Study On The Role Of NO And Cytoplasm Alkalization In Ethylene - Induced Stomatal Closure And Its Relationship

Ethylene is an important plant hormone, which plays various physiological roles in plant growth and development. However, up to data, the effect of ethylene on stomatal movement is rather contradictory. In the present work, role and relationship of cyctosolic alkalization and NO in ethylene-induced stomatal closure in Arabidopsis were studied by means of stomatal bioassay and laser scanning confocal microscopy.The main results are as follows:1.1-aminocyclopropane-l-carboxylic acid (ACC), which is immediate precursor of ethylene biosynthesis, significantly induced stomatal closure and NO production in guard cells of wild Arabidopsis. These effects of ACC were obviously inhibited by sodium tungstate (Na2WO4), an inhibitor of NO-generating enzyme nitrate reductase (NR), and by2-(4-Carboxyphenyl)-4,4,5,5-tetramethy limidazoline-l-oxyl-3-oxide (c-PTIO), a NO scavenger. Sodium nitroprusside (SNP), a NO-releasing compound, largely prevented the inhibitory effect of Na2WO4on ACC-induced stomatal closure, but not of c-PTIO. Upon ACC exposure, NR mutants Nia1-2and Nia2-5/Nial-2produced less NO in guard cells and their stomata less closed, whereas the stomata of mutant Nia2-1fully closed and NO level in its guard cells largely increased, like wild (WT). These results clearly show that NO mediates stomatal closure by ethylene in Arabidopsis, and ethylene-induced NO production is Nial-dependent.2. ACC exposure significantly induced the rise of cyctosolic pH in guard cells and stomatal closure in wild Arabidopsis, these effects were prevented by butyric acid, which is frequently applied as a modulator of guard cell cytosolic pH. However, methylamine, a weak alkalinizing agent, completely reversed the inhibitory effects of butyric acid on ACC-induced stomatal closure. These results indicate that cytosolic alkalization is involved in ethylene-induced stomatal closure in Arabidopsis.3. SNP largely reversed the inhibitory effect of weak acid butyric acid on ACC-induced stomatal closure in wild Arabidopsis, but methylamine did not reverse the inhibitory effects of Na2WO4and c-PTIO on ACC-induced stomatal closure. ACC-induced NO production in guard cells was significantly inhibited by butyric acid, but c-PTIO and Na2WO4had no effect on ACC-induced cytosolic alkalization in guard cells. ACC exposure did close the stomata of mutant Nia2-1, less closed the stomata of mutants Nial-2or Nia2-5/Nial-2, methylamine did not reverse the effect of ACC on stomatal aperture in NR mutants. In addition, ACC obviously rised guard cell cyctosolic pH in mutants Nial-2, Nia2-1and Nia2-5/Nial-2, like in WT. These results show that cytosolic alkalization act upstream of NO production during ethylene-induced stomatal closure in Arabidopsis.4. ACC neither induced cytosolic alkalization and NO production in guard cells, nor closed stomata in ethylene perception mutant etrl-1and etrl-3. However, both SNP and methylamine induced stomatal closure in etr1-1and etr1-3mutants, like in WT. The data show that ETR1indeed mediated ethylene-induced stomatal closere, cytosolic alkalization and NO production in Arabidopsis.5. ACC did not induce NO production and stomatal closere in ethylene pathway-related mutants ran1-1, ein2-1, ein2-5, ein3-1and arr2-4, whereas SNP exposure closed stomata of these mutants. The results indicate that RAN1, EIN2, EIN3and ARR2play an important role in ethylene-induced NO production and stomatal closere in Arabidopsis.In summary, cytosolic alkalization-mediated NO production are involved in ethylene-induced stomatal closere, and Nial, ETR1,RAN1, EIN2, EIN3and ARR2are essential for ethylene-induced NO production and stomatal closere.