Characterization of the tomato ARF gene family uncovers a multi-levels post-transcriptional regulation including alternative splicing.
ABSTRACT: BACKGROUND: The phytohormone auxin is involved in a wide range of developmental processes and auxin signaling is known to modulate the expression of target genes via two types of transcriptional regulators, namely, Aux/IAA and Auxin Response Factors (ARF). ARFs play a major role in transcriptional activation or repression through direct binding to the promoter of auxin-responsive genes. The present study aims at gaining better insight on distinctive structural and functional features among ARF proteins. RESULTS: Building on the most updated tomato (Solanum lycopersicon) reference genome sequence, a comprehensive set of ARF genes was identified, extending the total number of family members to 22. Upon correction of structural annotation inconsistencies, renaming the tomato ARF family members provided a consensus nomenclature for all ARF genes across plant species. In silico search predicted the presence of putative target site for small interfering RNAs within twelve Sl-ARFs while sequence analysis of the 5'-leader sequences revealed the presence of potential small uORF regulatory elements. Functional characterization carried out by transactivation assay partitioned tomato ARFs into repressors and activators of auxin-dependent gene transcription. Expression studies identified tomato ARFs potentially involved in the fruit set process. Genome-wide expression profiling using RNA-seq revealed that at least one third of the gene family members display alternative splicing mode of regulation during the flower to fruit transition. Moreover, the regulation of several tomato ARF genes by both ethylene and auxin, suggests their potential contribution to the convergence mechanism between the signaling pathways of these two hormones. CONCLUSION: All together, the data bring new insight on the complexity of the expression control of Sl-ARF genes at the transcriptional and post-transcriptional levels supporting the hypothesis that these transcriptional mediators might represent one of the main components that enable auxin to regulate a wide range of physiological processes in a highly specific and coordinated manner.
Project description:Auxin signaling regulates various auxin-responsive genes via two types of transcriptional regulators, Auxin Response Factors (ARF) and Aux/IAA. ARF transcription factors act as critical components of auxin signaling that play important roles in modulating various biological processes. However, limited information about this gene family in fruit crops is currently available. Herein, 47 ARF genes were identified in banana based on its genome sequence. Phylogenetic analysis of the ARFs from banana, rice, and Arabidopsis suggested that the ARFs could be divided into four subgroups, among which most ARFs from the banana showed a closer relationship with those from rice than those from Arabidopsis. Conserved motif analysis showed that all identified MaARFs had typical DNA-binding and ARF domains, but 12 members lacked the dimerization domain. Gene structure analysis showed that the number of exons in MaARF genes ranged from 5 to 21, suggesting large variation amongst banana ARF genes. The comprehensive expression profiles of MaARF genes yielded useful information about their involvement in diverse tissues, different stages of fruit development and ripening, and responses to abiotic stresses in different varieties. Interaction networks and co-expression assays indicated the strong transcriptional response of banana ARFs and ARF-mediated networks in early fruit development for different varieties. Our systematic analysis of MaARFs revealed robust tissue-specific, development-dependent, and abiotic stress-responsive candidate MaARF genes for further functional assays in planta. These findings could lead to potential applications in the genetic improvement of banana cultivars, and yield new insights into the complexity of the control of MaARF gene expression at the transcriptional level. Finally, they support the hypothesis that ARFs are a crucial component of the auxin signaling pathway, which regulates a wide range of physiological processes.
Project description:Light signaling and plant hormones, particularly ethylene and auxins, have been identified as important regulators of carotenoid biosynthesis during tomato fruit ripening. However, whether and how the light and hormonal signaling cascades crosstalk to control this metabolic route remain poorly elucidated. Here, the potential involvement of ethylene and auxins in the light-mediated regulation of tomato fruit carotenogenesis was investigated by comparing the impacts of light treatments and the light-hyperresponsive <i>high pigment-2</i> (<i>hp2</i>) mutation on both carotenoid synthesis and hormonal signaling. Under either light or dark conditions, the overaccumulation of carotenoids in <i>hp2</i> ripening fruits was associated with disturbed ethylene production, increased expression of genes encoding master regulators of ripening and higher ethylene sensitivity and signaling output. The increased ethylene sensitivity observed in <i>hp2</i> fruits was associated with the differential expression of genes encoding ethylene receptors and downstream signaling transduction elements, including the downregulation of the transcription factor <i>ETHYLENE RESPONSE FACTOR.E4</i>, a repressor of carotenoid synthesis. Accordingly, treatments with exogenous ethylene promoted carotenoid biosynthetic genes more intensively in <i>hp2</i> than in wild-type fruits. Moreover, the loss of <i>HP2</i> function drastically altered auxin signaling in tomato fruits, resulting in higher activation of the auxin-responsive promoter <i>DR5</i>, severe down-regulation of <i>AUXIN/INDOLE-3-ACETIC ACID</i> (<i>Aux/IAA</i>) genes and altered accumulation of <i>AUXIN RESPONSE FACTOR</i> (<i>ARF</i>) transcripts. Both tomato <i>ARF2</i> paralogues (<i>Sl-ARF2a</i> and <i>SlARF2b</i>) were up-regulated in <i>hp2</i> fruits, which agrees with the promotive roles played by these ARFs in tomato fruit ripening and carotenoid biosynthesis. Among the genes differentially expressed in <i>hp2</i> fruits, the additive effect of light treatment and loss of <i>HP2</i> function was particularly evident for those encoding carotenoid biosynthetic enzymes, ethylene-related transcription factors, Aux/IAAs and ARFs. Altogether, the data uncover the involvement of ethylene and auxin as part of the light signaling cascades controlling tomato fruit metabolism and provide a new link between light signaling, plant hormone sensitivity and carotenoid metabolism in ripening fruits.
Project description:Fruit ripening is a highly coordinated developmental process driven by a complex hormonal network. Ethylene is the main regulator of climacteric fruit ripening. However, a putative role of other key phytohormones in this process cannot be excluded. We previously observed an increasing level of auxin during the post-harvest ripening of the durian fruit, which occurred concomitantly with the rise in the climacteric ethylene biosynthesis. Herein, we connect the key auxin signaling component, auxin response factors (ARFs), with the regulatory network that controls fruit ripening in durian through the identification and functional characterization of a candidate ripening-associated ARF. Our transcriptome-wide analysis identified 15 ARF members in durian (DzARFs), out of which 12 were expressed in the fruit pulp. Most of these DzARFs showed a differential expression, but DzARF2A had a marked ripening-associated expression pattern during post-harvest ripening in Monthong, a commercial durian cultivar from Thailand. Phylogenetic analysis of DzARF2A based on its tomato orthologue predicted a role in ripening through the regulation of ethylene biosynthesis. Transient expression of DzARF2A in Nicotiana benthamiana leaves significantly upregulated the expression levels of ethylene biosynthetic genes, pointing to a ripening-associated role of DzARF2A through the transcriptional regulation of ethylene biosynthesis. Dual-luciferase reporter assay determined that DzARF2A trans-activates durian ethylene biosynthetic genes. We previously reported significantly higher auxin level during post-harvest ripening in a fast-ripening cultivar (Chanee) compared to a slow-ripening one (Monthong). DzARF2A expression was significantly higher during post-harvest ripening in the fast-ripening cultivars (Chanee and Phuangmanee) compared to that of the slow-ripening ones (Monthong and Kanyao). Thus, higher auxin level could upregulate the expression of DzARF2A during ripening of a fast-ripening cultivar. The auxin-induced expression of DzARF2A confirmed its responsiveness to exogenous auxin treatment in a dose-dependent manner, suggesting an auxin-mediated role of DzARF2A in fruit ripening. We suggest that high DzARF2A expression would activate ARF2A-mediated transcription of ethylene biosynthetic genes, leading to increased climacteric ethylene biosynthesis (auxin-ethylene crosstalk) and faster ripening. Hence, we demonstrated DzARF2A as a new component of the regulatory network possibly mediating durian fruit ripening through transcriptional regulation of ethylene biosynthetic genes.
Project description:Auxin response factors (ARFs) encode transcriptional factors that function in the regulation of plant development processes. A tomato ARF gene, SlARF5, was observed to be expressed at high levels in emasculated ovaries but maintained low expression levels in pollinated ovaries. The amiRNA SlARF5 lines exhibited ovary growth and formed seedless fruits following emasculation. These parthenocarpic fruits developed fewer locular tissues, and the fruit size and weight were decreased in transgenic lines compared to those of wild-type fruits. Gene expression analysis demonstrated that several genes involved in the auxin-signaling pathway were downregulated, whereas some genes involved in the gibberellin-signaling pathway were enhanced by the decreased SlARF5 mRNA levels in transgenic plants, indicating that SlARF5 may play an important role in regulating both the auxin- and gibberellin-signaling pathways during fruit set and development.
Project description:Auxin response factors (ARFs) play fundamental roles in modulating various biological processes including fruit development and abscission via regulating the expression of auxin response genes. Currently, little is known about roles of ARFs in litchi (<i>Litchi chinensis</i> Sonn.), an economically important subtropical fruit tree whose production is suffering from fruit abscission. In this study, a genome-wide analysis of ARFs was conducted for litchi, 39 ARF genes (<i>LcARFs</i>) were identified. Conserved domain analysis showed that all the LcARFs identified have the signature B3 DNA-binding (B3) and ARF (Aux_rep) domains, with only 23 members having the dimerization domain (Aux_IAA). The number of exons in LcARF genes ranges from 2 to 16, suggesting a large variation for the gene structure of <i>LcARFs</i>. Phylogenetic analysis showed that the 39 LcARFs could be divided into three main groups: class I, II, and III. In total, 23 <i>LcARFs</i> were found to be potential targets of small RNAs, with three conserved and one novel miRNA-<i>ARF</i> (miRN43-<i>ARF9</i>) regulatory pathways discovered in litchi. Expression patterns were used to evaluate candidate <i>LcARFs</i> involved in various developmental processes, especially in flower formation and organ abscission. The results revealed that most ARF genes likely acted as repressors in litchi fruit abscission, that is, <i>ARF2D/2E</i>, <i>7A/7B</i>, <i>9A/9B</i>, <i>16A/16B</i>, while a few <i>LcARFs</i>, such as <i>LcARF5A/B</i>, might be positively involved in this process. These findings provide useful information and resources for further studies on the roles of ARF genes in litchi growth and development, especially in the process of fruit abscission.
Project description:AUXIN RESPONSE FACTORS (ARFs) are plant-specific transcription factors (TFs) that couple perception of the hormone auxin to gene expression programs essential to all land plants. As with many large TF families, a key question is whether individual members determine developmental specificity by binding distinct target genes. We use DAP-seq to generate genome-wide in vitro TF:DNA interaction maps for fourteen maize ARFs from the evolutionarily conserved A and B clades. Comparative analysis reveal a high degree of binding site overlap for ARFs of the same clade, but largely distinct clade A and B binding. Many sites are however co-occupied by ARFs from both clades, suggesting transcriptional coordination for many genes. Among these, we investigate known QTLs and use machine learning to predict the impact of cis-regulatory variation. Overall, large-scale comparative analysis of ARF binding suggests that auxin response specificity may be determined by factors other than individual ARF binding site selection.
Project description:Auxin-response factors (ARFs) bind with specificity to TGTCTC auxin-response elements (AuxREs), which are found in promoters of primary/early auxin-response genes. Nine different ARFs have been analyzed for their capacity to activate or repress transcription in transient expression assays employing auxin-responsive GUS reporter genes. One ARF appears to act as a repressor. Four ARFs function as activators and contain glutamine-rich activation domains. To achieve transcriptional activation on TGTCTC AuxREs in transient expression assays, ARFs require a conserved dimerization domain found in both ARF and Aux/IAA proteins, but they do not absolutely require their DNA-binding domains. Our results suggest that ARFs can activate or repress transcription by binding to AuxREs directly and that selected ARFs, when overexpressed, may potentiate activation further by associating with an endogenous transcription factor(s) (e.g., an ARF) that is bound to AuxREs. Transfection experiments suggest that TGTCTC AuxREs are occupied regardless of the auxin status in cells and that these occupied AuxREs are activated when exogenous auxin is applied to cells or when ARF activators are overexpressed. The results provide new insight into mechanisms involved with auxin regulation of primary/early-response genes.
Project description:Auxin response factors (ARFs) are important transcription factors to relay auxin signaling. From the Genome Database for Rosaceae (GDR), we identified 17 peach ARF genes (PpARFs) encoding the proteins with three conserved domains. Their gene structure and functional domains were analyzed. Their transcriptional response to exogenous auxin treatment was tested and confirmed. We also expressed PpARF-GFP fusion reporters in tobacco leaves and observed their nuclear localization by fluorescence microscopy. It has been known that ARFs are widely involved in fruit development. We compared the expression pattern of all PpARFs in different tissues including the fruits at different developmental stages of two peach cultivars, "melting" and "stony hard". We found eight PpARFs were more highly expressed in the "melting" peaches compared to "stony hard" peaches, while three PpARFs were more highly expressed in "stony hard" peaches. Among them, the expression difference of PpARF4, PpARF7 and PpARF12 was large, and their function in regulating fruit development and fruit quality was discussed. Our work provides a basis for further exploring the mechanisms underlying auxin regulated peach fruit ripening.
Project description:Auxin response factors (ARFs) are involved in auxin-mediated transcriptional regulation in plants. In this study, we performed functional characterization of SlARF6A in tomato. SlARF6A is located in the nucleus and exhibits transcriptional activator activity. Overexpression of SlARF6A increased chlorophyll contents in the fruits and leaves of tomato plants, whereas downregulation of SlARF6A decreased chlorophyll contents compared with those of wild-type (WT) plants. Analysis of chloroplasts using transmission electron microscopy indicated increased sizes of chloroplasts in SlARF6A-overexpressing plants and decreased numbers of chloroplasts in SlARF6A-downregulated plants. Overexpression of SlARF6A increased the photosynthesis rate and accumulation of starch and soluble sugars, whereas knockdown of SlARF6A resulted in opposite phenotypes in tomato leaves and fruits. RNA-sequence analysis showed that regulation of SlARF6A expression altered the expression of genes involved in chlorophyll metabolism, photosynthesis and sugar metabolism. SlARF6A directly bound to the promoters of SlGLK1, CAB, and RbcS genes and positively regulated the expression of these genes. Overexpression of SlARF6A also inhibited fruit ripening and ethylene production, whereas downregulation of SlARF6A increased fruit ripening and ethylene production. SlARF6A directly bound to the SAMS1 promoter and negatively regulated SAMS1 expression. Taken together, these results expand our understanding of ARFs with regard to photosynthesis, sugar accumulation and fruit development and provide a potential target for genetic engineering to improve fruit nutrition in horticulture crops.
Project description:<h4>Background</h4>Auxin response factors (ARFs) in auxin signaling pathway are an important component that can regulate the transcription of auxin-responsive genes involved in almost all aspects of plant growth and development. To our knowledge, the comprehensive and systematic characterization of ARF genes has never been reported in Prunus sibirica, a novel woody biodiesel feedstock in China.<h4>Results</h4>In this study, we identified 14 PsARF genes with a perfect open reading frame (ORF) in P. sibirica by using its previous transcriptomic data. Conserved motif analysis showed that all identified PsARF proteins had typical DNA-binding and ARF domain, but 5 members (PsARF3, 8 10, 16 and 17) lacked the dimerization domain. Phylogenetic analysis of the ARF proteins generated from various plant species indicated that ARFs could be categorized into 4 major groups (Class I, II, III and IV), in which all identified ARFs from P. sibirica showed a closest relationship with those from P. mume. Comparison of the expression profiles of 14 PsARF genes in different developmental stages of Siberian apricot mesocarp (SAM) and kernel (SAK) reflected distinct temporal or spatial expression patterns for PsARF genes. Additionally, based on the expressed data from fruit and seed development of multiple plant species, we identified 1514 ARF-correlated genes using weighted gene co-expression network analysis (WGCNA). And the major portion of ARF-correlated gene was characterized to be involved in protein, nucleic acid and carbohydrate metabolic, transport and regulatory processes.<h4>Conclusions</h4>In summary, we systematically and comprehensively analyzed the structure, expression pattern and co-expression network of ARF gene family in P. sibirica. All our findings provide theoretical foundation for the PsARF gene family and will pave the way for elucidating the precise role of PsARF genes in SAM and SAK development.