The Functions Of Arabidopsis Heterotrimeric G-protein In ABA-, NO- And H₂O₂-Mediated Stomatal Movement

Abscisic Acid (ABA), Nitric oxide (NO) and Hydrogen Peroxide (H₂O₂) are key signal moleculars in plants responded to outside stimulas and involved in various plant signal transduction pathways. Complicated signal trasduction network exists in the processes of stomatal opening and closure, which includes ABA-, NO-, H₂O₂- and G-protein-mediated signal transduction pathways and their cross-talk. Stomata are considered model system for studying signal transduction mechanism in advanced plants. Research on signal transduction pathways and their regulation mechanisms in stomatal movement has scientific significance in learning the mechanism of plant adaption to the environment.The heterotrimeric G-protein is composed of three subunits:α,βandγ. Plant heterotrimeric G-protein mediated cell signal transduction is one of the most conservative signal transduction mechanisms in plant cells. There are a large number of reports about the mechanism of ABA-induced stomatal closure. And G protein'sαsubunit has been proved being involved in ABA-mediated stomatal closure movement. However, the function of the subunits of G-protein in stomatal movement signal transduction network remains unclear.In this study, wild type Arabidopsis Col and its deletion mutants of gpa1-4, agb1-2,αβ36 which are lack of the GPA1, AGB1 and GPA1/AGB1 protein respectively are used as experimental materials. Epidermal strip bioassay and fluorescence microscopy technology are involved in discussing the function of heterotrimeric G-protein in ABA-, NO- and H₂O₂- mediated signal transduction in guard cells. The main conclusions are as follows:1)Exogenous ABA induces stomatal closure in Arabidopsis wild type. gpa1-4 is insensitive in this process, which indicates GPA1 mediated ABA-induced stomatal closure. agb1-2 andαβ36 are sensitive to ABA which can reach the conclusion that AGB1 is not involved in this signal pathway.2)Exogenous ABA can inhibit wild type Arabidopsis'stomatal opening movement. In this process, gpa1-4 and agb1-2 are sensitive to ABA. Butαβ36 is insensitive which can indicate that GPA1 and AGB1 can work independently in this signal pathway. But this signal pathway will be inhibited lack of both of them. 3)Exogenous NO stimulates stomatal closure movement in wild type Arabidopsis. In this process, gpa1-4 and agb1-2 are sensitive to ABA, butαβ36 is insensitive to it. Therefore, it is concluded that both GPA1 and AGB1 protein are involved in this process. But this signal pathway will be hindered lack of both GPA1 and AGB1.4)Exogenous NO can inhibit stomatal opening movement in wild type Arabidopsis and has the same effect in the three G-protein mutants which can conclude that G protein is not involved or plays a negative regulation role in this process.5)Exogenous H₂O₂ can induce stomatal closure movement in wild type Arabidopsis. agb1-2 is sensitive to H₂O₂. But gpa1-4 andαβ36 are insensitive to H₂O₂. It is indicated that GPA1 protein is indispensable in this signal pathway.6)Exogenous H₂O₂ can inhibit stomatal opening movement in wild type Arabidopsis. gpa1-4 andαβ36 are insensitive to H₂O₂. But agb1-2 is sensitive to H₂O₂ as the wild type. Therefore it is concluded that GPA1 protein is indispensable in this signal pathway.7)ABA can induce NO and H₂O₂ generation which will lead to stomatal closure in wild type Arabidopsis. GPA1 protein is involved in the process of H₂O₂ generation induced by ABA. The process of ABA inducing NO in stomatal needs the existence of both GPA1 and AGB18)In wild type Arabidopsis, ABA can induce NO and H₂O₂ generation in stomatal which can inhibit stomatal opening movement. In this process, GPA1 protein is proved to be indispensable in ABA-induced H₂O₂ generation. And AGB1 protein is necessary in ABA-induced NO generation which inhibit stomatal opening movement.9)H₂O₂ can induce NO generation which lead to stomatal closure in wild type Arabidopsis. agb1-2 has similar performance as the wild type. However H₂O₂ can not induce NO generation in gpa1-4 andαβ36. Therefore, it is indicated that GPA1 protein positively regulates the signal pathway of H₂O₂-induced NO generation in stomatal.10)H₂O₂ can induce NO generation which inhibit stomatal opening movement in wild type Arabidopsis. H₂O₂ has similar function in agb1-2 as the wild type. However H₂O₂ can not induce NO generation which can inhibit stomatal opening movement in gpa1-4 andαβ36. Therefore, it can be concluded that GPA1 protein is indispensable in the signal pathway of H₂O₂-induced NO generation which inhibits stomatal opening. 11)There is no H₂O₂ generation in the process of NO-stimulated stomatal closure. gpa1-4, agb1-2 andαβ36 have the similar responses as the wild type Arabidopsis when treated by NO. So it is presumed G protein is not involved in this process.12)There is no H₂O₂ generation in the process of NO-inhibitd stomatal opening movement. gpa1-4, agb1-2 andαβ36 have the similar responses as the wild type Arabidopsis. Therefore it is presumed G protein is not involved in this process.13)Endogenous ABA can enhance NO and H₂O₂'s effect in stimulating stomatal closure movement in wild type Arabidopsis. However, endogenous ABA does not have the same effect in gpa1-4, agb1-2 andαβ36 mutants as in wild type Arabidopsis. It is presumed that both GPA1 and AGB1 protein are involved in this signal path way. And the two proteins act synergistically. Both of the two proteins are indispensable in this signal pathway.14)Endogenous ABA can enhance H₂O₂'s effect in inhibiting stomatal opening movement in wild type Arabidopsis. Endogenous ABA can weaken H₂O₂'s effect in inhibiting stomatal opening movement inαβ36 mutant. Therefore, it is presumed that both GPA1 and AGB1 protein can positively regulate in this signal path way. And the two proteins can act independently in this signal pathway. GPA1 and AGB1 protein are proved to be both involved in the signal path way of endogenous ABA enhancing NO's effect in inhibing stomatal opening movement. And they work synergistically in this signal pathway.