Structural and functional insights into the mechanism of action of plant boron transporters

AbstractBoron has essential roles in plant growth and development. BOR proteins are key in the active uptake and distribution of boron, and regulation of intracellular boron concentrations. However, their mechanism of action remains poorly studied. BOR proteins are members of the SLC4 family of transporters and thus homologues of well studied mammalian transporters including the human Anion Exchanger 1 (hAE1). Here we generated Arabidopsis thaliana BOR1 (AtBOR1) variants based i) on known disease causing mutations of hAE1 (S466R, A500R) and ii) a loss of function mutation (D311A) identified in the yeast BOR protein, ScBOR1p. The AtBOR1 variants express in yeast and localise to the plasma membrane, although both S466R and A500R exhibit lower expression than the WT AtBOR1 and D311A. The D311A, S466R and A500R mutations result in a loss of boron efflux activity in a yeast bor1p knockout strain. A. thaliana plants containing these three individual mutations exhibit substantially decreased growth phenotypes in soil under conditions of low boron. These data confirm an important role for D311 in the function of the protein and show that mutations equivalent to disease causing mutations in hAE1 have major effects in AtBOR1. We also obtained a low resolution cryo-EM structure of a BOR protein from Oryza sativa, OsBOR3 lacking the 30 C-terminal amino acids. This structure confirms the gate and core domain organisation previously observed for related proteins, and is strongly suggestive of an inward facing conformation.