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.

Involvement of the MetO/Msr System in Two Acer Species That Display Contrasting Characteristics during Germination.

The levels of methionine sulfoxide (MetO) and the abundances of methionine sulfoxide reductases (Msrs) were reported as important for the desiccation tolerance of Acer seeds. To determine whether the MetO/Msrs system is related to reactive oxygen species (ROS) and involved in the regulation of germination in orthodox and recalcitrant seeds, Norway maple and sycamore were investigated. Changes in water content, MetO content, the abundance of MsrB1 and MsrB2 in relation to ROS content and the activity of reductases depending on nicotinamide adenine dinucleotides were monitored. Acer seeds differed in germination speed-substantially higher in sycamore-hydration dynamics, levels of hydrogen peroxide, superoxide anion radicals (O2•-) and hydroxyl radicals (•OH), which exhibited peaks at different stages of germination. The MetO level dynamically changed, particularly in sycamore embryonic axes, where it was positively correlated with the levels of O2•- and the abundance of MsrB1 and negatively with the levels of •OH and the abundance of MsrB2. The MsrB2 abundance increased upon sycamore germination; in contrast, it markedly decreased in Norway maple. We propose that the ROS-MetO-Msr redox system, allowing balanced Met redox homeostasis, participates in the germination process in sycamore, which is characterized by a much higher speed compared to Norway maple.

Integration of MsrB1 and MsrB2 in the Redox Network during the Development of Orthodox and Recalcitrant Acer Seeds.

Two related tree species, Norway maple (Acer platanoides L.) and sycamore (Acer pseudoplatanus L.), produce desiccation-tolerant (orthodox) and desiccation-sensitive (recalcitrant) seeds, respectively. We compared the seeds of these two species to characterize the developmentally driven changes in the levels of peptide-bound methionine sulfoxide (MetO) and the abundance of methionine sulfoxide reductases (Msrs) B1 and B2, with respect to the cellular redox environment. Protein oxidation at the Met level was dynamic only in Norway maple seeds, and the reduced MsrB2 form was detected only in this species. Cell redox status, characterized by the levels of reduced and oxidized ascorbate, glutathione, and nicotinamide adenine dinucleotide (NAD)/phosphate (NADP), was clearly more reduced in the Norway maple seeds than in the sycamore seeds. Clear correlations between MetO levels, changes in water content and redox status were reported in orthodox Acer seeds. The abundance of Msrs was correlated in both species with redox determinants, mainly ascorbate and glutathione. Our data suggest that MsrB2 is associated with the acquisition of desiccation tolerance and that ascorbate might be involved in the redox pathway enabling the regeneration of Msr via intermediates that are not known yet.

Seasonal senescence of leaves and roots of Populus trichocarpa-is the scenario the same or different?

The remobilization and resorption of plant nutrients is considered as a crucial aspect of the seasonal senescence of plant organs. In leaves, the mechanisms responsible for the relocation of valuable compounds are well understood while the related processes in roots are still being debated. Some research indicates that remobilization in roots occurs, while other studies have not found evidence of this process. Considering that the total biomass of fine roots is equal to or greater than that of leaves, clarifying the conflicting reports and ambiguities may provide critical information on the circulation of chemical elements in forest ecosystems. This study provides new information concerning the basis for remobilization processes in roots by combining physiological data with gene expression and protein levels. We suggest that, as in leaves, molecular mechanisms involved in nitrogen (N) resorption are also activated in senescent roots. An analysis of N concentration indicated that N levels decreased during the senescence of both organs. The decrease was associated with an increase in the expression of a glutamine synthetase (GS) gene and a concomitant elevation in the amount of GS-one of the most important enzymes in N metabolism. In addition, significant accumulation of carbohydrates was observed in fine roots, which may represent an adaptation to unfavorable weather conditions that would allow remobilization to occur rather than a rapid death in response to ground frost or cold. Our results provide new insights into the senescence of plant organs and clarify contentious topics related to the remobilization process in fine roots.

Regulation of Gene Expression of Methionine Sulfoxide Reductases and Their New Putative Roles in Plants.

Oxidation of methionine to methionine sulfoxide is a type of posttranslational modification reversed by methionine sulfoxide reductases (Msrs), which present an exceptionally high number of gene copies in plants. The side-form general antioxidant function-specific role of each Msr isoform has not been fully studied. Thirty homologous genes of Msr type A (MsrA) and type B (MsrB) that originate from the genomes of Arabidopsis thaliana, Populus trichocarpa, and Oryza sativa were analyzed in silico. From 109 to 201 transcription factors and responsive elements were predicted for each gene. Among the species, 220 and 190 common transcription factors and responsive elements were detected for the MsrA and MsrB isoforms, respectively. In a comparison of 14 MsrA and 16 MsrB genes, 424 transcription factors and responsive elements were reported in both types of genes, with almost ten times fewer unique elements. The transcription factors mainly comprised plant growth and development regulators, transcription factors important in stress responses with significant overrepresentation of the myeloblastosis viral oncogene homolog (MYB) and no apical meristem, Arabidopsis transcription activation factor and cup-shaped cotyledon (NAC) families and responsive elements sensitive to ethylene, jasmonate, sugar, and prolamine. Gene Ontology term-based functional classification revealed that cellular, metabolic, and developmental process terms and the response to stimulus term dominated in the biological process category. Available experimental transcriptomic and proteomic data, in combination with a set of predictions, gave coherent results validating this research. Thus, new manners Msr gene expression regulation, as well as new putative roles of Msrs, are proposed.

Autophagy counteracts instantaneous cell death during seasonal senescence of the fine roots and leaves in Populus trichocarpa.

BACKGROUND: Senescence, despite its destructive character, is a process that is precisely-regulated. The control of senescence is required to achieve remobilization of resources, a principle aspect of senescence. Remobilization allows plants to recapture valuable resources that would otherwise be lost to the environment with the senescing organ. Autophagy is one of the critical processes that is switched on during senescence. This evolutionarily conserved process plays dual, antagonistic roles. On the one hand, it counteracts instantaneous cell death and allows the process of remobilization to be set in motion, while on the other hand, it participates in the degradation of cellular components. Autophagy has been demonstrated to occur in many plant species during the senescence of leaves and flower petals. Little is known, however, about the senescence process in other ephemeral organs, such as fine roots, whose lifespan is also relatively short. We hypothesized that, like the case of seasonal leaf senescence, autophagy also plays a role in the senescence of fine roots, and that both processes are synchronized in their timing. RESULTS: We evaluated which morphological and cytological symptoms are universal or unique in the senescence of fine roots and leaves. The results of our study confirmed that autophagy plays a key role in the senescence of fine roots, and is associated also with the process of cellular components degradation. In both organs, structures related to autophagy were observed, such as autophagic bodies and autophagosomes. The role of autophagy in the senescence of these plant organs was further confirmed by an analysis of ATG gene expression and protein detection. CONCLUSIONS: The present study is the first one to examine molecular mechanisms associated with the senescence of fine roots, and provide evidence that can be used to determine whether senescence of fine roots can be treated as another example of developmentally programmed cell death (dPCD). Our results indicate that there is a strong similarity between the senescence of fine roots and other ephemeral organs, suggesting that this process occurs by the same autophagy-related mechanisms in all plant ephemeral organs.

The effect of a doubled glutathione level on parameters affecting the germinability of recalcitrant Acer saccharinum seeds during drying.

Approximately 20% of plant species, including silver maple (Acer saccharinum L.), produce seeds that are sensitive to desiccation, which is reflected in their poor storage potential and viability. In the search for a compound that can improve seed recalcitrance, freshly harvested seeds were soaked in either 2.5 mM reduced glutathione (GSH) or water and desiccated to comparable water levels of 55-20%. We examined the impact of a doubled endogenous level of glutathione on the seed germination capacity, the activity of enzymes involved in glutathione metabolism, the cell membrane components and integrity, reactive oxygen species, and ascorbate levels. GSH treatment resulted in slower dehydration and a higher germination capacity. The increased glutathione was mainly consumed by glutathione S-transferase, leading to more efficient detoxification, and by dehydroascorbate reductase (DHAR), accelerating the ascorbate regeneration. As a result, the cellular environment became more reduced, and protection of the membrane structures was enhanced. The ameliorated membrane integrity was manifested via a lower electrolyte leakage and a lower lipid peroxide level despite the higher level of hydrogen peroxide (H2O2) detected in the GSH-treated seeds. The degradation of phospholipids (PLs) was less intense and related to the phosphatidylinositol (PI) level, which is the precursor of the phospholipase D cofactor, whereas in water-soaked seeds, PL degradation was promoted by H2O2. The germination capacity of the dehydrated seeds depended primarily on the level of H2O2, lipid hydroxyperoxides, electrolyte leakage, GSH, the half-cell reduction potential of glutathione, PI, and the activity of DHAR and γ-glutamylcysteine synthetase. Interestingly, H2O2 affected all of the parameters. The germination of GSH-boosted seeds was strongly impacted by the pool of ascorbate, the half-cell reduction potential of ascorbate, and the glutathione peroxidase activity. In general, germination was DHAR activity-dependent. A strong negative correlation was detected in the water-soaked seeds, whereas a strong positive correlation was detected in the GSH-treated seeds. The enhanced level of glutathione likely improved the efficiency of the ascorbate-glutathione cycle, confirming its effect on seed germinability after dehydration.

Invasive oaks escape pre-dispersal insect seed predation and trap enemies in their seeds.

Species introduced to habitats outside their native range often escape control by their natural enemies. Besides competing with native species, an alien species might also affect the native herbivores by introducing a new source of different quality food. Here, we describe the case of northern red oak (Quercus rubra) invasion in Europe. We collected data on insect (moth Cydia spp. and weevil Curculio spp.) seed predation of northern red oak in its native (USA, North America) and invasive (Poland, Europe) range, as well as for sessile oaks (Quercus petrea) in Europe. We also evaluated the quality of acorns as hosts for weevil larvae by collecting infested acorns and measuring weevil developmental success, and quantifying acorn traits such as seed mass, tannins, lipids and protein concentration. We used DNA barcoding to identify insects to the species level. The predation by moths was similar and very low in both species and in both ranges. However, red oaks escape pre-dispersal seed predation by weevils in Europe. Weevil infestation rates of northern red oak acorns in their invasive range were 10 times lower than that of sessile oaks, and also 10 times lower than that of red oaks in North America. Furthermore, even when weevils oviposited into northern red oaks, the larvae failed to develop, suggesting that the exotic host created a trap for the insect. This phenomenon might gradually decrease the local abundance of the seed predator, and further aid the invasion.

Age-related changes in protein metabolism of beech (Fagus sylvatica L.) seeds during alleviation of dormancy and in the early stage of germination.

The long-term storage of seeds generally reduces their viability and vigour. The aim of this work was to evaluate the effect of long-term storage on beech (Fagus sylvatica L.) seeds at optimal conditions, over 9 years, on the total and soluble protein levels and activity of proteolytic enzymes, including endopeptidases, carboxypeptidases and aminopeptidases, as well as free amino acid levels and protein synthesis, in dry seeds, after imbibition and during cold stratification leading to dormancy release and germination. The same analyses were conducted in parallel on seeds gathered from the same tree in the running growing season and stored under the same conditions for only 3 months. The results showed that germination capacity decreased from 100% in freshly harvested seeds to 75% in seeds stored for 9 years. The levels of total and soluble proteins were highest in freshly harvested seeds and decreased significantly during storage, these proportions were retained during cold stratification and germination of seeds. Significant differences between freshly harvested and stored seeds were observed in the activities of proteolytic enzymes, including endopeptidases, aminopeptidases and carboxypeptidases, and in the levels of free amino acids. The neosynthesis of proteins during dormancy release and in the early stage of seed germination was significantly weaker in stored seeds. These results confirm the importance of protein metabolism for seed viability and the consequences of its reduction during seed ageing.

The involvement of the mitochondrial peroxiredoxin PRXIIF in defining physiological differences between orthodox and recalcitrant seeds of two Acer species.

Norway maple (Acer platanoides L., orthodox) and sycamore (Acer pseudoplatanus L., recalcitrant) belong to the same genus and grow under similar climatic conditions, but their seeds differ in their tolerance to desiccation. The initial water content (WC) of the seeds used in this study was 50%, and they were dried to 40, 20 and 7%. The mitochondrial peroxiredoxin IIF (PRXIIF) was identified in seeds of both species by immunoblotting. Semiquantitative RT-PCR analyses indicated that the transcript level of PRXIIF in both types of seeds increased during different stages of desiccation and was higher in seeds of Norway maple than in sycamore. General proteome analyses showed important differences between orthodox and recalcitrant seeds. In sycamore seeds that had been desiccated to a 7% WC, the number of protein spots and the levels of those spots were lower than in desiccation-tolerant Norway maple seeds. Post-translational modifications of PRXIIF in seeds at a 50% WC were detected via 2D electrophoresis and subsequent western blot analysis. The detected shift in the pI values (± 0.3) in A. pseudoplatanus was possibly caused by phosphorylation because several potential phosphorylation sites were predicted in silico for that protein. The gene and amino acid sequences were obtained and aligned with known sequences of other plant PRXIIF genes and proteins. High values of sequence identity were noted between the PRXIIF protein sequences of Acer species, Populus trichocarpa Torr. & A. Gray and Arabidopsis thaliana (L.) Heynh. The involvement of PRXIIF in defining the physiological differences between desiccation-tolerant and desiccation-sensitive Acer seeds is discussed in the context of its role in mitochondrial redox homeostasis.