Report

COI1: An Arabidopsis Gene Required for Jasmonate-Regulated Defense and Fertility

See allHide authors and affiliations

Science  15 May 1998:
Vol. 280, Issue 5366, pp. 1091-1094
DOI: 10.1126/science.280.5366.1091

Abstract

The coi1 mutation defines an Arabidopsis gene required for response to jasmonates, which regulate defense against insects and pathogens, wound healing, and pollen fertility. The wild-type allele, COI1, was mapped to a 90-kilobase genomic fragment and located by complementation of coi1-1 mutants. The predicted amino acid sequence of the COI1 protein contains 16 leucine-rich repeats and an F-box motif. It has similarity to the F-box proteins Arabidopsis TIR1, human Skp2, and yeast Grr1, which appear to function by targeting repressor proteins for removal by ubiquitination.

Jasmonates (JAs), which include jasmonic acid and its cyclopentanone derivatives, are widely distributed throughout the plant kingdom. They are synthesized by the octadecanoic pathway from linolenic acid in undamaged tissues and (apparently) by a different pathway in wounded tissues. JAs affect a variety of processes in plants, including root growth, fruit ripening, senescence, pollen development, tuber formation, tendril coiling, and defense against insect pests and pathogens. They alter gene transcription, RNA processing, and translation (1).

Tomato plants respond to injury, such as that caused by chewing insects, by making proteinase inhibitor (pin) proteins that inhibit insect digestive proteases (2). JA is required for plant defenses against insect predation (3) and acts together with ethylene formed in the wounded tissues to regulate pin gene expression (4). In Arabidopsis, the functions of JA are defined by the triple mutant fad3-2 fad7-2 fad8, which is deficient in linolenic acid, the precursor to JA (5), and by the coi1 mutant (6) and other mutants (7) with reduced sensitivity to JA. The fad3-2 fad7-2 fad8 and coi1 mutants have deficient wound responses (8, 9) and high mortality from attack by chewing insects (8, 10), and they produce nonviable pollen (5,6). Treatment of plants with JA corrects these deficiencies in fad3-2 fad7-2 fad8, but not in coi1 (5,6, 8-10). The coi1 mutation therefore defines a gene,COI1, that functions in the JA signal pathway and is required for pollen development and defense against pests, and also for defense responses to pathogens (11). To investigate its function, we have used a map-based strategy to isolate COI1.

We genetically mapped coi1 in F2 plants from a cross between the coi1-1/coi1-1 mutant derived from Arabidopsis ecotype Columbia (6) and wild-type COI1/COI1 plants of ecotype Landsberg erecta. Tests on 188 plants for genetic linkage between the coi1 phenotype (12) and cleaved amplified polymorphic sequence (CAPS) markers (13) placed coi1-1 on chromosome 2 flanked by visible markers as and cer8 (Fig.1A). Further mapping locatedCOI1 on bacteria artificial chromosome (BAC) D25L8 (Fig.1B). The COI1 gene was mapped in D25L8 by screening DNA fragments for transient complementation of coi1-1 mutants transgenic for the COI1-dependent wound- and JA-responsive reporter gene, PThi2.1-GUS (Fig. 1, B to E) (14). Fragment 8ks was sufficient to complement coi1-1 (Fig. 1E); this fragment identified clones 1.8c and 1.9c in anArabidopsis cDNA library (15). The 1.8c cDNA complemented the coi1-1 mutant in the transient assay (Fig.1E).

Figure 1

Genetic and physical mapping ofArabidopsis DNA sequences that complementcoi1-1. (A) Genetic mapping of 460 plants with chromosome recombinations between visible markers as andcer-8 placed coi1-1 between CAPS markers C83 and B23 (24). C83 and B23 were used to screen YAC libraries (25). Numbers of detected chromosome recombinations between the marker and coi1-1 are denoted by r; subscripts L and R indicate markers derived from YAC left and right ends, respectively (open box indicates right end). (B) The closest markers flanking coi1-1, EG13C9 (right end) and YUP10A2 (left end), hybridized to BAC clone D25L8 (25). Restriction enzyme digestion fragments of D25L8 are named to indicate size (in kilobases) and are marked (+) if they complementedcoi1-1 in the transient transformation assay or (–) if they did not. (C) Map of 8ks and adjoining DNA. Thick line shows predicted coding sequences; introns are marked as open triangles; p1 (located outside of 8ks) and p2 (within 8ks) are PCR primers that amplify a 1.5-kb fragment with an internal recognition site for Xcm I (see Fig. 2B). Arrows indicate position and orientation of transcription of expressed sequences 1.8c and 1.9c identified by 8ks in a cDNA library with inserts controlled by the CaMV 35S promoter (15). (D) Plants transgenic for PThi2.1-GUS (14) were infiltrated with 10 μM methyl jasmonate (JA) or water (control), or wounded by gold particles fired from a particle gun. GUS activity could be detected in wounded and JA-treated wild-type plants, but not in controls or coi1-1 mutants, indicating thatCOI1 is required for JA- and wound-induced expression of the Thi2.1 gene. (E) coi1-1 plants transgenic for PThi2.1-GUS were bombarded (26) with gold particles coated with candidate COI1 DNA sequences. Histochemical staining revealed sites of transformation as blue spots of GUS activity, indicating complementation ofcoi1-1 by DNA from BAC D25L8, fragment 8ks, and cDNA 1.8c. Two leaves are shown for each complementation assay.

To confirm that 8ks complemented other JA responses in thecoi1-1 mutant, we used Agrobacterium tumefaciens–mediated transformation to produce stable transgenic plants (16). We could not achieve direct transformation ofcoi1-1 mutants by two different methods that efficiently transformed wild-type plants (16, 17). However, we could successfully transform fertile COI1/coi1-1plants with 8ks by in planta vacuum-infiltration transformation. About 25% of the untransformed progeny of these plants segregated ascoi1-1 mutants, as expected. However, of 79 seedlings selected from the same seed lot for kanamycin resistance through the neomycin phosphotransferase gene linked to 8ks on the transforming DNA, all exhibited wild-type growth inhibition in response to JA and produced fertile pollen. This indicated that the transgene, 8ks, had functionally complemented the coi1-1 mutation. Similar results indicated that coi1-1 was complemented by cDNA clone 1.8c but not by cDNA clone 1.9c, nor by the transformation vector. We determined the DNA sequence of the COI1 gene (18) and developed a CAPS marker to identify coi1-1 mutants that had been complemented by the 8ks transgene.

The DNA sequence of 8ks indicated a single open reading frame that corresponded to cDNA clone 1.8c (Fig. 1C). The corresponding sequence from the coi1-1 mutant deviated from that of the wild type by a single nucleotide change, G to A, at position +1401 relative to the translation start of 1.8c. This mutation created a polymorphism between DNA digested with Xcm I from wild-type plants and from thecoi1-1 mutant (Fig. 2B). Analysis of the 79 transgenic plants for this polymorphism revealed that 24 were homozygous for the coi1-1 mutation, one of which is T-36 (Fig. 2); 37 were heterozygous; and 18 were homozygous for the wild type. This agreed with the expected ratio of 1:2:1 (coi1-1: heterozygous:wild-type) in the 79 tested plants (P > 0.3), and confirms the functional complementation of the coi1-1 mutant by COI1sequences in 8ks.

Figure 2

Stable complementation of thecoi1-1 mutant by 8ks. (A) Seedlings of wild type, the coi1-1 mutant, and line T-36 (transgenic for 8ks) were grown on MS medium (control) or medium containing 50 μM JA (6). JA inhibited growth of seedlings of the wild type and T-36, but not the coi1-1 mutant. (B) A 1.5-kb fragment containing part of COI1 or thecoi1-1 mutant allele could be amplified by the PCR primers p1 (5′-GGTTCTCTTTAGTCTTTAC-3′) and p2 (5′-CAGACAACTATTTCGTTACC-3′) from chromosomal DNA but not from the transgene 8ks (Fig. 1C). Xcm I cleaved the 1.5-kb fragment from wild-type DNA at the recognition site CCA-9N-TGG, but not from the coi1-1 mutant, in which the Xcm I recognition site was altered by the G1401A mutation to CCA-9N-TGA. The 1.5-kb fragment from T-36 was not cleaved by Xcm I, indicating that this plant was homozygous for the coi1-1mutation and was therefore functionally complemented by the transgene 8ks.

The COI1 cDNA sequence predicted a 1779-nucleotide gene product coding for a 592–amino acid protein (Fig.3). Support for the designation of this open reading frame as COI1 came from the complementation experiments, described above, and from deviations of the sequences of three coi1 alleles (12) from the wild-type sequence. The DNA sequence of coi1-1 differed from the wild type by a single nucleotide that converted codon 467 (W) into a translation stop codon. In coi1-15 a 1-nucleotide deletion at codon 60 (C) caused a frame shift that introduced a translation stop at codon 75. In coi1-18 a 10-nucleotide deletion, replaced by a ∼3-kb insertion, altered the sequence from codon 358 (E) to a translation stop at codon 366. The three mutant alleles were therefore predicted to produce truncated proteins. Northern (RNA) blot analysis revealed that the COI1 transcript was expressed in similar amounts in untreated, wounded, and JA-treated tissues (10).

Figure 3

Derived amino acid sequence of COI1(27). A line over the sequence indicates regions with similarity to previously defined functional domains. The altered sequences of the mutant alleles coi1-1,coi1-15, and coi1-18 are indicated. TheCOI1 cDNA sequence has been deposited in GenBank (accession number AF036340). The COI1 genomic sequence was simultaneously determined in the Arabidopsis genome-sequencing project (accession number AF00210).

The deduced amino acid sequence of the COI1 protein contains a degenerate F-box motif (19) and 16 imperfect leucine-rich repeats (LRRs) (20), consensus xLxxaxxxCxxLxxaxa, where “a” is a hydrophobic amino acid (Fig. 3). Both of these motifs are involved in protein-protein interactions (20, 21). Database searches indicated that COI1 was related to three LRR-containing F-box proteins: Arabidopsis TIR1 (22), to which it has 34% identity, as well as human Skp2 and yeast Grr1 (19,21). TIR1 is required for response to auxin, and tir1mutants are fertile plants with reduced sensitivity to root growth inhibition by auxin. However, coi1 mutants are male sterile and show wild-type sensitivity to growth inhibition by auxin (10). Apparently, therefore, COI1 and TIR1 function in separate signal pathways. Both Skp2 and Grr1 regulate cell division, and Grr1 also regulates nutrient uptake (23). These and other F-box proteins function as receptors that selectively recruit repressor proteins into a complex required for the ubiquitination of substrates targeted for removal (21). We speculate that COI1 may be an F-box protein that recruits regulators of defense response and pollen development for modification by ubiquitination.

  • * Present address: Sainsbury Laboratory, John Innes Centre, Norwich, UK.

  • Present address: Biological Sciences, Wye College, Wye, Ashford, Kent, UK.

REFERENCES AND NOTES

View Abstract

Navigate This Article