We also checked if the DGKs are directly involved
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.
BR-stimulated PA formation via the DGK pathway might have many effects in regulation of cell metabolism. For example, PA originated from DGKs pathway plays important roles in activation of NADPH oxidases, thus turning on ROS signaling . PA is also connected to regulation of respiration processes and energy metabolism. The interconnection between the AOX respiratory pathway and PA was observed via PP2A that is activated by PA and plays a key role in mediating AOX protein abundance , . PA binds a range of other key proteins to change their activity , . Particularly, it binds glyceraldehyde-3-phosphate dehydrogenase that is one of the key enzymes in sugars glycolysis and energy metabolism . This indicates that PA is involved in regulation of both steps of energy producing pathways from SW033291 – cytosolic glycolysis and mitochondrial respiration, where AOX respiratory pathway protects COX from self-inhibition and ROS production.
In summary, we propose that DGKs are the essential part of BR-regulated mechanism of plant adaptation to salinity. From our unpublished results on maize under cold conditions and related papers aimed at an investigation on BR levels under salinity conditions , we know that plants accumulate BRs in response to abiotic stresses that might explain such dramatic effects of BRZ under salinity conditions. It might explain that BRZ treatment suppressed BR accumulation in response to salinity and this affected other BR-dependent processes. And for dgk mutants this effect was much more drastic than for WT plants. Thus, this suggests that functional DGK genes are critical for development of BR-dependent protective reaction against salinity stress.
Conclusions In summary, we clearly observed that dgk mutants had strongly suppressed germination rates in response to BRZ treatment under salinity conditions, while exogenously applied EBL partially reverted effects of the inhibitor. This demonstrates that blocking of EBL accumulation by BRZ makes dgk mutants highly sensitive to salinity, as opposed to WT plants. Dgk mutants also showed impaired intensity of alternative and cytochromes respiratory pathways in A. thaliana plants under salinity conditions that might indicate impaired cell capability to maintain stability and functional efficiency of mitochondria. Our results demonstrate that the PA production response to EBL treatment is also strongly reduced in dgk mutants, particularly dgk3, dgk1dgk2, dgk5dgk6 and dgk1 lines. To summarize, our results point at DGK-dependent regulation of seed germination, cell respiration processes and PA production as an essential part of the BR mechanism of adaptation to salinity in A. thaliana plants. This brings new data into disclosure of the BR-mediated mechanism of cell adaptation to salinity conditions. Further experiments are required for deeper investigation of phospholipid signaling, particularly DGK and PA involvement, in the regulation of BR-dependent response to stress action.