The Physiology And Molecular Mechanism Of Nitrate Regulated Adventitious Roots Formation In Apple Rootstock

The excessive nitrogen application is a widespread problem in apple orchards of China,which adversely affects root development.Studies have shown that nitrate has a dual regulation effect on the growth and development of adventitious and lateral roots of plants.At low concentrations of nitrate induce adventitious roots(ARs),while high concentrations have an inhibitory effect,but relevant studies in apples have not been reported.In view of this,in present study,typical apple dwarf rootstocks were used as test materials,under tissue culture conditions,the regulatory effects of different concentrations of nitrate on rootstock ARs were analyzed,transcriptome sequencing was performed,key genes were mined,and the genes involved in ARs were initially analyzed.The main results of the functions that occur are as follows:In order to identify the physiological and molecular mechanism that how high nitrate concentration inhibits AR formation in apple rootstock,stem cuttings of B9 apple rootstock were cultured on five different nitrate concentrations(C1=0,C2=9.4 m M/L,C3=18.7 m M/L,C4=37.5 m M/L and C5=75.1 m M/L)and 37.5 m M/L was identified as an ARs inhibiting concentration.Morphological and anatomical observations showed that high availability of nitrate inhibited AR formation.The endogenous concentrations of ABA,BR,GA3,IAA,JA and ZR were also elevated in response to high nitrate at most of the time points.Furthermore,m RNA and mi RNA regulated AR inhibition in the stem basal part of B9 apple rootstock.According to results,3686 genes were differentially expressed,from which 1797 genes(48.75 %)were up-regulated and 1889 genes(51.24 %)were down-regulated,we also identified,467 transcription factors belonging to 38 different transcription factor families.For the mi RNAs,205 Known mi RNAs belonging to 42 families and 438 novel mi RNA were identified that were differentially regulated by the high nitrate concentration.Go and KEGG analysis revealed that metabolic and cellular processes,and starch and sucrose metabolism and phenylalanine metabolism were significantly enriched in the stem basal parts of apple rootstock.Nitrate uptake and distribution is carried out by nitrate transporters(NRTs).In this work,we identified 84 Md NRT genes in the apple genome and analyzed them in terms of phylogenetic analysis,syntenic relationships,domain structure,cis-elements and motif analysis.Our results revealed that apple NRT family can be divided into three subfamilies;Md NRT1/PTR,Md NRT2 and Md NRT3 having 77,5,and 2 members,respectively.The syntenic relationships indicated that almost 48.8 % of the total NRT genes appeared from genome duplication.The composition of cis-elements suggests that most of the Md NRT genes might be involved in hormones,light,and stress responses.Interestingly,Md NRT2.1 gene has the strongest response to nitrate availability,advocating that Md NRT2.1 may have a significant role in the apple ARs formation and development.Furthermore,over-expression of 35 s Md NRT2.1 in tobacco increased root number(53.6 %),length(72.5 %),volume(54.6 %),and surface area(58.4 %)compared with wild type.Therefore,we concluded that Md NRT2.1 has direct role in ARs formation and development..On the contrary,the inhibited effects of high nitrate were also clearly visible on transgenic lines and wild type from low to high nitrate concentrations.This study highlights the inhibitory effect of high nitrate on AR formation and provides foundation for further work related with ARs formation and nitrate supply.