In Arabidopsis seeds, embryo growth is coordinated with endosperm breakdown. Mutants in the endosperm-specific gene ZHOUPI (ZOU), which encodes a unique basic helix-loop-helix (bHLH) transcription factor, have an abnormal endosperm that persists throughout seed development, significantly impeding embryo growth. Here we show that loss of function of the bHLH-encoding gene INDUCER OF CBP EXPRESSION 1 (ICE1) causes an identical endosperm persistence phenotype. We show that ZOU and ICE1 are co-expressed in the endosperm and interact in yeast via their bHLH domains. We show both genetically and in a heterologous plant system that, despite the fact that both ZOU and ICE1 can form homodimers in yeast, their role in endosperm breakdown requires their heterodimerization. Consistent with this conclusion, we confirm that ZOU and ICE1 regulate the expression of common target genes in the developing endosperm. Finally, we show that heterodimerization of ZOU and ICE1 is likely to be necessary for their binding to specific targets, rather than for their nuclear localization in the endosperm. By comparing our results with paradigms of bHLH function and evolution in animal systems we propose that the ZOU/ICE1 complex might have ancient origins, acquiring novel megagametophyte-specific functions in heterosporous land plants that were conserved in the angiosperm endosperm.

SUMMARYIn angiosperm seeds the embryo is embedded within the endosperm, which is in turn enveloped by the seed coat, making inter-compartmental communication essential for coordinated seed growth. In this context the basic helix-loop-helix domain transcription factor AtZHOUPI (AtZOU) fulfils a key role in both the lysis of the transient endosperm and in embryo cuticle formation in Arabidopsis thaliana. In maize (Zea mays), a cereal with a persistent endosperm, a single gene, ZmZOU, falls into the same phylogenetic clade as AtZOU. Its expression is limited to the endosperm where it peaks during the filling stage. In ZmZOU-RNA interference knock-down lines embryo size is slightly reduced and the embryonic suspensor and the adjacent embryo surrounding region show retarded breakdown. Ectopic expression of ZmZOU reduces stomatal number, possibly due to inappropriate protein interactions. ZmZOU forms functional heterodimers with AtICE/AtSCREAM and the closely related maize proteins ZmICEb and ZmICEc, but its interaction is more efficient with the ZmICEa protein, which shows sequence divergence and only has close homologues in other monocotyledonous species. Consistent with the observation that these complexes can trans-activate target gene promoters from Arabidopsis, ZmZOU partially complements the Atzou-4 mutant. However, structural, trans-activation and gene expression data support the hypothesis that ZmZOU and ZmICEa may have coevolved to form a functional complex unique to monocot seeds. This divergence may explain the reduced functionality of ZmZOU in Arabidopsis, and reflect functional specificities which are unique to the monocotyledon lineage.