The plant signalling molecule auxin provides positional information in a variety of developmental processes by means of its differential distribution (gradients) within plant tissues. Thus, cellular auxin levels often determine the developmental output of auxin signalling. Conceptually, transmembrane transport and metabolic processes regulate the steady-state levels of auxin in any given cell. In particular, PIN auxin-efflux-carrier-mediated, directional transport between cells is crucial for generating auxin gradients. Here we show that Arabidopsis thaliana PIN5, an atypical member of the PIN gene family, encodes a functional auxin transporter that is required for auxin-mediated development. PIN5 does not have a direct role in cell-to-cell transport but regulates intracellular auxin homeostasis and metabolism. PIN5 localizes, unlike other characterized plasma membrane PIN proteins, to endoplasmic reticulum (ER), presumably mediating auxin flow from the cytosol to the lumen of the ER. The ER localization of other PIN5-like transporters (including the moss PIN) indicates that the diversification of PIN protein functions in mediating auxin homeostasis at the ER, and cell-to-cell auxin transport at the plasma membrane, represent an ancient event during the evolution of land plants.

SUMMARYThe apical hook of dark-grown Arabidopsis seedlings is a simple structure that develops soon after germination to protect the meristem tissues during emergence through the soil and that opens upon exposure to light. Differential growth at the apical hook proceeds in three sequential steps that are regulated by multiple hormones, principally auxin and ethylene. We show that the progress of the apical hook through these developmental phases depends on the dynamic, asymmetric distribution of auxin, which is regulated by auxin efflux carriers of the PIN family. Several PIN proteins exhibited specific, partially overlapping spatial and temporal expression patterns, and their subcellular localization suggested auxin fluxes during hook development. Genetic manipulation of individual PIN activities interfered with different stages of hook development, implying that specific combinations of PIN genes are required for progress of the apical hook through the developmental phases. Furthermore, ethylene might modulate apical hook development by prolonging the formation phase and strongly suppressing the maintenance phase. This ethylene effect is in part mediated by regulation of PIN-dependent auxin efflux and auxin signaling.

Phytotropins such as 1-N-naphthylphthalamic acid (NPA) strongly inhibit auxin efflux, but the mechanism of this inhibition remains unknown. Auxin efflux is also strongly decreased by the vesicle trafficking inhibitor brefeldin A (BFA). Using suspension-cultured interphase cells of the BY-2 tobacco (Nicotiana tabacum L. cv Bright-Yellow 2) cell line, we compared the effects of NPA and BFA on auxin accumulation and on the arrangement of the cytoskeleton and endoplasmic reticulum (ER). The inhibition of auxin efflux (stimulation of net accumulation) by both NPA and BFA occurred rapidly with no measurable lag. NPA had no observable effect on the arrangement of microtubules, actin filaments, or ER. Thus, its inhibitory effect on auxin efflux was not mediated by perturbation of the cytoskeletal system and ER. BFA, however, caused substantial alterations to the arrangement of actin filaments and ER, including a characteristic accumulation of actin in the perinuclear cytoplasm. Even at saturating concentrations, NPA inhibited net auxin efflux far more effectively than did BFA. Therefore, a proportion of the NPA-sensitive auxin efflux carriers may be protected from the action of BFA. Maximum inhibition of auxin efflux occurred at concentrations of NPA substantially below those previously reported to be necessary to perturb vesicle trafficking. We found no evidence to support recent suggestions that the action of auxin transport inhibitors is mediated by a general inhibition of vesicle-mediated protein traffic to the plasma membrane.The polar transport of auxins (such as indole-3-acetic acid [IAA]) plays a crucial role in the regulation of growth and development in plants (Davies, 1995). Much experimental evidence supports the proposal by Rubery and Sheldrake (1974) and Raven (1975) that auxin transport polarity results from the differential permeabilities of each end of transporting cells to auxin anions (IAA Ϫ ) and undissociated auxin molecules (IAA; for review, see Goldsmith, 1977). IAA (a weak organic acid) is relatively lipophilic and can readily enter cells by diffusion from the more acidic extracellular space; the IAA Ϫ anion, on the other hand, is hydrophilic and does not cross membranes easily. As a consequence, auxins tend to accumulate in plant cells by a process of "anion trapping" and exit the symplast with the intervention of transmembrane auxin anion efflux carriers (Goldsmith, 1977). There is now overwhelming evidence that the differential efflux of IAA Ϫ anions from the two ends of auxin-transporting cells results from an asymmetric (polar) distribution of such carriers (Goldsmith, 1977;Lomax et al., 1995). Genes encoding putative auxin influx and efflux carriers have been identified from Arabidopsis and other species (for review, see Morris, 2000;Muday and DeLong, 2001;Friml and Palme, 2002). It has been shown that efflux carrier proteins, encoded by members of the PIN (PIN-FORMED) gene family, and possibly influx carriers (encoded by AUX1), are targeted to specific regions of the plasma membrane (PM) i...

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Summary Aluminium ions (Al) have been recognized as a major toxic factor for crop production in acidic soils. This study aimed to assess the impact of Al on the activity of phosphatidylcholine‐hydrolysing phospholipase C (PC‐PLC), a new member of the plant phospholipase family. We labelled the tobacco cell line BY‐2 and pollen tubes with a fluorescent derivative of phosphatidylcholine and assayed for patterns of fluorescently labelled products. Growth of pollen tubes was analysed. We observed a significant decrease of labelled diacylglycerol (DAG) in cells treated with AlCl3. Investigation of possible metabolic pathways that control DAG generation and consumption during the response to Al showed that DAG originated from the reaction catalysed by PC‐PLC. The growth of pollen tubes was retarded in the presence of Al and this effect was accompanied by the decrease of labelled DAG similar to the case of the BY‐2 cell line. The growth of pollen tubes arrested by Al was rescued by externally added DAG. Our observation strongly supports the role of DAG generated by PC‐PLC in the response of tobacco cells to Al.

Aluminum (Al) is a major factor that limits plant growth in acid soils. It causes a cessation of root growth and changes in root morphology suggesting a role of the root cytoskeleton as a target of Al-toxicity. Here we report a rapid effect of Al on the microtubular cytoskeleton of the suspension tobacco cell lines BY-2 and VBI-0. Viability studies showed that the cells were more sensitive to Al during exponential phase as compared to stationary cells. During the first hours of exposure, Al induced the formation of additional bundles of cortical microtubules (cMTs), whereas the thickness of the individual bundles decreased. Prolonged exposure resulted in disorientation of cMTs. These changes of cMTs preceded the decrease of cell viability by several hours and were accompanied by an increase in the levels of alpha-tubulin (in its tyrosinated form) and elements of the tubulin-folding chaperone CCT. These findings suggest that the microtubular cytoskeleton is one of the early targets of Al toxicity.

The results suggest that the ARP2/3 complex has a morphogenetic function related to cell wall synthesis and auxin transport.

Concurrently with cold-induced disintegration of microtubular structures in the cytoplasm, gradual tubulin accumulation was observed in a progressively growing proportion of interphase nuclei in tobacco BY-2 cells. This intranuclear tubulin disappeared upon rewarming. Simultaneously, new microtubules rapidly emerged from the nuclear periphery and reconstituted new cortical arrays, as was shown by immunofluorescence. A rapid exclusion of tubulin from the nucleus during rewarming was also observed in vivo in cells expressing GFP-tubulin. Nuclei were purified from cells that expressed GFP fused to an endoplasmic-reticulum retention signal (BY-2-mGFP5-ER), and green-fluorescent protein was used as a diagnostic marker to confirm that the nuclear fraction was not contaminated by nuclear-envelope proteins. These purified, GFP-free nuclei contained tubulin when isolated from cold-treated cells, whereas control nuclei were void of tubulin. Furthermore, highly conserved putative nuclear-export sequences were identified in tubulin sequences. These results led us to interpret the accumulation of tubulin in interphasic nuclei, as well as its rapid nuclear export, in the context of ancient intranuclear tubulin function during the cell cycle progression.