The Relationship Of Phosphalipase D, Hydrogen Peroxide And Nitric Oxide In Transducting ABA And Salt Signal

Salinity and drought have been the major threats affecting agricultural productivity. Salt stress elevates cellular Na+ level. To remove excess Na+ from the cytoplasm by the compartmentation of Na+ into the vacuole or exclusion of Na+ to the apoplast by Na+/H+ antiporters associated with vacuolar membrane (tonoplast) or plasma membrane respectively is crucial to improve salt tolerance.We report that NaC1 induced a transient increase in NO accumulation in maize leaves. NO induced the increase of vacuolar H+-ATPase and H+-PPase activities, along with an increase of Na+/H+ exchange activity, thereby increasing salt tolerance in maize. Phospholipase D (PLD) and its product phosphatidic acid (PA) may contribute to NO-induced H+-pump activation.Under drought stress, elevated abscisic acid (ABA) content and redistribution in plants induces stomatal closure and consequently reduces transpiration water loss to increase plant tolerance against drought. The signaling process of ABA-induced stomatal closure is a very complicated network and a large number of ABA signaling intermediates have been found in guard cells. Understanding relationship, interaction and "cross-talk" of these intermediates will help us manipulate ABA signaling transduction, decrease water loss through stomatal pores and enhance drought tolerance.Phospholipase D (PLD), hydrogen peroxide (H202) and nitric oxide (NO) are involved in ABA-induced stomatal closure. PLD-loss-function or blocked H202 and NO production impaired ABA-induced stomatal closure. Interaction and relationship of PLD, H202 and NO remain unclear. 12 PLD genes are in Arabidopsis. There is distinguishable biochemical and regulatory properties in different PLDs, which determines these PLDs to mediate different cell processes. The present work proves both PLDα1 and PLDδare involved in the signaling process of ABA-induced stomatal closure. Stomatal closure is insensitive to ABA treatment in pldαland pldδ. H₂O₂ promotes stomatal closure in pldα1, but fails to induce stomatal closure in pldδ. PLDal mediates H₂O₂ production in ABA signal and PLDδ responds to H₂O₂ function. Further studies suggest PLDal mediates H₂O₂ production via its involvement in ABA-activated NADPH oxidase activity. Enhanced NADPH oxidase response to ABA treatment was impaired in pldal. Exogenously added phosphatidic acid (PA), one of PLDal hydrolysis products, rescues activation of NADPH oxidase. PA interacting with ABI1 phosphatase 2C positively regulates stomatal closure. Our data that H₂O₂ production in ABI_R73A (the essential amino acid mutation for PA-ABI1 binding) response to ABA treatment suggests PLDal/PA-mediated NADPH oxidase activation and H₂O₂ production is independent in PA-ABI1 binding. The data suggest that PLDal-derived PA has two targets at least, ABI1 and NADPH oxidase, and PA interaction with both of the targets are required for mediating ABA response. Without PA binding to ABI1, ABI1 is presumed to be localized in the nucleus to inhibit ABA response. So, in ABI1_R734 mutant, even though the mutant still makes H₂O₂, H₂O₂ itself is insufficient to mediate ABA-promoted stomatal closure.PLDal and NO are required in the process of ABA-induced stomatal closure. The relationship between PLDal and NO in ABA-induced stomatal closure has also been studied in this work. NO production is insensitive to ABA treatment in pldal mutant. Exogenously applied 16:0, 18:0 and 18:1 PA promote NO production in pldal, suggesting a crucial role of PLDal/PA in ABA-induced NO production. Meanwhile, exogenous NO promotes stomatal closure inpldal. All these proofs indicate PLDα1/PA is upstream of NO function in the signal process of ABA-induced stomatal closure. PLDα1 is not involved in H₂O₂-induced NO production, which further proves both H₂O₂ and NO is downstream of PLDα1.Nitrate reductase (NR) is the main source for NO production in guard cell mediating ABA-induced stomatal closure. To address how PA regulates NO production, we cloned two Arabidopsis NR structural genes NIA1, NIA2 and expressed them in E.coli and tobacco via transient expression system. The study indicates two PA, 16:0 and 18:0 PA binds to NIA2, which is responsible for 90% of NR activity and promotes NIA2 activity.The effect of NO on PLD activity in vivo has also been investigated. The data suggests NO activates PLD activity at least in some specific cells such as mesophyll cell protoplasts. Thus, a working model depicting the relationship of ABA, PLDal, H202, NO and PLDδhas been proposed.