Localization and Dynamics of the Methionine Sulfoxide Reductases MsrB1 and MsrB2 in Beech Seeds.

Beech seeds are produced irregularly, and there is a need for long-term storage of these seeds for forest management practices. Accumulated reactive oxygen species broadly oxidize molecules, including amino acids, such as methionine, thereby contributing to decreased seed viability. Methionine oxidation can be reversed by the activity of methionine sulfoxide reductases (Msrs), which are enzymes involved in the regulation of many developmental processes and stress responses. Two types of Msrs, MsrB1 and MsrB2, were investigated in beech seeds to determine their abundance and localization. MsrB1 and MsrB2 were detected in the cortical cells and the outer area of the vascular cylinder of the embryonic axes as well as in the epidermis and parenchyma cells of cotyledons. The abundances of MsrB1 and MsrB2 decreased during long-term storage. Ultrastructural analyses have demonstrated the accumulation of these proteins in protein storage vacuoles and in the cytoplasm, especially in close proximity to the cell membrane. In silico predictions of possible Msr interactions supported our findings. In this study, we investigate the contribution of MsrB1 and MsrB2 locations in the regulation of seed viability and suggest that MsrB2 is linked with the longevity of beech seeds via association with proper utilization of storage material.

Masting in Fagus crenata and its influence on the nitrogen content and dry mass of winter buds.

In Fagus, full-mast seeding years are invariably followed by at least one non-mast year. Both flower and leaf primordia develop during the summer within the same winter buds. Flower bud initiation occurs when the N content of developing seeds is increasing rapidly. We hypothesized that competition for nitrogen (N) between developing seeds and buds limits flower primordium formation in mast years and, hence, limits seed production in years following mast years. We tested this hypothesis in three Fagus crenata Blume forests at elevations of 550, 900 and 1500 m. Bud N concentration (N con), amount of N per bud (N bud) and dry mass per bud (DM) were compared between a mast year (2005) and the following non-mast year (2006), and between winter buds containing both leaf and flower primoridia (BF), which were formed during the non-mast year, and winter buds containing leaf primordia only (BL), which were formed in both mast and non-mast years. In addition, leaf numbers per shoot corresponding to the analyzed buds were counted, and the effect of masting on litter production was analyzed by quantifying the amounts of litter that fell in the years 2004 to 2007. The dry mass and N content of BF formed in 2006 by trees at both 550 and 1500 m were 2.1-3.4-fold higher than the corresponding amounts in BL, although the numbers of leaves per current-year shoot in 2007 that developed from the two bud types in the same individuals did not differ significantly. These results indicate that more N and carbohydrate are expended in producing BF than in producing BL. The amount of litter from reproductive organs produced in the mast year was similar to the amount of leaf litter at 900 and 1500 m, but three times as much at 550 m. Leaf numbers per shoot were significantly lower at all elevations in the mast year than in the non-mast years (and the amount of leaf litter at 550 and 1500 m tended to be lower in the mast year than in the non-mast years. In conclusion, preferential allocation of resources to seeds in the mast year reduced the availability of resources for flower primordium formation, and this may have accounted for the poor seed production in the following non-mast year.

Functional characterization of a dehydrin protein from Fagus sylvatica seeds using experimental and in silico approaches.

A strong increase in the level of dehydrin/response ABA transcripts expression reported from the 14th week after flowering coincident with the accumulation of 26 and 44 kDa dehydrins in the embryonic axes of developing beech (Fagus sylvatica L.) seeds. Both transcript and protein levels were strongly correlated with maturation drying. These results suggest that the 44-kDa dehydrin protein is a putative dimer of dehydrin/response ABA protein migrating as a 26-kDa protein. Dehydrins and dehydrin-like proteins form large oligomeric complexes under native conditions and are shown as several spots differing in pI through isoelectrofocusing analyses. Detailed prediction of specific sites accessible for various post-translational modifications (PTMs) in the dehydrin/response ABA protein sequence revealed sites specific to acetylation, amidation, glycosylation, methylation, myristoylation, nitrosylation, O-linked ß-N-acetylglucosamination and Yin-O-Yang modification, palmitoylation, phosphorylation, sumoylation, sulfation, and ubiquitination. Thus, these results suggest that specific PTMs might play a role in switching dehydrin function or activity, water binding ability, protein-membrane interactions, transport and subcellular localization, interactions with targeted molecules, and protein stability. Despite the ability of two Cys residues to form a disulfide bond, -SH groups are likely not involved in dimer arrangement. His-rich regions and/or polyQ-tracts are potential candidates as spatial organization modulators. Dehydrin/response ABA protein is an intrinsically disordered protein containing low complexity regions. The lack of a fixed structure and exposition of amino acids on the surface of the protein structure enhances the accessibility to 40 predicted PTM sites, thereby facilitating dehydrin multifunctionality, which is discussed in the present study.

Non-reducing sugar levels in beech (Fagus sylvatica) seeds as related to withstanding desiccation and storage.

Levels of sucrose and raffinose family oligosaccharides (RFOs) (raffinose and stachyose) were determined in beech (Fagus sylvatica L.) seeds during development, maturation, desiccation and storage. An increase in RFOs and a marked decrease in the S:(R+St) ratio (i.e. mass ratio of sucrose to the sum of RFOs) were observed at the time of desiccation tolerance (DT) acquisition by seeds. In seeds stored at -10 degrees C through 1, 4, 7, and 12 years, changes in sucrose, raffinose and stachyose levels and in alpha-galactosidase activity were noted. The S/R+St ratio and alpha-galactosidase activity significantly increased in seeds after 7 and 12 years of storage, when a marked decrease in viability, measured as germination capacity, was recorded. Germination capacity was found to be strongly correlated with sucrose content, the S:(R+St) ratio, and alpha-galactosidase activity. A strong positive correlation was found between germination capacity and stachyose content. The results clearly indicated that the composition of RFOs in beech seeds is closely related to DT acquisition and seed viability during storage.

Two cDNA clones (FsDhn1 and FsClo1) up-regulated by ABA are involved in drought responses in Fagus sylvatica L. seeds.

Two abscisic acid (ABA)-responsive clones (FsDhn1 and FsClo1) were isolated from a cDNA library of ABA-treated seeds of Fagus sylvatica L. FsDhn1 codes for type-II late embryogenesis abundant (LEA) proteins, also known as dehydrins. The corresponding transcripts were ABA-induced and expressed when seeds were artificially dried. FsClo1 codes for a caleosin, a calcium-binding protein. By expressing FsCLO1 as a fusion protein in Escherichia coli, we obtained biochemical evidence of its ability to bind calcium, a second messenger involved in ABA signaling. This cDNA clone is seed specific, shows a LEA expression pattern and is also induced by ABA and dehydration treatments. The relationship of both genes with seed responses to ABA is discussed.

Immunolocalization of FsPK1 correlates this abscisic acid-induced protein kinase with germination arrest in Fagus sylvatica L. seeds.

An enzymatically active recombinant protein kinase, previously isolated and characterized in Fagus sylvatica L. dormant seeds (FsPK1), was used to obtain a specific polyclonal antibody against this protein. Immunoblotting and immunohistochemical analysis of FsPK1 protein in beech seeds showed a strong immunostaining in the nucleus of the cells located in the vascular tissue of the embryonic axis corresponding to the future apical meristem of the root. This protein kinase was found to accumulate in the seeds only when embryo growth was arrested by application of ABA, while the protein amount decreased during stratification, previously proved to alleviate dormancy, and no protein was detected at all when seed germination was induced by addition of GA(3). These results indicate that FsPK1 may be involved in the control of the embryo growth mediated by ABA and GAs during the transition from dormancy to germination in Fagus sylvatica seeds.

Overexpression of a protein phosphatase 2C from beech seeds in Arabidopsis shows phenotypes related to abscisic acid responses and gibberellin biosynthesis.

A functional abscisic acid (ABA)-induced protein phosphatase type 2C (PP2C) was previously isolated from beech (Fagus sylvatica) seeds (FsPP2C2). Because transgenic work is not possible in beech, in this study we overexpressed this gene in Arabidopsis (Arabidopsis thaliana) to provide genetic evidence on FsPP2C2 function in seed dormancy and other plant responses. In contrast with other PP2Cs described so far, constitutive expression of FsPP2C2 in Arabidopsis, under the cauliflower mosaic virus 35S promoter, produced enhanced sensitivity to ABA and abiotic stress in seeds and vegetative tissues, dwarf phenotype, and delayed flowering, and all these effects were reversed by gibberellic acid application. The levels of active gibberellins (GAs) were reduced in 35S:FsPP2C2 plants, although transcript levels of AtGA20ox1 and AtGA3ox1 increased, probably as a result of negative feedback regulation, whereas the expression of GASA1 was induced by GAs. Additionally, FsPP2C2-overexpressing plants showed a strong induction of the Responsive to ABA 18 (RAB18) gene. Interestingly, FsPP2C2 contains two nuclear targeting sequences, and transient expression assays revealed that ABA directed this protein to the nucleus. Whereas other plant PP2Cs have been shown to act as negative regulators, our results support the hypothesis that FsPP2C2 is a positive regulator of ABA. Moreover, our results indicate the existence of potential cross-talk between ABA signaling and GA biosynthesis.