Tree architecture: A strigolactone-deficient mutant reveals a connection between branching order and auxin gradient along the tree stem.

Due to their long lifespan, trees and bushes develop higher order of branches in a perennial manner. In contrast to a tall tree, with a clearly defined main stem and branching order, a bush is shorter and has a less apparent main stem and branching pattern. To address the developmental basis of these two forms, we studied several naturally occurring architectural variants in silver birch (Betula pendula). Using a candidate gene approach, we identified a bushy kanttarelli variant with a loss-of-function mutation in the BpMAX1 gene required for strigolactone (SL) biosynthesis. While kanttarelli is shorter than the wild type (WT), it has the same number of primary branches, whereas the number of secondary branches is increased, contributing to its bush-like phenotype. To confirm that the identified mutation was responsible for the phenotype, we phenocopied kanttarelli in transgenic BpMAX1::RNAi birch lines. SL profiling confirmed that both kanttarelli and the transgenic lines produced very limited amounts of SL. Interestingly, the auxin (IAA) distribution along the main stem differed between WT and BpMAX1::RNAi. In the WT, the auxin concentration formed a gradient, being higher in the uppermost internodes and decreasing toward the basal part of the stem, whereas in the transgenic line, this gradient was not observed. Through modeling, we showed that the different IAA distribution patterns may result from the difference in the number of higher-order branches and plant height. Future studies will determine whether the IAA gradient itself regulates aspects of plant architecture.

LATA1, a RING E3 ligase, modulates the tiller angle by affecting auxin asymmetric distribution and content in rice.

The tiller angle is an important agronomic trait that determines plant architecture and grain yield in rice (Oryza sativa L.). However, the molecular regulation mechanism of the rice tiller angle remains unclear. Here, we identified a rice tiller angle gene, LARGE TILLER ANGLE 1 (LATA1), using the MutMap approach. LATA1 encodes a C3H2C3-type RING zinc finger E3 ligase and the conserved region of the RING zinc finger is essential for its E3 activity. LATA1 was highly expressed in the root and tiller base and LATA1-GFP fusion protein was specifically localized to the nucleus. The mutation of LATA1 significantly reduced indole-3-acetic acid content and attenuated lateral auxin transport, thereby resulting in defective shoot gravitropism and spreading plant architecture in rice. Further investigations found that LATA1 may indirectly affect gravity perception by modulating the sedimentation rate of gravity-sensing amyloplasts upon gravistimulation. Our findings provide new insights into the molecular mechanism underlying the rice tiller angle and new genetic resource for the improvement of plant architecture in rice.

Transcriptional control of local auxin distribution by the CsDFB1-CsPHB module regulates floral organogenesis in cucumber.

Plant cystatins are cysteine proteinase inhibitors that play key roles in defense responses. In this work, we describe an unexpected role for the cystatin-like protein DEFORMED FLORAL BUD1 (CsDFB1) as a transcriptional regulator of local auxin distribution in cucumber (Cucumis sativus L.). CsDFB1 was strongly expressed in the floral meristems, floral primordia, and vasculature. RNA interference (RNAi)-mediated silencing of CsDFB1 led to a significantly increased number of floral organs and vascular bundles, together with a pronounced accumulation of auxin. Conversely, accompanied by a decrease of auxin, overexpression of CsDFB1 resulted in a dramatic reduction in floral organ number and an obvious defect in vascular patterning, as well as organ fusion. CsDFB1 physically interacted with the cucumber ortholog of PHABULOSA (CsPHB), an HD-ZIP III transcription factor whose transcripts exhibit the same pattern as CsDFB1 Overexpression of CsPHB increased auxin accumulation in shoot tips and induced a floral phenotype similar to that of CsDFB1-RNAi lines. Furthermore, genetic and biochemical analyses revealed that CsDFB1 impairs CsPHB-mediated transcriptional regulation of the auxin biosynthetic gene YUCCA2 and the auxin efflux carrier PIN-FORMED1, and thus plays a pivotal role in auxin distribution. In summary, we propose that the CsDFB1-CsPHB module represents a regulatory pathway for local auxin distribution that governs floral organogenesis and vascular differentiation in cucumber.

The cytochrome P450 CYP77A4 is involved in auxin-mediated patterning of the Arabidopsis thaliana embryo.

Metabolism often plays an important role in developmental control, in addition to supporting basal physiological requirements. However, our understanding of this interaction remains limited. Here, we performed quantitative phenome analysis of Arabidopsis thaliana cytochrome P450 mutants to identify a novel interaction between development and metabolism. We found that cyp77a4 mutants exhibit specific defects in cotyledon development, including asymmetric positioning and cup-shaped morphology, which could be rescued by introducing the CYP77A4 gene. Microscopy revealed that the abnormal patterning was detected at least from the 8-cell stage of the cyp77a4 embryos. We next analysed auxin distribution in mutant embryos, as the phenotypes resembled those of auxin-related mutants. We found that the auxin response pattern was severely perturbed in the cyp77a4 embryos owing to an aberrant distribution of the auxin efflux carrier PIN1. CYP77A4 intracellularly localised to the endoplasmic reticulum, which is consistent with the notion that this enzyme acts as an epoxidase of unsaturated fatty acids in the microsomal fraction. We propose that the CYP77A4-dependent metabolic pathway is an essential element for the establishment of polarity in plant embryos.

Altered Root Growth, Auxin Metabolism and Distribution in Arabidopsis thaliana Exposed to Salt and Osmotic Stress.

Salt and osmotic stress are the main abiotic stress factors affecting plant root growth and architecture. We investigated the effect of salt (100 mM NaCl) and osmotic (200 mM mannitol) stress on the auxin metabolome by UHPLC-MS/MS, auxin distribution by confocal microscopy, and transcript levels of selected genes by qRT-PCR in Arabidopsis thaliana ecotype Columbia-0 (Col-0) and DR5rev::GFP (DR5) line. During long-term stress (13 days), a stability of the auxin metabolome and a tendency to increase indole-3-acetic acid (IAA) were observed, especially during salt stress. Short-term stress (3 h) caused significant changes in the auxin metabolome, especially NaCl treatment resulted in a significant reduction of IAA. The data derived from auxin profiling were consistent with gene expressions showing the most striking changes in the transcripts of YUC, GH3, and UGT transcripts, suggesting disruption of auxin biosynthesis, but especially in the processes of amide and ester conjugation. These data were consistent with the auxin distribution observed in the DR5 line. Moreover, NaCl treatment caused a redistribution of auxin signals from the quiescent center and the inner layers of the root cap to the epidermal and cortical cells of the root elongation zone. The distribution of PIN proteins was also disrupted by salt stress; in particular, PIN2 was suppressed, even after 5 min of treatment. Based on our results, the DR5 line was more sensitive to the applied stresses than Col-0, although both lines showed similar trends in root morphology, as well as transcriptome and metabolome parameters under stress conditions.

A role for auxin signaling in the acquisition of longevity during seed maturation.

Seed longevity, the maintenance of viability during dry storage, is a crucial factor to preserve plant genetic resources and seed vigor. Inference of a temporal gene-regulatory network of seed maturation identified auxin signaling as a putative mechanism to induce longevity-related genes. Using auxin-response sensors and tryptophan-dependent auxin biosynthesis mutants of Arabidopsis thaliana L., the role of auxin signaling in longevity was studied during seed maturation. DII and DR5 sensors demonstrated that, concomitant with the acquisition of longevity, auxin signaling input and output increased and underwent a spatiotemporal redistribution, spreading throughout the embryo. Longevity of seeds of single auxin biosynthesis mutants with altered auxin signaling activity was affected in a dose-response manner depending on the level of auxin activity. Longevity-associated genes with promoters enriched in auxin response elements and the master regulator ABSCISIC ACID INSENSITIVE3 were induced by auxin in developing embryos and deregulated in auxin biosynthesis mutants. The beneficial effect of exogenous auxin during seed maturation on seed longevity was abolished in abi3-1 mutants. These data suggest a role for auxin signaling activity in the acquisition of longevity during seed maturation.

AUX1 acts upstream of PIN2 in regulating root gravitropism.

AUX1 and PIN2 auxin transporter are required for the asymmetric distribution of auxin for root gravitropic response. However, the relationship between AUX1 and PIN2 in root gravitropism is unclear. Here, we report that aux1-T mutant show stronger defects in root gravitropism than pin2-T, and aux1-T pin2-T double mutants display similar agravitropic phenotype to aux1-T. The gravity-induced asymmetric distribution of auxin responses could not be established in pin2-T, aux1-T and aux1-T pin2-T mutants; whereas aux1-T pin2-T double mutants showed similar auxin responses to aux1-T mutant. These findings support AUX1 plays a role in root gravitropism upstream of PIN2.

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.

What Has Been Seen Cannot Be Unseen-Detecting Auxin In Vivo.

Auxins mediate various processes that are involved in plant growth and development in response to specific environmental conditions. Its proper spatio-temporal distribution that is driven by polar auxin transport machinery plays a crucial role in the wide range of auxins physiological effects. Numbers of approaches have been developed to either directly or indirectly monitor auxin distribution in vivo in order to elucidate the basis of its precise regulation. Herein, we provide an updated list of valuable techniques used for monitoring auxins in plants, with their utilities and limitations. Because the spatial and temporal resolutions of the presented approaches are different, their combination may provide a comprehensive outcome of auxin distribution in diverse developmental processes.

Blue-light-induced PIN3 polarization for root negative phototropic response in Arabidopsis.

Root negative phototropism is an important response in plants. Although blue light is known to mediate this response, the cellular and molecular mechanisms underlying root negative phototropism remain unclear. Here, we report that the auxin efflux carrier PIN-FORMED (PIN) 3 is involved in asymmetric auxin distribution and root negative phototropism. Unilateral blue-light illumination polarized PIN3 to the outer lateral membrane of columella cells at the illuminated root side, and increased auxin activity at the illuminated side of roots, where auxin promotes growth and causes roots bending away from the light source. Furthermore, root negative phototropic response and blue-light-induced PIN3 polarization were modulated by a brefeldin A-sensitive, GNOM-dependent, trafficking pathway and by phot1-regulated PINOID (PID)/PROTEIN PHOSPHATASE 2A (PP2A) activity. Our results indicate that blue-light-induced PIN3 polarization is needed for asymmetric auxin distribution during root negative phototropic response.

The mechanism underlying asymmetric bending of lateral petals in Delphinium (Ranunculaceae).

During the process of flower opening, most petals move downward in the direction of the pedicel (i.e., epinastic movement). In most Delphinium flowers, however, their two lateral petals display a very peculiar movement, the mirrored helical rotation, which requires the twist of the petal stalk. However, in some lineages, their lateral petals also exhibit asymmetric bending that increases the degree of mirrored helical rotation, facilitating the formation of a 3D final shape. Notably, petal asymmetric bending is a novel trait that has not been noticed yet, so its morphological nature, developmental process, and molecular mechanisms remain largely unknown. Here, by using D. anthriscifolium as a model, we determined that petal asymmetric bending was caused by the localized expansion of cell width, accompanied by the specialized array of cell wall nano-structure, on the adaxial epidermis. Digital gene analyses, gene expression, and functional studies revealed that a class I homeodomain-leucine zipper family transcription factor gene, DeanLATE MERISTEM IDENTITY1 (DeanLMI1), contributes to petal asymmetric bending; knockdown of it led to the formation of explanate 2D petals. Specifically, DeanLMI1 promotes cell expansion in width and influences the arrangement of cell wall nano-structure on the localized adaxial epidermis. These results not only provide a comprehensive portrait of petal asymmetric bending for the first time but also shed some new insights into the mechanisms of flower opening and helical movement in plants.

Auxin distribution is differentially affected by nitrate in roots of two rice cultivars differing in responsiveness to nitrogen.

BACKGROUND AND AIMS: Although ammonium (NH4(+)) is the preferred form of nitrogen over nitrate (NO3(-)) for rice (Oryza sativa), lateral root (LR) growth in roots is enhanced by partial NO3(-) nutrition (PNN). The roles of auxin distribution and polar transport in LR formation in response to localized NO3(-) availability are not known. METHODS: Time-course studies in a split-root experimental system were used to investigate LR development patterns, auxin distribution, polar auxin transport and expression of auxin transporter genes in LR zones in response to localized PNN in 'Nanguang' and 'Elio' rice cultivars, which show high and low responsiveness to NO3(-), respectively. Patterns of auxin distribution and the effects of polar auxin transport inhibitors were also examined in DR5::GUS transgenic plants. KEY RESULTS: Initiation of LRs was enhanced by PNN after 7 d cultivation in 'Nanguang' but not in 'Elio'. Auxin concentration in the roots of 'Nanguang' increased by approx. 24 % after 5 d cultivation with PNN compared with NH4(+) as the sole nitrogen source, but no difference was observed in 'Elio'. More auxin flux into the LR zone in 'Nanguang' roots was observed in response to NO3(-) compared with NH4(+) treatment. A greater number of auxin influx and efflux transporter genes showed increased expression in the LR zone in response to PNN in 'Nanguang' than in 'Elio'. CONCLUSIONS: The results indicate that higher NO3(-) responsiveness is associated with greater auxin accumulation in the LR zone and is strongly related to a higher rate of LR initiation in the cultivar 'Nanguang'.

Asymmetric auxin distribution establishes a contrasting pattern of aerenchyma formation in the nodal roots of Zea nicaraguensis during gravistimulation.

Auxin distribution is essential for determining root developmental patterns. The formation of lateral roots and constitutive aerenchyma, which is a gas space developed through cell death, is regulated by auxin in rice (Oryza sativa). However, it is unclear whether the involvement of auxin in constitutive aerenchyma formation is conserved in other species. In this study, we found that constitutive aerenchyma formation was regulated by auxin in the nodal roots of Zea nicaraguensis, a wild relative of maize (Zea mays ssp. mays) grown naturally on frequently flooded coastal plains. Subsequent gravistimulation (root rotation) experiments showed opposite patterns of aerenchyma and lateral root formation. Lateral root formation on the convex side of rotated roots is known to be stimulated by a transient increase in auxin level in the pericycle. We found that aerenchyma formation was accelerated in the cortex on the concave side of the rotated nodal roots of Z. nicaraguensis. A cortex-specific expression analysis of auxin-responsive genes suggested that the auxin level was higher on the concave side than on the convex side. These results suggest that asymmetric auxin distribution underlies the regulation of aerenchyma and lateral root formation in the nodal roots of Z. nicaraguensis. As aerenchyma reduces the respiratory cost of the roots, constitutive aerenchyma on the concave side of the nodal root may balance resource allocation, thereby contributing to the uptake of water and nutrients by newly formed lateral roots. Our study provides insights into auxin-dependent asymmetric root patterning such as that of gravistimulation and hydropatterning response.

ERF1 inhibits lateral root emergence by promoting local auxin accumulation and repressing ARF7 expression.

Lateral roots (LRs) are crucial for plants to sense environmental signals in addition to water and nutrient absorption. Auxin is key for LR formation, but the underlying mechanisms are not fully understood. Here, we report that Arabidopsis ERF1 inhibits LR emergence by promoting local auxin accumulation with altered distribution and regulating auxin signaling. Loss of ERF1 increases LR density compared with the wild type, whereas ERF1 overexpression causes the opposite phenotype. ERF1 enhances auxin transport by upregulating PIN1 and AUX1, resulting in excessive auxin accumulation in the endodermal, cortical, and epidermal cells surrounding LR primordia. Furthermore, ERF1 represses ARF7 transcription, thereby downregulating the expression of cell-wall remodeling genes that facilitate LR emergence. Together, our study reveals that ERF1 integrates environmental signals to promote local auxin accumulation with altered distribution and repress ARF7, consequently inhibiting LR emergence in adaptation to fluctuating environments.

Auxin Distribution in Lateral Root Primordium Development Affects the Size and Lateral Root Diameter of Rice.

Lateral roots (LRs) occupy a large part of the root system and play a central role in plant water and nutrient uptake. Monocot plants, such as rice, produce two types of LRs: the S-type (short and thin) and the L-type (long, thick, and capable of further branching). Because of the ability to produce higher-order branches, the L-type LR formation contributes to efficient root system expansion. Auxin plays a major role in regulating the root system development, but its involvement in developing different types of LRs is largely unknown. Here, we show that auxin distribution is involved in regulating LR diameter. Dynamin-related protein (DRP) genes were isolated as causative genes of the mutants with increased L-type LR number and diameter than wild-type (WT). In the drp mutants, reduced endocytic activity was detected in rice protoplast and LRs with a decreased OsPIN1b-GFP endocytosis in the protoplast. Analysis of auxin distribution using auxin-responsive promoter DR5 revealed the upregulated auxin signaling in L-type LR primordia (LRP) of the WT and the mutants. The application of polar auxin transport inhibitors enhanced the effect of exogenous auxin to increase LR diameter with upregulated auxin signaling in the basal part of LRP. Inducible repression of auxin signaling in the mOsIAA3-GR system suppressed the increase in LR diameter after root tip excision, suggesting a positive role of auxin signaling in LR diameter increase. A positive regulator of LR diameter, OsWOX10, was auxin-inducible and upregulated in the drp mutants more than the WT, and revealed as a potential target of ARF transcriptional activator. Therefore, auxin signaling upregulation in LRP, especially at the basal part, induces OsWOX10 expression, increasing LR diameter.

Auxin Homeostasis and Distribution of the Auxin Efflux Carrier PIN2 Require Vacuolar NHX-Type Cation/H+ Antiporter Activity.

The Arabidopsis vacuolar Na+/H+ transporters (NHXs) are important regulators of intracellular pH, Na+ and K+ homeostasis and necessary for normal plant growth, development, and stress acclimation. Arabidopsis contains four vacuolar NHX isoforms known as AtNHX1 to AtNHX4. The quadruple knockout nhx1nhx2nhx3nhx4, lacking any vacuolar NHX-type antiporter activity, displayed auxin-related phenotypes including loss of apical dominance, reduced root growth, impaired gravitropism and less sensitivity to exogenous IAA and NAA, but not to 2,4-D. In nhx1nhx2nhx3nhx4, the abundance of the auxin efflux carrier PIN2, but not PIN1, was drastically reduced at the plasma membrane and was concomitant with an increase in PIN2 labeled intracellular vesicles. Intracellular trafficking to the vacuole was also delayed in the mutant. Measurements of free IAA content and imaging of the auxin sensor DII-Venus, suggest that auxin accumulates in root tips of nhx1nhx2nhx3nhx4. Collectively, our results indicate that vacuolar NHX dependent cation/H+ antiport activity is needed for proper auxin homeostasis, likely by affecting intracellular trafficking and distribution of the PIN2 efflux carrier.

Receptor-like kinase OsCR4 controls leaf morphogenesis and embryogenesis by fixing the distribution of auxin in rice.

Cell differentiation is a key event in organ development; it involves auxin gradient formation, cell signaling, and transcriptional regulation. Yet, how these processes are orchestrated during leaf morphogenesis is poorly understood. Here, we demonstrate an essential role for the receptor-like kinase OsCR4 in leaf development. oscr4 loss-of-function mutants displayed short shoots and roots, with tiny, crinkly, or even dead leaves. The delayed outgrowth of the first three leaves and seminal root in oscr4 was due to defects in plumule and radicle formation during embryogenesis. The deformed epidermal, mesophyll, and vascular tissues observed in oscr4 leaves arose at the postembryo stage; the corresponding expression pattern of proOsCR4:GUS in embryos and young leaves suggests that OsCR4 functions in these tissues. Signals from the auxin reporter DR5rev:VENUS were found to be altered in oscr4 embryos and disorganized in oscr4 leaves, in which indole-3-acetic acid accumulation was further revealed by immunofluorescence. OsWOX3A, which is auxin responsive and related to leaf development, was activated extensively and ectopically in oscr4 leaves, partially accounting for the observed lack of cell differentiation. Our data suggest that OsCR4 plays a fundamental role in leaf morphogenesis and embryogenesis by fixing the distribution of auxin.

Microfilament Depolymerization Is a Pre-requisite for Stem Cell Formation During In vitro Shoot Regeneration in Arabidopsis.

De novo shoot regeneration is widely used in fundamental studies and agricultural applications. Actin microfilaments are involved in many aspects of plant cell division, cell morphogenesis and cell signal transduction. However, the function of actin microfilaments during de novo shoot regeneration is poorly understood. Here, we investigated the organization of actin microfilaments during this process and found that stem cell formation was associated with microfilament depolymerization. Furthermore, inhibition of microfilament depolymerization by phalloidin treatment or downregulation of actin depolymerizing factors (ADFs) restrained stem cell initiation and shoot regeneration. Inhibition of ADF expression affected the architecture of microfilaments during stem cell formation, and the polar transport and distribution of auxin were also disrupted. Together, our results demonstrate that organization of the microfilament cytoskeleton play important roles in stem cell formation and shoot meristem induction during shoot regeneration.

Induction of somatic embryos in Arabidopsis requires local YUCCA expression mediated by the down-regulation of ethylene biosynthesis.

Somatic embryogenesis is an important experimental model for studying cellular and molecular mechanisms of early embryo development. Although it has long been known that removal of exogenous auxin from medium results in somatic embryogenesis, the mechanisms underlying the initiation of somatic embryos (SEs) are poorly understood. In this study, we showed that YUCCAs (YUCs) encoding key enzymes in auxin biosynthesis are required for SE induction in Arabidopsis. To identify other factors mediating SE initiation, we performed transcriptional profiling and gene expression analysis. The results showed that genes involved in ethylene biosynthesis and its responses were down-regulated during SE initiation. Ethylene level decreased progressively during SE initiation, whereas treatment with the metabolic precursor of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC), or mutation of ETHYLENE-OVERPRODUCTION1 (ETO1) disrupted SE induction, suggesting that ethylene plays a role in this process. Suppression of SE induction was also observed in the constitutive triple response 1 (ctr1) mutant, in which ethylene signaling was enhanced. These results indicate that down-regulation of not only ethylene biosynthesis, but also ethylene response is critical for SE induction. We further showed that ethylene disturbed SE initiation through inhibiting YUC expression that might be involved in local auxin biosynthesis and subsequent auxin distribution. Our results provide new information on the mechanisms of hormone-regulated SE initiation.