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

Abstract 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

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-β-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 loose meristem activity upon germination, until they become unable to sustain growth. We also show that the majority of cullin proteins is progressively neddylated during the late stages of seed maturation and becomes 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

Inhibition of Polycomb Repressive Complex2 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

“DAG1 and GAI shared functions in light-mediated seed germination”

Seed germination is the transition of the quiescent embryo into a new photosintetically active plant, and is defined by the radicle protrusion from the seed coat. It is controlled by multiple environmental and endogenous factors. Germination of seeds of Arabidopsis thaliana needs a pulse of red light and is mediated mainly by the photoreceptor phytochrome B (phyB). A key role is also played by phytohormones, such as abscissic acid (ABA) and gibberellin (GA), which play an antagonistic role. In fact, ABA inhibits this process, whereas GA triggers seed germination. PIL5 is a phytocrome-interacting bHLH protein and is the master repressor of light-mediated seed germination. PIL5 directly induces the expression of RGA and GAI, encoding two DELLA proteins, which are negative regulators of GA-mediated processes. We have previously shown that DAG1, a Dof transcription factor, also plays a central role in this light-mediated process. DAG1 acts dowstream of PIL5 and negatively regulates GA biosynthesis by directly repressing the AtGA3ox1 gene. We are currently investigating the functional and molecular relationship between RGA, GAI and DAG1 by using a combined genetic and molecular approach. Our results suggest that RGA and GAI have different functions with respect to DAG1 in repressing seed germination. In fact, our data shows that the expression level of DAG1 and GAI, but not of RGA, are mutually regulated in the seed. Moreover, GAI, similarly to DAG1, is likely to partecipate in the repression of the AtGA3ox1 gene. Interestingly, genetic analyses indicate that DAG1 and GAI could have also a function during embryogenesis, as the dag1gai-t6 double mutant is embryo-lethal. We conclude that DAG1 and GAI may cooperate in both seed germination and embryo development

Bottom ash-based geopolymer materials: Mechanical and environmental properties

The feasibility of geopolymer synthesis using incinerator bottom ash as the source of Si- and Al- containing phases was tested in the present study. Alkaline activation at moderate temperatures was used to force the rearrangement of the alumino-silicate matrix resulting in the production of a geopolymeric material. Different mixtures were investigated for geopolymer production, which differed in the Si/Al ratio (obtained upon addition of sodium silicate and metakaolin at varying proportions) and the NaOH concentration in the alkaline medium. The obtained geopolymeric materials were characterized in terms of physical and mechanical properties, mineralogical and microstructural characteristics as well as chemical durability. Wide variations in the main characteristics of the obtained products were observed, indicating a significant influence of the process variables on the evolution of the geopolymerization reactions. While the physical and mechanical properties correlated well with the Si/Al ratio, no clear trend was found with the NaOH concentration; however, although the strength development upon geopolymerization was clear, the experimental data suggested that the optimal Si/Al ratio was probably beyond the investigated range. SEM, FT-IR, TG and leaching test data showed that, although the onset of the geopolymer-formation reactions was clear, the degree of matrix restructuring was relatively poor, indicating the need for further investigation to promote the development of the geopolymerization reactions

The DOF protein DAG1 and the della protein GAI cooperate in negatively regulating the AtGA3ox1 gene

International audienceDear Editor, Seed germination is controlled by multiple endo­ genous and environmental factors, which are integrated to trigger this developmental process at the right time. Gibberellins (GAs) are known to induce this process, and the levels of GAs are modulated by light—one of the most important environmental factors affecting seed germina­ tion. The bHLH transcription factor PIL5 (PHYTOCHROME INTERACTING FACTOR 3­LIKE 5) is the master repressor of light­mediated seed germination in Arabidopsis (Oh et al., 2004). In seeds kept in the dark, PIL5 activates transcrip­ tion of the GAI (GA INSENSITIVE) gene (Peng et al., 1997; Oh et al., 2007), a DELLA transcriptional regulator that represses GA­mediated processes (Sun, 2011). GAI plays a role in many growth processes with both unique and overlapping functions with another DELLA protein: RGA (REPRESSOR OF ga1-3) (Dill and Sun, 2001). Also, the DOF transcription factor DAG1 (DOF AFFECTING GERMINATION1) acts in the light­mediated seed germination pathway downstream of PIL5: DAG1 expression is reduced in seeds irradiated for 24 h with red (R) light, and this reduction is dependent on PIL5 as, in pil5 mutant seeds, DAG1 expression is reduced irrespective of light conditions (Gabriele et al., 2010). Null mutant seeds dag1 need a fluence rate six times lower than wild­type to germinate (Papi et al., 2000, 2002); similarly, gai–t6rga28 double mutant seeds require less R light fluences than wild­type ones to germinate (Oh et al., 2007). To further clarify the role of DAG1 in light­mediated seed germination, we focus here on the functional rela­ tionship between DAG1 and GAI in the control of this process. We have recently demonstrated that DAG1 specifi­ cally represses AtGA3ox1 expression. In dag1 mutant seeds, only this GA biosynthetic gene was up­regulated, while the level of expression of AtGA3ox2 and AtGA2ox2 were unchanged compared to the wild­type (Gabriele et al., 2010). A very similar expression profile of AtGA3ox1 was shown by Oh et al. (2007) in gai–t6rga28 double mutant seeds. To verify whether GAI plays a role in the regulation of GA metabolic genes, and in particular of AtGA3ox1, we performed a quantitative RT–PCR (RT–qPCR) analysis on gai-t6 mutant seeds. The level of the AtGA3ox1 transcript was highly increased in the gai-t6 null mutant compared to the wild­type, both in seeds imbibed in the dark and those exposed to R light (Figure 1A), while expression of AtGA3ox2 and AtGA2ox2 was not significantly altered. Since—similarly to DAG1 inactivation—GAI inactivation specifically affected AtGA3ox1 expression, we decided to verify whether the presence of GAI is necessary for DAG1­ mediated repression of AtGA3ox1. In agreement with our hypothesis, promoter analysis of GAI­regulated genes revealed a significant enrichment of DOF­binding sites (Gallego­Bartolomé et al., 2011), suggesting that these transcription factors may mediate GAI activity. We used the dag1DAG1–HA (Gabriele et al., 2010) and the dag1gai-t6DAG1–HA lines, which overexpress DAG1 respectively in the dag1 and dag1gai-t6 mutant backgrounds. Both these lines expressed the DAG1–HA chimeric protein as revealed by immunoblot analysis (Supplemental Figure 1). As expected, the expression of AtGA3ox1 in dag1DAG1–HA seeds was highly reduced compared to wild­type both in the dark and under R light, due to overexpression of DAG1–HA, whereas AtGA3ox1 was strongly overexpressed in dag1gai-t6DAG1–HA seeds (Figure 1B and 1C), suggesting that both DAG1 and GAI are involved in the regulation of AtGA3ox1. Since inactivation of GAI makes DAG1 unable to repress AtGA3ox1 expression, we set to assess whether these two factors directly collaborate in regulating this GA biosynthetic gene. We performed chromatin immunopre­ cipitation (ChIP) assays using the GAI–MYC transgenic line constructed by Oh et al. (2007), and the dag1DAG1–HA line (Gabriele et al., 2010) as a positive control. Cross­linked and sonicated protein–DNA complexes were precipitated with anti­MYC and anti­HA antibodies, respectively. We amplified by real­time PCR (qPCR) three regions of the AtGA3ox1 promoter containing different numbers of cop­ ies of DOF­binding sites (0, 2, and 15) (Figure 1D). As a nega­ tive control, we performed the same assays without adding the antibody, or with both antibodies on wild­type seeds (Supplemental Figure 2). The relative amounts of precipi­ tated promoter fragments of AtGA3ox1 by DAG1–HA are higher than the negative control, and the enrichment of the target fragment is proportional to the number of DOF sites present in the region. By contrast, the enrichment of precipitated promoter fragments of AtGA3ox1 was ver

Selective inhibition of the PRC2 methyltransferase activity suppresses trimethylation of H3K27 in Arabidopsis seeds

Polycomb repressive complex 2 (PRC2) represents a transcriptional epigenetic repression system, and is responsible of trimethylating histone H3 at lysine 27 (H3K27me3) through its catalytic subunit (EZH2). In plants PRC2 controls diverse developmental processes, from fertilization to flowering. In Arabidopsis, it has been widely described the involvement of PRC2 during endosperm formation, and it was shown that lack of PRC2 lead to embryonic lethality and seed abortion. This has severely hampered studies on the function of PRC2 during seed development. Since in animals EZH2 overexpression and the consequent high level of H3K27me3 are hallmarks of several cancers, several PRC2 specific inhibitors have been designed. Curiously, a pharmacological approach has never been tested on plants, although it may provide a useful tool for the study of PRC2 in plants. Taking advantage of the homology of the catalytic subunit between animals and plants, we have assessed the efficacy of the UNC1999 inhibitor, previously reported to be highly effective in vitro, and of a new selective EZH2 inhibitor on Arabidopsis seeds. Our results showed a reduction of the H3K27me3 mark and a consistent increase in the expression levels of PRC2 targets, thus giving new opportunities to deepen the studies on PRC2

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.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 sug

A novel and atypical NF-KB pro-inflammatory program regulated by a CamKII-proteasome axis is involved in the early activation of Muller glia by high glucose

Background Diabetic retinopathy (DR) is a microvascular complication of diabetes with a heavy impact on the quality of life of subjects and with a dramatic burden for health and economic systems on a global scale. Although the pathogenesis of DR is largely unknown, several preclinical data have pointed out to a main role of Muller glia (MG), a cell type which spans across the retina layers providing nourishment and support for Retina Ganglion Cells (RGCs), in sensing hyper-glycemia and in acquiring a pro-inflammatory polarization in response to this insult. Results By using a validated experimental model of DR in vitro, rMC1 cells challenged with high glucose, we uncovered the induction of an early (within minutes) and atypical Nuclear Factor-kB (NF-kB) signalling pathway regulated by a calcium-dependent calmodulin kinase II (CamKII)-proteasome axis. Phosphorylation of proteasome subunit Rpt6 (at Serine 120) by CamKII stimulated the accelerated turnover of IkB alpha (i.e., the natural inhibitor of p65-50 transcription factor), regardless of the phosphorylation at Serine 32 which labels canonical NF-kB signalling. This event allowed the p65-p50 heterodimer to migrate into the nucleus and to induce transcription of IL-8, Il-1 beta and MCP-1. Pharmacological inhibition of CamKII as well as proteasome inhibition stopped this pro-inflammatory program, whereas introduction of a Rpt6 phospho-dead mutant (Rpt6-S120A) stimulated a paradoxical effect on NF-kB probably through the activation of a compensatory mechanism which may involve phosphorylation of 20S alpha 4 subunit. Conclusions This study introduces a novel pathway of MG activation by high glucose and casts some light on the biological relevance of proteasome post-translational modifications in modulating pathways regulated through targeted proteolysis