The Research On Salt Tolerance Mechanism Of Tetraploid Watermelon Seedling

Soil salinization is an important factor which limits the watermelon production in China,especially in northwest and coastal areas.Large amount of fertilizer application and frequent irrigation also easily cause soil secondary salinization.With the continuous expansion of watermelon cultivated in facilities,the soil salinity accumulated in facilities due to the sealing characteristics of facilities and improper management of fertilizer and water and other reasons,resulting in secondary salinization,which seriously restricts the high-quality,efficient and sustainable development of watermelon.Therefore,it is of great significance to understand the mechanism of salt tolerance of watermelon and to develop new watermelon varieties with salt tolerance.Polyploid watermelons were discovered to posses higher salt tolerance than their homozygous diploid ancestors,however,the specific mechanism of salt tolerance is still not clear.This study provides information at physiological level,absorption and transport of Na~+and K~+,transcriptome and DNA methylation for the mechanism of salt stress response in diploid and tetraploid watermelons,which should explain the mechanism of salt tolerance in polyploid watermelon.1.Morphological and cellular structures of diploid and tetraploid watermelon seedlingsThe results showed that the leaf length,width,circumference and area of tetraploid watermelon were 0.88-,1.24-,1.19-and 1.48 times of these in diploid watermelon seedlings,respectively.The tetraploid watermelon showed 1.14 times larger stem diameter than that of diploid watermelon seedlings.The total length,average diameter,projected area,surface area and volume of the tetraploid watermelon root were all larger than those of its homologous diploid watermelon seedlings.The palisade tissue cells of leaf of tetraploid watermelon were longer and larger,the content of chloroplast and mitochondria were more,the spongy tissue was loose and the gap was larger.The stem and root of diploid and tetraploid watermelon were penetrated by vascular bundles,compared with diploid watermelon,the tetraploid watermelon exhibited larger xylem cells,more sieve tubes and chaperones.2.Physiological and biochemical indexes of diploid and tetraploid watermelon seedlings under salt stressAfter the treatment,the tetraploid rootstock-grafted watermelon plants showed higher salt stress tolerance than the diploid plants,there were no significant differences in the physiological effects between the rootstocks with the same ploidy.The tetraploid rootstock-grafted watermelon plants exhibited higher biomass contents,photosynthesis parameters(Pn,E,C),chlorophyll fluorescence parameters(Fv/Fm,Fv/Fo,qP,ETR),antioxidant protection enzymes activies(SOD,POD,PAL),protective osmotic regulation substances contents(GSH,Pro)the diploid rootstock-grafted watermelon plants throughout the salt treatment process.while MDA and active oxygen content were found the opposite trend.The content of 9kinds of endogenous hormones(GA3,ZR,ABA,IAA,Br,Ipa,GA4,Ja-me,Dhzr)increased,which were observed significant higher in tetraploid rootstock-grafted watermelon plants than that of the seedlings with diploid ones in the roots.These results explained that the salt tolerance of tetraploid watermelon was related to its stem and root system,but not leaf.3.Changes of ion absorption and transport in diploid and tetraploid watermelon seedlings under salt stressAfter treatment with NaCl for 8 days,the Na~+concentrations increased,which were higher in the diploid rootstock-grafted watermelon plants than those in the tetraploid rootstock-grafted watermelon plants.The K~+concentrations decreased,which were lower in the diploid rootstock-grafted watermelon seedlings than in the tetraploid rootstock-grafted seedlings.The stem exhibited the highest Na~+and K~+concentrations,followed by leaves and roots.After 300 mmol/L NaCl stress,the Na~+flux increased in all the plants,while the K~+flux decreased in all seedlings,the Na~+and K~+fluxes of the root tips were higher in the tetraploid rootstock-grafted watermelon plants than in the diploid plants.4.Transcriptome analysis of diploid and tetraploid watermelon seedlings after salt stressAfter salt stress,the up-regulated genes in diploid leaves were significantly enriched in alternative splicing,endoplasmic reticulum protein synthesis and TCA cycle pathways;the down-regulated genes in diploid root were enriched in the lignin synthesis and the synthesis of secondary metabolites pathways;the up-regulated genes in diploid root were enriched in alternative splicing,endoplasmic reticulum protein synthesis routes.After salt stress,the up-regulated genes in tetraploid leaves showed the same pathway with diploid;the down-regulated genes in tetraploid stems were significantly enriched in the ribosomes,lignin biosynthesis and DNA replication process;the up-regulated genes in tetraploid stems were significantly enriched in the degradation of valine,leucine and isoleucine,porphyrin and chloroplast metabolism;the DEGs in tetraploid roots were enriched in the same paths with diploid roots.5.Genome-wide DNA methylation of diploid and tetraploid watermelon seedlings after salt stressThe results showed that after salt stress the diploid and tetraploid watermelon seedlings exhibited different DNA methylation mechanisms in response to salt stress,and DNA methylation in different contexts(CG,CHG,CHH)showed different pathways and tissue specificity in response to salt stress.After salt stress,the hypermethylated genes in diffenental methylation ragion(DMR)in diploid watermelon leaves were significantly enriched in ammonia acyl tRNA biosynthesis,RNA degradation pathways;the hypomethylated genes in diploid watermelon leaves were significantly enriched in the transcription factors,the ribosome biosynthesis pathway and so on;the hypomethylated genes in diploid watermelon root were significantly enriched in the cutin,suberine and wax biosynthesis,linoleic acid biosynthesis pathways.After salt stress,the hypermethylated genes in tetraploid watermelon leaves were significantly enriched in mRNA surveillance pathway,glycosylphosphatidylinositol(GPI)-anchor biosynthesis,RNA transport and other pathways.DNA methylation mediated salt stress response mechanism in tetraploid watermelon stem was complicated,many pathways were involved in.The hypermethylated and hypomethylated genes in tetraploid watermelon roots were significantly enriched in RNA transport,mRNA surveillance pathway and spliceosome.6.Associate analysis of transcriptome and DNA methylation data of diploid and tetraploid watermelon seedlings after salt stressThe salt stress response mechanism of diploid watermelon seedlings regulated by DNA methylation was as follows:the up-regulated expression of 8 genes in the root involoved in the metabolic pathway of linoleic acid were mediated by DNA hypomethylation.The salt stress response mechanism of tetraploid watermelon seedlings regulated by DNA methylation was as follows:the gene expression levels were upregulated by DNA hypermethylation in tetraploid seedlings,2 genes in the leaves were involved in cutin,suberine and wax biosynthesis,12 genes in the stems involved in the leucine,isoleucine,and valine biosynthesis and metabolism of pyruvic acid ester,3 genes in the roots involved in the alternative splicing.In addition,4 genes in the roots involved in the RNA transhipment,amino sugar and nucleotide sugar metabolic process were upregulated by DNA hypomethylation,these genes worked together to participate in tetraploid watermelon salt stress response.