NtPHYB1 Regulates The Functions Of Seed Photodormancy And Post-ripening

Photodormancy existed in newly harvested tobacco seeds,which could not germinate in darkness,but could release photodormancy after-ripening.Studies have shown that phytochrome regulates the germination of photosynthetic seed,but whether they are involved in regulating the function of photodormancy and after-ripening of seeds and the mechanism of action are still unclear.Using WT(K326),NtPHYB1^K326-GFP overexpression(B1,B3,B4)and NtPHYB1^K326-RNAi deficiency(B5,B6,B7)as materials,this study studied the germination differences of intact seeds,deseared seeds and in vitro embryos through anatomical experiments.On this basis,the sensitivity of three types of seeds to GA₃,paclobutrazole,abscisic acid and fluridinone was studied.The changes of endogenous GAs and ABA contents during the germination of newly harvested seeds were studied by liquid chromatography tandem mass spectrometry.The PHYB1 gene,GA and ABA metabolism and the expression levels of signaling genes(GA3ox2,GA2ox2,GAI,NCED6,CYP707A1,ABI3,ABI5)and germination-related genes(XTH2,TOC1)during the germination of newly harvested and after-ripening seeds were studied by real-time quantitative PCR.The results are as follows:(1)Under light condition,the expression of XTH2 was up-regulated and the expression of NCED6,ABI3,ABI5,GA2ox2 and TOC1 were down-regulated during the germination of wild-type newly harvested and after-ripening seeds.The expression of GA3ox2 and CYP707A1 in wild-type newly harvested seeds was up-regulated at the early stage of germination.Hormone quantitative analysis showed that the content of ABA decreased gradually during the germination of newly harvested seeds under light,while the content of active GAs increased first and then decreased.However,the content of ABA increased gradually and the content of active GAs had no significant change during the germination process in the dark.These results indicated that the expression and content of GA and ABA signals and metabolic genes were controlled during the germination process of photodormancy seeds,and then the ratio of ABA/GAs was regulated to regulate seed dormancy and germination.(2)After-ripening of seeds can promote the expression of GA3ox2,CYP707A1 and XTH2,and inhibit the expression of NCED6,ABI3,ABI5,GA2ox2,GAI and TOC1.In addition,seeds gradually lost their sensitivity to exogenous GA₃,ABA,PAC and FLU with the subsequent extension of after-ripening time.(3)NtPHYB1 can regulate photodormancy of newly harvested seeds in coordination with light.There was no significant difference in seed germination between newly harvested and after-ripening seeds under light,but the seed germination rate decreased significantly under dark.Exogenous addition of GA₃and after-ripening could promote the germination of seeds under darkness.Compared with the other two types of seeds,the germination rate of NtPHYB1^K326-RNAi seeds under darkness was lower.(4))The expression level of NCED6 in NtPHYB1^K326-RNAi seeds was significantly higher than that in wild-type and NtPHYB1^K326-GFP seeds,and both newly harvested and after-ripening seeds showed a similar pattern.NtPHYB1^K326-RNAi seeds were also more sensitive to exogenous ABA,PAC and Fl U than the other two types of seeds.These results suggested that PHYB1 may regulate the photodormancy and after-ripening of seeds by mediating ABA signaling and the expression of metabolic genes.(5))NtPHYB1 has the function of regulating after-ripening of tobacco seeds in coordination with temperature change.The natural temperature change environment is beneficial to the after-ripening of wild and overexpressed NtPHYB1^K326-GFP seeds,while the constant temperature environment is beneficial to the after-ripening of defective NtPHYB1^K326-RNAi seeds.(6)The desiccated seeds could germinate under light and darkness,but in vitro embryos could not germinate under both conditions.