Functional Connections Between Glucose Signaling Regulators AtRGS1 And AtHXK1 In Arabidopsis

Glucose affects plant growth and development as a metabolite and also through a hormone-like action. HEXOKINASE 1(AtHXK1) and Regulator of G-protein Signaling 1(AtRGS1) pathways, mediate D-glucose signaling in Arabidopsis. However, it is not known the degree, if any, that these pathways overlap. In this study, we show a complex signaling crosstalk between these pathways by epistasis analyses, glucose-regulated gene expression, and protein-protein interaction.The main results are as follows:1. It has been shown that glucose-dependent seedling development arrest is defective in either HXK1 or RGS1 loss-of-function mutants; here, we show that their double mutant hxk1-3/rgs1-2 show intermediate sensitivity to high concentration glucose instead of additive one, indicating that HXK1 communicates with RGS1 in this glucose dose dependent growth regulation.2. hxk1 mutant exhibited the opposite root elongation and leaf expansion phenotype to the rgs1 mutants, while the double mutant showed an intermediate phenotype, implying a feed-back relationship between AtRGS1 and AtHXK1 in controlling cell activity. This was confirmed by the opposite behavior in glucose promotion of root meristem activation. The intermediate response in the double mutant was not additive since the phenotype of the single mutants were opposite.3. Loss of AtRGS1 confers Arabidopsis seedlings salt tolerance, but loss of AtHXK1 results in salt hypersensitivity. Genetic ablation of both AtHXK1 and AtRGS1 completely rescues the salt hypersensitivity in AtHXK1 single mutant, suggesting that, for growth during the salt stress response, AtHXK1 is positive modulator, AtRGS1 is negative modulator epistatic to AtHXK1.4. AtRGS1 and HXK1 showed hierarchical relationships in regulation of TBL26, CA2 and CAB2 expression, suggesting collaboration between AtRGS1 and AtHXK1 in regulating gene expression. This unusual genetic relationship is born out in the developmental phenotypes of the mutants.5. Loss of AtHXK1 abrogated AtRGS1-dependent signaling as measured by TBL26 expression without changing the level of glucose activated AtRGS1 endocytosis, indicating that AtHXK1 acts downstream of AtRGS1 in sugar-activated AtRGS1 signaling.6. A deep screen for Arabidopsis G-protein interacting proteins yielded an uncharacterized conserved protein AtRHIP1 that interacts with both AtRGS1 and AtHXK1. As a predicted signaling component, RHIP1 provides a direct nexus in AtRGS1- and AtHXK1-dependent sugar sensing. The interactions of RHIP1 with both AtRGS1 and AtHXK1 were confirmed in plant cells by BiFC.7. AtRGS1 and AtHXK1 also have another indirect common protein interactor, VHA-B3, another V-ATPase B subunit isoforms, providing another nexus in AtRGS1- and AtHXK1-dependent sugar sensing. The interactions of VHA-B1/3 with both AtRGS1 and AtHXK1 were confirmed in plant cells by BiFC.8. Gene expression and seedling growth phenotype analyses of the rhip1 mutant and the RHIP1 over-expression lines confirmed a potential role of RHIP1 in AtRGS1 and AtHXK1 signaling.In conclusion, glucose signaling is a complex hierarchical relationship which is specific to the target gene and sugar phenotype and suggests that there are two glycolysis-independent glucose signaling sensors: AtRGS1 and AtHXK1 that weakly communicate with each other to fine tune the response to glucose.