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First published online 12 September 2007
doi: 10.1242/dev.011510

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The HECATE genes regulate female reproductive tract development in Arabidopsis thaliana

Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA 92093, USA.

View larger version (51K): [in this window] [in a new window]   Fig. 1. Fruit tissue structure and the HECATE genes of Arabidopsis. (A,B) Colorized longitudinal (A) and transverse (B) sections showing internal fruit tissues of wild-type Arabidopsis. Stigma (Sg), yellow; style (Sy), turquoise; septum (S), pink; transmitting tract (TT), blue; ovules (O), brown; ovule funiculus (F), green. (C) Diagrams of HEC1 (At5g67060), HEC2 (At3g50330) and HEC3 (At5g09750) showing the location of the bHLH domain. Positions of the T-DNA (not shown to scale) insertions in HEC1 and HEC3 are represented by triangles. The region of HEC2 used for creating the HEC2-RNAi construct is bracketed. (D) Alignment of the bHLH domains (underlined) of the HEC, INDEHISCENT (IND) (Liljegren et al., 2004) and the distantly related SPATULA (SPT) (Heisler et al., 2001) proteins. The alignment includes a region upstream of the bHLH domain, where the HECs and IND also show conservation. The asterisk marks an alanine that replaces the conserved glutamate carried by most other Arabidopsis bHLH proteins.

View larger version (133K): [in this window] [in a new window]   Fig. 2. RNA in situ analysis of HEC expression during gynoecium development in Arabidopsis. (A-C) Transverse sections through stage 8 gynoecia. HEC1, HEC2 and HEC3 are expressed in the developing septum (arrowheads). The HEC1 signal seen in the anthers at this stage (double arrowhead) was not seen for HEC2 or HEC3 and was not duplicated by the HEC1 GUS reporter (see Fig. S1E in the supplementary material). (D-F) Transverse sections through early stage 12 gynoecia. HEC1, HEC2 and HEC3 expression becomes localized to the developing transmitting tract (arrowheads). HEC1 and HEC2 are expressed in ovules (small double arrowheads) and HEC3 shows expression in the ovule funiculus (arrow). (G,H) Longitudinal sections through stage 8 and stage 11 flowers. HEC1 (G) and HEC3 (H) expression is evident in the developing septum and transmitting tract (arrowheads) and stigma (arrows). (I,J) Longitudinal and transverse sections through late stage 12 gynoecia. HEC3 continues to be strongly expressed in the transmitting tract (large arrowheads) and ovule funiculus (small arrowheads), but not in the stigma (arrow). HEC1 and HEC2 expression can no longer be detected at this time (data not shown). Scale bars: 50 µm.

View larger version (90K): [in this window] [in a new window]   Fig. 3. Mutations in Arabidopsis HEC genes result in reduced fertility. (A) Wild-type Col-0 fruit; arrowhead points to shadow cast by developing seed. (B) hec1 fruit show no obvious abnormal phenotype. (C) hec3 fruit are shorter and exhibit reduced fertility; arrowhead points to empty space that should be occupied by seed. (D) hec1 hec3 fruit are variable in size but generally short, with significantly reduced fertility. (E) HEC3p:HEC3 rescues hec1 hec3. (F) HEC2-RNAi hec1 hec3 fruit are completely sterile. (G-J) Pollen tubes stained with Aniline Blue 24 hours post-pollination. In the wild type (G) and hec1 (H), the bulk of the pollen tubes have nearly reached the base of the ovary (arrowhead). In hec3 (I), pollen tubes are fewer in number and have not penetrated as far as in the wild type or hec1 (arrowhead). In hec1 hec3 (J) there is a significant reduction in pollen tubes entering the ovary, and penetration along the apical-basal axis is greatly diminished (arrowhead). Scale bars: 1 mm in A-F; 0.1 mm in G-J.

View larger version (174K): [in this window] [in a new window]   Fig. 4. Loss of transmitting tract and stigma development in hec mutants. (A-G) Transverse sections of stage 14 Arabidopsis ovaries stained with Alcian Blue to reveal the transmitting tract (arrowheads) and with Fast Red as a counterstain. In the wild-type (Col-0) gynoecium (A), the ECM of the transmitting tract stains bright blue in the center of the septum. The hec1 transmitting tract (B) is essentially equivalent to wild type, but the hec3 transmitting tract (C) is noticeably smaller. Typical examples of hec1 hec3 transmitting tracts (D,E) are severely reduced in size within narrowed septa. Typical examples of HEC2-RNAi hec1 hec3 gynoecia (F,G) exhibit no blue staining at the transmitting tract, and have either only a few cells at the septum fusion point (F, arrow) or an unfused septum (G, arrow). (H-J) Transverse sections of stage 14 styles stained with Alcian Blue and Fast Red. The transmitting tract (arrowhead) is reduced for hec3 in the stylar region (I) in comparison with wild type (H). hec1 hec3 has very little transmitting tract in the style (J). (K-O) Scanning electron micrographs of stigma and style regions of stage 14 gynoecia. The stigma of hec1 hec3 gynoecia (L) are significantly less well developed than those of wild-type Col-0 (K). HEC2-RNAi hec1 hec3 gynoecia (M,N) lack any stigmatic development and have longer styles than wild type. Some fruit displayed a defect in apical fusion (N, arrow) similar to that of spt-2 (O, arrow; Ler background). Scale bars: 50 µm in A-J; 100 µm in K-O.

View larger version (111K): [in this window] [in a new window]   Fig. 5. The Arabidopsis HECs interact with SPT in yeast and are negatively regulated by ETT. (A-C) The HECs are still expressed (arrowheads) in the septum in the spt-2 mutant. HEC1 expression (A) was analyzed by crossing the HEC1p:HEC1:GUS line into spt-2, and HEC2 (B) and HEC3 (C) expression was analyzed directly by in situ hybridization. (D-F) The HECs are ectopically expressed in abaxial cell layers of the gynoecium (arrowheads) in ett-7. HEC1 expression (D) was analyzed by crossing the HEC1p:HEC1:GUS line into spt-2, and HEC2 (E) and HEC3 (F) expression was analyzed directly by in situ hybridization. Expression could also still be seen in the septum (arrows) for HEC1 (D) and HEC2 (E). (G) The HEC proteins do not homodimerize or heterodimerize with each other in a yeast two-hybrid system. Full-length HEC1 and HEC2 were used in both the bait and prey constructs. Full-length HEC3 was used as prey. However, an N-terminal deletion of HEC3 was used as the bait, as the full-length HEC3 bait construct activated the yeast reporter genes on its own (data not shown). The protein cruciferin was used as a negative control. Results were confirmed with the HIS3 reporter. (H) The HEC proteins form heterodimers with SPT in a yeast two-hybrid system. An N-terminal deletion of SPT was used as the prey construct (see Materials and methods). The protein cruciferin was used as a negative control. Results were confirmed with the HIS3 reporter. Scale bars: 50 µm.

View larger version (85K): [in this window] [in a new window]   Fig. 6. Overexpression of HEC genes in Arabidopsis. (A) Wild-type flower. (B) 35S::HEC1 flower. Ectopic stigmatic tissue on anthers and sepals (arrowhead; inset shows an enlarged view of the region indicated by the lower arrowhead). (C) 35S::HEC2 flower. Ectopic stigmatic tissue on sepals (arrowheads; inset shows an enlarged view of the region indicated by the left arrowhead). (D) 35S::HEC3 flower. Most floral organs have carpelloid tissue (arrowhead). (E) Wild-type fruit (apical). (F) Wild-type fruit (basal). Gynophore is bracketed. (G) 35S::HEC3 fruit. Note the enlarged stigma, reduced ovary and elongated gynophore (bracketed). (H) 35S::HEC1 fruit. Note the enlarged stigma, reduced ovary and elongated gynophore (bracketed). (I) 35S::HEC1 flower. The gynoecium has an enlarged stigma, a reduced ovary and an elongated gynophore. (J) 35S::HEC1 inflorescence. Primary shoot terminates in a stigma. Axillary shoots form carpelloid structures with overproliferation of stigmatic tissue. (K) 35S::HEC3 inflorescence. Flowers transformed into carpelloid stalks capped by stigmas. (L) 35S::HEC1 inflorescence. No floral development. Scale bars: 400 µm.