The ROP1 GTPase-based signaling network controls tip growth in Arabidopsis pollen tubes. Our previous studies imply that ROP1 might be directly activated by RopGEF1, which belongs to a plant-specific family of Rho gua...The ROP1 GTPase-based signaling network controls tip growth in Arabidopsis pollen tubes. Our previous studies imply that ROP1 might be directly activated by RopGEF1, which belongs to a plant-specific family of Rho guanine nucleotide exchange factors （RopGEFs） and in turn may be activated by an unknown factor through releasing RopGEFI＇s auto-inhibition. In this study, we found that RopGEF1 forms a complex with ROP1 and AtPRK2, a receptor-like protein kinase previously shown to interact with RopGEFs. AtPRK2 phosphorylated RopGEF1 in vitro and the atprkl,2,5 tri- ple mutant showed defective pollen tube growth, similar to the phenotype of the ropgef1,9,12,14 quadruple mutant. Overexpression of a dominant negative form of AtPRK2 （DN-PRK2） inhibited pollen germination in Arabidopsis and reduced pollen elongation in tobacco. The DN-PRK2-induced pollen germination defect was rescued by overexpressing a constitutively active form of RopGEF1, RopGEF1（90-457）, implying that RopGEF1 acts downstream of AtPRK2. Moreover, AtPRK2 increased ROP1 activity at the apical plasma membrane whereas DN-PRK2 reduced ROP1 activity. Finally, two mutations at the C-terminal putative phosphorylation sites of RopGEF1 （RopGEF1S460A and RopGEF1S480A） eliminated the function of RopGEF1 in vivo. Taken together, our results support the hypothesis that AtPRK2 acts as a positive regula- tor of the ROP1 signaling pathway most likely by activating RopGEF1 through phosphorylation.展开更多
The pollen receptor kinases (PRK) are critical regulators of pollen tube growth. The Arabidopsis genome encodes eight PRK genes, of which six are highly expressed in pollen tubes. The potential functions of AtPRK1 thr...The pollen receptor kinases (PRK) are critical regulators of pollen tube growth. The Arabidopsis genome encodes eight PRK genes, of which six are highly expressed in pollen tubes. The potential functions of AtPRK1 through AtPRK5, but not of AtPRK6,in pollen growth were analyzed in tobacco. Herein, AtPRK6 was cloned, and its function was identified. AtPRK6 was expressed specifically in pollen tubes. A yeast two-hybrid screen of AtPRK6 against 14 Arabidopsis Rop guanine nucleotide exchange factors (RopGEFs) showed that AtPRK6 interacted with AtRopGEF8 and AtRopGEF12. These interactions were confirmed in Arabidopsis mesophyll protoplasts. The interactions between AtPRK6 and AtRopGEF8/12 were mediated by the C-termini of AtRopGEF8/12 and by the juxtamembrane and kinase domain of AtPRK6, but were not dependent on the kinase activity. In addition, transient overexpression of AtPRK6::GFP in Arabidopsis protoplasts revealed that AtPRK6 was localized to the plasma membrane. Tobacco pollen tubes overexpressing AtPRK6 exhibited shorter tubes with enlarged tips. This depolarized tube growth required the kinase domain of AtPRK6 and was not dependent on kinase activity. Taken together, the results show that AtPRK6,through its juxtamembrane and kinase domains (KD), interacts with AtRopGEF8/12 and plays crucial roles in polarized growth of pollen tubes.展开更多
基金a MOST 973 project，National Institute of General Medical Research，DOE (DE-FG02-04ER15555,which supported F.C.and the biochemical experiments described in this work) to Z.Y.,and a National Science Foundation of China (31070274) to F.C
文摘The ROP1 GTPase-based signaling network controls tip growth in Arabidopsis pollen tubes. Our previous studies imply that ROP1 might be directly activated by RopGEF1, which belongs to a plant-specific family of Rho guanine nucleotide exchange factors （RopGEFs） and in turn may be activated by an unknown factor through releasing RopGEFI＇s auto-inhibition. In this study, we found that RopGEF1 forms a complex with ROP1 and AtPRK2, a receptor-like protein kinase previously shown to interact with RopGEFs. AtPRK2 phosphorylated RopGEF1 in vitro and the atprkl,2,5 tri- ple mutant showed defective pollen tube growth, similar to the phenotype of the ropgef1,9,12,14 quadruple mutant. Overexpression of a dominant negative form of AtPRK2 （DN-PRK2） inhibited pollen germination in Arabidopsis and reduced pollen elongation in tobacco. The DN-PRK2-induced pollen germination defect was rescued by overexpressing a constitutively active form of RopGEF1, RopGEF1（90-457）, implying that RopGEF1 acts downstream of AtPRK2. Moreover, AtPRK2 increased ROP1 activity at the apical plasma membrane whereas DN-PRK2 reduced ROP1 activity. Finally, two mutations at the C-terminal putative phosphorylation sites of RopGEF1 （RopGEF1S460A and RopGEF1S480A） eliminated the function of RopGEF1 in vivo. Taken together, our results support the hypothesis that AtPRK2 acts as a positive regula- tor of the ROP1 signaling pathway most likely by activating RopGEF1 through phosphorylation.
基金supported by the National Natural Science Foundation of China (31300247)
文摘The pollen receptor kinases (PRK) are critical regulators of pollen tube growth. The Arabidopsis genome encodes eight PRK genes, of which six are highly expressed in pollen tubes. The potential functions of AtPRK1 through AtPRK5, but not of AtPRK6,in pollen growth were analyzed in tobacco. Herein, AtPRK6 was cloned, and its function was identified. AtPRK6 was expressed specifically in pollen tubes. A yeast two-hybrid screen of AtPRK6 against 14 Arabidopsis Rop guanine nucleotide exchange factors (RopGEFs) showed that AtPRK6 interacted with AtRopGEF8 and AtRopGEF12. These interactions were confirmed in Arabidopsis mesophyll protoplasts. The interactions between AtPRK6 and AtRopGEF8/12 were mediated by the C-termini of AtRopGEF8/12 and by the juxtamembrane and kinase domain of AtPRK6, but were not dependent on the kinase activity. In addition, transient overexpression of AtPRK6::GFP in Arabidopsis protoplasts revealed that AtPRK6 was localized to the plasma membrane. Tobacco pollen tubes overexpressing AtPRK6 exhibited shorter tubes with enlarged tips. This depolarized tube growth required the kinase domain of AtPRK6 and was not dependent on kinase activity. Taken together, the results show that AtPRK6,through its juxtamembrane and kinase domains (KD), interacts with AtRopGEF8/12 and plays crucial roles in polarized growth of pollen tubes.
