Intracellular trafficking and proteolysis of the Arabidopsis auxin-efflux facilitator PIN2 are involved in root gravitropism

Root gravitropism describes the orientation of root growth along the gravity vector and is mediated by differential cell elongation in the root meristem. This response requires the coordinated, asymmetric distribution of the phytohormone auxin within the root meristem, and depends on the concerted activities of PIN proteins and AUX1 - members of the auxin transport pathway. Here, we show that intracellular trafficking and proteasome activity combine to control PIN2 degradation during root gravitropism. Following gravi-stimulation, proteasome-dependent variations in PIN2 localization and degradation at the upper and lower sides of the root result in asymmetric distribution of PIN2. Ubiquitination of PIN2 occurs in a proteasome-dependent manner, indicating that the proteasome is involved in the control of PIN2 turnover. Stabilization of PIN2 affects its abundance and distribution, and leads to defects in auxin distribution and gravitropic responses. We describe the effects of auxin on PIN2 localization and protein levels, indicating that redistribution of auxin during the gravitropic response may be involved in the regulation of PIN2 protein.     

生长素输出载体PIN家族研究进展

生长素极性运输调控植物的生长发育。生长素极性运输主要依赖3类转运蛋白: AUX/LAX、PIN和ABCB蛋白家族。生长素在细胞间流动的方向与PIN蛋白在细胞上的极性定位密切相关。PIN蛋白由1个中心亲水环和2个由中心亲水环隔开的疏水区组成。中心亲水环上含多个磷酸化位点,其为一些蛋白激酶的靶点。PIN蛋白受多方面调控,包...     

Complex regulation of Arabidopsis AGR1/PIN2-mediated root gravitropic response and basipetal auxin transport by cantharidin-sensitive protein phosphatases

Polar auxin transport, mediated by two distinct plasma membrane-localized auxin influx and efflux carrier proteins/complexes, plays an important role in many plant growth and developmental processes including tropic responses to gravity and light, development of lateral roots and patterning in embryogenesis. We have previously shown that the Arabidopsis AGRAVITROPIC 1/PIN2 gene encodes an auxin efflux component regulating root gravitropism and basipetal auxin transport. However, the regulatory mechanism underlying the function of AGR1/PIN2 is largely unknown. Recently, protein phosphorylation and dephosphorylation mediated by protein kinases and phosphatases, respectively, have been implicated in regulating polar auxin transport and root gravitropism. Here, we examined the effects of chemical inhibitors of protein phosphatases on root gravitropism and basipetal auxin transport, as well as the expression pattern of AGR1/PIN2 gene and the localization of AGR1/PIN2 protein. We also examined the effects of inhibitors of vesicle trafficking and protein kinases. Our data suggest that protein phosphatases, sensitive to cantharidin and okadaic acid, are likely involved in regulating AGR1/PIN2-mediated root basipetal auxin transport and gravitropism, as well as auxin response in the root central elongation zone (CEZ). BFA-sensitive vesicle trafficking may be required for the cycling of AGR1/PIN2 between plasma membrane and the BFA compartment, but not for the AGR1/PIN2-mediated root basipetal auxin transport and auxin response in CEZ cells.     

Flavonol‐mediated stabilization of PIN efflux complexes regulates polar auxin transport

The transport of auxin controls the rate, direction and localization of plant growth and development. The course of auxin transport is defined by the polar subcellular localization of the PIN proteins, a family of auxin efflux transporters. However, little is known about the composition and regulation of the PIN protein complex. Here, using blue‐native PAGE and quantitative mass spectrometry, we identify native PIN core transport units as homo‐ and heteromers assembled from PIN1, PIN2, PIN3, PIN4 and PIN7 subunits only. Furthermore, we show that endogenous flavonols stabilize PIN dimers to regulate auxin efflux in the same way as does the auxin transport inhibitor 1‐naphthylphthalamic acid (NPA). This inhibitory mechanism is counteracted both by the natural auxin indole‐3‐acetic acid and by phosphomimetic amino acids introduced into the PIN1 cytoplasmic domain. Our results lend mechanistic insights into an endogenous control mechanism which regulates PIN function and opens the way for a deeper understanding of the protein environment and regulation of the polar auxin transport complex.     

Evaluating auxin distribution in tomato (Solanum lycopersicum) through an analysis of the PIN and AUX/LAX gene families

The temporal and spatial control of auxin distribution has a key role in the regulation of plant growth and development, and much has been learnt about the mechanisms that influence auxin pools and gradients in vegetative tissues, particularly in Arabidopsis. For example polar auxin transport, mediated by PIN and AUX/LAX proteins, is central to the control of auxin distribution. In contrast, very little information is known about the dynamics of auxin distribution and the molecular basis of its transport within and between fruit tissues, despite the fact that auxin regulates many aspects of fruit development, which include fruit formation, expansion, ripening and abscission. In addition, functional information regarding the key regulators of auxin fluxes during both vegetative and reproductive development in species other than Arabidopsis is scarce. To address these issues, we have investigated the spatiotemporal distribution of auxin during tomato (Solanum lycopersicum) fruit development and the function of the PIN and AUX/LAX gene families. Differential concentrations of auxin become apparent during early fruit growth, with auxin levels being higher in internal tissues than in the fruit pericarp and the pattern of auxin accumulation depended on polar transport. Ten tomato PIN (SlPIN1 to 10) and five AUX/LAX (SlLAX1 to 5) genes were identified and found to display heterogeneous expression patterns, with tissue and developmental-stage specificity. RNAi-mediated co-silencing of SlPIN4 and SlPIN3 did not affect fruit development, which suggested functional redundancy of PIN proteins, but did lead to a vegetative phenotype, and revealed a role for these genes in the regulation of tomato shoot architecture.     

Functional expression and characterization of Arabidopsis ABCB,AUX 1 and PIN auxin transporters in Schizosaccharomyces pombe

Heterologous expression systems based on tobacco BY‐2 cells, Arabidopsis cell cultures, Xenopus oocytes, Saccharomyces cerevisiae, and human HeLa cells have been used to express and characterize PIN, ABCB (PGP), and AUX/LAX auxin transporters from Arabidopsis. However, no single system has been identified that can be used for effective comparative analyses of these proteins. We have developed an accessible Schizosaccharomyces pombe system for comparative studies of plant transport proteins. The system includes knockout mutants in all ABC and putative auxin transport genes and Gateway^®‐compatible expression vectors for functional analysis and subcellular localization of recombinant proteins. We expressed Arabidopsis ABCB1 and ABCB19 in mam1pdr1 host lines under the inducible nmt41 promoter. ABCB19 showed a higher ³H‐IAA export activity than ABCB1. Arabidopsis PIN proteins were expressed in a mutant lacking the auxin effluxer like 1 (AEL1) gene. PIN1 showed higher activity than PIN2 with similar protein expression levels. Expression of AUX1 in a permease‐deficient vat3 mutant resulted in increased net auxin uptake activity. Finally, ABCB4 expressed in mam1pdr1 displayed a concentration‐dependent reversal of ³H‐IAA transport that is consistent with its observed activity in planta. Structural modelling suggests that ABCB4 has three substrate interaction sites rather than the two found in ABCB19, thus providing a rationale for the observed substrate activation. Taken together, these results suggest that the S. pombe system described here can be employed for comparative analyses and subsequent structural characterizations of plant transport proteins.     

Dynamics in PIN2 auxin carrier ubiquitylation in gravity-responding Arabidopsis roots

Plant tropisms are decisively influenced by dynamic adjustments in spatiotemporal distribution of the growth regulators auxin. Polar auxin transport requires activity of PIN-type auxin carrier proteins, with their distribution at the plasma membrane significantly contributing to the directionality of auxin flow. Control of PIN protein distribution involves regulation of their endocytosis and further sorting into the lytic vacuole for degradation and recently, protein ubiquitylation has been demonstrated to control degradative sorting of plasma membrane proteins in plants.1-6 Here we show dynamic adjustments in PIN2 ubiquitylation in gravity-stimulated roots, a response that coincides with establishment of a lateral PIN2 expression gradient. Our results imply that perception and transduction of gravity signals triggers differential ubiquitylation of PIN2, which might feed back on the coordination of auxin distribution in root meristems.     

The exocyst complex contributes to PIN auxin efflux carrier recycling and polar auxin transport in Arabidopsis

In land plants polar auxin transport is one of the substantial processes guiding whole plant polarity and morphogenesis. Directional auxin fluxes are mediated by PIN auxin efflux carriers, polarly localized at the plasma membrane. The polarization of exocytosis in yeast and animals is assisted by the exocyst: an octameric vesicle‐tethering complex and an effector of Rab and Rho GTPases. Here we show that rootward polar auxin transport is compromised in roots of Arabidopsis thaliana loss‐of‐function mutants in the EXO70A1 exocyst subunit. The recycling of PIN1 and PIN2 proteins from brefeldin–A compartments is delayed after the brefeldin‐A washout in exo70A1 and sec8 exocyst mutants. Relocalization of PIN1 and PIN2 proteins after prolonged brefeldin‐A treatment is largely impaired in these mutants. At the same time, however, plasma membrane localization of GFP:EXO70A1, and the other exocyst subunits studied (GFP:SEC8 and YFP:SEC10), is resistant to brefeldin‐A treatment. In root cells of the exo70A1 mutant, a portion of PIN2 is internalized and retained in specific, abnormally enlarged, endomembrane compartments that are distinct from VHA‐a1‐labelled early endosomes or the trans‐Golgi network, but are RAB‐A5d positive. We conclude that the exocyst is involved in PIN1 and PIN2 recycling, and thus in polar auxin transport regulation.     

铝胁迫对拟南芥根尖ＡｔＰＩＮ２ 蛋白表达活性的影响

铝胁迫能影响根尖生长素的运输,这与生长素运输载体密切相关,PIN2作为根尖生长素的运输蛋白,其独特的组织定位可能诱导PIN2蛋白参与了铝调节生长素的运输过程。该研究以拟南芥PIN2缺失突变体( pin2)、PIN2□∷□GFP融合体及其野生型( WT)为材料,应用激光扫描共聚焦显微技术,研究铝处理对拟南芥根尖生长素运输蛋白PIN2的表达活性、蛋白在组织及亚细胞水平分布及其对铝内置化作用的影响。结果表明：短期铝处理或低铝浓度能明显增加拟南芥根尖细胞PIN2蛋白表达活性,而长期铝处理或高铝浓度抑制其表达活性;以100μmol?L-1 AlCl3处理4 h的蛋白表达活性最高。蛋白印迹反应发现,铝处理促进PIN2蛋白在细胞膜上累积,减少胞内囊泡中PIN2蛋白的含量;囊泡运输抑制剂( BFA)能抑制铝诱导PIN2蛋白的分配。铝胁迫增加拟南芥根尖细胞H2 O2累积,pin2的H2 O2累积量大于WT,而相对根长小于WT。 Morin染色结果显示,pin2的铝内置化显著小于WT。上述研究表明,PIN2蛋白在100μmol?L-1 AlCl3处理条件下活性最高,细胞膜累积程度加强,铝内置化能力增强,从而调节根系的生长发育。该研究结果进一步为铝抑制生长素的运输机制提供了理论基础。     

Polarization of PIN3-dependent auxin transport for hypocotyl gravitropic response in Arabidopsis thaliana

Gravitropism aligns plant growth with gravity. It involves gravity perception and the asymmetric distribution of the phytohormone auxin. Here we provide insights into the mechanism for hypocotyl gravitropic growth. We show that the Arabidopsis thaliana PIN3 auxin transporter is required for the asymmetric auxin distribution for the gravitropic response. Gravistimulation polarizes PIN3 to the bottom side of hypocotyl endodermal cells, which correlates with an increased auxin response at the lower hypocotyl side. Both PIN3 polarization and hypocotyl bending require the activity of the trafficking regulator GNOM and the protein kinase PINOID. Our data suggest that gravity-induced PIN3 polarization diverts the auxin flow to mediate the asymmetric distribution of auxin for gravitropic shoot bending.     

Subcellular trafficking of PIN auxin efflux carriers in auxin transport

The directional transport of the plant hormone auxin is a unique process mediating a wide variety of developmental processes. Auxin movement between cells depends on AUX1/LAX, PGP and PIN protein families that mediate auxin transport across the plasma membrane. The directionality of auxin flow within tissues is largely determined by polar, subcellular localization of PIN auxin efflux carriers. PIN proteins undergo rapid subcellular dynamics that is important for the process of auxin transport and its directionality. Furthermore, various environmental and endogenous signals can modulate trafficking and polarity of PIN proteins and by this mechanism change auxin distribution. Thus, the subcellular dynamics of auxin transport proteins represents an important interface between cellular processes and development of the whole plant. This review summarizes our recent contributions to the field of PIN trafficking and auxin transport regulation.     

Sterol-dependent endocytosis mediates post-cytokinetic acquisition of PIN2 auxin efflux carrier polarity

The polarization of yeast and animal cells relies on membrane sterols for polar targeting of proteins to the plasma membrane, their polar endocytic recycling and restricted lateral diffusion. However, little is known about sterol function in plant-cell polarity. Directional root growth along the gravity vector requires polar transport of the plant hormone auxin. In Arabidopsis, asymmetric plasma membrane localization of the PIN-FORMED2 (PIN2) auxin transporter directs root gravitropism. Although the composition of membrane sterols influences gravitropism and localization of two other PIN proteins, it remains unknown how sterols contribute mechanistically to PIN polarity. Here, we show that correct membrane sterol composition is essential for the acquisition of PIN2 polarity. Polar PIN2 localization is defective in the sterol-biosynthesis mutant cyclopropylsterol isomerase1-1 (cpi1-1) which displays altered sterol composition, PIN2 endocytosis, and root gravitropism. At the end of cytokinesis, PIN2 localizes initially to both newly formed membranes but subsequently disappears from one. By contrast, PIN2 frequently remains at both daughter membranes in endocytosis-defective cpi1-1 cells. Hence, sterol composition affects post-cytokinetic acquisition of PIN2 polarity by endocytosis, suggesting a mechanism for sterol action on establishment of asymmetric protein localization.     

Inositol trisphosphate-induced Ca2+ signaling modulates auxin transport and PIN polarity

The phytohormone auxin is an important determinant of plant development. Directional auxin flow within tissues depends on polar localization of PIN auxin transporters. To explore regulation of PIN-mediated auxin transport, we screened for suppressors of PIN1 overexpression (supo) and identified an inositol polyphosphate 1-phosphatase mutant (supo1), with elevated inositol trisphosphate (InsP(3)) and cytosolic Ca(2+) levels. Pharmacological and genetic increases in InsP(3) or Ca(2+) levels also suppressed the PIN1 gain-of-function phenotypes and caused defects in basal PIN localization, auxin transport and auxin-mediated development. In contrast, the reductions in InsP(3) levels and Ca(2+) signaling antagonized the effects of the supo1 mutation and disrupted preferentially apical PIN localization. InsP(3) and Ca(2+) are evolutionarily conserved second messengers involved in various cellular functions, particularly stress responses. Our findings implicate them as modifiers of cell polarity and polar auxin transport, and highlight a potential integration point through which Ca(2+) signaling-related stimuli could influence auxin-mediated development.     

Arabidopsis phosphatidylinositol monophosphate 5-kinase 2 is involved in root gravitropism through regulation of polar auxin transport by affecting the cycling of PIN proteins

Phosphatidylinositol monophosphate 5-kinase (PIP5K) catalyzes the synthesis of PI-4,5-bisphosphate (PtdIns(4,5)P(2)) by phosphorylation of PI-4-phosphate at the 5 position of the inositol ring, and is involved in regulating multiple developmental processes and stress responses. We here report on the functional characterization of Arabidopsis PIP5K2, which is expressed during lateral root initiation and elongation, and whose expression is enhanced by exogenous auxin. The knockout mutant pip5k2 shows reduced lateral root formation, which could be recovered with exogenous auxin, and interestingly, delayed root gravity response that could not be recovered with exogenous auxin. Crossing with the DR5-GUS marker line and measurement of free IAA content confirmed the reduced auxin accumulation in pip5k2. In addition, analysis using the membrane-selective dye FM4-64 revealed the decelerated vesicle trafficking caused by PtdIns(4,5)P(2) reduction, which hence results in suppressed cycling of PIN proteins (PIN2 and 3), and delayed redistribution of PIN2 and auxin under gravistimulation in pip5k2 roots. On the contrary, PtdIns(4,5)P(2) significantly enhanced the vesicle trafficking and cycling of PIN proteins. These results demonstrate that PIP5K2 is involved in regulating lateral root formation and root gravity response, and reveal a critical role of PIP5K2/PtdIns(4,5)P(2) in root development through regulation of PIN proteins, providing direct evidence of crosstalk between the phosphatidylinositol signaling pathway and auxin response, and new insights into the control of polar auxin transport.     

A dominant negative mutant of protein kinase CK2 exhibits altered auxin responses in Arabidopsis

Protein kinase CK2 is a pleiotropic Ser/Thr kinase, evolutionary conserved in eukaryotes. Studies performed in different organisms, from yeast to humans, have highlighted the importance of CK2 in cell growth and cell-cycle control. However, the signalling pathways in which CK2 is involved have not been fully identified. In plants, the phytohormone auxin is a major regulator of cell growth. Recent discoveries have demonstrated that differential distribution of within auxin plant tissues is essential for developmental processes, and that this distribution is dependent on polar auxin transport. We report here that a dominant-negative mutant of CK2 (CK2mut) in Arabidopsis thaliana shows phenotypic traits that are typically linked to alterations in auxin-dependent processes. However, CK2mut plants exhibit normal responses to exogenous indole-3-acetic acid (IAA) indicating that they are not affected in the perception of the hormone but upstream in the pathway. We demonstrate that mutant plants are not deficient in IAA but are impaired in its transport. Using genetic and pharmacological tools we show that CK2 activity depletion hinders correct formation of auxin gradients and leads to widespread changes in the expression of auxin-related genes. In particular, members of the auxin efflux carrier family (PINs), and the protein kinase PINOID, both key regulators of auxin fluxes, were misexpressed. PIN4 and PIN7 were also found mislocalized, with accumulation in endosomal bodies. We propose that CK2 functions in the regulation of auxin-signalling pathways, particularly in auxin transport.     

A PP6-type phosphatase holoenzyme directly regulates PIN phosphorylation and auxin efflux in Arabidopsis

The directional transport of the phytohormone auxin depends on the phosphorylation status and polar localization of PIN-FORMED (PIN) auxin efflux proteins. While PINIOD (PID) kinase is directly involved in the phosphorylation of PIN proteins, the phosphatase holoenzyme complexes that dephosphorylate PIN proteins remain elusive. Here, we demonstrate that mutations simultaneously disrupting the function of Arabidopsis thaliana FyPP1 (for Phytochrome-associated serine/threonine protein phosphatase1) and FyPP3, two homologous genes encoding the catalytic subunits of protein phosphatase6 (PP6), cause elevated accumulation of phosphorylated PIN proteins, correlating with a basal-to-apical shift in subcellular PIN localization. The changes in PIN polarity result in increased root basipetal auxin transport and severe defects, including shorter roots, fewer lateral roots, defective columella cells, root meristem collapse, abnormal cotyledons (small, cup-shaped, or fused cotyledons), and altered leaf venation. Our molecular, biochemical, and genetic data support the notion that FyPP1/3, SAL (for SAPS DOMAIN-LIKE), and PP2AA proteins (RCN1 [for ROOTS CURL IN NAPHTHYLPHTHALAMIC ACID1] or PP2AA1, PP2AA2, and PP2AA3) physically interact to form a novel PP6-type heterotrimeric holoenzyme complex. We also show that FyPP1/3, SAL, and PP2AA interact with a subset of PIN proteins and that for SAL the strength of the interaction depends on the PIN phosphorylation status. Thus, an Arabidopsis PP6-type phosphatase holoenzyme acts antagonistically with PID to direct auxin transport polarity and plant development by directly regulating PIN phosphorylation.