Functional Characterization Of Tulip Aquaporin Gene TgPIP1;3 And TgNIP2;1 In Response To Heat And Drought Stress

Tulip(Tulipa gesneriana L.)is an autumn bulbous flowers of the genus Tulipa of the family Liliaceae,which is widely used in flower beds and flower sea landscape and production of fresh cut flowers.As an autumn planted bulbous flower,tulips are suitable to be grown in cool area and are sensitive to heat condition.High temperatures not only shorten the flowering period of tulips,but also affect the development of tulip bulbs.Recently,tulip fresh-cut flowers are more and more popular,which show susceptibility to water loss stress after harvest,resulting in a rapid decline in the flower quality.Therefore,it is important to investigate the genes related to abiotic stress response in tulips and characterize the molecular mechanism of abiotic stress response in order to partially solve the problems of tulip industry.In this study,using the full-length transcriptome data of tulip,two aquaporin genes,TgPIP1;3 and TgNIP2;1,were screened and cloned.We analyzed the expression patterns and subcellular localization under high temperature and drought stress,and further investigated gene functions by heterologous overexpression and silencing in tulip.The results of the study are as follows:1.Both TgPIP1;3 and TgNIP2;1 were changed by high temperature and drought stress in tulip.Experimental results of RT-q PCR showed that the expression of TgPIP1;3and TgNIP2;1 were significantly up-regulated under heat stress.Upon drought stress treatment,TgPIP1;3 and TgNIP2;1 were significantly down-regulated in tulip flowers and roots,indicating that TgPIP1;3 and TgNIP2;1 may play different functions in tulip in response to heat and drought stresses.Subcellular localization assay showed that both TgPIP1;3 and TgNIP2;1 are localized to the cell membrane and nuclear membrane.2.TgPIP1;3 or TgNIP2;1 improved heat tolerance of Arabidopsis.Under heat stress condition,the leaf damage index of TgPIP1;3 and TgNIP2;1 overexpressed transgenic lines were significantly lower than that of the wild type,and the electrical conductivity and MDA content were also significantly lower than that of the wild type.Realtime q RT-PCR assay showed that the expression levels of six heat stress responsive genes,including HSFA2,HSP17.6,HSP70,HSP101,DREB2 A and DREB2 B,were significantly higher in TgPIP1;3 and TgNIP2;1 transgenic Arabidopsis than those in the wild type after heat stress treatment.All the results indicated that TgPIP1;3 and TgNIP2;1 improved heat tolerance in Arabidopsis.3.TgPIP1;3 and TgNIP2;1 function as negative regulator in response to drought stress.The cotyledon greening rate of TgPIP1;3 and TgNIP2;1 transgenic Arabidopsis was significantly slower than the wild type under low concentration of mannitol stress.Moreover,the survival rate of TgPIP1;3 and TgNIP2;1 transgenic Arabidopsis under high concentration of mannitol stress was significantly lower than the wild type.The results of physiological indicators showed that the cell membrane damage was significantly greater in the transgenic lines under mannitol stress.Additionally,TgPIP1;3 and TgNIP2;1 was silenced in tulip flowers by VIGS technology.The results showed that under drought stress,the water loss rate of tulip petals of silenced TgPIP1;3 or TgNIP2;1 was significantly slower than that of the TRV2 control,and the petal reactive oxygen content was also significantly lower than that of the control.4.Investigation of TgNIP2;1 promoter.Totally 1507 bp of TgNIP2;1 promoter was cloned,and cis-acting element analysis were performed.The results showed the hormone-responsive elements such as abscisic acid,gibberellin and growth hormone are present in the TgNIP2;1 promoter.Dual luciferase results showed that TgRNF144A-A and TgDi19-2a could bind to the promoter of TgNIP2;1 which might function as up-stream regulator.In conclusion,two tulip aquaporin genes,TgPIP1;3 and TgNIP2;1 were identified,which were significantly induced by heat and drought.The functions of two genes under heat and drought stress were characterized and two putative upstream regulators of TgNIP2;1 were identified.These results provided a theoretical basis for further analysis of the molecular mechanism of stress tolerance in tulip.