microRNA165 and 166 modulate response of the Arabidopsis root apical meristem to salt stress.

In plants, developmental plasticity allows for the modulation of organ growth in response to environmental cues. Being in contact with soil, roots are the first organ that responds to various types of soil abiotic stress such as high salt concentration. In the root, developmental plasticity relies on changes in the activity of the apical meristem, the region at the tip of the root where a set of self-renewing undifferentiated stem cells sustain growth. Here, we show that salt stress promotes differentiation of root meristem cells via reducing the dosage of the microRNAs miR165 and 166. By means of genetic, molecular and computational analysis, we show that the levels of miR165 and 166 respond to high salt concentration, and that miR165 and 166-dependent PHABULOSA (PHB) modulation is central to the response of root growth to this stress. Specifically, we show that salt-dependent reduction of miR165 and 166 causes a rapid increase in PHB expression and, hence, production of the root meristem pro-differentiation hormone cytokinin. Our data provide direct evidence for how the miRNA-dependent modulation of transcription factor dosage mediates plastic development in plants.

Highlight Induced Transcriptional Priming against a Subsequent Drought Stress in Arabidopsis thaliana.

In plants, priming allows a more rapid and robust response to recurring stresses. However, while the nature of plant response to a single stress can affect the subsequent response to the same stress has been deeply studied, considerably less is known on how the priming effect due to one stress can help plants cope with subsequent different stresses, a situation that can be found in natural ecosystems. Here, we investigate the potential priming effects in Arabidopsis plants subjected to a high light (HL) stress followed by a drought (D) stress. The cross-stress tolerance was assessed at the physiological and molecular levels. Our data demonstrated that HL mediated transcriptional priming on the expression of specific stress response genes. Furthermore, this priming effect involves both ABA-dependent and ABA-independent responses, as also supported by reduced expression of these genes in the aba1-3 mutant compared to the wild type. We have also assessed several physiological parameters with the aim of seeing if gene expression coincides with any physiological changes. Overall, the results from the physiological measurements suggested that these physiological processes did not experience metabolic changes in response to the stresses. In addition, we show that the H3K4me3 epigenetic mark could be a good candidate as an epigenetic mark in priming response. Overall, our results help to elucidate how HL-mediated priming can limit D-stress and enhance plant responses to stress.

New Inhibitors of the Human p300/CBP Acetyltransferase Are Selectively Active against the Arabidopsis HAC Proteins.

Histone acetyltransferases (HATs) are involved in the epigenetic positive control of gene expression in eukaryotes. CREB-binding proteins (CBP)/p300, a subfamily of highly conserved HATs, have been shown to function as acetylases on both histones and non-histone proteins. In the model plant Arabidopsis thaliana among the five CBP/p300 HATs, HAC1, HAC5 and HAC12 have been shown to be involved in the ethylene signaling pathway. In addition, HAC1 and HAC5 interact and cooperate with the Mediator complex, as in humans. Therefore, it is potentially difficult to discriminate the effect on plant development of the enzymatic activity with respect to their Mediator-related function. Taking advantage of the homology of the human HAC catalytic domain with that of the Arabidopsis, we set-up a phenotypic assay based on the hypocotyl length of Arabidopsis dark-grown seedlings to evaluate the effects of a compound previously described as human p300/CBP inhibitor, and to screen previously described cinnamoyl derivatives as well as newly synthesized analogues. We selected the most effective compounds, and we demonstrated their efficacy at phenotypic and molecular level. The in vitro inhibition of the enzymatic activity proved the specificity of the inhibitor on the catalytic domain of HAC1, thus substantiating this strategy as a useful tool in plant epigenetic studies.

Developmental Signals in the 21st Century; New Tools and Advances in Plant Signaling.

This special issue includes different research papers and reviews that studied the role of signaling cascades controlling both plant developmental processes and plant response mechanisms to biotic and abiotic stresses [...].

A PHABULOSA-Controlled Genetic Pathway Regulates Ground Tissue Patterning in the Arabidopsis Root.

In both animals and plants, development involves anatomical modifications. In the root of Arabidopsis thaliana, maturation of the ground tissue (GT)-a tissue comprising all cells between epidermal and vascular ones-is a paradigmatic example of these modifications, as it generates an additional tissue layer, the middle cortex (MC).1-4 In early post-embryonic phases, the Arabidopsis root GT is composed of one layer of endodermis and one of cortex. A second cortex layer, the MC, is generated by asymmetric cell divisions in about 80% of Arabidopsis primary roots, in a time window spanning from 7 to 14 days post-germination (dpg). The cell cycle regulator CYCLIN D6;1 (CYCD6;1) plays a central role in this process, as its accumulation in the endodermis triggers the formation of MC.5 The phytohormone gibberellin (GA) is a key regulator of the timing of MC formation, as alterations in its signaling and homeostasis result in precocious endodermal asymmetric cell divisions.3,6,7 However, little is known on how GAs are regulated during GT maturation. Here, we show that the HOMEODOMAIN LEUCINE ZIPPER III (HD-ZIPIII) transcription factor PHABULOSA (PHB) is a master regulator of MC formation, controlling the accumulation of CYCD6;1 in the endodermis in a cell non-autonomous manner. We show that PHB activates the GA catabolic gene GIBBERELLIN 2 OXIDASE 2 (GA2ox2) in the vascular tissue, thus regulating the stability of the DELLA protein GIBBERELLIN INSENSITIVE (GAI)-a GA signaling repressor-in the root and, hence, CYCD6;1 expression in the endodermis.

The DOF Transcription Factors in Seed and Seedling Development.

The DOF (DNA binding with one finger) family of plant-specific transcription factors (TF) was first identified in maize in 1995. Since then, DOF proteins have been shown to be present in the whole plant kingdom, including the unicellular alga Chlamydomonas reinhardtii. The DOF TF family is characterised by a highly conserved DNA binding domain (DOF domain), consisting of a CX2C-X21-CX2C motif, which is able to form a zinc finger structure. Early in the study of DOF proteins, their relevance for seed biology became clear. Indeed, the PROLAMIN BINDING FACTOR (PBF), one of the first DOF proteins characterised, controls the endosperm-specific expression of the zein genes in maize. Subsequently, several DOF proteins from both monocots and dicots have been shown to be primarily involved in seed development, dormancy and germination, as well as in seedling development and other light-mediated processes. In the last two decades, the molecular network underlying these processes have been outlined, and the main molecular players and their interactions have been identified. In this review, we will focus on the DOF TFs involved in these molecular networks, and on their interaction with other proteins.

From the Outside to the Inside: New Insights on the Main Factors That Guide Seed Dormancy and Germination.

The transition from a dormant to a germinating seed represents a crucial developmental switch in the life cycle of a plant. Subsequent transition from a germinating seed to an autotrophic organism also requires a robust and multi-layered control. Seed germination and seedling growth are multistep processes, involving both internal and external signals, which lead to a fine-tuning control network. In recent years, numerous studies have contributed to elucidate the molecular mechanisms underlying these processes: from light signaling and light-hormone crosstalk to the effects of abiotic stresses, from epigenetic regulation to translational control. However, there are still many open questions and molecular elements to be identified. This review will focus on the different aspects of the molecular control of seed dormancy and germination, pointing out new molecular elements and how these integrate in the signaling pathways already known.

Inhibition of Polycomb Repressive Complex 2 activity reduces trimethylation of H3K27 and affects development in Arabidopsis seedlings.

BACKGROUND: Polycomb repressive complex 2 (PRC2) is an epigenetic transcriptional repression system, whose catalytic subunit (ENHANCER OF ZESTE HOMOLOG 2, EZH2 in animals) is responsible for trimethylating histone H3 at lysine 27 (H3K27me3). In mammals, gain-of-function mutations as well as overexpression of EZH2 have been associated with several tumors, therefore making this subunit a suitable target for the development of selective inhibitors. Indeed, highly specific small-molecule inhibitors of EZH2 have been reported. In plants, mutations in some PRC2 components lead to embryonic lethality, but no trial with any inhibitor has ever been reported. RESULTS: We show here that the 1,5-bis (3-bromo-4-methoxyphenyl)penta-1,4-dien-3-one compound (RDS 3434), previously reported as an EZH2 inhibitor in human leukemia cells, is active on the Arabidopsis catalytic subunit of PRC2, since treatment with the drug reduces the total amount of H3K27me3 in a dose-dependent fashion. Consistently, we show that the expression level of two PRC2 targets is significantly increased following treatment with the RDS 3434 compound. Finally, we show that impairment of H3K27 trimethylation in Arabidopsis seeds and seedlings affects both seed germination and root growth. CONCLUSIONS: Our results provide a useful tool for the plant community in investigating how PRC2 affects transcriptional control in plant development.

In vivo Bioluminescence-Based Monitoring of Liver Metastases from Colorectal Cancer: An Experimental Model.

BACKGROUND: In this study we aimed to develop a new in vivo bioluminescence-based tool to monitor and to quantify colon cancer (CC) liver metastasis development. METHODS: HCT 116 cells were transducted with pLenti6/V5-DEST-fLuc for constitutive expression of firefly luciferase. Infection was monitored analyzing endogenous bioluminescence using the IVIS Lumina II In vivo Imaging System and a positive clone constitutively expressing luciferase (HCT 116-fLuc) was isolated. HCT 116-fLuc cells were left untreated or treated with 1 µM GDC-0449, a Hedgehog pharmacological inhibitor. Moreover, 1 x 106 HCT 116-fLuc cells were implanted via intra-splenic injection in nude mice. Bioluminescence was analyzed in these mice every 7 days for 5 weeks. After that, mice were sacrificed and bioluminescence was analyzed on explanted livers. RESULTS: We found that in vitro bioluminescence signal was significantly reduced when HCT 116-fLuc cells were treated with GDC-0449. Regarding in vivo data, bioluminescence sources consistent with hepatic anatomical localization were detected after 21 days from HCT 116-fLuc intrasplenic injection and progressively increased until the sacrifice. The presence of liver metastasis was further confirmed by ex-vivo bioluminescence analysis of explanted livers. CONCLUSIONS: Our in vitro results suggest that inhibition of Hedgehog pathway may hamper CC cell proliferation and impel for further studies. Regarding in vivo data, we set-up a strategy for liver metastasis visualization, that may allow follow-up and quantification of the entire metastatic process. This cost-effective technique would reduce experimental variability, as well as the number of sacrificed animals.

Abscisic acid inhibits hypocotyl elongation acting on gibberellins, DELLA proteins and auxin.

Hypocotyl elongation of Arabidopsis seedlings is influenced by light and numerous growth factors. Light induces inhibition of hypocotyl elongation (photomorphogenesis), whereas in the dark hypocotyl elongation is promoted (skotomorphogenesis). Abscisic acid (ABA) plays a major role in inhibition of hypocotyl elongation, but the molecular mechanism remains unclear. We investigated the effect of ABA during photo- and skotomorphogenesis, making use of appropriate mutants, and we show that ABA negatively controls hypocotyl elongation acting on gibberellin (GA) metabolic genes, increasing the amount of the DELLA proteins GAI and RGA, thus affecting GA signalling, and (ultimately) repressing auxin biosynthetic genes.

Genome-wide RNA-seq analysis indicates that the DAG1 transcription factor promotes hypocotyl elongation acting on ABA, ethylene and auxin signaling.

Hypocotyl elongation is influenced by light and hormones, but the molecular mechanisms underlying this process are not yet fully elucidated. We had previously suggested that the Arabidopsis DOF transcription factor DAG1 may be a negative component of the mechanism of light-mediated inhibition of hypocotyl elongation, as light-grown dag1 knock-out mutant seedlings show significant shorter hypocotyls than the wild type. By using high-throughput RNA-seq, we compared the transcriptome profile of dag1 and wild type hypocotyls and seedlings. We identified more than 250 genes differentially expressed in dag1 hypocotyls, and their analysis suggests that DAG1 is involved in the promotion of hypocotyl elongation through the control of ABA, ethylene and auxin signaling. Consistently, ChIP-qPCR results show that DAG1 directly binds to the promoters of WRKY18 encoding a transcription factor involved in ABA signaling, of the ethylene- induced gene ETHYLENE RESPONSE FACTOR (ERF2), and of the SMALL AUXIN UP RNA 67 (SAUR67), an auxin-responding gene encoding a protein promoting hypocotyl cell expansion.

The DAG1 transcription factor negatively regulates the seed-to-seedling transition in Arabidopsis acting on ABA and GA levels.

BACKGROUND: In seeds, the transition from dormancy to germination is regulated by abscisic acid (ABA) and gibberellins (GAs), and involves chromatin remodelling. Particularly, the repressive mark H3K27 trimethylation (H3K27me3) has been shown to target many master regulators of this transition. DAG1 (DOF AFFECTING GERMINATION1), is a negative regulator of seed germination in Arabidopsis, and directly represses the GA biosynthetic gene GA3ox1 (gibberellin 3-ß-dioxygenase 1). We set to investigate the role of DAG1 in seed dormancy and maturation with respect to epigenetic and hormonal control. RESULTS: We show that DAG1 expression is controlled at the epigenetic level through the H3K27me3 mark during the seed-to-seedling transition, and that DAG1 directly represses also the ABA catabolic gene CYP707A2; consistently, the ABA level is lower while the GA level is higher in dag1 mutant seeds. Furthermore, both DAG1 expression and protein stability are controlled by GAs. CONCLUSIONS: Our results point to DAG1 as a key player in the control of the developmental switch between seed dormancy and germination.

The COP9 SIGNALOSOME Is Required for Postembryonic Meristem Maintenance in Arabidopsis thaliana.

Cullin-RING E3 ligases (CRLs) regulate different aspects of plant development and are activated by modification of their cullin subunit with the ubiquitin-like protein NEDD8 (NEural precursor cell expressed Developmentally Down-regulated 8) (neddylation) and deactivated by NEDD8 removal (deneddylation). The constitutively photomorphogenic9 (COP9) signalosome (CSN) acts as a molecular switch of CRLs activity by reverting their neddylation status, but its contribution to embryonic and early seedling development remains poorly characterized. Here, we analyzed the phenotypic defects of csn mutants and monitored the cullin deneddylation/neddylation ratio during embryonic and early seedling development. We show that while csn mutants can complete embryogenesis (albeit at a slower pace than wild-type) and are able to germinate (albeit at a reduced rate), they progressively lose meristem activity upon germination until they become unable to sustain growth. We also show that the majority of cullin proteins are progressively neddylated during the late stages of seed maturation and become deneddylated upon seed germination. This developmentally regulated shift in the cullin neddylation status is absent in csn mutants. We conclude that the CSN and its cullin deneddylation activity are required to sustain postembryonic meristem function in Arabidopsis.

DOF AFFECTING GERMINATION 2 is a positive regulator of light-mediated seed germination and is repressed by DOF AFFECTING GERMINATION 1.

BACKGROUND: The transcription factor DOF AFFECTING GERMINATION1 (DAG1) is a repressor of the light-mediated seed germination process. DAG1 acts downstream PHYTOCHROME INTERACTING FACTOR3-LIKE 5 (PIL5), the master repressor, and negatively regulates gibberellin biosynthesis by directly repressing the biosynthetic gene AtGA3ox1. The Dof protein DOF AFFECTING GERMINATION (DAG2) shares a high degree of aminoacidic identity with DAG1. While DAG1 inactivation considerably increases the germination capability of seeds, the dag2 mutant has seeds with a germination potential substantially lower than the wild-type, indicating that these factors may play opposite roles in seed germination. RESULTS: We show here that DAG2 expression is positively regulated by environmental factors triggering germination, whereas its expression is repressed by PIL5 and DAG1; by Chromatin Immuno Precipitation (ChIP) analysis we prove that DAG1 directly regulates DAG2. In addition, we show that Red light significantly reduces germination of dag2 mutant seeds. CONCLUSIONS: In agreement with the seed germination phenotype of the dag2 mutant previously published, the present data prove that DAG2 is a positive regulator of the light-mediated seed germination process, and particularly reveal that this protein plays its main role downstream of PIL5 and DAG1 in the phytochrome B (phyB)-mediated pathway.

Independent and interactive effects of DOF affecting germination 1 (DAG1) and the Della proteins GA insensitive (GAI) and Repressor of ga1-3 (RGA) in embryo development and seed germination.

BACKGROUND: The transcription factor DOF AFFECTING GERMINATION1 (DAG1) is a repressor of seed germination acting downstream of the master repressor PHYTOCROME INTERACTING FACTOR3-LIKE 5 (PIL5). Among others, PIL5 induces the expression of the genes encoding the two DELLA proteins GA INSENSITIVE 1 (GAI) and REPRESSOR OF ga1-3 (RGA). RESULTS: Based on the properties of gai-t6 and rga28 mutant seeds, we show here that the absence of RGA severely increases dormancy, while lack of GAI only partially compensates RGA inactivation. In addition, the germination properties of the dag1rga28 double mutant are different from those of the dag1 and rga28 single mutants, suggesting that RGA and DAG1 act in independent branches of the PIL5-controlled germination pathway. Surprisingly, the dag1gai-t6 double mutant proved embryo-lethal, suggesting an unexpected involvement of (a possible complex between) DAG1 and GAI in embryo development. CONCLUSIONS: Rather than overlapping functions as previously suggested, we show that RGA and GAI play distinct roles in seed germination, and that GAI interacts with DAG1 in embryo development.

The Arabidopsis COP9 SIGNALOSOME INTERACTING F-BOX KELCH 1 protein forms an SCF ubiquitin ligase and regulates hypocotyl elongation.

The regulation of protein turnover by the ubiquitin proteasome system (UPS) is a major posttranslational mechanism in eukaryotes. One of the key components of the UPS, the COP9 signalosome (CSN), regulates 'cullin-ring' E3 ubiquitin ligases. In plants, CSN participates in diverse cellular and developmental processes, ranging from light signaling to cell cycle control. In this work, we isolated a new plant-specific CSN-interacting F-box protein, which we denominated CFK1 (COP9 INTERACTING F-BOX KELCH 1). We show that, in Arabidopsis thaliana, CFK1 is a component of a functional ubiquitin ligase complex. We also show that CFK1 stability is regulated by CSN and by proteasome-dependent proteolysis, and that light induces accumulation of the CFK1 transcript in the hypocotyl. Analysis of CFK1 knockdown, mutant, and overexpressing seedlings indicates that CFK1 promotes hypocotyl elongation by increasing cell size. Reduction of CSN levels enhances the short hypocotyl phenotype of CFK1-depleted seedlings, while complete loss of CSN activity suppresses the long-hypocotyl phenotype of CFK1-overexpressing seedlings. We propose that CFK1 (and its regulation by CSN) is a novel component of the cellular mechanisms controlling hypocotyl elongation.

Inactivation of the ELIP1 and ELIP2 genes affects Arabidopsis seed germination.

Light regulates Arabidopsis seed germination through the phyB/PIL5 (PHYTOCHROME INTERACTING FACTOR 3-LIKE 5) transduction pathway, and we have previously shown that the Dof transcription factor DOF AFFECTING GERMINATION1 (DAG1) is a component of this pathway. By means of microarray analysis of dag1 and wild type developing siliques, we identified the EARLY LIGHT-INDUCED PROTEIN1 and 2 (ELIP1 and ELIP2) genes among those deregulated in the loss-of-function dag1 mutant. We analysed seed germination of elip single and double mutants, of elip dag1 double mutants as well as of elip1 elip2 dag1 triple mutant under different environmental conditions. We show that ELIP1 and ELIP2 are involved in opposite ways in the control of this developmental process, in particular under abiotic (light, temperature, salt) stress conditions.

The Dof protein DAG1 mediates PIL5 activity on seed germination by negatively regulating GA biosynthetic gene AtGA3ox1.

We have previously shown that inactivation of the gene encoding the Arabidopsis thaliana transcription factor DOF AFFECTING GERMINATION 1 (DAG1) renders seed germination more sensitive to both phytochrome B (phyB) and gibberellins (GA). dag1 mutant seeds require less red (R) light fluence and a lower GA concentration than WT to germinate. Here, we show that inactivation of the gene PHYTOCHROME INTERACTING FACTOR 3-LIKE 5 (PIL5) results in down-regulation of DAG1. Inactivation of PIL5 in the dag1 mutant background further increased the germination potential of dag1 mutant seeds, supporting the suggestion that DAG1 is under the positive control of PIL5. Germination of dag1phyB seeds showed a reduced requirement of gibberellins as compared with phyB mutant seeds, both in the presence and in the absence of GA biosynthesis. Furthermore, the GA biosynthetic gene AtGA3ox1 is upregulated in dag1 seeds as compared with the WT, and DAG1 actually binds to the AtGA3ox1 promoter, as shown by chromatin immunoprecipitation experiments. Expression analysis at different time points confirms that AtGA3ox1 is directly regulated by DAG1, while suggesting that DAG1 is not a direct regulatory target of PIL5. Our data indicate that in the phyB pathway leading to seed germination, DAG1 negatively regulates GA biosynthesis and suggest that DAG1 acts downstream of PIL5. In addition, the analysis of hypocotyls of dag1 and phyB mutant plantlets, of plantlets overexpressing phyB in the dag1 mutant, as well as of dag1phyB double mutant suggests that DAG1 may act as a negative regulatory element downstream of phyB also in hypocotyl elongation.