An improved auxin-inducible degron system preserves native protein levels and enables rapid and specific protein depletion.

Rapid perturbation of protein function permits the ability to define primary molecular responses while avoiding downstream cumulative effects of protein dysregulation. The auxin-inducible degron (AID) system was developed as a tool to achieve rapid and inducible protein degradation in nonplant systems. However, tagging proteins at their endogenous loci results in chronic auxin-independent degradation by the proteasome. To correct this deficiency, we expressed the auxin response transcription factor (ARF) in an improved inducible degron system. ARF is absent from previously engineered AID systems but is a critical component of native auxin signaling. In plants, ARF directly interacts with AID in the absence of auxin, and we found that expression of the ARF PB1 (Phox and Bem1) domain suppresses constitutive degradation of AID-tagged proteins. Moreover, the rate of auxin-induced AID degradation is substantially faster in the ARF-AID system. To test the ARF-AID system in a quantitative and sensitive manner, we measured genome-wide changes in nascent transcription after rapidly depleting the ZNF143 transcription factor. Transcriptional profiling indicates that ZNF143 activates transcription in cis and regulates promoter-proximal paused RNA polymerase density. Rapidly inducible degradation systems that preserve the target protein's native expression levels and patterns will revolutionize the study of biological systems by enabling specific and temporally defined protein dysregulation.

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.

Surface-localized glycoproteins act through class C ARFs to fine-tune gametophore initiation in Physcomitrium patens.

Arabinogalactan proteins are functionally diverse cell wall structural glycoproteins that have been implicated in cell wall remodeling, although the mechanistic actions remain elusive. Here, we identify and characterize two AGP glycoproteins, SLEEPING BEAUTY (SB) and SB-like (SBL), that negatively regulate the gametophore bud initiation in Physcomitrium patens by dampening cell wall loosening/softening. Disruption of SB and SBL led to accelerated gametophore formation and altered cell wall compositions. The function of SB is glycosylation dependent and genetically connected with the class C auxin response factor (ARF) transcription factors PpARFC1B and PpARFC2. Transcriptomics profiling showed that SB upregulates PpARFC2, which in turn suppresses a range of cell wall-modifying genes that are required for cell wall loosening/softening. We further show that PpARFC2 binds directly to multiple AuxRE motifs on the cis-regulatory sequences of PECTIN METHYLESTERASE to suppress its expression. Hence, our results demonstrate a mechanism by which the SB modulates the strength of intracellular auxin signaling output, which is necessary to fine-tune the timing of gametophore initials formation.

Transcriptional repressor RST1 controls salt tolerance and grain yield in rice by regulating gene expression of asparagine synthetase.

Salt stress impairs nutrient metabolism in plant cells, leading to growth and yield penalties. However, the mechanism by which plants alter their nutrient metabolism processes in response to salt stress remains elusive. In this study, we identified and characterized the rice (Oryza sativa) rice salt tolerant 1 (rst1) mutant, which displayed improved salt tolerance and grain yield. Map-based cloning revealed that the gene RST1 encoded an auxin response factor (OsARF18). Molecular analyses showed that RST1 directly repressed the expression of the gene encoding asparagine synthetase 1 (OsAS1). Loss of RST1 function increased the expression of OsAS1 and improved nitrogen (N) utilization by promoting asparagine production and avoiding excess ammonium (NH4+) accumulation. RST1 was undergoing directional selection during domestication. The superior haplotype RST1Hap III decreased its transcriptional repression activity and contributed to salt tolerance and grain weight. Together, our findings unravel a synergistic regulator of growth and salt tolerance associated with N metabolism and provide a new strategy for the development of tolerant cultivars.

Expansion and innovation in auxin signaling: where do we grow from here?

The phytohormone auxin plays a role in almost all growth and developmental responses. The primary mechanism of auxin action involves the regulation of transcription via a core signaling pathway comprising proteins belonging to three classes: receptors, co-receptor/co-repressors and transcription factors. Recent studies have revealed that auxin signaling can be traced back at least as far as the transition to land. Moreover, studies in flowering plants have highlighted how expansion of the gene families encoding auxin components is tied to functional diversification. As we review here, these studies paint a picture of auxin signaling evolution as a driver of innovation.

Evolution and function analysis of auxin response factors reveal the molecular basis of the developed root system of Zygophyllum xanthoxylum.

BACKGROUND: As a xerophytic shrub, forming developed root system dominated with lateral roots is one of the effective strategies for Zygophyllum xanthoxylum to adapt to desert habitat. However, the molecular mechanism of lateral root formation in Z. xanthoxylum is still unclear. Auxin response factors (ARFs) are a master family of transcription factors (TFs) in auxin-mediated biological processes including root growth and development. RESULTS: Here, to determine the relationship between ARFs and root system formation in Z. xanthoxylum, a total of 30 potential ZxARF genes were first identified, and their classifications, evolutionary relationships, duplication events and conserved domains were characterized. 107 ARF protein sequences from alga to higher plant species including Z. xanthoxylum are split into A, B, and C 3 Clades, consisting with previous studies. The comparative analysis of ARFs between xerophytes and mesophytes showed that A-ARFs of xerophytes expanded considerably more than that of mesophytes. Furthermore, in this Clade, ZxARF5b and ZxARF8b have lost the important B3 DNA-binding domain partly and completely, suggesting both two proteins may be more functional in activating transcription by dimerization with AUX/IAA repressors. qRT-PCR results showed that all A-ZxARFs are high expressed in the roots of Z. xanthoxylum, and they were significantly induced by drought stress. Among these A-ZxARFs, the over-expression assay showed that ZxARF7c and ZxARF7d play positive roles in lateral root formation. CONCLUSION: This study provided the first comprehensive overview of ZxARFs and highlighted the importance of A-ZxARFs in the lateral root development.

Genome-wide identification and expression analysis of ARF gene family in embryonic development of Korean pine (Pinus koraiensis).

BACKGROUND: The Auxin Responsive Factor (ARF) family plays a crucial role in mediating auxin signal transduction and is vital for plant growth and development. However, the function of ARF genes in Korean pine (Pinus koraiensis), a conifer species of significant economic value, remains unclear. RESULTS: This study utilized the whole genome of Korean pine to conduct bioinformatics analysis, resulting in the identification of 13 ARF genes. A phylogenetic analysis revealed that these 13 PkorARF genes can be classified into 4 subfamilies, indicating the presence of conserved structural characteristics within each subfamily. Protein interaction prediction indicated that Pkor01G00962.1 and Pkor07G00704.1 may have a significant role in regulating plant growth and development as core components of the PkorARFs family. Additionally, the analysis of RNA-seq and RT-qPCR expression patterns suggested that PkorARF genes play a crucial role in the development process of Korean pine. CONCLUSION: Pkor01G00962.1 and Pkor07G00704.1, which are core genes of the PkorARFs family, play a potentially crucial role in regulating the fertilization and developmental process of Korean pine. This study provides a valuable reference for investigating the molecular mechanism of embryonic development in Korean pine and establishes a foundation for cultivating high-quality Korean pine.

Unraveling the role of PlARF2 in regulating deed formancy in Paeonia lactiflora.

MAIN CONCLUSION: PlARF2 can positively regulate the seed dormancy in Paeonia lactiflora Pall. and bind the RY cis-element. Auxin, a significant phytohormone influencing seed dormancy, has been demonstrated to be regulated by auxin response factors (ARFs), key transcriptional modulators in the auxin signaling pathway. However, the role of this class of transcription factors (TFs) in perennials with complex seed dormancy mechanisms remains largely unexplored. Here, we cloned and characterized an ARF gene from Paeonia lactiflora, named PlARF2, which exhibited differential expression levels in the seeds during the process of seed dormancy release. The deduced amino acid sequence of PlARF2 had high homology with those of other plants and contained typical conserved Auxin_resp domain of the ARF family. Phylogenetic analysis revealed that PlARF2 was closely related to VvARF3 in Vitis vinifera. The subcellular localization and transcriptional activation assay showed that PlARF2 is a nuclear protein possessing transcriptional activation activity. The expression levels of dormancy-related genes in transgenic callus indicated that PlARF2 was positively correlated with the contents of PlABI3 and PlDOG1. The germination assay showed that PlARF2 promoted seed dormancy. Moreover, TF Centered Yeast one-hybrid assay (TF-Centered Y1H), electrophoretic mobility shift assay (EMSA) and dual-luciferase reporter assay analysis (Dual-Luciferase) provided evidence that PlARF2 can bind to the 'CATGCATG' motif. Collectively, our findings suggest that PlARF2, as TF, could be involved in the regulation of seed dormancy and may act as a repressor of germination.

Auxin response factors fine-tune lignin biosynthesis in response to mechanical bending in bamboo.

Lignin contributes to plant mechanical properties during bending loads. Meanwhile, phytohormone auxin controls various plant biological processes. However, the mechanism of auxin's role in bending-induced lignin biosynthesis was unclear, especially in bamboo, celebrated for its excellent deformation stability. Here, we reported that auxin response factors (ARF) 3 and ARF6 from Moso bamboo (Phyllostachys edulis (Carrière) J. Houz) directly regulate lignin biosynthesis pathway genes, and affect lignin biosynthesis in bamboo. Auxin and lignin exhibited asymmetric distribution patterns, and auxin promoted lignin biosynthesis in response to bending loads in bamboo. Employing transcriptomic and weighted gene co-expression network analysis approach, we discovered that expression patterns of ARF3 and ARF6 strongly correlated with lignin biosynthesis genes. ARF3 and ARF6 directly bind to the promoter regions of 4-coumarate: coenzyme A ligase (4CL3, 4CL7, and 4CL9) or caffeoyl-CoA O-methyltransferase (CCoAOMT2) genes, pivotal to lignin biosynthesis, and activate their expressions. Notably, the efficacy of this binding hinges on auxin levels. Alternation of the expressions of ARF3 and ARF6 substantially altered lignin accumulation in transgenic bamboo. Collectively, our study shed light on bamboo lignification genetics. Auxin signaling could directly modulate lignin biosynthesis genes to impact plant lignin content.

Auxin homeostasis is maintained by sly-miR167-SlARF8A/B-SlGH3.4 feedback module in the development of locular and placental tissues of tomato fruits.

The locular gel, produced by the placenta, is important for fruit flavor and seed development in tomato. However, the mechanism underlying locule and placenta development is not fully understood yet. Here, we show that two SlARF transcription factors, SlARF8B and SlARF8A (SlARF8A/B), promote the development of locular and placenta tissues. The expression of both SlARF8A and SlARF8B is repressed by sly-microRNA167 (sly-miR167), allowing for the activation of auxin downstream genes. In slarf8a, slarf8b, and slarf8a/b mutants, the auxin (IAA) levels are decreased, whereas the levels of inactive IAA conjugates including IAA-Ala, IAA-Asp, and IAA-Glu are increased. We further find that SlARF8B directly inhibits the expression of SlGH3.4, an acyl acid amino synthetase that conjugates the amino acids to IAA. Disruption of such auxin balance by the increased expression of SlGH3.4 or SlGH3.2 results in defective locular and placental tissues. Taken together, our findings reveal an important regulatory module constituted by sly-miR167-SlARF8A/B-SlGH3.4 during the development of locular and placenta tissues of tomato fruits.

The maize transcription factor CCT regulates drought tolerance by interacting with Fra a 1, E3 ligase WIPF2, and auxin response factor Aux/IAA8.

Plants are commonly exposed to abiotic stressors, which can affect their growth, productivity, and quality. Previously, the maize transcription factor ZmCCT was shown to be involved in the photoperiod response, delayed flowering, and quantitative resistance to Gibberella stalk rot. In this study, we demonstrate that ZmCCT can regulate plant responses to drought. ZmCCT physically interacted with ZmFra a 1, ZmWIPF2, and ZmAux/IAA8, which localized to the cell membrane, cytoplasm, and nucleus, respectively, both in vitro and in vivo in a yeast two-hybrid screen in response to abiotic stress. Notably, ZmCCT recruits ZmWIPF2 to the nucleus, which has strong E3 self-ubiquitination activity dependent on its RING-H2 finger domain in vitro. When treated with higher indole-3-acetic acid/abscisic acid ratios, the height and root length of Y331-ΔTE maize plants increased. Y331-ΔTE plants exhibited increased responses to exogenously applied auxin or ABA compared to Y331 plants, indicating that ZmCCT may be a negative regulator of ABA signalling in maize. In vivo, ZmCCT promoted indole-3-acetic acid biosynthesis in ZmCCT-overexpressing Arabidopsis. RNA-sequencing and DNA affinity purification-sequencing analyses showed that ZmCCT can regulate the expression of ZmRD17, ZmAFP3, ZmPP2C, and ZmARR16 under drought. Our findings provide a detailed overview of the molecular mechanism controlling ZmCCT functions and highlight that ZmCCT has multiple roles in promoting abiotic stress tolerance.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

The auxin response factor (ARF) gene family in Cyclocarya paliurus: genome-wide identification and their expression profiling under heat and drought stresses.

Auxin response factors (ARFs), as the main components of auxin signaling, play a crucial role in various processes of plant growth and development, as well as in stress response. So far, there have been no reports on the genome-wide identification of the ARF transcription factor family in Cyclocarya paliurus, a deciduous tree plant in the family Juglaceae. In this study, a total of 34 CpARF genes were identified based on whole genome sequence, and they were unevenly distributed on 16 chromosomes, with the highest distribution on chromosome 6. Domain analysis of CpARF proteins displayed that 31 out of 34 CpARF proteins contain a typical B3 domain (DBD domain), except CpARF12/ CpARF14/CpARF31, which all belong to Class VI. And 20 CpARFs (58.8%) contain an auxin_IAA binding domain, and are mainly distributed in classes I, and VI. Phylogenetic analysis showed that CpARF was divided into six classes (I-VI), each containing 4, 4, 1, 8, 4, and 13 members, respectively. Gene duplication analysis showed that there are 14 segmental duplications and zero tandem repeats were identified in the CpARF gene family of the C. paliurus genome. The Ka/Ks ratio of duplicate gene pairs indicates that CpARF genes are subjected to strong purification selection pressure. Synteny analysis showed that C. paliurus shared the highest homology in 74 ARF gene pairs with Juglans regia, followed by 73, 51, 25, and 11 homologous gene pairs with Populus trichocarpa, Juglans cathayensis, Arabidopsis, and rice, respectively. Promoter analysis revealed that 34 CpARF genes had cis-elements related to hormones, stress, light, and growth and development except for CpARF12. The expression profile analysis showed that almost all CpARF genes were differentially expressed in at least one tissue, and several CpARF genes displayed tissue-specific expression. Furthermore, 24 out of the 34 CpARF genes have significantly response to drought stress (P < 0.05), and most of them (16) being significantly down-regulated under moderate drought treatment. Meanwhile, the majority of CpARF genes (28) have significantly response to drought stress (P < 0.05), and most of them (26) are significantly down-regulated under severe drought treatment. Furthermore, 32 out of the 34 CpARF genes have significantly response to high, middle, and low salt stress under salt treatment (P < 0.05). Additionally, subcellular localization analysis confirmed that CpARF16 and CpARF32 were all localized to nucleus. Thus, our findings expand the understanding of the function of CpARF genes and provide a basis for further functional studies on CpARF genes in C. paliurus. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01474-1.

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.

The Impact of Tobamovirus Infection on Root Development Involves Induction of Auxin Response Factor 10a in Tomato.

Plant viruses cause systemic diseases that severely impair plant growth and development. While the accumulation of viruses in the root system has long been established, little is known as to how viruses affect root architecture. Here, we examined how the emerging tobamovirus, tomato brown rugose fruit virus (ToBRFV), alters root development in tomato. We found that ToBRFV and tobacco mosaic virus both invaded root systems during the first week of infection. ToBRFV infection of tomato plants resulted in a significant decrease in root biomass and elongation and root-to-shoot ratio and a marked suppression of root branching. Mutation in RNA-dependent RNA polymerase 6 increased the susceptibility of tomato plants to ToBRFV, resulting in severe reduction of various root growth parameters including root branching. Viral root symptoms were associated with the accumulation of auxin response factor 10a (SlARF10a) transcript, a homolog of Arabidopsis ARF10, a known suppressor of lateral root development. Interestingly, loss-of-function mutation in SlARF10a moderated the effect of ToBRFV on root branching. In contrast, downregulation of sly-miR160a, which targets SlARF10a, was associated with constitutive suppression root branching independent of viral infection. In addition, overexpression of a microRNA-insensitive mutant of SlARF10a mimicked the effect of ToBRFV on root development, suggesting a specific role for SlARF10a in ToBRFV-mediated suppression of root branching. Taken together, our results provide new insights into the impact of tobamoviruses on root development and the role of ARF10a in the suppression of root branching in tomato.

Spatially activated conserved auxin-transcription factor regulatory module controls de novo root organogenesis in rice.

MAIN CONCLUSION: This study reveals that the process of crown root development and auxin-induced de novo root organogenesis during in vitro plantlet regeneration share a common auxin-OsWOX10 regulatory module in rice. In the fibrous-type root system of rice, the crown roots (CR) are developed naturally from the shoot tissues. Generation of robust auxin response, followed by activation of downstream cell fate determinants and signaling pathways at the onset of crown root primordia (CRP) establishment is essential for new root initiation. During rice tissue culture, embryonic calli are induced to regenerate shoots in vitro which undergo de novo root organogenesis on an exogenous auxin-supplemented medium, but the mechanism underlying spatially restricted root organogenesis remains unknown. Here, we reveal the dynamics of progressive activation of genes involved in auxin homeostasis and signaling during initiation and outgrowth of rice crown root primordia. By comparative global dataset analysis, we identify the crown root primordia-expressed genes whose expression is also regulated by auxin signaling. In-depth spatio-temporal expression pattern analysis shows that the exogenous application of auxin induces a set of key transcription factors exclusively in the spatially positioned CRP. Further, functional analysis of rice WUSCHEL-RELATED HOMEOBOX 10 (OsWOX10) during in vitro plantlet regeneration from embryogenic calli shows that it promotes de novo root organogenesis from regenerated shoots. Expression of rice OsWOX10 also induces adventitious roots (AR) in Arabidopsis, independent of homologous endogenous Arabidopsis genes. Together, our findings reveal that a common auxin-transcription factor regulatory module is involved in root organogenesis under different conditions.

LBD18 and IAA14 antagonistically interact with ARF7 via the invariant Lys and acidic residues of the OPCA motif in the PB1 domain.

MAIN CONCLUSION: LBD18 and IAA14 antagonistically interact with ARF7 through the electrostatic faces in the ARF7PB1 domain, modulating ARF7 transcriptional activity. Auxin Response Factor 7 (ARF7)/ARF19 control lateral root development by directly activating Lateral Organ Boundaries Domain 16 (LBD16)/LBD18 genes in Arabidopsis. LBD18 upregulates ARF19 expression by binding to the ARF19 promoter. It also interacts with ARF7 through the Phox and Bem1 (PB1) domain to enhance the ARF7 transcriptional activity, forming a dual mode of positive feedback loop. LBD18 competes with the repressor indole-3-acetic acid 14 (IAA14) for ARF7 binding through the PB1 domain. In this study, we examined the molecular determinant of the ARF7 PB1 domain for interacting with LBD18 and showed that the electronic faces in the ARF7 PB1 domain are critical for interacting with LBD18 and IAA14/17. We used a luminescence complementation imaging assay to determine protein-protein interactions. The results showed that mutation of the invariant lysine residue and the OPCA motif in the PB1 domain in ARF7 significantly reduces the protein interaction between ARF7 and LBD18. Transient gene expression assays with Arabidopsis protoplasts showed that IAA14 suppressed transcription-enhancing activity of LBD18 on the LUC reporter gene fused to the ARF19 promoter harboring an auxin response element, but mutation of the invariant lysine residue and OPCA motif in the PB1 domain of IAA14 reduced the repression capability of IAA14 for transcription-enhancing activity of LBD18. We further showed that the same mutation in the PB1 domain of IAA14 reduces its repression capability, thereby increasing the LUC activity induced by both ARF7 and LBD18 compared with IAA14. These results suggest that LBD18 competes with IAA14 for ARF7 binding via the electrostatic faces of the ARF7 PB1 domain to modulate ARF7 transcriptional activity.

To bind or not to bind: how AUXIN RESPONSE FACTORs select their target genes.

Most plant growth and development processes are regulated in one way or another by auxin. The best-studied mechanism by which auxin exerts its regulatory effects is through the nuclear auxin pathway (NAP). In this pathway, Auxin Response Factors (ARFs) are the transcription factors that ultimately determine which genes become auxin regulated by binding to specific DNA sequences. ARFs have primarily been studied in Arabidopsis thaliana, but recent studies in other species have revealed family-wide DNA binding specificities for different ARFs and the minimal functional system of the NAP system, consisting of a duo of competing ARFs of the A and B classes. In this review, we provide an overview of key aspects of ARF DNA binding such as auxin response elements (TGTCNN) and tandem repeat motifs, and consider how structural biology and in vitro studies help us understand ARF DNA preferences. We also highlight some recent aspects related to the regulation of ARF levels inside a cell, which may alter the DNA binding profile of ARFs in different tissues. We finally emphasize the need to study minimal NAP systems to understand fundamental aspects of ARF function, the need to characterize algal ARFs to understand how ARFs evolved, how cutting-edge techniques can increase our understanding of ARFs, and which remaining questions can only be answered by structural biology.

Plasmodesmata callose binding protein 2 contributes to the regulation of cambium/phloem formation and auxin response during the tissue reunion process in incised Arabidopsis stem.

Plants are exposed to a variety of biotic and abiotic stresses, including wounding at the stem. The healing process (tissue reunion) begins immediately after stem wounding. The plant hormone auxin plays an important role during tissue reunion. In decapitated stems, auxin transport from the shoot apex is reduced and tissue reunion does not occur but is restored by application of indole-3-acetic acid (IAA). In this study, we found that plasmodesmata callose binding protein 2 (PDCB2) affects the expansion of the cambium/phloem region via changes in auxin response during the process of tissue reunion. PDCB2 was expressed in the cortex and endodermis on the incised side of stems 1-3 days after incision. PDCB2-knockout plants showed reduced callose deposition at plasmodesmata and DR5::GUS activity in the endodermis/cortex in the upper region of the incision accompanied by an increase in size of the cambium/phloem region during tissue reunion. In addition, PIN(PIN-FORMED)3, which is involved in lateral auxin transport, was induced by auxin in the cambium/phloem and endodermis/cortex in the upper part of the incision in wild type, but its expression of PIN3 was decreased in pdcb2 mutant. Our results suggest that PDCB2 contributes to the regulation of cambium/phloem development via auxin response.

Distinct modes of manipulation of rice auxin response factor OsARF17 by different plant RNA viruses for infection.

Plant auxin response factor (ARF) transcription factors are an important class of key transcriptional modulators in auxin signaling. Despite the well-studied roles of ARF transcription factors in plant growth and development, it is largely unknown whether, and how, ARF transcription factors may be involved in plant resistance to pathogens. We show here that two fijiviruses (double-stranded RNA viruses) utilize their proteins to disturb the dimerization of OsARF17 and repress its transcriptional activation ability, while a tenuivirus (negative-sense single-stranded RNA virus) directly interferes with the DNA binding activity of OsARF17. These interactions impair OsARF17-mediated antiviral defense. OsARF17 also confers resistance to a cytorhabdovirus and was directly targeted by one of the viral proteins. Thus, OsARF17 is the common target of several very different viruses. This suggests that OsARF17 plays a crucial role in plant defense against different types of plant viruses, and that these viruses use independently evolved viral proteins to target this key component of auxin signaling and facilitate infection.