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