Physiological,biochemical And Molecular Responses Of Melon To Microplastics Exposure Environment

Microplastics（MPs）are plastic particles with a diameter of less than5 mm.As an emerging type of pollutant,its impact on the ecological environment has attracted widespread attention.The use of a large amount of plastic in agriculture and its residues in the environment make this situation particularly prominent,with significant potential ecological risks.The impact of MPs on plants is multifaceted.It can affect the physical and chemical properties of soil,biological communities,and material cycling,control root development,and form complex pollution with other pollutants.MPs pollution delays the germination process of seeds,hinders plant growth and development,inhibits photosynthetic pigment production,interferes with nutrient metabolism in plants,causes oxidative damage,and even produces genotoxicity.Cucumis melo L.is an important Cucurbitaceae plant with high economic value,which is widely cultivated in various countries around the world.China is the world’s largest producer and consumer of melon,and using plastic greenhouse and plastic film mulching for cultivation has become the main mode.The long-term adoption of this model will inevitably lead to the accumulation of soil MPs.Currently,there are no relevant research reports on the impact of MPs on melon.In this paper,PVC particles with different sizes were selected to simulate the microplastic exposure environment（MEE）.The effects of different particle sizes and concentrations of MPs on seed germination and plant growth of melon and their physiological and biochemical mechanisms were analyzed.Combined with transcriptome sequencing,the differential expression of melon genes induced by MPs was analyzed to explore the possible molecular mechanism.The main conclusions are as follows.1.Effects of MEE on seed germination and physiological and biochemical metabolism of melonThree kinds of PVC-MPs particles with different particle sizes were tested during the germination of melon seeds.The results showed that only 75μM MPs particles significantly inhibited the germination rate of melon seeds at a medium concentration(2-4 g·kg^-1),and 13μM and 225μM had no significant effect.75μM MPs particles can significantly inhibited seed germination potential and germination index under all treatment concentrations.There were significant differences between different concentrations.The effect of MPs on melon seed germination was significantly correlated with particle size and treatment concentration.The morphology of germinating seeds was also significantly influenced by MPs.At a concentration of 1-4 g·kg^-1,the number of young roots increased and the number of root tips decreased.The content of H₂O₂and O₂^-changed significantly during seed germination,but the trend of change was not significantly correlated with treatment time.The content of MDA in the early stage of treatment was lower than that in the control,and significantly increased at the 6th day,which was higher than that in the control.POD activity significantly increased at the late stage of treatment（the 4th day）;CAT activity increased first and then decreased with the increase of treatment time.The soluble sugar and starch content decreased significantly from 0 to 1 d.On the 6th day,the amylase activity of the treatment group increased,but the starch and soluble sugar content continued to decrease.2.Effects of MEE on growth and physiological and biochemical metabolism of melon seedlingsWhen the concentration of MPs was 1 g·kg^-1,the fresh weight and dry weight of melon seedlings were significantly inhibited,significantly lower than the control,and the trend of change was similar.The correlation between fresh weight and plant height was weak.At this treatment concentration has a negative impact on root morphology;the root length,root surface area,root volume,root tip number and root number were significantly reduced.However,root activity enhanced to a certain extent.The content of ascorbic acid in leaves decreased significantly after treatment with MPs at different concentrations.The change trend of proline,malondialdehyde,soluble sugar,and soluble protein content did not show a linear relationship with concentration.However,the turning point of content change mostly occurred at a concentration of 2-4 g·kg^-1.The effects of MPs on chlorophyll and carotenoid content showed the same trend.The turning points of chlorophyll fluorescence parameters also mostly occurred at concentrations of 2-4 g·kg^-1.CAT activity continued to decrease with increasing MPs concentration.The activity of SOD and POD,and the content of H₂O₂did not show a trend of change related to concentration.The variation trend of O₂^-content and production rate presents an inverted V shape,with its peak value appearing at a concentration of 2-4 g·kg^-1.The changes of CAT,POD,and SOD in roots were similar to those in leaves,and their activities were significantly higher at 2 g·kg^-1and 8 g·kg^-1than in the control.The content of H₂O₂decreased and decreased with the increase of MPs concentration.The O₂^-content and production rate were significantly higher at 2 g·kg^-1and 8g·kg^-1than the control.The peak value of both concentrations appeared at8 g·kg^-1.The accumulation of active oxygen species in roots reaches its maximum at a concentration of 2-4 g·kg^-1.3.Differentially expressed genes responding to MEE in melon seedlingsThe differentially expressed genes induced by MPs in melon seedlings mainly involve pathways such as stress resistance,signal transduction,hormone metabolism,and plant pathogen interactions.KEGG and GO analysis showed that most genes were enriched in the phenylpropane metabolic pathway.Most of the key genes and transcription factors that regulate the biosynthesis and metabolism of phenylpropane and flavonoids were significantly down-regulated.Such as MAPK signal transduction,plant hormone and plant pathogen interactions,and other related genes.Basic metabolism related genes such as active oxygen metabolism and photosynthesis were also affected to varying degrees.Some genes related to antioxidant enzymes and photosynthesis were also downregulated.MYB and C4H were significantly involved in the response of melon seedling roots to MPs.4.Analysis of Cm C4H family and transgenic verification of its interaction with MYBCinnamate 4-hydroxylase（C4H）is a rate-limiting enzyme in phenylpropane metabolism and plays an important role in the regulation of lignin synthesis.A total of 6 C4H family members were identified in melon genome,all of which were hydrophilic proteins.The results of subcellular localization showed that most of Cm C4H members were located in the plasma membrane,while Cm C4H2 and Cm C4H6 were located in vacuoles.Sequence analysis showed that all Cm C4H family members contain 2-3 exons,and encoded proteins with 13 conservative motifs.Phylogenetic analysis showed that Cm C4H can be divided into two major categories,which were closely related to cucumber C4H.The analysis of cis-acting elements showed that most of Cm C4H members contained hormone response elements,light response elements and stress response elements.The treatment of Cm MYB transformed Arabidopsis plants with MPs resulted in the upregulation of Cm C4H members and Cm MYB,except for Cm C4H5.Upregulation of Cm C4H expression by introducing MYB may help melon plants cope with MPs stress.