Cloning And Functional Study Of Maize Sugar Transproter ZmSWEET4a

As the main product of photosynthesis,sugar transport and distribution play an important role in plant growth and development,as well as the interaction between plants and pathogens.SWEETs(Sugars Will eventually be exported transporters)are types of important genes,which encode sugar transporters.They participate in the regulation of sugar transport and partitioning in plants,involved in many important physiological processes of plant growth and development,and interactions between plants and pathogens.Pathogens can change the direction of plant sugar transport through SWEET proteins to make plants susceptible,and the functional deficient mutant of SWEET genes could be disease resistant.Therefore,research to decipher the function of SWEET genes is of great significance for breeding.It has been reported that members of the SWEET4 subfamily play critical roles in growth and development,and responsive to the injection of pathogens in Arabidopsis and grape.In the present study,through evolutionary analysis,members of the SWEET4 subfamily in Zea mays were identified.And SWEET4 a gene in maize was selected as a candidate.The following studies were mainly carried out:1.By constructing the phylogenetic tree of SWEETs gene from maize and other monocot and dicot species.Results showed that SWEET genes were evolutionally conserved and the phylogenetic tree can be divided into four sub-branches.2.The gene chip data showed that ZmSWEET4 a was expressed in all surveyed tissues of maize,and the expression level was highest in leaves.The fluorescence quantitative PCR results showed that ZmSWEET4 a was expressed in all five tissues,including root,stem,leaf,tassel and silk,and the expression level was highest in the leaves,which was basically consistent with the result of chip data.3.Plant stress defense signaling pathway related hormones(salicylic acid,jasmonic acid and ethylene)were further applied to detect the induced expression level of ZmSWEET4 a.Result exhibited that the expression level of ZmSWEET4 a was highly induced by ethylene and salicylic acid,however,no significant changes were observed under the treatment of jasmonic acid.4.Subsequently,the full-length CDS sequence of ZmSWEET4 a was cloned.The gene was 759 bp long,encoding 252 amino acids.In addition,subcellular localization analysis by maize protoplast transformation system showed that ZmSWEET4 a protein was expressed in cell membrane and nucleus,indicating that ZmSWEET4 a is a nuclear and membrane co-locatable protein.5.The overexpression recombinant vector of ZmSWEET4 a gene was constructed and transformed into Arabidopsis thaliana by agrobacterium-mediated method.Finally,the transgenic lines were successfully obtained.Results showed that overexpressing ZmSWEET4 a gene in Arabidopsis can increase aboveground and underground biomass.6.The functional complementation of yeast hexose transport defective strain EBY4000 confirmed that ZmSWEET4 a could transport glucose,fructose and galactose.As a result,diverse exogenous monosaccharides were added during the growth period of transgenic and wild type Arabidopsis.The results showed that exogenous application of fructose and glucose further promoted the germination rate and leaf development of transgenic lines overexpressing ZmSWEET4 a,compared with those of wild type Arabidopsis.In addition,overexpressed ZmSWEET4 a in Arabidopsis promoted the accumulation of starch in leaves.7.In order to decipher the roles of ZmSWEET4 a gene in response to pathogens,we first used Pseudomonas savastanoi pv.phaseolicola strain,NPS3121,to infect the wild type and transgenic Arabidopsis thaliana.Wild type Arabidopsis had non-host resistance to the bacteria.Phenotypic analysis,Trypan blue and DAB staining all indicated overexpression of ZmSWEET4 a in Arabidopsis promoted the susceptibility to NPS3121,while the wild type had no obvious symptoms.8.We compared the expression level of genes related to plant defense response before and after pathogen infection.The results implied that ZmSWEET4 a may response to Pseudomonas savastanoi pv.phaseolicola NPS3121 through ethylene dependent pathway.9.Finally,the bacterial wilt pathogen Pantoea stev artii subsp stewartii of maize was selected.The transgenic and wild type Arabidopsis was infected by injection.Similar results were obtained as above: overexpressing ZmSWEET4 a gene increased the susceptibility of transgenic Arabidopsis,and this process may be related to ethylene dependent pathway.In conclusion,our results indicated that ZmSWEET4 a gene encoded nuclear and membrane localized sugar transport.ZmSWEET4 a could transport diverse monosaccharide and played an important role in plant growth and development.Furthermore,ZmSWEET4 a may be involved in regulating the interaction between plant and pathogens through ethylene dependent pathways,confering plant disease susceptibility.