Dormancy cycling in seeds: mechanisms and regulation

The life cycle of most plants starts, and ends, at the seed stage. In most species mature seeds are shed and dispersed on the ground. At this stage of its life cycle the seed may be dormant and will, by definition, not germinate under favourable conditions (Bewley, 1997).

Seasonal dormancy cycling is a characteristic found in plant seeds. Being able to cycle in and out of dormancy allows the seed to survive decades or even centuries, allowing germination to be spread over time, but only when optimal conditions are available, not only for germination but especially for seedling establishment. In this thesis we have attempted to further elucidate the mechanisms behind dormancy, germination and dormancy cycling.

Sisymbrium officinale seeds need nitrate and light to start germination (Chapter 2, 3, 4, 6). Nitrate acts in part by reducing the abscisic acid (ABA) levels (a plant hormone that elevates dormancy levels). The action of light and nitrate can also be reached by applying gibberellins (GAs) to the seeds (Chapter 2, 3, 4, 6). GAs are capable of inducing enzymes that hydrolyze the ensdosperm walls (Debeaujon and Koornneef, 2000; Chen and Bradford, 2000; Nonogaki et al., 2000; Manz et al., 2005) In  this way GAs could be involved in lowering the physical restrictions imposed by the resistance of the seed coat and the endosperm. On the other hand, GAs may also increase the embryo growth potential.

For successful survival of the dormant seed, metabolic activity is reduced to avoid rapid depletion of reserves. The metabolic activity of the seed was measured using electron paramagnetic resonance (EPR), with TEMPONE as a spin probe, and the respiratory activity was measured with the Q2-test (Chapter 2).We showed that primary dormancy was accompanied by hardly any metabolic or respiratory activity, and this increased considerably when dormancy was broken by nitrate. However, when the light pulse was not given and the seeds had become secondary dormant the metabolic activity slowed down.

Regulation of dormancy is tightly linked with abiotic stress factors from the environment. The regulation and survival of the seed under stress conditions is largely dependent on the composition of the cytoplasm. We tested this by EPR, using carboxyl-proxyl (CP) spin probe (Chapter 4). The primary dormant and sub-dormant seeds possessed a higher viscosity than the germinating seeds. The viscosity of secondary dormant seeds appeared intermediate; however, the ease at which the vitrified water melted was similar to that of primary dormant seeds. As a result of the differences in viscosity, the temperature of vitrified water melting differed between the different dormancy states. The changes in cytoplasmic viscosity and vitrified water melting may be linked to changes in metabolism and the content of high molecular weight compounds.

As membranes are the primary target for temperature perception, they are often implicated in regulating dormancy. Therefore, Hilhorst (1998) put forward a hypothesis in which changes in responsiveness to dormancy breaking factors like nitrate and light was a function of cellular membrane fluidity. In Chapter 3 we indeed showed that dormancy is a function of membrane fluidity. Primary dormant seeds of Sisymbrium officinale appeared to have very rigid membranes, whereas breaking dormancy increased membrane fluidity considerably. However, when sub-dormant seeds became secondary dormant membrane fluidity decreased again, but not to the rigidity seen in primary dormant seeds. One of the most common ways in which cells control membrane fluidity is by homeoviscous adaptation with the help of desaturases. Desaturase involvement in changes in membrane fluidity due to changes in dormancy was tested in Chapter 3 (using Sisymbrium officinale) and Chapter 5 (using Arabidopsis thaliana). Here we found that although desaturase activity may change the membrane fluidity or influence the germination/dormancy phenotype, the two are not linked, unless the effects of these enzymes are very local within the seed. Finally, in Chapter 7, we presented a new model in which a membrane anchored dormancy related protein/transcription factor is activated by changes in membrane fluidity. The activated form is transported to the nucleus, where it starts the germination process, which includes changes in metabolism and mobilization of storage reserves.