Low-Temperature-Induced Regulatory Network Rewiring Via WRKY Regulators In Banana Peel

Low-temperature storage is an effective means to extend the shelf life of fruits after harvest,but can lead to chilling injury in vegetables and fruits,especially in tropic fruits.Plants have evolved mechanisms to cope with low-temperature stress at the physiological and molecular levels.While much work has gone into uncovering the mechanisms for adaptive lowtemperature tolerance in model plants,such as the classical ICE1-CBFCOR signaling module,how tropical plant species,such as banana and mango,respond to low temperature and why they easily appear chilling injuries,is poorly understood at the molecular level.Banana(Musa spp.)fruits,as typical tropical fruits,are not cold tolerant,and appear chilling injury in a short-time low temperature storage,leading to serious economic loss.In this study,we choose banana fruit as a model tropic plant and systematically characterized the changes in chromatin accessibility,histone modifications,distal cis-regulatory elements,transcription factor binding,and expression levels in banana peels in response to low temperature.Main researches are following:(1)using RNA-seq to understanding the changes of transcription in low temperature.The results showed that low temperature upregulated the expression level of a large number of genes,such as PPO1,LOX5 and PLD1,which were closely associated with browning formation.GO and KEGG showed that upregulated genes were related to phospholipid degradation and oxidation.(2)we further analyzed how chromatin accessibility and histone modifications influenced the expression level of cold-induced genes.The results showed that low temperature increased chromatin accessibility and the enrichment of active histone modifications.For example,H3K4me3,H3K9 ac and H3K27 ac were mainly enriched in TSS region.In addition,we also found that H3K27 ac and H3K9 ac were not hallmark for active enhancer in banana peel.(3)Then,the analysis of histone modification in each cluster showed that upregulated clusters showed an increase in active histone marks,which were consistent with the higher transcription level of the associated genes.Interestingly,H3K27me3 hardly influenced the expression level of differently expressed genes,although its level was affected by environmental stress.Notably,upregulated browning-related genes(BRGs)such as GSDL11 experienced an increase in active histone modifications.(4)Considering a part of DNA hyposensitive sites were located in intergenic region,we investigated distal candidate enhancers.Enhancer activity at these sites was demonstrated by dual luciferase assay.At the same time,these enhancers showed cold-specific and time-specific function,and were involved in the regulation of BRGs expression levels.(5)Motif activity and transcription factor footprint analysis in functional regions showed that WRKY transcription factor had important functions in banana peel in response to low temperature.when compared with banana peel in RT,differently expressed WRKY regulators were significantly induced in low temperature.These results suggested that WRKY regulators palyed a core role in response to low temperature in cold sensitive fruits and were important for peel browning formation.(6)The analysis for the binding sites of WRKY regulators by DAPseq showed that WRKY regulators were involved in the regulation of banana peel in response to low temperature.WRKY regulators directly bound to the promoter region and participate in the regulation of MaCBF1.Also,a large number of BRGs like MaPLA1β2 and MaGDE1,were also bound by WRKYs as well as by a WRKY-targeted enhancer,and showed an increased expression level.These revealed BRGs are directly or indirectly bound by WRKYs at low temperature,along with increased expression level.(7)We over-expressed WRKY regulators in banana peel.Compared with the control,WRKY over-expressed groups promoted browning formation,and the expression level of 7 BRGs were significantly induced.(8)We reconstructed the regulation network of cold-induced genes with five core WRKY regulators by intergrating DAP-seq,DNase-seq and RNA-seq datasets.Regulation network showed 10 regulation patterns including 5,802 regulation interactions.Of these interactions,18 BRGs were targeted by at least one WRKY regulator as well as a distal enhancer,and most of them showed similar expression pattern like five WRKY regulators.In summary,dynamic patterns of cold-induced transcripts are generally accompanied by concordant chromatin accessibility and histone modification changes.These upregulated genes were enriched in WRKY binding sites in their promoters and/or active enhancers,which could form enhancer-promoter interactions that appear to regulate critical browning pathways,including phospholipid degradation,oxidation,and cold tolerance.Meanwhile,we reconstruct regulatory network with core WRKYs and find a large number of BRGs are directly or indirectly regulated by at least one WRKY as well as increased expression level.Our results illustrate the transcriptional reprogramming via WRKYs during banana peel browning at low temperature,and provide an extensive resource for studying gene regulation in tropical plants in response to cold stress,and potential targets for improving cold tolerance and shelf-life of tropical fruits.