Study Of Nucleoporin MdNup54/62 Regulation Flowering And Heat Stress Response In Apple

The difficulty of‘Fuji'apple flowering is the key problem restricting the development of apple industry in China.And in recent years,with the global warming,the problems of warmer winter and extreme high temperature further hindered the development of apple industry in China.Thus strengthening the research on the molecular mechanism of apple flowering and heat stress response are important,as well as to explore the molecular mechanism of temperature affecting flowering.Studies have shown that Nucleoporins(Nups)which control large molecules such as proteins and RNA material in and out of the nucleus play an important regulatory role in plant flowering and abiotic stress,etc.In this study,we use‘Nagafu No.2'as the material.And the sequence characteristics,expression characteristics and interaction proteins of apple MdNup54 and MdNup62 were analyzed by bioinformatics analysis,Yeast two-hybrid,luciferase complementary,genetic transformation,and other experimental techniques.And the mechanism of MdNup62 in flowering and high temperature stress response was also studied.The main results are as follows:(1)We systematically identified and analyzed the expression of apple Nups.And we identified 38 apple Nups,only lacking Nup58 compared with Arabidopsis Nups.Phylogenetic tree analysis showed that the clustering of apple Nups and their homologous sequences in Arabidopsis were the closest,indicating that the plant Nups were well conserved.Moreover,apple Nups lack a common conserved domain and consist of Nup62,Nup93 andNup107 subcomplexes,which form the nuclear pore complex NPC.In the tissue-specific expression analysis,11 apple Nups,including MdNup54,and MdNup62,had the highest expression levels in flower buds,suggesting that they might play a role in apple flowering.Additionally,auxin treatment inhibited apple flowering,and the expression levels of MdNup54,MdNup62,MdNup93,MdNup96,MdNup133,MdNup160 and MdHOS1 in flower buds were significantly inhibited by auxin treatment,indicating that these seven genes were involved in apple flowering.(2)We screened interacting proteins of apple MdNup54 and MdNup62.Respectively,a total of 139 and 62 potential interacting proteins of MdNup54 and MdNup62 were identified by yeast two-hybrid screening of apple flower bud c DNA library.And yeast two-hybrid verification showed that MdNup54 interacted with Md MYB11,Md KNAT4,Md KNAT6,and MdHSP70.Additionally,MdNup62 interacts with MdHSFA1a/b/d/e,MdHSFA9a/b,MdNup54,and MdNup62.Further,the interaction between MdNup62 and MdHSFA1d,MdHSFA9b,MdNup54 were verified by the fluorescence complementation assay,and the interaction between MdNup54 and Md MYB11,Md KNAT4,Md KNAT6,MdHSP70 were also verified.Moreover,the interaction between MdNup62 and MdHSFA9b was verified by pull-down experiment.(3)Apple MdNup62 was involved in flowering and heat stress response pathways.MdNup62 was highly expressed during apple flower bud physiological differentiation stage,and was induced by high temperature.Subcellular localization verified that MdNup62 was located in the nuclear pore.And MdNup62 overexpressed lines had obvious early flowering phenotype,and the expression levels of Arabidopsis flowering genes At FT,At LFY,and At AP1 were significantly increased.However,MdNup62-OE Arabidopsis lines could not tolerate high temperature,and the expression of HSPs(At HSP101,At HSP22.0-ER,At HSP21,At HSP70T-2)were significantly decreased.And the levels of O^2-,H₂O₂,and MDA were significantly increased,while the activities of SOD,POD,and CAT were decreased.Moreover,under high temperature treatment,MdNup62-OE tomato lines were also not resistant to high temperature.These results suggested that MdNup62 involved in flowering and heat stress response.(4)Apple Heat shock factors,MdHSFA1d and MdHSFA9b,were involved in flowering and heat stress response pathways.And we found that MdHSFA1d and MdHSFA9b have high expression level during apple flower bud physiological differentiation stage by q RT-PCR.Subcellular localization showed that MdHSFA1d and MdHSFA9b were both located in the cytoplasm and nucleus.The same as MdNup62,both of MdHSFA1d and MdHSFA9b overexpressed lines significantly promoted early flowering in Arabidopsis,and the expression levels of flowering genes At FT,At LFY,and At SOC1 were significantly increased.However,under high temperature treatment,MdHSFA1d-OE and MdHSFA9b-OE lines significantly enhanced the plant's high temperature tolerance,and the expression level of downstream HSPs(At HSP101,At HSP22.0-ER,At HSP21,At HSP70T-2)were significantly increased.In conclusion,apple MdNup54 and MdNup62 can directly interact with non-nucleopore members such as transcription factors and functional proteins.MdNup62 is involved in regulating flowering and heat stress response by interacting with MdHSFA1d and MdHSFA9b.These results enriched the molecular mechanism of apple flowering and heat stress response,and provided clues for the study of the molecular mechanism of temperature affecting flowering.