Dissection Of Mechanisms Underlying The Enhanced Stress Tolerance In Autotetraploid Poncirus Trifoliata And Functional Identification Of Two Stress Responsive Genes

Trifoliate orange(Poncirus trifoliata(L.)Raf.),one of the most important rootstocks in citrus,has better cold tolerance and disease resistance.However,its drought and salt tolerance are relatively weak,which severely constrains its utilization in citrus industry.Increasing studies revealed that polyploid plants could confer enhanced stress tolerance,indicating that ploidy-breeding might be an effective approach to breed stress-resistant citrus germplasms.Additionally,as the core of the modern breeding objectives,genetic engineering can also be used in breeding new cultivars with better stress resistance.So,it is of vital importance for citrus to investigate polyploid germplasms and study key stress-related genes.Based on these backgrounds,this study screened some autotetraploid trifoliate oranges and investigated their drought and salt tolerance.Then,with RNA-seq,we revealed the mechanisms underlying the enhanced stress tolerance of tetraploids,and studied two important stress-responsive genes from the RNA-seq data.The main results are described as below:1.Firstly,based on morphological screening,FCM(flow cytometry)and chromosome counting,we screened 75 tetraploid plants from about 20,000 natural trifoliate orange seedlings(probability of tetraploid occurrence: 0.375%).Whole genomic SNP analysis confirmed that the obtained tetraploid plants were all autotetraploids.By comparing the tetraploids with diploids,we found that tetraploid plants had significantly different phenotypes,including higher plant height,wider and thicker leaves,reduced stomatal density,and enlarged tissue cells.After drought and salt treatments,by observing phenotypes and measuring related indicators,we found that tetraploids had significantly enhanced drought and salt tolerance in comparison with diploids.2.To reveal the mechanisms underlying the enhanced drought tolerance in tetraploids,we conducted RNA-seq using both diploid and tetraploid leaves before and after drought treatment.By GO enrichment analysis,we found that some important stress-related pathways were differently enriched between diploids and tetraploids,especially antioxidant-related pathways,in which related genes were only enriched in tetraploids but not in diploids.By comparing the antioxidant capacity between diploidsand tetraploids,we found that the expression of antioxidant enzyme genes and enzyme activities in tetraploids were higher than diploids under drought stress,resulting in reduced ROS accumulation in tetraploids.By KEGG pathway enrichment analysis,we found that “starch and sucrose metabolism” pathway was significantly enriched among the DEGs between diploids and tetraploids under drought stress.One vacuole invertase gene(VINV)was dramatically induced by drought and had significantly increased expression levels in tetraploids.In line with the gene expression,tetraploids had lower sucrose content but higher glucose content compared with diploids.Above all,our study demonstrates that the enhanced ROS scavenging and osmotic adjustment capacity contribute to the enhanced drought tolerance of autotetraploid trifoliate oranges.3.To dissect the mechanisms underlying the enhanced salt tolerance of tetraploids,we conducted RNA-seq using both leaves and roots of tetraploids and diploids before and after salt treatment.By GO analysis with screened DEGs,we found that many DEGs were related to stresses.KEGG enrichment analysis indicated that the enriched pathways of DEGs in leaves and roots were different.In leaves,“plant hormone signal transduction” pathway was significantly enriched,in which genes related to auxin,brassinosteriod,cytokinine,and jasmonic acid were more highly expressed in tetraploids than diploids.In addition,one POD gene had higher expression levels in tetraploids than diploids,leading to improved ROS scavenging ability in tetraploid leaves.And in roots,KEGG analysis revealed that “starch and sucrose metabolism” and “arginine and proline metabolism” pathways played pivotal roles.Further investigations indicated that tetraploids accumulated more soluble sugar and proline than diploids under salt stress.In addition,one antioxidant enzyme gene(APX)had higher expression in tetraploid roots than diploid,providing tetraploid roots with enhanced ROS scavenging ability.So,this study reveals that the different defense mechanisms of leaves and roots synergistically contribute to the enhanced salt tolerance in tetraploids compared with diploids.4.From the RNA-seq data for drought stress,we screened one LEA family gene(Ptr LEA7)and investigated its function.Ptr LEA7,with a 420 bp full-length sequence,encodes 139 amino acids and belongs to LEA?4 subfamily.The expression of this gene is induced by dehydration,cold and ABA treatments.The subcellular localization indicatedthat this protein was located in both cytoplasm and nucleus.With the transgenic plants over-expressing Ptr LEA7 in both tobacco and trifoliate orange,we conducted drought treatment and found that transgenic lines had enhanced drought tolerance,indicating that this gene positively regulates drought tolerance.Furthermore,we found that expression of antioxidant enzyme genes and catalase(CAT)activity were increased,and ROS accumulation was decreased in transgenic trifoliate oranges than wild type,indicating that Ptr LEA7 positively regulates drought tolerance by enhancing antioxidant capacity.5.From the RNA-seq data for salt stress,we screened one R2R3-MYB family transcription factor(Ptr MYB3),which has a 756 bp full-length sequence and encodes 139 amino acids.Ptr MYB3 protein is located in nucleus and has transcriptional activation activity.The expression of Ptr MYB3 is induced by salt,dehydration and cold stresses.With VIGS(virus induced gene silencing)technique,this gene was silenced in trifoliate orange.After salt treatment,we found that Ptr MYB3-silenced lines had significantly enhanced salt tolerance.Further investigations indicated that genes related to amylase,pectin methylesterase(PME)and peroxidase(POD)had higher expression levels in the silenced lines than wild type.LUC assay confirmed that Ptr MYB3 was a transcriptional repressor,indicating that Ptr MYB3 might negatively regulate salt tolerance by suppressing the expression of related genes.