Seed germination is the first developmental process in the life cycle of a plant.
This process is regulated by external factors such as light and temperature, but also by phytohormones like gibberellins (GA) and abscisic acid (ABA). These two plant hormones have antagonistic effects on germination. In fact, GAs promote this process while ABA inhibits it. As regards environmental factors, light plays a predominant role because it also detects the seed’s position in the soil. Phytochrome B (phyB) is the photoreceptor of red light, i.e. the main photoreceptor involved in seed germination.
The mechanisms that regulate this process on a molecular level were studied in the plant model Arabidopsis thaliana. The main regulator is PIL5 (Phytochrome Interacting Factor3-Like5), a transcription factor that represses germination in the absence of light (Oh et al., 2006). PIL5 acts by activating other repressors further down, such as RGA and GAI, that belong to the DELLA protein family, and others that are still unknown (Oh et al., 2007).
In the presence of light, PIL5 interacts with phyB and is degraded so as to facilitate germination. DAG1 is a Dof-type transcription factor (DNA binding with One Finger) involved in the process of seed germination. In fact, dag1 mutant seeds show an increase in the potential for germination (Papi et al., 2000) and require less light and less gibberellins to germinate (Papi et al., 2002).
In order to prove that DAG1 could belong to the regulation pathway of seed germination, mediated by PIL5, a genetic-molecular study was conducted to understand if DAG1 was regulated by PIL5, and also what its molecular targets were (Gabriele et al., 2010).
Understanding the transduction pathway that regulates the seed germination process is of particular interest not just from the point of view of basic research, but more so for a practical comparison.
Indeed, understanding the molecular mechanisms that regulate this process can help optimise the seed germination conditions of agronomic plants.
Embryogenesis begins with the formation of the zygote and ends when all the embryonic structures are formed (heart stage). Subsequently, the embryo grows through both division and cellular differentiation and reserve substances such as lipids, carbohydrates and proteins accumulate within it.
The seed maturation phase comes to an end with dehydration, the accumulation of reserve products slows down dramatically and the size of the seed decreases. The mature dehydrated embryo thus enters into a state of dormancy characterised by various structural and metabolic cellular changes, such as the blocking of protein synthesis and the reduction in the number of mitochondria with a consequent decrease in cellular respiration.
Dormancy has been defined as the inability of an undamaged and living seed to germinate even in favourable environmental conditions (Bewley, 1997). The state of dormancy can last years before, in optimal conditions, seed germination is induced.
Dehydration and dormancy are essential stages because they give the seed the remarkable ability to be resistant to physical and chemical agents while maintaining its germinative power.
Seed germination is a distinct and successive process in relation to dormancy. It starts with the rehydration (imbibition) of the seed and the resumption of metabolic activities. Germination is considered complete when the embryonic radicles break through the integuments of the seed. The growth that follows is at the expense of the reserves accrued during maturation of the seed until the development of photosynthetic organs guarantees autotrophic autonomy.
Seeds are equipped with very sophisticated “biosensors” that enable them to monitor the surrounding environment in order to determine when environmental conditions are optimal for their growth and development. The presence of light, detected by the photoreceptor phyB, is the most important condition.
The signal transduction pathway that regulates this process has been partially explained in recent years (Oh et al., 2006; 2007).
Figure 1. Molecular model of the seed germination process in Arabidopsis thaliana
FIG.1 - CLICK ZOOM IN - open in new window
Seed germination is repressed in the absence of light through the action of PIL5, the principal negative regulator. PIL5, in turn, activates other negative regulators, including RGA (REPRESSOR OF Ga1-3) and GAI (GA- INSENSITIVE), together with the DELLA protein, known as repressors of the GA signalling pathway (Silverstone et al., 2001).
Nevertheless, PIL5 exercises its negative action on germination even while acting indirectly on the regulation of GA and ABA biosynthesis. In fact, in the dark, PIL5 inhibits the biosynthesis of GA while inducing the biosynthesis of ABA. Consequently the germination of the seeds is inhibited.
PIL5 is a transcription factor capable of interacting with the phytochrome phyB in its active form, that is, in the presence of light. Following this interaction, PIL5 becomes phosphorylated and begins protein degradation; therefore the effect on the biosynthesis of GA and ABA is reversed and germination is induced (Fig. 1).
DAG1 is a Dof-type transcription factor previously characterised as a germination repressor (Papi et al., 2000). In fact, dag1 mutant seeds require less red light and less gibberellins to germinate compared with control seeds (Papi et al., 2002).
It is interesting therefore to verify the hypothesis that DAG1 is a component of the regulation pathway of the seed germination process, mediated by phyB-pil5.
According to the model given in figure 1, other elements further down from pil5, other than RGA and GAI, are yet to be identified. In order to understand if dag1 could be one of these regulators, we took both a genetic and molecular approach in our laboratory.
The double mutants dag1 phyB and dag1 pil5 were produced and the analysis of their germination properties revealed that DAG1 is under the control of pil5 which induces its expression in the dark, albeit indirectly.
Furthermore, it was also interesting to verify what was in turn regulated by DAG1 within that pathway.
We analysed the expression of genes in the metabolism of GA both in dag1 mutant seeds and in wild control seeds. This showed that DAG1 is necessary to suppress the expression of AtGA2ox1, one of the biosynthetic genes.
The activity of DAG1 is therefore necessary to reduce the level of GA in the dark so that seed germination is inhibited.
In addition, since DAG1 is a transcription factor, we proved that its effect on the biosynthetic gene GA, AtGA3ox1, is direct, i.e. if DAG1 identifies sequences of Dof-type links (CTTT/AAAG) on the promoter of AtGA3ox1.
Mutant dag1 plants expressing the protein DAG1-HA were produced, and a Ch-IP (Chromatin Immunoprecipitation) was expressed using seeds derived from these transgenic plants.
With this technique, it is possible to immunoprecipitate a protein of interest (DAG1-HA) with the DNA with which it interacts in a specific way. In this way we demonstrated that dag1 directly regulates the gene AtGA3ox1 by interacting with the Dof sequences present on its promoter.
Previous work by our group had shown that the DAG1 gene is expressed in the vascular tissue of all adult plants and in the seedling since germination, i.e. from when the embryonic radicles appear from the integument of the seed, while its expression is absent during embryogenesis (Gualberti et al., 2002).
Our recent data on germination and the regulation of AtGA3ox1 on the part of DAG1 helped us verify the expression of DAG1 also in the seed during imbibition that precedes germination.
Using seeds derived from plants expressing the reporter gene GUS under the control of the DAG1 promoter, we demonstrated that DAG1 is expressed in the imbibed seed (Fig. 2).
Seeds from the plants pDAG1::GUS were imbibed for 12 or 24 hours and then analysed in the stereo microscope Zeiss Lumar V.12 after histochemical staining.
The study and understanding of the seed germination process is of particular interest not just for basic research but especially for the potential applications in agronomic plants.
In fact, this process, which represents the first phase of development in the life cycle of a plant, is finely regulated as much by environmental factors as by endogenous factors.
Our studies have helped to further explain the molecular mechanisms that regulate this process by inserting the DAG1 transcription factor in this transduction pathway and demonstrating its role as a repressor of germination through negative regulation of the biosynthesis of GA.
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