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. 2015 Oct 1;142(19):3343-50.
doi: 10.1242/dev.120444. Epub 2015 Aug 20.

Arabidopsis HECATE genes function in phytohormone control during gynoecium development

Christoph Schuster  1 , Christophe Gaillochet  2 , Jan U Lohmann  1
Affiliations

Arabidopsis HECATE genes function in phytohormone control during gynoecium development

Christoph Schuster et al. Development. .

Abstract

The fruit, which develops from the fertilised gynoecium formed in the innermost whorl of the flower, is the reproductive organ and one of the most complex structures of an angiosperm plant. Phytohormones play important roles during flower and fruit patterning, morphogenesis and growth, and there is emerging evidence for a cross-talk between different classes of plant hormones throughout these processes. Here, we show that the bHLH transcription factors HECATE 1 (HEC1), HEC2 and HEC3, which have previously been identified as essential components of transmitting tract formation, affect both auxin and cytokinin responses during reproductive tissue development. We find that HEC1 interacts with SPATULA (SPT) to control carpel fusion and that both transcription factors restrict sensitivity to cytokinin in the gynoecium. In addition, HEC1 is tightly integrated into the auxin-signalling network at the levels of biosynthesis, transport and transcriptional response. Based on this data, we propose that HEC1 acts as a local modulator of auxin and cytokinin responses to control gynoecium development in Arabidopsis.

Keywords: Arabidopsis; Auxin; Cytokinin; Gynoecium development; HECATE; Phytohormones; SPATULA.

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Figures

Fig. 1.
Fig. 1.
HEC1 and SPT genetically interact to control gynoecium development. (A,B) Longitudinal (A) and cross section view (B) of a wild-type Arabidopsis gynoecium. Stigma (Sm), style (Sy), ovary (Ov), valve (V), replum (R), septum (Sp), transmitting tract (TT), ovules (O), lateral (L) and medial (M) regions are shown. (C) Stage 17 fruits of hec1,2,3, spt and hec1,2,3 spt mutant plants. The apical part of spt fruits is unfused, and this phenotype is dramatically enhanced in hec1,2,3 spt quadruple mutant (arrowheads). Scale bars: 200 µm in A; 1 mm in C. See also supplementary material Fig. S1.
Fig. 2.
Fig. 2.
Development of the hec1,2,3, spt and hec1,2,3 spt mutant gynoecia. (A-D) Stage 9-10 gynoecia of wild type (A), hec1,2,3 (B), spt (C) and hec1,2,3 spt (D) mutant plants. Whereas the gynoecial tube in the medial regions of wild-type plants (A) is extending, hec1,2,3 (B) and spt (C) mutant gynoecia show a retarded growth in this region (arrowheads). This phenotype is enhanced in hec1,2,3 spt mutants (D). (E-H) Stage 11 gynoecia of wild type (E), hec1,2,3 (F), spt (G) and hec1,2,3 spt (H) mutant plants. Stigmatic papillae (arrow) appear at the top of the wild-type gynoecium (E), but are absent in hec1,2,3 (F). Carpel fusion defects of the hec1,2,3 spt quadruple mutant become more prominent (H). (I-P) Stage 12 (I-L) and stage 13 (M-P) gynoecia of wild type (I,M), hec1,2,3 (J,N), spt (K,O) and hec1,2,3 spt (L,P). Note that developing ovules are visible externally in the quadruple mutant (L,P). Scale bars: 50 µm in A-H; 100 µm in I-L; 200 µm in M-P.
Fig. 3.
Fig. 3.
Loss of HEC function leads to impaired auxin signalling. (A-D) Wild-type (A), hec1,2,3 (B) and p16:iaaL (C) plants. Overexpressing the bacterial iaaL gene mimics the hec1,2,3 fruit phenotype. The overexpression phenotype correlates with the expression level of the iaaL transgene; plants with strong iaaL expression show distinct fruit phenotypes, whereas weak transgene expression does not cause any phenotypic alterations (D). (E) mRNA expression levels of YUC4 in dissected inflorescences (Infl) and gynoecia at stage 10-11 and stage 12 of wild type and hec1,2,3. **P<0.01. Error bars: s.d. of two (D) or three (E) biological replicates. (F-K) pDR5:3xYFP-NLS expression in the developing gynoecium of wild type (F-H) and hec1,2,3 mutants (I-K) at stage 8-9 (F,I), stage 9-10 (G,J), and stage 10 (H,K). In wild type, four regions of local auxin response are connected to a ring-like structure at the onset of stage 10 (F-H, insets), but hec1,2,3 mutants fail to establish this radial symmetry (I-K). l: lateral regions; m: medial regions. Arrows in K indicate retarded growth in the medial region. F-K, n≥9. Scale bars: 0.5 cm (A-C) and 20 µm (F-K). See also supplementary material Figs S2 and S3.
Fig. 4.
Fig. 4.
HEC1 controls PIN expression. (A-B) mRNA expression levels of PIN1 and PIN3 in inflorescences of pAlcA:GUS and pAlcA:HEC1 plants after ethanol induction (A) or ethanol induction and auxin (IAA) treatment (B) measured by qRT-PCR. (C) Expression of PIN1 and PIN3 in gynoecia of wild type and hec1,2,3 at multiple developmental stages measured by qRT-PCR. (D-F) pPIN1:PIN1-GFP activity in stage 10 fruits of wild type (D) and p35S:HEC1 (E). In contrast to wild type (D and F; n=23), p35S:HEC1 show ubiquitous PIN1-GFP expression (E and F; n=27). (G-I) Reduction of pPIN1:PIN1-GFP expression at stage 9-10 in the lateral part (l) of hec1,2,3 gynoecia (H and I, n=9 plants with 3 gynoecia imaged) compared with wild type (G and I, same sample size as hec1,2,3). Image analysis revealed a significant difference in the lateral:medial (l/m) PIN1-GFP intensity ratio (***P=3.5×10−6, GFP signal threshold=4σ). (J) HEC1 mRNA expression in wild type stage 8. (K,L) ChIP experiment against PIN1 (K) and PIN3 (L) using a stable p35S:HEC1-GFP line. HSF1 served as negative control. (M) HEC1 mRNA expression in mock and IAA-treated wild-type inflorescences. Error bars: s.d. of three (C,K-M) or four (A,B) biological replicates. *P<0.05; **P<0.01. Scale bars: 50 µm (D,E) and 20 µm (G,H,J). See also supplementary material Fig. S4.
Fig. 5.
Fig. 5.
HEC1-mediated stem cell over-proliferation is independent of auxin transport and concentration. (A-D) Transgenic plants expressing GUS (A), iaaM (B), iaaL (C) or HEC1 (D) under the control of the CLV3 promoter in a pin1 mutant background. All pCLV3:HEC1-pin1 T1 lines showed enlarged meristems, while pCLV3:GUS-pin1 controls, pCLV3:iaaM-pin1 and pCLV3:iaaL-pin1 T1 lines did not (E). Scale bars: 1 mm. See also supplementary material Fig. S5.
Fig. 6.
Fig. 6.
hec and spt mutants are hypersensitive to cytokinin. (A-H) Scanning electron microscopy of wild-type (A,E), spt (B,F), hec1,2 (C,G) and hec1,2,3 (D,H) fruits after mock (A-D) or cytokinin (50 µM BA) (E-H) treatment. Cytokinin treatment of spt, hec1,2 and hec1,2,3 mutants lead to apically unfused fruits showing ectopic tissue proliferation (F-H), whereas fruits of wild-type plants do not display any phenotypic alterations after treatment compared with mock controls (A,E). SEM images show stage 17b fruits, except panel B (stage 15). (I-L) hec1,2,3 mutant gynoecia stage 11-12 (I,J) and stage 13 (K,L) after mock (I,K) and cytokinin (J,L) treatment. The arrowhead in J indicates the extensions at the top of the gynoecium. Scale bars: 200 µm (A-H) and 100 µm (I-L). See also supplementary material Fig. S6.
Fig. 7.
Fig. 7.
Hypothetical model of HEC gene function during gynoecium development. HEC1 interacts with SPT to control carpel fusion, and both transcription factors buffer auxin and cytokinin signals during gynoecium development. This might involve type-A ARRs, which antagonise cytokinin function. HEC1 stimulates auxin biosynthesis and directly activates the expression of PIN1 and PIN3 auxin efflux transporters and thus ultimately regulates auxin distribution during early stages of gynoecium development. Interestingly, the SPT-IND complex binds to the promoter of the PID gene that modulates PIN polarisation. This highlights how combinatorial effects of related bHLH transcription factors regulate distinct components of the auxin signalling machinery. Finally, HEC1 itself is tightly integrated into the auxin signalling network, and its spatial expression seems to be partly controlled by auxin-dependent activation and ETT mediated repression. Cross-talk between auxin and cytokinin pathways is an important feature of shoot meristem control and might also play a role in the developing gynoecium and fruit.
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