PtomiR476a Targets PPR-RFL Transcripts To Regulate Wood Formation In Populus Tomentosa

Chinese white poplar（Populus tomentosa Carr.）,which is an important economic tree in China,is the main source of papermaking and construction.Secondary xylem of woody plants（including Populus tomentosa）is the biological basis of wood production.Studies have shown that the formation of vascular tissues depends on a series of continuous activities of cambial cells,and is regulated and influenced by many endogenous factors and external environmental factors.Although the research on the regulation of wood development by endogenous hormone signals and transcription factors has made great progress in recent years,it is still unclear whether energy state signals affect the development of secondary xylem.As the main site of aerobic respiration of eukaryotic cells,mitochondria significantly affect energy metabolism and thus participate in the regulation of cell behaviors,such as proliferation,differentiation and apoptosis.Recent studies have shown that mitochondrial signal transduction may play an important role in plant growth and development,response to external stimuli and adaptability to environmental changes.Mitochondrial dysfunction can lead to changes in auxin signaling pathway.However,the specific mechanism of mitochondria regulate plant development through auxin remains unclear.MicroRNA（miRNA）is an endogenous small single-stranded non-coding RNA with 16-29 nucleotides（nt）in length,and they are involved in post-transcriptional regulation in plants and animals.A large number of studies have shown that miRNA plays important roles in the processes of plant growth and development.A poplar specific microRNA PtoMIR476a was screened in the preliminary study of our team.Through the genetically modified（GM）technology,the overexpression transgenic plants of PtoMIR476a and its downstream target gene PtoRFL26 were obtained in Populus tomentosa.We observed that overexpression of PtoMIR476a resulted in a significant increase in the number of stem cambium cell layers,and a significant decrease in the proportion of xylem,indicating that the microRNA may participate in the regulation of microtubule secondary growth and development.In this dissertation,the biological function of PtomiR476a in regulating the secondary vascular tissue of stems has been studied.The specific results are as follows:1.PtomiR476a was found to be constitutively expressed in all tissues of Populus tomentosa by qRT-PCR and GUS staining,but it had the relative higher expression levels in stem.Compared with wild-type plants,overexpression of PtoMIR476a resulted in an increase in the number of cambial cell layers,a decrease in the number of xylem cell layers,an increase in the areas of wood elements and vascular cells,and an increase in the density of vascular cells.The results suggested that overexpression of PtoMIR476a may inhibit the differentiation of cambium into xylem,and promote the expansion of wood elements and vascular cells;2.Bioinformatics analysis and degradome sequencing assay showed that the members of 11 PENTATRICOPEPTIDE REPEAT（PPR）-Restorer of Fertility Like（RFL）protein families were potential target genes of PtomiR476a.QRT-PCR analysis showed that PtoRFL26 was the most significantly down-regulated target gene among the 11 PPR members in the transgenic plants of overexpression Restorer of Fertility Like.Therefore,PtoRFL26 was selected for for further study;3.The expression pattern analysis showed that PtoRFL26 was mainly expressed in stems,and further confirmed that PtoRFL26 was highly expressed in cambium by online website tool AspWood（http://aspwood.popgenie.org）and in situ hybridization analysis.Compared with the wild type poplar,overexpression of PtoRFL26 reduced the number of cambial cell layers,and increased the number of xylem cell layers.Meanwhile,the number of xylem fiber cells and vessel cells was also decreased significantly.It is suggested that overexpression of PtoRFL26 may promote the differentiation of cambium into xylem and inhibit the expansion of wood elements and vessel cells;4.Semi quantitative analysis showed that overexpression of PtoMIR476a and PtoRFL26 affected the expression of many mitochondrial-related genes.Furthermore,the content of NADH/NAD⁺,ATP and H₂O₂ in transgenic Populus tomentosa was detected and measured.It was found that the content of NADH/NAD⁺and ATP in transgenic Populus tomentosa of overexpression of PtoMIR476a was significantly higher than that in wild type,while the content of H₂O₂ was significantly lower.Correspondingly,the transgenic Populus tomentosa plants overexpressing PtoRFL26showed the opposite trend.The above results showed that overexpression of PtoMIR476a promoted the metabolism of mitochondria,while overexpression of PtoRFL26 inhibited or destroyed the normal metabolism of mitochondria;5.In order to analyze the mechanism of PtomiR476a/PtoRFL26 influencing the development of secondary vascular tissues,transcriptome sequencing of wild-type and overexpression of PtoMIR476a transgenic plants was carried out.The results showed that transcription factors WOX4/5/11,AIL1 and CYCD3.1 which related to cambium development were significantly up-regulated in the transgenic plants of overexpression of PtoMIR476a.Meanwhile,qRT-PCR analysis further validated the results of transcriptome sequencing;6.Previous studies have shown that mitochondrial dysfunction can affect auxin signaling pathway.The expression levels of auxin-related genes were analyzed by qRT-PCR analysis in wild-type and transgenic plants.The results showed that auxin synthesis and transport related genes were up-regulated in over-expressed PtoMIR476a transgenic plants,while the expression of these genes was significantly down-regulated in over-expressed PtoRFL26 transgenic plants.At the same time,the expression of auxin transporter-related genes was significantly down-regulated and the auxin content was reduced by 15.3%after treatment of wild-type plants with the mitochondrial inhibitor rotenone.Furthermore,DR5-YFP reporter gene was transferred into poplar,and fluorescence detection showed that auxin signal was accumulated in cambium and adjacent areas,and auxin signals in cambium wold disappear after rotenone treatment.These results indicate that the disorder of mitochondrial function may affect the synthesis and transport of auxin,and subsequently affect the development of secondary vascular tissue in poplar stems.In summary,the results of this dissertation preliminarily confirmed that PtomiR476a can target PtoRFL26,which can disrupt the auxin signal pathway by regulating part of the coding gene of complex I in mitochondria,and further affect the secondary growth of stem vascular tissues in Populus tomentosa.From the above experiments,we can conclude that mitochondria play a regulatory role in the wood formation of Populus tomentosa.These results provide new ideas for the study of wood formation,and may provide a new way for the improvement of forest species.