Shedding light on flower development

short communication Plant Signaling & Behavior 6:4, 471-476; April 2011; ©2011 Landes Bioscience

Shedding light on flower development

Phytochrome B regulates gynoecium formation in association with the transcription factor SPATULA Julia Foreman,1 James N. White,1 Ian A. Graham,2 Karen J. Halliday1 and Eve-Marie Josse1,* Institute of Molecular Plant Sciences; School of Biological Sciences; University of Edinburgh; Edinburgh, Scotland, UK; 2Centre for Novel Agricultural Products; Department of Biology; University of York; York, UK

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Key words: flower development, gynoecium, SPATULA, phytochrome, auxin Abbreviations: bHLH, basic helix-loop-helix; PIF, phytochrome interacting factor; NPA, N-1-naphthylphthalamic acid; GA, gibberellic acid; wt, wild-type

Accurate development of the gynoecium, the female reproductive organ, is necessary to achieve efficient fertilization. In Arabidopsis, the correct patterning of the apical-basal axis of the gynoecium requires the establishment of a morphogenic gradient of auxin. This allows the production of specialized tissues, whose roles consist of attracting pollen, allowing pollen tube growth and protecting the ovules within the ovaries. Mutations in the bHLH transcription factor SPATULA (SPT) are known to impair the development of the apical tissues of the gynoecium. Here, we show that the spt phenotype is rescued by the removal of phytochrome B, and discuss how light signaling may control flower development.

Introduction In Arabidopsis, the gynoecium, the female reproductive organ, is a highly specialized organ resulting from the congenital fusion of two carpels, forming a hollow cylinder. A fully developed gynoecium consists of a short basal gynophore on which sits the large ovary, within which the ovules develop. The ovary is divided into two compartments by a septum, and is extended apically by a short style and a stigma. The stigmatic tissue is designed to trap the pollen and during fertilization, the pollen tubes germinate on the stigma and grow through the transmitting tract that develops within the style and the septum, before swerving laterally to eventually reach the mature ovules.1 Several regulatory mechanisms are involved in the formation of the gynoecium and its apical-basal specification. The current dogma implies the formation of a morphogenic gradient of auxin, where an auxin maximum on the apical side of the gynoecium is needed to promote the formation of the style and stigma. Progressively diminishing levels of auxin towards the basal side of the gynoecium specify the ovaries area and eventually the gynophore at the basal side, where auxin concentration reaches a minimum.2,3 A large number of transcription factors have been described to take part in gynoecium patterning via the mediation of auxin-related processes, including ETTIN (ETT), STYLISH (STY), SPATULA (SPT), HECATE (HEC) and SEUSS (SEU) (reviewed in refs. 1, 3 and 4).

Mutations in the SPATULA (SPT ) gene impair the development of the apical tissues of the gynoecium, as the carpels fail to fuse properly, disrupting the formation of the transmitting tract, the style and the stigma.5-7 This results in a reduced frequency of fertilization and low seed production.8 Several lines of evidence have linked SPT and the establishment of the morphogenic auxin gradient throughout the basal-apical axis of the gynoecium: indeed, spt apical phenotype can be rescued by the application of N-1-naphthylphthalamic acid (NPA), a polar auxin transport inhibitor, suggesting that SPT activity may result in disrupting auxin transport. Furthermore, the auxin-response factor ETTIN (ETT) is crucial for both the setup and the interpretation of the auxin gradient, and has been shown to mainly act by restricting SPT expression.2 In addition to its role in gynoecium patterning, SPT has also been shown to be involved in defining the fate of the apical meristem during the very early stages of flower development.6 SPT is a basic helix-loop-helix transcription factor, belonging to the Phytochrome Interacting Factors/PIF-Like (PIF/PIL) family, where almost all members have been shown to regulate different aspects of light development.7,9 Phytochromes are red/ far-red light photoreceptors which, upon red light activation, change into an active conformation and rapidly migrate into the nucleus, where they bind members of the PIF family, leading to the de-repression of PIFs-controlled transcription. While PIF1, PIF3, PIF4, PIF5, PIF6 and PIF7 bind to the phytochromes directly,10-14 PIL factors lack the ability to bind phytochromes

*Correspondence to: Eve-Marie Josse; Email: [email protected] Submitted: 12/13/10; Accepted: 12/13/10 DOI: 10.4161/psb.6.4.14496 www.landesbioscience.com

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Figure 1. Effect of hormone treatment on gynoecium development. Light microscopy images of the apical extremity of Col (A–D) and spt-11 (E–H) gynoecia dissected from stage 12 flowers. The early buds were treated as indicated and the flowers were left to develop for 7 days. Bar = 200 μm (inset: whole gynoecium, bar = 500 μm).

directly.11 They however can form heterodimers with true PIFs, and modulate their function.15,16 SPT belongs to this later PIL category.11 SPT has been shown, together with PIF1, to control seed germination in response to both cold and light treatment.17,18 One mechanism through which SPT and PIF1 act is by regulating gibberellic acid (GA) biosynthetic genes in the developing seed.14,17-19 GA is a phytohormone triggering cell expansion, and is required for seed germination, as well as growth at many stages throughout the plant life. Of particular interest, GA is necessary for the development of the fruit post-fertilization. In this instance, it was recently shown that auxin promotes GA metabolism in fertilized ovules, and that constitutive GA signaling is sufficient to trigger parthenocarpy, independently of the fertilization event.20 SPT is also involved in seedling development: while spt mutants present large cotyledons, an overexpression of SPT leads to the development of a long hypocotyl and very small cotyledons when grown in red light, resembling a phytochrome B (phyB)-null mutant.17 Additionally, SPT also controls leaf size in a similar manner, especially under colder conditions.21,22 The possibility of a role for SPT in phytochrome signaling, as well as its dramatic action in gynoecium development, led

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us to investigate whether phyB could influence SPT-dependent gynoecium development. Results In spt monogenic mutants, the gynoecium develops abnormally, with unfused carpels at the upper-most side and reduced stigmatic papillae. However, when polar auxin transport is inhibited by NPA treatment, the spt gynoecium presents a morphology similar to an untreated wt gynoecium 2,6 (Fig. 1). We have previously shown that SPT regulates GA biosynthesis in the seed,17 and it has been demonstrated that, post-fertilization, an auxin signal is able to trigger GA production in the developing fruit.20 We therefore set out to determine whether the spt gynoecium phenotype could be affected or rescued by GA treatment. Figure 1 shows that GA treatment of a wt gynoecium does not affect its formation (Fig. 1C), but is unable to rescue spt-11 gynoecium development (Fig. 1G). However, GA treatment does not prevent spt-11 phenotypic rescue by NPA treatment (Fig. 1H). This suggests that, when controlling gynoecium development, SPT is not targeting GA biosynthesis. Since SPT belongs to the same clade as the light regulated PIF proteins, we next decided to test whether SPT function during gynoecium formation was phytochrome-dependent. Null phyB


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Figure 2. phyB mutation complements the spt gynoecium, silique and seed phenotypes. (A) Light microscopy images of the apical extremity of Ler, spt-2, phyB-1, spt-2phyB-1, Col, spt-11, phyB-9 and spt-11phyB-9 gynoecia dissected from stage 11 flowers. Bar = 100 μm. (inset: whole gynoecium, bar = 200 μm). (B) Length of dry siliques produced by the same plants grown in long days at 22°C (n = 20). (C) Seed area measured from seeds harvested from the siliques measured in (B). Error bars represent standard error (n = 200).

mutant fruits develop normally, both pre- and post-fertilization (Fig. 2A–C), the final silique’s size being only slightly longer in a phyB-1 mutant (Ler ecotype), but not in a phyB-9 mutant (Col ecotype) (Fig. 2B). Both the Ler spt-2 and the Col null allele spt-11 present the previously described gynoecium development defect6,21 (Fig. 2A). However, in a phyB null background, this phenotype is fully rescued (Fig. 2A). Additionally, the phyB mutation rescues the spt short silique phenotype (Fig. 2B) as well as spt larger seed size (Fig. 2C). This clearly demonstrates that, for gynoecium and fruit development, the function of SPT is phyB-dependent. SPT function has been shown to be involved at different stages of the gynoecium development: indeed, on one hand, SPT

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promotes carpel development early during flower formation,6 and on the other hand SPT is required for normal tract formation and apical fusion during gynoecium development.5-7 We therefore set out to observe the phyB-dependence of the spt gynoecium phenotype through a number of developmental stages (Fig. 3). While both Col gynoecium and phyB-9 gynoecium apices are fused throughout development, spt-11 gynoecium presents a lack of carpel fusion at the apical pole as early as we could observe (stage 8). Interestingly, the spt-11 phyB-9 double mutant is very similar to a spt-11 mutant at these early developmental stages. Fusion of the carpels and rescue of the spt phenotype only occurred by stage 11 of gynoecium development. This suggests that the phyB


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mutation is only able to rescue SPT function at a later developmental stage, when the basal-apical axis of the gynoecium is being defined. Discussion Phytochrome’s paramount role in controlling numerous aspects of the plant life, from germination and early development to plant architecture and flowering, have been studied at length in the past years.23-25 However, this is the first report of a role for phyB in flower development. A large body of evidence has shown that, under the regulation of the transcription factor STYLISH (STY), auxin forms a morphogenetic gradient within the gynoecium, which is believed to be interpreted by ETT, SPT and HECATE (HEC), leading to the formation of a series of different structures along the apicalbasal axis of the gynoecium, in an auxin concentration-dependent manner.2,26-29 Meanwhile, numerous recent studies have investigated the relationship between phyB and phytohormones, with a large focus on auxin pathways. Indeed, phytochrome signaling has been shown to influence auxin pathways at different levels: auxin production, auxin distribution and sensitivity to auxin signals (reviewed in ref. 30). Phytochromes have recently been shown to directly control auxin production. Active phyB reduces auxin production via the concurrent activation of (SUPERROOT 2) SUR2, a suppressor of auxin biosynthesis and the inhibition of TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1), an enhancer of auxin biosynthesis.30-33 Conversely, it has been shown that reduced levels of phyB, triggered by shade conditions, set off the opposite response, with an elevation of IAA production.30,33 Strikingly, mutations in TAA1, together with its homolog TRYPTOPHAN AMINOTRANSFERASE RELATED 2 (TAR2) lead to the production of a gynoecium presenting an apicalized phenotype, with reduced or nonexistent valve area and an over-abundance of stigmatic tissue,34 showing that the integrity of the TAA1-dependent branch of auxin biosynthesis is essential for a correct patterning of the gynoecium. Interestingly, the expression pattern of both TAA1 and TAR2 in the gynoecium coincides with SPT expression, suggesting a causal link between SPT and auxin production, with SPT either directly responding to or being involved in auxin production.35 Here, we show that in a phyB-null mutant, the spt gynoecium phenotype is rescued. This suggest that, in a spt mutant, where the auxin gradient fails to either be set-up or interpreted, reducing phyB levels could result in a modification of auxin production, participating to local changes in auxin concentration throughout the gynoecium, and resulting in a rescue of the spt phenotype. Additionally, polar auxin transport and light signaling have been functionally linked in numerous studies, mainly looking at seedling development, shoot-root communication and the control of branching. Indeed, phytochrome mutants have reduced sensitivity to NPA-induced hypocotyl growth inhibition, suggesting that polar auxin transport requires functional phytochrome action.36 Similarly, phytochrome mutants show reduced shoot-root auxin transport,37 as well as reduced auxin-dependent

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branching.25 At the molecular level, phytochromes were shown to influence both the expression and the localization of a handful of auxin transporter involved in polar auxin transport.37-40 This means that the absence of phyB in a spt background could impair auxin transport through the gynoecium in a NPAmimicking way, resulting in the establishment of the auxin gradient needed for the correct specification of the gynoecium apex, and a rescue of the spt phenotype. Eventually, both light and auxin pathways share common targets and are highly integrated, especially during the shade avoidance response, where neighboring vegetation produce a far-red light rich environment, which depletes the active phyB pool (reviewed in refs. 30 and 41). There, phyB depletion leads to the accumulation of PIF family members, resulting in the induction of the expression of a number of transcription factors. These include the SPT homologs LONG HYPOCOTYL IN FAR-RED (HFR1), PIL1 and PIL2,42-44 as well as the more distant relatives PHYTOCHROME RAPIDLY REGULATED1 (PAR1) and PAR2.45,46 Interestingly, both HFR1 and PAR1/2 have been shown to suppress the transcription of a number of auxin signaling targets including members of the SAUR and the Aux/IAA family, suggesting that shade conditions lead to a de-repression of auxin signaling.43,45,46 Moreover, PIF4 was also shown to regulate auxin-mediated signaling pathways in response to high temperature.47 Additionally, members of the PIF/PIL family are known to regulate each other’s expression44 and have highly redundant functions.12,48 Taken together, these results offer the possibility that SPT could regulate auxin signaling by targeting shade-induced genes like HFR1 and PIL1. In this case, SPT function could therefore be supplemented in a phyB-null mutant by the action of members of the PIF family like PIF4 and PIF5 that are stabilized. In this context, it is also interesting to notice that SPT is able to heterodimerize with a wide range of bHLH transcription factors, showing interaction in yeast-2-hybrid experiments with HEC1/2/3, 28 as well as with PIF1 and PIF4 (Bou-Torrent and Martinez-Garcia, personal communication). This therefore offers the possibility that in the gynoecium, SPT and PIF4 could dimerize, leading to an increase in PIF4 stability, and an induction of shade related genes. Alternatively, as the spt phenotype could result from a decrease in auxin sensitivity, 2 the de-repression of auxin signaling when phyB levels are depleted could increase the general sensitivity for auxin, rescuing the gynoecium development in a spt mutant. In conclusion, we show here that the spt phenotype is rescued equally by NPA addition and by a phyB mutation: this is correlative evidence that phyB could be acting on auxin production, distribution or sensitivity within the gynoecium to promote the establishment of the basal-apical axis of the developing flower. This work demonstrates a role for phyB in the control of flower development, and shows a cooperative function for the PIF3homologue SPT and phyB in this developmental process. Future work, however, will be necessary to identify the exact point(s) of interaction between phyB and SPT signaling leading to the establishment of the auxin gradient within the gynoecium.


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Figure 3. phyB mutation does not complement the spt early phenotype. Time series of gynoecium development: Col, spt-11, phyB-9 and spt-11phyB-9 flowers from stage 8 to 16 were dissected and their gynoecium was observed by light microscopy. Bar = 100 μm.

Materials and Methods Lines and growth conditions. Both Landsberg erecta (Ler) and Columbia-0 (Col) accessions of Arabidopsis thaliana were used. The spt-2 and phyB-1 mutants (Ler alleles) as well as the spt11 and phyB-9 mutants (Col alleles) were described previously in references 6, 21 and 49. spt-2 phyB-1 and spt-11 phyB-9 were obtained by cross-pollination of their respective parents and were selected via PCR and sequencing methods. Plants were grown in a (2:1) soil-sand mixture under long days conditions (16:8) at 22°C under 100 μmol.m-2.s-1 of white light. Hormone treatments. All siliques, flowers and large buds were removed, and the inflorescences were dipped 2 days in a

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row in the indicated solution of hormone (NPA or GA 3) or water (mock) prepared in 0.01% silwet L-77 (Lehle seeds, VIS-02). Flowers were then observed 7 days later. Light microscopy and size measurements. Unstained plant material was dissected and viewed with a Leica MZ 16 F microscope. Silique length (n = 20) and seed area (n = 200) were measured using the ImageJ software (http://rsbweb.nih.gov/ij/). Gynoecium development stages were defined as published in references 1 and 50. Acknowledgments

This work was supported by the UK Biotechnology and Biological Sciences Research Council (B.B.S.R.C.) grants BBE0003631 to K.J.H. and BBE0005411 to I.A.G.


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