Introduction BRs are well known class of steroid

Introduction BRs are well known class of steroid phytohormones involved in the regulation of key processes of plant growth and development [1], [2], [3], [4], [5]. A lot of attention is driven to BRs in the last decade because of their undeniable functions in promoting SB 203580 hydrochloride reactions of plants to a range of environmental abiotic stressors [6], [7]. Although a great progress has been made the molecular mechanisms of their biological action remain to be elucidated. One of the prospective directions is linkage of BRs with cell signaling systems, particularly phospholipid signaling. Phospholipid signaling involves a lot of enzymes like phospholipases, DGKs that convert major phospholipids of cell membranes into minor ones (PA or diacylglycerol (DAG)) which serve as second messengers under variety of stress conditions [8]. PA exhibits a close linkage with BR signaling, particularly, it regulates intracellular localization of protein phosphatase 2A protein (PP2A) that regulates BRs-dependent transcriptional factors [9]. It is known that the PA level is promoted in response to BRs treatment [10], [11]. However, little is known about pathways of PA generation in this process. PA is one of the most abundant lipid messengers in phospholipid signaling and its effects may be strictly dependent on the site or path of production [12]. There are two major pathways of PA formation in plant cells – by phospholipase D (PLD) that directly converts phospholipids into PA or by phospholipase C (PLC) that generates DAG which immediately can be converted to PA by DGK. Interestingly, PA molecular species from both pathways can differ; it depends on the type of phospholipid that was used by PLD or PLC. Each plant phospholipid has its own set of fatty acids (saturated or unsaturated) that determines which PA species will be produced from this phospholipid. Different PA species might have special properties for binding to specific enzymes. The A. thaliana model system has 7 DGK isozymes structurally classified into 3 groups [13]. Each group has its own properties and can be involved into regulation of specific cell processes [13]. The role of DGKs in PA production and its function in cell metabolism was previously described for barley roots in response to high salinity and osmotic stress [14], and for A. thaliana in regulation of cold stress signaling [15]. We have recently found that EBL is involved in the regulation of plant growth, an alternative respiratory pathway and PA production in Brassica napus plants under high salinity [11]. With the use of a specific inhibitor to DGKs – R59022 – we have shown that DGKs could be involved in the formation of PA molecules in response to high salinity and BR treatment [11]. The idea of the current work is to demonstrate the involvement of DGKs in response to EBL treatment and salinity using diacylglycerol kinase knockouts that might further reveal the biological function of BRs in the response to stress effectors and their linkage with stimulation of growth, mitochondria homeostasis and phospholipid signaling inhibited by stress factor. We hypothesize that both high salinity and EBL could regulate expression of dgk genes and accumulation of BRs is a required step in the induction of salinity tolerance in A. thaliana. According to our hypothesis, a reduction of the intracellular level of BRs with the use of BRZ would interfere with gene regulation dependent on both BRs and salinity. In this case, if salinity promotes BR levels, BRZ will affect this, and if both BRs and salinity regulate common genes BRZ will also affect this process.
Experimental
Results
Discussion As was mentioned in the introduction, DGKs indeed are important enzymes of the complex lipid signaling network producing the key lipid messenger - PA. However, little is known about the role of DGKs in PA formation during hormonal signaling, particularly BRs. It has already been shown that different DGK genes are upregulated by BRs treatment [20]. This may point at an essential role of DGKs as PA-producing enzyme in BRs-dependent signaling and biological action.