The Function And Mechanism Of The Putrescine Synthesis Pathway In Potato Freezing Tolerance

Potato(Solanum tuberosum L.)is the most important non-grain crop in the world and plays an important role in ensuring food security.However,potato is frost sensitive and doesn't possess cold acclimated capacity,hence there is a serious threat of frost stress for potato production in different regions of China.Although previous studies in our laboratory have shown that polyamine metabolic pathway might play an important role in potato freezing tolerance,the mechanism underlying potato freezing tolerance is still unclear.This study reveals that the synthesis of putrescine in the polyamine pathway has an important effect on cold acclimated freezing tolerance of potato.The main results are shown as follows:1.The expression of genes and the content of main metabolites in the polyamine pathway were determined in two series of potato genotypes with distinct freezing tolerance.The results showed that the arginine decarboxylase gene ADC1 and its downstream product putrescine were specifically up-regulated in freezing-tolerant genotypes after exposure to low temperature.Therefore,the synthesis of putrescine may play an important role in potato freezing tolerance.2.Exogenous putrescine was applied to the freezing sensitive potato genotype E-potato 3(E3),and freezing tolerance of the plants treated with putrescine and control plants was tested.The results of phenotypic observation,injury scores and electrolyte leakage measurements showed that there was no significant difference in freezing tolerance between the putrescine treated plants and control plants.But after cold acclimation of 4 °C for 14 d and subsequent freezing treatment of-2 °C for 8 d,the injury scores leakage electrolyte of the putrescine treated plants were lower than the wild type.And after cold acclimation,electrolyte leakage of the putrescine treated plants were lower than the wild type at-3 °C and-4 °C,therefore freezing tolerance of the putrescine treated plants was significantly stronger than that of the control plants after coldacclimation.This study proved for the first time that putrescine could enhance the cold acclimated freezing tolerance of potato.3.Cloning of potato arginine decarboxylase gene ADC1 showed that there were two alleles in freezing-resistant genotype(S.acale,Aca)and freezing-sensitive genotype(S.tuberosum,Tub).The length of ADC1 CDS was 2163 bp,encoding 720 aa,and the identity of CDS and amino acid were 98.79% and 99.38%,respectively.Cluster analysis of its protein sequences from different species showed that two critical active sites K and C existed in N-terminal and C-terminal respectively were conserved in ADCs,except in three Brassica napus ADCs.The conservative of critical sites indicates that ADC has important biological functions in plants.The 2500 bp upstream sequence of ADC1 CDS region was cloned,and predicted to contain various responsive cis-elements in ADC1 promoter,such as ABRE element for ABA response?LTR element for low temperature and p-box for light and so on,and the variation of sequence and elements in 600-1000 bp upstream of ATG in ADC1 might related to the difference responsive to cold in different genotypes.Meanwhile the expression of ADC1 was highest in leaf and flower,and ADC1 could respond to ABA,drought and salt stress.These results suggest that ADC1 may have multiple functions in potato.4.To confirm the role of ADC1 in potato freezing tolerance,SaADC1 was transformed into the freezing sensitive variety E3.The expression of ADC1 and putrescine content in the overexpressing lines OE-ADC1-1 and OE-ADC1-2 were significantly higher than those in the control lines before and after cold acclimation.Phenotypic observation,injurey scores and electrolyte leakage measurements showed that there was no significant difference in freezing tolerance between transgenic lines and the wild type without cold acclimation,but the freezing tolerance of transgenic plants was significantly stronger than that of wild plants after cold acclimation.These results proved for the first time that ADC1 could enhanced cold acclimated freezing tolerance in potato.5.To study the mechanism of putrescine synthesis pathway affecting freezing tolerance,the expression of CBFs was detected in putrescine-treated plants and SaADC1 overexpression lines.It was found that the expression of four CBFs in potato was enhanced in varying degrees compared with the control.And the expression of four CORs genes downstream of CBFs was also enhanced in transgenic lines.In this study,it is proved ADC1-mediated putrescine synthesis may enhance the cold acclimated freezing tolerance by enhancing the expression of related genes in CBF pathway in plants for the first time.In order to explore whether the pathway of putrescine synthesis affects ABA signaling pathway,the ABA content in putrescine-treated plants and SaADC1 overexpressing lines were detected.The results showed that there was no significant change in ABA content compared with the control.Therefore,the pathway of putrescine synthesis does not affect the level of ABA.However,there are several ABRE elements in the promoter of ADC1,and ABA induces the expression of ADC1.It is speculated that ABA signaling pathway may affect putrescine synthesis.By using Nicotiana benthamiana transient expression system,seven AREB/ABF/ABI5 transcription factors were found to interact with ADC1 promoter to enhance the expression of the reporter gene.Moreover,AREB3 and ABI5 were induced by low temperature and ABA.Thus,it was supposed that low temperature affected the expression of ADC1 through ABA signaling pathway in potatoes,which was related to AREB3 and ABI5.6.In order to further study the regulatory network of putrescine synthesis pathway on potato freezing tolerance,RNA-Seq of OE-ADC1-1 was carried out in this study.The enrichment analysis of differentially expressed genes showed that the enrichment pathway was mainly related to substance transport and hormone signal transduction after4 °C for 1 hour,while it was mainly related to substance metabolism after 4 °C for 2 days.Therefore,ADC1 can affect not only the signal pathway,but also the metabolism of substances in vivo,which may be related to the cold acclimated freezing tolerance of potato.Further analysis showed that the expression of five fatty acid desaturases in the "oxidoreductase activity" pathway and three glutathione S-transferase genes in the "Glutathione metabolism" pathway increased significantly in OE-ADC1-1 after 4 °C for1 hour,suggesting that the putrescine synthesis pathway affected the oxidation-reductionhomeostasis of potato under low temperature.This study revealed the function and mechanism of putrescine biosynthesis pathway on potato freezing tolerance,which lays a theoretical foundation for further research.Besides,this study also provides genetic resources for improving frost tolerance of cultivated potato.