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Plant Cell. Jul 2004; 16(7): 1925–1937.
PMCID: PMC514171

Arabidopsis RADICAL-INDUCED CELL DEATH1 Belongs to the WWE Protein–Protein Interaction Domain Protein Family and Modulates Abscisic Acid, Ethylene, and Methyl Jasmonate Responses

Abstract

Experiments with several Arabidopsis thaliana mutants have revealed a web of interactions between hormonal signaling. Here, we show that the Arabidopsis mutant radical-induced cell death1 (rcd1), although hypersensitive to apoplastic superoxide and ozone, is more resistant to chloroplastic superoxide formation, exhibits reduced sensitivity to abscisic acid, ethylene, and methyl jasmonate, and has altered expression of several hormonally regulated genes. Furthermore, rcd1 has higher stomatal conductance than the wild type. The rcd1-1 mutation was mapped to the gene At1g32230 where it disrupts an intron splice site resulting in a truncated protein. RCD1 belongs to the (ADP-ribosyl)transferase domain–containing subfamily of the WWE protein–protein interaction domain protein family. The results suggest that RCD1 could act as an integrative node in hormonal signaling and in the regulation of several stress-responsive genes.

INTRODUCTION

The production of reactive oxygen species (ROS) is a common feature of plant responses to abiotic and biotic stress. ROS production triggers a network of signaling events leading to several outputs, including stress tolerance, acclimation, and cell death (Dat et al., 2000; Mittler, 2002; Vranovà et al., 2002). ROS are also involved in the regulation of the closure of stomata, gravitropism in the root tip, and root branching (Joo et al., 2001; Foreman et al., 2003; Kwak et al., 2003). In all these processes, increased ROS production is functionally coupled to the action of plant hormones such as ethylene (ET), salicylic acid (SA), jasmonic acid (JA), and abscisic acid (ABA).

The use of Arabidopsis thaliana mutants has been instrumental in the dissection of hormone signaling networks and their interactions. For example, upon ozone (O3) exposure, the radical-induced cell death1 (rcd1) mutant accumulates SA and ET, which together promote continuous ROS production and cell death, whereas methyl jasmonate (MeJA) acts antagonistically to ET inhibiting lesion spread (Overmyer et al., 2000, 2003; Langebartels and Kangasjärvi, 2004). At the transcriptional level, these hormones act both synergistically and antagonistically in regulating stress-induced gene expression (Tuominen et al., 2004). JA and ET together are required for induction of PDF1.2 expression, which is inhibited by SA. Conversely, mutant phenotypes indicate that PR1 expression is induced by SA and inhibited by JA (Turner et al., 2002). In the hypersensitive response-like lesions1 mutant, induction of PR1 expression requires both SA and ET, and induction of PDF1.2 expression requires all three hormones SA, ET, and JA (Devadas et al., 2002), indicating further levels of complexity in hormone interactions. Hence, it is likely that the timing, location, and levels of the various hormones determine the interaction (antagonistic or synergistic) and output responses.

Hormones regulate stress-induced gene expression via modulation of each other's synthesis and signaling. For example, ET production increases the biosynthesis of ABA (Grossmann, 2003). The balance between these two hormones is maintained through subsequent inhibition of ET biosynthesis by ABA and by mutually antagonistic interactions between ABA and ET signaling (Fedoroff, 2002). This antagonistic interaction between ABA and ET was elucidated with the ET mutants ein2 and ctr1, which act as a suppressor and an enhancer, respectively, of the ABA-insensitive abi1 (Beaudoin et al., 2000; Ghassemian et al., 2000). Accordingly, ein2 overproduces ABA (Ghassemian et al., 2000). Several ABA mutants together with constitutive ET signaling mutants also appear to be insensitive to glucose, and ABA has been established as a regulator of glucose signaling through double mutant analysis. ET also modulates glucose signaling through the regulation of ABA (León and Sheen, 2003).

ABA is involved in responses to several abiotic stresses and is a well-known regulator of stomatal closure. In stomata, ABA induces ROS production (Pei et al., 2000). Recently, direct genetic evidence for the requirement of ROS in ABA-induced stomatal closure was shown with mutants of the plasma membrane NADPH oxidase catalytic subunits atrbohF and atrbohD atrbohF, which exhibited impaired ABA-induced stomatal closing (Kwak et al., 2003).

As a potential interactor between hormones and ROS formation, the O3-sensitive rcd1 mutant (Overmyer et al., 2000) offers a tool to study hormonal interactions in relation to ROS production addressing the importance of the subcellular site of ROS action. We report here the map-based identification of RCD1, followed by genetic and physiological analyses for the elucidation of processes in which the RCD1 protein is involved. RCD1 encodes a protein of unknown function that belongs to a family of proteins bearing WWE protein–protein interaction domains. RCD1 was found to be involved in a subset of responses to several plant hormones, including ABA, ET, and MeJA.

RESULTS

rcd1 Is Sensitive to Apoplastic ROS but Tolerant to Chloroplastic ROS

Mutant rcd1 plants were initially isolated on the basis of their enhanced sensitivity to extracellular ROS generated by O3 exposure. In addition to O3 sensitivity, which has been described previously (Overmyer et al., 2000), rcd1 plants also have several other distinctive phenotypes, including smaller, more erect rosettes, altered leaf shape, and earlier flowering (Figure 1A), which all cosegregate with O3 sensitivity (Table 1). To differentiate whether the codominant ozone-sensitive phenotype of rcd1 was a result of haplo-insufficience or a gain of function, triploid rcd1/RCD1/RCD1 plants were produced and tested for ozone sensitivity. The O3 sensitivity of the triploid plants was similar to heterozygous rcd1/RCD1 plants (data not shown), implying that rcd1 confers O3 sensitivity by a gain of function. Tables 1 and and22 indicate that both the morphological and ROS sensitivity phenotypes are conferred either by two very tightly linked genes or by the same gene. Because rcd1 is sensitive to apoplastic ROS (Overmyer et al., 2000), we tested whether rcd1 is also sensitive to ROS produced inside the cell with plants grown on MS plates containing 1 μM herbicide paraquat, which causes generation of excess ROS within the chloroplast (Mehler, 1951). Unexpectedly, rcd1 was more tolerant to paraquat than Columbia-0 (Col-0); it was able to germinate and develop true leaves, whereas Col-0 germinated but quickly bleached and died thereafter (Figure 1B). Also surprisingly, whereas the sensitivity to apoplastic ROS (i.e., O3 or exogenous superoxide) segregated as a codominant trait (Overmyer et al., 2000; Table 1), the tolerance to paraquat and the mutant habitus morphology cosegregated as simple recessive traits (Table 2). These unique genetics are characteristic of not only the rcd1-1 allele. A second rcd1 allele, rcd1-2, isolated in a screen for paraquat tolerant mutants, is also sensitive to O3 (Fujibe et al., 2004).

Figure 1.
rcd1-1 Phenotypes.
Table 1.
The Segregation of rcd1-1 Habitus and O3 Sensitivity Phenotypes
Table 2.
The Segregation of rcd1-1 Habitus and Paraquat Tolerance Phenotypes

rcd1 Maps to the Gene At1g32230

Linkage was initially established on chromosome 1 (Overmyer et al., 2000). Genetic mapping was used (Figure 2A; Methods) to define rcd1-1 to an interval on the BACs F3C3 and F27G20 containing seven open reading frames, six of which matched the published Arabidopsis genome sequence perfectly. The gene At1g32230 harbored a single C-to-T transition, typical to ethyl methanesulfonate mutagenesis, on the antisense strand resulting in a G-to-A transition at the GT splice site of the third exon–intron junction. The nucleotide sequence of At1g32230 from three nonallelic O3-sensitive mutants isolated in the same screen was identical to the published Col-0 genome sequence, indicating that the base change resulted from the ethyl methanesulfonate mutagenesis and was not present in the original seed population.

Figure 2.
RCD1 Map Position and Arabidopsis SIMILAR TO RCD1 Gene Family.

Complementation tests confirmed that the mutant phenotype is conferred by the mutation observed. Expression of the rcd1-1 cDNA under the 35S promoter of Cauliflower mosaic virus (CaMV) in Col-0 increased the tolerance of the transgenic lines to paraquat (Figure 1B) and sensitivity to O3 (Figure 1C). The use of CaMV 35S promoter to overexpress the mutant cDNA in Col-0 resulted in significantly higher transcript abundance for the mutant rcd1-1 cDNA than the wild-type RCD1 cDNA (data not shown), which is also clearly visible as a difference in the paraquat resistance between the transgenic plants that contain one copy (lines 10-3 and 17-9) or two copies (lines 7 and 3) of the 35S-regulated rcd1-1 cDNA (Figure 1B). Thus, overexpression of the mutant protein results in a dominant-like complementation of a recessive paraquat resistance because of the higher abundance of the mutant protein in the transgenic complementation lines. The F1 progeny from a cross between rcd1-1 and rcd1-2 also bears both paraquat and O3 phenotypes and leaf morphology of rcd1 (Fujibe et al., 2004; data not shown), indicating a lack of genetic complementation of both phenotypes in these two independently isolated mutants. These results confirm that the phenotypes are associated with the mutation in At1g32230.

RCD1 Belongs to a Subfamily of WWE Domain–Containing Proteins

The RCD1 protein coding sequence matches a RIKEN Arabidopsis full-length cDNA sequence (AY142655) except for an insertion of three bases (CAG) resulting in an extra glutamine residue at Q475. RCD1 is identical to CEO1, which was identified as a Col-0 cDNA that complements an oxidative stress–sensitive yeast strain (Belles-Boix et al., 2000). It is also 95% identical with a Landsberg erecta (Ler) protein published as ATP8 (Lin and Heaton, 2001), which was identified in a yeast two-hybrid screen using turnip crinkle virus movement protein as bait. The difference between RCD1 and ATP8 sequences is mostly attributable to a deletion in Ler resulting in a 17–amino acid shorter protein.

The predicted RCD1 protein has a molecular weight of 66 kD and a pI of 6.46. RCD1 is presumed to be targeted to the nucleus based on two potential nuclear localization signals, GKKRKRA at position 18 and KRRRL at position 54. Database searches identified an Arabidopsis paralog (At2g35510) of RCD1 with 76% similarity, named as SRO1 (for SIMILAR TO RCD ONE1). Other related genes are a rice ortholog (Oryza sativa; unknown protein AC35157.2) with 53% similarity to RCD1 and four shorter Arabidopsis proteins with 43 to 49% similarity to RCD1 (At1g23550, At1g70440, At3g47720, and At5g62520, which will be referred to as SRO2, SRO3, SRO4, and SRO5, respectively).

Aravind (2001) classified RCD1 in a novel subfamily of proteins, involved in ubiquitin and ADP-ribose conjugation systems, that contain a conserved globular domain called the WWE domain (PF02825; IPR004170) predicted to mediate specific protein–protein interactions. RCD1 (F3C3.1_At_10801372) was the only plant protein that was originally used by Aravind (2001) to define the WWE domain. The WWE domain is present in RCD1, SRO1, and the rice ortholog AC35157.2 but is missing from the shorter (SRO2 to SRO5) proteins. RCD1 and the SRO proteins also contain a region (amino acids 317 to 416 of RCD1) similar to the catalytic domain of poly(ADP-ribose)polymerase (PARP signature, pfam00644) (Figure 2B.) The same domain also has 46 to 47% similarity to three polyADP-ribose metabolism enzymes of Caenorhabditis elegans (PME-1, PME-5, and PME-6; Gagnon et al., 2002). PSI-BLAST searches identified from different organisms several ADP-ribose–related proteins that have similarity with this region. PSI-BLAST analyses also identified nine mammalian proteins of unknown function that have a low, 15 to 20% overall amino acid identity with RCD1, but all have both WWE and PARP domains with similar domain architecture as in RCD1 (data not shown).

RCD1 Expression and Intron Splicing in the Wild Type and rcd1-1

RT-PCR indicated that in the wild-type Col-0, RCD1 intron splicing was in agreement with the predicted At1g32230 structure and the published CEO1 and RIKEN full-length cDNAs (data not shown). In rcd1-1, two types of transcripts were found by RT-PCR amplification and sequence analysis. The 2.7-kb transcript represents mRNA where the next (GT) in intron III downstream of the mutated splice acceptor site has been used in the splicing of intron III. This misspliced rcd1-1 mRNA is only 11 bp longer than the wild-type mRNA. In rcd1-1, a shorter, 1.4-kb transcript is present at approximately the same expression level as the 2.7-kb transcript. In the 1.4-kb transcript, intron splicing has occurred from the 5′ end of exon IV into exon III so that exon III is completely missing (data not shown). Both misspliced rcd1-1 transcript types result in loss of reading frame and a premature stop codon. Similarly, rcd1-2 (Fujibe et al., 2004) has a missense mutation causing a premature stop codon just a few amino acids upstream of the rcd1-1 mutation (K. Yamamoto, personal communication) creating virtually the same truncated protein as rcd1-1.

In the wild-type Col-0, RCD1 had a constitutive level of expression in stems, leaves, buds, and young flowers, whereas in old flowers, siliques and roots transcript levels were low (Figure 3A). In rcd1-1, expression of the gene was slightly elevated compared with the wild type, and in contrast with Col-0, was also expressed in old flowers and roots (Figure 3B). In siliques of rcd1-1, only the shorter transcript was present in low abundance. Exposure to O3 (250 nL L−1) for 7 h increased RCD1 mRNA levels in Col-0 only approximately twofold to threefold (Figure 3C), whereas the CHIB and PR5, which are known to be O3 inducible, were induced approximately fourfold to sevenfold.

Figure 3.
Expression of RCD1 and Defense Response Marker Genes.

ET- and ABA-Regulated Genes Have Altered Expression in rcd1-1

Yeast two-hybrid analysis with the RCD1 (CEO1) protein identified several transcription factors, for example DREB2A, as RCD1 interacting partners (Belles-Boix et al., 2000), which suggests that RCD1 might be involved in regulating gene expression by affecting the function or activity of these transcription factors. Microarray analysis was used to identify genes regulated by RCD1 under normal growth conditions. Of the 6500 genes analyzed, nine had consistent and statistically significantly higher, and two lower basal mRNA levels in rcd1-1 in all hybridizations performed (Table 3). Two of the upregulated genes, encoding for 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase and alternative oxidase (AOX1A), are regulated by ET (Simons et al., 1999; Petruzzelli et al., 2000), and three are related to carbohydrate metabolism. The two downregulated genes (RD29A/LTI78 and KIN2) are involved in cold and dehydration stress and are regulated by ABA (Nordin et al., 1993; Foster and Chua, 1999). For an independent evaluation and to expand the microarray data, a dot blot macroarray containing 92 defense- and stress-regulated genes was hybridized with 33P-labeled cDNA probe. Two of the upregulated or downregulated genes in the microarray (AOX1A and RD29A/LTI78) were also present on the macroarray and behaved consistently in both arrays (Table 3). According to the macroarrays, a larger group of cold- and ABA-regulated genes had reduced basal transcript levels in rcd1-1. When the response of RAB18 to ABA was analyzed with RNA gel blot analysis, the induction of the gene by ABA was considerably lower in rcd1-1 than in Col-0 (Figure 4).

Figure 4.
Expression of RAB18 in ABA-Treated Col-0 and rcd1-1.
Table 3.
A Microarray Containing ~6500 Different Genes Was Hybridized with RNA Isolated from Nonstressed Col-0 and the rcd1-1 Mutant

rcd1-1 Has Higher Stomatal Conductance

Impaired expression of ABA-related genes prompted us to analyze the stomatal conductance of rcd1-1. The ABA-insensitive abi2 was used as a reference. In clean air, rcd1-1 had ~40% higher stomatal conductance than Col-0 (Figure 5A) but lower than in abi2 (Figure 5B, 0 h). O3 induced differential decrease in stomatal conductance in the accessions analyzed (Figure 5B); 2 h of O3 decreased conductance in Col-0 by 40% but only by 20% in rcd1-1. After 4 h, rcd1-1 had conductance similar to Col-0, whereas abi2 had only the level present in Ler preceding exposure (Figure 5B). The higher stomatal conductance in abi2 and rcd1-1 also had an effect on transpiration; the weight loss of detached leaves was considerably higher in abi2 and slightly but statistically significantly higher in rcd1-1 than in the corresponding wild types (Figure 5C).

Figure 5.
rcd1-1 Has Higher Stomatal Conductance.

Cold Acclimation Is Not Affected in rcd1-1

Because rcd1-1 had a lower basal level and exhibited impaired induction of some cold- and ABA-responsive genes, we investigated the cold acclimation capability of the mutant. Frost tolerance of both nonacclimated plants and plants exposed for 2 d to exogenous ABA or to 4°C was tested by controlled freezing of excised leaves. When ion leakage was measured as an indicator of freezing-induced tissue damage, a similar increase at −6°C in both nonacclimated rcd1-1 and Col-0 indicated that the mutation in RCD1 does not affect nonacclimated freezing tolerance (Figure 6). Acclimating plants with ABA (Figure 6A) or at 4°C (Figure 6B) before exposure to freezing temperatures equally decreased freezing-induced tissue damage in both genotypes, indicating that the capability to cold acclimate is not significantly impaired in rcd1-1. However, ABA- and cold-acclimated as well as nonacclimated rcd1-1 at −6 and −8°C showed somewhat higher electrolyte leakage than Col-0, which might indicate a slightly increased freezing sensitivity for rcd1-1.

Figure 6.
rcd1-1 Has a Normal Cold Acclimation.

RCD1 Is Involved in ET and MeJA Responses

Because stress hormone–related gene expression showed alterations in rcd1-1 (Table 3; Overmyer et al., 2000), we characterized its sensitivity to ET, JA, and SA. In the triple response assay, hypocotyl length in rcd1-1 did not differ from Col-0 wild type (Figure 7A). Thus, in this generally accepted marker for reduced ET sensitivity, rcd1 cannot be regarded as ET insensitive. However, exogenously applied ET precursor ACC induced the ET-responsive gene CHIB in rcd1-1 to the same extent as in the ET-insensitive ein2, and when in Col-0 considerably higher CHIB transcript accumulation was evident (Figure 7B). The induction of ET biosynthetic genes (ACC oxidase, ACO1, and ACC synthase, AT-ACS6) and ET evolution were also increased in rcd1-1 (Table 3; Overmyer et al., 2000), which is consistent with loss of feedback regulation of the biosynthesis because of insensitivity to the hormone.

Figure 7.
Triple Response Assay and RNA Dot Blot Analysis of ET and Jasmonate Marker Gene Expression.

The responsiveness of rcd1-1 plants to JA was also altered. We have previously shown that rcd1-1 can sense MeJA normally in an assay for inhibition of root growth (Overmyer et al., 2000). However, in MeJA-treated rcd1-1, expression of VSP1 was lower than in Col-0 but not as low as in the MeJA-insensitive mutant jar1 (Figure 7C). Interestingly, induction of VSP1 expression by MeJA in rcd1-1 was comparable to that in ein2. Induction of PR-1 expression by SA was not affected in rcd1-1 (data not shown).

rcd1-1 Exhibits Moderate Insensitivity to Glucose

ABA, ET, and glucose signaling are known to interact (León and Sheen, 2003). Because rcd1-1 has reduced sensitivity to ET and ABA, its response to glucose was tested. Compared with Col-0, rcd1-1 exhibited moderate glucose insensitivity; it was able to germinate normally and develop green cotyledons on 2% glucose when Col-0 germinated but development of its cotyledons was repressed (Figure 8). The ET-insensitive ein2 displayed a glucose-hypersensitive phenotype. On 4% glucose, the development of rcd1-1 seedlings was deferred, although less than in Col-0, and 6% glucose inhibited the development of both Col-0 and rcd1-1 seedlings in a similar manner (data not shown). Interestingly, the rcd1-1 mutation partially suppressed the glucose-hypersensitive phenotype of ein2 in the rcd1 ein2 double mutant.

Figure 8.
rcd1-1 Seedling Development Is Partially Insensitive to Glucose.

DISCUSSION

Specificity of ROS Generation in the Apoplast and Chloroplast

Plants respond to different ROS by activation of specific sets of genes, depending on the type of ROS, and/or their subcellular site of production. For example, in the Arabidopsis mutant flu, singlet oxygen generated in the plastid activated different sets of genes than O2·− and H2O2 (op den Camp et al., 2003). Although O3 and paraquat produce the same ROS (O2·− and H2O2), rcd1 is sensitive to O3 but resistant to paraquat. The differential sensitivity most likely results from the different subcellular location of ROS production; paraquat acts in the chloroplast (Mehler, 1951), whereas O3 generates O2·− and H2O2 within the apoplast (Kangasjärvi et al., 1994).

Evidence from several experiments suggests the presence of an apoplastic superoxide dismutase (SOD) responsible for the dismutation of O2·− into H2O2 in the cell wall (Ogawa et al., 1997; Schinkel et al., 1998). The accumulation of O2·− in the apoplast of rcd1-1 (Overmyer et al., 2000) could be an indication of a lower apoplastic SOD activity, which could be compensated by higher expression of SODs and ascorbate peroxidases (APX) delivered to other subcellular compartments (e.g., to the chloroplast). This kind of compensation maintaining stable total SOD activity has been seen in SOD antisense and overexpression lines (Kliebenstein et al., 1998) and would explain the increased paraquat tolerance in the rcd1 mutants. Accordingly, rcd1 has higher mRNA levels for the chloroplastic FeSOD1 (Overmyer et al., 2000) and for the thylakoid-bound APX (Fujibe et al., 2004). Furthermore, UV-B irradiation–induced oxidative stress increased the transcript levels of the chloroplastic FeSOD, CuZnSOD, and APX in rcd1-2 (Fujibe et al., 2004).

Is rcd1 an Ozone Sensitive or Hormone Mutant?

The higher stomatal conductance in rcd1 raises the question whether its sensitivity to O3 could simply result from a higher influx of O3 and not necessarily from a higher sensitivity to O3 per se. Xanthine/xanthine oxidase (X/XO), a stomata-independent extracellular superoxide generating system, triggered about the same level of primary cell death in both rcd1-1 and Col-0 (Overmyer et al., 2000). After infiltration, X/XO produces a strong burst of O2·− that can cause direct damage. Accordingly, in the X/XO-infiltrated plants, the primary damage, presumably a direct result of the apoplastic ROS production from X/XO, was more or less the same in both rcd1-1 and Col-0. However, in Col-0, the ROS-dependent propagation of lesions did not take place, which suggests a role for RCD1 in the regulation of ROS-related lesion propagation.

It has been shown that the lesion propagation in O3 damage is under hormonal control (Overmyer et al., 2000; Rao et al., 2000, 2002; Moeder et al., 2002; Kanna et al., 2003). Both jasmonate-insensitive (jar1 and coi1) and ET-overproducing mutants (eto1 and eto2) are O3 sensitive as a result of lesion propagation, whereas the ET- or SA-insensitive mutants (etr1, ein2, npr1, and NahG) are O3 tolerant. Furthermore, the mutants and accessions that have first been described as O3 sensitive have also turned out to be partially JA insensitive (oji1, rcd1, and Cvi-0) or ET overproducers (rcd1) (Overmyer et al., 2000; Rao et al., 2000; Kanna et al., 2003). Moreover, the endogenous ROS production triggered by O3 is ET dependent (Overmyer et al., 2000; Moeder et al., 2002; Wohlgemuth et al., 2002; Kanna et al., 2003). Thus, it is conceivable that in several instances O3 sensitivity is a result of altered interactions between hormonal signaling pathways, which regulate the propagation of cell death through endogenous ROS production (Overmyer et al., 2003). Accordingly, O3 sensitivity, or the lesion propagation in rcd1, is more a hormone balance–related runaway process; thus, it might be more accurate to classify rcd1 as a hormonal mutant instead of an O3-sensitive mutant. However, some of the phenotypic characters of rcd1, such as the leaf and rosette morphology and faster life cycle, are still visible in double mutants where rcd1 has been crossed with ET-, SA-, and JA-insensitive mutants (etr1, ein2, npr1, NahG, and jar1; K. Overmyer, M. Brosché, and J. Kangasjärvi, unpublished data). Thus, the question as to whether all the developmental phenotypes conferred by rcd1 are related to hormonal interactions is still an open question and requires further studies.

Interactions among ABA, ET, and ROS

In the triple response assay, rcd1-1 did not display ET insensitivity compared with ein2 (Figure 7A). By contrast, ET and JA gene expression responses in rcd1-1 and ein2 plants were similarly compromised in comparison to Col-0. Thus, RCD1 seems to affect only a subset of ET responses. The upregulation of ACC oxidase in rcd1-1 correlates with the higher basal level of ET and the fact that rcd1-1 overproduces ET in response to O3 (Overmyer et al., 2000). Based on this misregulation of ET biosynthesis, RCD1 has been proposed to be a regulator of ET production (Wang et al., 2002). ET and jasmonates have mutually antagonistic interactions (Tuominen et al., 2004); thus, the altered JA sensitivity in rcd1 might be secondary to the increased ET synthesis, which might downregulate JA responses.

Both microarray and macroarray data showed downregulation of cold- and ABA-regulated genes and upregulation of ET-regulated genes in rcd1-1. A further indication for the involvement of RCD1 in relation to both ABA and ET is through the response of rcd1-1 to glucose (Figure 8), in which rcd1-1 resembled ABA insensitive and constitutive ET signaling or ET overproducing mutants (León and Sheen, 2003). As described above, rcd1-1 overproduces ET and has similar defects in the ET and JA marker genes by ACC and JA as ein2 but bears the opposite glucose sensitivity phenotype, insensitivity rather than hypersensitivity. Furthermore, partial suppression of ein2 glucose hypersensitivity in the rcd1 ein2 double mutant (Figure 8) demonstrates the same epistatic relationship between ABA and ET first observed in the double mutants aba2 etr1 and aba2 ein2 that exhibit the glucose-insensitive phenotype of aba2 (León and Sheen, 2003). Mutation in a recently described ABI8 causes a similar partial suppression of ein2 glucose hypersensitivity in the abi8 ein2 double mutant (Brocard-Gifford et al., 2004). At least in the sugar response, the primary effect of the rcd1-1 mutation seems to be more likely in ABA than ET responses. This also implies that the control of ET biosynthesis by RCD1 may involve ABA.

RCD1 Has a Function in Responses to Drought

One of the RCD1 partner proteins identified by Belles-Boix et al. (2000), DREB2A, is a transcription factor that binds specifically to DRE/CRT, a cis-acting element involved in gene expression in response to drought and low-temperature stress (Yamaguchi-Shinozaki and Shinozaki, 1994; Stockinger et al., 1997; Liu et al., 1998; Sakuma et al., 2002). Array analysis indicated that one of the target genes for DREB2a, RD29A/LTI78, had lower steady state transcript levels in the rcd1 mutant (Table 3). Recently, Narusaka et al. (2003) reported the interdependency of the DRE and ABRE (ABA-responsive) elements in the promoter of the RD29A/LTI78 gene, which contains a single copy of ABRE (ACGTGG/TC), which alone is insufficient for ABA responsiveness (Skriver et al., 1991). However, the expression of RD29A/LTI78 is strongly induced by exogenous ABA, suggesting that the DRE/CRT-core motif could act as a coupling element of ABRE. Because the truncated rcd1-1 protein lacks the C-terminal domain that interacts (Belles-Boix et al., 2000) with DREB2A, the reduced ABA sensitivity of rcd1-1 could result from the mutant protein's inability to activate (or derepress) DREB2A, whose association with the DRE/CRT would be necessary for certain ABRE-dependent ABA responses, such as induction of RD29A/LTI78.

rcd1-1 Separates ABA Induction of Cold Acclimation–Related Genes from Cold Acclimation

In spite of the reduced sensitivity of RAB18 expression to ABA, rcd1-1 mutant was still able to cold acclimate with both ABA and 4°C treatments. Consequently, ABA-induced expression of cold-induced genes and ABA-induced development of freezing tolerance appear to lie in separate branches of the ABA signal transduction network. Several ABA-dependent and independent pathways exist in response to cold, drought, and salt stress (Ishitani et al., 1997; Heino and Palva, 2003), and rcd1-1 is a further example of this. To give rise to the desired output, various signaling pathways have to converge at some point in the signaling networks. The calcium binding protein CBL1 (calcineurin B-like 1) has been identified as one of these integration points in response to cold, drought, and salt stress (Albrecht et al., 2003; Cheong et al., 2003). Plants overexpressing CBL1 are salt and drought tolerant but freezing sensitive, and loss of CBL1 function makes the plants freezing tolerant and drought and salt sensitive (Albrecht et al., 2003). In a similar way, it is probable that RCD1 plays a role in some branches of ABA signaling and in response to dehydration stress, but it is not involved in ABA-induced cold acclimation and freezing tolerance.

Potential Mode of Action of RCD1

In addition to the gene expression analysis presented in Figures 3 and and4,4, RCD1 expression was investigated by searching publicly available microarray experiments. In the abiotic stress and ABA experiments (Seki et al., 2002a, 2002b), available in the RIKEN Arabidopsis Genome Encyclopedia (http://rarge.gsc.riken.go.jp/microarray/microarray_data.pl), RCD1 was induced twofold to fourfold, which is similar to induction by O3. The nuclear localization signal sequences in RCD1 suggest nuclear localization for the protein. These, along with the fact that the interacting partners identified for RCD1 (Belles-Boix et al., 2000) are either known or putative transcription factors, supports the role of RCD1 as a regulator of signal transduction. The domain composition of RCD1 suggests that it does not bind DNA but interacts with other proteins through its N-terminal WWE protein–protein interaction domain (Aravind, 2001) and/or its C-terminal end (Belles-Boix et al., 2000).

In an in vitro binding assay, RCD1 did not hybridize with itself (Lin and Heaton, 2001), suggesting that it functions as a monomer. The downregulation of cold- and ABA-regulated genes in the mutant could be a manifestation of the interaction between RCD1 and its partner proteins not taking place correctly. One possible example for this could be activation of RCD1 by protein X binding to the WWE domain, followed by the binding of the complex to its targets through the C terminus. The RCD1 and SRO proteins have upstream of their C-terminal protein interaction domain a PARP signature [the catalytic domain of poly(ADP-ribose)polymerase] that binds NAD+ and attaches the ADP-ribose-moiety from NAD+ to the target molecule. This suggests that the function of the RCD1 (and SRO1) protein could be related to ADP ribosylation. Poly(ADP-ribosyl)ation by PARPs is a reversible posttranslational modification involved in the regulation of a variety of processes (Smith, 2001). However, RCD1 does not have the conserved zinc-finger domains typical of a classical PARP; thus, it most likely has functions distinct from the classical PARP. The PARP domain protein family also contains a distinct, but less-known subfamily of mono(ADP-ribosyl)transferases that modify their targets by the addition of a single ADP-ribose unit (Smith, 2001; Corda and Di Girolamo, 2003). In animals, mono-ADP-ribosylation of intracellular components by intracellular mono-ADP-ribosyltransferases is just becoming an established process involved in the regulation of protein activity, although so far only genes coding for the extracellular mono-ADP-ribosyltransferases have been identified (Corda and Di Girolamo, 2003). It could be that RCD1 is one of the predicted (Corda and Di Girolamo, 2003) intracellular plant mono-ADP-ribosyltransferases and inactivates/activates its target proteins by ADP-ribosylation. An intriguing connection is that some bacterial toxins, for example the cholera toxin, are ADP-ribose transferases that increase the activation of trimeric G-protein by ADP-ribosylation. Involvement of G-proteins has been implied in both ABA signaling and in the regulation of ROS production in pathogen responses (Assmann, 2002).

RCD1 has been identified in four separate stress-related circumstances (Belles-Boix et al., 2000; Overmyer et al., 2000; Lin and Heaton, 2001; Fujibe et al., 2004) and displays several pleiotropic phenotypes, most of which can be related to hormonal responses. The alterations in hormonal responses in the rcd1 mutant suggest that RCD1 could be an integrative node in hormonal signaling and be involved in the hormonal regulation of several specific stress-responsive genes with a biochemical mechanism that has not been described in plants previously.

METHODS

Plant Material and Growth Conditions

Arabidopsis thaliana was grown on 1:1 mixture of peat and vermiculite with subirrigation. Seeds were vernalized for 3 to 5 d. Growth conditions were 23°C/19°C (day/night), 70%/90% relative humidity, under a 12-h photoperiod with 150 μmol m−2 s−1 irradiance in controlled growth chambers (Weiss Bio1300; Weiss Umweltstechnik, Reiskirchen-Lindenstruth, Germany) or growth rooms under similar conditions. For specific stress or hormone experiments performed on plants grown in vitro on plates, slightly different conditions may have been used and are listed under the respective experiments. Ozone exposures were started at 9:00 am with 250 nL L−1 for 6 h (sensitivity tests) or 7 h (gene expression analysis) as described (Kiiskinen et al., 1997). Seeds of various Arabidopsis ecotypes and mutants were obtained from the ABRC (http://www.arabidopsis.org/abrc/) and the Nottingham Arabidopsis Stock Centre (http://nasc.nott.ac.uk/).

Triploid Plant Production

For the production of RCD1/RCD1/rcd1 triploid plants, rcd1/rcd1 gl1/gl1 plants were the pollen recipient in a cross with a known tetraploid line in the co1/co1 gi1/gi1 GL1/GL1 Col-1 background (obtained from the ABRC; stock number CS3432). Only plants identified as true crosses based on the presence of trichomes in the F1 were used for analysis.

Positional Cloning of rcd1-1

Visual identification of the recessive rcd1 habitus was used to select 2000 homozygous rcd1/rcd1 individuals from more than 10,000 F2 progeny of rcd1 × Ler cross. The plants were genotyped with simple sequence length polymorphic and cleaved-amplified polymorphic sequence markers. Candidate genes were amplified from rcd1-1 using Pfu polymerase (Promega, Madison, WI) and sequenced with internal primers. BLAST and PSI-BLAST searches (Altschul et al., 1997) of the nonredundant protein database (National Center for Biotechnology Information) were performed to find homologs of RCD1.

Genetic Complementation

RCD1 and rcd1-1 cDNAs were prepared from leaf total RNA by RT-PCR according to the manufacturer's instructions (One-Step RT-PCR; Qiagen, Hilden, Germany) using gene-specific primers (5′-TTACAATCCACCTGCACCTTC-3′ and 5′-ATGGAAGCCAAGATCGTCA-3′) and Hot Start Taq DNA polymerase (Promega). PCR products were cloned into pGEMT-Easy vector (Promega), confirmed by sequencing, cloned (NotI) into pART27 binary vector (Gleave, 1992), and introduced into Agrobacterium tumefaciens strain C58C1pGV2260 by electroporation. Plants were transformed using the floral dip method (Clough and Bent, 1998). Kanamycin-resistant T1 plants were confirmed by PCR and DNA gel blot analyses. As a complementation test, surface-sterilized T2 seeds were germinated on 1% agar MS plates containing 1.0 μM paraquat. To determine O3 sensitivity, 21- to 28-d-old T2 plants were exposed to O3 for 4 h with 300 nL L−1. Cell death was measured by ion leakage from rosette leaves as described in Overmyer et al. (2000).

Reverse Transcriptase–Mediated PCR

Total RNA from rcd1-1 leaves was used for reverse transcription with FirstChoice RLM-RACE kit (Ambion, Austin, TX) according to the manufacturer's instructions. 5′-RACE PCR was in two steps using nested gene-specific primers (5′-TTACAATCCACCTGCACCTCCTCATGGTCT-3′ and 5′-ATCTCCTTCGACTTTGGCTGGTTTTGA-3′) and nested primers from the 5′ RACE adaptor provided in the kit. Annealing temperatures for the reactions were 60°C (outer) and 65°C (inner). For sequencing, the PCR products were cloned into pGEMT-Easy vector.

Gene Expression Analyses

RNA was isolated according to Carpenter and Simon (1998) or using the Qiagen RNEasy plant kit (Qiagen). Gene-specific DNA probes were labeled with α-32P using Ready-To-Go DNA labeling beads kit (Amersham Biosciences, Buckinghamshire, UK). RNA gel blot and dot blot hybridizations and validation of the specificity and sensitivity of the dot blot hybridizations are described in Overmyer et al. (2000) and Tuominen et al. (2004). The microarray containing ~6500 genes was hybridized with probes prepared from 23-d-old Col-0 and rcd1-1 RNA. Six biological repeats (each 5 to 10 plants) were pooled into pairs of two, each of the three repeats were labeled with cy3 and cy5 and with the dyes swapped for a total of six hybridizations. The image analysis was with GenePixPro 5.0 (Axon Instruments, Union City, CA). Visually bad spots or areas and low intensity spots were excluded. Low intensity spots were determined as spots where <55% of the pixels had intensity above the background +1 sd in either channel. The GenePixPro 5.0 data was imported into GeneSpring 6.0 (Silicon Genetics, Redwood City, CA) and normalized with the Lowess method. The background subtracted median intensities were used for calculations.

Expression of 92 stress- and defense-related genes was characterized in samples collected from 3-week-old plants with macroarrays described in Overmyer et al. (2000) and Tuominen et al. (2004). Gene expression was quantified by hybridization of a 33P-labeled cDNA probe prepared from each mRNA sample and normalized by division with the mean expression level of two constitutively expressed genes, ACT2 (At3g18780) and ACT8 (At1g49240).

Hormone Treatments

For ABA treatment, sterilized seeds were sown on medium containing half-strength MS salts (Sigma-Aldrich, St. Louis, MO), 0.1% Mes, 2% sucrose, and 0.8% Bacto agar, pH 5.7. After vernalization for 3 d at 4°C, the plates were incubated in controlled growth chambers (Sanyo, Sakata, Japan) with 22°C temperature and 70% relative humidity under a 12-h photoperiod (130 μmol m−2 s−1). At 4 d, seedlings were transferred to 12-well plates containing 1.5 mL of the same medium. At 14 d, 200 μL of ABA, ACC, and/or MeJA solutions was added to the plates. Plants were harvested after 48 h.

Triple Response Assay and Sensitivity to Glucose

For triple response assay, surface-sterilized seeds were sown on MS with 0, 1, or 3 μM ACC, vernalized for 5 d at 4°C, and incubated in a growth chamber with 22°C temperature and 70% relative humidity in darkness for 3 d. To assess glucose sensitivity, seeds were sown on MS plates supplemented with 0, 2, 4, or 6% glucose and incubated in the same conditions except for constant light for 4 d.

Stomatal Conductance and Water Loss

Leaf stomatal conductance to water vapor was measured with an AP4 steady state diffusion porometer (Delta-T, Cambridge, UK). Three healthy leaves from five different plants were analyzed for each genotype and time point in two replicate measurements. Transpiration from detached leaves was measured as described by Leung et al. (1997).

Freezing Tolerance

Plants grown on agar plates were acclimated by applying ABA to the growth media to a final concentration of 60 μM or keeping the plants at 4°C in a controlled growth chamber (Sanyo). To test freezing tolerance, at least four rosettes were cut above the root, and their freezing tolerance was tested as described in Tamminen et al. (2001).

Accession Numbers for Data Deposited in a Public Repository

Seeds of the rcd1-1 mutant have been deposited to the Nottingham Arabidopsis Stock Centre with the stock number N9354. For the cDNA microarray analysis, the complete protocols, array design, experiment design, and raw data files are available from the ArrayExpress Database (http://www.ebi.ac.uk/arrayexpress/) with the accession number E-MEXP-77. The nucleotide sequence of the RCD1 gene containing the rcd1-1 mutation has been deposited in GenBank with the accession number AY578788 and two cDNA sequences of the splice variants of rcd1-1 with accession numbers AY578789 and AY578790.

Acknowledgments

We are grateful to Mirva Lehtinen and Amy Warren for technical assistance, Mika Korva and Leena Laakso for plant care, and Olevi Kull (Tartu University, Estonia) for the use of the AP4 porometer. We gratefully acknowledge the Monsanto Company for access to SNP and INDEL polymorphism information (Jander et al., 2002), which was used in genetic marker development, the national Finnish DNA Microarray Centre (Turku Centre for Biotechnology) for the manufacturing, Eva-Mari Aro (Department of Biology, University of Turku, Finland) for providing the cDNA arrays used, and Kotaro T. Yamamoto (Hokkaido University, Japan) for his generous gift of rcd1-2 seeds and his invaluable discussions. This work was supported financially by the Scientific Council of Research of Environment and Natural Resources in Finland (Grants 43671, 52336, and 37995 to J.K. and postdoctoral fellowship 41615 to H.T.) and by the Finnish Centre of Excellence Programme (2000 to 2005). E.B.-B. was the recipient of a predoctoral Human Capital and Mobility fellowship from the European Union (41SF6694).

Notes

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.plantcell.org) is: Jaakko Kangasjärvi (if.iknisleh@ivrajsagnak.okkaaj).

Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.021832.

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