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

MF59®-Adjuvanted H5N1 Vaccine Induces Immunologic Memory and Heterotypic Antibody Responses in Non-Elderly and Elderly Adults

Pathogenic avian influenza virus (H5N1) has the potential to cause a major global pandemic in humans. Safe and effective vaccines that induce immunologic memory and broad heterotypic response are needed.Healthy adults aged 18-60 and > 60 years (n = 313 and n = 173, respectively) were randomized (1:1) to receive two primary and one booster injection of 7.5 microg or 15 microg doses of a subunit MF59-adjuvanted H5N1 (A/Vietnam/1194/2004) (clade 1) vaccine. Safety was monitored until 6 months after booster. Immunogenicity was assessed by hemagglutination inhibition (HI), single radial hemolysis (SRH) and microneutralization assays (MN). Mild injection-site pain was the most common adverse reaction. No serious adverse events relating to the vaccine were reported. The humoral immune responses to 7.5 microg and 15 microg doses were comparable. The rates for seroprotection (HI>40; SRH>25 mm(2); MN > or = 40) after the primary vaccination ranged 72-87%. Six months after primary vaccination with the 7.5 microg dose, 18% and 21% of non-elderly and elderly adults were seroprotected; rates increased to 90% and 84%, respectively, after the booster vaccination. In the 15 microg group, seroprotection rates among non-elderly and elderly adults increased from 25% and 62% after primary vaccination to 92% and 88% after booster vaccination, respectively. A heterologous immune response to the H5N1/turkey/Turkey/05 strain was elicited after second and booster vaccinations.Both formulations of MF59-adjuvanted influenza H5N1 vaccine were well tolerated. The European Union requirement for licensure for pre-pandemic vaccines was met by the lower dose tested. The presence of cross-reactive antibodies to a clade 2 heterologous strain demonstrates that this vaccine may be appropriate for pre-pandemic programs.(ClinicalTrials.gov) NCT00311480

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

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