Genome-wide Expression Profiling Of D.longan,and Identification And Expression Analysis Of NAC TF During Somatic Embryogenesis Under Drought Stress Conditions

Precursor RNAs undergo extensive processing to become mature RNAs.RNA transcripts are subjected to 5′capping,3′-end processing,splicing,and modification;they also form dynamic secondary structures during co-transcriptional and post-transcriptional processing.Like coding RNAs,non-coding RNAs（nc RNAs）undergo extensive processing.Transcriptome studies have revealed roles for co-transcriptional and post-transcriptional RNA processing in regulating gene expression and coordinating plant development and plant-environment interactions.The transcriptional and post-transcriptional levels regulate plant responses under stress conditions.Drought stress causes changes in plants’morphological,physiological,biochemical,and molecular characteristics.The response to drought in different plants may vary from avoidance,tolerance,and escape to recovery from stress.This response is genetically programmed and regulated in a complex yet synchronized manner.The crucial genetic regulations mediated by non-coding RNAs（nc RNAs）have emerged as game-changers in modulating the plant responses to drought and other abiotic stresses.The nc RNAs interact with their targets to form potentially subtle regulatory networks that control multiple genes to determine the overall response of plants.Many long and small drought-responsive nc RNAs have been identified and characterized.In this study,whole-transcriptome analysis of embryogenic callus of longan treated with drought was performed,and the miRNA,lncRNA,m RNA,circRNA and ce RNA network after drought stress were analyzed.Micro RNAs（miRNAs）are essential non-coding RNAs involved in almost all biological processes,including biotic and abiotic stress in plants.Longan is a model crop for studying embryogenesis,fruit development,and stress tolerance.To better understand the molecular basis and to light on complex regulatory networks involved in longan drought tolerance,genome-wide profiling of small RNAs from longan embryogenic callus under control and drought stress was undertaken using BGISeq 2000 platform.The expression profiles of the miRNAs from drought treated（PEG 5%and PEG 7.5%）and control（CK）libraries generated from high-throughput sequencing were compared.We pick out 186 known miRNAs and 129 novel miRNAs.Among them,118 miRNAs were differentially expressed in all libraries.miRNA targets exposed that the genes primarily encrypted transcription factors,and are involved in sulfur metabolism,amino acid,signal transduction,and secondary metabolism pathways.The transport,metabolism,and signaling pathways genes are involved in defense mechanisms and drought tolerance.qRT-PCR analyses authenticate the expression levels of drought-responsive miRNAs and their possible targets.Fourteen miRNAs such as miR159a＿1,miR167a-5p,miR390a-5p,miR393h,miR395a＿5,miR397a＿3,miR398b,miR399f＿3,Dl-miR12,Dl-miR38,Dl-miR39 were up-regulated under drought stress,involved in different pathways,and four miRNAs were down-regulated.miR159,miR164,miR393,miR395,miR171,and miR166 play a central role in drought tolerance.Our results suggest that the differentially expressed miRNAs had significant roles in the circadian clock,stress adaptation,plant hormone signal transduction,and secondary metabolism network.The plant genome can produce long non-coding RNAs（lncRNAs）,some of which have been identified as essential regulators of gene expression.We found 548 differentially expressed lncRNAs in CK-PEG 5%,2340 differentially expressed lncRNAs in CK-PEG 7.5%,and 2315 lncRNAs in PEG 5%-PEG7.5%.We found 106 DE lncRNAs,and 289 differentially expressed m RNAs in all categories.In CK-PEG 7.5%,LTCONS＿00026116,LTCONS＿00001494,LTCONS＿00057672 were highly up-regulated,LTCONS＿00058846,LTCONS＿00047032,were highly down-regulated lncRNAs.The number of significantly differentially expressed lncRNAs in PEG 5%-PEG 7.5%was greater.Most DEGs were involved in the cellular and metabolic process,cellular,anatomical entity,binding,and catalytic activity.The results indicated that the co-expressed DE lncRNA-m RNAs involved in the Ras signaling pathway,NOD-like receptor signaling pathway,Neurotrophin signaling pathway,and MAPK signaling pathway.From DE lncRNA-DE m RNA network analyses,we found that 23 DE lncRNAs interacted with 30 DEm RNAs in CK-PEG 5%,30 DE lncRNAs were linked with 39 DE m RNAs in CK-PEG 7.5%,35 DE lncRNAs-DE m RNAs in PEG 5%-PEG 7.5%.These genes are related to BHLH TF,sugar carrier protein,ubiquitin-conjugating enzyme,LOB domain-containing protein,heat shock protein（HSP）,Ankyrin repeat-containing protein（ITN1）,Beta carbonic anhydrase（BCA）,and fatty-acid-binding protein.CircRNAs play an important role in miRNA function and transcriptional control.We identified circRNAs and analyzed their expression under drought stress.We identified 3136 differentially expressed circRNAs in CK-PEG 5%,4603 differentially expressed circRNAs in CK-PEG 7.5%,and 2973 circRNAs in PEG 5%-PEG 7.5%.We performed GO and KEGG enrichment analyses to predict the functions of differentially expressed circRNAs.Most host genes for circRNAs were involved in Fatty acid degradation,metabolism and biosynthesis,lipid and carbohydrate metabolism,signal transduction,and translation.For ce RNA analyses,227 miRNAs and 11338 transcripts were used to calculate the interaction relationship between ce RNA and target RNA,10differential ce RNAs with most relationships in the ce RNA interaction network were selected,and the target m RNAs related to pectin acetylesterase,succinate dehydrogenase,pentatricopeptide containing protein,serine-threonine protein kinase,vacuolar protein sorting associated protein.In ce RNA-miRNA differential interaction,We found four miRNAs（miR167d-5p,miR156a-5p,miR319a-3p,miR396a-5p.）,three lncRNAs（LTCONS＿00051044,LTCONS＿00002874,LTCONS＿00017579）and two m RNAs that are annotated as Pectin acetylesterase,and serine/threonine-protein kinase.The NAM,ATAF1/2,and CUC2 form a huge plant-specific gene family of NAC TFs involved in the growth,development,and regulation of biotic and abiotic stress responses.We performed a comprehensive analysis of the NAC transcription factor family in longan,and 114 NAC genes were found(2^ndgeneration genome).We investigated the NAC gene family exploring the phylogeny,domain conservation,intron/exon,motifs,cis-regulatory elements,protein-protein interaction,and expression profiles of RNA-seq samples in different tissues and early somatic embryogenesis of longan.Phylogenetic analysis showed that the genes with similar gene structure and motif distribution were clustered in the same group.Cis-element identification indicates the possible role of NAC genes in biological and physiological processes.Protein-protein interaction identified the Dl NACs homologous with Arabidopsis proteins.We further investigated the expression pattern of Dl NAC genes in different tissues（pulp,stem,large fruit,young fruit,and flower）during somatic embryogenesis at embryogenic callus（EC）,incomplete compact pro-embryogenic cultures（ICp EC）,and globular embryos（GE）stages.The qRT-PCR results showed that the Dl NAC genes were expressed higher at EC and GE stages compared with the ICp EC stage.Dl NAC6,Dl NAC42,Dl NAC47,Dl NAC49,Dl NAC81,Dl NAC56,Dl NAC2,and Dl NAC31 show expression in different tissues and different stages of SE.Our results provide insight into the evolution,diversity,and characterization of NAC genes in the longan and explain their biological roles and molecular mechanisms in plants.Drought and salinity stress are common environmental stress conditions that adversely affect plant development and crop production.Plants respond to abiotic stress by triggering numerous abiotic stress-responsive genes,including transcription factors,which activate several downstream signaling pathways and adaptive networks.Effect of drought and salinity on growth,proline,Hydrogen peroxide（H₂O₂）,lipid peroxidation（MDA）,and antioxidant enzyme activities in longan embryogenic callus were evaluated.Callus tissues were cultured on MS medium containing different concentration of PEG（2.5%,5%,7.5%,10%,PEG 6000）,Na Cl（50m M,100m M,150m M,and 200m M）and control.Under drought stress,proline content,H₂O_2,and MDA content were increased.Antioxidant activities such as SOD,POD,and CAT were also increased in drought stress compared to control.While on salt stress,proline and H₂O₂contents were decreased with increased salt concentration,while MDA contents were increased.The antioxidant activities were also decreased under salt stress in high concentrations while increasing at a lower concentrations.The NAC transcription factor family members contribute to abiotic stress-induced signal transduction in plants.The expression level of Dl NACs,under drought and salt stress,was analyzed through a quantitative real-time polymerase chain reaction.Seventeen Dl NACs were significantly up-regulated under both treatments.Upon drought stress Dl NAC2-1,Dl NAC73-1,Dl NAC83-3,Dl NAC100-1,Dl NAC100-5,Dl NAC101-1,Dl NAC102 were highly expressed at PEG 10%.Dl NAC35-1/2/3,Dl NAC42-1,Dl NAC71-1,Dl NAC72 genes had high expression at PEG5%.These analyses provide a comprehensive view of longan NAC genes in response to drought and salt stress.From whole transcriptome analyses of longan under drought stress,we found miR164b,miR164a＿4,miR156c＿1,miR2275a-3p＿2,miR2275,miR2275a＿1,miR2275a-3p＿2 were differentially expressed miRNAs,and target Dl NAC TF genes including Dl NAC98-2,Dl NAC22-1,Dl NAC42-1,Dl NAC79-2,Dl NAC100-3,Dl NAC100-4,Dl NAC79-3 under drought stress.miR164b was up-regulated while other were down-regulated in all categories.17 DElncRNAs were found that targetd NAC genes in longan under drougt stress.Upon which 4 novel NAC genes（MTCONS＿00016968,MTCONS＿00020293,MTCONS＿00039868,MTCONS＿00044886）and other were known Dl NACs.11 circRNAs target Dl NAC TF genes.Dl NAC72,Dl NAC41-2,Dl NAC17,Dl NAC82,Dl NAC83-4,Dl NAC8-2,Dl NAC101-4,Dl NAC101-5,Dl NAC100-6,Dl NAC100-7 were targeted by these circRNAs.We conclude that NAC TF had significant response upon drought stress.