Study On Involment Of Reactive Oxygen Species In Root Development Of Maize Under Zinc Deficiency

Zinc（Zn）is one of the essential micronutrients for plant growth and development.The contents of reactive oxygen species（ROS）including O₂^·-and H₂O₂ increase in plants under Zn deficiency stress with the enhanced ROS formation and the lower ROS scavenging ability.However,less is known about whether excessive levels of ROS mediate root development and growth under Zn deficient condition.The maize variety Zhengdan 958 was chosed to grow with nutrient solution culture.Roots and shoots were sampled at 10 and 15 days of transplant after the treatments（DAT）of 0（–Zn）and 1μM（+Zn）supply.Exogenous addition of H₂O₂ and its scavenger（N,N-dimethylthiourea,DMTU）,and a caltlase inhibitor（aminotriazole,AT）were condcted.The morphological and histological features of roots were observed.The distribution of ROS in roots were anlyzed by chemical staining and fluorescent probe technology.The differential expression of related genes and proteins in root were analyzed through proteomics and transcriptomics methods.The objective of this study was to reveal possible roles of ROS in mediating root development and growth of Zn deficient maize plants.The main results are summarized as followings:Zinc deficiency affected maize growth and physiological response significantly.Without Zn application,maize shoots became dwarf and young leaves showed yellow.The chlorophyll content was decreased and photosynthesis parameters such as net photosynthesis rate（Pn）,stomatal conductance（Gs）,and transpiration rate（Tr）were inhibited by Zn deficiency stress.At 10 DAT,Zn limitation had no significant effect on root growth and biomass.At 15 DAT,insufficient Zn supply reduced root biomass,the length of crown roots and total number of crown roots and lateral roots.Zn deficiency decreased significantly Zn concentrations in plants while increased the contents of Cu,Mn and Mo in roots and the contents of P,Ca,Mg and Cu in shoots.Zinc deficiency affected the accumulation and distribution of ROS in maize roots.Zn deficiency resulted in an increase of O₂^·-and H₂O₂ in crown roots and lateral roots at 10 and 15 DAT.The high ROS levels were in the apical meristem and elongation zones of crown roots,and in the apical meristem of lateral roots.The treatment of exogenous H₂O₂ and catalase enzyme inhibitor AT increased the accumulation of H₂O₂ in crown roots and lateral roots in Zn-sufficient or Zn-deficient maize seedlings.The concentrattion of H₂O₂ in crown roots and lateral roots was decreased with the exogenous aadition of H₂O₂ scavenger DMTU.H₂O₂ mediated the growth and development of maize roots under zinc deficiency stress.The exogenous addition of H₂O₂ scavenger DMTU did not affect lateral root development while reduce the inhibition of crown root growth by Zn deficiency.In contrast,exogenous addition of H₂O₂ and catalase inhibitor AT inhibited crown root growth even under Zn adeqate supply.Anatomical observation showed that Zn deficiency inhibited the length of elongation zone and mature zone in crown roots,while increased with the treatment of exogenous DMTU.Exogenous AT and H₂O₂ addition significantly inhibited the length of the meristem zone and the elongation zone of crown roots with Zn supply.Transcriptomic response of maize roots under zinc deficiency stress.Transcriptome data found 179 and 533 differentially expressed genes between Zn deficient and sufficient conditions in roots at 10 and 15 DAT,respectively.Under Zn deficient stress at 10 DAT,124 and 55 genes were up-regulated and down-regulated,while at 15 DAT,173 and 360 genes were up-regulated and down-regulated,respectively.On the basis of Kyoto Encyclopedia of Genes and Genomes（KEGG）enrichment analyses,the up-regulated genes at 10 DAT were signicantly enriched in cysteine and methionine metabolism,amino acid biosynthesis,secondary metabolite synthesis,sulfur metabolism and other pathways,while the down-regulated genes were signicantly enriched in phenylpropanoid biosynthesis,glutathione metabolism,benzoxazine biosynthesis,starch and sucrose metabolism,and other pathways.The up-regulated genes at 15 DAT were only significantly enriched in sesquiterpenoid and triterpenoid biosynthesis and the down-regulated genes were enriched in phenylpropanoid biosynthesis,biosynthesis of secondary metabolites and metabolic pathways,etc.Proteomic response of maize roots under zinc deficiency stress.A total of 116differentially expressed proteins were found in roots at DAT 10 in proteome data,among which 55 proteins were up-regulated and 61 proteins were down-regulated in roots without Zn addition.At 15 DAT,113 differentially expressed proteins were identified in Zn deficient roots compared with the control,and 23 and 90 proteins were up-regulated and down-regulated,respectively.KEGG pathway analysis indicated that the up-regulated proteins at 10 DAT were significantly enriched in cysteine and methionine metabolism,linoleic acid metabolism,phenylpropanoid biosynthesis,metabolic pathways and other pathways,while the down-regulated proteins were significantly enriched in amino sugar and nucleotide sugar metabolism,glutathione metabolism,cyanoamino acid metabolism and C5-branched dibasic acid metabolism.At 15 DAT,the significantly enriched KEGG pathways mapped by the up-regulated proteins were carbon metabolism,propanoate metabolism,sulfur metabolism and beta-alanine metabolism.The KEGG pathways enriched with down-regulated proteins were mainly involved in ribosome,glycolysis/gluconeogenesis,carbon metabolism,glycine,serine and threonine metabolism and pyruvate metabolism.The down-regulation of several genes associated with cell wall extension,such as cellulose synthase,xyloglucan endtransglucosylase/hydrolase and beta expansin 8,were found in the transcriptome and proteome data of Zn-deficient root,and the down-regulated beta expansin 8 was verified by PRM analysis.In summary,Zn deficiency stress caused high H₂O₂ and O₂^·-levels in roots and decreased crown root elongation.Exogenous addtion of H₂O₂ scavenger DMTU reduced the content of H₂O₂ in roots and the inhibitory effect of Zn deficieny on root elongation.Under Zn supply,the addition of H₂O₂ and catalase inhibitors AT resluted in short crown roots.It is suggested that hydrogen peroxide might involve in the inhibition of crown root development in maize plants subjected to Zn deficiency stress,whcih may account for the down-regultion of genes related to cell wall extension through transcriptomic and proteomic analysis.