Apple Glucose Sensor MdHXK1 Improves Salt Tolerance By Interacting With And Phosphorylating Na~+/H~+ Exchanger MdNHX1

Saline soil is the main environmental factor for limiting the growth of apple fruit trees,which has a serious impact on the fruit production and development in China.Through long-term evolution,plants have formed a series of salt stress adaptation mecha nisms.Among them,the cationic/proton transporter plays a key role in mai-ntaining the ionic balance in the cells.Na⁺/H⁺exchanger proteins play an important role in maintaining low concentrations of Na⁺in cells to avoid ion toxicity.The SOS1 protein,localized on the plasma membrane,mediates the outflow of Na⁺through the classical Salt-overly-sensitive?SOS?pathway.The Na⁺/H⁺exchanger proteins,which is located on the vacuole,separate Na⁺into the vacuole and reduces the toxic effects of Na⁺on the organelle in the cytoplasm.However,few studies on the regulation mechanism of Na⁺/H⁺exchanger proteins have been reported.As the main product of photosynthesis,sugars are not only the main energy substrates of the plant,but also provide the carbon skeleton for the synthesis of lipids,proteins and nucleic acids.Thus sugars play a very important basic role in plant growth and development.In addition,sugars are the major osmoregulation substances for plants to cope with environmental stress.In recent years,with the genetics studies,cell biology and a series of functional analysis show that sugar not only involves in the catalytic pathway,but also act as a signal molecule like plant hormone regulating plant growth and development through the sugar signal pathway.The hexokinase HXK1 is thought to be a conserved glucose sensor.It is a bifunctional enzyme with both catalytic activity and signal transduction function.HXK1 can regulate the plants growth and development by integrating nutrition,light signals and hormonal signals.However,the role of HXK1 in response to abiotic stress remains to be further studied.Here,we found that Md HXK1 interacted with a Na⁺/H⁺exchanger protein MdNHX1 in apple,and revealed the mechanism of sugar signal regulation of plant salt tolerance.1.Exogenous addition of glucose can improve plant salt tolerance,and this process depends on the hexokinase kinase MdHXK1Exogenous addition of 4%glucose significantly improved the salt tolerance of in vitro shoot cultures of apple‘Gala',while the hexokinase kinase inhibitor Glucosamine counteracted the role of glucose;Inhibition of Md HXK1 using TRV transient infection system reduced the tolerance of apple leaves to NaCl.The MdHXK1 overexpressing apple calli,35S::Md HXK1,and the catalytic site mutations of MdHXK1,35S::Md HXK1^S177A and35S::Md HXK1^G104D,were obtained and showed that Md HXK1 was mainly dependent on its signal pathway under high glucose condition.2.MdHXK1 interactes with Na⁺/H⁺exchanger protein MdNHX1 through directly phosphorylationThe result of gel-shift experiments showed that glucose could induce phosphorylation oMdNHX1.LC-MS/MS and the specific phosphorylated Antibody test showed that the phosphorylation site was serine at position 275 Ser²⁷⁵.3.MdHXK1 affects protein stability and Na⁺/H⁺exchange activity of MdNHX1The cell-free degradation assay showed that the degradation rate of MdNHX1-GST prote-in in Md HXK1-GFP overexpression apple callus was lower than that of GFP empty vector,while the degradation rate of MdNXX1-GST in Md HXK1 antisense expression apple callus was significantly higher than that of GFP.The results showed that MdHXK1 increased the protein stability of MdNXX1.The experiments of protoplast transient expression and nhx1 yeast mutant complementary showed that co-expression of MdHXK1 and MdNHX1increased the Na⁺/H⁺exchange activity in apple callus and yeast cel s.4.MdHXK1 improves plant salt tolerance depending on MdNHX1Overexpression of Md HXK1 increased the salt tolerance of transgenic apple plants.Inhi--bition the expression of MdNHX1 using Agrobacterium rhizogenes in the Md HXK1overexpression plant decreased salt tolerance.Meanwhile,TRV transient infection system was used to inhibite the expression of MdNHX1 in MdHXX1 overexpressing plant leaves,which also reduced the tolerance of leaves to NaCl.