Arabidopsis thaliana L. ecotype Col-0 was either grown in axenic culture on MS medium  supplemented with 2% sucrose or cultured in soil in the greenhouse. Arabidopsis plants were transformed using Agrobacterium tumefaciens pGV3101 under vacuum infiltration as described . Cold stress: Plants were grown for 3 weeks in sterile culture on MS plates (14 h light, 21°C) and then transferred to 4°C at the beginning of the light period. Samples were harvested and frozen in liquid nitrogen prior to RNA-extraction. Drought stress: Plants were grown as above and transferred from petridishes to filter paper (whatman 3 MM). For salt stress experiments plants were grown hydroponically as described before  and NaCl concentration was adjusted to 200 mM at the start of the experiments.
Total RNA was isolated from seedlings, mature leaves, stems and other organs with phenol following LiCl precipitation, separation and transfer to nylon membranes as described . Labeling with α32P-dATP was performed with Hexalabel DNA labeling Kit (MBI, Fermentas). Hybridization was performed at 65°C in 0.25 M sodium phosphate pH 7.2, 7% SDS, 1 mM EDTA and 1% BSA for 16 h using the cDNAs of AtOCTs and actin as a probe. Filters were washed twice with 2 × SSC/0.1% SDS and 0.2 × SSC/0.1% SDS for 20 min at 65°C and exposed to X-ray films
Green fluorescent Protein (GFP) fusion
The RT-PCR amplified ORFs of AtOCTs were cloned behind the CaMV 35S promoter in front to GFP5 (S65T). Restricition sites used were for AtOCT1(At1g73220), AtOCT2(At1g79360), AtOCT5(At1g79410) SacI/BamHI, for AtOCT6(At1g16370), AtOCT3(At1g16390) BamHI/BspHI and AtOCAT4 (At3g20660) KpnI/BspHI, The linker between the AtOCTs and GFP was 7–8 amino acids (WGIQGDII for AtOCT1, AtOCT2, and AtOCT5, WGAGAGV for AtOCT6 and AtOCT3 and YGAGAGVfor AtOCT4). The primers used were AtOCT1 ATG/SacI 5'-ggggagctcATGGAACCTTCAAAACAAGAAG-3', AtOCT1 BamHI 5'-cccggatccccCAAGTAATCATGATTGTTTCG-3', AtOCT2 ATG/SacI 5'-aaagagctcATGGCAGAACCAACTCAG-3', AtOCT2 BamHI 5'-cccggatccccCATGCAATGACATTATTAACG-3, AtOCT3 ATG/BamHI 5'-tttggatccATGGCCGACTCGACTCG-3, AtOCT3 BspHI 5'-cctcatgactcctgcgccagcacccCAACCAATAAATTGTCTTTTTGC-3', AtOCT4 ATG/KpnI 5'-aaaaaggtaccATGGAATCTCCGGAGGATAG-3, AtOCT4 BspHI 5'-ccctcatgactcctgcgccagcaccaTAACATATTACTTCTCCTCTTTC-3, AtOCT5 ATG/SacI 5'-tttgagctcATGGCGGATTCGTTGGC-3, AOCT5 BamHI 5'-cccggatccccCAGCAACTATGGCTAGTC-3' AtOCT6 ATG/BamHI 5'-tttggatccATGGCTGATCCAATATCAG-3', AtOCT6 BspHI 5'-aaatcatgactcctgcgccagcacccCAGCAAACATGGCTGG-3',
Promoter glucuronidase (GUS) fusion
Promoter GUS construct consist of promoter including first 21–24 bases of the ORFs and were fused translationally to GUS. The fragments were cloned in pBluescript SK (-) (Stratagene, La Jolla, USA) confirmed by sequencing. Subsequently the total promoter constructs were cloned in frame to uidA (GUS) of pCB308 . The length of the fragments were: POCT2-2210 bp, POCT3-1553 bp, POCT4-900 bp, POCT5-1873 bp, POCT6-1683 bp, and the following primers with restriction sites were used.
P-GUS OCT2f BcuI 5'-gggactagtTACCTCTGCTCAGTTGG-3'
P-GUS OCT2r SmaI 5'-gggAGCGGCTGAGTTGGTTCTG-3'
P-GUS OCT3f BcuI 5'-gggactagtTTTCTTGATTCGATTTTGAGC-3'
P-GUS OCT3r SmaI 5'-gggAGAAGCGGCCGAGTCGAGTC-3'
P-GUS OCT4f BcuI 5'-gggactagtAAGCGTAAGAGGACGCTC-3'
P-GUS OCT4r SmaI 5'-gggTTTCTATCCTCCGGAGATTCC-3'
P-GUS OCT5f BamHI 5'-gggggatccGATGTATATGTGTGTAGAGAGAG-3'
P-GUS OCT5r SmaI 5'-gggGCCATGGTTGCTTACTTTGATCG-3'
P-GUS OCT6f BcuI 5'-gggactagtTTTGGAGTAAGAATTGGTTTG-3'
P-GUS OCT6r SmaI 5'-gggGGTTCTGATATTGGATCAGCC-3'
Transient transformation of the protoplasts with polyethylene glycol was performed according to the protocol of Negrutiu et al . Transient GFP expression was monitored 24 h after transformation. Vacuoles were released from protoplast by creating an osmotic shock by adding water (1:1 to the protoplast suspension) and escaping vacuoles were monitored immediately.
Histochemical localization of GUS activity
Histochemical assays for β-glucuronidase activity were performed as previously described. . Briefly, for the fresh sections, tissues were embedded in 5% low melting agarose, and agar blocks were cut (75 – 150 μm) with razor blades using a vibratome (Leica, Germany).