A Comparative Study On The Physiological And Transcriptomic Mechanisms Of Different Rooting Types In Mulberry Hardwood Cuttings

Adventitious root（AR）formation is a critical process in vegetative propagation of many horticultural and forestry crops,it is a bottleneck for cutting propagation.The initiation and induction of root primordium are the bottleneck of AR formation.The formation of ARs in hardwood cuttings of conventional mulberry was mainly based on callus-rooting.However,after treatment with indole-3-butyric acid（IBA）,the cuttings were changed to the cortex-rooting type with high rooting efficiency under appropriate conditions.But the regulation of IBA-induced changes in rooting form is still unknown.In order to explore the differences between the two rooting types of mulberry"Yu711"hardwood cuttings and the role of IBA on cuttings,this paper conducted a comparative study on the three developmental stages（stage1-induction period,stage2-expansion period,stage3-rooting period）of the two rooting types by means of physiological index determination,RNA-Seq and Small RNA-Seq.And we further analyzed the physiological and transcriptome changes in the early phases（54h after planting）of AR formation in cortex-rooting hardwood cuttings of mulberry.The main results and conclusions of the two rooting types during the three stages are as follows:1.The content of soluble sugar,soluble protein and starch in the two rooting types of cuttings decreased,which meant that the AR formation involves a strong conversion of substance and energy.In addition,the content of starch in the cortex-rooting cuttings was always lower than that in the control,indicating that IBA promoted the metabolism of starch in the cortex-rooting cuttings.Compared with the callus-rooting type,the content of auxin（indole-3-acetic acid,IAA）in the cortex-rooting type showed more obvious fluctuations,and the content of zeatin（ZA）was higher than that in the control,indicating that IBA may promote the synthesis of ZA and IAA in the cortex-rooting cuttings during AR formation.The activities of peroxidase（POD）,polyphenol oxidase（PPO）and indoleacetic acid oxidase（IAAO）in the two rooting types showed a trend of increasing gradually during the whole development period,and the cortex-rooting type was always higher than the callus-rooting type.On the contrary,the content of H₂O₂ and soluble phenols in the two rooting types showed a trend of gradual decline,while the cortex-rooting type was always lower than the callus-rooting type.These results showed that IBA increased the activity of rooting enzymes in cortex-rooting cuttings,reduced the production of toxic substances such as reactive oxygen species,and made cortex-rooting easier to root than callus-rooting cuttings.2.Compared with callus-rooting,there were 264 common DEGs in cortex-rooting cuttings at three developmental stages,and these genes were clustered into 5 clusters with different expression patterns.Compared with the callus-rooting type,the genes encoding POD and PPO in the cortex-rooting cuttings were significantly up-regulated in stage3,which was consistent with the fact that the activity of POD and PPO in the this stage was significantly higher than that in the control group.This result indicated that POD and PPO were the key factors to regulate the AR formation in the cuttings.The Plant-pathogen interactions,mitogen-activated protein kinase（MAPK）and plant hormone signal transduction pathways in cortex-rooting cuttings were activated on the whole,while the circadian rhythm pathway was inhibited,indicating that IBA activated the stress response and inhibited the response to light of cortex-rooting cuttings.In addition,these pathways affect each other and jointly regulate AR formation by sharing BAK1（brassinosteroid insensitive1-associated receptor kinase1）,FLS2（flagellin-sensing2）and other proteins.3.The results of Small RNA-Seq showed that there were 6 miRNAs that were differentially expressed in cortex-rooting cuttings at three developmental stages compared with the callus-rooting types,namely mi R319a-3p,mi R162-3p,mi R396b,mi R164a＿4,mi R170-5p and mi R171b-3p＿3.And the target genes of these 6 miRNAs were annotated into multiple KEGG pathways.Conjoint analysis of Small RNA-seq and RNA-seq showed that,compared with callus-rooting type,cortex-rooting type had three differentially expressed negative correlation target pairs at three developmental stages,namely mi R395a＿5·RPM1（disease resistance protein RPM1）,mi R396b·PXL1（phloem intercalated with xylem-like 1）and mi R172c＿2·fls H（filamentation-temperature-sensitive protein H）.They regulated physiological activities such as cell division and stress response during AR formation.In addition,no matter the cortex-rooting type or the callus-rooting type,there were many differences in the expression of miRNA·m RNA in the comparison groups between adjacent developmental stages,indicating that different amounts and types of miRNA·m RNA pairs were involved in the regulation at different developmental stages.It can be seen that the regulation of miRNA·m RNA pairs on AR formation of mulberry hardwood cuttings is complex.The primary results and conclusions of the study on the early phases of AR formation of cortex-rooting cuttings are as follows:4.The gene expression level of TCA cycle in the glucose metabolism pathway increased,and the effect of sucrose conversion to other forms of soluble sugar was inhibited,resulting in the decrease of soluble sugar content in the cuttings during the early phases of AR formation.The high level of energy and substrate produced by respiration improves the synthesis capacity of proteins.The degradation pathway of proteins and amino acids is inhibited,which leads to the increase of soluble protein content in cuttings.5.The increase in auxin transporter expression triggered auxin accumulation in cortex-rooting cuttings,which led to the peak of IAA at 6 hours after planting.In the following 48h,IAA decreased from"pool"to"long-term reduction"to promote the formation of ARs.The content of ZA also decreased to the homeostasis level after rapid induction,and the genes involved in cytokinin degradation,transport and signal transduction showed a phase-specific expression pattern.Ethylene and jasmonic acid may play a positive role in the induction stage of AR formation of mulberry hardwood cutting rather than in the initial stage.The activities of peroxidase and catalase were persistently increasing,indole-3-acetic acid oxidase was maintained at a higher stable-state from 0h,while the activities of polyphenol oxidase fluctuated with soluble phenolics and IAA levels.6.The comparative transcriptome of cortex-rooting cuttings identified a set of conserved genes in the early phases of AR formation that were differentially regulated at-6（control）,0 and 54 h AP（hours after planting,h AP）.They were separated into five clusters with distinct biological functions.Considerable conserved genes were assigned to pathways of sugar metabolism,MAPK,and hormone signaling.The gene co-expression network analysis revealed three major co-expressed modules involved in stress responses,starch metabolism,and circadian rhythm.Based on the above two research directions,the conclusion is drawn that IBA has a positive effect on the ARs formation of mulberry hardwood cuttings.Pathways such as energy metabolism,plant hormone signal transduction,plant-pathogen interaction,stress response and circadian rhythm play an important role in regulating the AR formation of mulberry cuttings.Mi R395a＿5·RPM1,mi R396b·PXL1,mi R172c＿2·fls H were involved in the regulation of IBA-induced ARs formation of mulberry hardwood cuttings.