New facts about callose events in the young ovules of some sexual and apomictic species of the Asteraceae family.

Callose (ß-1,3-glucan) is one of the cell wall polymers that plays an important role in many biological processes in plants, including reproductive development. In angiosperms, timely deposition and degradation of callose during sporogenesis accompanies the transition of cells from somatic to generative identity. However, knowledge on the regulation of callose biosynthesis at specific sites of the megasporocyte wall remains limited and the data on its distribution are not conclusive. Establishing the callose deposition pattern in a large number of species can contribute to full understanding of its function in reproductive development. Previous studies focused on callose events in sexual species and only a few concerned apomicts. The main goal of our research was to establish and compare the pattern of callose deposition during early sexual and diplosporous processes in the ovules of some Hieracium, Pilosella and Taraxacum (Asteraceae) species; aniline blue staining technique was used for this purpose. Our findings indicate that callose deposition accompanies both meiotic and diplosporous development of the megaspore mother cell. This suggests that it has similar regulatory functions in intercellular communication regardless of the mode of reproduction. Interestingly, callose deposition followed a different pattern in the studied sexual and diplosporous species compared to most angiosperms as it usually began at the micropylar pole of the megasporocyte. Here, it was only in sexually reproducing H. transylvanicum that callose first appeared at the chalazal pole of the megasporocyte. The present paper additionally discusses the occurrence of aposporous initial cells with callose-rich walls in the ovules of diploid species.

Comparative proteomic analysis provides new insights into regulation of microspore embryogenesis induction in winter triticale (× Triticosecale Wittm.) after 5-azacytidine treatment.

Effective microspore embryogenesis (ME) requires substantial modifications in gene expression pattern, followed by changes in the cell proteome and its metabolism. Recent studies have awakened also interest in the role of epigenetic factors in microspore de-differentiation and reprogramming. Therefore, demethylating agent (2.5-10 µM 5-azacytidine, AC) together with low temperature (3 weeks at 4 °C) were used as ME-inducing tiller treatment in two doubled haploid (DH) lines of triticale and its effect was analyzed in respect of anther protein profiles, expression of selected genes (TAPETUM DETERMINANT1 (TaTPD1-like), SOMATIC EMBRYOGENESIS RECEPTOR KINASE 2 (SERK2) and GLUTATHIONE S-TRANSFERASE (GSTF2)) and ME efficiency. Tiller treatment with 5.0 µM AC was the most effective in ME induction; it was associated with (1) suppression of intensive anabolic processes-mainly photosynthesis and light-dependent reactions, (2) transition to effective catabolism and mobilization of carbohydrate reserve to meet the high energy demand of cells during microspore reprograming and (3) effective defense against stress-inducing treatment, i.e. protection of proper folding during protein biosynthesis and effective degradation of dysfunctional or damaged proteins. Additionally, 5.0 µM AC enhanced the expression of all genes previously identified as being associated with embryogenic potential of microspores (TaTPD1-like, SERK and GSTF2).

Impact of Ascorbate-Glutathione Cycle Components on the Effectiveness of Embryogenesis Induction in Isolated Microspore Cultures of Barley and Triticale.

Enhanced antioxidant defence plays an essential role in plant survival under stress conditions. However, excessive antioxidant activity sometimes suppresses the signal necessary for the initiation of the desired biological reactions. One such example is microspore embryogenesis (ME)-a process of embryo-like structure formation triggered by stress in immature male gametophytes. The study focused on the role of reactive oxygen species and antioxidant defence in triticale (×Triticosecale Wittm.) and barley (Hordeum vulgare L.) microspore reprogramming. ME was induced through various stress treatments of tillers and its effectiveness was analysed in terms of ascorbate and glutathione contents, total activity of low molecular weight antioxidants and activities of glutathione-ascorbate cycle enzymes. The most effective treatment for both species was a combination of low temperature and exogenous application of 0.3 M mannitol, with or without 0.3 mM reduced glutathione. The applied treatments induced genotype-specific defence responses. In triticale, both ascorbate and glutathione were associated with ME induction, though the role of glutathione did not seem to be related to its function as a reducing agent. In barley, effective ME was accompanied by an accumulation of ascorbate and high activity of enzymes regulating its redox status, without direct relation to glutathione content.

In search of female sterility causes in the tetraploid and pentaploid cytotype of Pilosella brzovecensis (Asteraceae).

Within the agamic Pilosella complex, apomixis (asexual reproduction through seed) involves apospory, parthenogenesis, and autonomous endosperm development. Observations of reproductive biology in P. brzovecensis throughout four growing seasons in the garden have shown that both tetraploid and pentaploid plants of this species do not produce viable seeds and reproduce exclusively vegetatively by underground stolons. The reasons for the seed development failure were unknown, therefore our research focused on the analysis of reproductive events in the ovules of this taxon. We found that apospory was initiated in the ovules of both cytotypes. Multiple aposporous initial (AI) cells differentiated in close proximity to the megaspore mother cell (MMC) and suppressed megasporogenesis at the stage of early prophase I. However, none of the AI cells was able to further develop into a multi-nucleate aposporous embryo sac (AES) due to the inhibition of mitotic divisions. It was unusual that callose was accumulated in the walls of AI cells and its synthesis was most likely associated with a response to the dysfunction of these cells. Callose is regarded as the isolating factor and its surprising deposition in the ovules of P. brzovecensis may signal disruption of reproductive processes that cause premature termination of the aposporous development pathway and ultimately lead to ovule sterility. The results of our embryological analysis may be the basis for undertaking advanced molecular studies aimed at fully understanding of the causes of female sterility in P. brzovecensis.

ß-1,3-Glucanases and chitinases participate in the stress-related defence mechanisms that are possibly connected with modulation of arabinogalactan proteins (AGP) required for the androgenesis initiation in rye (Secale cereale L.).

This work presents the biochemical, cytochemical and molecular studies on two groups of PR proteins, ß-1,3-glucanases and chitinases, and the arabinogalactan proteins (AGP) during the early stages of androgenesis induction in two breeding lines of rye (Secale cereale L.) with different androgenic potential. The process of androgenesis was initiated by tillers pre-treatments with low temperature, mannitol and/or reduced glutathione and resulted in microspores reprogramming and formation of androgenic structures what was associated with high activity of ß-1,3-glucanases and chitinases. Some isoforms of ß-1,3-glucanases, namely several acidic isoforms of about 26 kDa; appeared to be anther specific. Chitinases were well represented but were less variable. RT-qPCR revealed that the cold-responsive chitinase genes Chit1 and Chit2 were expressed at a lower level in the microspores and whole anthers while the cold-responsive Glu2 and Glu3 were not active. The stress pre-treatments modifications promoted the AGP accumulation. An apparent dominance of some AGP epitopes (LM2, JIM4 and JIM14) was detected in the androgenesis-responsive rye line. An abundant JIM13 epitopes in the vesicles and inner cell walls of the microspores and in the cell walls of the anther cell layers appeared to be the most specific for embryogenesis.