Response Of Melon To Saline-alkali Stress At Morphological,Physiological,Biochemical And Transcriptome Levels

Salinization has adverse effects on plant growth and physiological and biochemical metabolism,and it is also one of the main factors restricting facility cultivation of melon.Under the saline-alkaline environment,the quality of melon becomes worse and the yield decreases,which seriously affects the economic value of its cultivation.This problem is becoming more and more serious with the increase of continuous cropping years and the application of large amounts of fertilizer.In this paper,melon cultivar Xinyinhui was used as experimental material.The effects of salinization on seed germination and seedling growth of melon were observed by using NaCl,NaHCO₃ alone and NaCl+NaHCO₃ combined treatments to simulate salinization environment and Holange nutrient solution as control.The concentration of NaCl treatment was 0,50,100,150,200,250 mmol/L for seeds,0,50,100,200 mmol/L for seedlings;the concentration of NaHCO₃ treatment was:seed:0,30,60,90,120 mmol/L;seedling:0,30,60,90 mmol/L;the concentration of NaCl+NaHCO3 composite treatment was 0+0,25+12.5,50+25 and100+50 mmol./L.The physiological and biochemical indexes of Muskmelon under saline-alkali stress were determined.At the same time,transcriptome sequencing of muskmelon leaves treated with NaCl and NaHCO3 for 0 h,6 h and 48 h was carried out to explore the molecular response of muskmelon seedlings to saline-alkali stress.The results will provide theoretical basis for melon production in saline-alkaline soil.The main results are as follows:（1）50 mmol/L of NaCl treatment could promote the germination of melon seeds,and the germination rate,germination speed,radicle length,hypocotyl length and fresh weight were higher than those of the control,while more than 100 mmol/L of NaCl inhibited seed germination.（2）NaHCO₃ inhibited seed germination obviously,and the inhibiting effect was proportional to the treatment concentration and time.Morphological indexes such as radicle length,hypocotyl length and fresh weight decreased with the increase of treatment concentration.（3）The inhibitory effects of NaCl and NaHCO₃ combined treatments were similar in concentration dependence.Saline-alkali stress had a significant effect on active oxygen metabolism during the germination of melon seeds.Within 48 hours after treatment,SOD activity of melon seeds increased significantly,CAT and POD activities increased-decreased-increased,MDA content increased first and then decreased,proline content continued to increase,soluble sugar content continued to decline.（4）50 mmol/L of NaCl could promote the growth of melon seedlings,plant height,root length and fresh weight were higher than those of the control.More than 100 mmol/L of NaCl had adverse effects on plant height,fresh weight,root length and stem diameter.Leaves of seedlings were yellowing and wilting,root growth stagnated or even atrophied.With the prolongation of treatment time,high concentration of NaCl would eventually cause lethal effects on seedlings.NaHCO3 treatment also inhibited the growth of melon seedlings,plant height,root length and fresh weight were lower than the control.Under 90 mmol/L of NaHCO₃,leaves yellowed and roots withered in melon seedlings.The growth of melon seedlings was inhibited by combined treatment of NaCl and NaHCO₃.The inhibition increased with the increase of stress concentration and stress time.（5）After NaCl and NaHCO₃ combined treatment,the content of H₂O₂in the leaves of melon seedlings showed a trend of increasing,decreasing and increasing.SOD activity increased first and then decreased.CAT activity showed a trend of decreasing increasing,and decreasing.POD activity decreased first and then increased.The content of MDA showed a trend of increasing,decreasing and increasing.Pro content continue to rise.There was no significant difference in soluble sugar content contrasted with control.The contents of chlorophyll a,chlorophyll b,carotenoids and total chlorophyll in photosynthesis related indicators increased first and then decreased.The actual photochemistry efficiency Y（II）decreased first and then increased,which was significantly different from the control.The maximum photochemical efficiency of PS II did not change significantly.The overall qP showed a downward trend,while NPQ showed an upward trend;Y（NPQ）and Y（NO）showed an upward trend first and then a downward trend.（6）Transcriptome sequencing was performed in the simulated saline-alkali stress group treated with 50 mmol/L NaCl+25 mmol/L NaHCO₃ and the untreated control group.with high-throughput sequencing technology.The sampling points were 6 h and 48 h.in this study,12 samples were sequenced and 74.69 GB clean bases and 250.39 MB clean reads were obtained.The average output of each sample was 6.22 GB clean bases and20.86 MB clean reads,and the percentage of Q30 base was more than 90%.Clean Reads of each sample were compared to the reference genome sequence,and the efficiency was 90.32%-93.19%.In addition,474 new genes were found.Based on the results of comparison,we found that there were about 52,000 single nucleotide polymorphisms（SNP）loci in each sample on average,and most of them were SNP loci in gene region.At the same time,we also found that the proportion of heterozygous SNP loci in this melon variety was very small,only about 2.8%.In addition,transcriptome data analysis showed that there were 12 alternative splicing（AS）types in each sample,and AS types were same under saline-alkali stress and control conditions.However,the number of AS events in saline-alkali stress samples was significantly higher than that in normal culture conditions,and the number of TSS and TTS was the largest,indicating that exon 3’and 5’shear played a dominant role in saline-alkali stress response.（7）Through the analysis of gene expression patterns,370differentially expressed genes,including 216 up-regulated genes and 154down-regulated genes,were found in control and treatment groups（H6 vs T6）at 6 h.There were 325 DEGs in 48 h control and treatment group（H48vs T48）,including 91 up-regulated genes and 234 down-regulated genes,and 77 genes were expressed in H6 vs T6 and H48 vs T48.Through GO classification annotation of DEGs,it was found that in the early stage of salt-alkali stress（6 h）,the function of cell membrane and transcription factors were mainly affected.melon was resistant to salt-alkali stress,and in the late stage of salt-alkali stress（48 h）,the function of cells was affected,which further produced toxic effects on melon.KEGG enrichment analysis of DEGs showed that salt-alkali stress mainly affected the metabolism of melon,and the proportion of involved pathways in all KEGG pathways was 70.28%（H6 vs T6）and 68.76%（H48 vs T48）.The first 20 pathways of significant enrichment in H6 vs T6 and H48 vs T48 groups were analyzed.It was found that the adaptability of melon to saline-alkali stress was improved mainly through metabolism,plant hormone signal transduction and circadian rhythm changes in the early stage of saline-alkali stress.With the prolongation of saline-alkali stress time,the degree of damage of melon was aggravated and DNA damage was caused,which seriously affected the growth and development of melon.Finally,the results of transcriptome sequencing were verified by fluorescence quantitative experiment.The expression of 18 DEGs at 0,6,12,24 and 48hours after saline-alkali stress was analyzed,which confirmed the reliability and accuracy of transcriptome data.