TOL proteins mediate vacuolar sorting of the borate transporter BOR1 in <i>Arabidopsis thaliana</i>

<p>Boron (B) is an essential micronutrient for plants; however, it shows cytotoxicity at high concentrations. A borate transporter BOR1 is required for efficient transport of B toward the root stele in <i>Arabidopsis thaliana</i>. BOR1 shows polar localization in the plasma membrane of various root cells toward the stele-side under B limitation. To avoid over-accumulation of B, BOR1 in the plasma membrane is rapidly internalized and transported into the vacuole for proteolysis after high-B supply in an ubiquitination-dependent manner. Although BOR1 has been predicted to be transported into multi-vesicular bodies/late endosomes (MVB/LEs) via the endosomal sorting complex required for transport (ESCRT) machinery, experimental evidence was absent so far. In this study, we investigated the intracellular localization of BOR1 by visualizing endomembrane compartments, and tested the involvement of ESCRT-0-like proteins TOM1-LIKEs (TOLs) in the vacuolar sorting of BOR1. Under low-B conditions, a large portion of cytoplasmic BOR1 was localized in the <i>trans</i>-Golgi networks/early endosomes (TGN/EEs) labeled with VHA-a1 subunit. Pharmacological interference of endosomal recycling using brefeldin A-induced colocalization of BOR1 with RabA5D, which labels recycling vesicles associated with the TGN. On the other hand, under high-B conditions, BOR1 was localized in the inside of TOL5-positive MVB/LEs. To examine the roles of TOL proteins in intracellular trafficking of BOR1, we analyzed BOR1-GFP localization in the TOL quintuple mutant (<i>tolQ; tol2-1tol3-1tol5-1tol6-1tol9-1</i>) after high-B supply. In the <i>tolQ</i> mutant, vacuolar sorting of BOR1 was delayed, while the polar localization of BOR1 was not disturbed. Taken together, BOR1 is constantly transported to the TGN/EE by endocytosis and recycled to the plasma membrane likely via RabA5D-positive endomembrane compartments under low-B conditions. On the other hand, BOR1 is transported to the vacuole via TOL5-positive MVB/LEs under high-B conditions. TOL proteins are required for sorting of ubiquitinated BOR1 into MVB/LE for vacuolar degradation.</p

Improved tolerance to boron deficiency by enhanced expression of the boron transporter BOR2

<div><p></p><p>Boron (B) cross-links the pectin polysaccharide rhamnogalacturonan II (RG-II) and thus is important for cell wall structure in plants. B deficiency is an agricultural problem that causes significant losses of crop productivity worldwide. To address this, B deficiency-tolerant plants have been generated using B transporters. With the goal of further improving plant tolerance to low-B conditions, we generated transgenic <i>Arabidopsis thaliana</i> (L.) Heynh. with enhanced expression of BOR2, a B transporter that promotes cross-linking of RG-II and root elongation under low B supply. We generated a DNA construct containing the cauliflower mosaic virus 35S RNA promoter, a native promoter of <i>BOR2</i>, a <i>BOR2</i> gene and green fluorescent protein (GFP) (<i>Pro35S-BOR2:BOR2-GFP</i>), and obtained three independent transgenic lines with relatively high levels of BOR2-GFP expression. In the transgenic lines, BOR2-GFP was expressed mainly in the lateral root caps in the meristem zone and in the epidermal cells in the elongation zone, similar to its expression when driven only by its native promoter. In the <i>Pro35S-BOR2:BOR2-GFP</i> lines, BOR2-GFP was also expressed in various cells in the maturation zone of roots and epidermal cells of shoots, where the expression was hardly detectable in <i>ProBOR2:BOR2-GFP</i> lines. The transgenic lines were cultured under various B concentrations on solid media and it was found that root growth of three lines and shoot growth of two lines were enhanced compared to wild-type plants under low-B conditions. This finding established that enhanced expression of BOR2 leads to improved root growth under low-B conditions. We also examined growth of the transgenic lines under hydroponic conditions. One of the three lines showed better growth and fertility under a low-B condition, while the wild type did not set seeds under the same condition, suggesting the potential utility of BOR2 expression in agricultural applications.</p></div