Role Of PTPs In Light/Dark-regulated Stomatal Movement And Relationship Between PTPs And H₂O₂,NO

It was proved that PTPs (protein tyrosine phosphatases) played a veryimportant role in the signal transduction of animal cell, but the PTKs (protein tyrosinekinases) didn't be found in plant cell at that time. So people ignored to study thephosphorylation and dephosphorylation at tyrosine site in plant cell, and knew littleabout the function of PTPs in plant cell. Until recently there are some articles show thatPTPs really exist in some plant species, and more than 20 PTPs encoding genes wereidentified in Arabidopsis thaliana genome. These genes are very important in plant celldivision, growing, development, stress resistance, and so on. However, up to now, therewas little information about the role and the mechanism of PTPs in signalingtransduction of guard cell. In order to study the effects and the mechanism of PTPs,H₂O₂ (hydrogen peroxide)and NO (nitric oxide) on light/dark-regulated stomatalmovement in Vacia faba, we use the laser-scanning confocal microscopy (LSCM)technique, the epidermal strip bioassay and enzyme activity inspect method to integratehere the intricate network of signaling pathway in guard cells and to develop themechanisms of stomatal movement.The main results are as followed:1. PAO (phenylarsine oxide), a specific inhibitor of PTPs, has no effect on stomatalaperture in light, but promote stomatal open significantly in dark. Extraneous H₂O₂ andSNP have no effect on stomatal aperture in dark, but promote stomatal closuresignificantly in light. These results indicated that both PTPs activity and the level ofH₂O₂, NO were low in light but high in dark, and all of them participated the process oflight/dark-regulated stomatal movement.2. ASA (ascorbic acid, H₂O₂ scavenger), CAT (catlase, H₂O₂ scavenger), DPI(diphenyleneiodonium chloride, an inhibitor of superoxide generating enzyme NADPHoxidase) or PAO has no effect on stomatal aperture in light, but promote stomatal opensignificantly in dark. The result indicated that PAO probably regulated the level of H₂O₂in guard cells and stomatal movement by inhibiting PTPs activity, and CAT, ASA andDPI proberbly regulated PTPs activity and stomatal movement by decreasing the levelof H₂O₂ in guard cells. 3. Compared with H₂O₂ treatment alone, the combination treatment of PAO, ASAand CAT with H₂O₂ can improve the stomatal aperture, but the combination treatment ofDPI with H₂O₂ can't do it in light. The result indicated that (1) Like ASA and CAT, PAOstarted the elimination mechanism to decrease level of H₂O₂, and then promoted stomaopen by inhibiting PTPs activity, but DPI has not that effect. Considered the effects andthe result that PAO promote stoma open in dark, we can concluded that PAO promotedstoma open through inhibiting PTPs activity and thereby eliminating H₂O₂ rather thaninhibiting H₂O₂ generation. (2) Like PAO, ASA and CAT probably decreased theactivity of PTPs through eliminating H₂O₂, and then promoted stoma open, but DPI hasnot that effect. Considered this effect and the result that ASA, CAT and DPI promotestoma open in dark, we can knew that ASA and CAT promoted stoma open througheliminating H₂O₂, and DPI through inhibiting H₂O₂ generation.4. Utilizing the specific fluorescent indicator of H₂O₂ and PTPs activity detectiontechnique, we found that (1) The endogenous H₂O₂ level and PTPs activity were low inlight but high in dark. It shown light decrease but dark improve the endogenous H₂O₂level and PTPs activity in guard cell, and light/dark regulated stomatal movementthrough regulating the endogenous H₂O₂ level and PTPs activityof guard cell. (2) PAOdecreased distinctly not only endogenous H₂O₂ in dark but also exogenous H₂O₂ in light.And exogenous H₂O₂ improve the PTPs activity significantly in light. On the contrary,ASA, CAT and DPI decrease it significantly in dark. The results indicate that PTPs canexactly enhance the endogenous H₂O₂ level in guard cells, and H₂O₂ can also enhancePTPs activity.5. cPTIO (NO scavenger), L-NAME (NO synthase inhibitor) and PAO has noeffect on stomatal aperture in light, but they promoted stoma open significently in dark.This result indicated that PAO regulated the level of NO in guard cells and stomatalmovement by inhibiting PTPs activity, cPTIO and L-NAME regulate PTPs activity andstomatal movement by decreasing the level of NO in guard cells.6. Compared with SNP alone, the combination treatment of PAO and cPTIO withSNP can improve the stomatal aperture in light, but the combination treatment ofL-NAME with SNP cann't do it. The result indicated that (1) Like cPTIO, PAO startedthe elimination mechanism to decrease level of NO, and then promoted stoma open byinhibiting PTPs activity, but L-NAME has not that effect. Considered this result andPAO promoted stoma open in dark, we can knew that PAO promoted stoma open by inhibiting PTPs activity to eliminate NO rather than to inhibit NO generation. (2) LikePAO, cPTIO proberbly decreased the activity of PTPs by eliminating NO, and thenpromoted stoma open, but L-NAME has not that effect. Considered this effect and theresult that cPTIO and L-NAME promote stoma open in dark, we can see that cPTIOpromoted stoma open by eliminating NO, and L-NAME promoted stoma open byinhibiting NO generation.7. Utilizing the specific fluorescent indicator of NO and PTPs activity detectiontechnique, we found that (1) The endogenous NO level and PTPs activity were low inlight but high in dark. It shown light decrease but dark improve the endogenous NOlevel and PTPs activity in guard cell, and light/dark regulated stomatal movementthrough regulating the endogenous NO and PTPs activity in guard cell. (2) PAOdecreased distinctly not only endogenous NO level in dark but also the NO levelelevated by SNP in guard cells in light. SNP improve the PTPs activity significantly inlight. On the contrary, cPTIO and L-NAME decrease it significantly in dark. The resultsindicate that PTPs can exactly enhance the endogenous NO level in guard cell, and NOcan also enhance PTPs activity.