FILAMENTOUS FLOWER controls lateral organ development by acting as both an activator and a repressor

Abstract

Background: The YABBY (YAB) family of transcription factors participate in a diverse range of processes that include leaf and floral patterning, organ growth, and the control of shoot apical meristem organisation and activity. How these disparate functions are regulated is not clear, but based on interactions with the LEUNIG-class of co-repressors, it has been proposed that YABs act as transcriptional repressors. In the light of recent work showing that DNA-binding proteins associated with the yeast co-repressor TUP1 can also function as activators, we have examined the transcriptional activity of the YABs.

Results: Of the four Arabidopsis YABs tested in yeast, only FILAMENTOUS FLOWER (FIL) activated reporter gene expression. Similar analysis with Antirrhinum YABs identified the FIL ortholog GRAMINIFOLIA as an activator. Plant-based transactivation assays not only confirmed the potential of FIL to activate transcription, but also extended this property to the FIL paralog YABBY3 (YAB3). Subsequent transcriptomic analysis of lines expressing a steroid-inducible FIL protein revealed groups of genes that responded either positively or negatively to YAB induction. Included in the positively regulated group of genes were the polarity regulators KANADI1 (KAN1), AUXIN RESPONSE FACTOR 4 (ARF4) and ASYMMETRIC LEAVES1 (AS1). We also show that modifying FIL to function as an obligate repressor causes strong yab loss-of-function phenotypes.

Conclusions: Collectively these data show that FIL functions as a transcriptional activator in plants and that this activity is involved in leaf patterning. Interestingly, our study also supports the idea that FIL can act as a repressor, as transcriptomic analysis identified negatively regulated FIL-response genes. To reconcile these observations, we propose that YABs are bifunctional transcription factors that participate in both positive and negative regulation. These findings fit a model of leaf development in which adaxial/abaxial patterning is maintained by a regulatory network consisting of positive feedback loops.

Figure 1

YAB transcriptional activity in yeast. Three independently transformed yeast strains expressing Arabidopsis YABs (FIL, YAB2, YAB3, YAB5) or Antirrhinum YABs (GRAM, AmYAB2, PROL) fused to the GAL4 DNA-binding domain (BD) were assayed for MEL1 reporter activity using an X-α-gal plate assay. Colour change after 4 h (+++), after 16 h (++), or no colour change after 24 h (−) are shown.

Figure 2

Transcriptional activities of vegetatively expressed YAB proteins. (A) Outline of constructs used for transactivation assays. AD, GAL4 activation domain; BD, GAL4-DNA binding domain; YAB, vegetatively expressed YABs (FIL, YAB2, YAB3, YAB5); SRDX, repressive domain (see text for details); UAS, BD binding site; LUC, Firefly Luciferase; rLUC, Renilla Luciferase. (B) YAB transcriptional activation assays with Gal4 BD and BD-AD used as negative and positive control respectively. rLuc was used as an internal control to determine the relative bioluminescence for each sample (ratio Luc/rLuc). Numbers in the shaded boxes indicate fold activation as calculated by dividing total Luc activity of samples by the baseline values arising from the 35S_pro::BD construct. Asterisks indicate significant differences (Student's t-test; p < 0.05) and error bars indicate SEM.

Figure 3

Vegetative phenotypes associated with steroid-induced constitutive activation of FIL. (A,B) A 35S_pro::FIL:GR plant grown on media without DEX (A) or with DEX (B). Inset shows close-up view of an epinastic leaf. (C) 35S_pro::FIL plants displaying an intermediate phenotype. (D-F) Scanning electron micrographs showing the adaxial (D, F) or abaxial (E) surface of mature leaves of 35S_pro::FIL:GR plants grown on media without DEX (D,E) or with DEX (F). (G,H) Histochemical staining for YAB3:GUS activity in yab3-2/35S_pro::FIL:GR plants grown in the absence of DEX (G) or with DEX (H). Arrows indicate prolonged GUS activity in the first true leaves to emerge following germination. (I) Section through a histochemically stained leaf shown in (H) viewed by dark field optics. Arrowheads indicate adaxial accumulation of GUS activity. (J-L) Twenty one day-old fil yab3 yab5 (abbreviated as fy3y5) triple mutants (J) and fy3y5/35S_pro::FIL:GR (K,L) lines grown on media with DEX (J, L) or without DEX (K) under short days. Scale bar: 1 mm in (A-C,G,H, J-L), 100 μm in (D-F, I).

Figure 4

FIL-response genes that areimmediate targets of FIL. (A) Fold change in expression of FIL-response genes in ten-day-old 35S_pro::FIL:GR seedlings following a 4 h DEX or DEX/CHX treatment. Brackets indicate genes that display significant transcriptional responses to both treatments and hence mark direct targets of FIL. (B) Response of selected positively regulated FIL-response genes in 10 day-old FIL_pro::FIL:GR seedlings following a 4 h DEX treatment. (C,D) Induction of abaxial polarity regulators in 10 day-old 35S_pro::FIL:GR (C) or 35S_pro::YAB3:GR (D) seedlings following a 4 h DEX treatment. (E) Expression of KAN1, ARF4 and AS1 in fil single and yab double, triple and quadruple mutants. Expression in a minimum of three biological replicates was determined using quantitative real-time RT-PCR and normalized first to a housekeeping gene and then to mock treatment controls. Asterisks mark significant differences determined by a Student’s t-test (one asterisk, 0.01<p<0.05; two asterisks, 0.001<p<0.005) and error bars are SEM. The grey line marks the expression level expected if there is no response to treatment.

Figure 5

Phenotypes induced by adominant negative FIL:SRDX construct. (A,B) 35S_pro^I>>FIL plants grown on media without DEX (A) or with DEX (B). (C,D) 35S_pro^I>>FIL:SRDX plants grown on media without DEX (C) or with DEX (D). (E) A fil yab3/FIL_pro::FIL plant displaying full complementation. Inset: fil yab3 double mutant plant. (F) fil yab3/FIL_pro::FIL:SRDX seedlings showing narrow cotyledons that are sometimes bifurcated (asterisk). Cotyledons of fil yab3 (G) and fil yab3 yab5 mutant seedlings (H). (I) fil yab3/FIL_pro::FIL:SRDX plant with needle-like leaves. (J) fil yab3 yab5 triple mutant plant with narrow and needle-like leaves. (K) Histochemical staining for YAB3:GUS activity in a fil yab3/FIL_pro::FIL:SRDX plant. YAB3 promoter activity is detected throughout young radial leaves. Inset: fil yab3/FIL_pro::FIL stained for GUS activity. (L) A fil/+ yab3/FIL_pro::FIL:SRDX plant with a fil yab3 mutant leaf phenotype. (M-R) Scanning electron micrograph showing the abaxial epidermis of wildtype (M), fil yab3 (N), fil yab3 FIL_pro::FIL (O) and fil/+ yab3/FIL_pro::FIL:SRDX (P) leaves. Note that the larger cell morphology in (N,P) is due to leaf adaxialisation. (Q,R) SEM showing epidermal cell morphology of fil yab3 yab5 needle leaves (Q) and those of fil yab3/FIL_pro::FIL:SRDX plants (R). Scale bars are 5 mm in (E,L) and inset in (A); 2 mm for (A-D, F-H); 1 mm for (I,J); 200 μm for (K) and inset in (K); 100 μm in (M-R).

Figure 6

Model for YAB functionduring the early stagesof leaf development. Adaxial-abaxial patterning is established during the early stages of leaf development and is closely associated with the onset of FIL/YAB3 expression. FIL/YAB3 maintain KAN1 and ARF4 expression through direct positive regulation, which in turn establishes a positive feedback loop. As well as acting as positive regulators, YABs associate with transcriptional co-repressors, forming a repressive complex that potentially targets adaxial-promoting factors. It is likely that this regulatory network is confined to the early stages of leaf development, as at later stages FIL/YAB3 expression is present at high levels in the margins of the growing lamina, but KAN1 expression is not readily detectable.