Regulation of floral organ abscission in Arabidopsis thaliana

Abstract

Abscission is a developmental program that results in the active shedding of infected or nonfunctional organs from a plant body. Here, we establish a signaling pathway that controls abscission in Arabidopsis thaliana from ligand, to receptors, to downstream effectors. Loss of function mutations in Inflorescence Deficient in Abscission (IDA), which encodes a predicted secreted small protein, the receptor-like protein kinases HAESA (HAE) and HAESA-like 2 (HSL2), the Mitogen-Activated Protein Kinase Kinase 4 (MKK4) and MKK5, and a dominant-negative form of Mitogen-Activated Protein Kinase 6 (MPK6) in a mpk3 mutant background all have abscission-defective phenotypes. Conversely, expression of constitutively active MKKs rescues the abscission-defective phenotype of hae hsl2 and ida plants. Additionally, in hae hsl2 and ida plants, MAP kinase activity is reduced in the receptacle, the part of the stem that holds the floral organs. Plants overexpressing IDA in a hae hsl2 background have abscission defects, indicating HAE and HSL2 are epistatic to IDA. Taken together, these results suggest that the sequential action of IDA, HAE and HSL2, and a MAP kinase cascade regulates the programmed separation of cells in the abscission zone.

Fig. 1.

MKK4 and MKK5 regulate floral organ abscission. (A) Representative siliques from Col-0 (wild type) and MKK4-MKK5RNAi plants at flower position 10. The floral organs of the position 10 flower of the Col-0 have abscised, but the flower from MKK4-MKK5RNAi plant displays an abscission defective phenotype. (B and E) Sepal (Se), petal (Pt) and stamen (St) AZ morphology of Col-0 (B) and MKK4-MKK5RNAi (E) flowers. The AZ (flanked by brackets) consists of the small dense cells at the base of the floral organs. (C, D, F, and G) SEM of floral organ AZs from Col-0 (C and D) and MKK4-MKK5RNAi (F and G) flowers from position 4 (C and F) and 10 (D and G). The floral organs were forcibly removed from the Col-0 position 4 (C) and from the MKK4-MKK5RNAi position 4 (F) and 10 (G) flowers. The AZ fracture plane created by forcible removal of the floral organs is observed as broken cells. The surface of AZ from Col-0 position 10 flowers (D) display the characteristic enlarged and rounded cells of the abscission scar that normally develops during abscission. (H and I) GUS reporter gene expression of MKK4 (H) and MKK5 (I) in flowers and siliques. (J) Petal breakstrength of MKK4-MKK5RNAi plants from flowers at different positions. Two petals from a flower were assayed from seven flowers (n = 14 per each position). Error bars indicate standard error. The force required to remove the petals could only be measured at positions 2–6 from Col-0 plants. (Scale bars: A, H, and I, 0.5 cm; B and E, 100 μm; C, D, F, and G, 50 μm.)

Fig. 2.

HAE and HSL2 regulate floral organ abscission. (A) Representative siliques from Col-0 (wild type), hae, hsl2, and hae hsl2 plants at flower position 10. The floral organs of the position 10 flower of the Col-0, and single mutant hae or hsl2 plants have abscised, but the flower from hae hsl2 plants display an abscission defective phenotype. (B) Sepal (Se) and stamen (St) AZ morphology of a representative hae hsl2 flower. The AZ (flanked by brackets) consists of the small dense cells at the base of the floral organs. (C and D) SEM of floral organ AZs from hae hsl2 flowers from position 4 (C) and 10 (D). The floral organs were forcibly removed from the hae hsl2 position 4 and 10 flowers. The AZ fracture plane created by forcible removal of the floral organs is observed as broken cells. (E) Reporter gene expression of HAE and HSL2. Expression analysis using HAE or HSL2 promoter:GUS plants at flower stage 17 demonstrates that HAE and HSL2 expression in flowers is restricted to the AZ. (F) Petal breakstrength of Col-0, hae, hsl2, and hae hsl2 from flowers at different positions from 15 flowers (n = 13 per each position). The error bars indicate standard deviation. The force required to remove the petals could only be measured at positions 2, 4, and 6 from Col-0, hae, and hsl2 plants. (Scale bars: A, 0.5 cm; B, 100 μm; C and D, 50 μm.)

Fig. 3.

MPK6 and MPK3 are required for floral organ abscission. (A) (Upper) An in-gel MAP kinase assay of protein extracted from the receptacles of Col-0, hae hsl2, and ida-2 flowers at positions 6–8. (Lower) Western blot, using a MPK6 antibody of the same protein samples, showing equivalent amounts of protein were loaded for each sample. (B) Representative siliques at flower position 10 of the MPK6^KR or MPK6^AF transgenes expressed in Col-0 and mpk3 plants. The abscission defective phenotype is only observed in the transgenic mpk3 plants. (C) Sepal (Se), petal (Pt), and stamen (St) AZ morphology of MPK6^KR in mpk3 flowers. The AZ (flanked by brackets) at the base of the floral organs is normal in the transgenic plants. (D and E) SEM of the floral organ AZs from MPK6^KR in mpk3 flowers from position 4 (D) and 10 (E). The floral organs were forcibly removed from the flowers. (F and G) GUS reporter gene expression of MPK3 (F) and MPK6 (G) in flowers and siliques. Scale bars: B, F, and G, 0.5 cm; C–E, 50 μm.)

Fig. 4.

IDA and HAE HSL2 regulate abscission via a MAPK signaling cascade. Representative siliques from Col-0, ida-2, hae hsl2, GVG-MKK4^DD ida-2 and GVG-MKK4^DD hae hsl2 plants at flower position 10. The abscission defective phenotype of ida-2 or hae hsl2 is rescued by the GVG-MKK4^DD transgene.

Fig. 5.

HAE and HSL2 are epistatic to IDA. (A) Representative siliques from Col-0, hae hsl2, IDAOE, and IDAOE hae hsl2 plants at flower position 10. The flowers from the hae hsl2 double mutant plants display an abscission defective phenotype, and overexpression of IDA in the Col-0 background shows premature abscission and the production of a white substance in receptacle (indicated by the arrow). The hae hsl2 double mutant masks the IDA overexpression phenotype and maintains the abscission defective phenotype. (Scale bar: 0.5 cm.) (B) Schematic diagram of the role of IDA, HAE HSL2, and a MAPK cascade in the regulation of floral organ abscission.