Involvement Of The Environmental Sensing Signal Molecule Hydrogen Peroxide In The Regulation Of Potato Tuberization And Its Molecular Mechanism

Tuber induction and formation of potato is the most pivotal event in their growth,development and determination of yield,which involves the interaction of multiple environmental factors with plant hormones and signal molecules,as well as the regulations of this interaction on large number of key genes and multiple signal transduction and metabolic pathways.Reactive oxygen species（ROS）have dual functions acting as signal molecules and strong oxidants in plant cells,and hydrogen peroxide（H₂O₂）is the main form of ROS,can act as an important second messenger in the signal transduction pathways and trigger physiological,biochemical and gene expression changes in plants to produce corresponding traits and enhance adaptability.The plasma membrane NADPH oxidase,also known as respiratory burst oxidase homolog（Rboh）,is a key enzyme for ROS production.Under normal conditions,excessive ROS can be scavenged by various antioxidative defense mechanisms.ascorbic acid（AsA）is one of the universal non-enzymatic antioxidants having substantial potential of not only scavenging ROS,but also modulating a number of fundamental functions in plants both under stress and non-stress conditions.ROS and ascorbic acid（AsA）both are the most abundant oxidants and antioxidants in plant cells,respectively,which jointly participate in the regulation of the cellular redox status.However,it is little known that whether and how ROS and AsA are involved in the induction and formation of potato tubers.In this study,diploid potato germplasms were used to study the effects of H₂O₂ and AsA on the induction and formation of potato tubers,and the following results were obtained:1.Screening of tuberization ability of diploid potato germplasms and regulation of sucrose on tuber formation and expression of the key genesA total of 102 diploid potato germplasms were cultured in vitro under normal growth condition for 3 months,the tuberization ability of these plantlets were investigated,and five diploid potato germplasms with higher tuberization ability were screened out.The results of treating the five diploid materials with different concentrations of sucrose showed that enhancement of sucrose concentration could shift to an earlier date of tuber formation and significantly increase the tuberization frequency of the diploid potato materials,and the appropriate concentration was 6%to 10%.The expression analysis of 10 tuberization-related genes during tuber formation indicted that sucrose could induce tuber formation by regulating the expression of the tuberization-related genes.Among them,Solanum tuberosum self-prunning 6A（StSP6A）is a key gene controlling tuber formation of potato,and the increase of sucrose concentration can lead to the significant upregulation of StSP6A expression,indicating that StSP6A plays a key role in the sucrose-induced tuber formation in potato.2.H₂O₂ participates induction and formation of potato tubers by activating tuberization-related signal transduction pathwaysROS accumulation was observed by histochemical staining,and H₂O₂ levels and superoxide anion O₂^·-production rate were measured in stolons/tubers of potato.The results showed that there was an obvious accumulation of ROS(including H₂O₂,O₂^·-and total ROS)during tuber induction and formation in stolons/tubers,especially in the hook-like subapical part of stolons prior to tuberization detected by staining observation and quantitative measurement.Furthermore,exogenous H₂O₂ treatment significantly enhanced percentage of tuber formation.In addition,expression analysis of 9 key tuberization-related genes demonstrated that the H₂O₂-induced tuberization could be associated with H₂O₂-controlled regulation of these tuberization-and signaling pathway-related genes,especially StSP6A,which was dramatically up-regulated during early stage of tuber induction and H₂O₂ treatment.Knockdown of StSP6A seriously inhibited the formation of tubers induced by exogenous H₂O₂.These findings indicate that H₂O₂ accumulation in stolons might plays an important role acting as a signaling molecule to initiate tuber induction,H₂O₂-induced tuber formation is triggered by regulating the tuberization-related gene expression and activating signal transduction pathways,and StSP6A is a pivotal player in H₂O₂-induced tuber formation in potato.3.Genome-wide identification of Rbohgene family and expression analysis during tuber formation in potatoIn the present study,genome-wide identification,bioinformatics and expression profile analysis of Rbohgene family were performed,and expression pattern of each member of StRbohgene family during different stages of tuber formation was analyzed in potato.The results showed that a total of 8 Rbohgene family members were identified in potato,encoding 745 to1461 amino acids.The StRbohgene family members were distributed on 6 chromosomes and divided into 5 subfamilies（II-Ⅵ）.The results of gene structure and protein domain analysis showed that they had obvious subfamily characteristics.The expression patterns of StRbohgene family members during different stages of potato tuberization showed that StRbohs had significant differences in expression patterns and levels at different stages of tuber formation.StRbohA was the most significantly up-regulated gene,suggesting that StRbohA may be involved in the induction and formation of potato tubers by mediating the production of ROS.4.Cloning of StRbohA gene and effect of StRbohA overexpression on tuberization in diploid potato plantsBased on the above results,the StRbohA gene,which was significantly increased during tuber induction and formation,was cloned,overexpression vector was constructed,and genetic transformation was carried out in potato.A total of 8 positive lines were obtained.Analysis of StRbohA gene expression in 8 overexpressed lines showed that StRbohA expression in 7 lines was significantly higher than that in wild-type（WT）.The analysis of tuberization ability of the overexpressed lines showed that the overexpressed plantlets shifted to an earlier date of tuber formation and their tuberization frequency was significantly higher than that of WT.The results from measuring the contents of H₂O₂ and sucrose in the leaves of the four overexpressed lines showed that contents of H₂O₂ and sucrose in the overexpressed plantlets significantly increased as compared with WT.These results indicated that overexpression of StRbohA could significantly increase the content of H₂O₂ in leaves of the overexpressed plantlets,and may further induce tuber formation by increasing sucrose content.5.Effect of exogenous ascorbic acid on potato tuber formation and its possible molecular mechanismThe effect of exogenous AsA treatment on potato tuber formation and the expression of tuberization-related genes,as well as the influence of knockout of the key gene StSP6A on the AsA-induced tuberization were investigated in the present study.The results showed that exogenous AsA treatments with 1 to 5 m M could significantly induce tuber formation,and exogenous AsA treatments also significantly affected the expression of those tuberization-related genes,especially StSP6A,which was significantly up-regulated during the early stage of tuber formation and AsA treatment.Knockout of StSP6A resulted in seriously inhibition of the effect of exogenous AsA-induced tuberizations in potato.These findings indicate that AsA-induced tuber formation is triggered by regulating the tuberization-related gene expression,and StSP6A plays key role in AsA-induced tuber formation in potato.In conclusion,sucrose,H₂O₂,the main form of ROS,and AsA were all involved in tuber induction and formation of diploid potato.They may regulate tuberization-related gene network through different pathways,further control tuber induction and formation,and StSP6A plays a key role in regulating the tuberization-related gene network.These findings could lay a theoretical foundation for a deeper understanding of molecular mechanism of tuber formation and subsequent precise molecular breeding in potato.