Molecular Mechanism By Which Apple Sucrose Transporter MdSUT2.2 Involves In Regulating Sugar Content In Response To Drought And Salt Stresses

Apple is an important fruit tree in northern China,and it ranks first in the world in both output and export volume.Abiotic stresses such as drought,low temperature,salt and nutrient deficits have become the main factors which limit fruit production and reduce fruit quality.It seriously restricts the sustainable and safe development of fruit trees industry.After plants recognize the adversity signal,the signal is transmitted between cells and the whole plant.The transmission of stress signals often leads to changes in gene expression at the cellular level.It in turn affects the metabolism and development of the entire plant.Higher plants change at the molecular and cellular levels to adapt to environmental stress.The physiological metabolism of sugar in plants has important and multiple functions in the process of growth and development.Sugar is the main photosynthesis product and the main component of plant phloem transport,which regulate plant growth and development.Under abiotic stress conditions,sugar can regulate osmotic potential in plants,which maintain cell osmotic pressure and protect cells.Sugar transporters act as sugar transporters and regulate sugar content in plants.However,the research on the role of sugar transporters in apples is not very clear.The function of sucrose transporter MdSUT2.2 was studied in apple.The study found that:1.We report that MdSUT2.2 in apple was a homolog of the Arabidopsis sucrose transporter AtSUT2.Ectopic expression of MdSUT2.2 in Arabidopsis decreased sucrose sensitivity in germination and seeding stage and increased sucrose transport activity.In addition,our results showed that MdSUT2.2 impacted on plant growth by accelerating vegetative growth and promoting early flowering in Arabidopsis.Overexpression of MdSUT2.2 significantly improved abiotic stress tolerance including NaCl,ABA,and mannitol in apple calli and Arabidopsis.Together,these findings provide evidence that the apple sucrose transporter MdSUT2.2 is involved in abiotic stress resistance and the regulation of plant growth and development.2.We tested the expression profile of several sugar transporter and amylase genes in response to ABA treatment.MdSUT2.2 and MdAREB2 were isolated and genetically transformed into apple（Malus domestica）to investigate their roles in ABA-induced sugar accumulation.The MdAREB2 transcription factor was found to bind to the promoters of the sugar transporter and amylase genes and activate their expression.Both MdAREB2 and MdSUT2.2 transgenic plants produced more soluble sugars than controls.Furthermore,MdAREB2 promoted the accumulation of sucrose and soluble sugars in an MdSUT2.2-dependent manner.Our results demonstrate that the ABA-responsive transcription factor MdAREB2 directly activates the expression of amylase and sugar transporter genes to promote soluble sugar accumulation,suggesting a mechanism by which ABA regulates sugar accumulation in plants.3.An ABA-inducible transcription factor gene,MdAREB2,was identified in apple.Transgenic analysis was performed to characterize its function in ABA sensitivity.Overexpression of the MdAREB2 gene increased ABA sensitivity in the transgenic apple compared with the wild-type（WT）control.In addition,it was found that the protein MdAREB2 was phosphorylated at a novel site Thr411 in response to ABA.A yeast two-hybridization screen of an apple cDNA library demonstrated that a protein kinase,MdCIPK22,interacted with MdAREB2.Their interaction was further verified with Pull Down and Co-IP assays.A series of transgenic analyses in apple calli and plantlets showed that MdCIPK22 was required for ABA-induced phosphorylation at Thr411 of the MdAREB2protein and enhanced its stability and transcriptional activity.Finally,it was found that MdCIPK22 increased ABA sensitivity in an MdAREB2-dependent manner.4.The specific phosphorylation site of sucrose transporter was identified with mass spectrometry.Transgenic analyses were performed to characterize their biological function.It was found that overexpression of sucrose transporter gene MdSUT2.2 promoted sugar accumulation and drought tolerance.MdSUT2.2 protein was phosphorylated at Ser³⁸¹ site in response to drought.A DUALmembrane system using MdSUT2.2 as bait through an apple cDNA library got a protein kinase MdCIPK22.BiFC,pull-down and co-immunoprecipitation assays further demonstrated that MdCIPK22 interacted with MdSUT2.2.A series of transgenic analysis showed that MdCIPK22 was required for the drought-induced phosphylation at Ser³⁸¹ site of MdSUT2.2 protein,and that it enhanced the stability and transport activity of MdSUT2.2 protein.Finally,it was found that MdCIPK22 promoted sugar accumulation and improved drought tolerance in an MdSUT2.2-dependent manner.5.Overexpression of MdSUT2.2 gene increased salt resistance in the transgenic apple,compared with the WT contro‘Gala’.In addition,it is found that protein MdSUT2.2 was phosphorylated at Ser254 site in response to salt.A DUALmembrane yeast hybridization system through an apple cDNA library demonstrated that a protein kinase MdCIPK13interacted with MdSUT2.2.A series of transgenic analysis in apple calli showed that MdCIPK13 was required for the salt-induced phosphylation of MdSUT2.2 protein and enhanced its stability and transport activity.Finally,it was found that MdCIPK13 improved salt resistance in an MdSUT2.2-dependent manner.6.MdSOS2L1 was functionally characterized with a role in the regulation of malate excretion from roots and the inhibition of Cd uptake into roots.The DUAL membrane system using MdSOS2L1 as bait through an apple cDNA library obtained a malate transporter MdALMT14.BiFC,pull-down and co-immunoprecipitation assays further demonstrated that MdSOS2L1 interacted with MdALMT14.A series of transgenic analyses showed that MdSOS2L1 was required for the Cd²⁺-induced phosphorylation at the Ser³⁵⁸ site of MdALMT14 proteins and that this modification enhanced the stability of MdALMT14protein.Finally,the results showed that MdSOS2L1 enhances Cd²⁺tolerance in an MdALMT14-dependent manner.In summary,the present study sheds light on the current understanding of the roles of the MdSOS2L1-MdALMT14 complex in physiological responses to Cd toxicities in plants.Our results showed that moderate drought or salt stress or ABA treatment could promote the accumulation of soluble sugars in plants or fruits,and thus improve the fruit’s flavor quality in the production of fruit trees and other fruit crops.The studies showed that apple sugar transporter MdSUT2.2 played an important role in the ABA-induced soluble sugar accumulation.MdAREB2 is a key transcription factor for the expression of MdSUT2.2 and activation of MdSUT2.2.These results indicate that ABA or drought-induced protein kinase MdCIPK22 can phosphorylate the transcription factor MdAREB2,enabling it to efficiently activate the expression of amylase and sugar transporter genes and increasing the stress resistance.In addition,protein kinases MdCIPK22 and MdCIPK13 can interact with MdSUT2.2 which was phosphorylated.Phosphorylation of proteins improves the stability of sugar transporters,promotes the accumulation of soluble sugars in plants,alters the osmotic potential of plants,and increases the resistance of plants.In fruit trees,the results reveal that moderate abiotic stress can promote fruit quality,thus providing theoretical guidance for the development of new cultivation techniques to improve fruit quality.The development of relevant research in apples helps to broaden the understanding of the molecular mechanisms of the adversity response of plants（especially woody fruit trees for many years）,and has important theoretical significance.At the same time,it is also a stress-resistant cultivation and fruit tree industry of apple trees and other fruit trees in China.Sustainable development provides technological support.