Polar transport of the plant hormone auxin is controlled by PIN-and ABCB/PGP-efflux catalysts. PIN polarity is regulated by the AGC protein kinase, PINOID (PID), while ABCB activity was shown to be dependent on interaction with the FKBP42, TWISTED DWARF1 (TWD1). Using co-immunoprecipitation (co-IP) and shotgun LC-MS/MS analysis, we identified PID as a valid partner in the interaction with TWD1. Invitro and yeast expression analyses indicated that PID specifically modulates ABCB1-mediated auxin efflux in an action that is dependent on its kinase activity and that is reverted by quercetin binding and thus inhibition of PID autophosphorylation. Triple ABCB1/PID/TWD1 co-transfection in tobacco revealed that PID enhances ABCB1-mediated auxin efflux but blocks ABCB1 in the presence of TWD1. Phospho-proteomic analyses identified S634 as a key residue of the regulatory ABCB1 linker and a very likely target of PID phosphorylation that determines both transporter drug binding and activity. In summary, we provide evidence that PID phosphorylation has a dual, counter-active impact on ABCB1 activity that is coordinated by TWD1-PID interaction.

Plant architecture is influenced by the polar, cell-to-cell transport of auxin that is primarily provided and regulated by plasma membrane efflux catalysts of the PIN-FORMED and B family of ABC transporter (ABCB) classes. The latter were shown to require the functionality of the FK506 binding protein42 TWISTED DWARF1 (TWD1), although underlying mechanisms are unclear. By genetic manipulation of TWD1 expression, we show here that TWD1 affects shootward root auxin reflux and, thus, downstream developmental traits, such as epidermal twisting and gravitropism of the root. Using immunological assays, we demonstrate a predominant lateral, mainly outward-facing, plasma membrane location for TWD1 in the root epidermis characterized by the lateral marker ABC transporter G36/PLEIOTROPIC DRUG-RESISTANCE8/PENETRATION3. At these epidermal plasma membrane domains, TWD1 colocalizes with nonpolar ABCB1. In planta bioluminescence resonance energy transfer analysis was used to verify specific ABC transporter B1 (ABCB1)-TWD1 interaction. Our data support a model in which TWD1 promotes lateral ABCB-mediated auxin efflux via protein-protein interaction at the plasma membrane, minimizing reflux from the root apoplast into the cytoplasm.

The rate, polarity, and symmetry of the flow of the plant hormone auxin are determined by the polar cellular localization of PIN-FORMED (PIN) auxin efflux carriers. Flavonoids, a class of secondary plant metabolites, have been suspected to modulate auxin transport and tropic responses. Nevertheless, the identity of specific flavonoid compounds involved and their molecular function and targets in vivo are essentially unknown. Here we show that the root elongation zone of agravitropic pin2/eir1/ wav6/agr1 has an altered pattern and amount of flavonol glycosides. Application of nanomolar concentrations of flavonols to pin2 roots is sufficient to partially restore root gravitropism. By employing a quantitative cell biological approach, we demonstrate that flavonoids partially restore the formation of lateral auxin gradients in the absence of PIN2. Chemical complementation by flavonoids correlates with an asymmetric distribution of the PIN1 protein. pin2 complementation probably does not result from inhibition of auxin efflux, as supply of the auxin transport inhibitor N-1-naphthylphthalamic acid failed to restore pin2 gravitropism. We propose that flavonoids promote asymmetric PIN shifts during gravity stimulation, thus redirecting basipetal auxin streams necessary for root bending.The plant hormone auxin (3-indolyl acetic acid, IAA) 5 controls virtually all plant developmental and physiological processes. In roots, the differential growth response associated with gravity stimulation (gravitropism) occurs in the elongation zone (1, 2) and is a result of the asymmetric distribution of auxin to the lower side of epidermal cells (3). In these tissues accumulating auxin, cell elongation is inhibited and the root tip bends downwards. This cell-to-cell or polar auxin transport (PAT) is determined by the asymmetric cellular localization of auxin inand efflux components of the ABCB/PGP/MDR, AUX1/LAX, and PIN-FORMED (PIN) family (4 -7). Although ABCBs are apparently involved in long-range auxin transport and movements of auxin out of apical regions (8 -10), AUX1 and PIN2/ EIR1/WAV6AGR1 have been demonstrated to channel auxin from the lateral root cap basipetally to the expanding epidermal cells (11-13).The regulation of auxin transport during root gravitropic responses is still largely unclear. Among various possible mechanisms, the localized synthesis and directed transport of flavonoids, plant-specific phenylpropanoid compounds, have been shown to modulate the rate of the gravity response (14,15). A number of lines of experimentation have suggested that flavonoids may act as non-essential auxin transport inhibitors (16 -20). This is mainly based on the finding that flavonoids displace binding of synthetic auxin transport inhibitors, like N-1-naphthylphthalamic acid (NPA), a herbicide (Naptalam), in vitro (31, 50 -52). Moreover, roots of transparent testa (tt) Arabidopsis mutant with manipulated flavonoid levels exhibit altered gravitropic curvature and auxin transport, which are restored to the wild-type level by exogenous...

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Protein phosphorylation plays a key role in the regulation of polar auxin transport. 1 The AGC protein kinase, PINOID (PID), 2 has been shown to phosphorylate the hydrophilic loop of PIN proteins in vivo leading to apical PIN targeting.3 It appears that PID together with protein phosphatase PP2A/RCN1 act as a binary switch to control the balance of phosphorylation and dephosphorylation determining PIN polarity and controlling auxin flows. 4,5 Employing different phospho-proteomics approaches, plant ABCB proteins have recently shown to be phosphorylated in a so-called regulatory linker domain in analogy to their mammalian orthologs.6-9 Subsequently, the photoreceptor kinase, PHOTROPIN1 (phot1), was shown to phosphorylate ABCB19 and inhibit its auxin efflux activity. 10 Recently, PID was identified as a valid interaction partner with TWD1 by co-immunoprecipitation and shotgun LC-MS/MS analysis.11 In-vitro and yeast expression data indicated that PID specifically modulates ABCB1-mediated auxin efflux, which is dependent on its kinase activity. ABCB1/PID co-transfection in N. benthamiana revealed that PID phosphorylates at S634 of the linker in the absence of TWD1 enhancing ABCB1-mediated auxin efflux. PID had a negative impact on ABCB1 in triple ABCB1/PID/TWD1 co-transfection. This suggests that PID determines ABCB1 activity by means of protein phosphoryation in an action that is controlled by TWD1-PID interaction.However, it is an open question if altered PIN polarity is indeed directly caused by PIN phosphorylation or not simply the Plant growth and development is determined by intracellular and intercellular auxin gradients that are controlled at first hand by auxin efflux catalysts of the aBCB/PGP and Pin families. aBCB transport activity was shown to be counter-actively regulated by protein phosphorylation by the aGC protein kinase, PinoiD (PiD), that is coordinated by interaction with the immunophilin-like FKBP42, tWiStED DWarF1 (tWD1). in contrast, PiD was shown to determine Pin polarity, however, the direct impact of PiD on Pin activity has yet not been tested. Co-expression in yeast indicates that PiD had no effect on Pin1,2 alone but specifically inhibits interactive aBCB1-Pin1/Pin2 auxin efflux in an action that is dependent on its kinase activity. Pin1-PiD co-transfection in n. benthamiana revealed that PiD blocks Pin1-mediated auxin efflux without changing Pin1 location. in summary, these data provide evidence that PiD phosphorylation does not only determine Pin polarity but also has a direct impact on transport activity of the activity of the binary Pin-aBCB1 complex.