Introduction 2002 2+ 1996 1997 3 2001 3  3 2004 3 2002 2003 CVP2 2004 3 2002 fry1 Arabidopsis 3 2001 3 3 2+ 3 4 1995 3 4 2+ 1997 4  2+ 2+ 1988 Arabidopsis thaliana AtIpk2α AtIpk2β 2002 2003 Arabidopsis 3 3 5 4 2002 2003 2005 2007 2005 AtIpk2β arg82 ARG82/IPK2 AtIpk2β AtIpk2β Materials and methods AtIpk2β arg82 S. cerevisiae arg82/Ipk2 http://www.web.uni-frankfurt.de/fb15/mikro/euroscarf/ ARG82 IPK2 kanMX4 AtIpk2β AtIpk2β 600 600 A 600 . AtIpk2β Arabidopsis Arabidopsis 2002 AtIpk2β- 1 Actin 1 Table 1 Gene-specific primers used in this study Primer name Primer sequence For yeast expression vector pYX-F 5′-CTCCCATGGTAATGCTCAAAGTCCCTG-3′ pYX-R 5′-GATTGTCGACCTAGCGCCCGTTCTC-3′ For real-time RT-PCR AtIPK2β-F  5′-CCACGGTTTCGTTGGGGTTC-3′ AtIPK2β-R  5′-GTTACAACCGCCATAAACCTCTG-3′ AtActin2-F  5′-CATCCTCCGTCTTGACCTTGC-3′ AtActin2-R  5′-CAAACGAGGGCTGGAACAAG-3′ For RT-PCR NtActin-F  5′-TTACGCCCTTCCTCATGCAATT-3′ NtActin-R  5′-GGCGCCACCACCTTGATCTTC-3′ AY562132-F 5′-GTAGCATTGTTGGTGGTGGTGTG-3′ AY562132-R 5′- ACCGTGGAGCAGTCAATGGAAG-3′ AY554169-F 5′- ACAGTCGTGAGCATTCCCAAC-3′ AY554169-R 5′-CCAAACCTTCTGTGCTACCTC-3′ AY554170-F 5′-CCGGAGTGAAGGGGATGG-3′ AY554170-R 5′-CAAGCAATGTGAATGGTATGTGAG-3′ NtERD10B-F 5′-CAATTTAGTGCAGGCCAGGC-3′ NtERD10B-R 5′-GGTCCATGGTGGCCAGGAAG-3′ NtERD10C-F 5′-GGGTAGCGCAAACGTGGAG-3′ NtERD10C-R 5′-CTTTTCCCTCAGCCTCGTGC-3′ Vector construction and plant transformation AtIpk2β Xba Sal Agrobacterium tumefaciens Nicotiana tabacum Agrobacteria. Salt stress tolerance tests 2 AtIpk2β −2 −1 −2 −1 + + 2  −2 −1 Oxidative stress experiments g A 532 A 600 2 2 1987 Enzyme assays and protein determination 2 2 2 g 2004 1976 A 290 2 2 A 240 2 2 2 2 A 560 dl Whole plant drought tolerance test Wild type and transgenic plant seeds were surface sterilized as described above. Seedlings germinated on MS medium were transplanted to soil. After two months, healthy plants of the same size and age were pooled into two groups. For the first group (12 individual plants, each grown in a 6.5-inch pot), plants were irrigated with 60 g/l PEG-6000 to simulate drought stress. After 21 days, the plants were photographed. Simultaneously, plants of the second group were subjected to drought stress by omitting watering. After three weeks, the plants were re-hydrated and observed for recovery. Photos were taken 21 days after initiation of the stress treatment. The relative humidity was maintained at ∼60%. The experiments were repeated twice with three replicates in each experiment. Freezing tolerance test Three-week-old seedlings of wild type and transgenic plants grown on MS medium were cultured at 4°C for one day under long day condition (16-h light/8-h dark). After cold acclimation, the plants were left at −20°C for 1 or 2 h, transferred immediately to 4°C for another 12 h (overnight). Subsequently, plants were kept in the greenhouse at 25°C and observed further. Photographs were taken seven days after initiation of the recovery growth in the greenhouse. Determination of proline concentration 1973 Western blot analysis Arabidopsis 2 1986 g 1976 E. coli Stress gene expression analysis using reverse transcription PCR 1 Nicotiana tabacum ACTIN 1 Statistical analysis t P Results AtIpk2β S. cerevisiae ARG82/IPK2 2000 S. cerevisiae arg82/Ipk2 arg82 2000 AtIpk2β 2002 2003 AtIpk2β arg82 1 AtIpk2β Arabidopsis AtIpk2β AtIpk2β arg82 1 1 2002 2003 AtIpk2β 1 Fig. 1 AtIpk2β S. cerevisiae arg82 a AtIpk2β arg82 arg82 arg82 AtIpk2β b 600 arg82 arg82 arg82 arg82 AtIpk2β arg82 arg82  AtIpk2β Arabidopsis AtIpk2β Arabidopsis AtIpk2β AtIpk2β Arabidopsis AtIpk2β AtIpk2β 2 Nicotiana tabacum Agrobacterium tumefaciens AtIpk2β 0 35S:AtIpk2β AtIpk2β Arabidopsis 2 Fig. 2 a AtIpk2β Ocs ocs b Arabidopsis AtIpk2β  AtIpk2β AtIpk2β 3 AtIpk2β 3 3 3 3 2007 1998 3 Fig. 3 a c d e f g P t  4 4 4 + + 4 4 AtIpk2β 4 AtIpk2β Fig. 4 AtIpk2β a b c P P t d + e f g P t  Transgenic plants showed increased tolerance to osmotic, drought, freezing temperature and oxidative stress AtIpk2β 2006 AtIpk2β 5 5 5 Fig. 5 a b c d P t  1997 5 4 AtIpk2β AtIpk2β 2 2 6 2 2 6 Fig. 6 a 2 2 2 2  b c P t d 2 2 2  P P t  AtIpk2β 6 6 AtIpk2β 6 2004 7 2 2 7 7 7 AtIpk2β Fig. 7 a b d 2 2  P P t e  Increased stress responsive gene expression in transgenic tobacco plants 2004 AtIpk2β AtIpk2β 7 7 Discussion 3 3 2000 1999 2000 2002 2003 2005 S. cerevisiae 2002 Drosophila 3 2 2 2002 3 2+ 4 AtIpk2β Arabidopsis 3 4 2000 Arabidopsis AtIpk2β Arabidopsis 2002 Arabidopsis AtIpk2β 35S::AtIpk2β atipk2β AtIpk2β AtNHX1 + + Arabidopsis 2001 + + AtNHX1 + + 4 AtNHX1 2004 + + + − 2002 + + 2+ 2005 AtNHX1 AtIpk2β 2+ 1989 1982 2000 5 Arabidopsis 1999 1995 AtNHX1 2001 AtIpk2β 2002 7 2 2 7 AtIpk2β Arabidopsis Chlamydomonas 2004 1999 2000 2000 AtIpk2β AtIpk2β 1997 1997 1996 2002 3 2001 2001 3 2+  AtIpk2β 2+ 2006 2005 + + 2001 2002 2003 2006 2004 2005 2004 AtIpk2β AtIpk2β Electronic supplementary material Below is the link to the electronic supplementary material.
 Quantitative real-time RT-PCR and Western blot analyses of AtIpk2b in wild type Arabidopsis seedlings in response to different stresses. Fourteen-day old seedlings grown on MS agar plates were treated with 300-mM NaCl or 500-mM mannitol, or kept at 4°C for 24 h. (A) Quantitative real-time RT-PCR analysis of AtIpk2b transcript level. The experiments were repeated three times independently and the average calculated. Error bars represent standard error of the mean. (B) Western blot analysis of AtIpk2b expression level. Coomassie Bright Blue stained gel is shown below the Western blot to demonstrate equal protein loading. (TIF 457 kb)