Functional Characterization Of MiR828a Involved In The Negative Regulation Of Lignin Biosynthesis In Poplar

Poplar is one of the widely cultivated woody plants throughout the world and the most dominant woody trees species used for timber,other commercial purposes,ecological and environmental protection in China.It is also used as a model organism for genetic studies and molecular mechanism of wood plants.Populus is a fast growing woody tree with excellent wood quality.The main components of wood include lignin,cellulose,and hemicellulose.Among them,lignin is the one of the most important components affecting wood properties.However,many mechanisms in the physiological process of poplar such as lignin biosynthesis and secondary growth formation are still needed to understand clearly.Therefore,it is significantly important to elucidate the molecular mechanism of lignin biosynthesis in poplar.MicroRNA?miRNAs?are endogenous small single-stranded non-coding RNAs with a length of about 20-22?nt?nucleotides,which interfere the expression of target genes by degrading their target mRNAs or by repressing their translation.Numerous studies have shown that miRNAs play a critical role in plant growth and development.Moreover,miRNA regulates the biosynthesis of polysaccharides and lignin,which are major components of cell wall.It has been verified that miR828 can regulate lignin biosynthesis in citrus during granulation process,lignin and H₂O₂ accumulation in wounding of sweet potato.Is miR828 involved the regulation of lignin biosynthesis in poplar wood development?It is hypothesized that miR828 might play an important role in wood formation of polar.Therefore,the biological function of miR828 in poplar was identified in this study.The main results are as follows:?1?Based on the bioinformatics analysis,there are two family members in poplar miR828;miR828a,andmiR828b.The precursor sequences and mature sequences of miR828a and miR828b are the same nucleotides.Consequently,miR828a was prioritized to investigate in this study.miR828a consists of 156 nt and binds to the non-coding region on chromosome number CM000348.2 of poplar.The secondary structure of miR828a can be folded into a typical stem-loop structure analyzed by the RNA fold web server.It has a minimum free energy of-57.3 kcal/mol and the mature sequences located on the 5?end arm of its secondary structure.?2?Tissue expression analysis via quantitative real-time PCR?qRT-PCR?showed that the expression of miR828a was specifically expressed in root and stem base xylem and phloem,suggesting that it might be involved in the regulation of growth and development in poplar.To analyze the expression pattern of it,we cloned about a 1576bp promoter fragment of the upstream of the miR828a precursor sequences of P.tomentosa and fused with a GUS reporter gene.Agrobacterium tumefaciens was used to transform the poplar plants and transgenic plants were analyzed by GUS staining method,the results showed that miR828a expressed in all vascular tissues of the root,stem,and petiole.These results showed that miR828a might be involved in secondary wall biosynthesis in poplar.?3?To experimentally verify the biological function of miR828a,overexpression and knockdown of miR828a were constructed into plant expression vector and transformed into poplar.In the transgenic plants,plant height and stem diameter dramatically decreased when miR828a was overexpressed,while the opposite features of phenotypes were found when miR828a was knockdown.Furthermore,we also observed the cell wall components of stem tissues were measured and the result showed that the overexpression of miR828a had decreased lignin content,whereas the knockdown of miR828a had increased compared with wild type.But no significant effect on xylem level was found in both miR828a-overexpressed and miR828a-knockdown transgenic plants,however,higher glucan level was found in these transgenic plants.These results indicated that miR828a negatively regulates the lignin biosynthesis in poplar.?4?To determine the direct target genes of miR828a,the mature sequence of miR828a was compared with the poplar genome sequences at the whole genome level.As a result,nineteen candidate target sites of miR828a were recognized in the poplar genome might be recognized by miR828a.Seven of them are encoded in MYB genes and the rest of twelve are encoded in other genes,among which three target genes MYB011,MYB129,and MYB171 possess a higher scores.Moreover,tissue expression analysis showed that the expression of MYB011 and MYB129 presented different expression patterns with miR828a,especially the expression of MYB011 are higher than MYB129.Therefore,it is assumed that MYB011 may be the key target gene of miR828 in poplar.?5?To validate that MYB011 is a target gene of miR828a,the expression of MYB011 was measured in both miR828a-overexpressed and miR828a-knockdown transgenic plants by using qRT-PCR.The down-regulation of MYB011 was found in miR828a-overexpressed transgenic plants and the up-regulation of MYB011 was found in miR828a-knockdown transgenic plants.Besides,the degradome reads confirmed that cleavage of MYB011 mRNA by miR828a binding site.Moreover,to examine the function of MYB011,we constructed the MYB011 into a plant expression vector driven by CaMV 35S promoter.The MYB011-overexpressed transgenic plants showed that the increased plant height and the secondary wall thickness in xylem,and possessed lateral shoot.In addition,increased lignification was observed in MYB011-transgenic plants.Collectively,in this paper,a poplar miRNA gene miR828a was cloned by using transgenic technology and biochemical methods.It was proved that miR828a negatively regulated the expression of target gene MYB011,and subsequently decreased plant height,stem diameter,secondary thickness in xylem and lignin content.This study can provide a new strategy for molecular breeding to improve the wood properties and wood quality of poplar in the future.