Function Of Heat Shock Transcription Factor HSFAs And HSFCs Subfamily Genes In Perennial Ryegrass

Perennial ryegrass(Lolium perenne)is an important cool-season grass species,and is widely cultivated in landscaping,sport field and ecological management.However,perennial ryegrass is sensitive to high temperature and cannot survive in the summer season in the central and southern regions of China.Natural variations of stress tolerance of perennial ryegrass varieties and mechanisms of stress tolerance are of great interest for breeders.HSFs play a central role in regulating plant responses to heat stress.In this thesis,the physiological and molecular regulatory networks of perennial ryegrass under abiotic stress have been illustrated.The function and regulation mechanisms of HSFs in the heat stress response of perennial ryegrass have been characterized,which provided theoretical basis and technical support for the breeding of heat resistant germplasm of perennial ryegrass.These results provided new clues to partially solve the summer stress tolerance of perennial ryegrass.The important research results are as follows:(1)Abiotic stress screening of perennial ryegrass and transcriptional network analysis: The tolerance of 17 perennial ryegrass varieties under abiotic stress was identified.The physiological and molecular regulation networks of perennial ryegrass under drought,heat and cold stresses were characterized.The results showed that 17 varieties exhibited contrasting tolerances to drought,heat and cold stresses.Tolerant varieties had higher proline contents and antioxidant enzyme activities than sensitive ones after stress treatments.RNA sequencing analysis showed that the functions of commonly regulated unigenes by drought,heat and cold were mainly related to CHO metabolism,oxidative pentose phosphate,metal handling,hormone metabolism and lipid metabolism.Enriched pathways by drought stress include biodegradation of Xenobiotics,N-metabolism,CHO metabolism,oxidative pentose phosphate.Enriched pathways by heat stress include tetrapyrrole synthesis,photosynthesis,polyamine metabolism,S-assimilation,CHO metabolism,biodegradation of Xenobiotics,while CHO metabolism,glyoxylate cycle,biodegradation of Xenobiotics,polyamine metabolism,glycolysis were overrepresented after cold treatment.Based on the transcriptome data of perennial ryegrass after drought,heat and cold stresses,we constructed the regulatory network of perennial ryegrass under abiotic stresses.The results showed HSFs play a central role in regulating plant responses to abiotic stresses.(2)LpHSFCs positively regulate plant heat tolerance in perennial ryegrass: Heat stress induced LpHSFC1 b and LpHSFC2 b were cloned from perennial ryegrass leaves.Sequence analysis results showed that they all have typical conserved domains(DBD,OD and NLS)of HSF protein.Subcellular localization experiments showed that they are located in the nucleus.Ectopic expression of LpHSFC1 b and LpHSFC2 b in Arabidopsis significant improved plant thermotolerances.Transgenic Arabidopsis had decreased electrolyte leakage,malondialdehyde content and chlorosis/necrosis severity indices,and increased expression of heat stress responsive genes.(3)Genome-wide identification and expression analysis of the HSFs gene family in perennial ryegrass: Totally 17 LpHSFs were identified from the genome and transcriptome data of perennial ryegrass by using bioinformatics methods,of which 10 belonged to class HSFA,5 to class HSFB and 2 to class HSFC.Based on phylogenetic tree analysis,all LpHSFs were classified and named according to the orthologue genes in rice.The analysis of genetic structure,conserved domain and cis-acting elements showed that highly conserved organization,particularly within 3 subgroups,and most LpHSFs contained multiple phyhormone and stress responsive elements.The analysis of interaction network indicated that LpHSF genes were mainly related to transcription factor activity,transcription,DNA-dependent,signal transduction,nucleic acid binding,response to abiotic or biotic stimulus,RNA,hormone metabolism,signalling and stress.Expressions profiles of LpHSFs were validated by RNA-seq and q RT-PCR analysis showed LpHSFs could be responsive to abiotic stresses,particularly heat stress.The results indicated LpHSFs play a central role in regulating plant responses to heat stress and growth.(4)LpHSFA3 enhances heat stress tolerance by regulating LpHSFA2 in perennial ryegrass: Heat-induced LpHSFA3 and LpHSFA2 a were cloned from perennial ryegrass leaves.Sequence analysis results showed that they all have typical conserved domains(DBD,OD,NLS,and AHA)of HSF protein.LpHSFA3 and LpHSFA2 a were transformed into Arabidopsis plants and perennial ryegrass protoplasts,respectively.Ectopic overexpression of LpHSFA3 and LpHSFA2 a in Arabidopsis improved thermotolerance as evidenced by reduced electrolyte leakage and increased survival rate.Heat stress responsive genes were activated in LpHSFA3 and LpHSFA2 a transgenic Arabidopsis plants and perennial ryegrass protoplast.LpHSFA3 and LpHSFA2 a were localized in nucleus and functioned as transcription factors.LpHSFA3 binds to the HSE region in LpHSFA2 a promoter and constitutively activates the expression of LpHSFA2 a.These results indicated that transcription factor LpHSFA3 functions as positive regulator of LpHSFA2 a which provided novel evidence to understand the regulatory networks of plant heat stress response.(5)Optimization of protoplast transformation and genetic transformation system of perennial ryegrass: Using 10-12 d seedling leaves of perennial ryegrass' Shining Star' variety,we established an efficient protocol protoplast transformation.Based on this system,we established a protocol that can quickly screen and confirm heat stress candidate genes.In addition,we screened 5 perennial ryegrass varieties for callus induction rate,multiplication rate,regeneration rate and transformation rate.We preliminarily established a protocol for perennial ryegrass 'Shining Star' variety callus regeneration and genetic transformation.These results showed that perennial ryegrass exhibited divergent stress tolerances,and LpHSFs functioned as the key regulators during the abiotic stress responses in perennial ryegrass.LpHSFA3 functions as positive regulator of LpHSFA2 a through binding to the promoter region of LpHSFA2 a to improve thermotolerance of perennial ryegrass.LpHSFC1 b and LpHSFC2 b also functioned as positive regulators during heat stress responses in perennial ryegrass.These data characterized the mechanisms of thermotolerance of perennial ryegrass.Establishment of perennial ryegrass gene transformation protocol provided effective approach for breeding of new germplasms of perennial ryegrass.