Plants from Bolesław calamine population
In metallicolous population in the male lineage, degenerations of microsporocytes, tetrads of microspores, microspores, tapetum cells (microsporogenesis) and pollen grains at all stages of their development (microgametogenesis) were found in 40% of analyzed flower buds (only a part of anthers were disrupted) leading to decreasing the pollen production by the plant and to reduction of viable pollen grains (87%) (Tab. 1; Fig. 2a-d,).
Developmental disturbances and necrotic processes in megasporogenesis were found only in 2% of analyzed flower buds. During female gametophyte development and at the stage of mature embryo sac, degenerations were observed in 12% of ovules (Tab. 2). They included degenerations of whole female gametophytes at different stages of their development or degenerations of single cells as egg cells, synergids. In some ovaries development of two ovules was not synchronous. Ovules inhibited in their development were eliminated from further development. In 25% of analyzed ovules strong shortening of mature embryo sac was observed, of which almost half degenerated (Fig. 2e, f). In some ovules (7%) female gametophyte were not formed. All these degenerations were usually observed only in one of two ovules in the ovary. In 10% of ovules aging, mature, unfertilized embryo sac started to degenerate (Fig. 2g). Majority of embryos developed regularly according to the Onagrad type, degenerations occurred in 15% of analyzed ovules. They included especially degenerations at young stages (at zygote/proembryo stages). In late stages of embryogenesis only few groups of degenerated cells were observed in the embryo tissues.
Degenerations of whole flower buds were very low (2%), in some pistils a part of stigma cells degenerated (Fig. 2h), degenerations of ovules in flowers and young siliculas (Fig. 2i) were found in 4%.
In 55% of fruits two-seed siliculas developed, one seed in 30%, empty siliculas in 15% of analyzed fruits (Fig. 2i-l; Tab. 3).
Dithizone staining showed the presence (red coloration) of heavy metals in flowers and fruits in specimens from calamine population (Fig. 2m, n).
II. Distribution of un/low- and high-methyl-esterified HG in cell walls
Low-esterified HG, detected by LM19 antibody were distributed in the same pattern in cell walls of embryos in plants from mountain and calamine populations (Fig. 3a-c). The distribution of high-esterified HG, detected by LM20 antibody in cell walls of embryos differed between populations. High-esterified HG were present only in embryos from the calamine population, and their distribution was not continuous in the cell wall. They were identified locally, as spots (Fig. 3d-f).
III. Costs of tolerance measured by reduced plant fertility
In the male lineage, pollen viability was reduced to 87% and the differences between calamine and mountain populations were statistically significant. Also degenerations in anthers during development occurred with higher frequency in calamine population (39% vs 19%) (Tab. 1).
In the female lineage, the frequency of degenerations in flowers, ovules at different stages of female gametophyte development in plants from calamine population exceeded the frequency of these processes in plants from mountain population (14% vs 7%) and the differences were statistically significant (Tab. 2).
The number of seeds in the silicula was variable in both populations. In calamine population formation of normal two-seed siliculas was higher (55%) than in plants from mountain population (41%) and the differences were statistically significant between two populations (Tab. 3).