Regulatory Mechanism Of Adventitious Root Development On The Hypocotyls Of Pepper(Capsicum Annuum L.)Seedlings

Cutting propagation is an efficient method of vegetative propagation and regeneration.With the development of seedling grafting technique,cutting propagation is becoming widely used in pepper(Capsicum annuum L.).Adventitious rooting is a critical event for regeneration of cuttings,which influences the efficiency and quality of cutting propagation.In this study,we performed a genome-wide transcript analysis of rooting zone at 0,24 and 120 h after planting.Differentially expressed genes(DEGs)and regulatory pathways were identified and analyzed.In addition,we investigated the role of GRAS gene family in adventitious rooting from pepper hypocotyl cuttings.Furthermore,the physiological and molecular mechanisms of IBA-induced adventitious rooting were analyzed.The main results were as follows:1.We sequenced the transcriptome of the rooting zone at 0,24 and 120 h after planting and generated6.84-7.21 giga base clean bases.A total of 4991,6658 and 4128 DEGs were identified in AR0 vs.AR24,AR0 vs.AR120,and AR24 vs.AR120 comparisons respectively,among which 1321 DEGs were in common among all three pairwise comparison groups.In the induction phase,genes encoding auxin efflux carrier,auxin influx carrier,zeatin O-glucosyltransferase,BR-6-oxidase,ACC oxidase,ethylene receptor,serine/threonine-protein kinase,polygalacturonase,?-xylosidase,pectinesterase,and expansin were significantly up-regulated,indicating that signal transduction,cell wall degradation and modification were involved in the induction phase of pepper adventitious rooting.However,genes encoding DNA polymerase,RNA polymerase,translation initiation factor,ribosomal protein,histone,microtubule-associated protein,cytoskeleton-regulatory protein,cyclin,cytokinesis-regulatory protein,cell division control protein,cellulose synthase,UDP-glucosyltransferase,extensin,and ion transport protein were significantly up-regulated in the initiation phase,suggesting that DNA replication,transcription and translation,cytoskeleton remodeling,cell division,cell wall biosynthesis,and ion transport were important events in the initiation phase of adventitious rooting.These results revealed that adventitious rooting is a complicated organogenesis process regulated by diverse metabolic and signalling pathways.2.Based on the pepper whole-genome sequences,we systematically investigated the members of GRAS gene family and elucidated their phylogenetic relationships,tissue-specific transcription patterns,and responses to drought and salt stresses.The results showed that 54 Ca GRAS genes were identified in pepper.These Ca GRAS genes were distributed on all chromosomes except for the 11 th.The number of exons ranged from 1 to 3,and isoelectric points ranged from 4.97 to 9.13.Phylogenetic analysis showed that Ca GRAS genes were clustered into eight phylogenetic groups.Additionally,the expression of Ca GRAS genes exhibited a variety of patterns in different pepper tissues,among which 34 genes had higher expression abundance in roots,eight genes in stems,three genes in mature leaves and eight genes in shoot tips.Moreover,most Ca GRAS genes were responsive to drought and salt stresses.Some genes including Ca GRAS11,Ca GRAS30,Ca GRAS40,Ca GRAS44 and Ca GRAS50 were greatly induced by both drought and salt stresses.Furthermore,time-course transcription profiles revealed that 12 Ca GRAS genes were involved in adventitious rooting process,among which Ca GRAS1,Ca GRAS2,Ca GRAS21,Ca GRAS34 and Ca GRAS49 were probably important regulators of adventitious rooting from pepper hypocotyl cuttings.3.In order to investigate the mechanism of IBA-induced adventitious rooting from pepper hypocotyl cuttings,we detected the contents of glucose,fructose,sucrose,total soluble sugars and starch,and activity of cell wall invertase in both basal and upper 5 mm segments of hypocotyls.The results indicated that exogenous IBA could enhance the adventitious root number in a dose-dependent manner.Compared with the control without IBA,cuttings treated with 150 ?M IBA showed maximum adventitious root number,rooting zone length and average root length.In addition,the transcriptional levels of Ca ARL1 and Ca Cyc B1 were greatly enhanced in 150 ?M IBA-treated cuttings.Both the basal and upper segments from IBA-treated cuttings could form adventitious root primordial,thus the length of rooting zone was greatly increased.Furthermore,we found that IBA treatment could up-regulate the transcriptional levels of Ca CWI and Ca STP,and enhance the contents of carbohydrates and activity of cell wall invertase.Therefore,it is conferred that IBA treatment could increase sugar availability at the site of root formation to meet the demand for energy and carbon skeleton,thereby promoting adventitious root development on the hypocotyls of pepper seedlings.