Functional Analysis Of Arabidopsis Inositol Polyphosphate Kinase Gene (AtIPK2?) In Flowering Time Regulation And Seedling De-etiolation

Inositol polyphosphate kinase is an important component in inositol phosphate signaling pathway,it phosphorylates Ins(1,4,5)P3 to Ins(1,4,5,6)P4 and Ins(1,3,4,5,6)P5.Inositol polyphosphate kinases in plants are homologous with yeast IPK2 and IPMK in mammals.IPK2 is involved in stabilizing transcriptional complex Mcml-ArgR in the arginine metabolic pathway.IPMK not only acts as component of transcriptional complexes,but also participates in histone modification.Arabidopsis has two homologous IPK2 including AtIPK2a and AtIPK2?,which are highly identical in protein sequences and structures.They are both located in nucleus and cytoplasm.We have reported that AtIPK2f? regulates the expression of genes responsible for axillary shoot branching through auxin signaling pathway,and AtIPK2a is functional redundantly wirh AtIPK2?,both of which are involved in pollen development,pollen tube guidance and embryogenesis.All of these studies showed that AtIPK2? has multiple functions in Arabidopsis development.The timely floral transition of plants in suitable environment is required for reproductive growth and fertilization.The intricate regulation network of plants is evolved for responding different environmental conditions and inducing floral transition.The time of floral transition is represented by flowering time.There are mainly five signaling pathways according to the researches,which are photoperiod pathway,temperature pathways including vernalization pathway and ambient temperature pathway,autonomous pathway,age pathway and GA pathway.All of these pathways consist of many protein factors and transduce the signals of flowering time to the downstream regulators including CONSTANS(CO),FLOWERING LOCUS C(FLC)and integrators such as FLOWERING LOCUS T(FT)and SUPPRESSOR OF OVEREXPRESSION OF CO 1(SOC1).These factors are transcriptional and post-transcriptional modulated,and the histone modification is important to transcriptional regulation.Researches revealed that many proteins,small RNAs,non-coding RNAs and other molecules are related to the histone modification in the recent years.The complexity of the regulation suggests that more factors are still waiting to be discovered.Light is not only the energy source but also acts as an important signal for plant growth and development.It is the early signal in photoperiod pathway and plays a key role in seedling de-etiolation,which is also called photomorphogenesis of seedlings.There are photoreceptors absorbing different wavelength of light in plants,which includes Phytochromes,Cryptochrome and UVR8.They response to the light and mediates different signaling pathways.These pathways interact with each other to form a network.Years of studies about photomorphogenesis revealed many factors,among which there are factors play important roles in floral transition and other developmental processes.There are some reports about the functions of AtIPK2? in the recent years,but its mechanism in transcriptional regulation and roles in other developmental processes are still unknown.In this study,we discovered that the AtIPK2? is functional in both of the flowering time regulation and seedling photomorphogenesis,these functions with possible regulation mechanisms were analyzed by various biological methods.The main results are the following:1.AtIPK2? is involved in the regulation of flowering time in Arabidopsis.The atipk2? mutant flowers earlier and the overexpression lines flower later compared to the wide-type.AtIPK2a is functional redundantly to AtIPK2?.Plants overexpressing AtIPK2a also flowers later.2.AtIPK2? regulates flowering time through photoperiod pathway.The key gene CO in photoperiod pathway is down regulated in AtIPK2? overexpressed plants.The transcription levels of circadian central genes CCA1,LHY,TOC1 and the related gene GI in photoperiod pathway are affected in atipk2? mutant,while the circadian rhythms of transcription are unaffected.3.The N terminus of AtIPK2? shows intrinsic transcriptional activity.Truncations of N terminus are autoactive in yeast,and the catalytic sites are not required in the domain.The N terminus of AtIPK2a possesses the identical intrinsic transcriptional activity as AtIPK2p-N.4.AtIPK2? regulates the expression of FLC and FT by binding to the chromatin.The expression of FLC and FT in AtIPK2? overexpressed plants is down regulated.AtIPK2? plays a role in the upstream of FLC and FT.The N terminus of AtIPK2?interacts with specific DNA sequences of FLC and FT in yeast one-hybrid system,and ChIP experiments showed that AtIPK2? binds to the FLC and FT chromatin in the transgenic plants.5.Regulation of FLC and FT mRNA levels by AtIPK2? is promoted by AtIPK2a.The flowering time of transgenic plants overexpressing AtIPK2? and AtIPK2a simultaneously is later than AtIPK2? overexpression line AtIPK2?-Myc.RT-qPCR of HA-AtIPK2?/AtIPK2?-Myc overexpression plants showed that the mRNA levels of FLC and FT are lower.The DNA sequences interacting with AtIPK2?-N are able to bind to AtIPK2a-N,and the interaction of FLC chromatin with AtIPK2p is enhanced by AtIPK2a in HA-AtIPK2?/AtIPK2?-Myc compared to AtIPK2?-Myc transgenic plants.6.AtIPK2? interacts with FVE and is involved in the regulation of H3K27me3 and H3Ac.AtIPK2? associates with FVE in yeast,Arabidopsis protoplast and transgenic plants.ChIP experiments showed that AtIPK2? binds to the FLC and FT chromatin with FVE in the transgenic Arabidopsis,and the relative levels of H3K27me3 and H3Ac are affected in atipk2p mutant.7.The function of AtIPK2? in the histone modification on FLC and FT loci is enhanced by AtIPK2a.AtIPK2? binds to AtIPK2a,which stabilizes the interaction between AtIPK2? and FVE in yeast.The relative levels of histone modification on FLC and FT loci in HA-AtIPK2?/AtIPK2?-Myc are higher than AtIPK2?-Myc transgenic plants.8.The expression of AtIPK2? is regulated by light.There are elements responding to light in the promoter region of AtIPK2?,and the detection of PAtIPK2?::GUS transgenic plants showed that the transcription of GUS gene is induced by different light.The expression level of AtIPK2? is changed in photoreceptor mutants compared to the wide-type plants.9.AtIPK2? is involved in signaling pathway mediated by far-red/red light photoreceptors.The hypocotyl lengths of atipk2? mutant are longer under continuous far-red light,and the de-etiolation of cotyledons is promoted by pulsed far-red light or daily pulsed red light compared to the wide-type seedlings.The chlorophyll level responding to the early red light signal is declined in atipk2? mutant.10.AtIPK2? is functional in the signaling pathway mediated by blue light.The anthocyanin level in atipk2? mutant is down regulated under continuous blue light,and the[Ca2+]cyt level is decreased in the root tips detected by Fluo 3-AM.In summary,we analyzed the functions of AtIPK2?in the regulation of flowering time and seedling photomorphogenesis.We revealed that the mechanism of AtIPK2?in modulating the transcription levels of FLC and FT.This work is helpful to deeply understand the functions and molecular mechanism of AtIPK2? in Arabidopsis development.It also lays foundations for discovering more functions of inositol phosphate signaling pathway in plants.