A Gain-of-Function Mutation in the Arabidopsis Disease Resistance gene rpp Confers Sensitivity to Low TemperaturelIwIIOAl Xiaozhen Huang, Jianyong Li, Fei Bao, Xiaoyan Zhang, and Shuhua Yang' State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China How plants adapt to low temperature is not well understood. To identify components involved in low-temperature signaling, we characterized the previously isolated chilling-sensitive2 mutant(chs2)of Arabidopsis(Arabidopsis thaliana). This mutant grey normally at 22.C but showed phenotypes similar to activation of defense responses when shifted to temperatures below 16C. hese phenotypes include yellowish and collapsed leaves, increased electrolyte leakage, up-regulation of PATHOGENESIS RELATEd genes, and accumulation of excess hydrogen peroxide and salicylic acid(SA). Moreover, the chs2 mutant was seedling lethal when germinated at or shifted for more than 3 d to low temperatures of 4C to 12C. Map-based cloning revealed that a single amino acid substitution occurred in the TIR-NB-LRR (for Toll /Interleukin-1 receptor-nucleotide-binding Leucine-rich repeat)-type resistance(R)protein RPP4(for Recognition of Peronospora parasitica), which causes a deregulation of the R protein in a temperature-dependent manner. The chs2 mutation led to an increase in the mutated RPP4 mRNA transcript, activation of defense responses, and an induction of cell death at low temperatures. In addition, a chs2 intragenic suppressor, in which the mutation occurs in the conserved NB domain, abolished defense responses at lower temperatures Genetic analyses of chs2 in combination with known SA pathway and immune ng mutants indicate that the chs2 onferred temperature sensitivity requires ENHANCED DISEASE SUSCEPTIBILITY UIREd FOR Mla12 RESIStance and SUPPRESSOR OF G2 ALLELE OF skpl but does not require PHYTOALEXIN CIENT4, NONEXPRESSOR OF PR GENESI, or SA. This study reveals that an activated TIR-NB-LRR protein has a large impact on temperature sensitivity in plant growt For optimal growth and survival, plants have evolved 1992, 2004), and DREB/CBF-independent pathways unique and sophisticated defense mechanisms against have been identified as important for cold responses as multiple stresses, both abiotic and biotic. Cold stress well (Xin and Browse, 1998; Dong et al., 2006; Lee et al has a significant limiting effect on the geographic 2006; Xin et al. 2007; Zhu et al., 2008) location of plants and on crop prodt luctivity (G Plants have evolved at least two layers of defense 1990). It can disrupt cellular homeostasis by altering mechanisms against pathogens. One of them is medi- the fatty acid composition of membrane lipids, which ated by resistance(r)proteins. Interaction of an R can deactivate membrane proteins and uncouple ma protein with a specific pathogen avirulence protein for physiological processes(Los and Murata, 200 triggers the hypersensitive response(HR), which is a Plants respond and adapt to cold stress in many form of programmed cell death that limits path- biochemical and physiological processes. A numb ogen growth and spread(Scheel, 1998). Most of the of genes are involved in the DREB/CBF(for DRE- characterized R proteins encode proteins with nucle- binding protein/C-repeat-binding factor)-dependent otide-binding Leu-rich repeat(NB-LRR) domains. A pathway to control cold acclimation(Gilmour et al well-conserved ARC (for After the Nb domain, f-1,R protein, and and these two domains are often referred to as the nb. Foundation of China (grant nos. 30670181, 3077202, and 90817007) ARC domain. The NB-LRR proteins can be grouped the National Key Basic Research Program of (grant no. into two main classes based on their N-terminal struc- cultural of China for trans- ture, which has either a Toll/Interleukin-l receptor 2003) The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy The Arabidopsis(Arabidopsis thaliana)RPP5(for Rec- describedintheiNstructionsforAuthors(www.plantphysiol.org)is: ognition of Peronospora parasitica) locus in Columbia ShuhuaYang(yangshuhua@cau.edu.cn) Col)is composed of seven TIR-NB-LRR class r genes, Iw The online version of this article contains Web-only data. includins PP4 and SNC1(for Suppressor of nprI IOA] Open Access articles can be viewed online without a sub- constitutive 1)genes(Noel et al., 1999). RPP4 plays an important role in resistance to Hyaloperonospora www.plantphysiol.org/cgi/doi/10.1104/pp.110.157610 parasitica through multiple signaling components, in- PlantPhysiologyOctober2010,Vol.154,Pp.796-809,www.plantphysiol.org@2010AmericanSocietyofPlantBiologists
A Gain-of-Function Mutation in the Arabidopsis Disease Resistance Gene RPP4 Confers Sensitivity to Low Temperature1[W][OA] Xiaozhen Huang, Jianyong Li, Fei Bao, Xiaoyan Zhang, and Shuhua Yang* State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China How plants adapt to low temperature is not well understood. To identify components involved in low-temperature signaling, we characterized the previously isolated chilling-sensitive2 mutant (chs2) of Arabidopsis (Arabidopsis thaliana). This mutant grew normally at 22C but showed phenotypes similar to activation of defense responses when shifted to temperatures below 16C. These phenotypes include yellowish and collapsed leaves, increased electrolyte leakage, up-regulation of PATHOGENESIS RELATED genes, and accumulation of excess hydrogen peroxide and salicylic acid (SA). Moreover, the chs2 mutant was seedling lethal when germinated at or shifted for more than 3 d to low temperatures of 4C to 12C. Map-based cloning revealed that a single amino acid substitution occurred in the TIR-NB-LRR (for Toll/Interleukin-1 receptor- nucleotide-binding Leucine-rich repeat)-type resistance (R) protein RPP4 (for Recognition of Peronospora parasitica4), which causes a deregulation of the R protein in a temperature-dependent manner. The chs2 mutation led to an increase in the mutated RPP4 mRNA transcript, activation of defense responses, and an induction of cell death at low temperatures. In addition, a chs2 intragenic suppressor, in which the mutation occurs in the conserved NB domain, abolished defense responses at lower temperatures. Genetic analyses of chs2 in combination with known SA pathway and immune signaling mutants indicate that the chs2- conferred temperature sensitivity requires ENHANCED DISEASE SUSCEPTIBILITY1, REQUIRED FOR Mla12 RESISTANCE, and SUPPRESSOR OF G2 ALLELE OF skp1 but does not require PHYTOALEXIN DEFICIENT4, NONEXPRESSOR OF PR GENES1, or SA. This study reveals that an activated TIR-NB-LRR protein has a large impact on temperature sensitivity in plant growth and survival. For optimal growth and survival, plants have evolved unique and sophisticated defense mechanisms against multiple stresses, both abiotic and biotic. Cold stress has a significant limiting effect on the geographic location of plants and on crop productivity (Guy, 1990). It can disrupt cellular homeostasis by altering the fatty acid composition of membrane lipids, which can deactivate membrane proteins and uncouple major physiological processes (Los and Murata, 2004). Plants respond and adapt to cold stress in many biochemical and physiological processes. A number of genes are involved in the DREB/CBF (for DREbinding protein/C-repeat-binding factor)-dependent pathway to control cold acclimation (Gilmour et al., 1992, 2004), and DREB/CBF-independent pathways have been identified as important for cold responses as well (Xin and Browse, 1998; Dong et al., 2006; Lee et al., 2006; Xin et al., 2007; Zhu et al., 2008). Plants have evolved at least two layers of defense mechanisms against pathogens. One of them is mediated by resistance (R) proteins. Interaction of an R protein with a specific pathogen avirulence protein triggers the hypersensitive response (HR), which is a form of programmed cell death that limits pathogen growth and spread (Scheel, 1998). Most of the characterized R proteins encode proteins with nucleotide-binding Leu-rich repeat (NB-LRR) domains. A well-conserved ARC (for Apaf-1, R protein, and CED4) domain is found just after the NB domain, and these two domains are often referred to as the NBARC domain. The NB-LRR proteins can be grouped into two main classes based on their N-terminal structure, which has either a Toll/Interleukin-1 receptor (TIR) domain or a coiled-coil domain (Meyers et al., 2003). The Arabidopsis (Arabidopsis thaliana) RPP5 (for Recognition of Peronospora parasitica5) locus in Columbia-0 (Col) is composed of seven TIR-NB-LRR class R genes, including RPP4 and SNC1 (for Suppressor of npr1-1, constitutive 1) genes (Noel et al., 1999). RPP4 plays an important role in resistance to Hyaloperonospora parasitica through multiple signaling components, in- 1 This work was supported by the National Natural Science Foundation of China (grant nos. 30670181, 3077202, and 90817007), the National Key Basic Research Program of China (grant no. 2009CB119100), and the Ministry of Agricultural of China for transgenic research (grant no. 2008ZX08009–003). * Corresponding author; e-mail yangshuhua@cau.edu.cn. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Shuhua Yang (yangshuhua@cau.edu.cn). [W] The online version of this article contains Web-only data. [OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.110.157610 796 Plant Physiology, October 2010, Vol. 154, pp. 796–809, www.plantphysiol.org 2010 American Society of Plant Biologists
Function of a Mutant RPP4 in Response to Chilling cluding DETACHMENT 9(DTH9: Mayda et al., these findings support an extensive signaling network 2000), ENHANCED DISEASE SUSCEPTIBILITYI between cold stress and defense re EDSl; Aarts et al, 1998), PHYTOALEXIN DEFI Here, we report the investigation of a cold-sensitive CIENT4(PAD4; Glazebrook et al., 1996), NONEX- mechanism of chilling-sensitive2(chs2) in Arabidopsis PRESSOR OF PR GENES1(NPRl; Cao et al., 1997), The chs2 mutant exhibits HR-like cell death and con- NON-RACE-SPECIFIC DISEASE RESISTANCE1(NDRI; sequent lethality under cold stress. Map-based cloning Century et al., 1995), Phenylalanine Ammonium Lyase revealed that CHS2 encodes the TIR-NB-LRR-type R (PAL; Mauch-Mani and Slusarenko, 1996), aur PphB protein RPP4. An amino acid substitution in the nb. SUSCEPTIBLE2(PBS2) and PBS3(Warren et al RC region leads to a temperature-dependent gain-of- and REQUIRED FOR Mla12 RESISTANCE(RAR1; of an activated r gene in cold response, suggesting a Austin et al., 2002), RPS5(Warren et al., 1998), and contribution of defense responses to temperature sensi SALICYLIC ACID INDUCTION-DEFICIENT1(SIDI), tivity. SID2, and salicylic acid(SA; McDowell et al., 2000; van der Biezen et al., 2002). In addition, RPP4 mediates disease resistance and basal defense against h. parasitica RESULTS through the transcription factor AtWRKY70(Knoth Morphological Phenotypes of the Chilling-Sensitive et al., 2007). SNCI confers disease resistance and suppresses plant growth in a temperature-dependent Mutant chs2 nanner when activated(Stokes and Richards, 2002; The chs2-1 and chs2-2 mutants were isolated as Zhang et al., 2003; Yang and Hua, 2004; Zhu et al chilling sensitive from an ethane methyl sulfonate 2010). The RPP5 locus r genes are coordinately regu (EMS)-mutagenized pool of Arabidopsis(Schneider lated by transcriptional activation and RNa silencing et al, 1995). We further characterized the mutant Yi and Richards, 2007) phenotypes of these two alleles. They resembled the Although the initial stimuli of cold and biotic wild type when grown in soil at 22 C; however, the tresses are obviously different, in many cases these leaves of these two mutants turned yellow and wilted signals are integrated into a unified scheme and trig 3 d after being shifted to low temperature of 4oC to ger a common set of responses. For instance, cold and 12C, and they eventually, died(Fig. 1 A). When defense responses are shown to share common targets, planted on Murashige and Skoog(MS)plates directly such as PATHOGENESIS-RELATED(PR) genes, which at 4C. the chs2 seedlings died shortly after germina- not only play a role in pathogen resistance but also ar tion(Fig. 1C). Given that these two alleles sh induced by cold stress and promote freezing tolerance similar phenotypes, we chose chs2-2(referred as chs2 (Snider et al. 2000; Seo et al., 2008 ). Furthermore, cold hereafter) for further studies and defense responses share common regulators, such To get a better understanding of chs2 in response to as the SUMO E3 ligase SIZ1 (for SAP and Mizl; Lee chilling, we examined the phenotypes of chs2 plants by et al, 2007; Miura et al. 2007), AtSR1/CAMTA3(for shifting them to cold conditions at different growth stages either in soil or on agar plates. The 22.C-grown Arabidopsis signal responsive/Calmodulin-binding chs2 plants at every developmental stage tested were transcription activator 3; Doherty et al., 2009; Du et al., 2009), and the transcriptional repressor of DREB ypersensitive to cold stress both in soil and on MS (Fi et al, 2009). In addition, defense responses induced by ing the rosette leaves, cauline leaves, stems, flowers a number of r genes are modulated by temperature, including Mi in tomato(Solanum lycopersicum; Hwang quickly after cold exposure( Fig. 1B). It is noteworthy et al. 2000), N in tobacco(Nicotiana tabacum that the mutant grown at 16C to 18 C showed dwarf stature with curly chlorotic leaves and short inflores- et al., 2004), and RESISTANCE TO POWDERY MIL.- cence internodes(Fig. 1E). with decreased tempera- TIVE4, SNC1, and the RPPl-like TIR-NB-LRR cluster ture, the chs2 mutant plants showed more severe in Arabidopsis(Xiao et al., 2003; Yang and Hua, 2004, growth defects, and they were lethal when tempera- Zhou et al. 2008; Alcazar et al. 2009). A recent study ture was below 12C(Fig. 1F). Therefore, the chs2 revealed that the Nb-lrr proteins function as tem- mutant is sensitive to low temperature throughout plant development, with lower temperature causing perature-sensitive components in plant immune re- more severe growth defects sponses(Zhu et al., 2010). Some of the defense signaling components, such as PAD4, EDSI, and SA, are also regulated by temperature(Clarke et al., 2004; Physiological Characteristics of chs2 at Yang and Hua, 2004). Moreover, the plasma mem Low Temperatures brane-bound NAC transcription factor NTL6 is pro- Leakage of ions from cell membranes is a good teolytically activated by cold and in turn enters the ndex to measure chilling sensitivity in plants(lyons, nucleus, thereby inducing defense responses by bind- 1973). We carried out ion leakage assays to determine ing to the promoter of Pr genes(Seo et al., 2010). All of the extent of chilling injury to chs2 plants. No obvious Plant Ph Vol.154,2010
cluding DETACHMENT 9 (DTH9; Mayda et al., 2000), ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1; Aarts et al., 1998), PHYTOALEXIN DEFICIENT4 (PAD4; Glazebrook et al., 1996), NONEXPRESSOR OF PR GENES1 (NPR1; Cao et al., 1997), NON-RACE-SPECIFIC DISEASE RESISTANCE1 (NDR1; Century et al., 1995), Phenylalanine Ammonium Lyase (PAL; Mauch-Mani and Slusarenko, 1996), avrPphB SUSCEPTIBLE2 (PBS2) and PBS3 (Warren et al., 1999), SUPPRESSOR OF G2 ALLELE OF skp1 (SGT1b) and REQUIRED FOR Mla12 RESISTANCE (RAR1; Austin et al., 2002), RPS5 (Warren et al., 1998), and SALICYLIC ACID INDUCTION-DEFICIENT1 (SID1), SID2, and salicylic acid (SA; McDowell et al., 2000; van der Biezen et al., 2002). In addition, RPP4 mediates disease resistance and basal defense against H. parasitica through the transcription factor AtWRKY70 (Knoth et al., 2007). SNC1 confers disease resistance and suppresses plant growth in a temperature-dependent manner when activated (Stokes and Richards, 2002; Zhang et al., 2003; Yang and Hua, 2004; Zhu et al., 2010). The RPP5 locus R genes are coordinately regulated by transcriptional activation and RNA silencing (Yi and Richards, 2007). Although the initial stimuli of cold and biotic stresses are obviously different, in many cases these signals are integrated into a unified scheme and trigger a common set of responses. For instance, cold and defense responses are shown to share common targets, such as PATHOGENESIS-RELATED (PR) genes, which not only play a role in pathogen resistance but also are induced by cold stress and promote freezing tolerance (Snider et al., 2000; Seo et al., 2008). Furthermore, cold and defense responses share common regulators, such as the SUMO E3 ligase SIZ1 (for SAP and Miz1; Lee et al., 2007; Miura et al., 2007), AtSR1/CAMTA3 (for Arabidopsis signal responsive/Calmodulin-binding transcription activator 3; Doherty et al., 2009; Du et al., 2009), and the transcriptional repressor of DREB protein DEAR1 (for DREB and EAR protein 1; Tsutsui et al., 2009). In addition, defense responses induced by a number of R genes are modulated by temperature, including Mi in tomato (Solanum lycopersicum; Hwang et al., 2000), N in tobacco (Nicotiana tabacum; Someya et al., 2004), and RESISTANCE TO POWDERY MILDEW8, SUPPRESSOR OF SALICYLIC ACID INSENSITIVE4, SNC1, and the RPP1-like TIR-NB-LRR cluster in Arabidopsis (Xiao et al., 2003; Yang and Hua, 2004; Zhou et al., 2008; Alcazar et al., 2009). A recent study revealed that the NB-LRR proteins function as temperature-sensitive components in plant immune responses (Zhu et al., 2010). Some of the defense signaling components, such as PAD4, EDS1, and SA, are also regulated by temperature (Clarke et al., 2004; Yang and Hua, 2004). Moreover, the plasma membrane-bound NAC transcription factor NTL6 is proteolytically activated by cold and in turn enters the nucleus, thereby inducing defense responses by binding to the promoter of PR genes (Seo et al., 2010). All of these findings support an extensive signaling network between cold stress and defense responses. Here, we report the investigation of a cold-sensitive mechanism of chilling-sensitive2 (chs2) in Arabidopsis. The chs2 mutant exhibits HR-like cell death and consequent lethality under cold stress. Map-based cloning revealed that CHS2 encodes the TIR-NB-LRR-type R protein RPP4. An amino acid substitution in the NBARC region leads to a temperature-dependent gain-offunction phenotype. This study reveals the involvement of an activated R gene in cold response, suggesting a contribution of defense responses to temperature sensitivity. RESULTS Morphological Phenotypes of the Chilling-Sensitive Mutant chs2 The chs2-1 and chs2-2 mutants were isolated as chilling sensitive from an ethane methyl sulfonate (EMS)-mutagenized pool of Arabidopsis (Schneider et al., 1995). We further characterized the mutant phenotypes of these two alleles. They resembled the wild type when grown in soil at 22C; however, the leaves of these two mutants turned yellow and wilted 3 d after being shifted to low temperature of 4C to 12C, and they eventually died (Fig. 1A). When planted on Murashige and Skoog (MS) plates directly at 4C, the chs2 seedlings died shortly after germination (Fig. 1C). Given that these two alleles showed similar phenotypes, we chose chs2-2 (referred as chs2 hereafter) for further studies. To get a better understanding of chs2 in response to chilling, we examined the phenotypes of chs2 plants by shifting them to cold conditions at different growth stages either in soil or on agar plates. The 22C-grown chs2 plants at every developmental stage tested were hypersensitive to cold stress both in soil and on MS plates (Fig. 1, B–D). All parts of the chs2 plants including the rosette leaves, cauline leaves, stems, flowers, and siliques became yellow, collapsed, and then died quickly after cold exposure (Fig. 1B). It is noteworthy that the mutant grown at 16C to 18C showed dwarf stature with curly chlorotic leaves and short inflorescence internodes (Fig. 1E). With decreased temperature, the chs2 mutant plants showed more severe growth defects, and they were lethal when temperature was below 12C (Fig. 1F). Therefore, the chs2 mutant is sensitive to low temperature throughout plant development, with lower temperature causing more severe growth defects. Physiological Characteristics of chs2 at Low Temperatures Leakage of ions from cell membranes is a good index to measure chilling sensitivity in plants (Lyons, 1973). We carried out ion leakage assays to determine the extent of chilling injury to chs2 plants. No obvious Function of a Mutant RPP4 in Response to Chilling Plant Physiol. Vol. 154, 2010 797
Huang et al. 1. Cold sensitivity of chs2 mu- A chs2-1 chs2-2 ants.A, Phenotypes of wild-type nd chs2 plants grown in soil at 22°C chs2 for 4 weeks (top row), cold treated at 4 C for 10 d(middle row followed by 22C for 3 d(bottom rowl plants grown in soil at 22 C for 6 (top)followed by cold treatment 4C (10 d) at 4C for 10 d(bottom). C and D directly geminated on MS plates and grown at 4C for 3 months(C)or grown at 22 C for 3 weeks and then trans. ferred to 4C for an additional 10 d (D) 4C(10d) 2c0 ypes of chs2 plants grown on MS plates at the indicated C temperatures for 4 weeks. Images are Col chs2 ve plants 16°C changes in ion leakage were detected in wild-type roplasts in chs2 are severely damaged under cold leaves during cold treatment. However, ion leakage of conditions chs2 plants increased drastically following cold treat The chloroplast morphology in cold-treated chs2 ment(Fig. 2A). This result indicates that the cell pla plants was further examined using transmission elec- membranes of chs2 leaves are severely injured under tron microscopy. The mature chloroplasts of the chs2 old stress, which is in agreement with the cold- and wild-type plants at 22C exhibited crescent-shaped sensitive phenotype of chs2 and well-developed thylakoid membranes. Chloro- Free Pro is an osmolyte considered to protect plants plasts in cold-treated wild-type plants were similar to gainst cold stress (Xin and Browse, 1998; Nanjo et al those in plants without cold treatment, but with larger 1999). We investigated if the cold sensitivity of chs 2 is starch granules, which is a normal response to cold accompanied by reduced Pro levels. Indeed, the Pro stress. In contrast, cold-treated chs2 chloroplasts were content in chs2 was much lower than in the wild type smaller and more spherical than those in the wild-type when treated at 4C for 6 d(Fig ) suggesting that plants, and they contained fewer internal thylakoid less Pro accumulation in chs2 might at least partly membranes. Moreover, the starch grains in cold-treated account for its cold sensitivity. chs2 chloroplasts were either absent or reduced in size and number. The mutant chloroplasts also appeared to plasts Are Damaged in chs2 Plants under contain more plastoglobuli than wild-type chloroplasts (Fig. 3B). Thus, cold stress causes serious damage to the Because the chs2 plants exhibited yellow leaves leaves chloroplasts in chs2 plants We then determined whether light had an effect on under cold stress(Fig. 1), we measured the chlorophyll cold-induced phenotypes of chs2. Although the cold content in the chs2 mutant. The levels of chlorophyll induced phenotype of chs 2 was significantly delayed in a and chlorophyll b in cold-treated chs2 plants were the dark( Supplemental Fig SlA), the plants eventually pproximately 42% and 50% of those in the wild-type died. Accordingly, the degradation of chlorophyll a plants, respectively(Fig 3A), implying that the chi and b was also delayed in the dark(Supplemental Fig Plant Physiol. Vol. 154, 2010
changes in ion leakage were detected in wild-type leaves during cold treatment. However, ion leakage of chs2 plants increased drastically following cold treatment (Fig. 2A). This result indicates that the cell membranes of chs2 leaves are severely injured under cold stress, which is in agreement with the coldsensitive phenotype of chs2. Free Pro is an osmolyte considered to protect plants against cold stress (Xin and Browse, 1998; Nanjo et al., 1999). We investigated if the cold sensitivity of chs2 is accompanied by reduced Pro levels. Indeed, the Pro content in chs2 was much lower than in the wild type when treated at 4C for 6 d (Fig. 2B), suggesting that less Pro accumulation in chs2 might at least partly account for its cold sensitivity. Chloroplasts Are Damaged in chs2 Plants under Cold Stress Because the chs2 plants exhibited yellow leaves under cold stress (Fig. 1), we measured the chlorophyll content in the chs2 mutant. The levels of chlorophyll a and chlorophyll b in cold-treated chs2 plants were approximately 42% and 50% of those in the wild-type plants, respectively (Fig. 3A), implying that the chloroplasts in chs2 are severely damaged under cold conditions. The chloroplast morphology in cold-treated chs2 plants was further examined using transmission electron microscopy. The mature chloroplasts of the chs2 and wild-type plants at 22C exhibited crescent-shaped and well-developed thylakoid membranes. Chloroplasts in cold-treated wild-type plants were similar to those in plants without cold treatment, but with larger starch granules, which is a normal response to cold stress. In contrast, cold-treated chs2 chloroplasts were smaller and more spherical than those in the wild-type plants, and they contained fewer internal thylakoid membranes. Moreover, the starch grains in cold-treated chs2 chloroplasts were either absent or reduced in size and number. The mutant chloroplasts also appeared to contain more plastoglobuli than wild-type chloroplasts (Fig. 3B). Thus, cold stress causes serious damage to the chloroplasts in chs2 plants. We then determined whether light had an effect on cold-induced phenotypes of chs2. Although the coldinduced phenotype of chs2 was significantly delayed in the dark (Supplemental Fig. S1A), the plants eventually died. Accordingly, the degradation of chlorophyll a and b was also delayed in the dark (Supplemental Fig. Figure 1. Cold sensitivity of chs2 mutant plants. A, Phenotypes of wild-type Col and chs2 plants grown in soil at 22C for 4 weeks (top row), cold treated at 4C for 10 d (middle row), followed by 22C for 3 d (bottom row). B, Phenotypes of wild-type Col and chs2 plants grown in soil at 22C for 6 weeks (top) followed by cold treatment at 4C for 10 d (bottom). C and D, Phenotypes of Col and chs2 plants directly geminated on MS plates and grown at 4C for 3 months (C) or grown at 22C for 3 weeks and then transferred to 4C for an additional 10 d (D). E, Phenotypes of wild-type Col and chs2 plants grown in soil at 16C for 7 weeks. F, Phenotypes of chs2 plants grown on MS plates at the indicated temperatures for 4 weeks. Images are of representative plants. Huang et al. 798 Plant Physiol. Vol. 154, 2010
Function of a Mutant RPP4 in Response to Chilling A100 of normal chloroplast function and by the overgenera tion of ros in the chloroplasts chs2 When subjected to low temperature, plants accu 8o Col/CHS2: chs2 mulate excess H,O2(O'Kane et al. 1996), which in turn induces the expression of genes associated with oxi- dative stress(Iba, 2002; Mittler et al, 2004; Rizhsky et al., 2004). More H,O, accumulation in chs2 was observed under cold conditions(Fig. 4A). Therefore, we examined the expression of several genes encoding ROS-detoxification enzymes, including copper/zinc superoxide dismutase(CSD), ascorbate peroxidase Days of treatment (APX), and catalase( CAT), in cold-treated chs2 plants No obvious differences in expression of CSDl, APXI or CatI were detected between wild-type and chs2 plants at 22%C. In contrast, the expression of these genes was substantially elevated in chs 2 plants relative to wild-type plants under cold stress( Supplemental Fig S2B). The zinc-finger protein ZAT12 plays a cru- 5400 cial role in oxidative and abiotic stress signaling (Rizhsky et al., 2004; Davletova et al., 2005). In add tion, ferritin protein nanocages are essential for pro- ①200 tecting cells against oxidative damage(Ravet et al 2009). We found that ZAT12 and FERI were also lificantly up-regulated in cold-treated chs 2 plant Figure 2. Physiological analysis of chs2 mutant plants. A, lon leakage quent impairment of oxidative signaling. and c rev 22C relative to wild-type plants(Supplemental Fig. $2 Therefore, the chilling sensitivity of chs2 might result from an imbalance of ros detoxification assay in chs 2 plants. Plants grown at 22C for 3 weeks were then treated at 4C for the indicated times. The data represent means of three licates+ sD. Similar results were observed in three independent The Expression of Cold-Regulated Genes Is Not Affected weeks were treated at 4C for 6 d. The data represent means of a a u experiments.B, Pro content in chs 2 plants. Plants grown at 22C for plicate t sD. *P<0.01(t test), significant difference from Col We further examined whether the chs2 mutation Similar results were observed in three independent experiments. FW, affects the induction of cold-regulated genes. The Fresh weight CBFl to CBF3 genes were rapidly induced in chs2 and wild-type plants 3 h after exposure to cold, and their target genes RD29A and COR47 were substan- SIB demonstrate that light accelerates tially induced at 6 to 12 h after cold treatment. No phenotype, but low temperature significant difference in expression of these genes was type in the absence of light. observed between chs2 and wild-type plants( Supple- mental Fig S3). Therefore, the chs 2 gene appears not to affect the CBF pathway The chs2 Mutation Causes Reactive Oxidative Species Accumulation and Imbalanced reactive Oxidative chs2 Constitutively Activates Defense Responses under Species-Scavenging Network under Cold Stress Low temperature can perturb electron transport Leaves in cold-treated chs2 plants turned yellow, lost chains and cause the production of reactive oxidative turgor pressure, and collapsed (Fig. 1), resembling the species(ROS; Fryer et al., 2002; Hideg et al. 2002; pathogen-induced HR cell death response. Extensive Pfannschmidt et al. 2003). Therefore, we examined cell death did occur in cold-treated chs 2 plants but not hydrogen peroxide(H, o,accumulation in chs2 plants n wild-type plants, as revealed by trypan blue stain- nder cold conditions by 3, 3'-diaminobenzidine ng(Fig. 4B ). Furthermore, PRI and PR2 were highly (DAB)staining. Strong staining was detected in cold- expressed in chs2 plants under cold stress(Fig. 4C) treated chs2 plants but not in wild-type plants(Fig Consistently, cold-treated chs2 plants harboring a PRI 4A), indicating that the mutant plants accumulated GuS construct showed stronger staining of GUS than more H2O2 than the wild-type plants. Under light, wild-type PRl: Gus transgenic plants(Fig. 4D) chloroplast is the main site of ROS generation; consis- Because high PR gene expression is often associated tently, daB precipitates were mostly present in the with elevated levels of SA, the endogenous SA levels chloroplasts. Therefore, the chs 2-induced phenotypes in chs 2 were examined. The levels of both free SA and under cold stress might be caused by the impairment total SA in chs 2 were comparable to those in wild-type Plant Ph Vol.154,2010
S1B). These results demonstrate that light accelerates the chs2-conferred phenotype, but low temperature triggers this phenotype in the absence of light. The chs2 Mutation Causes Reactive Oxidative Species Accumulation and Imbalanced Reactive Oxidative Species-Scavenging Network under Cold Stress Low temperature can perturb electron transport chains and cause the production of reactive oxidative species (ROS; Fryer et al., 2002; Hideg et al., 2002; Pfannschmidt et al., 2003). Therefore, we examined hydrogen peroxide (H2O2) accumulation in chs2 plants under cold conditions by 3,3#-diaminobenzidine (DAB) staining. Strong staining was detected in coldtreated chs2 plants but not in wild-type plants (Fig. 4A), indicating that the mutant plants accumulated more H2O2 than the wild-type plants. Under light, chloroplast is the main site of ROS generation; consistently, DAB precipitates were mostly present in the chloroplasts. Therefore, the chs2-induced phenotypes under cold stress might be caused by the impairment of normal chloroplast function and by the overgeneration of ROS in the chloroplasts. When subjected to low temperature, plants accumulate excess H2O2 (O’Kane et al., 1996), which in turn induces the expression of genes associated with oxidative stress (Iba, 2002; Mittler et al., 2004; Rizhsky et al., 2004). More H2O2 accumulation in chs2 was observed under cold conditions (Fig. 4A). Therefore, we examined the expression of several genes encoding ROS-detoxification enzymes, including copper/zinc superoxide dismutase (CSD), ascorbate peroxidase (APX), and catalase (CAT), in cold-treated chs2 plants. No obvious differences in expression of CSD1, APX1, or CAT1 were detected between wild-type and chs2 plants at 22C. In contrast, the expression of these genes was substantially elevated in chs2 plants relative to wild-type plants under cold stress (Supplemental Fig. S2B). The zinc-finger protein ZAT12 plays a crucial role in oxidative and abiotic stress signaling (Rizhsky et al., 2004; Davletova et al., 2005). In addition, ferritin protein nanocages are essential for protecting cells against oxidative damage (Ravet et al., 2009). We found that ZAT12 and FER1 were also significantly up-regulated in cold-treated chs2 plants relative to wild-type plants (Supplemental Fig. S2B). Therefore, the chilling sensitivity of chs2 might result from an imbalance of ROS detoxification and consequent impairment of oxidative signaling. The Expression of Cold-Regulated Genes Is Not Affected in chs2 We further examined whether the chs2 mutation affects the induction of cold-regulated genes. The CBF1 to CBF3 genes were rapidly induced in chs2 and wild-type plants 3 h after exposure to cold, and their target genes RD29A and COR47 were substantially induced at 6 to 12 h after cold treatment. No significant difference in expression of these genes was observed between chs2 and wild-type plants (Supplemental Fig. S3). Therefore, the chs2 gene appears not to affect the CBF pathway. chs2 Constitutively Activates Defense Responses under Cold Conditions Leaves in cold-treated chs2 plants turned yellow, lost turgor pressure, and collapsed (Fig. 1), resembling the pathogen-induced HR cell death response. Extensive cell death did occur in cold-treated chs2 plants but not in wild-type plants, as revealed by trypan blue staining (Fig. 4B). Furthermore, PR1 and PR2 were highly expressed in chs2 plants under cold stress (Fig. 4C). Consistently, cold-treated chs2 plants harboring a PR1: GUS construct showed stronger staining of GUS than wild-type PR1:GUS transgenic plants (Fig. 4D). Because high PR gene expression is often associated with elevated levels of SA, the endogenous SA levels in chs2 were examined. The levels of both free SA and total SA in chs2 were comparable to those in wild-type Figure 2. Physiological analysis of chs2 mutant plants. A, Ion leakage assay in chs2 plants. Plants grown at 22C for 3 weeks were then treated at 4C for the indicated times. The data represent means of three replicates 6 SD. Similar results were observed in three independent experiments. B, Pro content in chs2 plants. Plants grown at 22C for 3 weeks were treated at 4C for 6 d. The data represent means of four replicates 6 SD. * P , 0.01 (t test), significant difference from Col. Similar results were observed in three independent experiments. FW, Fresh weight. Function of a Mutant RPP4 in Response to Chilling Plant Physiol. Vol. 154, 2010 799
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, 2010
plants grown at 22C. 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 constitutively 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 mapping population. Given that the chs2 mutation is dominant, 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 molecular 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-NBLRR-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 finding 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 morphology 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. Wildtype Col and chs2 plants were grown at 22C for 3 weeks and then treated at 4C 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 independent experiments. B, Transmission electron microscopy 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
Function of a Mutant RPP4 in Response to Chilling A 22c 4°C 4. chs2 constitutively activates Col chs2 Col chs2 nts were grown at 3 weeks and then treated at are of representative plants from one of three independent experiments. A H,O, accumulation in chs2 plants Cold-induced cell death in chs2 nts. Detached leaves were stained ages are of representative plant of Pri and Pr2 in wild- type and chs 2 plants by real-time Rt- PCR. The data represent means of three eplicates sD. Similar results were observed in three ind ments. D, GUS analysis of PRi in ch B plants. PR1: GUS transgenic plants were crossed with chs2 plants. The F2 homozygous lines were used for GUS chs2 under cold conditions. Thre treated at 4C for 6 d. The data repre nt means of three replicates +sD. Similar results were observed in three sosN PR1:GUS/Col 22°C223d22°c6d 2℃22c-3d22C6d PR1: GUS/chs2 E Free Sa Total SA 60 10 20 22°C 4C PRI gene expression and the cell death phenotype proteins, and mutations in or close to this conserved were significantly suppressed in chs2-s1(Fig 5, E and motif might abrogate the activity of NB-LRR proteins F). This mutation was mapped to the original RPP4( Bendahmane et al., 2002) locus. Sequencing analysis revealed a second point mutation of e to K at amino acid position 300 in chs 2- sl, which resides close to the walker b/Kinase 2 motif RPP4 Expression in chs2 at Different Conditions of the rPp4 nB domain(Fig. 5B). This motif was To elucidate the physiological function of RPP4, we shown to be important for the function of NB-LRR examined its organ-specific expression in Arabidopsis Plant Ph Vol.154,2010
PR1 gene expression and the cell death phenotype were significantly suppressed in chs2-s1 (Fig. 5, E and F). This mutation was mapped to the original RPP4 locus. Sequencing analysis revealed a second point mutation of E to K at amino acid position 300 in chs2- s1, which resides close to the Walker B/Kinase 2 motif of the RPP4 NB domain (Fig. 5B). This motif was shown to be important for the function of NB-LRR proteins, and mutations in or close to this conserved motif might abrogate the activity of NB-LRR proteins (Bendahmane et al., 2002). RPP4 Expression in chs2 at Different Conditions To elucidate the physiological function of RPP4, we examined its organ-specific expression in Arabidopsis. Figure 4. chs2 constitutively activates defense responses to cold. Wild-type Col and chs2 plants were grown at 22C for 3 weeks and then treated at 4C for 6 d. For A, B, and E, 20 plants were tested for each genotype. Images are of representative plants from one of three independent experiments. A, H2O2 accumulation in chs2 plants stained by DAB. Bar = 20 mm. B, Cold-induced cell death in chs2 plants. Detached leaves were stained with trypan blue. Bar = 100 mm. Images are of representative plants. C, Expression of PR1 and PR2 in wildtype and chs2 plants by real-time RTPCR. The data represent means of three replicates 6 SD. Similar results were observed in three independent experiments. D, GUS analysis of PR1 in chs2 plants. PR1:GUS transgenic plants were crossed with chs2 plants. The F2 homozygous lines were used for GUS staining analysis. Images are of representative plants. E, SA accumulation in chs2 under cold conditions. Threeweek-old 22C-grown plants were treated at 4C for 6 d. The data represent means of three replicates 6 SD. Similar results were observed in three independent experiments. FW, Fresh weight. Function of a Mutant RPP4 in Response to Chilling Plant Physiol. Vol. 154, 2010 801
Huang et al. ased cloning of CHS2 on chromosome IV. Positions of the markers used for mapping are indi- cated above the line. The correspond ChromosomeⅣ ng nucleotide positions are numbered 9400 9450 9500 9550 9600k in kilobases below the line. the num- ber of recombinants is indicated in rentheses. Predicted genes are 45 kb shown by arrows indicating the direc- tion of transcription. B, A schematic a4g16845at4g16855 4g16900at4g16940at4g16960 CHS2 gene. Boxes and lines indicate Genes exons and introns. res ely. The nucleotide substitutions in chs2 and (RPP4) (SNC1 chs2-s1 are shown. C, Complementa- tion of the chs2 mutant. wild-type col B chs2 chs2, and Col transformed with a ge chs2-1(C-T) TAG nomic clone containing the mutat CHS2/RPP4 chs2( Col/CHS2: chs 2) were grown at 22 C for 2 weeks(left)and then treated chs2-S1 (G-A) at 4C for 10 d(right). D, Screenin of the chs2 suppressor chs2-s1. EMS. C 22°C D ed chs2 p at 22C for 2 weeks and then treated at chs2-s 4C for 10d. E, PRI gene expression in Col, chs 2, Col/CHS2: chs2, and chs2 si plants treated at 4C for 6 d by real- time RT-PCR. The data represen means of three replicates t SD. Similar results were observed in three inde. pendent experiments. F, Trypan blue staining of the leaves from chs2, Col CHS2: chs, and chs2-s1 plants Bar 32 100m. chs2 Col/CHS2: chs2 chs2-s1 Transgenic plants harboring a GUS plants(Fig. 6C). However, we found that RPP4 in the driven by the RPP4 promoter were d and wild-type Col background was induced by benzothia- analyzed. GUS staining revealed that RPP4 was ex- diazole(an Sa analog) and cold. Strikingly, cold stress pressed at low levels in leaves, stems, flowers, and dramatically enhanced the induction of the mutated siliques, and it was barely expressed in roots(Fig 6A). RPP4 in chs2(Fig. 6C; Supplemental Fig. S4A). Cold This result is in agreement with public data from induced overexpression could be a consequence of Genevestigator(https://www.genevestigator.com/gv/feedbackregulationuponRgeneactivationwhich index. jsp)and was validated by quantitative reverse might account for the phenotypes of chs2 mutants transcription(rTr-PCr analvsis under cold stress RPP4 was expressed at relatively low levels in the To test if overexpression of wild-type RPP4 would plants, consistent with the low steady-state expression recapitulate the chs2 phenotype, we generated trans- levels of R genes under normal conditions. However, genic lines expressing wild-type RPP4 driven either R genes can be induced by certain stimuli such as by its native promoter(RPP4: RPP4)or by the cauli athogens and SA. Therefore, we investigated whether flower mosaic virus 35S promoter(35S: RPP4), and we RPP4 expression was responsive to various stimuli. The lyzed their phenotypes under cold conditions. In expression of RPP4 was not induced by the oxidative terestingl ither the rPp RPP4 nor 35S: RPP4 ducer methyl viologen in either wild-type Col or chs2 transgenic line, in which RPP4 was indeed overex Plant Physiol. Vol. 154, 2010
Transgenic plants harboring a GUS reporter gene driven by the RPP4 promoter were generated and analyzed. GUS staining revealed that RPP4 was expressed at low levels in leaves, stems, flowers, and siliques, and it was barely expressed in roots (Fig. 6A). This result is in agreement with public data from Genevestigator (https://www.genevestigator.com/gv/ index.jsp) and was validated by quantitative reverse transcription (RT)-PCR analysis (Fig. 6B). RPP4 was expressed at relatively low levels in the plants, consistent with the low steady-state expression levels of R genes under normal conditions. However, R genes can be induced by certain stimuli such as pathogens and SA. Therefore, we investigated whether RPP4 expression was responsive to various stimuli. The expression of RPP4 was not induced by the oxidative inducer methyl viologen in either wild-type Col or chs2 plants (Fig. 6C). However, we found that RPP4 in the wild-type Col background was induced by benzothiadiazole (an SA analog) and cold. Strikingly, cold stress dramatically enhanced the induction of the mutated RPP4 in chs2 (Fig. 6C; Supplemental Fig. S4A). Coldinduced overexpression could be a consequence of feedback regulation upon R gene activation, which might account for the phenotypes of chs2 mutants under cold stress. To test if overexpression of wild-type RPP4 would recapitulate the chs2 phenotype, we generated transgenic lines expressing wild-type RPP4 driven either by its native promoter (RPP4:RPP4) or by the cauli- flower mosaic virus 35S promoter (35S:RPP4), and we analyzed their phenotypes under cold conditions. Interestingly, neither the RPP4:RPP4 nor 35S:RPP4 transgenic line, in which RPP4 was indeed overexFigure 5. Map-based cloning of CHS2. A, A genetic map of the CHS2 locus on chromosome IV. Positions of the markers used for mapping are indicated above the line. The corresponding nucleotide positions are numbered in kilobases below the line. The number of recombinants is indicated in parentheses. Predicted genes are shown by arrows indicating the direction of transcription. B, A schematic diagram of the genomic structure of the CHS2 gene. Boxes and lines indicate exons and introns, respectively. The nucleotide substitutions in chs2 and chs2-s1 are shown. C, Complementation of the chs2 mutant. Wild-type Col, chs2, and Col transformed with a genomic clone containing the mutated chs2 (Col/CHS2:chs2) were grown at 22C for 2 weeks (left) and then treated at 4C for 10 d (right). D, Screening of the chs2 suppressor chs2-s1. EMSmutagenized chs2 plants were grown at 22C for 2 weeks and then treated at 4C for 10 d. E, PR1 gene expression in Col, chs2, Col/CHS2:chs2, and chs2- s1 plants treated at 4C for 6 d by realtime RT-PCR. The data represent means of three replicates 6 SD. Similar results were observed in three independent experiments. F, Trypan blue staining of the leaves from chs2, Col/ CHS2:chs2, and chs2-s1 plants. Bar = 100 mm. Huang et al. 802 Plant Physiol. Vol. 154, 2010
Function of a Mutant RPP4 in Response to Chilling A C Figure 6. Expression of RPP4 and SNCI o Col A and B, Expression of RPP4 by GUS stai and by real-time PCR(B). Total RNA wa from various tissues. The data represent means of hree replicates sD C and D, Expression of RPP4 (C)and SNC1( D)under various treatments. Total RNA was extracted from plants treated 4C), methyl viologen(MV; 5 AM), or be Mock MV BTH Cold diazole(BTH: 0.5 mM) for 24 h. E and F D sion of RPP4(E)and SNCI( F)in 2-week-old 22.grown plants(Col, chs2, RPP4: RPP4, 355. RPP4, snc]-1, and bon1-1)by real esent means of three replicates +sD P<0.01 (t test), significant difference from n nI CoL. All experiments were repeated three times with similar results. G. Phenot ants(Col, chs 2, RPP4: RPP4, 3.5S: RPP4, snc1-1 Mock MV BTH Cold and bon]-1)grown in soil at 22C for 4 weeks and then cold treated at 4C for 10 d E F RPP4: RPP4 35S: RPP4 pressed(Fig. 6E), exhibited chs2-like phenotypes at transcript than did cold-treated Col plants(Fig. 6D 4oC(Fig. 6G). Therefore, the chs2-conferred pheno- Supplemental Fig $4B) types are not simply caused by constitutive expression To determine whether up-regulation of SNCl also of RPP4 but rather by the amino acid substitution in contributes to the chs2 phenotype, we tested the cold chs2. All of these data indicate that chs2 is a gain-of- sensitivity of sncl-I and bonl-1 plants, in which SNCI function mutant and that cold-induced overexpression is activated or derepressed(Yang and Hua, 2004; L of the mutated RPP4 gene is required for the chs2 et al., 2007; Fig 6F). Neither of them showed a chs2-like phenotype lethal phenotype at cold stress(Fig. 6G). In addition, we transformed the CHS2 chs2 clone into sncl-1l loss- of-function mutant plants. All 10 independent trans- chs2-Induced Chilling Sensitivity Is Independent genic lines displayed a chs2-like chilling-sensitive of SNcl phenotype(data not shown), indicating that the chs2 mutation confers a chs2 phenotype independent of Since the RPP5 locus R genes are coordinately SNCI regulated (Yi and Richards, 2007), we examined the expression of SNC1, a close homolog of RPP4, in the chs2-Induced Chilling Sensitivity Is Independent of SA chs2 mutant. Similar expression patterns of SNC1 induction were found in wild-type Col and chs2 plants (Fig. 6D). SNCI expression was induced by benzothia Because chs2 plants accumulated high levels of free diazole and cold stress in both genotypes. Moreover, SA and total SA after cold treatment(Fig. 4E), we then chs2 plants accumulated higher levels of the SNC1 determined whether activation of the Sa pathway is Plant Ph Vol.154,2010
pressed (Fig. 6E), exhibited chs2-like phenotypes at 4C (Fig. 6G). Therefore, the chs2-conferred phenotypes are not simply caused by constitutive expression of RPP4 but rather by the amino acid substitution in chs2. All of these data indicate that chs2 is a gain-offunction mutant and that cold-induced overexpression of the mutated RPP4 gene is required for the chs2 phenotype. chs2-Induced Chilling Sensitivity Is Independent of SNC1 Since the RPP5 locus R genes are coordinately regulated (Yi and Richards, 2007), we examined the expression of SNC1, a close homolog of RPP4, in the chs2 mutant. Similar expression patterns of SNC1 induction were found in wild-type Col and chs2 plants (Fig. 6D). SNC1 expression was induced by benzothiadiazole and cold stress in both genotypes. Moreover, chs2 plants accumulated higher levels of the SNC1 transcript than did cold-treated Col plants (Fig. 6D; Supplemental Fig. S4B). To determine whether up-regulation of SNC1 also contributes to the chs2 phenotype, we tested the cold sensitivity of snc1-1 and bon1-1 plants, in which SNC1 is activated or derepressed (Yang and Hua, 2004; Li et al., 2007; Fig. 6F). Neither of them showed a chs2-like lethal phenotype at cold stress (Fig. 6G). In addition, we transformed the CHS2:chs2 clone into snc1-11 lossof-function mutant plants. All 10 independent transgenic lines displayed a chs2-like chilling-sensitive phenotype (data not shown), indicating that the chs2 mutation confers a chs2 phenotype independent of SNC1. chs2-Induced Chilling Sensitivity Is Independent of SA and NPR1 Because chs2 plants accumulated high levels of free SA and total SA after cold treatment (Fig. 4E), we then determined whether activation of the SA pathway is Figure 6. Expression of RPP4 and SNC1 in chs2. A and B, Expression of RPP4 by GUS staining (A) and by real-time PCR (B). Total RNA was extracted from various tissues. The data represent means of three replicates 6 SD. C and D, Expression of RPP4 (C) and SNC1 (D) under various treatments. Total RNA was extracted from plants treated with cold (4C), methyl viologen (MV; 5 mM), or benzothiadiazole (BTH; 0.5 mM) for 24 h. E and F, Expression of RPP4 (E) and SNC1 (F) in 2-week-old 22C-grown plants (Col, chs2, RPP4:RPP4, 35S: RPP4, snc1-1, and bon1-1) by real-time PCR. The data represent means of three replicates 6 SD. * P , 0.01 (t test), significant difference from Col. All experiments were repeated three times with similar results. G, Phenotypes of the plants (Col, chs2, RPP4:RPP4, 35S:RPP4, snc1-1, and bon1-1) grown in soil at 22C for 4 weeks and then cold treated at 4C for 10 d. Function of a Mutant RPP4 in Response to Chilling Plant Physiol. Vol. 154, 2010 803
Huang et al. necessary for the chs2 phenotype by crossing chs2 with 8), indicating that NPRI is dispensable for the chs2- the SA-deficient mutant sid2-2(Wildermuth et al. con 2001 ). The chs2 sid2 double mutants exhibited chilling sensitivity and extensive cell death phenotypes similar chs2-Induced Chilling Sensitivity Requires Multiple to those of chs2(Fig. 7, A and C). As expected, the mutants were reduced to a wild-type level una double levels of sa and total sa in the chs2 sid2 Signaling Components r cold To assess whether defense signaling components stress(Fig. 8). Therefore, the chs2-conferred chilling-(including EDSl, PAD4, SGTIb, and RARI) are in- sensitive phenotype does not require SA volved in the chs2-mediated temperature signaling NPRI is a master regulator of SA signaling and plant pathway, we first examined RPP4 expression in eds1-2 immunity( Cao et al., 1994). To examine the require( Col; Bartsch et al. 2006), pad4-1 rage et al., 1999), ment for NPRI in chs2-mediated signaling, a chs2 npr1 rar1-20(Muskett et al. 2002), and sgt1b/eta3(Gray double mutant was generated and then characterized et aL., 2003)mutants. RPP4 expression was slightly he loss of NPRI function, while significantly reduc- down-regulated by edsI and pad4 but not by rarl or ing PRl expression, did not abrogate the chs2-medi t1b(Supplemental Fig. S5). We also generated dou- ated cold-sensitive morphology, cell death, or the ble mutants of chs2 with eds1-1(Parker et al., 1996), accumulation of SA at low temperature(Figs. 7 and pad4-1, rar1-20, and sgt1bleta3 for further analyses chs2 chs2 eds chs2 pad4B chs2 rar1 chs2 sgt1b chs2 sid2 chs2 npr1 c Col chs2 chs2 eds chs2 pad4 1500 chs2 rar1 chs2 sgt1b chs2 sid2 chs2 npr1 1200 3 chs2 chs2 eds chs2 pad4 chs2 rar1 chs2 sgt1b chs2 sid2 chs2 npr1 Figure 7. Phenotypes of the chs 2 double mutants under cold conditions. Three-week-old 22C-grown plants were treated at 4C for 6d(C-E), 14 d(A), or 5 weeks(B). A and B, Growth phenotypes of the double mutants under cold conditions. Representative plants are shown. C, Trypan blue staining of the leaves from the double mutants. Bar =100 um. Note that the photographs of 4( reated Col and chs 2 plants stained with trypan blue are identical to those shown in Figure 2B. D, DAB staining of the leaves from the double mutants. Bar =100 um. E, PRi gene expression in the double mutants by real-time PCR. The data represent means of three replicates sD. *P<0.01 (t test), significant difference from chs2. All experiments were repeated three times with similar Plant Physiol. Vol. 154, 2010
necessary for the chs2 phenotype by crossing chs2 with the SA-deficient mutant sid2-2 (Wildermuth et al., 2001). The chs2 sid2 double mutants exhibited chilling sensitivity and extensive cell death phenotypes similar to those of chs2 (Fig. 7, A and C). As expected, the levels of SA and total SA in the chs2 sid2 double mutants were reduced to a wild-type level under cold stress (Fig. 8). Therefore, the chs2-conferred chillingsensitive phenotype does not require SA. NPR1 is a master regulator of SA signaling and plant immunity (Cao et al., 1994). To examine the requirement for NPR1 in chs2-mediated signaling, a chs2 npr1 double mutant was generated and then characterized. The loss of NPR1 function, while significantly reducing PR1 expression, did not abrogate the chs2-mediated cold-sensitive morphology, cell death, or the accumulation of SA at low temperature (Figs. 7 and 8), indicating that NPR1 is dispensable for the chs2- conferred phenotype. chs2-Induced Chilling Sensitivity Requires Multiple Signaling Components To assess whether defense signaling components (including EDS1, PAD4, SGT1b, and RAR1) are involved in the chs2-mediated temperature signaling pathway, we first examined RPP4 expression in eds1-2 (Col; Bartsch et al., 2006), pad4-1 (Jirage et al., 1999), rar1-20 (Muskett et al., 2002), and sgt1b/eta3 (Gray et al., 2003) mutants. RPP4 expression was slightly down-regulated by eds1 and pad4 but not by rar1 or sgt1b (Supplemental Fig. S5). We also generated double mutants of chs2 with eds1-1 (Parker et al., 1996), pad4-1, rar1-20, and sgt1b/eta3 for further analyses. Figure 7. Phenotypes of the chs2 double mutants under cold conditions. Three-week-old 22C-grown plants were treated at 4C for 6 d (C–E), 14 d (A), or 5 weeks (B). A and B, Growth phenotypes of the double mutants under cold conditions. Representative plants are shown. C, Trypan blue staining of the leaves from the double mutants. Bar = 100 mm. Note that the photographs of 4Ctreated Col and chs2 plants stained with trypan blue are identical to those shown in Figure 2B. D, DAB staining of the leaves from the double mutants. Bar = 100 mm. E, PR1 gene expression in the double mutants by real-time PCR. The data represent means of three replicates 6 SD. * P , 0.01 (t test), significant difference from chs2. All experiments were repeated three times with similar results. Huang et al. 804 Plant Physiol. Vol. 154, 2010
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.154,2010
Because 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 background. Among 211 F2 progeny, all 12 lines of chs2 eds1 and 25 lines of chs2/+ eds1 showed wild-type-like morphology at 4C (Fig. 7A). Extensive cell death, elevated PR1 expression, and accumulation of H2O2 and SA under cold conditions were also totally suppressed 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 sensitivity 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 4C (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 16C to 18C (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 compromised 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 exhibits yellowish leaves, increased ion leakage, damaged chloroplasts, ROS accumulation, extensive cell death, and consequent lethality at chilling temperatures (below 12C). To our surprise, all the morphological 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. Chloroplast morphological change and ROS accumulation are observed in mutants showing cell death phenotypes (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 nucleotidebinding 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 conditions. Three-week-old 22C-grown plants were treated at 4C 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
