Gravitropism of Arabidopsis thaliana Roots Requires the Polarization of PIN2 toward the Root Tip in Meristematic Cortical Cells

In the root, the transport of auxin from the tip to the elongation zone, referred to here as shootward, governs gravitropic bending. Shootward polar auxin transport, and hence gravitropism, depends on the polar deployment of the PIN-FORMED auxin efflux carrier PIN2. In Arabidopsis thaliana, PIN2 has the expected shootward localization in epidermis and lateral root cap; however, this carrier is localized toward the root tip (rootward) in cortical cells of the meristem, a deployment whose function is enigmatic. We use pharmacological and genetic tools to cause a shootward relocation of PIN2 in meristematic cortical cells without detectably altering PIN2 polarization in other cell types or PIN1 polarization. This relocation of cortical PIN2 was negatively regulated by the membrane trafficking factor GNOM and by the regulatory A1 subunit of type 2-A protein phosphatase (PP2AA1) but did not require the PINOID protein kinase. When GNOM was inhibited, PINOID abundance increased and PP2AA1 was partially immobilized, indicating both proteins are subject to GNOM-dependent regulation. Shootward PIN2 specifically in the cortex was accompanied by enhanced shootward polar auxin transport and by diminished gravitropism. These results demonstrate that auxin flow in the root cortex is important for optimal gravitropic response.     

Jasmonic acid modulates xylem development by controlling polar auxin transport in vascular tissues

Development of xylem cells is affected by environmental stresses such as drought and oxidative stress, and recent findings suggested that jasmonic acid (JA) mediates this process through interaction with other phytohormones such as cytokinin. In this study, we showed that polar auxin transport regulated by PIN3 and PIN7 is involved in the JA-mediated xylem development in vascular tissues. The mutant plants that lack the activity of PIN3 and PIN7 responsible for the auxin transport developed extra xylems in vascular tissues such as the JA-treated wild-type plants. Visualization of auxin response and xylem development in the roots treated with NPA, an inhibitor of polar auxin transport, suggested that disruption of polar auxin transport is involved in the xylem phenotype of pin3 pin7 double mutants. We also found that cytokinin increases expressions of PIN3 and PIN7 responsible for the auxin transport while JA decreases only PIN7. These suggested that PIN7-mediated polar auxin transport system modulates xylem development in response to JA. The finding that JA affects auxin distribution in root vascular tissues further supported this. Collectively, these suggest that JA promotes xylem development by disrupting auxin transport in vascular tissues, and the auxin efflux genes, more especially PIN7 whose expression is suppressed by JA mediates this process.     

Combined in silico/in vivo analysis of mechanisms providing for root apical meristem self-organization and maintenance

Background and Aims

The root apical meristem (RAM) is the plant stem cell niche which provides for the formation and continuous development of the root. Auxin is the main regulator of RAM functioning, and auxin maxima coincide with the sites of RAM initiation and maintenance. Auxin gradients are formed due to local auxin biosynthesis and polar auxin transport. The PIN family of auxin transporters plays a critical role in polar auxin transport, and two mechanisms of auxin maximum formation in the RAM based on PIN-mediated auxin transport have been proposed to date: the reverse fountain and the reflected flow mechanisms.

Methods

The two mechanisms are combined here in in silico studies of auxin distribution in intact roots and roots cut into two pieces in the proximal meristem region. In parallel, corresponding experiments were performed in vivo using DR5::GFP Arabidopsis plants.

Key Results

The reverse fountain and the reflected flow mechanism naturally cooperate for RAM patterning and maintenance in intact root. Regeneration of the RAM in decapitated roots is provided by the reflected flow mechanism. In the excised root tips local auxin biosynthesis either alone or in cooperation with the reverse fountain enables RAM maintenance.

Conclusions

The efficiency of a dual-mechanism model in guiding biological experiments on RAM regeneration and maintenance is demonstrated. The model also allows estimation of the concentrations of auxin and PINs in root cells during development and under various treatments. The dual-mechanism model proposed here can be a powerful tool for the study of several different aspects of auxin function in root.     

Narciclasine modulates polar auxin transport in Arabidopsis roots

Plant development displays an exceptional plasticity and adaptability that involves the dynamic, asymmetric distribution of the phytohormone auxin. Polar auxin flow, which requires transport facilitators of the PIN family, largely contributes to the establishment and maintenance of auxin gradients and mediates multiple developmental processes. Here, we report the effects of narciclasine (NCS), an Amaryllidaceae alkaloid isolated from Narcissus tazetta bulbs, on postembryonic development of Arabidopsis roots. Arabidopsis seedlings grown on NCS showed defects in root gravitropism which correlates with a reduction in auxin transport in roots. Expressions of auxin transport genes were affected and the polar localization of PIN2 protein was altered under NCS treatment. Taken together, we propose that NCS modulates auxin transport gene expression and PIN2 localization, and thus affects auxin transport and auxin distribution necessary for postembryonic development of Arabidopsis roots.     

Light plays an essential role in intracellular distribution of auxin efflux carrier PIN2 in Arabidopsis thaliana

Background

Light plays a key role in multiple plant developmental processes. It has been shown that root development is modulated by shoot-localized light signaling and requires shoot-derived transport of the plant hormone, auxin. However, the mechanism by which light regulates root development is not largely understood. In plants, the endogenous auxin, indole-3-acetic acid, is directionally transported by plasma-membrane (PM)-localized auxin influx and efflux carriers in transporting cells. Remarkably, the auxin efflux carrier PIN proteins exhibit asymmetric PM localization, determining the polarity of auxin transport. Similar to PM-resident receptors and transporters in animal and yeast cells, PIN proteins undergo constitutive cycling between the PM and endosomal compartments. Auxin plays multiple roles in PIN protein intracellular trafficking, inhibiting PIN2 endocytosis at some concentrations and promoting PIN2 degradation at others. However, how PIN proteins are turned over in plant cells is yet to be addressed.

Methodology and Principle Findings

Using laser confocal scanning microscopy, and physiological and molecular genetic approaches, here, we show that in dark-grown seedlings, the PM localization of auxin efflux carrier PIN2 was largely reduced, and, in addition, PIN2 signal was detected in vacuolar compartments. This is in contrast to light-grown seedlings where PIN2 was predominantly PM-localized. In light-grown plants after shift to dark or to continuous red or far-red light, PIN2 also accumulated in vacuolar compartments. We show that PIN2 vacuolar targeting was derived from the PM via endocytic trafficking and inhibited by HY5-dependent light signaling. In addition, the ubiquitin 26S proteasome is involved in the process, since its inhibition by mutations in COP9 and a proteasome inhibitor MG132 impaired the process.

Conclusions and Significance

Collectively, our data indicate that light plays an essential role in PIN2 intracellular trafficking, promoting PM-localization in the presence of light and, on the other hand, vacuolar targeting for protein degradation in the absence of light. Based on these results, we postulate that light regulation of root development is mediated at least in part by changes in the intracellular distribution of auxin efflux carriers, PIN proteins, in response to the light environment.     

The PIN1 family gene PvPIN1 is involved in auxin-dependent root emergence and tillering in switchgrass

Switchgrass (Panicum virgatum L.; family Poaceae) is a warm-season C4 perennial grass. Tillering plays an important role in determining the morphology of aboveground parts and the final biomass yield of switchgrass. Auxin distribution in plants can affect a variety of important growth and developmental processes, including the regulation of shoot and root branching, plant resistance and biological yield. Auxin transport and gradients in plants are mediated by influx and efflux carriers. PvPIN1, a switchgrass PIN1-like gene that is involved in regulating polar transport, is a putative auxin efflux carrier. Neighbor-joining analysis using sequences deposited in NCBI databases showed that the PvPIN1gene belongs to the PIN1 family and is evolutionarily closer to the Oryza sativa japonica group. Tiller emergence and development was significantly promoted in plants subjected toPvPIN1 RNA interference (RNAi), which yielded a phenotype similar to that of wild-type plants treated with the auxin transport inhibitor TIBA (2,3,5-triiodobenzoic acid). A transgenic approach that inducedPvPIN1 gene overexpression or suppression altered tiller number and the shoot/root ratio. These data suggest that PvPIN1plays an important role in auxin-dependent adventitious root emergence and tillering.     

Systems Analysis of Auxin Transport in the Arabidopsis Root Apex

Auxin is a key regulator of plant growth and development. Within the root tip, auxin distribution plays a crucial role specifying developmental zones and coordinating tropic responses. Determining how the organ-scale auxin pattern is regulated at the cellular scale is essential to understanding how these processes are controlled. In this study, we developed an auxin transport model based on actual root cell geometries and carrier subcellular localizations. We tested model predictions using the DII-VENUS auxin sensor in conjunction with state-of-the-art segmentation tools. Our study revealed that auxin efflux carriers alone cannot create the pattern of auxin distribution at the root tip and that AUX1/LAX influx carriers are also required. We observed that AUX1 in lateral root cap (LRC) and elongating epidermal cells greatly enhance auxin’s shootward flux, with this flux being predominantly through the LRC, entering the epidermal cells only as they enter the elongation zone. We conclude that the nonpolar AUX1/LAX influx carriers control which tissues have high auxin levels, whereas the polar PIN carriers control the direction of auxin transport within these tissues.     

The involvement of auxin in root architecture plasticity in Arabidopsis induced by heterogeneous phosphorus availability

Homogeneous low phosphorus availability was reported to regulate root architecture in Arabidopsis via auxin, but the roles of auxin in root architecture plasticity to heterogeneous P availability remain unclear. In this study, we employed auxin biosynthesis-, transport- and signalling-related mutants. Firstly, we found that in contrast to low P (LP) content in the whole medium, primary root (PR) growth of Arabidopsis was partially rescued in the medium divided into two parts: upper with LP and lower with high P (HP) content or in the reverse arrangement. The down part LP was more effective to arrest PR growth as well as to decrease density of lateral roots (DLR) than the upper LP, and effects were dependent on polar auxin transport. Secondly, we verified that auxin receptor TIR1 was involved in the responses of PR growth and lateral root (LR) development to P supply and loss of function of TIR1 inhibited LR development. Thirdly, effects of heterogeneous P on LRD in the upper part of PR was dependent on PIN2 and PIN4, and in the down part on PIN3 and PIN4, whereas density of total LRs was dependent on auxin transporters PIN2 and PIN7. Finally, heterogeneous P availability altered the accumulation of auxin in PR tip and the expression of auxin biosynthesisrelated genes TAA1, YUC1, YUC2, and YUC4. Taken together, we provided evidences for the involvement of auxin in root architecture plasticity in response to heterogeneous phosphorus availability in Arabidopsis.     

Negative gravitropic response of roots directs auxin flow to control root gravitropism

Root tip is capable of sensing and adjusting its growth direction in response to gravity, a phenomenon known as root gravitropism. Previously, we have shown that negative gravitropic response of roots (NGR) is essential for the positive gravitropic response of roots. Here, we show that NGR, a plasma membrane protein specifically expressed in root columella and lateral root cap cells, controls the positive root gravitropic response by regulating auxin efflux carrier localization in columella cells and the direction of lateral auxin flow in response to gravity. Pharmacological and genetic studies show that the negative root gravitropic response of the ngr mutants depends on polar auxin transport in the root elongation zone. Cell biology studies further demonstrate that polar localization of the auxin efflux carrier PIN3 in root columella cells and asymmetric lateral auxin flow in the root tip in response to gravistimulation is reversed in the atngr1;2;3 triple mutant. Furthermore, simultaneous mutations of three PIN genes expressed in root columella cells impaired the negative root gravitropic response of the atngr1;2;3 triple mutant. Our work revealed a critical role of NGR in root gravitropic response and provided an insight of the early events and molecular basis of the positive root gravitropism.     

Auxin and cytokinin control formation of the quiescent centre in the adventitious root apex of arabidopsis

Background and Aims

Adventitious roots (ARs) are part of the root system in numerous plants, and are required for successful micropropagation. In the Arabidopsis thaliana primary root (PR) and lateral roots (LRs), the quiescent centre (QC) in the stem cell niche of the meristem controls apical growth with the involvement of auxin and cytokinin. In arabidopsis, ARs emerge in planta from the hypocotyl pericycle, and from different tissues in in vitro cultured explants, e.g. from the stem endodermis in thin cell layer (TCL) explants. The aim of this study was to investigate the establishment and maintenance of the QC in arabidopsis ARs, in planta and in TCL explants, because information about this process is still lacking, and it has potential use for biotechnological applications.

Methods

Expression of PR/LR QC markers and auxin influx (LAX3)/efflux (PIN1) genes was investigated in the presence/absence of exogenous auxin and cytokinin. Auxin was monitored by the DR5::GUS system and cytokinin by immunolocalization. The expression of the auxin-biosynthetic YUCCA6 gene was also investigated by in situ hybridization in planta and in AR-forming TCLs from the indole acetic acid (IAA)-overproducing superroot2-1 mutant and its wild type.

Key Results

The accumulation of auxin and the expression of the QC marker WOX5 characterized the early derivatives of the AR founder cells, in planta and in in vitro cultured TCLs. By determination of PIN1 auxin efflux carrier and LAX3 auxin influx carrier activities, an auxin maximum was determined to occur at the AR tip, to which WOX5 expression was restricted, establishing the positioning of the QC. Cytokinin caused a restriction of LAX3 and PIN1 expression domains, and concomitantly the auxin biosynthesis YUCCA6 gene was expressed in the apex.

Conclusions

In ARs formed in planta and TCLs, the QC is established in a similar way, and auxin transport and biosynthesis are involved through cytokinin tuning.     

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.     

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。     

Role of the Arabidopsis PIN6 Auxin Transporter in Auxin Homeostasis and Auxin-Mediated Development

Plant-specific PIN-formed (PIN) efflux transporters for the plant hormone auxin are required for tissue-specific directional auxin transport and cellular auxin homeostasis. The Arabidopsis PIN protein family has been shown to play important roles in developmental processes such as embryogenesis, organogenesis, vascular tissue differentiation, root meristem patterning and tropic growth. Here we analyzed roles of the less characterised Arabidopsis PIN6 auxin transporter. PIN6 is auxin-inducible and is expressed during multiple auxin–regulated developmental processes. Loss of pin6 function interfered with primary root growth and lateral root development. Misexpression of PIN6 affected auxin transport and interfered with auxin homeostasis in other growth processes such as shoot apical dominance, lateral root primordia development, adventitious root formation, root hair outgrowth and root waving. These changes in auxin-regulated growth correlated with a reduction in total auxin transport as well as with an altered activity of DR5-GUS auxin response reporter. Overall, the data indicate that PIN6 regulates auxin homeostasis during plant development.     

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.     

Differential Auxin-Transporting Activities of PIN-FORMED Proteins in Arabidopsis Root Hair Cells

The Arabidopsis (Arabidopsis thaliana) genome includes eight PIN-FORMED (PIN) members that are molecularly diverged. To comparatively examine their differences in auxin-transporting activity and subcellular behaviors, we expressed seven PIN proteins specifically in Arabidopsis root hairs and analyzed their activities in terms of the degree of PIN-mediated root hair inhibition or enhancement and determined their subcellular localization. Expression of six PINs (PIN1–PIN4, PIN7, and PIN8) in root hair cells greatly inhibited root hair growth, most likely by lowering auxin levels in the root hair cell by their auxin efflux activities. The auxin efflux activity of PIN8, which had not been previously demonstrated, was further confirmed using a tobacco (Nicotiana tabacum) cell assay system. In accordance with these results, those PINs were localized in the plasma membrane, where they likely export auxin to the apoplast and formed internal compartments in response to brefeldin A. These six PINs conferred different degrees of root hair inhibition and sensitivities to auxin or auxin transport inhibitors. Conversely, PIN5 mostly localized to internal compartments, and its expression in root hair cells rather slightly stimulated hair growth, implying that PIN5 enhanced internal auxin availability. These results suggest that different PINs behave differentially in catalyzing auxin transport depending upon their molecular activity and subcellular localization in the root hair cell.Auxin plays a critical role in plant development and growth by forming local concentration gradients. Local auxin gradients, created by the polar cell-to-cell movement of auxin, are implicated in primary axis formation, root meristem patterning, lateral organ formation, and tropic movements of shoots and roots (for recent review, see Vanneste and Friml, 2009). The cell-to-cell movement of auxin is achieved by auxin influx and efflux transporters such as AUXIN-RESISTANT1 (AUX1)/LIKE-AUX1 for influx and PIN-FORMED (PIN) and the P-glycoprotein (PGP) of ABCB (ATP-binding cassette-type transporter subfamily B) for efflux. Since diffusive efflux of the natural auxin indole-3-acetic acid (IAA; pKa = 4.75) is not favorable and PINs are localized in the plasma membrane in a polar manner, PINs act as rate-limiting factors for cellular auxin efflux and polar auxin transport through the plant body. These PINs'' properties explain why representative physiological effects of auxin transport are associated with PINs.Auxin flows from young aerial parts all the way down to the root tip columella in which an auxin maximum is formed for root stem cell maintenance and moves up toward the root differentiation zone through root epidermal cells, where a part of it travels back to the root tip via cortical cells (Blilou et al., 2005). This directional auxin flow is supported by the polar localization of PINs: PIN1, PIN3, and PIN7 at the basal side of stele cells (Friml et al., 2002a, 2002b; Blilou et al., 2005), PIN4 at the basal side in root stem cells (Friml et al., 2002a), and PIN2 at the upper side of root epidermis and at the basal side of the root cortex (Luschnig et al., 1998; Müller et al., 1998). Another interesting aspect of PIN-mediated auxin transport is the dynamics in directionality of auxin flow due to environmental stimuli-directed changes of subcellular PIN polarity, as exemplified for PIN3, whose subcellular localization changes in response to the gravity vector (Friml et al., 2002b).An intriguing question is how different PIN proteins have different subcellular polarities, which might be attributable to PIN-specific molecular properties, cell-type-specific factors, or both. The different PIN subcellular polarities in different cell types seemingly indicate that cell-type-specific factors are involved in polarity. In the case of PIN1, however, both classes of factors appear to affect its subcellular localization because when expressed under the PIN2 promoter, PIN1 localizes to the upper or basal side of root epidermal cells, depending on the GFP insertion site of the protein (Wiśniewska et al., 2006). A recent study demonstrated that the polar targeting of PIN proteins is modulated by phosphorylation/dephosphorylation of the central hydrophilic loop of PINs, which is mediated by PINOID (PID; a Ser/Thr protein kinase)/PP2A phosphatase (Michniewicz et al., 2007). The central hydrophilic domain of PINs might provide the molecule-specific cue for PIN polarity, together with as yet unknown cell-specific factors. Different recycling behaviors of PINs, which show variable sensitivities to brefeldin A (BFA), also imply different molecular characters among PIN species. Most PIN1 proteins are internalized by BFA treatment, whereas considerable amounts of PIN2 remain in the plasma membrane in addition to internal accumulation after BFA treatment. Recycling and basal polar targeting of PIN1 is dependent on the BFA-sensitive guanine nucleotide exchange factor for adenosyl ribosylation factors (ARF GEFs), GNOM, which is the major target of BFA. In contrast, apical targeting and recycling of PIN2 is independent of GNOM and controlled by BFA-resistant ARF GEFs (Geldner et al., 2003; Kleine-Vehn and Friml, 2008).In contrast to their distinct subcellular localizations, the differential auxin-transporting activities of PINs remain to be studied. The divergent primary structures of PIN proteins are not only indicative of differential subcellular polarity, but also would represent their differential catalytic activities for auxin transport. The auxin efflux activities of Arabidopsis (Arabidopsis thaliana) PINs have been demonstrated using Arabidopsis and heterologous systems: PIN1 and PIN5 in Arabidopsis cells (Petrásek et al., 2006; Mravec et al., 2009); PIN2, PIN3, PIN4, PIN6, and PIN7 in tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells (Lee and Cho, 2006; Petrásek et al., 2006; Mravec et al., 2008); PIN1, PIN2, PIN5, and PIN7 in yeast (Saccharomyces cerevisiae) cells (Petrásek et al., 2006; Blakeslee et al., 2007; Mravec et al., 2009; Yang and Murphy, 2009); and PIN1, PIN2, and PIN7 in HeLa cells (Petrásek et al., 2006; Blakeslee et al., 2007). Among the eight Arabidopsis PIN members, PIN1, PIN2, PIN3, PIN4, PIN6, and PIN7, which share a similar molecular structure in terms of the presence of a long central loop (hereafter called long-looped PINs; Fig. 1A; Supplemental Fig. S1), have been shown to catalyze auxin efflux at the cellular level. On the other hand, PIN5 and PIN8 possess a very short putative central loop (hereafter called short-looped PINs). Although PIN5 was recently shown to be localized in the endoplasmic reticulum (ER) and proposed to transport auxin metabolites into the ER lumen, its cellular function regarding its intracellular auxin-transporting activity has not been shown, and the auxin-transporting activity of PIN8 has yet to be demonstrated. In spite of the same transport directionality (auxin efflux) and similar molecular structures, the long-looped PINs exhibit sequence divergence not only in their central loop, but also in certain residues of the transmembrane domains. This structural divergence of long-looped PINs might be indicative of their differential auxin-transporting activities, which have not yet been quantitatively compared.Open in a separate windowFigure 1.Differential activities of PINs in the Arabidopsis root hair. A, Two distinctive PIN groups with different central hydrophilic loop sizes. Topology of PIN proteins was predicted by four different programs as described in Supplemental Figure S1. Numbers above indicate the number of transmembrane helices for each N- and C-terminal region, and numbers below indicate the number of amino acid residues of the central hydrophilic domain. B, Representative root images of control (Cont; Columbia-0) and root-hair-specific PIN-overexpressing (PINox; ProE7:PIN-GFP or ProE7:PIN [−]) plants. Bar = 100 μm for all. C, Root hair lengths of control and PINox plants. Six to 12 independent transgenic lines (average = 8.3), and 42 to 243 roots (average = 86.8) and 336 to 2,187 root hairs (average = 727.8) per construct, were observed for the estimation of root hair length. Data represent means ± se. The root hair lengths of PIN5ox lines were significantly longer than those of the control (P = 0.016 for PIN5ox; P < 0.0001 for PIN5-GFP1ox and PIN5-GFP2ox).To comparatively assess the cytological behaviors and molecular activities of different PIN members, it would be favorable to use a single assay system that provides a consistent cellular environment and enables quantitative estimation of PIN activity. In previous studies, we adopted the root hair single cell system to quantitatively assay auxin-transporting or regulatory activities of PINs, PGPs, AUX1, and PID (Lee and Cho, 2006; Cho et al., 2007a). Root hair growth is proportional to internal auxin levels in the root hair cell. Therefore, auxin efflux inhibits and auxin influx enhances root hair growth (Cho et al., 2007b; Lee and Cho, 2008). In addition, the use of a root-hair-specific promoter (Cho and Cosgrove, 2002; Kim et al., 2006) for expression of auxin transporters enables the transporters'' biological effect to be pinpointed to only the root hair cell, thus excluding probable non-cell-autonomous effects that could be caused by the general expression of auxin transporters.In this study, we expressed five long-looped PINs (PIN1, PIN2, PIN3, PIN4, and PIN7) and two short-looped PINs (PIN5 and PIN8) in root hair cells and compared their auxin-transporting activities and cytological dynamics. To directly measure the radiolabeled auxin-transporting activities of PIN5 and PIN8, we used an additional assay system, tobacco suspension cells. Our data revealed that PINs have differential molecular activities and pharmacological responses and that the short-looped and long-looped PINs have different subcellular localizations.     

GmPIN-dependent polar auxin transport is involved in soybean nodule development

To overcome nitrogen deficiency, legume roots establish symbiotic interactions with nitrogen-fixing rhizobia that are fostered in specialized organs (nodules). Similar to other organs, nodule formation is determined by a local maximum of the phytohormone auxin at the primordium site. However, how auxin regulates nodule development remains poorly understood. Here, we found that in soybean, (Glycine max), dynamic auxin transport driven by PIN-FORMED (PIN) transporter GmPIN1 is involved in nodule primordium formation. GmPIN1 was specifically expressed in nodule primordium cells and GmPIN1 was polarly localized in these cells. Two nodulation regulators, (iso)flavonoids trigger expanded distribution of GmPIN1b to root cortical cells, and cytokinin rearranges GmPIN1b polarity. Gmpin1abc triple mutants generated with CRISPR-Cas9 showed the impaired establishment of auxin maxima in nodule meristems and aberrant divisions in the nodule primordium cells. Moreover, overexpression of GmPIN1 suppressed nodule primordium initiation. GmPIN9d, an ortholog of Arabidopsis thaliana PIN2, acts together with GmPIN1 later in nodule development to acropetally transport auxin in vascular bundles, fine-tuning the auxin supply for nodule enlargement. Our findings reveal how PIN-dependent auxin transport modulates different aspects of soybean nodule development and suggest that the establishment of auxin gradient is a prerequisite for the proper interaction between legumes and rhizobia.

In soybean, nodule primordium formation involves GmPIN1-mediated polar auxin transport within primordium cells, and nodule enlargement involves the collaboration of GmPIN9d and GmPIN1-dependent auxin transport within nodule vasculature.     

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.