Electrophysiological study of Arabidopsis ABCB4 and PIN2 auxin transporters: Evidence of auxin activation and interaction enhancing auxin selectivity

Abstract

Polar auxin transport through plant tissue strictly requires polarly localized PIN proteins and uniformly distributed ABCB proteins. A functional synergy between the two types of membrane protein where their localizations overlap may create the degree of asymmetric auxin efflux required to produce polar auxin transport. We investigated this possibility by expressing ABCB4 and PIN2 in human embryonic kidney cells and measuring whole‐cell ionic currents with the patch‐clamp technique and CsCl‐based electrolytes. ABCB4 activity was 1.81‐fold more selective for Cl− over Cs+ and for PIN2 the value was 2.95. We imposed auxin gradients and determined that ABCB4 and PIN2 were 12‐fold more permeable to the auxin anion (IAA−) than Cl−. This measure of the intrinsic selectivity of the transport pathway was 21‐fold when ABCB4 and PIN2 were co‐expressed. If this increase occurs in plants, it could explain why asymmetric PIN localization is not sufficient to create polar auxin flow. Some form of co‐action or synergy between ABCB4 and PIN2 that increases IAA− selectivity at the cell face where both occur may be important. We also found that auxin stimulated ABCB4 activity, which may contribute to a self‐reinforcement of auxin transport known as canalization.

Systems approaches reveal that ABCB and PIN proteins mediate co-dependent auxin efflux

“…However, an experimental proof of these different scenarios has been hampered by the fact that members of the ABCB and PIN families are functionally redundant and show overlapping transport activities with each other, meaning they function concertedly (Geisler et al, 2017). While results from heterologous expression systems have provided support for both independent and interactive ABCB and PIN transport (Bouchard et al, 2006;Blakeslee et al, 2007;Geisler et al, 2017;Deslauriers and Spalding, 2021), results from these may be affected by the presence of endogenous transporters and do not necessarily represent the activity in planta (Yesilirmak and Sayers, 2009). In this study, we used a systems approach to investigate potential ABCB-PIN regulatory interactions.…”

“…( 1) and (2) in the "Supplemental Methods") and present results demonstrating how our conclusions are affected by changing these parameter values (as detailed below). Scenario III is supported by results from heterologous expression systems, which have suggested both independent and interactive ABCB and PIN transport (Bouchard et al, 2006;Blakeslee et al, 2007;Geisler et al, 2017;Deslauriers and Spalding, 2021). However, these heterologous results may be affected by the presence of endogenous transporters in such systems, therefore, motivating further examination of these scenarios in planta using a systems approach.…”

“…The complexity in auxin transport activity is increased further when considering potential functional interactions between the ABCB and PIN proteins. Though a clear proof of such direct interactions has not yet been shown (Geisler et al, 2017), some synergy in activity between PINs and ABCBs is supported at both genetic (Mravec et al, 2008) and cellular levels (Blakeslee et al, 2007;Deslauriers and Spalding, 2021). More specifically, in heterologous systems, co-expression of PIN1 and either ABCB1 or ABCB19 appears to enhance auxin efflux activity, pairing PIN2 with ABCB1 and ABCB19 does not (Blakeslee et al, 2007).…”

“…In addition, co-expression of ABCB4 and PIN1 appears to result in net cellular efflux, while co-expression of ABCB4 and PIN2 results in net cellular influx (Blakeslee et al, 2007). Furthermore, a recent electrophysiology study suggested that ABCB4 and PIN2 produce a synergistic efflux that is approximately double that of either protein alone (Deslauriers and Spalding, 2021). In summary, there is indication for a synergistic and antagonistic interference of ABCB-PIN-mediated auxin transport; however, due to a possible interference of PINs or ABCBs with endogenous transporter homologs/ orthologs in homologous or heterologous expression systems, respectively, the individual role of ABCBs and PINs has not been dissected.…”

Loss of Multiple ABCB Auxin Transporters Recapitulates the Major twisted dwarf 1 Phenotypes in Arabidopsis thaliana

“…Plant ABCB transporters appear to be selective for aromatic and aliphatic organic acids, with enhanced specificity evident when ABCBs, PINs, and TWD1 co-occur ( Geisler et al, 2005 ; Bouchard et al, 2006 ; Titapiwatanakun et al, 2009 ; Yang and Murphy, 2009 ; Bailly et al, 2012 , 2013 ; Deslauriers and Spalding, 2021 ). Recently, a C-terminal proline residue (P1008 in ABCB1) present in a subgroup of ABCB transporters that have been associated with auxin transport (ABCB1, 19, 6, 20, 4, 21, 15–18, 22) was identified ( Hao et al, 2020 ).…”