Study On The Lateral Root Development Regulated By MiR319a In Poplar

Root is a fundamentally important composition of plant organs.Root system is essential for plant growth and survival due to its role in water and nutrient acquisition and anchorage by plants.Root architecture is mainly composed of primary root,lateral root and adventitious root.The primary roots grow from the embryo which is thicker and play a key role in the early development of the root system.The lateral roots grow from the existing root tissue and can develop into a highly organized,spatially separated and intertwined complex root system.Adventitious roots are roots that grow from non-root tissues,which show irregular shapes and play an important role in adapting to the environment and regeneration of plants.In dicotyledons,the primary root and lateral roots determine the structure of the root system,especially the lateral roots,which play a decisive role in the growth and development of the plant in the middle and late stages.Poplar is a widely planted economic forest in the world.It is an important raw material for papermaking,wood furniture,etc.It can also function in the development of emerging green energy as a renewable bioenergy.Therefore,it is of great significance to study the development of poplar roots.The development of plant roots is regulated by a variety of internal and external factors,including environmental factors,endogenous hormones,and small RNA(miRNA).MicroRNAs(miRNAs)are a class of approximately 21-nucleotide(nt)small non-coding regulatory RNAs that regulate endogenous gene expression by targeting mRNAs for cleavage or translational repression in eukaryotes.MiRNAs are widely distributed in the plant genome and are involved in physiological processes such as morphogenesis,growth and development,hormone perception and signal transduction,and response to external abiotic stress in plant organs.MiR319 is a highly conserved miRNA in plants.It has been reported that miR319 is involved in the regulation of leaf,shoot and flower organs development.However,it remains largely unknown that whether miR319 a affect roots growth process and the molecular mechanism about root regulation.Here we use Populus tomentosa as research material to investigate the role of miR319 a on lateral root development.The detail findings are as follows: 1.miR319 a participates in root development and affects lateral root formation in Populus tomentosa.We generated transgenic plants which overexpressed GUS protein fusion with miR319 a promoter.GUS staining analysis showed that miR319 a is expressed in all organs,including lateral roots.Phenotype observation showed that overexpression of miR319 a in poplar leaded to fewer lateral roots whereas reducing the expression of miR319 a level produced more lateral roots.These results indicated that miR319 a might play negative role in lateral root development.2.The expressions of TCPs(targets of miR319a)are correlated with lateral root formation in Populus tomentosa and TCP19 functions positively in lateral root formation.It has been reported that TEOSINTE BRANCHED 1,CYCLOIDEA,PCF transcription factors(TCPs)are the targets genes of miR319.According to previous studies,there are six TCPs which are targeted by miR319 a in Populus tomentosa.QRT-PCR,degradome sequencing and qRT-PCR assay suggested that all these TCPs were regulated by miR319 a in roots of WT and transgenic poplar.Especially,compared with other TCPs,TCP19 was regulated significantly and had the highest expression levels in roots.Then,we generated TCP19 overexpressing plants and the results showed that TCP19 positively regulated number and density of lateral roots.3.Analysis of transcription activation activity and tissue expression of TCP19.TCP19 was fused with the yeast expression vector pGBKT7,and transferred into yeast cells to observe the transcription activation activity by color reaction,and the results showed that TCP19 had a transcription inhibitory activity.The relative expression level of TCP19 in various tissues of wild-type plants revealed that TCP19 had relatively high expression levels in roots and leaves and low expression in stems.4.miR319 a regulates lateral root formation via influencing auxin signaling.The previous researches demonstrated that auxin was involved in all stages of lateral root development.DR5(the auxin response reporter gene)was introduced to observe the distribution of auxin in the lateral roots of wild and transgenic poplar.From the results,we could find that overexpression of miR319 a repressed the accumulation of auxin,while the knockdown of miR319 a promoted its accumulation.5.The genes associated with auxin metabolism and signaling are regulated by miR319 a.According to the above results,it's concluded that miR319a/TCP19 module was involved in the regulation of lateral root growth via auxin signal pathway.To further verify the results,qRT-PCR analysis was used to test the expression of genes which participate in auxin synthesis,transport,and response.We found that the expression level of auxin transport genes(PIN1,PIN2,PIN5 a,PIN5b)and auxin response genes(IAA3.1,IAA3.2)had significantly changes in transgenic poplar compared with wild type.6.IAA3.2 negatively regulate the lateral root development.Although substantial studies have revealed that the overexpression of IAA3 inhibits plant roots growth,the function of IAA3.2 in poplar is to be revealed.Through genetic transformation we obtained IAA3.2 over-expressing transgenic plants,which produced decreased lateral roots number and density compared to wide type.This further confirms that the regulation of TCP19 to IAA3.2 could control lateral root development.7.TCP19 can directly bind to the promoter of IAA3.2 in vivo.To explore the mechanism of miR319a/TCP19 affecting on lateral root development,promoter analysis was used to analyze the promoter sequences of auxin transport and response genes.With the help of PlantCare(website tool),we found that there were two TCPs binding sites in the promoter region of IAA3.2.Effector-reporter and ChIP-qPCR assay demonstrated TCP19 had the ability to directly bind to the promoter of IAA3.2 in vivo.Taken together,we can make a conclusion that miR319 a plays negative roles in lateral root development via targeting TCPs in post-transcriptional level.The regulation of miR319a/TCP19 module is achieved by auxin-mediated signaling pathway and TCP19 can directly binds to the promoter of IAA3.2.All these results demonstrated that miR319 a plays an important role in lateral root and may provide a new method for us to improve poplar quality.