WAV E3 ubiquitin ligases mediate degradation of IAA32/34 in the TMK1-mediated auxin signaling pathway during apical hook development.

Auxin regulates plant growth and development through downstream signaling pathways, including the best-known SCFTIR1/AFB-Aux/IAA-ARF pathway and several other less characterized "noncanonical" pathways. Recently, one SCFTIR1/AFB-independent noncanonical pathway, mediated by Transmembrane Kinase 1 (TMK1), was discovered through the analyses of its functions in Arabidopsis apical hook development. Asymmetric accumulation of auxin on the concave side of the apical hook triggers DAR1-catalyzed release of the C-terminal of TMK1, which migrates into the nucleus, where it phosphorylates and stabilizes IAA32/34 to inhibit cell elongation, which is essential for full apical hook formation. However, the molecular factors mediating IAA32/34 degradation have not been identified. Here, we show that proteins in the CYTOKININ INDUCED ROOT WAVING 1 (CKRW1)/WAVY GROWTH 3 (WAV3) subfamily act as E3 ubiquitin ligases to target IAA32/34 for ubiquitination and degradation, which is inhibited by TMK1c-mediated phosphorylation. This antagonistic interaction between TMK1c and CKRW1/WAV3 subfamily E3 ubiquitin ligases regulates IAA32/34 levels to control differential cell elongation along opposite sides of the apical hook.

Intrinsic disorder and conformational coexistence in auxin coreceptors.

AUXIN/INDOLE 3-ACETIC ACID (Aux/IAA) transcriptional repressor proteins and the TRANSPORT INHIBITOR RESISTANT 1/AUXIN SIGNALING F-BOX (TIR1/AFB) proteins to which they bind act as auxin coreceptors. While the structure of TIR1 has been solved, structural characterization of the regions of the Aux/IAA protein responsible for auxin perception has been complicated by their predicted disorder. Here, we use NMR, CD and molecular dynamics simulation to investigate the N-terminal domains of the Aux/IAA protein IAA17/AXR3. We show that despite the conformational flexibility of the region, a critical W-P bond in the core of the Aux/IAA degron motif occurs at a strikingly high (1:1) ratio of cis to trans isomers, consistent with the requirement of the cis conformer for the formation of the fully-docked receptor complex. We show that the N-terminal half of AXR3 is a mixture of multiple transiently structured conformations with a propensity for two predominant and distinct conformational subpopulations within the overall ensemble. These two states were modeled together with the C-terminal PB1 domain to provide the first complete simulation of an Aux/IAA. Using MD to recreate the assembly of each complex in the presence of auxin, both structural arrangements were shown to engage with the TIR1 receptor, and contact maps from the simulations match closely observations of NMR signal-decreases. Together, our results and approach provide a platform for exploring the functional significance of variation in the Aux/IAA coreceptor family and for understanding the role of intrinsic disorder in auxin signal transduction and other signaling systems.

SlPP2C2 interacts with FZY/SAUR and regulates tomato development via signaling crosstalk of ABA and auxin.

Abscisic acid (ABA) signaling interacts frequently with auxin signaling when it regulates plant development, affecting multiple physiological processes; however, to the best of our knowledge, their interaction during tomato development has not yet been reported. Here, we found that type 2C protein phosphatase (SlPP2C2) interacts with both flavin monooxygenase FZY, an indole-3-acetic acid (IAA) biosynthetic enzyme, and small auxin upregulated RNA (SAUR) of an IAA signaling protein and regulates their activity, thereby affecting the expression of IAA-responsive genes. The expression level of SlPP2C2 was increased by exogenous ABA, IAA, NaCl, or dehydration treatment of fruits, leaves, and seeds, and it decreased in imbibed seeds. Manipulating SlPP2C2 with overexpression, RNA interference, and CRISPR/Cas9-mediated genome editing resulted in pleiotropic changes, such as morphological changes in leaves, stem trichomes, floral organs and fruits, accompanied by alterations in IAA and ABA levels. Furthermore, the RNA-seq analysis indicated that SlPP2C2 regulates the expression of auxin-/IAA-responsive genes in different tissues of tomato. The results demonstrate that SlPP2C2-mediated ABA signaling regulates the development of both vegetative and reproductive organs via interaction with FZY/SAUR, which integrates the cross-talk of ABA and auxin signals during development and affects the expressions of development-related genes in tomato.

Transient efflux inhibition improves plant regeneration by natural auxins.

Plant genome editing and propagation are important tools in crop breeding and production. Both rely heavily on the development of efficient in vitro plant regeneration systems. Two prominent regeneration systems that are widely employed in crop production are somatic embryogenesis (SE) and de novo shoot regeneration. In many of the protocols for SE or shoot regeneration, explants are treated with the synthetic auxin analog 2,4-dichlorophenoxyacetic acid (2,4-D), since natural auxins, such as indole-3-acetic acid (IAA) or 4-chloroindole-3-acetic acid (4-Cl-IAA), are less effective or even fail to induce regeneration. Based on previous reports that 2,4-D, compared to endogenous auxins, is not effectively exported from plant cells, we investigated whether efflux inhibition of endogenous auxins could convert these auxins into efficient inducers of SE in Arabidopsis immature zygotic embryos (IZEs). We show that natural auxins and synthetic analogs thereof become efficient inducers of SE when their efflux is transiently inhibited by co-application of the auxin transport inhibitor naphthylphthalamic acid (NPA). Moreover, IZEs of auxin efflux mutants pin2 or abcb1 abcb19 show enhanced SE efficiency when treated with IAA or efflux-inhibited IAA, confirming that auxin efflux reduces the efficiency of Arabidopsis SE. Importantly, in contrast to the 2,4-D system, where only 50-60% of the embryos converted to seedlings, all SEs induced by transport-inhibited natural auxins converted to seedlings. Efflux-inhibited IAA, like 2,4-D, also efficiently induced SE from carrot suspension cells, whereas IAA alone could not, and efflux-inhibited 4-Cl-IAA significantly improved de novo shoot regeneration in Brassica napus. Our data provides new insights into the action of 2,4-D as an efficient inducer of plant regeneration but also shows that replacing this synthetic auxin for efflux-inhibited natural auxin significantly improves different types of plant regeneration, leading to a more synchronized and homogenous development of the regenerated plants.

Biophysical characterization of chloride intracellular channel 6 (CLIC6).

Chloride intracellular channels (CLICs) are a family of proteins that exist in soluble and transmembrane forms. The newest discovered member of the family CLIC6 is implicated in breast, ovarian, lung gastric, and pancreatic cancers and is also known to interact with dopamine-(D(2)-like) receptors. The soluble structure of the channel has been resolved, but the exact physiological role of CLIC6, biophysical characterization, and the membrane structure remain unknown. Here, we aimed to characterize the biophysical properties of this channel using a patch-clamp approach. To determine the biophysical properties of CLIC6, we expressed CLIC6 in HEK-293 cells. On ectopic expression, CLIC6 localizes to the plasma membrane of HEK-293 cells. We established the biophysical properties of CLIC6 by using electrophysiological approaches. Using various anions and potassium (K+) solutions, we determined that CLIC6 is more permeable to chloride-(Cl-) as compared to bromide-(Br-), fluoride-(F-), and K+ ions. In the whole-cell configuration, the CLIC6 currents were inhibited after the addition of 10 µM of IAA-94 (CLIC-specific blocker). CLIC6 was also found to be regulated by pH and redox potential. We demonstrate that the histidine residue at 648 (H648) in the C terminus and cysteine residue in the N terminus (C487) are directly involved in the pH-induced conformational change and redox regulation of CLIC6, respectively. Using qRT-PCR, we identified that CLIC6 is most abundant in the lung and brain, and we recorded the CLIC6 current in mouse lung epithelial cells. Overall, we have determined the biophysical properties of CLIC6 and established it as a Cl- channel.

An essential signaling function of cytoplasmic NELFB is independent of RNA polymerase II pausing.

The four-subunit negative elongation factor (NELF) complex mediates RNA polymerase II (Pol II) pausing at promoter-proximal regions. Ablation of individual NELF subunits destabilizes the NELF complex and causes cell lethality, leading to the prevailing concept that NELF-mediated Pol II pausing is essential for cell proliferation. Using separation-of-function mutations, we show here that NELFB function in cell proliferation can be uncoupled from that in Pol II pausing. NELFB mutants sequestered in the cytoplasm and deprived of NELF nuclear function still support cell proliferation and part of the NELFB-dependent transcriptome. Mechanistically, cytoplasmic NELFB physically and functionally interacts with prosurvival signaling kinases, most notably phosphatidylinositol-3-kinase/AKT. Ectopic expression of membrane-tethered phosphatidylinositol-3-kinase/AKT partially bypasses the role of NELFB in cell proliferation, but not Pol II occupancy. Together, these data expand the current understanding of the physiological impact of Pol II pausing and underscore the multiplicity of the biological functions of individual NELF subunits.

AefR, a TetR Family Transcriptional Repressor, Regulates Several Auxin Responses in Pseudomonas syringae Strain PtoDC3000.

The plant hormone indole-3-acetic acid (IAA), also known as auxin, plays important roles in plant growth and development, as well as in several plant-microbe interactions. IAA also acts as a microbial signal and in many bacteria regulates metabolism, stress responses, and virulence. In the bacterial plant pathogen Pseudomonas syringae pv. tomato strain DC3000 (PtoDC3000), exposure to IAA results in large-scale transcriptional reprogramming, including the differential expression of several known virulence genes. However, how PtoDC3000 senses and responds to IAA and what aspects of its biology are regulated by IAA is not understood. To investigate the mechanisms involved in perceiving and responding to IAA, we carried out a genetic screen for mutants with altered responses to IAA. One group of mutants of particular interest carried disruptions in the aefR gene encoding a TetR family transcriptional regulator. Gene expression analysis confirmed that the aefR mutants have altered responses to IAA. Thus, AefR is the first demonstrated auxin response regulator in PtoDC3000. We also investigated several aspects of PtoDC3000 biology that are regulated by both AefR and IAA, including antibiotic resistance, motility, and virulence. The observation that the aefR mutant has altered virulence on Arabidopsis, suggests that the sector of the IAA response regulated by aefR is important during pathogenesis. Our findings also provide evidence that AefR plays a role in coordinating changes in gene expression during the transition from early to late stages of infection. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genome-wide characterization, functional analysis, and expression profiling of the Aux/IAA gene family in spinach.

BACKGROUND: The auxin/indole-3-acetic acid (Aux/IAA) gene family is a crucial element of the auxin signaling pathway, significantly influencing plant growth and development. Hence, we conducted a comprehensive investigation of Aux/IAAs gene family using the Sp75 and Monoe-Viroflay genomes in spinach. RESULTS: A total of 24 definitive Aux/IAA genes were identified, exhibiting diverse attributes in terms of amino acid length, molecular weight, and isoelectric points. This diversity underscores potential specific roles within the family, such as growth regulation and stress response. Structural analysis revealed significant variations in gene length and molecular weight. These variations indicate distinct roles within the Aux/IAA gene family. Chromosomal distribution analysis exhibited a dispersed pattern, with chromosomes 4 and 1 hosting the highest and lowest numbers of Aux/IAA genes, respectively. Phylogenetic analysis grouped the identified genes into distinct clades, revealing potential evolutionary relationships. Notably, the phylogenetic tree highlighted specific gene clusters suggesting shared genetic ancestry and potential functional synergies within spinach. Expression analysis under NAA treatment unveiled gene-specific and time-dependent responses, with certain genes exhibiting distinct temporal expression patterns. Specifically, SpoIAA5 displayed a substantial increase at 2 h post-NAA treatment, while SpoIAA7 and SpoIAA9 demonstrated continuous rises, peaking at the 4-hour time point. CONCLUSIONS: These observations indicate a complex interplay of gene-specific and temporal regulation in response to auxin. Moreover, the comparison with other plant species emphasized both shared characteristics and unique features in Aux/IAA gene numbers, providing insights into the evolutionary dynamics of this gene family. This comprehensive characterization of Aux/IAA genes in spinach not only establishes the foundation for understanding their specific functions in spinach development but also provides a valuable resource for experimental validation and further exploration of their roles in the intricate network of auxin signaling pathways.

Genome-wide identification and expression pattern analysis of the Aux/IAA (auxin/indole-3-acetic acid) gene family in alfalfa (Medicago sativa) and the potential functions under drought stress.

BACKGROUND: Auxin/induced-3-acetic acid (Aux/IAA) is an important plant hormone that affects plant growth and resistance to abiotic stresses. Drought stress is a vital factor in reducing plant biomass yield and production quality. Alfalfa (Medicago sativa L.) is the most widely planted leguminous forage and one of the most economically valuable crops in the world. Aux/IAA is one of the early responsive gene families of auxin, playing a crucial role in response to drought stress. However, the characteristics of the Aux/IAA gene family in alfalfa and its potential function in response to drought stress are still unknown. RESULT: A total of 41 Aux/IAA gene members were identified in alfalfa genome. The physicochemical, peptide structure, secondary and tertiary structure analysis of proteins encoded by these genes revealed functional diversity of the MsIAA gene. A phylogenetic analysis classified the MsIAA genes into I-X classes in two subgroups. And according to the gene domain structure, these genes were classified into typical MsIAA and atypical MsIAA. Gene structure analysis showed that the MsIAA genes contained 1-4 related motifs, and except for the third chromosome without MsIAAs, they were all located on 7 chromosomes. The gene duplication analysis revealed that segmental duplication and tandem duplication greatly affected the amplification of the MsIAA genes. Analysis of the Ka/Ks ratio of duplicated MsAux/IAA genes suggested purification selection pressure was high and functional differences were limited. In addition, identification and classification of promoter cis-elements elucidated that MsIAA genes contained numerous elements associated to phytohormone response and abiotic stress response. The prediction protein-protein interaction network showed that there was a complex interaction between the MsAux/IAA genes. Gene expression profiles were tissue-specific, and MsAux/IAA had a broad response to both common abiotic stress (ABA, salt, drought and cold) and heavy metal stress (Al and Pb). Furthermore, the expression patterns analysis of 41 Aux/IAA genes by the quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that Aux/IAA genes can act as positive or negative factors to regulate the drought resistance in alfalfa. CONCLUSION: This study provides useful information for the alfalfa auxin signaling gene families and candidate evidence for further investigation on the role of Aux/IAA under drought stress. Future studies could further elucidate the functional mechanism of the MsIAA genes response to drought stress.

Non-canonical AUX/IAA protein IAA33 competes with canonical AUX/IAA repressor IAA5 to negatively regulate auxin signaling.

The phytohormone auxin controls plant growth and development via TIR1-dependent protein degradation of canonical AUX/IAA proteins, which normally repress the activity of auxin response transcription factors (ARFs). IAA33 is a non-canonical AUX/IAA protein lacking a TIR1-binding domain, and its role in auxin signaling and plant development is not well understood. Here, we show that IAA33 maintains root distal stem cell identity and negatively regulates auxin signaling by interacting with ARF10 and ARF16. IAA33 competes with the canonical AUX/IAA repressor IAA5 for binding to ARF10/16 to protect them from IAA5-mediated inhibition. In contrast to auxin-dependent degradation of canonical AUX/IAA proteins, auxin stabilizes IAA33 protein via MITOGEN-ACTIVATED PROTEIN KINASE 14 (MPK14) and does not affect IAA33 gene expression. Taken together, this study provides insight into the molecular functions of non-canonical AUX/IAA proteins in auxin signaling transduction.

Exploring the synergistic effects of indole acetic acid (IAA) and compost in the phytostabilization of nickel (Ni) in cauliflower rhizosphere.

Heavy metals (HMs) contamination, owing to their potential links to various chronic diseases, poses a global threat to agriculture, environment, and human health. Nickel (Ni) is an essential element however, at higher concentration, it is highly phytotoxic, and affects major plant functions. Beneficial roles of plant growth regulators (PGRs) and organic amendments in mitigating the adverse impacts of HM on plant growth has gained the attention of scientific community worldwide. Here, we performed a greenhouse study to investigate the effect of indole-3-acetic acid (IAA @ 10- 5 M) and compost (1% w/w) individually and in combination in sustaining cauliflower growth and yield under Ni stress. In our results, combined application proved significantly better than individual applications in alleviating the adverse effects of Ni on cauliflower as it increased various plant attributes such as plant height (49%), root length (76%), curd height and diameter (68 and 134%), leaf area (75%), transpiration rate (36%), stomatal conductance (104%), water use efficiency (143%), flavonoid and phenolic contents (212 and 133%), soluble sugars and protein contents (202 and 199%), SPAD value (78%), chlorophyll 'a and b' (219 and 208%), carotenoid (335%), and NPK uptake (191, 79 and 92%) as compared to the control. Co-application of IAA and compost reduced Ni-induced electrolyte leakage (64%) and improved the antioxidant activities, including APX (55%), CAT (30%), SOD (43%), POD (55%), while reducing MDA and H2O2 contents (77 and 52%) compared to the control. The combined application also reduced Ni uptake in roots, shoots, and curd by 51, 78 and 72% respectively along with an increased relative production index (78%) as compared to the control. Hence, synergistic application of IAA and compost can mitigate Ni induced adverse impacts on cauliflower growth by immobilizing it in the soil.

Exogenous indole-3-acetic acid promotes the plant growth and accumulation of selenium in grapevine under selenium stress.

To alleviate the selenium (Se) stress in fruit trees and improve its accumulation, the effects of exogenous indole-3-acetic acid (IAA) on the growth and Se accumulation of grapevine under Se stress were studied. The application of exogenous IAA increased the biomass of grapevine, and the concentration of exogenous IAA had a regression relationship with the biomass. The root and shoot biomass were the maximum at 60 mg L- 1 IAA, increasing by 15.61% and 23.95%, respectively, compared with the control. Exogenous IAA also increased the photosynthetic pigments and the activities of superoxide dismutase and peroxidase in grapevine. Moreover, exogenous IAA increased the contents of total Se, organic Se, and inorganic Se, and the concentration of exogenous IAA had a regression relationship with the total Se content. The highest contents of root total Se and shoot total Se were accumulated at 90 mg L- 1 IAA, increasing by 29.94% and 55.77% respectively,. In addition, the correlation and path analyses revealed that the carotenoid content and root total Se content were closely associated with the shoot total Se content. Therefore, the application of exogenous IAA can alleviate the stress of Se to grape and promote its uptake and the most effective amount for the uptake of Se is 90 mg L- 1 IAA.

Genome-wide identification of Aux/IAA gene family in white clover (Trifolium repens L.) and functional verification of TrIAA18 under different abiotic stress.

BACKGROUND: White clover (Trifolium repens L.) is an excellent leguminous cool-season forage with a high protein content and strong nitrogen-fixing ability. Despite these advantages, its growth and development are markedly sensitive to environmental factors. Indole-3-acetic acid (IAA) is the major growth hormone in plants, regulating plant growth, development, and response to adversity. Nevertheless, the specific regulatory functions of Aux/IAA genes in response to abiotic stresses in white clover remain largely unexplored. RESULTS: In this study, we identified 47 Aux/IAA genes in the white clover genome, which were categorized into five groups based on phylogenetic analysis. The TrIAAs promoter region co-existed with different cis-regulatory elements involved in developmental and hormonal regulation, and stress responses, which may be closely related to their diverse regulatory roles. Collinearity analysis showed that the amplification of the TrIAA gene family was mainly carried out by segmental duplication. White clover Aux/IAA genes showed different expression patterns in different tissues and under different stress treatments. In addition, we performed a yeast two-hybrid analysis to investigate the interaction between white clover Aux/IAA and ARF proteins. Heterologous expression indicated that TrIAA18 could enhance stress tolerance in both yeast and transgenic Arabidopsis thaliana. CONCLUSION: These findings provide new scientific insights into the molecular mechanisms of growth hormone signaling in white clover and its functional characteristics in response to environmental stress.

Indole-3 acetic acid negatively regulates rose black spot disease resistance through antagonizing the salicylic acid signaling pathway via jasmonic acid.

MAIN CONCLUSION: IAA cooperates with JA to inhibit SA and negatively regulates rose black spot disease resistance. Black spot disease caused by the fungus Marssonina rosae is the most prevalent and severe ailment in rose cultivation, leading to the appearance of black spots on leaves and eventual leaf fall, significantly impacting the utilization of roses in gardens. Salicylic acid (SA) and jasmonic acid (JA) are pivotal hormones that collaborate with indole-3 acetic acid (IAA) in regulating plant defense responses; however, the detailed mechanisms underlying the induction of black spot disease resistance by IAA, JA, and SA remain unclear. In this study, transcript analysis was conducted on resistant (R13-54) and susceptible (R12-26) lines following M. rosae infection. In addition, the impact of exogenous interference with IAA on SA- and JA-mediated disease resistance was examined. The continuous accumulation of JA, in synergy with IAA, inhibited activation of the SA signaling pathway in the early infection stage, thereby negatively regulating the induction of effective resistance to black spot disease. IAA administration alleviated the inhibition of SA on JA to negatively regulate the resistance of susceptible strains by further enhancing the synthesis and accumulation of JA. However, IAA did not contribute to the negative regulation of black spot resistance when high levels of JA were inhibited. Virus-induced gene silencing of RcTIFY10A, an inhibitor of the JA signaling pathway, further suggested that IAA upregulation led to a decrease in disease resistance, a phenomenon not observed when the JA signal was inhibited. Collectively, these findings indicate that the IAA-mediated negative regulation of black spot disease resistance relies on activation of the JA signaling pathway.

CaIAA2-CaARF9 module mediates the trade-off between pepper growth and immunity.

To challenge the invasion of various pathogens, plants re-direct their resources from plant growth to an innate immune defence system. However, the underlying mechanism that coordinates the induction of the host immune response and the suppression of plant growth remains unclear. Here we demonstrate that an auxin response factor, CaARF9, has dual roles in enhancing the immune resistance to Ralstonia solanacearum infection and in retarding plant growth by repressing the expression of its target genes as exemplified by Casmc4, CaLBD37, CaAPK1b and CaRROP1. The expression of these target genes not only stimulates plant growth but also negatively impacts pepper resistance to R. solanacearum. Under normal conditions, the expression of Casmc4, CaLBD37, CaAPK1b and CaRROP1 is active when promoter-bound CaARF9 is complexed with CaIAA2. Under R. solanacearum infection, however, degradation of CaIAA2 is triggered by SA and JA-mediated signalling defence by the ubiquitin-proteasome system, which enables CaARF9 in the absence of CaIAA2 to repress the expression of Casmc4, CaLBD37, CaAPK1b and CaRROP1 and, in turn, impeding plant growth while facilitating plant defence to R. solanacearum infection. Our findings uncover an exquisite mechanism underlying the trade-off between plant growth and immunity mediated by the transcriptional repressor CaARF9 and its deactivation when complexed with CaIAA2.

The IAA17.1/HSFA5a module enhances salt tolerance in Populus tomentosa by regulating flavonol biosynthesis and ROS levels in lateral roots.

Auxin signaling provides a promising approach to controlling root system architecture and improving stress tolerance in plants. However, how the auxin signaling is transducted in this process remains unclear. The Aux indole-3-acetic acid (IAA) repressor IAA17.1 is stabilized by salinity, and primarily expressed in the lateral root (LR) primordia and tips in poplar. Overexpression of the auxin-resistant form of IAA17.1 (IAA17.1m) led to growth inhibition of LRs, markedly reduced salt tolerance, increased reactive oxygen species (ROS) levels, and decreased flavonol content. We further identified that IAA17.1 can interact with the heat shock protein HSFA5a, which was highly expressed in roots and induced by salt stress. Overexpression of HSFA5a significantly increased flavonol content, reduced ROS accumulation, enhanced LR growth and salt tolerance in transgenic poplar. Moreover, HSFA5a could rescue the defective phenotypes caused by IAA17.1m. Expression analysis showed that genes associated with flavonol biosynthesis were altered in IAA17.1m- and HAFA5a-overexpressing plants. Furthermore, we identified that HSFA5a directly activated the expression of key enzyme genes in the flavonol biosynthesis pathway, while IAA17.1 suppressed HSFA5a-mediated activation of these genes. Collectively, the IAA17.1/HSFA5a module regulates flavonol biosynthesis, controls ROS accumulation, thereby modulating the root system of poplar to adapt to salt stress.

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.

PagKNAT2/6b promotes shoot branching by attenuating auxin-strigolactone signalling in poplar.

Shoot branching from axillary bud (AB) directly determines plant architecture. However, the mechanism through which AB remains dormant or emerges to form branches as plants grow remains largely unknown. Here, the auxin-strigolactone (IAA-SL) pathway was first shown to regulate shoot branching in poplar, and we found that PagKNAT2/6b could modulate this pathway. PagKNAT2/6b was expressed mainly in the shoot apical meristem and AB and was induced by shoot apex damage. PagKNAT2/6b overexpressing poplar plants (PagKNAT2/6b OE) exhibited multiple branches that mimicked the branching phenotype of nontransgenic plants after decapitation treatment, while compared with nontransgenic controls, PagKNAT2/6b antisense transgenic poplar and Pagknat2/6b mutant lines exhibited a significantly decreased number of branches after shoot apex damage treatment. In addition, we found that PagKNAT2/6b directly inhibits the expression of the key IAA synthesis gene PagYUC6a, which is specifically expressed in the shoot apex. Moreover, overexpression of PagYUC6a in the PagKNAT2/6b OE background reduced the number of branches after shoot apex damage treatment. Overall, we conclude that PagKNAT2/6b responds to shoot apical injury and regulates shoot branching through the IAA-SL pathway. These findings may provide a theoretical basis and candidate genes for genetic engineering to create new forest tree species with different crown types.

Sextuple knockouts of a highly conserved and coexpressed AUXIN/INDOLE-3-ACETIC ACID gene set confer shade avoidance-like responses in Arabidopsis.

AUXIN/INDOLE-3-ACETIC ACIDs are transcriptional repressors for auxin signalling. Aux/IAAs of Arabidopsis thaliana display some functional redundancy. The IAA3/SHY2 clade (IAA1, IAA2, IAA3 and IAA4) show strong sequence similarity, but no higher-order mutants have been reported. Here, through CRISPR/Cas9 genome editing, we generated loss-of-function iaa1/2/3/4 mutants. The quadruple mutants only exhibited a weak phenotype. Thus, we additionally knocked out IAA7/AXR2 and IAA16, which are coexpressed with IAA1/2/3/4. Remarkably, under white light control conditions, the iaa1/2/3/4/7/16 mutants exhibited a shade avoidance-like phenotype with over-elongated hypocotyls and petioles and hyponastic leaves. The sextuple mutants were highly sensitive to low light intensity, and the hypocotyl cells of the mutants were excessively elongated. Transcriptome profiling and qRT-PCR analyses revealed that the sextuple mutation upregulated IAA19/MSG2 and IAA29, two shared shade/auxin signalling targets. Besides, genes encoding cell wall-remodelling proteins and shade-responsive transcription regulators were upregulated. Using dual-luciferase reporter assays, we verified that IAA2/IAA7 targeted the promoters of cell wall-remodelling genes to inhibit their transcription. Our work indicates that the IAA1/2/3/4/7/16 gene set is required for the optimal integration of auxin and shade signalling. The mutants generated here should be valuable for exploring the complex interactions among signal sensors, transcription activators and transcription repressors during hormone/environmental responses.

A gain-of-function mutation in BnaIAA13 disrupts vascular tissue and lateral root development in Brassica napus.

Rapeseed (Brassica napus) is an important oilseed crop worldwide. Plant vascular tissues are responsible for material transport and provide mechanical support. The lateral roots (LRs) absorb sufficient water and nutrients. The genetic basis of vascular tissues and LRs development in rapeseed remains unknown. This study characterized an EMS-mutagenized rapeseed mutant, T16, which showed dwarf stature, reduced LRs, and leaf wilting. Scanning electron microscopy observations showed that the internode-cell shortened. Observations of the tissue sections revealed defects in the development of vascular bundles in the stems and petioles. Genetic analysis revealed that the phenotypes of T16 were controlled by a single semi-dominant nuclear gene. Map-based cloning and genetic complementarity confirmed that BnaA03.IAA13 is the functional gene, a G-to-A mutation in second exon changed the glycine at the 79th position to glutamic acid, disrupting the conserved degron motif VGWPP. Transcriptome analysis in roots and stems showed that auxin and cytokinin signaling pathways were disordered in T16. Evolutionary analysis showed that AUXIN/INDOLE-3-ACETIC ACID was conserved during plant evolution. The heterozygote of T16 significantly reduced the plant height while maintaining other agronomic traits. Our findings provide novel insights into the regulatory mechanisms of vascular tissues and LRs development, and provide a new germplasm resource for rapeseed breeding.