The PIN-FORMED (PIN) protein family of auxin transporters

Abstract

The PIN-FORMED (PIN) proteins are secondary transporters acting in the efflux of the plant signal molecule auxin from cells. They are asymmetrically localized within cells and their polarity determines the directionality of intercellular auxin flow. PIN genes are found exclusively in the genomes of multicellular plants and play an important role in regulating asymmetric auxin distribution in multiple developmental processes, including embryogenesis, organogenesis, tissue differentiation and tropic responses. All PIN proteins have a similar structure with amino- and carboxy-terminal hydrophobic, membrane-spanning domains separated by a central hydrophilic domain. The structure of the hydrophobic domains is well conserved. The hydrophilic domain is more divergent and it determines eight groups within the protein family. The activity of PIN proteins is regulated at multiple levels, including transcription, protein stability, subcellular localization and transport activity. Different endogenous and environmental signals can modulate PIN activity and thus modulate auxin-distribution-dependent development. A large group of PIN proteins, including the most ancient members known from mosses, localize to the endoplasmic reticulum and they regulate the subcellular compartmentalization of auxin and thus auxin metabolism. Further work is needed to establish the physiological importance of this unexpected mode of auxin homeostasis regulation. Furthermore, the evolution of PIN-based transport, PIN protein structure and more detailed biochemical characterization of the transport function are important topics for further studies.

Figure 1

Simplified cladogram of the Plantae supergroup illustrates the distribution of PIN sequences within the group. Species with complete, fully assembled genomes containing PIN sequences are shown as green arrows, and those lacking it as yellow arrows, above their respective lineages. Phylogenetic relationships were revised according to literature (Glaucophyta - red-green algae [57], Mesostigmatales/Chlorokybales [58], Streptophyte algae [20], bryophytes [59,60], and vascular plants (Embryophyta) [61]). The dotted lines indicate branching events where the consensus about branching order is not well established yet. Arrows indicate the following species. Angiosperms: Arabidopsis thaliana; Oryza sativa; Populus trichocarpa; Vitis vinifera. Lycopodiopsida (club mosses): Selaginella moellendorffii. Bryophyta (mosses): Physcomitrella patens. Chlorophyta (green algae): Chlamydomonas reinhardtii; Ostreococcus tauri; Micromonas pusilla. Rhodophyta (red algae): Cyanidioschyzon merolae.

Figure 2

The predicted structure of PIN proteins. The sequence shown is derived from AtPIN7; the positions marked in yellow are invariant in sequences of all 'long' PINs, the positions marked in red are invariant in sequences of all PINs.

Figure 3

Cladogram of PIN proteins. The protein sequences of PINs were obtained from a repository of genomic sequences [62] and were aligned by the package MAFFT (program mafft-linsi, default setting) [63]; the non-homologous parts of the hydrophilic loop were edited out. The cladogram was computed by MrBayes [64] with parameters: lset = invgamma; ngammacat = 6; prset aamodelpr = fixed(wag). The computation was run for 5,000,000 generations, sampled every 100 generations and the first 10,000 generations were discarded. The sequences are divided into different groups according to the sequence similarity of the hydrophilic loop. All members of group 5 have a similar sequence in the hydrophilic loop but subgroup 5a has a site for phosphorylation by PINOID kinase whereas subgroup 5b lacks it. Species abbreviations: At, Arabidopsis thaliana; Alyr, Arabidopsis lyrata; Bradi, Brachypodium distachyon; Cpap, Carica papaya; Glyma, Glycine maxima; Mtru, Medicago truncatula; Osat, Oryza sativa; Ppat, Physcomitrella patens; Ptri, Populus trichocarpa; Smoel, Selaginella moellendorffii; Sb, Sorghum bicolor; Vvin, Vitis vinifera; Zm, Zea mays.

Figure 4

Typical genomic organization of the AtPIN genes using AtPIN4 as the example. Exons are displayed as black squares and introns as white squares with the positions of exon/intron borders marked.

Figure 5

Expression map of Arabidopsis thaliana PIN genes compiled from both promoter activity data and protein localization. Each PIN gene-expression domain is marked out by a colored line (see key in upper right corner. The organs depicted are (a) flower; (b) embryo (late globular stage); (c) stem; (d) rosette leaf; (e) mature part of the primary root; (f) lateral root primordium (stage 5); (g) root tip. The figure is based on the data from [11,12,14,22,23,65,66]. Note that PIN5 expression is not depicted, as it is expressed weakly throughout the aerial part of the plant with maxima in the hypocotyl, the guard cells of stomata, and cauline leaves [13,65].

Figure 6

Schematic diagram of an idealized plant cell and the role of specific PIN proteins in auxin management at the cellular level. The low pH in the apoplast (the region outside the cell membrane comprising the plant cell wall) is maintained by the activity of the plasma membrane H⁺-ATPase. In the acidic environment of the apoplast, a relatively high proportion of auxin molecules stay protonated (un-ionized; indole-acetic acid (IAA)) and these can enter the cell directly via passive diffusion. In its ionized (dissociated) form (IAA^- + H⁺), auxin cannot cross membranes by passive diffusion; it needs to be actively transported by carriers. Ionized auxin molecules can enter cells via active transport by auxin-influx carriers. In the relatively higher pH of the cytoplasm, auxin molecules undergo almost complete dissociation. The asymmetric positioning of the auxin-efflux carriers from the 'long' PIN subfamily at the plasma membrane then determines the direction of auxin efflux from the cell. Localization of AtPIN5 (from the 'short' PIN subfamily) at the membranes of the endoplasmic reticulum leads to compartmentalization of auxin into the lumen of the endoplasmic reticulum, where it undergoes metabolic conversion. PM, plasma membrane; ER, endoplasmic reticulum; GA, Golgi apparatus.

Figure 7

Auxin distribution and PIN-dependent auxin-transport routes in the Arabidopsis thaliana root tip. Auxin distribution (depicted as a blue gradient) has been inferred from DR5 activity and indole-acetic acid (IAA) immunolocalization. The localization of auxin transporters is based on immunolocalization studies and on in vivo observations of proteins tagged with green fluorescent protein. Arrows indicate auxin flow mediated by a particular PIN transporter.

Figure 8

Examples of pin loss-of-function phenotypes. (a-d, f) pin1 mutants can have (a) fused leaves, (b) pin-like inflorescence, (c, d) defective flowers and (f) three cotyledons in the seedling. (e) pin2 mutant showing agravitropic root growth. (g) Fused, cup-shaped cotyledons of triple-mutant pin1,3,4 seedling. (h) No apical-basal patterning in a triple-mutant pin1,3,4,7 embryo.