Received 8 Oct. 2004 Accepted 21 Feb. 2005 Supported by the Targeted Research Initiative Program of the Oklahoma Agricultural Experimental Station and the Oklahoma Wheat Research Foundation. *Author for correspondence. Tel: +1 785 532 1124; Fax: +1 785 532 6167; E-mail: <gbai@agron.ksu.edu>. Journal of Integrative Plant Biology Formerly Acta Botanica Sinica 2005, 47 (7): 839-848 http://www.blackwell-synergy.com http://www.chineseplantscience.com Nylon Filter Arrays Reveal Differential Expression of Expressed Sequence Tags in Wheat Roots Under Aluminum Stress Kai XIAO 1, 2 , Gui-Hua BAI 2* and Brett F CARVER 3 (1. College of Agronomy, Hebei Agricultural University, Baoding 071001, China; 2. USDA/ARS and Department of Agronomy, Kansas State University, Manhattan, KS 66506, USA; 3. Department of Plant and Soil Science, Oklahoma State University, Stillwater, OK 74078, USA) Abstract: To enrich differentially expressed sequence tags (ESTs) for aluminum (Al) tolerance, cDNA subtraction libraries were generated from Al-stressed roots of two wheat (Triticum aestivum L.) near- isogenic lines (NILs) contrasting in Al-tolerance gene(s) from the Al-tolerant cultivar Atlas 66, using sup- pression subtractive hybridization (SSH). Expression patterns of the ESTs were investigated with nylon filter arrays containing 614 cDNA clones from the subtraction library. Gene expression profiles from macroarray analysis indicated that 25 ESTs were upregulated in the tolerant NIL in response to Al stress. The result from Northern analysis of selected upregulated ESTs was similar to that from macroarray analysis. These highly expressed ESTs showed high homology with genes involved in signal transduction, oxidative stress alleviation, membrane structure, Mg 2+ transportation, and other functions. Under Al stress, the Al-tolerant NIL may possess altered structure or function of the cell wall, plasma membrane, and mitochondrion. The wheat response to Al stress may involve complicated defense-related signaling and metabolic pathways. The present experiment did not detect any induced or activated genes involved in the synthesis of malate and other organic acids in wheat under Al-stress. Key words: aluminum tolerance; differential gene expression; near-isogenic lines; suppression subtractive hybridization (SSH); wheat. Aluminum (Al 3+ ) toxicity occurs in approximately 30% of arable land worldwide and is a major limiting factor for crop production in acidic soils (Carver et al. 1995; Von Uexkull et al. 1995). Aluminum may alter the cation-exchange capacity of cell walls (Horst 1995), change the potential of the cell membrane, affect up- take of Ca 2+ and/or Mg 2+ , induce oxidative stress via lipid peroxidation, replace Mg 2+ or Fe 3+ in cellular reactions, interfere with signal transduction (Jones et al. 1995), and directly bind to DNA and/or RNA. These interactions are manifested as inhibited root growth and stunted shoot growth (Carver et al. 1995). The exuda- tion of organic compounds, such as malate, oxalate, or citrate, that are capable of chelating Al 3+ into non-toxic complexes, was proposed as one of the important mechanisms to relieve Al 3+ toxicity in plants (Delhaize et al. 1993; Basu et al. 1994; Delhaize and Ryan 1995; Horst 1995; Kochian 1995; Ryan et al. 1995; Pellet et al. 1996; Zhang et al. 2001; Tang et al. 2002). Other mechanisms may also exist, especially in light of poly- genic control of Al 3+ tolerance in wheat (Carver et al. 1995). So far, though more than 20 Al-induced genes have been reported from a range of plant species. The mo- lecular mechanism of Al tolerance in plants remains largely unknown. In wheat, those earlier studies on gene expression in response to abiotic stress mostly used differential screening or differential display methods.