Physiological And Molecular Responses Of The Dwarfing And The Interactions Of Cd/Zn In Dwarf Polish Wheat(Triticum Polonicum L.)

Polish Wheat（Triticum polonicum L.,2n=4x=28,AABB）,which have large seeds,high thousand seed weight,dwarfing genes,and accumulate high contents of zinc（Zn）and iron（Fe）are important resource materials for wheat production and genetic improvement.Dwarf Polish Wheat（Triticum polonicum L.,2n=4x=28,AABB,DPW）,collected from Tulufan,Xinjiang,China,is a spontaneous mutant dwarf accession of T.polonicum,and shows high tolerance to cadmium（Cd）and Zn.It is a desirable material for studying the mechanism of the dwarfing and the interactions between Cd and Zn.It has shown that the dwarfing gene of DPW is located on 4BS,which is a recessive single gene or locus,the dwarfing mechanism of DPW is still unknown.Moreover,previous studies of Cd and Zn focused on the interactions between metals,the molecular mechanism is unknown.Therefore,the aims of this study were to understand physiological and molecular responses of the dwarfing and the interactions of Cd and Zn using RNA sequence（RNA-Seq）,isobaric tags for relative and absolute quantification（iTRAQ）,phenotypic characterization,determination of metal content,subcellular distribution of metals,and quantitative real time PCR（qRT-PCR）analysis.The main results are summarized as below:1.The dwarfism of DPW was not associated with the size of seeds and the type of leaves.Rht-dp was an abscisic acid-（ABA）,cytokinin-（CK）,brassinolide-（BR）,auxin-（IAA）,and photoperiod-insensitive dwarfing gene.The stems of elongation under photoperiod treatments were analyzed using RNA-Seq and iTRAQ to understand the dwarfing mechanism of Rht-dp.A total of 108 genes and 12 proteins were considered as dwarfism-related differentially expressed genes（DEGs）and differentially expressed proteins（DEPs）,respectively.Most of these DEGs were involved in flavonoid metabolism,cellulose metabolism,and auxin transport.Most of these DEPs were found to be involved in flavonoid metabolism,S-adenosyl-methionine（SAM）metabolisms,cellulose metabolism,cell wall plasticity and cell expansion.We also analyzed the distribution and content of lignin during stem elongation.The results indicated that the significantly lower syringyl lignin（S type）content in DPW than that in High Polish Wheat（Triticum polonicum L.,2n=4x=28,AABB,HPW）caused the different lignin content between DPW and HPW.Meanwhile,the content of cellulose in DPW was significantly lower than that in HPW;the content of flavonoid in DPW was significantly higher than that in HPW.The size of longitudinal cells in DPW was significantly smaller than that in HPW.2.Two days after Cd,Zn,and Cd+Zn treatments,the metal content determination results showed that Zn inhibited the uptake and the transport of Cd,Cd inhibited the uptake of Zn and had no effect on the transport of Zn from roots to shoots.ITRAQ analysis of roots showed that Cd,Zn,and Cd+Zn stresses changed the expressions of 205,302,and 190DEPs respectively.Among these,53 DEPs were altered significantly in response to all these stresses,whereas 58,131,and 47 DEPs were altered in response to Cd,Zn,and Cd+Zn,respectively.61 DEPs were induced in response to both Cd and Zn stresses;33DEPs were induced in response to both Cd and Cd+Zn stresses;and 57 DEPs were induced in response to both Zn and Cd+Zn stresses.A number of DEPs that mainly involved in oxidation-reduction,glutathione（GSH）metabolism,SAM metabolism,and sucrose metabolism were induced in response to Cd stress,but not Cd+Zn stress.Meanwhile,a number of DEPs that mainly involved in sucrose metabolism,organic acid metabolism and oxidation-reduction were induced in response to Zn stress but not Cd+Zn stress.Other DEPs induced in response to Cd,Zn,or Cd+Zn stresses,involved in ribosome biogenesis,DNA metabolism,and protein folding and modification.3.Five days after Cd,Zn,and Cd+Zn treatments,the metal content determination results showed that Zn inhibited the Cd uptake in roots,and promoted the Cd transport from roots to shoots;however,Cd only inhibited the Zn uptake in roots.RNA-Seq analysis of roots showed that the expressions of 1,269,820,and 1,254 DEGs were affected by Cd,Zn,and Cd+Zn,respectively.Only 381 DEGs were co-induced by these three treatments;763DEGs were induced by Cd;172 DEGs were induced by Zn;42 DEGs were induced by Cd and Zn;83 DEGs were induced by Cd and Cd+Zn;225 DEGs were induced by Zn and Cd+Zn;565 DEGs were induced by Cd+Zn.Several metal transporters,such as Cd-transporting ATPase and plant Cd resistance,were specifically regulated by Cd+Zn.Other metal-related genes,such as ATP-binding Cassette（ABC）transporter,metal chelator,nicotianamine synthase（NAS）,vacuolar iron transporters（VIT）,metal-nicotianamine transporter YSL（YSL）,and nitrate transporter（NRT）,were regulated by Cd or/and Cd+Zn.4.RNA-Seq analysis found that several nitrate（NO₃^–）metabolism–related genes were involved in Cd/Zn interactions in roots of DPW.Compared with lack of NO₃^–,supply of NO₃^–enhanced the Cd uptake,and reduced the Zn uptake and their transports.Under Cd+Zn stress,supply of NO₃^–significantly inhibited the Zn uptake.Under lack or supply of NO₃^–,Zn inhibited the Cd uptake and promoted its transport.Under supply of NO₃^–,the Cd uptake was stronger inhibited by Zn;Cd promoted the Zn uptake.Under Cd stress,supply of NO₃^–increased the Cd content in soluble fraction of roots,decreased the content in cell wall of shoots;under Zn stress,supply of NO₃^–enhanced the Zn content in cell wall of roots,reduced the content in soluble fraction of roots and all fractions of shoots.Under Cd+Zn stress,supply of NO₃^–increased the Cd content in soluble fraction of roots,decreased the content in cell wall of shoots;decreased the Zn content in all fractions of roots and in organelle and soluble fraction of shoots.Under Cd stress,supply of NO₃^–enhanced the ¹⁵NO₃^–content in roots and shoots;under Zn stress,supply of NO₃^–reduced the ¹⁵NO₃^–content in roots;under Cd+Zn stress,supply of NO₃^–reduced the ¹⁵NO₃^–content in shoots.5.30 ABC genes in DPW were characterized from the transcriptome data and classified into seven subfamilies（ABCA-ABCG）.Among them,24 ABC transporters were newly found in wheat.The expressions of 13 ABC genes in roots and shoots under different metal stresses were analyzed.All these genes were differentially regulated by Cd（except ABCE2,ABCF4,and ABCF6 in roots）.Meanwhile,these genes were also differentially regulated by other metals including Zn,Fe,copper（Cu）,magnesium（Mg）,and nickel（Ni）.