Influence of bisphenol A on growth and metabolism of Vicia faba ssp. minor seedlings depending on lighting conditions

The number of necrotic seedlings, chlorophyll fluorescence, photosynthetic activities and necrotic areas in leaf discs

Macroscopic analyses showed that BPA damaged seedlings under both DK (Supplementary Fig. S1A) and DK/LT (Supplementary Fig. S1B) conditions.

Chl fluorescence (R.F.I.Ch.) in the leaf discs of Ctrl cultivated for 72 h in DK (Supplementary Fig. S2A) and DK/LT (Supplementary Fig. S2D) measured using the images (Supplementary Fig. S2A′,D′) under blue LT were approximately 200 a.u. (Fig. 1). Under the same conditions, this parameter was reduced (p ≤ 0.05) by approximately 12.5% and 25% (Fig. 1) with 30 mg L⁻¹ BPA (Supplementary Fig. S2B′) and 120 mg L⁻¹ BPA (Supplementary Fig. S2C′) treatment, respectively. However, in DK/LT (Supplementary Fig. S2E′,F′), the R.F.I.Ch. values were similar (p > 0.05) (average 175 a.u.) compared to the Ctrl (Fig. 1).

Red fluorescence intensity of chlorophyll (R.F.I.Ch.) in 1-cm-leaf discs of 2-month-old V. faba ssp. minor cultured for 72 h in the darkness or in the darkness/light under Ctrl (0 mg BPA) conditions or with 30 mg and 120 mg L⁻¹ BPA. Bars indicate ± SE of the results from three biological replicates. Identically labelled columns indicate results that are not significantly different at p ≤ 0.05 for darkness and darkness/light groups, separately.

In the stems in the DK/LT in Ctrl, 30 and 120 mg L⁻¹ BPA, the values obtained for F_v/F_m PSII quantum yield (Fig. 2A), steady-state PSII quantum yield (QY; Fig. 2B), steady-state nonphotochemical quenching (NPQ; Fig. 2C) and photochemical quenching (qP; Fig. 2D) were similar. Only the empirical parameter, i.e., R_fd, used to assess plant vitality differed (Fig. 2E). There were similar R_fd values for 30 mg L⁻¹ BPA and Ctrl and reduced in 120 mg L⁻¹ BPA (p ≤ 0.05) by approximately 25% (p ≤ 0.05).

The values of chlorophyll a (photosystem II, PSII) fluorescence parameter of V. faba ssp. minor seedlings cultured under Ctrl (0 mg BPA) conditions or with 30 or 120 mg L⁻¹ BPA darkness/light for 72 h. Ratio of variable to maximum fluorescence—the quantum efficiency of open PSII centers, (Fv/Fm, A); maximum fluorescence yield, quantum yield in light-adapted steady-state, (Fm); steady-state PSII quantum yield in light (QY, B); steady-state non-photochemical quenching in light, (NPQ, C); photochemical quenching, (qP, D); empiric parameter used to assess plant vitality (R_fd, E). Identically labelled columns indicate results that are not significantly different at p ≤ 0.05.

Upon treatment with 30 mg L⁻¹ BPA, 13% and 7% of seedlings under DK and DK/LT conditions, respectively, were necrotic, similar results were observed in the Ctrl group (9% and 10%, respectively). Upon treatment with 120 mg L⁻¹ BPA, the number of necrotic seedlings was on average six and fivefold increased (p ≤ 0.05) under DK and DK/LT conditions, respectively, compared to both Ctrl and 30 mg L⁻¹ BPA (Fig. 3A). Thus, in the 120 mg L⁻¹ BPA series, necrotic areas were identified in the arbitrarily distinguished apical, subapical, subbasal and basal zones of stems (Supplementary Fig. S3A, Is′–IVs′; Supplementary Fig. S4A, Is′–IVs′) and roots (Supplementary Fig. S3A, Ir′–IVr′; Supplementary Fig. S4A, Ir′–IVr′) of seedlings compared with the Ctrl series (Supplementary Fig. S3A, Is–IVs and Supplementary Fig. S4A, Is–IVs; Supplementary Fig. S4A, Ir–IVr and Supplementary Fig. S4A, Ir–IVr, respectively). Under both DK (Supplementary Fig. S3) and DK/LT (Supplementary Fig. S4) conditions, dark spots on stems (Supplementary Fig. S4A, Is′–IIIs′; Supplementary Fig. S4A, Is′, IIs′ and IVs′) and roots (Supplementary Fig. S3A, IIr′, IIIr′; Supplementary Fig. S4A, Ir′, IIIr′, IVr′) and injuries resembling mechanical damage (Supplementary Fig. S3A, IVs′ and Supplementary Fig. S4A, IIIs′; Supplementary Fig. S3A, Ir′, IVr′; Supplementary Fig. S4A, IIr′) were observed (p ≤ 0.05).

Percentage of necrotic seedlings (A) and necrotic areas in leaf discs of 2-month-old plants (B) of V. faba ssp. minor cultured for 72 h in darkness or darkness/light under Ctrl (0 mg BPA) conditions or with 30 or 120 mg L⁻¹ BPA. Identically labelled columns indicate results that are not significantly different at p ≤ 0.05 for darkness and darkness/light groups, separately.

Necrotic changes were noted in approximately 30% and 20% (Fig. 3B) area of the leaf discs (Supplementary Fig. S2A–F) in the DK (Supplementary Fig. S2B; Fig. 3B) and DK/LT (Supplementary Fig. S2E; Fig. 3B), groups respectively. These levels were increased (p ≤ 0.05) by approximately 28% and 14%, respectively, upon 30 mg L⁻¹ BPA treatment compared with Ctrl (Supplementary Fig. S2A; Fig. 3B). Treatment with 120 mg L⁻¹ BPA enhanced the necrotic areas by approximately 40 and 240% (p ≤ 0.05) in the DK (Supplementary Fig. S2C) and DK/LT (Supplementary Fig. S2F) groups, respectively, compared to Ctrl.

Cell viability in roots and stems of seedlings

Analyses of the viability of particular cells in roots and stems revealed three different cell populations (Supplementary Fig. S5a–f): cells with green nuclei, representing 95% living cells (Supplementary Fig. S5A,B); cells with orange-red nuclei, representing dying-dead cells and dead cells with damaged and fragmented nuclei (Supplementary Fig. S5C,D); and cells without nuclei (Supplementary Fig. S5E,F). The latter population was observed in the apical, subapical, subbasal and basal necrotic zones in roots and stems (Supplementary Figs. S1A,B; S3 and S4). The observations also showed that the living and necrotic regions in roots and stems were clearly separated (Supplementary Fig. S5F).

Length, volume, fresh and dry mass of root and stem seedlings

BPA influenced the length, volume, fresh and dry weights of roots and stems (Supplementary Fig. S1A,B; Fig. 4A–D) of V. faba ssp. minor seedlings cultured under DK and DK/LT conditions.

Length (A), volume (B) as well as fresh (C) and dry (D) weight of whole roots and stems of V. faba ssp. minor seedlings cultured for 72 h in the darkness and in the light under Ctrl (0 mg BPA) conditions or with 30 or 120 mg BPA L⁻¹. Identically labelled columns indicate results that are not significantly different at p ≤ 0.05 for darkness and darkness/light groups, separately.

The lengths of roots in the Ctrl and 30 mg L⁻¹ BPA treatments were similar (p > 0.05) in both the DK and DK/LT groups and were on average 6.5 cm and 5.5 cm, respectively. However, upon exposure to 120 mg L⁻¹ BPA, the roots in the DK and DK/LT treatments were reduced by approximately 30% (p ≤ 0.05) compared to those in the other treatments. The lengths of stems in the Ctrl and 30 mg L⁻¹ BPA groups were also similar (p > 0.05) in both the DK and DK/LT groups and were on average 7.5 cm and 2.5 cm, respectively. However, upon exposure to 120 mg L⁻¹ BPA, the stems in both the DK and DK/LT groups were reduced by approximately 25% (p ≤ 0.05) compared to those in the other groups. Regardless of BPA variants, the average length of the stems in the DK was threefold greater (p ≤ 0.05) than that in the DK/LT (Fig. 4A).

The volumes of roots in the DK groups exposed to 30 and 120 L⁻¹ mg BPA were on average 300 mm³ and 20% reduced (p ≤ 0.05) compared with those in the Ctrl. However, in the DK/LT groups, the volumes of roots were similar to those in the Ctrl (p > 0.05) and were on average 600 mm³ and twofold increased (p ≤ 0.05) than those cultivated in the DK. The volumes of stems in the DK groups treated with 30 and 120 mg L⁻¹ BPA were similar (p > 0.05) and were on average 320 mm³ and 20% reduced (p ≤ 0.05) compared to Ctrl. In contrast, the volumes of stems in the DK/LT exposed to the same conditions were on average 240 mm³ and approximately 60% reduced (p ≤ 0.05) compared to Ctrl (Fig. 4B).

The FW of roots in DK in 120 mg L⁻¹ BPA was approximately 210 mg and by approximately 20% (p ≤ 0.05) reduced, compared to Ctrl plants and those exposed to 30 mg L⁻¹ BPA. In the DK/LT group, the FWs in all variants were similar (p > 0.05) and were 270 mg on average. The FWs of stems in the DK and DK/LT groups exposed to 30 and 120 mg L⁻¹ BPA were similar (p > 0.05) and were 340 mg and 220 mg on average, respectively. These values were also reduced (p ≤ 0.05) by approximately 15% and 10%, respectively, compared to Ctrl (Fig. 4C).

DWs of roots in the DK in all series were similar (p > 0.05) at 15 mg on average. In the DK/LT groups exposed to Ctrl and 30 mg L⁻¹ BPA, similar (p > 0.05) values were also obtained, on average 18 mg. However, these values were increased by approximately 20% (p ≤ 0.05) compared to the plants exposed to 120 mg L⁻¹ BPA. The DWs of stems in the DK in all series were similar (p > 0.05), 25 mg on average. In the DK/LT groups, the DWs of the plants exposed to 30 and 120 mg L⁻¹ BPA were also similar, 15.5 mg on average, and these values were reduced (p ≤ 0.05) by approximately 30% compared to Ctrl (Fig. 4D).

Phenol and peroxide levels in plant materials and treatment solutions

The amount of total phenols, which is expressed as the amount of gallic acid as a marker, increased (p ≤ 0.05) from approximately 235 and 50 µg mL⁻¹ in the Ctrl group to approximately 330 and 90 µg mL⁻¹ with 120 mg L⁻¹ BPA treatment in one-third of roots in the DK and DK/LT groups, respectively. Under the same conditions, the level of total phenols in stems decreased (p ≤ 0.05) from approximately 340 and 350 µg mL⁻¹ in the Ctrl group to approximately 270 and 170 µg mL⁻¹ with 120 mg L⁻¹ BPA treatment in the DK and DK/LT groups, respectively (Fig. 5A). In treatment solution from the DK group, the amount of phenols increased (p ≤ 0.05) from approximately 25 µg mL⁻¹ with Ctrl and 30 mg L⁻¹ BPA treatments to approximately 55 µg mL⁻¹ with 120 mg L⁻¹ BPA treatment. However, in the DK/LT group, the amount of phenols increased (p ≤ 0.05) from approximately 55 µg mL⁻¹ with Ctrl and 30 mg L⁻¹ BPA treatments to approximately 65 µg mL⁻¹ with 120 mg L⁻¹ BPA treatment (Fig. 5B).

Amounts polyphenols (A,B) and hydrogen peroxide (C,D) in the one-third of apical parts of roots and stems (A,C) and in the treatment solutions (B,D) of V. faba ssp. minor seedlings cultured in the darkness and in the darkness/light under Ctrl (0 mg BPA) conditions or with 30 and 120 mg L⁻¹ BPA for 72 h. Identically labelled columns indicate results that are not significantly different at p ≤ 0.05 for darkness and darkness/light groups, separately.

Measurements of peroxides (PXs) calculated based on hydrogen peroxide showed similar amounts per gram of FW in Ctrl roots and those cultured with 30 mg L⁻¹ BPA in both lighting conditions (p > 0.05; approximately 23 µg g⁻¹ FW). However, upon treatment with 120 mg L⁻¹ BPA, the levels increased (p ≤ 0.05) by 45% and 65% in the DK and DK/LT groups, respectively. In stems of plants in the DK group under Ctrl conditions, approximately 23 µg g⁻¹ FW PX was observed. PX levels were reduced (p ≤ 0.05) by approximately 20% and 40% respectively, upon treatment with 30 and 120 mg L⁻¹ BPA. However, in the DK/LT conditions, the amount of PXs in Ctrl was approximately 8.5 µg g⁻¹ FW, and PX levels were reduced (p ≤ 0.05) by approximately 20% and 40%, respectively, in the other two series. Moreover, PX levels in the latter series were on average threefold reduced (p ≤ 0.05) compared with those in the Ctrl group (Fig. 5C). In treatment solution from the DK group, the amount of PXs increased (p ≤ 0.05) from approximately 2 µg mL⁻¹ with Ctrl by approximately 15% and 30% upon treatment with 30 and 120 mg L⁻¹ BPA treatment. However, in the DK/LT group, the amount of PXs in Ctrl, 30 and 20 mg L⁻¹ BPA treatments were similar (p > 0.05) and were approximately 2 µg mL⁻¹ (Fig. 5D).

Quinone levels in plant materials and treatment solutions

The amount of 1,4-BQ, one of five types of the studied QSs, in roots decreased both in 30 mg L⁻¹ BPA and in 120 mg L⁻¹ BPA variants. In the former in the DK and DK/LT it decreased by approximately 30% and 20%, respectively and in the latter by approximately 70% and 40% compared to Ctrl conditions, where the amounts were about 34 and 24 µg g⁻¹ FW.

The amount of 1,4-BQ in stems in the DK and DK/LT groups increased (p ≤ 0.05) from approximately 4 and 15 µg g⁻¹ FW, respectively, with Ctrl treatment to approximately 80% and 40%, respectively, with 30 mg L⁻¹ BPA treatment. The amount of 1,4-BQ in stems treated with 120 mg L⁻¹ BPA did not increase in the DK group and increased in DK/LT by approximately 65% in the DK/LT group. In the treatment solutions, in the Ctrl and 30 mg L⁻¹ BPA treatment in the DK group, approximately 3.5 µg g⁻¹ FW of 1,4-BQ was noted. While in 120 mg L⁻¹ BPA treatment its level increased (p ≤ 0.05) by approximately 25% (Fig. 6A,A′).

Amounts of quinones 1,4-bezoquinone (A,A′), tetrachloro-1,4-benzoquinone (B,B′), tetrabromo-1,4-benzoquinone (C,C′), 1,4-naphthoquinone (D,D′) and 1,2-naphtohoquinone-4-sulfonic acid (E,E′) in one-third of the apical parts of roots and stems (A–E) and the treatment solutions (A′–E′) of V. faba ssp. minor seedlings cultured in the dark and light under Ctrl (0 mg BPA) conditions or with 30 and 120 mg L⁻¹ BPA for 72 h. Identically labelled columns indicate results that are not significantly different at p ≤ 0.05 for roots and stems (A–E) as well as for the darkness and darkness/light groups (A–E′), separately.

The amount of TCh-1,4-BQ in roots in DK was not affected by BPA and was on average 75 µg g⁻¹ FW. In the DK/LT group, the amount of TCh-1,4-BQ decreased (p ≤ 0.05) by 60% and 180% upon exposure to 30 and 120 mg L⁻¹ BPA compared with the approximate value of 230 µg g⁻¹ FW observed under Ctrl conditions. In stems in the DK group, the amount of TCh-1,4-BQ was reduced (p ≤ 0.05) by 200% upon exposure to 30 mg L⁻¹ BPA, but the result was not statistically significant (p > 0.05). In addition, exposure to 120 mg L⁻¹ BPA resulted in a 75% reduction in TCh-1,4-BQ levels. In the DK/LT group, the amount of TCh-1,4-BQ decreased (p ≤ 0.05) by approximately 100% upon exposure to 120 mg L⁻¹ BPA from the baseline value of approximately 140 µg g⁻¹ FW under Ctrl conditions (Fig. 6B,B′).

The amount of TB-1,4-BQ in roots in the DK group increased (p ≤ 0.05) by approximately 20% and approximately 50% in plants exposed to 30 mg L⁻¹ and 120 mg L⁻¹ BPA, respectively, compared to Ctrl. In the DK/LT group, the amount of TB-1,4-BQ fluctuated depending on the BPA concentration and decreased from approximately 260 µg g⁻¹ FW upon Ctrl treatment by approximately 50% and 55% in plants exposed to 30 and 120 mg L⁻¹ BPA, respectively. In the stems in the DK group, the amount of TB-1,4-BQ was similar among all treatments (average 106 µg g⁻¹ FW). However, in the DK/LT group, the amount of this quinone was reduced (p ≤ 0.05) by approximately 40% upon treatment with 30 and 120 mg L⁻¹ BPA. In the treatment solution, the amount of TB-1,4-BQ in the DK group treated with 30 mg L⁻¹ and 120 mg L⁻¹ BPA increased (p ≤ 0.05) by approximately 45% and 85%, respectively, compared to Ctrl (Fig. 6C,C′).

The amount of 1,4-NQ in roots in the DK group exposed to Ctrl conditions was approximately 19.5 mg⁻¹ FW. Upon exposure to 30 and 120 mg L⁻¹ BPA, the levels were reduced (p ≤ 0.05) by approximately 155% and 46% compared to Ctrl, respectively. In the DK/LT group, approximately 2 mg⁻¹ FW of 1,4-NQ was observed. Upon treatment with 30 BPA mg L⁻¹, similar (p > 0.05) levels were observed, whereas the levels were reduced (p ≤ 0.05) on average by 150% upon treatment with 120 mg L⁻¹ BPA compared to Ctrl. In the stems in the DK group, the amount of 1,4-NQ was increased (p ≤ 0.05) by approximately 150% and by 80% in plants exposed to 30 and 120 mg L⁻¹ BPA, respectively, compared to Ctrl (Fig. 6D).

In the treatment solutions, 1,4-NQ levels (approximately 2 mg⁻¹ FW) were similar (p > 0.05) in the DK groups exposed to 30 mg L⁻¹ BPA or Ctrl conditions. However, upon treatment with120 mg L⁻¹ BPA, these levels increased (p ≤ 0.05) by approximately 50%. In the DK/LT group, similar levels of this QS were noted upon exposure to 30 and 120 mg L⁻¹ BPA (p > 0.05). On average, these values were reduced by approximately 40% compared to Ctrl (7 mg⁻¹ FW; Fig. 6D′).

The levels of 1,2-NQ-4-SA in roots in the DK group were similar with all treatments (average 17 mg⁻¹ FW). However, in the DK/LT group, approximately 50 mg⁻¹ FW of this QS was recorded under Ctrl conditions. Upon exposure to 30 and 120 mg g L⁻¹ BPA, similar levels of 1,2-NQ-4-SA (p > 0.05) (average 30 mg⁻¹ FW) were observed. However, these levels were reduced (p ≤ 0.05) by approximately 60% compared to Ctrl. In stems in the DK group, the amounts of 1,2-NQ-4-SA were similar (average 19 µg g⁻¹ FW). However, in the DK/LT group exposed to Ctrl and 120 mg L⁻¹ BPA treatment, similar values (60 µg g⁻¹ FW) were obtained. Moreover, these values were increased (p ≤ 0.05) by approximately 35% compared to treatment with 30 mg BPA mg L⁻¹ BPA (Fig. 6E).

In the treatment solution, similar 1,2-NQ-4-SA levels were noted in the DK group exposed to Ctrl and 30 mg L⁻¹ BPA (average 4 µg g⁻¹ FW). However, upon exposure to 120 mg L⁻¹ BPA, these levels increased (p ≤ 0.05) by approximately 70%. In the DK/LT group exposed to Ctrl and 30 mg L⁻¹ BPA, similar, 2-NQ-4-SA levels were noted (average 3 µg g⁻¹ FW). However, upon exposure to 120 mg L⁻¹ BPA, the levels increased (p ≤ 0.05) by approximately 100% (Fig. 6E′).

Sugar and protein levels in plant material

The levels of soluble sugars (SOS; Fig. 7A) in one-third of each apical part of roots in the Ctrl in the DK and DK/LT were approximately 20 mg and 11 mg g⁻¹ FW, respectively, and the difference was significant (p ≤ 0.05). Upon treatment with 30 and 120 mg L⁻¹ BPA, SOS levels were reduced (p ≤ 0.05) by approximately 40% and approximately 60%, respectively, in the DK group and by approximately 25% and approximately 50% (p ≤ 0.05), respectively, in the DK/LT group compared to Ctrl. However, average SOS levels in roots in the DK group were 14.5 mg g⁻¹ FW after exposure to BPA, and these levels were approximately twofold greater (p ≤ 0.05) than those in the DK/LT. SOS levels in one-third of each of the apical parts of stems in the DK and DK/LT groups exposed to Ctrl conditions were approximately 12 mg and 5 mg g⁻¹ FW, respectively. The difference was significant (p ≤ 0.05). In the DK group, upon treatment with 30 and 120 mg L⁻¹ BPA, SOS levels were increased by approximately 40% (p ≤ 0.05) and reduced (p ≤ 0.05) by approximately 10%, respectively. However, in the DK/LT group, similar SOS levels in response to treatment with 30 and 120 mg L⁻¹ BPA were noted compared with Ctrl conditions. However, on average, 13.0 mg g⁻¹ FW SOS was noted in the stems of the DK group, and this value was twofold greater (p ≤ 0.05) that that noted in the DK/LT group.

Amounts of soluble (A), storage (B), cell wall-bound (C) sugars and proteins (D) in one-third of the apical parts of roots and stems of V. faba ssp. minor seedlings cultured for 72 h in darkness or darkness/light under Ctrl (0 mg BPA) conditions or with 30 and 120 mg L⁻¹ BPA. Identically labelled columns indicate results that are not significantly different at p ≤ 0.05, separately.

The levels of storage sugars (STS; Fig. 7B) in one-third of each apical part of roots in the Ctrl and 30 mg L⁻¹ BPA treatments in both the DK and DK/LT groups were similar at 3.5 mg g⁻¹ FW (p > 0.05) on average. However, upon treatment with 120 mg L⁻¹ BPA in the DK group, this parameter was increased by approximately 25%. However, in the DK/LT group, this parameter was reduced (p ≤ 0.05) by 50% compared to Ctrl and 30 mg L⁻¹ BPA treatments. The levels of STSs in one-third of each apical part of stems subject to Ctrl and 30 mg L⁻¹ BPA treatment both in the DK and DK/LT groups were similar at 4 mg g⁻¹ FW on average. However, upon treatment with 120 mg L⁻¹ BPA in the DK group, this parameter was increased (p ≤ 0.05) by approximately 20%. However, in the DK/LT group, this parameter was reduced (p ≤ 0.05) by approximately 20% compared to Ctrl and 30 mg L⁻¹ BPA treatments in both the DK and DK/LT groups.

The amounts of cell wall-bound sugar (CWS; Fig. 7C) in one-third of each apical part of roots in the DK and DK/LT groups subject to Ctrl conditions were approximately 3.5 mg and 6 mg g⁻¹ FW, respectively. The difference was significant (p ≤ 0.05). Similar CWS levels were noted between 30 mg L⁻¹ BPA and Ctrl in the DK group were noted (p > 0.05). However, in the DK/LT group, CWS levels were reduced (p ≤ 0.05) by approximately 50% compared to those in the Ctrl in the DK and DK/LT groups, respectively. CWS levels (Fig. 7C) in one-third of each apical part of stems in Ctrl in the DK and DK/LT groups were approximately 3.0 and 4.5 mg g⁻¹ FW, respectively. The difference was significant (p ≤ 0.05). CWS levels in the DK and DK/LT groups exposed to 30 mg L⁻¹ BPA treatment were similar to those noted induced with 120 L⁻¹ mg BPA and Ctrl treatments (p > 0.05).

The amounts of total proteins in one-third of each apical part of roots in Ctrl in the DK and DK/LT were approximately 13 mg and 11 mg g⁻¹ FW, respectively, and the difference was not significant (p > 0.05). The protein levels in both the 30 and 120 mg L⁻¹ BPA groups were reduced (p ≤ 0.05) by approximately 30% compared to Ctrl in both the DK and DK/LT groups. On average, protein levels in the DK and DK/LT groups were similar (p > 0.05). The total amounts of proteins in one-third of each apical part of stems in the DK and DK/LT groups with Ctrl treatment were approximately 10 and 16 mg g⁻¹ FW, respectively, and the difference was significant (p ≤ 0.05). Protein levels in the DK and DK/LT groups exposed to 30 and 120 mg L⁻¹ BPA treatment were similar (p > 0.05) to Ctrl. Protein levels in stems in both BPA treatments in the DK group were reduced (p ≤ 0.05) by approximately 30% compared to Ctrl.

BPA levels in culture solutions and plant materials

In the DK and DK/LT groups exposed to Ctrl conditions, approximately 450 µg and 530 µg BPA was present in the remaining culture solutions, respectively (Fig. 8A). BPA levels were increased (p ≤ 0.05) by approximately 50% upon exposure to 30 mg L⁻¹ BPA in both the DK and DK/LT groups. However, upon exposure to 120 mg L⁻¹ BPA, BPA levels were increased by approximately 120% and approximately 60% (p ≤ 0.05) in the DK and DK/LT groups, respectively, compared to Ctrl. When BPA levels induced by Ctrl conditions in the DK group were subtracted from the data obtained upon exposure to 30 and 120 mg L⁻¹ BPA, the BPA levels were approximately 420 µg and 1600 µg, respectively. In the DK/LT group, the levels were approximately 420 µg and 1000 µg, respectively. Approximately 750 µg and 3000 µg of BPA were added to the culture solutions upon treatment with 30 and 120 mg L⁻¹, respectively. After subtracting the amounts of BPA determined in the culture solutions upon treatment, in the DK groups with 30 and 120 mg L⁻¹ BPA treatment, approximately 50% of BPA disappeared. However, in the DK/LT groups treated with 30 and 120 mg L⁻¹ BPA, approximately 50% and approximately 70% of BPA, respectively, disappeared.

Amount of BPA in the treatment solutions (A) as well as in one-third of the apical parts of in roots and stems (B) of the V. faba ssp. minor seedlings cultured for 72 h in the darkness and in the darkness/light under Ctrl (0 mg) conditions or with 30 and 120 mg L⁻¹ BPA. Identically labelled columns indicate results that are not significantly different at p ≤ 0.05 for darkness and darkness/light groups, separately.

In the roots and stems of the DK and DK/LT groups subject to Ctrl and 30 mg L⁻¹ BPA treatment, the amounts of BPA were similar and were approximately 2.5 µg g⁻¹ FW on average. Upon treatment with 120 mg L⁻¹ BPA, BPA levels were increased fivefold compared with (p ≤ 0.05) that in the other two BPA treatments (Fig. 8B). In the DK/LT group exposed to Ctrl conditions, BPA was not found in roots or stems. Upon treatment with 30 mg L⁻¹ BPA, BPA levels were approximately 25 µg and 15 µg g⁻¹ FW, respectively. However, upon 120 mg L⁻¹ BPA treatment, these levels were two- and threefold reduced (p ≤ 0.05) in roots and stems, respectively, compared to 30 mg L⁻¹ BPA (Fig. 8B).

Correlation analysis

Pearson’s correlations analyses between increasing BPA concentrations versus length, volume, FW and DW of roots and stems as well as versus amounts of sugars and proteins, versus Chlls, versus necrosis and phenols, versus peroxides and gallic acids, versus necrosis and peroxides, versus peroxides and endogenous amounts of BPA, versus amount of quinones, necrosis, versus phenols and quinones of seedlings cultured in the DK and DK/LT were performed.

To facilitate identification of correlative patterns, we conducted a systematic analysis of Pearson’s correlation coefficients between some of the quantitative readouts. The results were visualised using the conditional formatting of MS Excel (Fig. 9A–D).

(A) Pearson correlation coefficients (r_x,y) for increasing concentrations of BPA versus length, volume, fresh weight (FW), dry weight (DW) of roots and stems, and amounts of soluble, storage and cell wall-bounded sugars and proteins in roots and stems in the dark (DK) and the dark/light (DK/LT) groups. (B) Pearson correlation coefficients (r_x,y) for increasing concentrations of BPA verus fluorescence of Chlls in leaf discs; for number of necrotic seedlings versus phenols from roots, stems and treatment solutions; for peroxides from roots, stems and treatment solutions versus gallic acid from roots, stems and treatment solutions;. In the darkness (DK) and in the darkness/light (DK/LT) groups. (C) Pearson correlation coefficients (r_x,y) for increasing concentrations of BPA versus number of necrotic seedlings (necrosis), the amount of peroxides in roots, stems and treatment solutions as well as for amounts of peroxides in roots, stems and treatment solutions versus numbers of necrotic seedlings. In the darkness (DK) and in the darkness/light (DK/LT) groups. (D) Pearson correlation coefficients (r_x,y) for increasing concentrations of BPA versus the amount of phenols (gallic acid); for applied increasing concentrations of BPA versus endogenous BPA; for applied increasing concentrations of BPA versus quinones in roots, stems and treatment solutions; for amounts of gallic acid in roots, stems and treatment solutions versus quinones in roots, stems and treatment solutions; for endogenous (End) BPA in roots and stems and treatment solutions versus quinones in roots and stems and treatment solutions. In the darkness (DK) and in the darkness/light (DK/LT) groups.

Increasing concentrations of BPA (0, 30 and 120 mg L⁻¹) versus length, volumes, FW and DW of roots and stems of the seedlings cultured in the DK and DK/LT were negatively statistically significant (values lower than 0.4). Similar correlations were observed in roots of the seedlings cultured in the DK, versus soluble, cell wall-bounded sugars and proteins and in the DK/LT, versus the amounts of storage, cell wall-bounded sugars and proteins. In roots in the DK and stems in the DK/LT versus storage sugars and in the DK versus proteins the correlations were positively statistically significant (values greater than 0.4). In the other cases of these groups the correlations were statistically not significant (Fig. 9A).

In leaf discs, the Pearson’s correlation coefficients (r_x,y) for increasing concentrations of BPA versus Chlls fluorescence in the DK were negatively statistically significant while in the DK/LT were not significant. The relationships of number of necrotic seedlings cultured in DK and DK/LT in increasing concentration of BPA versus amount of phenols in roots and treatment solution or in stems were respectively positively and negatively significant. The similar correlations were observed in roots and stems in the DK and DK/LT and in the treatment solutions in the DK peroxides versus gallic acid. In the DK/LT the correlations between peroxides and gallic were not significant (Fig. 9B).

In roots, Pearson’s correlation coefficients (r_x,y) for increasing concentrations of BPA versus number of necrotic seedlings and amount of peroxides in the DK and DK/LT and amount of peroxides in treatment solution in the DK were statistically significant, while in the DK/LT were statistically not significant, however in stems in the DK and DK/LT the relationships versus peroxides were negatively statistically significant. In roots in the DK and DK/LT and treatment solution in the DK/LT, the relationships of the amounts of peroxides versus number of necrotic seedlings were positively significant. For stems the same relationships were negatively significant (Fig. 9C).

In roots and treatment solution or in stems in the DK and DK/LT, the Pearson’s correlation coefficients (r_x,y) for increasing concentrations of BPA versus gallic acid were respectively positively and negatively significant. In roots and stems in the DK and in treatment solution in the DK and DK/LT versus endogenous BPA the relationships were positively significant, while versus roots and stems were negatively significant (Fig. 9D).

In roots, Pearson’s correlation coefficients of seedlings growing in increasing concentrations of BPA versus amount of quinones such as 1,4-BQ and TCh-1,4-BQ in the DK as well as 1,4-BQ, TCh-1,4-BQ, TB-1,4-BQ and 1,4-NQ in the DK/LT were negatively significant. In other cases of these groups the relationships were not significant. In stems in the DK versus 1,4-BQ, TCh-1,4-BQ, TB-1,4-BQ and 1,2-NQ-4-SA and in stems in the DK/LT versus 1,4-BQ and 1,4-NQ the relations were positively significant while versus TCh-1,4-BQ and TB-1,4-BQ the relations were negatively significant. In the other cases in these groups the relationships were statistically not significant (Fig. 9D). In treatment solutions in the DK, the relationships of increasing concentrations of BPA versus amounts of quinones such as 1,4-BQ and in the DK/LT versus TB-1,4-BQ and 1,4-NQ were negatively significant while in DK versus amounts of TCh-1,4-BQ, TB-1,4-BQ and 2-NQ-4-SA and in the DK/LT versus amounts of 1,2-NQ-4-SA the relationships were positively significant. In the other cases in these groups the relationships were statistically not significant (Fig. 9D).

In roots of seedlings growing in increasing concentrations of BPA in the DK the relationships between amounts of gallic acid versus the amounts of quinones such as 1,4-BQ and 1,4-NQ, and in the DK/LT versus all studied quinones were negatively significant while in DK versus TB-1,4-BQ the relationships were positively significant. In the other cases in these groups the relationships were statistically not significant. In stems of seedlings growing in increasing concentrations of BPA in the DK the relationships between the amount of gallic acid versus amount of all studied quinones and in the DK/LT versus amounts of 1,4-BQ and 1,4-NQ were negatively significant while in DK/LT versus TB-1,4-BQ and TB-1,4-BQ were positively significant. In the other cases in these groups the relationships were statistically not significant (Fig. 9D).

In roots of seedlings growing in increasing concentrations of BPA in the DK the relationships between amount of gallic acid versus the amount of all studied quinones in treatment solution and in the DK/LT versus TCh-1,4-BQ, TB-1,4-BQ and 1,2-NQ-4-SA were positively significant while versus 1,4-BQ and 1,4-NQ were negatively significant. In stems in the DK the relationships between amount of gallic acid versus the amount of 1,4-BQ, TCh-1,4-BQ, TB-1,4-BQ and 1,4-NQ and in the DK/LT versus TCh-1,4-BQ, TB-1,4-BQ and 1,2-NQ-4-SA were negatively significant while in the DK versus 1,2-NQ-4-SA and in the DK/LT versus 1,4-BQ and 1,4-NQ relationships were positively significant (Fig. 9D).

In roots of seedlings growing in increasing concentrations of BPA in the DK the relationships between the amount of endogenous BPA versus the amounts of 1,4-BQ and TCh-1,4-BQ and in the DK/LT versus all studied quinones were negatively significant. While in the DK the positively significant relationships were noticed between the amount of endogenous BPA versus the amount of 1,2-NQ-4-SA. In the other cases in these groups the relationships were statistically not significant. In stems in the DK, the relationships between the amount of endogenous BPA versus of the amounts of 1,4-BQ, TCh-1,4-BQ, TB-1,4-BQ and 1,2-NQ-4-SA and in the DK/LT versus 1,4-NQ were positively significant. The relationships between the amount of endogenous BPA in the DK/LT versus of the amounts of TB-1,4-BQ, TB-1,4-BQ and 1,2-NQ-4-SA were negatively significant. In the other cases in these groups the relationships were statistically not significant (Fig. 9D).

In roots in the DK, the relationships between the amount of endogenous BPA in treatment solutions of seedlings growing in increasing concentrations of BPA versus all five studied quinones were positively significant. In the DK/LT versus the amounts of TB-1,4-BQ and 1,4-NQ were negatively significant. In the other cases of these groups the relationships were statistically not significant. In stems in the DK the relationships between the amount of endogenous BPA in treatment solutions of seedlings growing in increasing concentrations of BPA versus all five studied quinones were positively significant. While in DK/LT versus of the amount of 1,4-BQ, TB-1,4-BQ and 1,4-NQ were negatively significant. In the other cases in these groups the relationships were statistically not significant (Fig. 9D).