Sl-IAA27 gene expression is induced during arbuscular mycorrhizal symbiosis in tomato and in Medicago truncatula

Aux/IAA genes play a pivotal role in auxin transcriptional regulation. Their functions were mainly studied in Arabidopsis through analysis of gain-of-function mutants. In the tomato, the Solanaceae reference species, different studies on Sl-IAA down-regulated lines showed specific role for Sl-IAA genes. Our recent work revealed that the Sl-IAA 27 gene displays a distinct behavior compared with most Aux/IAA genes, being down-regulated by auxin. Interestingly, the silencing of Sl-IAA27 leads to altered chlorophyll accumulation in leaves, reduced fertilization, altered fruit development and altered root formation. Here we report that IAA27 could be a key auxin signaling gene involved in AM in tomato and also in Medicago model plant. Indeed both Sl-IAA27 and its closest homolog in Medicago truncatula, Mt-IAA27, are overexpressed in mycorrhized roots. These data are in line with the putative role of auxin in arbuscular mycorrhization

Sl-IAA27 regulates strigolactone biosynthesis and mycorrhization in tomato (var. MicroTom)

- Root colonization by arbuscular mycorrhizal (AM) fungi is a complex and finely tuned process. Previous studies have shown that, among other plant hormones, auxin plays a role in this process but the specific involvement of Aux/IAAs, the key regulators of auxin responses, is still unknown. -In this study, we addressed the role of the tomato Sl-IAA27 during AM symbiosis by using Sl-IAA27-RNAi and pSL-IAA27::GUS stable tomato lines. - The data show that Sl-IAA27 expression is up-regulated by the AM fungus and that silencing of Sl-IAA27 has a negative impact on AM colonization. Sl-IAA27-silencing resulted in down-regulation of three genes involved in strigolactone synthesis, NSP1, D27 and MAX1, and treatment of Sl-IAA27-silenced plants with the strigolactone analog GR24 complemented their mycorrhizal defect phenotype. - Overall, the study identified an Aux/IAA gene as a new component of the signaling pathway controlling AM fungal colonization in tomato. This gene is proposed to control strigolactone biosynthesis via the regulation of NSP1

Auxin Perception Is Required for Arbuscule Development in Arbuscular Mycorrhizal Symbiosis

Most land plant species live in symbiosis with arbuscular mycorrhizal fungi. These fungi differentiate essential functional structures called arbuscules in root cortical cells from which mineral nutrients are released to the plant. We investigated the role of microRNA393 (miR393), an miRNA that targets several auxin receptors, in arbuscular mycorrhizal root colonization. Expression of the precursors of the miR393 was down-regulated during mycorrhization in three different plant species: Solanum lycopersicum, Medicago truncatula, and Oryza sativa. Treatment of S. lycopersicum, M. truncatula, and O. sativa roots with concentrations of synthetic auxin analogs that did not affect root development stimulated mycorrhization, particularly arbuscule formation. DR5-GUS, a reporter for auxin response, was preferentially expressed in root cells containing arbuscules. Finally, overexpression of miR393 in root tissues resulted in down-regulation of auxin receptor genes (transport inhibitor response1 and auxin-related F box) and underdeveloped arbuscules in all three plant species. These results support the conclusion that miR393 is a negative regulator of arbuscule formation by hampering auxin perception in arbuscule-containing cells

Genome-wide identification, phylogenetic analysis, expression profiling, and protein-protein interaction properties of TOPLESS gene family members in tomato

Members of the TOPLESS gene family emerged recently as key players in gene repression in several mechanisms, especially in auxin perception. The TOPLESS genes constitute, in ‘higher-plant’ genomes, a small multigenic family comprising four to 11 members. In this study, this family was investigated in tomato, a model plant for Solanaceae species and fleshy fruits. Six open reading frames predicted to encode topless-like proteins (SlTPLs) containing the canonical domains (LisH, CTLH, and two WD40 repeats) were identified in the tomato genome. Nuclear localization was confirmed for all members of the SlTPL family with the exception SlTPL6, which localized at the cytoplasm and was excluded from the nucleus. SlTPL genes displayed distinctive expression patterns in different tomato organs, with SlTPL1 showing the highest levels of transcript accumulation in all tissues tested except in ripening fruit where SlTPL3 and SlTPL4 were the most prominently expressed. To gain insight into the specificity of the different TOPLESS paralogues, a protein–protein interaction map between TOPLESS and auxin/indole-3-acetic acid (Aux/IAA) proteins was built using a yeast two-hybrid approach. The PPI map enabled the distinction of two patterns: TOPLESS isoforms interacting with the majority of Aux/IAA, and isoforms with limited capacity for interaction with these protein partners. Interestingly, evolutionary analyses of the TOPLESS gene family revealed that the highly expressed isoforms (SlTPL1, SlTPL3, and SlTPL4) corresponded to the three TPL-related genes undergoing the strongest purifying selection, while the selection was much weaker for SlTPL6, which was expressed at a low level and encoded a protein lacking the capacity to interact with Aux/IAAs

Genome-Wide Identification, Functional Analysis and Expression Profiling of the Aux/IAA Gene Family in Tomato

Auxin is a central hormone that exerts pleiotropic effects on plant growth including the development of roots, shoots, flowers and fruit. The perception and signaling of the plant hormone auxin rely on the cooperative action of several components,among which auxin/indole-3-acetic acid (Aux/IAA) proteins play a pivotal role. In this study, we identified and comprehensively analyzed the entire Aux/IAA gene family in tomato (Solanum lycopersicum), a reference species for Solanaceae plants, and the model plant for fleshy fruit development. Functional characterization using a dedicated single cell system revealed that tomato Aux/IAA proteins function as active repressors of auxin-dependent gene transcription, with, however, different Aux/IAA members displaying varying levels of repression. Phylogenetic analysis indicated that the Aux/IAA gene family is slightly contracted in tomato compared with Arabidopsis, with a lower representation of non-canonical proteins. Sl-IAA genes display distinctive expression pattern in different tomato organs and tissues, and some of them display differential responses to auxin and ethylene, suggesting that Aux/IAAs may play a role in linking both hormone signaling pathways. The data presented here shed more light on Sl-IAA genes and provides new leads towards the elucidation of their function during plant development and in mediating hormone cross-talk

Coupling Complete Blood Count and Steroidomics to Track Low Doses Administration of Recombinant Growth Hormone: An Anti-Doping Perspective

Growth Hormone (GH) under its human recombinant homologue (rhGH), may be abused by athletes to take advantage of its well-known anabolic and lipolytic properties; hence it is prohibited in sports by the World Anti-Doping Agency. Due to the rapid turnover of rhGH, anti-doping screening tests have turned to monitor two endocrine biomarkers (IGF-I and P-III-NP), but unfortunately, they show population-wise variability, limiting the identification rate of rhGH users. Previous studies have evidenced the numerous effects of GH on human physiology, especially in hematopoiesis and steroidogenesis. In this work, aiming to discover novel physiological rhGH biomarkers, we analyzed the complete blood count and the steroidomics profile of healthy, physically active, young males treated either with EPO + rhGH or EPO + placebo. The time-trends of these two physiological routes have been analyzed through geometric trajectory analysis (GTA) and OPLS-DA. Individuals supplemented with micro-doses of rhGH exhibited different leukopoietic and steroidal profiles compared to the control population, suggesting a role of the rhGH in both pathways. In the article, hypotheses on the observed differences are discussed according to the most recent literature and compared to results in animal models. The use of leukopoietic and steroidal biomarkers together with endocrine biomarkers (IGF-1 and P-III-NP) allows to correctly classify over 98% of samples with no false positives, miss-classifying only one single sample (false negative) over a total of 56; a promising result, if compared to the current rhGH detection strategies

Phenotypes Associated with Down-Regulation of Sl-IAA27 Support Functional Diversity Among Aux/IAA Family Members in Tomato

The phytohormone auxin is known to regulate several aspects of plant development, and Aux/IAA transcription factors play a pivotal role in auxin signaling. To extend our understanding of the multiple functions of Aux/IAAs further, the present study describes the functional characterization of Sl-IAA27, a member of the tomato Aux/IAA gene family. Sl-IAA27 displays a distinct behavior compared with most Aux/IAA genes regarding the regulation of its expression by auxin, and the Sl-IAA27-encoded protein harbors a unique motif of unknown function also present in Sl-IAA9 and remarkably conserved in monocot and dicot species. Tomato transgenic plants underexpressing the Sl-IAA27 gene revealed multiple phenotypes related to vegetative and reproductive growth. Silencing of Sl-IAA27 results in higher auxin sensitivity, altered root development and reduced Chl content in leaves. Both ovule and pollen display a dramatic loss of fertility in Sl-IAA27 down-regulated lines,and the internal anatomy of the flower and the fruit are modified, with an enlarged placenta in smaller fruits. In line with the reduced Chl content in Sl-IAA27 RNA interference(RNAi) leaves, genes involved in Chl synthesis display lower expression at the level of transcript accumulation. Even though Sl-IAA27 is closely related to Sl-IAA9 in terms of sequence homology and the encoded proteins share common structural features, the data indicate that the two genes regulate tomato fruit initiation and development in a distinct manner

The tomato SlIAA15 is involved in trichome formation and axillary shoot development

The Aux/IAA genes encode a large family of short-lived proteins known to regulate auxin signalling in plants. Functional characterization of SlIAA15, a member of the tomato (Solanum lycopersicum) Aux/IAA family, shows that the encoded protein acts as a strong repressor of auxin-dependent transcription. The physiological significance of SlIAA15 was addressed by a reverse genetics approach, revealing that SlIAA15 plays multiple roles in plant developmental processes. The SlIAA15 down-regulated lines display lower trichome number, reduced apical dominance with associated modified pattern of axillary shoot development, increased lateral root formation and decreased fruit set. Moreover, the leaves of SlIAA15-inhibited plants are dark green and thick, with larger pavement cells, longer palisade cells and larger intercellular space of spongy mesophyll cells. The SlIAA15-suppressed plants exhibit a strong reduction in type I, V and VI trichome formation, suggesting that auxin-dependent transcriptional regulation is required for trichome initiation. Concomitant with reduced trichome formation, the expression of some R2R3 MYB genes, putatively involved in the control of trichome differentiation, is altered. These phenotypes uncover novel and specialized roles for Aux/IAAs in plant developmental processes, clearly indicating that members of the Aux/IAA gene family in tomato perform both overlapping and specific functions

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