Auxin/indole-3-acetic Acid Research Articles

Overexpression of Auxin/Indole-3-Acetic Acid Gene TrIAA27 Enhances Biomass, Drought, and Salt Tolerance in Arabidopsis thaliana

White clover (Trifolium repens L.) is an important forage and aesthetic plant species, but it is susceptible to drought and heat stress. The phytohormone auxin regulates several aspects of plant development and alleviates the effects of drought stress in plants, including white clover, by involving auxin/indole acetic acid (Aux/IAA) family genes. However, Aux/IAA genes and the underlying mechanism of auxin-mediated drought response remain elusive in white clover. To extend our understanding of the multiple functions of Aux/IAAs, the current study described the characterization of a member of the Aux/IAA family TrIAA27 of white clover. TrIAA27 protein had conserved the Aux/IAA family domain and shared high sequence similarity with the IAA27 gene of a closely related species and Arabidopsis. Expression of TrIAA27 was upregulated in response to heavy metal, drought, salt, NO, Ca2+, H2O2, Spm, ABA, and IAA treatments, while downregulated under cold stress in the roots and leaves of white clover. TrIAA27 protein was localized in the nucleus. Constitutive overexpression of TrIAA27 in Arabidopsis thaliana led to enhanced hypocotyl length, root length, plant height, leaf length and width, and fresh and dry weights under optimal and stress conditions. There was Improved photosynthesis activity, chlorophyll content, survival rate, relative water content, endogenous catalase (CAT), and peroxidase (POD) concentration with a significantly lower electrolyte leakage percentage, malondialdehyde (MDA) content, and hydrogen peroxide (H2O2) concentration in overexpression lines compared to wild-type Arabidopsis under drought and salt stress conditions. Exposure to stress conditions resulted in relatively weaker roots and above-ground plant growth inhibition, enhanced endogenous levels of major antioxidant enzymes, which correlated well with lower lipid peroxidation, lower levels of reactive oxygen species, and reduced cell death in overexpression lines. The data of the current study demonstrated that TrIAA27 is involved in positively regulating plant growth and development and could be considered a potential target gene for further use, including the breeding of white clover for higher biomass with improved root architecture and tolerance to abiotic stress.

Network analysis of metabolomics, transcriptome and hormones reveals propionic acid-mediated novel survival strategy against drought in wheat.

Propionic acid (PA), a low-molecular-weight organic acid, is crucial to plant life metabolism. However, the regulatory mechanism of PA-mediated drought resistance in wheat remains largely unknown. Herein, we reported on a regulatory network of PA-mediated drought resistance in wheat using integrated transcriptome and metabolomics analysis and verified genes associated with drought resistance. Compared to the water-treated group, the application of PA alleviated the damage of drought by increasing plant water content, antioxidant enzyme activities and decreasing the malondialdehyde level (MDA). Transcriptome and metabolomics analysis revealed that PA triggered upregulation of key genes and metabolites, including TaBCAT, TaALDH6A1, TaALDH7A1, TaCHI, TaFLS, chrysin, and galangin, which were involved in valine, leucine and isoleucine degradation or flavonoid biosynthesis, respectively. In addition, the expression of genes encoding auxin-related transcription factors (TFs) strikingly increased, such as auxin/indoleacetic acid (AUX/IAA) and auxin response factor (ARF). Moreover, PA activated abscisic acid (ABA) and indole-3-acetic acid (IAA) signalling pathways. Taken together, our findings suggest that PA promotes energy metabolism and antioxidant activities to confer wheat drought resistance by introducing comprehensive and systemic effects of valine, leucine and isoleucine degradation flavonoid biosynthesis. Furthermore, activated AUX/IAA and ARF TFs might serve vital roles in drought resistance via modulating IAA signalling. This study provides novel insights into PA-mediated crop resistance and the improvement of the agroecological environment.

Metabolism of 2,4-D in plants: comparative analysis of metabolic detoxification pathways in tolerant crops and resistant weeds.

The commercialization of 2,4-D (2,4-dichlorophenoxyacetic acid) latifolicide in 1945 marked the beginning of the selective herbicide market, with this active ingredient playing a pivotal role among commercial herbicides due to the natural tolerance of monocots compared with dicots. Due to its intricate mode of action, involving interactions within endogenous auxin signaling networks, 2,4-D was initially considered a low-risk herbicide to evolve weed resistance. However, the intensification of 2,4-D use has contributed to the emergence of 2,4-D-resistant broadleaf weeds, challenging earlier beliefs. This review explores 2,4-D tolerance in crops and evolved resistance in weeds, emphasizing an in-depth understanding of 2,4-D metabolic detoxification. Nine confirmed 2,4-D-resistant weed species, driven by rapid metabolism, highlight cytochrome P450 monooxygenases in Phase I and glycosyltransferases in Phase II as key enzymes. Resistance to 2,4-D may also involve impaired translocation associated with mutations in auxin/indole-3-acetic acid (Aux/IAA) co-receptor genes. Moreover, temperature variations affect 2,4-D efficacy, with high temperatures increasing herbicide metabolism rates and reducing weed control, while drought stress did not affect 2,4-D efficacy. Research on 2,4-D resistance has primarily focused on non-target-site resistance (NTSR) mechanisms, including 2,4-D metabolic detoxification, with limited exploration of the inheritance and genetic basis underlying these traits. Resistance to 2,4-D in weeds is typically governed by a single gene, either dominant or incompletely dominant, raising questions about gain-of-function or loss-of-function mutations that confer resistance. Future research should unravel the physiological and molecular-genetic basis of 2,4-D NTSR, exploring potential cross-resistance patterns and assessing fitness costs that may affect future evolution of auxin-resistant weeds. © 2024 Society of Chemical Industry.

A dose-dependent bimodal switch by homologous Aux/IAA transcriptional repressors

Combinatorial interactions between different regulators diversify and enrich the chance of transcriptional regulation in eukaryotic cells. However, a dose-dependent functional switch of homologous transcriptional repressors has rarely been reported. Here, we show that SHY2, an auxin/indole-3-acetic acid (Aux/IAA) repressor, exhibits a dose-dependent bimodal role in auxin-sensitive root-hair growth and gene transcription in Arabidopsis, whereas other Aux/IAA homologs consistently repress the auxin responses. The co-repressor (TOPLESS [TPL])-binding affinity of a bimodal Aux/IAA was lower than that of a consistently repressing Aux/IAA. The switch of a single amino acid residue in the TPL-binding motif between the bimodal form and the consistently repressing form switched their TPL-binding affinity and transcriptional and biological roles in auxin responses. Based on these data, we propose a model whereby competition between homologous repressors with different co-repressor-binding affinities could generate a bimodal output at the transcriptional and developmental levels.

Genome-Wide Identification, Expression, and Interaction Analysis of the Auxin Response Factor and AUX/IAA Gene Families in Vaccinium bracteatum.

Auxin, a plant hormone, plays diverse roles in the modulation of plant growth and development. The transport and signal transduction of auxin are regulated by various factors involved in shaping plant morphology and responding to external environmental conditions. The auxin signal transduction is primarily governed by the following two gene families: the auxin response factor (ARF) and auxin/indole-3-acetic acid (AUX/IAA). However, a comprehensive genomic analysis involving the expression profiles, structures, and functional features of the ARF and AUX/IAA gene families in Vaccinium bracteatum has not been carried out to date. Through the acquisition of genomic and expression data, coupled with an analysis using online tools, two gene family members were identified. This groundwork provides a distinguishing characterization of the chosen gene families in terms of expression, interaction, and response in the growth and development of plant fruits. In our genome-wide search of the VaARF and VaIAA genes in Vaccinium bracteatum, we identified 26 VaARF and 17 VaIAA genes. We analyzed the sequence and structural characteristics of these VaARF and VaIAA genes. We found that 26 VaARF and 17 VaIAA genes were divided into six subfamilies. Based on protein interaction predictions, VaIAA1 and VaIAA20 were designated core members of VaIAA gene families. Moreover, an analysis of expression patterns showed that 14 ARF genes and 12 IAA genes exhibited significantly varied expressions during fruit development. Two key genes, namely, VaIAA1 and VaIAA20, belonging to a gene family, play a potentially crucial role in fruit development through 26 VaARF-IAAs. This study provides a valuable reference for investigating the molecular mechanism of fruit development and lays the foundation for further research on Vaccinium bracteatum.

Genome-Wide Analysis of the Auxin/Indoleacetic Acid (Aux/IAA) Gene Family in Autopolyploid Sugarcane (Saccharum spontaneum).

The auxin/indoleacetic acid (Aux/IAA) family plays a central role in regulating gene expression during auxin signal transduction. Nonetheless, there is limited knowledge regarding this gene family in sugarcane. In this study, 92 members of the IAA family were identified in Saccharum spontaneum, distributed on 32 chromosomes, and classified into three clusters based on phylogeny and motif compositions. Segmental duplication and recombination events contributed largely to the expansion of this superfamily. Additionally, cis-acting elements in the promoters of SsIAAs involved in plant hormone regulation and stress responsiveness were predicted. Transcriptomics data revealed that most SsIAA expressions were significantly higher in stems and basal parts of leaves, and at nighttime, suggesting that these genes might be involved in sugar transport. QRT-PCR assays confirmed that cold and salt stress significantly induced four and five SsIAAs, respectively. GFP-subcellular localization showed that SsIAA23 and SsIAA12a were localized in the nucleus, consistent with the results of bioinformatics analysis. In conclusion, to a certain extent, the functional redundancy of family members caused by the expansion of the sugarcane IAA gene family is related to stress resistance and regeneration of sugarcane as a perennial crop. This study reveals the gene evolution and function of the SsIAA gene family in sugarcane, laying the foundation for further research on its mode of action.

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

BackgroundThe 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.ResultsA 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.ConclusionsThese 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.

Endogenous Hormone Levels and Transcriptomic Analysis Reveal the Mechanisms of Bulbil Initiation in Pinellia ternata.

Pinellia ternata is a medicinal plant that has important pharmacological value, and the bulbils serve as the primary reproductive organ; however, the mechanisms underlying bulbil initiation remain unclear. Here, we characterized bulbil development via histological, transcriptomic, and targeted metabolomic analyses to unearth the intricate relationship between hormones, genes, and bulbil development. The results show that the bulbils initiate growth from the leaf axillary meristem (AM). In this stage, jasmonic acid (JA), abscisic acid (ABA), isopentenyl adenosine (IPA), and salicylic acid (SA) were highly enriched, while indole-3-acetic acid (IAA), zeatin, methyl jasmonate (MeJA), and 5-dexoxystrigol (5-DS) were notably decreased. Through OPLS-DA analysis, SA has emerged as the most crucial factor in initiating and positively regulating bulbil formation. Furthermore, a strong association between IPA and SA was observed during bulbil initiation. The transcriptional changes in IPT (Isopentenyltransferase), CRE1 (Cytokinin Response 1), A-ARR (Type-A Arabidopsis Response Regulator), B-ARR (Type-B Arabidopsis Response Regulator), AUX1 (Auxin Resistant 1), ARF (Auxin Response Factor), AUX/IAA (Auxin/Indole-3-acetic acid), GH3 (Gretchen Hagen 3), SAUR (Small Auxin Up RNA), GA2ox (Gibberellin 2-oxidase), GA20ox (Gibberellin 20-oxidase), AOS (Allene oxide synthase), AOC (Allene oxide cyclase), OPR (Oxophytodienoate Reductase), JMT (JA carboxy l Methyltransferase), COI1 (Coronatine Insensitive 1), JAZ (Jasmonate ZIM-domain), MYC2 (Myelocytomatosis 2), D27 (DWARF27), SMAX (Suppressor of MAX2), PAL (Phenylalanine Ammonia-Lyase), ICS (Isochorismate Synthase), NPR1 (Non-expressor of Pathogenesis-related Genes1), TGA (TGACG Sequence-specific Binding), PR-1 (Pathogenesis-related), MCSU (Molybdenium Cofactor Sulfurase), PP2C (Protein Phosphatase 2C), and SnRK (Sucrose Non-fermenting-related Protein Kinase 2) were highly correlated with hormone concentrations, indicating that bulbil initiation is coordinately controlled by multiple phytohormones. Notably, eight TFs (transcription factors) that regulate AM initiation have been identified as pivotal regulators of bulbil formation. Among these, WUS (WUSCHEL), CLV (CLAVATA), ATH1 (Arabidopsis Thaliana Homeobox Gene 1), and RAX (Regulator of Axillary meristems) have been observed to exhibit elevated expression levels. Conversely, LEAFY demonstrated contrasting expression patterns. The intricate expression profiles of these TFs are closely associated with the upregulated expression of KNOX(KNOTTED-like homeobox), suggesting a intricate regulatory network underlying the complex process of bulbil initiation. This study offers a profound understanding of the bulbil initiation process and could potentially aid in refining molecular breeding techniques specific to P. ternata.

Elucidating the role of exogenous melatonin in mitigating alkaline stress in soybeans across different growth stages: a transcriptomic and metabolomic approach

BackgroundSoybean (Glycine max), a vital grain and oilseed crop, serves as a primary source of plant protein and oil. Soil salinization poses a significant threat to soybean planting, highlighting the urgency to improve soybean resilience and adaptability to saline stress. Melatonin, recently identified as a key plant growth regulator, plays crucial roles in plant growth, development, and responses to environmental stress. However, the potential of melatonin to mitigate alkali stress in soybeans and the underlying mechanisms remain unclear.ResultsThis study investigated the effects of exogenous melatonin on the soybean cultivar Zhonghuang 13 under alkaline stress. We employed physiological, biochemical, transcriptomic, and metabolomic analyses throughout both vegetative and pod-filling growth stages. Our findings demonstrate that melatonin significantly counteracts the detrimental effects of alkaline stress on soybean plants, promoting plant growth, photosynthesis, and antioxidant capacity. Transcriptomic analysis during both growth stages under alkaline stress, with and without melatonin treatment, identified 2,834 and 549 differentially expressed genes, respectively. These genes may play a vital role in regulating plant adaptation to abiotic stress. Notably, analysis of phytohormone biosynthesis pathways revealed altered expression of key genes, particularly in the ARF (auxin response factor), AUX/IAA (auxin/indole-3-acetic acid), and GH3 (Gretchen Hagen 3) families, during the early stress response. Metabolomic analysis during the pod-filling stage identified highly expressed metabolites responding to melatonin application, such as uteolin-7-O-(2''-O-rhamnosyl)rutinoside and Hederagenin-3-O-glucuronide-28-O-glucosyl(1,2)glucoside, which helped alleviate the damage caused by alkali stress. Furthermore, we identified 183 differentially expressed transcription factors, potentially playing a critical role in regulating plant adaptation to abiotic stress. Among these, the gene SoyZH13_04G073701 is particularly noteworthy as it regulates the key differentially expressed metabolite, the terpene metabolite Hederagenin-3-O-glucuronide-28-O-glucosyl(1,2)glucoside. WGCNA analysis identified this gene (SoyZH13_04G073701) as a hub gene, positively regulating the crucial differentially expressed metabolite of terpenoids, Hederagenin-3-O-glucuronide-28-O-glucosyl(1,2)glucoside. Our findings provide novel insights into how exogenous melatonin alleviates alkali stress in soybeans at different reproductive stages.ConclusionsIntegrating transcriptomic and metabolomic approaches, our study elucidates the mechanisms by which exogenous melatonin ameliorates the inhibitory effects of alkaline stress on soybean growth and development. This occurs through modulation of biosynthesis pathways for key compounds, including terpenes, flavonoids, and phenolics. Our findings provide initial mechanistic insights into how melatonin mitigates alkaline stress in soybeans, offering a foundation for molecular breeding strategies to enhance salt-alkali tolerance in this crop.

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

BackgroundAuxin/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.ResultA 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.ConclusionThis 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.

Large-scale analysis of the ARF and Aux/IAA gene families in 406 horticultural and other plants

The auxin response factor (ARF) and auxin/indole-3-acetic acid (Aux/IAA) family of genes are central components of the auxin signaling pathway and play essential roles in plant growth and development. Their large-scale analysis and evolutionary trajectory of origin are currently not known. Here, we identified the corresponding ARF and Aux/IAA family members and performed a large-scale analysis by scanning 406 plant genomes. The results showed that the ARF and Aux/IAA gene families originated from charophytes. The ARF family sequences were more conserved than the Aux/IAA family sequences. Dispersed duplications were the common expansion mode of ARF and Aux/IAA families in bryophytes, ferns, and gymnosperms; however, whole-genome duplication was the common expansion mode of the ARF and Aux/IAA families in basal angiosperms, magnoliids, monocots, and dicots. Expression and regulatory network analyses revealed that the Arabidopsis thaliana ARF and Aux/IAA families responded to multiple hormone, biotic, and abiotic stresses. The APETALA2 and serum response factor-transcription factor gene families were commonly enriched in the upstream and downstream genes of the ARF and Aux/IAA gene families. Our study provides a comprehensive overview of the evolutionary trajectories, structural functions, expansion mechanisms, expression patterns, and regulatory networks of these two gene families.

Characterization and Transcriptome Analysis Reveal Exogenous GA3 Inhibited Rosette Branching via Altering Auxin Approach in Flowering Chinese Cabbage

Branching is an important agronomic trait that is conducive to plant architecture and yield in flowering Chinese cabbage. Plant branching is regulated by a complex network mediated by hormones; gibberellin (GA) is one of the important hormones which is involved in the formation of shoot branching. Research on the regulatory mechanism of GA influencing rosette branch numbers is limited for flowering Chinese cabbage. In this study, the exogenous application of 600 mg/L GA3 effectively inhibited rosette branching and promoted internode elongation in flowering Chinese cabbage. RNA-Seq analysis further found that these DEGs were significantly enriched in ‘the plant hormone signal transduction’ pathways, and auxin-related genes were significantly differentially expressed between MB and MB_GA. The upregulation of auxin (AUX) and the upregulation of auxin/indole-3-acetic acid (AUX/IAA), as well as the downregulation of SMALL AUXIN-UPREGULATED RNA (SAUR), were found in the negative regulation of the rosette branching. The qRT-PCR results showed that the expression of AUX/IAA and SAUR from IAA gene family members were consistent with the results of transcriptome data. Phytohormone profiling by targeted metabolism revealed that endogenous auxin contents were significantly increased in MB_GA. Transcriptome and metabolome analysis clarified the main plant hormones and genes underlying the rosette branching in flowering Chinese cabbage, confirming that auxin could inhibit rosette branching. In this regard, the results present a novel angle for revealing the mechanism of gibberellin acting on the branching architecture in flowering Chinese cabbage.

Genome-Wide Identification and Analysis of the Aux/IAA Gene Family in Panax ginseng: Evidence for the Role of PgIAA02 in Lateral Root Development.

Panax ginseng C. A. Meyer (Ginseng) is one of the most used traditional Chinese herbal medicines, with its roots being used as the main common medicinal parts; its therapeutic potential has garnered significant attention. AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) is a family of early auxin-responsive genes capable of regulating root development in plants through the auxin signaling pathway. In the present study, 84 Aux/IAA genes were identified from the ginseng genome and their complexity and diversity were determined through their protein domains, phylogenetic relationships, gene structures, and cis-acting element predictions. Phylogenetic analyses classified PgIAA into six subgroups, with members in the same group showing greater sequence similarity. Analyses of interspecific collinearity suggest that segmental duplications likely drove the evolution of PgIAA genes, followed by purifying selection. An analysis of cis-regulatory elements suggested that PgIAA family genes may be involved in the regulation of plant hormones. RNA-seq data show that the expression pattern of Aux/IAA genes in Ginseng is tissue-specific, and PgIAA02 and PgIAA36 are specifically highly expressed in lateral, fibrous, and arm roots, suggesting their potential function in root development. The PgIAA02 overexpression lines exhibited an inhibition of lateral root growth in Ginseng. In addition, yeast two-hybrid and subcellular localization experiments showed that PgIAA02 interacted with PgARF22/PgARF36 (ARF: auxin response factor) in the nucleus and participated in the biological process of root development. The above results lay the foundation for an in-depth study of Aux/IAA and provide preliminary information for further research on the role of the Aux/IAA gene family in the root development of Ginseng.

Transcriptome profiling of indole-3-butyric acid–induced adventitious root formation in softwood cuttings of walnut

Inducing adventitious root (AR) formation in mature walnut species (Juglans L.) is challenging. However, the AR formation of mature trees can be improved by rejuvenation. In rejuvenated cuttings, exogenous indole-3-butyric acid (IBA) is essential for AR formation, and the underlying mechanism is still not well understood. Therefore, we utilized transcriptome sequencing to investigate the mechanism of IBA-induced AR formation. Our results revealed that, in comparison to the control group, IBA treatment (9 mmol · L-1) significantly increased the endogenous indole-3-acetic acid (IAA) content, leading to an enhanced rooting rate. We performed RNA sequencing to identify differentially expressed genes (DEGs) between the IBA-treated and control (CK) groups at 1, 2, 3, and 5 days after cutting (DAC). The results showed that, compared to the control cuttings, there were 1 539, 889, 785, and 984 up-regulated genes and 2 791, 2 936, 3 017, and 1 752 down-regulated genes, at 1, 2, 3, and 5 DAC, respectively. Analysis of RNA-seq data revealed that G-type ATP-binding cassette 36/37 (ABCG36/37) and ATP-binding cassette subfamily D 1 (ABCD1), associated with IBA transport, were down-regulated in the rejuvenation cuttings. In contrast, PIN-FORMED (PIN) and PINOID (PID), associated with auxin efflux, were up-regulated. We identified 49 auxin/indole-3-acetic acid (AUX/IAA)-encoding genes, including IAA1, IAA3, IAA5, IAA6, IAA8, IAA11, IAA12, IAA19, and IAA20, which were up-regulated at 1–5 DAC in the rejuvenated cuttings. This study highlights that the overexpression of JrWOX5/11 in poplar significantly enhance AR growth, as evidenced by increased root length, surface area, volume, and quantity. Moreover, the co-expression network analysis involving JrWOX11 and JrWOX5 in walnut cuttings elucidates complex genetic interactions, underscoring their pivotal role in the formation of AR. Our data supported the following molecular mechanism of IBA-induced adventitious root formation. Firstly, IBA is converted to free IAA in peroxisomes. Then, the highly concentrated IAA in the procambium and parenchyma cells induces WUSCHEL-related homeobox 11 (WOX11) expression at two days. Finally, WOX11 acts redundantly to up-regulate WOX5, initiating the development of root primordia cells.

Synthetically derived BiAux modulates auxin co-receptor activity to stimulate lateral root formation.

The root system plays an essential role in plant growth and adaptation to the surrounding environment. The root clock periodically specifies lateral root prebranch sites (PBS), where a group of pericycle founder cells (FC) is primed to become lateral root founder cells and eventually give rise to lateral root primordia or lateral roots (LRs). This clock-driven organ formation process is tightly controlled by modulation of auxin content and signaling. Auxin perception entails the physical interaction of TRANSPORT INHIBITOR RESPONSE 1 (TIR1) or AUXIN SIGNALING F-BOX (AFBs) proteins with AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) repressors to form a co-receptor system. Despite the apparent simplicity, the understanding of how specific auxin co-receptors are assembled remains unclear. We identified the compound bis-methyl auxin conjugated with N-glucoside, or BiAux, in Arabidopsis (Arabidopsis thaliana) that specifically induces the formation of PBS and the emergence of LR, with a slight effect on root elongation. Docking analyses indicated that BiAux binds to F-box proteins, and we showed that BiAux function depends on TIR1 and AFB2 F-box proteins and AUXIN RESPONSE FACTOR 7 activity, which is involved in FC specification and LR formation. Finally, using a yeast (Saccharomyces cerevisiae) heterologous expression system, we showed that BiAux favors the assemblage of specific co-receptors subunits involved in LR formation and enhances AUXIN/INDOLE-3-ACETIC ACID 28 protein degradation. These results indicate that BiAux acts as an allosteric modulator of specific auxin co-receptors. Therefore, BiAux exerts a fine-tune regulation of auxin signaling aimed to the specific formation of LR among the many development processes regulated by auxin.

Temporal control of the Aux/IAA genes BnIAA32 and BnIAA34 mediates Brassica napus dual shade responses.

Precise responses to changes in light quality are crucial for plant growth and development. For example, hypocotyls of shade-avoiding plants typically elongate under shade conditions. Although this typical shade-avoidance response (TSR) has been studied in Arabidopsis (Arabidopsis thaliana), the molecular mechanisms underlying shade tolerance are poorly understood. Here we report that B. napus (Brassica napus) seedlings exhibit dual shade responses. In addition to the TSR, B. napus seedlings also display an atypical shade response (ASR), with shorter hypocotyls upon perception of early-shade cues. Genome-wide selective sweep analysis indicated that ASR is associated with light and auxin signaling. Moreover, genetic studies demonstrated that phytochrome A (BnphyA) promotes ASR, whereas BnphyB inhibits it. During ASR, YUCCA8 expression is activated by early-shade cues, leading to increased auxin biosynthesis. This inhibits hypocotyl elongation, as young B. napus seedlings are highly sensitive to auxin. Notably, two non-canonical AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) repressor genes, BnIAA32 and BnIAA34, are expressed during this early stage. BnIAA32 and BnIAA34 inhibit hypocotyl elongation under shade conditions, and mutations in BnIAA32 and BnIAA34 suppress ASR. Collectively, our study demonstrates that the temporal expression of BnIAA32 and BnIAA34 determines the behavior of B. napus seedlings following shade-induced auxin biosynthesis.

Exogenous GR24 Inhibits Strawberry Tillering by Affecting the Phytohormone Signaling and Sugar Metabolism Pathways

Tillering is an important part in strawberry growth, and strawberries can reproduce nutritionally through stolons to generate genetically stable offspring. However, excessive tillering during the fruit-growing stage can negatively impact fruit yield and quality. In this study, different concentrations of exogenous rac-GR24 (GR24) are used to treat the strawberry plants. It was found that GR24 effectively inhibited the sprouting of strawberry stolons, while promoting the growth of the stems and leaves. Among the treatments, the most effective concentration was found to be 5 μmol/L GR24. This treatment resulted in a decrease in the glucose content in the strawberry crowns and also caused changes in the contents of two endogenous phytohormones, gibberellic acid (GA3) and trans-zeatin riboside (tZR). Transcriptome data further suggested that exogenous GR24 may inhibit strawberry plant tillering by affecting various phytohormone signaling pathways and the sugar metabolism pathway. In 5 μmol/L GR24-treated plants, the expression level of type-B response regulator (B-ARR) was down-regulated and the expression level of CYTOKININ RESPONSE 1 (CRE1), histidine-containing phosphotransfer protein (AHP), and type-A response regulator (A-ARR) were up-regulated, suggesting the inhibition of the cytokinin (CTK) signaling pathway. The down-regulation of auxin (AUX) and auxin response factor (ARF), as well as the up-regulation of auxin/indole-3-acetic acid (AUX/IAA), led to the inhibition of the indole-3-acetic acid (IAA) signaling pathway. Additionally, the up-regulation of pyrabactin resistance 1/ pyrabactin resistance 1-like (PYR/PYL), non-fermenting 1-related protein kinase 2 (SnRK2), and ABRE binding factors (ABF) and the down-regulation of protein phosphatase 2C (PP2C) were observed in the up-regulated abscisic acid (ABA) signaling pathways. In the sugar metabolism pathway, the up-regulation of invertase (INV), hexokinase (HK), and fructokinase (FRK) and the down-regulation of trehalase (TREH) and beta-amylase (BMY) led to a decreased glucose synthesis and an increased glucose consumption. Therefore, GR24 can effectively inhibit strawberry plant tillering through these pathways, making it an effective reagent for tillering inhibition.

Identification of an Aux/IAA regulator for flesh firmness using combined GWAS and bulked segregant RNA-Seq analysis in watermelon

Watermelon is a highly cultivated fruit crop renowned for its quality properties of fruit flesh. Among various quality factors, fruit flesh firmness is a crucial quality parameter influencing the fruit texture, shelf life and its commercial value. The auxin/indole-3-acetic acid (Aux/IAA) plays a significant role in fruit development and ripening of non-climacteric fruits. However, the regulatory mechanism of Aux/IAA in controlling fruit flesh firmness and ripening in watermelon remains unknown. In this study, we employed an integrative approach combining genome-wide association study (GWAS) and bulked segregant RNA-Seq analysis (BSR-Seq) to identify an overlapping candidate region between 12 776 310 and 12 968 331 bp on chromosome 6, underlying an auxin-responsive gene (Aux/IAA) associated with flesh firmness in watermelon. Transcriptome analysis, followed by real-time quantitative reverse transcription PCR (qRT-PCR), confirmed that the expression of Aux/IAA was consistently higher in fruits with high flesh firmness. The sequence alignment revealed a single base mutation in the coding region of Aux/IAA. Furthermore, the concomitant Kompetitive/Competitive allele-specific PCR (KASP) genotyping data sets for F2 population and germplasm accessions identified Aux/IAA as a strong candidate gene associated with flesh firmness. Aux/IAA was enriched in the plant hormone signal transduction pathway, involving cell enlargement and leading to low flesh firmness. We determined the higher accumulation of abscisic acid (ABA) in fruits with low flesh firmness than hard flesh. Moreover, overexpression of Aux/IAA induced higher flesh firmness with an increased number of fruit flesh cells while reducing ABA content and flesh cell sizes. Additionally, the allelic variation in Aux/IAA for soft flesh firmness was found to exist in Citrullus mucosospermus and gradually fixed into Citrullus lanatus during domestication, indicating that soft flesh firmness was a domesticated trait. These findings significantly enhanced our understanding of watermelon fruit flesh firmness and consequently the watermelon fruit quality.

Genome-Wide Characterization of the Aux/IAA Gene Family in Orchardgrass and a Functional Analysis of DgIAA21 in Responding to Drought Stress.

Drought stress is an important factor that reduces plant biomass production and quality. As one of the most important economic forage grasses, orchardgrass (Dactylis glomerata) has high drought tolerance. Auxin/indole-3-acetic acid (Aux/IAA) is one of the early responsive gene families of auxin and plays a key role in the response to drought stress. However, the characteristics of the Aux/IAA gene family in orchardgrass and their potential function in responding to drought stress remain unclear. Here, 30 Aux/IAA members were identified in orchardgrass. Segmental duplication may be an important driving force in the evolution of the Aux/IAA gene family in orchardgrass. Some Aux/IAA genes were induced by IAA, drought, salt, and temperature stresses, implying that these genes may play important roles in responding to abiotic stresses. Heterologous expression in yeast revealed that DgIAA21 can reduce drought tolerance. Similarly, the overexpression of DgIAA21 also reduced drought tolerance in transgenic Arabidopsis, which was supported by lower total chlorophyll content and relative water content as well as higher relative electrolyte leakage and malondialdehyde content (MDA) than Col-0 plants under drought conditions. The results of this study provided valuable insight into the function of DgIAAs in response to drought stress, which can be further used to improve forage grass breeding programs.

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.