| In plants, abscisic acid (ABA) is an important hormone that can response to abiotic stress. As the main plant hormone and important signaling molecule, ABA plays a protective role in response to abiotic stresses including drought, salinity and cold. ABA can control important cellular processes including regulation of seed maturation, seed dormancy and germination, root growth and flowering. Several plant respiratory burst oxidase homolog (RBOH) genes, homologous to mammalian NADPHoxidase subunit gp91phox were identified. There are 10 RBOH genes exist in the Arabidopsis genome, from AtrbohA to AtrbohJ. It is also now established that the major sources of reactive oxygen species (ROS) can be plasma membrane-localized NADPH oxidase which can generate superoxide in ABA signaling during stomatal closure. The Arabidopsis AtrbohD and AtrbohF are highly expressed in Arabidopsis guard cells, leading to ABA-induced ROS elevation and stomatal closure. Our recent data showed that PLDal(Phospholipase Dα1) and its lipid product PA(Phosphatidic Acid) played an important role in ABA-induced stomatal closure. But whether PLDα1 and PA is required for the ABA activation of specific Atrboh to mediate ROS generation and stomatal closure, PLDal-derived PA interacts directly with specific AtrbohD and AtrbohF and activates NADPH oxidase activity and ROS production in guard cell for ABA signal transduction have not been reported.In light of these observations, the present study was undertaken to address whether and how PLDal and PA regulated directly the AtrbohD (F). We investigated the role and relationship between PLDal, PA and AtrbohD (F) in ABA-induced stomatal closure and ROS generation in guard cells in Arabidopsis using molecular, physiological and biochemical approaches. The results are as follows.The full-length,5’and 3’ cDNA fragment sequences of Arabidopsis(ecotype Columbia) Rboh genes AtrbohD and AtrbohF were amplified from leaf cDNA using genetic specific primer pairs by RT-PCR techniques, respectively. These cDNA sequences were inserted into the T vector for sequencing. The sequence analysis results showed that the amplified target fragments were correct completely and contain intact ORF frame. For protein expression in E. coli, AtrbohD and AtrbohF cDNAs were cloned into a pET-28a vector with the Histidine tag (His-tag). Fusion proteins were expressed in Escherichia coli strain BL21 and purified with Ni-affinity agarose. Purified proteins were used for immunoblotting and the analysis of protein-lipid interaction using the fat western-blot assay in Vitro. Both Atrboh D and AtrbohF bound to native PA from egg yolk in the N terminal, but not to phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), diacylglycerol (DAG) or phosphatidylinositol polyphosphates (PIPS) as compared with empty vector control. For PA species available, Atrboh D and AtrbohF bound to saturated PA such as dioleoyl PA (di 18:1 PA), dilinoleoyl PA (di18:2 PA), dipalmitoyl-dioleoyl PA (16:0-18:1 PA), dipalmitoyl-dilinoleoyl PA (16:0-18:2 PA), distearoyl-dilinoleoyl PA (18:0-18:2 PA), but not to unsaturated PA such as dipalmitoyl PA (di 16:0 PA) or distearoyl PA (di 18:0 PA). In the bound PA species, AtbohD displayed stronger binding than did AtrbohF. The lipid-AtrbohD/F interaction was further determined using an ELISA-based assay. Both AtrbohD and AtrbohF interacted with 16:0-18:2 PA in a dose dependent manner, but the binding of AtrbohF was weaker than AtrbohD. Increases in AtrbohD (F) concentrations had no effect on PC-coated plates. These results suggest that AtrbohD (F) specially binds to PA.To identify the protein region involved in PA binding, serial deletion mutants of AtrbohD (F) were generated and expressed in Escherichia coli. The resultes show purified proteins were used for immunoblotting and the analysis of protein-lipid interaction using the fat western-blot and liposome assay in Vitro. The results show that the PA binding motifs in AtrbohD and AtrbohF were mapped to residues 100 to 330 aa and 104 to 341 aa, respectively. Further deletions were conducted to map the PA binding motif. The results indicate that amino acid residues 140 to 160 aa are required for PA binding. Using site mutantion analysis, The data suggest that R(149,150,156,157) are important residues involved in AtrbohD binding to PA. We next determined the effect of these basic amino acid residues on PA and AtrbohD (F) interaction in guard cells protoplasts (GCP). We expressed the PA binding and no-PA binding fragment of AtrbohD 160 (1-160 aa) and AtrbohF 171 (1-171 aa) tagged with Hemagglutinin tag (HA) in AtrbohD-null mutant GCPs to coprecipitate PA. Fluorescent lipid pull-down methods confirm the PA-Atrboh interaction in guard cells and suggest the residues R(149,150,156,157) are important to PA-AtrbohD binding.To identify the role of PLDal and its lipid product PA in ABA-induced ROS production and stomatal closure, we next analyzed their relationship between PLDα1, PA and AtrbohD (F) using the epidermal strips from rosette leaves in pldα1, atrbohD and atrbohF mutants. Exogenously applied ABA,16:0-18:2 PA and H2O2, stomatal apertures were measured in guard cells. The data indicate PA-AtrbohD (F) interaction regulates ABA-induced stomatal closure. Exogenously applied ABA and 16:0-18:2 PA using Laser scanning confocal assay for ROS production. The results show the interaction between PA-AtrbohD (F) is important and requires for the ABA activation of the production of ROS in guard cells.These results indicate that PLDα1-derived PA interacts directly with AtrbohD (F). The binding region and important residues involved in AtrbohD binding to PA are identified. PA-Atrbohs interaction is essential to ABA-mediated ROS generation and stomatal closure. PA-Atrbohs interaction suggests a new regulatory mechanism in the signaling process of ABA-induced stomatal closure. |
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