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5, 1.0, 2.5, 5.0, 10 and 20?mm GABA under 300?mm NaCl stress, and 0, 12, 24, 48, 72?h under 300?mm NaCl and the same time ranges under a combination of 300?mm NaCl and 10?mm GABA, and the different nitrogen sources, GABA, proline, alanine and (NH4)2SO4, at 0.25 and 10?mm, respectively, under the treatment of 300?mm NaCl for 12?h. H2O2 was detected in situ by using 3,3??-diaminobenzidine (DAB) as described by Romero-Puertas et?al. (2004). Whole seedlings from the above treatments were immersed in the solution of 10?mmol?L?1 2-(N-morpholino) ethanesulfonic acid (MES; pH 6.5, 0.1% DAB) under vacuum for 5?min and incubated for 8?h in the dark at room temperature, before being placed in the light until a brown dot appeared. After decolouring in boiling INPP5D ethanol, the seedlings were photographed. DAB staining in the leaves was quantitated using selleck chemical Adobe Photoshop software as follows. For each designated treatment, the margin of each leaf was marked by lasso tool, and then the value of lightness shown in straight alpha channel tool was converted to percentage in curve tool. Finally, the colouration intensity was expressed as percentage of 100% colouration obtained at the treatment of 0?mm GABA or 0?h. Three to four replicates of DAB staining experiments showed similar results obtained each time, and the data shown were representative of these replicated experiments. The plant tissues were ground to be a fine powder in liquid nitrogen, and about 0.1?g of the frozen homogenate was extracted using methanol and lanthanum chloride, and endogenous GABA content was measured by GABase method (Zhang & Bown 1997). http://www.selleckchem.com/products/RO4929097.html All the determinations were representative of three separate experiments and six technical replicates. Total RNA of roots was extracted with Trizol reagent (Invitrogen, Carlsbad, CA, USA). Poly(A)+ RNA was isolated from the total RNA with the polyATract mRNA isolation system kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. The subtractive library was constructed with ds cDNA of 300?mm NaCl (Driver) and 300?mm NaCl?+?10?mm GABA (Tester) based on BD PCR-Select cDNA Subtract Kit (Clontech, Mountain View, CA, USA) according to the protocol provided by the manufacturer. Positive clones were sequenced with an ABI3730 at the Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai Songon) in China. Nucleotide sequences and translated sequences were analysed for homology in GenBank Release 165.0 using the BLAST program (NCBI, Rockville Pike, Bethesda, MD, USA). E-values <0.001 from BLAST were considered to have significant similarities. Selected ESTs were amplified by PCR using two primers (T7 promoter primer and SP6 promoter primer) complementary to the sequence of the pGEM-T vector flanking both sides of the cDNA inserts. The PCR products were diluted to a final concentration of 0.1?g??L?1, denatured with NaOH (0.
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