INSTYTUT DENDROLOGII

The role of selected proteins in the acquisition and breaking of deep physiological dormancy in seeds of Acer platanoides L. and Fagus sylvatica L.

Seeds of many forest tree species in temperate climate are characterized by dormancy. Norway maple (Acer platanoides L.) and European beech (Fagus sylvatica L.) seeds have deep physiological dormancy, located in mature embryos. It is an internal mechanism that protects seeds against germination in unfavourable conditions for subsequent growth and development of seedlings. Seed dormancy could be broken by stratification of hydrated seeds for several weeks in 3°C temperature. Dormancy status is regulated by a variety of factors, e.g. antagonistic activity of abscisic acid (inhibitor of germination) and gibberellins (promotors of germination), according to the hormone imbalance theory. However, the mechanisms associated with the acquisition and status of dormancy are still not fully understood. This study aims to contribute to better understanding of the mechanisms that regulate dormancy acquisition and braking in seeds of long-lived trees with deep physiological dormancy.

Different approaches were used to estimate the changes associated with the regulation of dormancy acquisition and breaking: (1) proteomics, (2) protein immunodetection, (3) transcriptomics, (4) protein immunolocalization and (5) flow cytometry.

Results of this study indicate that different proteins were associated with embryo development during morphogenesis and maturation. Proteins associated with processing of genetic information (glycine-rich protein and proteosomal protein), metabolism of methionine (reductase of methionine sulfoxide), cell processes (a-tubulin) and defence (ascorbate peroxidase) were differentially abundant during those phases. In the 17th week after flowering the phase of embryo maturation began, during  which seeds enter dormancy. At the end of embryo maturation phase the proteins such as ABI5 (associated with abscisic acid pathway) and RGL2 (gibberellins pathway) were accumulated, which suggests their participation in dormancy acquisition. Moreover, Norway maple embryos in the last phase of maturation contained large numbers of nuclei with 4C DNA. This is probably connected with embryo readiness to start activity after dormancy breaking.

Dormancy breaking during cold stratification is associated with changes in hormonal signalling pathways of abscisic acid and gibberellins that cause germination of seeds. The ABI5 and 14-3-3 proteins are regulated by the cold stratification, which reduces their levels and stops inhibitory influence of abscisic acid. Analysis of transcription (Real-Time-PCR) of mRNA of ABI5 and 14-3-3 proteins during stratification showed that the transcripts’ level decreased faster than proteins’ level. The cold stratification reduced the amount of repressor protein – RGL2, and thus allowed seed germination.

The ABI5 and RGL2 proteins changed the localization in the embryo during seed stratification. Changes were noted on the tissue, cell and organelle levels. Location of the proteins changed from the apical meristem to cortex. The accumulation of ABI5 and RGL2 proteins in the apical meristem in the first weeks of stratification is probably associated with maintaining the dormancy. The RGL2 was found in nucleoli at the beginning of stratification, what reflects the intense transcriptional activity in this period, related to the maintenance of dormancy. The results indicate the differences in regulating the dormancy of the examined species. That likely results from two species belonging to different seed categories associated with tolerance to desiccation and storage – orthodox (tolerant, Norway maple) and intermediate (sensitive, beech).

This work confirmed the hormonal theory of dormancy, which states that dormancy is regulated by the imbalance between inhibitors and promotors of germination. It was also showed that 17 week after flowering is a crucial moment for seed to acquisition of the dormancy. The results showed differences in the depth of dormancy between the two species, which resulted probably from differences in seed desiccation tolerance.