Minisymposium 13: Seed Biology

Abs # 25004: Nitric oxide as an endogenous regulator of seed physiology

Presenter:	Bethke, Paul C, pcbethke@nature.berkeley.edu
Authors	Bethke, Paul C (A)   Beligni, Veronica  (C)   Badger, Murray  (B)   Jacobsen, John V (D)   Jones, Russell L (A)
Affiliations:	(A): University of California at Berkeley (B): Australian National University (C): Scripps Research Institute (D): CSIRO Plant Industry

Nitric oxide (NO) is a highly reactive, gaseous free radical that readily diffuses though cell membranes. Chemical reactions involving NO are utilized in biological systems for signal transduction, and NO production can either contribute to or lessen the deleterious effects of reactive oxygen species (ROS). In barely aleurone cells, gibberellin perception triggers a series of events that culminate in ROS-induced programmed cell death (PCD). We have shown that incubation of GA-treated aleurone layers with the NO donors SNP and SNAP delays PCD without affecting the GA-induced synthesis and secretion of alpha-amylase. We have expanded these finding to show that NO donors effectively break dormancy of barley grain and Arabidopsis seeds. Grain of the barley cultivars Proctor and Sloop showed <10% germination when imbibed in the light. Imbibing grain in the light with SNP resulted in approximately 40% germination, whereas virtually no grain germinated when imbibed with the NO scavenger cPTIO. Arabidopsis seeds that are largely dormant in the absence of stratification (<10%germination) show 90-95% germination when imbibed in the presence of SNP or when stratified. These data suggest that endogenous NO may regulate aspects of seed physiology, and they prompted us to examine mechanisms for NO synthesis in seeds and grain. NO can be produced enymatically, for example, by NO synthase or nitrate reductase, or nonenzymatically from nitrite at low pH. We show that the incubation medium around barley aleurone layers is ideally suited for the nonenzymatic production of NO from nitrite. Medium pH is 3.5 or less, and antioxidants in the form of phenolics are abundant. Addition of nitrite to aleurone layer medium results in an immediate increase in NO production as measured by mass spectrometry. cPTIO abolished the NO signal, and addition of pH 5.5 buffer greatly reduced NO production. We show that low micromolar concentrations of nitrite produce NO at the rate that delays PCD. Nitrite-dependent NO production rates increase when phenolics such as catechin or tannic acid are added to aleurone layer incubation medium. We have also used an NO-reactive fluorescent probe to measure NO production by Arabidopsis seeds, and these data will be discussed.