Floral organ abscission is regulated by a positive feedback loop

Abstract

Abscission is the process by which plants shed unwanted organs, either as part of a natural developmental program or in response to environmental stimuli. Studies in Arabidopsis thaliana have elucidated a number of the genetic components that regulate abscission of floral organs, including a pair of related receptor-like protein kinases, HAESA and HAESA-like 2 (HAE/HSL2) that regulate a MAP kinase cascade that is required for abscission. HAE is transcriptionally up-regulated in the floral abscission zone just before cell separation. Here, we identify AGAMOUS-like 15 (AGL15; a MADS-domain transcription factor) as a putative regulator of HAE expression. Overexpression of AGL15 results in decreased expression of HAE as well as a delayed abscission phenotype. Chromatin immunoprecipitation experiments indicate that AGL15 binds the HAE promoter in floral receptacles. AGL15 is then differentially phosphorylated through development in floral receptacles in a MITOGEN-ACTIVATED PROTEIN KINASE KINASE 4/5-dependent manner. MAP kinase phosphorylation of AGL15 is necessary for full HAE expression, thus completing a positive feedback loop controlling HAE expression. Together, the network components in this positive feedback loop constitute an emergent property that regulates the large dynamic range of gene expression (27-fold increase in HAE) observed in flowers when the abscission program is initiated. This study helps define the mechanisms and regulatory networks involved in a receptor-mediated signaling pathway that controls floral organ abscission.

Keywords: abscission; protein phosphorylation; signal transduction; transcription factor; transcriptional regulation.

Fig. 1.

HAE is expressed in abscission zones and is required along with HSL2 for floral organ abscission. (A) WT inflorescence photographed from above with floral position numbered. (B) Same flowers from inflorescence in A arranged in floral position order with both floral stage and position indicated. (C) hae hsl2 double mutant does not abscise its floral organs. Arrows indicate abnormally attached floral organs. (D and E) White light photograph (D) and photograph (E) of YFP fluorescence of HAE-promoter-HAE-YFP plants expressing HAE–YFP specifically in abscission zones of flowers that are about to abscise and after abscission has occurred. The arrows indicate the position of the abscission zone. (F) HAE transcript expression is induced shortly before abscission in stamen abscission zones. Floral stages of stamen abscission zones are indicated in which s15a, s15b, and s15c are progressively older subdivisions of stage 15 flowers (16).

Fig. 2.

AGL15 is transcriptionally increased through the process of abscission and is predicted to bind the HAE promoter. (A) The HAE promoter has five AGL15 binding sites (red) and five W boxes (green). (B) AGL15 is transcriptionally up-regulated immediately before abscission in stamen abscission zones (16).

Fig. 3.

Overexpression of AGL15 results in reduced HAE expression in floral receptacles. (A and B) Strong overexpression of AGL15–double-HA tag (AGL15-DHA) results in Col-0 plants that do not abscise normally (lines 2 and 3), whereas medium overexpression results in plants that resemble WT Col-0 (line 1). Rubisco is shown as a loading control. (C) HAE expression in stage-15 receptacles. HAE expression was reduced fivefold in plants that strongly overexpress AGL15. *P < 0.005. n = 3; SEM error bars are shown. (D) HAE expression in floral receptacles. Single or double agl15, agl18 mutants do not have altered HAE expression (n = 5). (E) agl15 agl18 double mutants abscise statistically earlier than WT, whereas single mutants do not. *P < 0.05 (t test). n = 12; SEM error bars are shown.

Fig. 4.

AGL15 binds to the HAE promoter in floral receptacles. (A) Enrichment of HAE promoter DNA from 35S–AGL15 ChIP. ChIP on s15 receptacles from 35S–AGL15-DHA indicate that AGL15 binds the HAE promoter in vivo. *P = 0.03 (t test). n = 3; SEM error bars are shown. (B) Enrichment of HAE promoter DNA from native promoter AGL15 ChIP. ChIP on floral receptacles of AGL15p:AGL15-DHA plants indicate AGL15 can interact with the HAE promoter in floral receptacles at native expression levels. *P < 0.05 (t test). n = 4; SEM error bars are shown.

Fig. 5.

AGL15 is differentially phosphorylated in floral receptacles, and AGL15 phosphorylation requires the MKK4/5 cascade. (A) AGL15 in floral receptacles produced by the 35S promoter runs as two bands on a 12% gel. Calf intestinal phosphatase treatment converts the two bands into one band. Rubisco is shown as a loading control. (B) AGL15 produced by its own promoter runs as a single band in receptacles from young unopened flowers (stage 10) and runs as three bands in floral receptacles from stage-12 to -16 flowers on 10% SDS/PAGE gels containing 25 µM Phos-tag. The fast-migrating AGL15 isoform is arbitrarily labeled AGL15 A, and the two slower-migrating species are labeled AGL15 B and C. (C) Same protein extracts as in B run on a 10% SDS/PAGE gel without Phos-tag produce only a single band for AGL15. (D) F₁ plants from a cross between AGL15p:AGL15-DHA x MKK4/5 RNAi (both dominant) have less phosphorylated AGL15 in floral receptacles than AGL15p:AGL15-DHA plants on 10% SDS/PAGE gels with 25 µM Phos-tag. In particular, AGL15 isoform B is greatly reduced. (E) Same protein extracts in D run on 10% SDS/PAGE gels without Phos-tag. (F) AGL15p:AGL15-DHA plants cannot be differentiated from WT Col-0 plants. (G) MKK4/5 RNAi plants do not abscise their floral organs (8). (H) AGL15p:AGL15-DHA x MKK4/5 RNAi F₁ plants have delayed abscission, but are not completely defective in abscission. The plants abscise after stage-16 flowers, whereas WT Col-0 abscises after stage-15 flowers. The above Western blots were repeated at least three times with the same result.

Fig. 6.

MKK4/5 positively regulates HAE expression. (A) HAE expression in stage-15 receptacles. HAE expression is reduced 3.5-fold in stage-15 floral receptacles from MKK4/5 RNAi plants. *P < 0.05 (statistical significance, t test; n = 5; SEM error bars are shown). (B) Gene expression following MKK4 active induction. HAE expression is increased twofold in seedlings that transiently express MKK4 active under the control of the dexamethasone-inducible system. The HAE coexpressed gene and player in abscission, EVR, is also increased (26). *P < 0.05 (statistical significance, t test; SEM error bars are shown). (C) Proposed core positive feedback loop controlling abscission. IDA peptides trigger HAE/HSL2 to activate a MAPK cascade consisting of MKK4/5 and MPK3/6, which then phosphorylates AGL15 that binds the HAE promoter. MPK3/6 phosphorylation relieves AGL15 repression of HAE expression, leading to production of HAE transcript and later HAE protein.