Auxin transport inhibitors block PIN1 cycling and vesicle trafficking

Polar transport of the phytohormone auxin mediates various processes in plant growth and development, such as apical dominance, tropisms, vascular patterning and axis formation. This view is based largely on the effects of polar auxin transport inhibitors. These compounds disrupt auxin efflux from the cell but their mode of action is unknown. It is thought that polar auxin flux is caused by the asymmetric distribution of efflux carriers acting at the plasma membrane. The polar localization of efflux carrier candidate PIN1 supports this model. Here we show that the seemingly static localization of PIN1 results from rapid actin-dependent cycling between the plasma membrane and endosomal compartments. Auxin transport inhibitors block PIN1 cycling and inhibit trafficking of membrane proteins that are unrelated to auxin transport. Our data suggest that PIN1 cycling is of central importance for auxin transport and that auxin transport inhibitors affect efflux by generally interfering with membrane-trafficking processes. In support of our conclusion, the vesicle-trafficking inhibitor brefeldin A mimics physiological effects of auxin transport inhibitors.     

生长素调控极性运输载体PIN2质膜丰度与降解

主要观察了外源生长素长时间(8 h)处理对拟南芥生长素极性输出载体PIN2-GFP质膜丰度的影响,低温、KCN处理和生长素受体突变对生长素调控质膜PIN2-GFP丰度的影响,以及液泡H+-ATPase抑制剂ConA对野生型和生长素受体四突变体tir1afb1,2,3液泡中PIN2-GFP积累的影响.结果表明:外源生长素通过其受体TIR1/AFB介导的信号途径下调质膜PIN2-GFP的丰度,促进PIN2-GFP的内吞、胞内运输和液泡降解.暗示生长素通过TIR1/AFB介导的信号途径反馈调控质膜PIN2-GFP的水平,从而阻止了胞内生长素的过多输出.     

AtSNX1 defines an endosome for auxin-carrier trafficking in Arabidopsis

Polarized cellular distribution of the phytohormone auxin and its carriers is essential for normal plant growth and development. Polar auxin transport is maintained by a network of auxin influx (AUX) and efflux (PIN) carriers. Both auxin transport and PIN protein cycling between the plasma membrane and endosomes require the activity of the endosomal GNOM; however, intracellular routes taken by these carriers remain largely unknown. Here we show that Arabidopsis thaliana SORTING NEXIN 1 (AtSNX1) is involved in the auxin pathway and that PIN2, but not PIN1 or AUX1, is transported through AtSNX1-containing endosomes. We demonstrate that the snx1-null mutant exhibits multiple auxin-related defects and that loss of function of AtSNX1 severely enhances the phenotype of a weak gnom mutant. In root cells, we further show that AtSNX1 localizes to an endosomal compartment distinct from GNOM-containing endosomes, and that PIN2 accumulates in this compartment after treatment with the phosphatidylinositol-3-OH kinase inhibitor wortmannin or after a gravity stimulus. Our data reveal the existence of a novel endosomal compartment involved in PIN2 endocytic sorting and plant development.     

An ARF-GEF acting at the Golgi and in selective endocytosis in polarized plant cells

Circumstantial evidence suggests that intracellular membrane trafficking pathways diversified independently in the plant kingdom, but documented examples are rare. ARF-GEFs (guanine-nucleotide exchange factors for ADP-ribosylation factor GTPases) are essential for vesicular trafficking in all eukaryotic kingdoms, but of the eight ARF-GEF families, only the ancestral BIG and GBF types are found in plants. Whereas fungal and animal GBF proteins perform conserved functions at the Golgi, the Arabidopsis thaliana GBF protein GNOM is thought to act in only the process of recycling from endosomes. We now show that the related Arabidopsis GBF protein GNOM-LIKE1 (GNL1) has an ancestral function at the Golgi but is also required for selective internalization from the plasma membrane in the presence of brefeldin A (BFA). We identified gnl1 mutants that accumulated biosynthetic and recycling endoplasmic reticulum markers in enlarged internal compartments. Notably, in the absence of functional GNL1, Golgi stacks were rendered sensitive to the selective ARF-GEF inhibitor BFA, which caused them to fuse with the endoplasmic reticulum. Furthermore, in BFA-treated gnl1 roots, the internalization of a polar plasma-membrane marker, the auxin efflux carrier PIN2, was selectively inhibited. Thus, GNL1 is a BFA-resistant GBF protein that functions with a BFA-sensitive ARF-GEF both at the Golgi and in selective endocytosis, but not in recycling from endosomes. We propose that the evolution of endocytic trafficking in plants was accompanied by neofunctionalization within the GBF family, whereas in other kingdoms it occurred independently by elaboration of additional ARF-GEF families.     

Lateral relocation of auxin efflux regulator PIN3 mediates tropism in Arabidopsis

Long-standing models propose that plant growth responses to light or gravity are mediated by asymmetric distribution of the phytohormone auxin. Physiological studies implicated a specific transport system that relocates auxin laterally, thereby effecting differential growth; however, neither the molecular components of this system nor the cellular mechanism of auxin redistribution on light or gravity perception have been identified. Here, we show that auxin accumulates asymmetrically during differential growth in an efflux-dependent manner. Mutations in the Arabidopsis gene PIN3, a regulator of auxin efflux, alter differential growth. PIN3 is expressed in gravity-sensing tissues, with PIN3 protein accumulating predominantly at the lateral cell surface. PIN3 localizes to the plasma membrane and to vesicles that cycle in an actin-dependent manner. In the root columella, PIN3 is positioned symmetrically at the plasma membrane but rapidly relocalizes laterally on gravity stimulation. Our data indicate that PIN3 is a component of the lateral auxin transport system regulating tropic growth. In addition, actin-dependent relocalization of PIN3 in response to gravity provides a mechanism for redirecting auxin flux to trigger asymmetric growth.     

Enhanced gravi- and phototropism in plant mdr mutants mislocalizing the auxin efflux protein PIN1

Many aspects of plant growth and development are dependent on the flow of the hormone auxin down the plant from the growing shoot tip where it is synthesized. The direction of auxin transport in stems is believed to result from the basal localization within cells of the PIN1 membrane protein, which controls the efflux of the auxin anion. Mutations in two genes homologous to those encoding the P-glycoprotein ABC transporters that are especially abundant in multidrug-resistant tumour cells in animals were recently shown to block polar auxin transport in the hypocotyls of Arabidopsis seedlings. Here we show that the mdr mutants display faster and greater gravitropism and enhanced phototropism instead of the impaired curvature development expected in mutants lacking polar auxin transport. We find that these phenotypes result from a disruption of the normal accumulation of PIN1 protein along the basal end of hypocotyl cells associated with basipetal auxin flow. Lateral auxin conductance becomes relatively larger as a result, enhancing the growth differentials responsible for tropic responses.     

Generation of cell polarity in plants links endocytosis, auxin distribution and cell fate decisions

Dynamically polarized membrane proteins define different cell boundaries and have an important role in intercellular communication-a vital feature of multicellular development. Efflux carriers for the signalling molecule auxin from the PIN family are landmarks of cell polarity in plants and have a crucial involvement in auxin distribution-dependent development including embryo patterning, organogenesis and tropisms. Polar PIN localization determines the direction of intercellular auxin flow, yet the mechanisms generating PIN polarity remain unclear. Here we identify an endocytosis-dependent mechanism of PIN polarity generation and analyse its developmental implications. Real-time PIN tracking showed that after synthesis, PINs are initially delivered to the plasma membrane in a non-polar manner and their polarity is established by subsequent endocytic recycling. Interference with PIN endocytosis either by auxin or by manipulation of the Arabidopsis Rab5 GTPase pathway prevents PIN polarization. Failure of PIN polarization transiently alters asymmetric auxin distribution during embryogenesis and increases the local auxin response in apical embryo regions. This results in ectopic expression of auxin pathway-associated root-forming master regulators in embryonic leaves and promotes homeotic transformation of leaves to roots. Our results indicate a two-step mechanism for the generation of PIN polar localization and the essential role of endocytosis in this process. It also highlights the link between endocytosis-dependent polarity of individual cells and auxin distribution-dependent cell fate establishment for multicellular patterning.     

拟南芥PIN2介导的生长素极性运输调控植物根向地性

主要观察了拟南芥生长素输出载体PIN2及其介导的极性运输、生长素诱导合成对根尖生长素不对称分布和根向地性反应的影响.结果表明:拟南芥PIN2基因突变、诱导内源生长素IAA及用抑制剂NPA或TIBA抑制生长素极性运输都严重影响了根尖生长素不对称分布的形成,最终抑制植物根向地性反应,暗示PIN2介导的生长素极性运输和生长素不对称分布在根向地性反应中起着关键性调控作用.从这些研究结果可进一步理解生长素调控植物根向地性的分子机理.     

Efflux-dependent auxin gradients establish the apical-basal axis of Arabidopsis

Axis formation occurs in plants, as in animals, during early embryogenesis. However, the underlying mechanism is not known. Here we show that the first manifestation of the apical-basal axis in plants, the asymmetric division of the zygote, produces a basal cell that transports and an apical cell that responds to the signalling molecule auxin. This apical-basal auxin activity gradient triggers the specification of apical embryo structures and is actively maintained by a novel component of auxin efflux, PIN7, which is located apically in the basal cell. Later, the developmentally regulated reversal of PIN7 and onset of PIN1 polar localization reorganize the auxin gradient for specification of the basal root pole. An analysis of pin quadruple mutants identifies PIN-dependent transport as an essential part of the mechanism for embryo axis formation. Our results indicate how the establishment of cell polarity, polar auxin efflux and local auxin response result in apical-basal axis formation of the embryo, and thus determine the axiality of the adult plant.     

Identification of auxin responsive genes in Arabidopsis by cDNA array

The plant hormone auxin influences a variety of developmental and physiological processes. But the mechanism of its action is quite unclear. In order to identify and analyze the expression of auxin responsive genes, a cDNA array approach was used to screen for genes with altered expression from Arabidopsis suspension culture after IAA treatment and was identified 50 differentially expressed genes from 13824 cDNA clones. These genes were related to signal transduction, stress responses, senescence, photosynthesis, protein biosynthesis and transportation. The results provide the molecular evidence that auxin influences a variety of physiological processes and pave a way for further investigation of the mechanism of auxin action. Furthermore,we found that the expression of a ClpC (regulation subunit of Clp protease) was repressed by exogenous auxin, but increased in dark-induced senescing leaves. This suggests that ClpC may be a senescence-associated gene and can be regulated by auxin.     

过氧化氢对拟南芥生长素信号传导相关蛋白的影响

本论文通过添加外源过氧化氢来探究其对植物生长素信号的影响。以GUS基因作为报告基因,研究拟南芥中各生长素相关蛋白(生长素含量标志蛋白DR5、输入蛋白AUX1、输出蛋白PIN1和PIN2)对过氧化氢的响应。结果表明过氧化氢处理后拟南芥呈植株变小、根变短和不定根数目增多的表型;与对照相比,GUS染色后各生长素相关蛋白的表达下降,这表明过氧化氢对植物生长素的合成与运输起一定的抑制作用。在过氧化氢处理的基础上,添加抗氧化物质发现植株表型有明显的恢复,GUS染色也表明相关蛋白的表达量均增加;另外,0.0001 ?mol/L的外源IAA也能恢复过氧化氢处理后拟南芥的表型和生长素相关蛋白的表达。综上所述,过氧化氢主要通过抑制植物体内的生长素信号来调控植物的生长发育。     

Auxin promotes Arabidopsis root growth by modulating gibberellin response

The growth of plant organs is influenced by a stream of the phytohormone auxin that flows from the shoot apex to the tip of the root. However, until now it has not been known how auxin regulates the cell proliferation and enlargement that characterizes organ growth. Here we show that auxin controls the growth of roots by modulating cellular responses to the phytohormone gibberellin (GA). GA promotes the growth of plants by opposing the effects of nuclear DELLA protein growth repressors, one of which is Arabidopsis RGA (for repressor of gal-3). GA opposes the action of several DELLA proteins by destabilizing them, reducing both the concentration of detectable DELLA proteins and their growth-restraining effects. We also show that auxin is necessary for GA-mediated control of root growth, and that attenuation of auxin transport or signalling delays the GA-induced disappearance of RGA from root cell nuclei. Our observations indicate that the shoot apex exerts long-distance control on the growth of plant organs through the effect of auxin on GA-mediated DELLA protein destabilization.     

Regulation of phyllotaxis by polar auxin transport

The regular arrangement of leaves around a plant's stem, called phyllotaxis, has for centuries attracted the attention of philosophers, mathematicians and natural scientists; however, to date, studies of phyllotaxis have been largely theoretical. Leaves and flowers are formed from the shoot apical meristem, triggered by the plant hormone auxin. Auxin is transported through plant tissues by specific cellular influx and efflux carrier proteins. Here we show that proteins involved in auxin transport regulate phyllotaxis. Our data indicate that auxin is transported upwards into the meristem through the epidermis and the outermost meristem cell layer. Existing leaf primordia act as sinks, redistributing auxin and creating its heterogeneous distribution in the meristem. Auxin accumulation occurs only at certain minimal distances from existing primordia, defining the position of future primordia. This model for phyllotaxis accounts for its reiterative nature, as well as its regularity and stability.     

A novel sensor to map auxin response and distribution at high spatio-temporal resolution

Auxin is a key plant morphogenetic signal but tools to analyse dynamically its distribution and signalling during development are still limited. Auxin perception directly triggers the degradation of Aux/IAA repressor proteins. Here we describe a novel Aux/IAA-based auxin signalling sensor termed DII-VENUS that was engineered in the model plant Arabidopsis thaliana. The VENUS fast maturing form of yellow fluorescent protein was fused in-frame to the Aux/IAA auxin-interaction domain (termed domain II; DII) and expressed under a constitutive promoter. We initially show that DII-VENUS abundance is dependent on auxin, its TIR1/AFBs co-receptors and proteasome activities. Next, we demonstrate that DII-VENUS provides a map of relative auxin distribution at cellular resolution in different tissues. DII-VENUS is also rapidly degraded in response to auxin and we used it to visualize dynamic changes in cellular auxin distribution successfully during two developmental responses, the root gravitropic response and lateral organ production at the shoot apex. Our results illustrate the value of developing response input sensors such as DII-VENUS to provide high-resolution spatio-temporal information about hormone distribution and response during plant growth and development.     

A small GTP-binding protein dissociates from synaptic vesicles during exocytosis.

Low-molecular-weight GTP-binding proteins are strong candidates for regulators of membrane traffic. In yeast, mutations in the sec4 or ypt1 genes encoding small GTP-binding proteins inhibit constitutive membrane flow at the plasma membrane or Golgi complex, respectively. It has been suggested that membrane fusion-fission events are regulated by cycling of small GTP-binding proteins between a membrane-bound and free state, but although most of these small proteins are found in both soluble and tightly membrane-bound forms, there is no direct evidence to support such cycling. In rat brain a small GTP-binding protein, rab3A, is exclusively associated with synaptic vesicles, the secretory organelles of nerve terminals. Here we use isolated nerve terminals to study the fate of rab3A during synaptic vesicle exocytosis. We find that rab3A dissociates quantitatively from the vesicle membrane after Ca2(+)-dependent exocytosis and that this dissociation is partially reversible during recovery after stimulation. These results are direct evidence for an association-dissociation cycle of a small GTP-binding protein during traffic of its host membrane.     

CO2 regulator SLAC1 and its homologues are essential for anion homeostasis in plant cells

The continuing rise in atmospheric [CO2] is predicted to have diverse and dramatic effects on the productivity of agriculture, plant ecosystems and gas exchange. Stomatal pores in the epidermis provide gates for the exchange of CO2 and water between plants and the atmosphere, processes vital to plant life. Increased [CO2] has been shown to enhance anion channel activity proposed to mediate efflux of osmoregulatory anions (Cl- and malate(2-)) from guard cells during stomatal closure. However, the genes encoding anion efflux channels in plant plasma membranes remain unknown. Here we report the isolation of an Arabidopsis gene, SLAC1 (SLOW ANION CHANNEL-ASSOCIATED 1, At1g12480), which mediates CO2 sensitivity in regulation of plant gas exchange. The SLAC1 protein is a distant homologue of bacterial and fungal C4-dicarboxylate transporters, and is localized specifically to the plasma membrane of guard cells. It belongs to a protein family that in Arabidopsis consists of four structurally related members that are common in their plasma membrane localization, but show distinct tissue-specific expression patterns. The loss-of-function mutation in SLAC1 was accompanied by an over-accumulation of the osmoregulatory anions in guard cell protoplasts. Guard-cell-specific expression of SLAC1 or its family members resulted in restoration of the wild-type stomatal responses, including CO2 sensitivity, and also in the dissipation of the over-accumulated anions. These results suggest that SLAC1-family proteins have an evolutionarily conserved function that is required for the maintenance of organic/inorganic anion homeostasis on the cellular level.