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Keywords:

  • Botrytis cinerea;
  • disease resistance;
  • ETHYLENE RESPONSE FACTOR (ERF);
  • ethylene signaling;
  • RELATED TO AP2 2 (RAP2.2)

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • Ethylene plays a crucial role in plant resistance to necrotrophic pathogens, in which ETHYLENE RESPONSE FACTORs (ERFs) are often involved.
  • Here, we evaluated the role of an ERF transcription factor, RELATED TO AP2 2 (RAP2.2), in Botrytis resistance and ethylene responses in Arabidopsis. We analyzed the resistance of transgenic plants overexpressing RAP2.2 and the T-DNA insertion mutant to Botrytis cinerea. We assessed its role in the ethylene signaling pathway by molecular and genetic approaches.
  • RAP2.2-overexpressing transgenic plants showed increased resistance to B. cinerea, whereas its T-DNA insertion mutant rap2.2-3 showed decreased resistance. Overexpression of RAP2.2 in ethylene insensitive 2 (ein2) and ein3 ein3-like 1 (eil1) mutants restored their resistance to B. cinerea. Both ethylene and Botrytis infection induced the expression of RAP2.2 and the induction was disrupted in ein2 and ein3 eil1 mutants. We identified rap2.12-1 as a T-DNA insertion mutant of RAP2.12, the closest homolog of RAP2.2. The hypocotyls of rap2.2-3 rap2.12-1 double mutants showed ethylene insensitivity. The constitutive triple response in constitutive triple response1 (ctr1) was partially released in the rap2.2-3 rap2.12-1 ctr1 triple mutants.
  • Our findings demonstrate that RAP2.2 functions as an important regulator in Botrytis resistance and ethylene responses.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Plants are continually challenged by various pathogens throughout their lifetime. Most pathogens are grouped into biotrophs and necrotrophs according to their manners of infection; necrotrophs kill host plants and derive nutrients from dead or dying tissues, and many of them are capable of infecting a broad spectrum of plant species (Glazebrook, 2005; Laluk & Mengiste, 2010). The necrotrophic fungus Botrytis cinerea is one of the most destructive pathogens. It causes gray mould disease on > 200 crop plants, leading to massive losses in crop production (Williamson et al., 2007). Despite its devastating impact on agricultural productivity, our knowledge about plant resistance to B. cinerea remains limited.

Ethylene (ET) is an important phytohormone with multiple biological roles. It regulates diverse plant developmental processes, ranging from seedling emergence to organ senescence. It also acts as a stress hormone, regulating plant responses to biotic and abiotic stresses (Johnson & Ecker, 1998; Bleecker & Kende, 2000; Wang et al., 2002). Many ET signaling components have been identified over the last two decades and the ET signaling pathway is well established in the model plant Arabidopsis thaliana (Wang et al., 2002; Stepanova & Alonso, 2009; Yoo et al., 2009). ET is perceived by histidine kinase receptors, leading to deactivation of a negative regulator CONSTITUTIVE TRIPLE RESPONSE1 (CTR1), a Raf-like protein kinase. The release of CTR1 suppression on ET signaling results in the activation of ETHYLENE INSENSITIVE2 (EIN2) and downstream EIN3/EIN3-LIKEs (EILs) transcription factors (Kieber et al., 1993; Chao et al., 1997; Hua & Meyerowitz, 1998; Alonso et al., 1999; Guo & Ecker, 2003; Huang et al., 2003). EIN2 is a transmembrane protein with homology to NRAMP metal ion transporters, and acts as a positive regulator of the ET responses. EIN3 and its homolog EILs are plant-specific transcription factors that regulate a set of ET-responsive genes by binding to the primary ethylene response element (PERE) in promoter regions (Solano et al., 1998). During plant resistance to necrotrophic pathogens, ET plays a crucial role synergistically with jasmonic acid (JA), as demonstrated by genetic approaches (Glazebrook, 2005; van Loon et al., 2006; Grant & Jones, 2009). Both ein2 and the ein3 eil1 double mutant, two ET-insensitive mutants, showed increased susceptibility to necrotrophic B. cinerea (Thomma et al., 1999; Alonso et al., 2003).

In Arabidopsis, ETHYLENE RESPONSE FACTORs (ERFs) are often involved in plant stress responses, regulating downstream ET-responsive genes via the GCC-box elements in promoters (Ohme-Takagi & Shinshi, 1995). Several ERFs have been identified as regulators in Botrytis resistance (Gutterson & Reuber, 2004; Nakano et al., 2006). ORA59 is one that has been well studied: transgenic plants overexpressing ORA59 showed enhanced resistance to B. cinerea, whereas those silencing ORA59 showed impaired resistance. ORA59 positively regulates the defense marker gene PLANT DEFENSIN1.2 (PDF1.2) expression in vivo by binding two functional GCC-box elements in PDF1.2 promoter (Pre et al., 2008; Zarei et al., 2011). Furthermore, ORA59 expression is induced by JA and ET synergistically and the induction requires both the JA and ET signaling pathways, indicating that ORA59 acts as an integrator between the JA and ET signaling pathways (Pre et al., 2008). ERF1 is another ERF involved in plant resistance to B. cinerea. Overexpression of ERF1 resulted in constitutive expression of ET-responsive genes, PDF1.2 and BASIC CHITINASE, and enhanced Botrytis resistance in Arabidopsis (Solano et al., 1998; Berrocal-Lobo et al., 2002). Moreover, the PERE cis-element in ERF1 promoter is recognized by EIN3, and ERF1 is also capable of activating PDF1.2 expression in vivo (Solano et al., 1998; Pre et al., 2008; Wehner et al., 2011). This evidence suggests that ERF1 serves as an intermediate transcription regulator between EIN3 and the downstream ET-responsive genes. Like ORA59, ERF1 expression is induced rapidly by JA and ET synergistically and both signaling pathways are required for the induction (Lorenzo et al., 2003).

The ERF transcription factor RELATED TO AP2 2 (RAP2.2) is involved in plant response to hypoxia, during which it’s regulated by the N-end rule pathway of protein degradation (Hinz et al., 2010; Gibbs et al., 2011). Here, we present that RAP2.2 plays important roles in plant resistance to necrotrophic B. cinerea and the hypocotyl response to ET. The expression of RAP2.2 was induced by Botrytis infection and ET treatment, and the induction was disrupted in the ET-insensitive mutants, ein2 and ein3 eil1. Our genetic results showed that transgenic plants overexpressing RAP2.2 were more resistant to B. cinerea than the wildtype Arabidopsis plants, whereas the T-DNA insertion mutant rap2.2-3 was more susceptible. Moreover, overexpression of RAP2.2 in the ein2 or ein3 eil1 background restored the resistance to B. cinerea. In addition, we identified rap2.12-1 as a T-DNA insertion mutant of RAP2.12, the closest homolog of RAP2.2. The seedlings of rap2.2-3 rap2.12-1 double mutants showed ET insensitivity in hypocotyls. And the rap2.2-3 rap2.12-1 ctr1 triple mutants showed a partial release of the constitutive triple response in hypocotyls. Our results revealed that RAP2.2 acts as an important regulator in Botrytis resistance and ET responses, in addition to a previously reported role in hypoxia tolerance.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant growth condition

All plants used in this study were in Arabidopsis thaliana (L.) Heynh Columbia-0 (Col-0) background. Plants were grown under long-day conditions as previously described (Wu et al., 2011). For the triple response assay, surface-sterilized seeds were placed on Murashige and Skoog (MS) plates supplemented with 10 μM 1-aminocyclopropane-1-carboxylic acid (ACC). Seeds were kept in darkness at 4°C for 3 d and 22°C for another 3 d, after which the lengths of hypocotyls and roots were measured.

Hormone treatments

For the ethylene gas treatment, 3-d-old etiolated seedlings grown on MS plates were put into the sealed containers with or without 10 μl l−1 ethylene gas. Seedlings were harvested for RNA extraction at 0, 3 and 6 h after the treatment. For the hormone treatments, 4-wk-old soil-grown plants were sprayed with 100 μM ACC, 100 μM JA, or the respective solvent dimethyl sulfoxide (DMSO; 0.1% water solution), and kept under the normal growth condition. Fully expanded rosette leaves were harvested for RNA extraction at 0, 1, 3 and 6 h after the treatment. For the assessment of RAP2.2 expression in ET signaling pathway mutants, rosette leaves from 2-wk-old soil-grown plants were harvested for RNA extraction.

Identification of the rap2.2-3 and rap2.12-1 mutant alleles and generation of transgenic Arabidopsis plants

The seeds of T-DNA insertion alleles were requested from the Arabidopsis Biological Resource Center (ABRC, http://abrc.osu.edu). Homozygous mutants were genotyped by PCR using gene-specific and T-DNA-specific primers (LBa1: 5′-TGG TTC ACG TAG TGG GCC ATC G-3′). The following gene-specific primers were used for genotyping: RP1 (5′-TGA CTC GTA ATG GCT GTT ATG TG-3′) and LP1 (5′-GTG TAA CAC AAA CAC GCC CAT AG-3′) for rap2.2-3 (SALK_010265); RP2 (5′-CAC ACA AAT CAA ACC TCA TGC-3′) and LP2 (5′-TTT TGT GCC TAT GTT AAA ACA TTT TC-3′) for rap2.12-1 (SALK_019873) mutant. The gene expression levels in mutant plants were identified using quantitative reverse transcription polymerase chain reaction (RT-PCR).

In order to generate transgenic plants overexpressing RAP2.2, the overexpression construct was generated as previously described (Liu et al., 2008). The full-length coding sequence (CDS) was amplified from Arabidopsis cDNA by RT-PCR with primers carrying Xho I and Sac I sites (underlined): 5′-CTT CAC CTC GAG ATG TGT GGA GGA GCT ATA AT-3′ and 5′-CTT CAC GAG CTC TCA AAA GTC TCC TTC CAG CAT GA-3′. The amplified fragments were cloned into pGEM-T-vector (Promega), and confirmed by sequencing. RAP2.2 CDS was released by digesting the resulting plasmid with Xho I/Sac I and cloned into the same restriction sites of the binary vector pJIM19-HA-FLAG. The resulting pJIM19-RAP2.2 plasmid was then transformed into Agrobacterium tumefaciens strain GV3101 and introduced into the wildtype, ein2 and ein3 eil1 Arabidopsis plants as previously described (Qin et al., 2005). Seeds from transformed plants were harvested and sown on MS plates supplemented with 50 mg l−1 hygromycin (Sigma-Aldrich). T2-generation transgenic lines were screened for possible single locus of T-DNA insertion according to a 3 : 1 (hygromycin-resistant/hygromycin-sensitive) segregation ratio. Homozygous T3-generation transgenic plants were used for phenotype and expression assays.

Plant inoculation with B. cinerea

The Botrytis cinerea strain was provided by Dr Dingzhong Tang and described in previous studies (Ferrari et al., 2003; Tang et al., 2007). B. cinerea was cultured on potato dextrose agar (PDA) plates for 10 d at 22°C in the dark. Conidia were collected by rinsing the cultural plates with potato dextrose broth (PDB) medium. The spore suspension was adjusted to the appropriate concentration, and sprayed onto 4-wk-old soil-grown plants in a fine mist. The inoculated plants were covered with transparent plastic films to maintain high humidity and incubated in a growth chamber with a 12 h light : 12 h dark cycle. The disease symptoms were photographed and the decay rates were assessed at the indicated time points. As an indicator of Botrytis growth in planta, the transcriptional abundance of the BcActin A gene was determined by quantitative RT-PCR.

Quantitative RT-PCR

RNA extraction and cDNA synthesis were performed as previously reported (Guo et al., 2009). Briefly, total RNA was extracted from plant tissues using TRIzol (Invitrogen), followed by DNase I (TaKaRa, Japan) treatment. For each sample, 1 μg of RNA was used for reverse transcription using the Superscript II Reverse Transcriptase Kit (Invitrogen). The resulting cDNA was subjected to quantitative RT-PCR as previously described (Xing et al., 2010). To ensure the specificity of the reaction, the PCR products were assessed by melting curve and gel electrophoresis. Gene expression levels were standardized to the expression levels of AtUBQ10. For each sample, at least three independent biological replicates were performed. The relative expression levels of each gene were calculated using the 2−ΔΔCT method (Livak & Schmittgen, 2001). The primers for quantitative RT-PCR are listed in the Supporting Information, Table S1.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

RAP2.2 is a positive regulator in Botrytis resistance

The RAP2.2 gene (At3g14230) encodes an ERF transcription factor. By using a high-throughput yeast two-hybrid screening system in our recent study, we identified RAP2.2, as well as seven other transcription factors, to interact with a Mediator complex subunit Med25/PFT1 (Ou et al., 2011). As Med25/PFT1 is required for Arabidopsis resistance to the necrotrophic fungal pathogen B. cinerea (Kidd et al., 2009), it raises an interesting question about whether RAP2.2 is also involved in Botrytis resistance. To clarify this question, we obtained its T-DNA insertion line, SALK_010265 (designated as rap2.2-3), from ABRC. Homozygous plants were identified by PCR using primers flanking the insertion site, and the genomic region around the insertion site was cloned for sequencing (Fig. 1a,b). The result showed that the rap2.2-3 allele carries a T-DNA insertion in the 5′-UTR region of RAP2.2 (Fig. 1a). The expression levels of RAP2.2 in homozygous mutant plants were reduced to approximately one-quarter of that in the wildtype (Fig. 1c). We also generated an overexpression construct in which RAP2.2 was driven by the cauliflower mosaic virus (CaMV) 35S promoter and transformed this construct into the wildtype plants (Fig. 1d). Two transgenic lines (35S:RAP2.2#96 and 35S:RAP2.2#53) with constitutive expression of RAP2.2 at high levels were chosen for further analyses (Fig. 1e).

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Figure 1. The identification of T-DNA insertion mutant rap2.2-3 and generation of 35S:RAP2.2 transgenic Arabidopsis thaliana plants. (a) The identification of the T-DNA insertion mutant SALK_010265. The mutant was designated as rap2.2-3. Untranslated regions (UTRs) are represented as closed boxes and exons as open boxes. LP1 and RP1 primers are designed for genotyping. P1 and P2 primers are designed for quantitative reverse transcription polymerase chain reaction (RT-PCR). (b) Genotyping of the wildtype, heterozygous and homozygous plants with primers LP1, RP1 and LBa1 on the T-DNA. A band of 1052 bp was able to be amplified from homozygous lines. (c) The expression level of RAP2.2 in rap2.2-3 determined by quantitative RT-PCR, using 4-wk-old wildtype and rap2.2-3 plants. RAP2.2 expression levels were standardized to AtUBQ10, and the value in the wildtype was set at 1.0. (d) The scheme of RAP2.2 overexpression construct. Hyg, hygromycin; 35S-P, the cauliflower mosaic virus (CaMV) 35S promoter; HA, hemagglutinin tag; Ter, terminator; LB, left border; RB, right border. (e) The expression levels of RAP2.2 in the 35S:RAP2.2#96 and 35S:RAP2.2#53 transgenic plants determined by quantitative RT-PCR, using 4-wk-old wildtype and 35S:RAP2.2 plants. RAP2.2 expression levels were standardized to AtUBQ10 and the value in the wildtype was set at 1.0. Error bars in (c) and (e) represent the SD from three biological replicates, each containing at least 20 plants. RAP2.2 expression levels that differ significantly (< 0.05) from those in the wildtype are marked with asterisks.

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To assess the resistance to B. cinerea, 4-wk-old wildtype, rap2.2-3, 35S:RAP2.2#96 and 35S:RAP2.2#53 plants were inoculated by spraying with Botrytis spore suspension (1 × 105 spores ml−1), as previously described (Dhawan et al., 2009). In the wildtype plants, the infection caused necrotic lesions in leaf tissue at 2 d after inoculation (DAI) and chlorosis at 4 DAI (Fig. 2a). In the rap2.2-3 plants, the lesions and chlorosis were visible as early as 2 DAI. The infection continued to spread and caused leaf maceration at 4 DAI. By contrast, the disease symptoms caused by B. cinerea were very limited in the 35S:RAP2.2#96 and 35S:RAP2.2#53 plants (Fig. 2a). To monitor in planta fungal growth in the inoculated plants, we determined the abundance of transcripts of the fungal Actin A gene (BcActin A) by quantitative RT-PCR. As shown in Fig. 2(b), the BcActin A transcripts were detected at 1 DAI in the wildtype plants and their abundance increased as infection advanced at 2 and 4 DAI. During Botrytis infection, the transcripts of BcActin A were more abundant in the rap2.2-3 plants than in the wildtype. However, fewer BcActin A transcripts accumulated in the 35S:RAP2.2#96 and 35S:RAP2.2#53 plants, and the abundance of transcripts did not increase at 2 and 4 DAI, suggesting that fungal growth ceased in the transgenic plants overexpressing RAP2.2.

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Figure 2. RAP2.2 plays an important role in plant resistance to Botrytis cinerea. (a) Disease symptoms of the wildtype, rap2.2-3 and 35S:RAP2.2#96 and 35S:RAP2.2#53 Arabidopsis thaliana plants at 0, 2 and 4 d after inoculation with 1 × 105Botrytis spores ml−1. (b) The expression levels of BcActin A gene determined by quantitative reverse transcription polymerase chain reaction (RT-PCR), using 4-wk-old wildtype, rap2.2-3 and 35S:RAP2.2 plants inoculated with 1 × 105Botrytis spores ml−1. BcActin A expression levels were standardized to AtUBQ10, and the value in the wildtype at 1 d was set at 1.0. (c) Disease symptoms of the wildtype, rap2.2-3 and 35S:RAP2.2#96 and 35S:RAP2.2#53 plants at 7 and 12 d after inoculation with a higher concentration of 3.5 × 105Botrytis spores ml−1. (d) Percentages of decayed plants after inoculation with 3.5 × 105Botrytis spores ml−1. The plants were scored as decayed when their centers were completely rotten. Error bars represent the SD from three biological replicates and each replicate contained at least 30 plants for each genotype. The percentages that differ significantly (< 0.05) from those in the wildtype are marked with asterisks. (e) and (f) The expression levels of PDF1.2 and ChiB determined by quantitative RT-PCR, using 4-wk-old wildtype, rap2.2-3, 35S:RAP2.2#53 and ein2 plants at 0 and 2 d after inoculation with 1 × 105Botrytis spores ml−1. Gene expression levels were standardized to AtUBQ10, and the values in the wildtype at 0 d were set at 1.0. The results at 0 d are shown at the top left. Error bars in (b), (e) and (f) represent the SD from three biological replicates, each containing at least 20 plants. The expression levels that differ significantly (< 0.05) from those in the wildtype are marked with asterisks.

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To clearly establish the role of RAP2.2 in Botrytis resistance, a higher inoculum (3.5 × 105 spores ml−1) was used to challenge 4-wk-old wildtype, rap2.2-3 and 35S:RAP2.2 plants. When the plants were incubated for extended periods under the condition suitable for disease development, the infection continued to macerate the wildtype plants. Plant resistance was determined by the percentage of decayed plants. At 7 DAI, the percentages of completely decayed plants were 33% in wildtype, 71% in rap2.2-3, 17% in 35S:RAP2.2#96 and 10% in 35S:RAP2.2#53. At 12 DAI, 85% of the wildtype plants were completed decayed, and almost all the rap2.2-3 plants were dead. However, there were still many 35S:RAP2.2 (29% of #96 and 64% of #53) surviving from fungal infection at the end of disease assay (Fig. 2c,d). These results showed that rap2.2-3 exhibited less resistance to B. cinerea than the wildtype plants, whereas the 35S:RAP2.2 plants exhibited greater resistance, suggesting that RAP2.2 expression levels are positively correlated with plant resistance to the necrotrophic B. cinerea.

Furthermore, the transcripts of two well-characterized marker genes, PDF1.2 and ChiB, were measured in the wildtype rap2.2-3, 35S:RAP2.2#53 and ein2 plants after inoculation with B. cinerea. Both marker genes were highly induced in the wildtype plants at 2 DAI, whereas their expression levels were much lower in the ein2 mutants (Fig. 2e,f). In the rap2.2-3 plants, the expression levels of PDF1.2 and ChiB were lower than those in the wildtype at 2 DAI; whereas in the 35S:RAP2.2#53 plants, their expression levels were higher than those in the wildtype. This result is consistent with the Botrytis resistance of rap2.2-3 mutants and 35S:RAP2.2 transgenic plants. Taken together, the disease assay results showed that knockdown of RAP2.2 expression by T-DNA insertion impaired the plant resistance to B. cinerea, whereas its overexpression enhanced it. Thus, RAP2.2 acts as a positive regulator required for Botrytis resistance in Arabidopsis.

Induction of RAP2.2 by ET relies on EIN2 and EIN3/EIL1

Both ET and JA play important roles in plant resistance to necrotrophic pathogens. These two phytohormones synergistically induce the expression of ERF1 and ORA59, two ERF transcription factors involved in the resistance to B. cinerea (Berrocal-Lobo et al., 2002; Lorenzo et al., 2003; Pre et al., 2008). As RAP2.2 is also an ERF transcription factor and involved in Botrytis resistance, we tested whether RAP2.2 expression was regulated by ET and JA treatments as well. Four-week-old wildtype Arabidopsis plants were used for a hormone treatment experiment. In the plants treated with the ET precursor ACC, the expression level of RAP2.2 increased substantially at 1 h and the induction persisted until 6 h (Fig. 3a). This result is consistent with a previous study, in which RAP2.2 was induced by ACC at 2 h (Hinz et al., 2010). However, RAP2.2 expression levels did not change after JA treatment at the indicated time points. The defense gene PDF1.2, a well characterized marker of the ET and JA signaling pathways, was induced by both ET and JA as expected (Fig. 3b). This result indicates that RAP2.2 expression is regulated by ET but not by JA in Arabidopsis, which is different from ORA59 or ERF1.

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Figure 3. RAP2.2 expression is induced by ethylene (ET). (a, b) The expression levels of RAP2.2 and PDF1.2 under hormone treatments, determined by quantitative reverse transcription polymerase chain reaction (RT-PCR). RNA was extracted from 4-wk-old soil-grown wildtype Arabidopsis thaliana plants treated with 0.1% dimethyl sulfoxide solution (DMSO; white columns), 100 μM 1-aminocyclopropane-1-carboxylic acid (ACC; gray columns) or 100 μM jasmonic acid (JA; black columns) at 0, 1, 3 and 6 h. Gene expression levels were standardized to AtUBQ10, and the values in the wildtype at 0 h were set at 1.0. (c, d) The expression levels of RAP2.2 and PDF1.2 in the wildtype, ein2 and ein3 eil1 plants after ET gas treatment, determined by quantitative RT-PCR. RNA was extracted from 3-d-old wildtype (white columns), ein2 (gray columns) and ein3 eil1 (black columns) seedlings treated with 10 μl l−1 of ET gas for 0 h, 3 h and 6 h. Gene expression levels were standardized to AtUBQ10, and the values in the wildtype at 0 h were set at 1.0. Error bars represent the SD from three biological replicates. Each replicate in (a) and (b) contained at least 20 plants and each in (c) and (d) contained at least 60 seedlings. A one-way ANOVA was used to assess the statistical significance of the differences in expression levels. Different letters (a–d) indicate significant differences between each other (P < 0.05).

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To further investigate how RAP2.2 is regulated by the ET signaling pathway, another hormone treatment experiment was conducted on the wildtype plants and ET-insensitive mutants. Three-day-old wildtype, ein2 and ein3 eil1 etiolated seedlings were treated with ET gas and the expression levels of RAP2.2 were assessed by quantitative RT-PCR. As shown in Fig. 3(c), RAP2.2 expression was induced rapidly and transiently by ET gas treatment at 3 h. However, in both ein2 and ein3 eil1 seedlings, the expression level of RAP2.2 was less than one-half of that in the wildtype at 0 h, and remained unchanged at 3 h after ET treatment. Although RAP2.2 expression displayed a delayed induction in ein3 eil1 at 6 h, its expression level was just comparable with the basal level in the wildtype at 0 h. And the induction of PDF1.2 expression by ET treatment was also disrupted in ein2 and ein3 eil1 (Fig. 3d). This result showed that the induction of RAP2.2 expression by ET was severely disrupted in these ET-insensitive mutants, indicating that the induction is dependent on EIN2 and EIN3/EIL1. We also examined the expression levels of RAP2.2 in other ET signaling pathway mutants. The expression level of RAP2.2 was higher in the constitutive triple response mutant ctr1, but lower in the ET-insensitive mutant etr1, than in the wildtype (Fig. S1). In conclusion, these results indicate that RAP2.2 expression is induced by ET, which relies on the integrity of the ET signaling pathway.

Induction of RAP2.2 by Botrytis infection relies on the ET signaling pathway

We further tested whether RAP2.2 expression is induced by Botrytis infection, and, if so, whether this induction is also dependent on the ET signaling pathway. Four-week-old wildtype, ein2 and ein3 eil1 plants were inoculated with Botrytis spore suspension (1 × 105 spores ml−1), and the expression levels of RAP2.2 were determined by quantitative RT-PCR. In the wildtype plants, RAP2.2 expression was induced at 1 DAI and the induction persisted thereafter. But in either ein2 or ein3 eil1 mutants, its expression didn’t show any change (Fig. 4a). During fungal infection, PDF1.2, as a positive control, was induced prominently in the wildtype plants, but its expression levels were much lower in these two ET-insensitive mutants (Fig. 4b). This result indicates that RAP2.2 expression is induced by Botrytis infection in Arabidopsis and the ET signaling pathway is required for this induction.

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Figure 4.  Induction of RAP2.2 by Botrytis infection relies on the ethylene (ET) signaling pathway. The expression levels of RAP2.2 (a) and PDF1.2 (b) in the Arabidopsis thaliana wildtype, ein2 and ein3 eil1 plants after inoculation with Botrytis cinerea, determined by quantitative reverse transcription polymerase chain reaction (RT-PCR). RNA was extracted from 4-wk-old soil-grown wildtype (white columns), ein2 (gray columns) and ein3 eil1 (black columns) plants treated with Botrytis spore suspension (1 × 105 spores ml−1) at 0, 1, 2 and 4 d. Gene expression levels were standardized to AtUBQ10, and the values in the wildtype at 0 d were set at 1.0. Error bars represent the SD from three biological replicates, each containing at least 20 plants. A one-way ANOVA was used to assess the statistical significance of the differences in RAP2.2 expression levels. Different letters (a–c) indicate significant differences between each other (P < 0.05).

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Overexpression of RAP2.2 in ein2 or ein3 eil1 restored their resistance to B. cinerea

These results demonstrate that the RAP2.2 induction by ET or Botrytis infection is dependent on EIN2 and EIN3/EIL1. It suggests that RAP2.2 possibly functions downstream of the EIN2-EIN3 cascade in the ET signaling pathway, and thus constitutive expression of RAP2.2 could bypass the need for upstream ET signaling components. To test this hypothesis, we first overexpressed RAP2.2 in the ein2 mutant background and selected two transgenic lines, ein2;35S:RAP2.2#17 and #30, with constitutively high levels of RAP2.2 expression for disease assay. Four-week-old ein2 mutants and two transgenic lines were inoculated with Botrytis spore suspension (2 × 105 spores ml−1). As shown in Fig. 5(a), the ein2 mutant plants displayed severe disease symptoms as previously reported (Thomma et al., 1999). But the disease symptoms in ein2;35S:RAP2.2 transgenic plants developed much more slowly. At 7 DAI, 79% of ein2 mutant plants were completely decayed, compared with a decay rate of 56% in #17 and 31% in #30 transgenic lines. At 12 DAI, nearly all the ein2 plants were dead, whereas the percentages of decayed plants were 83% in #17 and 70% in #30 transgenic lines, significantly lower than in the ein2 mutants (Fig. 5c). Moreover, the ein2;35S:RAP2.2#30 transgenic line had a lower decay rate than the #17 line, which was correlated with the expression levels of RAP2.2 in the transgenic lines (Fig. 5b). Quantitative RT-PCR result showed that fewer BcActin A transcripts accumulated in two ein2;35S:RAP2.2 transgenic plants than in the ein2 mutants (Fig. 5b). These results indicate that overexpression of RAP2.2 in the ein2 mutant background restores the resistance to B. cinerea.

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Figure 5. Overexpression of RAP2.2 in ein2 restores the resistance to Botrytis cinerea. (a) Disease symptoms of the ein2, ein2;35S:RAP2.2#17 and ein2;35S:RAP2.2#30 Arabidopsis thaliana plants at 0, 7 and 12 d after inoculation with 2 × 105Botrytis spores ml−1. (b) The expression levels of BcActin A gene determined by quantitative reverse transcription polymerase chain reaction (RT-PCR), using 4-wk-old ein2 (black columns) and ein2;35S:RAP2.2 (#17, dark gray columns; #30, light gray columns) plants inoculated with 2 × 105Botrytis spores ml−1. Gene expression levels were standardized to AtUBQ10, and the value in ein2 at 1 d was set at 1.0. Error bars represent the SD from three biological replicates, each containing at least 20 plants. The expression levels that differ significantly (< 0.05) from those in ein2 are marked with asterisks. The expression levels of RAP2.2 in the ein2 and ein2;35S:RAP2.2 plants are shown at the top left. (c) Percentages of decayed plants after inoculation with 2 × 105Botrytis spores ml−1. Error bars represent the SD from three biological -replicates, each replicate containing at least 30 plants for each genotype. The percentages that differ significantly (< 0.05) from those in ein2 are marked with asterisks.

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Next, we overexpressed RAP2.2 in the ein3 eil1 double mutant background. The expression levels of RAP2.2 in ein3 eil1;35S:RAP2.2#7 and #16 increased considerably (Fig. 6b). We selected these two transgenic lines for inoculation with B. cinerea (2 × 105 spores ml−1). The disease symptoms developed much more slowly in the ein3 eil1;35S:RAP2.2 transgenic plants than in the ein3 eil1 mutants (Fig. 6a). At 7 DAI, 65% of ein3 eil1 mutant plants were decayed, whereas only 24% of #7 and 9% of #16 transgenic plants were decayed. At 12 DAI, almost all the ein3 eil1 plants were completely dead, whereas the decay rates were 73% in #7 and 55% in #16 transgenic lines, which were significantly lower than in the ein3 eil1 mutants (Fig. 6c). Besides, the ein3 eil1;35S:RAP2.2#16 plants, which had a higher expression level of RAP2.2 than the #7 plants, showed lower decay rates during Botrytis infection. The decay rates in the ein3 eil1 mutants and the ein3 eil1;35S:RAP2.2 transgenic plants were also correlated with the abundance of BcActin A transcripts, as determined by quantitative RT-PCR (Fig. 6b). These results indicate that overexpression of RAP2.2 in the ein3 eil1 mutant background restores plant resistance to B. cinerea.

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Figure 6. Overexpression of RAP2.2 in ein3 eil1 restores the resistance to Botrytis cinerea. (a) Disease symptoms of the ein3 eil1, ein3 eil1;35S:RAP2.2#7 and ein3 eil1;35S:RAP2.2#16 Arabidopsis thaliana plants at 0, 7 and 12 d after inoculation with 2 × 105Botrytis spores ml−1. (b) The expression levels of BcActin A gene determined by quantitative RT-PCR, using 4-wk-old ein3 eil1 (black columns) and ein3 eil1;35S:RAP2.2 (#7, dark grey columns; #16, light grey columns) plants inoculated with 2 × 105Botrytis spores ml−1. Gene expression levels were standardized to AtUBQ10, and the value in ein3 eil1 at 1 d was set at 1.0. Error bars represent the SD from three biological replicates, each containing at least 20 plants. The expression levels that differ significantly (< 0.05) from those in ein3 eil1 are marked with asterisks. The expression levels of RAP2.2 in the ein3 eil1 and ein3 eil1;35S:RAP2.2 plants are shown at the top left. (c) Percentages of decayed plants after inoculation with 2 × 105Botrytis spores ml−1. Error bars represent the SD from three biological replicates, each replicate containing at least 30 plants for each genotype. The percentages that differ significantly (< 0.05) from those in ein3 eil1 are marked with asterisks.

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Our results showed that overexpression of RAP2.2 in the ein2 and ein3 eil1 mutants restored their resistance to B. cinerea, indicating that RAP2.2 functions downstream of the ET signaling pathway during Botrytis resistance.

RAP2.2 and RAP2.12 are involved in the triple response to ET

The results in the previous section indicate that RAP2.2 plays an important role in Botrytis resistance via the ET signaling pathway. To test whether RAP2.2 is involved in other ET responses, we assessed the sensitivity of rap2.2-3 etiolated seedlings to ACC treatment, for example, the triple response. However, rap2.2-3 did not show any significant difference from the wildtype under the 10 μM ACC treatment (Fig. 7a). This is probably because of the functional redundancy between RAP2.2 and RAP2.12; the latter is the closest homolog of RAP2.2 in the group VII ERF transcription factors (Nakano et al., 2006). To test this possibility, a T-DNA insertion line SALK_019873 (designated as rap2.12-1) was identified as a knockdown mutant of RAP2.12 (Fig. S2). The rap2.2-3 rap2.12-1 double mutant was generated for the triple response assay. Under 10 μM ACC treatment, two single mutants, rap2.2-3 and rap2.12-1, showed no significant differences in the triple response from the wildtype plants. But the rap2.2-3 rap2.12-1 double mutants exhibited ET insensitivity in hypocotyls. Although they were shorter than those of ein2 (8.11 ± 1.40 mm) under ACC treatment, the hypocotyl lengths of rap2.2-3 rap2.12-1 double mutants (2.37 ± 0.40 mm) were significantly longer than those of the wildtype (1.57 ± 0.21 mm, < 0.001; Fig. 7a,b). The root lengths of rap2.2-3 rap2.12-1 showed no significant differences from those of the wildtype (Fig. S3). This result indicates that the hypocotyls of rap2.2-3 rap2.12-1 are more insensitive to ET than the wildtype.

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Figure 7. RAP2.2 and RAP2.12 play redundant roles in the hypocotyl response to ethylene (ET). (a) Phenotypes of the Arabidopsis thaliana wildtype, rap2.2-3, rap2.12-1, rap2.2-3 rap2.12-1 and ein2 plants grown on MS plates containing 10 μM 1-aminocyclopropane-1-carboxylic acid (ACC) in the dark for 3 d (bar, 1 mm). (b) Hypocotyl lengths of the wildtype, rap2.2-3, rap2.12-1, rap2.2-3 rap2.12-1 and ein2 mutants (control, open columns; ACC, closed columns). The data represent means ± SD from at least 25 seedlings per genotype. A two-way ANOVA was used to assess the statistical significance of the differences in hypocotyl lengths. Different letters (A–B and a–c) indicate significant differences between each other (P < 0.05). The experiment was repeated three times with similar results. (c) Phenotypes of ctr1, rap2.2-3 ctr1, rap2.12-1 ctr1 and rap2.2-3 rap2.12-1 ctr1 grown on MS plates in the dark for 3 d (bar, 1 mm). (d) Hypocotyl lengths of ctr1, rap2.2-3 ctr1, rap2.12-1 ctr1 and rap2.2-3 rap2.12-1 ctr1 mutants. The data represent means ± SD from at least 25 seedlings per genotype. A one-way ANOVA was used to assess the statistical significance of the differences in hypocotyl lengths. Different letters (a–c) indicate significant differences between each other (P < 0.05). The experiment was repeated three times with similar results.

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CTR1 is a crucial negative regulator in the ET signaling pathway, as ctr1 exhibits a constitutive triple response (Kieber et al., 1993). To further demonstrate the biological roles of RAP2.2 and RAP2.12 in the ET responses, we generated the rap2.2-3 rap2.12-1 ctr1 triple mutant and analyzed the constitutive triple response. As expected, the hypocotyls of ctr1 displayed the typical constitutive triple response and were significantly shorter (2.70 ± 0.39 mm) than those of the wildtype (Fig. S4a). The hypocotyl lengths of the two double mutants rap2.2-3 ctr1 (3.19 ± 0.35 mm) and rap2.12-1 ctr1 (3.36 ± 0.62 mm) were significantly greater than those of ctr1. Moreover, the hypocotyl lengths of the rap2.2-3 rap2.12-1 ctr1 triple mutants (4.8 ± 0.36 mm) were much greater than those of ctr1 and two double mutants (< 0.001; Fig. 7c,d). The root lengths of these mutants were comparable (Fig. S4b), indicating that the root response to ET did not change. This result shows that the constitutive triple response of ctr1 was partially released in the hypocotyls of the rap2.2-3 rap2.12-1 ctr1 triple mutants.

Taken together, these results demonstrate that RAP2.2 and RAP2.12 function in the hypocotyl response to ET and act as downstream regulators in the ET signaling pathway.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

RAP2.2 is involved in hypoxia tolerance in Arabidopsis, as indicated by the improved survival rate under hypoxia of the transgenic plants overexpressing RAP2.2, compared with the reduced survival rate in the T-DNA insertion mutants (Hinz et al., 2010). Here, this study adds two major aspects to our understanding of the biological roles of RAP2.2. First, RAP2.2 plays an important role in plant resistance to the necrotrophic fungal pathogen B. cinerea. Secondly, RAP2.2 acts as a positive regulator in the hypocotyl response to ET. Since ET plays important roles in both Botrytis resistance and hypoxia tolerance, these diverse functions conducted by RAP2.2 are probably functionally connected. It suggests that RAP2.2 might serve as a general transcriptional regulator in the ET signaling pathway and participate in multiple ET-mediated responses.

In A. thaliana, ERFs are involved in various stress responses. For example, ERF1 and ORA59 regulate plant disease resistance to necrotrophic pathogens (Berrocal-Lobo et al., 2002; Lorenzo et al., 2003; Pre et al., 2008), whereas RAP2.2, RAP2.12, HYPOXIA RESPONSIVE1 and 2 (HRE1 and HRE2) function in hypoxia tolerance (Hinz et al., 2010; Licausi et al., 2010, 2011). Besides, some of these ERFs are involved in the ET signaling pathway. ERF1 is a positive regulator in plant resistance to necrotrophic pathogens. In addition to enhanced disease resistance, the ERF1-overexpressing plants also exhibited activated ET responses in hypocotyls and roots of etiolated seedlings, as well as a dwarf phenotype resembling that of ctr1 in adult plants (Solano et al., 1998; Berrocal-Lobo et al., 2002). HRE1 and HRE2 belong to the same group VII ERFs with RAP2.2. The transgenic plants overexpressing HRE1 showed improved anoxia tolerance compared with the wildtype plants, whereas the hre1 hre2 double mutants showed reduced tolerance (Licausi et al., 2010). A further study showed that the transgenic seedlings silencing HRE1 displayed exaggerated apical hook curvatures under 2 μM ACC treatment, indicating a negative role in the ET responses (Yang et al., 2011). The data from our study demonstrate that RAP2.2 is involved in the resistance to B. cinerea, as the transgenic plants overexpressing RAP2.2 showed increased resistance, whereas the T-DNA insertion mutant rap2.2-3 showed decreased resistance (Fig. 2). The involvement of RAP2.2 in the ET signaling pathway is also demonstrated by the molecular and genetic evidence (Figs 3, 5–7). Taken together with the finding of its function in hypoxia tolerance (Hinz et al., 2010), these data suggest that RAP2.2 perhaps acts as a general regulator in the ET signaling pathway and plays dual roles in Botrytis resistance and hypoxia tolerance.

Although RAP2.2 functions in both Botrytis resistance and hypoxia tolerance, it is likely that RAP2.2 is under different regulatory mechanisms during these two stress responses. During Botrytis resistance, RAP2.2 expression is induced by Botrytis infection in leaf tissues and the induction relies on the ET signaling pathway (Fig. 4). Knockdown of RAP2.2 in rap2.2-3 impaired Botrytis resistance whereas overexpression of RAP2.2 enhanced plant resistance. Furthermore, the transgenic plants with higher levels of RAP2.2 expression were more resistant to B. cinerea (Figs 2, 5, 6). These results indicate that during Botrytis resistance, RAP2.2 is under transcriptional regulation via the ET signaling pathway and the expression level of RAP2.2 is important for plant resistance. Our results also showed that RAP2.2 was induced by ET treatment, further demonstrating that RAP2.2 expression is regulated by the ET signaling pathway (Fig. 3). This result is consistent with a previous report, in which RAP2.2 expression was induced by ET in shoots but not in roots (Hinz et al., 2010). It was shown that RAP2.2 expression in shoots was induced by ET and darkness rather than by hypoxia itself (Hinz et al., 2010; Licausi et al., 2010), suggesting a different mechanism underlying RAP2.2 function in hypoxia tolerance.

Recently, RAP2.12, the closest homolog of RAP2.2, has been studied in detail with its biological role in hypoxia tolerance (Licausi et al., 2011). The N-end rule pathway of protein degradation serves as a sensor of low concentration of oxygen in plant cells. Under aerobic conditions, RAP2.12 is dedicated to the N-end rule pathway of protein degradation via the oxygen-dependent oxidation of its terminal cysteine residue. Under hypoxia conditions, cysteine oxidation is prevented and RAP2.12 accumulates in nuclei to activate hypoxia-responsive gene expression (Licausi et al., 2011). In addition, all of the group VII ERFs, including RAP2.2, are regulated by this ubiquitin-dependent N-end rule pathway of protein degradation (Gibbs et al., 2011). This evidence therefore suggests that RAP2.2 is under post-transcriptional regulation in the hypoxia stress response. In the natural environment, hypoxia stress often happens in plant roots where many hypoxia-responsive genes are either exclusively or predominantly expressed (Bailey-Serres & Voesenek, 2008; Licausi, 2011). For example, both RAP2.2 and RAP2.12 are constitutively expressed at high levels in roots, whereas HRE1 and HRE2 are induced by hypoxia with much higher expression levels in roots than in shoots (Hinz et al., 2010; Licausi et al., 2010, 2011). It seems that the post-transcriptional regulation of these ERFs via the N-end rule pathway of protein degradation facilitates a quick response to hypoxia stress in plant roots. Because RAP2.2 is ET-inducible in shoots but constitutively expressed in roots (Fig. 3a; Hinz et al., 2010), and RAP2.2 is involved in the ET response in hypocotyls but not in roots (Fig. 7), it is possible that RAP2.2 plays different biological roles in shoots and roots. In roots, where hypoxia is often experienced, RAP2.2 is constitutively expressed and is under the post-transcriptional regulation via the N-end rule pathway of protein degradation (Hinz et al., 2010; Gibbs et al., 2011; Licausi et al., 2011). In shoots, where necrotrophic pathogens are encountered, RAP2.2 is also regulated in transcription abundance via the ET signaling pathway (Figs 3, 4).

PDF1.2 is a well-characterized marker gene in plant resistance to B. cinerea; its basal expression levels are elevated in the transgenic plants overexpressing ERF1 or ORA59 compared with the wildtype plants (Solano et al., 1998; Pre et al., 2008). However, the PDF1.2 basal expression level in the 35S:RAP2.2 transgenic plants was comparable with that in the wildtype, but after inoculation with B. cinerea, PDF1.2 was induced more prominently in the 35S:RAP2.2 transgenic plants (Fig. 2e). Taking into consideration the fact that PDF1.2 expression was reduced in rap2.2-3 mutants (Fig. 2e), it seems that RAP2.2 is necessary but insufficient for PDF1.2 induction. There are two possible reasons for this phenomenon. One is that the post-transcriptional regulation of RAP2.2 stability also functions during plant resistance to B. cinerea. However, plant cells under pathogen attack usually accumulate reactive oxygen intermediates and are unlikely to experience oxygen shortage (Nimchuk et al., 2003; Glazebrook, 2005). Thus, during Botrytis resistance, the N-end rule pathway of protein degradation might not work on RAP2.2 protein stability in the same way as in hypoxia tolerance. It should be investigated whether another post-transcriptional regulation mechanism is involved in RAP2.2 function during Botrytis resistance, such as protein degradation mediated by the putative E3 ubiquitin ligase SINAT2 (Welsch et al., 2007). The other possibility is that RAP2.2 requires other partners to fulfill its function in plant resistance, which might be absent or inactive in the absence of Botrytis infection. For instance, PFT1 is an interesting candidate for the future study as a RAP2.2 molecular partner, as PFT1 was identified as an interacting protein of RAP2.2 in our previous study and they are both involved in Botrytis resistance in Arabidopsis (Kidd et al., 2009; Ou et al., 2011).

In the natural environment, plants continually undergo various biotic and abiotic stresses. To survive from these stresses, plants have evolved elaborate signaling networks with frequent crosstalk to regulate both disease resistance and abiotic stresses tolerance (Fujita et al., 2006). WRKY33 is a positive regulator of plant resistance to B. cinerea, as the knockout mutants compromise the resistance to B. cinerea, whereas WRKY33 overexpression enhances the resistance (Zheng et al., 2006). Interestingly, the knockout mutant wrky33 also exhibited enhanced sensitivity to hypoxia, suggesting that WRKY33 is probably involved in the signaling crosstalk between biotic and abiotic stress responses (Hwang et al., 2011). In this study, our results demonstrate that RAP2.2 functions as an important regulator in Botrytis resistance. Given its role in hypoxia tolerance, RAP2.2 might act as a crosstalk node of signaling networks, regulating plant responses to biotic and abiotic stresses. RAP2.2 belongs to the group VII ERFs in A. thaliana, four members of which are involved in hypoxia stress response in Arabidopsis (Nakano et al., 2006; Hinz et al., 2010; Licausi et al., 2010, 2011; Gibbs et al., 2011). Some members of group VII ERFs in other plant species, including JERF3, CaPF1 and HvRAF, are involved in the response to other stresses, such as salt, cold and bacterial pathogens (Wang et al., 2004; Yi et al., 2004; Jung et al., 2007). This evidence suggests that the group VII ERFs are involved in plant responses to both biotic and abiotic stresses.

In conclusion, our results demonstrate that RAP2.2 plays an important role in plant resistance to B. cinerea and the hypocotyl response to ET. As it plays dual roles in Botrytis resistance and hypoxia tolerance, RAP2.2 might serve as a general transcription regulator in the ET signaling pathway and a signaling crosstalk node between biotic and abiotic stress responses.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank Dr Hongwei Guo (Peking University, China) for kindly providing etr1-1, ctr1-1, ein2-5 and ein3-1 eil1-1; Dr Dingzhong Tang (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, China) for the B. cinerea strain; and Dr Haiyang Wang (Cornell University, USA) for the pJIM19-HA-FLAG construct. We are also grateful to Dr Caihong Yu, Mr Xinyan Zhang (Peking University, China) and Ms Qiujing Shen (Genetics and Developmental Biology, Chinese Academy of Sciences, China) for their technical assistance. The work was supported by the National Transgenic Research Project (grant no. 2011ZX08009-003-001).

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Fig. S1 The expression levels of RAP2.2 in the wildtype, ctr1, etr1 and ein2 plants.

Fig. S2 The identification of the T-DNA insertion mutant rap2.12-1.

Fig. S3 Root lengths of the wild-type, rap2.2-3, rap2.12-1, rap2.2-3 rap2.12-1 and ein2 mutants under 10 μM ACC treatment.

Fig. S4 Hypocotyl (a) and root (b) lengths of ctr1, rap2.2-3 ctr1, rap2.12-1 ctr1, rap2.2-3 rap2.12-1 ctr1 and the wildtype plants after 3 d incubation in the dark.

Table S1 Primers for quantitative RT-PCR

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