Polar Auxin Transport Inhibitor Research Articles

The AP2 transcription factor BARE RECEPTACLE regulates floral organogenesis via auxin pathways in woodland strawberry.

During flower development, different floral organs are formed to ensure fertilization and fruit set. Although the genetic networks underlying flower development are increasingly well understood, less is known about the mechanistic basis in different species. Here, we identified a mutant of woodland strawberry (Fragaria vesca), bare receptacle (bre), which produces flowers with greatly reduced carpels and other floral organs. Genetic analysis revealed that BRE encodes an APETALA2 (AP2) transcription factor. BRE was highly expressed in floral meristems and floral organ primordia. BRE could directly bind the GCC-box motif in the YUCCA (YUC) auxin biosynthesis genes FveYUC4 and FveYUC2 and promote their expression. The yuc4 mutant had fewer floral organs, and the bre yuc4 double mutant had similar numbers of petals and carpels to bre. Auxin homeostasis and distribution were severely disrupted in bre. Although auxin application or FveYUC4 overexpression did not rescue the bre phenotypes, bre was hypersensitive to treatment with the polar auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). In addition, BRE was able to directly bind and regulate the expression of five other auxin pathway genes. Overall, these results demonstrate that BRE is required for floral organogenesis, particularly carpel initiation, and acts through the auxin pathway in strawberry.

Target of rapamycin coordinates auxin are involved in exogenous melatonin regulated low temperature tolerance in cucumber seedlings

Low temperature (LT) is an important environmental factor affecting the growth and yield of plants. Melatonin (MT) can effectively enhance the LT tolerance of cucumber. This study found that LT stress induced the expression of Comt1 (caffeic acid O-methyltransferase 1), with the highest expression being about 2-times that of the control. Meanwhile, the content of MT was found to be roughly 63.16% of that in the control samples. Compared with LT treatment alone, exogenous MT pretreatment upregulated the expression levels of TOR (Target of rapamycin), PIN1 (Pin-formed 1), and YUC4 (YUCCA 4), with maximum upregulations reaching approximately 66.67%, 79.32%, and 42.86%, respectively. These results suggest that MT may modulate the tolerance of cucumber seedlings to LT stress by regulating the expression of TOR, PIN1, and YUC4. In addition, co-treatment with AZD-8055 (a TOR inhibitor) or NPA (N-1-naphthylphthalamic acid, an auxin polar transport inhibitor) and MT attenuated MT-induced resistance to LT stress, leading to higher levels of reactive oxygen species (ROS), reduced antioxidant defense capacity, and increased damage to the membrane system in cucumber seedlings. Concurrently, the content of osmoregulatory substances and the photosynthesis decreased. These results demonstrate that both TOR and auxin were required for MT to alleviate LT-induced damage in cucumber. In summary, the present study demonstrates that TOR and auxin signaling synergistically contribute to alleviating LT damage in cucumber seedlings by exogenous MT. These findings help us understand the function of MT and provide insights into the regulatory network of MT that regulates the LT tolerance of plants.

Auxin regulates bulbil initiation by mediating sucrose metabolism in Lilium lancifolium.

Lily bulbils, which serve as advantageous axillary organs for vegetative propagation, have not been extensively studied in terms of the mechanism of bulbil initiation. The functions of auxin and sucrose metabolism have been implicated in axillary organ development, but their relationship in regulating bulbil initiation remains unclear. In this study, exogenous indole-3-acetic acid (IAA) treatment increased the endogenous auxin levels at leaf axils and significantly decreased bulbil number, whereas treatment with the auxin polar transport inhibitor N-1-naphthylphthalamic acid (NPA), which resulted in a low auxin concentration at leaf axils, stimulated bulbil initiation and increased bulbil number. A low level of auxin caused by NPA spraying or silencing of auxin biosynthesis genes YUCCA FLAVIN MONOOXYGENASE-LIKE 6 (LlYUC6) and TRYPTOPHAN AMINOTRANSFERASERELATED 1 (LlTAR1) facilitated sucrose metabolism by activating the expression of SUCROSE SYNTHASES 1 (LlSusy1) and CELL WALL INVERTASE 2 (LlCWIN2), resulting in enhanced bulbil initiation. Silencing LlSusy1 or LlCWIN2 hindered bulbil initiation. Moreover, the transcription factor BASIC HELIX-LOOP-HELIX 35 (LlbHLH35) directly bound the promoter of LlSusy1, but not the promoter of LlCWIN2, and activated its transcription in response to the auxin content, bridging the gap between auxin and sucrose metabolism. In conclusion, our results reveal that an LlbHLH35-LlSusy1 module mediates auxin-regulated sucrose metabolism during bulbil initiation.

Auxin and hydrogen peroxide (H2O2) interaction differentially regulate seedling growth, Na+/K+ ratio and H2S homeostasis accompanying NaCl stress in etiolated sunflower (Helianthus annuus L. cv. Microgreen) seedling roots and cotyledons

Although H2S has been reported to modulate auxin and H2O2 signaling during abiotic stress, no reports decipher the interactive roles of exogenous auxin and H2O2 on the regulation of endogenous H2S homeostasis. In the present work, auxin (IAA) and hydrogen peroxide (H2O2) interaction appear to be crucial in regulating Na+/K+ homeostasis, recovery in seedling growth and H2S accumulation in NaCl-stressed sunflower seedlings. Interestingly, impairment in endogenous IAA transport (TIBA application) also impacts H2S accumulation in the roots and cotyledons. IAA supplementation is effective in reducing Na+/K+ ratio and electrolytic leakage in roots, which reverses in the presence of TIBA (polar auxin transport inhibitor) application. IAA and H2O2-mediated modulation of H2S content corroborates with l-DES activity in seedling roots and cotyledons. In contrast to cotyledons, roots exhibited a reduction in H2S content in the presence of treatments (IAA and H2O2). H2O2 supplementation seems to partially circumvent the changes caused by the disruption of auxin transport. Thus, we hypothesize that auxin might imply H2O2 as a crucial signaling component in the regulation of H2S levels in sunflower tissues. To sum up, IAA and H2O2 interaction regulates root growth, LR proliferation, Na+/K+ ratio, and H2S metabolism in sunflower seedling roots.

Cytokinin and indole-3-acetic acid crosstalk is indispensable for silicon mediated chromium stress tolerance in roots of wheat seedlings

The rising heavy metal contamination of soils imposes toxic impacts on plants as well as other life forms. One such highly toxic and carcinogenic heavy metal is hexavalent chromium [Cr(VI)] that has been reported to prominently retard the plant growth. The present study investigated the potential of silicon (Si, 10 µM) to alleviate the toxicity of Cr(VI) (25 µM) on roots of wheat (Triticum aestivum L.) seedlings. Application of Si to Cr(VI)-stressed wheat seedlings improved their overall growth parameters. This study also reveals the involvement of two phytohormones, namely auxin and cytokinin and their crosstalk in Si-mediated mitigation of the toxic impacts of Cr(VI) in wheat seedlings. The application of cytokinin alone to wheat seedlings under Cr(VI) stress reduced the intensity of toxic effects of Cr(VI). In combination with Si, cytokinin application to Cr(VI)-stressed wheat seedlings significantly minimized the decrease induced by Cr(VI) in different parameters such as root-shoot length (10.8% and 13%, respectively), root-shoot fresh mass (11.3% and 10.1%, respectively), and total chlorophyll and carotenoids content (13.4% and 6.8%, respectively) with respect to the control. This treatment also maintained the regulation of proline metabolism (proline content, and P5CS and PDH activities), ascorbate-glutathione (AsA-GSH) cycle and nutrient homeostasis. The protective effect of Si and cytokinin against Cr(VI) stress was minimized upon supplementation of an inhibitor of polar auxin transport- 2,3,5-triiodobenzoic acid (TIBA) which suggested a potential involvement of auxin in Si and cytokinin-mediated mitigation of Cr(VI) toxicity. The exogenous addition of a natural auxin – indole-3-acetic acid (IAA) confirmed auxin is an active member of a signaling cascade along with cytokinin that aids in Si-mediated Cr(VI) toxicity alleviation as IAA application reversed the negative impacts of TIBA on wheat roots treated with Cr(VI), cytokinin and Si. The results of this research are also confirmed by the gene expression analysis conducted for nutrient transporters (Lsi1, CCaMK, MHX, SULT1 and ZIP1) and enzymes involved in the AsA-GSH cycle (APX, GR, DHAR and MDHAR). The overall results of this research indicate towards possible induction of a crosstalk between cytokinin and IAA upon Si supplementation which in turn stimulates physiological, biochemical and molecular changes to exhibit protective effects against Cr(VI) stress. Further, the information obtained suggests probable employment of Si, cytokinin and IAA alone or combined in agriculture to maintain plant productivity under Cr(VI) stress and data regarding expression of key genes can be used to develop new crop varieties with enhanced resistance against Cr(VI) stress together with its reduced load in seedlings.

A unique mutation in PIN-FORMED1 and a genetic pathway for reduced sensitivity of Arabidopsis roots to N-1-naphthylphthalamic acid.

The small chemical N-1-naphthylphthalamic acid (NPA) has long been used as a polar auxin transport inhibitor. Recent biochemical and structural investigations have revealed that this molecule competes with the auxin IAA (indole-3-acetic acid) inside the PIN-FORMED auxin efflux carriers. However, the existence of any mutations in PIN family proteins capable of uncoupling the docking of IAA from NPA remains unclear. We report that Arabidopsis thaliana seedlings overexpressing SMALL AUXIN UP RNA 41 were hypersensitive to NPA-induced root elongation inhibition. We mutagenized this line to improve the genetic screening efficiency for NPA hyposensitivity mutants. Using bulked segregation analysis and mapping-by-sequencing assessment of these mutants, we identified a core genetic pathway for NPA-induced root elongation inhibition, including genes required for auxin biosynthesis, transportation, and signaling. To evaluate specific changes of auxin signaling activity in mutant roots before and after NPA treatment, the DR5::GFP/DR5::YFP markers were introduced and observed. Most importantly, we discovered a unique mutation in the PIN1 protein, substituting a proline residue with leucine at position 584, leading to a loss of NPA sensitivity while keeping the auxin efflux capacity. Transforming the null mutant pin1-201 with the PIN1::PIN1P584L -GFP fusion construct rescued the PIN1 function and provided NPA hyposensitivity. The proline residue is predicted to be adjacent to a hinge in the middle region of the ninth transmembrane helix of PIN1 and is conserved from moss to higher plants. Our work may bring new insights into the engineering of NPA-resistant PINs for auxin biology studies.

Phytochrome B1/B2 and auxin transport are involved in the regulation of shoot: root ratio by far-red radiation in tomato

Plants possess a set of photoreceptors to perceive changes in the light spectrum. Phytochromes (PHY) B1/B2 sense changes in red: far red ratios and are involved in mediating the shade-avoidance responses (SAR) in tomato. Far red (FR) increases the fraction of dry mass partitioning to the shoot at the expense of the root in tomato, but the control of this response has not yet been explained. We studied the role of phytochromes and auxin transport in the regulation of shoot: root ratio. We hypothesized that a loss-of-function mutation in phyB1/B2 leads to a strong increase in shoot: root ratio, similar to the effect of reduced R: FR ratio in wildtype plants. We also hypothesized that the phyB1/B2 double mutation suppresses shoot: root ratio responses when exposed to a reduced R: FR ratio. Furthermore, we hypothesized that the increased shoot: root ratio is linked to the changes in auxin transport between shoot and root. To test these hypotheses, we conducted an experiment in a climate chamber where both wildtype and phyB1/B2 double mutant tomato plants (Solanum lycopersicum cv. Moneymaker) were grown for 21 days with 0, 55 or 85 μmol m−2 s−1 FR in a background of white + red LED light at 150 μmol m−2 s−1. On the 14th day, auxin polar transport inhibitor 1-N-naphthylphthalamic acid (NPA) was applied at the shoot-root junction. PhyB1/B2 mutants showed a higher shoot: root ratio than the wildtype plants. Both wildtype and phyB1/B2 mutants responded to FR with an increase in shoot: root ratio. Blocking auxin transport from shoot to root led to an increase in shoot: root ratio for both genotypes under all light conditions. These results suggest that, similar to other SAR responses like stem elongation, the response of shoot: root ratio to additional FR also involves the regulation by phytochromes, possibly via affecting auxin transport between shoot and root.

LIPID TRANSFER PROTEIN4 regulates cotton ceramide content and activates fiber cell elongation.

Cell elongation is a fundamental process for plant growth and development. Studies have shown lipid metabolism plays important role in cell elongation; however, the related functional mechanisms remain largely unknown. Here, we report that cotton (Gossypium hirsutum) LIPID TRANSFER PROTEIN4 (GhLTP4) promotes fiber cell elongation via elevating ceramides (Cers) content and activating auxin-responsive pathways. GhLTP4 was preferentially expressed in elongating fibers. Over-expression and down-regulation of GhLTP4 led to longer and shorter fiber cells, respectively. Cers were greatly enriched in GhLTP4-overexpressing lines and decreased dramatically in GhLTP4 down-regulating lines. Moreover, auxin content and transcript levels of indole-3-acetic acid (IAA)-responsive genes were significantly increased in GhLTP4-overexpressing cotton fibers. Exogenous application of Cers promoted fiber elongation, while NPA (N-1-naphthalic acid, a polar auxin transport inhibitor) counteracted the promoting effect, suggesting that IAA functions downstream of Cers in regulating fiber elongation. Furthermore, we identified a basic helix-loop-helix transcription factor, GhbHLH105, that binds to the E-box element in the GhLTP4 promoter region and promotes the expression of GhLTP4. Suppression of GhbHLH105 in cotton reduced the transcripts level of GhLTP4, resulting in smaller cotton bolls and decreased fiber length. These results provide insights into the complex interactions between lipids and auxin-signaling pathways to promote plant cell elongation.

Expression of CsSCL1 and Rooting Response in Chestnut Leaves Are Dependent on the Auxin Polar Transport and the Ontogenetic Origin of the Tissues.

The mechanisms underlying the de novo regeneration of adventitious roots are still poorly understood, particularly in trees. We developed a system for studying adventitious rooting (AR) at physiological and molecular levels using leaves excised from chestnut microshoots of the same genotype but with two distinct ontogenetic origins that differ in rooting competence. Leaves were treated with auxin and N-1-naphthyl-phthalamic acid (NPA), an inhibitor of auxin polar transport (PAT). The physiological effects were investigated by recording rooting rates and the number and quality of the roots. Molecular responses were examined by localizing and monitoring the changes in the expression of CsSCL1, an auxin-inducible gene in juvenile and mature shoots during AR. The rooting response of leaves was ontogenetic-stage dependent and similar to that of the donor microshoots. Initiation of root primordia and root development were inhibited by application of NPA, although its effect depended on the timing of application. CsSCL1 was upregulated by auxin only in rooting-competent leaves during the novo root organogenesis, and the expression was reduced by NPA. The inhibitory effect on gene expression was detected during the reprograming of rooting competent cells towards root initials in response to auxin, indicating that PAT-mediated upregulation of CsSCL1 is required in the initial steps of AR in chestnut leaves. The localized expression of CsSCL1 in the quiescent center (QC) also suggests a role for this gene in the maintenance of meristematic competence and root radial patterning.

Comparative transcriptome analysis reveals the regulatory effects of exogenous auxin on lateral root development and tanshinone accumulation in Salvia miltiorrhiza.

The physiological and transcriptome analysis revealed that auxin was a positive regulator of lateral root development and tanshinone accumulation in Salvia miltiorrhiza. Roots of S. miltiorrhiza are widely used as medicinal materials in China, and the root morphology and content of bioactive compounds [such as phenolic acids and diterpenoid quinones (tanshinones)] are the main factors to determine the quality of this herb. Auxin regulates root development and secondary metabolism in many plant species, but little is known about its function in S. miltiorrhiza. In this study, S. miltiorrhiza seedlings were treated (exogenous application) with the auxin indole-3-acetic acid (IAA) and the polar auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) to investigate the regulatory roles of auxin in S. miltiorrhiza. The results indicated that exogenous IAA promoted both lateral root development and tanshinones biosynthesis in S. miltiorrhiza. The NPA application suppressed the lateral root development but showed no obvious effects on tanshinones accumulation. Based on the RNA-seq analysis, expressions of genes related to auxin biosynthesis and signaling transduction were altered in both treated groups. Coincidental with the enhanced content of tanshinones, transcripts of several key enzyme genes in the tanshinones biosynthetic pathway were stimulated after the exogenous IAA application. The expression profiles of seven common transcription factor domain-containing gene families were analyzed, and the results implied that some AP2/ERF genes were probably responsible for the auxin-induced lateral root development in S. miltiorrhiza. These findings shed new light on the regulatory roles of auxin on root development and bioactive compounds biosynthesis in S. miltiorrhiza, and lay the groundwork for future research into the detailed molecular mechanism underlying these biological functions.

SlGH3.15, a member of the GH3 gene family, regulates lateral root development and gravitropism response by modulating auxin homeostasis in tomato

Multiple Gretchen Hagen 3 (GH3) genes have been implicated in a range of processes in plant growth and development through their roles in maintaining hormonal homeostasis. However, there has only been limited study on the functions of GH3 genes in tomato (Solanum lycopersicum). In this work, we investigated the important function of SlGH3.15, a member of the GH3 gene family in tomato. Overexpression of SlGH3.15 led to severe dwarfism in both the above- and below-ground sections of the plant, accompanied by a substantial decrease in free IAA content and reduction in the expression of SlGH3.9, a paralog of SlGH3.15. Exogenous supply of IAA negatively affected the elongation of the primary root and partially restored the gravitropism defects in SlGH3.15-overexpression lines. While no phenotypic change was observed in the SlGH3.15 RNAi lines, double knockout lines of SlGH3.15 and SlGH3.9 were less sensitive to treatments with the auxin polar transport inhibitor. Overall, these findings revealed important roles of SlGH3.15 in IAA homeostasis and as a negative regulator of free IAA accumulation and lateral root formation in tomato.

Integration of root architecture, root nitrogen metabolism, and photosynthesis of ‘Hanfu’ apple trees under the cross-talk between glucose and IAA

Sugars and auxin have important effects on almost all phases of plant life cycle, which are so fundamental to plants and regulate similar processes. However, little is known about the effect of cross-talk between glucose and indole-3-acetic acid (IAA) on growth and development of apple trees. To examine the potential roles of glucose and IAA in root architecture, root nitrogen (N) metabolism and photosynthetic capacity in ‘Hanfu’ (Malus domestica), a total of five treatments was established: single application of glucose, IAA, and auxin polar transport inhibitor (2, 3, 5-triiodobenzoic acid, TIBA), combined application of glucose with TIBA and that of glucose with IAA. The combined application of glucose with IAA improved root topology system and endogenous IAA content by altering the mRNA levels of several genes involved in root growth, auxin transport and biosynthesis. Moreover, the increased N metabolism enzyme activities and levels of genes expression related to N in roots may suggest higher rates of transformation of nitrate (NO3--N) into amino acids application of glucose and IAA. Contrarily, single application of TIBA decreased the expression levels of auxin transport gene, hindered root growth and decreased endogenous IAA content. Glucose combined with TIBA application effectively attenuated TIBA-induced reductions in root topology structure, photosynthesis and N metabolism activity, and mRNA expression levels involved in auxin biosynthesis and transport. Taken together, glucose application probably changes the expression level of auxin synthesis and transport genes, and induce the allocation of endogenous IAA in root, and thus improves root architecture and N metabolism of root in soil with deficit carbon.

МОРФОГЕНЕЗ IN VITRO В КАЛЛУСАХ ЯЧМЕНЯ: ВЛИЯНИЕ НАФТИЛФТАЛАМОВОЙ КИСЛОТЫ

Morphogenesis in vitro in barley calli of the cv. Steptoe and its ABA-deficient mutant AZ34 under the influence of an inhibitor of polar auxin transport, N-1-naphthylphthalamic acid (NPA), was studied by light microscopy The formation of «polymeric» embryoids (polyembryoids), characterized by the presence of multiple, often fused (fasciated) organs in varying degrees, in calli of cv. Steptoe and the formation of fasciated roots in calli of AZ34 were revealed. The distribution of IAA in calli was studied by the immunolocalization method. It was found that the most intense staining for IAA in both genotypes was found in fasciated roots. The important role of the polar transport of auxins in the regulation of plant morphogenesis in vitro has been confirmed.

Position of meristems and the angles of the cell division plane regulate the uniqueness of lateral organ shape.

Leaf meristem is a cell proliferative zone present in the lateral organ primordia. In this study, we examined how cell proliferative zones in primordia of planar floral organs and polar auxin transport inhibitor (PATI)-treated leaf organs differ from those of non-treated foliage leaves of Arabidopsis thaliana, with a focus on the accumulation pattern of ANGUSTIFOLIA3 (AN3) protein, a key element for leaf meristem positioning. We found that PATI-induced leaf shape changes were correlated with cell division angle but not with meristem positioning/size or AN3 localisation. In contrast, different shapes between sepals and petals compared with foliage leaves were associated with both altered meristem position, due to altered AN3 expression patterns, and different distributions of cell division angles. A numerical simulation showed that meristem position majorly affected the final shape but biased cell division angles had a minor effect. Taken together, these results suggest that the unique shapes of different lateral organs depend on the position of the meristem in the case of floral organs and cell division angles in the case of leaf organs with different auxin flow.

Mapping of the Classical Mutation rosette Highlights a Role for Calcium in Wound-Induced Rooting.

Removal of the root system induces the formation of new roots from the remaining shoot. This process is primarily controlled by the phytohormone auxin, which interacts with other signals in a yet unresolved manner. Here, we study the classical tomato mutation rosette (ro), which lacks shoot-borne roots. ro mutants were severely inhibited in formation of wound-induced roots (WiRs) and had reduced auxin transport rates. We mapped ro to the tomato ortholog of the Arabidopsis thaliana BIG and the mammalians UBR4/p600. RO/BIG is a large protein of unknown biochemical function. In A. thaliana, BIG was implicated in regulating auxin transport and calcium homeostasis. We show that exogenous calcium inhibits WiR formation in tomato and A. thaliana ro/big mutants. Exogenous calcium antagonized the root-promoting effects of the auxin indole-3-acetic-acid but not of 2,4-dichlorophenoxyacetic acid, an auxin analog that is not recognized by the polar transport machinery, and accumulation of the auxin transporter PIN-FORMED1 (PIN1) was sensitive to calcium levels in the ro/big mutants. Consistent with a role for calcium in mediating auxin transport, both ro/big mutants and calcium-treated wild-type plants were hypersensitive to treatment with polar auxin transport inhibitors. Subcellular localization of BIG suggests that, like its mammalian ortholog, it is associated with the endoplasmic reticulum. Analysis of subcellular morphology revealed that ro/big mutants exhibited disruption in cytoplasmic streaming. We suggest that RO/BIG maintains auxin flow by stabilizing PIN membrane localization, possibly by attenuating the inhibitory effect of Ca2+ on cytoplasmic streaming.

The Germin-like protein OsGER4 is involved in promoting crown root development under exogenous jasmonic acid treatment in rice.

In rice (Oryza sativa L.), crown roots (CRs) have many important roles in processes such as root system expansion, water and mineral uptake, and adaptation to environmental stresses. Phytohormones such as auxin, cytokinin, and ethylene are known to control CR initiation and development in rice. However, the role of jasmonic acid (JA) in CR development remained elusive. Here, we report that JA promotes CR development by regulating OsGER4, a rice Germin-like protein. Root phenotyping analysis revealed that exogenous JA treatment induced an increase in CR number in a concentration-dependent manner. A subsequent genome-wide association study and gene expression analyses pinpointed a strong association between the Germin-like protein OsGER4 and the increase in CR number under exogenous JA treatment. The ProGER4::GUS reporter line showed that OsGER4 is a hormone-responsive gene involved in various stress responses, mainly confined to epidermal and vascular tissues during CR primordia development and to vascular bundles of mature crown and lateral roots. Notable changes in OsGER4 expression patterns caused by the polar auxin transport inhibitor NPA support its connection to auxin signaling. Phenotyping experiments with OsGER4 knockout mutants confirmed that this gene is required for CR development under exogenous JA treatment. Overall, our results provide important insights into JA-mediated regulation of CR development in rice.

Overexpression of a SHORT-ROOT transcriptional factor enhances the auxin mediated formation of adventitious roots and lateral roots in poplar trees

SHORT-ROOT (SHR) defines root stem cells and maintains radial patterning, but its involvement in adventitious root (AR) formation has not been reported. In this study, we showed that PtSHR2 was transcriptionally upregulated by excision before the formation of AR and responded dynamically to auxin. PtSHR2 overexpression (SHR2BOE) in hybrid poplars resulted in an increased number of ARs with an initial delay. Despite a lower endogenous content in the stems than in wild-type plants, indole-3-acetic acid (IAA) content at the SHR2BOE basal stem increased rapidly after cutting and reached a higher maximum than in wild-type plants, which was accompanied by a more sustained and stronger induction of AR formation marker genes. In addition, the higher auxin content in SHR2BOE ARs resulted in more and longer lateral roots (LRs). Application of auxin abolished the early delay in the formation of AR and largely other AR phenotypes of SHR2BOE plants, whereas the polar auxin transport inhibitor N-1-naphthylphthalamic acid completely inhibited both AR and LR abnormalities. Since the enhanced rooting ability of SHR2BOE stem cuttings in hydroponics was clearly confirmed, our results suggest a novel role of poplar SHR2 as a positive regulator during the organogenesis of AR and LR by affecting local auxin homeostasis.

Structural insights into auxin recognition and efflux by Arabidopsis PIN1

Polar auxin transport is unique to plants and coordinates their growth and development1,2. The PIN-FORMED (PIN) auxin transporters exhibit highly asymmetrical localizations at the plasma membrane and drive polar auxin transport3,4; however, their structures and transport mechanisms remain largely unknown. Here, we report three inward-facing conformation structures of Arabidopsis thaliana PIN1: the apo state, bound to the natural auxin indole-3-acetic acid (IAA), and in complex with the polar auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). The transmembrane domain of PIN1 shares a conserved NhaA fold5. In the substrate-bound structure, IAA is coordinated by both hydrophobic stacking and hydrogen bonding. NPA competes with IAA for the same site at the intracellular pocket, but with a much higher affinity. These findings inform our understanding of the substrate recognition and transport mechanisms of PINs and set up a framework for future research on directional auxin transport, one of the most crucial processes underlying plant development.

A novel chemical inhibitor of polar auxin transport promotes shoot regeneration by local enhancement of HD-ZIP III transcription.

De novo shoot organogenesis is a prerequisite for numerous applications in plant research and breeding but is often a limiting factor, for example, in genome editing approaches. Class III homeodomain-leucine zipper (HD-ZIP III) transcription factors have been characterized as crucial regulators of shoot specification, however up-stream components controlling their activity during shoot regeneration are only partially identified. In a chemical genetic screen, we isolated ZIC2, a novel activator of HD-ZIP III activity. Using molecular, physiological and hormone transport analyses in Arabidopsis and sunflower (Helianthus annuus), we examined the molecular mechanism by which the drug promotes HD-ZIP III expression. ZIC2-dependent upregulation of HD-ZIP III transcription promotes shoot regeneration in Arabidopsis and is accompanied by the induction of shoot specifying factors WUS and RAP2.6L and a subset of cytokinin biosynthesis enzymes. ZIC2's effect on HD-ZIP III expression and regeneration is based on its ability to limit polar auxin transport. We further provide evidence that chemical modulation of auxin efflux can enhance de novo shoot formation in the regeneration recalcitrant species sunflower. Activation of HD-ZIP III transcription during shoot regeneration depends on the local distribution of auxin and chemical modulation of auxin transport can be used to overcome poor shoot organogenesis in tissue culture.

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.