Auxin Response Factors promote organogenesis by chromatin-mediated repression of the pluripotency gene SHOOTMERISTEMLESS

Abstract

Specification of new organs from transit amplifying cells is critical for higher eukaryote development. In plants, a central stem cell pool maintained by the pluripotency factor SHOOTMERISTEMLESS (STM), is surrounded by transit amplifying cells competent to respond to auxin hormone maxima by giving rise to new organs. Auxin triggers flower initiation through Auxin Response Factor (ARF) MONOPTEROS (MP) and recruitment of chromatin remodelers to activate genes promoting floral fate. The contribution of gene repression to reproductive primordium initiation is poorly understood. Here we show that downregulation of the STM pluripotency gene promotes initiation of flowers and uncover the mechanism for STM silencing. The ARFs ETTIN (ETT) and ARF4 promote organogenesis at the reproductive shoot apex in parallel with MP via histone-deacetylation mediated transcriptional silencing of STM. ETT and ARF4 directly repress STM, while MP acts indirectly, through its target FILAMENTOUS FLOWER (FIL). Our data suggest that - as in animals- downregulation of the pluripotency program is important for organogenesis in plants.

Fig. 1

ETT and ARF4 contribute to initiation of reproductive primordia. a, b Flower initiation defect of the hypomorph mp-S319 mutant compared to wild type (WT) and mp-S319 ett arf4. a Phenotype; Scale bar = 1 mm. b Quantification of flowers formed; ***P-value = 0.0001, one-tailed Mann–Whitney U test. n = 11 (WT) n = 11 (mp-S319) n = 9 (mp-S319 ett arf4). Box and whisker plot: Lower vertical bar = sample minimum. Lower box = lower quartile. Black Line = median. Upper box = upper quartile. Upper vertical bar = sample maximum. c Expression of the pluripotency genes SHOOTMERISTEMLESS (STM; top) and CLAVATA3 (CLV3; bottom) in inflorescence apices assayed by in situ hybridization. The black arrow points to the region where STM expression is downregulated in the incipient primordium. Scale bar = 50 µm. d Relative expression of the STM class I KNOX gene and the STM target IPT7 in trimmed inflorescence apices normalized over that of the UBQ10 housekeeping gene. Shown are mean ± SEM of three experiments. Source data are provided as a Source Data file

Fig. 2

ETT and ARF4 act in a pathway parallel to MP. a Phenotypes of 10-day-old wild type (WT), ett arf4, mp-12 null mutant and mp-12 ett arf4 seedlings. Scale bar = 1 mm. Two phenotypic classes of mp-12 seedlings are indicated (I, II). b Top: Close-up view of mp-12 ett arf4 seedlings shown in a. Scale bar = 500 um. Two phenotypic classes of mp-12 ett arf4 seedlings are indicated (III, IV). Bottom: Meristem of a class IV mp-12 ett arf4 seedling viewed from above (Scale bar = 200 μm). c Number of mp-12 or mp-12 ett arf4 seedlings belonging to the phenotypic classes (I–IV) shown in a and b. The error bars are proportional to the standard error of the pooled percentage computed using binomial distribution. ***P-value = 0.00001 for both mp-12 ett arf4 III and IV class phenotype relative to mp-12 class III and IV mutants; two-tailed Mann–Whitney U test. n = 114 and 51 for mp-12 and mp-12 ett arf4. Source data are provided as a Source Data file. d Scanning electron microscopy images of representative 10-day-old WT, mp-12 and class III mp-12 ett arf4 seedlings. Scale bar = 200 μm. e STM expression patterns by in situ hybridization in 7-day-old seedlings. Scale bar = 50 μm. f Relative expression of STM and the STM target IPT7 in 10-day-old seedlings normalized over that of UBQ10. Shown are mean ± SEM of three experiments. Source data are provided as a Source Data file. g Expression of pSTM:GUS in matureWT and mp-12 ett arf4 embryos. Scale bar = 50 µm

Fig. 3

STM downregulation is important for flower initiation. a, b Primordium initiation defect of mp-S319 compared to mp-S319 without (Mock) and with conditional increase in STM activity upon activation of p35S:STM-GR with the synthetic steroid dexamethasone (Dex). a Phenotype; white arrow points to developing flowers. Scale bar = 1 mm. b Quantification of flowers initiated. Box and whisker plot: Lower vertical bar = sample minimum. Lower box = lower quartile. Black Line = median. Upper box = upper quartile. Upper vertical bar = sample maximum. NS P-value = 0.11, *P-value = 0.024 one-tailed Mann–Whitney U test. n = 15 and 11 (WT mock and dex) n = 8 and 5 (mp-S319 mock and dex). Source data are provided as a Source Data file. c, d Primordium initiation defect of mp-S319 ett arf4 compared to mp-S319 ett arf4 plants in which STM expression is knocked down using an artificial microRNA, amiRSTM. c Phenotype; white arrow points to developing flowers. Scale bar = 1 mm. d Quantification of flowers initiated. Box and whisker plot: Lower vertical bar = sample minimum. Lower box = lower quartile. Black Line = median. Upper box = upper quartile. Upper vertical bar = sample maximum. NS P-value = 0.99; ***P-value = 0.00001 one-tailed Mann–Whitney U test. n = 11(arf4), n = 13 (arf4 p35S:amiRSTM), n = 8 (mp-S319 ett arf4), n = 15 (mp-S319 ett arf4 p35S:amiRSTM). arf4 is indistinguishable from the wild type. Source data are provided as a Source Data file

Fig. 4

ETT binds the STM locus in vivo. a Phylogenetic shadowing of the STM locus. Top: five conserved domains (A–E) tested. Below: ARF binding sites and locus architecture. Blue ovals, conserved Auxin Response Factor core elements (TGTC); red triangles, ETT binding sites (TGTCAT); black boxes, exons; grey boxes, introns; black line, upstream intergenic region. Bottom: mVista plot. Grey shaded areas correspond to conserved regions tested. b, c Chromatin immunoprecipitation (ChIP) to test ETT (b) or MP (c) binding to conserved regions of the STM locus. TA3 served as negative control. An MP-bound region of the FIL locus served as positive control for MP ChIP (c) and as negative control for ETT ChIP (b). ChIP was performed under identical conditions in the wild type (WT). Shown are mean ± SEM of three experiments. Source data are provided as a Source Data file

Fig. 5

The MP target FIL promotes flower initiation together with ETT/ARF4. a Flower initiation phenotypes of wild type (WT), ett arf4, fil-8, young and old fil-8 ett arf4 and auxin transport inhibitor (NPA) treated fil ett arf4 plants. NPA (500 nM), was applied from 11 days of age until bolting. Scale bar = 1 mm. b Quantification of lateral organs formed in the absence and presence of NPA. Box and whisker plot: Lower vertical bar = sample minimum. Lower box = lower quartile. Black Line = median. Upper box = upper quartile. Upper vertical bar = sample maximum. P-values ( ± NPA): NS P = 0.11 (WT), NS P = 0.73 (fil/ + ett arf4) and *P = 0.006 (fil ett arf4), one-tailed Mann–Whitney U test. n = 7 (WT mock) n = 5 (WT NPA) n = 4 (fil/ + ett arf4 mock), n = 4 (fil/ + ett arf4 NPA) n = 5 (fil ett arf4 Mock) n = 5 (fil ett arf4 NPA). See supplementary Fig. 7a for images of mock and NPA treated control plants. Source data are provided as a Source Data file. c Relative expression of STM, IPT7 and FIL normalized over that of UBQ10 in the genotypes indicated. Shown are mean ± SEM of one representative of three experiments. Source data are provided as a Source Data file. d Chromatin immunoprecipitation (ChIP) to test FIL binding to conserved regions of the STM locus. Top: STM locus; green triangles, conserved FIL binding sites (AATNATAA). TA3 served as negative control. ChIP was performed under identical conditions in the WT. Shown are mean ± SEM of three experiments. Source data are provided as a Source Data file

Fig. 6

Leaf phenotypes of plants with reduced FIL, ETT and ARF4 activity. a Representative images of leaf phenotypes of wild type (WT), fil-8, ett arf4, fil/+ ett arf4 and fil-8 ett arf4. I-III indicate the different phenotypic classes observed. I simple leaves; II lobed leaves; III divided leaves and leaves bearing ectopic meristems (inset). Scale bars: 1 cm and 1 mm (inset). b Quantification of number of leaves in each phenotype class shown in a. The error bars are proportional to the standard error of the pooled percentage computed using binomial distribution *** P < 0.00001, two-tailed Mann–Whitney U test relative to ett arf4. n = 87 and 81 for WT and fil-8. n = 104, 148 and 64 for ett arf4, fil/+ ett arf4 and fil ett arf4. Source data are provided as a Source Data file. c Relative expression of STM and IPT7 in fully expanded adult leaves normalized over that of UBQ10. Shown are mean ± SEM of three experiments. Source data are provided as a Source Data file

Fig. 7

STM repression by FIL and ETT/ARF4 relies on reduced histone H3 acetylation. a, b Flower initiation phenotypes of wild type (WT), hda19, the hypomorph mp-S319 mutant and mp-S319 hda19. a Phenotype; Scale bar = 1 mm. b Quantification of flowers formed; ***P = 0.00024, one-tailed Mann–Whitney U test. n = 11 (WT) n = 11 (mp-S319) n = 9 (mp-S319 hda19). c Relative expression of STM and IPT7 in trimmed inflorescences normalized over that of UBQ10. Shown are mean ± SEM of three experiments. d Chromatin immunoprecipitation (ChIP) to test HDA19 binding to conserved regions of the STM locus in inflorescences. TA3 served as negative control. ChIP was performed under identical conditions in the WT. Shown are mean ± SEM of three experiments. e Anti-histone 3 lysine 27 acetylation (H3K27ac) compared to H3 ChIP at the STM locus in expanded leaves of WT, fil/+ ett arf4 and fil ett arf4. TA3 served as negative control. ChIP was performed under identical conditions in the WT. Shown are mean ± SEM of three experiments. b–e Source data are provided as a Source Data file. f Model for the repression of STM in incipient reproductive primordia. See text for details