Genetic and environmental manipulation of Arabidopsis hybridization barriers uncover antagonistic functions in endosperm cellularization

Speciation by reproductive isolation can occur by hybridization barriers acting in the endosperm of the developing seed. The nuclear endosperm is a nutrient sink, accumulating sugars from surrounding tissues, and undergoes coordinated cellularization, switching to serve as a nutrient source for the developing embryo. Tight regulation of cellularization is therefore vital for seed and embryonic development. Here we show that hybrid seeds from crosses between Arabidopsis thaliana as maternal contributor and A. arenosa or A. lyrata as pollen donors result in an endosperm based post-zygotic hybridization barrier that gives rise to a reduced seed germination rate. Hybrid seeds display opposite endosperm cellularization phenotypes, with late cellularization in crosses with A. arenosa and early cellularization in crosses with A. lyrata. Stage specific endosperm reporters display temporally ectopic expression in developing hybrid endosperm, in accordance with the early and late cellularization phenotypes, confirming a disturbance of the source-sink endosperm phase change. We demonstrate that the hybrid barrier is under the influence of abiotic factors, and show that a temperature gradient leads to diametrically opposed cellularization phenotype responses in hybrid endosperm with A. arenosa or A. lyrata as pollen donors. Furthermore, different A. thaliana accession genotypes also enhance or diminish seed viability in the two hybrid cross-types, emphasizing that both genetic and environmental cues control the hybridization barrier. We have identified an A. thaliana MADS-BOX type I family single locus that is required for diametrically opposed cellularization phenotype responses in hybrid endosperm. Loss of AGAMOUS-LIKE 35 significantly affects the germination rate of hybrid seeds in opposite directions when transmitted through the A. thaliana endosperm, and is suggested to be a locus that promotes cellularization as part of an endosperm based mechanism involved in post-zygotic hybrid barriers. The role of temperature in hybrid speciation and the identification of distinct loci in control of hybrid failure have great potential to aid the introduction of advantageous traits in breeding research and to support models to predict hybrid admixture in a changing global climate.