According to our results germination
According to our results, germination of transgene dgks lines didn’t differ under optimal conditions and 50 mM NaCl, while 100 mM NaCl slightly affected germination rates of dgk mutants. Exogenous application of EBL even diminished the effect of 100 mM NaCl on dgk mutants. The critical effect on germination of dgks lines has been achieved with BRZ treatment (Fig. 1). BRZ is widely characterized as efficient inhibitor of BR biosynthesis in A. thaliana . Under control conditions BRZ rather significantly inhibited germination of all dgks lines (up to 20%) while WT plants were unaffected at all. Under salinity conditions BRZ strongly prohibited germination rate of dgk plants, particularly, under 100 mM NaCl germination rate dropped to 8% (in dgk3) and didn’t exceed 26% (in dgk5dgk6). Interestingly to note, that BRZ in the same concentration didn’t affect germination rate of WT plants under 100 mM NaCl. Simultaneous application of EBL and BRZ led to significant (2–5 folds) restoration of germination rates of all tested dgks lines. This may be evidence that loosing of functional dgk Ruxolitinib phosphate australia is critical for salinity resistance in plants with diminished level of endogenous BRs. So, in absence of sufficient level of BRs all dgk mutants demonstrated high sensitivity to salinity, but not WT plants. Also, we checked the influence of higher concentrations of BRZ on WT plants. Concentrations of 5 µM of BRZ under 75 mM NaCl significantly affected WT germination (50% decrease) but this effect was fully reverted with application of exogenous EBL (Sup. Fig. 3). This indicates that WT plants can be suppressed with BRZ treatment, but mutations in DGK genes makes mutants more sensitive to salinity conditions under lowered levels of endogenous BRs. The possible explanation could be that BRs may influence critical cell processes that are involved in metabolism reorganization to salinity action and regulated with DGK and PA formation. And those steps are ineffective because of mutated DGKs and impaired PA production. Also we need to admit the fact that BRs regulate expression of DGK genes that may make WT plants more resistant than dgk mutants where mutated genes could be a part of adaptation strategy to salinity conditions. We investigated respiration activity of dgk mutants under optimal and salinity conditions to elucidate the role of mitochondria in response to EBL and BRZ treatment that might be one of the key processes that maintain energy homeostasis during stress action. According to our results, all dgk mutants demonstrated impaired total respiration rates in response to EBL and BRZ treatment under salinity conditions. Also, AOX and COX respiratory pathways of dgk mutants were more sensitive to salinity conditions and BRZ treatment comparing to WT plants (Fig. 3, Fig. 4). These findings indicate the role of DGK isoforms in BR-mediated regulation of AOX and COX respiratory pathways. These results may explain the low germination rate of mutant plants in response to BRZ treatment under salinity conditions. Insufficient AOX activity may be the reason of ineffective functions of the COX pathway that leads to low ATP generation and reactive oxygen species production (ROS) . We also checked if the DGKs are directly involved in the PA formation process in response to EBL treatment. This could point at many PA-binding proteins and regulation of PA-dependent processes that are involved in maintaining of cell respiration and BRs signaling. dgk3, dgk1dgk2, dgk5dgk6 and dgk1 lines indeed showed significant decrease in PA production in response to EBL treatment comparing to WT plants that proves involvement of these isozymes in BR-stimulated PA formation (Fig. 5). Interestingly to note, that basal levels of PA do not differ in WT and dgk mutants under control conditions (Sup. Fig. 5). Also, with the use of the specific inhibitor R59022 for DGKs we showed a decrease in PA formation in response to EBL treatment in both WT and dgk mutants (Fig. 5). This strongly indicates that in dgk mutants remaining functional DGKs are also involved in PA formation in response to EBL treatment.