Screening And Functional Studies Of Phloem Translocation Carriers Of An L-valine-phenazine-1-carboxylic Acid Conjugate

Plant amino acid transporters play important roles in the uptake and phloem translocation of amino acid.With the help of amino acid transport carries,amino acid conjugates of pesticides can improve the phloem translocation of agrochemical parent ingredients,allowing for reduction of usage,and hence decrease threats to the environments.A large number of amino acid-fungicide conjugates have been synthesized in previous studies and L-Val-PCA（L-valine-phenazine-1-carboxylic acid conjugate）exhibited a better phloem mobility.However,it is still unclear for the phloem translocation carriers of L-Val-PCA.This study investigated the characteristics of L-Val-PCA uptake,screened and identified the phloem translocation carriers,and validated the phloem-translocation functions of carriers,which could lay foundation for utilization of the transporters and further molecular design of amino acid-fungicide conjugates.The main study findings as followed:1.It has been found that L-Val-PCA uptake by castor bean seedling cotyledons is closely related to time,p H,and concentration using leaf disc method.The uptake concentrations of L-Val-PCA in the cotyledons within 1-5 h gradually increased with the increase of the incubation time.The gradual decreases of the uptake concentrations of L-Val-PCA appeared with the decrease of p H values（5.6,6,7,and 8）.The L-Val-PCA concentrations gradually increased the L-Val-PCA incubation concentrations（0.025,0.05,0.1,0.15,0.2,and 1 mmol/L）.Furthermore,the uptake concentrations of L-Val-PCA were decreasing after energy inhibitor CCCP was added into various concentrations（0.025,0.05,0.1,0.15 and 0.2 mmol/L）of L-Val-PCA solutions,which demonstrates that L-Val-PCA uptake includes active transport process.2.It has been found that three transporters Rc AAP2,Rc ANT7 and Rc LHT1 have a similar expression pattern after L-Val-PCA or L-Val solution treatments of castor bean cotyledons for various times（1,3,and 6 h）using RT-q PCR analysis of the relative expression levels of sixty-two Ricinus amino acid transporters,and the expression levels of Rc AAP2 were significantly up-regulated 2.05-fold and 1.38-fold at 1 h after L-Val-PCA or L-Val solution treatments,the expression levels Rc ANT7 were up-regulated 1.33-fold and 1.18-fold,and the expression levels of Rc LHT1 were up-regulated 1.77-fold and1.31-fold.3.Rc AAP2,Rc ANT7,and Rc LHT1 belong to the amino acid transporter superfamily by Pfam analysis and SMART database blast.Phylogenetic analysis reveals that Rc AAP2,Rc ANT7 and Rc LHT1 have high homology with the AAP6,AVT6A and LHT1 protein subfamily.The three-dimensional structures of three transporters were modeled using the Phyre2 database.Secondary structure analysis suggests that three transporters have 11 transmembrane domains,and are membrane bound.4.Three plant binary vectors p ART27-Rc AAP2-e GFP,p ART27-Rc ANT7-e GFP and p ART27-Rc LHT1-e GFP were constructed by In-Fusion method.Rc AAP2,Rc ANT7 and Rc LHT1 were observed in the plasma membrane of mesophyll cells and phloem cells by laser confocal microscopy at 72 h after Agrobacterium-mediated transformation of these three vectors into Nicotiana benthamiana and Ricinus communis.5.Three yeast expression vectors p YES2-Rc AAP2,p YES2-Rc ANT7,p YES2-Rc LHT1 were constructed by In-Fusion method and transformed into Saccharomyces cerevisiae W303a by lithium acetate transformation method.The growths of yeast cells on the SC/-Ura medium with various concentrations of L-Val-PCA were obviously inhibited after expression of Rc AAP2,Rc ANT7 and Rc LHT1 for 72 h under the induction of galactose.That suggests three transporters can facilitate L-Val-PCA uptake by yeast cells.6.The overexpression and RNAi systems that directly used for phloem translocation functional study of transporters were constructed in the model plant Ricinus using vacuum agroinfiltration strategy.The parameters were also optimized.The Agrobacterium suspension density OD_600nmwas 1.2,vacuum infiltration time was 20 plus 20 min （infiltration for 20 min at 0.09 MPa,returned to atmospheric pressure,and then infiltration for another 20 min after pressure was quickly adjusted to 0.09 MPa）,expression time was 72 h, the test concentration of L-Val-PCA was 40μmol/L.Three overexpression vectors p ART27-Rc AAP2,p ART27-Rc ANT7 and p ART27-Rc LHT1 and three RNAi vectors p ART27-Rc AAP2-SA,p ART27-Rc ANT7-SA and p ART27-Rc LHT1-SA were constructed by In-Fusion method.The L-Val-PCA phloem sap concentrations were significantly increased 1.93-fold,2.11-fold and 2.10-fold after overexpression of Rc AAP2,Rc ANT7 and Rc LHT1 for 72 h,and significantly decreased 2.20-fold,1.38-fold,and 1.41-fold after RNAi of Rc AAP2,Rc ANT7 and Rc LHT1 for 72 h.That indicates Rc AAP2,Rc ANT7 and Rc LHT1 play important roles in the phloem translocation of L-Val-PCA.