Molecular Mechanisms Of Regulation Of The Environmental Signaling Molecule NO And Its Signal Transduction Components On The Potato Tuberization

Potato is an important staple crop due to its richness in starch,protein and other nutrients,and its tuber tuberization is very complicated,which is a result of the interaction between external environment and internal factors.To investigate the relationship between NO and tuberization,we used the diploid potato as the material and added different concentrations of NO donor SNP in MS medium for potato tuberization to determine the most suitable SNP concentration for tuberization.We then analyzed the promoter of StSP6A that responded most strongly to SNP-induced tuberization,and we selected the MYC gene family as a potential regulator of the promoter for further identification.The expression of its family members during SNP-induced tuberization was analyzed.Subsequently,StSP6A was knocked out and we sequenced the transcriptome of stolon samples from sp6a null-mutants and wildtype plants to analyze the differentially expressed genes associated with tuberization,and we selected StFPF as a candidate gene and analyzed its function in tuberization.Finally,we found that stress can enhance gene editing efficiency during our experiments,and because of this we designed experiments to investigate the efficiency of gene editing under salt and osmotic stress conditions,with the following main findings:1.Screening appropriate concentration of SNP for tuberization and its regulation of expression of key genes for tuberizationThe results showed that SNP at 0.25 mM significantly increased the number of tubers,the weight of individual tuber and the total weight of tubers,and the expression analysis of 10 tuberization-related genes during tuberization induced by SNP at 0.25 mM showed that NO induced tuber formation by regulating the expression of tuberization-related genes.Among them,StSP6A is a key gene controlling potato tuber formation.The addition of SNP can significantly up-regulate the expression of StSP6A,while the knock-out of StSP6A can eliminate the effect of exogenous NO-induced potato tuberization,indicating that StSP6A plays a key role in NO-induced potato tuber formation.2.Genome-wide identification of potato MYC gene family and analysis of expression patterns during SNP-induced tuberizationTo analyze the promoter of StSP6A,we selected a high number of MYC binding sites and performed genome-wide identification of the MYC gene family,bioinformatics analysis and analysis of its expression pattern under SNP-induced tuberization.The results showed that a total of 14 MYC family members were identified in potato,encoding amino acids 402-702 aa and distributed on six chromosomes.Analysis based on the expression pattern of MYC family in different tissues showed significant differences in the expression levels of each member.The expression pattern was analyzed during SNP-induced tuberization,in which the expression of StMYC5 and StMYC7 was significantly up-regulated at 6 and 10 d,indicating that StMYC5 and StMYC7 may be involved in the NO-induced tuberization.Overexpression of StMYC7 in potato can significantly improve the efficiency of potato tuberization,indicating that StMYC7 may have a promoting effect on potato tuberization.3.Analysis of differentially expressed genes involved during stolon developmentRNA-seq expression analysis of stolon from wild-type diploid potatoes at the three tuberization stages and the corresponding times of sp6a null-mutants showed that there were fewer differentially expressed genes from the non-hooked to the hooked stage of stolon,with a total of 167 DEGs（146 up-regulated and 21 down-regulated）mainly involved in:signal transduction（CML,MAPKKK17₁8,MKS1,MYC2）,hormones（GA20ox,JAZ,JAR1₄₆,ACS1₂₆）and cell wall（TCH4）-related genes.There were more differentially expressed genes from hooked stage to tuber initiation stage,with a total of 3479 DEGs（1755 up-regulated and 1724 down-regulated）,mainly related to:sugar metabolism,phytohormones,antioxidants,signal transduction,cell wall,photoperiod,and rhythm control.4.The StSP6A co-expression gene StFPF can promote potato tuberizationThrough RNA-seq analysis and gene microarray results from the database,we found a gene co-expressed with StSP6A,StFPF,and we identified its homologs in potato.We analyzed the expression pattern of StFPF during tuberization,which showed that the expression of StFPFl and StFPF2 was significantly higher at the tuberization.We then genetically transformed these two genes and phenotypically characterized the transformed plants,and the results showed that StFPF1 and StFPF2 could slightly increase the potato tuber yields,and StFPF2 could significantly improve the tuberization efficiency of the sp6a mutant lines.5.Salt and osmotic stresses can improve genome editing efficiencyIn this work,we investigated the effect of NaCl and mannitol stress on the efficiency of Agrobacterium-mediated genome editing,and the results showed that the efficiency of root regeneration decreased significantly with increasing stress,while we rapidly screened transformant root lines using a GFP reporter gene,and based on the sequencing results,we found that all transgenic roots obtained under NaCl concentrations higher than 20 mM or mannitol concentrations higher than 100 mM carried mutations.Analysis of the mutation types revealed that the highest probability of chimeric mutations ranged from 62.50%to 100%,and the nullmutations were also present.Among them,the highest probability of null-mutations was found at a NaCl concentration of 10 mM,with a frequency of 37.5%.Also,we tested the potential off-target sites and we did not detect any off-target mutations.Our study shows that salt and osmotic stress can improve the efficiency of CRISPR/Cas9mediated genome editing and does not lead to off-targeting.In summary,the signaling molecule NO and its signal transduction components are involved in the induction and formation of diploid potato tubers,with StSP6A playing a key role in regulating the tuber-related gene network.These findings provide a theoretical basis for a deeper understanding of the molecular mechanism of potato tuber formation and for subsequent precision molecular breeding.