Differential expression of a novel gene in response to coronatine, methyl jasmonate, and wounding in the Coi1 mutant of Arabidopsis

Abstract

Coronatine is a phytotoxin produced by some plant-pathogenic bacteria. It has been shown that coronatine mimics the action of methyl jasmonate (MeJA) in plants. MeJA is a plant-signaling molecule involved in stress responses such as wounding and pathogen attack. In Arabidopsis thaliana, MeJA is essential for pollen grain development. The coi1 (for coronatine-insensitive) mutant of Arabidopsis, which is insensitive to coronatine and MeJA, produces sterile male flowers and shows an altered response to wounding. When the differential display technique was used, a message that was rapidly induced by coronatine in wild-type plants but not in coi1 was identified and the corresponding cDNA was cloned. The coronatine-induced gene ATHCOR1 (for A. thaliana coronatine-induced) is expressed in seedlings, mature leaves, flowers, and siliques but was not detected in roots. The expression of this gene was dramatically reduced in coi1 plants, indicating that COI1 affects its expression. ATHCOR1 was rapidly induced by MeJA and wounding in wild-type plants. The sequence of ATHCOR1 shows no strong homology to known proteins. However, the predicted polypeptide contains a conserved amino acid sequence present in several bacterial, animal, and plant hydrolases and includes a potential ATP/GTP-binding-site motif (P-loop).

Figure 1

Northern analysis of total RNA extracted from flowers and seedlings of wild-type (wt) and coi1 mutant seedlings (coi) growing in MS or in MS containing 1 μm coronatine (Cor) for 4 h, hybridized with TGCOPP9–280. The display probe detected a major transcript of about 1.3 kb that is induced by coronatine in wild-type but not coi1 seedlings. Transcripts of similar molecular weight are observed in lower levels in seedlings and flowers of the wild type but not in male-sterile flowers of coi1. The position of the 16S ribosomal band is indicated.

Figure 2

Sequence of ATHCOR1 cDNA (AF021244) and its predicted protein. The 5′ end of the untranslated sequence obtained from mRNAs by RACE is underlined. The first guanosine residue was not found in the genomic clone (not shown) and was interpreted as the mRNA cap. Amino acids in bold represent a possible N-glycosylation site and the bold, underlined sequence represents a potential ATP-/GTP-binding site motif A (P-loop).

Figure 3

Protein alignments between regions of the translated peptide from ATHCOR1 with two conserved domains (A and B) found in different hydrolases. SHAF, Similar to human-activating factor acetylhydrolase from C. elegans, U64598; PAF, platelet-activating factor acetylhydrolase from Cavia porcellus, JC5021; HDH, haloacetate dehalogenase from Moraxella sp., A44856; Mtu, unknown Mycobacterium tuberculosis Z95389; AtsEH, Arabidopsis epoxide hydrolase, D16628; GlyEH, soybean epoxide hydrolase, D63781; and DLH, dienelactone hydrolase from Synechocystis sp. dienelactone hydrolase, D90904. Black boxes indicate conserved amino acids at the minimum of 50%. Gray boxes indicate changes by similar residues. The percentage of similarity between ATHCOR1 and each of the sequences is presented. The consensus for the putative ATP-/GTP-binding site described in the literature (Saraste et al., 1990) is indicated by an asterisk. Sequences were aligned by the CLUSTAL W program (Thompson et al., 1994) and shaded by the BOXSHADE program (used at the Bioinformatics Group WWW site at the Swiss Institute for Experimental Cancer Research, Lousanne, Switzerland).

Figure 4

Time-course induction of total RNA extracted from seedlings of wild-type Arabidopsis growing in MS medium (Control) or MS containing 1 μm coronatine (Cor) or 10 μm MeJA. ATHCOR1 was rapidly induced by MeJA after 30 min of induction. A weaker induction of ATHCOR1 was observed with coronatine treatment. A background hybridization with the 28S ribosomal band is shown.

Figure 5

A, Northern analysis of total RNA extracted from different organs of wild-type (wt) and coi1 (coi1) mutant plants. ATHCOR1 is normally expressed in seedlings (S), young (L) and mature (M) leaves, flowers (F), and siliques (Si), but it could not be detected in roots (R). Very low levels of the corresponding transcript were detected in coi1 tissues. B, Total RNA stained with ethidium bromide before being transferred onto the membrane.

Figure 6

A, Northern analysis of total RNA extracted from leaves of wild-type (wt) and coi1 (coi1) mutant plants after wounding. ATHCOR1 was highly induced in wild-type leaves by wounding. Its expression peaked about 30 min after leaves were injured and decreased to normal levels after the first 4 h. The same pattern of induction was observed in the leaves of coi1, although at much lower levels. B, Total RNA stained with ethidium bromide before being transferred onto the membrane.