A downstream mediator in the growth repression limb of the jasmonate pathway

Abstract

Wounding plant tissues initiates large-scale changes in transcription coupled to growth arrest, allowing resource diversion for defense. These processes are mediated in large part by the potent lipid regulator jasmonic acid (JA). Genes selected from a list of wound-inducible transcripts regulated by the jasmonate pathway were overexpressed in Arabidopsis thaliana, and the transgenic plants were then assayed for sensitivity to methyl jasmonate (MeJA). When grown in the presence of MeJA, the roots of plants overexpressing a gene of unknown function were longer than those of wild-type plants. When transcript levels for this gene, which we named JASMONATE-ASSOCIATED1 (JAS1), were reduced by RNA interference, the plants showed increased sensitivity to MeJA and growth was inhibited. These gain- and loss-of-function assays suggest that this gene acts as a repressor of JA-inhibited growth. An alternative transcript from the gene encoding a second protein isoform with a longer C terminus failed to repress jasmonate sensitivity. This identified a conserved C-terminal sequence in JAS1 and related genes, all of which also contain Zim motifs and many of which are jasmonate-regulated. Both forms of JAS1 were found to localize to the nucleus in transient expression assays. Physiological tests of growth responses after wounding were consistent with the fact that JAS1 is a repressor of JA-regulated growth retardation.

Figure 1.

Screen for MeJA Insensitivity in Plants Transformed with Jasmonate-Regulated cDNAs. Root growth of transgenic plants in the absence and presence of MeJA (25 μM). Control plants were either wild type, displaying MeJA-inhibited root growth, or coi1-1, displaying insensitivity to MeJA. The intermediate phenotype of the At5g13220.3 overexpressing line is indicated with an encircled 7. (A) Photographic image of 7-d-old seedlings. Bar = 5 mm. (B) Primary root lengths of 10-d-old seedlings. −, no MeJA in growth medium; +, MeJA (25 μM) in growth medium (n = 10; error bars indicate sd).

Figure 2.

RNAi and Overexpression Constructs for At5g13220 Differentially Affect Sensitivity to MeJA. (A) Seeds were germinated on agar containing either no MeJA (left plate) or MeJA (25 μM; right plate) for 7 d, at which time they were photographed. Wild-type and a coi1-1 segregating seeds were used as controls for MeJA sensitivity. (B) Dry weight of aerial tissues for plants grown in the absence or presence of MeJA (25 μM). Plants were grown for 21 d prior to harvest (n = 12; error bars indicate sd). Ri7, RNAi-7; Ri9, RNAi-9; OE4A, overexpression line 4A; OE4B, overexpression line 4B. (C) Transcript levels relative to EIF4A1 for At5g13220 (all transcript forms) in wild-type and transgenic plants. Quantitative PCR was used to measure relative transcript abundance in 4-week-old soil-grown plants (n = 3; error bars indicate sd). Transcript abundance was not normalized to EIF4A1 transcript abundance.

Figure 3.

Overexpression of At5g13220.3 Affects the Activity of JA Biosynthesis Enzymes, AOS, OPR3, and VSP2 Transcript Levels, and JA Synthesis in Response to Wounding. (A) Extracts of resting leaves were assayed for their ability to oxygenate the jasmonate precursor α-linolenic acid (18:3) in vitro (n = 4; error bars indicate sd). (B) Relative transcript levels in resting leaves for two JA biosynthesis genes (AOS and OPR3) and for the VSP2 gene (n = 3; error bars indicate sd). (C) JA measured in leaves before and after wounding. The wound-induced level of JA at 1.5 h was significantly lower in plants overexpresssing the At5g13320.3 transcript than in wild-type plants (*P = 0.035; n = 3; error bars indicate sd). FWT, fresh weight.

Figure 4.

Effects of MeJA and Wounding on At5g13220 Transcript Levels and of MeJA Treatment on At5g13220/coi1-1 Plants. (A) Mechanical wounding activates At5g13220 expression in wild-type plants but not in the JA biosynthesis mutant aos or the JA perception mutant coi1-1. Plants (5 weeks old) were wounded on the leaf apices (see Methods). (B) MeJA treatment increases transcript levels. Plants (5 weeks old) were sprayed with a suspension of MeJA (100 μM in water). For all experiments, total RNA (15 μg) preparations were analyzed by RNA gel blotting. (C) Copy number of transcripts for At5g13220.1 and At5g13220.3 normalized to 100 transcripts for EIF4A1 (n = 6; error bars indicate se). U, unwounded leaves; W, leaves wounded and harvested after 1 h. (D) Effect of crossing At5g13220.3 overexpressing plants into a background lacking a functional COI1 gene. Plants were grown in the presence (top panel) or absence (bottom panel) of MeJA (25 μM) for 21 d. Bar = 1 cm.

Figure 5.

Cellular Localization of GFP Coupled to At5g13220 Gene Products. (A) C-terminal sequences encoded by two alternative transcripts of the At5g13220 gene fused to GFP. Only the C-terminal amino acids of the At5g13220 gene products are indicated. (B) Fluorescence of onion cells expressing GFP constructs. (C) GFP fluorescence of the At5g13220.3-GFP fusion. (D) 4′,6-Diamidino-2-phenylindole (DAPI) fluorescence of cells expressing the At5g13220.3-GFP fusion. (E) GFP fluorescence of the At5g13220.1-GFP fusion. (F) DAPI fluorescence of cells expressing the At5g13220.1-GFP fusion.

Figure 6.

Overexpression of At5g13220.3 (JAS1.3) Reduces Wound-Induced Growth Inhibition. Wild-type, RNAi-7, and OE4A plants as well as the aos mutant were wounded four times on two leaves at 3-d intervals prior to sampling. Plants were 23 d old at the time of the first wound. (A) Number of leaves over 1 mm long (n = 8; error bars indicate sd). U, unwounded; W, wounded. (B) Dry mass of plants from the same experiment (n = 8; error bars indicate sd). The reduction in dry mass as a result of wounding for each genotype is given as a percentage. (C) Visual phenotypes of plants at a similar stage to those used for (A) and (B).