The transcription factors BEL1 and SPL are required for cytokinin and auxin signaling during ovule development in Arabidopsis

Abstract

Hormones, such as auxin and cytokinin, are involved in the complex molecular network that regulates the coordinated development of plant organs. Genes controlling ovule patterning have been identified and studied in detail; however, the roles of auxin and cytokinin in ovule development are largely unknown. Here we show that key cytokinin pathway genes, such as isopentenyltransferase and cytokinin receptors, are expressed during ovule development. Also, in a cre1-12 ahk2-2 ahk3-3 triple mutant with severely reduced cytokinin perception, expression of the auxin efflux facilitator PIN-FORMED 1 (PIN1) was severely reduced. In sporocyteless/nozzle (spl/nzz) mutants, which show a similar phenotype to the cre1-12 ahk2-2 ahk3-3 triple mutant, PIN1 expression is also reduced. Treatment with the exogenous cytokinin N(6)-benzylaminopurine also altered both auxin distribution and patterning of the ovule; this process required the homeodomain transcription factor BELL1 (BEL1). Thus, this article shows that cytokinin regulates ovule development through the regulation of PIN1. Furthermore, the transcription factors BEL1 and SPL/NZZ, previously described as key regulators of ovule development, are needed for the auxin and cytokinin signaling pathways for the correct patterning of the ovule.

Figure 1.

Analysis of the Cytokinin Pathway during Ovule Development. Ovule stages as in Schneitz et al. (1995). (A) Schematic representation of the cytokinin pathway and the genes analyzed in this article. (B) to (E) GUS expression in IPT1pro:GUS ovules from stage 2-III to stage 3-VI. (F) to (H) GFP expression in TCSpro:GFP ovules from stage 2-III to ovule stage 3-V. (I) to (L) GUS expression in AHK2pro:GUS ovules from stage 1-I to stage 3-V. (M) to (P) GUS expression CRE1pro:GUS ovules from stage 1-I to stage 3-V. (Q) Scheme of ovule development from stage 1-II to stage 3-V. chal, chalaza; f, funiculus; fg, female gametophyte; ii, inner integument; oi, outer integument; n, nucellus. Bars = 20 μm.

Figure 2.

Role of Cytokinin in Ovule Development. (A) Wild-type ovule at stage 3-V. The dotted line indicates the female gametophyte. (B) cre1-12 ahk2-2 ahk3-3 ovule at stage 3-V. The female gametophyte arrested at stage FG1 (arrowhead). (C) cre1-12 ahk2-2 ahk3-3 finger-like structure. (D) pin1-5 ovule at stage 3-V. The female gametophyte arrested at stage FG1 (arrowhead). (E) pin1-5 finger-like structure. (F) Wild-type ovule expressing PIN1pro:PIN1-GFP. (G) cre1-12/+ ahk2-2/+ ahk3-3/+ ovule expressing PIN1pro:PIN1-GFP. (H) cre1-12 ahk2-2 ahk3-3 triple mutant ovule expressing PIN1pro:PIN1-GFP. (I) spl ovule expressing PIN1pro:PIN1-GFP. (F) to (I) Pictures taken using the bright field (left) and the dark field (right). The dotted line shows the ovule profile. chal, chalaza; f, funiculus; fg, female gametophyte; ii, inner integument; nu, nucellus; oi, outer integument. Bars = 20 μm.

Figure 3.

Analysis of BAP-Treated Ovules. (A) Wild-type ovule at stage 2-III mock-treated, expressing PIN1pro:PIN1-GFP 1 d after treatment (1D). (B) Wild-type ovule at stage 2-III BAP-treated, expressing PIN1pro:PIN1-GFP 1 d after treatment. Arrow indicates ectopic PIN1 expression. (C) spl ovule at stage 2-III BAP-treated, expressing PIN1pro:PIN1-GFP 1 d after treatment. (D) In situ hybridization with SPL/NZZ probe, on wild-type mock-treated ovule (2D, 2 d after treatment). (E) In situ hybridization with SPL/NZZ probe on wild-type BAP-treated ovule 2 d after treatment. (F) In situ hybridization with SPL/NZZ probe on cre1-12 ahk2-2 ahk3-3 triple mutant ovule. (G) Quantitative SPL/NZZ expression analysis in wild-type BAP-treated plants and cre1-12 ahk2-2 ahk3-3 triple mutant flowers by real-time RT-PCR. (A) to (C) Pictures were taken using bright field (left) and dark field (right). The dotted line shows the ovule’s profile. f, funiculus; fg, female gametophyte; ii, inner integument; nu, nucellus; oi, outer integument. Bars = 20 μm.

Figure 4.

Effect on Ovule Development after 2 d of BAP Treatment. (A) Wild-type ovule 2 d (2D) after BAP treatment. (B) cre1-12 ovule 2 d after BAP treatment. (C) Wild-type ovule of PIN1pro:PIN1-GFP plants 2 d after BAP treatment. (D) and (E) DR5rev-pro:GFP ovule 2 d after the mock treatment (D) or BAP treatment (E). Arrow indicates ectopic DR5rev:GFP signal in the CK-IS. (F) pin1-5 ovule 2 d after BAP treatment. f, funiculus; ii, inner integument; nu, nucellus; oi, outer integument. Bars = 20 μm.

Figure 5.

BEL1 Expression Is Regulated by Cytokinin. (A) Wild-type ovule, stage 2-III. (B) bel1-1 ovule, stage 2-III. (C) Wild-type ovule, stage 2-III, 2 d (2D) after BAP treatment. (D) In situ hybridization on wild-type ovule with BEL1 probe. (E) In situ hybridization on wild-type ovule treated with BAP using the BEL1 probe 2 d after treatment. (F) In situ hybridization on cre1-12 ahk2-2 ahk3-3 triple mutant ovule with BEL1 probe. (G) Quantitative BEL1 expression analysis by real-time RT-PCR. Wild-type mock-treated or BAP-treated 2 d after treatment, wild-type and cre1-12 ahk2-2 ahk3-3 triple mutant flowers, cre1-12 single mutant, and ahk2-2 ahk3-3 double mutant 2 d after mock treatment or BAP treatment. (H) to (J) WUSpro:WUS-GUS activity in wild-type ovule (H), bel1-1 ovule (I), and in a wild-type ovule 2 d after BAP treatment (J). The ovules are at stage 2-III/3-I. The white arrowhead indicates ectopic WUSpro:WUS-GUS expression in the aberrant structures of the ovules ([I] to [J]). (K) PIN1pro:PIN1-GFP in bel1-1 ovule. (L) DR5rev-pro:GFP in bel1-1 ovule. The ovule is at stage 2-III. (M) bel1-1 ovule treated with NPA. The arrowhead indicates the region where the bel1-1 structure is formed. chal, chalaza; f, funiculus; ii, inner integument; nu, nucellus; oi, outer integument. Bars = 20 μm.

Figure 6.

BEL1 and SPL Integrate Hormonal Signaling in Ovules. (A) In wild-type ovules, cytokinin activates WUS, which promotes the expression of SPL in the nucellus (nu) (Sieber et al., 2004) and BEL1 in the chalaza (chal). PIN1 is activated in the nucellus by SPL and repressed in the chalaza by BEL1, which in turn represses WUS (Brambilla et al., 2007). f, funiculus. (B) In spl mutant ovules, PIN1 is not expressed, leading to a premature block of female gametophyte development and phenocopies the pin1-5 mutant. (C) In the bel1 mutant, PIN1 is upregulated and is also expressed in the chalaza region, where normally it is not present. (D) In finger-like bel1 spl double mutant ovules, PIN1 is not expressed in the ovule. (E) The application of exogenous NPA to the bel1 mutant triggers the formation of finger-like ovules, because the inhibition of the auxin flux by NPA treatment avoids the formation of the aberrant structures typical for bel1 ovules.