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Arabidopsis RADICAL-INDUCED CELL DEATH1 belongs to the WWE protein-protein interaction domain protein family and modulates abscisic acid, ethylene, and methyl jasmonate responses - PubMed

. 2004 Jul;16(7):1925-37.

doi: 10.1105/tpc.021832. Epub 2004 Jun 18.

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Arabidopsis RADICAL-INDUCED CELL DEATH1 belongs to the WWE protein-protein interaction domain protein family and modulates abscisic acid, ethylene, and methyl jasmonate responses

Reetta Ahlfors et al. Plant Cell. 2004 Jul.

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.

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Figures

Figure 1.
Figure 1.

rcd1-1 Phenotypes. (A) Habitus of 4-week-old Col-0 and homozygous rcd1-1/rcd1-1 plants. (B) Paraquat tolerance of 2-week-old seedlings grown in 1.0 μM paraquat. A single copy (10-3 and 17-9) or multiple copies (7 and 3) of the rcd1-1 cDNA transgene confer tolerance to paraquat when expressed under the CaMV 35S promoter in the paraquat sensitive wild-type Col-0. (C) Ozone sensitivity of 3-week-old plants. Cell death (measured as ion leakage) after a 4-h 250 nL L−1 O3 exposure in the Col-0 wild type, mutant rcd1-1, and the transgenic lines 17-9, 10-3, and 3 expressing the rcd1-1 cDNA in Col-0.

Figure 2.
Figure 2.

RCD1 Map Position and Arabidopsis SIMILAR TO RCD1 Gene Family. (A) Positional cloning of rcd1. Flanking (UFOa and F23M19-M1) and additional markers in between were used to identify recombinant offspring from the mapping cross between Ler (RCD1/RCD1) and Col (rcd1/rcd1). (B) Structure of RCD1-like family gene transcripts. Black boxes, untranslated regions; gray boxes, exons. The position of the rcd1-1 mutation at the end of exon III (and within the PARP-like domain in the encoded proteins) is indicated with an asterisk. Proteins encoded by SIMILAR TO RCD2 (SRO2) to SRO5 do not contain the WWE domain.

Figure 3.
Figure 3.

Expression of RCD1 and Defense Response Marker Genes. (A) and (B) RNA gel blot analysis of RCD1 expression in various tissues of Col-0 (A) and rcd1-1 (B). B, buds; F, flowers; OF, old flowers; SQ, siliques; CL, cauline leaves; ST, stem; YL, young leaves; OL, old leaves; R, root. (C) Expression of RCD1, PR5, and CHIB in Col-0 exposed to 250 nL L−1 O3 for 7 h. Samples were harvested after 0.5, 1, 2, 4.5, and 8 h. C, clean air control; O3, ozone. The blots were reprobed with 18S rDNA to equalize for loading differences.

Figure 4.
Figure 4.

Expression of RAB18 in ABA-Treated Col-0 and rcd1-1. RNA gel blot hybridization analysis of RAB18 expression in rosette leaves treated with 0, 10, and 60 μM ABA. The signal intensity was normalized with 18S rDNA for loading differences.

Figure 5.
Figure 5.

rcd1-1 Has Higher Stomatal Conductance. (A) Stomatal conductance of 24-d-old rcd1-1 and Col-0 wild type. (B) Stomatal conductance of 24-d-old rcd1-1, abi2, and corresponding wild-type plants during a 6-h O3 treatment of 250 nL L−1 from 9:00 to 15:00 h. The error bars in (A) and (B) indicate standard deviation of two to three leaves from each of five plants. (C) Water loss from detached leaves of rcd1-1, abi2, and corresponding wild types. Weight of leaves cut from four 21-d-old plants were monitored for 2 h. The error bars in (C) (shown when wider than the symbol) indicate standard deviation for the four plants from which the leaves were cut and weighed together. Water loss from rcd1-1 differs significantly from the Col-0 wild type (analysis of variance, P < 0.0005).

Figure 6.
Figure 6.

rcd1-1 Has a Normal Cold Acclimation. Cold-induced tissue damage in Col-0 and rcd1-1 after 2 d of treatment with 60 μM ABA (A) or low temperature (4°C) (B). Damage was measured as ion leakage. The error bars indicate standard deviation of three replicates (four plants each). NA, nonacclimated control.

Figure 7.
Figure 7.

Triple Response Assay and RNA Dot Blot Analysis of ET and Jasmonate Marker Gene Expression. (A) Triple response assay. Col-0, rcd1-1, ein2, and rcd1 ein2 seedlings were grown in darkness for 3 d in the presence of the indicated concentrations of ACC, and the hypocotyl lengths were measured. (B) CHIB expression in 14-d-old in vitro grown Col-0, rcd1-1, and ein2 treated with the indicated concentration of ACC for 48 h. Controls were treated with an equal amount of water. (C) VSP1 expression in 14-d-old in vitro grown Col-0, rcd1-1, jar1, and ein2 treated with the indicated concentration of MeJA for 48 h. Controls were treated with an equal amount of water.

Figure 8.
Figure 8.

rcd1-1 Seedling Development Is Partially Insensitive to Glucose. Three-day-old seedlings of rcd1-1, Col-0, ET insensitive, and glucose hypersensitive ein2 and rcd1 ein2 double mutant on MS media supplemented with 2 and 4% glucose.

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