Auxin Response Factor Transcription Research Articles

Abolishing ARF8A activity promotes disease resistance in tomato

Auxin response factors (ARFs) are a family of transcription factors that regulate auxin-dependent developmental processes. Class A ARFs function as activators of auxin-responsive gene expression in the presence of auxin, while acting as transcriptional repressors in its absence. Despite extensive research on the functions of ARF transcription factors in plant growth and development, the extent, and mechanisms of their involvement in plant resistance, remain unknown. We have previously reported that mutations in the tomato AUXIN RESPONSE FACTOR8 (ARF8) genes SlARF8A and SlARF8B result in the decoupling of fruit development from pollination and fertilization, leading to partial or full parthenocarpy and increased yield under extreme temperatures. Here, we report that fine-tuning of SlARF8 activity results in increased resistance to fungal and bacterial pathogens. This resistance is mostly preserved under fluctuating temperatures. Thus, fine-tuning SlARF8 activity may be a potent strategy for increasing overall growth and yield.

Identification of ARF transcription factor gene family and its defense responses to bacterial infection and salicylic acid treatment in sugarcane.

Auxin response factor (ARF) is a critical regulator in the auxin signaling pathway, involved in a variety of plant biological processes. Here, gene members of 24 SpapARFs and 39 SpnpARFs were identified in two genomes of Saccharum spontaneum clones AP85-441 and Np-X, respectively. Phylogenetic analysis showed that all ARF genes were clustered into four clades, which is identical to those ARF genes in maize (Zea mays) and sorghum (Sorghum bicolor). The gene structure and domain composition of this ARF family are conserved to a large degree across plant species. The SpapARF and SpnpARF genes were unevenly distributed on chromosomes 1-8 and 1-10 in the two genomes of AP85-441 and Np-X, respectively. Segmental duplication events may also contribute to this gene family expansion in S. spontaneum. The post-transcriptional regulation of ARF genes likely involves sugarcane against various stressors through a miRNA-medicated pathway. Expression levels of six representative ShARF genes were analyzed by qRT-PCR assays on two sugarcane cultivars [LCP85-384 (resistant to leaf scald) and ROC20 (susceptible to leaf scald)] triggered by Acidovorax avenae subsp. avenae (Aaa) and Xanthomonas albilineans (Xa) infections and salicylic acid (SA) treatment. ShARF04 functioned as a positive regulator under Xa and Aaa stress, whereas it was a negative regulator under SA treatment. ShARF07/17 genes played positive roles against both pathogenic bacteria and SA stresses. Additionally, ShARF22 was negatively modulated by Xa and Aaa stimuli in both cultivars, particularly LCP85-384. These findings imply that sugarcane ARFs exhibit functional redundancy and divergence against stressful conditions. This work lays the foundation for further research on ARF gene functions in sugarcane against diverse environmental stressors.

Genome-wide identification and expression analysis of the Auxin-Response factor (ARF) gene family in Medicago sativa under abiotic stress

BackgroundAlfalfa (Medicago sativa) is the most widely planted legume forage and one of the most economically valuable crops in the world. The periodic changes in its growth and development and abiotic stress determine its yield and economic benefits. Auxin controls many aspects of alfalfa growth by regulating gene expression, including organ differentiation and stress response. Auxin response factors (ARF) are transcription factors that play an essential role in auxin signal transduction and regulate the expression of auxin-responsive genes. However, the function of ARF transcription factors is unclear in autotetraploid-cultivated alfalfa.ResultA total of 81 ARF were identified in the alfalfa genome in this study. Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were analyzed, identifying that ARF genes are mainly involved in transcriptional regulation and plant hormone signal transduction pathways. Phylogenetic analysis showed that MsARF was divided into four clades: I, II, III, and IV, each containing 52, 13, 7, and 9 genes, respectively. The promoter region of the MsARF gene contained stress-related elements, such as ABRE, TC-rich repeats, MBS, LTR. Proteins encoded by 50 ARF genes were localized in the nucleus without guide peptides, signal peptides, or transmembrane structures, indicating that most MsARF genes are not secreted or transported but only function in the nucleus. Protein structure analysis revealed that the secondary and tertiary structures of the 81 MsARF genes varied. Chromosomal localization analysis showed 81 MsARF genes were unevenly distributed on 25 chromosomes, with the highest distribution on chromosome 5. Furthermore, 14 segmental duplications and two sets of tandem repeats were identified. Expression analysis indicated that the MsARF was differentially expressed in different tissues and under various abiotic stressors. The quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that the expression profiles of 23 MsARF genes were specific to abiotic stresses such as drought, salt, high temperature, and low temperature, as well as tissue-specific and closely related to the duration of stress.ConclusionThis study identified MsARF in the cultivated alfalfa genome based on the autotetraploid level, which GO, KEGG analysis, phylogenetic analysis, sequence characteristics, and expression pattern analysis further confirmed. Together, these findings provide clues for further investigation of MsARF functional verification and molecular breeding of alfalfa. This study provides a novel approach to systematically identify and characterize ARF transcription factors in autotetraploid cultivated alfalfa, revealing 23 MsARF genes significantly involved in response to various stresses.

AUXIN RESPONSIVE FACTOR8 regulates development of the feeding site induced by root-knot nematodes in tomato.

Root-knot nematodes (RKN) from the genus Meloidogyne induce the dedifferentiation of root vascular cells into giant multinucleate feeding cells. These feeding cells result from an extensive reprogramming of gene expression, and auxin is known to be a key player in their development. However, little is known about how the auxin signal is transmitted during giant cell development. Integrative analyses combining transcriptome and small non-coding RNA datasets with the specific sequencing of cleaved transcripts identified genes targeted by miRNAs in tomato (Solanum lycopersicum) galls. The two auxin-responsive transcription factors ARF8A and ARF8B, and their miRNA167 regulators, were identified as robust gene-miRNA pair candidates to be involved in the tomato response to M. incognita. Spatiotemporal expression analysis using promoter-β-glucuronidase (GUS) fusions showed the up-regulation of ARF8A and ARF8B in RKN-induced feeding cells and surrounding cells. The generation and phenotyping of CRISPR (clustered regularly interspaced palindromic repeats) mutants demonstrated the role of ARF8A and ARF8B in giant cell development and allowed the characterization of their downstream regulated genes.

Identification and transcriptome data analysis of ARF family genes in five Orchidaceae species

The Orchidaceae is a large family of perennial herbs especially noted for the exceptional diversity of specialized flowers. Elucidating the genetic regulation of flowering and seed development of orchids is an important research goal with potential utility in orchid breeding programs. Auxin Response Factor (ARF) genes encode auxin-responsive transcription factors, which are involved in the regulation of diverse morphogenetic processes, including flowering and seed development. However, limited information on the ARF gene family in the Orchidaceae is available. In this study, 112 ARF genes were identified in the genomes of 5 orchid species (Apostasia shenzhenica, Dendrobium catenatum, Phalaenopsis aphrodite, Phalaenopsis equestris and Vanilla planifolia,). These genes were grouped into 7 subfamilies based on their phylogenetic relationships. Compared with the ARF family in model plants, such as Arabidopsis thaliana and Oryza sativa, one group of ARF genes involved in pollen wall synthesis has been lost during evolution of the Orchidaceae. This loss corresponds with absence of the exine in the pollinia. Through mining of the published genomic and transcriptomic data for the 5 orchid species: the ARF genes of subfamily 4 may play an important role in flower formation and plant growth, whereas those of subfamily 3 are potentially involved in pollen wall development. the study results provide novel insights into the genetic regulation of unique morphogenetic phenomena of orchids, which lay a foundation for further analysis of the regulatory mechanisms and functions of sexual reproduction-related genes in orchids.

Genome-wide investigation of ARF transcription factor gene family and its responses to abiotic stress in Coix (Coix lacryma-jobi L.).

Auxin response factor (ARF) is an important transcription factor that regulates the expression of auxin-responsive genes by direct binding to their promoters, which play a central role in plant growth, development, and response to abiotic stresses. The availability of the entire Coix (Coix lacryma-jobi L.) genome sequence provides an opportunity to investigate the characteristics and evolutionary history of the ARF gene family in this medicine and food homology plant for the first time. In this study, a total of 27 ClARF genes were identified based on the genome-wide sequence of Coix. Twenty-four of the 27 ClARF genes were unevenly distributed on 8 chromosomes except Chr 4 and 10, and the remaining three genes (ClARF25-27) were not assigned to any chromosome. Most of the ClARF proteins were predicted to be localized to the nucleus, except ClARF24, which was localized to both the plasma membrane and nucleus. Twenty-seven ClARFs were clustered into six subgroups based on the phylogenetic analysis. Duplication analysis showed that segmental duplication, rather than tandem duplications promoting the expansion of the ClARF gene family. Synteny analysis showed that purifying selection might have been a primary driving force in the development of the ARF gene family in Coix and other investigated cereal plants. The prediction of the cis element of the promoter showed that 27 ClARF genes contain several stress response elements, suggesting that ClARFs might be involved in the abiotic stress response. Expression profile analysis shows that 27 ClARF genes were all expressed in the root, shoot, leaf, kernel, glume, and male flower of Coix with varying expression levels. Furthermore, qRT-PCR analyses revealed that the majority of ClARFs members were upregulated or downregulated in response to hormone treatment and abiotic stress. The current study expands our understanding of the functional roles of ClARFs in stress responses and provides basic information for the ClARF genes.

Cooperative action of separate interaction domains promotes high-affinity DNA binding of Arabidopsis thaliana ARF transcription factors

The signaling molecule auxin coordinates many growth and development processes in plants, mainly through modulating gene expression. Transcriptional response is mediated by the family of auxin response factors (ARF). Monomers of this family recognize a DNA motif and can homodimerize through their DNA-binding domain (DBD), enabling cooperative binding to an inverted binding site. Most ARFs further contain a C-terminal PB1 domain that is capable of homotypic interactions and mediating interactions with Aux/IAA repressors. Given the dual role of the PB1 domain, and the ability of both DBD and PB1 domain to mediate dimerization, a key question is how these domains contribute to DNA-binding specificity and affinity. So far, ARF-ARF and ARF-DNA interactions have mostly been approached using qualitative methods that do not provide a quantitative and dynamic view on the binding equilibria. Here, we utilize a DNA binding assay based on single-molecule Förster resonance energy transfer (smFRET) to study the affinity and kinetics of the interaction of several Arabidopsis thaliana ARFs with an IR7 auxin-responsive element (AuxRE). We show that both DBD and PB1 domains of AtARF2 contribute toward DNA binding, and we identify ARF dimer stability as a key parameter in defining binding affinity and kinetics across AtARFs. Lastly, we derived an analytical solution for a four-state cyclic model that explains both the kinetics and the affinity of the interaction between AtARF2 and IR7. Our work demonstrates that the affinity of ARFs toward composite DNA response elements is defined by dimerization equilibrium, identifying this as a key element in ARF-mediated transcriptional activity.

Genomic identification of ARF transcription factors and expression analysis in Cannabis sativa L

Auxin response factors (ARFs) participate in the regulation of auxin signaling by binding directly to the auxin response elements (AuxREs) in the promoter regions of the downstream auxin response genes during plant development. To date, ARF genes in Cannabis sativa L. (cannabis) have not been characterized. The present study identified 21 ARF gene family members based on the genome-wide database of cannabis. Systematic analysis of these CsARFs included analysis of the gene structures, conserved domains, possible isoforms, phylogeny, cis-elements, and expression levels in various tissues and subspecies of cannabis. Currently, the function of the ARF genes in the growth and development of cannabis is largely unknown. To uncover this function, the expression patterns of the CsARF genes were analyzed in various tissues and subspecies of cannabis in detail. Combination of the results of the evolutionary tree and expression patterns of CsARFs, the functions of five CsARFs (CsARF7, CsARF8, CsARF11, CsARF19, and CsARF20) were predicted, and these genes may be involved in the development of cannabis flower. In addition, six CsARFs (CsARF3, CsARF5, CsARF7, CsARF8, CsARF10, and CsARF15) may be involved in the development of cannabis root and stem. Ultimately, CsARF13 may be involved in the development of cannabis leaf. The present study provides basic information on the CsARF genes and paves the way for further exploration of the function and mechanisms involved in the regulation of the growth and development of cannabis.

The auxin-responsive transcription factor SlDOF9 regulates inflorescence and flower development in tomato.

Understanding the mechanisms underlying differentiation of inflorescence and flower meristems is essential towards enlarging our knowledge of reproductive organ formation and to open new prospects for improving yield traits. Here, we show that SlDOF9 is a new modulator of floral differentiation in tomato. CRISPR/Cas9 knockout strategy uncovered the role of SlDOF9 in controlling inflorescence meristem and floral meristem differentiation via the regulation of cell division genes and inflorescence architecture regulator LIN. Tomato dof9-KO lines have more flowers in both determinate and indeterminate cultivars and produce more fruit upon vibration-assisted fertilization. SlDOF9 regulates inflorescence development through an auxin-dependent ARF5-DOF9 module that seems to operate, at least in part, differently in Arabidopsis and tomato. Our findings add a new actor to the complex mechanisms underlying reproductive organ differentiation in flowering plants and provide leads towards addressing the diversity of factors controlling the transition to reproductive organs.

Chinese Cherry (Cerasus pseudocerasus Lindl.) ARF7 Participates in Root Development and Responds to Drought and Low Phosphorus

In this paper, an auxin-responsive transcription factor, CpARF7, was isolated from the roots of Chinese cherry (Cerasus pseudocerasus Lindl. Cv. “Manao Hong”). CpARF7 is highly homologous to AtARF7 or AtARF19 in Arabidopsis, and PavARF1 or PavARF14 in sweet cherry. However, in the phenotype of transgenic tomatoes, the root morphology changed, the main root elongated, and the lateral root increased. Both drought treatment and low-phosphorus conditions can elongate the roots of transgenic tomatoes. In addition, the drought resistance and low-phosphorus tolerance of the transgenic lines are improved, and the POD, SOD, and CAT activities under drought and low-phosphorus environments are increased. There is an effect on the tomato somatotropin suppressor gene, SlIAAs, in which SlIAA1/14/19/29 are up-regulated and SlIAA2/11/12/16 are down-regulated. These results indicate that CpARF7 plays an essential regulatory role in root formation and abiotic stress response, and deepens the understanding of auxin-responsive genes in root growth and abiotic stress.

Transcriptome analysis of peach fruit under 1-MCP treatment provides insights into regulation network in melting peach softening

Abstract Peach (Prunus persica L.) displays distinguish texture phenotype during postharvest, which could be classified into three types, including melting flesh (MF), non-melting flesh, and stony-hard. MF peach would soften rapidly with an outbreak of ethylene production, which causes huge wastage during fruit transportation and storage. 1-Methylcyclopropene (1-MCP) was used to alleviate fruit softening. In this study, we performed RNA-sequencing on two MF peach cultivars (‘YuLu’ and ‘Yanhong’) after 1-MCP treatment to identify the candidate genes participating in peach fruit softening. A total of 167 genes were identified by weighted gene coexpression network analysis and correlation analysis that could respond to 1-MCP treatment and might be related to softening. Among them, 5 auxin-related genes including 2 indole-3-acetic acid, 1 auxin response transcription factor and 2 small auxin-up RNAs, and 4 cell wall modifying genes (PpPG1, PpPG2, PpPG24 and PpPMEI) were characterized as key genes participating in MF peach softening. Furthermore, 2 transcription factors, which belong to HD-ZIP and MYB, were predicted as candidates regulating softening process by constructing a transcriptional network of these 4 cell wall modifying genes combined with expression pattern analysis, of which HD-ZIP could trans-activate the promoter of PpPG1.

Corrigendum to: Genome-Wide Transcript Profiling Reveals an Auxin-Responsive Transcription Factor, OsAP2/ERF-40, Promoting Rice Adventitious Root Development

Corrigendum to: Genome-Wide Transcript Profiling Reveals an Auxin-Responsive Transcription Factor, OsAP2/ERF-40, Promoting Rice Adventitious Root Development

Genome-Wide Identification and Expression Analysis of the Aux/IAA and Auxin Response Factor Gene Family in Medicago truncatula.

Aux/IAA and auxin response transcription factor (ARF) genes are key regulators of auxin responses in plants. A total of 25 MtIAA and 40 MtARF genes were identified based on the latest updated Medicago truncatula reference genome sequence. They were clustered into 10 and 8 major groups, respectively. The homologs among M. truncatula, soybean, and Arabidopsis thaliana shared close relationships based on phylogenetic analysis. Gene structure analysis revealed that MtIAA and MtARF genes contained one to four concern motifs and they are localized to eight chromosomes, except chromosome 6 without MtARFs. In addition, some MtIAA and MtARF genes were expressed in all tissues, while others were specifically expressed in specific tissues. Analysis of cis-acting elements in promoter region and expression profiles revealed the potential response of MtIAA and MtARF genes to hormones and abiotic stresses. The prediction protein–protein interaction network showed that some ARF proteins could interact with multiple Aux/IAA proteins, and the reverse is also true. The investigation provides valuable, basic information for further studies on the biological functions of MtIAA and MtARF genes in the regulation of auxin-related pathways in M. truncatula.

Genome-Wide Identification of ARF Transcription Factor Gene Family and Their Expression Analysis in Sweet Potato.

Auxin response factors (ARFs) are a family of transcription factors that play an important role of auxin regulation through their binding with auxin response elements. ARF genes are represented by a large multigene family in plants; however, to our knowledge, the ARF gene family has not been well studied and characterized in sweet potatoes. In this study, a total of 25 ARF genes were identified in Ipomea trifida. The identified ItrARF genes’ conserved motifs, chromosomal locations, phylogenetic relationships, and their protein characteristics were systemically investigated using different bioinformatics tools. The expression patterns of ItfARF genes were analyzed within the storage roots and normal roots at an early stage of development. ItfARF16b and ItfARF16c were both highly expressed in the storage root, with minimal to no expression in the normal root. ItfARF6a and ItfARF10a exhibited higher expression in the normal root but not in the storage root. Subsequently, ItfARF1a, ItfARF2b, ItfARF3a, ItfARF6b, ItfARF8a, ItfARF8b, and ItfARF10b were expressed in both root types with moderate to high expression for each. All ten of these ARF genes and their prominent expression signify their importance within the development of each respective root type. This study provides comprehensive information regarding the ARF family in sweet potatoes, which will be useful for future research to discover further functional verification of these ItfARF genes.

Elicitation of solid callus cultures of Salvia miltiorrhiza Bunge with salicylic acid and a synthetic auxin (1-naphthaleneacetic acid)

The presented study analyses the influence of salicylic acid (SA) and the synthetic auxin 1-naphthaleneacetic acid (NAA) on total tanshinone level and on dihydrotanshinone (DHT), cryptotanshinone (CT), tanshinine I (TI) and tanshinone IIA (TIIA) level in Salvia miltiorrhiza callus cultures growing on solid Murashige and Skoog (MS) medium. The influence of SA and NAA was evaluated at 10-day intervals throughout a 80-day treatment period. SA was applied at 0.1, 0.2 and 0.4 mM, and NAA at 2.69, 13.43, 26.85 and 40.28 μM. DHT, CT, TI and TIIA concentrations were measured using HPLC. NAA did not increase the concentration of any tanshinone. SA increased content in a concentration- and time-dependent manner; however, the yields were relatively low, possibly due to the metabolic specificity of S. miltiorrhiza cultivars in Poland. Total tanshinone concentration reached 226.38 ± 37.33 μg g−1 DW after 50 days of 0.4 mM SA elicitation. After 50 days of SA elicitation, the following maximum tanshinone concentrations were observed for 0.4 mM SA: DHT (71.58 ± 12.72 μg g−1 DW), CT (108.54 ± 18.29 μg g−1 DW), TI (29.50 ± 4.13 μg g−1 DW) and TIIA (16.75 ± 2.74 μg g−1 DW). To account for these observed differences in tanshinone biosynthesis, the distribution of SA and auxin responsive cis-active motif in the proximal promoters of the mevalonic acid, methylerythritol-4-phosphate and tanshinone-precursor biosynthesis pathway genes was evaluated in A. thaliana and S. miltiorrhiza. Our findings indicate that the SA-responsive cis-active elements have a much broader distribution than those recognized by auxin-responsive transcription factors.

Microrna Regulation of Nodule Zone-Specific Gene Expression In Soybean

Microrna Regulation of Nodule Zone-Specific Gene Expression In Soybean

Genome-wide analysis of ARF transcription factors reveals HcARF5 expression profile associated with the biosynthesis of β-ocimene synthase in Hedychium coronarium.

Herein, 37 ARF genes were identified and analyzed in Hedychium coronarium and HcARF5 showed a potential role in the regulation of HcTPS3. Auxin is an important plant hormone, implicated in various aspects of plant growth and development processes especially in the biosynthesis of various secondary metabolites. Auxin response factors (ARF) belong to the transcription factors (TFs) gene family and play a crucial role in transcriptional activation/repression of auxin-responsive genes by directly binding to their promoter region. Nevertheless, whether ARF genes are involved in the regulatory mechanism of volatile compounds in flowering plants is largely unknown. β-ocimene is a key floral volatile compound synthesized by terpene synthase 3 (HcTPS3) in Hedychium coronarium. A comprehensive analysis of H. coronarium genome reveals 37 candidate ARF genes in the whole genome. Tissue-specific expression patterns of HcARFs family members were assessed using available transcriptome data. Among them, HcARF5 showed a higher expression level in flowers, and significantly correlated with the key structural β-ocimene synthesis gene (HcTPS3). Furthermore, transcript levels of both genes were associated with the flower development. Under hormone treatments, the response of HcARF5 and HcTPS3, and the emission level of β-ocimene contents were evaluated. Subcellular and transcriptional activity assay showed that HcARF5 localizes to the nucleus and possesses transcriptional activity. Yeast one-hybrid (Y1H) and dual-luciferase assays revealed that HcARF5 directly regulates the transcriptional activity of HcTPS3. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that HcARF5 interacts with scent-related HcIAA4, HcIAA6, and HcMYB1 in vivo. Overall, these results indicate that HcARF5 is potentially involved in the regulation of β-ocimene synthesis in H. coronarium.

Aux/IAA and ARF Gene Families in Salix suchowensis: Identification, Evolution, and Dynamic Transcriptome Profiling During the Plant Growth Process.

The phytohormone auxin plays a pivotal role in the regulation of plant growth and development, including vascular differentiation and tree growth. The auxin/indole-3-acetic acid (Aux/IAA) and auxin response transcription factor (ARF) genes are key components of plant auxin signaling. To gain more insight into the regulation and functional features of Aux/IAA and ARF genes during these processes, we identified 38 AUX/IAA and 34 ARF genes in the genome of Salix suchowensis and characterized their gene structures, conserved domains, and encoded amino acid compositions. Phylogenetic analysis of some typical land plants showed that the Aux/IAA and ARF genes of Salicaceae originated from a common ancestor and were significantly amplified by the ancestral eudicot hexaploidization event and the “salicoid” duplication that occurred before the divergence of poplar and willow. By analyzing dynamic transcriptome profiling data, some Aux/IAA and ARF genes were found to be involved in the regulation of plant growth, especially in the initial plant growth process. Additionally, we found that the expression of several miR160/miR167-ARFs was in agreement with canonical miRNA–ARF interactions, suggesting that miRNAs were possibly involved in the regulation of the auxin signaling pathway and the plant growth process. In summary, this study comprehensively analyzed the sequence features, origin, and expansion of Aux/IAA and ARF genes, and the results provide useful information for further studies on the functional involvement of auxin signaling genes in the plant growth process.

Architecture of DNA elements mediating ARF transcription factor binding and auxin-responsive gene expression in Arabidopsis

The hormone auxin controls many aspects of the plant life cycle by regulating the expression of thousands of genes. The transcriptional output of the nuclear auxin signaling pathway is determined by the activity of AUXIN RESPONSE transcription FACTORs (ARFs), through their binding to cis-regulatory elements in auxin-responsive genes. Crystal structures, in vitro, and heterologous studies have fueled a model in which ARF dimers bind with high affinity to distinctly spaced repeats of canonical AuxRE motifs. However, the relevance of this "caliper" model, and the mechanisms underlying the binding affinities in vivo, have remained elusive. Here we biochemically and functionally interrogate modes of ARF-DNA interaction. We show that a single additional hydrogen bond in Arabidopsis ARF1 confers high-affinity binding to individual DNA sites. We demonstrate the importance of AuxRE cooperativity within repeats in the Arabidopsis TMO5 and IAA11 promoters in vivo. Meta-analysis of transcriptomes further reveals strong genome-wide association of auxin response with both inverted (IR) and direct (DR) AuxRE repeats, which we experimentally validated. The association of these elements with auxin-induced up-regulation (DR and IR) or down-regulation (IR) was correlated with differential binding affinities of A-class and B-class ARFs, respectively, suggesting a mechanistic basis for the distinct activity of these repeats. Our results support the relevance of high-affinity binding of ARF transcription factors to uniquely spaced DNA elements in vivo, and suggest that differential binding affinities of ARF subfamilies underlie diversity in cis-element function.

An auxin-responsive transcription factor AbLBD1 promotes the development of lateral roots and reduces the biosynthesis of tropane alkaloids in Atropa belladonna

Atropa belladonna produces anticholinergic tropane alkaloids (TAs), such as hyoscyamine and scopolamine, which are synthesized in its lateral roots. Yet, the regulation of lateral root growth and TA biosynthesis remain poorly understood. In this study, an auxin-responsive transcription factor (AbLBD1), encoding a lateral organ boundaries domain protein (LBD), was functionally characterized from A. belladonna. Overexpression of AbLBD1 effectively promoted the initiation of lateral roots and adventitious roots of A. belladonna. The root biomass of AbLBD1-overexpressing lines was 1.48 to 2.45 fold higher that of the control. However, the contents of hyoscyamine and scopolamine were markedly reduced in AbLBD1-overexpressing lines. Gene expression analysis showed that the expression levels of TAs biosynthesis genes, namely AbPMT, AbPYKS, AbCYP82M3, and AbH6H, were greatly decreased in AbLBD1-overpressing lines. In conclusion, AbLBD1 plays a positive role in regulating lateral root development but a negative one in regulating TAs biosynthesis in A. belladonna. This study proved for the first time that AbLBD1 was a key auxin response regulator in root, which promotes initiation of lateral root and inhibits the biosynthesis of tropane alkaloids.