Auxin Response Factor Proteins Research Articles

Comprehensive analyses of the ARF gene family in cannabis reveals their potential roles in regulating cannabidiol biosynthesis and male flower development.

Cannabidiol (CBD), as an important therapeutic property of the cannabis plants, is mainly produced in the flower organs. Auxin response factors (ARFs) are play a crucial role in flower development and secondary metabolite production. However, the specific roles of ARF gene family in cannabis remain unknown. In this study, various bioinformatics analysis of CsARF genes were conducted using online website and bioinformatics, quantitative real time PCR technology was used to investigate the expression patterns of the CsARF gene family in different tissues of different cannabis varieties, and subcellular localization analysis was performed in tobacco leaf. In this study, 22 CsARF genes were identified and found to be unevenly distributed across 9 chromosomes of the cannabis genome. Phylogenetic analysis revealed that the ARF proteins were divided into 4 subgroups. Duplication analysis identified one pair of segmental/whole-genome duplicated CsARF, and three pairs of tandemly duplicated CsARF. Collinearity analysis revealed that two CsARF genes, CsARF4 and CsARF19, were orthologous in both rice and soybean. Furthermore, subcellular localization analysis showed that CsARF2 was localized in the nucleus. Tissue-specific expression analysis revealed that six genes were highly expressed in cannabis male flowers, and among these genes, 3 genes were further found to be highly expressed at different developmental stages of male flowers. Meanwhile, correlation analysis between the expression level of CsARF genes and CBD content in two cultivars 'H8' and 'Y7' showed that the expression level of CsARF13 was negatively correlated with CBD content, while the expression levels of six genes were positively correlated with CBD content. In addition, most of CsARF genes were responsive to IAA treatment. Our study laid a foundation for the further studies of CsARFs function in cannabis, and provides candidate genes for breeding varieties with high CBD yield in cannabis production.

The PpIAA5-ARF8 Module Regulates Fruit Ripening and Softening in Peach

Fruit ripening and softening are important physiological processes in fruit quality formation, and auxin is involved in regulating the ripening and softening process in peach fruit. Little research has been reported on the role of Aux/IAA (auxin/indole-3-acetic acid)-ARF (auxin response factor) protein interactions in the ripening process of peach fruit. The transcriptomics and RT–qPCR results revealed that PpIAA5 expression increased before ripening in peach fruits. Overexpression of PpIAA5 significantly represses the expression of peach fruit ripening- and softening-related genes PpPG and PpACO1 in peach fruit tissues using transient transformation. A yeast library and yeast two-hybrid screen yielded PpARF8, a protein that interacts with PpIAA5. The interaction relationship was further established using a bimolecular fluorescence complementation assay. Transient overexpression of PpARF8 in peach fruit tissues promoted the expression of PpPA, PpPG, and PpACO1. Furthermore, a tomato transient transformation assay validated that the PpARF8 gene promotes fruit ripening and softening. Taken together, our results suggest that the PpIAA5-ARF8 signaling module can affect the ripening and softening of peach fruits.

Unraveling the Guardians of Growth: A Comprehensive Analysis of the Aux/IAA and ARF Gene Families in Populus simonii.

The auxin/indole-3-acetic acid (Aux/IAA) and auxin response factor (ARF) genes are two crucial gene families in the plant auxin signaling pathway. Nonetheless, there is limited knowledge regarding the Aux/IAA and ARF gene families in Populus simonii. In this study, we first identified 33 putative PsIAAs and 35 PsARFs in the Populus simonii genome. Analysis of chromosomal location showed that the PsIAAs and PsARFs were distributed unevenly across 17 chromosomes, with the greatest abundance observed on chromosomes 2. Furthermore, based on the homology of PsIAAs and PsARFs, two phylogenetic trees were constructed, classifying 33 PsIAAs and 35 PsARFs into three subgroups each. Five pairs of PsIAA genes were identified as the outcome of tandem duplication, but no tandem repeat gene pairs were found in the PsARF family. The expression profiling of PsIAAs and PsARFs revealed that several genes exhibited upregulation in different tissues and under various stress conditions, indicating their potential key roles in plant development and stress responses. The variance in expression patterns of specific PsIAAs and PsARFs was corroborated through RT-qPCR analysis. Most importantly, we instituted that the PsIAA7 gene, functioning as a central hub, exhibits interactions with numerous Aux/IAA and ARF proteins. Furthermore, subcellular localization findings indicate that PsIAA7 functions as a protein localized within the nucleus. To conclude, the in-depth analysis provided in this study will contribute significantly to advancing our knowledge of the roles played by PsIAA and PsARF families in both the development of P. simonii tissue and its responses to stress. The insights gained will serve as a valuable asset for further inquiries into the biological functions of these gene families.

Genome-wide survey, molecular evolution and expression analysis of Auxin Response Factor (ARF) gene family indicating their key role in seed number per pod in pigeonpea (C. cajan L. Millsp.)

Auxin Response Factors (ARF) are a family of transcription factors that mediate auxin signalling and regulate multiple biological processes. Their crucial role in increasing plant biomass/yield influenced this study, where a systematic analysis of ARF gene family was carried out to identify the key proteins controlling embryo/seed developmental pathways in pigeonpea. A genome-wide scan revealed the presence of 12 ARF genes in pigeonpea, distributed across the chromosomes 1, 3, 4, 8 and 11. Domain analysis of ARF proteins showed the presence of B3 DNA binding, AUX response, and IAA domains. Majority of them are of nuclear origin, and do not exhibit the level of genomic expansion as observed in Glycine max (51 members). The duplication events seem to range from 31.6 to 42.3 million years ago (mya). Promoter analysis revealed the presence of multiple cis-acting elements related to stress responses, hormone signalling and other development processes. The expression atlas data highlighted the expression of CcARF8 in hypocotyl, bud and flower whereas, CcARF7 expression was significantly high in pod. The real-time expression of CcARF2, CcARF3 and CcARF18 was highest in genotypes with high seed number indicating their key role in regulating embryo development and determining seed set in pigeonpea.

Structure, function and evolution of the auxin response factor protein family

Structure, function and evolution of the auxin response factor protein family

AUXIN RESPONSE FACTOR protein accumulation and function.

Auxin is a key regulator of plant developmental processes. Its effects on transcription are mediated by the AUXIN RESPONSE FACTOR (ARF) family of transcription factors. ARFs tightly control specific auxin responses necessary for proper plant growth and development. Recent research has revealed that regulated ARF protein accumulation and ARF nucleo-cytoplasmic partitioning can determine auxin transcriptional outputs. In this review, we explore these recent findings and consider the potential for regulated ARF accumulation in driving auxin responses in plants.

Advances in structure and function of auxin response factor in plants.

Auxin is a crucial phytohormone that has various effects on the regulators of plant growth and development. Auxin signal transduction is mainly controlled by two gene families: auxin response factor (ARF) and auxin/indole-3-acetic acid (Aux/IAA). ARFs are plant-specific transcription factors that bind directly to auxin response elements in the promoters of auxin-responsive genes. ARF proteins contain three conserved regions: a conserved N-terminal B3 DNA-binding domain, a variable intermediate middle region domain that functions in activation or repression, and a C-terminal domain including the Phox and Bem1p region for dimerization, similar to the III and IV elements of Aux/IAA, which facilitate protein-protein interaction through homodimerization of ARF proteins or heterodimerization of ARF and Aux/IAA proteins. In the two decades following the identification of the first ARF, 23 ARF members have been identified and characterized in Arabidopsis. Using whole-genome sequencing, 22, 25, 23, 25, and 36 ARF genes have been identified in tomato, rice, wheat, sorghum, and maize, respectively, in addition to which the related biofunctions of some ARFs have been reported. ARFs play crucial roles in regulating the growth and development of roots, leaves, flowers, fruits, seeds, responses to biotic and abiotic stresses, and phytohormone signal crosstalk. In this review, we summarize the research progress on the structures and functions of ARFs in Arabidopsis, tomato, and cereal crops, to provide clues for future basic research on phytohormone signaling and the molecular design breeding of crops.

Regulation of AUXIN RESPONSE FACTOR condensation and nucleo-cytoplasmic partitioning

Auxin critically regulates plant growth and development. Auxin-driven transcriptional responses are mediated through the AUXIN RESPONSE FACTOR (ARF) family of transcription factors. ARF protein condensation attenuates ARF activity, resulting in dramatic shifts in the auxin transcriptional landscape. Here, we perform a forward genetics screen for ARF hypercondensation, identifying an F-box protein, which we named AUXIN RESPONSE FACTOR F-BOX1 (AFF1). Functional characterization of SCFAFF1 revealed that this E3 ubiquitin ligase directly interacts with ARF19 and ARF7 to regulate their accumulation, condensation, and nucleo-cytoplasmic partitioning. Mutants defective in AFF1 display attenuated auxin responsiveness, and developmental defects, suggesting that SCFAFF1 -mediated regulation of ARF protein drives aspects of auxin response and plant development.

Protein Levels of Several Arabidopsis Auxin Response Factors Are Regulated by Multiple Factors and ABA Promotes ARF6 Protein Ubiquitination.

The auxin response factor (ARF) transcription factors are a key component in auxin signaling and play diverse functions in plant growth, development, and stress response. ARFs are regulated at the transcript level and posttranslationally by protein modifications. However, relatively little is known regarding the control of ARF protein levels. We expressed five different ARFs with an HA (hemagglutinin) tag and observed that their protein levels under the same promoter varied considerably. Interestingly, their protein levels were affected by several hormonal and environmental conditions, but not by the auxin treatment. ABA (abscisic acid) as well as 4 °C and salt treatments decreased the levels of HA-ARF5, HA-ARF6, and HA-ARF10, but not that of HA-ARF19, while 37 °C treatment increased the levels of the four HA-ARFs, suggesting that the ARF protein levels are regulated by multiple factors. Furthermore, MG132 inhibited the reduction of HA-ARF6 level by ABA and 4 °C treatments, suggesting that these treatments decrease HA-ARF6 level through 26S proteasome-mediated protein degradation. It was also found that ABA treatment drastically increased HA-ARF6 ubiquitination, without strongly affecting the ubiquitination profile of the total proteins. Together, these results reveal another layer of control on ARFs, which could serve to integrate multiple hormonal and environmental signals into the ARF-regulated gene expression.

A network of transcriptional repressors modulates auxin responses.

The regulation of signalling capacity, combined with the spatiotemporal distribution of developmental signals themselves, is pivotal in setting developmental responses in both plants and animals1. The hormone auxin is a key signal for plant growth and development that acts through the AUXIN RESPONSE FACTOR (ARF) transcription factors2-4. A subset of these, the conserved class A ARFs5, are transcriptional activators of auxin-responsive target genes that are essential for regulating auxin signalling throughout the plant lifecycle2,3. Although class A ARFs have tissue-specific expression patterns, how their expression is regulated is unknown. Here we show, by investigating chromatin modifications and accessibility, that loci encoding these proteins are constitutively open for transcription. Through yeast one-hybrid screening, we identify the transcriptional regulators of the genes encoding class A ARFs from Arabidopsis thaliana and demonstrate that each gene is controlled by specific sets of transcriptional regulators. Transient transformation assays and expression analyses in mutants reveal that, in planta, the majority of these regulators repress the transcription of genes encoding class A ARFs. These observations support a scenario in which the default configuration of open chromatin enables a network of transcriptional repressors to regulate expression levels of class A ARF proteins and modulate auxin signalling output throughout development.

The auxin response factor gene family in wheat (Triticum aestivum L.): Genome-wide identification, characterization and expression analyses in response to leaf rust

Auxin response factors (ARFs) are plant-specific transcription factors that bind explicitly to Auxin Response Elements and mediate downstream expression of auxin responsive genes involved at various stages of plant growth and development. This study identified 52 ARF genes located within the wheat genome. Based on phylogenetic analysis, the identified ARF proteins of wheat were classified into four classes (class I, II, III and IV). Synteny analysis revealed the orthologous relation with other monocots. The maximum numbers of predicted ARF genes were mapped on wheat chromosome 3B Ten conserved motifs of ARF was determined on all the identified sequences. Based on amino acid composition of the middle region of the ARF proteins, nine ARF proteins were transcription activators and the remaining were transcription repressors. The ARF genes of wheat contained 1–17 introns and deduced proteins of the genes having similar pattern of intron-exon distribution clustered within the same clade in the phylogenetic tree. TaARF1, TaARF4, TaARF7, TaARF34, TaARF39 were predicted to be targeted by the stress responsive miRNA Tae-miRNA160. The identified proteins were mostly localized within the nucleus and had sequence specific DNA binding activity. They were annotated in regulation of expression of early auxin responsive genes in auxin signaling pathway. Thirty-one TaARF proteins interacted and exhibited co-expressed regulation with other TaARF and Aux/IAA proteins. Increased expression of TaARF1 and TaARF23 at 120 h post inoculation (hpi) and 72 hpi in resistant and susceptible isolines respectively, specifies their roles in providing better resistance to the plant during leaf-rust infection.

Genome-wide characterization and expression analysis of the auxin response factor (ARF) gene family during melon (Cucumis melo L.) fruit development

ARFs in plants mediate auxin signaling transduction and regulate growth process. To determine genome-wide characterization of ARFs family in melon (Cucumis melo L.), ARFs were identified via analysis of information within the melon genomic database, and bioinformatic analyses were performed using various types of software. Based on different treatment methods involving dipping with the growth regulator Fengchanji No. 2 and artificial pollination, Jingmi No. 11 melon was used as the test material, and melon plants with unpollinated ovaries served as controls. The expression of ARFs during the early development of melon was analyzed via qRT-PCR. Seventeen genes that encode ARF proteins were identified in the melon genome for the first time. The expression of these ARFs differed in different tissues. The expression levels of CmARF2, CmARF16-like, CmARF18-like2, and CmARF19-like were especially high in melon fruits. The expression of ARFs during the early development of melon fruits differed in response to the different treatments, which suggested that CmARF9, CmARF16-like, CmARF19-like, CmARF19, CmARF1, CmARF2, CmARF3, and CmARF5 may be associated with melon fruit growth during early development. Interestingly, the increase in the transverse diameter of fruits treated with growth regulators was significantly greater than that of fruits resulting from artificial pollination, while the increase in the longitudinal diameter of the fruits resulting from artificial pollination was significantly greater.

A simple and sensitive SYBR Gold-based assay to quantify DNA-protein interactions.

A simple, accessible, and inexpensive assay to quantify the strength of DNA-protein interactions was developed. The assay relies on capturing DNA-protein complexes using an affinity resin that binds tagged, recombinant proteins. Sequential washes with filtration spin cups and centrifugation remove non-specific interactions in a gentle, uniform manner and a final elution isolates specific DNA-protein complexes. SYBR Gold nucleic acid stain is added to the eluted product and the fluorescence intensity accurately quantifies the amount of captured DNA, ultimately illustrating the relative strength of the DNA-protein interaction. The major utility of the assay resides in the versatility and quantitative nature of the SYBR Gold:nucleic acid interaction, eliminating the need for customized or labeled oligos and permitting relatively inexpensive quantification of binding capacity. The assay also employs DNA-protein complex capture by the very common purification tag, 6xHis, but other tags could likely be utilized. Further, SYBR Gold fluorescence is compatible with a wide variety of instruments, including UV transilluminators, a staple to any molecular biology laboratory. This assay was used to compare the binding capacities of different auxin response factor (ARF) transcription factors to various dsDNA targets, including the classical AuxRE motif and several divergent sequences. Results from dose-response assays suggest that different ARF proteins might show distinct comparative affinities for AuxRE variants, emphasizing that specific ARF-AuxRE binding strengths likely contribute to the complex and fine-tuned cellular auxin response.

Evolution of the Auxin Response Factors from charophyte ancestors.

Auxin is a major developmental regulator in plants and the acquisition of a transcriptional response to auxin likely contributed to developmental innovations at the time of water-to-land transition. Auxin Response Factors (ARFs) Transcription Factors (TFs) that mediate auxin-dependent transcriptional changes are divided into A, B and C evolutive classes in land plants. The origin and nature of the first ARF proteins in algae is still debated. Here, we identify the most ‘ancient’ ARF homologue to date in the early divergent charophyte algae Chlorokybus atmophyticus, CaARF. Structural modelling combined with biochemical studies showed that CaARF already shares many features with modern ARFs: it is capable of oligomerization, interacts with the TOPLESS co-repressor and specifically binds Auxin Response Elements as dimer. In addition, CaARF possesses a DNA-binding specificity that differs from class A and B ARFs and that was maintained in class C ARF along plants evolution. Phylogenetic evidence together with CaARF biochemical properties indicate that the different classes of ARFs likely arose from an ancestral proto-ARF protein with class C-like features. The foundation of auxin signalling would have thus happened from a pre-existing hormone-independent transcriptional regulation together with the emergence of a functional hormone perception complex.

Nucleo-cytoplasmic Partitioning of ARF Proteins Controls Auxin Responses in Arabidopsis thaliana.

The phytohormone auxin plays crucial roles in nearly every aspect of plant growth and development. The auxin response factor (ARF) transcription factor family regulates auxin-responsive gene expression and exhibits nuclear localization in regions of high auxin responsiveness. Here we show that the ARF7 and ARF19 proteins accumulate in micron-sized assemblies within the cytoplasm of tissues with attenuated auxin responsiveness. We found that the intrinsically disordered middle region and the folded PB1 interaction domain of ARFs drive protein assembly formation. Mutation of a single lysine within the PB1 domain abrogates cytoplasmic assemblies, promotes ARF nuclear localization, and results in an altered transcriptome and morphological defects. Our data suggest a model in which ARF nucleo-cytoplasmic partitioning regulates auxin responsiveness, providing a mechanism for cellular competence for auxin signaling.

The auxin response factor gene family in allopolyploid Brassica napus.

Auxin response factor (ARF) is a member of the plant-specific B3 DNA binding superfamily. Here, we report the results of a comprehensive analysis of ARF genes in allotetraploid Brassica napus (2n = 38, AACC). Sixty-seven ARF genes were identified in B. napus (BnARFs) and divided into four subfamilies (I–IV). Sixty-one BnARFs were distributed on all chromosomes except C02; the remaining were on Ann and Cnn. The full length of the BnARF proteins was highly conserved especially within each subfamily with all members sharing the N-terminal DNA binding domain (DBD) and the middle region (MR), and most contained the C-terminal dimerization domain (PBI). Twenty-one members had a glutamine-rich MR that may be an activator and the remaining were repressors. Accordingly, the intron patterns are highly conserved in each subfamily or clade, especially in DBD and PBI domains. Several members in subfamily III are potential targets for miR167. Many putative cis-elements involved in phytohormones, light signaling responses, and biotic and abiotic stress were identified in BnARF promoters, implying their possible roles. Most ARF proteins are likely to interact with auxin/indole-3-acetic acid (Aux/IAA) -related proteins, and members from different subfamilies generally shared many common interaction proteins. Whole genome-wide duplication (WGD) by hybridization between Brassica rapa and Brassica oleracea and segmental duplication led to gene expansion. Gene loss following WGD is biased with the An-subgenome retaining more ancestral genes than the Cn-subgenome. BnARFs have wide expression profiles across vegetative and reproductive organs during different developmental stages. No obvious expression bias was observed between An- and Cn-subgenomes. Most synteny-pair genes had similar expression patterns, indicating their functional redundancy. BnARFs were sensitive to exogenous IAA and 6-BA treatments especially subfamily III. The present study provides insights into the distribution, phylogeny, and evolution of ARF gene family.

Three Auxin Response Factors Promote Hypocotyl Elongation.

The hormone auxin regulates growth largely by affecting gene expression. By studying Arabidopsis (Arabidopsis thaliana) mutants deficient in AUXIN RESPONSE FACTORS (ARFs), we have identified three ARF proteins that are required for auxin-responsive hypocotyl elongation. Plants deficient in these factors have reduced responses to environmental conditions that increase auxin levels, including far-red-enriched light and high temperature. Despite having decreased auxin responses, the ARF-deficient plants responded to brassinosteroid and gibberellin, indicating that different hormones can act partially independently. Aux/IAA proteins, encoded by IAA genes, interact with ARF proteins to repress auxin response. Silencing expression of multiple IAA genes increased hypocotyl elongation, suggesting that Aux/IAA proteins modulate ARF activity in hypocotyls in a potential negative feedback loop.

Genome-wide characterization and expression pattern of auxin response factor (ARF) gene family in soybean and common bean

Auxin response factors (ARFs) encode one of the most abundant groups of auxin-mediated response transcription factors in higher plants and play a major role in various biological processes. The success of whole genome sequencing allows for comprehensive phylogenetic analysis of ARF genes in plants. Here, we identified 80 ARF genes belonging to five different groups in legume species, soybean (Glycine max, 55) and common bean (Phaseolus vulgaris, 25), based on phylogenetic analysis and supported by motif analysis. There is no member ARF of common bean in group V which contained two members from soybean, GmARF12 and GmARF55. The duplication events among two species were also observed by using Ks values. A majority of the ARF genes (96.1 %; 38 GmARFs and 56.0 %, 14 PvARFs) were segmentally duplicated while only two and one of ARF G. max and P. vulgaris genes were tandemly duplicated, respectively. In addition, expression profiling indicated that ARF genes in soybean and common bean perform various functions in plant growth and development, excluding flowering development. Furthermore, diversity of cis-elements in promoter regions also revealed differential expression of ARF proteins.

A Review of Auxin Response Factors (ARFs) in Plants.

Auxin is a key regulator of virtually every aspect of plant growth and development from embryogenesis to senescence. Previous studies have indicated that auxin regulates these processes by controlling gene expression via a family of functionally distinct DNA-binding auxin response factors (ARFs). ARFs are likely components that confer specificity to auxin response through selection of target genes as transcription factors. They bind to auxin response DNA elements (AuxRE) in the promoters of auxin-regulated genes and either activate or repress transcription of these genes depending on a specific domain in the middle of the protein. Genetic studies have implicated various ARFs in distinct developmental processes through loss-of-function mutant analysis. Recent advances have provided information on the regulation of ARF gene expression, the role of ARFs in growth and developmental processes, protein–protein interactions of ARFs and target genes regulated by ARFs in plants. In particular, protein interaction and structural studies of ARF proteins have yielded novel insights into the molecular basis of auxin-regulated transcription. These results provide the foundation for predicting the contributions of ARF genes to the biology of other plants.

Genome-wide identification of the auxin response factor (ARF) gene family and expression analysis of its role associated with pistil development in Japanese apricot (Prunus mume Sieb. et Zucc)

Auxin has been widely implicated in various aspects of plant growth and development, including flower development. In order to further elucidate the role of auxin during flower development, especially on the pistil development process, auxin response factors (ARFs), an important component in auxin signalling pathway, were studied in the early flower buds of Japanese apricot (Prunus mume Sieb. et Zucc). In this study, a comprehensive overview of the ARF gene family in Japanese apricot is presented, including the chromosomal locations, phylogenetic relationships, gene structures, the domain and nuclear localization analysis. Seventeen Japanese apricot genes that encode ARF proteins (PmARFs) have been identified based on the genome sequence of Japanese apricot. Comparison of the expression of some PmARF genes between perfect and imperfect flower buds in Japanese apricot suggests that PmARFs, especially the PmARF13 and PmARF17 gene may be required for pistil development and function in Japanese apricot. These results will be useful for future functional analyses of the ARF family genes in plants.