Identification And Analysis Of Periodical-and Age-related MiRNAs In Cambium Of Ginkgo Biloba

Ageing is a universal property,often accompanied by telomere attrition,epigenetic alterations,loss of proteostasis and somatic mutations in yeast or animal cells.In plants,many woody and herbaceous perennials can live for many years,however,relatively little is known about age-related changes in long-lived perennials.Vascular cambium forms a continuous cylinder of meristematic cells in the stem,producing secondary xylem to the inside and secondary phloem to the outside.Cambial activity is a complex and dynamic process,strongly affected by many fectors and regulated by different signals,including the environmental conditions and several endogenous factors.So far,many studies focused on the periodicity of cambial activity and little is known about the age-related regulations.Ginkgo biloba,a fascinating gymnosperm,is the only living representative of the order Ginkgoales,which probably originated about 280 million years ago.It often has a long lifespan,being considered as a long-lived perennial.Hundreds of G.biloba plants ageing over 1000 years still exist in China.In the present study,on account of the cambial cells division and differentiation abilities,which are closely related to tree lifespan,using cytology,molecular biology,genetics and bio informatics technologies,we took G.biloba as experimental materials and the main results are as follows.?1?According to the phenological period and changes in the cambial cell layers and sizes,we divided the periodicity of cambial activity into dormant?September to erly-February?,reactive?mid-February to April?,active?late-April to October?,and transformed?November?stages.In the dormant stage,the cambium was composed of 4-6 layers of cells with thick walls,and the width of cambium zone was 58.79 ± 11.67?m The number of cell layers peaked at 10-12 in the active stage and the width of cambium zone was 104.58 ± 9.77 ?m We also found that the cambial activity was sensitive to temperature.?2?Through the observation of cambial cell ultrastructure,we found that the thickness of cell walls were thinnest at the active stage.The tangential walls?T walls?were 0.56 ± 0.10 ?m and radial walls?R walls?were 0.23±0.03 ?m.At the dormant stage,T walls were 0.90 ±0.33?m and R walls were 0.66 ±0.11 ?m.In addition,there were many starch granules at dormant in phloem while almost no starch granules were observed at active stage.?3?We sampled G.biloba of different ages in May based on the study of the periodicity of cambial activity.Totally,we sampled 34 female trees to obtain tree ring cores and through the ring analysis,we identified the tree ages range from 15 to 1353 years.The ring width decreased sharply in the first 200 years,with a slightly change over 200 years old.?4?We finally chose 9 trees for further study and divided them into three groups as 20 years?15Y,20Y and 22Y,as young trees?,200 years?193Y,211Y and 236Y,as older trees?and 600 years?538Y,553Y and 667Y,as oldest trees?.Through physiological index measurement,we found that IAA concentration and the activity of SOD,POD decreased in old trees.The content of ABA and H2O2 increased significantly with age.?5?We further used the cambium samples from the 9 selected trees to perform sRNA sequencing.Over 11,409,789 raw reads were obtained from each library.After removing low-quality reads,adapter sequences,poly A/T/C/G sequences and other artifacts,over 11,237,460 high-quality clean reads between 18 nt and 30 nt in length were obtained for further analyses.?6?After using a BLASTN search for sequence similarity in the miRBase database,276 known and 356 novel miRNAs were obtained in G.biloba cambium.After differential analyses,we found that the expression levels of 89,67 and 17 miRNAs were significantly changed between VC20 and VC200,VC20 and VC600,VC200 and VC600.Furthermore,hierarchical cluster analysis indicated that these differentially expressed miRNAs can be grouped into 8 clusters.Among them,there are 30 miRNAs with the expression level increased with age and 35 miRNAs showed decreased expression levels with age.?7?Through degradome sequencing,a total of 16,378,280 degradome tags were obtained.After alignment to GeneBank and Rfam,we obtained cDNA_sense tags and blast them with miRbase.As a result,a total of 83 targets of 54 miRNAs were identified.?8?The conjoint analyses of transcriptome,sRNA and degradome showed that the targets of 13 miRNAs such as tcc-miR172d,osa-miR528-5p,aly-miR165a-3p were associated with cell division and differentiation.The expression levels of these miRNAs were higher in VC200 and VC600 than that in VC20.Conversely,their corresponding targets displayed lower expression in old trees.In addition,miRNA-mRNA interaction and degradome sequencing demonstrated the cleavage events of miR166 and HD-ZIP ?,showing that miR166 and its targets are involved in cambial activity.?9?miRNA-mRNA interaction networks showed that the targets of miR160 and miR167 participate in IAA signaling and they expressed lower in old trees.Degradome sequencing demonstrated the cleavage events of miR160 and Gb₂2507,Gb₃3804,Gb₃9786.miRNA-mRNA interactions associated with CTK were also found in the present study.CTK.response regulators?Gb₁5501,Gb₁5884?and receptor?Gb₁3091?showed lower expression levels in old trees while the corresponding miRNAs expressed increased in VC200 and VC600.?10?14 targets of 12 miRNAs were annotated in secondary metabolism pathway,including miR396,novel₆7 etc.The expreesion of these miRNAs increased in old trees while their corresponding target genes expreesed decreased with age.Degradome sequencing demonstrated the cleavage events of miR396b-5p and Gb₂0206,Gb₁1915,miR156 and Gb₂3724.?11?We further detected 8 developmental-and stress response-related miRNAs in cambial dormancy-activity transition.We found that miR159 and miR166 were highly expressed during the reactive stage,while the expression levels of miR164 and miR319 significantly increased during the active stage.Conversely,miR482 and miR2950 were highly expressed during the dormant stage,suggesting that these miRNAs were involved in the regulation of the cambial activity transition-In addition,6 cell division related genes were selected to perform qRT-PCR at more ages?form 3 to 667 years old?and found that cell cyclin related gene?Gb₁1201?,CTK response reulator 3?Gb₀7568?and auxin responsive family protein?Gb₂0590?showed strongly age-dependent changes.