The influence of anther locular fluid on exine self-assembly, investigated by in vivo transplantation experiments.

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conclusionExperimental transplantation of microspores and manipulation of locular fluid, in vivo, confirm a complex interplay between physicochemical processes and gene expression in shaping the 3-D ultrastructure of the developing exine. We aimed to understand the underlying mechanisms of development of the exine, the outer layer of the pollen wall, one of the most complex cell walls in plants. Control of the processes involved remained obscure until it became clear that the stages observed coincided, in essence, with the sequence of micellar self-assembling mesophases. To test this, a series of in vitro experiments were undertaken earlier (Gabarayeva et al., Ann Bot 123:1205-1218, 2019;Gabarayeva et al., New Phytol 225:1956-1973, 2020), in which exine-like patterns were generated in colloidal mixtures by self-assembly, without any genomic participation. The results of those experiments, carried out "in a vial", have shown that physicochemical interactions, phase separation and self-assembly are capable of generating exine-like patterns. The aim of the new experiments described here, conducted in living plants, was to alter the environment within the anther locule, observing any effects on the processes of exine ontogeny, and to see whether physicochemical interactions play the important role, suggested by in vitro experiments. In the first experiment, early microspore tetrads of Borago officinalis were transplanted into the anthers of Cucurbita maxima. In the second experiment, a surfactant mixture was injected into Cucurbita anthers to alter the environment of self-assembly. After several days, anthers were fixed and studied with TEM. The results confirm our earlier finding from in vitro studies, that-although gene expression in developing microspores and the anther is of fundamental importance-physicochemical forces also play a significant role in exine development. It is the interplay between controls that underpins the vast morphological diversity observed in sporoderms.