Auxin Sensitivity Research Articles

The auxin-like effect of substrate and auxin sensitivity of plant modulate the PGPR activity of Lysinibacillus pinottii sp. nov. PB211

The auxin-like effect of substrate and auxin sensitivity of plant modulate the PGPR activity of Lysinibacillus pinottii sp. nov. PB211

MCTP controls nucleocytoplasmic partitioning of AUXIN RESPONSE FACTORs during lateral root development.

The plant hormone auxin orchestrates almost all aspects of plant growth and development. AUXIN RESPONSE FACTORs (ARFs) control the transcription of auxin-responsive genes, forming cytoplasmic condensates to modulate auxin sensitivity and diversify auxin response regulation. However, the dynamic control of ARF distribution across different subcellular compartments remains largely obscure. Here, we show that three MULTIPLE C2 DOMAIN AND TRANSMEMBRANE REGION PROTEINs (MCTPs), MCTP3, MCTP4, and MCTP6, control ARF nucleocytoplasmic partitioning and determine lateral root development. MCTP3/4/6 are highly expressed in lateral roots and specifically interact with ARF7 and ARF19 to dissolve their cytoplasmic condensates. This promotes ARF nuclear localization in lateral root primordia and enhances auxin signaling during lateral root formation. Our findings confer MCTP as a key switch to modulate auxin responses and outline an MCTP-ARF signaling cascade that is crucial for the establishment of the plant root system.

Auxin co-receptor IAA17/AXR3 controls cell elongation in Arabidopsis thaliana root solely by modulation of nuclear auxin pathway.

The nuclear TIR1/AFB-Aux/IAA auxin pathway plays a crucial role in regulating plant growth and development. Specifically, the IAA17/AXR3 protein participates in Arabidopsis thaliana root development, response to auxin and gravitropism. However, the mechanism by which AXR3 regulates cell elongation is not fully understood. We combined genetical and cell biological tools with transcriptomics and determination of auxin levels and employed live cell imaging and image analysis to address how the auxin response pathways influence the dynamics of root growth. We revealed that manipulations of the TIR1/AFB-Aux/IAA pathway rapidly modulate root cell elongation. While inducible overexpression of the AXR3-1 transcriptional inhibitor accelerated growth, overexpression of the dominant activator form of ARF5/MONOPTEROS inhibited growth. In parallel, AXR3-1 expression caused loss of auxin sensitivity, leading to transcriptional reprogramming, phytohormone signaling imbalance and increased levels of auxin. Furthermore, we demonstrated that AXR3-1 specifically perturbs nuclear auxin signaling, while the rapid auxin response remains functional. Our results shed light on the interplay between the nuclear and cytoplasmic auxin pathways in roots, revealing their partial independence but also the dominant role of the nuclear auxin pathway during the gravitropic response of Arabidopsis thaliana roots.

MicroRNA156 conditions auxin sensitivity to enable growth plasticity in response to environmental changes in Arabidopsis

MicroRNAs (miRNAs) play diverse roles in plant development, but whether and how miRNAs participate in thermomorphogenesis remain ambiguous. Here we show that HYPONASTIC LEAVES 1 (HYL1)—a key component of miRNA biogenesis—acts downstream of the thermal regulator PHYTOCHROME INTERACTING FACTOR 4 in the temperature-dependent plasticity of hypocotyl growth in Arabidopsis. A hyl1-2 suppressor screen identified a dominant dicer-like1 allele that rescues hyl1-2’s defects in miRNA biogenesis and thermoresponsive hypocotyl elongation. Genome-wide miRNA and transcriptome analysis revealed microRNA156 (miR156) and its target SQUAMOSA PROMOTER-BINDING-PROTEIN-LIKE 9 (SPL9) to be critical regulators of thermomorphogenesis. Surprisingly, perturbation of the miR156/SPL9 module disengages seedling responsiveness to warm temperatures by impeding auxin sensitivity. Moreover, miR156-dependent auxin sensitivity also operates in the shade avoidance response at lower temperatures. Thus, these results unveil the miR156/SPL9 module as a previously uncharacterized genetic circuit that enables plant growth plasticity in response to environmental temperature and light changes.

Dopamine Inhibits Arabidopsis Growth through Increased Oxidative Stress and Auxin Activity

Like some bacterial species and all animals, plants synthesize dopamine and react to its exogenous applications. Despite dopamine’s widespread presence and activity in plants, its role in plant physiology is still poorly understood. Using targeted experimentation informed by the transcriptomic response to dopamine exposure, we identify three major effects of dopamine. First, we show that dopamine causes hypersensitivity to auxin indole-3-acetic acid by enhancing auxin activity. Second, we show that dopamine increases oxidative stress, which can be mitigated with glutathione. Third, we find that dopamine downregulates iron uptake mechanisms, leading to a decreased iron content—a response possibly aimed at reducing DA-induced oxidative stress. Finally, we show that dopamine-induced auxin sensitivity is downstream of glutathione biosynthesis, indicating that the auxin response is likely a consequence of DA-induced oxidative stress. Collectively, our results show that exogenous dopamine increases oxidative stress, which inhibits growth both directly and indirectly by promoting glutathione-biosynthesis-dependent auxin hypersensitivity.

Divergent regulation of auxin responsive genes in root-knot and cyst nematodes feeding sites formed in Arabidopsis.

Cysts (CNs) and root-knot nematodes (RKNs) induce specialized feeding cells, syncytia, and giant cells (GCs), respectively, within plant roots. The plant tissues around the GCs usually by respond forming a root swelling called a gall that contains the GCs. The ontogenesis of feeding cells is different. GC formation is a process of new organogenesis from vascular cells, which are still not well characterized, that differentiate into GCs. In contrast, syncytia formation involves the fusion of adjacent cells that have already differentiated. Nonetheless, both feeding sites show an auxin maximum pertinent to feeding site formation. However, data on the molecular divergences and similarities between the formation of both feeding sites regarding auxin-responsive genes are still scarce. We studied genes from the auxin transduction pathways that are crucial during gall and lateral root (LR) development in the CN interaction by using promoter-reporter (GUS/LUC)transgenic lines, as well as loss of function lines of Arabidopsis. The promoters pGATA23 and several deletions of pmiR390a were active in syncytia, as were in galls, but pAHP6 or putative up-stream regulators as ARF5/7/19 were not active in syncytia. Additionally, none of these genes seemed to play a key role during cyst nematode establishment in Arabidopsis, as the infection rates in loss of function lines did not show significant differences compared to control Col-0 plants. Furthermore, the presence of only canonical AuxRe elements in their proximal promoter regions is highly correlated with their activation in galls/GCs (AHP6, LBD16), but those promoters active in syncytia (miR390, GATA23) carry AuxRe overlapping core cis-elements for other transcription factor families (i.e., bHLH, bZIP). Strikingly, in silico transcriptomic analysis showed very few genes upregulated by auxins common to those induced in GCs and syncytia, despite the high number of upregulated IAA responsive genes in syncytia and galls. The complex regulation of auxin transduction pathways, where different members of the auxin response factor (ARF) family may interact with other factors, and the differences in auxin sensitivity, as indicated by the lower induction of the DR5 sensor in syncytia than galls, among other factors, may explain the divergent regulation of auxin responsive genes in the two types of nematode feeding sites.

Mitigation of gravitropic bending of snapdragon cut flowers through SNP treatment via ROS asymmetry elimination and auxin sensitivity reduction

Snapdragons (Antirrhinum majus L.) are an important fresh-cut flower in the international flower market. During storage and transportation, snapdragons often undergo a gravitropic response that has a substantial adverse effect on their ornamental and market value. The gravitropic response is a complex physiological and biochemical process that is affected by signals such as auxins and reactive oxygen species (ROS). In this study, we found that the pretreatment of exogenous nitric oxide (NO) donor sodium nitroprusside (SNP, 0.2 mM) effectively inhibited the gravitropic bending of snapdragon cut flowers after horizontal placement. The gravitropic stimulus induced higher ROS (O2−∙ and H2O2) accumulation in the lower side compared to the upper side of the bending zone of the control flower stems. Compared to the control, a substantially higher accumulation of ROS was induced through the SNP treatment, correlating with the increased activity of catalase (CAT) and ascorbate peroxidase (APX), as well as the enhanced expression of AmAPX1 and AmCAT1. However, unlike the control, no ROS asymmetric distribution was found for the SNP-treated flowers. Four free auxins, IAA, IBA, ME-IAA and ICA, were detected in the flower stems, among which, only IBA showed asymmetric distribution after the gravitropic stimulus. An IBA content that was approximately 26% higher was detected in the lower side compared to the upper side of the control flower at 2 h, but no difference was found for the SNP-treated flowers. A higher expression of the AmPIN3 gene at the lower sides compared to the upper sides of the control may be correlated with the transient IBA asymmetric distribution. Interestingly, an expression of AmIAA2 and AmSUR50 that was around 2-fold was detected in the lower side compared to the upper side of the control, indicating that higher auxin sensitivity may be induced in the lower side. However, the expression of AmIAA2 and AmSUR50 was reduced by the SNP treatment, implying that high ROS levels may reduce auxin sensitivity. Furthermore, a higher AmExp1 and AmH+–ATPase1 expression in the lower side compared to the upper side was found for the control but not for the SNP-treated flowers, indicating the two genes may be involved in the bending of the flower stems. The above results reveal that SNP treatment eliminates the asymmetric distribution of ROS and reduced auxin sensitivity of snapdragon flower stems, thereby eliminating their asymmetric growth and further inhibiting their gravitropic response.

Auxin sensitivity improves production of rosmarinic acid in transformed hairy roots of <em>Lavandula angustifolia</em>

Hairy root culture is a promising approach to improve production of plant secondary metabolites. The genes, which are located in T-DNA of a root-inducing plasmid, regulate auxin sensitivity of hairy roots. Therefore, this study was aimed to improve the growth and rosmarinic acid production of Lavandula angustifolia hairy roots. Lateral branches of hairy roots were transferred to ½ MS and ½ B5 liquid media. To assess auxin sensitivity, indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) with four different concentrations (0, 0.1, 0.5 and 1 mg/l) were also applied. The growth of hairy roots in ½ MS medium was two-fold higher than in ½ B5 medium. In addition, both auxins were found to significantly improve the growth of hairy roots whereas non-transformed roots stopped growing in the presence of the auxins. The highest dry weight and rosmarinic acid production of hairy roots were obtained from ½ MS medium supplemented with IBA irrespective of its concentration. As a result, the hairy roots grown in ½ MS medium supplemented with IBA produced the maximum amount of rosmarinic acid (7.98 mg/g dry weight of hairy roots). This first report of rosmarinic acid production in L. angustifolia hairy roots provides new insights into the auxin sensitivity of L. angustifolia hairy roots.

INOSITOL (1,3,4) TRIPHOSPHATE 5/6 KINASE1-dependent inositol polyphosphates regulate auxin responses in Arabidopsis.

The combinatorial phosphorylation of myo-inositol results in the generation of different inositol phosphates (InsPs), of which phytic acid (InsP6) is the most abundant species in eukaryotes. InsP6 is also an important precursor of the higher phosphorylated inositol pyrophosphates (PP-InsPs), such as InsP7 and InsP8, which are characterized by a diphosphate moiety and are also ubiquitously found in eukaryotic cells. While PP-InsPs regulate various cellular processes in animals and yeast, their biosynthesis and functions in plants has remained largely elusive because plant genomes do not encode canonical InsP6 kinases. Recent work has shown that Arabidopsis (Arabidopsis thaliana) INOSITOL (1,3,4) TRIPHOSPHATE 5/6 KINASE1 (ITPK1) and ITPK2 display in vitro InsP6 kinase activity and that, in planta, ITPK1 stimulates 5-InsP7 and InsP8 synthesis and regulates phosphate starvation responses. Here we report a critical role of ITPK1 in auxin-related processes that is independent of the ITPK1-controlled regulation of phosphate starvation responses. Those processes include primary root elongation, root hair development, leaf venation, thermomorphogenic and gravitropic responses, and sensitivity to exogenously applied auxin. We found that the recombinant auxin receptor complex, consisting of the F-Box protein TRANSPORT INHIBITOR RESPONSE1 (TIR1), ARABIDOPSIS SKP1 HOMOLOG 1 (ASK1), and the transcriptional repressor INDOLE-3-ACETIC ACID INDUCIBLE 7 (IAA7), binds to anionic inositol polyphosphates with high affinity. We further identified a physical interaction between ITPK1 and TIR1, suggesting a localized production of 5-InsP7, or another ITPK1-dependent InsP/PP-InsP isomer, to activate the auxin receptor complex. Finally, we demonstrate that ITPK1 and ITPK2 function redundantly to control auxin responses, as deduced from the auxin-insensitive phenotypes of itpk1 itpk2 double mutant plants. Our findings expand the mechanistic understanding of auxin perception and suggest that distinct inositol polyphosphates generated near auxin receptors help to fine-tune auxin sensitivity in plants.

The regeneration factors ERF114 and ERF115 regulate auxin-mediated lateral root development in response to mechanical cues

Plants show an unparalleled regenerative capacity, allowing them to survive severe stress conditions, such as injury, herbivory attack, and harsh weather conditions. This potential not only replenishes tissues and restores damaged organs but can also give rise to whole plant bodies. Despite the intertwined nature of development and regeneration, common upstream cues and signaling mechanisms are largely unknown. Here, we demonstrate that in addition to being activators of regeneration, ETHYLENE RESPONSE FACTOR 114 (ERF114) and ERF115 govern developmental growth in the absence of wounding or injury. Increased ERF114 and ERF115 activity enhances auxin sensitivity, which is correlated with enhanced xylem maturation and lateral root formation, whereas their knockout results in a decrease in lateral roots. Moreover, we provide evidence that mechanical cues contribute to ERF114 and ERF115 expression in correlation with BZR1-mediated brassinosteroid signaling under both regenerative and developmental conditions. Antagonistically, cell wall integrity surveillance via mechanosensory FERONIA signaling suppresses their expression under both conditions. Taken together, our data suggest a molecular framework in which cell wall signals and mechanical strains regulate organ development and regenerative responses via ERF114- and ERF115-mediated auxin signaling.

The co-chaperone HOP participates in TIR1 stabilisation and in auxin response in plants.

HOP (HSP70‐HSP90 organising protein) is a conserved family of co‐chaperones well known in mammals for its role in the folding of signalling proteins associated with development. In plants, HOP proteins have been involved in the response to multiple stresses, but their role in plant development remains elusive. Herein, we describe that the members of the HOP family participate in different aspects of plant development as well as in the response to warm temperatures through the regulation of auxin signalling. Arabidopsis hop1 hop2 hop3 triple mutant shows different auxin‐related phenotypes and a reduced auxin sensitivity. HOP interacts with TIR1 auxin coreceptor in vivo. Furthermore, TIR1 accumulation and auxin transcriptional response are reduced in the hop1 hop2 hop3 triple mutant, suggesting that HOP's function in auxin signalling is related, at least, to TIR1 interaction and stabilisation. Interestingly, HOP proteins form part of the same complexes as SGT1b (a different HSP90 co‐chaperone) and these co‐chaperones synergistically cooperate in auxin signalling. This study provides relevant data about the role of HOP in auxin regulation in plants and uncovers that both co‐chaperones, SGT1b and HOP, cooperate in the stabilisation of common targets involved in plant development.

Reduced auxin signalling through the cyclophilin gene DIAGEOTROPICA impacts tomato fruit development and metabolism during ripening.

Auxin is an important hormone playing crucial roles during fruit growth and ripening; however, the metabolic impact of changes in auxin signalling during tomato (Solanum lycopersicum L.) ripening remains unclear. Here, we investigated the significance of changes in auxin signalling during different stages of fruit development by analysing changes in tomato fruit quality and primary metabolism using mutants with either lower or higher auxin sensitivity [diageotropica (dgt) and entire mutants, respectively]. Altered auxin sensitivity modifies metabolism, through direct impacts on fruit respiration and fruit growth. We verified that the dgt mutant plants exhibit reductions in fruit set, total fruit dry weight, fruit size, number of seeds per fruit, and fresh weight loss during post-harvest. Sugar accumulation was associated with delayed fruit ripening in dgt, probably connected with reduced ethylene levels and respiration, coupled with a lower rate of starch degradation. In contrast, despite exhibiting parthenocarpy, increased auxin perception (entire) did not alter fruit ripening, leading to only minor changes in primary metabolism. By performing a comprehensive analysis, our results connect auxin signalling and metabolic changes during tomato fruit development, indicating that reduced auxin signalling led to extensive changes in sugar concentration and starch metabolism during tomato fruit ripening.

Transcriptome Analysis and Genes Function Verification of Root Development of Paeonia suffruticosa under Sandy Loam Cultivation

Relatively poor in vitro rooting has limited the large-scale commercial production of tree peony. In this study, on the basis of transcriptome sequencing, differentially expressed genes and the associated metabolic pathways were identified in tree peony roots at different stages of root formation under sandy loam cultivation. A total of 31.63 Gb raw data were generated and 120,188 unigenes (mean length of 911.98 bp) were annotated according to six databases (NR, NT, GO, KEGG, COG, and Swiss-Prot). Analyses of the ungerminated root primordium period, induced root primordium period, and root formation period detected 8,232, 6,907, and 10,687 differentially expressed genes related to 133, 132, and 133 metabolic pathways, respectively. Two significantly differentially expressed genes (Unigene13430_All and CL10096.Contig1_All) were associated with the auxin pathway. The full-length Unigene13430_All coding sequence (843 bp) encoded 280 amino acids, whereas the full-length CL10096.Contig1_All coding sequence (1,470 bp) encoded 489 amino acids. Unigene13430_All and CL10096.Contig1_All were identified as IAA gene family members and were respectively named PsIAA27 and PsARF19. The qRT-PCR analysis and functional verification indicated that the expressions of PsARF19 and PsIAA27 in tree peony seedlings, cuttings and grafted seedlings were significant different. PsARF19 promoted root development, it might be a regulatory gene related to the formation of tree peony roots, while PsIAA27 inhibited lateral root development, and it might be involved in controlling auxin sensitivity during root formation. The results of this study may form the basis of future investigations on the mechanism mediating peony root formation. The transcriptome data will be an excellent resource for researchers interested in characterizing the rooting-related tree peony genes.

Microrna Regulation of Nodule Zone-Specific Gene Expression In Soybean

Microrna Regulation of Nodule Zone-Specific Gene Expression In Soybean

Small compounds targeting tyrosine phosphorylation of Scaffold Protein Receptor for Activated C Kinase1A (RACK1A) regulate auxin mediated lateral root development in Arabidopsis

ABSTRACT Receptor for activated C kinase 1 (RACK1) is WD-40 type scaffold protein, conserved in all eukaryote organisms. Many reports implicated RACK1 in plant hormone signal transduction pathways including in auxin and diverse stress signaling pathways; however, the precise molecular mechanism of its role is not understood. Previously, a group of small compounds targeting the Arabidopsis RACK1A functional site-Tyr248 have been developed. Here, the three different small compounds are used to elucidate the role of RACK1A in auxin mediated lateral root development. Through monitoring the auxin response in the architecture of lateral roots and auxin reporter assays, a small molecule- SD29-12 was found to stabilize the auxin induced RACK1A Tyr248 phosphorylation, thereby stimulating auxin signaling and inducing lateral roots formation. In contrast, two other compounds, SD29 and SD29-14, inhibited auxin induced RACK1A Tyr248 phosphorylation resulting in the inhibition of auxin sensitivity and alternation in the lateral roots formation. Taken together, auxin induced RACK1A Tyr248 phosphorylation is found to be the critical regulatory mechanism for auxin-mediated lateral root development. This work leads to the molecular understanding of the role RACK1A plays in the auxin induced lateral root development signaling pathways. The auxin signal stimulating compound has the potential to be used as auxin-based root inducing bio-stimulant.

Complex-Type N-Glycans Influence the Root Hair Landscape of Arabidopsis Seedlings by Altering the Auxin Output.

Roots supply plants with nutrients and water, besides anchoring them in the soil. The primary root with its lateral roots constitutes the central skeleton of the root system. In particular, root hairs increase the root surface, which is critical for optimizing uptake efficiency. During root-cell growth and development, many proteins that are components of, e.g., the cell wall and plasma membrane are constitutively transported through the secretory system and become posttranslationally modified. Here, the best-studied posttranslational modification is protein N-glycosylation. While alterations in the attachment/modification of N-glycans within the ER lumen results in severe developmental defects, the impact of Golgi-localized complex N-glycan modification, particularly on root development, has not been studied in detail. We report that impairment of complex-type N-glycosylation results in a differential response to synthetic phytohormones with earlier and increased root-hair elongation. Application of either the cytokinin BAP, the auxin NAA, or the ethylene precursor ACC revealed an interaction of auxin with complex N-glycosylation during root-hair development. Especially in gntI mutant seedlings, the early block of complex N-glycan formation resulted in an increased auxin sensitivity. RNA-seq experiments suggest that gntI roots have permanently elevated nutrient-, hypoxia-, and defense-stress responses, which might be a consequence of the altered auxin responsiveness.

Coordination between MIDASIN 1-mediated ribosome biogenesis and auxin modulates plant development.

Ribosomes are required for plant growth and development, and ribosome biogenesis-deficient mutants generally display auxin-related phenotypes. Although the relationship between ribosome dysfunction and auxin is known, many aspects of this subject remain to be understood. We previously reported that MIDASIN 1 (MDN1) is an essential pre-60S ribosome biogenesis factor (RBF) in Arabidopsis. In this study, we further characterized the aberrant auxin-related phenotypes of mdn1-1, a weak mutant allele of MDN1. Auxin response is disturbed in both shoots and roots of mdn1-1, as indicated by the DR5:GUS reporter. By combining transcriptome profiling analysis and reporter gene detection, we found that expression of genes involved in auxin biosynthesis, transport, and signaling is changed in mdn1-1. Furthermore, MDN1 deficiency affects the post-transcriptional regulation and protein distribution of PIN-FORMED 2 (PIN2, an auxin efflux facilitator) in mdn1-1 roots. These results indicate that MDN1 is required for maintaining the auxin system. More interestingly, MDN1 is an auxin-responsive gene, and its promoter can be targeted by multiple AUXIN RESPONSE FACTORs (ARFs), including ARF7 and ARF19, in vitro. Indeed, in arf7 arf19, the auxin sensitivity of MDN1 expression is significantly reduced. Together, our results reveal a coordination mechanism between auxin and MDN1-dependent ribosome biogenesis for regulating plant development.

Low Red Light:Far Red Light Inhibits Branching by Promoting Auxin Signaling

The branching trait is influenced by various environmental signals, including the Red light:Far Red light (R:FR), an indicator of competition and an inhibitor of axillary bud growth. Branch development is influenced by an array of hormones, including auxin which indirectly suppresses bud growth as a consequence of auxin transport and signaling in the main stem. The suppressive effect of auxin sourced from the shoot apex and transported basipetally is a major mechanism contributing to apical dominance, a form of correlative inhibition where superior growing points restrict the growth of those lower on the plant. The current study shows that increased apical dominance is a mechanism that suppresses the branching of plants grown in a low R:FR. The elevated apical dominance was not due to increased levels of the natural auxin IAA, but was associated with enhanced expression of auxin-induced genes both in the presence and absence of exogenous auxin. A direct test of bud sensitivity to auxin confirmed that a low R:FR promoted auxin responsiveness leading to reduced bud growth. Thus, the low R:FR enhancement of auxin sensitivity is a mechanism that contributes to the reduced branching phenotype of plants growing in competitive environments.

Genetic control of tracheid properties in Norway spruce wood

Through the use of genome-wide association studies (GWAS) mapping it is possible to establish the genetic basis of phenotypic trait variation. Our GWAS study presents the first such effort in Norway spruce (Picea abies (L). Karst.) for the traits related to wood tracheid characteristics. The study employed an exome capture genotyping approach that generated 178 101 Single Nucleotide Polymorphisms (SNPs) from 40 018 probes within a population of 517 Norway spruce mother trees. We applied a least absolute shrinkage and selection operator (LASSO) based association mapping method using a functional multi-locus mapping approach, with a stability selection probability method as the hypothesis testing approach to determine significant Quantitative Trait Loci (QTLs). The analysis has provided 30 significant associations, the majority of which show specific expression in wood-forming tissues or high ubiquitous expression, potentially controlling tracheids dimensions, their cell wall thickness and microfibril angle. Among the most promising candidates based on our results and prior information for other species are: Picea abies BIG GRAIN 2 (PabBG2) with a predicted function in auxin transport and sensitivity, and MA_373300g0010 encoding a protein similar to wall-associated receptor kinases, which were both associated with cell wall thickness. The results demonstrate feasibility of GWAS to identify novel candidate genes controlling industrially-relevant tracheid traits in Norway spruce.

TDIF regulates auxin accumulation and modulates auxin sensitivity to enhance both adventitious root and lateral root formation in poplar trees.

Of six TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF)-encoding genes in poplar, PtTDIF1 is predominantly expressed in adventitious roots (ARs), and the other five PtTDIFs are preferentially expressed in lateral roots (LRs). Upon auxin application, expression of all PtTDIFs declined in ARs but transiently increased in LRs. Both exogenous TDIF peptides and overexpression of PtTDIFs in poplar positively regulated the initiation and elongation of LRs, and overexpression of PtTDIFs also increased the number of ARs. As visualized by the auxin-responsive marker DR5:GUS, TDIF had differential impacts on the auxin signaling activity in ARs and LRs, which was corroborated by the free indole-3-acetic acid (IAA) measurements in them. Shoot tips of PtTDIF2- and PtTDIFL2-overexpressing (together as PtTDIFsOE) trees revealed an enhanced IAA biosynthetic capacity, and removal of the aerial tissues dramatically diminished the root phenotypes of micro-propagated PtTDIFsOE trees. Furthermore, PtTDIFsOE poplars displayed an increased sensitivity for exogenous IAA, and N-1-naphthylphthalamic acid (NPA) completely blocked the TDIF-induced AR and LR formation. In PtTDIFsOE roots, several auxin-related LR initiation markers such as GATA23, LBD16 and LBD29 were transcriptionally upregulated, further supporting that TDIF regulates LR organogenesis by strengthening the spatiotemporal auxin cues and that dynamic interplays between hormones govern root branching and developmental plasticity in tree species.