Recent support for the pH gradient-dependent mechanism of PAT came from experiments demonstrating that transgenic Arabidopsis plants overexpressing the H+-pyrophosphatase AVP1 (for Arabidopsis vacuolar pyrophosphatase) have a decreased apoplastic pH and exhibit an increased transport of IAA from the shoot tip to the root. Conversely, in the avp1 loss-of-function mutant, apoplastic pH was more alkaline and basipetal IAA transport was reduced (). These results show that apoplastic pH has an impact on PAT activity, which is in accordance with chemiosmotic model (; ).
This classical chemiosmotic model was strengthened 30 years later by the identification and characterization of proteins for auxin efflux carriers (PIN family) and auxin influx carriers (AUX1 family) (; ; ). The corresponding mutant phenotypes were found to be impaired in auxin transport, and both AUX1 and PIN proteins have been shown to be asymmetrically localized at the cell membrane in accordance with known directions of auxin flux (; ; ; ; , ).
A chemiosmotic model for polar auxin transport proposes that auxin uptake is driven by the proton motive force across the plasma membrane, while auxin efflux is driven by the membrane potential (). The first step in polar transport is auxin influx. Auxin enters plant cells nondirectionally via passive diffusion of the protonated form (IAAH) across the phospholipid bilayer or via secondary active transport of the dissociated form (IAA-) through a 2H+-IAA-symporter. Once IAA enters the cytosol, which has a pH of approximately 7.2, nearly all of it dissociates to the anionic form. Because the membrane is impermeable to the anion, auxin accumulates inside the cell or along membrane surfaces unless it is exported by transport proteins on the plasma membrane. According to the chemiosmotic model, transport of IAA- out of the cell is driven by the negative membrane potential inside the cell.
1. As proposed by the chemiosmotic hypothesis with regard to the efflux carrier components (; ), all PIN proteins display asymmetric (polar) localization at cell membranes in particular cell types, correlating well with the known directions of auxin flow (). PIN1 is expressed from the earliest developmental stages on, first in proembryo cells where it is plasma membrane localized in a non-polar manner, then becoming polarized to the basal (lower) side of provascular cells around the early globular stage (; ). Later, it marks future vascular cells from the tips of developing cotyledons towards the root pole with polarity pointing towards the root pole (). A similar pattern of PIN1 can be also found postembryonically in the root stele, and in the xylem parenchyma cells of more differentiated vasculature in the aerial parts of the plant (; ; ). In the outer cell layers of the embryo and shoot apical meristem, highly dynamic PIN1 expression is associated with forming shoot-derived organ primordia with polarity pointing toward the apex of these organs (; ; ). PIN2 was found to be expressed only postembryonically in the root tip, with the protein localized predominantly at the basal (towards the root apex) side of young cortex cells and at the apical (towards the shoot apex) side of epidermis and lateral root cap cells (; ). PIN3 protein is localized pre-dominantly to the inner baso-lateral side of shoot endodermis (starch sheath) cells or root pericycle cells and symmetrically in the columella cells of the root tip (). PIN4 expression was found from the middle globular stage on, in cells of the central root meristem, with the protein polarity pointing towards the quiescent center (QC) cells but without a clearly defined polarity in the quiescent cells themselves (; ). The expression of PIN7 has been detected during embryogenesis in suspensor cells with polar protein localization at early stages pointing towards the proembryo and switching to the opposite side away from the proembryo during post-globular stages (; ). Postembryonically, PIN7 exhibits a similar pattern to PIN3, in the columella and stele cells of the root meristem ().
The presumption that auxin flow may require carrier-mediated cell-to-cell transport came from the observation that known inhibitors of polar auxin transport such as 2,3,5-tri-iodobenzoic acid (TIBA) increase the accumulation of labeled IAA in maize coleoptile cells, which suggested that TIBA blocked efflux, rather then influx of auxin (). This finding, together with other growing evidence about auxin flow, suggested that auxin-specific carrier proteins may exist, and led to the formulation of the chemiosmotic model for PAT (; ; ). In the fairly acidic environment of the cell wall (pH 5.5), some IAA exists in its protonated form (IAAH). Such a neutral, lipophilic molecule is able to pass the plasma membrane via simple diffusion. In the more alkaline cytosolic environment (pH 7.0) most IAA undergoes deprotonation giving rise to polar IAA− anions. Charged, deprotonated IAA− cannot easily depart from the cell, which consequently leads to the accumulation of IAA molecules inside the cell. IAA− can leave the cell only by active efflux, presumably mediated by specific efflux carriers. An asymmetric, cellular distribution of these carriers within each cell would explain the unidirectional (polar) feature of auxin flow. In other words, auxin efflux carriers being predominantly localized to one side of the cell would provide auxin transport in that direction (). In addition, the existence of specific auxin influx facilitating the uptake of IAA into cells was also proposed and demonstrated (; ).
Recently, a role for the nitrate transporter NRT1.1 in auxin influx has been demonstrated in heterologous system, providing an explanation for its ability to alter LR formation depending on the nitrogen status of the plant (Krouk et al., ). Interestingly, NRT1.1 acts as a transceptor as it is also involved in the perception/transduction of the nitrate signal (Ho et al., ). Further understanding of the auxin transport function of NRT1.1 is of great interest as this provides a direct mechanism for developmental effects of auxin in response to nutrient status of the soil.
As per chemiosmotic polar diffusion hypothesis, the term first coined by Goldsmith () based on the famous work of Rubery and Sheldrake () and Raven () cellular IAA movement is facilitated by combined activities of auxin influx and efflux carriers. IAA is a weak acid (pKa 4.75) and at mildly acidic apoplastic pH, only a small portion of IAA (IAAH ~15%) is able to passively diffuse inside the cell but the majority (85%) of IAA remains in its dissociated form (IAA−) and would require a carrier for its active uptake across the cell (Figure ). Inside the cell, at pH 7.0, all IAA remains in its polar IAA− form and would require auxin efflux carriers (Zazímalová et al., ). Chemiosmotic hypothesis also predicted that the polarity of auxin movement is provided by asymmetric localization of auxin carriers.
Thus, the functional characterization of a number of the PGP proteins suggested that in addition to the proton gradient-driven movement of auxin across the plasma membrane, as described by the chemiosmotic hypothesis, an energized transport of auxin by ABC transporter family proteins operates in plants. It seems that PINs and PGPs define two functionally distinct auxin efflux systems (), but it is hard to imagine that their roles in planta will be entirely independent. Indeed, an extensive and complicated functional interaction between PIN- and PGP-based transport systems has been recently demonstrated, but the developmental role and exact molecular basis of such interactions have been not fully clarified ().
The chemiosmotic hypothesis gained significant support from genetic studies in Arabidopsis thaliana that led to the identification and characterization of molecular components of auxin influx (AUX1/LAX family) and auxin efflux (PIN family) (; ; ), as well as the identification of several PGP proteins from the ABC transporter superfamily, which are also involved in transport of auxin across the plasma membrane (; ). Many of the molecular components of polar auxin transport (PAT), in particular PIN proteins, show asymmetric localization at the cell membrane within auxin transport-competent cells as predicted by the chemiosmotic hypothesis.
The directionality of auxin flow is an important characteristic that distinguishes PAT from other transport processes in plants. A substantial body of evidence has demonstrated that, indeed, the polarity of PAT is a crucial feature in auxin-mediated plant development (reviewed by ; ). As mentioned, the chemiosmotic hypothesis proposed that the polarity of auxin flow is determined by the polar, subcellular localization of auxin efflux carriers (; ). In fact, the polar subcellular localization of PIN auxin efflux carriers correlates with the known or predicted directions of auxin flow, supporting this hypothesis ( and ; reviewed by ; ). In addition, the direct manipulation of PIN polarity has a clear impact on the direction of auxin transport, confirming that cellular PIN positioning is a determining factor in PAT directionality (; . Because comparable data for AUX1 and PGP proteins are lacking so far, the issue of PAT directionality is, at the moment, mostly about how the polar subcellular localization of PIN proteins is controlled.
The identification of substances that can inhibit auxin flow (; ) established that cellular auxin efflux is crucial for auxin transport and provided tools for further studies into the physiological importance of this process. Studies using these inhibitors combined with auxin transport experiments led, in the middle 1970s, to the formulation of the chemiosmotic hypothesis, which proposed a mechanism by which auxin could move from cell to cell. It postulated that auxin is transported into and out of the cell through the action of specific carrier proteins (; ). Importantly, it also proposed that the strictly controlled directionality of auxin flow may be a result of the asymmetric cellular localization of auxin efflux carriers.