| Sorghum(Sorghum bicolor L.)stands for the fifth largest grain crop around the world,widely applying in the fields of food,feed,brewing and biofuel directions.Starch is the main component of sorghum grains and the main storage form of carbohydrates,directly influencing the performances of yield and quality of sorghum grains via the biosynthesis and accumulation.Although the technology system of starch synthesis beyond plant species is already constructed by humans,plant-source,especially grain-crop-source starch still serves as the major supplies for various applications.Thus,mechanism dissection related to the transcriptional regulation of starch biosynthesis is beneficial to expand the theoretical basis of starch biosynthesis in sorghum grains,as well as to provide theoretical guidelines and gene resources for the molecular improvement of the yield and quality in sorghum.In the present study,transcriptomic sequencing(RNA-seq)was performed among 20 tissues collected from the whole plant of BTx623,a sequenced line of sorghum.Related genes involved in the starch biosynthesis in sorghum grains were identified via RNA-seq of 20 tissues.Then the expression patterns of these genes were profiled,and the transcription factors(TFs)highly expressed in sorghum grains were also screened.Three TFs of SbNAC38,SbNAC68,and SbNAC107 belonging to NAC family were identified via the co-expression profiling between TFs and the identified related genes.Followingly,the experiments of sub-cellular localization,self-activation activity detection were performed to discover the features of SbNAC38/68/107.Some other research of transient over-expression,promoter activity detection,EMSA assays were carried out to reveal the important function of SbNAC38/68/107 in regulating target genes involved in the starch biosynthesis pathway of sorghum grains.The main findings are as follows:1.The genes related to sorghum starch biosynthesis are highly conservedThrough BLAST analysis,a total of 27 related genes involved in starch biosynthesis in sorghum endosperm were identified across the whole sorghum genome,including 6 for ADP glucose pyrophosphorylase(AGPase),10 for starch synthase(SS),4 for starch branching enzyme(SBE),2 for both starch debranching enzyme(DBE)and starch phosphorylase(SP),and 1 for Brittle-1(BT1).Results of protein sequence alignment showed that functional enzymes involved in sorghum starch biosynthesis possessed the same conserved domains and motifs as those reported in maize and Arabidopsis,tending to cluster in the same clade via phylogenetic analysis.Results of RAN-seq and qRT-PCR indicated that 27 related genes could be divided into two groups of Group I and Group II.Group I covered 15 genes that highly expressed in sorghum grains,and Group Ⅱ contained the rest 12 that mainly expressed in other tissues with similar levels.Results of cis-elements detection showed that the cis-elements responding to light signal,biotic and abiotic stresses,phytohormones,and some other TFs were generally distributed within the promoter regions of 27 genes.2.Co-expression of SbNAC38/68/107 with sorghum SBRGsGene expression analysis among tissues was carried out via RNA-seq.A total of 1708 tissue specific genes were detected,including 416 in grains.At the same time,a total of 141 tissue specific TFs were detected across all tissues,among which 80 TFs specifically expressed in grains and endosperm,and 26 TFs presented high transcription levels in grains.These TFs belonged toll TF families,such as ERF,NAC,bZIP,bHLH.Co-expression analysis showed that 26 grain specific TFs exhibited co-expression trends with 15 starch biosynthesis genes highly expressed in sorghum grains,with correlation coefficients>0.6.Besides,the identified TFs of SbNAC38/68/107 that highly expressed in grains also exhibited significant co-expression with these 15 genes with correlation coefficients>0.6.3.Cloning and functional features of SbNAC38/68/107Results of gene cloning,qRT-PCR assay,and functional characterization indicated that SbNAC38/68/107 all highly expressed in sorghum grains,and the corresponding encoded proteins were all located in the nucleus.Besides,no self-activating activity was detected for SbNAC68 via yeast system,while both SbNAC38 and SbNAC107 possessed self-activation activity in yeast system.What’s more,the coding sequence of 21 bp for the core activating domain of SbNAC38 located between the positions of 896 bp to 906 bp of SbNAC38,while the corresponding coding sequence of 18 bp located between 895 bp and 912 bp along the sequence of SbNAC107.4.SbNAC38/68/107 tend to regulate the transcription of sorghum SBRGs by binding to CACGCAA motifResults of protoplast co-transformation analysis showed that both SbNAC38/107 could significantly activate the promoter of SbGBSSI,while all of SbNAC38/68/107 could significantly activate the promoter of SbBtl.Results from overexpression of SbNAC68 and SbNAC107 in leaf protoplasts indicated that SbNAC68 could significantly promote the transcription of sorghum starch biosynthesis related genes,i.e.,SbSSⅠ,SbSSV;SbAGPLS1,SbAGPLS4,SbGBSSⅠ,and SbGBSSⅡa,while inhibit the transcription of SbAGPS2,SbISAl,SbSSⅡc,SbSBEⅡb and SbSSⅢb.SbNAC107 could significantly improve the transcription levels of sorghum starch biosynthesis related genes,including SbSSⅠ,SbSSⅡb,SbAGPLS1,SbAGPLS4 and SbGBSSⅠ,while inhibit both of SbAGPS2 and SSⅢb.Results of EMSA assay showed that all of SbNAC38/68/107 could directly bind to the motif of CACGCAA.Among these three NAC TFs,SbNAC38 could directly combine with this motif within the promoters of SbSSⅠ,SbSBEI,and SbAGPS1;SbNAC68 could directly bind with this motif within the promoters of SbGBSSⅠ,SbSSⅡa,SbAGPLS1,SbSBEI,SbAGPS1;while SbNAC107 could directly bind with this motif within the promoters of SbGBSSⅠ.SbSSⅡa,and SbSSⅠ.In summary,the integrated results of the present study implied that SbNAC3 8/68/107 served as key regulatory TFs and participated in the transcriptional regulation of diverse related genes,jointly affecting the starch biosynthesis and accumulation in sorghum grains. |
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