| With the increasing demand for maize yield and utilization,new maize germplasm resources are urgently needed to be developed.Tropical and subtropical maize are rich in genetic variation,thus the introducing,collecting,and utilizing of these germplasm is an effective way to broaden germplasm genetic resource and accelerate the breeding process.However,due to the sensitivity of photoperiod,the reproductive growth of tropical and subtropical maize under temperate conditions is greatly limited.Therefore,the discovery of hub genes and the regulation of network mechanisms involved in the photoperiod pathways will contribute to the development and utilization of germplasm resources in China,which represent significantly theoretical and practical value.In this study,we constructed two near isogenic lines obtaining two photoperiod-related candidate genes Zm CCT and Zm PIF3,respectively,from the crossing of CML288 and Huangzao4.Firstly,i TRAQ-based proteome,m RNA-Seq and mi RNA-seq technology were used to explore the photoperiod regulation mechanism mediated by Zm CCT under long-day conditions and interaction network with other biological pathways.At the same time,we obtained the Zm PIF3near-isogenic line by further fine mapping.We analyzed the temporal and spatial expression of Zm PIF3 in Huangzao 4 and near-isogenic lines by q RT-PCR,and concluded Zm PIF3 as one induced gene to regulate flowering time,which were further verified by in situ hybridization analysis.Two-hybrid technique and CO-IP-MS combined analysis were used to screen out two proteins interacting with Zm PIF3,which provided a studing basis for further study on the photoperiod interaction network mechanism mediated by Zm PIF3.Finally,we used the emerging Hi-C technology to initially explore the differences in long-range chromatin interaction between tropical and temperate materials at the genome-wide level,unveiling new sight for understanding the epigenetics differences between the two maize germplasms.In conclusion,this study basically showed the molecular regulation mechanism of photoperiod sensitivity at different molecular levels by multi-omics joint analysis.The main findings are as follows:1.Based on our previous study,we mapped and identified one photoperiod sensitive candidate gene,Zm PIF3,located within 90 kb between the molecular markers S769 and S777 by further fine mapping.By observing the phenotypic differences between the NIL and Huangzao4,we found that in Zhengzhou(LD),the PIF3 NIL was 1.37 days later than the Huangzao4,and the pollen period was 2.07 days later.However,in Hainan(SD),there is no difference in the flowering time between NIL and Huangzao4.The results indicated that the Zm PIF3 gene from CML288 can inhibit maize tasseling and pollen period under long-day conditions.By q RT-PCR quantification,we found that the expression of Zm PIF3 in CML288 and PIF3-NIL was significantly lower than that in Huangzao 4 under LD conditions;the expression difference between the three lines was not significant under SD conditions,suggesting that Zm PIF3 gene may be a flowering promoting factor.In addition,two candidate interaction proteins of Zm PIF3,GRMZM2G162529(phosphoribulokinase precursor)and GRMZM2G474769(MYB family transcription factor),were found by yeast two-hybrid and CO-IP-mass spectrometry to further understand Zm PIF3 in the maize photoperiod sensitive regulatory network.2.Under long-day conditions,we collected the fresh leaves at the three-leaf and sixleaf stages from the LP-sensitive near-isogenic line NIL the insensitive inbred line Huangzao 4,as the experimental materials.Totally,5259 of LP-responsive proteins were identified using the i TRAQ-lable method,of which,943 proteins were identified with differential accumulation level in both of Huangzao4 and NIL.Then the bioinformatics tool WEGO(GO annotation)and the Cytoscape(v3.0.2)plug-in Clue GO + Cluepedia v2.1(GO-KEGG network)were used for functional enrichment analysis of differentially accumulated proteins(DEPs).We found that DEPs are mainly enriched in 20 functional categories,including protein processing in the endoplasmic reticulum,splice,ribosome,glyoxylic acid,dicarboxylic acid metabolism,L-malate dehydrogenase activity and RNA transport.By function annotation,14 proteins related to circadian rhythm or flowering time were identified in this study,and the sugars produced by plant photosynthesis are also analyzed as one key regulation factor and metabolites response to LP condition.3.Using high-throughput m RNA sequencing(RNA-seq)and i TRAQ-lable combined analysis methods,the same materials,Huangzao4 and NIL,were used to analyze the changes in transcription and protein levels during the transition stage from vegetative growth to reproductive growth.The results showed that 58.9% and 55.8% of DEPs in NIL and Huangzao4 were associated with biological processes,including five major GO categories: metabolic processes,response to stimuli,transport,plant development and reproduction,and cell development.Differential expressed genes(DEGs)are mainly involved in phosphorus ion transport and photosynthesis processes.Importantly,we found an extremely weak correlation between m RNA expression and protein accumulation(R2=0.002~0.02),demonstrating that post-transcriptional regulation is involved in photoperiod-dependent floral transition.4.To further understand the role of post-transcriptional regulatory mechanisms in the photoperiod pathway,mi RNA-Seq sequencing was used to identify mi RNAs related to photoperiod sensitive responses in maize under long-day conditions.The results showed that 113 and 134 known mi RNAs showed significantly different expression levels between two inbred lines in maize leaves and SAM,respectively.Among them,the expression level of mi R399 d in Huangzao4 was much higher than that of other mi RNAs in leaves and shoot apex merism(SAM).Meanwhile,we also found that the sucrose content in the near isogenic lines(NILs)was significantly higher than that in Huangzao4.By the external supplement with 6% sucrose,we found that the seedlings grown in the medium containing exogenous high sucrose showed lower expression levels of mi R399,which suggested that sucrose inhibited the expression of mi R399 in vivo.Therefore,it is speculated that the excessive accumulation of endogenous sucrose in the near isogenic line may also repress the expression of mi R399.In combination with previous proteome results,carbohydrates may also be involved in the regulation of floral transient,so these results may suggest that mi R399 can participate in the photoperiod regulation network in maize in response to long-day conditions by responding to sucrose accumulation.In addition,mi RNA array technology was used to screen for mi RNAs that respond to external sucrose regulation in Arabidopsis.Similarly,the mi R399 planted in medium with high sucrose was also expressed at lower level than the seedling grown under normal condition.The results showed that the expression of mi R399 s was inhibited by high concentrations of exogenous sucrose in Arabidopsis and maize.This study lay the foundation for a better understanding of the relationship between sucrose,mi R399 s and photoperiod sensitivity.5.To study the differences in epigenetic regulation between tropical and temperate germplasm,the high-resolution chromatin capture technique(Hi-C)were used between the tropical maize inbred line CML288 and the temperate maize inbred line D132.A spatial three-dimensional model of the chromosome structure in the nucleus of the maize shows that the A/B components are spatially separated from each other.By comparing the large-scale chromatin interaction differences between D132 and CML288,we found that approximately 8% of the compartments were switched and approximately 60% of the topological domain(TAD)was reorganization in CML288 compared to D132.Through the RNA-Seq technique,we found that the transcription levels of a large number of genes have changed significantly in both the compartment transition and the TAD reconstruction region.Furthermore,using the combined analysis of Hi-C and Chip-Seq with antibody H3K4me3,we found that changes in histone modifications may result in differences in chromatin interactions between D132 and CML288.This study firstly reveals the differences in genome-wide chromatin structure between tropical and temperate maize inbred lines,providing a studying reference for comparative Hi-C analysis among other species. |
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