Introduction:
Calamine flora, colonizing areas polluted with heavy metals, is an excellent model for studying the microevolution of species/populations, speciation and metallophyte origin, the genetic structure of populations (Hildebrandt et al. 2006; Kuta et al. 2012; Słomka et al. 2011; Wąsowicz et al. 2014; Wierzbicka and Rostański 2002), physiological adaptation to harsh conditions (Słomka et al. 2008), colonization and seed banks (Grodzińska et al. 2001), as well as the biology of reproduction (Bothe & Słomka 2017; Izmaiłow et al. 2015).
Embryological characteristics are important traits to evaluate metallophyte adaptation/tolerance. Impaired sexual reproduction of plants colonizing polluted areas leading to reduced plant fertility and seed set, could be considered as costs of adaptation. The more disturbances in the reproductive processes and degenerations, the lower the tolerance. This can be inferred from published studies on the reproduction of angiosperm’s taxa representing different metallophyte status from Polish post-industrial areas: e.g. Armeria maritima ,Capsella bursa-pastoris , Cardaminopsis arenosa ,Cirsium arvense , Chondrilla luncea , Echium vulgare,Vicia cracca or Viola tricolor and other violets (Czapik 2002; Izmaiłow 2000; Izmaiłow & Biskup 2003; Izmaiłow et al. 2015; Kościńska-Pająk 2000; Kwiatkowska & Izmaiłow 2014; Siuta et al. 2005; Słomka et al. 2012, 2017, 2018).
There is a lack of embryological data on B. laevigata , a facultative metallophyte (plants can grow in metal-enriched soil/substrate and also in unpolluted areas) which is a member of European calamine flora. The species has been widely studied, including its distribution, taxonomic and systematic position, karyotype analysis, ploidy level (diploid vs tetraploid), genetic diversity and genetic structure of European populations (e.g. Manton 1937; Olowokudejo 1992; Tremetsberger et al. 2002).
In Poland (Eastern Europe), B. laevigata (2n=18) (Babs-Kostecka et al. 2014; Skalińska 1950), a glacial relict, occurs in the Tatra Mountains and on over 100-year-old Zn-Pb waste-heap in Bolesław, an isolated location in an anthropogenic, postindustrial site with elevated concentrations of heavy metals. Recently, a new location on a lowland in Central Poland has been discovered (Przemyski & Piwowarczyk 2012). Until now, the studies on Polish populations of B. laevigata have focused on: mycorrhizal status, adaptation and tolerance to heavy metal stress, concentrations of heavy metals in plant organs and the genetic structure of populations (e.g. Antosiewicz 1995; Babst-Kostecka et al. 2014; Godzik 1993; Gołda & Wojciechowska 1979; Orłowska et al. 2002; Wąsowicz et al. 2014; Wierzbicka & Pielichowska 2004; Wierzbicka et al. 2017). B. laevigata was also used for restoriation in post-industrial areas (Rostański 2014).
Plants that live in heavy metal-enriched habitats take metal ions from the rhizosphere and transport them into plant cell/tissue/organs. In cytosol, free metals are harmful as they cause oxidative stress or damage to organelles. To deal with this negative impact, plants have to bind metals and detoxify them. In the evolution of adaptation, plants have developed two main strategies (cell level) that allow them to survive in such extreme conditions: (1) metal accumulation, in some species hyperaccumulation, and (2) metal exclusion (e.g. Baker 1981; Hall 2002; Prasad 2004). The cell wall is the main structure that can be actively modified under internal or external factors/stress and plays a crucial role in defense strategies. In the metal exclusion strategy when metals are taken from the environment, transported through the apoplast, the cell wall is the barrier that stops metals entering the cell symplast. In metal accumulation strategy, metals entering the cell are sequestrated in the cell wall. In particular, homogalacturonan, a major component of the plant primary walls plays a crucial role in the binding and accumulation of heavy metals in the cell wall. However, other compounds, such as other polysaccharides, proteins, amino acids or phenolics, can also take part in metal detoxification (le Gall et al. 2015; Krzesłowska 2011).
The aim of this study was to (1) estimate the frequency of developmental disturbances and degenerations in male and female lineages ofBiscutella laevigata as the costs of adaptation/tolerance to areas polluted with heavy metal; (2) how the un/low- and high-methyl-esterified homogalacturonan (HG) recognized by LM19 and LM20 antibodies, respectively, are distributed in the cell wall of mature embryos as a defense response to toxic metals.