Boron (B) is an essential element in plants but is toxic when it accumulates to high levels. In root cells of Arabidopsis (Arabidopsis thaliana), the borate exporter BOR1 is polarly localized in the plasma membrane toward the stele side for directional transport of B. Upon high-B supply, BOR1 is rapidly internalized and degraded in the vacuole. The polar localization and B-induced vacuolar sorting of BOR1 are mediated by endocytosis from the plasma membrane. To dissect the endocytic pathways mediating the polar localization and vacuolar sorting, we investigated the contribution of the clathrin adaptor protein, ADAPTOR PROTEIN2 (AP2) complex, to BOR1 trafficking. In the mutants lacking mor s-subunits of the AP2 complex, the polar localization and constitutive endocytosis of BOR1 under low-B conditions were dramatically disturbed. A coimmunoprecipitation assay showed association of the AP2 complex with BOR1, while it was independent of YxxF sorting motifs, which are in a cytosolic loop of BOR1. A yeast twohybrid assay supported the interaction of the AP2 complex m-subunit with the C-terminal tail but not with the YxxF motifs in the cytosolic loop of BOR1. Intriguingly, lack of the AP2 subunit did not affect the B-induced rapid internalization/vacuolar sorting of BOR1. Consistent with defects in the polar localization, the AP2 complex mutants showed hypersensitivity to B deficiency. Our results indicate that AP2-dependent endocytosis maintains the polar localization of BOR1 to support plant growth under low-B conditions, whereas the B-induced vacuolar sorting of BOR1 is mediated through an AP2-independent endocytic pathway.

The hexactinellids are a diverse group of predominantly deep sea sponges that synthesize elaborate fibrous skeletal systems of amorphous hydrated silica. As a representative example, members of the genus Euplectella have proved to be useful model systems for investigating structure-function relationships in these hierarchically ordered siliceous network-like composites. Despite recent advances in understanding the mechanistic origins of damage tolerance in these complex skeletal systems, the details of their synthesis have remained largely unexplored. Here, we describe a previously unidentified protein, named "glassin," the main constituent in the water-soluble fraction of the demineralized skeletal elements of Euplectella. When combined with silicic acid solutions, glassin rapidly accelerates silica polycondensation over a pH range of 6-8. Glassin is characterized by high histidine content, and cDNA sequence analysis reveals that glassin shares no significant similarity with any other known proteins. The deduced amino acid sequence reveals that glassin consists of two similar histidine-rich domains and a connecting domain. Each of the histidine-rich domains is composed of three segments: an amino-terminal histidine and aspartic acid-rich sequence, a proline-rich sequence in the middle, and a histidine and threonine-rich sequence at the carboxyl terminus. Histidine always forms HX or HHX repeats, in which most of X positions are occupied by glycine, aspartic acid, or threonine. Recombinant glassin reproduces the silica precipitation activity observed in the native proteins. The highly modular composition of glassin, composed of imidazole, acidic, and hydroxyl residues, favors silica polycondensation and provides insights into the molecular mechanisms of skeletal formation in hexactinellid sponges.T he hexactinellids are a circumglobal group of predominantly deep sea sponges. Exhibiting a diverse array of morphologies, the skeletal systems of hexactinellids, which are composed of amorphous hydrated silica (the other silica-forming sponge class is the Demospongiae), range in structural complexity from loose aggregations of individual skeletal elements (spicules) to complex, hierarchically ordered lattices. Hexactinellids have evolved the ability to colonize either rocky substrates or soft sediments and exhibit remarkable skeletal modifications to accomplish these feats (1). For example, the sediment-dwelling hexactinellids produce bundles of long fibrillar anchor spicules that form robust holdfast structures. These anchor spicules exhibit surprising flexibility and damage tolerance and have served as useful model systems for investigating high performance silica-based organic-inorganic biological composites (2-4). In one representative genus, Euplectella (Fig. 1), the constituent spicules are assembled into a highly regular cylindrical lattice that exhibits multiple levels of structural hierarchy spanning from the nanoscale to the macroscale. The lattice is composed of two interpenetrating grid-like networks of no...

Boron (B) is an essential micronutrient for plants but is toxic when accumulated in excess. The plant BOR family encodes plasma membrane-localized borate exporters (BORs) that control translocation and homeostasis of B under a wide range of conditions. In this study, we examined the evolutionary divergence of BORs among terrestrial plants and showed that the lycophyte Selaginella moellendorffii and angiosperms have evolved two types of BOR (clades I and II). Clade I includes AtBOR1 and homologs previously shown to be involved in efficient transport of B under conditions of limited B availability. AtBOR1 shows polar localization in the plasma membrane and high-B-induced vacuolar sorting, important features for efficient B transport under low-B conditions, and rapid down-regulation to avoid B toxicity. Clade II includes AtBOR4 and barley Bot1 involved in B exclusion for high-B tolerance. We showed, using yeast complementation and B transport assays, that three genes in S. moellendorffii, SmBOR1 in clade I and SmBOR3 and SmBOR4 in clade II, encode functional BORs. Furthermore, amino acid sequence alignments identified an acidic di-leucine motif unique in clade I BORs. Mutational analysis of AtBOR1 revealed that the acidic di-leucine motif is required for the polarity and high-B-induced vacuolar sorting of AtBOR1. Our data clearly indicated that the common ancestor of vascular plants had already acquired two types of BOR for low- and high-B tolerance, and that the BOR family evolved to establish B tolerance in each lineage by adapting to their environments.

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