Auxin Response Factor Transcription Research Articles

BnHO1, a haem oxygenase-1 gene from Brassica napus, is required for salinity and osmotic stress-induced lateral root formation

In this report, a rapeseed (Brassica napus) haem oxygenase-1 gene BnHO1 was cloned and sequenced. It shared high homology with Arabidopsis HY1 proteins, and encodes a 32.6 kDa protein with a 54-amino-acid transit peptide, predicting the mature protein of 25.1 kDa. The mature BnHO1 expressed in Escherichia coli exhibits haem oxygenase (HO) activity. Furthermore, the application of lower doses of NaCl (10 mM) and polyethylene glycol (PEG) (2%) mimicked the inducible effects of naphthylacetic acid and the HO-1 inducer haemin on the up-regulation of BnHO1 and subsequent lateral root (LR) formation. Contrasting effects were observed when a higher dose of NaCl or PEG was applied. The above inducible and inhibitory responses were blocked significantly when the HO-1 inhibitor zinc protoporphyrin IX (ZnPPIX) or haemin was applied, both of which were reversed by the application of carbon monoxide or ZnPPIX, respectively. Moreover, the addition of ZnPPIX at different time points during LR formation indicated that BnHO1 might be involved in the early stages of LR formation. The auxin response factor transcripts and the auxin content in seedling roots were clearly induced by lower doses of salinity or osmotic stress. However, treatment with the inhibitor of polar auxin transport N-1-naphthylphthalamic acid prevented the above inducible responses conferred by lower doses of NaCl and PEG, which were further rescued when the treatments were combined with haemin. Taken together, these results suggested a novel role of the rapeseed HO-1 gene in salinity and osmotic stress-induced LR formation, with a possible interaction with auxin signalling.

Genome-wide analysis of auxin response factor (ARF) gene family from tomato and analysis of their role in flower and fruit development

Auxin response transcription factors have been widely implicated in auxin-mediated responses during various developmental processes ranging from root and shoot development to flower and fruit development in plants. In order to use them for improvement of agronomic traits related to fruit, we need to have better understanding of their role during fruit development. In this study, 17 SlARF genes have been identified from tomato (Solanum lycopersicum), using various publically available tomato EST databases. Phylogenetic analysis of the 23 AtARF and 17 SlARF proteins results in formation of three major classes and a total of 14 sister pairs, including seven SlARF-AtARF, four SlARF-SlARF and three AtARF-AtARF sister pairs, providing insights into various orthologous relationships between AtARFs and SlARFs. Further, search for orthologs of these SlARFs resulted in identification of nine, ten, four and three ARF genes from potato, tobacco, N. benthemiana and pepper, respectively. A phylogenetic analysis of these genes, along with their orthologs from Solanaceae species, suggests the presence of a common set of the ARF genes in this family. Comparison of the expression of these SlARF genes in wild type and rin mutant provides an insight into their role during different stages of flower and fruit development. This study suggests that ARF genes may play diverse role during flower and fruit development. Comprehensive data generated here will provide a platform for identification of ARF genes and elucidation of their function during reproductive development stages in Solanaceae in general and fruit development in tomato, in particular.

Translation reinitiation and development are compromised in similar ways by mutations in translation initiation factor eIF3h and the ribosomal protein RPL24

BackgroundWithin the scanning model of translation initiation, reinitiation is a non-canonical mechanism that operates on mRNAs harboring upstream open reading frames. The h subunit of eukaryotic initiation factor 3 (eIF3) boosts translation reinitiation on the uORF-containing mRNA coding for the Arabidopsis bZip transcription factor, AtbZip11, among others. The RPL24B protein of the large ribosomal subunit, which is encoded by SHORT VALVE1, likewise fosters translation of uORF-containing mRNAs, for example mRNAs for auxin response transcription factors (ARFs).ResultsHere we tested the hypothesis that RPL24B and eIF3h affect translation reinitiation in a similar fashion. First, like eif3h mutants, rpl24b mutants under-translate the AtbZip11 mRNA, and the detailed spectrum of translational defects in rpl24b is remarkably similar to that of eif3h. Second, eif3h mutants display defects in auxin mediated organogenesis and gene expression, similar to rpl24b. Like AtbZip11, the uORF-containing ARF mRNAs are indeed undertranslated in eif3h mutant seedlings.ConclusionWe conclude that, similar to eIF3h, RPL24B bolsters the reinitiation competence of uORF-translating ribosomes. Coordination between eIF3 and the large ribosomal subunit helps to fine-tune translation of uORF-containing mRNAs and, in turn, to orchestrate plant development.

Phenotypic Plasticity of Adventitious Rooting in Arabidopsis Is Controlled by Complex Regulation of AUXIN RESPONSE FACTOR Transcripts and MicroRNA Abundance

The development of shoot-borne roots, or adventitious roots, is indispensable for mass propagation of elite genotypes. It is a complex genetic trait with a high phenotypic plasticity due to multiple endogenous and environmental regulatory factors. We demonstrate here that a subtle balance of activator and repressor AUXIN RESPONSE FACTOR (ARF) transcripts controls adventitious root initiation. Moreover, microRNA activity appears to be required for fine-tuning of this process. Thus, ARF17, a target of miR160, is a negative regulator, and ARF6 and ARF8, targets of miR167, are positive regulators of adventitious rooting. The three ARFs display overlapping expression domains, interact genetically, and regulate each other's expression at both transcriptional and posttranscriptional levels by modulating miR160 and miR167 availability. This complex regulatory network includes an unexpected feedback regulation of microRNA homeostasis by direct and nondirect target transcription factors. These results provide evidence of microRNA control of phenotypic variability and are a significant step forward in understanding the molecular mechanisms regulating adventitious rooting.

Regulation of preprocambial cell state acquisition by auxin signaling inArabidopsisleaves

The principles underlying the formation of veins in the leaf have long intrigued developmental biologists. In Arabidopsis leaves, files of anatomically inconspicuous subepidermal cells that will elongate into vein-forming procambial cells selectively activate ATHB8 gene expression. The biological role of ATHB8 in vein formation and the molecular events that culminate in acquisition of the ATHB8 preprocambial cell state are unknown, but intertwined pathways of auxin transport and signal transduction have been implicated in defining paths of vascular strand differentiation. Here we show that ATHB8 is required to stabilize preprocambial cell specification against auxin transport perturbations, to restrict preprocambial cell state acquisition to narrow fields and to coordinate procambium formation within and between veins. We further show that ATHB8 expression at preprocambial stages is directly and positively controlled by the auxin-response transcription factor MONOPTEROS (MP) through an auxin-response element in the ATHB8 promoter. We finally show that the consequences of loss of ATHB8 function for vein formation are masked by MP activity. Our observations define, at the molecular level, patterning inputs of auxin signaling in vein formation.

Auxin enters the matrix—assembly of response machineries for specific outputs

The basic mechanism of auxin as a modulator of gene expression is now well understood. Interactions among three components are required for this process. Auxin is first perceived by its receptor, which then promotes degradation of inhibitors of auxin response transcription factors. These in turn are released from inhibition and modify expression of target genes. How this simple signaling pathway is able to regulate a diverse range of auxin responses is not as well understood, however a clue lies in the existence of large gene families for all components. Recent data indicates that diversification of gene expression patterns, protein activity, and protein-protein interactions among components establishes a matrix of response machineries that generates specific outputs from the generic auxin signal.

Characterization of OsIAA1 gene, a member of rice Aux/IAA family involved in auxin and brassinosteroid hormone responses and plant morphogenesis

Aux/IAA and auxin response factor (ARF) are two important families that have been well recognized for their roles in auxin-mediated responses. Aux/IAA proteins are short-lived transcriptional regulators that mediate the auxin responses through interaction with ARF transcription factors. Although quite a few members of the Aux/IAA family have been functionally characterized in dicotyledonous plants such as Arabidopsis, but relatively limited information is available in important crops such as rice. This work focused on isolation and characterization of a member of Aux/IAA family in rice named OsIAA1. The results indicated that OsIAA1 was constitutively expressed in all the tissues and organs investigated. The expression of this gene was induced by various phytohormones including IAA, 2,4-D, kinetin, 24-epibrassinolide, and jasmonic acid. Over-expression of OsIAA1 in rice resulted in reduced inhibition of root elongation to auxin treatment, but increased sensitivity to 24-epiBL treatment. In addition, the OsIAA1-overexpression transgenic plants showed distinctive morphological changes such as decreased plant height and loose plant architecture. Protein interaction analysis suggested that OsIAA1 may act through interaction with OsARF1. T-DNA insertion mutant of OsARF1 showed reduced sensitivity to BR treatment, resembling the phenotype of OsIAA1-overexpression plants. In addition, expression patterns of some genes responsive to brassinosteroid and auxin were changed in the OsIAA1-overexpression plants. These data suggested that OsIAA1 may play important roles in the cross-talk of auxin and brassinosteroid signaling pathways and plant morphogenesis.

Comprehensive expression profiling analysis of OsIAA gene family in developmental processes and in response to phytohormone and stress treatments

Auxin is one of the most important phytohormones and exerts pleiotropic effects on plant growth and development. Aux/IAA and auxin response factor (ARF) are two important protein families that are well recognized for their roles in auxin-mediated responses. Aux/IAA proteins are short-lived transcriptional regulators that mediate auxin responses through interaction with ARF transcription factors. Here, we systematically compared the genomic organization of the two families in rice. The expression profiles of both families were compared to show possible association of the two gene families at expression level, which would help reveal their synergistic relationships and functions. The expression profile analysis of Aux/IAA and ARF genes in 30 organs/tissues (collected from an entire life cycle of rice) suggested that Aux/IAA and ARF genes are expressed in very diverse patterns. In general, the genes showing similar expression patterns tended to be in the same phylogenetic subgroup even though their expression patterns were not always the same. Hierarchical cluster analysis revealed that there are eight pairs of IAA and ARF genes, with each pair showing highly correlated expression. The expression levels of the IAA gene family were also checked under various hormone treatments including abscisic acid, kinetin, gibberellin, jasmonic acid, auxin and brassinolide. The results indicated that most of the IAA genes respond to at least one of the treatments. Furthermore, DNA chip and real-time PCR results show that many genes in these families were responsive to various abiotic stresses, indicating an interaction between plant growth and abiotic stress. The effect of abiotic stress on plant growth and auxin distribution was further confirmed with the root growth of DR5-GUS transgenic rice under mannitol treatment, in which a close association between mannitol-induced changes of auxin distribution and root growth was observed.

Patterning and Polarity in Seed Plant Shoots

Leaves and stems are ultimately derived from the shoot apical meristem (SAM); leaves arise from the peripheral zone of the SAM and stem tissue is derived from both the peripheral and central zones of the SAM. Both the peripheral and central regions of the SAM are formed during embryogenesis when the basic body plan of the plant is established. Interplay between points of maximal concentration of auxin and specific patterns of transcription of both auxin-responsive transcription factors and other patterning genes subdivide the embryo along both the apical-basal and central-peripheral axes. Differential gene expression along these axes leads to the differentiation of tissues, lateral organs, meristems, and boundary regions, each with varying responsiveness to auxin. Subsequent shoot growth and development is a reiteration of basic patterning processes established during embryogenesis.

Identification of photoperception and light signal transduction pathways in citrus

Studies employing model species have elucidated several aspects of photoperception and light signal transduction that control plant development. However, the information available for economically important crops is scarce. Citrus genome databases of expressed sequence tags (EST) were investigated in order to identify genes coding for functionally characterized proteins responsible for light-regulated developmental control in model plants. Approximately 176,200 EST sequences from 53 libraries were queried and all bona fide and putative photoreceptor gene families were found in citrus species. We have identified 53 orthologs for several families of transcriptional regulators and cytoplasmic proteins mediating photoreceptor-induced responses although some important Arabidopsis phytochrome- and cryptochrome-signaling components are absent from citrus sequence databases. The main gene families responsible for phototropin-mediated signal transduction were present in citrus transcriptome, including general regulatory factors (14-3-3 proteins), scaffolding elements and auxin-responsive transcription factors and transporters. A working model of light perception, signal transduction and response-eliciting in citrus is proposed based on the identified key components. These results demonstrate the power of comparative genomics between model systems and economically important crop species to elucidate several aspects of plant physiology and metabolism.

TheArabidopsisSTV1 Protein, Responsible for Translation Reinitiation, Is Required for Auxin-Mediated Gynoecium Patterning

Ribosomal protein L24 (RPL24) is implicated in translation reinitiation of polycistronic genes. A newly isolated Arabidopsis thaliana short valve1 (stv1) mutant, in which one of the RPL24-encoding genes, RPL24B, is deleted, shows specific defects in the apical-basal patterning of the gynoecium, in addition to phenotypes induced by ribosome deficiency. A similar gynoecium phenotype is caused by mutations in the auxin response factor (ARF) genes ETTIN (ETT) and MONOPTEROS (MP), which have upstream open reading frames (uORFs) in their 5'-transcript leader sequences. Gynoecia of a double mutant of stv1 and a weak ett mutant allele are similar to those of a strong ett allele, and transformation with a uORF-eliminated ETT construct partially suppressed the stv1 gynoecium phenotype, implying that STV1 could influence ETT translation through its uORFs. Analyses of 5'-leader-reporter gene fusions showed that the uORFs of ETT and MP negatively regulate the translation of the downstream major ORFs, indicating that translation reinitiation is an important step for the expression of these proteins. Taken together, we propose that perturbation of translation reinitiation of the ARF transcripts causes the defects in gynoecium patterning observed in the stv1 mutant.

Auxin response factors ARF6 and ARF8 promote jasmonic acid production and flower maturation

Pollination in flowering plants requires that anthers release pollen when the gynoecium is competent to support fertilization. We show that in Arabidopsis thaliana, two paralogous auxin response transcription factors, ARF6 and ARF8, regulate both stamen and gynoecium maturation. arf6 arf8 double-null mutant flowers arrested as infertile closed buds with short petals, short stamen filaments, undehisced anthers that did not release pollen and immature gynoecia. Numerous developmentally regulated genes failed to be induced. ARF6 and ARF8 thus coordinate the transition from immature to mature fertile flowers. Jasmonic acid (JA) measurements and JA feeding experiments showed that decreased jasmonate production caused the block in pollen release, but not the gynoecium arrest. The double mutant had altered auxin responsive gene expression. However, whole flower auxin levels did not change during flower maturation, suggesting that auxin might regulate flower maturation only under specific environmental conditions, or in localized organs or tissues of flowers. arf6 and arf8 single mutants and sesquimutants (homozygous for one mutation and heterozygous for the other) had delayed stamen development and decreased fecundity, indicating that ARF6 and ARF8 gene dosage affects timing of flower maturation quantitatively.

MicroRNA-Directed Regulation of ArabidopsisAUXIN RESPONSE FACTOR17Is Essential for Proper Development and Modulates Expression of Early Auxin Response Genes

The phytohormone auxin plays critical roles during plant growth, many of which are mediated by the auxin response transcription factor (ARF) family. MicroRNAs (miRNAs), endogenous 21-nucleotide riboregulators, target several mRNAs implicated in auxin responses. miR160 targets ARF10, ARF16, and ARF17, three of the 23 Arabidopsis thaliana ARF genes. Here, we describe roles of miR160-directed ARF17 posttranscriptional regulation. Plants expressing a miRNA-resistant version of ARF17 have increased ARF17 mRNA levels and altered accumulation of auxin-inducible GH3-like mRNAs, YDK1/GH3.2, GH3.3, GH3.5, and DFL1/GH3.6, which encode auxin-conjugating proteins. These expression changes correlate with dramatic developmental defects, including embryo and emerging leaf symmetry anomalies, leaf shape defects, premature inflorescence development, altered phyllotaxy along the stem, reduced petal size, abnormal stamens, sterility, and root growth defects. These defects demonstrate the importance of miR160-directed ARF17 regulation and implicate ARF17 as a regulator of GH3-like early auxin response genes. Many of these defects resemble phenotypes previously observed in plants expressing viral suppressors of RNA silencing and plants with mutations in genes important for miRNA biogenesis or function, providing a molecular rationale for phenotypes previously associated with more general disruptions of miRNA function.

The PLETHORA Genes Mediate Patterning of the Arabidopsis Root Stem Cell Niche

A small organizing center, the quiescent center (QC), maintains stem cells in the Arabidopsis root and defines the stem cell niche. The phytohormone auxin influences the position of this niche by an unknown mechanism. Here, we identify the PLETHORA1 ( PLT1) and PLT2 genes encoding AP2 class putative transcription factors, which are essential for QC specification and stem cell activity. The PLT genes are transcribed in response to auxin accumulation and are dependent on auxin response transcription factors. Distal PLT transcript accumulation creates an overlap with the radial expression domains of SHORT-ROOT and SCARECROW, providing positional information for the stem cell niche. Furthermore, the PLT genes are activated in the basal embryo region that gives rise to hypocotyl, root, and root stem cells and, when ectopically expressed, transform apical regions to these identities. Thus, the PLT genes are key effectors for establishment of the stem cell niche during embryonic pattern formation.

The tobacco bZIP transcription factor BZI-1 binds the GH3 promoter in vivo and modulates auxin-induced transcription.

Summary In order to establish the biological function of the tobacco basic leucine zipper (bZIP) transcription factor BZI-1 in hormone signalling, we have analysed transgenic plants which were altered with respect to the protein level or the activation potential of BZI-1. Overexpression of a dominant-negative derivative of BZI-1, lacking the N-terminal activation domain, resulted in plants displaying reduced internode size, enhanced lateral shoot formation and small, curly leaves. The response to auxin monitored with reference to root organogenesis, epinastic leaf curvature and transcription of the auxin-induced GH3 gene was reduced. In vitro, BZI-1 specifically binds to ACGT elements (ACEs) present in the GH3 promoter. In vivo, binding to the GH3 promoter was confirmed by chromatin immunoprecipitation (ChIP). Overexpression of BZI-1 in transgenic plants did not lead to a significant activation of the GH3 target gene. In contrast, plants expressing a VP16 (Herpes simplex virion protein 16)-BZI-1 fusion protein showed enhanced auxin-induced GH3 transcription. However, VP16-BZI-1 is insufficient to trigger GH3 expression independently of the auxin stimulus. Whereas auxin responsiveness has been shown to be mediated by ARF (auxin response factor) transcription factors, we discuss a function of BZI-1 assisting in fine-tuning of auxin-induced transcription.

Protein phosphorylation in the delivery of and response to auxin signals.

The importance of reversible protein phosphorylation in regulation of plant growth and development has been amply demonstrated by decades of research. Here we discuss recent studies that suggest roles for protein phosphorylation in regulation of both auxin responses and polar auxin transport. Specific kinases act at auxin-requiring steps in floral and embryonic development, and at the junction(s) between light and auxin signaling pathways in hypocotyl elongation and phototropism responses. New evidence for rapid mitogen-activated protein kinase (MAPK) activation by auxin treatment suggests that MAPK cascade(s) might mediate cellular responses to auxin. Protein phosphorylation also may play a crucial role in regulating the activity or turnover of auxin-responsive transcription factors. Auxin transport is modulated by phosphorylation, and protein phosphatase activity is involved in regulation of auxin transport streams in roots. Although the regulatory circuits have not been fully elucidated, these studies suggest that protein phosphorylating and dephosphorylating enzymes perform key functions in auxin biology. In some cases, these enzymes act at the intersections between auxin signaling and other signaling pathways.

Regulation of both light- and auxin-mediated development by theArabidopsis IAA3/SHY2 gene

Light affects plant growth and development throughout the life cycle. However, light signals do not function autonomously but should be integrated with endogenous developmental factors such as the plant hormone auxin to specify correct developmental decisions. We have previously reported that theArabidopsis shy2-1D mutation alters various light responses, including highly photomorphogenic development in darkness. Here we show that the mutation also alters various auxin responses, including constitutive formation of lateral roots and reduced auxin sensitivity in inhibition of hypocotyl and root growth. The mutation is a gain of function mutation occuring in theIAA3 gene, one of theAux/IAA family genes encoding putative transcription factors of auxin-responsive genes. These results suggest that IAA3/SHY2 may play important roles in both light-and auxin-mediated development Considering that Aux/IAA proteins and auxin response transcription factors interact with one another, we propose that IAA3/SHY2 may integrate light signals into auxin-mediated developmental responses.

Biochemical characterization of recombinant polypeptides corresponding to the predicted βαα fold in Aux/IAA proteins

The plant hormone indoleacetic acid (IAA or auxin) transcriptionally activates a select set of early genes. The Auxl IAA class of early auxin-responsive genes encodes a large family of short-lived, nuclear proteins. Aux/IAA polypeptides homo-and heterodimerize, and interact with auxin-response transcription factors (ARFs) via C-terminal regions conserved in both protein families. This shared region contains a predicted βαα motif similar to the prokaryotic β-Ribbon DNA binding domain, which mediates both protein dimerization and DNA recognition. Here, we show by circular dichroism spectroscopy and by chemical cross-linking experiments that recombinant peptides corresponding to the predicted βαα region of three Aux/IAA proteins from Arabidopsis thaliana contain substantial α-helical secondary structure and undergo homo- and heterotypic interactions in vitro. Our results indicate a similar biochemical function of the plant βαα domain and suggest that the βαα fold plays an important role in mediating combinatorial interactions of Aux/IAA and ARF proteins to specifically regulate secondary gene expression in response to auxin.

Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements.

A highly active synthetic auxin response element (AuxRE), referred to as DR5, was created by performing site-directed mutations in a natural composite AuxRE found in the soybean GH3 promoter. DR5 consisted of tandem direct repeats of 11 bp that included the auxin-responsive TGTCTC element. The DR5 AuxRE showed greater auxin responsiveness than a natural composite AuxRE and the GH3 promoter when assayed by transient expression in carrot protoplasts or in stably transformed Arabidopsis seedlings, and it provides a useful reporter gene for studying auxin-responsive transcription in wild-type plants and mutants. An auxin response transcription factor, ARF1, bound with specificity to the DR5 AuxRE in vitro and interacted with Aux/IAA proteins in a yeast two-hybrid system. Cotransfection experiments with natural and synthetic AuxRE reporter genes and effector genes encoding Aux/IAA proteins showed that overexpression of Aux/IAA proteins in carrot protoplasts resulted in specific repression of TGTCTC AuxRE reporter gene expression.