Regulation Of Auxin Transport Research Articles

The bHLH transcription factor SPATULA regulates root growth by controlling the size of the root meristem

BackgroundThe Arabidopsis thaliana gene SPATULA (SPT), encoding a bHLH transcription factor, was originally identified for its role in pistil development. SPT is necessary for the growth and development of all carpel margin tissues including the style, stigma, septum and transmitting tract. Since then, it has been shown to have pleiotropic roles during development, including restricting the meristematic region of the leaf primordia and cotyledon expansion. Although SPT is expressed in roots, its role in this organ has not been investigated.ResultsAn analysis of embryo and root development showed that loss of SPT function causes an increase in quiescent center size in both the embryonic and postembryonic stem cell niches. In addition, root meristem size is larger due to increased division, which leads to a longer primary root. spt mutants exhibit other pleiotropic developmental phenotypes, including more flowers, shorter internodes and an extended flowering period. Genetic and molecular analysis suggests that SPT regulates cell proliferation in parallel to gibberellic acid as well as affecting auxin accumulation or transport.ConclusionsOur data suggest that SPT functions in growth control throughout sporophytic growth of Arabidopsis, but is not necessary for cell fate decisions except during carpel development. SPT functions independently of gibberellic acid during root development, but may play a role in regulating auxin transport or accumulation. Our data suggests that SPT plays a role in control of root growth, similar to its roles in above ground tissues.

Rapid apoplastic pH measurement in Arabidopsis leaves using a fluorescent dye

In plants, the extracellular space (apoplast) is one of the main places where exchange of molecules occurs between cells. Not only is this compartment involved in the storage of multiple metabolites and ions, including calcium and protons, but it also plays a role in the transmission of signaling molecules for cell-to-cell communication. It has recently been shown multiple times that these two aspects are linked and can influence each other. In particular, apoplast pH was shown as a primary regulator of auxin (IAA) transport in Arabidopsis thaliana. To prove the role of apoplastic pH, we have developed a protocol for apoplastic fluid extraction from Arabidopsis leaves, followed by pH determination using the 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) fluorescent dye. This technique successfully allows one to monitor apoplastic pH variations among different plant lines and to link changes in apoplastic pH to cellular responses in the plant.

Abscisic acid accumulation modulates auxin transport in the root tip to enhance proton secretion for maintaining root growth under moderate water stress

Maintenance of root growth is essential for plant adaptation to soil drying. Here, we tested the hypothesis that auxin transport is involved in mediating ABA's modulation by activating proton secretion in the root tip to maintain root growth under moderate water stress. Rice and Arabidopsis plants were raised under a hydroponic system and subjected to moderate water stress (-0.47 MPa) with polyethylene glycol (PEG). ABA accumulation, auxin transport and plasma membrane H(+)-ATPase activity at the root tip were monitored in addition to the primary root elongation and root hair density. We found that moderate water stress increases ABA accumulation and auxin transport in the root apex. Additionally, ABA modulation is involved in the regulation of auxin transport in the root tip. The transported auxin activates the plasma membrane H(+)-ATPase to release more protons along the root tip in its adaption to moderate water stress. The proton secretion in the root tip is essential in maintaining or promoting primary root elongation and root hair development under moderate water stress. These results suggest that ABA accumulation modulates auxin transport in the root tip, which enhances proton secretion for maintaining root growth under moderate water stress.

Mechanical Regulation of Auxin-Mediated Growth

The phytohormone auxin is a primary regulator of growth and developmental pattern formation in plants. Auxin accumulates at specific sites (e.g., organ primordia) and induces localized growth within a tissue. Auxin also mediates developmental responses to intrinsic and external physical stimuli; however, exactly how mechanics influences auxin distribution is unknown. Here we show that mechanical strain can regulate auxin transport and accumulation in the tomato shoot apex, where new leaves emerge and rapidly grow. Modification of turgor pressure, application of external force, and artificial growth induction collectively show that the amount and intracellular localization of the auxin efflux carrier PIN1 are sensitive to mechanical alterations. In general, the more strained the tissue was, the more PIN1 was present per cell and the higher the proportion localized to the plasma membrane. Modulation of the membrane properties alone was sufficient to explain most of the mechanical effects. Our experiments support the hypothesis that the plasma membrane acts as a sensor of tissue mechanics that translates the cell wall strain into cellular responses, such as the intracellular localization of membrane-embedded proteins. One implication of this fundamental mechanism is the mechanical enhancement of auxin-mediated growth in young organ primordia. We propose that growth-induced mechanical strain upregulates PIN1 function and auxin accumulation, thereby promoting further growth, in a robust positive feedback loop.

A ROP GTPase-Dependent Auxin Signaling Pathway Regulates the Subcellular Distribution of PIN2 in Arabidopsis Roots

PIN-FORMED (PIN) protein-mediated auxin polar transport is critically important for development, pattern formation, and morphogenesis in plants. Auxin has been implicated in the regulation of polar auxin transport by inhibiting PIN endocytosis, but how auxin regulates this process is poorly understood. Our genetic screen identified the Arabidopsis SPIKE1 (SPK1) gene whose loss-of-function mutations increased lateral root density and retarded gravitropic responses, as do pin2 knockout mutations. SPK1 belongs to the conserved DHR2-Dock family of Rho guanine nucleotide exchange factors. The spk1 mutations induced PIN2 internalization that was not suppressed by auxin, as did the loss-of-function mutations for Rho-like GTPase from Plants 6 (ROP6)-GTPase or its effector RIC1. Furthermore, SPK1 was required for auxin induction of ROP6 activation. Our results have established a Rho GTPase-based auxin signaling pathway that maintains PIN2 polar distribution to the plasma membrane via inhibition of its internalization in Arabidopsis roots. Our findings provide new insights into signaling mechanisms that underlie the regulation of the dynamic trafficking of PINs required for long-distance auxin transport and that link auxin signaling to PIN-mediated pattern formation and morphogenesis.

ABP1 and ROP6 GTPase Signaling Regulate Clathrin-Mediated Endocytosis in Arabidopsis Roots

The dynamic spatial and temporal distribution of the crucial plant signaling molecule auxin is achieved by feedback coordination of auxin signaling and intercellular auxin transport pathways. Developmental roles of auxin have been attributed predominantly to its effect on transcription; however, an alternative pathway involving AUXIN BINDING PROTEIN1 (ABP1) has been proposed to regulate clathrin-mediated endocytosis in roots and Rho-like GTPase (ROP)-dependent pavement cell interdigitation in leaves. In this study, we show that ROP6 and its downstream effector RIC1 regulate clathrin association with the plasma membrane for clathrin-mediated endocytosis, as well as for its feedback regulation by auxin. Genetic analysis revealed that ROP6/RIC1 acts downstream of ABP1 to regulate endocytosis. This signaling circuit is also involved in the feedback regulation of PIN-FORMED 1 (PIN1) and PIN2 auxin transporters activity (via its constitutive endocytosis) and corresponding auxin transport-mediated processes, including root gravitropism and leave vascular tissue patterning. Our findings suggest that the signaling module auxin-ABP1-ROP6/RIC1-clathrin-PIN1/PIN2 is a shared component of the feedback regulation of auxin transport during both root and aerial development.

Lysine 63 -linked ubiquitylation of PIN2 auxin carrier protein governs hormonally controlled adaptation of Arabidopsis root growth

Cross-talk between plant cells and their surroundings requires tight regulation of information exchange at the plasma membrane (PM), which involves dynamic adjustments of PM protein localization and turnover to modulate signal perception and solute transport at the interface between cells and their surroundings. In animals and fungi, turnover of PM proteins is controlled by reversible ubiquitylation, which signals endocytosis and delivery to the cell's lytic compartment, and there is emerging evidence for related mechanisms in plants. Here, we describe the fate of Arabidopsis PIN2 protein, required for directional cellular efflux of the phytohormone auxin, and identify cis- and trans-acting mediators of PIN2 ubiquitylation. We demonstrate that ubiquitin acts as a principal signal for PM protein endocytosis in plants and reveal dynamic adjustments in PIN2 ubiquitylation coinciding with variations in vacuolar targeting and proteolytic turnover. We show that control of PIN2 proteolytic turnover via its ubiquitylation status is of significant importance for auxin distribution in root meristems and for environmentally controlled adaptations of root growth. Moreover, we provide experimental evidence indicating that PIN2 vacuolar sorting depends on modification specifically by lysine(63)-linked ubiquitin chains. Collectively, our results establish lysine(63)-linked PM cargo ubiquitylation as a regulator of polar auxin transport and adaptive growth responses in higher plants.

Transcription factor WRKY23 assists auxin distribution patterns during Arabidopsis root development through local control on flavonol biosynthesis

Gradients of the plant hormone auxin, which depend on its active intercellular transport, are crucial for the maintenance of root meristematic activity. This directional transport is largely orchestrated by a complex interaction of specific influx and efflux carriers that mediate the auxin flow into and out of cells, respectively. Besides these transport proteins, plant-specific polyphenolic compounds known as flavonols have been shown to act as endogenous regulators of auxin transport. However, only limited information is available on how flavonol synthesis is developmentally regulated. Using reduction-of-function and overexpression approaches in parallel, we demonstrate that the WRKY23 transcription factor is needed for proper root growth and development by stimulating the local biosynthesis of flavonols. The expression of WRKY23 itself is controlled by auxin through the Auxin Response Factor 7 (ARF7) and ARF19 transcriptional response pathway. Our results suggest a model in which WRKY23 is part of a transcriptional feedback loop of auxin on its own transport through local regulation of flavonol biosynthesis.

The role of flavonoids in root-rhizosphere signalling: opportunities and challenges for improving plant-microbe interactions

The flavonoid pathway produces a diverse array of plant compounds with functions in UV protection, as antioxidants, pigments, auxin transport regulators, defence compounds against pathogens and during signalling in symbiosis. This review highlights some of the known function of flavonoids in the rhizosphere, in particular for the interaction of roots with microorganisms. Depending on their structure, flavonoids have been shown to stimulate or inhibit rhizobial nod gene expression, cause chemoattraction of rhizobia towards the root, inhibit root pathogens, stimulate mycorrhizal spore germination and hyphal branching, mediate allelopathic interactions between plants, affect quorum sensing, and chelate soil nutrients. Therefore, the manipulation of the flavonoid pathway to synthesize specifically certain products has been suggested as an avenue to improve root-rhizosphere interactions. Possible strategies to alter flavonoid exudation to the rhizosphere are discussed. Possible challenges in that endeavour include limited knowledge of the mechanisms that regulate flavonoid transport and exudation, unforeseen effects of altering parts of the flavonoid synthesis pathway on fluxes elsewhere in the pathway, spatial heterogeneity of flavonoid exudation along the root, as well as alteration of flavonoid products by microorganisms in the soil. In addition, the overlapping functions of many flavonoids as stimulators of functions in one organism and inhibitors of another suggests caution in attempts to manipulate flavonoid rhizosphere signals.

Genetic regulation of polar auxin transport and its role in control of shoot morphogenesis

The effects of auxin on plant morphogenesis are related to its differential distribution within different plant tissues. Tissue-specific auxin synthesis and its influx into and efflux from cells are shown to be of importance for establishing the auxin gradients. The paper deals with control of these processes. Much attention is given to genetic control of auxin efflux and to the influence of genes regulating this process on plant morphogenesis.

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.

Strigolactones' ability to regulate root development may be executed by induction of the ethylene pathway

The newly defined phytohormones strigolactones (SLs) were recently shown to act as regulators of root development. Their positive effect on root-hair (RH) elongation enabled examination of their cross talk with auxin and ethylene. Analysis of wild-type plants and hormone-signaling mutants combined with hormonal treatments suggested that SLs and ethylene regulate RH elongation via a common regulatory pathway, in which ethylene is epistatic to SLs. The SL and auxin hormonal pathways were suggested to converge for regulation of RH elongation; this convergence was suggested to be mediated via the ethylene pathway, and to include regulation of auxin transport.

Inositol Trisphosphate-Induced Ca 2+ 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.

Redox regulation of auxin signaling and plant development in Arabidopsis

Thioredoxin (NTR/TRX) and glutathione (GSH/GRX) are the two major systems which play a key role in the maintenance of cellular redox homeostasis. They are essential for plant development, cell division or the response to environmental stresses. In a recent article,1 we studied the interplay between the NADP-linked thioredoxin and glutathione systems in auxin signaling genetically, by associating TRX reductase (ntra ntrb) and glutathione biosynthesis (cad2) mutations. We show that these two thiol reduction pathways interfere with developmental processes. This occurs through modulation of auxin activity as shown by genetic analyses of loss of function mutations in a triple ntra ntrb cad2 mutant. The triple mutant develops almost normally at the rosette stage but fails to generate lateral organs from the inflorescence meristem, producing almost naked stems that are reminiscent of mutants affected in PAT (polar auxin transport) or biosynthesis. The triple mutant exhibits other defects in processes regulated by auxin, including a loss of apical dominance, vasculature defects and reduced secondary root production. Furthermore, it has lower auxin (IAA) levels and decreased capacity for PAT, suggesting that the NTR and glutathione pathways influence inflorescence meristem development through regulation of auxin transport and metabolism.

The heterozygous abp1/ABP1 insertional mutant has defects in functions requiring polar auxin transport and in regulation of early auxin‐regulated genes

AUXIN-BINDING PROTEIN 1 (ABP1) is not easily accessible for molecular studies because the homozygous T-DNA insertion mutant is embryo-lethal. We found that the heterozygous abp1/ABP1 insertion mutant has defects in auxin physiology-related responses: higher root slanting angles, longer hypocotyls, agravitropic roots and hypocotyls, aphototropic hypocotyls, and decreased apical dominance. Heterozygous plants flowered earlier than wild-type plants under short-day conditions. The length of the main root, the lateral root density and the hypocotyl length were little altered in the mutant in response to auxin. Compared to wild-type plants, transcription of early auxin-regulated genes (IAA2, IAA11, IAA13, IAA14, IAA19, IAA20, SAUR9, SAUR15, SAUR23, GH3.5 and ABP1) was less strongly up-regulated in the mutant by 0.1, 1 and 10 μm IAA. Surprisingly, ABP1 was itself an early auxin-up-regulated gene. IAA uptake into the mutant seedlings during auxin treatments was indistinguishable from wild-type. Basipetal auxin transport in young roots was slower in the mutant, indicating a PIN2/EIR1 defect, while acropetal transport was indistinguishable from wild-type. In the eir1 background, three of the early auxin-regulated genes tested (IAA2, IAA13 and ABP1) were more strongly induced by 1 μm IAA in comparison to wild-type, but eight of them were less up-regulated in comparison to wild-type. Similar but not identical disturbances in regulation of early auxin-regulated genes indicate tight functional linkage of ABP1 and auxin transport regulation. We hypothesize that ABP1 is involved in the regulation of polar auxin transport, and thus affects local auxin concentration and early auxin gene regulation. In turn, ABP1 itself is under the transcriptional control of auxin.

Flavonoid Production Is Effectively Regulated by RNAi Interference of Two Flavone Synthase Genes from Glycine max

Flavonoids are a group of secondary metabolites found in many higher plants. The multiple roles of their flavone subclass include protection against UV damage, regulation of auxin transport, and modulation of flower color. In soybean (Glycine max), flavone synthase II (FNS II) is the key enzyme responsible for flavone biosynthesis. Two FNS II genes from soybean cultivar Hefeng 47 were cloned according to basic local alignment search tool (BLAST) contexts using flavone synthase sequences reported in other species. These were named GmFNSII-1 and GmFNSII-2. Sequence alignments showed that the cDNA of GmFNSII-1 was identical to that of CYP93B16, whereas GmFNSII-2 was clearly distinct. Functional assays in yeast (Schizosaccharomyces pombe) suggested that these two enzymes could convert (2S)-naringenin into apigenin. The two GmFNSII genes had similar tissue-specific expression patterns, but GmFNSII-2 was significantly expressed in the roots after treatment with 0.4 M glucose. This demonstrates that the gene plays an important role in the response to defense signals in soybean. RNA interference-mediated suppression of those GmFNSII genes effectively regulated flavone and isoflavone production in hairy roots that arose from soybean cotyledons transformed with Agrobacterium rhizogenes (ATCC15834). Our study also highlights some of the challenges associated with metabolic engineering of plant natural products.

Glucose Attenuation of Auxin-Mediated Bimodality in Lateral Root Formation Is Partly Coupled by the Heterotrimeric G Protein Complex

BackgroundAuxin and glucose are both essential elements in normal root development. The heterotrimeric G protein complex in Arabidopsis thaliana, defined as containing alpha (AtGPA1), beta (AGB1), and gamma (AGG) subunits and a GTPase accelerating protein called Regulator of G Signaling 1 protein (AtRGS1), are involved in glucose signaling and regulate auxin transport.Methodology/Principal FindingsA systems approach was used to show that formation of lateral roots, a process requiring coordinated cell division followed by targeted cell expansion, involves a signaling interaction between glucose and auxin. We dissected the relationship between auxin and glucose action using lateral root formation as the biological context. We found that auxin and glucose act synergistically to yield a complex output involving both stimulatory and antagonist glucose effects on auxin responsiveness. Auxin-induced, lateral-root formation becomes bimodal with regard to auxin dose in the presence of glucose. This bimodality is mediated, in part, by the G protein complex defined above.Conclusion/SignificanceAuxin and glucose are essential signals controlling the rate of cell proliferation and expansion in roots. Auxin promotes the formation of lateral roots and is consequently essential for proper root architecture. Glucose affects the activation state of the heterotrimeric G protein complex which regulates auxin distribution in the root. The bimodality of auxin-induced, lateral-root formation becomes prominent in the presence of glucose and in roots lacking the G protein complex. Bimodality is apparent without added glucose in all loss-of-function mutants for these G protein components, suggesting that the heterotrimeric G protein complex attenuates the bimodality and that glucose inhibits this attenuation through the complex. The bimodality can be further resolved into the processes of lateral root primordia formation and lateral root emergence, from which a model integrating these signals is proposed.

Flavonoid accumulation in Arabidopsis thaliana root galls caused by the obligate biotrophic pathogen Plasmodiophora brassicae

Three different flavonoids-naringenin, quercetin and kaempferol-accumulate in root galls of Arabidopsis thaliana after infection with the obligate biotrophic pathogen Plasmodiophora brassicae. In addition, high-performance liquid chromatography and thin layer chromatography analysis indicated that these flavonoids and their glycosides were induced in galls rather than in healthy roots. The transcripts of selected genes involved in the biosynthesis of flavonoids were up-regulated during the time course of the disease. Some, such as chalcone synthase and chalcone isomerase, were up-regulated at both times investigated in this study, whereas up-regulation was observed only at later times for others, such as a flavonol synthase-like gene. Plants with mutations in different flavonoid biosynthesis genes were slightly more tolerant to clubroot at low infection pressure. However, flavonoid treatment of either leaves or roots did not reduce gall development. The possibility that flavonoids might influence auxin levels by regulating auxin transport or auxin degradation in roots was investigated by measuring auxin levels and response in roots of flavonoid-deficient mutants and the wild-type after inoculation with P. brassicae, as well as the antioxidative potential of flavonoids in the peroxidase-catalysed degradation of indole-3-acetic acid. In addition, the auxin transport rate from the shoots to the roots was measured in infected wild-type or flavonoid mutant plants compared with controls. In conclusion, our results indicate a role of flavonoids in the modulation of auxin efflux in root galls.

Arabidopsis cryptochrome-1 restrains lateral roots growth by inhibiting auxin transport

Cryptochromes are blue-light photoreceptors that control many aspects of plant development. In this study, cryptochrome mutants of Arabidopsis were examined to assess the role of cryptchrome-1 (CRY1) in lateral roots growth. When grown in blue light for 12 d, mutant seedlings ( cry1) showed increased growth of lateral roots, while CRY1-overexpressing transgenic seedlings (CRY1ox) exhibited a marked decrease. Lateral roots growth of CRY1ox could be stimulated by auxin, but expression of PIN1 (efflux carrier of polar auxin transport) was strongly reduced. Contrary, the cry1 mutation showed the opposite effect, indicating that blue light and the auxin-signaling pathway interact in lateral roots growth of Arabidopsis. The free IAA content in CRY1ox roots was half of that in wild type and cry1 mutant roots. Moreover, the content of flavonoids (quercetin, kaempferol, isorhamnetin), which act as endogenous negative regulators of auxin transport, increased in CRY1ox seedlings. Taken together, these results suggest that Arabidopsis CRY1 restrains lateral roots growth by inhibiting auxin transport.

Arabidopsis N-MYC DOWNREGULATED-LIKE1, a positive regulator of auxin transport in a G protein-mediated pathway.

Root architecture results from coordinated cell division and expansion in spatially distinct cells of the root and is established and maintained by gradients of auxin and nutrients such as sugars. Auxin is transported acropetally through the root within the central stele and then, upon reaching the root apex, auxin is transported basipetally through the outer cortical and epidermal cells. The two Gbetagamma dimers of the Arabidopsis thaliana heterotrimeric G protein complex are differentially localized to the central and cortical tissues of the Arabidopsis roots. A null mutation in either the single beta (AGB1) or the two gamma (AGG1 and AGG2) subunits confers phenotypes that disrupt the proper architecture of Arabidopsis roots and are consistent with altered auxin transport. Here, we describe an evolutionarily conserved interaction between AGB1/AGG dimers and a protein designated N-MYC DOWNREGULATED-LIKE1 (NDL1). The Arabidopsis genome encodes two homologs of NDL1 (NDL2 and NDL3), which also interact with AGB1/AGG1 and AGB1/AGG2 dimers. We show that NDL proteins act in a signaling pathway that modulates root auxin transport and auxin gradients in part by affecting the levels of at least two auxin transport facilitators. Reduction of NDL family gene expression and overexpression of NDL1 alter root architecture, auxin transport, and auxin maxima. AGB1, auxin, and sugars are required for NDL1 protein stability in regions of the root where auxin gradients are established; thus, the signaling mechanism contains feedback loops.