Changes to the proteome and targeted metabolites of xylem sap in Brassica oleracea in response to salt stress N. FERNANDEZ-GARCIA, 1 M. HERNANDEZ, 1 J. CASADO-VELA 2 *, R. BRU, 3 F. ELORTZA, 2 P. HEDDEN 4 & E. OLMOS 1 1 Department of Abiotic Stress and Plant Pathology. CEBAS-CSIC. P.O. Box 164. Murcia, 2 Proteomics Platform, CIC bioGUNE, CIBERehd, ProteoRed. Bizkaia Technology Park, Building 800, 48160, Bizkaia, 3 Proteomics and Functional Genomics Group. ProteoRed. Departamento de Agroquímica y Bioquímica, Facultad de Ciencias, Universidad de Alicante, Spain and 4 Department of Plant Science. Rothamsted Research, Harpenden AL5 2JQ, Herts, UK ABSTRACT Root-to-shoot signalling via xylem sap is an important mechanism by which plants respond to stress. This signal- ling could be mediated by alteration in the concentrations of inorganic and/or organic molecules. The effect of salt stress on the contents of xylem sap in Brassica olarecea has been analysed by mass spectrometry in order to quantify these changes. Subcellular location of arabinogalactan proteins (AGPs) by immunogold labelling and peroxidase isozymes was also analysed by isoelectrofocusing. The xylem sap metabolome analysis demonstrated the presence of many organic compounds such as sugars, organic acids and amino acids. Of these, amino acid con- centrations, particularly that of glutamine, the major amino acid in the sap, were substantially reduced by salt stress. The xylem sap proteome analysis demonstrated the accumula- tion of enzymes involved in xylem differentiation and ligni- fication, such as cystein proteinases, acid peroxidases, and a putative hydroxycinnamoyl-CoA:shikimate hydroxycin- namoyl transferase under salt stress. The peroxidase isozyme pattern showed that salt stress induced a high accumulation of an acid isoform. These results suggest that xylem differentiation and lignification is induced by salt stress. The combination of different methods to analyse the xylem sap composition provides new insights into mechanisms in plant develop- ment and signalling under salt stress. Key-words: arabinogalactan proteins; cystein proteinases; lignification; peroxidases; programmed cell death; phi cells; xylem differentiation. INTRODUCTION The movement of solutes from roots to the aerial parts of the plant is accomplished by the tracheary elements of the xylem, which was traditionally considered as the main conduit for water and minerals. Structurally the xylem is a complex tissue containing at least tracheary elements and parenchyma cells and other cell-types that function as supporting cells (Evert 2007). On the other hand, sieve tubes of phloem are the main pathway to transport the photosynthetic assimilates from leaves to fruits, roots and buds. Probably for these reasons, xylem sap analysis has focused mainly on the mineral contents. However, xylem sap con- tains also organic solutes, including carbohydrates, amino acids, organic acids, hormones and proteins (Satoh 2006). Transport via the xylem could supply signal molecules other than water and nutrient from the root system and that modification in the concentrations of these signals under abiotic stress can play important roles in plant adaptation to stress (Dodd 2005). Differences in the concentrations of organic compounds in xylem sap from stressed and unstressed plants have been found for a number of species (Gollan, Schurr & Schulze 1992; Patonnier, Peltier & Marigo 1999; Chen et al. 2001; De Sousa & Sodek 2003; Albacete et al. 2009). Xylem sap analysis under salt stress focused mainly on the role of hormone signalling mediated by absci- sic acid (ABA) (Wolf,Jeschke & Hartung 1990;Zhao,Munns & King 1991; Gomez-Cadenas et al. 1998; Chen et al. 2001; Albacete et al. 2009). However, detailed studies of other organic compounds such as sugars,organic acids,amino acids or proteins in xylem sap and the quantification of changes under salt stress are scarce in the literature. In the last 5 years, there have been considerable advances in xylem sap analysis using proteomic approaches. The xylem sap proteomes of Zea mays, Brassica napus, Glycine max, Vitis vinifera and Populus have been studied using separation in 2D gel electrophoresis and mass spectromet- ric identification (Kehr, Buhtz & Giavalisco 2005; Alvarez et al. 2006; Djordjevic et al. 2007; Agüero et al. 2008; Aki et al. 2008; Dafoe & Constabel 2009). Xylem sap pro- teome analysis shows similar groups of proteins present in the different species investigated (Buhtz et al. 2004; Dafoe & Constabel 2009). These groups of proteins were classified as proteins involved in cell wall metabolism and remodelling (glycosyl hydrolases, arabino-furanosidases, Correspondence: E. Olmos. Fax: +34 968 396213; e-mail: eolmos@ cebas.csic.es *Current address: Translational Oncology Unit, Centro Nacional de Biotecnología, lab. 35. C/ Darwin 3, Madrid, Spain. Plant, Cell and Environment (2011) 34, 821–836 doi: 10.1111/j.1365-3040.2011.02285.x © 2011 Blackwell Publishing Ltd 821