Molecular Mechanisms Of Alternative Polyadenylation On Regulation Of Aluminum Toxicity In Chinese Fir

Chinese fir is an important timber species in southern China,which is of great significance to social and economic development.In recent years,over-cultivation has increased soil acidification,causing the release of insoluble alumina in the soil,forming toxic soluble aluminum salts,and slowing plant growth and development.Chinese fir is the most important economic tree species in southern China.Studying its molecular mechanism in response to aluminum poisoning is of great significance to China’s forestry security.However,the current research on the response of Chinese fir to aluminum toxicity stress has focused on physiological and biochemical levels and a few experiments on transcription levels.Studies on post-transcriptional levels of aluminum toxicity responses in Chinese fir have not been reported.Alternative polyadenylation is an important post-transcriptional regulation,which regulates gene expression at the post-transcriptional level by regulating the expression of mRNA isoforms with different length.It has been reported that alternative polyadenylation is involved in regulating plant stress,growth and development.However,the alternative polyadenylation related research has not been carried out in gymnosperms.This study integrates molecular biology and bioinformatics to conduct research on the alternative polyadenylation of Chinese fir seedlings under aluminum toxicity and control conditions.It has been shown initially that alternative polyadenylation is involved in regulating the response of Chinese fir to aluminum toxicity.Our study shows:（1）Phenotypic experiments showed that 4 mM AlCl₃inhibited the root morphogenesis of Chinese fir seedlings.It was found that in the treatment time of 32 h,the culture medium containing different concentrations of aluminum ions could inhibit the relative elongation ratio of Chinese fir roots,and the roots of Chinese fir seedlings treated with aluminum were soft and curved compared with the control.（2）PAS-seq analysis showed that most genes contained more than two poly（A）sites（66.57%and 63.77%）under the control and aluminum treatment conditions;the genome-wide results also verified that aluminum toxicity caused global polyadenylation sites profile changes（p-value<2.2e-16）.By counting the number and proportion of PAC and PAT in different regions of the gene and performing t-test tests,the results showed that aluminum toxicity significantly affected the distribution of PAT in the intergenic region,3’UTR region and intron region（p-value=0.0136,0.0198 and 0.0046,respectively）,but the distribution of PAC ratios in different gene regions was not significantly changed.（3）Aluminum stress caused changes in the base use of FUE and CE element sequences in the upstream intron region of Poly（A）signals.Data analysis revealed that under aluminum treatment,the use of A bases in FUE and CE regions decreased.The use of T bases increased in the CE region.However,there was no significant difference in the frequency of base usage in the intergenic region,3’UTR,5’UTR and CDS regions under both control and treatments.（4）Using DESeq2 software to analyze the differential expression of PAC to count the regional distribution and proportion of DE-PAC.The results show that 9.5%（8278）of PACs were differential expressed,of which 4938 PACs were up-regulated and 3340 PACs were down-regulated.The DE-PAC mainly occurred in the 3’UTR and intergenic regions as expected.However,there were more up-regulated PACs than that of down-regulated PACs.GO enrichment analysis found that up-regulated PACs containing genes were mainly enriched in important GO categories such as organic acid metabolism,response to stimuli and gene expression.The down-PAC gene was also enriched in some of same GO categories,but the drug binding and kinase activity were uniquely down-regulated.（5）Alternative polyadenylation is involved in the growth and development of Chinese fir under aluminum toxic stress.By calculating a total of 733 switching genes,GO enrichment analysis found that the main enrichment was terms including molecular metabolism,organic acid metabolism,gene expression and response to stress.At the same time,we found genes related to hormones and stress such as ABI5,GST and so on were enriched in the aluminum toxicity stress associated poly（A）site switching genes.