Function of a Mutant RPP4 in Response to Chilling C 2c→4C chs 2 cold-induced lethality at 4.C(Fig. 7, A and B).In accordance with the morphological phenotype, cell death and HO accumulation were abolished in chs2 060 rar1-20(Fig. 7, C and D). Cold-induced PRI expression was partially suppressed in the chs2 rar1-20 double mutant(Fig. 7E). In addition, levels of Sa in chs2 rarl 20 were restored to wild-type levels( Fig 8). Therefore, the chs2-conferred phenotype requires rari chs2 sgtlb double mutant plants largely resembled wild-type plants 3 to 6 d after cold treatment, when chs2 started to exhibit a chilling defect. However, olonged cold tment(1-2 weeks) resulted in lightly yellow leaves in chs2 sgtlb(Fig 7A). Moreover Total SA chs2 sgt1b showed dwarfism with curly and chlorotic 80 leaves after cold treatment for 5 weeks(Fig. 7B), which is characteristic of chs 2 grown at 16C to 18C(Fig. 1E) The cell death ph and ho were partially suppressed by the sgtlb mutation(Fig 7, C and D). PR expression was partial COGA nised in chs2 sgtlb plants(Fig. 7E). In addition accumulation in chs2 sgtlb was drastically reduced to one-fourth level compared with chs2(Fig. 8). Taken together, these data indicate that the chs 2 phenotype is partially dependent on SGT1b Figure 8. SA accumulation in the double mutants under cold con ons. Three-week-old 22C-grown plants were tr 4°Cfor6d. DISCUSSION Shown are mean values of free and total SA genotypes of three replicates t sD. Similar resu in different The Chilling Sensitivity of chs2 Is a Result of Activated three independent experimen Defense Responses In this study, we characterized a previously reported Because eds1-1 is in the Wassilewskija accession, chilling-sensitive mutant, chs2. This chs2 mutant ex which does not contain the rPP4 gene, we compared hibits yellowish leaves, increased ion leakage, dam the phenotypes of multiple chs 2 /EDSland chs2 eds1 aged chloroplasts, ROS accumulation, extensive cell lines from the F2 population of chs2 crossed with death, and consequent lethality at chilling tempera eds1-1 to eliminate potential effects of mixed back tures(below 12C). To our surprise, all the morpho- ground. Among 211 F2 progeny, all 12 lines of chs2 edsI logical and cell death phenotypes of chs2 under cold and 25 lines of chs2/+ edsl showed wild-type-like conditions are a result of the up-regulation of defense mulation of H,O2 ro甲m甲 ccumulation and SA under cold conditions were also totally sup- are observed in mutants showing cell death pheno- pressed in these chs2 edsI and chs2/+edsl lines(Figs. 7 types(Tanaka et al., 2003 Dong et al., 2007; Hirashima and 8). Moreover, all 14 chs2 EDSI lines and 26 chs2/ et al. 2009). The accumulation of excess H,O2 in chs2 +EDSI lines out of 211 F2 progeny we analyzed is likely due to programmed cell death induced by uniformly resembled chs2 phenotypes (data not the activated RPP4 gene This finding reveals a great shown). These results indicate that chs2 chilling sensi- impact of defense responses on cold sensitivity in tivity is dependent on EDSI plant growth and survival The chs2 pad4 double mutant resembled the chs chs2 mutants contain a gain-of-function mutation mutant in terms of morphology under cold, although (S389F)in the TIR-NB-LRR-type r gene RPP4. The the cold-induced lethal phenotype of chs2 pad4 was $389F mutation is located in the omain delayed slightly compared with the chs2 mutant(Fig. RPP4. The plant NB-ARC domain has been shown to 7, A and B) Cell death, H2O2 accumulation, and PRI be responsible for ATP binding and hydrolysi gene expression in the chs2 pad4 double mutant were Tameling et al., 2002; Ueda et al., 2006). The NB-ARC all comparable to those in chs2 under cold stress(Figs. domain serves as a molecular switch for R protein 7 and 8). Therefore, the chs2-conferred phenotypes are activity, and its action is dependent on its nucleotide argely independent of Pad4 binding state(ATP/ADP). Some R protein mutations RARI and SGTlb were previously identified as affecting the ATP-binding domain will inactivate the regulators of various R genes(Austin et al., 2002; protein(Dinesh-Kumar et al. 2000; Tao et al., 2000; Muskett et al., 2002). rar1-20 completely suppressed Howles et al. 2005; Ueda et al. 2006; van Ooijen et al Plant Ph Vol.154,2010Because eds1-1 is in the Wassilewskija accession, which does not contain the RPP4 gene, we compared the phenotypes of multiple chs2/EDS1and chs2 eds1 lines from the F2 population of chs2 crossed with eds1-1 to eliminate potential effects of mixed back￾ground. Among 211 F2 progeny, all 12 lines of chs2 eds1 and 25 lines of chs2/+ eds1 showed wild-type-like morphology at 4
C (Fig. 7A). Extensive cell death, elevated PR1 expression, and accumulation of H2O2 and SA under cold conditions were also totally sup￾pressed in these chs2 eds1 and chs2/+ eds1 lines (Figs. 7 and 8). Moreover, all 14 chs2 EDS1 lines and 26 chs2/ +EDS1 lines out of 211 F2 progeny we analyzed uniformly resembled chs2 phenotypes (data not shown). These results indicate that chs2 chilling sensi￾tivity is dependent on EDS1. The chs2 pad4 double mutant resembled the chs2 mutant in terms of morphology under cold, although the cold-induced lethal phenotype of chs2 pad4 was delayed slightly compared with the chs2 mutant (Fig. 7, A and B). Cell death, H2O2 accumulation, and PR1 gene expression in the chs2 pad4 double mutant were all comparable to those in chs2 under cold stress (Figs. 7 and 8). Therefore, the chs2-conferred phenotypes are largely independent of PAD4. RAR1 and SGT1b were previously identified as regulators of various R genes (Austin et al., 2002; Muskett et al., 2002). rar1-20 completely suppressed chs2 cold-induced lethality at 4
C (Fig. 7, A and B). In accordance with the morphological phenotype, cell death and H2O2 accumulation were abolished in chs2 rar1-20 (Fig. 7, C and D). Cold-induced PR1 expression was partially suppressed in the chs2 rar1-20 double mutant (Fig. 7E). In addition, levels of SA in chs2 rar1- 20 were restored to wild-type levels (Fig. 8). Therefore, the chs2-conferred phenotype requires RAR1. chs2 sgt1b double mutant plants largely resembled wild-type plants 3 to 6 d after cold treatment, when chs2 started to exhibit a chilling defect. However, prolonged cold treatment (1–2 weeks) resulted in slightly yellow leaves in chs2 sgt1b (Fig. 7A). Moreover, chs2 sgt1b showed dwarfism with curly and chlorotic leaves after cold treatment for 5 weeks (Fig. 7B), which is characteristic of chs2 grown at 16
C to 18
C (Fig. 1E). The cell death phenotype and H2O2 accumulation were partially suppressed by the sgt1b mutation (Fig. 7, C and D). PR expression was partially compro￾mised in chs2 sgt1b plants (Fig. 7E). In addition, SA accumulation in chs2 sgt1b was drastically reduced to one-fourth level compared with chs2 (Fig. 8). Taken together, these data indicate that the chs2 phenotype is partially dependent on SGT1b. DISCUSSION The Chilling Sensitivity of chs2 Is a Result of Activated Defense Responses In this study, we characterized a previously reported chilling-sensitive mutant, chs2. This chs2 mutant ex￾hibits yellowish leaves, increased ion leakage, dam￾aged chloroplasts, ROS accumulation, extensive cell death, and consequent lethality at chilling tempera￾tures (below 12
C). To our surprise, all the morpho￾logical and cell death phenotypes of chs2 under cold conditions are a result of the up-regulation of defense responses through the activated R gene RPP4. Chlo￾roplast morphological change and ROS accumulation are observed in mutants showing cell death pheno￾types (Tanaka et al., 2003; Dong et al., 2007; Hirashima et al., 2009). The accumulation of excess H2O2 in chs2 is likely due to programmed cell death induced by the activated RPP4 gene. This finding reveals a great impact of defense responses on cold sensitivity in plant growth and survival. chs2 mutants contain a gain-of-function mutation (S389F) in the TIR-NB-LRR-type R gene RPP4. The S389F mutation is located in the NB-ARC1 domain of RPP4. The plant NB-ARC domain has been shown to be responsible for ATP binding and hydrolysis (Tameling et al., 2002; Ueda et al., 2006). The NB-ARC domain serves as a molecular switch for R protein activity, and its action is dependent on its nucleotide￾binding state (ATP/ADP). Some R protein mutations affecting the ATP-binding domain will inactivate the protein (Dinesh-Kumar et al., 2000; Tao et al., 2000; Howles et al., 2005; Ueda et al., 2006; van Ooijen et al., Figure 8. SA accumulation in the double mutants under cold condi￾tions. Three-week-old 22
C-grown plants were treated at 4
C for 6 d. Shown are mean values of free and total SA amount in different genotypes of three replicates 6 SD. Similar results were observed in three independent experiments. Function of a Mutant RPP4 in Response to Chilling Plant Physiol. Vol. 154, 2010 805