New clothes for the jasmonic acid receptor COI1: delayed abscission, meristem arrest and apical dominance

Abstract

In a screen for delayed floral organ abscission in Arabidopsis, we have identified a novel mutant of CORONATINE INSENSITIVE 1 (COI1), the F-box protein that has been shown to be the jasmonic acid (JA) co-receptor. While JA has been shown to have an important role in senescence, root development, pollen dehiscence and defense responses, there has been little focus on its critical role in floral organ abscission. Abscission, or the detachment of organs from the main body of a plant, is an essential process during plant development and a unique type of cell separation regulated by endogenous and exogenous signals. Previous studies have indicated that auxin and ethylene are major plant hormones regulating abscission; and here we show that regulation of floral organ abscission is also controlled by jasmonic acid in Arabidopsis thaliana. Our characterization of coi1-1 and a novel allele (coi1-37) has also revealed an essential role in apical dominance and floral meristem arrest. In this study we provide genetic evidence indicating that delayed abscission 4 (dab4-1) is allelic to coi1-1 and that meristem arrest and apical dominance appear to be evolutionarily divergent functions for COI1 that are governed in an ecotype-dependent manner. Further characterizations of ethylene and JA responses of dab4-1/coi1-37 also provide new information suggesting separate pathways for ethylene and JA that control both floral organ abscission and hypocotyl growth in young seedlings. Our study opens the door revealing new roles for JA and its interaction with other hormones during plant development.

Figure 1. Isolation and Phenotypic, Molecular and Genetic Analysis of Novel dab Mutants.

(A) Close-up of WS and dab4-1 (coi1-37) inflorescences. Arrows indicate the flower position 7 in WS and 16 in dab4-1 (coi1-37). (B) Revealed fracture planes of the petal abscission zones were examined using scanning electron microscopy. Images were taken after petals were forcibly removed for each position. Flower positions as indicated are shown. Scale bar, 10 µm. (C) The force to remove a petal from each flower position was measured in dab4-1 (coi1-37) and WS. (D) Diagram showing location of mutations for coi1-37, coi1-21 and coi1-1. Primer locations for RT-PCR are denoted with arrows. (E) 1,537 bp deletion in coi1-37 was examined using PCR and confirmed by sequencing the amplicons from both coi1-37 and WS. (F) Expression of DAB4/COI1 was examined in Fl (Flower), St (Stem), Lf (Leaf), and Rt (root) using RT-PCR. gD denotes genomic DNA amplicon using the same primer set. UBQ10 was used as a loading control. (G-I) Genetic complementation of coi1-37 with coi1-21 and coi1-1. coi1-21-/- and coi1-1−/− were crossed to dacoi1-37+/−. Test cross analysis was performed to examine the delayed floral organ abscission phenotype was examined in the test cross population of (G) coi1-21−/− x coi1-37+/− and (H) coi1-37−/− x coi1-17+/− compared to WS (I).

Figure 2. Additional Functions of COI1 (DAB4) Governed by Specific Ecotype.

(A) Pollen viability was examined in coi1-37 (dab4-1). Pollen grains of coi1-37 were stained with Alexander's stain in the anther (left) and removed from anther (right). (B) Germination of coi1-37 pollen in the anther. Anthers were isolated from floral position 2 (after anthesis in wildtype) and examined with fluorescence microscopy. (C) Leaves of coi1-37 compared to WS. Dark green and epinastic leaf growth are specific to WS ecotype of dab4-1. (D) Comparison of WS and coi1-37 plants at 8 weeks after germination showing stronger apical dominance and continued proliferous growth in coi1-37. (E) coi1-37 at 12 weeks after germination showing strong apical dominance and continued growth and flowering. (F) Comparison of 116d old WS to coi1-1 outcrossed to WS. The coi1-1 (WS) mutant acquired increased apical dominance and epinastic leaf growth. Arrow indicates epinastic and dark leaf growth in coi1-1 (WS).

Figure 3. Inflorescence Meristem of coi1-37 (dab4-1) is Indeterminate.

(A) Inflorescence meristems were examined with SEM at different developmental stages after germination in WS and coi1-37. Arrow shows that WS inflorescence meristem is arresting while coi1-37 meristem is still proliferating even at 87d after germination. SEM of wild type at “after-arrest” is missing since it already arrested completely. Scale bar, 10 µm. (B) Comparison of transcript levels of select genes from 58d old inflorescence meristem-enriched tissue from WS and dab4-1 (coi1-37).

Figure 4. JA Signaling and Biosynthesis Regulate Floral Organ Abscission.

42d-old plants were treated with either 200 µM meJA or 1 ppm ethylene. Air treated plants were used as controls. Arrows indicate the last floral positions with petals still attached. (A–C) Responses of WS to (A) air, (B) meJA and (C) ethylene. (D–F) Responses of coi1-37 to (D) air, (E) meJA and (F) ethylene. coi1-37 shows accelerated floral organ abscission only with ethylene treatment as indicated with arrows. (G–I) Responses of Col to (G) air, (H) meJA and (I) ethylene. (J–L) Responses of aos to (J) air, (K) meJA and (L) ethylene. Both applications accelerated floral organ abscission. (M–O) Responses of ein2-1 to (M) air, (N) meJA and (O) ethylene. ein2-1 shows accelerated floral organ abscission only to meJA, but unresponsive to ethylene.

Figure 5. Dose-responses of Dark Grown Seedlings to Ethylene and JA.

Dark-grown seedlings were treated with the indicated concentration of ethylene for 3 days and the length of the hypocotyls were measured to determine the ethylene-dose responses (A,C,E). Data was also normalized (B,D,F) to determine the relative responsiveness to ethylene at various dosages. The error bars represent standard deviation. Responses at a particular concentration of ethylene were compared using a t test and considered statistically significant if P<0.05. (A-B) Ethylene dose-responses of WS and coi1-37 seedlings in the absence of applied meJA. * indicates significant difference from WS (P <0.05). (C-D) The ethylene dose-responses of WS in the absence and presence of 10 µM meJA. * indicates significant difference from untreated control (P <0.05). (E-F) The ethylene dose-responses of coi1−37 in the absence and presence of 10 µM meJA.

Figure 6. Proposed Model of Regulation of Floral Organ Abscission, Inflorescence Meristem Maintenance, and Senescence by COI1.

Working model of floral organ abscission. Based on the progression, in one model, floral organ abscission can be divided into 4 phases. Phase 1, pre-abscission phase where abscission zones (organ boundaries) are established in the early development, Phase 2 where abscission cells acquire competence to abscission signals while structurally dissolution of middle lamellae is observed, Phase 3 where abscission cells are activated when cell wall loosening is occurring, and Phase 4 where cell repair is observed in the post abscission trans-differentiation phase. Previously studied regulators in floral organ abscission are included in this model: DAB4/COI1. BOP1/2 (BLADE ON PETIOLES 1/2), HWS (HAWAIIAN SKIRT), PGs (Polygalacturonases). PMEs (Pectin methyltransferases), EXP (Expansins), IDA (INFLORESCENCE DEFICIENT IN ABSCISSION) and NEV (NEVERSHED).