Auxin production in the endosperm drives seed coat development in Arabidopsis
- Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, Sweden
Figures
Figure 1 with 2 supplements
Fertilization activates auxin and GA signaling in the seed coat.
(A–C) DR5v2::VENUS expression before (A) and one and three days after pollination (B–C). (D–E) R2D2 auxin sensor before (D) and 16 hr after pollination (HAP) (E). (F–H) RGA::GFP:RGA reporter before …
Figure 1—figure supplement 1
Effect of GA treatments on GA and auxin reporter lines.
(A) The GFP signal derived from RGA::GFP:RGA is depleted following application of GA3. (B–C) Activity of the auxin reporters DR5v2 (B) and R2D2 (C) remains unchanged after GA3 treatment. See also Fig…
Figure 1—figure supplement 2
Ovule size and cell measurements.
(A) Relative area of 1 DAP WT seeds compared to 2 DAE ovules. (B) Integument cell number did not change following fertilization, indicating that seed coat growth is driven by cell expansion rather …
Figure 2 with 1 supplement
GA and auxin are sufficient to drive seed coat development.
(A–B) Scoring of vanillin staining of ovules after 2,4-D, GA3 and mock treatments, as categorized in (B). Numbers on top indicate total ovules counted. Bar indicates 20 µm. (C) Ovule area three days …
Figure 2—figure supplement 1
Activity of the BAN promoter in unfertilized ovules.
(A–B) BAN::GFP reporter is expressed in the inner integument cells of unfertilized ovules. No expression is observed in cells of the female gametophyte. Bars indicate 20 µm.
Figure 3 with 1 supplement
Auxin is necessary for seed coat development.
(A) Opened silique of an axr1 axl-1/+ mutant showing aborting white seeds (red arrows) that undergo full seed coat development. (B) Opened silique of a wei8 tar1 tar2-1/+ mutant showing seeds that …
Figure 3—figure supplement 1
Seed size is affected in auxin mutants.
Area of 2 DAE ovules (A), 3 DAP (B) and 5 DAP (C) seeds was determined for WT, axr1/+ axl-1 auxin signalling mutant and wei8 tar1 tar2-1/+ and wei8 tar1 tar2-2/+ auxin biosynthesis mutants. Sample …
Figure 4 with 1 supplement
Auxin production in the central cell triggers seed coat development.
(A–B) Ovule development at 5 DAE: (A) WT and (B) DD65::TAA1; DD65::YUC6. (C) Vanillin-stained ovules of DD65::TAA1; DD65::YUC6 at 5 DAE. Bars indicate 20 µm. (D) Scoring of vanillin staining in WT …
Figure 4—figure supplement 1
Auxin reporters in the DD65::TAA1 DD65::YUC6 transgenic lines.
(A–D) DR5v2 (A–B) and R2D2 (C–D) activity in WT (A,C) and auxin over-producing line (B,D). All samples taken at 5 DAE. Bars indicate 20 µm.
Figure 5 with 1 supplement
agl62 seeds fail to develop a seed coat.
(A–C) Endosperm cellularization as determined by Feulgen staining in agl62 (A), agl62 hal (B) and hal (C) seeds at 3 DAP. Inlay in (A) shows cellularized endosperm (arrow indicates cell wall). (D) …
Figure 5—figure supplement 1
Seed abortion in agl62 expressing AGL62::PGP10.
Seed abortion rate of five independent T1 transgenic lines expressing AGL62::PGP10 in the agl62/+ mutant background. The full horizontal line indicates the expected seed abortion level in agl62/+ …
Figure 6 with 2 supplements
PRC2 and auxin work in the same pathway during seed coat development.
(A–C) Vanillin-stained WT ovule at 5 DAE (A), WT seed at 1 DAP (B) and vrn2/+ emf2/+ autonomous seed at 5 DAE (C). (D) Percentage of vanillin-stained ovules per silique at 6 DAE in WT and mutant …
Figure 6—figure supplement 1
Relative expression of genes involved in GA biosynthesis (GA20ox2), GA catabolism (GA2ox2 and GA2ox6) and GA signaling (RGA) between vrn2 emf2/+ and WT ovules.
Data show signal log ratios between vrn2 emf2/+ and WT ovules at 4 DAE, as indicated in Table 1—source data 1. Expression differences are statistically significant (q < 0.01).
Figure 6—figure supplement 2
Expression of the RGA reporter in unfertilized ovules.
RGA::GFP:RGA activity at 2 DAE in WT (A) and swn/+ clf/+ ovules (B). Red staining is PI. Bars indicate 20 µm.
Figure 7 with 1 supplement
PcG activity is reduced in the developing seed coat.
(A–I) Expression of MSI1:MSI1::GFP (A–C), SWN:SWN::GFP (D–F) and LHP1:LHP1::GFP (G–I) reporters in 2 DAE ovules (A,D and G), 1 DAP seeds (B,E and G) and 2,4-D treated ovules at 3 DAE (C, F and I). …
Figure 7—figure supplement 1
Expression of PcG reporters in unfertilized ovules.
(A–F) Expression of MSI1:MSI1::GFP (A–B), SWN:SWN::GFP (C–D) and LHP1:LHP1::GFP (E–F) reporters at anthesis (A,C and E) and mock-treated ovules at 3 DAE (B, D and F). Red staining is PI. Bars …
Figure 8
Model for the post-fertilization mechanism driving seed coat development.
Before fertilization (left panel) sporophytic PRC2 represses seed coat developmental pathways. The fertilization of the central cell (middle panel) results in the production of auxin that is …
Tables
Table 1
Significantly enriched biological processes for genes commonly upregulated in fertilized WT and autonomous vrn2 emf2/+ seeds, compared to unfertilized WT ovules (p-value<0.05).
GO-term | p-value | Number of genes | Description |
|---|---|---|---|
GO:0016043 | 2,12E-10 | 63 | cell organization and biogenesis |
GO:0006412 | 2,17E-09 | 66 | translation |
GO:0009058 | 1,46E-07 | 96 | biosynthetic process |
GO:0042545 | 1,04E-04 | 10 | cell wall modification |
GO:0009753 | 6,58E-04 | 10 | response to jasmonic acid |
GO:0009739 | 1,80E-03 | 8 | response to gibberellin |
GO:0009605 | 1,94E-03 | 16 | response to external stimulus |
GO:0008361 | 1,98E-03 | 10 | regulation of cell size |
GO:0009861 | 2,09E-03 | 10 | jasmonic acid and ethylene-dependent systemic resistance |
GO:0009611 | 2,60E-03 | 12 | response to wounding |
GO:0016049 | 5,22E-03 | 9 | cell growth |
GO:0006694 | 7,68E-03 | 4 | steroid biosynthetic process |
GO:0007155 | 8,49E-03 | 4 | cell adhesion |
GO:0009723 | 1,02E-02 | 8 | response to ethylene |
GO:0009733 | 1,46E-02 | 12 | response to auxin |
GO:0007267 | 1,81E-02 | 3 | cell-cell signaling |
GO:0007276 | 1,96E-02 | 6 | gamete generation |
GO:0009698 | 2,51E-02 | 7 | phenylpropanoid metabolic process |
GO:0009813 | 2,60E-02 | 4 | flavonoid biosynthetic process |
GO:0009812 | 3,53E-02 | 4 | flavonoid metabolic process |
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Table 1—source data 1
Seed and ovule transcriptome data.
This table includes absolute and relative gene expression values for the microarray data of WT vs. vrn2/- emf2/+ ovules and seeds, and for the mRNAseq data of WT vs. agl62/+ seeds.
- https://doi.org/10.7554/eLife.20542.004
Table 2
Primer sequences.
Used for | Gene | Sequence 5’ - > 3’ * | |
|---|---|---|---|
RT-qPCR | AT5G58230 (MSI1) | fw | GCCCAAGTTCAGCTTCCTCT |
rv | TTTGTACCTTTCCAGTTGCACA | ||
AT4G02020 (SWN) | fw | CAACTCCTCTGGACGAATCAAG | |
rv | TCTGTTTTCCAAACCCTCGAGTC | ||
AT4G16845 (VRN2) | fw | TCATTCTCACAGAGTCCAGCC | |
rv | AGTCATCAAGCATCTGGCGAT | ||
AT5G51230 (EMF2) | fw | CGCACTTGATTTGGTGCTGG | |
rv | TGTTCATGGTTCGGGCATCA | ||
AT2G23380 (CLF) | fw | AAGTACTGCGGTTGCCCAAA | |
rv | ACATTCCCGATCTGCAGCAA | ||
Cloning | AT1G15550 (GA3ox1) | fw | GGGGACAAGTTTGTACAAAAAAGCAGGCTGCAAGATGCCTGCTATGTTA |
rv | GGGGACCACTTTGTACAAGAAAGCTGGGTATCTAATCATTCTTCTCTGTGATTTCT | ||
AT1G61720 (promBAN) Gateway | fw | GGGGACAAGTTTGTACAAAAAAGCAGGCTGAGCTCTAACAGAACCTTACTGTAACACT | |
rv | GGGGACCACTTTGTACAAGAAAGCTGGGTACTAGTGAGTCTGGTCCATGGTTGTA | ||
AT1G61720 (promBAN) In-Fusion | fw | CCATGGCCGCGGGATATCAGATTCTTAGGTGAAGACAAG | |
rv | CGCTGAATGATTCATGATTGTACTTTTGAAATTACAG | ||
GFP | fw | ATGAATCATTCAGCGAAAACC | |
rv | CTTCACCTAAGAATCCATCTAGTAACATAGATGACA | ||
AT1G10680 (promPGP10) | fw | GGGGACAAGTTTGTACAAAAAAGCAGGCTGGCGTTGCGTATAATCCGTT | |
rv | GGGGACCACTTTGTACAAGAAAGCTGGGTTTTCACTTTTGGATATGGAGAGA | ||
At5G60440 (promAGL26) | fw | TAAGCAGAGCTCGAATTGCATCTCGGCAATGAC | |
rv | TGCTTAACTAGTTTTTAGTGATATTTGAGAAGCT | ||
AT1G10680 (PGP10) | fw | GGGGACAAGTTTGTACAAAAAAGCAGGCTATGCAACCGTCAAATGATCCAG | |
rv | GGGGACCACTTTGTACAAGAAAGCTGGGTTTAAGGATGATGGCGCTGC | ||
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*Primer adapters are underlined
