| Due to the increasing amount of plastic waste,microplastics from weathered plastics are widely distributed in various environments,including the atmosphere,farmland,forests,and lakes,attracting more public attention.The microplastics accumulated in the soil can be absorbed by plants and affect their growth and development.Previous studies have been studied extensively on the response of plants to microplastic stress,mainly focusing on herbaceous plants,such as model plants,important crops,and horticultural plants.However,the woody plant response to microplastic stress is still unclear.This study used the woody plant Ginkgo biloba as the experimental material to explore the effects on changes of morphological,physiological,biochemical,and transcriptional regulation under microplastic stress.The main results are as follows.（1）In the concentration dose experiment,G.biloba seedlings were treated by 50 mg/L,100 mg/L,and 300 mg/L microplastics and morphological,physiological and biochemical data were measured.The results showed that compared to lower microplastic concentrations（50 mg/L and 100 mg/L）treatment,higher concentration（300 mg/L）resulted in more markedly morphological,physiological and biochemical changes of seedling roots and leaves.（2）During 300 mg/L microplastics exposure treatment,plant samples were collected at 12 hours,24 hours,3 days,7 days,and 14 days after treatment,and their morphological,physiological and biochemical indicators were measured and analyzed.The results showed that short-term（12 hours,24 hours,and 3 days）treatments had no significant effects.However,under long-term（7 and 14 days）treatment,root and leaf morphology,secondary metabolite accumulation,antioxidant enzyme activity and leaf photosynthesis were markedly affected.The results showed decreased leaf area and root length and increased lateral root number and length.The total flavonoids in leaves,soluble sugars and soluble proteins in roots were increased,while the content of soluble sugars,soluble proteins in leaves,and total flavonoids in roots were decreased.The result of micronucleus test showed that 300 mg/L exerted an apparent genetic toxicity on seedling roots.（3）The transcriptome result showed that 3940 DEGs were enriched in G.biloba seedling root among the control group（Control,0 day treatment）,1 day treatment（MP1d）and 7 days treatment（MP7 d）.In KEGG enrichment analysis of MP1d vs Control,DEGs are mainly enriched in "alpha-Linolenic acid metabolism","MAPK signaling pathway-plant","Phenylpropanoid biosynthesis","Plant-pathogen interaction","Phenylalanine metabolism",etc.In the MP7 d vs Control group,DEGs are mainly enriched in "Taurine and hypotaurine metabolism","Phenylpropanoid biosynthesis","Stilbenoid,diarylheptanoid and gingerol biosynthesis","Ubiquinone and other terpenoid-quinone biosynthesis",etc.DEG analysis in the phenylpropanoid pathway revealed that microplastics treatment significantly changed flavonoid synthesis related gene expression,such as PAL,C4H,CHS,COMT,LAC,DFR,and F3’H.For example,most of the key enzyme genes LAC are significantly down regulated in lignin pathway,while most of the key genes involved in flavonoid synthesis such as CHS,CHI,and F3’H are up regulated significantly.Interestingly,the expression of some genes related to lignin and flavonoids displayed an opposite trend,The qRT-PCR results further verified the expression patterns of these genes mentioned above.（4）Arabidopsis of wild type,flavonoid synthesis gene overexpressed plants,and mutant plants were treated by microplastics stress to verify if flavonoids were involved in the response.NBT and PI staining results showed that CHS overexpressed plants had less oxidative damage in their roots and leaves,and their root cell vitality was significantly higher than that of wild type plants.The mutant plants showed a severe oxidative damage,and the root cell vitality was significantly lower than that of the wild type.These results indicated that flavonoids were involved in the response of Arabidopsis to microplastic stress and can significantly reduce the damage by microplastics stress.In conclusion,long-term microplastic exposure can exert significant morphological,physiological,biochemical,and transcriptional regulation effects on G.biloba.Flavonoids play an important role in microplastic-stress resistance.These findings provide a reference for studying the effects of increased microplastic pollution in the environment on plants. |
