Transmembrane kinase 1-mediated auxin signal regulates membrane-associated clathrin in Arabidopsis roots

Clathrin-mediated endocytosis (CME) is the major endocytic pathway in eukaryotic cells that directly regulates abundance of plasma membrane proteins. Clathrin triskelia are composed of clathrin heavy chains (CHCs) and light chains (CLCs), and the phytohormone auxin differentially regulates membrane-associated CLCs and CHCs, modulating the endocytosis and therefore the distribution of auxin efflux transporter PIN-FORMED2 (PIN2). However, the molecular mechanisms by which auxin regulates clathrin are still poorly understood. Transmembrane kinase (TMKs) family proteins are considered to contribute to auxin signaling and plant development; it remains unclear whether they are involved in PIN transport by CME. We assessed TMKs involvement in the regulation of clathrin by auxin, using genetic, pharmacological, and cytological approaches including live-cell imaging and immunofluorescence. In tmk1 mutant seedlings, auxin failed to rapidly regulate abundance of both CHC and CLC and to inhibit PIN2 endocytosis, leading to an impaired asymmetric distribution of PIN2 and therefore auxin. Furthermore, TMK3 and TMK4 were shown not to be involved in regulation of clathrin by auxin. In summary, TMK1 is essential for auxin-regulated clathrin recruitment and CME. TMK1 therefore plays a critical role in the establishment of an asymmetric distribution of PIN2 and an auxin gradient during root gravitropism.     

Rabdosinate调节生长素极性运输蛋白PIN1、PIN3和PIN4抑制拟南芥幼苗根生长

利用3种拟南芥生长素极性运输外运载体突变体及4种转基因株系研究了二萜rabdosinate抑制拟南芥幼苗主根及侧根生长的作用机制。结果显示,60~80 μmol·L^-1的rabdosinate显著抑制野生型拟南芥幼苗主根生长及侧根形成,而对突变体pin1、pin2和pin3主根未显示明显的抑制效应,对侧根的抑制减弱;发现rabdosinate （60~80 μmol·L^-1）引起生长素报告株系根尖DR5活性升高,并增加融合蛋白PIN1-GFP丰度以及减少PIN3-GFP和PIN4-GFP的丰度。推断rabdosinate可通过增加PIN1丰度促进了根部生长素向顶运输,而减少PIN3丰度降低根尖部生长素的横向转运,引起了生长素在根尖部的累积及生长素浓度梯度的改变,进而抑制幼苗主根生长及侧根发育。     

Clathrin regulates blue light‐triggered lateral auxin distribution and hypocotyl phototropism in Arabidopsis

Phototropism is the process by which plants grow towards light in order to maximize the capture of light for photosynthesis, which is particularly important for germinating seedlings. In Arabidopsis, hypocotyl phototropism is predominantly triggered by blue light (BL), which has a profound effect on the establishment of asymmetric auxin distribution, essential for hypocotyl phototropism. Two auxin efflux transporters ATP‐binding cassette B19 (ABCB19) and PIN‐formed 3 (PIN3) are known to mediate the effect of BL on auxin distribution in the hypocotyl, but the details for how BL triggers PIN3 lateralization remain poorly understood. Here, we report a critical role for clathrin in BL‐triggered, PIN3‐mediated asymmetric auxin distribution in hypocotyl phototropism. We show that unilateral BL induces relocalization of clathrin in the hypocotyl. Loss of clathrin light chain 2 (CLC2) and CLC3 affects endocytosis and lateral distribution of PIN3 thereby impairing BL‐triggered establishment of asymmetric auxin distribution and consequently, phototropic bending. Conversely, auxin efflux inhibitors N‐1‐naphthylphthalamic acid and 2,3,5‐triiodobenzoic acid affect BL‐induced relocalization of clathrin, endocytosis and lateralization of PIN3 as well as asymmetric distribution of auxin. These results together demonstrate an important interplay between auxin and clathrin function that dynamically regulates BL‐triggered hypocotyl phototropism in Arabidopsis.     

AUXIN UP-REGULATED F-BOX PROTEIN1 regulates the cross talk between auxin transport and cytokinin signaling during plant root growth

Plant root development is mediated by the concerted action of the auxin and cytokinin phytohormones, with cytokinin serving as an antagonist of auxin transport. Here, we identify the AUXIN UP-REGULATED F-BOX PROTEIN1 (AUF1) and its potential paralog AUF2 as important positive modifiers of root elongation that tether auxin movements to cytokinin signaling in Arabidopsis (Arabidopsis thaliana). The AUF1 mRNA level in roots is strongly up-regulated by auxin but not by other phytohormones. Whereas the auf1 single and auf1 auf2 double mutant roots grow normally without exogenous auxin and respond similarly to the wild type upon auxin application, their growth is hypersensitive to auxin transport inhibitors, with the mutant roots also having reduced basipetal and acropetal auxin transport. The effects of auf1 on auxin movements may be mediated in part by the misexpression of several PIN-FORMED (PIN) auxin efflux proteins, which for PIN2 reduces its abundance on the plasma membrane of root cells. auf1 roots are also hypersensitive to cytokinin and have increased expression of several components of cytokinin signaling. Kinematic analyses of root growth and localization of the cyclin B mitotic marker showed that AUF1 does not affect root cell division but promotes cytokinin-mediated cell expansion in the elongation/differentiation zone. Epistasis analyses implicate the cytokinin regulator ARR1 or its effector(s) as the target of the SKP1-Cullin1-F Box (SCF) ubiquitin ligases assembled with AUF1/2. Given the wide distribution of AUF1/2-type proteins among land plants, we propose that SCF(AUF1/2) provides additional cross talk between auxin and cytokinin, which modifies auxin distribution and ultimately root elongation.     

A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis.

The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis.     

磷酸肌醇激酶FAB1调控拟南芥根毛伸长

FAB1/PIKfyve是介导PI(3,5)P₂ (磷脂酰肌醇3,5-二磷酸)生物合成的磷酸肌醇激酶。在动物和酵母(Saccharomyces cerevisiae)中, PI(3,5)P₂参与调控胞内膜运输, 但在植物中的研究较少。该文通过分析拟南芥(Arabidopsis thaliana) FAB1的T-DNA插入突变体的表型解析PI(3,5)P₂的生物学功能。拟南芥FAB1基因家族包含FAB1A、FAB1B、FAB1C和FAB1D四个基因。研究发现, fab1a/b呈现雄配子体致死的表型。利用遗传杂交获得fab1b/c/d三突变体, 发现FAB1B、FAB1C和FAB1D功能缺失导致根毛相比野生型变短, 经FAB1特异性抑制剂YM201636处理后的野生型中也观察到相似的短根毛表型。此外, fab1b/c/d三突变体中DR5转录水平降低。同时, 外源施加生长素类似物2,4-D和NAA能部分恢复fab1b/c/d植株短根毛的表型, 但fab1b/c/d突变体对生长素转运抑制剂(1-NOA和TIBA)的敏感性与野生型相似。此外, FAB1B/C/D功能缺失使根毛中ROS的含量减少且影响肌动蛋白的表达。上述结果表明, FAB1B/C/D通过调控生长素分布、ROS含量和肌动蛋白的表达影响拟南芥根毛伸长。     

Why hypocotyl extension mutants need to be characterized at the cell level: a case study of axr3-1

Since the discovery of auxin, a debate has taken place as to whether the auxin distribution in elongating organs can account for the distinctive cell elongation profiles that have been found. In an attempt to address this important issue, the elongation profiles of cells have been compared in the hypocotyls of wild-type and auxin-hypersensitive axr3-1 Arabidopsis Columbia ecotype seedlings. Clear differences in cell elongation profiles were found in the two types of seedling, whether they were light- or dark-grown. However, it was not possible unambiguously to ascribe the cell elongation profile differences to the proposition that the axr3-1 mutation causes the hypocotyl to be hypersensitive to auxin. The possibility that the abnormal hypocotyl elongation profile of the mutant was a secondary effect, consequent on a more fundamental effect of the axr3-1 mutation, is considered. It is clear from this study that cell elongation and its control needs to be studied at the cell, and not the organ, level. To characterize a mutant as having a short, or long, hypocotyl is inadequate. To determine which factors control the timing and the magnitude of cell elongation requires the demonstration of correlations between the growth rate of cells and their content of regulating substances or their sensitivity to that substance. Studies of the cell elongation profiles of the many hypocotyl length mutants could also be a very effective means of probing the co-ordination of root and shoot elongation.     

BYPASS1-LIKE regulates lateral root initiation via exocytic vesicular trafficking-mediated PIN recycling in Arabidopsis

Auxin and auxin-mediated signaling pathways are known to regulate lateral root development. Although exocytic vesicle trafficking plays an important role in recycling the PIN-FORMED (PIN) auxin efflux carriers and in polar auxin transport during lateral root formation, the mechanistic details of these processes are not well understood. Here, we demonstrate that BYPASS1-LIKE (B1L) regulates lateral root initiation via exocytic vesicular trafficking-mediated PIN recycling in Arabidopsis thaliana. b1l mutants contained significantly more lateral roots than the wild type, primarily due to increased lateral root primordium initiation. Furthermore, the auxin signal was stronger in stage I lateral root primordia of b1l than in those of the wild type. Treatment with exogenous auxin and an auxin transport inhibitor indicated that the lateral root phenotype of b1l could be attributed to higher auxin levels and that B1L regulates auxin efflux. Indeed, compared to the wild type, C-terminally green fluorescent protein-tagged PIN1 and PIN3 accumulated at higher levels in b1l lateral root primordia. B1L interacted with the exocyst, and b1l showed defective PIN exocytosis. These observations indicate that B1L interacts with the exocyst to regulate PIN-mediated polar auxin transport and lateral root initiation in Arabidopsis.     

phot1 inhibition of ABCB19 primes lateral auxin fluxes in the shoot apex required for phototropism

It is well accepted that lateral redistribution of the phytohormone auxin underlies the bending of plant organs towards light. In monocots, photoreception occurs at the shoot tip above the region of differential growth. Despite more than a century of research, it is still unresolved how light regulates auxin distribution and where this occurs in dicots. Here, we establish a system in Arabidopsis thaliana to study hypocotyl phototropism in the absence of developmental events associated with seedling photomorphogenesis. We show that auxin redistribution to the epidermal sites of action occurs at and above the hypocotyl apex, not at the elongation zone. Within this region, we identify the auxin efflux transporter ATP-BINDING CASSETTE B19 (ABCB19) as a substrate target for the photoreceptor kinase PHOTOTROPIN 1 (phot1). Heterologous expression and physiological analyses indicate that phosphorylation of ABCB19 by phot1 inhibits its efflux activity, thereby increasing auxin levels in and above the hypocotyl apex to halt vertical growth and prime lateral fluxes that are subsequently channeled to the elongation zone by PIN-FORMED 3 (PIN3). Together, these results provide new insights into the roles of ABCB19 and PIN3 in establishing phototropic curvatures and demonstrate that the proximity of light perception and differential phototropic growth is conserved in angiosperms.     

Gene expression profile of zeitlupe/lov kelch protein1 T-DNA insertion mutants in Arabidopsis thaliana: Downregulation of auxin-inducible genes in hypocotyls

Elongation of hypocotyl cells has been studied as a model for elucidating the contribution of cellular expansion to plant organ growth. ZEITLUPE (ZTL) or LOV KELCH PROTEIN1 (LKP1) is a positive regulator of warmth-induced hypocotyl elongation under white light in Arabidopsis, although the molecular mechanisms by which it promotes hypocotyl cell elongation remain unknown. Microarray analysis showed that 134 genes were upregulated and 204 genes including 15 auxin-inducible genes were downregulated in the seedlings of 2 ztl T-DNA insertion mutants grown under warm conditions with continuous white light. Application of a polar auxin transport inhibitor, an auxin antagonist or an auxin biosynthesis inhibitor inhibited hypocotyl elongation of control seedlings to the level observed with the ztl mutant. Our data suggest the involvement of auxin and auxin-inducible genes in ZTL-mediated hypocotyl elongation.     

Expression of TWISTED DWARF1 lacking its in‐plane membrane anchor leads to increased cell elongation and hypermorphic growth

Plant growth is achieved predominantly by cellular elongation, which is thought to be controlled on several levels by apoplastic auxin. Auxin export into the apoplast is achieved by plasma membrane efflux catalysts of the PIN‐FORMED (PIN) and ATP‐binding cassette protein subfamily B/phosphor‐glycoprotein (ABCB/PGP) classes; the latter were shown to depend on interaction with the FKBP42, TWISTED DWARF1 (TWD1). Here by using a transgenic approach in combination with phenotypical, biochemical and cell biological analyses we demonstrate the importance of a putative C‐terminal in‐plane membrane anchor of TWD1 in the regulation of ABCB‐mediated auxin transport. In contrast with dwarfed twd1 loss‐of‐function alleles, TWD1 gain‐of‐function lines that lack a putative in‐plane membrane anchor (HA–TWD1‐C_t) show hypermorphic plant architecture, characterized by enhanced stem length and leaf surface but reduced shoot branching. Greater hypocotyl length is the result of enhanced cell elongation that correlates with reduced polar auxin transport capacity for HA–TWD1‐C_t. As a consequence, HA–TWD1‐C_t displays higher hypocotyl auxin accumulation, which is shown to result in elevated auxin‐induced cell elongation rates. Our data highlight the importance of C‐terminal membrane anchoring for TWD1 action, which is required for specific regulation of ABCB‐mediated auxin transport. These data support a model in which TWD1 controls lateral ABCB1‐mediated export into the apoplast, which is required for auxin‐mediated cell elongation.     

The auxin response of actin is altered in the rice mutantYin-Yang

Summary The rice mutantYin-Yang has been selected during a screen for resistance to cytoskeletal drugs and is characterized by alterations in epidermal cell length and a precocious onset of gravitropism. The elongation response of coleoptile segments to auxin does not reveal changes of auxin sensitivity inYin-Yang. However, in contrast to the wild type, cell elongation inYin-Yang is highly sensitive to the actin-polymerisation blocker cytochalasin D. This increased sensitivity to cytochalasin D requires optimal concentrations of auxin to become manifest. The auxin response of actin microfilaments in epidermal cells differs between wild type and mutant. In the wild type, the longitudinal microfilament bundles become loosened in response to auxin. In the mutant, these bundles disintegrate partially and are replaced by a network of short filaments surrounding the nucleus. Several aspects of the mutant phenotype can be mimicked in the wild type by treatment with cytochalasin D. The mutant phenotype is discussed in terms of signal-dependent changes of actin dynamics and the putative role of actin during cell elongation.Abbreviations CD cytochalasin D - EPC ethyl-N-phenylcarbamate     

拟南芥金属蛋白酶FtSH4通过生长素与活性氧调控叶片衰老

植物金属蛋白酶Ft SH基因家族在拟南芥(Arabidopsis thaliana)中有12个成员,目前各基因的功能还不清楚。该文利用细胞生物学和遗传学方法初步分析了拟南芥FtSH4在叶片衰老中的功能。ftsh4-4突变体叶片中H_2O_2含量及细胞死亡率增加,叶绿素含量降低;此外,突变体中过氧化物酶基因表达上调,过氧化物酶活性增加,出现早衰表型。外源抗氧化剂As A、内源和外源生长素能够通过降低ftsh4-4体内H_2O_2含量、过氧化物酶基因的表达及过氧化物酶活性,恢复ftsh4-4叶片的衰老表型。ftsh4-4突变体中生长素响应因子基因ARF2和ARF7上调表达,外源生长素和抗氧化剂能够降低ARF2和ARF7的表达,并且ARF2突变能够降低ftsh4-4的H_2O_2含量并恢复其早衰表型。以上结果表明,FtSH4基因通过生长素与活性氧在调控植物叶片衰老中起重要作用。     

Root gravitropism requires lateral root cap and epidermal cells for transport and response to a mobile auxin signal

Re-orientation of Arabidopsis seedlings induces a rapid, asymmetric release of the growth regulator auxin from gravity-sensing columella cells at the root apex. The resulting lateral auxin gradient is hypothesized to drive differential cell expansion in elongation-zone tissues. We mapped those root tissues that function to transport or respond to auxin during a gravitropic response. Targeted expression of the auxin influx facilitator AUX1 demonstrated that root gravitropism requires auxin to be transported via the lateral root cap to all elongating epidermal cells. A three-dimensional model of the root elongation zone predicted that AUX1 causes the majority of auxin to accumulate in the epidermis. Selectively disrupting the auxin responsiveness of expanding epidermal cells by expressing a mutant form of the AUX/IAA17 protein, axr3-1, abolished root gravitropism. We conclude that gravitropic curvature in Arabidopsis roots is primarily driven by the differential expansion of epidermal cells in response to an influx-carrier-dependent auxin gradient.     

A loss-of-function mutation in the nucleoporin AtNUP160 indicates that normal auxin signalling is required for a proper ethylene response in Arabidopsis

As part of a continuing effort to elucidate mechanisms that regulate the magnitude of ethylene signalling, an Arabidopsis mutant with an enhanced ethylene response was identified. Subsequent characterization of this loss-of-function mutant revealed severe hypocotyl shortening in the presence of saturating ethylene along with increased expression in leaves of a subset of ethylene-responsive genes. It was subsequently determined by map-based cloning that the mutant (sar1-7) represents a loss-of-function mutation in the previously described nucleoporin AtNUP160 (At1g33410, SAR1). In support of previously reported results, the sar1-7 mutant partially restored auxin responsiveness to roots of an rce1 loss-of-function mutant, indicating that AtNUP160/SAR1 is required for proper expression of factors responsible for the repression of auxin signalling. Analysis of arf7-1/sar1-7 and arf19-1/sar1-7 double mutants revealed that mutations affecting either ARF7 or ARF19 function almost fully blocked manifestation of the sar1-7-dependent ethylene hypersensitivity phenotype, suggesting that ARF7- and ARF19-mediated auxin signalling is responsible for regulating the magnitude of and/or competence for the ethylene response in Arabidopsis etiolated hypocotyls. Consistent with this, addition of auxin to ethylene-treated seedlings resulted in severe hypocotyl shortening, reminiscent of that seen for other eer (enhanced ethylene response) mutants, suggesting that auxin functions in part synergistically with ethylene to control hypocotyl elongation and other ethylene-dependent phenomena.     

The unconventional prefoldin RPB5 interactor mediates the gravitropic response by modulating cytoskeleton organization and auxin transport in Arabidopsis

Gravity-induced root curvature involves the asymmetric distribution of the phytohormone auxin. This response depends on the concerted activities of the auxin transporters such as PIN-FORMED (PIN) proteins for auxin efflux and AUXIN RESISTANT 1 (AUX1) for auxin influx. However, how the auxin gradient is established remains elusive. Here we identified a new mutant with a short root, strong auxin distribution in the lateral root cap and an impaired gravitropic response. The causal gene encoded an Arabidopsis homolog of the human unconventional prefoldin RPB5 interactor (URI). AtURI interacted with prefoldin 2 (PFD2) and PFD6, two β-type PFD members that modulate actin and tubulin patterning in roots. The auxin reporter DR5_rev:GFP showed that asymmetric auxin redistribution after gravistimulation is disordered in aturi-1 root tips. Treatment with the endomembrane protein trafficking inhibitor brefeldin A indicated that recycling of the auxin transporter PIN2 is disrupted in aturi-1 roots as well as in pfd mutants. We propose that AtURI cooperates with PFDs to recycle PIN2 and modulate auxin distribution.     

The growth of tomato (Lycopersicon esculentum Mill.) hypocotyls in the light and in darkness differentially involves auxin.

Light and auxin antagonistically regulate hypocotyl elongation. We have investigated the physiological interactions of light and auxin in the control of tomato (Lycopersicon esculentum Mill.) hypocotyl elongation by studying the auxin-insensitive mutant diageotropica (dgt). The length of the hypocotyls of the dgt mutant is significantly reduced when compared to the wild type line Ailsa Craig (AC) in the dark and under red light, but not under the other light conditions tested, indicating that auxin sensitivity is involved in the elongation of hypocotyls only in these conditions. Similarly, the auxin transport inhibitor naphthylphthalamic [correction of naphtylphtalamic] acid (NPA) differentially affects elongation of dark- or light-grown hypocotyls of the MoneyMaker (MM) tomato wild type. Using different photomorphogenic mutants, we demonstrate that at least phytochrome A, phytochrome B1 and, to a much lesser extent [correction of extend], cryptochrome 1, are necessary for a switch from an auxin transport-dependent elongation of hypocotyls in the dark to an auxin transport-independent elongation in the light. Interestingly, the dgt mutant and NPA-treated seedlings exhibit a looped phenotype only under red light, indicating that the negative gravitropism of hypocotyls also differentially involves auxin in the various light conditions.