-K. (2001). Plant salt tolerance. Trends in Plant Science, 6(2), 66-71. doi:10.1016/s1360-1385(00)01838-

Environmental-dependent proline accumulation in plants living on gypsum soils

[EN] Biosynthesis of proline¿or other compatible solutes¿is a conserved response of all organisms to different abiotic stress conditions leading to cellular dehydration. However, the biological relevance of this reaction for plant stress tolerance mechanisms remains largely unknown, since there are very few available data on proline levels in stress-tolerant plants under natural conditions. The aim of this work was to establish the relationship between proline levels and different environmental stress factors in plants living on gypsum soils. During the 2-year study (2009¿2010), soil parameters and climatic data were monitored, and proline contents were determined, in six successive samplings, in ten taxa present in selected experimental plots, three in a gypsum area and one in a semiarid zone, both located in the province of Valencia, in south-east Spain. Mean proline values varied significantly between species; however, seasonal variations within species were in many cases even wider, with the most extreme differences registered in Helianthemum syriacum (almost 30 lmol g-1 of DW in summer 2009, as compared to ca. 0.5 in spring, in one of the plots of the gypsum zone). Higher proline contents in plants were generally observed under lower soil humidity conditions, especially in the 2009 summer sampling preceded by a severe drought period. Our results clearly show a positive correlation between the degree of environmental stress and the proline level in most of the taxa included in this study, supporting a functional role of proline in stress tolerance mechanisms of plants adapted to gypsum. However, the main trigger of proline biosynthesis in this type of habitat, as in arid or semiarid zones, is water deficit, while the component of ¿salt stress¿ due to the presence of gypsum in the soil only plays a secondary role.This work has been supported by the Spanish Ministry of Science and Innovation (Project CGL2008-00438/BOS), with contribution from the European Regional Development Fund.Boscaiu, M.; Bautista Carrascosa, I.; Lidón Cerezuela, AL.; Llinares Palacios, JV.; Lull, C.; Donat-Torres, M.; Mayoral García-Berlanga, O.... (2013). Environmental-dependent proline accumulation in plants living on gypsum soils. Acta Physiologiae Plantarum. 35:2193-2204. https://doi.org/10.1007/s11738-013-1256-3S2193220435Alvarado JJ, Ruiz JM, López-Cantarero I, Molero J, Romero L (2000) Nitrogen metabolism in five plant species characteristic of gypsiferous soils. J Plant Physiol 156:612–616Ashraf M, Foolad MR (2007) Roles of glycine betaine and proline in improving plant abiotic stress resistance. Environ Exp Bot 59:206–216Bates LS, Waldren RP, Teare ID (1973) Rapid determination of free proline for water stress studies. Plant Soil 39:205–207Briens M, Larher F (1982) Osmoregulation in halophytic higher plants: a comparative study of soluble carbohydrates, polyols, betaines and free proline. Plant, Cell Environ 5:287–292Burriel F, Hernando V (1947) Nuevo método para determinar el fósforo asimilable en los suelos. Anales de Edafología y Fisiología Vegetal 9:611–622Caballero I, Olano JM, Loidi J, Escudero A (2003) Seed bank structure along a semi-arid gypsum gradient in Central Spain. J Arid Environ 55:287–299Escudero A, Carnes LF, Pérez García F (1997) Seed germination of gypsophytes and gypsovags in semi-arid central Spain. J Arid Environ 36:487–497Escudero A, Somolinos RC, Olano JM, Rubio A (1999) Factors controlling the establishment of Helianthemum squamatum, an endemic gypsophite of semi-arid Spain. J Ecol 87:290–302FAO (1990) Management of gypsiferous soils. FAO Soils Bull 62Ferriol M, Pérez I, Merle H, Boira H (2006) Ecological germination requirements of the aggregate species Teucrium pumilum (Labiatae) endemic to Spain. Plant Soil 284:205–216Flowers TJ, Colmer TD (2008) Salinity tolerance in halophytes. New Phytol 179:945–963Flowers TJ, Troke PF, Yeo AR (1977) The mechanism of salt tolerance in halophytes. Ann Rev Plant Physiol 28:89–121Gil R, Lull C, Boscaiu M, Bautista I, Lidón A, Vicente O (2011) Soluble carbohydrates as osmolytes in several halophytes from a Mediterranean salt marsh. Not Bot Horti Agrobo 39(2):9–17Grigore MN, Boscaiu M, Vicente O (2011) Assessment of the relevance of osmolyte biosynthesis for salt tolerance of halophytes under natural conditions. Eur J Plant Sci Biotech 5:12–19Hare PD, Cress WA, Van Standen J (1998) Dissecting the roles of osmolyte accumulation during stress. Plant Cell Environ 21:535–553Keeney DR, Nelson DW (1982) Nitrogen inorganic forms. In: Page AL et al (eds) Methods of soil analysis, part 2: chemical and microbiological properties. Soil Science Society of America, Madison, pp 643–698Knudsen D, Peterson GA, Pratt PF (1982) Lithium, Sodium and Potassium. In: Page AL et al (eds) Methods of soil analysis, part 2: chemical and microbiological properties. Soil Science Society of America, Madison, pp 225–246Kuo S (1996) Phosphorus. In: Spark DL (ed) Methods of soil analysis: chemical methods, part 3. Soil Science Society of America, Madison, pp 869–919Martens H, Maes T (1989) Multivariate calibration. Wiley, New York, pp 97–108Martínez-Duro E, Ferrandis P, Escudero A, Luzuriaga AL, Herranz JM (2010) Secondary old-field succession in an ecosystem with restrictive soils: does time from abandonment matter? Appl Veg Sci 13:234–248Meyer SE (1986) The ecology of gypsophile endemism in the eastern Mojave desert. Ecology 67:1303–1313Meyer SE, García-Moya E (1989) Plant community patterns and soil moisture regime in gypsum grasslands of north central Mexico. J Arid Environ 16:147–155Meyer SE, García-Moya E, Lagunes-Espinoza LC (1992) Topographic and soil surface effects on gypsophile plant community patterns in central Mexico. J Veg Sci 3:429–438Moruno F, Soriano P, Vicente O, Boscaiu M, Estrelles E (2011) Opportunistic germination behaviour of Gypsophila (Caryophyllaceae) in two priority habitats from semi-arid Mediterranean steppes. Not Bot Horti Agrobo 39(1):18–23Mota JF, Sánchez Gómez P, Merlo Calvente ME, Catalán Rodríguez P, Laguna Lumbreras E, de la Cruz Rot M, Navarro Reyes FB, Marchal Gallardo F, Bartolomé Esteban C, Martínez Labarga JM, Sainz Ollero H, Valle Tendero F, Serra Laliga L, Martínez Hernández F, Garrido Becerra JA, Pérez García FJ (2009) Aproximación a la checklist de los gipsófitos ibéricos. Anales de Biología 31:71–80Murakeözy ÉP, Nagy Z, Duhazé C, Bouchereau A, Tuba Z (2003) Seasonal changes in the levels of compatible osmolytes in three halophytic species of inland saline vegetation in Hungary. J Plant Physiol 160:395–401Nelson DW, Sommers LE (1982) Total carbon, organic carbon, and organic matter. In: Page AL et al (eds) Methods of soil analysis, part 2: chemical and microbiological properties. Soil Science Society of America, Madison, pp 539–577Palacio S, Escudero A, Montserrat-Martí G, Maestro M, Milla R, Albert M (2007) Plants living on gypsum: beyond the specialist model. Ann Bot 99:333–343Parsons RF (1977) Gypsophily in plants—a review. Am Midl Nat 96:1–20Pueyo Y, Alados CL, Maestro M, Komac B (2007) Gypsophile vegetation patterns under a range of soil properties induced by topographical position. Plant Ecol 189:301–311Rivas-Martínez S, Rivas-Sáenz S (2009) Worldwide Bioclimatic Classification System. Phytosociological Research Center, Complutense University of Madrid, Spain. http://www.globalbioclimatics.org/ . Accessed 15 Nov 2012Romão RL, Escudero A (2005) Gypsum physical soil crusts and the existence of gypsophytes in semi-arid central Spain. Plant Ecol 181:127–137Rubio A, Escudero A (2000) Small-scale spatial soil-plant relationship in semi-arid gypsum environment. Plant Soil 220:139–150Ruíz JM, López-Cantarero I, Rivero RM, Romero L (2003) Sulphur phytoaccumulation in plant species characteristic of gypsiferous soils. Int J Phytorem 5:203–210Szabados L, Savouré A (2010) Proline: a multifunctional amino acid. Trends Plant Sci 15:89–97Szabados L, Kovács H, Zilberstein A, Bouchereau A (2011) Plants in extreme environments: importance of protective compounds in stress tolerance. Adv Bot Res 57:105–150Tecator Application Note (1984) AN 5226: Determination of ammonium in 2 M KCl soil extracts by FIAstar 5000. AN 5201: Determination of the sum of nitrate and nitrite in water by FIAstar 5000. (Adapted for 2 M KCl soil extracts)Tipirdamaz R, Gagneul D, Duhazé C, Aïnouche A, Monnier C, Özkum D, Larher F (2006) Clustering of halophytes from an inland salt marsh in Turkey according to their ability to accumulate sodium and nitrogenous osmolytes. Environ Exp Bot 57:139–153Verheye WH, Boyadgiev TG (1997) Evaluating the land use potential of gypsiferous soils from field pedogenic characteristics. Soil Use Manage 13:97–103Vicente O, Boscaiu M, Naranjo MA, Estrelles E, Bellés JM, Soriano P (2004) Responses to salt stress in the halophyte Plantago crassifolia (Plantaginaceae). J Arid Environ 58:463–481Yancey PH (2005) Organic osmolytes as compatible, metabolic and counteracting cytoprotectants in high osmolarity and other stresses. J Exp Biol 208:2819–283