A Barley Efflux Transporter Operates in a Na⁺-Dependent Manner, as Revealed by a Multidisciplinary Platform

Abstract

Plant growth and survival depend upon the activity of membrane transporters that control the movement and distribution of solutes into, around, and out of plants. Although many plant transporters are known, their intrinsic properties make them difficult to study. In barley (Hordeum vulgare), the root anion-permeable transporter Bot1 plays a key role in tolerance to high soil boron, facilitating the efflux of borate from cells. However, its three-dimensional structure is unavailable and the molecular basis of its permeation function is unknown. Using an integrative platform of computational, biophysical, and biochemical tools as well as molecular biology, electrophysiology, and bioinformatics, we provide insight into the origin of transport function of Bot1. An atomistic model, supported by atomic force microscopy measurements, reveals that the protein folds into 13 transmembrane-spanning and five cytoplasmic a-helices. We predict a trimeric assembly of Bot1 and the presence of a Na + ion binding site, located in the proximity of a pore that conducts anions. Patch-clamp electrophysiology of Bot1 detects Na + -dependent polyvalent anion transport in a Nernstian manner with channel-like characteristics. Using alanine scanning, molecular dynamics simulations, and transport measurements, we show that conductance by Bot1 is abolished by removal of the Na + ion binding site. Our data enhance the understanding of the permeation functions of Bot1.

Boron toxicity in higher plants: an update

“…Other alternative explanations for their antagonist role have been proposed: (i) B reduces the uptake of chloride and vice-versa (Yermiyahu et al 2008); (ii) B affects the activity of specific membrane components, regulating the functions of specific aquaporin isoforms and ATPase as possible components of the salinity tolerance mechanism (Martinez-Ballesta et al 2008); (iii) some B effluxer are regulated by Na + ions. Indeed, it has been recently demonstrated that the Na + -dependent barley efflux pump BOT1 permits an efficient exclusion of B from plant cells back to the soil which may create an energy barrier to drive borate anions efflux, thus ameliorating B toxicity (Nagarajan et al 2016). To confirm the antagonist role of B and NaCl, it has been observed that the supplementation of B enhances crop salt tolerance and improves the plant yield in saline soils (El-Hamdaoui et al 2003;Bonilla et al 2004), suggesting such an ameliorative role of B as a useful strategy to mitigate the negative effect of salinity.…”

Insights into the Mechanisms Underlying Boron Homeostasis in Plants

“…A human BOR-like transporter, BTR1/SLC4A11, was characterized as an electrogenic, voltage-regulated Na + -coupled B(OH) 4 - cotransporter by electrophysiology in human embryonic kidney (HEK) 293 cells ( Park et al, 2004 ). HvBot1, a BOR homolog in barley that is involved in excess B tolerance, is a uniporter with high affinity for borate anion in Xenopus oocytes and patch-clamped proteoliposomes ( Nagarajan et al, 2016 ). Therefore, BOR homologs likely function as borate anion uniporters driven by the negative membrane potential of plant cells.…”

Polar Localization of the NIP5;1 Boric Acid Channel Is Maintained by Endocytosis and Facilitates Boron Transport in Arabidopsis Roots

“…The Arabidopsis borate exporter BOR1 is required for boron translocation to the xylem and preferential distribution of boron to young, developing shoot tissues (Noguchi et al, 1997;Takano et al, 2001Takano et al, , 2002. A recent electrophysiological study of barley (Hordeum vulgare) Bot1, an At-BOR1 homolog required for high-boron tolerance in barley, suggested that Hv-Bot1 is a channel-like anion transporter with a high affinity for borate (Nagarajan et al, 2015).…”