3. The effect of the chs2 mutation on chlorophyll a chlorophyll b ast development under cold stress. Wild- Col and chs2 plants were grown at 22.C for 3 weeks and then treated at 4c for 10 d.a Chlorophyll content of Col and chs2 seedlings The data P<0.01(t test), significant difference from Col. 400 1 Similar results were observed in three indepen- dent experiments. B, Transmission electron mi- )200 ids from chs 2 plants. Bar=5um (top row) and 2 um(bottom row). Images are of representative plants. 22C 4°C chs2 chs2 plants grown at 22C. However, cold-treated chs2 the chs2 phenotype was caused by the chs 2 mutation, a lants accumulated approximately 22- and 65-fold 12-kb genomic fragment including the complete chs2 higher levels of SA and total SA, respectively, than gene under the control of its own promoter( CHS2: chs 2) wild-type plants(Fig. 4E). Thus, chs 2 plants constitu- was transformed into wild-type Col. Thirty-two out of tively activate defense responses under cold stress 35 Tl-independent transgenic lines showed all the chs2-conferred phenotypes under cold stress, including A Mutation in RPP4 Is Responsible for the seedling lethality(Fig. 5C), high ion leakage(Fig. 2A) Chilling-Sensitive Phenotype elevated PRI expression(Fig. 5E), and extensive cell death( Fig. 5F). These data indicate that mutated chs2 The chs 2 mutant was previously shown to contain a recapitulates all the chs 2-conferred phenotypes and dominant mutation in a single nuclear locus(Schneider therefore that CHS2 is RPP4. RPP4 encodes a TIR-NB et al., 1995). To identify the chs2 mutation, chs2-2 was LRR-classr protein with high similarity to SNC1(74% crossed with Landsberg erecta (Ler) to generate a map amino acid identity and 78% similarity). The Ser-389 ping population. Given that the chs2 mutation is dom- residue in chs2 is very close to the putative GxP or inant, wild-type-looking seedlings were chosen for GLPL motif in the ARC domain, which is conserved mapping from the segregating F2 population after many NB-LRR Proteins(Rafiqi et al, 2009). This find cold treatment. The chs2 mutation was init itially mapped ing hence supports the importance of the ARC domain to the middle of chromosome IV. Approximately 3,000 or the normal activity of plants were then selected for fine mapping. The chs2 mutation was narrowed to a 145-kb region containing The chs2-51 Mutation Suppresses the Chilling Sensitivity the RPP5 cluster region(Fig 5A). To identify the mo- of chs? lecular lesion in chs 2-2, all of the annotated genes in this region were amplified from chs2-2 and sequenced. Only To further confirm that the mutation in rpp is one nucleotide substitution of C to T was found in the responsible for the chs2 phenotype, we carried out a second exon of At4g16860(RPP4 or ColA) in chs2-2 genetic suppressor screen in the chs2 background. M2 resulting in a Ser-to-Phe change at residue 389(Fig 5B). plants derived from EMS-mutagenized chs2 seeds The same mutation was found in chs2-1 were screened for mutants displaying wild-type mor- The chs2 mutant is a dominant mutation, suggesting phology under cold stress. One such mutant, named a gain-of-function substitution. To determine whether chs2-sl(for chs2 suppressor 1), was isolated(Fig. 5D) Plant Physiol. Vol. 154, 2010plants grown at 22
C. However, cold-treated chs2 plants accumulated approximately 22- and 65-fold higher levels of SA and total SA, respectively, than wild-type plants (Fig. 4E). Thus, chs2 plants constitu￾tively activate defense responses under cold stress. A Mutation in RPP4 Is Responsible for the Chilling-Sensitive Phenotype The chs2 mutant was previously shown to contain a dominant mutation in a single nuclear locus (Schneider et al., 1995). To identify the chs2 mutation, chs2-2 was crossed with Landsberg erecta (Ler) to generate a map￾ping population. Given that the chs2 mutation is dom￾inant, wild-type-looking seedlings were chosen for mapping from the segregating F2 population after cold treatment. The chs2 mutation was initially mapped to the middle of chromosome IV. Approximately 3,000 plants were then selected for fine mapping. The chs2 mutation was narrowed to a 145-kb region containing the RPP5 cluster region (Fig. 5A). To identify the mo￾lecular lesion in chs2-2, all of the annotated genes in this region were amplified from chs2-2 and sequenced. Only one nucleotide substitution of C to T was found in the second exon of At4g16860 (RPP4 or ColA) in chs2-2, resulting in a Ser-to-Phe change at residue 389 (Fig. 5B). The same mutation was found in chs2-1. The chs2 mutant is a dominant mutation, suggesting a gain-of-function substitution. To determine whether the chs2 phenotype was caused by the chs2 mutation, a 12-kb genomic fragment including the complete chs2 gene under the control of its own promoter (CHS2:chs2) was transformed into wild-type Col. Thirty-two out of 35 T1-independent transgenic lines showed all the chs2-conferred phenotypes under cold stress, including seedling lethality (Fig. 5C), high ion leakage (Fig. 2A), elevated PR1 expression (Fig. 5E), and extensive cell death (Fig. 5F). These data indicate that mutated chs2 recapitulates all the chs2-conferred phenotypes and therefore that CHS2 is RPP4. RPP4 encodes a TIR-NB￾LRR-class R protein with high similarity to SNC1 (74% amino acid identity and 78% similarity). The Ser-389 residue in chs2 is very close to the putative GxP or GLPL motif in the ARC domain, which is conserved in many NB-LRR proteins (Rafiqi et al., 2009). This find￾ing hence supports the importance of the ARC domain for the normal activity of R proteins. The chs2-s1 Mutation Suppresses the Chilling Sensitivity of chs2 To further confirm that the mutation in RPP4 is responsible for the chs2 phenotype, we carried out a genetic suppressor screen in the chs2 background. M2 plants derived from EMS-mutagenized chs2 seeds were screened for mutants displaying wild-type mor￾phology under cold stress. One such mutant, named chs2-s1 (for chs2 suppressor 1), was isolated (Fig. 5D). Figure 3. The effect of the chs2 mutation on chloroplast development under cold stress. Wild￾type Col and chs2 plants were grown at 22
C for 3 weeks and then treated at 4
C for 10 d. A, Chlorophyll content of Col and chs2 seedlings. The data represent means of four replicates 6 SD. * P , 0.01 (t test), significant difference from Col. Similar results were observed in three indepen￾dent experiments. B, Transmission electron mi￾croscopy of plastids from chs2 plants. Bar = 5 mm (top row) and 2 mm (bottom row). Images are of representative plants. Huang et al. 800 Plant Physiol. Vol. 154, 2010