Discussion:
Analysis of the embryological traits of Biscutella laevigata , a facultative metallophyte, from heap and mountain populations clearly indicates that the costs of tolerance, measured as the success of sexual reproduction, are not high. Plants colonizing the Bolesław calamine heap develop fruits and seeds to an amount allowing the population to be maintained. Plants accumulated metals in their generative organs (flowers), as was demonstrated by histochemical tests, but they developed defense mechanisms, including the biosynthesis of high-esterified homogalacturonans in the cell wall of embryos. The fraction of pectins is probably involved in the reactions of resistance to metals that retain noxious ions in the apoplast of the new generation (embryo). The cell wall, apoplastic compartment, is a well-known extracellular site involved in the detoxification of metal ions harmful to the cell (le Gall et al. 2015; Krzesłowska 2011).
Disturbances in female and male lineages and degeneration processes occurred with higher frequency in plants growing in the calamine population than in the mountain population. Are these changes only a result of the heavy metal presence, or are they also influenced by other factors? On post-industrial areas, secondary enriched with heavy metals by industrial and mining activities, other factors might influence plant physiological processes and reproduction, including: very low nutrient content, skeletal soil structure with low water retention, strong insolation and eolian erosion resulting from strong winds in open dump sites. To survive these harsh environmental conditions, plants must overcome heavy metal, drought, low/high temperatures, nutrient and salt stresses. Species spontaneously colonizing contaminated sites undergo micro-evolutionary processes, which enable the survival of individuals adapted to local conditions and the beneficial traits / genes to be passed on to their offspring (Wierzbicka & Rostański 2002; Wierzbicka et al. 2017).
The frequency of disturbances and degenerations in male and female lineages are good indicators of the costs of tolerance/adaptation (Izmaiłow et al. 2015). The flowering plant life cycle is divided into haploid and diploid generations. The haploid phase, extremely reduced and consisting of only a few cells, is dependent on the sporophytic generation for nutrition and protection (Ge et al. 2010). All processes of sexual reproduction (male and female meiosis, gametophyte development, double fertilization and embryo development) are not only dependent on genotype or species, but also on external conditions. The taxon in an early stage of colonization is poorly adapted to environmental stress and disturbances, with degenerations in particular being much more frequent than in metallophytes, which are better adapted to harsh conditions. However, several traits related to the biology of reproduction are conservative, regardless of the environmental factors, e.g. type of cytokinesis, pattern of embryo sac development, embryogenesis model or type of seeds (Izmaiłow et al. 2015; Siwek 2007). From the research conducted on B. laevigata, it follows that the simultaneous type of cytokinesis in anthers, three-celled pollen grains, a monosporic embryo sac developing according to the Polygonum type, the Onagrad type of embryo development, nucellar endosperm, and additional embryo sacs in an ovule, were conservative and the same in plants from the mountain and calamine populations. Necrosis and degenerations found in male and female lineages of B. laevigata from the calamine population could be a result of the same evolutionary strategies that were observed in Armeria maritima s.l., another metallophyte of calamine flora (author’s observations, unpublished). These processes reduced the pollen viability and seed set. This was also reflected in the frequency of one-seed siliculas. Selective abortion of seeds is a well-known phenomenon in flowering plants. Developing embryos compete and the weakest are aborted. Moreover, the mother plant may set the level of abortion. The abortion of viable embryos leads to higher offspring quality (de Jong & Klinghamer 2005). We observed in B. laevigata from the calamine population that some gametophytes (female or male) were also excluded from further development and this could lead to better quality of offspring. The presence of unfertilized mature and properly developed female gametophytes at a stage of aging indicates that the reduced fertility of B. laevigata , a self-incompatible species (Leducq et al. 2010; Young et al. 2012), might also result from a lack or insufficient number of pollinators.
Quantitative analysis of metals in specimens from metallicolous populations indicates that they are present in generative organs (flowers and fruits), although metals are generally at a much lower level than in vegetative tissues (e.g. Dhiman et al. 2017; Godzik 1993; Mesjasz-Przybyłowicz et al. 2001). In plants from metallicolous sites, metal uptake, distribution and deposition depend on the strategy that plants have evolved. Specimens could accumulate (even hyperaccumulate) metals in the aerial part or exclude them. Metals are quickly detoxified by binding with many molecules (e.g. phytochelatines), localized in vacuoles, sequestered in cell walls, deposited in aging leaves or in trichomes, for the protection of organelles and biochemical reactions against free toxic heavy metal ions in cytosol (Antonovics 1971; Ernst et al. 1992; Prasad 2004).
The reproductive processes depend on how the plant fights ionic stress and other extreme conditions, how much energy and resources it devotes to it and how much energy remains to produce flowers, fruits and seeds. Metal ions, along with water, mineral salts and assimilates, are transported to generative structures, where they might directly affect the haploid cells of gametophyte phase or embryos, even when they are present in small doses (Kranner and Colville 2011; Sperotto et al. 2014; Walker & Waters 2011). The gametophyte phase of ontogenesis is more sensitive to abiotic stress (metal, salt, hot/cold temperature) than the sporophyte, therefore adaptations develop first on the sporophyte level, and then in the cells of the germlines (Hedhly 2011; Kazan and Lyons 2016; Kwiatkowska & Izmaiłow 2014; Słomka et al. 2012; Sun et al. 2004; Zinn et al. 2010).