Auxin Movement Research Articles

Cellular mechanism of polarized auxin transport on fruit shape determination revealed by time-lapse live imaging.

Polarized auxin transport regulates fruit shape determination by promoting anisotropic cell growth. Angiosperms produce organs with distinct shape resultant from adaptive evolution. Understanding the cellular basis underlying the development of plant organ has been a central topic in plant biology as it is key to unlock the mechanisms leading to the diversification of plants. Variations in the location of synthesis, polarized auxin transport (PAT) have been proposed to account for the development of diverse organ shapes, but the exact cellular mechanism has yet to be elucidated. The Capsella rubella develops a perfect heart-shaped fruit from an ovate shape gynoecium that is tightly linked to the localized auxin synthesis in the valve tips and provides a unique opportunity to address this question. In this study, we studied auxin movement in the fruits and the cellular effect of N-1-Naphthylphthalamic Acid (NPA) on the fruitshape determination by constructing the pCrPIN3:PIN3:GFP reporter and live-imaging. We found PAT in the valve epidermis is in congruent with fruit shape development and NPA treatment disrupts the heat-shaped fruit development mainly by repressing cell anisotropic growth with minor effect on division. As the Capsella fruit is unusually big in size, we also included a detailed step-by-step protocol on how to conduct live-imaging experiment. We further test the utility of this protocol by conducting a live-imaging analysis of the gynophore in Arachis hypogaea. Collectively, the results of this study elucidated the mechanism on how auxin signal was translated into instructions guiding cell growth during organ shape determination. In addition, the description of the detailed live-imaging protocol will encourage further studies of the cellular mechanisms underlying shape diversification in angiosperms.

Low-oxygen-induced root bending is altered by phytoglobin1 through mediation of ethylene response factors (ERFs) and auxin signaling.

phytoglobin1 positively regulates root bending in hypoxic Arabidopsis roots through regulation of ethylene response factors and auxin transport. Hypoxia-induced root bending is known to be mediated by the redundant activity of the group VII ethylene response factors (ERFVII) RAP2.12 and HRE2, causing changes in polar auxin transport (PAT). Here, we show that phytoglobin1 (Pgb1), implicated in hypoxic adaptation through scavenging of nitric oxide (NO), can alter root direction under low oxygen. Hypoxia-induced bending is exaggerated in roots over-expressing Pgb1 and attenuated in those where the gene is suppressed. These effects were attributed to Pgb1 repressing both RAP2.12 and HRE2. Expression, immunological and genetic data place Pgb1 upstream of RAP2.12 and HRE2 in the regulation of root bending in oxygen-limiting environments. The attenuation of slanting in Pgb1-suppressing roots was associated with depletion of auxin activity at the root tip because of depression in PAT, while exaggeration of root bending in Pgb1-over-expressing roots with the retention of auxin activity. Changes in PIN2 distribution patterns, suggestive of redirection of auxin movement during hypoxia, might contribute to the differential root bending responses of the transgenic lines. In the end, Pgb1, by regulating NO levels, controls the expression of 2 ERFVIIs which, in a cascade, modulate PAT and, therefore, root bending.

Parallel auxin transport via PINs and plasmodesmata during the Arabidopsis leaf hyponasty response

Key messageThe leaf hyponasty response depends on tip-to-petiole auxin transport. This transport can happen through two parallel pathways: active trans-membrane transport mediated by PIN proteins and passive diffusion through plasmodesmata.A plant’s ability to counteract potential shading by neighboring plants depends on transport of the hormone auxin. Neighbor sensing at the leaf tip triggers auxin production. Once this auxin reaches the abaxial petiole epidermis, it causes cell elongation, which leads to leaf hyponasty. Two pathways are known to contribute to this intercellular tip-to-petiole auxin movement: (i) transport facilitated by plasma membrane-localized PIN auxin transporters and (ii) diffusion enabled by plasmodesmata. We tested if these two modes of transport are arranged sequentially or in parallel. Moreover, we investigated if they are functionally linked. Mutants in which one of the two pathways is disrupted indicated that both pathways are necessary for a full hyponasty response. Visualization of PIN3-GFP and PIN7-GFP localization indicated PIN-mediated transport in parallel to plasmodesmata-mediated transport along abaxial midrib epidermis cells. We found plasmodesmata-mediated cell coupling in the pin3pin4pin7 mutant to match wild-type levels, indicating no redundancy between pathways. Similarly, PIN3, PIN4 and PIN7 mRNA levels were unaffected in a mutant with disrupted plasmodesmata pathway. Our results provide mechanistic insight on leaf hyponasty, which might facilitate the manipulation of the shade avoidance response in crops.

Decoding the Auxin Matrix: Auxin Biology Through the Eye of the Computer.

The plant hormone auxin is certainly the most studied developmental regulator in plants. The many functions of auxin during development, from the embryo to the root and shoot construction, are mediated by an ever-growing collection of molecular regulators, with an overwhelming degree of both ubiquity and complexity that we are still far from fully understanding and that biological experiments alone cannot grasp. In this review, we discuss how bioinformatics and computational modeling approaches have helped in recent years to explore this complexity and to push the frontiers of our understanding of auxin biology. We focus on how analysis of massive amounts of genomic data and construction of computational models to simulate auxin-regulated processes at different scales have complemented wet experiments to increase the understanding of how auxin acts in the nucleus to regulate transcription and how auxin movement between cells regulates development at the tissular scale.

Regulation of PIN-FORMED Protein Degradation.

Auxin action largely depends on the establishment of auxin concentration gradient within plant organs, where PIN-formed (PIN) auxin transporter-mediated directional auxin movement plays an important role. Accumulating studies have revealed the need of polar plasma membrane (PM) localization of PIN proteins as well as regulation of PIN polarity in response to developmental cues and environmental stimuli, amongst which a typical example is regulation of PIN phosphorylation by AGCVIII protein kinases and type A regulatory subunits of PP2A phosphatases. Recent findings, however, highlight the importance of PIN degradation in reestablishing auxin gradient. Although the underlying mechanism is poorly understood, these findings provide a novel aspect to broaden the current knowledge on regulation of polar auxin transport. In this review, we summarize the current understanding on controlling PIN degradation by endosome-mediated vacuolar targeting, autophagy, ubiquitin modification and the related E3 ubiquitin ligases, cytoskeletons, plant hormones, environmental stimuli, and other regulators, and discuss the possible mechanisms according to recent studies.

Hydraulic flux-responsive hormone redistribution determines root branching.

Plant roots exhibit plasticity in their branching patterns to forage efficiently for heterogeneously distributed resources, such as soil water. The xerobranching response represses lateral root formation when roots lose contact with water. Here, we show that xerobranching is regulated by radial movement of the phloem-derived hormone abscisic acid, which disrupts intercellular communication between inner and outer cell layers through plasmodesmata. Closure of these intercellular pores disrupts the inward movement of the hormone signal auxin, blocking lateral root branching. Once root tips regain contact with moisture, the abscisic acid response rapidly attenuates. Our study reveals how roots adapt their branching pattern to heterogeneous soil water conditions by linking changes in hydraulic flux with dynamic hormone redistribution.

Leaf vein patterning is regulated by the aperture of plasmodesmata intercellular channels.

To form tissue networks, animal cells migrate and interact through proteins protruding from their plasma membranes. Plant cells can do neither, yet plants form vein networks. How plants do so is unclear, but veins are thought to form by the coordinated action of the polar transport and signal transduction of the plant hormone auxin. However, plants inhibited in both pathways still form veins. Patterning of vascular cells into veins is instead prevented in mutants lacking the function of the GNOM (GN) regulator of auxin transport and signaling, suggesting the existence of at least one more GN-dependent vein-patterning pathway. Here we show that in Arabidopsis such a pathway depends on the movement of auxin or an auxin-dependent signal through plasmodesmata (PDs) intercellular channels. PD permeability is high where veins are forming, lowers between veins and nonvascular tissues, but remains high between vein cells. Impaired ability to regulate PD aperture leads to defects in auxin transport and signaling, ultimately leading to vein patterning defects that are enhanced by inhibition of auxin transport or signaling. GN controls PD aperture regulation, and simultaneous inhibition of auxin signaling, auxin transport, and regulated PD aperture phenocopies null gn mutants. Therefore, veins are patterned by the coordinated action of three GN-dependent pathways: auxin signaling, polar auxin transport, and movement of auxin or an auxin-dependent signal through PDs. Such a mechanism of tissue network formation is unprecedented in multicellular organisms.

Brachytic2 mutation is able to counteract the main pleiotropic effects of brown midrib3 mutant in maize

Maize is the basis of nutrition of domesticated herbivores and one of the most promising energy crops. The presence of lignin in the cell wall, tightly associated to carbohydrates, prevents the physical access of enzymes such as cellulase, limiting the carbohydrate degradability and consequently the energy value. To increase the utilization of the biomass cellulose content, the challenge of breeding programs is to lower or modify the lignin components. In maize several mutations are able to modify the lignin content and in particular the mutation in brown midrib3 (bm3) gene appeared as one of the most promising in breeding programs. Unfortunately this mutation has several negative pleiotropic effects on various important agronomic traits such as stay green, lodging and susceptibility to several infections.The maize Brachyitic 2 (br2) gene encodes for a putative protein involved in polar movement of auxins. br2 mutant plants are characterized by shortening of lower stalk internodes, unusual stalk strength and tolerance to wind lodging, darker leaves persisting longer in the active green state in comparison to wild type plants, suggesting a possible utilization of br2 plants to counteract the negative effects of the bm3 mutation. In this work, we report the generation and a preliminary characterization of the double mutant bm3 br2, suggesting the potential use of this new genetic material to increase biomass cellulose utilization.

The mechanism of auxin transport in lateral root spacing

The mechanism of auxin transport in lateral root spacing

Genome-wide identification of polar auxin transporter gene families reveals a possible new polar auxin flow in inverted cuttings of Populus yunnanensis.

Inverted cuttings of Populus yunnanensis are characterized by enlarged stems and dwarfed new shoots, and phytohormones play a crucial role in the response to inversion. The polar auxin transport (PAT) system is distinct from the transport systems of other hormones and is controlled by three major transporter gene families: pin-formed (PIN), auxin-resistant/like aux (AUX/LAX) and ATP-binding cassette transporters of the B class (ABCB). Here, we identified these three families in P. trichocarpa, P. euphratica and P. yunnanensis through a genome-wide analysis. The Populus PIN, AUX/LAX and ABCB gene families comprised 15, 8 and 31 members, respectively. Most PAT genes in Populus and Arabidopsis were identified as clear sister pairs, and some had unique motifs. Transcriptome profiling revealed that the expression of most PAT genes was unrelated to cutting inversion and that only several genes showed altered expression when cuttings were inverted. The auxin content difference at positions was opposite in upright and inverted cutting bodies during rooting, which obeyed the original plant polarity. However, during plant growth, the two direction types exhibited similar auxin movements in the cutting bodies, and the opposite auxin changes were observed in new shoots. Four PAT genes with a positive response to cutting inversion, PyuPIN10, PyuPIN11, PyuLAX6 and PyuABCB27, showed diverse expression patterns between upright and inverted cuttings during rooting and plant growth. Furthermore, PAT gene expression retained its polarity, which differs from the results found for auxin flow during plant growth. The inconformity indicated that a new downward auxin flow in addition to the old upward flow might be established during the growth of inverted cuttings. Some highly polar PAT genes were involved in the maintenance of original auxin polarity, which might cause the enlarged stems of inverted cuttings. This work lays a foundation for understanding the roles of auxin transport in plant responses to inversion.

Future Leaders to Watch – Andrea Paterlini

ABSTRACTFuture Leaders to Watch is a series of interviews with the first authors of a selection of papers published in Biology Open, helping early-career researchers promote themselves alongside their papers. Andrea Paterlini is author of ‘Uncharted routes: exploring the relevance of auxin movement via plasmodesmata’, published in BiO. Andrea is a PhD student in the groups of Yka Helariutta and Ottoline Leyser at the Sainsbury Laboratory, University of Cambridge, investigating the structure, function and regulation of plasmodesmata, the small pores that connect plant cells.

Uncharted routes: exploring the relevance of auxin movement via plasmodesmata.

ABSTRACTAuxin is an endogenous small molecule with an incredibly large impact on growth and development in plants. Movement of auxin between cells, due to its negative charge at most physiological pHs, strongly relies on families of active transporters. These proteins import auxin from the extracellular space or export it into the same. Mutations in these components have profound impacts on biological processes. Another transport route available to auxin, once the substance is inside the cell, are plasmodesmata connections. These small channels connect the cytoplasms of neighbouring plant cells and enable flow between them. Interestingly, the biological significance of this latter mode of transport is only recently starting to emerge with examples from roots, hypocotyls and leaves. The existence of two transport systems provides opportunities for reciprocal cross-regulation. Indeed, auxin levels influence proteins controlling plasmodesmata permeability, while cell–cell communication affects auxin biosynthesis and transport. In an evolutionary context, transporter driven cell–cell auxin movement and plasmodesmata seem to have evolved around the same time in the green lineage. This highlights a co-existence from early on and a likely functional specificity of the systems. Exploring more situations where auxin movement via plasmodesmata has relevance for plant growth and development, and clarifying the regulation of such transport, will be key aspects in coming years.This article has an associated Future Leader to Watch interview with the author of the paper.

Drought Disrupts Auxin Localization in Abscission Zone and Modifies Cell Wall Structure Leading to Flower Separation in Yellow Lupine.

Drought causes the excessive abscission of flowers in yellow lupine, leading to yield loss and serious economic consequences in agriculture. The structure that determines the time of flower shedding is the abscission zone (AZ). Its functioning depends on the undisturbed auxin movement from the flower to the stem. However, little is known about the mechanism guiding cell–cell adhesion directly in an AZ under water deficit. Therefore, here, we seek a fuller understanding of drought-dependent reactions and check the hypothesis that water limitation in soil disturbs the natural auxin balance within the AZ and, in this way, modifies the cell wall structure, leading to flower separation. Our strategy combined microscopic, biochemical, and chromatography approaches. We show that drought affects indole-3-acetic acid (IAA) distribution and evokes cellular changes, indicating AZ activation and flower abortion. Drought action was manifested by the accumulation of proline in the AZ. Moreover, cell wall-related modifications in response to drought are associated with reorganization of methylated homogalacturonans (HG) in the AZ, and upregulation of pectin methylesterase (PME) and polygalacturonase (PG)—enzymes responsible for pectin remodeling. Another symptom of stress action is the accumulation of hemicelluloses. Our data provide new insights into cell wall remodeling events during drought-induced flower abscission, which is relevant to control plant production.

Disruption of the Auxin Gradient in the Abscission Zone Area Evokes Asymmetrical Changes Leading to Flower Separation in Yellow Lupine.

How auxin transport regulates organ abscission is a long-standing and intriguing question. Polar auxin transport across the abscission zone (AZ) plays a more important role in the regulation of abscission than a local concentration of this hormone. We recently reported the existence of a spatiotemporal sequential pattern of the indole-3-acetic acid (IAA) localization in the area of the yellow lupine AZ, which is a place of flower detachment. In this study, we performed analyses of AZ following treatment with an inhibitor of polar auxin transport (2,3,5-triiodobenzoic acid (TIBA)). Once we applied TIBA directly onto the AZ, we observed a strong response as demonstrated by enhanced flower abscission. To elucidate the molecular events caused by the inhibition of auxin movement, we divided the AZ into the distal and proximal part. TIBA triggered the formation of the IAA gradient between these two parts. The AZ-marker genes, which encode the downstream molecular components of the inflorescence deficient in abscission (IDA)-signaling system executing the abscission, were expressed in the distal part. The accumulation of IAA in the proximal area accelerated the biosynthesis of abscisic acid and ethylene (stimulators of flower separation), which was also reflected at the transcriptional level. Accumulated IAA up-regulated reactive oxygen species (ROS) detoxification mechanisms. Collectively, we provide new information regarding auxin-regulated processes operating in specific areas of the AZ.

The canalization hypothesis - challenges and alternatives.

The 'canalization hypothesis' was suggested 50years ago by Tsvi Sachs to account for the formation of vascular strands in response to wounding or auxin application. The hypothesis proposes that positive feedback between auxin movement through a cell and the cell's auxin conductivity leads to the gradual selection of narrow 'canals' of polar auxin transport that will differentiate into vascular strands. Though the hypothesis has provided an invaluable conceptual framework to understand the patterned formation of vascular strands, evidence has been accumulating that seems to be incompatible with the hypothesis. We suggest that the challenging evidence is incompatible with current interpretations of the hypothesis but not with the concept at the core of the hypothesis' original formulation.

Directionality of Plasmodesmata-Mediated Transport in Arabidopsis Leaves Supports Auxin Channeling

The plant hormone auxin serves as central regulator of growth and development. Auxin transporters in the plasma membrane are assumed to define tissue-level patterns of auxin distribution [1, 2]. However, auxin is small enough to diffuse through the plasmodesmata that connect neighboring cells [3], presenting an alternative pathway, whose contribution to auxin transport remained largely unexplored [4]. Here, photoactivation microscopy [5, 6] was used to measure the capacity for small-molecule diffusion in the epidermis of Arabidopsis thaliana leaves. In the elongated epidermis cells covering the midrib and petiole, the plasmodesmata-mediated cell-wall permeability was found to be several times higher in the longitudinal than in the transverse direction. The physiological relevance of this asymmetry was tested through quantification of the shade-avoidance response, which depends on auxin transport from the leaf tip to the petiole in the abaxial side of the leaf [7], with the hypothesis that directionality of diffusion supplements transporter-mediated auxin movement [8]. Triggering the response by auxin application at the tip led to stronger leaf movement in wild-type plants than in gsl8 mutants [9], which lack the callose synthase necessary to establish directionality. The results match the predictions of a mathematical model of auxin transport based on the permeabilities measured in wild-type and mutant plants. It is concluded that plasmodesmata permeability can be selectively modulated within a plant cell and that the conferred directionality in diffusion can influence the tissue-specific distribution patterns of small molecules, like auxin. VIDEO ABSTRACT.

CRK5 Protein Kinase Contributes to the Progression of Embryogenesis of Arabidopsis thaliana.

The fine tuning of hormone (e.g., auxin and gibberellin) levels and hormone signaling is required for maintaining normal embryogenesis. Embryo polarity, for example, is ensured by the directional movement of auxin that is controlled by various types of auxin transporters. Here, we present pieces of evidence for the auxin-gibberellic acid (GA) hormonal crosstalk during embryo development and the regulatory role of the Arabidopsis thaliana Calcium-Dependent Protein Kinase-Related Kinase 5 (AtCRK5) in this regard. It is pointed out that the embryogenesis of the Atcrk5-1 mutant is delayed in comparison to the wild type. This delay is accompanied with a decrease in the levels of GA and auxin, as well as the abundance of the polar auxin transport (PAT) proteins PIN1, PIN4, and PIN7 in the mutant embryos. We have previously showed that AtCRK5 can regulate the PIN2 and PIN3 proteins either directly by phosphorylation or indirectly affecting the GA level during the root gravitropic and hypocotyl hook bending responses. In this manuscript, we provide evidence that the AtCRK5 protein kinase can in vitro phosphorylate the hydrophilic loops of additional PIN proteins that are important for embryogenesis. We propose that AtCRK5 can govern embryo development in Arabidopsis through the fine tuning of auxin-GA level and the accumulation of certain polar auxin transport proteins.

PIN-driven auxin transport emerged early in streptophyte evolution.

PIN-FORMED (PIN) transporters mediate directional, intercellular movement of the phytohormone auxin in land plants. To elucidate the evolutionary origins of this developmentally crucial mechanism, we analysed the single PIN homologue of a simple green alga Klebsormidium flaccidum. KfPIN functions as a plasma membrane-localized auxin exporter in land plants and heterologous models. While its role in algae remains unclear, PIN-driven auxin export is probably an ancient and conserved trait within streptophytes.

AtCRK5 Protein Kinase Exhibits a Regulatory Role in Hypocotyl Hook Development during Skotomorphogenesis.

Seedling establishment following germination requires the fine tuning of plant hormone levels including that of auxin. Directional movement of auxin has a central role in the associated processes, among others, in hypocotyl hook development. Regulated auxin transport is ensured by several transporters (PINs, AUX1, ABCB) and their tight cooperation. Here we describe the regulatory role of the Arabidopsis thaliana CRK5 protein kinase during hypocotyl hook formation/opening influencing auxin transport and the auxin-ethylene-GA hormonal crosstalk. It was found that the Atcrk5-1 mutant exhibits an impaired hypocotyl hook establishment phenotype resulting only in limited bending in the dark. The Atcrk5-1 mutant proved to be deficient in the maintenance of local auxin accumulation at the concave side of the hypocotyl hook as demonstrated by decreased fluorescence of the auxin sensor DR5::GFP. Abundance of the polar auxin transport (PAT) proteins PIN3, PIN7, and AUX1 were also decreased in the Atcrk5-1 hypocotyl hook. The AtCRK5 protein kinase was reported to regulate PIN2 protein activity by phosphorylation during the root gravitropic response. Here it is shown that AtCRK5 can also phosphorylate in vitro the hydrophilic loops of PIN3. We propose that AtCRK5 may regulate hypocotyl hook formation in Arabidopsis thaliana through the phosphorylation of polar auxin transport (PAT) proteins, the fine tuning of auxin transport, and consequently the coordination of auxin-ethylene-GA levels.

The Arabidopsis ATP-BINDING CASSETTE Transporter ABCB21 Regulates Auxin Levels in Cotyledons, the Root Pericycle, and Leaves.

The phytohormone auxin plays significant roles in regulating plant growth and development. In Arabidopsis, a subset of ATP-BINDING CASSETTE subfamily B (ABCB) transporters participate in polar movement of auxin by exclusion from and prevention of reuptake at the plasma membrane. A previous analysis identified ABCB21 as a conditional auxin uptake/efflux transporter that regulates cellular auxin levels, but clear physiological roles for ABCB21 in planta remain unknown. Here we show that ABCB21 maintains the acropetal auxin transport stream by regulating auxin levels in the pericycle. Loss of ABCB21 reduces rootward auxin transport and delays lateral root emergence. In seedling shoots, ABCB21 regulates mobilization of auxin from the photosynthetic cotyledons that is important for phototropic bending. In rosette leaves ABCB21 contributes to lateral auxin distribution. These results support a primary role for ABCB21 in regulating auxin distribution supplementary to the primary ABCB auxin transporters ABCB1 and 19.