| Plant hormone gibberellin（GA）plays an important role in the growth of plants,which has a regulatory role in the growth and development of plant roots,stems,leaves,flowers and fruits.Long noncoding RNA（lncRNA）can regulate the flowering,reproductive development of plants and participates in the regulation of plant abiotic stress.In plants,studies on lncRNA in response to gibberellin have been reported in poplar.However,there are few reports on IncRNA in maize.In this study,we used water and gibberellin solution to treat maize in order to explore the distribution,function and regulatory network of gibberellin-responsive lncRNA in maize.Moreover,we analyzed the function of gibberellin-responsive lncRNA（GIBBERELLIN RESPONSIVE lncRNA2,GARR2）,and the transcriptional regulatory network of lncRNA GARR2 was also surveyed.In previous research,water treatment was used as a control,and different concentrations of gibberellin GA3 solution was used to treat both normal plant height WT and dwarf plants D11 with a nuclear background of Mo 17.The results showed that both the normal plant height and the dwarf plants were sensitive to GA3 application.Sampling water-and GA3-treatment of the second leaves of normal plant height and dwarf plants were subjected to strand-specific sequencing of ribosomal-depleted RNA libraries to discover lncRNA that responded to gibberellin.Then,we analyzed the function and regulatory network of gibberellin-responsive lncRNA（GIBBERELLINRESPONSIVE lncRNA2,GARR2）.1.Identification of gibberellin-responsive IncRNA in maize leaf tissue.A total of 9933 lncRNAs were identified in the normal plant height plant WT and the dwarf material D11.Among them,7241,8802,7755,and 7273 lncRNAs were found in the water-treated WT,10-4 M GA3-treated WT,the water-treated D11 and the 10-4 M GA3-treated D11,respectively.Compared with the water-treated WT plant,2044 specific lncRNAs were found in the GA3-treated WT.Compared with water-treated D11,a total of 862 specific lncRNAs were discovered in GA3-treated D11.A total of 169 common gibberellin-responsive lncRNAs were discovered through the intersection of 2044 and 862 lncRNAs specifically discovered in GA3-treated WT and D11.The analysis of lncRNA differential expression showed that compared with the water-treated WT,22 differentially expressed lncRNAs were discovered in the GA3-treated WT.Compared with the water-treated D11,a total of 34 differentially expressed lncRNAs were discovered in the GA3-treated D11.Further detailed analysis of the identified lncRNAs showed that 28 lncRNAs were putative miRNA precursors.LncRNA TCONS00058380 was the precursor of known miRNAs zma-miR528a and zma-miR528b.We also revealed the trans and target genes of differentially expressed lncRNA.Results showed that there were 13 cis target genes of differentially expressed lncRNA in WT.In D11,there were 19 cis target genes of differentially expressed lncRNA.These target genes encoded glycosyltransferases and dehydrogenases,transcription factors NAC and bZIP members,etc.The interaction network analysis of target genes showed that four cis target genes of differentially expressed lncRNA（TCONS00061140,TCONS0006726,TCONS00038874,TCONS00042125）were functionally interlaced.2.Functional validation of gibberellin-responsive lncRNA（GIBBERELLIN RESPONSIVE lncRNA2,GARR2）.The CRISPR-Cas9 technique was used to edit the sites of lncRNA GARR2.The sequencing results showed that there were two edited events of lncRNA GARR2.Compared with the transgenic receptor,one transgenic line exhibited single cytosine C insertion in the target site of lncRNA GARR2.Compared with the transgenic receptor,the other transgenic line displayed four nucleotides（TTCC）deletion in the target site of lncRNA GARR2.The traits such as seedling height,length of the second leaf sheath,kernel length,and kernel width of the transgenic receptor and transgenic lines were measured.Results indicated that compared with the transgenic receptor,seedling height and the length of the second leaf sheath were increased in lncRNA GARR2 transgenic lines with single cytosine C insertion.Compared with the transgenic receptor,kernel length and width of the corresponding transgenic lines were decreased.For the other transgenic lines with four nucleotides（TTCC）deletion in the target site of lncRNA GARR2,the length of the second leaf sheath was increased relative to transgenic receptor.3.Survey the transcriptional regulatory network of gibberellin-responsive lncRNA（GIBBERELLIN RESPONSIVE lncRNA2,GARR2）.At V3 stage（three expanded leaves）,the second leaf sheath of the transgenic receptor and lncRNA GARR2 edited lines with single cytosine C insertion was harvested for transcriptome analysis to reveal the transcriptional regulatory network of lncRNA GARR2.The results showed that compared with transgenic receptors,a total of 2711 differentially expressed genes were found in lncRNA GARR2 edited lines,of which 1616 genes were up-regulated and 1095 genes were down-regulated.KEGG pathway analysis showed that the differentially expressed genes were mainly enriched in the plant hormone signal transduction pathway.And,the differentially expressed genes were enriched in the pathway related to the synthesis,metabolism and signal transduction of the plant hormones gibberellin and auxin.Some differentially expressed genes enriched in gibberellin and auxin pathways were analyzed by qRT-PCR.A total of 24 differentially expressed genes related to the gibberellin pathway were selected for qRT-PCR analysis.Among them,14 genes were up-regulated and 10 genes were down-regulated.A total of 6 differentially expressed genes related to the auxin pathway were selected for qRT-PCR analysis.Among them,5 genes were up-regulated and one gene was down-regulated.The differentially expressed genes were enriched in the gibberellin and auxin pathways,indicating that the transcriptional regulation of lncRNA GARR2 may be related to the gibberellin and auxin pathways.In this study,gibberellin-responsive long noncoding RNA were discovered through the strand-specific sequencing of ribosomal-depleted RNA libraries of maize leaf tissues.And,the CRISPR-Cas9 technique was used to obtain gibberellin-responsive lncRNA GARR2-edited lines,which were adopted for the functional validation of IncRNA GARR2.In addition,the transcriptional regulatory network of gibberellin-responsive lncRNA GARR2 was revealed by transcriptome sequencing.Results reported here are helpful to enrich the theoretical basis of gibberellin-mediated regulatory cascade at noncoding RNA hierarchy,and provide novel targets for the improvement of plant architecture with GA-driven knowledge. |
