Expression of TWISTED DWARF1 lacking its in‐plane membrane anchor leads to increased cell elongation and hypermorphic growth

Abstract

SUMMARYPlant growth is achieved predominantly by cellular elongation, which is thought to be controlled on several levels by apoplastic auxin. Auxin export into the apoplast is achieved by plasma membrane efflux catalysts of the PIN-FORMED (PIN) and ATP-binding cassette protein subfamily B/phosphor-glycoprotein (ABCB/PGP) classes; the latter were shown to depend on interaction with the FKBP42, TWISTED DWARF1 (TWD1). Here by using a transgenic approach in combination with phenotypical, biochemical and cell biological analyses we demonstrate the importance of a putative C-terminal in-plane membrane anchor of TWD1 in the regulation of ABCB-mediated auxin transport. In contrast with dwarfed twd1 loss-of-function alleles, TWD1 gainof-function lines that lack a putative in-plane membrane anchor (HA-TWD1-C t ) show hypermorphic plant architecture, characterized by enhanced stem length and leaf surface but reduced shoot branching. Greater hypocotyl length is the result of enhanced cell elongation that correlates with reduced polar auxin transport capacity for HA-TWD1-C t . As a consequence, HA-TWD1-C t displays higher hypocotyl auxin accumulation, which is shown to result in elevated auxin-induced cell elongation rates. Our data highlight the importance of C-terminal membrane anchoring for TWD1 action, which is required for specific regulation of ABCB-mediated auxin transport. These data support a model in which TWD1 controls lateral ABCB1-mediated export into the apoplast, which is required for auxin-mediated cell elongation.

Master and servant: Regulation of auxin transporters by FKBPs and cyclophilins

“…Notably, while TWD1 appears to increase the functionality of ABCBs and their presence on the plasma membrane (Fig. 2), [9,64], DGT seems to decrease the functionality of PINs and their presence of on the plasma membrane [10]. Thus, it appears that FKBPs and cyclophilins, belonging to different subgroups of the immunophilin protein superfamily, serve as regulators ("servants") of functionally distinct, primary or secondary energized, transporters of auxin ("masters") (Box 2), respectively.…”

“…The twd1/ucu2/fkbp42 mutants is marked by a characteristic twisting of the root epidermal cell files and displays a defect in root gravitropism, an auxin-transport regulated phenotype [8,9,62,63] (Table 1). Over-expression of a mutated version of TWD1 lacking its membrane anchor also results in a hypermorphic ("giant") growth phenotype caused by enhanced cell elongation, which is probably due to altered apoplastic auxin concentrations caused by enhanced auxin transport [64] (see Section 4). All attempts to demonstrate PPIase activity in TWD1 have failed [62,65], probably because only 3 out of the 11 key residues inside the hydrophobic pocket of its FK506-binding domain involved in PPIase function are conserved [55] (see Fig.…”

“…The unusual FK506-binding domain in TWD1 was instead found to participate in protein interaction with ABCB-type auxin transporters and is apparently involved in the functional regulation of these transporters [44] (see Section 4). TWD1 also possesses a tetratricopeptide repeat domain (common [131] Regulation and PM-to-ER trafficking of ABCB-type auxin transporters [8,9] Z. mais [133] Interaction with ABCB transporters on PM (via FKB-domain) [8,62], ABCC transporters on tonoplast (via TPR domain) [66], with HSP90 (TPR domain) [62,65] and calmodulin (calmodulin-binding domain) [66] Over-expression of TWD1 lacking its membrane anchor results in hypermorphic growth [64] FKBP72/PAS1 A. thaliana [69] Unclear (C-terminal membrane anchor), nuclear [68,134] Low, inhibited by FK506 and rapamycin [134] Up-regulated cell division, leaf fusions, short hypocotyls, sterile [69] O. sativa [131] Chaperon during translocation of NAC-like transcription factor (AtFAN) into nucleus [68] Z. mais [133] Regulation of very long fatty acid elongation [70] -DGT/CypA/ROC1/CYP1/CYP2/ P. patens [10] Nuclear and cytoplasmic [10,90], phloem sieve elements [79] Significant [77,78], inhibited by CsA [78] Regulates growth [90], gene expression [84,90,92], patterned cell division [88], phloem function [79], ROS balance in root apical meristem [87] A. [137] in many large immunophilins) that was shown to interact with vacuolar ABC transporters of the C subclass and HSP90 [65,66], as w...…”

“…1). Furthermore, TWD1 contains a unique C-terminal in-plane membrane anchor domain [64,67]. The functional importance of HSP90 and calmodulin binding to TWD1 is not known but analogy to human FKBP38 and other multi-domain FKBPs suggests that both HSP90 and calmodulin might act as modulators of TWD1.…”

“…Both, the interaction with the very long fatty acid elongase complex and that with the NAC transcription factor, respectively, is mediated by the C-terminal region of PAS1 that contains a calmodulin-binding domain [53]. The Cterminal domain of PAS1 also contains a membrane anchor domain similar to that in TWD1 [64,67,71] that seems to be important for the cellular distribution of PAS1 and its exclusion from the nucleus except during cell division [71]. Another Arabidopsis FKBP, FKBP12, interacts with AtFIP37, a DNA-binding protein involved in mRNA splicing, cell cycle regulation and embryo development [72,73] although the developmental importance of this interaction is unclear.…”