Characterization Of GA-responsive Coding And Long Non-coding RNA In Maize

Phytohormone gibberellin?GA?orchestrates various facets of biological processes,such as seed germination,plant architecture determinant,and floral organ development.During the first'Green Revolution',semi-dwarf traits of varieties mainly attribute to changes in GA biosynthesis and signaling.Thus,elucidation of GA regulatory cascade bears both theoretical and applied sig-nificance.GA-responsive coding and long non-coding RNAs have been reported in some plants,including muskmelon,grape,and poplar.However,GA-responsive coding and long non-coding RNAs in cereal crops,such as maize remains elusive.Herein,we tried to characterize GA-respon-sive coding and long non-coding RNAs in maize.Two types of plants,one was plant with normal height?hereinafter named as wild-type WT?,the other was dwarf plants?hereinafter referred as D11?,were selected from advanced backcross population?BC5?with Mo 17 nuclear background.These two types of plants were treated with distilled water?as control?and gibberellin GA3 solution with various concentration?10-4 M,10-5 M,10-6 M?.Results showed that compared to water-treated samples,length of the second leaf sheath of GA3-treated WT and D11 was significantly increased.With the guidance of gibberellin treatment outcomes,we harvested the second leaf sheath of water and 10-4 M GA3-treated WT and D11for the construction of Ribosomal-depleted RNA library,which was used for the following high-throughput sequencing to identify GA-responsive coding and long non-coding RNAs in maize.In this study,function of some isolated GA-responsive coding and long non-coding RNAs was preliminarily surveyed.1?GA-responsive coding RNA:Compared to water-treated plants,390 transcripts were dif-ferentially expressed in GA3-treated WT,including 271 up-regulated and 119 down-regulated tran-scripts.These transcripts were responsible for vesicle transport and second metabolisms biosyn-thesis.In relative to water-treated samples,6995 transcripts were differentially expressed in GA3-treated D11,including 2686 up-regulated and 4309 down-regulated transcripts.These transcripts were involved in plasmid and second metabolisms biosynthesis.There were eleven differentially expressed transcripts relevant for GA pathway,including one CPS-,two KS-,one KO-,four GA2ox-,one GID1-,and two DELLA-coding transcripts.Totally,213 differentially expressed transcripts,including 38 consistently up-regulated and 56 consistently down-regulated transcripts were detected by both GA3-treated WT and D11.Co-expression analysis showed that these com-mon 213 differentially expressed transcripts shaped a complicated regulatory network.2?GA-responsive long non-coding RNA:A total of 10247,9948,10444,and 8359 were identified in water-treated WT,water-treated D11,GA3-treated WT,and GA3-treated D11,respec-tively.More than 88%were classified into intergenic IncRNA.Nearly 9%lncRNA were generated through the transcription of anti-sense strand.The remaining IncRNAs were produced by the tran-scription of sense strand.Compared to water-treated plants,21 IncRNAs were differentially ex-pressed in GA3-treated WT,including 11 up-regulated and 10 down-regulated lncRNAs.There were fourteen cis target genes of differentially expressed IncRNAs.In relative to water-treated samples,428 IncRNAs were differentially expressed in GA3-treated D11,including 172 up-regu-lated and 256 down-regulated lncRNAs.There were 13 trans and 206 cis?111 upstream and 95 downstream?target genes of differentially expressed IncRNAs.One target gene encodes GA-cat-abolic enzyme GA2ox6 and its corresponding IncRNA was TCONS 00031061.LncRNA TCONS₀0031061 and its target GA2ox6 were in maize chromosome 6 with a distance of 208 bp.Four lncRNAs,namely TCONS 00107636,TCONS 00025659,TCONS 00044580,and TCONS 00064738,were detected by both GA3-treated WT and D11.Of these four IncRNAs,sequence of 2809 bp TCONS₀0025659 showed the highest similar with B73 reference resource.We could not find any sequence change between TCONS₀0025659 and B73 reference sequences in full 2809 bp interval.Integrated RT-PCR and sequencing results confirmed the identity of TCONS 00025659.Additionally,three GA-responsive elements were found in 1.5 kb upstream of TCONS 00025659.LncRNA TCONS₀0025659 was expressed in maize root,leaf blade,leaf sheath,and kernel.DNA-probe based in situ hybridization indicated that GFP signal was observed in maize root tip tissue.DAPI-staining results are also helpful for the following TCONS 00025659 cellular location.TCONS 00025659 possessed high similar with Gypsy/DIRS1 retro-transposon indicative of the possible retro-transposon origin of lncRNA_TCONS 00025659.Normal height and dwarf plants from advanced backcross population were employed for the identification of GA-responsive coding and long non-coding RNAs in maize.Features of some GA-responsive coding and long non-coding RNAs were preliminarily characterized.Results from this study will enrich our understanding of GA-mediated regulatory cascade,and provide promis-ing coding and long non-coding RNA targets for the future work.