1. A synergid-independent attraction system allows fertilization recovery
Figure1. Fertilization recovery is achieved by redundant pollen tube attractants
- ccg, myb98 homozygous mutant
- gcs1(hap2)mutant pollen ← CRISPER-Cas9
Figure2. Synergid-independent attractants salvage seed production
- MYB98::H2B-TdTomato
- immunostaining
- pollen-tube-attraction ability of synergid-depleted ovules competition
2. Central-cell-expressed peptides SAL1 and SAL2 attract pollen tubes
Figure3. Central-cell-expressed SAL1 and SAL2 have pollen-tube-attraction activity
- CRPs(cysteine rich peptides) downregulated in ccg ovules but upregulated in myb98 ovules
↪︎ SAL1(AT3G48231), SAL1a(AT2G33233), SAL2(AT4G14276), SAL2a(AT5G52605) ← His-tag
- expressing the gemomic GFP-fusion constructs
- application of purified SALs
- peptide-binding assay
3. SAL1 and SAL2 attract pollen tubes upon deficiency of synergid-derived attractants
Figure4. Central-cell-expressed attractants increase the fertility of myb98 mutant
- genetic rescue assay ← MYB98 promoter, DD22 promoter
- a significant increase in the ratio of pollen tube targeted ovules to GFP positive ovules
Figure5. SALs are redistributed to the integument and micropylar region in myb98 mutant ovule
- SAL1-GFP, SAL2-GFP were directionally secreted to the micropyle and apoplast of the nearby sporophytic integuments
- myb98 gSAL1-GFP, myb98 gSAL2-GFP 「seed set&pollen-tube-attraction ↑」
- triple mutant myb98/sal1/2 「seed set&pollen-tube-attraction ↓」
4. SAL1 and SAL2 promote fertilization recovery upon double fertilization failure and elimination of both synergids
Figure6. SALs promote fertilization recovery upon consumption of both synergids
- gcs1 x gSAL1-GFP, gcs1 x gSAL2-GFP were directionally secreted to the micropyle
- Immunostaining of the ovules : sal1/2 x WT 32HAP, WT x WT 32HAP, WT x gcs1 32HAP, WT ovule, MYB98::DTA-GFP ovule, A.lyrate 32HAP
Figure7. knockout of SAL1 and SAL2 disrupts central-cell-dependent fertilization recovery
- sal1/2, sal1/1a/2/2a mutants ← CRISPR-Cas9
- gcs1-pollen-tube-inseted sal1/2 ovules < gcs1-inserted WT ovules
