Arabidopsis class I KNOTTED-like homeobox proteins act downstream in the IDA-HAE/HSL2 floral abscission signaling pathway

Abstract

Floral organ abscission in Arabidopsis thaliana is regulated by the putative ligand-receptor system comprising the signaling peptide INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) and the two receptor-like kinases HAESA and HAESA-LIKE2. The IDA signaling pathway presumably activates a MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) cascade to induce separation between abscission zone (AZ) cells. Misexpression of IDA effectuates precocious floral abscission and ectopic cell separation in latent AZ cell regions, which suggests that negative regulators are in place to prevent unrestricted and untimely AZ cell separation. Through a screen for mutations that restore floral organ abscission in ida mutants, we identified three new mutant alleles of the KNOTTED-LIKE HOMEOBOX gene BREVIPEDICELLUS (BP)/KNOTTED-LIKE FROM ARABIDOPSIS THALIANA1 (KNAT1). Here, we show that bp mutants, in addition to shedding their floral organs prematurely, have phenotypic commonalities with plants misexpressing IDA, such as enlarged AZ cells. We propose that BP/KNAT1 inhibits floral organ cell separation by restricting AZ cell size and number and put forward a model whereby IDA signaling suppresses BP/KNAT1, which in turn allows KNAT2 and KNAT6 to induce floral organ abscission.

Figure 1.

Phenotypes of ida and bp Mutants. (A) and (B) C24 wild-type (WT) plants undergo flower abscission shortly after anthesis, which defines position 1 when counting flowers along the primary inflorescence (arrowhead). At position 7 (left image in [B]) turgid petals and senescent sepals are still attached to the receptacle of the flower. The AZ region at position 12 (arrow) after floral organs have abscised is shown on the right in (B). (C) to (F) ida-1 (C24; [C] and [D]) and ida-2 (Col; [E] and [F]) retain floral organs indefinitely. Position 7 (left in [D] and [F]) and position 12 (right in [D] and [F]). (G) Cartoon illustrating suppression screen of ida-1. Revertant mutants portraying normal abscission could either be mutated downstream inhibitors (blunt arrowheads) in the IDA signaling pathway or constitutively active components (arrowheads) of the pathway. (H) and (I) Line 49 with downward-pointing siliques and abscised floral organs by position 9 (arrowhead). AZ with floral organs attached at position 7 (left in [I]) and abscised at position 12 (right in [I]). (J) and (K) bp-3 mutant with downward-pointing siliques and abscised floral organs by position 7 (left in [K]) and an enlarged AZ region at position 12 (arrow, right in [K]) (L) and (M) Segregants from line 49 backcrossed to the C24 wild type with downward-pointing siliques and abscised floral organs by position 7 (left in [M]) and an enlarged AZ region at position 12 (arrow, right in [M]). (N) pBS (i.e., the force required to remove the petals from the flower) of line 49 compared with ida-1 and C24 wild-type (wt) flowers (n = 15, bars = sd). (O) and (P) Scanning electron micrographs of fracture planes of petal AZ (delineated in gray) and the entire AZ region of mature siliques of ida-1 (O) and line 49 (P). Arabic numerals indicate floral positions along the inflorescence. m, mature siliques; f, filament AZ; p, petal AZ; s, sepal AZ. Note that for mature siliques of line 49, it is not possible to distinguish the petal and sepal AZs from each other. Bars = 30 μm for positions 4 to 12 and 100 μm in mature siliques.

Figure 2.

BP/KNAT1 Involvement in Floral Abscission. (A) BP/KNAT1_pro:GUS expression during floral organ abscission. Arabic numerals indicate floral positions along the inflorescence. (B) pBS of bp-3 and bp-10 mutants compared with Col wild-type (wt) and C24 wild-type flowers, respectively (n = 15, bars = sd). (C) to (E) Mature siliques stained with the Yariv reagent β-GLcY, resulting in a red precipitate. (F) to (H) Mature siliques stained with the negative control α-GLcY.

Figure 3.

Floral AZ Scanning Electron Micrographs of bp-3 and 35S:IDA (A) Fracture planes of petal AZ (delineated in gray) of the wild type (wt), bp-3, and 35S:IDA at given floral positions. From position 6 onward, it is not possible to distinguish the different AZs for 35S:IDA plants, and consequently the whole AZ region is shown for all positions older than position 6. (B) Entire AZ region of the wild type and bp-3 at given positions. Note that from position 8 onward in bp-3, it is not possible to distinguish the petal and sepal AZs from each other and the AZ region expands into the pedicel. The extended AZ region is delineated in gray. (C) Entire AZ region of mature siliques. For bp-3 and 35S:IDA, it is not possible to indicate the different AZs. f, filament AZ; p, petal AZ; s, sepal AZ. bp-3 displays a larger sepal AZ region on the abaxial side of the pedicel (arrowhead in [B] and [C]). Bars = 30 μm for petal AZ fracture planes and 100 μm for the entire AZ region.

Figure 4.

Genetic Interaction of BP/KNAT1, HAE, and HSL2. (A) hae hsl2 mutants with deficiency in floral abscission (B) to (G) bp mutants rescue the abscission defect of hae hsl2. bp-3 hae hsl2 main inflorescence (B), silique at position 12 (D), and AZ from mature silique (F); bp ida-1 hae hsl2 main inflorescence (C), silique at position 12 (E), and AZ from mature silique (G). (H) pBS of bp-3 hae hsl2 compared with hae hsl2 and Col wild type (wt). (n = 15, bars = sd). [See online article for color version of this figure.]

Figure 5.

Phenotypes of knat2 and knat6 in bp-3 and 35S:IDA. (A) Phenotype of knat2 knat6 with organs attached at position 15 (arrow). (B) to (H) Genetic interaction phenotypes of knat2 and knat6 with bp-3. bp-3 knat2 ([B] and [E]), bp-3 knat6 ([C] and [F]), and bp-3 knat2 knat6 ([D], [G], and [H]). Inactivation of knat6 but not knat2 partially rescues the silique orientation phenotype of bp-3 but not the enlarged AZ region. In bp-3 knat2 knat6 triple mutants, siliques display wild-type orientation and a normal-sized AZ region with attached floral organs (arrow in [D]). (I) and (J) Overexpression phenotypes of 35S:IDA are not apparent in the knat2 knat6 mutant background. Note floral organs attached in 35S:IDA knat2 knat6 (arrow). (K) to (N) Siliques at position 7 have attached floral organs in 35S:IDA knat2 knat6 plants and normal-sized AZs ([K] and [M]) compared with 35S:IDA ([L] and [N]). (O) Mature 35S:IDA knat2 knat6 AZ with enlarged AZ region. [See online article for color version of this figure.]

Figure 6.

KNAT2 and KNAT6 Expression in ida-1, hae hsl2, and bp during Floral Abscission. (A) KNAT2 promoter-driven GUS expression during floral organ abscission in the C24 wild type. (B) KNAT6 promoter-driven GUS expression during floral organ abscission in the Ws wild type. (C) KNAT2 promoter-driven GUS expression in ida-2, hae hsl2, and bp-3 AZs for flowers at position 10. GUS activity at the base of wild-type (wt) floral buds was unaltered in the ida-2 and hae hsl2 mutant backgrounds. (D) KNAT6 promoter-driven GUS expression in ida-2, hae hsl2, and bp-9 (Ragni et al., 2008) AZs. GUS activity in the vasculature of emerging wild-type (wt) lateral roots was unaltered in the ida-2 and hae hsl2 mutant backgrounds. Arabic numerals indicate floral positions along the inflorescence.

Figure 7.

Overexpression of KNAT2 and KNAT6 in ida-1. (A) 35S:KNAT6 (Col) inflorescence. (B) and (C) 35S:KNAT6 (Col) and 35S:KNAT2 (Ler) rescue the abscission defect of ida-1. (D) pBS of 35S:KNAT2 ida-1 compared with ida-1 and Ler wild type (wt). (n = 15, bars = sd). [See online article for color version of this figure.]

Figure 8.

Models of IDA Signaling. (A) to (C) IDA can be imagined to bind the extracellular LRRs of a HAE homodimer (A), a HSL2 homodimer (B), or a HAE-HSL2 heterodimer (C) and to cause autophosphorylation (P) of the kinase receptor domains. The protein phosphatase KAPP dephosphorylates HAE and returns the receptor to an off state (Stone et al., 1994). (D) The signal from IDA-HAE/HSL2 is suggested to be transduced via a MAPK cascade that includes MKK4, MKK5, MPK3, and MPK6. Genetic evidence suggests that BP/KNAT1 acts as a negative downstream component of this signaling pathway by restricting cell wall degradation and consequently cell separation and the expression of KNAT2 and KNAT6. Upon activation of the IDA signaling pathway, the BP/KNAT1 restriction of KNAT2 and KNAT6 is elevated, and these transcription factors can act as positive regulators of floral organ separation. In addition, BP/KNAT1 is a positive regulator of EVR, which influences cell elongation by a separate parallel pathway (Leslie et al., 2010).