Isolation of molecular markers from the barley endosperm coenocyte and the surrounding nucellus cell layers

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Abstract
The cereal endosperm develops from a coenocyte to a cellular storage organ through formation of nucleo-cytoplasmic domains and cell wall deposition in the interzones between these domains. During its early stages, the endosperm develops in close contact with nucellus, the sporophytic tissue which gives rise to the megagametophyte. Owing to the positioning of the two tissues deeply within the ovary, neither cell types have been easily accessible for molecular studies. In this paper we report for the first time the cloning of molecular markers for the barley endosperm coenocyte and the nucellus. The novel END1 and NUC1 cDNAs were isolated by differential screening of a cDNA library from 5 DAP (days after pollination) ovaries using a positive probe from hand-dissected embryo sacs with adhering nucellus and testa cell layers, and a negative probe from pericarp. In situ and northern blot hybridization data show that END1 transcripts are asymmetrically distributed in teh endosperm coenocyte limited to an area over the nucellar projection. In the cellular endosperm, END1 transcripts are present in modified aleurone cells and a few layers of ventral starchy endosperm cells. The second clone, NUC1, hybridizes to transcripts in the nucellus before fertilization and in autolyzing nucellus cells after fertilization. At later stages, after the disappearance of nucellus, NUC1 transcripts are present in the nucellar epidermis and in the lateral cells of the nucellar projection. This work provide tools for future elucidation of the genes specifying endosperm histogenesis.