Fertility loss in senescing Arabidopsis ovules is controlled by the maternal sporophyte via a NAC transcription factor triad

1. ovule degeneration proceeds in distinct successive senescence stages

- ovule senescence stage (intact~stage3)

- ovules in pistil (4DAE~6DAE)

 

2. the cells of the gametophyte degenerate sequentially during ovule senescence

- pDD33>>HTA6-GFP : antipodals

- pDD45::NLS-tdTomato : egg cells

- pDD27::NLS-GFP : synergid and central cells

- CC present > Regular SC nucleus

 

3. integument degeneration displays hallmarks of developmentally controlled PCD

- dPCD-associated genes

→ pACT11

→ pCEP1

→ pRNS3

→ pBFN1

 

4. NAP, SHYG, and ORE1 are up-regulated in senescing ovules

- 19,513 genes to be expressed in ovules (out of 27,416)

→ 272 DEGs were sequentially up-regulated from 2 to 4 DAE 

 

- 78 TF encoding genes were differentially expressed during ovule senescence

→ 18 TF genes were sequentially up-regulated from 2 to 4 DAE

→ NAP, SHYG, ORE1

→ pNAP, pSHYG, pORE1 : all 3 promoter-reporter constructs expressing NLS-GFP-GUS were expressed in senescing ovules

 

5. NAP, SHYG, and ORE1 are positive regulators of unfertilized ovule senescence

- nap shyg ore1 triple mutant(;3x mut) showed the strongest delay in ovule senescence

- triple CRISPR-generated mutant phenocopied the triple T-DNA mutant(;3x CRISPR)

 

6. the functional life span of nap shyg-1 ore1 ovules is significantly extended

- a grafting based method

→scions : pRPS5A::NLS-GFP

→stock : Col-0 and 3xmut (2DAE~6DAE)

 

- proliferating endosperm nuclei expressing the paternally inherited pRPS5A::NLS-GFP provided a straightforward readout for successful fertilization and initiation fo seed development

 

- wlid type > 3x mut