Mature seed physiology: identification and characterization of novel apoplastic structures implementing the biophysical properties of the seed coat

Seeds are a major evolutionary land plant innovation that has contributed to the striking spread and diversity of angiosperms. However, in contrast to the plant vegetative phase, during which indeterminate development allows adaption to an ever-changing environment, embryo survival only relies on the pre-established highly resistant mature seed state. This is notably accomplished by a protective maternal dead seed coat shielding the living tissues from the outer environment. In Arabidopsis, the seed coat biophysical properties achieve a remarkable balancing act: they limit oxygen diffusion, avoiding premature fatal oxidation of living tissues, while allowing some level of oxidation necessary to release seed dormancy to promote germination during the favorable season. Despite their importance for seed physiology and plant survival, the biophysical properties of the testa remain poorly understood. A seed coat cuticle tightly associated with the endosperm surrounding the embryo was shown to promote seed viability and dormancy by limiting seed oxidation and water uptake. Intriguingly, this cuticle was recently shown to be defective, i.e. more permeable in transparent testa (tt) seeds, deficient in synthesis of flavonoids rather than in cuticular components. tt mutant seeds were previously also known to exhibit low dormancy and viability, and although flavonoids can act as antioxidants, their contribution to normal seed physiology remains poorly understood. Here we show that the seed coat cuticle is tightly associated with a previously unidentified tannic cell wall, originating from inner integument 1 (ii1) seed coat cells, forming two fused layers; we show that this bilayer remains attached to the endosperm upon testa rupture. Furthermore, and surprisingly, we found that tannic cell walls are major constituents of the mature seed coat where they are important components of remaining ii1 cells and the brown pigmented layer. Moreover, we report that tannic cell walls are the rupture points during the seed coat rupture event. We propose that the tannic cell wall represents a biophysical innovation sustaining the seed's key physiological properties