CrAGL62 and potential paternally expressed gene CrbZIP contribute to paternal-excess postzygotic barriers in citrus.

Interploidy hybridization in plants is constrained by a postzygotic barrier. Triploid production via 2x × 4x interploidy hybridization is an important strategy for obtaining seedless cultivar in some fruit tree crops including citrus. However, as the molecular mechanisms of postzygotic barrier in citrus remain unclear, triploid breeding efficiency is limited. Here, we compared seed development in citrus 2x × 4x and 2x × 2x crosses. Embryo and endosperm developed normally in 2x × 2x crosses, while endosperm cellularization completely failed in 2x × 4x crosses, resulting in embryo arrest at the globular stage, demonstrating that impaired endosperm cellularization causes triploid embryo abortion. Transcriptomic analysis of laser microdissection (LMD)-captured endosperm at pre- and post-cellularization stages revealed extensive transcriptional misregulation in 2x × 4x crosses, including persistent upregulation of type I MADS-box genes (CrAGLs) and the potential paternally expressed gene CrbZIP. Consistently, functional analyses showed that overexpression of CrAGL62, CrAGL80a, or CrbZIP delayed endosperm cellularization in precocious Mini-citrus (Fortunella hindsii), while cragl62 mutants exhibited severely impaired cellularization. Our findings showed CrAGL62 and CrbZIP as key regulators of endosperm cellularization in citrus, providing potential targets for modulating endosperm development to improve citrus triploid breeding efficiency.