Defence mechanisms in trees are not well understood. We assessed whether distribution of iron ions and their co-localisation with reactive oxygen species in Pinus sylvestris root cells reflect differential preferences of the pathogens Heterobasidion annosum sensu stricto, H. parviporum and H. abietinum to the host. Strains of H. annosum s.s. characterised by a greater preference for P. sylvestris induced accumulation of superoxide (O(2)(-)) in host cells 6 h after inoculation, whereas two peaks in accumulation of O(2)(-) (after 4 and 48 h) were observed after infection with strains of the pathogens H. parviporum and H. abietinum, which have a lower preference for P. sylvestris. Moreover, strains of H. annosum s.s. caused increased production of hydrogen peroxide (H(2)O(2)) in P. sylvestris cells, in contrast with strains of the other two species (H. parviporum and H. abietinum). Following inoculation with H. annosum s.s. strains, H(2)O(2) was correlated negatively with O(2)(-) and correlated positively with ferrous iron (Fe(2+)). Co-localisation of Fe(3+) with H(2)O(2) may suggest that they are involved in inducing hypersensitive responses and eventually cell death in roots inoculated with H. annosum s.s. strains, in contrast with H. parviporum, in which other mechanisms operate when the host is parasitised.
http://www.eje.cz riod of time (Šefrová & Laštůvka, 2001). The range of C. ohridella continues to expand; in 2003, it reached southern Sweden (Rämert et al., 2011), and a recent report indicates that this insect is in Kazakhstan (Gninenko et al., 2017). Its main host, horse chestnut Aesculus hippocastanum L., was introduced into central Europe in XVI c. (Ravazzi & Caudullo, 2016). Currently, trees of this species are common throughout the continent. They occur in both urban and rural environments and are commonly planted in lines or alleys. This species has high decorative value, especially during the fl owering period, and its chestnuts that fall to the ground attract the attention of children and, as described by Gwiazdowicz & Wiśniewski (2011) are also of interest to senior citizens. Escin, the mixture of triterpenoid saponins found in the tissues of A. hippocastanum, is used to treat chronic venous insuffi ciency, haemorrhoids and post-operative oedema (Sirtori, 2001). Escin also exhibits anti-cancer effects (Cheong et al., 2018). Fujimura et al.
Physiological changes in host plants in response to the broad spectrum of fungal modes of infection are still not well understood. The current study was conducted to better understand the infection of in vitro cultures of Pinus sylvestris L. seedlings by three trophically diverse fungal species, Fusarium oxysporum E. F. Sm. & Swingle, Trichoderma harzianum Rifai and Hebeloma crustuliniforme (Bull.) Quél. Biochemical methods and microscopy were utilized to determine (i) which factors (apoplastic and cellular pH, reactive oxygen species, glutathione and cell death) play a role in the establishment of pathogenic, saprotrophic and mycorrhizal fungi, and (ii) whether cell death is a common response of conifer seedling tissues when they are exposed to trophically diverse fungi. Establishment of the pathogen, F. oxysporum, was observed more frequently in the meristematic region of root tips than in the elongation zone, which was in contrast to T. harzianum and H. crustuliniforme. Ectomycorrhizal (ECM) hyphae, however, were occasionally observed in the studied root zone and caused small changes in the studied factors. Colonization of the meristematic zone occurred due to host cell death. Independently of the zone, changes in cellular pH resulting in an acidic cytoplasm conditioned the establishment of F. oxysporum. Additionally, cell death was negatively correlated with hydrogen peroxide (H2O2) in roots challenged by a pathogenic fungus. Cell death was the only factor uniquely associated with the colonization of host roots by a saprotrophic fungus. The mechanism may differ, however, between the zones since apoplastic pH was negatively correlated with cell death in the elongation zone, whereas in the meristematic zone, none of the studied factors explained cell death. Colonization by the ECM fungus, H. crustuliniforme, was associated with a decreasing number of cells with acidic apoplast and by production of H2O2 in the elongation zone resulting in cell death. Saprotrophic and ECM fungi had a greater effect on cell acidification in the meristematic zone than the pathogenic fungus.
Physiological dormancy is a characteristic of the seeds of plants that are exposed to adverse harsh environmental conditions. Many tree seeds from the northern hemisphere have acquired deep dormancy as an adaptation to the winter period. Such kinds of dormancy can be removed by cold stratification. This physiological process is regulated through abscisic and gibberellic acids signal transduction, in which ABI5, 14-3-3 and RGL2 have a negative response to cold stratification and cause dormancy breaking. Our study is, to our knowledge, the first to report tissue localisation of ABI5 and RGL2 in deeply physiologically dormant seeds. Localisation of these proteins differs in time (weeks of stratification) and space (anatomy of the embryo root). Studies showed that changes occurred on three levels: (1) tissue, as the fluorescence signal throughout the weeks of stratification was localised in different regions of the embryo axes, and these changes were associated with changes in development regulation of the individual regions; (2) cell, either in nucleus or in cytoplasm, involving regulation of gene expression, and synthesis and inactivation in cytoplasm; and (3) organelle, specifically in nuclei/nucleoli, indicating transcription regulation of the specific genes. At the end of stratification, when dormancy is broken, ABI5 and RGL2 were not noticeable in the cells of the apical meristem. ABI5 likely blocked germination through inhibition of meristem activity, whereas RGL2 through blocking of procambial cell differentiation. We speculate that similar molecular and cellular mechanisms exist among other seeds characterised by physiological dormancy.
The dormancy and the growth of trees in temperate climates are synchronized with seasons. Preparation for dormancy and its proper progression are key for survival and development in the next season. Using a unique approach that combined microscopy and proteomic methods, we investigated changes in Norway spruce (Picea abies (L.) H. Karst.) embryonic shoots during four distinct stages of dormancy in natural weather conditions. We identified 13 proteins that varied among dormancy stages, and were linked to regulation of protein level; functioning of chloroplasts and other plastids; DNA and RNA regulation; and oxidative stress. We also found a group of five proteins, related to cold hardiness, that did not differ in expression among stages of dormancy, but had the highest abundancy level. Ultrastructure of organelles is tightly linked to their metabolic activity, and hence may indicate dormancy status. The observed ultrastructure during endodormancy was stable, whereas during ecodormancy, the structural changes were dynamic and related mainly to nucleus, plastids and mitochondria. At the ultrastructural level, the lack of starch and the presence of callose in plasmodesmata in all regions of embryonic shoot were indicators of full endodormancy. At the initiation of ecodormancy, we noted an increase in metabolic activity of organelles, tissue-specific starch hyperaccumulation and degradation. However, in proteomic analysis, we did not find variation in expression of proteins related to starch degradation or to symplastic isolation of cells. The combination of ultrastructural and proteomic methods gave a more complete picture of vegetative bud dormancy than either of them applied separately. We found some changes at the structural level, but not their analogues in the proteome. Our study suggests a very important role of plastids' organization and metabolism, and their protection in the course of dormancy and during the shift from endo- to ecodormancy and the acquisition of growth competence.
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