Auxin transport through non-hair cells sustains root-hair development.

The plant hormone auxin controls root epidermal cell development in a concentration-dependent manner. Root hairs are produced on a subset of epidermal cells as they increase in distance from the root tip. Auxin is required for their initiation and continued growth, but little is known about its distribution in this region of the root. Contrary to the expectation that hair cells might require active auxin influx to ensure auxin supply, we did not detect the auxin-influx transporter AUX1 in root-hair cells. A high level of AUX1 expression was detected in adjacent non-hair cell files. Non-hair cells were necessary to achieve wild-type root-hair length, although an auxin response was not required in these cells. Three-dimensional modelling of auxin flow in the root tip suggests that AUX1-dependent transport through non-hair cells maintains an auxin supply to developing hair cells as they increase in distance from the root tip, and sustains root-hair outgrowth. Experimental data support the hypothesis that instead of moving uniformly though the epidermal cell layer, auxin is mainly transported through canals that extend longitudinally into the tissue

De-Novo Discovery of Differentially Abundant Transcription Factor Binding Sites Including Their Positional Preference

Transcription factors are a main component of gene regulation as they activate or repress gene expression by binding to specific binding sites in promoters. The de-novo discovery of transcription factor binding sites in target regions obtained by wet-lab experiments is a challenging problem in computational biology, which has not been fully solved yet. Here, we present a de-novo motif discovery tool called Dispom for finding differentially abundant transcription factor binding sites that models existing positional preferences of binding sites and adjusts the length of the motif in the learning process. Evaluating Dispom, we find that its prediction performance is superior to existing tools for de-novo motif discovery for 18 benchmark data sets with planted binding sites, and for a metazoan compendium based on experimental data from micro-array, ChIP-chip, ChIP-DSL, and DamID as well as Gene Ontology data. Finally, we apply Dispom to find binding sites differentially abundant in promoters of auxin-responsive genes extracted from Arabidopsis thaliana microarray data, and we find a motif that can be interpreted as a refined auxin responsive element predominately positioned in the 250-bp region upstream of the transcription start site. Using an independent data set of auxin-responsive genes, we find in genome-wide predictions that the refined motif is more specific for auxin-responsive genes than the canonical auxin-responsive element. In general, Dispom can be used to find differentially abundant motifs in sequences of any origin. However, the positional distribution learned by Dispom is especially beneficial if all sequences are aligned to some anchor point like the transcription start site in case of promoter sequences. We demonstrate that the combination of searching for differentially abundant motifs and inferring a position distribution from the data is beneficial for de-novo motif discovery. Hence, we make the tool freely available as a component of the open-source Java framework Jstacs and as a stand-alone application at http://www.jstacs.de/index.php/Dispom

The Role of Phe82 and Phe351 in Auxin-Induced Substrate Perception by TIR1 Ubiquitin Ligase: A Novel Insight from Molecular Dynamics Simulations

It is well known that Auxin plays a key role in controlling many aspects of plant growth and development. Crystal structures of Transport inhibitor response 1 (TIR1), a true receptor of auxin, were very recently determined for TIR1 alone and in complexes with auxin and different synthetic analogues and an Auxin/Indole-3-Acetic Acid (Aux/IAA) substrate peptide. However, the dynamic conformational changes of the key residues of TIR1 that take place during the auxin and substrate perception by TIR1 and the detailed mechanism of these changes are still unclear. In the present study, various computational techniques were integrated to uncover the detailed molecular mechanism of the auxin and Aux/IAA perception process; these simulations included molecular dynamics (MD) simulations on complexes and the free enzyme, the molecular mechanics Poisson Boltzmann surface area (MM-PBSA) calculations, normal mode analysis, and hydrogen bond energy (HBE) calculations. The computational simulation results provided a reasonable explanation for the structure-activity relationships of auxin and its synthetic analogues in view of energy. In addition, a more detailed model for auxin and Aux/IAA perception was also proposed, indicating that Phe82 and Phe351 played a pivotal role in Aux/IAA perception. Upon auxin binding, Phe82 underwent conformational changes to accommodate the subsequent binding of Aux/IAA. As a result, auxin enhances the TIR1-Aux/IAA interactions by acting as a “molecular glue”. Besides, Phe351 acts as a “fastener” to further improve the substrate binding. The structural and mechanistic insights obtained from the present study will provide valuable clues for the future design of promising auxin analogues

Characterization of the Tomato ARF Gene Family Uncovers a Multi-Levels Post-Transcriptional Regulation Including Alternative Splicing

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 59-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

Expression of auxin-binding protein1 during plum fruit ontogeny supports the potential role of auxin in initiating and enhancing climacteric ripening

Auxin-binding protein1 (ABP1) is an active element involved in auxin signaling and plays critical roles in auxin-mediated plant development. Here, we report the isolation and characterization of a putative sequence from Prunus salicina L., designated PslABP1. The expected protein exhibits a similar molecular structure to that of well-characterized maize-ABP1; however, PslABP1 displays more sequence polarity in the active-binding site due to substitution of some crucial amino-acid residues predicted to be involved in auxin-binding. Further, PslABP1 expression was assessed throughout fruit ontogeny to determine its role in fruit development. Comparing the expression data with the physiological aspects that characterize fruit-development stages indicates that PslABP1 up-regulation is usually associated with the signature events that are triggered in an auxin-dependent manner such as floral induction, fruit initiation, embryogenesis, and cell division and elongation. However, the diversity in PslABP1 expression profile during the ripening process of early and late plum cultivars seems to be due to the variability of endogenous auxin levels among the two cultivars, which consequently can change the levels of autocatalytic ethylene available for the fruit to co-ordinate ripening. The effect of auxin on stimulating ethylene production and in regulating PslABP1 was investigated. Our data suggest that auxin is involved in the transition of the mature green fruit into the ripening phase and in enhancing the ripening process in both auxin- and ethylene-dependent manners thereafter

The maize root stem cell niche: a partnership between two sister cell populations

Using transcript profile analysis, we explored the nature of the stem cell niche in roots of maize (Zea mays). Toward assessing a role for specific genes in the establishment and maintenance of the niche, we perturbed the niche and simultaneously monitored the spatial expression patterns of genes hypothesized as essential. Our results allow us to quantify and localize gene activities to specific portions of the niche: to the quiescent center (QC) or the proximal meristem (PM), or to both. The data point to molecular, biochemical and physiological processes associated with the specification and maintenance of the niche, and include reduced expression of metabolism-, redox- and certain cell cycle-associated transcripts in the QC, enrichment of auxin-associated transcripts within the entire niche, controls for the state of differentiation of QC cells, a role for cytokinins specifically in the PM portion of the niche, processes (repair machinery) for maintaining DNA integrity and a role for gene silencing in niche stabilization. To provide additional support for the hypothesized roles of the above-mentioned and other transcripts in niche specification, we overexpressed, in Arabidopsis, homologs of representative genes (eight) identified as highly enriched or reduced in the maize root QC. We conclude that the coordinated changes in expression of auxin-, redox-, cell cycle- and metabolism-associated genes suggest the linkage of gene networks at the level of transcription, thereby providing additional insights into events likely associated with root stem cell niche establishment and maintenance

Role of the Arabidopsis PIN6 auxin transporter in auxin homeostasis and auxin-mediated development

Plant-specific PIN-formed (PIN) efflux transporters for the plant hormone auxin are required for tissue-specific directional auxin transport and cellular auxin homeostasis. The Arabidopsis PIN protein family has been shown to play important roles in developmental processes such as embryogenesis, organogenesis, vascular tissue differentiation, root meristem patterning and tropic growth. Here we analyzed roles of the less characterised Arabidopsis PIN6 auxin transporter. PIN6 is auxin-inducible and is expressed during multiple auxin–regulated developmental processes. Loss of pin6 function interfered with primary root growth and lateral root development. Misexpression of PIN6 affected auxin transport and interfered with auxin homeostasis in other growth processes such as shoot apical dominance, lateral root primordia development, adventitious root formation, root hair outgrowth and root waving. These changes in auxin-regulated growth correlated with a reduction in total auxin transport as well as with an altered activity of DR5-GUS auxin response reporter. Overall, the data indicate that PIN6 regulates auxin homeostasis during plant development.Christopher I. Cazzonelli, Marleen Vanstraelen, Sibu Simon, Kuide Yin, Ashley Carron-Arthur, Nazia Nisar, Gauri Tarle, Abby J. Cuttriss¤, Iain R. Searle, Eva Benkova, Ulrike Mathesius, Josette Masle, Jiří Friml, Barry J. Pogso

Using RNA-Seq for gene identification, polymorphism detection and transcript profiling in two alfalfa genotypes with divergent cell wall composition in stems

<p>Abstract</p> <p>Background</p> <p>Alfalfa, [<it>Medicago sativa </it>(L.) sativa], a widely-grown perennial forage has potential for development as a cellulosic ethanol feedstock. However, the genomics of alfalfa, a non-model species, is still in its infancy. The recent advent of RNA-Seq, a massively parallel sequencing method for transcriptome analysis, provides an opportunity to expand the identification of alfalfa genes and polymorphisms, and conduct in-depth transcript profiling.</p> <p>Results</p> <p>Cell walls in stems of alfalfa genotype 708 have higher cellulose and lower lignin concentrations compared to cell walls in stems of genotype 773. Using the Illumina GA-II platform, a total of 198,861,304 expression sequence tags (ESTs, 76 bp in length) were generated from cDNA libraries derived from elongating stem (ES) and post-elongation stem (PES) internodes of 708 and 773. In addition, 341,984 ESTs were generated from ES and PES internodes of genotype 773 using the GS FLX Titanium platform. The first alfalfa (<it>Medicago sativa</it>) gene index (MSGI 1.0) was assembled using the Sanger ESTs available from GenBank, the GS FLX Titanium EST sequences, and the <it>de novo </it>assembled Illumina sequences. MSGI 1.0 contains 124,025 unique sequences including 22,729 tentative consensus sequences (TCs), 22,315 singletons and 78,981 pseudo-singletons. We identified a total of 1,294 simple sequence repeats (SSR) among the sequences in MSGI 1.0. In addition, a total of 10,826 single nucleotide polymorphisms (SNPs) were predicted between the two genotypes. Out of 55 SNPs randomly selected for experimental validation, 47 (85%) were polymorphic between the two genotypes. We also identified numerous allelic variations within each genotype. Digital gene expression analysis identified numerous candidate genes that may play a role in stem development as well as candidate genes that may contribute to the differences in cell wall composition in stems of the two genotypes.</p> <p>Conclusions</p> <p>Our results demonstrate that RNA-Seq can be successfully used for gene identification, polymorphism detection and transcript profiling in alfalfa, a non-model, allogamous, autotetraploid species. The alfalfa gene index assembled in this study, and the SNPs, SSRs and candidate genes identified can be used to improve alfalfa as a forage crop and cellulosic feedstock.</p